Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma.

Next, we performed fluorescence in situ hybridization (FISH) on cell blocks obtained from all the primary CC cell lines evaluable (i.e., a total of 11 tumors). In agreement with RT-PCR data demonstrating high levels of c-MYC transcripts, we found c-MYC amplification in 9 out of 11 (82%) of the CC cell lines ( Table 1 and Figure 1B ). Moreover, in western blot experiments, c-Myc protein levels were increased in cell lines harboring HUWE1 mutation when compared to HUWE1 wild-type cell lines ( Figure 1C ). Next, we investigated the in vitro effects of GS-626510 on the growth of the 11 primary CC cell lines using flow cytometric-based assays as described in the Methods . As shown in Figure 2 , after incubation with varying concentrations of GS-626510 we found a progressive, dose-response decrease in cell proliferation in all CC tested, with IC 50 values ranging from 3 to 57 nM. On the basis of previously published evidence from our group as well as others in other tumor models [ 19 , 20 ], this is within the therapeutic range of activity of BET inhibitors (i.e., plasma/serum concentration) necessary to achieve a therapeutic effect in vivo . To evaluate further the potential correlation between HUWE1, c-MYC, and sensitivity of CC to GS-626510 we transiently transfected CVX8 cells with HUWE1 siRNA as well as control siRNA as described in the methods section. Validation of HUWE1 mRNA silencing in tumor cells was confirmed by q-real-time PCR experiments ( Figure 3A ). As shown in Figure 3A , HUWE1 silencing consistently increased c-MYC mRNA level and accordingly, c-Myc protein expression level was found significantly higher in the cells with silenced HUWE1 than in the control matched cells ( Figure 3B ). Silencing of HUWE1 in all CC cell lines tested (i.e., CVX4, CVX7, CVX5, and CVX8) significantly enhanced cell growth when compared to control cells (p = 0.03). Importantly, as demonstrated in Figure 3C , primary tumor transfected with HUWE1 siRNA become significantly more proliferative when compared to the matched tumor cells transfected with siRNA control after 96 hours. After 72 hours of GS-626510 exposure, IC 50 values of both HUWE1 siRNA and negative control siRNA transfected CVX8 cells were evaluated through flow cytometric-based assay as described in Methods . As shown in Figure 3D , CVX8 cells transfected with HUWE1 siRNA exhibited a significantly lower IC 50 than the control cells (i.e., IC 50 =10 nM and 85 nM, respectively) (p = 0.006). We next evaluated the impact of GS-626510 in vivo in xenograft models generated by injecting subcutaneously CVX8 cells into female CB17/lcrHsd-Prkd/scid mice. Twice daily oral doses of GS-626510, 10 mg/kg, were well tolerated with no clear impact on body weight compared with vehicle control. As shown in Figure 4A , mice undergoing 33 days of twice-daily oral treatment with GS-626510 (10 mg/kg) exhibited a significantly slower rate of tumor growth, compared to vehicle control. This difference was statistically significant starting on dosing day 23 (p-value = 0.004). Furthermore, the overall survival was significantly prolonged in the experimentally treated group when compared to animals treated with vehicle control (Log Rank Mantel-Cox test p=0.004) ( Figure 4B ).

To assess/validate c-MYC and HUWE1 transcript expression in CC we performed real-time PCR in a total of 23 tumor samples including 12 fresh frozen CC and 11 primary CC cell lines as well as 18 normal tissue controls obtained from cervix/uterus and ovaries collected from patients undergoing hysterectomies for benign conditions (i.e., endometriosis and uterine leiomyoma). We found c-MYC expression in fresh-frozen tumors and primary CC cell lines (mean ± SEM relative mRNA values = 7.06 ± 1.17) to be significantly higher when compared to normal tissues (mean ± SEM = 2.96 ± 0.97) (p = 0.01) ( Figure 1B ). In contrast, HUWE1 transcript expression was not significantly different when compared to tissue controls in most of the tumors.

