Enhanced oxidative phosphorylation in Retinoblastoma tumors is dependent on depleted Hexokinase1 and lack of AMPKα activation

Enhanced oxidative phosphorylation in Retinoblastoma tumors is dependent on depleted Hexokinase1 and lack of AMPKα activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced oxidative phosphorylation in Retinoblastoma tumors is dependent on depleted Hexokinase1 and lack of AMPKα activation Vishnu Suresh Babu, Ashwin Mallipatna, Deepak S.A, Gagan Dudeja, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1779138/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lack of retinoblastoma protein (Rb) causes aggressive intraocular retinal tumors in children. Recently, Rb tumors have been shown to have a distinctly altered metabolic phenotype, such as reduced expression of glycolytic pathway proteins alongside altered pyruvate and fatty acid levels. In this study, we demonstrate that loss of Hexokinase 1(HK1) in tumor cells rewires their metabolism allowing enhanced oxidative phosphorylation-dependent energy production. We show that rescuing HK1 or RB1 in these retinoblastoma cells reduced cancer hallmarks such as proliferation, invasion, spheroid formation and increased their sensitivity to chemotherapy drugs. Induction of HK1 was accompanied by a metabolic shift of the cells to glycolysis and a reduction in mitochondrial mass. Cytoplasmic HK1 bound Liver Kinase B1 (LKB1) and phosphorylated AMP-activated kinase-α (AMPKα Thr172 ), thereby reducing mitochondria-dependent energy production. We validated these findings in tumor samples from Rb patients compared to age-matched healthy retina. HK1 or RB1 expression in Rb-/- cells led to reduction in their respiratory capacity and glycolytic proton flux. HK1 overexpression reduced tumor burden in an intraocular tumor xenograft model. AMPKα activation by AICAR also enhanced the tumoricidal effects of chemotherapeutic drug topotecan in vivo . Therefore, enhancing HK1 or AMPKα activity can reprogram cancer metabolism and sensitize retinoblastoma tumors to lower doses of existing treatments, a potential therapeutic modality for retinoblastoma. Retinoblastoma Hexokinase-1 AMPKα glycolysis oxidative phosphorylation cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction: Retinoblastoma (Rb) is the most common intraocular malignancy of childhood, caused by the inactivation of the RB1 gene ( 1 ). Inactivation or deletion of the functional RB1 gene deregulates proliferative control and facilitates Rb tumorigenesis ( 2 ), but it has diverse roles beyond cell cycle control that are still being unraveled. The RB1 gene encodes for the retinoblastoma protein (pRb) that serves as a prototype of transcriptional repression and cell cycle regulation by preferential binding on E2F genes ( 3 ) ( 4 ). Canonically, pRb protein represses E2F transcription factors directly by binding to its promoter and transactivation domain, causing a reciprocal induction and inhibition of cell cycle-specific transcription ( 5 ). Thus, cancers with significant loss of functional pRb proteins unleash the E2F transcriptional factors to enhance uncontrolled cell proliferation and provide a subsequent advantage to tumor progression ( 6 ) ( 7 ). In addition, non-canonical functions of pRb proteins involve mechanistic regulation of histone modifications, chromosome integrity and cellular metabolism ( 8 , 9 ) and loss of functional pRb proteins has been attributed to the alteration of these pathways in cancers ( 10 – 12 ). Despite exploring the loss of pRb protein consequences in other cancers, significant gaps exist in discerning the canonical and non-canonical role of pRb loss in Rb tumor progression. Reprogramming of metabolic circuits in tumors favours their growth, with the changes in individual metabolic pathways frequently correlating with enhanced glycolysis and nucleotide biosynthesis( 13 ). RB1 is associated with altered metabolism, an important emerging cancer hallmark( 14 ). However, the metabolic reprogramming due to loss of RB1 is likely dependent on the photoreceptor precursor cell of origin( 15 – 18 ). The photoreceptors continuously require high amounts of energy for the visual cycle operation, that is met primarily by glycolysis( 19 , 20 ). Photoreceptor mitochondria adapt to different energy requirements and mitochondrial turnover is essential for photoreceptor function and health( 21 ). The interaction between essential cellular signaling pathways( 9 ) altered due to Rb1 loss and changes in metabolism that provide energy and growth cues for these retinal tumors are not fully understood. Recently, we have identified a number of cell cycle and metabolic genes in Rb tumors and in Rb null cellular models ( 22 ). In particular, we identified Hexokinase 1 (HK1) to be significantly reduced while the E2F family member E2F2 was significantly upregulated. HK1 is a key regulator of glycolytic flux and catalyses the first step in the conversion of glucose to pyruvate, an essential process. Therefore, we investigated various critical effectors of metabolic pathways to dissect how RB1 controls retinal tumor metabolism. Our results reveal that loss of Rb and consequently Hexokinase 1 (HK1), reprograms tumor metabolic pathways to enhance ATP generation using mitochondrial oxidative phosphorylation (OXPHOS) instead of glycolysis. We further exploit our observations for therapeutic purposes and demonstrate the utility of targeting metabolism for Rb tumor therapy. Results: RB1 complementation induces HK1 and reduces E2F2 levels to regulate key cancer hallmarks. In our previous study, we reported low expression of HK1 and high expression of E2F2 in Rb tumors ( 22 ). We found that ectopic expression of RB1 led to the induction of HK1 protein and transcript in Rb null WERI-Rb1 cells. We further validated our findings, showing increased HK1 and diminished E2F2 protein in Y79 and GL1-Rb1 (patient derived primary Rb-/- cells), under RB1 complementation (Figure S1A, B respectively). Similarly, HK1 transcript was increased in Y79 (Figure S1C), and GL1-Rb1 cells (Figure S1D), while reducing E2F2 transcript levels in these cells (Figure S1C, D). Next, we studied how HK1 and E2F2 ablation by shRNA expression or their overexpression affects cancer hallmarks. RB1- dependent mechanisms are often linked to cell cycle anomalies ( 5 ) rather than metabolic alterations in ocular tumors. Therefore, we tested if key cancer hallmarks including cell proliferation, invasion, and migration, were affected by ectopic modulation of HK1 , in comparison to E2F2 and RB1 . Overexpression of HK1 phenocopied RB1 complementation in both WERI-Rb1 and GL1-Rb1 cells by reducing proliferation (Fig. 1 A, C) while HK1 knockdown exhibited an increase in proliferation (Fig. 1 A, C) compared to controls. However, E2F2 overexpression increased cell proliferation while E2F2 knockdown alone and in combination with RB1 complementation, reduced cell proliferation at 96hour (Fig. 1 B). RB1 overexpression significantly reduced invasion in transwell assays compared to controls (Fig. 1 D, E, F). HK1 overexpression phenocopied RB1 individually and in combination, reducing invasion whereas HK1 knockdown increased invasion (Fig. 1 G, H, I). E2F2 overexpression increased invasion and migration while E2F2 knockdown exhibited a reduction in invasive phenotype (Fig. 1 J, K, L). To assess whether HK1 and E2F2 are involved in 3-D spheroid formation and chemosensitivity of WERI-Rb1 cells, we transfected RB1 , HK1 and E2F2 overexpression and their knockdown constructs in WERI-Rb1 cells. Tumor spheroids from RB1 overexpressing cells formed small irregular clusters compared to large, tight spheroids in control cells (Fig. 1 M, N). HK1 overexpressing cells formed small irregular clusters while HK1 knockdown formed tight spheroids (Fig. 1 O, P, D; P < 0.0001). E2F2 overexpressed cells developed tight large spheroids, while E2F2 knockdown alone and in combination with RB1 overexpression exhibited a reduction in spheroid size (Fig. 1 Q, R). Ectopic expression of RB1 , HK1 and their combination effectively sensitized the cells to lower doses of chemotherapeutic drug topotecan compared to the E2F2 overexpression (Fig. 1 S). Collectively, our data identified an increase in tumor hallmarks upon E2F2 overexpression in Rb null state and reduction in tumor hallmarks upon HK1 and RB1 complementation in WERI-Rb1 cells (Fig. 1 T, U). Hexokinase1 induction inhibits cancer growth in-vivo We then tested how E2F2 and HK1 modulation in retinoblastoma cells affected their tumorigenic potential in a rabbit intraocular xenograft tumor model. Orthotopic sub-retinal transplantation of E2F2 overexpressing (n = 4) and control (n = 4) WERI-Rb1 cells (1.5x10 6 cells per injection) in rabbit eyes caused retinal tumors measurable at 6 and 8 weeks, while HK1 overexpressing cells (n = 4) did not form tumors in rabbit retina as shown by optical coherence tomography (Fig. 2 A, B). E2F2 tumors also had a larger area compared to control tumors ( P = 0.0106;) which is reflected in the histological analysis of the retinal sections (Fig. 2 C). RB1 and HK1 drive the tumor energy-sensing circuitry independent of canonical RB1-E2F2 cell cycle regulation Since HK1 represents a critical node of the cellular metabolic network, while E2F2 is a less studied member of the E2F family, we investigated the intracellular status of metabolic signaling proteins upon modulation of RB1, E2F2 and HK1. E2F2 overexpression and knockdown did not induce HK1 proteins and similarly, HK1 modulation did not affect E2F2 protein levels (Fig. 3 A), indicating that they are independently regulated by Rb. Ectopic expression of RB1 and HK1 phosphorylated AMPKα, while E2F2 overexpression did not alter AMPKα phosphorylation (Fig. 3 A), indicating that the HK1 and E2F2 regulated circuit modules are exclusive. Complementing RB1 and HK1 induced phosphorylation and deactivation of ACC (Fig. 3 A). LKB1 and HIF1α were increased in RB1 and HK1 overexpressing cells. However, RB1 or HK1 expression did not alter total ACC and total AMPKα levels (Fig. 3 A). Elevated expression of HK1 along with LKB1 likely led to the activation of AMPKα while HK1 knockdown did not activate AMPKα or increase LKB1 even in the presence of RB1 . Thus, Rb induces HK1 which leads to LKB1 elevation subsequently activating AMPKα and regulating cellular metabolism. Similar results were also obtained in Y79 cells transduced with RB1 , E2F2 and HK1 vectors (Figure S2 A). Throughout the experiments, E2F1 levels are enhanced depending on absence of Rb, which serves as an internal control for the experiments. However, E2F2 modulation alone, in absence of RB1 , did not affect HK1 gene expression, further confirming an alternate signal cascade controlled by Rb and HK1 (Fig. 3 B). We found significant high expression of HIF1α in RB1 complemented cells, while HK1 complemented cells did not show any significant effect on HIF1α gene expression (Fig. 3 C), contrasting to its protein levels (Fig. 3 A). However, Rb and HIF1α are known to interact whereas in other cellular contexts. HK1 and HIF1α are known to associate ( 23 , 24 ). Our cell cycle analysis shows cell populations increased in the G1/S phase with a low distribution of cells in the G2/M phase in RB1 overexpressing cells, indicating a reduction in cell division (Figure S2B). Unlike RB1 complementation, HK1 over expression did not show any cell cycle arrest at the G1/S phase with adequate distribution of cells in the G2/M phase, although total numbers of cells reduce across phases. However, higher proportions of cells were observed at the S phase and G2/M phase upon E2F2 overexpression, indicative of rapid cell division. Immunohistochemial analysis revealed low HK1 and phospho-AMPKα signals in Rb tissues ( n = 25) compared to pediatric retina (n = 2) (Figure D,E), which validates our in vitro observation in patient tissues. Thus, HK1 expression even in absence of RB1 can actuate a halt in cancer hallmarks and alter metabolic circuits in Rb cells but in a manner distinct from canonical cell functions governed by RB1 and E2F2 (Figure S2B). HK1 induction rewires cellular metabolism Since HK1 catalyzes the initial rate limiting step of glycolysis ( 25 ) and the allosteric regulation of other glycolytic genes, including the PKM2 mediated metabolic switch from glycolysis to OXPHOS ( 26 ), we investigated additional glycolysis and glucose uptake related genes. RB1 increased HK2 & GLUT1 levels (Fig. 4 A) while PKM2 levels were reduced and PFKP remained unaffected (Fig. 4 B). Strikingly, the levels of HK2, GLUT1, PKM2 and PFKP were not altered by HK1 overexpression, indicating that the activation of AMPKα and inhibition of ACC is specifically controlled by HK1. Also, E2F2 modulation alone did not change levels of any of these proteins. In addition, gene expression profiling of RB1 complemented cells shows significantly increased expression of glycolytic genes HK2 (Hexokinase 2) and G6PC (Glucose 6 phosphate catalytic subunit 1), whereas HK1 complemented cells shows elevated expression of G6PC gene, while HK2 gene expression remain unaltered (Fig. 4 C). Collectively, our findings reveal a novel energy sensing circuit in Rb tumors, that can be altered by HK1 complementation (Fig. 4 D). HK1 binds to LKB1, but not AMPK α To elucidate the molecular interactions underlying the metabolic alterations induced by RB1 and HK1 complementation, we performed co-immunoprecipitation (co-IP) assays. We found no direct interaction between Rb & HK1 or Rb & AMPKα, whereas we observed an interaction between Rb and HIF1α (Fig. 5 A, S3A). Therefore, we hypothesize that the induction of HK1 is likely due to RB1 mediated transcriptional activation, but the effect on AMPKα is mediated through HK1. HK1 co-IP did not show an interaction between HK1 and Rb, although HK1 was bound to both HIF1α and LKB1 (Fig. 5 B, S3B). HK1 interaction with VDAC1 served as the positive control ( 27 ) and HK1 did not bind to E2F1 or E2F2. LKB1 interaction with HK1 was confirmed using anti-LKB1 co-IP (Fig. 5 D, S3D), but LKB1 did not bind to HIF1α. We confirmed the interaction of Rb-HIF1α, HIF1α-HK1 by performing a reverse co-IP using anti-HIF1α (Fig. 5 C, S3C), but the fractions of HIF1α bound to HK1 and Rb appear to be distinct. Interaction of Rb and HK1 with binding partners at distinct sub-cellular locations Altered distribution of HK1 and Rb in subcellular compartments can disrupt cell signaling and affect metabolism ( 28 , 29 ). Therefore, we assessed the binding partners of Rb and HK1 in subcellular fractions of control and RB1 complemented WERI-Rb1 cells. Co-IP from subcellular fractions revealed that HIF1α bound Rb in the nuclear fraction (Fig. 6 A, S3E), while it bound HK1 in the cytoplasmic fraction (Fig. 6 B). The cytoplasmic HK1 expression was higher under RB1 overexpression compared to controls and it interacted with LKB1 exclusively in the cytoplasm (Fig. 6 B, S3F). LKB1 is an upstream kinase that phosphorylates AMPKα ( 30 ) which, in turn phosphorylates and inhibits ACC ( 31 ). In the mitochondrial fraction, HK1 bound VDAC1, while none of the other proteins tested were found to interact (Fig. 6 C, S3G). A schematic shows the interaction of targets identified in subcellular fractions (Fig. 6 D). Using promoter binding analysis, we found that the HK1 promoter has binding sites for E2F1 , E2F2 and HIF1α (Figure S3H), it is possible that the transcriptional activation of HK1 is mediated via direct promoter binding by RB1 and HIF1α or relieving E2F mediated repression. While RB1 is known to directly induce HIF1α ( 24 ), E2F1 and E2F2 also have binding sites in the HIF1α promoter (Figure S3G), making a case for derepression as a possible mechanism HK1 and Rb expression alters mitochondrial respiration Growing evidence suggests that perturbing HK1-VDAC1 interactions in the mitochondria affects mitochondrial membrane potential and glutamine transport( 32 , 33 ). We investigated the status of active mitochondria in cells using the Mito-tracker dye. We observed lesser mitochondrial density in RB1 and HK1 complemented cells compared to controls ( P < 0.001), while HK1 knockdown showed higher mitochondrial density. HK1 overexpression led to a reduction in active mitochondria (Fig. 7 A, B). This could be a direct result of HK1 induced activated AMPKα driven mitophagy ( 34 ), thereby triggering a metabolic checkpoint and energy restriction. Since cellular energy production through OXPHOS is dependent on mitochondrial status ( 35 ), the lack of RB1 and HK1 increases mitochondrial activity in retinoblastoma tumors. To validate our findings, we used Seahorse XFp metabolic flux assays that revealed low basal and induced mitochondrial respiration upon ectopic expression of RB1 and HK1 either alone or in combination (Fig. 7 C). Notably, the mitochondrial respiration was high in HK1 knockdown cells and was suppressed by RB1 . Spare respiratory capacity was reduced significantly in RB1 and HK1 expressing cells (Fig. 7 D; P = 0.003) indicating curtailed OXPHOS dependence. Consequently, both basal and compensatory glycolytic proton efflux rates (glycoPER) in cells expressing RB1 and HK1 were significantly elevated, indicating their metabolic shift towards glycolysis. However, the glycolytic capacity of HK1 ablated cells was significantly lower compared to control (Fig. 7 E, F), indicating that tumor cells have low dependence on glycolysis. AMPKα activation halts retinal tumor growth. We tested the possibility of targeting the altered metabolic phenotype of retinoblastoma tumors by modulating AMPKα using activators AICAR (adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside) and Metformin while using the inhibitor dorsomorphin as a control. Treatment with AICAR significantly activated AMPKα levels in WERI-Rb1 cells (Figure S4A) and reduced cell proliferation (Figure S4B), invasion (Figure S4C) and migration (Figure S4D), while dorsomorphin treatment enhanced cell proliferation, invasion and migration (Figure S4A-C). AICAR treatment effectively shrunk 7-day-spheroids after 48hours (Fig. 8 A) indicated by the reduction in spheroid area (Fig. 8 B). Dorsomorphin treatment did not exhibit any effects on the 7-day old spheroids. Metformin, an alternative AMPKα activator, also reduced the size of tumor spheroids by 40% percent. However, AICAR was more effective than metformin (Figure S4E, F) possibly due to their mode of exerting AMPKα dependent effects. In clinical treatment regimens, chemotherapeutic drugs are typically used in combinations to reduce the toxic bystander effects from each drug type as well as prevent tumors from developing resistance to monotherapy. Therefore, a metabolic blockade specific to tumor cells may prove to be an effective method of enhancing the lethality of currently used chemotherapeutic drugs at a lower dosage. We observed an additive effect of AICAR with topotecan (FICi = 0.75; Fig. 8 C) in inducing tumor cell death by a checkerboard drug interaction assay. Dorsomorphin exerted an antagonistic effect when combined with Topotecan in a similar assay (FICi = 1.25; Figure S4G). Treatment of AICAR IC 50 was effective in reducing basal mitochondrial respiration (Figure S4, I, J), maximal respiration (Figure S4J) and spare respiration (Figure S4K) in Rb null cells. These observations were comparable to the OCR measurements obtained with HK1 overexpression (Fig. 7 C, D). Next, we allowed intraocular tumors to develop in rabbit eyes for 6 weeks followed by treatment with topotecan IC 100 , IC 50 monotherapy and combined treatment with topotecan IC 50 and AICAR IC 50 for two weeks. The status of tumors was analyzed by OCT imaging (Fig. 8 D, E, F, G). The combination therapy showed effective reduction of tumor area compared to control tumors, indicating the additive nature of AICAR with low dose topotecan (Fig. 8 G, H; P = 0.0001). Sham treated; tumor-containing eyes show large tumors with distorted retinal architecture in histological analysis post enucleation (Fig. 8 I). High dose topotecan (Topotecan IC 100 ) effectively reduced tumor size while damaging remnant retinal layers (Fig. 8 I). Topotecan IC 50 dose was less effective in reducing tumor size. However, the combination of topotecan IC 50 and AICAR IC 50 was most effective in reducing tumor size while maintaining the retinal structure (Fig. 8 I), indicating reduced bystander retinal toxicity. Discussion: Retinoblastoma patient tumor samples and cellular models have reduced expression of glycolytic genes such as HK1 and metabolites such as pyruvate( 22 ). HK1 catalyses the first step in glycolysis, thereby controlling glycolytic flux( 36 ). HK1 has been found to be frequently overexpressed in tumors( 37 ) and associated with oncogenes like KRAS( 38 ) mutants, providing for the tumor cells’ high energy demand. However, HK1 levels are significantly reduced in retinoblastoma tumors and ectopically expressing RB1 in RB1 -null cells transcriptionally induced HK1 expression and altered the cellular metabolism. While RB1 regulated both HK1 and the E2F family member E2F2 , we consistently observed that the metabolic rewiring of cells in the presence of RB1 was dependent only on HK1 , but not E2F2 , providing evidence of unique, modular, signalling networks regulated by RB1 . This also highlights the potential for developing treatment modalities targeting multiple mechanisms that can potentially be more effective. Complementing RB1 or HK1 led to activation of AMPKα , which in turn phosphorylated and inactivated the critical regulator of fatty acid metabolism, ACC ( 39 – 41 ). This shifted the metabolic profile of cells back to a glycolysis dependence, consequently causing a reduction in mitochondrial respiration and mass. Notably, cytoplasmic LKB1 levels depended on exclusively on HK1 induction and HK1 physically bound with both LKB1 and HIF1α in the cell cytoplasm. However, the HK1 pools bound to LKB1 , HIF1α and mitochondrial VDAC were distinct. Therefore, HK1 bound LKB1 could be a key signalling complex for energy sensing, leading to AMPKα activation. Photoreceptors rely on glycolysis for rapid energy production( 42 ), while whole retina depends on both glycolysis and mitochondrial OXPHOS for its functional and structural requirements to provide vision( 43 ). Interestingly, FDG PET/CT plays minimal role in routine diagnostic evaluation of intraocular tumors( 44 ), possibly due to the lack of HK1 causing insufficient labelled glucose analogue uptake( 45 ), highlighting the importance of HK1 in the photoreceptors. The retinoblastoma tumor cells arise from photoreceptor precursor cells ( 46 ) and lose their photoreceptor-related functions and gene expression profile( 22 ). Photoreceptors rely on aerobic glycolysis for rapid energy production ( 20 ), consuming almost 80–96% of the glucose to produce lactate ( 43 ), although these cells also employ mitochondrial OXPHOS for functional and structural requirements during the visual cycle. Lipids are essential to replace the outer segments of rods and cones ( 47 ), which possibly explains why catabolic processes of fatty acids are restricted in these cells. Therefore, since Rb tumors do not need to produce outer segment disks, the lack of HK1 leads to an active ACC, which can catalyze malonyl CoA generation, increasing biosynthesis of long-chain fatty acids to fuel β-oxidation. We found that a shift back to glycolysis, an ATP restrictive catabolic process, caused by both RB1 and HK1 , led to reduced cell proliferation, invasion, tumorigenesis and enhanced sensitivity to chemotherapeutic drugs. Though several chemotherapy strategies are successful in various cancer treatments( 48 ), controlling the bystander toxicity of chemotherapeutic drugs is still an unmet clinical requirement. Currently, retinoblastoma tumors are treated using different combination doses and durations of melphalan, topotecan, carboplatin, vincristine & etoposide( 49 ) which often cause bystander toxicity or reduction in efficacy with multiple cycles of therapy required( 50 , 51 ). Importantly, ectopic expression of HK1 in retinoblastoma cells prevented tumor formation in a rabbit intraocular xenograft model, whereas E2F2 expressing cells formed large tumors. Further, activation of AMPKα using an intraocular injection of AICAR (IC 50 ) in combination with topotecan (IC 50 ) reduced established intraocular tumors in 2 weeks with reduced retinal damage on histopathology compared to topotecan (IC 100 ). We noted that the direct AMPKα activator AICAR reduced tumorsphere formation more efficiently than Metformin. Since Metformin mediates its action on AMPKα via LKB1 ( 52 ), which is reduced in Rb null cells, it is less effective than AICAR, which directly activates AMPKα. Therefore, reprogramming the metabolism of retinoblastoma tumors towards glycolysis may be an effective strategy to sensitize tumors to lower doses of current therapies. This adjuvant strategy may be particularly useful in salvaging vision and in refractory cases. HK1/AMPKα axis also serves as a potential drug target for various advanced cancers lacking functional Rb proteins, which includes small cell lung cancer ( 53 ), prostate cancer ( 54 ), glioblastoma ( 55 ) and leukemia ( 56 ). We provide evidence for a new treatment modality for Rb depleted cancers, based on uncovering a transcriptional and cellular signaling network driven by HK1 and AMPKα in retinoblastoma cells highlighting their unique, context-dependent metabolic reprogramming capacity. Materials & Methods: 1.Clinical samples The study was conducted by the Declaration of Helsinki principles under a protocol approved by the institutional ethics committee of Narayana Nethralaya (EC Ref no: C/2013/03/02). Informed written consent was received from the parents before inclusion in the study. The GL1-Rb1 line was developed from an enucleated tumor specimen obtained from the right eye of a 3-year-old female retinoblastoma subject (AJCC staging-cT4b). The tumor tissue was enzymatically dissociated using dispase & trypsin and the cells were cultured in RPMI 1640 media supplemented with 10% FBS, 1% pen strep and a 10ng cocktail of EGF, VEGF and FGF. A biphasic population of primary Rb cells was observed during the first two weeks, comprising of retinoblastoma tumorspheres adherent to feeder fibroblasts and suspension clusters of single-cell retinoblastoma. Over four weeks, the tumorspheres detach from the fibroblast and form an unusual chain of suspension cells. These cells were cultured separately and cell population doubling time was calculated from the exponential growth phase curve. For immunohistochemistry validations, we have used additional Rb subjects (n = 25) of the age range 0.2-4 years and pediatric controls (n = 2) of the age range (0.2–0.3 years). Clinical and histopathology details are mentioned in Supplementary Table S1. 2. Cell lines: WERI-Rb1 and Y79 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The WERI-Rb1 and Y79 cells were cultured in RPMI 1640 medium (Gibco, Cat #11875093) supplemented with 10% FBS and 1% Pen Strep (Penicillin –Streptomycin) and maintained at 37ºC in a humidified atmosphere of 5% CO 2, with intermittent shaking in an upright T25 flask. 3. Gene expression analysis: Total RNA was isolated from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. 1µg of RNA was reverse transcribed using Bio-Rad iScript cDNA synthesis kit (cat# 1708890) and quantitative real-time PCR was performed using Kappa Sybr Fast qPCR kit (cat# KK4601) using Bio-Rad CFX96 system. Relative mRNA expression levels were quantified using the ΔΔC(t) method. Results were normalized to housekeeping human β-actin. Details of primers used are described in Table S2. 4. Histopathology & light microscopy Paraffin-embedded specimens of rabbit xenograft eyes (n = 4 eyes per group) were used. 4µm paraffin sections were dewaxed at 60°C, rehydrated in decreasing concentration of ethanol. Slides were stained with hematoxylin & eosin according to standard procedures. Brightfield images were captured using Axioplan 2; (Carl Zeiss, Oberkochen, Germany) 5. Lentiviral plasmids and vectors: We constructed a lentiviral plasmid expressing the RB1 gene in the pCL20 backbone. We purchased commercially available overexpression plasmids for E2F2 (cat#TOLH-1508827, Transomics Technologies Inc, USA) and HK1 (cat#TOLH-1505162, Technologies Inc, USA) in pLX304 lentiviral backbone having CMV promoter. The corresponding shRNA constructs for E2F2 and HK1 were in the pZIP lentiviral backbone containing CMV promoter (Transomics Technologies Inc, USA) and the target sequences are available in Table S3. Lentiviral transduction was used for RB1 , E2F2, HK1 overexpression and knock-down in cell lines using the previously described protocol ( 57 ). Lentivirus was produced in HEK 293T cells, and the media supernatant was concentrated by centrifugation. 1x10 6 WERI-Rb1 cells in free serum-free media were transduced with 50x concentrated lentiviral preparations of RB1 , E2F2, HK1 & their knock-down viruses in 6 well plates for 4 hours, with intermittent shaking at every 30 minutes. The specific gene expression efficiencies were determined using RT-PCR after 72 hrs. 6. Western blotting: For Western blot analysis, cells were lysed in RIPA buffer (20mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 0.08% Sodium Deoxycholate, 1% NP40 supplemented with 1 tablet of protease inhibitor (Complete ultra mini-tablet, Roche) and phosphatase inhibitor (PhosStop tablet, Roche). 20µg of total protein was loaded per lane and were separated by SDS-PAGE. The separated proteins on the gel were transferred onto PVDF membrane and were probed for specific antibodies against Rb (cat# 9390; Cell signaling) phospho-Rb (cat# 8516, Cell signaling) E2F2 (ab209662; Abcam), HK1(cat# 2024; Cell signaling), HK2 (cat#2867; Cell signaling), PKM2 (cat#4053; Cell signaling), LDHA (cat#3582; Cell signaling), HIF1α (cat#14179, Cell signaling), LKB1 (cat#3050; Cell signaling), AMPKα1 + α2 (ab80039; Abcam), phospho-AMPKα (cat#2535; Cell signaling), ACC (cat#3662; Cell signaling), phospho-ACC (cat#3661; Cell signaling), α-Tubulin (cat# 3873; Cell signaling) and GAPDH (cat#5174; Cell signaling) at 1:1000 dilution in 5%BSA in 1xTBST, overnight at 4°C. After 4 washes with 1x TBST for 10 minutes, membranes were incubated with HRP-conjugated anti-mouse (cat#7076; Cell signaling) or anti-rabbit antibodies (cat#7074; Cell signaling) at 1:2000 dilution for 2 h. Images were visualized using the Image Quant LAS 500 system (GE Healthcare Life Sciences, USA). 