Haploinsufficiency of miR-143 and miR-145 reveal targetable dependencies in resistant del(5q) myelodysplastic syndrome

Haploinsufficiency of miR-143 and miR-145 reveal targetable dependencies in resistant del(5q) myelodysplastic syndrome | 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 Haploinsufficiency of miR-143 and miR-145 reveal targetable dependencies in resistant del(5q) myelodysplastic syndrome Aly Karsan, Nadia Gharaee, Joanna Wegrzyn-Woltosz, Grace Cole, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4339623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Feb, 2025 Read the published version in Leukemia → Version 1 posted 9 You are reading this latest preprint version Abstract Myelodysplastic syndromes (MDS) are stem cell disorders characterized by ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Chromosomal alterations are frequent in MDS, with interstitial deletion of chromosome 5q (del(5q)) being the most common. Lenalidomide is the current first-line treatment for del(5q) MDS and its efficacy relies on degradation of CK1α which is encoded by the CSNK1A1 gene located in the commonly deleted region (CDR) of chromosome 5q. However, lenalidomide-resistance is common, often secondary to loss-of-function mutations in TP53 or RUNX1 . The CDR in del(5q) harbors several genes, including noncoding miRNAs, the loss of which contribute to disease phenotypes. miR-143 and miR-145 are located within the del(5q) CDR, but precise understanding of their role in human hematopoiesis and in the pathogenesis of del(5q) MDS is lacking. Here we provide evidence that deficiency of miR-143 and miR-145 plays a role in clonal expansion of del(5q) MDS. We show that insulin-like growth factor 1 receptor (IGF-1R) is a direct target of both miR-143 and miR-145 . Our data demonstrate that IGF-1R inhibition reduces proliferation and viability of del(5q) cells in vitro and in vivo , and that lenalidomide-resistant del(5q) MDS cells depleted of either TP53 or RUNX1 are sensitive to IGF-1R inhibition. Resistant del(5q) MDS-L cells, as well as primary MDS marrow cells, are also sensitive to targeting of IGF-1R-related dependencies in del(5q) MDS, which include the Abl and MAPK signaling pathways. This work thus provides potential new therapeutic avenues for lenalidomide-resistant del(5q) MDS. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis that lead to bone marrow failure or progression to AML. 1–3 MDS is characterized by multiple cytogenetic and molecular defects, which result in an extremely heterogeneous phenotype, making design of molecular-targeted therapies a challenge. 4 Interstitial deletion of the long arm of chromosome 5 is a common genetic aberration seen in MDS. 9 MDS with isolated del(5q) is characterized by macrocytic anemia and hypolobulated megakaryocytes. 6,7 The immunomodulatory drug lenalidomide (LEN) has shown great efficacy in del(5q) MDS patients, leading to improved blood counts and survival. 8,9 However, 30–40% of del(5q) MDS patients are refractory to LEN in the first-line setting, and at least half of primary responders become resistant within two years. 8,10 LEN functions by Cereblon-mediated CK1α degradation, the protein product of the CSNK1A1 gene. 11 CSNK1A1 is located on the CDR of del(5q) MDS which makes del(5q) cells particularly sensitive to further degradation of the protein by LEN. We and others have shown that LEN-resistance is most frequently associated with loss-of-function mutations in TP53 , and we have provided direct evidence that TP53 and RUNX1 mutations inhibit LEN-induced cell death in the context of CSNK1A1 haploinsufficiency 12,13 . Mutation of either of these two genes inhibits differentiation of del(5q) MDS cells into megakaryocytes, which is required for LEN-induced death of del(5q) cells. 13 miR-143 and miR-145 , both located within the CDR, have reduced expression in del(5q) MDS. 14,15 Deficiency of miR-145 leads to activation of the TGFβ and TIRAP pathways. 15–17 However, most experiments have been conducted in mouse models and very little is known about how miR-143/miR-145 haploinsufficiency affects human hematopoiesis. We hypothesized that haploinsufficiency and subsequent derepression of miR-143/miR-145 targets may support the clonal expansion seen in del(5q) MDS. 18,19 As miR-143 and miR-145 are transcribed as a single primary miRNA transcript and both of these miRNAs have been characterized as tumor suppressor genes in solid tumors 20 , we examined the role of combined haploinsufficiency of these miRNAs in primary human CD34 + cells. In this study, we show that depletion of these two miRNAs results in clonal expansion of human hematopoietic stem/progenitor cells (HSPC), and that targeting pathways activated by miR-143/miR-145 haploinsufficiency reveals a CK1α-independent vulnerability of del(5q) MDS that remains even when these cells are resistant to LEN. Specifically, we show that IGF-1R signaling is activated with deficiency of miR-143/miR-145 and pharmacologic inhibition of IGF-1R signaling is able to target del(5q) myeloid cells that are resistant to LEN. In addition, we demonstrate that LEN-resistant del(5q) myeloid cells are sensitive to inhibition of pathways regulated by IGF-1R including the Abl and MAPK signaling pathways, providing potential novel therapeutic approaches for del(5q) MDS. Material and Methods Cell lines, human cord blood cells and patient MDS cells The leukemic cell lines KG1a, KG1, UT7, K562, U937, and THP1 were purchased from the American Type Culture Collection. The MDS-L cell line, was provided by K. Tohyama (Kawasaki Medical School, Okayama, Japan). 21 Human umbilical cord blood cells were obtained with consent under a protocol approved by the Research Ethics Board (REB) of the University of British Columbia (UBC). Primary umibilical cord blood cells and MDS samples were obtained, following patient consent, from the Hematology Cell Bank of British Columbia under a protocol approved by the University of British Columbia Research Ethics Board, as well as by institutional review boards at the Royal Adelaide Hospital, and Moffitt Cancer Center. Lentiviral production and transduction The miR-143/miR-145 decoy was generated by cloning tandem repeats of sequences complementary to miR-143 (MIMAT0000435) and miR-145 (MIMAT0000437) into the 3’UTR of GFP in the lentiviral vector pLL3.7. Mature miRNAs were expressed using the pCCL.PPT.MNDU3.PGK.GFP lentiviral vector. 22,23 ShRNA (MISSION shRNA, Sigma-Aldrich, Burlington, MA, US) were expressed from pLKO.1. VSV-G pseudotyped lentiviral particles (5–10 x 10 9 /ml) were used to transduce purified cord blood CD34 + cells or MDS-L cells as previously described. 24 qRT-PCR For experiments done with primary human cells, miRNA expression was assayed as previously described. 19 For experiments done with cell lines, miRNA expression was assayed using TaqMan assays with U47 snoRNA as a reference gene, per manufacturer's directions (assays #002249 (miR-143-3p), #002278 (miR-145-5p), and #001223 (U47), Thermo Fisher Scientific, Waltham, MA, US). Gene expression was analyzed using SYBR green master mix (Applied Biosystems, cat #4368708, Waltham, MA, US). Biotin-Based RNA pulldown assay Biotin-based pulldown assays were carried out as previously described (see Supplemental Methods for details). 21,22 Luciferase assay The Dual-Luciferase assay kit was obtained from Promega (Madison, WI, US). Wildtype (WT) and miR-143/miR-145 binding site-mutated IGF-1R 3’-UTR (135 bp) were cloned downstream of a luciferase reporter in the pSGG vector. Reporter constructs along with either vectors overexpressing the miRNAs or empty vector were transfected into HEK293 cells. Luciferase activity was assessed 48 hrs post-transfection according to the manufacturer’s protocol. Immunoblotting Protein lysates were obtained by lysing cells in cold RIPA buffer (50mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% SDS), in the presence of PMSF, sodium orthovanadate, protease and phosphatase inhibitors (Sigma-Aldrich, Burlington, MA, US). Immunoblots used the following antibodies: IGF-1R (sc-772; Santa Cruz, Dallas, TX), GAPDH (G8795; Sigma-Aldrich, Burlington, MA, US). Growth, survival, and proliferation assays The IGF-1R small molecule inhibitor BMS-536924 (cat #S1012), Lenalidomide (cat #S1029), Imatinib (cat #S2475), Trametinib (cat #S2673), Temsirolimus (cat #S1044) and Everolimus (cat #sS1120) were purchased from Selleck Chemicals (Houston, TX, US) and resuspended in DMSO. Doses for Imatinib, Trametinib, Temsirolimus and Everolimus were calculated to be similar to serum drug concentrations in patients receiving drug for cancer-related indications 23–27 . MDS-L cell expansion in liquid culture was determined based on manual Trypan blue counts. Annexin V (BD Biosciences, Frank Lakes, NJ, US) and propidium iodide (Sigma-Aldrich, Burlington, MA, US) staining was performed according to the manufacturers' instructions. For BrdU assays, cells were pulsed with 10 mM BrdU for 1.5 h at 37°C, and then fixed/stained with anti-BrdU antibody according to manufacturer’s instructions (FITC or APC BrdU Flow kit; BD Biosciences, Franklin Lakes, NJ, US). Colony-forming cell (CFC) assays Clonogenic assays were performed by plating 0.5–1 x 10 3 /ml human CD34 + HSPC or MDS-L cells in MethoCult H4434 (STEMCELL Technologies, Vancouver, BC, Canada); colonies were scored after 10–14 days. CFC assays colonies were scored as erythroid, myeloid or mixed erythroid-myeloid by morphological criteria; for replating assays, cells were scraped into PBS, then washed and replated into fresh methylcellulose. Xenotransplantation All transplants were performed on sublethally irradiated (315 cGy, 137 Cs) 8-12-week-old female immunodeficient mice. CD34 + HSPC xenotransplantation was performed by intrafemoral injection of 1–2 x 10 5 CD34 + cells into NOD/SCID-IL2Rγ ( NSG) mice, while MDS-L cells (10 6 ) were injected into the tail vein of NOD/Rag1/IL2Rγ (NRG) mice producing human IL-3, GM-CSF, and SCF (NRG-3GS). In vivo delivery of the IGF-1R small molecule inhibitor was performed as previously described: 25 BMS-536924 was dissolved in DMSO (10 mM) and further diluted in 10% solutol (Sigma-Aldrich, Burlington, MA, US). Animals were injected intraperitoneally with BMS-536924 (40 mg/kg) 3 times weekly starting at 2 weeks post-transplant. Mice were bred and maintained at the BC Cancer Research Center Animal Resources Facility (Vancouver, BC, Canada). Animal experimental protocols were approved by the UBC Animal Care Committee. Flow