MYC-driven Cancers Research Articles

Abstract 2662: Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world. Current treatments for HCC have limited efficacy. First-line HCC therapy atezolizumab with bevacizumab increased patient 6-month PFS from 37% to 55% relative to sorafenib, a multi-kinase inhibitor currently serving as a standard-of-care treatment. Although immunotherapies provide survival benefits, reported patient response and disease control rates were 27% and 74%, respectively, highlighting the need for novel therapeutic agents that can re-establish responsiveness to immunotherapies and enhance their therapeutic effects. Revolution Medicines has developed a class of selective mTORC1 inhibitors, termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-6272 is a representative bi-steric inhibitor that exhibits potent and selective (>30-fold) inhibition of mTORC1 over mTORC2. Several lines of evidence suggest that inhibition of the mTORC1 pathway may result in synthetic lethality of MYC-driven HCC. Thus, we hypothesized that MYC-driven HCC tumors are sensitive to mTORC1 inhibition and p-4EBP1 suppression. A single dose of RMC-6272 induced HCC regression in 80% of the Lap-tTA/Tet-O-MYC mouse model of MYC-driven HCC, whereas sorafenib did not cause tumor regression. Mechanistically, RMC-6272 inhibited the phosphorylation of mTORC1 downstream substrates S6k and 4EBP1 and depleted MYC protein levels. Given that the MYC oncogene enables immune evasion in HCC, we hypothesized that by reducing MYC, RMC-6272 can re-establish anti-tumor immune surveillance and responsiveness to α-PD-1 immunotherapy. Indeed, RMC-6272 induced tumor recruitment of dendritic cells, T cells, B cells, and natural killer (NK) cells, producing similar anti-tumor immunity as observed upon MYC genetic inactivation. Moreover, combining RMC-6272 with α-PD-1 resulted in deeper tumor regression and a more durable response as compared to either RMC-6272 or α-PD-1 alone in the MYC-driven HCC mouse model. By immunohistochemical staining and analysis, RMC-6272 in combination with α-PD-1, but not either agent alone, promoted tumor recruitment of CD4+ T cells and NK cells, immune cell degranulation, and the release of perforins and granzymes in MYC-driven HCC tumors. Our results show that the bi-steric mTORC1 inhibitor RMC-6272 synergizes with α-PD-1 to induce immune activation and the release of perforins and granzymes, resulting in sustained tumor regression in a MYC-driven HCC mouse model. The bi-steric mTORC1 inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952). These preclinical data highlight a prospective anti-cancer therapeutic opportunity for mTORC1 selective inhibitors in combination with immune checkpoint inhibitors in MYC-driven cancers. Citation Format: Wadie D. Mahauad-Fernandez, Yu C. Yang, Ian Lai, Jangho Park, Lilian Yao, James W. Evans, Jacqueline A. Smith, Mallika Singh, Dean W. Felsher. Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2662.

Abstract 1467: RUVBL1 controls protein synthesis and tumor progression via MYC-dependent EEF1A1 expression

Abstract Dysregulation of MYC family proto-oncogene is a hallmark of tumorigenesis in various cancers (e.g., in Ewing sarcoma [EwS]) so identifying additional MYC mechanisms and dependencies that can be targeted is of critical therapeutic value. Particularly, the epigenetic regulations that dictate the oncogenic MYC signaling and its downstream mechanisms remain unclear. In this study, we conducted a CRISPR interference (CRISPRi) library screen of 3,669 sgRNAs that targeted the transcription start sites of 728 epigenetic-related genes, and identified the requirement of the NuA4 (Nucleosome acetyltransferase of histone H4) complex in EwS. Subsequently, we developed a custom CRISPR-knockout (KO) library targeting all 18 members of the NuA4 complex at 25 sgRNA/gene. These orthogonal CRISPR screens revealed an essential role of RUVBL1 (an ATPase component of NuA4) in EwS. Suppression of RUVBL1 led to the loss of histone H4 acetylation (mass spectrometry), attenuated in vivo tumor growth (xenograft modeling), and ablated MYC signaling (RNA-seq). While the expression of MYC protein was unaffected by RUVBL1-KO, we observed a drastic reduction of the chromatin-associated MYC (ChIP-seq) in the sgRUVBL1 transduced cells, highlighting the pivotal role of RUVBL1 in MYC chromatin binding and target gene transactivation. Mechanistically, we discovered a RUVBL1/MYC co-regulated gene EEF1A1 (a translation elongation factor) to be essential for the survival and protein synthesis of EwS cells. We also found that RUVBL1 interacts with MYC and recruits KAT5 (a histone acetyltransferase in NuA4) to modify the histone H4K8 and H4K12 for acetylation, which are required for the expression of EEF1A1. Furthermore, we conducted a high-density CRISPR gene body scan of 194 sgRNAs that tiled the coding exons of RUVBL1 at ~7.1 bp/sgRNA (i.e., ~2.4 aa/sgRNA). Using this unbiased approach and combined with 3D structural modeling, we uncovered the critical role of a previously unrecognized lysine residue K108 in RUVBL1, which locates at the center of the RUVBL1 hexameric ring structure, to mediate the MYC interaction and MYC target gene expression. Based on these observations, we rationalized a combinational approach to block the oncogenic MYC signaling via simultaneously targeting RUVBL1 (by CB-6644; inhibits RUVBL1’s ATPase activity) and MYC (by JQ1; suppresses MYC expression), and revealed an improved therapeutic index in both established EwS cell lines and the patient-derived EwS samples. In conclusion, our results identified RUVBL1 as a novel vulnerability in EwS, which controls the amplitude of MYC signaling via tuning the chromatin binding of MYC. This notion can be exploited to synergize with other MYC-targeting therapeutics for an improved EwS therapy. We also expect this mechanism to be broadly applicable to other MYC-driven cancers. Citation Format: Mingli Li, Lu Yang, Sheela Pokharel, Anthony Chan, Nicole Mattson, Qiao Liu, Xiaobao Xu, Chun-Wei Chen. RUVBL1 controls protein synthesis and tumor progression via MYC-dependent EEF1A1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1467.