7. Co-immunoprecipitation: WERI-Rb1 cells (2x10 6 cells) were washed with ice-cold PBS and then lysed in a solution containing 10 mM Tris at pH 8, 170 mM NaCl, 0.5% NP40, and protease inhibitors for 30 min on ice. Cell lysates were removed by centrifugation and the supernatants were incubated with antibodies for Rb, HK1, LKB1, and HIF1α and IgG (as control) overnight at 4°C for their respective immunoprecipitations and with protein G–Sepharose for a further 2hr. Beads were washed four times with 1 ml of wash buffer (containing 200 mM Tris at pH 8.0, 100 mM NaCl and 0.5% NP-40). For subcellular fraction immunoprecipitation, WERI-Rb1 cells (4x10 6 ) per condition were used. Cytoplasmic & mitochondrial fractions were separated using extraction buffers provided with a cytochrome-c release kit (Abcam, ab65311). The nuclear fraction was lysed in a solution containing 10 mM Tris at pH 8, 170 mM NaCl, 0.5% NP40 with protease inhibitors for 30 min on ice. The respective cellular fractions were incubated with respective primary antibodies for immunoprecipitations. Bound proteins were eluted with SDS sample buffer and separated on SDS PAGE or NuPAGE Novex 4–12% Bis-Tris gels before immunoblotting with specific antibodies. 8. Cell proliferation assay: WERI-Rb1 cells were transduced with lentiviruses for control, RB1 , E2F2 & HK1 overexpression and used for the proliferation assay. 10000 WERI-Rb1 & GL1-Rb1 cells were seeded in 24 well plates for proliferation assay. Cell viability was determined once every 24hours for 4 consecutive days using trypan blue cell staining and cell counting using a hemocytometer. In treatment models, 10000 cells were seeded onto 24 well plates and treated with dorsomorphin (0.1mM) and AICAR (100µM) for up to 96hours. Mock treated (0.1% DMSO) cells were used as control. The cell viability was determined using a trypan blue assay. The experiments were performed in three experimental repeats in triplicates for different experimental conditions. Data were expressed as mean ± SD of triplicate experiments. 9. Cell migration & invasion assays: Cell migration & invasion assays were performed in 24-well transwell plates with cell culture inserts (BD Falcon). Post 72hours of transductions in WERI-Rb1 cells, for invasion assays, 15000 WERI-Rb1 cells in 150µl 0% RPMI media were seeded in transwell insert coated with 1% matrigel & incubated for 48 hours. The bottom chamber was filled with 600µl of 10% RPMI media. After 48-hour incubation, cells on the insert were removed using a cotton swab. Migrated cells on the lower surface of the insert membrane were fixed with 4% PFA and stained with 0.1% crystal violet. Images were captured at brightfield using Olympus CKX53 microscope. Cells were further lysed using 10% SDS and absorbance of crystal violet was measured at 595 nm using a microplate reader. For migration assay, the cells that migrated to the bottom chamber at 48hours were counted using trypan blue cell staining and cell counting using a hemocytometer. For drug treatments, 15000 WERI-Rb1 cells were seeded in 0% RPMI media in the transwell insert coated with 1% matrigel and the cells were further treated with dorsomorphin (0.1mM) and AICAR (100µM) for 48hours. Invasive and migrated cells were quantified using a 0.1% crystal violet staining protocol. Data were expressed as replicate data points ± SD of triplicate experiments. 10. Colony formation/ Tumor spheroid assay: The spheroid formation assays were carried out on a low attachment U-bottom 96 well plate (BRAND® 96-well microplate, Sigma Aldrich). Single-cell suspension of 500 cells in 10% RPMI medium was loaded in each well of a 96 well plate followed by centrifugation for 1000rpm for 1 min to facilitate cell aggregation. The cells were cultured at 37°C in a 90% humidified incubator with 5% CO 2 for 7 days for the generation of tight and regular tumor spheroids. For drug treatments, the 7-day-old spheroids were treated with 100µl of freshly prepared medium containing Topotecan (10nM), Dorsomorphin (0.1mM), Metformin (10mM) and AICAR (100µM) for 48hours. Mock treatment was used as a control. Spheroids were imaged using the EVOS FL imaging system, Invitrogen. ImageJ 2.1 software was used for spheroid area measurements. Data were expressed as replicate data points ± SD of triplicate experiments. 11. Chemosensitivity assay: Cell viability after chemotherapeutic drug topotecan IC 50 (10nM) treatment for 48hours was determined by Presto Blue cell viability reagent (Invitrogen) as per manufactures protocol. In brief, WERI-Rb1 cells (5x10 3 ) were plated into 96-well plates (Eppendorf, Sigma Aldrich) and incubated overnight. Cells were treated with topotecan IC 50 (10nM) for 48 hours. Four hours before the end of treatment, presto-blue reagent (Invitrogen) was added and incubated for 2 hours followed by measurement of fluorescence (540 nm excitation/590 nm emissions). The chemo-sensitivity of all treated cells was determined across conditions and compared against control mock-treated cells (considered as 100% viable). Data were expressed as mean ± SD of triplicate experiments. 12. Cell cycle analysis: WERI-Rb1 cells post 72hours transduction, were further synchronized in 0% RPM1 medium for 24hrs. After synchronization, the cells were trypsinized and fixed in 70% ice-cold ethanol overnight at 4°C and stained with propidium iodide (PI) solution (1µg/µl) and 0.125% RNase A (Sigma Aldrich) at room temperature for 15 minutes. Approximately 10000 cells were gated per condition and were analyzed using BD FACS Canto II and FACSDiva software. 13. Mitochondrial Stress assay: Cells were seeded onto an XFp 8-well flux plate (Seahorse Bioscience) precoated with poly-L-lysine. The cell density of 4000 cells/ 150ul per well were seeded and centrifuged at 500rpm to encourage adhesion to the plate and form an evenly dispersed monolayer. Cells were then incubated at 37°C non-CO 2 conditions and further processed using the XFp Extracellular Flux Analyzer as per the manufacturer’s protocols. Mitochondrial function was measured as OCR after injections of 0.5 µM oligomycin, 1 µM FCCP, 1 µM antimycin A and 1 µM rotenone, according to the manufacturer's instructions. Determinants of mitochondrial function (basal respiration, maximal respiration, spare respiratory and ATP production) were calculated using the formulas according to manufactures protocol. For measuring mitochondrial respiration in therapeutic models, the WERI-Rb1 cells were pre-treated with 0.1% DMSO, AICAR (100µM) and AICAR (100µM) and Topotecan (10nM) for 48hours and further re-seeded a cell density of 5x10^3 WERI-Rb1 cells onto poly-L-lysine coated 8 well flux plates for measuring mitochondrial respiration and energetics. All measurements are normalized to a total number of cells using Presto-blue cell viability reagent (Invitrogen) post mitochondrial stress assay. Data were analyzed using Seahorse XFp Wave Software (Version 2.4) and expressed as replicate data points ± SD of triplicate experiments. 14. Glycolytic Rate Assay For glycolytic rate analysis in WERI-Rb1 cells of different conditions, the cells were seeded onto a poly-L-lysine coated XFp 8-well flux plate in Seahorse XF glycolytic assay medium. Then, ECAR baseline readings were recorded using the Seahorse XFp analyzer and the following injections were done with 4 µM Rot/AA and 50 mM 2-deoxyglucose (2-DG) respectively. PER, glycoPER, basal glycolysis, basal proton efflux rate and compensatory glycolysis, were calculated using the manufacturer’s formula. 15. Immunofluorescence/ Mito Tracker green: 5x10 3 WERI-Rb1 cells per transduced condition were seeded on 96 well plates (Eppendorf) precoated with poly-L-lysine. The cells were stained with 1:5000 dilution of Mito Tracker green in 10% RPMI media and Hoechst 33342 for 15minutes. The images were captured using ImageXpress High content confocal system (Molecular device) and the mitochondrial intensity was calculated using MetaXpress software (Molecular device). 16. Checkerboard microdilution assay: WERI-Rb1 cells were seeded at a concentration of 5×10 3 cells/well in a 96-well plate and cultured at 37°C in a humidified 5% CO2 incubator before the assay. IC50 values of topotecan (10nM), AICAR (100µM) & Dorsomorphin (0.1mM) were determined using cell viability assays and were further diluted to various fractions of IC50 (IC50/2, IC50/4, IC50/8). To test the interaction of the drugs, each fractional concentration of AICAR or Dorsomorphin was tested against topotecan with varying IC50 fraction values for 48hours. The percentage of viable cells in each condition was determined using presto-blue viability assay and was represented as a heat map. The fractional inhibitory concentration (FIC) of AICAR or Dorsomorphin with topotecan was calculated and interpreted as per the standard procedures. The Fractional Inhibitory Concentration Index (FICi) was determined by combined FIC value for AICAR or Dorsomorphin and topotecan (FICi = FIC of AICAR or Dorsomorphin + FIC of topotecan). FICi value of ≤ 0.5 was considered synergistic; a value of > 0.5–1 indicated an additive effect of the two drugs; and a FICi value of > 1 displayed the antagonism of the two drugs. Data were expressed as heatmap indicating the mean of triplicate experiments. 17. Rabbit intraocular tumor xenograft model: Experimentation on rabbits was performed by the statement for the use of animals in ophthalmic and vision research approved by the Association for Research in Vision and Ophthalmology. New Zealand rabbits (n = 28; 4 eyes per group) with a mean initial weight of around 3 kg were used in this study. Immunosuppression was attained with daily subcutaneous injections of cyclosporine A (CsA; 15mg/kg/day for week 1 till week 6, followed by 10mg/kg/day for week 7 till week 10). All the animals received subretinal injection of cultured WERI-Rb1 cells (1.5x10 6 in 30 µl volume) in each eye. After the development of vitreous seeds, the animals were grouped into Group A, B, C, D, E, F and G, those receiving HK1 or E2F2overexpressionn, no treatment, Vehicle (PBS), Topotecan at IC 50 (25µg) and IC 100 (50µg), combined AICAR IC 50 (15µg)/topotecan IC 50 (25µg) via intravitreal injection (in 50 µl volume). The intravitreal doses of drugs were determined by assessment of in-vitro cytotoxicity profile and extrapolation to arrive at IC 50 & IC 100 concentration in the rabbit vitreous as described previously ( 58 , 59 ). The tumor growth was monitored for 3 weeks post-treatment. Daily cage-side observations were performed on all animals to monitor their health and ocular abnormalities. 18. Statistical analysis: Statistical analysis was performed using GraphPad Prism 8. Data are presented as mean ± s.d unless indicated otherwise, and P < 0.05 was considered statistically significant. For all representative images, results were reproduced at least three times in independent experiments. For all quantitative data, the statistical test used is indicated in the legends. A statistical ‘decision tree’ is shown in Figure S5. Declarations Acknowledgements: The authors thank Dr G Kumarmanickavel and Dr Swaminathan Sethu for their expertise and assistance throughout all aspects of our study. Competing interests: SH receives personal fees for scientific advice to Astra-Zeneca, Cellprothera and Merck; unrestricted research grant from Pfizer, outside the content of this work. The other authors have no competing interests. Author contributions: VSB and AG designed the experiments and wrote the manuscript. VSB performed cell line experiments. AM, GD and RS provided human tissue samples and helped interpret correlations with clinical data. DSA, SG, RK and NG performed the analyses and assisted with figure preparation and wrote relevant methods. NKV, LR and ABV developed the intra-ocular tumour model and performed animal experiments. SSC and SSB edited the manuscript. SH edited the manuscript, helped with data analysis and scientific guidance. ABV prepared the animal experiment data and wrote relevant methods. Additional information: Supplementary data figures and tables are available for this paper. Original uncropped western blots are also provided as single Supplementary data file for this study. Data availability statement: All data and analyses in this study are available from the corresponding author upon reasonable request. Ethics statement : The study was conducted by the Declaration of Helsinki principles under a protocol approved by the institutional ethics committee of Narayana Nethralaya (EC Ref no: C/2013/03/02). Informed written consent was received from the parents before inclusion in the study. Funding statement: The authors thank Narayana Nethralaya Foundation for funding research support to VSB, APN, RK, AG. 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Tables Table 1: Clinical and histopathological details of samples used in the study ID Sex Laterality Age at presentation (months) Clinical Risk IIRC Group AJCC Staging P1 M Bilateral 15 months Advanced Group E cT3 P2 F Unilateral 20 months Advanced Group E cT3 P3 M Unilateral 24 months Non-advanced Group E CT2 P4 F Bilateral 4 months Advanced Group E cT3 P5 M Bilateral 30 months Advanced Group E cT3 P6 F Bilateral 21 months Advanced Group D cT3 P7 F Unilateral 28 months Non-advanced Group D cT2 P8 M Unilateral 20 months Non-advanced Group D cT2 P9 M Unilateral 21 months Non-advanced Group D cT2 P10 F 23 months Bilateral Advanced Group E cT3b P11 F 24month Unilateral Advanced Group E cT3b P12 M 36 months Bilateral Advanced Group E cT3b P13 F 33 months Unilateral Advanced Group E cT3a P14 M 36 months Unilateral Advanced Group E cT3b P15 M 48 months Unilateral Advanced Group E cT3b P16 F 33 months Unilateral Non-advanced Group D cT2b P17 F 14 months Bilateral Non-advanced Group D cT2b P18 M 11 months Unilateral Advanced Group E cT3b P19 M 3 months Unilateral Advanced Group E cT3b P20 M 33 months Unilateral Advanced Group E cT3a P21 F 45months Bilateral Advanced Group E cT3b P22 M 7 months Bilateral Non-advanced Group D cT2a P23 M 30 months Bilateral Non-advanced Group D cT2b P24 F 14months Unilateral Non-advanced GroupD cT2b P25 M 11 months Unilateral Non-advanced Group D cT2b C1 M 2 months Multiple organ dysfunction ( No ocular complications) C2 F 12 months No ocular complications Additional Declarations (Not answered) Supplementary Files CDDSupplementarydatanew.pdf Supplementary Data Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amutha","middleName":"Barathi","lastName":"Veluchamy","suffix":""},{"id":125299785,"identity":"c904920a-33ca-42a2-89e9-fd40fbd8d61e","order_by":17,"name":"Arkasubhra Ghosh","email":"data:image/png;base64,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","orcid":"","institution":"GROW Research Laboratory","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Arkasubhra","middleName":"","lastName":"Ghosh","suffix":""}],"badges":[],"createdAt":"2022-06-21 06:01:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1779138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1779138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24630595,"identity":"ded45497-e374-4257-9a4f-a878647e111c","added_by":"auto","created_at":"2022-08-01 20:32:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1340496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRB1 and HK1 expression inhibits cancer hallmarks while E2F2 enhances cancer hallmarks\u003c/strong\u003e. Viable cell count of WERI-Rb1 cells to assess cell proliferation at 24hour, 48hour, 72hour \u0026amp; 96hour, transduced with RB1 overexpression construct alongside A) E2F2 and B) HK1 overexpression and knockdown constructs. C) Viable cell count to assess cell proliferation of primary retinoblastoma cells, modulated with RB1 overexpression, HK1 overexpression, and knockdown constructs, at 24hr, 48hr,72hr and 96hr. D) Crystal violet staining of transwell insert membrane to show invasiveness of WERI-Rb1 cells at 48hours, transduced with RB1 overexpression constructs. E) Crystal violet OD reading at 570nm to assess invasive RB1 complemented cells, F) Invasive RB1 \u0026amp; E2F2 modulated cells. G) Crystal violet staining of transwell insert membrane to show invasiveness of WERI-Rb1 cells at 48hours, transduced with RB1 \u0026amp; HK1 constructs.