cytometry Human cell engraftment was monitored by flow cytometry on a FACS Calibur, LSRFortessa or AriaII cell sorter (BD Biosciences, Franklin Lakes, NJ, US) (see Supplemental Methods for details). IPA and GSEA analysis Predicted targets of mir-143 and miR-145 from TargetScan (Version 6.2; http://targetscan.org ) 26 were subjected to pathway annotation analysis using Ingenuity Pathway Analysis (IPA) software (QIAGEN Bioinformatics). Gene Set Enrichment Analysis (GSEA) was used to analyze published MDS gene expression data for MIR143 and MIR14 5 expression using the continuous phenotype label method 28,29 . Sequencing analysis A target hybridization capture library was constructed and subjected to Illumina sequencing as per routine clinical protocol. The target space includes all recurrently mutated myeloid genes. The generated libraries were aligned against GRCh37 with variants called on samtools mpileup (version 0.1.18) 30 , using VarScan2 (version 2.3.6) 31 , ONCOCNV (version 6.6) 32 , and GenomonSV (version 0.5.0) packages. Variants were manually curated as per ACMG guidelines. Statistical analysis All statistics were calculated in Prism (GraphPad, La Jolla, CA, US). Results are shown as mean ± standard error of the mean (SEM) unless otherwise indicated. Statistical significance was determined using Welch’s t test for unpaired data, with multiple testing correction (Holm-Sidak method) when appropriate. Data from paired samples were compared using the ratio t test. The log-rank (Cox test) was used to compare differences between survival curves. P values below 0.05 and FDR values below 0.25 were considered to be statistically significant. Results Depletion of miR-143 and miR-145 supports clonal expansion of human HSPC To examine the role of miR-143 and miR-145 in human cells, we first confirmed that their expression was lower in CD34 + cells from del(5q) MDS patient marrow compared to MDS marrow diploid at chromosome 5q, as previously reported 15 (Fig. 1 A). Expression of both miRNAs was higher in the CD34 + stem/progenitor cell fraction compared to the more differentiated CD34 − cells, suggesting a role for miR-143 and miR-145 in HSPC (Fig. 1 B). To model miR-143/miR-145 depletion in human HSPC, we knocked down miRNA expression using lentiviral decoy constructs in CD34 + HSPCs (Supplemental Fig. 1A, B). In functional assays for hematopoietic progenitor activity, the miR-143/miR-145 decoy-transduced cells produced more colonies than vector-transduced cells in both primary and serial replating progenitor assays (Fig. 1 C). The effect on colony formation was seen in the more-primitive erythroid (BFU-E) with a trend towards an increase in the primitive mix (CFU-GEMM) colonies (Supplemental Fig. 1C). The decoy-transduced cell cultures had enhanced viability, as estimated by Annexin V and propidium iodide (PI) staining (Fig. 1 D). Thus, depletion of miR-143 and miR-145 enhances viability of CD34 + HSPC and promotes clonogenic potential in vitro . To determine the impact of miR-143 and miR-145 depletion on hematopoiesis in vivo , we injected CD34 + HSPC, two days post-lentiviral transduction with either the decoy or control vectors, into the femurs of sublethally-irradiated NSG mice. Human lymphomyeloid reconstitution in the marrow of recipient mice was monitored by serial marrow aspiration at weeks 3, 8, 20 and 34 post-transplant (Fig. 1 E). Between 8 and 34 weeks post-transplant, decoy-transduced cell populations showed increased marrow chimerism compared to vector-transduced cells (Fig. 1 F). This growth advantage included both myeloid and lymphoid GFP + populations (Fig. 1 F). In contrast, the proportions of myeloid and lymphoid cells in the non-transduced (GFP − ) human cell compartment in the same mice was similar between the two groups of transplants (Fig. 1 G), consistent with miRNA depletion providing a cell intrinsic competitive advantage in vivo . These findings support a role for haploinsufficiency of miR-143 and miR-145 in the clonal expansion of malignant cells in del(5q) MDS. miR-143 and miR-145 target IGF-1R in human hematopoietic progenitor cells We have previously determined which pathways are targeted by both miRNAs using Ingenuity Pathway Analysis (IPA) 33 on the non-redundant set of predicted targets of miR-143 and miR-145 16 . Here we used gene expression data from a previously-published cohort of MDS patients 34 , ranked samples on the basis of either the expression of miR-143 or the expression of miR-145 , and performed Gene Set Enrichment Analysis (GSEA) 28 to determine which BIOCARTA pathways were differentially upregulated with low expression of miR-143 and miR-145 (Supplemental Table 1). The IGF-1 signaling pathway was the only pathway common to both analyses (Fig. 2 A). In addition, the IGF-1 receptor (IGF-1R) was predicted to be a target of both miR-143 and miR-145 based on the miRNA-target prediction algorithm, TargetScan (Supplemental Table 2). 35 To validate the predicted interaction between miR-143/miR-145 and the 3’-UTR of IGF-1R mRNA, we performed luciferase assays using the wildtype (WT) or mutated IGF-1R 3’UTR containing mutations in the predicted binding sites, cloned downstream of a luciferase reporter. Overexpression of miR-143 or miR-145 markedly reduced luciferase activity in cells with the WT IGF-1R 3’UTR but not the mutated IGF-1R 3’UTR (Fig. 2 B). Furthermore, an miRNA pulldown assay demonstrated that biotinylated miR-143 and miR-145 transfected into del(5q) MDS-L cells directly bound to IGF-1R mRNA, but not to insulin receptor ( INSR ) mRNA, confirming the specificity of the interaction (Supplemental Fig. 2, Fig. 2 C). In addition, surface expression of IGF-1R was higher in del(5q) myeloid cell lines compared to myeloid cell lines diploid for chromosome 5q (Fig. 2 D). To confirm that the miR-143/145-IGF-1R interaction is functionally relevant in primary hematopoietic cells, we transduced CD34 + HSPC with control or miRNA-decoy vectors. Surface IGF-1R expression was increased in miRNA-decoy cells, particularly in the CD34 + HSPC fraction compared to the differentiated CD34 − cells (Fig. 2 E). Conversely, overexpression of each of the miRNAs in the MDS-L del(5q) cell line led to decreased protein expression of IGF-1R (Fig. 2 F). MDS-L cells overexpressing either miRNA had reduced colony formation in CFC assays, as well as reduced proliferation and a trend towards reduced viability (Supplemental Fig. 3). These data suggest that miR-143 and miR-145 regulate cell progenitor activity and cell proliferation in primitive human hematopoietic cells through the regulation of IGF-1R expression. IGF-1R inhibition reduces progenitor activity of miR-143 / miR-145 -haplodeficient del(5q) cells To determine which effects of miR-143/miR-145 deficiency are mediated by IGF-1R, we first asked whether specific inhibition of IGF-1R signaling in del(5q) MDS-L cells could abrogate cell expansion in vitro . To address this, we treated MDS-L cells with a small molecule IGF-1R inhibitor, BMS-536924, at various concentrations. At all tested doses of the inhibitor, there was a significant increase in cell death by 48 h of treatment (Fig. 3 A). Proliferation was also significantly reduced, in a dose- and time-dependent manner (Fig. 3 B). Cells treated with inhibitor appeared to mainly target cycling cells as cells were less likely to be in S-phase with more cells in G 0 /G 1 of the cell cycle compared to controls (Supplemental Fig. 4). At the highest dose, cells were virtually eliminated following 3 days of treatment (Supplemental Fig. 5). Consistent with the above, MDS-L cells pre-treated with an intermediate concentration (1 µM) of IGF-1R inhibitor for 48 h had reduced output in CFC assays (Fig. 3 C). To extend this result to primary cells, CD34 + HSPC were transduced with either miRNA-decoy or control vector, treated with BMS-536924 (1 µM) and subjected to CFC assays. As observed previously, miRNA-decoy-transduced cells had increased colony output (Figs. 2 E, 3 D). However, IGF-1R inhibition reduced colony formation only in decoy-transduced cells, but had no effect on vector-transduced cells (Fig. 3 D), potentially implicating a differential effect in del(5q) MDS cells compared to normal cells. Next, we used a complementary genetic approach, by targeting IGF-1R through lentiviral-delivered shRNA to reduce its expression in the del(5q) MDS-L cell line. Knockdown of IGF-1R resulted in reduced IGF-1R protein expression compared to the control shRNA-transduced cells (Fig. 3 E). Cells transduced with sh IGF-1R had reduced colony formation in clonogenic assays compared to controls (Fig. 3 F). Together, these studies suggest that pharmacologic or genetic inhibition of IGF-1R signaling reduces cell expansion observed with loss of miR-143 and miR-145 , consistent with the hypothesis that derepression of IGF-1R is functionally relevant in human del(5q) MDS. To determine whether targeting IGF-1R would prolong survival in mice xenografted with human del(5q) MDS-L cells, we used a xenograft model in which NRG-3GS immunodeficient mice expressing three human growth factors were transplanted with MDS-L cells. To determine the specific effect of IGF-1R depletion in this model, we transplanted MDS-L cells transduced with sh IGF-1R or shControl into NRG-3GS mice. Depletion of IGF-1R resulted in significantly prolonged survival of xenografted mice compared to the controls (median survival 57 days vs. 45 days, P = 0.0004) (Fig. 3 H). To confirm that pharmacological inhibition of IGF-1R would have the same effect, we xenografted parental MDS-L cells into NRG-3GS mice, and injected engrafted mice intraperitoneally with BMS-536924 (40 mg/kg) 3 times weekly starting at 2 weeks post-transplant. As with IGF-1R knockdown, chemical inhibition of IGF-1R resulted in significant improvement in median survival, compared to controls (52 days vs 44 days, P = 0.0016) (Fig. 3 I). Thus, either genetic knockdown or pharmacologic inhibition of IGF-1R inhibits the expansion of del(5q) MDS-L cells in a preclinical model of haploinsufficiency for miR-143 and miR-145. IGF-1R inhibition bypasses LEN resistance in del(5q) MDS cells Given that a significant proportion of del(5q) MDS patients do not respond to LEN or become resistant over time, we attempted to determine whether IGF-1R might be a relevant target in LEN-resistant del(5q) cells. To address this, we examined the efficacy of IGF-1R inhibitor, BMS-536924, along with LEN on four del(5q) and three non-del(5q) myeloid cell lines by measuring apoptosis and proliferation. Targeting IGF-1R reduced the viability of all del(5q) cell lines (Fig. 4 A), including those known to be resistant to LEN (KG1, HL60). 