Abstract 2361: MYC-driven breast cancer tumorigenesis is dependent on normal mitochondrial function

Abstract While the transcription factor MYC is amplified in all breast cancer subtypes, nearly 60% of patients with triple-negative tumors have elevated MYC copy number and expression. Patients with triple-negative breast cancer (TNBC) typically have increased metastasis, decreased response to therapies, and poor outcomes, highlighting MYC’s causal association with disease aggressiveness and low survival rates. Unfortunately, MYC is not considered directly pharmacologically tractable. Therefore, we sought to identify collateral “genetic dependencies,” downstream of oncogenic MYC. Using comparative genome-scale CRISPR/Cas9 screening in isogenic human mammary epithelial cells (HMECs), we have identified two mitochondrial membrane transporter genes, TIMM17A and MTCH2, specifically required for MYC-dependent proliferation and survival. Since MYC is suspected to drive metabolic reprogramming in cancers, we assessed how MYC affected mitochondrial protein content by quantitative mass spectrometry. This revealed an increase in N-acetylaspartate (NAA) in HMECs with MYC amplification compared to other genetic backgrounds. Interestingly, increased NAA levels are dependent on TIMM17A and MTCH2 presence. Loss of either of these genes results in cell death coupled with decreased NAA. NAA supplementation in the media of MYC-HMECs following loss of TIMM17A or MTCH2 can rescue the cell death. Importantly, NAA supplementation in cells with guides targeting GFP (negative control) or general essential gene RPL11 did not show increased cell growth/viability - showing that the NAA supplementation is specifically compensating for loss of mitochondrial transport function in MYC-HMECs. Based on these data, it appears that MYC-amplified TNBCs are uniquely dependent on TIMM17A and MTCH2 mitochondrial transporter function because they drive metabolic reprogramming resulting in addiction to N-acetylaspartate production. To conclude these studies, we are evaluating the dependence on mitochondrial transport and NAA synthesis in MYC-amplified TNBCs by assessing the function of TIMM17A and MTCH2 as MYC-specific genetic dependencies in patient derived xenografts of TNBC and determining if TNBCs are addicted to increased NAA synthesis by overexpressing aspartoacylase to breakdown NAA. Successful completion of this work will provide novel drug targets required for survival of aggressive MYC-amplified breast cancers. Citation Format: Sydney M. Moyer, Nina Ilic, Sydney Gang, Taylor E. Arnoff, William C. Hahn. MYC-driven breast cancer tumorigenesis is dependent on normal mitochondrial function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2361.