\u0026nbsp;H) Invasive RB1 \u0026amp; HK1 modulated cells. I) Trypan blue cell count of RB1 complemented WERI-Rb1 cells at 48hours in the lower transwell compartment. J) Crystal violet staining of transwell insert membrane to show invasiveness of WERI-Rb1 cells at 48hours, transduced with RB1 \u0026amp; E2F2 constructs. K) Crystal violet staining of transwell insert membrane to show invasiveness of WERI-Rb1 cells at 48hours, transduced with RB1 \u0026amp; E2F2 constructs.\u0026nbsp;L) Invasive RB1 \u0026amp; E2F2 modulated cells. M) Spheroid forming capability of RB1 complemented WERI-Rb1 cells for 7 days compared to control. N) Scatter plot showing the spheroid size of \u003cem\u003eRB1\u003c/em\u003e complemented cells compared to control. O) \u003cem\u003eRB1\u0026amp; HK1 \u003c/em\u003emodulated cells for 7 days. P) Spheroid size under RB1-HK1 constructs. Q) \u003cem\u003eRB1 \u0026amp; E2F2 \u003c/em\u003emodulated cells for 7 days. R) Spheroid size under RB1-E2F2 constructs. S) Chemosensitivity assay showing 25% cell viability under \u003cem\u003eRB1\u003c/em\u003e overexpression and \u003cem\u003eHK1\u003c/em\u003e overexpression combination at 96hour using10nM topotecan treatment compared to 50% cell viability in control. T) Schematic representation showing \u003cem\u003eRB1\u003c/em\u003e null state with upregulation of \u003cem\u003eE2F2\u003c/em\u003e and cancer hallmark property. \u003cem\u003eHK1\u003c/em\u003e is downregulated at this state. U) Schematic representation showing \u003cem\u003eRB1\u003c/em\u003e overexpression state showing downregulation of \u003cem\u003eE2F2\u003c/em\u003e and cancer hallmarks. \u003cem\u003eHK1\u003c/em\u003e expression is upregulated at this state.\u0026nbsp;Results represent representative of three independent experiments. Values represent mean ± s.d. Unpaired two-sided Student’s t-test was used for statistical analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/3c856d0a01f97693fab40387.jpg"},{"id":24630881,"identity":"06120527-581f-471c-8479-de62e8e85c9d","added_by":"auto","created_at":"2022-08-01 20:37:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":907450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHexokinase1 induction inhibits cancer growth in-vivo. \u003c/strong\u003eA) OCT images of retinoblastoma xenograft in rabbit retina at week 06 and week 08 post-injection of Control WERI-Rb1 cells (n=4 eyes), E2F2 overexpressed WERI-Rb1 cells (n=4 eyes), and HK1 overexpressed WERI-Rb1 cells (n=4 eyes). White arrows heads indicate tumors developed at week 6 \u0026amp; week 8 in control and E2F2 overexpression condition. Yellow arrowheads indicate no tumor development in HK1 overexpression conditions. B) Scatter plot shows mean Rb tumor area at week 8 in rabbit xenograft models. Values represent mean tumor area ± s.d. Mann-Whitney analysis was used for statistical analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. C) H\u0026amp;E staining of rabbit xenograft tumor sections showing control, HK1 \u0026amp;\u0026nbsp;E2F2 overexpression condition. E2F2 overexpression and control conditions show large tumors with disrupted retinal layers. HK1 overexpression did not develop any tumors and maintains an intact retina\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/1df0bd8e5dfcd8afb4c853a0.jpg"},{"id":24630880,"identity":"6a3114d2-48da-4bf4-94e0-8352b39f4b1c","added_by":"auto","created_at":"2022-08-01 20:37:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1213396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRB1 and HK1 drive the tumor energy-sensing circuitry independent of canonical RB1-E2F2 cell cycle regulation.\u003c/strong\u003e A) Western blot showing protein expression of candidate targets involved in energy sensing circuits and cell cycle in WERI-Rb1. Gene expression of B) \u003cem\u003eHK1\u003c/em\u003e and \u003cem\u003eE2F2\u003c/em\u003e C) \u003cem\u003eRB1 \u003c/em\u003eand \u003cem\u003eHIF1α \u003c/em\u003ein WERI-Rb1 cells modulated with RB1, E2F2 and HK1 overexpression and knockdown constructs. D) IHC profile of Rb, E2F2, HK1, AMPKα and phospho-AMPKα in Rb tissues (n=25) and paediatric retina (n=2). E) IHC scores of Rb, E2F2, HK1, AMPKα and phospho-AMPKα in Rb tissues and pediatric retina.\u0026nbsp;Values represent mean ± SEM. Unpaired two-sided Student’s t-test was used for statistical analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. OE=Overexpression, KD= Knockdown.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/0af4d482824f3e9bc0922179.jpg"},{"id":24630598,"identity":"ba738e46-bce5-48db-8f15-5eabe2452fb6","added_by":"auto","created_at":"2022-08-01 20:32:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":580024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHK1 induction rewires cellular metabolism\u003c/strong\u003e. Western blot showing protein levels of glycolysis associated proteins. A) HK2 and GLUT1; GAPDH as loading control B) PKFP, PKM2, and LDHA; α-tubulin as loading control C) Gene expression of \u003cem\u003eHK2\u003c/em\u003e and \u003cem\u003eG6PC \u003c/em\u003egenes in WERI-Rb1 cells modulated with RB1, E2F2, and HK1 constructs. D) Schematic showing metabolic rewiring mediated by RB1 \u0026amp; HK1. Values represent mean ± s.d. Unpaired two-sided Student’s t-test was used for statistical analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/16ac43a3d0fdf7f0adedbc0c.jpg"},{"id":24630883,"identity":"6f4fcf01-7057-43da-9f78-7aa7e5ba2a69","added_by":"auto","created_at":"2022-08-01 20:37:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":578348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation reveals binding partners of Rb and HK1. \u003c/strong\u003eCo-IP was performed on whole-cell lysates of Control and RB1 Overexpressed WERI-Rb1 cells using - A) anti-Rb antibodies B) anti-HK1 antibodies C) anti-HIF1α antibodies D) anti-LKB1 antibodies E) IgG antibodies.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/806f9c565621e992f66bc595.jpg"},{"id":24630603,"identity":"2e8c6d1f-7b13-46d6-a792-e4af412e1164","added_by":"auto","created_at":"2022-08-01 20:32:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":535828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of Rb and HK1 with binding partners at distinct sub-cellular locations. \u003c/strong\u003eA) Co-IP was performed on nuclear fraction lysates of Control and \u003cem\u003eRB1\u003c/em\u003e Overexpressed WERI-Rb1 cells, using anti-RB \u0026amp; anti-HK1 antibodies. B) Cytoplasmic fraction lysates of Control and \u003cem\u003eRB1\u003c/em\u003e Overexpressed WERI-Rb1 cells C) mitochondrial fraction lysates of Control and \u003cem\u003eRB1 \u003c/em\u003eOverexpressed WERI-Rb1 cells. D) Schematic showing the interaction of key molecular players in retinoblastoma subcellular fractions that mediate a metabolic switch.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/434ada5d54e1b15544bc8554.jpg"},{"id":24630897,"identity":"f4297c7e-9129-4c91-8c32-77751b5cea04","added_by":"auto","created_at":"2022-08-01 20:42:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1078141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHK1 and Rb expression alters mitochondrial respiration\u003c/strong\u003e. A) Immunofluorescence showing the mitochondrial mass in control and RB1 overexpression. WERI-Rb1 cells at 96hour post-transduction and stained with Mito-tracker green and Hoescht 33322 for 10 minutes before confocal imaging. White arrows indicate high mitochondrial mass, while red arrows indicate lesser mitochondrial mass, Scale bar= 25um. B) Mean mitochondrial IF intensity was calculated from 12 wells per condition using HCS and MetaXpress software. C) Seahorse XFp Mito-stress assay showing Basal OCR and Maximal OCR, D) Spare respiration capacity. Seahorse XFp glycolytic rate assay showing E) Basal glycoPER, F) Compensatory glycoPER. Values represent mean ± s.d. Unpaired two-sided Student’s t-test was used for statistical analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/c741a3fee0c5276cc08bb723.jpg"},{"id":24630601,"identity":"00f50715-c80e-4399-9f67-15e751e26bee","added_by":"auto","created_at":"2022-08-01 20:32:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1178311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMPKα activation inhibits cancer growth in-vitro and in-vivo. \u003c/strong\u003eA) Tumor spheroids treated with dorsomorphin, AICAR and chemotherapeutic drug topotecan. B) Scatter plot represents the area of tumor spheroids under AICAR and dorsomorphin treatments. C) Checkerboard assay to assess drug interactions between topotecan and AICAR. D) OCT images of rabbit retina showing tumor size and location in PBS treated control group (n=4 eyes). E) Treatment with Topotecan IC50 dose (n=4 eyes). F) Treatment with Topotecan IC100 dose (n=4eyes). G) Treatment with Topotecan IC50 and AICAR IC50 dose (n=4 eyes).H) Bar graph represents the pre-treatment and post treatment tumor area using control (PBS), topotecan and combination therapy. I) H\u0026amp;E staining of rabbit Rb tumor sections from control (PBS), topotecan IC50, topotecan IC100 and combination therapy of AICAR IC50 \u0026amp; topotecan IC50. Scalebar =100µm. Values represents mean ± s.d. Unpaired two-sided Student’s t-test was used for statistical analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/e8b37246d88fbeb58fa2c4f8.jpg"},{"id":26061658,"identity":"f1c2836b-b99c-431d-b526-183cbdb44a0b","added_by":"auto","created_at":"2022-09-05 11:03:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1854316,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/0f61a8ad-c101-46ba-a37c-c7d26863c131.pdf"},{"id":24630896,"identity":"5f11eb6c-a293-4588-91b0-aa6d0e805f13","added_by":"auto","created_at":"2022-08-01 20:42:30","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1774080,"visible":true,"origin":"","legend":"Supplementary Data","description":"","filename":"CDDSupplementarydatanew.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1779138/v1/5298ab0b54d43067c8423709.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Enhanced oxidative phosphorylation in Retinoblastoma tumors is dependent on depleted Hexokinase1 and lack of AMPKα activation","fulltext":[{"header":"Introduction:","content":"\u003cp\u003eRetinoblastoma (Rb) is the most common intraocular malignancy of childhood, caused by the inactivation of the \u003cem\u003eRB1\u003c/em\u003e gene (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Inactivation or deletion of the functional \u003cem\u003eRB1\u003c/em\u003e gene deregulates proliferative control and facilitates Rb tumorigenesis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), but it has diverse roles beyond cell cycle control that are still being unraveled. The \u003cem\u003eRB1\u003c/em\u003e gene encodes for the retinoblastoma protein (pRb) that serves as a prototype of transcriptional repression and cell cycle regulation by preferential binding on E2F genes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Canonically, pRb protein represses E2F transcription factors directly by binding to its promoter and transactivation domain, causing a reciprocal induction and inhibition of cell cycle-specific transcription (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Thus, cancers with significant loss of functional pRb proteins unleash the E2F transcriptional factors to enhance uncontrolled cell proliferation and provide a subsequent advantage to tumor progression (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In addition, non-canonical functions of pRb proteins involve mechanistic regulation of histone modifications, chromosome integrity and cellular metabolism (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and loss of functional pRb proteins has been attributed to the alteration of these pathways in cancers (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Despite exploring the loss of pRb protein consequences in other cancers, significant gaps exist in discerning the canonical and non-canonical role of pRb loss in Rb tumor progression. Reprogramming of metabolic circuits in tumors favours their growth, with the changes in individual metabolic pathways frequently correlating with enhanced glycolysis and nucleotide biosynthesis(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). \u003cem\u003eRB1\u003c/em\u003e is associated with altered metabolism, an important emerging cancer hallmark(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, the metabolic reprogramming due to loss of \u003cem\u003eRB1\u003c/em\u003e is likely dependent on the photoreceptor precursor cell of origin(\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The photoreceptors continuously require high amounts of energy for the visual cycle operation, that is met primarily by glycolysis(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Photoreceptor mitochondria adapt to different energy requirements and mitochondrial turnover is essential for photoreceptor function and health(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe interaction between essential cellular signaling pathways(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) altered due to Rb1 loss and changes in metabolism that provide energy and growth cues for these retinal tumors are not fully understood.\u003c/p\u003e \u003cp\u003eRecently, we have identified a number of cell cycle and metabolic genes in Rb tumors and in Rb null cellular models (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In particular, we identified Hexokinase 1 \u003cem\u003e(HK1)\u003c/em\u003e to be significantly reduced while the E2F family member E2F2 was significantly upregulated. \u003cem\u003eHK1\u003c/em\u003e is a key regulator of glycolytic flux and catalyses the first step in the conversion of glucose to pyruvate, an essential process. Therefore, we investigated various critical effectors of metabolic pathways to dissect how \u003cem\u003eRB1\u003c/em\u003e controls retinal tumor metabolism. Our results reveal that loss of Rb and consequently Hexokinase 1 (HK1), reprograms tumor metabolic pathways to enhance ATP generation using mitochondrial oxidative phosphorylation (OXPHOS) instead of glycolysis. We further exploit our observations for therapeutic purposes and demonstrate the utility of targeting metabolism for Rb tumor therapy.