21 In contrast, survival of non-del(5q) cell lines was not affected by the addition of BMS-536924 (Fig. 4 B). We have previously shown that loss of function mutations in TP53 or RUNX1 cause LEN resistance. 13 To further assess the efficacy of IGF-1R inhibition in the context of LEN resistance we used CRISPR/Cas9 MDS-L cells targeted for TP53 or RUNX1 that are resistant to LEN 13 . Despite LEN resistance, BMS-536924 caused significant cell death and reduced the clonogenic output of TP53- or RUNX1 -targeted MDS-L cells (Fig. 4 C, D). CSNK1A1 haploinsufficiency is required for LEN-induced cell death in del(5q) MDS cells 11,36 . As miR-143/miR-145 function is likely independent of CSNK1A1 , we sought to determine if IGF-1R inhibition is effective via a pathway that is distinct from LEN by using the OCI-AML3 cell line that expresses diploid copies of CSNK1A1 , miR-143 and miR-145 . The OCI-AML3 cell line was transduced with the miR-143 / miR-145 decoy construct or empty vector, or shRNAs against CSNK1A1 or a control shRNA, and colony output of these cells was measured in the presence of either LEN, BMS-536924, or vehicle (Supplemental Fig. 6). miR-143/miR-145 decoy-transduced cells had decreased clonogenic output with BMS-536924 treatment while LEN had no effect (Fig. 4 E). In contrast, cells transduced with sh CSNK1A1 , had reduced colony number with LEN treatment but BMS-536924 had no effect (Fig. 4 F). These results verify that IGF-1R inhibition acts via a mechanism distinct from LEN/CK1α to suppress del(5q) MDS cells. Del(5q) MDS cells targeting TP53 or RUNX1 are sensitive to Abl or MAPK inhibitors IGF-1R is frequently overexpressed in different cancers, but results of clinical trials with IGF-1 or IGF-1R have not shown convincing efficacy, resulting in the lack of further development of these compounds. 28,29,30 . We thus sought to determine whether targeting IGF-1R-related pathways also exposed vulnerabilities in del(5q) MDS cells. Abl is involved in autoregulation of IGF-1R activity 40,41 . Interestingly, our GSEA analysis showed the BIOCARTA_GLEEVEC geneset, containing genes involved in Abl signaling, to be the top differentially upregulated pathway in MDS patients with low miR-143 or miR-145 (Supplemental Table 2, Fig. 5 A). We also examined whether other pathways downstream of IGF-1R were differentially upregulated with miR-143 and miR-145 haploinsufficiency. We performed GSEA analysis on previously published RNA expression data of MDS samples 34 , and showed the BIOCARTA_MAPK and BIOCARTA_MTOR pathways to both be significantly activated in patients with low expression of either miR-143 or miR-145 (Fig. 5 B, C). In contrast, the PI3K/Akt pathway was not activated with low expression of miR-143/miR-145 (Supplemental Fig. 7). These results suggest that the Abl, MAPK and mTOR signaling pathways may provide a potential therapeutic target for LEN-resistant del(5q) MDS. Given that Abl, MAPK and MTOR signaling pathways were differentially upregulated with low expression of miR-143/miR-145 , we sought to determine the dependency of del(5q) cells on these pathways. The Abl inhibitor, imatinib, reduced the viability and colony output of LEN-resistant MDS-L cells (Fig. 5 D-F). Additionally, we assessed the effect of FDA approved inhibitors of MAPK and MTOR signaling pathways. Trametinib (MEK1/2 inhibitor), everolimus and temsirolimus (mTOR inhibitors) reduced the viability of del(5q) KG1a cells significantly (Fig. 5 G), but only trametinib induced significant cell death in MDS-L cells (Fig. 5 H). As trametinib was the only effective inhibitor in both del(5q) cell lines, we evaluated the sensitivity of LEN-resistant MDS-L cells to this inhibitor. Trametinib treatment resulted in reduced viability of LEN-resistant MDS-L cells, similar to wild-type control cells, in cells deficient for either TP53 or RUNX1 (Fig. 5 I, J). LEN-resistant del(5q) MDS patients cells are sensitive to inhibition of Abl or MAPK pathways To further validate our findings, we assessed the efficacy of trametinib on ex vivo cultured cells from del(5q) MDS patients that were either sensitive or resistant to LEN. Mutational analysis on these nine del(5q) MDS patient samples identified pathogenic variants of TP53 in one sample (Supplemental Table 3). All samples regardless of their sensitivity to LEN were sensitive to both imatinib and trametinib (Fig. 6 A), while CD34 + HSPC were not sensitive to the tested inhibitors (Fig. 6 B). These results suggest that targeting Abl or MAPK signaling pathways holds therapeutic promise for LEN-resistant del(5q) MDS patients. Discussion We and others have previously shown that the del(5q) MDS phenotype is partially mediated by miRNA haploinsufficiency in mouse models 16,28,29 . Here, we identify activation of the IGF-1R pathway as a consequence of depletion of miR-143 and miR-145 in human hematopoietic cells. We confirmed that IGF-1R is a direct target of both miR-143 and miR-145 using luciferase and RNA pulldown assays. With three lines of evidence - IGF-1R knockdown by shRNA, IGF-1R depletion by overexpression of miR-143 and/or miR-145 , and chemical inhibition of IGF-1R we demonstrate that targeting IGF-1R results in reduced viability, proliferation and colony output. In complementary experiments, we show both in vitro and in vivo that depletion of miR-143 and miR-145 results in derepression of IGF-1R signaling and clonal expansion, and further that inhibition of IGF-1R rescues the phenotype of miRNA depletion in functional assays. We further provide evidence that IGF-1R inhibition is able to bypass LEN-resistance. Importantly, our data identify two FDA-approved drugs that interact with the IGF-1R signaling pathway, namely Abl kinase AND MEK inhibitors, that could be repurposed for LEN-resistant del(5q) MDS. IGF-1 signaling has been reported to play a role in growth and self-renewal of both hematopoietic and embryonic stem cells 43,44 . IGF-1 signaling also contributes to progression of several cancers including AML and MDS 26,35,36 , and bone marrow cells of some MDS patients overexpress IGF-1R compared to normal bone marrow cells 45 . Although various IGF1/IGF-1R inhibitors have undergone clinical trials in solid tumors, these trials did not examine potential biomarkers to identify susceptible cases, and this may have been the reason for the failure of these trials 28,28,36 . The IGF-1 signaling pathway involves a complex network that is regulated at multiple levels. In particular, Abl and IGF-1R are involved in an autocrine signaling loop 40,41 . GSEA analysis revealed that genes of the Abl signaling pathway (BIOCARTA_GLEEVEC) are activated with deficiency of miR-143 and miR-145 . Signals secondary to IGF-1R signaling are also activated in many cancers including acute lymphoblastic leukemia, breast and prostate cancers, and act to promote tumor cell viability and proliferation 48–51 . Our analysis demonstrated that MAPK and mTOR signaling pathways which act downstream of IGF-1R are activated in MDS samples with low expression of miR-143 and miR-145 . Importantly, we show that LEN-resistant del(5q) cells are sensitive to either imatinib or trametinib, FDA-approved inhibitors of Abl and MAPK pathways, respectively. In contrast, mTOR inhibitors while effective in one del(5q) cell line, were not able to kill a second del(5q) cell line. Activation of the MAPK pathway is documented to be involved in progression and drug resistance of many cancers including AML and MDS, which has encouraged preclinical and clinical trials 52–57 . Here we show that both imatinib and trametinib are able to reduce the viability of LEN resistant MDS-L cells and primary MDS samples. Haploinsufficiency of CSKN1A1 in MDS cells with del(5q) renders these cells sensitive to LEN 13,14 . We demonstrated that sensitivity of del(5q) MDS cells to inhibition of IGF-1R, MAPK and Abl signaling are dependent on haploinsufficiency of miR-143/miR-145 and independent of CSNK1A1 haploinsufficiency, implicating a novel parallel dependency in del(5q) MDS cells. Our findings suggest that targeting Abl and MAPK signaling pathways by repurposing FDA-approved drugs may be a novel therapeutic option for LEN-resistant MDS. Overall, our findings suggest two distinct, coexisting vulnerabilities in del(5q) MDS, both of which target haploinsufficiency of genes within the CDR of del(5q). Haploinsufficiency of CSKN1A1 in MDS cells with del(5q) renders these cells sensitive to lenalidomide. In contrast sensitivity to IGF-1R inhibition in del(5q) MDS cells is secondary to haploinsufficiency of miR-143 and miR-145 . Our finding that lenalidomide-resistant del(5q) MDS cell lines are sensitive to IGF-1R, ABL or MEK inhibition provides alternative approaches, through a coexisting but independent vulnerability, for therapeutic trials in del(5q) MDS patients who are resistant to lenalidomide. Declarations Acknowledgements This work was funded by grants from the Canadian Institutes of Health Research (PJT-183924 and PJT-162131) the Terry Fox Research Institute Program Project Grant (1074) and the Leukemia and Lymphoma Society of Canada. AK is the recipient of the John Auston BC Cancer Foundation Clinical Investigator Award, and is a Tier 1 Canada Research Chair in Blood Cancers. Author Contributions AK conceived the project. NG, JWW and AK designed experiments. NG, JWW, GC, VSA, PU, DD, AK and MF performed experiments. NG, JB, RS and AK analyzed data. DH, MK, OC, RK and EP provided primary del(5q) MDS patient samples. NG, JWW and AK wrote the manuscript. All authors reviewed the final manuscript. Competing Interests The authors declare no competing financial interests. Data Availability Statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 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(A)\u003c/strong\u003e Expression of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e measured by qRT-PCR in CD34\u003csup\u003e+\u003c/sup\u003e cells from MDS samples with del(5q) or diploid at chromosome 5q (n = 8). \u003cstrong\u003e(B)\u003c/strong\u003e Expression of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e measured by qRT-PCR in CD34\u003csup\u003e+\u003c/sup\u003e and CD34\u003csup\u003e- \u003c/sup\u003ecells isolated from the same cord blood specimens (n = 3) \u003cstrong\u003e(C)\u003c/strong\u003e Primary and secondary CFC assays performed on CD34\u003csup\u003e+\u003c/sup\u003e HSPC transduced with miRNA decoys or vector control (n = 3) \u003cstrong\u003e(D)\u003c/strong\u003e Proportion of viable cells at 48h post-transduction with either miRNA decoys or vector control, as determined by negativity for Annexin V and propidium iodide following \u003cem\u003ein vitro\u003c/em\u003e culture of CD34\u003csup\u003e+\u003c/sup\u003e HSPC (n = 4).