Study approval was obtained from the Institutional Review Board at Yale University, and all patients signed consent prior to tissue collection according to the institutional guidelines and in accordance with the Declaration of Helsinki. A total of 41 samples including 12 fresh frozen CC tissues, 11 primary CC cell lines (cell lines characteristics and tissue source are described in Table 1 ) and 18 normal control tissue samples (from gynecologic organs including normal cervix, uterus, and ovaries) were evaluated in this study. Primary cell lines were established from patients at the time of initial staging surgery or tumor recurrence after sterile processing of fresh tumor biopsy samples and authenticated by whole-exome sequencing (WES) at the Yale Center for Genome Analysis, as previously described [ 4 ]. Briefly, tumors were processed by mechanical disruption in an enzymatic solution of 0.14% collagenase type I (Sigma) and 0.01% DNase (Sigma) in RPMI 1640 (Gibco, Life Technologies, Grand Island, NY). The resulting solution was incubated while stirring for 45 minutes at room temperature. The samples were then washed once with RPMI 1640 10% FBS, once with PBS (Gibco, Life Technologies, Grand Island, NY) and plated in cell culture dishes as a monolayer in serum-free keratinocyte medium, supplemented with 5 ng/ml epidermal growth factor and 35 to 50 μg/mL bovine pituitary extract (Invitrogen, Grand Island, NY), 1% anti-mycotic amphotericin B (Invitrogen, Carlsbad, CA), and 1% penicillin/streptomycin (Gibco, Life Technologies, Grand Island, NY). Cells were incubated at 37° C in a humidified atmosphere of 95% air/5% CO 2 . Tumors were staged according to the International Federation of Gynecology and Obstetrics staging system. All revived cell lines were used within 20 passages and cultured for less than 6 months. Primary CC cell lines with limited passages were used in the experiments listed below and corresponding cell blocks were analyzed for c-Myc and BRD4 surface expression by fluorescent in situ hybridization (FISH). GS-626510 was obtained from Gilead Sciences Inc., Foster City, CA through an MTA. Briefly, the compound was dissolved in DMSO as a 10 mM stock solution and diluted in culture medium immediately before use. GS-626510 is a novel, orally available BET inhibitor that binds with high affinity and specificity to BET family bromodomains (BRD2, BRD3, BRD4, and BRDT with Kd in the order of nM) [ 19 ]. RNA was extracted from 12 fresh frozen CC samples, 11 primary CC cell lines and 18 normal control tissue samples (gynecologic organs including normal cervix, uterus, and ovaries obtained from benign hysterectomy specimens). RNA extraction was performed using AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. Total RNA (5 μg) was reverse-transcribed using Superscript III (Invitrogen, Carlsbad, CA). Quantitative PCR was carried out to evaluate the expression level of c-MYC (c-MYC, Assay ID: Hs00905030_m1, Applied Biosystems) and HUWE1 (HUWE1, Assay ID: Hs00948075_m1, Applied Biosystems) in all samples with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol. The expression level of BRD4 (BRD4, Assay ID: Hs04188087_m1, Applied Biosystems) was also evaluated in the 11 CC cell lines. Each reaction was run in duplicate. The internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Assay ID: Hs99999905_ml, Applied Biosystems), was used to normalize variations in cDNA quantities from different samples. The comparative threshold cycle (Ct) method was used for the calculation of amplification fold as specified by the manufacturer. Analyses were performed using SDS software 2.2.2 (Applied Biosystems/Life Technologies). To determine dose-response, cells were aliquoted into 6-well microtiter plates at 100,000 cells per well. After 24 hours, the cells were treated with scalar amounts of drug ranging from 0.5 nM to 5 μM. Three days after treatment, the contents of each well were harvested in their entirety and stained with propidium iodide (Sigma Life Sciences, St. Louis, MO) (2 