\u003c/p\u003e"},{"header":"Results:","content":"\u003cp\u003e \u003cb\u003eRB1 complementation induces HK1 and reduces E2F2 levels to regulate key cancer hallmarks.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our previous study, we reported low expression of HK1 and high expression of E2F2 in Rb tumors (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We found that ectopic expression of \u003cem\u003eRB1\u003c/em\u003e led to the induction of HK1 protein and transcript in Rb null WERI-Rb1 cells. We further validated our findings, showing increased HK1 and diminished E2F2 protein in Y79 and GL1-Rb1 (patient derived primary Rb-/- cells), under \u003cem\u003eRB1\u003c/em\u003e complementation (Figure S1A, B respectively). Similarly, \u003cem\u003eHK1\u003c/em\u003e transcript was increased in Y79 (Figure S1C), and GL1-Rb1 cells (Figure S1D), while reducing \u003cem\u003eE2F2\u003c/em\u003e transcript levels in these cells (Figure S1C, D).\u003c/p\u003e \u003cp\u003eNext, we studied how \u003cem\u003eHK1\u003c/em\u003e and \u003cem\u003eE2F2\u003c/em\u003e ablation by shRNA expression or their overexpression affects cancer hallmarks. \u003cem\u003eRB1-\u003c/em\u003edependent mechanisms are often linked to cell cycle anomalies (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) rather than metabolic alterations in ocular tumors. Therefore, we tested if key cancer hallmarks including cell proliferation, invasion, and migration, were affected by ectopic modulation of \u003cem\u003eHK1\u003c/em\u003e, in comparison to \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e. Overexpression of \u003cem\u003eHK1\u003c/em\u003e phenocopied \u003cem\u003eRB1\u003c/em\u003e complementation in both WERI-Rb1 and GL1-Rb1 cells by reducing proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C) while \u003cem\u003eHK1\u003c/em\u003e knockdown exhibited an increase in proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C) compared to controls. However, E2F2 overexpression increased cell proliferation while E2F2 knockdown alone and in combination with RB1 complementation, reduced cell proliferation at 96hour (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). \u003cem\u003eRB1\u003c/em\u003e overexpression significantly reduced invasion in transwell assays compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E, F). \u003cem\u003eHK1\u003c/em\u003e overexpression phenocopied \u003cem\u003eRB1\u003c/em\u003e individually and in combination, reducing invasion whereas \u003cem\u003eHK1\u003c/em\u003e knockdown increased invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H, I). \u003cem\u003eE2F2\u003c/em\u003e overexpression increased invasion and migration while \u003cem\u003eE2F2\u003c/em\u003e knockdown exhibited a reduction in invasive phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, K, L). To assess whether HK1 and E2F2 are involved in 3-D spheroid formation and chemosensitivity of WERI-Rb1 cells, we transfected \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eHK1\u003c/em\u003e and \u003cem\u003eE2F2\u003c/em\u003e overexpression and their knockdown constructs in WERI-Rb1 cells. Tumor spheroids from \u003cem\u003eRB1\u003c/em\u003e overexpressing cells formed small irregular clusters compared to large, tight spheroids in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, N). \u003cem\u003eHK1\u003c/em\u003e overexpressing cells formed small irregular clusters while \u003cem\u003eHK1\u003c/em\u003e knockdown formed tight spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO, P, D; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). \u003cem\u003eE2F2\u003c/em\u003e overexpressed cells developed tight large spheroids, while \u003cem\u003eE2F2\u003c/em\u003e knockdown alone and in combination with \u003cem\u003eRB1\u003c/em\u003e overexpression exhibited a reduction in spheroid size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ, R). Ectopic expression of \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eHK1\u003c/em\u003e and their combination effectively sensitized the cells to lower doses of chemotherapeutic drug topotecan compared to the \u003cem\u003eE2F2\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS). Collectively, our data identified an increase in tumor hallmarks upon E2F2 overexpression in Rb null state and reduction in tumor hallmarks upon HK1 and RB1 complementation in WERI-Rb1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eT, U).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHexokinase1 induction inhibits cancer growth in-vivo\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe then tested how E2F2 and HK1 modulation in retinoblastoma cells affected their tumorigenic potential in a rabbit intraocular xenograft tumor model. Orthotopic sub-retinal transplantation of \u003cem\u003eE2F2\u003c/em\u003e overexpressing (n\u0026thinsp;=\u0026thinsp;4) and control (n\u0026thinsp;=\u0026thinsp;4) WERI-Rb1 cells (1.5x10\u003csup\u003e6\u003c/sup\u003e cells per injection) in rabbit eyes caused retinal tumors measurable at 6 and 8 weeks, while \u003cem\u003eHK1\u003c/em\u003e overexpressing cells (n\u0026thinsp;=\u0026thinsp;4) did not form tumors in rabbit retina as shown by optical coherence tomography (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). \u003cem\u003eE2F2\u003c/em\u003e tumors also had a larger area compared to control tumors (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0106;) which is reflected in the histological analysis of the retinal sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRB1 and HK1 drive the tumor energy-sensing circuitry independent of canonical RB1-E2F2 cell cycle regulation\u003c/h2\u003e \u003cp\u003eSince HK1 represents a critical node of the cellular metabolic network, while E2F2 is a less studied member of the E2F family, we investigated the intracellular status of metabolic signaling proteins upon modulation of \u003cem\u003eRB1, E2F2\u003c/em\u003e and \u003cem\u003eHK1. E2F2\u003c/em\u003e overexpression and knockdown did not induce HK1 proteins and similarly, \u003cem\u003eHK1\u003c/em\u003e modulation did not affect E2F2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that they are independently regulated by Rb. Ectopic expression of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e phosphorylated AMPKα, while \u003cem\u003eE2F2\u003c/em\u003e overexpression did not alter AMPKα phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that the HK1 and E2F2 regulated circuit modules are exclusive. Complementing \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e induced phosphorylation and deactivation of ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). LKB1 and HIF1α were increased in \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e overexpressing cells. However, \u003cem\u003eRB1\u003c/em\u003e or \u003cem\u003eHK1\u003c/em\u003e expression did not alter total ACC and total AMPKα levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Elevated expression of HK1 along with LKB1 likely led to the activation of AMPKα while \u003cem\u003eHK1\u003c/em\u003e knockdown did not activate AMPKα or increase LKB1 even in the presence of \u003cem\u003eRB1\u003c/em\u003e. Thus, Rb induces HK1 which leads to LKB1 elevation subsequently activating AMPKα and regulating cellular metabolism. Similar results were also obtained in Y79 cells transduced with \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e vectors (Figure S2 A). Throughout the experiments, E2F1 levels are enhanced depending on absence of Rb, which serves as an internal control for the experiments. However, \u003cem\u003eE2F2\u003c/em\u003e modulation alone, in absence of \u003cem\u003eRB1\u003c/em\u003e, did not affect \u003cem\u003eHK1\u003c/em\u003e gene expression, further confirming an alternate signal cascade controlled by Rb and HK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We found significant high expression of \u003cem\u003eHIF1α\u003c/em\u003e in \u003cem\u003eRB1\u003c/em\u003e complemented cells, while \u003cem\u003eHK1\u003c/em\u003e complemented cells did not show any significant effect on \u003cem\u003eHIF1α\u003c/em\u003e gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), contrasting to its protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, Rb and HIF1α are known to interact whereas in other cellular contexts. HK1 and HIF1α are known to associate (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Our cell cycle analysis shows cell populations increased in the G1/S phase with a low distribution of cells in the G2/M phase in \u003cem\u003eRB1\u003c/em\u003e overexpressing cells, indicating a reduction in cell division (Figure S2B). Unlike \u003cem\u003eRB1\u003c/em\u003e complementation, \u003cem\u003eHK1\u003c/em\u003e over expression did not show any cell cycle arrest at the G1/S phase with adequate distribution of cells in the G2/M phase, although total numbers of cells reduce across phases. However, higher proportions of cells were observed at the S phase and G2/M phase upon \u003cem\u003eE2F2\u003c/em\u003e overexpression, indicative of rapid cell division. Immunohistochemial analysis revealed low HK1 and phospho-AMPKα signals in Rb tissues ( n\u0026thinsp;=\u0026thinsp;25) compared to pediatric retina (n\u0026thinsp;=\u0026thinsp;2) (Figure D,E), which validates our \u003cem\u003ein vitro\u003c/em\u003e observation in patient tissues. Thus, \u003cem\u003eHK1\u003c/em\u003e expression even in absence of \u003cem\u003eRB1\u003c/em\u003e can actuate a halt in cancer hallmarks and alter metabolic circuits in Rb cells but in a manner distinct from canonical cell functions governed by RB1 and E2F2 (Figure S2B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHK1 induction rewires cellular metabolism\u003c/h2\u003e \u003cp\u003eSince HK1 catalyzes the initial rate limiting step of glycolysis (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and the allosteric regulation of other glycolytic genes, including the PKM2 mediated metabolic switch from glycolysis to OXPHOS (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), we investigated additional glycolysis and glucose uptake related genes. \u003cem\u003eRB1\u003c/em\u003e increased HK2 \u0026amp; GLUT1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) while PKM2 levels were reduced and PFKP remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Strikingly, the levels of HK2, GLUT1, PKM2 and PFKP were not altered by \u003cem\u003eHK1\u003c/em\u003e overexpression, indicating that the activation of AMPKα and inhibition of ACC is specifically controlled by HK1. Also, \u003cem\u003eE2F2\u003c/em\u003e modulation alone did not change levels of any of these proteins. In addition, gene expression profiling of \u003cem\u003eRB1\u003c/em\u003e complemented cells shows significantly increased expression of glycolytic genes \u003cem\u003eHK2\u003c/em\u003e (Hexokinase 2) and \u003cem\u003eG6PC\u003c/em\u003e (Glucose 6 phosphate catalytic subunit 1), whereas \u003cem\u003eHK1\u003c/em\u003e complemented cells shows elevated expression of \u003cem\u003eG6PC\u003c/em\u003e gene, while \u003cem\u003eHK2\u003c/em\u003e gene expression remain unaltered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Collectively, our findings reveal a novel energy sensing circuit in Rb tumors, that can be altered by \u003cem\u003eHK1\u003c/em\u003e complementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHK1 binds to LKB1, but not AMPK\u003c/b\u003eα\u003c/p\u003e \u003cp\u003eTo elucidate the molecular interactions underlying the metabolic alterations induced by \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e complementation, we performed co-immunoprecipitation (co-IP) assays. We found no direct interaction between Rb \u0026amp; HK1 or Rb \u0026amp; AMPKα, whereas we observed an interaction between Rb and HIF1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, S3A). Therefore, we hypothesize that the induction of HK1 is likely due to \u003cem\u003eRB1\u003c/em\u003e mediated transcriptional activation, but the effect on AMPKα is mediated through HK1. HK1 co-IP did not show an interaction between HK1 and Rb, although HK1 was bound to both HIF1α and LKB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, S3B). HK1 interaction with VDAC1 served as the positive control (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and HK1 did not bind to E2F1 or E2F2. LKB1 interaction with HK1 was confirmed using anti-LKB1 co-IP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, S3D), but LKB1 did not bind to HIF1α. We confirmed the interaction of Rb-HIF1α, HIF1α-HK1 by performing a reverse co-IP using anti-HIF1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, S3C), but the fractions of HIF1α bound to HK1 and Rb appear to be distinct.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInteraction of Rb and HK1 with binding partners at distinct sub-cellular locations\u003c/h2\u003e \u003cp\u003eAltered distribution of HK1 and Rb in subcellular compartments can disrupt cell signaling and affect metabolism (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Therefore, we assessed the binding partners of Rb and HK1 in subcellular fractions of control and \u003cem\u003eRB1\u003c/em\u003e complemented WERI-Rb1 cells. Co-IP from subcellular fractions revealed that HIF1α bound Rb in the nuclear fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, S3E), while it bound HK1 in the cytoplasmic fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The cytoplasmic HK1 expression was higher under \u003cem\u003eRB1\u003c/em\u003e overexpression compared to controls and it interacted with LKB1 exclusively in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, S3F). LKB1 is an upstream kinase that phosphorylates AMPKα (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) which, in turn phosphorylates and inhibits ACC (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In the mitochondrial fraction, HK1 bound VDAC1, while none of the other proteins tested were found to interact (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, S3G). A schematic shows the interaction of targets identified in subcellular fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Using promoter binding analysis, we found that the \u003cem\u003eHK1\u003c/em\u003e promoter has binding sites for \u003cem\u003eE2F1\u003c/em\u003e, \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eHIF1α\u003c/em\u003e (Figure S3H), it is possible that the transcriptional activation of \u003cem\u003eHK1\u003c/em\u003e is mediated via direct promoter binding by \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHIF1α\u003c/em\u003e or relieving E2F mediated repression. While \u003cem\u003eRB1\u003c/em\u003e is known to directly induce \u003cem\u003eHIF1α\u003c/em\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), \u003cem\u003eE2F1\u003c/em\u003e and \u003cem\u003eE2F2\u003c/em\u003e also have binding sites in the \u003cem\u003eHIF1α\u003c/em\u003e promoter (Figure S3G), making a case for derepression as a possible mechanism\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHK1 and Rb expression