\u003cstrong\u003e (E)\u003c/strong\u003e Schematic of experimental design. CD34\u003csup\u003e+\u003c/sup\u003e HSPC transduced with either \u003cem\u003emiR-143/miR-145\u003c/em\u003e decoy or control vectors were injected into sublethally-irradiated NSG mice. At the indicated time points post-transplant, engraftment was measured by flow cytometry of bone marrow aspirates (vector control, n = 3; miR-decoy, n = 6). \u003cstrong\u003e(F-G)\u003c/strong\u003e Engraftment of total human blood cells (human CD45\u003csup\u003e+\u003c/sup\u003e), myeloid cells (hCD45\u003csup\u003e+\u003c/sup\u003eCD33\u003csup\u003e+\u003c/sup\u003e) and lymphoid cells (hCD45\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003eCD20\u003csup\u003e+\u003c/sup\u003e) was measured in the (G)\u003cstrong\u003e \u003c/strong\u003eGFP\u003csup\u003e+\u003c/sup\u003e (transduced), and the (H)\u003cstrong\u003e \u003c/strong\u003eGFP\u003csup\u003e-\u003c/sup\u003e (untransduced) fractions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/ae768e7e4f1304a9838d3fbc.png"},{"id":56521842,"identity":"a0f28301-aa1f-4715-8ee5-ce494435892f","added_by":"auto","created_at":"2024-05-15 09:00:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":317947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003emiR-143\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emiR-145\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e target IGF-1R. (A)\u003c/strong\u003e GSEA enrichment plots for the IGF1 pathway based on either \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145\u003c/em\u003e expression. Descriptive statistics from GSEA are shown: NES, normalized enrichment score; NOM p-value, nominal p-value; FDR, false discovery rate. \u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e Luciferase activity of cells transfected with either WT or mutated \u003cem\u003eIGF-1R\u003c/em\u003e 3’UTR along with \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145\u003c/em\u003e (n = 3). \u003cstrong\u003e(C)\u003c/strong\u003e qRT-PCR following \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145 pulldown\u003c/em\u003e in MDS-L cells after transfection with Biotin-miR-143 or Biotin-miR-145 (n = 8). \u003cstrong\u003e(D)\u003c/strong\u003e Cell surface expression of IGF-1R using flow cytometry of myeloid cell lines with del(5q) or diploid at chromosome 5q (performed in triplicate for each cell line). \u003cstrong\u003e(E)\u003c/strong\u003e Normalized total IGF-1R expression in CD34\u003csup\u003e+\u003c/sup\u003e and CD34\u003csup\u003e-\u003c/sup\u003e cord blood cells transduced with miR-143/miR-145 decoy or control vector (n = 6). \u003cstrong\u003e(F)\u003c/strong\u003e Western blot for IGF-1R and GAPDH as a loading control in the del(5q) cell line MDS-L following lentiviral expression of miR-143 (miR-143OE), miR-145 (miR-145OE), or a vector containing a non-targeting sequence.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/9b4b4e6e23a4fb61af0af7e1.png"},{"id":56521843,"identity":"470a1c09-9ce7-4b33-94b6-843d18d80c38","added_by":"auto","created_at":"2024-05-15 09:00:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":482485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting IGF-1R reverses the effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emiR-143/miR-145\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e depletion in del(5q) cells. (A)\u003c/strong\u003e Effect of IGF-1R inhibition on MDS-L cell survival, as measured by Annexin V staining (n = 4). \u003cstrong\u003e(B)\u003c/strong\u003e Effect of IGF-1R inhibition on MDS-L cell proliferation, as measured by BrdU incorporation (n = 4). \u003cstrong\u003e(C)\u003c/strong\u003e Colony forming activity of MDS-L cells treated with IGF-1R inhibitor or vehicle (DMSO) (n = 4). \u003cstrong\u003e(D)\u003c/strong\u003e Progenitor activity, as measured by CFC assay, of CD34\u003csup\u003e+\u003c/sup\u003e HSPC depleted of \u003cem\u003emiR-143/miR-145\u003c/em\u003e using a decoy vector or control vector, and treated with IGF-1R inhibitor (BMS-536924, 1mM) or vehicle (DMSO) (n = 3). \u003cstrong\u003e(E)\u003c/strong\u003e Western blot for IGF-1R, and GAPDH as a loading control, in sh\u003cem\u003eIGF-1R\u003c/em\u003e-transduced or shControl-transduced MDS-L cells. \u003cstrong\u003e(F) \u003c/strong\u003eColony-forming activity of sh\u003cem\u003eIGF-1R\u003c/em\u003e-transduced MDS-L cells in semisolid medium (n = 6). \u003cstrong\u003e(G)\u003c/strong\u003e Schematic of experimental design. On the left, NRG-3GS mice were sublethally irradiated, then transplanted with MDS-L cells transduced with either sh\u003cem\u003eIGF-1R\u003c/em\u003e or shControl. On the right, NRG-3GS mice were sublethally irradiated, then transplanted with parental MDS-L cells that were allowed to engraft for 2 weeks prior to beginning intraperitoneal injections with either inhibitor (BMS-536924, 40 mg/kg body weight) or vehicle (2.5% DMSO:10% Solutol v/v) three times a week. (\u003cstrong\u003eH\u003c/strong\u003e) Survival curves of mice engrafted with MDS-L cells transduced with sh\u003cem\u003eIGF-1R \u003c/em\u003eor control vector (n = 8). (\u003cstrong\u003eI\u003c/strong\u003e) Survival curves of mice treated with IGF-1R inhibitor or vehicle (control n = 9; IGFR1 inhibitor n = 10).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/851b8ccdf70ed5d106a69d97.png"},{"id":56522532,"identity":"700ab78f-8e6a-4a22-b950-286deb788e2f","added_by":"auto","created_at":"2024-05-15 09:08:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":224868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDel(5q) myeloid cell lines resistant to lenalidomide are sensitive to IGF-1R inhibition. \u003c/strong\u003eApoptosis was measured by Annexin V labeling, at 48 and 144 hours of culture in the presence of vehicle (DMSO), LEN (1mM) or IGF-1R inhibitor (BMS-536924, 1mM) in del(5q) cells\u003cstrong\u003e (A)\u003c/strong\u003e and diploid chromosome 5q cells \u003cstrong\u003e(B). \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003e(C) \u003c/strong\u003eLEN-resistantMDS-L cells that were knocked out (KO) for \u003cem\u003eTP53\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e along with WT MDS-L cells were treated with LEN (1mM) or IGF-1R inhibitor (BMS-536924, 1mM) and apoptosis was measured by Annexin V staining. \u003cstrong\u003e(D) \u003c/strong\u003eColony forming activity of LEN-resistantMDS-L cells following treatment with LEN (1mM) or IGF-1R inhibitor (BMS-536924, 1mM). \u003cstrong\u003e(E)\u003c/strong\u003e Colony output of OCI-AML3 cells (diploid for chromosome 5q) transduced with \u003cem\u003emiR-143/miR-145\u003c/em\u003e decoy was measured in the presence of LEN (1mM) or IGF1-R inhibitor(BMS-536924, 1mM). \u003cstrong\u003e(F)\u003c/strong\u003e Clonogenic activity of OCI-AML3 cells transduced with shCSNK1A1 constructs was examined in the presence of LEN (1mM) or IGF-1R inhibitor (BMS-536924, 1mM).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/ae223d6b9192e309cc00ce72.png"},{"id":56523082,"identity":"5162d497-ce13-49f7-83ae-8a1051549247","added_by":"auto","created_at":"2024-05-15 09:16:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":416222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edel(5q) MDS cells depleted of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e or \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRUNX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e are sensitive to Abl or MAPK inhibitors.. \u003c/strong\u003eGSEA enrichment plots based on \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145 \u003c/em\u003eexpression for the BIOCARTA_GLEEVEC \u003cstrong\u003e(A), \u003c/strong\u003eBIOCARTA_MAPK \u003cstrong\u003e(B),\u003c/strong\u003e and BIOCARTA_MTOR \u003cstrong\u003e(C)\u003c/strong\u003e pathways in MDS patient CD34\u003csup\u003e+\u003c/sup\u003e bone marrow cells . \u003cstrong\u003e(D) \u003c/strong\u003eImatinib dose-response curves for LEN-resistant and WT MDS-L cells following treatment for 48h, as measured by AlamarBlue assay. \u003cstrong\u003e(E) \u003c/strong\u003eProportion of apoptotic cells as measured by Annexin V staining for LEN-resistant and WT MDS-L cells following 144h treatment with imatinib, LEN or vehicle (DMSO). \u003cstrong\u003e\u0026nbsp;(F) \u003c/strong\u003eColony output of LEN-resistant and WT MDS-L cells in the presence of imatinib (10 mM), LEN (1 mM) or vehicle (DMSO).\u003cstrong\u003e \u003c/strong\u003eEffect of inhibitors of pathways downstream of IGF-1R including everolimus (mTOR inhibitor, 0.1mM), temsirolimus (mTOR inhibitor, 0.5mM) and trametinib (MEK1/2 inhibitor, 0.03mM) or LEN (1 mM) or vehicle (DMSO), on del(5q) cell lines: KG1a cells \u003cstrong\u003e(G)\u003c/strong\u003e and MDS-L \u003cstrong\u003e(H)\u003c/strong\u003e.\u003cstrong\u003e (I) \u003c/strong\u003eTrametinib dose-response curves for LEN-resistant and WT MDS-L cells following treatment for 48h, as measured by AlamarBlue assay. \u003cstrong\u003e(J)\u003c/strong\u003e Proportion of apoptotic cells as measured by Annexin V staining for LEN-resistant and WT MDS-L cells following 144h treatment with trametinib (0.03 mM), LEN (1 mM) or vehicle (DMSO). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/2832509dbf4fc7324d494651.png"},{"id":56521846,"identity":"b5abb00c-ac29-46ab-a1d0-88256562a919","added_by":"auto","created_at":"2024-05-15 09:00:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":228187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEN-resistant MDS is sensitive to Abl and MEK inhibitors. \u003c/strong\u003eViability of bone marrow cells from del(5q) MDS patients \u003cstrong\u003e(A)\u003c/strong\u003e, or normal CD34\u003csup\u003e+\u003c/sup\u003e HSPC \u003cstrong\u003e(B)\u003c/strong\u003e, following treatment with Imatinib (10 mM), Trametinib (0.015 mM), LEN (1 mM), or vehicle (DMSO) for 48h, as measured by Annexin V/7AAD staining.