μL of 500 μg/mL stock solution in PBS), to be counted by flow cytometry. The number of viable cells in each well was normalized to the number of viable cells in the control well. The IC 50 of each cell line was then determined by comparing the log base 10 of drug concentration in each well to the percentage of viable cells using a non-parametric 3 parameter regression. All IC 50 data were calculated using Prism 8 software (GraphPad Prism Software Inc., San Diego, CA). All experiments were completed at least three times. Cells were seeded in 6-well plates and subjected to transfection when 80% confluent. HUWE1 siRNA and negative control siRNA were purchased from Ambion®, Life Technologies™. Briefly, the siRNA was incubated with Lipofectamine™ RNAiMAX reagent (Invitrogen, CA, USA) in OptiMEM™ medium for 20 minutes, then added to a monolayer of cells in Keratinocytes-SFM without antibiotics. Cells were either incubated for 48, 72 or 120 hours and RNA was extracted or twenty-four hours after the transfection, cells were treated with scalar amounts of GS-626510 ranging from 0.5 nM to 1 μM, for cell viability assay. Cells were seeded at 1,000,000 cells/well/2 mL into 6-well plates. After overnight incubation, cells were treated with siRNA negative control or siRNA HUWE1, as described above. After 48, 72 and 120 hours incubation, cells were washed three times with ice-cold PBS, mechanically scraped, and lysed for 30 minutes on ice with 400 μL of radioimmunoprecipitation assay buffer (RIPA) (50 mmol/L Tris–HCl pH 8, 150 mmol/L NaCl, Triton X-100 1%, Na deoxycholate 0.5%, SDS 0.1%, MgCl 5 mmol/L in H2O) supplemented with Protease and Phosphatase Inhibitor (cat#78430, Thermo Fisher Scientific). Analogously, proteins were obtained from primary CC cell lines, untreated. Protein levels were quantified with the Micro BCA Protein Assay Kit (#23225, Thermo Scientific), and equal amounts (30 μg) were resolved using SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were washed with TBS (140 mmol/L NaCl, 50 mmol/L Tris-HCl; pH 7.2) containing 0.1%Tween20 (TBST) and 5% skimmed milk to block nonspecific protein binding. Membranes were incubated with an antibody against c-Myc (1:1000, 5605S, Cell Signaling Technology), or GAPDH (1:1000, 2118S, Cell Signaling Technology) in 5% BSA in TBST overnight at 4°C, washed three times with TBST, and then incubated with an HRP-linked secondary antibody (1:2000, 7074S, Cell Signaling Technology) in 5% nonfat dry milk in TBST for 1 hour at room temperature, before washing three times in TBST. Signals were detected with western blotting detection reagents (Thermo Scientific). Bands were then visualized and the blots developed using a Q10 enhanced chemiluminescent system (GEL Logic 1500). The cell line CVX8 was expanded in T-150 tissue culture flasks. The cells were collected and washed in PBS. Two groups of 6 female CB17/lcrHsd-Prkd/scid mice each were injected with CVX8 tumor cells (7 × 10 6 cells in 0.3 mL of PBS with 50% Matrigel® (BD Biosciences)) subcutaneously into the lower abdomen area. Mice were triaged into treatment groups when mean tumor burden was 0.120–0.250 cm 3 . Dosing (vehicle PO or GS-626510 10 mg/kg BID, PO) began upon reaching target size and was delivered to the CVX8 xenografts for 33 days (every day), then the animals were followed for overall survival for additional 7 weeks. Tumor measurements and mouse weights were recorded at least two times weekly and reported for each mouse (individually identified). Tumor volumes were calculated using the formula 0.5 × (width 2 × height). At the end of the study, the animals were humanely euthanized. All mice were housed and treated in accordance with the policies set forth by the Institutional Animal Care and Use Committee (IACUC) at Yale University. GraphPad Prism version 8 (GraphPad Software, Inc. San Diego, CA) was used to determine statistical significance by the student’s t-test. Differences in all comparisons were considered statistically significant at p-values < 0.05.