alters mitochondrial respiration\u003c/h2\u003e \u003cp\u003eGrowing evidence suggests that perturbing HK1-VDAC1 interactions in the mitochondria affects mitochondrial membrane potential and glutamine transport(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). We investigated the status of active mitochondria in cells using the Mito-tracker dye. We observed lesser mitochondrial density in \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e complemented cells compared to controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while \u003cem\u003eHK1\u003c/em\u003e knockdown showed higher mitochondrial density. \u003cem\u003eHK1\u003c/em\u003e overexpression led to a reduction in active mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). This could be a direct result of HK1 induced activated AMPKα driven mitophagy (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), thereby triggering a metabolic checkpoint and energy restriction. Since cellular energy production through OXPHOS is dependent on mitochondrial status (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), the lack of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e increases mitochondrial activity in retinoblastoma tumors. To validate our findings, we used Seahorse XFp metabolic flux assays that revealed low basal and induced mitochondrial respiration upon ectopic expression of \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e either alone or in combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Notably, the mitochondrial respiration was high in \u003cem\u003eHK1\u003c/em\u003e knockdown cells and was suppressed by \u003cem\u003eRB1\u003c/em\u003e. Spare respiratory capacity was reduced significantly in \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) indicating curtailed OXPHOS dependence. Consequently, both basal and compensatory glycolytic proton efflux rates (glycoPER) in cells expressing \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e were significantly elevated, indicating their metabolic shift towards glycolysis. However, the glycolytic capacity of \u003cem\u003eHK1\u003c/em\u003e ablated cells was significantly lower compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F), indicating that tumor cells have low dependence on glycolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAMPKα activation halts retinal tumor growth.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe tested the possibility of targeting the altered metabolic phenotype of retinoblastoma tumors by modulating AMPKα using activators AICAR (adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside) and Metformin while using the inhibitor dorsomorphin as a control. Treatment with AICAR significantly activated AMPKα levels in WERI-Rb1 cells (Figure S4A) and reduced cell proliferation (Figure S4B), invasion (Figure S4C) and migration (Figure S4D), while dorsomorphin treatment enhanced cell proliferation, invasion and migration (Figure S4A-C). AICAR treatment effectively shrunk 7-day-spheroids after 48hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) indicated by the reduction in spheroid area (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Dorsomorphin treatment did not exhibit any effects on the 7-day old spheroids. Metformin, an alternative AMPKα activator, also reduced the size of tumor spheroids by 40% percent. However, AICAR was more effective than metformin (Figure S4E, F) possibly due to their mode of exerting AMPKα dependent effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn clinical treatment regimens, chemotherapeutic drugs are typically used in combinations to reduce the toxic bystander effects from each drug type as well as prevent tumors from developing resistance to monotherapy. Therefore, a metabolic blockade specific to tumor cells may prove to be an effective method of enhancing the lethality of currently used chemotherapeutic drugs at a lower dosage. We observed an additive effect of AICAR with topotecan (FICi\u0026thinsp;=\u0026thinsp;0.75; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) in inducing tumor cell death by a checkerboard drug interaction assay. Dorsomorphin exerted an antagonistic effect when combined with Topotecan in a similar assay (FICi\u0026thinsp;=\u0026thinsp;1.25; Figure S4G). Treatment of AICAR IC\u003csub\u003e50\u003c/sub\u003e was effective in reducing basal mitochondrial respiration (Figure S4, I, J), maximal respiration (Figure S4J) and spare respiration (Figure S4K) in Rb null cells. These observations were comparable to the OCR measurements obtained with \u003cem\u003eHK1\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003eNext, we allowed intraocular tumors to develop in rabbit eyes for 6 weeks followed by treatment with topotecan IC\u003csub\u003e100\u003c/sub\u003e, IC\u003csub\u003e50\u003c/sub\u003e monotherapy and combined treatment with topotecan IC\u003csub\u003e50\u003c/sub\u003e and AICAR IC\u003csub\u003e50\u003c/sub\u003e for two weeks. The status of tumors was analyzed by OCT imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, E, F, G). The combination therapy showed effective reduction of tumor area compared to control tumors, indicating the additive nature of AICAR with low dose topotecan (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, H; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001). Sham treated; tumor-containing eyes show large tumors with distorted retinal architecture in histological analysis post enucleation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI). High dose topotecan (Topotecan IC\u003csub\u003e100\u003c/sub\u003e) effectively reduced tumor size while damaging remnant retinal layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI). Topotecan IC\u003csub\u003e50\u003c/sub\u003e dose was less effective in reducing tumor size. However, the combination of topotecan IC\u003csub\u003e50\u003c/sub\u003e and AICAR IC\u003csub\u003e50\u003c/sub\u003e was most effective in reducing tumor size while maintaining the retinal structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI), indicating reduced bystander retinal toxicity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion:","content":"\u003cp\u003eRetinoblastoma patient tumor samples and cellular models have reduced expression of glycolytic genes such as \u003cem\u003eHK1\u003c/em\u003e and metabolites such as pyruvate(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). \u003cem\u003eHK1\u003c/em\u003e catalyses the first step in glycolysis, thereby controlling glycolytic flux(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). \u003cem\u003eHK1\u003c/em\u003e has been found to be frequently overexpressed in tumors(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and associated with oncogenes like KRAS(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) mutants, providing for the tumor cells\u0026rsquo; high energy demand. However, \u003cem\u003eHK1\u003c/em\u003e levels are significantly reduced in retinoblastoma tumors and ectopically expressing \u003cem\u003eRB1\u003c/em\u003e in \u003cem\u003eRB1\u003c/em\u003e-null cells transcriptionally induced \u003cem\u003eHK1\u003c/em\u003e expression and altered the cellular metabolism. While \u003cem\u003eRB1\u003c/em\u003e regulated both \u003cem\u003eHK1\u003c/em\u003e and the E2F family member \u003cem\u003eE2F2\u003c/em\u003e, we consistently observed that the metabolic rewiring of cells in the presence of \u003cem\u003eRB1\u003c/em\u003e was dependent only on \u003cem\u003eHK1\u003c/em\u003e, but not \u003cem\u003eE2F2\u003c/em\u003e, providing evidence of unique, modular, signalling networks regulated by \u003cem\u003eRB1\u003c/em\u003e. This also highlights the potential for developing treatment modalities targeting multiple mechanisms that can potentially be more effective.\u003c/p\u003e \u003cp\u003eComplementing \u003cem\u003eRB1\u003c/em\u003e or \u003cem\u003eHK1\u003c/em\u003e led to activation of \u003cem\u003eAMPKα\u003c/em\u003e, which in turn phosphorylated and inactivated the critical regulator of fatty acid metabolism, \u003cem\u003eACC\u003c/em\u003e(\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). This shifted the metabolic profile of cells back to a glycolysis dependence, consequently causing a reduction in mitochondrial respiration and mass. Notably, cytoplasmic \u003cem\u003eLKB1\u003c/em\u003e levels depended on exclusively on \u003cem\u003eHK1\u003c/em\u003e induction and \u003cem\u003eHK1\u003c/em\u003e physically bound with both \u003cem\u003eLKB1\u003c/em\u003e and \u003cem\u003eHIF1α\u003c/em\u003e in the cell cytoplasm. However, the \u003cem\u003eHK1\u003c/em\u003e pools bound to \u003cem\u003eLKB1\u003c/em\u003e, \u003cem\u003eHIF1α\u003c/em\u003e and mitochondrial \u003cem\u003eVDAC\u003c/em\u003e were distinct. Therefore, \u003cem\u003eHK1\u003c/em\u003e bound \u003cem\u003eLKB1\u003c/em\u003e could be a key signalling complex for energy sensing, leading to \u003cem\u003eAMPKα\u003c/em\u003e activation.\u003c/p\u003e \u003cp\u003ePhotoreceptors rely on glycolysis for rapid energy production(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), while whole retina depends on both glycolysis and mitochondrial OXPHOS for its functional and structural requirements to provide vision(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Interestingly, FDG PET/CT plays minimal role in routine diagnostic evaluation of intraocular tumors(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), possibly due to the lack of \u003cem\u003eHK1\u003c/em\u003e causing insufficient labelled glucose analogue uptake(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), highlighting the importance of HK1 in the photoreceptors. The retinoblastoma tumor cells arise from photoreceptor precursor cells (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) and lose their photoreceptor-related functions and gene expression profile(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Photoreceptors rely on aerobic glycolysis for rapid energy production (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), consuming almost 80\u0026ndash;96% of the glucose to produce lactate (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), although these cells also employ mitochondrial OXPHOS for functional and structural requirements during the visual cycle. Lipids are essential to replace the outer segments of rods and cones (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), which possibly explains why catabolic processes of fatty acids are restricted in these cells. Therefore, since Rb tumors do not need to produce outer segment disks, the lack of HK1 leads to an active ACC, which can catalyze malonyl CoA generation, increasing biosynthesis of long-chain fatty acids to fuel β-oxidation.\u003c/p\u003e \u003cp\u003eWe found that a shift back to glycolysis, an ATP restrictive catabolic process, caused by both \u003cem\u003eRB1\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e, led to reduced cell proliferation, invasion, tumorigenesis and enhanced sensitivity to chemotherapeutic drugs. Though several chemotherapy strategies are successful in various cancer treatments(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), controlling the bystander toxicity of chemotherapeutic drugs is still an unmet clinical requirement. Currently, retinoblastoma tumors are treated using different combination doses and durations of melphalan, topotecan, carboplatin, vincristine \u0026amp; etoposide(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) which often cause bystander toxicity or reduction in efficacy with multiple cycles of therapy required(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Importantly, ectopic expression of \u003cem\u003eHK1\u003c/em\u003e in retinoblastoma cells prevented tumor formation in a rabbit intraocular xenograft model, whereas \u003cem\u003eE2F2\u003c/em\u003e expressing cells formed large tumors. Further, activation of AMPKα using an intraocular injection of AICAR (IC\u003csub\u003e50\u003c/sub\u003e) in combination with topotecan (IC\u003csub\u003e50\u003c/sub\u003e) reduced established intraocular tumors in 2 weeks with reduced retinal damage on histopathology compared to topotecan (IC\u003csub\u003e100\u003c/sub\u003e). We noted that the direct AMPKα activator AICAR reduced tumorsphere formation more efficiently than Metformin. Since Metformin mediates its action on AMPKα via LKB1 (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), which is reduced in Rb null cells, it is less effective than AICAR, which directly activates AMPKα.\u003c/p\u003e \u003cp\u003eTherefore, reprogramming the metabolism of retinoblastoma tumors towards glycolysis may be an effective strategy to sensitize tumors to lower doses of current therapies. This adjuvant strategy may be particularly useful in salvaging vision and in refractory cases. HK1/AMPKα axis also serves as a potential drug target for various advanced cancers lacking functional Rb proteins, which includes small cell lung cancer (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), prostate cancer (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), glioblastoma (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) and leukemia (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). We provide evidence for a new treatment modality for Rb depleted cancers, based on uncovering a transcriptional and cellular signaling network driven by \u003cem\u003eHK1\u003c/em\u003e and \u003cem\u003eAMPKα\u003c/em\u003e in retinoblastoma cells highlighting their unique, context-dependent metabolic reprogramming capacity.\u003c/p\u003e "},{"header":"Materials \u0026 Methods:","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003cp\u003e1.Clinical samples\u003c/p\u003e \u003cp\u003e The study was conducted by the Declaration of Helsinki principles under a protocol approved by the institutional ethics committee of Narayana Nethralaya (EC Ref no: C/2013/03/02). Informed written consent was received from the parents before inclusion in the study. The GL1-Rb1 line was developed from an enucleated tumor specimen obtained from the right eye of a 3-year-old female retinoblastoma subject (AJCC staging-cT4b). The tumor tissue was enzymatically dissociated using dispase \u0026amp; trypsin and the cells were cultured in RPMI 1640 media supplemented with 10% FBS, 1% pen strep and a 10ng cocktail of EGF, VEGF and FGF. A biphasic population of primary Rb cells was observed during the first two weeks, comprising of retinoblastoma tumorspheres adherent to feeder fibroblasts and suspension clusters of single-cell retinoblastoma. Over four weeks, the tumorspheres detach from the fibroblast and form an unusual chain of suspension cells. These cells were cultured separately and cell population doubling time was calculated from the exponential growth phase curve. For immunohistochemistry validations, we have used additional Rb subjects (n\u0026thinsp;=\u0026thinsp;25) of the age range 0.2-4 years and pediatric controls (n\u0026thinsp;=\u0026thinsp;2) of the age range (0.2\u0026ndash;0.3 years). Clinical and histopathology details are mentioned in Supplementary Table S1.