\u003c/p\u003e","description":"","filename":"Fig6new2.png","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/981abe275535b533b1116e93.png"},{"id":77205435,"identity":"eb23e2d3-9f8c-4ebf-ab38-480a55566787","added_by":"auto","created_at":"2025-02-26 08:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3076127,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/3d64a903-a074-4d53-9ff7-d8924f97b6b5.pdf"},{"id":56521847,"identity":"4491fa72-5482-43e0-b92c-5c9ce52af1e1","added_by":"auto","created_at":"2024-05-15 09:00:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1828869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementalData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4339623/v1/6ad64f9cb58a9c89892773c9.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Haploinsufficiency of miR-143 and miR-145 reveal targetable dependencies in resistant del(5q) myelodysplastic syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis that lead to bone marrow failure or progression to AML.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e MDS is characterized by multiple cytogenetic and molecular defects, which result in an extremely heterogeneous phenotype, making design of molecular-targeted therapies a challenge.\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eInterstitial deletion of the long arm of chromosome 5 is a common genetic aberration seen in MDS.\u003csup\u003e9\u003c/sup\u003e MDS with isolated del(5q) is characterized by macrocytic anemia and hypolobulated megakaryocytes.\u003csup\u003e6,7\u003c/sup\u003e The immunomodulatory drug lenalidomide (LEN) has shown great efficacy in del(5q) MDS patients, leading to improved blood counts and survival.\u003csup\u003e8,9\u003c/sup\u003e However, 30\u0026ndash;40% of del(5q) MDS patients are refractory to LEN in the first-line setting, and at least half of primary responders become resistant within two years.\u003csup\u003e8,10\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eLEN functions by Cereblon-mediated CK1α degradation, the protein product of the \u003cem\u003eCSNK1A1\u003c/em\u003e gene.\u003csup\u003e11\u003c/sup\u003e \u003cem\u003eCSNK1A1\u003c/em\u003e is located on the CDR of del(5q) MDS which makes del(5q) cells particularly sensitive to further degradation of the protein by LEN. We and others have shown that LEN-resistance is most frequently associated with loss-of-function mutations in \u003cem\u003eTP53\u003c/em\u003e, and we have provided direct evidence that \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eRUNX1\u003c/em\u003e mutations inhibit LEN-induced cell death in the context of \u003cem\u003eCSNK1A1\u003c/em\u003e haploinsufficiency\u003csup\u003e12,13\u003c/sup\u003e. Mutation of either of these two genes inhibits differentiation of del(5q) MDS cells into megakaryocytes, which is required for LEN-induced death of del(5q) cells.\u003csup\u003e13\u003c/sup\u003e \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e, both located within the CDR, have reduced expression in del(5q) MDS.\u003csup\u003e14,15\u003c/sup\u003e Deficiency of \u003cem\u003emiR-145\u003c/em\u003e leads to activation of the TGFβ and TIRAP pathways.\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e However, most experiments have been conducted in mouse models and very little is known about how \u003cem\u003emiR-143/miR-145\u003c/em\u003e haploinsufficiency affects human hematopoiesis.\u003c/p\u003e \u003cp\u003eWe hypothesized that haploinsufficiency and subsequent derepression of \u003cem\u003emiR-143/miR-145\u003c/em\u003e targets may support the clonal expansion seen in del(5q) MDS.\u003csup\u003e18,19\u003c/sup\u003e As \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e are transcribed as a single primary miRNA transcript and both of these miRNAs have been characterized as tumor suppressor genes in solid tumors\u003csup\u003e20\u003c/sup\u003e, we examined the role of combined haploinsufficiency of these miRNAs in primary human CD34\u003csup\u003e+\u003c/sup\u003e cells. In this study, we show that depletion of these two miRNAs results in clonal expansion of human hematopoietic stem/progenitor cells (HSPC), and that targeting pathways activated by \u003cem\u003emiR-143/miR-145\u003c/em\u003e haploinsufficiency reveals a CK1α-independent vulnerability of del(5q) MDS that remains even when these cells are resistant to LEN. Specifically, we show that IGF-1R signaling is activated with deficiency of \u003cem\u003emiR-143/miR-145\u003c/em\u003e and pharmacologic inhibition of IGF-1R signaling is able to target del(5q) myeloid cells that are resistant to LEN. In addition, we demonstrate that LEN-resistant del(5q) myeloid cells are sensitive to inhibition of pathways regulated by IGF-1R including the Abl and MAPK signaling pathways, providing potential novel therapeutic approaches for del(5q) MDS.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines, human cord blood cells and patient MDS cells\u003c/h2\u003e \u003cp\u003eThe leukemic cell lines KG1a, KG1, UT7, K562, U937, and THP1 were purchased from the American Type Culture Collection. The MDS-L cell line, was provided by K. Tohyama (Kawasaki Medical School, Okayama, Japan).\u003csup\u003e21\u003c/sup\u003eHuman umbilical cord blood cells were obtained with consent under a protocol approved by the Research Ethics Board (REB) of the University of British Columbia (UBC). Primary umibilical cord blood cells and MDS samples were obtained, following patient consent, from the Hematology Cell Bank of British Columbia under a protocol approved by the University of British Columbia Research Ethics Board, as well as by institutional review boards at the Royal Adelaide Hospital, and Moffitt Cancer Center.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLentiviral production and transduction\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003emiR-143/miR-145\u003c/em\u003e decoy was generated by cloning tandem repeats of sequences complementary to \u003cem\u003emiR-143\u003c/em\u003e (MIMAT0000435) and \u003cem\u003emiR-145\u003c/em\u003e (MIMAT0000437) into the 3\u0026rsquo;UTR of GFP in the lentiviral vector pLL3.7. Mature miRNAs were expressed using the pCCL.PPT.MNDU3.PGK.GFP lentiviral vector.\u003csup\u003e22,23\u003c/sup\u003eShRNA (MISSION shRNA, Sigma-Aldrich, Burlington, MA, US) were expressed from pLKO.1. VSV-G pseudotyped lentiviral particles (5\u0026ndash;10 x 10\u003csup\u003e9\u003c/sup\u003e/ml) were used to transduce purified cord blood CD34\u003csup\u003e+\u003c/sup\u003e cells or MDS-L cells as previously described.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eFor experiments done with primary human cells, miRNA expression was assayed as previously described.\u003csup\u003e19\u003c/sup\u003e For experiments done with cell lines, miRNA expression was assayed using TaqMan assays with U47 snoRNA as a reference gene, per manufacturer's directions (assays #002249 (miR-143-3p), #002278 (miR-145-5p), and #001223 (U47), Thermo Fisher Scientific, Waltham, MA, US). Gene expression was analyzed using SYBR green master mix (Applied Biosystems, cat #4368708, Waltham, MA, US).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiotin-Based RNA pulldown assay\u003c/h3\u003e\n\u003cp\u003eBiotin-based pulldown assays were carried out as previously described (see Supplemental Methods for details).\u003csup\u003e21,22\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase assay\u003c/h2\u003e \u003cp\u003eThe Dual-Luciferase assay kit was obtained from Promega (Madison, WI, US). Wildtype (WT) and \u003cem\u003emiR-143/miR-145\u003c/em\u003e binding site-mutated \u003cem\u003eIGF-1R\u003c/em\u003e 3\u0026rsquo;-UTR (135 bp) were cloned downstream of a luciferase reporter in the pSGG vector. Reporter constructs along with either vectors overexpressing the miRNAs or empty vector were transfected into HEK293 cells. Luciferase activity was assessed 48 hrs post-transfection according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eProtein lysates were obtained by lysing cells in cold RIPA buffer (50mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% SDS), in the presence of PMSF, sodium orthovanadate, protease and phosphatase inhibitors (Sigma-Aldrich, Burlington, MA, US). Immunoblots used the following antibodies: IGF-1R (sc-772; Santa Cruz, Dallas, TX), GAPDH (G8795; Sigma-Aldrich, Burlington, MA, US).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGrowth, survival, and proliferation assays\u003c/h2\u003e \u003cp\u003eThe IGF-1R small molecule inhibitor BMS-536924 (cat #S1012), Lenalidomide (cat #S1029), Imatinib (cat #S2475), Trametinib (cat #S2673), Temsirolimus (cat #S1044) and Everolimus (cat #sS1120) were purchased from Selleck Chemicals (Houston, TX, US) and resuspended in DMSO. Doses for Imatinib, Trametinib, Temsirolimus and Everolimus were calculated to be similar to serum drug concentrations in patients receiving drug for cancer-related indications\u003csup\u003e23\u0026ndash;27\u003c/sup\u003e. MDS-L cell expansion in liquid culture was determined based on manual Trypan blue counts. Annexin V (BD Biosciences, Frank Lakes, NJ, US) and propidium iodide (Sigma-Aldrich, Burlington, MA, US) staining was performed according to the manufacturers' instructions. For BrdU assays, cells were pulsed with 10 mM BrdU for 1.5 h at 37\u0026deg;C, and then fixed/stained with anti-BrdU antibody according to manufacturer\u0026rsquo;s instructions (FITC or APC BrdU Flow kit; BD Biosciences, Franklin Lakes, NJ, US).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eColony-forming cell (CFC) assays\u003c/h2\u003e \u003cp\u003eClonogenic assays were performed by plating 0.5\u0026ndash;1 x 10\u003csup\u003e3\u003c/sup\u003e /ml human CD34\u003csup\u003e+\u003c/sup\u003e HSPC or MDS-L cells in MethoCult H4434 (STEMCELL Technologies, Vancouver, BC, Canada); colonies were scored after 10\u0026ndash;14 days. CFC assays colonies were scored as erythroid, myeloid or mixed erythroid-myeloid by morphological criteria; for replating assays, cells were scraped into PBS, then washed and replated into fresh methylcellulose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eXenotransplantation\u003c/h2\u003e \u003cp\u003eAll transplants were performed on sublethally irradiated (315 cGy, \u003csup\u003e137\u003c/sup\u003eCs) 8-12-week-old female immunodeficient mice. CD34\u003csup\u003e+\u003c/sup\u003e HSPC xenotransplantation was performed by intrafemoral injection of 1\u0026ndash;2 x 10\u003csup\u003e5\u003c/sup\u003e CD34\u003csup\u003e+\u003c/sup\u003e cells into NOD/SCID-IL2Rγ \u003cb\u003e(\u003c/b\u003eNSG) mice, while MDS-L cells (10\u003csup\u003e6\u003c/sup\u003e) were injected into the tail vein of NOD/Rag1/IL2Rγ (NRG) mice producing human IL-3, GM-CSF, and SCF (NRG-3GS). \u003cem\u003eIn vivo\u003c/em\u003e delivery of the IGF-1R small molecule inhibitor was performed as previously described:\u003csup\u003e25\u003c/sup\u003e BMS-536924 was dissolved in DMSO (10 mM) and further diluted in 10% solutol (Sigma-Aldrich, Burlington, MA, US). Animals were injected intraperitoneally with BMS-536924 (40 mg/kg) 3 times weekly starting at 2 weeks post-transplant. Mice were bred and maintained at the BC Cancer Research Center Animal Resources Facility (Vancouver, BC, Canada). Animal experimental protocols were approved by the UBC Animal Care Committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eHuman cell engraftment was monitored by flow cytometry on a FACS Calibur, LSRFortessa or AriaII cell sorter (BD Biosciences, Franklin Lakes, NJ, US) (see Supplemental Methods for details).