Cervical cancer (CC) is the third most common cancer in women, causing an estimated 530,232 cases and 275,008 deaths worldwide [ 1 ]. While most of cervical cancer-related deaths take place in women living in low-income countries, it is estimated that 13,170 women will be diagnosed with cervical cancer in the United States in 2019 and over 4,000 women will die of the disease [ 2 ]. The management of advanced/persistent or recurrent cervical cancer no longer amenable to control with gold standard treatments (i.e., surgery or radiation) has not improved significantly with the progress of modern chemotherapy and disseminated carcinoma of the cervix remains a discouraging clinical entity with a 1year survival rate between 10% and 15% [ 3 ]. A deeper understanding of the molecular pathogenesis of cervical cancer as well as the development of novel and more effective treatment modalities remains an unmet medical need. Comprehensive whole-exome sequencing (WES) studies from our laboratory [ 4 ], the Cancer Genome Atlas Network (TCGA) [ 5 ], and others [ 6 ] have recently demonstrated that the mutational profile of CC consists of alterations in multiple genes including but not limited to PIK3CA, ERBB2 and GNAS and a widespread APOBEC cytidine-deaminase-mutagenesis-pattern (TCW-motif) in both adenocarcinoma (ACC) and squamous-cell-carcinoma (SCC) of the cervix. Gain of function mutations in multiple oncogenes and onco-suppressor genes including HER2/neu, CCNE, and STK11 were also identified with frequent derangements noted in multiple pathways including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle pathways. In comprehensive molecular studies, alterations in the HUWE1 gene, (also named ARF-BP1, E3Histone, HectH9, LASU1, Mule, and Ureb1), an E3 ubiquitin ligase that targets multiple substrates including c-MYC, were detected in 30% of the fresh frozen CC with an additional 17% of the samples demonstrating amplifications in the segment of chromosome 8 containing c-MYC [ 4 ]. Importantly, in a set of 15 primary CC cell lines sequenced in this study, 47% and 20% harbored HUWE1 and c-MYC alterations, respectively [ 4 ]. While the function(s) of HUWE1 mutations in cervical cancer remains poorly understood, alterations in this gene have previously been reported in multiple human tumors including lung, breast and colorectal carcinomas [ 7 , 8 ]. In this regard, controversial mechanisms have been proposed in different tumors with some papers reporting HUWE1 as a potential oncogene [ 9 , 10 ] while others as an onco-suppressor gene [ 11 , 12 ]. Importantly, Inoue and colleagues have recently reported that selective HUWE1 deletion in the skin epithelium augmented the formation of highly proliferative skin tumors [ 8 ]. This phenotype was largely rescued by genetic deletion of c-MYC, suggesting that HUWE1-mediated tumor suppression is strongly dependent on its regulation of c-MYC [ 8 ]. c-MYC amplifications have been implicated in cell self-renewal, survival and tumorigenesis in many human cancers [ 13 , 14 ] and accordingly, targeting c-MYC-addicted tumors with c-MYC specific inhibitors is currently underway against a variety of human tumors in Phase I/II clinical trials ( ClinicalTrials.gov identifier (NCT number): NCT02314052 ; NCT02110563 ). In this regard, since development of direct c-MYC inhibitors or binders is challenging, preclinical studies are currently validating the inhibition of c-MYC using an alternative strategy (i.e., targeting bromodomain and extra-terminal (BET) domain such as BRD2, BRD3, BRD4, and BRDT) with BET inhibitors, which is followed by genome downregulation of c-MYC and c-MYC-dependent target genes [ 15 – 18 ]. In this study, we investigated HUWE1 and c-MYC expression in fresh-frozen CC and the potential activity of the novel BET bromodomain inhibitor GS-626510 (Gilead Science Inc.) against primary WES CC cultures and CC xenografts.