\u003c/p\u003e \u003cp\u003e2. Cell lines:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWERI-Rb1 and Y79 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The WERI-Rb1 and Y79 cells were cultured in RPMI 1640 medium (Gibco, Cat #11875093) supplemented with 10% FBS and 1% Pen Strep (Penicillin \u0026ndash;Streptomycin) and maintained at 37\u0026ordm;C in a humidified atmosphere of 5% CO\u003csub\u003e2,\u003c/sub\u003e with intermittent shaking in an upright T25 flask.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e3. Gene expression analysis:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTotal RNA was isolated from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer\u0026rsquo;s protocol. 1\u0026micro;g of RNA was reverse transcribed using Bio-Rad iScript cDNA synthesis kit (cat# 1708890) and quantitative real-time PCR was performed using Kappa Sybr Fast qPCR kit (cat# KK4601) using Bio-Rad CFX96 system. Relative mRNA expression levels were quantified using the ΔΔC(t) method. Results were normalized to housekeeping human β-actin. Details of primers used are described in Table S2.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e4. Histopathology \u0026amp; light microscopy\u003c/p\u003e \u003cp\u003eParaffin-embedded specimens of rabbit xenograft eyes (n\u0026thinsp;=\u0026thinsp;4 eyes per group) were used. 4\u0026micro;m paraffin sections were dewaxed at 60\u0026deg;C, rehydrated in decreasing concentration of ethanol. Slides were stained with hematoxylin \u0026amp; eosin according to standard procedures. Brightfield images were captured using Axioplan 2; (Carl Zeiss, Oberkochen, Germany)\u003c/p\u003e \u003cp\u003e5. Lentiviral plasmids and vectors:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe constructed a lentiviral plasmid expressing the \u003cem\u003eRB1\u003c/em\u003e gene in the pCL20 backbone. We purchased commercially available overexpression plasmids for \u003cem\u003eE2F2\u003c/em\u003e (cat#TOLH-1508827, Transomics Technologies Inc, USA) and \u003cem\u003eHK1\u003c/em\u003e (cat#TOLH-1505162, Technologies Inc, USA) in pLX304 lentiviral backbone having CMV promoter. The corresponding shRNA constructs for \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eHK1\u003c/em\u003e were in the pZIP lentiviral backbone containing CMV promoter (Transomics Technologies Inc, USA) and the target sequences are available in Table S3. Lentiviral transduction was used for \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eE2F2, HK1\u003c/em\u003e overexpression and knock-down in cell lines using the previously described protocol (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Lentivirus was produced in HEK 293T cells, and the media supernatant was concentrated by centrifugation. 1x10\u003csup\u003e6\u003c/sup\u003e WERI-Rb1 cells in free serum-free media were transduced with 50x concentrated lentiviral preparations of \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eE2F2, HK1\u003c/em\u003e \u0026amp; their knock-down viruses in 6 well plates for 4 hours, with intermittent shaking at every 30 minutes. The specific gene expression efficiencies were determined using RT-PCR after 72 hrs.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e6. Western blotting:\u003c/p\u003e \u003cp\u003eFor Western blot analysis, cells were lysed in RIPA buffer (20mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 0.08% Sodium Deoxycholate, 1% NP40 supplemented with 1 tablet of protease inhibitor (Complete ultra mini-tablet, Roche) and phosphatase inhibitor (PhosStop tablet, Roche). 20\u0026micro;g of total protein was loaded per lane and were separated by SDS-PAGE. The separated proteins on the gel were transferred onto PVDF membrane and were probed for specific antibodies against Rb (cat# 9390; Cell signaling) phospho-Rb (cat# 8516, Cell signaling) E2F2 (ab209662; Abcam), HK1(cat# 2024; Cell signaling), HK2 (cat#2867; Cell signaling), PKM2 (cat#4053; Cell signaling), LDHA (cat#3582; Cell signaling), HIF1α (cat#14179, Cell signaling), LKB1 (cat#3050; Cell signaling), AMPKα1\u0026thinsp;+\u0026thinsp;α2 (ab80039; Abcam), phospho-AMPKα (cat#2535; Cell signaling), ACC (cat#3662; Cell signaling), phospho-ACC (cat#3661; Cell signaling), α-Tubulin (cat# 3873; Cell signaling) and GAPDH (cat#5174; Cell signaling) at 1:1000 dilution in 5%BSA in 1xTBST, overnight at 4\u0026deg;C. After 4 washes with 1x TBST for 10 minutes, membranes were incubated with HRP-conjugated anti-mouse (cat#7076; Cell signaling) or anti-rabbit antibodies (cat#7074; Cell signaling) at 1:2000 dilution for 2 h. Images were visualized using the Image Quant LAS 500 system (GE Healthcare Life Sciences, USA).\u003c/p\u003e \u003cp\u003e7. Co-immunoprecipitation:\u003c/p\u003e \u003cp\u003eWERI-Rb1 cells (2x10\u003csup\u003e6\u003c/sup\u003e cells) were washed with ice-cold PBS and then lysed in a solution containing 10 mM Tris at pH 8, 170 mM NaCl, 0.5% NP40, and protease inhibitors for 30 min on ice. Cell lysates were removed by centrifugation and the supernatants were incubated with antibodies for Rb, HK1, LKB1, and HIF1α and IgG (as control) overnight at 4\u0026deg;C for their respective immunoprecipitations and with protein G\u0026ndash;Sepharose for a further 2hr. Beads were washed four times with 1 ml of wash buffer (containing 200 mM Tris at pH 8.0, 100 mM NaCl and 0.5% NP-40). For subcellular fraction immunoprecipitation, WERI-Rb1 cells (4x10\u003csup\u003e6\u003c/sup\u003e) per condition were used. Cytoplasmic \u0026amp; mitochondrial fractions were separated using extraction buffers provided with a cytochrome-c release kit (Abcam, ab65311). The nuclear fraction was lysed in a solution containing 10 mM Tris at pH 8, 170 mM NaCl, 0.5% NP40 with protease inhibitors for 30 min on ice. The respective cellular fractions were incubated with respective primary antibodies for immunoprecipitations. Bound proteins were eluted with SDS sample buffer and separated on SDS PAGE or NuPAGE Novex 4\u0026ndash;12% Bis-Tris gels before immunoblotting with specific antibodies.\u003c/p\u003e \u003cp\u003e8. Cell proliferation assay:\u003c/p\u003e \u003cp\u003eWERI-Rb1 cells were transduced with lentiviruses for control, \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eE2F2\u003c/em\u003e \u0026amp; \u003cem\u003eHK1\u003c/em\u003e overexpression and used for the proliferation assay. 10000 WERI-Rb1 \u0026amp; GL1-Rb1 cells were seeded in 24 well plates for proliferation assay. Cell viability was determined once every 24hours for 4 consecutive days using trypan blue cell staining and cell counting using a hemocytometer. In treatment models, 10000 cells were seeded onto 24 well plates and treated with dorsomorphin (0.1mM) and AICAR (100\u0026micro;M) for up to 96hours. Mock treated (0.1% DMSO) cells were used as control. The cell viability was determined using a trypan blue assay. The experiments were performed in three experimental repeats in triplicates for different experimental conditions. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of triplicate experiments.\u003c/p\u003e \u003cp\u003e9. Cell migration \u0026amp; invasion assays:\u003c/p\u003e \u003cp\u003eCell migration \u0026amp; invasion assays were performed in 24-well transwell plates with cell culture inserts (BD Falcon). Post 72hours of transductions in WERI-Rb1 cells, for invasion assays, 15000 WERI-Rb1 cells in 150\u0026micro;l 0% RPMI media were seeded in transwell insert coated with 1% matrigel \u0026amp; incubated for 48 hours. The bottom chamber was filled with 600\u0026micro;l of 10% RPMI media. After 48-hour incubation, cells on the insert were removed using a cotton swab. Migrated cells on the lower surface of the insert membrane were fixed with 4% PFA and stained with 0.1% crystal violet. Images were captured at brightfield using Olympus CKX53 microscope. Cells were further lysed using 10% SDS and absorbance of crystal violet was measured at 595 nm using a microplate reader. For migration assay, the cells that migrated to the bottom chamber at 48hours were counted using trypan blue cell staining and cell counting using a hemocytometer. For drug treatments, 15000 WERI-Rb1 cells were seeded in 0% RPMI media in the transwell insert coated with 1% matrigel and the cells were further treated with dorsomorphin (0.1mM) and AICAR (100\u0026micro;M) for 48hours. Invasive and migrated cells were quantified using a 0.1% crystal violet staining protocol. Data were expressed as replicate data points\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of triplicate experiments.\u003c/p\u003e \u003cp\u003e10. Colony formation/ Tumor spheroid assay:\u003c/p\u003e \u003cp\u003eThe spheroid formation assays were carried out on a low attachment U-bottom 96 well plate (BRAND\u0026reg; 96-well microplate, Sigma Aldrich). Single-cell suspension of 500 cells in 10% RPMI medium was loaded in each well of a 96 well plate followed by centrifugation for 1000rpm for 1 min to facilitate cell aggregation. The cells were cultured at 37\u0026deg;C in a 90% humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e for 7 days for the generation of tight and regular tumor spheroids. For drug treatments, the 7-day-old spheroids were treated with 100\u0026micro;l of freshly prepared medium containing Topotecan (10nM), Dorsomorphin (0.1mM), Metformin (10mM) and AICAR (100\u0026micro;M) for 48hours. Mock treatment was used as a control. Spheroids were imaged using the EVOS FL imaging system, Invitrogen. ImageJ 2.1 software was used for spheroid area measurements. Data were expressed as replicate data points\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of triplicate experiments.\u003c/p\u003e \u003cp\u003e11. Chemosensitivity assay:\u003c/p\u003e \u003cp\u003eCell viability after chemotherapeutic drug topotecan IC\u003csub\u003e50\u003c/sub\u003e (10nM) treatment for 48hours was determined by Presto Blue cell viability reagent (Invitrogen) as per manufactures protocol. In brief, WERI-Rb1 cells (5x10\u003csup\u003e3\u003c/sup\u003e) were plated into 96-well plates (Eppendorf, Sigma Aldrich) and incubated overnight. Cells were treated with topotecan IC\u003csub\u003e50\u003c/sub\u003e (10nM) for 48 hours. Four hours before the end of treatment, presto-blue reagent (Invitrogen) was added and incubated for 2 hours followed by measurement of fluorescence (540 nm excitation/590 nm emissions). The chemo-sensitivity of all treated cells was determined across conditions and compared against control mock-treated cells (considered as 100% viable). Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of triplicate experiments.\u003c/p\u003e \u003cp\u003e12. Cell cycle analysis:\u003c/p\u003e \u003cp\u003eWERI-Rb1 cells post 72hours transduction, were further synchronized in 0% RPM1 medium for 24hrs. After synchronization, the cells were trypsinized and fixed in 70% ice-cold ethanol overnight at 4\u0026deg;C and stained with propidium iodide (PI) solution (1\u0026micro;g/\u0026micro;l) and 0.125% RNase A (Sigma Aldrich) at room temperature for 15 minutes. Approximately 10000 cells were gated per condition and were analyzed using BD FACS Canto II and FACSDiva software.\u003c/p\u003e \u003cp\u003e13. Mitochondrial Stress assay:\u003c/p\u003e \u003cp\u003eCells were seeded onto an XFp 8-well flux plate (Seahorse Bioscience) precoated with poly-L-lysine. The cell density of 4000 cells/ 150ul per well were seeded and centrifuged at 500rpm to encourage adhesion to the plate and form an evenly dispersed monolayer. Cells were then incubated at 37\u0026deg;C non-CO\u003csub\u003e2\u003c/sub\u003e conditions and further processed using the XFp Extracellular Flux Analyzer as per the manufacturer\u0026rsquo;s protocols. Mitochondrial function was measured as OCR after injections of 0.5 \u0026micro;M oligomycin, 1 \u0026micro;M FCCP, 1 \u0026micro;M antimycin A and 1 \u0026micro;M rotenone, according to the manufacturer's instructions. Determinants of mitochondrial function (basal respiration, maximal respiration, spare respiratory and ATP production) were calculated using the formulas according to manufactures protocol. For measuring mitochondrial respiration in therapeutic models, the WERI-Rb1 cells were pre-treated with 0.1% DMSO, AICAR (100\u0026micro;M) and AICAR (100\u0026micro;M) and Topotecan (10nM) for 48hours and further re-seeded a cell density of 5x10^3 WERI-Rb1 cells onto poly-L-lysine coated 8 well flux plates for measuring mitochondrial respiration and energetics. All measurements are normalized to a total number of cells using Presto-blue cell viability reagent (Invitrogen) post mitochondrial stress assay. Data were analyzed using Seahorse XFp Wave Software (Version 2.4) and expressed as replicate data points\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of triplicate experiments.\u003c/p\u003e \u003cp\u003e14. Glycolytic Rate Assay\u003c/p\u003e \u003cp\u003eFor glycolytic rate analysis in WERI-Rb1 cells of different conditions, the cells were seeded onto a poly-L-lysine coated XFp 8-well flux plate in Seahorse XF glycolytic assay medium. Then, ECAR baseline readings were recorded using the Seahorse XFp analyzer and the following injections were done with 4 \u0026micro;M Rot/AA and 50 mM 2-deoxyglucose (2-DG) respectively. PER, glycoPER, basal glycolysis, basal proton efflux rate and compensatory glycolysis, were calculated using the manufacturer\u0026rsquo;s formula.\u003c/p\u003e \u003cp\u003e15. Immunofluorescence/ Mito Tracker green:\u003c/p\u003e \u003cp\u003e5x10\u003csup\u003e3\u003c/sup\u003e WERI-Rb1 cells per transduced condition were seeded on 96 well plates (Eppendorf) precoated with poly-L-lysine. The cells were stained with 1:5000 dilution of Mito Tracker green in 10% RPMI media and Hoechst 33342 for 15minutes. The images were captured using ImageXpress High content confocal system (Molecular device) and the mitochondrial intensity was calculated using MetaXpress software (Molecular device).\u003c/p\u003e \u003cp\u003e16. Checkerboard microdilution assay:\u003c/p\u003e \u003cp\u003eWERI-Rb1 cells were seeded at a concentration of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well in a 96-well plate and cultured at 37\u0026deg;C in a humidified 5% CO2 incubator before the assay. IC50 values of topotecan (10nM), AICAR (100\u0026micro;M) \u0026amp; Dorsomorphin (0.1mM) were determined using cell viability assays and were further diluted to various fractions of IC50 (IC50/2, IC50/4, IC50/8). To test the interaction of the drugs, each fractional concentration of AICAR or Dorsomorphin was tested against topotecan with varying IC50 fraction values for 48hours. The percentage of viable cells in each condition was determined using presto-blue viability assay and was represented as a heat map. The fractional inhibitory concentration (FIC) of AICAR or Dorsomorphin with topotecan was calculated and interpreted as per the standard procedures. The Fractional Inhibitory Concentration Index (FICi) was determined by combined FIC value for AICAR or Dorsomorphin and topotecan (FICi\u0026thinsp;=\u0026thinsp;FIC of AICAR or Dorsomorphin\u0026thinsp;+\u0026thinsp;FIC of topotecan). FICi value of \u0026le;\u0026thinsp;0.5 was considered synergistic; a value of \u0026gt;\u0026thinsp;0.5\u0026ndash;1 indicated an additive effect of the two drugs; and a FICi value of \u0026gt;\u0026thinsp;1 displayed the antagonism of the two drugs. Data were expressed as heatmap indicating the mean of triplicate experiments.