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIPA and GSEA analysis\u003c/h2\u003e \u003cp\u003ePredicted targets of mir-143 and miR-145 from TargetScan (Version 6.2; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://targetscan.org\u003c/span\u003e\u003cspan address=\"http://targetscan.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e26\u003c/sup\u003e were subjected to pathway annotation analysis using Ingenuity Pathway Analysis (IPA) software (QIAGEN Bioinformatics). Gene Set Enrichment Analysis (GSEA) was used to analyze published MDS gene expression data for MIR143 and MIR14\u003cem\u003e5\u003c/em\u003e expression using the continuous phenotype label method\u003csup\u003e28,29\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSequencing analysis\u003c/h2\u003e \u003cp\u003eA target hybridization capture library was constructed and subjected to Illumina sequencing as per routine clinical protocol. The target space includes all recurrently mutated myeloid genes. The generated libraries were aligned against GRCh37 with variants called on samtools mpileup (version 0.1.18)\u003csup\u003e30\u003c/sup\u003e, using VarScan2 (version 2.3.6)\u003csup\u003e31\u003c/sup\u003e, ONCOCNV (version 6.6)\u003csup\u003e32\u003c/sup\u003e, and GenomonSV (version 0.5.0) packages. Variants were manually curated as per ACMG guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistics were calculated in Prism (GraphPad, La Jolla, CA, US). Results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) unless otherwise indicated. Statistical significance was determined using Welch\u0026rsquo;s t test for unpaired data, with multiple testing correction (Holm-Sidak method) when appropriate. Data from paired samples were compared using the ratio t test. The log-rank (Cox test) was used to compare differences between survival curves. P values below 0.05 and FDR values below 0.25 were considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDepletion of miR-143 and miR-145 supports clonal expansion of human HSPC\u003c/h2\u003e \u003cp\u003eTo examine the role of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e in human cells, we first confirmed that their expression was lower in CD34\u003csup\u003e+\u003c/sup\u003e cells from del(5q) MDS patient marrow compared to MDS marrow diploid at chromosome 5q, as previously reported\u003csup\u003e15\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Expression of both miRNAs was higher in the CD34\u003csup\u003e+\u003c/sup\u003e stem/progenitor cell fraction compared to the more differentiated CD34\u003csup\u003e\u0026minus;\u003c/sup\u003e cells, suggesting a role for \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e in HSPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To model \u003cem\u003emiR-143/miR-145\u003c/em\u003e depletion in human HSPC, we knocked down miRNA expression using lentiviral decoy constructs in CD34\u003csup\u003e+\u003c/sup\u003e HSPCs (Supplemental Fig.\u0026nbsp;1A, B). In functional assays for hematopoietic progenitor activity, the \u003cem\u003emiR-143/miR-145\u003c/em\u003e decoy-transduced cells produced more colonies than vector-transduced cells in both primary and serial replating progenitor assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The effect on colony formation was seen in the more-primitive erythroid (BFU-E) with a trend towards an increase in the primitive mix (CFU-GEMM) colonies (Supplemental Fig.\u0026nbsp;1C). The decoy-transduced cell cultures had enhanced viability, as estimated by Annexin V and propidium iodide (PI) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Thus, depletion of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e enhances viability of CD34\u003csup\u003e+\u003c/sup\u003e HSPC and promotes clonogenic potential \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the impact of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e depletion on hematopoiesis \u003cem\u003ein vivo\u003c/em\u003e, we injected CD34\u003csup\u003e+\u003c/sup\u003e HSPC, two days post-lentiviral transduction with either the decoy or control vectors, into the femurs of sublethally-irradiated NSG mice. Human lymphomyeloid reconstitution in the marrow of recipient mice was monitored by serial marrow aspiration at weeks 3, 8, 20 and 34 post-transplant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Between 8 and 34 weeks post-transplant, decoy-transduced cell populations showed increased marrow chimerism compared to vector-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). This growth advantage included both myeloid and lymphoid GFP\u003csup\u003e+\u003c/sup\u003e populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In contrast, the proportions of myeloid and lymphoid cells in the non-transduced (GFP\u003csup\u003e\u0026minus;\u003c/sup\u003e) human cell compartment in the same mice was similar between the two groups of transplants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), consistent with miRNA depletion providing a cell intrinsic competitive advantage \u003cem\u003ein vivo\u003c/em\u003e. These findings support a role for haploinsufficiency of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e in the clonal expansion of malignant cells in del(5q) MDS.\u003c/p\u003e \u003cp\u003e \u003cb\u003emiR-143\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003emiR-145\u003c/b\u003e \u003cb\u003etarget IGF-1R in human hematopoietic progenitor cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe have previously determined which pathways are targeted by both miRNAs using Ingenuity Pathway Analysis (IPA)\u003csup\u003e33\u003c/sup\u003e on the non-redundant set of predicted targets of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e16\u003c/sup\u003e. Here we used gene expression data from a previously-published cohort of MDS patients\u003csup\u003e34\u003c/sup\u003e, ranked samples on the basis of either the expression of \u003cem\u003emiR-143\u003c/em\u003e or the expression of \u003cem\u003emiR-145\u003c/em\u003e, and performed Gene Set Enrichment Analysis (GSEA)\u003csup\u003e28\u003c/sup\u003e to determine which BIOCARTA pathways were differentially upregulated with low expression of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e (Supplemental Table\u0026nbsp;1). The IGF-1 signaling pathway was the only pathway common to both analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, the IGF-1 receptor (IGF-1R) was predicted to be a target of both \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e based on the miRNA-target prediction algorithm, TargetScan (Supplemental Table\u0026nbsp;2).\u003csup\u003e35\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the predicted interaction between \u003cem\u003emiR-143/miR-145\u003c/em\u003e and the 3\u0026rsquo;-UTR of \u003cem\u003eIGF-1R\u003c/em\u003e mRNA, we performed luciferase assays using the wildtype (WT) or mutated \u003cem\u003eIGF-1R\u003c/em\u003e 3\u0026rsquo;UTR containing mutations in the predicted binding sites, cloned downstream of a luciferase reporter. Overexpression of \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145\u003c/em\u003e markedly reduced luciferase activity in cells with the WT \u003cem\u003eIGF-1R\u003c/em\u003e 3\u0026rsquo;UTR but not the mutated \u003cem\u003eIGF-1R\u003c/em\u003e 3\u0026rsquo;UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, an miRNA pulldown assay demonstrated that biotinylated \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e transfected into del(5q) MDS-L cells directly bound to \u003cem\u003eIGF-1R\u003c/em\u003e mRNA, but not to insulin receptor (\u003cem\u003eINSR\u003c/em\u003e) mRNA, confirming the specificity of the interaction (Supplemental Fig.\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition, surface expression of IGF-1R was higher in del(5q) myeloid cell lines compared to myeloid cell lines diploid for chromosome 5q (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To confirm that the \u003cem\u003emiR-143/145-IGF-1R\u003c/em\u003e interaction is functionally relevant in primary hematopoietic cells, we transduced CD34\u003csup\u003e+\u003c/sup\u003e HSPC with control or miRNA-decoy vectors. Surface IGF-1R expression was increased in miRNA-decoy cells, particularly in the CD34\u003csup\u003e+\u003c/sup\u003e HSPC fraction compared to the differentiated CD34\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Conversely, overexpression of each of the miRNAs in the MDS-L del(5q) cell line led to decreased protein expression of IGF-1R (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). MDS-L cells overexpressing either miRNA had reduced colony formation in CFC assays, as well as reduced proliferation and a trend towards reduced viability (Supplemental Fig.\u0026nbsp;3). These data suggest that \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e regulate cell progenitor activity and cell proliferation in primitive human hematopoietic cells through the regulation of \u003cem\u003eIGF-1R\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIGF-1R inhibition reduces progenitor activity of\u003c/b\u003e \u003cb\u003emiR-143\u003c/b\u003e\u003cb\u003e/\u003c/b\u003e\u003cb\u003emiR-145\u003c/b\u003e\u003cb\u003e-haplodeficient del(5q) cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine which effects of \u003cem\u003emiR-143/miR-145\u003c/em\u003e deficiency are mediated by IGF-1R, we first asked whether specific inhibition of IGF-1R signaling in del(5q) MDS-L cells could abrogate cell expansion \u003cem\u003ein vitro\u003c/em\u003e. To address this, we treated MDS-L cells with a small molecule IGF-1R inhibitor, BMS-536924, at various concentrations. At all tested doses of the inhibitor, there was a significant increase in cell death by 48 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Proliferation was also significantly reduced, in a dose- and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Cells treated with inhibitor appeared to mainly target cycling cells as cells were less likely to be in S-phase with more cells in G\u003csub\u003e0\u003c/sub\u003e/G\u003csub\u003e1\u003c/sub\u003e of the cell cycle compared to controls (Supplemental Fig.