In this study, we preclinically characterize the expression of HUWE1 and c-MYC in multiple whole-exome sequenced primary CC cell lines, and evaluate the activity of a novel oral BET inhibitor (i.e., GS-626510, Gilead Science Inc., Foster City, CA), that may reversibly bind the BET bromodomains proteins BRD2, BRD3, BRD4, and BRDT and prevent protein-protein interaction between BET proteins and acetylated histones and transcription factors [ 17 , 19 , 20 ], against multiple primary CC cell lines. We provide the first evidence that HUWE1 inactivation increases c-Myc expression and cell proliferation in primary CC cell lines and that GS-626510 is endowed with remarkable in vitro and in vivo activity against primary CC cell lines as well as CC xenografts. In our recent WES comprehensive genetic analysis [ 4 ], we found amplification of a segment of chromosome 8 containing c-MYC in 17% of CC samples and mutations in HUWE1, an E3 ubiquitin that targets c-MYC, Cdc6, histones, and the proapoptotic molecule p53 for degradation [ 7 ] were detected in an additional 11 (16%) of the tumors. Furthermore, mutations in the FBXW7 gene, another ubiquitin ligase known to regulate a network of proteins with central roles in cell division, cell growth, and differentiation, including c-MYC [ 21 , 22 ] were detected in an additional 12% of CC. The frequent alterations in HUWE1, FBXW7, and c-MYC found in CC strongly suggest the HUWE1/FBXW7/c-MYC pathway as an attractive target for precision therapeutics in CC patients no longer responsive to standard treatment modalities. Consistent with this hypothesis, and since selective HUWE1 deletion in the skin epithelium has been recently demonstrated to cause increased formation of highly proliferative skin tumors, and a phenotype dependent on its regulation of c-MYC [ 8 ], we sought to investigate the effect mediated by silencing HUWE1 on c-MYC expression in primary fully sequenced CC cell lines and the potential activity of the novel BET bromodomain inhibitor GS-626510 (Gilead Science Inc.) against primary cervical tumor cultures and xenografts. Our results demonstrated c-MYC gene amplification and high c-MYC mRNA expression in the majority (82%) of the primary CC cell lines suggesting that a significant number of CC may be addicted to overactivation of the c-MYC/HUWE1/FBXW7 pathway. Consistent with this view, FISH and western blot experiments confirmed c-MYC gene amplification and high c-Myc protein expression in 9/11 of primary CC cell lines available to this study suggesting that this transcription factor plays an important role in the mechanisms driving proliferation and survival of CC. Taken together, these data confirm and extend previous WES studies from our group [ 4 ], the TCGA Network and others demonstrating common gain of function mutation in c-MYC and loss of function in HUWE1 and FBXW7 in CC patients [ 4 – 6 ]. Importantly, we found the silencing of HUWE1 to significantly upregulate c-MYC expression as well as to induce rapid cell proliferation in all CC cell lines tested. These data are in agreement with several independent recent studies by other research groups, which reported that manipulation of HUWE1 levels may be sufficient to produce dramatic changes in c-Myc ubiquitination in human prostate tumors [ 23 – 25 ]. Next, we preclinically investigated the therapeutic potential of GS-626510 against multiple well-characterized (i.e., WES sequenced) primary CC cell lines. We found the oral BET inhibitor to significantly inhibit tumor cell proliferation in all CC cell lines tested. Of interest, we found that the activity of the BET inhibitor was not affected by the presence or absence of hot spot mutations in the PIK3CA gene, a genetic feature identified by WES in many CC [ 4 – 6 ] and that we previously found to correlate with resistance to anti-HER2/neu targeted agents including trastuzumab and pan-c-erb-inhibitors (i.e., afatinib) in endometrial cancer cell lines [ 26 , 27 ]. Importantly, the novel BET inhibitor was also effective in vivo in decreasing tumor growth and prolonging the survival of mice harboring CC. In this regard, it is worth noting that no evidence of acute or chronic toxicity was detected in the treated animals as demonstrated by the lack of significant variation in behavior or body weights when compared to the mice in the control group. In spite of the limitation of using only one BET inhibitor and one xenograft model, these results, taken together, demonstrate promising activity of GS-626510 against CC cell lines harboring a deranged c-MYC/HUWE1 pathway both in vitro as well as in vivo . In conclusion, using a collection of genetically well-characterized CC cell lines we demonstrated that GS-626510, a novel BET-inhibitor, has impressive activity against CC primary tumors as well as CC xenografts. The silencing of HUWE1 by siRNA significantly increased c-Myc expression as well as CC cell proliferation and enhanced the in vitro sensitivity to GS-626510. Finally, GS-626510 demonstrated activity and was well tolerated at the therapeutic doses used in our in vivo experiments against CC xenografts. Clinical studies with BET-inhibitors in CC patients harboring disease resistant to standard salvage chemotherapy are warranted.