\u003c/p\u003e \u003cp\u003e17. Rabbit intraocular tumor xenograft model:\u003c/p\u003e \u003cp\u003eExperimentation on rabbits was performed by the statement for the use of animals in ophthalmic and vision research approved by the Association for Research in Vision and Ophthalmology. New Zealand rabbits (n\u0026thinsp;=\u0026thinsp;28; 4 eyes per group) with a mean initial weight of around 3 kg were used in this study. Immunosuppression was attained with daily subcutaneous injections of cyclosporine A (CsA; 15mg/kg/day for week 1 till week 6, followed by 10mg/kg/day for week 7 till week 10). All the animals received subretinal injection of cultured WERI-Rb1 cells (1.5x10\u003csup\u003e6\u003c/sup\u003e in 30 \u0026micro;l volume) in each eye. After the development of vitreous seeds, the animals were grouped into Group A, B, C, D, E, F and G, those receiving HK1 or E2F2overexpressionn, no treatment, Vehicle (PBS), Topotecan at IC\u003csub\u003e50\u003c/sub\u003e (25\u0026micro;g) and IC\u003csub\u003e100\u003c/sub\u003e (50\u0026micro;g), combined AICAR IC\u003csub\u003e50\u003c/sub\u003e (15\u0026micro;g)/topotecan IC\u003csub\u003e50\u003c/sub\u003e (25\u0026micro;g) via intravitreal injection (in 50 \u0026micro;l volume). The intravitreal doses of drugs were determined by assessment of in-vitro cytotoxicity profile and extrapolation to arrive at IC\u003csub\u003e50\u003c/sub\u003e \u0026amp; IC\u003csub\u003e100\u003c/sub\u003e concentration in the rabbit vitreous as described previously (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). The tumor growth was monitored for 3 weeks post-treatment. Daily cage-side observations were performed on all animals to monitor their health and ocular abnormalities.\u003c/p\u003e \u003cp\u003e18. Statistical analysis:\u003c/p\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d unless indicated otherwise, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. For all representative images, results were reproduced at least three times in independent experiments. For all quantitative data, the statistical test used is indicated in the legends. A statistical \u0026lsquo;decision tree\u0026rsquo; is shown in Figure S5.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e The authors thank Dr G Kumarmanickavel and Dr Swaminathan Sethu for their expertise and assistance throughout all aspects of our study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e SH receives personal fees for scientific advice to Astra-Zeneca, Cellprothera and Merck; unrestricted research grant from Pfizer, outside the content of this work. The other authors have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e VSB and AG designed the experiments and wrote the manuscript. VSB performed cell line experiments. AM, GD and RS provided human tissue samples and helped interpret correlations with clinical data. DSA, SG, RK and NG performed the analyses and assisted with figure preparation and wrote relevant methods. NKV, LR and ABV developed the intra-ocular tumour model and performed animal experiments. SSC and SSB edited the manuscript. SH edited the manuscript, helped with data analysis and scientific guidance. ABV prepared the animal experiment data and wrote relevant methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information:\u003c/strong\u003e Supplementary data figures and tables are available for this paper. Original uncropped western blots are also provided as single Supplementary data file for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e All data and analyses in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e: The study was conducted by the Declaration of Helsinki principles under a protocol approved by the institutional ethics committee of Narayana Nethralaya (EC Ref no: C/2013/03/02). Informed written consent was received from the parents before inclusion in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement:\u003c/strong\u003e The authors thank Narayana Nethralaya Foundation for funding research support to VSB, APN, RK, AG. Research internship of VSB in NKV lab was supported by NKV\u0026rsquo;s Start-Up Grant, LKC Medicine, Nanyang Technological University Singapore (L0412290). The funders had no role in design, data collection, analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFriend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323(6089):643\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarshall AE, Roes MV, Passos DT, DeWeerd MC, Chaikovsky AC, Sage J, et al. RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression. Mol Cell Biol. 2019;39(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDyson NJ. RB1: a prototype tumor suppressor and an enigma. Genes Dev. 2016;30(13):1492\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoel S, DeCristo MJ, McAllister SS, Zhao JJ. 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Rb suppresses human cone-precursor-derived retinoblastoma tumours. Nature. 2014;514(7522):385\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajagopal R, Zhang S, Wei X, Doggett T, Adak S, Enright J, et al. Retinal de novo lipogenesis coordinates neurotrophic signaling to maintain vision. JCI Insight. 2018;3(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGadea E, Thivat E, Planchat E, Morio B, Durando X. Importance of metabolic changes induced by chemotherapy on prognosis of early-stage breast cancer patients: a review of potential mechanisms. Obesity reviews: an official journal of the International Association for the Study of Obesity. 2012;13(4):368\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan HS, Gallie BL, Munier FL, Beck Popovic M. Chemotherapy for retinoblastoma. Ophthalmology clinics of North America. 2005;18(1):55\u0026ndash;63, viii.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrennan RC, Qaddoumi I, Mao S, Wu J, Billups CA, Stewart CF, et al. Ocular Salvage and Vision Preservation Using a Topotecan-Based Regimen for Advanced Intraocular Retinoblastoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2017;35(1):72\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim LS, Dalvin LA, Ancona-Lezama D, Yu MD, Jabbour P, Shields CL. Retinoblastoma vascular perfusion and intra-arterial chemotherapy cycle requirements. Clinical \u0026amp; experimental ophthalmology. 2019;47(9):1164\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, Depinho RA, et al. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science. 2005;310(5754):1642\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArakawa S, Yoshida T, Shirasawa M, Takayanagi D, Yagishita S, Motoi N, et al. RB1 loss induced small cell lung cancer transformation as acquired resistance to pembrolizumab in an advanced NSCLC patient. Lung cancer. 2021;151:101\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma A, Tovey JC, Ghosh A, Mohan RR. AAV serotype influences gene transfer in corneal stroma in vivo. Experimental eye research. 2010;91(3):440\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBacklund LM, Nilsson BR, Liu L, Ichimura K, Collins VP. Mutations in Rb1 pathway-related genes are associated with poor prognosis in anaplastic astrocytomas. British journal of cancer. 2005;93(1):124\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatnaik MM, Lasho T, Howard M, Finke C, Ketterling RL, Al-Kali A, et al. Biallelic inactivation of the retinoblastoma gene results in transformation of chronic myelomonocytic leukemia to a blastic plasmacytoid dendritic cell neoplasm: shared clonal origins of two aggressive neoplasms. Blood cancer journal. 2018;8(9):82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC, et al. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nat Protoc. 2018;13(12):2991\u0026ndash;3017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogan CM, Kaczmarek JV, Pierce JM, Chen SC, Boyd KL, Calcutt MW, et al. Evaluation of intravitreal topotecan dose levels, toxicity and efficacy for retinoblastoma vitreous seeds: a preclinical and clinical study. Br J Ophthalmol. 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaniels AB, Pierce JM, Chen SC. Complete preclinical platform for intravitreal chemotherapy drug discovery for retinoblastoma: Assessment of pharmacokinetics, toxicity and efficacy using a rabbit model. MethodsX. 2021;8:101358.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: \u003c/strong\u003eClinical and histopathological details of samples used in the study\u003c/p\u003e\n\u003ctable width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e\u003cstrong\u003eID\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003eLaterality\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u003cstrong\u003eAge at presentation (months)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Risk\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e\u003cstrong\u003eIIRC Group\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003eAJCC Staging\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003eBilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e15 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e20 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eP3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"83\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e24 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNon-advanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003eCT2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eP4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"83\"\u003e\n\u003cp\u003eBilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e4 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eP5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"83\"\u003e\n\u003cp\u003eBilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e30 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003eBilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e21 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eP7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"83\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e28 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNon-advanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eGroup D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eP8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"83\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e20 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNon-advanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eGroup D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eP9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"83\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e21 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNon-advanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eGroup D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e23 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eBilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e24month\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e36 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eBilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e33 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eAdvanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT3a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e36 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd 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width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e14months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNon-advanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroupD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT2b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eP25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e11 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003eUnilateral\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNon-advanced\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003eGroup D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ecT2b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e2 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eMultiple organ dysfunction ( No ocular complications)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eC2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e12 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eNo ocular complications\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Retinoblastoma, Hexokinase-1, AMPKα, glycolysis, oxidative phosphorylation, cancer","lastPublishedDoi":"10.21203/rs.3.rs-1779138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1779138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLack of retinoblastoma protein (Rb) causes aggressive intraocular retinal tumors in children. Recently, Rb tumors have been shown to have a distinctly altered metabolic phenotype, such as reduced expression of glycolytic pathway proteins alongside altered pyruvate and fatty acid levels. In this study, we demonstrate that loss of Hexokinase 1(HK1) in tumor cells rewires their metabolism allowing enhanced oxidative phosphorylation-dependent energy production. We show that rescuing \u003cem\u003eHK1\u003c/em\u003e or \u003cem\u003eRB1\u003c/em\u003e in these retinoblastoma cells reduced cancer hallmarks such as proliferation, invasion, spheroid formation and increased their sensitivity to chemotherapy drugs. Induction of HK1 was accompanied by a metabolic shift of the cells to glycolysis and a reduction in mitochondrial mass. Cytoplasmic HK1 bound Liver Kinase B1 (LKB1) and phosphorylated AMP-activated kinase-α (AMPKα \u003csup\u003eThr172\u003c/sup\u003e), thereby reducing mitochondria-dependent energy production. We validated these findings in tumor samples from Rb patients compared to age-matched healthy retina. \u003cem\u003eHK1\u003c/em\u003e or \u003cem\u003eRB1\u003c/em\u003e expression in Rb-/- cells led to reduction in their respiratory capacity and glycolytic proton flux. \u003cem\u003eHK1\u003c/em\u003e overexpression reduced tumor burden in an intraocular tumor xenograft model. AMPKα\u003cem\u003e \u003c/em\u003eactivation by\u003cem\u003e \u003c/em\u003eAICAR also enhanced the tumoricidal effects of chemotherapeutic drug topotecan \u003cem\u003ein vivo\u003c/em\u003e. Therefore, enhancing HK1 or AMPKα activity can reprogram cancer metabolism and sensitize retinoblastoma tumors to lower doses of existing treatments, a potential therapeutic modality for retinoblastoma.\u003c/p\u003e","manuscriptTitle":"Enhanced oxidative phosphorylation in Retinoblastoma tumors is dependent on depleted Hexokinase1 and lack of AMPKα activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-01 20:32:27","doi":"10.21203/rs.3.rs-1779138/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"583b58e2-5df7-43af-8b5f-ed176568bfa9","owner":[],"postedDate":"August 1st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-05T11:03:13+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-01 20:32:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1779138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1779138","identity":"rs-1779138","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}