\u0026nbsp;4). At the highest dose, cells were virtually eliminated following 3 days of treatment (Supplemental Fig.\u0026nbsp;5). Consistent with the above, MDS-L cells pre-treated with an intermediate concentration (1 \u0026micro;M) of IGF-1R inhibitor for 48 h had reduced output in CFC assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo extend this result to primary cells, CD34\u003csup\u003e+\u003c/sup\u003e HSPC were transduced with either miRNA-decoy or control vector, treated with BMS-536924 (1 \u0026micro;M) and subjected to CFC assays. As observed previously, miRNA-decoy-transduced cells had increased colony output (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, IGF-1R inhibition reduced colony formation only in decoy-transduced cells, but had no effect on vector-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), potentially implicating a differential effect in del(5q) MDS cells compared to normal cells. Next, we used a complementary genetic approach, by targeting \u003cem\u003eIGF-1R\u003c/em\u003e through lentiviral-delivered shRNA to reduce its expression in the del(5q) MDS-L cell line. Knockdown of \u003cem\u003eIGF-1R\u003c/em\u003e resulted in reduced \u003cem\u003eIGF-1R\u003c/em\u003e protein expression compared to the control shRNA-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Cells transduced with sh\u003cem\u003eIGF-1R\u003c/em\u003e had reduced colony formation in clonogenic assays compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Together, these studies suggest that pharmacologic or genetic inhibition of IGF-1R signaling reduces cell expansion observed with loss of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e, consistent with the hypothesis that derepression of \u003cem\u003eIGF-1R\u003c/em\u003e is functionally relevant in human del(5q) MDS.\u003c/p\u003e \u003cp\u003eTo determine whether targeting IGF-1R would prolong survival in mice xenografted with human del(5q) MDS-L cells, we used a xenograft model in which NRG-3GS immunodeficient mice expressing three human growth factors were transplanted with MDS-L cells. To determine the specific effect of \u003cem\u003eIGF-1R\u003c/em\u003e depletion in this model, we transplanted MDS-L cells transduced with sh\u003cem\u003eIGF-1R\u003c/em\u003e or shControl into NRG-3GS mice. Depletion of \u003cem\u003eIGF-1R\u003c/em\u003e resulted in significantly prolonged survival of xenografted mice compared to the controls (median survival 57 days vs. 45 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). To confirm that pharmacological inhibition of IGF-1R would have the same effect, we xenografted parental MDS-L cells into NRG-3GS mice, and injected engrafted mice intraperitoneally with BMS-536924 (40 mg/kg) 3 times weekly starting at 2 weeks post-transplant. As with \u003cem\u003eIGF-1R\u003c/em\u003e knockdown, chemical inhibition of IGF-1R resulted in significant improvement in median survival, compared to controls (52 days vs 44 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0016) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Thus, either genetic knockdown or pharmacologic inhibition of IGF-1R inhibits the expansion of del(5q) MDS-L cells in a preclinical model of haploinsufficiency for miR-143 and miR-145.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIGF-1R inhibition bypasses LEN resistance in del(5q) MDS cells\u003c/h2\u003e \u003cp\u003eGiven that a significant proportion of del(5q) MDS patients do not respond to LEN or become resistant over time, we attempted to determine whether IGF-1R might be a relevant target in LEN-resistant del(5q) cells. To address this, we examined the efficacy of IGF-1R inhibitor, BMS-536924, along with LEN on four del(5q) and three non-del(5q) myeloid cell lines by measuring apoptosis and proliferation. Targeting IGF-1R reduced the viability of all del(5q) cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), including those known to be resistant to LEN (KG1, HL60).\u003csup\u003e21\u003c/sup\u003e In contrast, survival of non-del(5q) cell lines was not affected by the addition of BMS-536924 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe have previously shown that loss of function mutations in \u003cem\u003eTP53\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e cause LEN resistance.\u003csup\u003e13\u003c/sup\u003e To further assess the efficacy of IGF-1R inhibition in the context of LEN resistance we used CRISPR/Cas9 MDS-L cells targeted for \u003cem\u003eTP53\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e that are resistant to LEN\u003csup\u003e13\u003c/sup\u003e. Despite LEN resistance, BMS-536924 caused significant cell death and reduced the clonogenic output of \u003cem\u003eTP53-\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e-targeted MDS-L cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). \u003cem\u003eCSNK1A1\u003c/em\u003e haploinsufficiency is required for LEN-induced cell death in del(5q) MDS cells\u003csup\u003e11,36\u003c/sup\u003e. As \u003cem\u003emiR-143/miR-145\u003c/em\u003e function is likely independent of \u003cem\u003eCSNK1A1\u003c/em\u003e, we sought to determine if IGF-1R inhibition is effective via a pathway that is distinct from LEN by using the OCI-AML3 cell line that expresses diploid copies of \u003cem\u003eCSNK1A1\u003c/em\u003e, \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e. The OCI-AML3 cell line was transduced with the \u003cem\u003emiR-143\u003c/em\u003e/\u003cem\u003emiR-145\u003c/em\u003e decoy construct or empty vector, or shRNAs against \u003cem\u003eCSNK1A1\u003c/em\u003e or a control shRNA, and colony output of these cells was measured in the presence of either LEN, BMS-536924, or vehicle (Supplemental Fig.\u0026nbsp;6). \u003cem\u003emiR-143/miR-145\u003c/em\u003e decoy-transduced cells had decreased clonogenic output with BMS-536924 treatment while LEN had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In contrast, cells transduced with sh\u003cem\u003eCSNK1A1\u003c/em\u003e, had reduced colony number with LEN treatment but BMS-536924 had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results verify that IGF-1R inhibition acts via a mechanism distinct from LEN/CK1α to suppress del(5q) MDS cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDel(5q) MDS cells targeting\u003c/b\u003e \u003cb\u003eTP53\u003c/b\u003e \u003cb\u003eor\u003c/b\u003e \u003cb\u003eRUNX1\u003c/b\u003e \u003cb\u003eare sensitive to Abl or MAPK inhibitors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIGF-1R is frequently overexpressed in different cancers, but results of clinical trials with IGF-1 or IGF-1R have not shown convincing efficacy, resulting in the lack of further development of these compounds. \u003csup\u003e28,29,30\u003c/sup\u003e. We thus sought to determine whether targeting IGF-1R-related pathways also exposed vulnerabilities in del(5q) MDS cells. Abl is involved in autoregulation of IGF-1R activity\u003csup\u003e40,41\u003c/sup\u003e. Interestingly, our GSEA analysis showed the BIOCARTA_GLEEVEC geneset, containing genes involved in Abl signaling, to be the top differentially upregulated pathway in MDS patients with low \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145\u003c/em\u003e (Supplemental Table\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We also examined whether other pathways downstream of IGF-1R were differentially upregulated with \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e haploinsufficiency. We performed GSEA analysis on previously published RNA expression data of MDS samples\u003csup\u003e34\u003c/sup\u003e, and showed the BIOCARTA_MAPK and BIOCARTA_MTOR pathways to both be significantly activated in patients with low expression of either \u003cem\u003emiR-143\u003c/em\u003e or \u003cem\u003emiR-145\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). In contrast, the PI3K/Akt pathway was not activated with low expression of \u003cem\u003emiR-143/miR-145\u003c/em\u003e (Supplemental Fig.\u0026nbsp;7). These results suggest that the Abl, MAPK and mTOR signaling pathways may provide a potential therapeutic target for LEN-resistant del(5q) MDS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that Abl, MAPK and MTOR signaling pathways were differentially upregulated with low expression of \u003cem\u003emiR-143/miR-145\u003c/em\u003e, we sought to determine the dependency of del(5q) cells on these pathways. The Abl inhibitor, imatinib, reduced the viability and colony output of LEN-resistant MDS-L cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). Additionally, we assessed the effect of FDA approved inhibitors of MAPK and MTOR signaling pathways. Trametinib (MEK1/2 inhibitor), everolimus and temsirolimus (mTOR inhibitors) reduced the viability of del(5q) KG1a cells significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), but only trametinib induced significant cell death in MDS-L cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). As trametinib was the only effective inhibitor in both del(5q) cell lines, we evaluated the sensitivity of LEN-resistant MDS-L cells to this inhibitor. Trametinib treatment resulted in reduced viability of LEN-resistant MDS-L cells, similar to wild-type control cells, in cells deficient for either \u003cem\u003eTP53\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLEN-resistant del(5q) MDS patients cells are sensitive to inhibition of Abl or MAPK pathways\u003c/h2\u003e \u003cp\u003eTo further validate our findings, we assessed the efficacy of trametinib on \u003cem\u003eex vivo\u003c/em\u003e cultured cells from del(5q) MDS patients that were either sensitive or resistant to LEN. Mutational analysis on these nine del(5q) MDS patient samples identified pathogenic variants of \u003cem\u003eTP53\u003c/em\u003e in one sample (Supplemental Table\u0026nbsp;3). All samples regardless of their sensitivity to LEN were sensitive to both imatinib and trametinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), while CD34\u003csup\u003e+\u003c/sup\u003e HSPC were not sensitive to the tested inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that targeting Abl or MAPK signaling pathways holds therapeutic promise for LEN-resistant del(5q) MDS patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe and others have previously shown that the del(5q) MDS phenotype is partially mediated by miRNA haploinsufficiency in mouse models\u003csup\u003e16,28,29\u003c/sup\u003e. Here, we identify activation of the IGF-1R pathway as a consequence of depletion of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e in human hematopoietic cells. We confirmed that \u003cem\u003eIGF-1R\u003c/em\u003e is a direct target of both \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e using luciferase and RNA pulldown assays. With three lines of evidence - \u003cem\u003eIGF-1R\u003c/em\u003e knockdown by shRNA, \u003cem\u003eIGF-1R\u003c/em\u003e depletion by overexpression of \u003cem\u003emiR-143\u003c/em\u003e and/or \u003cem\u003emiR-145\u003c/em\u003e, and chemical inhibition of IGF-1R we demonstrate that targeting IGF-1R results in reduced viability, proliferation and colony output. In complementary experiments, we show both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e that depletion of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e results in derepression of IGF-1R signaling and clonal expansion, and further that inhibition of IGF-1R rescues the phenotype of miRNA depletion in functional assays. We further provide evidence that IGF-1R inhibition is able to bypass LEN-resistance. Importantly, our data identify two FDA-approved drugs that interact with the IGF-1R signaling pathway, namely Abl kinase AND MEK inhibitors, that could be repurposed for LEN-resistant del(5q) MDS.\u003c/p\u003e \u003cp\u003eIGF-1 signaling has been reported to play a role in growth and self-renewal of both hematopoietic and embryonic stem cells\u003csup\u003e43,44\u003c/sup\u003e. IGF-1 signaling also contributes to progression of several cancers including AML and MDS\u003csup\u003e26,35,36\u003c/sup\u003e, and bone marrow cells of some MDS patients overexpress IGF-1R compared to normal bone marrow cells\u003csup\u003e45\u003c/sup\u003e. Although various IGF1/IGF-1R inhibitors have undergone clinical trials in solid tumors, these trials did not examine potential biomarkers to identify susceptible cases, and this may have been the reason for the failure of these trials\u003csup\u003e28,28,36\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe IGF-1 signaling pathway involves a complex network that is regulated at multiple levels. In particular, Abl and IGF-1R are involved in an autocrine signaling loop\u003csup\u003e40,41\u003c/sup\u003e. GSEA analysis revealed that genes of the Abl signaling pathway (BIOCARTA_GLEEVEC) are activated with deficiency of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e. Signals secondary to IGF-1R signaling are also activated in many cancers including acute lymphoblastic leukemia, breast and prostate cancers, and act to promote tumor cell viability and proliferation\u003csup\u003e48\u0026ndash;51\u003c/sup\u003e. Our analysis demonstrated that MAPK and mTOR signaling pathways which act downstream of IGF-1R are activated in MDS samples with low expression of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e. Importantly, we show that LEN-resistant del(5q) cells are sensitive to either imatinib or trametinib, FDA-approved inhibitors of Abl and MAPK pathways, respectively. In contrast, mTOR inhibitors while effective in one del(5q) cell line, were not able to kill a second del(5q) cell line. Activation of the MAPK pathway is documented to be involved in progression and drug resistance of many cancers including AML and MDS, which has encouraged preclinical and clinical trials\u003csup\u003e52\u0026ndash;57\u003c/sup\u003e. Here we show that both imatinib and trametinib are able to reduce the viability of LEN resistant MDS-L cells and primary MDS samples.\u003c/p\u003e \u003cp\u003eHaploinsufficiency of \u003cem\u003eCSKN1A1\u003c/em\u003e in MDS cells with del(5q) renders these cells sensitive to LEN\u003csup\u003e13,14\u003c/sup\u003e. We demonstrated that sensitivity of del(5q) MDS cells to inhibition of IGF-1R, MAPK and Abl signaling are dependent on haploinsufficiency of \u003cem\u003emiR-143/miR-145\u003c/em\u003e and independent of \u003cem\u003eCSNK1A1\u003c/em\u003e haploinsufficiency, implicating a novel parallel dependency in del(5q) MDS cells. Our findings suggest that targeting Abl and MAPK signaling pathways by repurposing FDA-approved drugs may be a novel therapeutic option for LEN-resistant MDS.\u003c/p\u003e \u003cp\u003eOverall, our findings suggest two distinct, coexisting vulnerabilities in del(5q) MDS, both of which target haploinsufficiency of genes within the CDR of del(5q). Haploinsufficiency of \u003cem\u003eCSKN1A1\u003c/em\u003e in MDS cells with del(5q) renders these cells sensitive to lenalidomide. In contrast sensitivity to IGF-1R inhibition in del(5q) MDS cells is secondary to haploinsufficiency of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e. Our finding that lenalidomide-resistant del(5q) MDS cell lines are sensitive to IGF-1R, ABL or MEK inhibition provides alternative approaches, through a coexisting but independent vulnerability, for therapeutic trials in del(5q) MDS patients who are resistant to lenalidomide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was funded by grants from the Canadian Institutes of Health Research (PJT-183924 and PJT-162131) the Terry Fox Research Institute Program Project Grant (1074) and the Leukemia and Lymphoma Society of Canada. AK is the recipient of the John Auston BC Cancer Foundation Clinical Investigator Award, and is a Tier 1 Canada Research Chair in Blood Cancers.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eAK conceived the project. NG, JWW and AK designed experiments. NG, JWW, \u0026nbsp;GC, VSA, PU, DD, AK and MF performed experiments. NG, JB, RS and AK analyzed data. DH, MK, OC, RK and EP provided primary del(5q) MDS patient samples. NG, JWW and AK wrote the manuscript. All authors reviewed the final manuscript.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAvailability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGarcia-Manero G, Chien KS, Montalban-Bravo G. Myelodysplastic syndromes: 2021 update on diagnosis, risk stratification and management. \u003cem\u003eAm J Hematol\u003c/em\u003e. 2020;95(11):1399-1420. doi:10.1002/ajh.25950\u003c/li\u003e\n\u003cli\u003eSperling AS, Gibson CJ, Ebert BL. 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Rigosertib in combination with azacitidine in patients with myelodysplastic syndromes or acute myeloid leukemia: Results of a phase 1 study. \u003cem\u003eLeuk Res\u003c/em\u003e. 2020;94:106369. doi:10.1016/j.leukres.2020.106369\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4339623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4339623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMyelodysplastic syndromes (MDS) are stem cell disorders characterized by ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Chromosomal alterations are frequent in MDS, with interstitial deletion of chromosome 5q (del(5q)) being the most common. Lenalidomide is the current first-line treatment for del(5q) MDS and its efficacy relies on degradation of CK1α which is encoded by the \u003cem\u003eCSNK1A1\u003c/em\u003e gene located in the commonly deleted region (CDR) of chromosome 5q. However, lenalidomide-resistance is common, often secondary to loss-of-function mutations in \u003cem\u003eTP53\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e. The CDR in del(5q) harbors several genes, including noncoding miRNAs, the loss of which contribute to disease phenotypes. \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e are located within the del(5q) CDR, but precise understanding of their role in human hematopoiesis and in the pathogenesis of del(5q) MDS is lacking. Here we provide evidence that deficiency of \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e plays a role in clonal expansion of del(5q) MDS. We show that insulin-like growth factor 1 receptor (IGF-1R) is a direct target of both \u003cem\u003emiR-143\u003c/em\u003e and \u003cem\u003emiR-145\u003c/em\u003e. Our data demonstrate that IGF-1R inhibition reduces proliferation and viability of del(5q) cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and that lenalidomide-resistant del(5q) MDS cells depleted of either \u003cem\u003eTP53\u003c/em\u003e or \u003cem\u003eRUNX1\u003c/em\u003e are sensitive to IGF-1R inhibition. Resistant del(5q) MDS-L cells, as well as primary MDS marrow cells, are also sensitive to targeting of IGF-1R-related dependencies in del(5q) MDS, which include the Abl and MAPK signaling pathways. This work thus provides potential new therapeutic avenues for lenalidomide-resistant del(5q) MDS.\u003c/p\u003e","manuscriptTitle":"Haploinsufficiency of miR-143 and miR-145 reveal targetable dependencies in resistant del(5q) myelodysplastic syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-15 08:59:55","doi":"10.21203/rs.3.rs-4339623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-06-12T10:42:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-11T15:12:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-05-20T03:13:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-10T20:08:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-08T23:53:56+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-05-08T23:22:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-29T11:08:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-29T11:08:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2024-04-29T01:38:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4a7eaec0-15c1-462c-9252-1f3059a8caa5","owner":[],"postedDate":"May 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-26T08:11:48+00:00","versionOfRecord":{"articleIdentity":"rs-4339623","link":"https://doi.org/10.1038/s41375-025-02537-2","journal":{"identity":"leukemia","isVorOnly":false,"title":"Leukemia"},"publishedOn":"2025-02-25 05:00:00","publishedOnDateReadable":"February 25th, 2025"},"versionCreatedAt":"2024-05-15 08:59:55","video":"","vorDoi":"10.1038/s41375-025-02537-2","vorDoiUrl":"https://doi.org/10.1038/s41375-025-02537-2","workflowStages":[]},"version":"v1","identity":"rs-4339623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4339623","identity":"rs-4339623","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}