Abstract 5479: Discovery and evaluation of GT19630, a c-Myc/GSPT1 cereblon E3 ligase modulator (CELMoD), for targeting Myc-driven blood cancers and small cell lung cancers (SCLC)

Abstract c-Myc is an oncogenic transcriptional factor driving tumor initiation, progression and poor prognosis in 80% of all tumor types, especially in B-cell malignancies and small cell lung cancer (SCLC) with Myc genomic alterations. Myc dysregulation have been directly linked to the poor clinical outcome in these cancers. Therefore, it is highly warranted to discover and develop novel Myc therapeutical agents for targeting Myc driven cancers. Here we described GT19630, a GSPT1/Myc CELMoD. GT19630 was discovered through an SAR effort for c-Myc degrader by using c-Myc ELISA and Western blot assays in c-Myc driven HL60 AML cells. GT19630 selectively degraded c-Myc proteins in HL60 cells (IC50=1.5 nM) as compared to growth-factor regulated c-Myc erythroid progenitor cells (TF-1 cells) with IC50=52.5 nM. GT19630 also selectively inhibited HL60 cell proliferation (IC50= 0.33 nM), as compared to its IC50 (26.2 nM) in GM-CSF-TF-1 proliferation, as well as in bone marrow colony-forming cell assays (myeloid=40.2 nM), suggesting>100X selectivity for HL60 cells over normal blood cells. Through the SAR for Myc degrader, GT19630 has been evolved as a leading molecule sharing chemical properties to CELMoDs. Therefore, this compound was further evaluated by proteomics and western blot, GT19630 was confirmed to selective degradation of CELMoD targets, GSPT1/GSPT2 (translation termination factor G1 to S phase transition proteins 1 and 2) with IC90<1 nM and CK1 alpha (IC90<10 nM), but not IKZF1/Ikaros. In silico modeling of GT19630 was performed in the DDB1−CRBN−CC-885−GSPT1 complex and confirmed the docking similarity with CELMoDs. Further, GT19630 inhibited the cell proliferation with IC50<10 nM in 74% B-cell malignant cell lines (20/27) bearing deregulated c-Myc and in 79% of SCLC cell lines (4/19) carrying deregulated Myc (c-Myc, N-Myc or L-Myc) tested. Moreover, GT19630 completely degraded Myc proteins in AML, lymphoma and multiple myeloma (c-Myc) and SCLC (c-Myc and N-Myc) xenograft tumors at the lowest dose of 1.0 mg/kg and induced complete tumor regression (lowest dose=0.3 mg/kg) tested. Furthermore, this compound eradicated lymphoma cells in Daudi-induced liquid lymphoma mouse models. In addition, GT19630, as a potent GSPT1/Myc CELMoD, demonstrated an even-driven pharmacology in vivo and induced complete tumor regression with a dosing regimen of 3 day on/7 day off. Remarkably, GT19630 selectively degraded Myc proteins in HL60 and DMS114 SCLC xenograft tumors as compared to a much less potency at degrading c-Myc in rat spleen. Finally, GT19630 demonstrated favorable PK and safety profiles (an 8-fold safety therapeutic windows) with no effect on myeloid lineages in rats at the dose of 6 mg/kg for 14 days, indicating GT19630 lacks myelosuppression as reported for other CELMoDs. Currently, GT19630 has been advanced into IND enabling stage. Citation Format: Liandong Ma, Youzhi Tong, Zhaohui Yang, Qianxiang Zhou, Hongha Yan, Ru Xu, Jie Chen, Jie Pan, Huiyuan Wang, Jiangwei Li, Dong Chen, Xiang Cai, Jie Qu, Yini Wang, Jun Qin, Yuki Nishida, Michael Andreeff, Qilnli Guo, Yuki Nishida, Michael Andreeff. Discovery and evaluation of GT19630, a c-Myc/GSPT1 cereblon E3 ligase modulator (CELMoD), for targeting Myc-driven blood cancers and small cell lung cancers (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5479.

Abstract 1728: Single cell RNA-seq analysis reveals a role of pro-inflammatory tumor-associated macrophages in driving prostate cancer progression

Abstract Genomic amplification and increased expression of C-MYC are found in high-grade intraepithelial neoplasm (HGPIN) and primary prostate adenocarcinoma. What role C-MYC plays in the initiation and progression of prostate cancer remains unclear. A genetically engineered mouse model with a high level of human C-MYC protein expression under a probasin promoter (Hi-Myc model) was developed that captures prostate cancer progression from hyperplasia at 6-week-old, to HGPIN at 3-6 month-old, and ultimately to invasive adenocarcinoma at 8-month-old. Applying single-cell RNA sequencing (scRNA-seq) technology, we have carefully studied the temporal transcriptional change in MYC-expressing tumor-initiating cells and the tumor-infiltrating immune cells to understand the cell-intrinsic and -extrinsic mechanisms by which C-MYC promotes the transition from HGPIN to invasive prostate cancer. We have identified a particular type of luminal cells, Prom1+ L1 cells, as the likely tumor initiating cells in the Hi-Myc model. The MYC-expressing L1 tumor initiating cells were more abundant and cycling more often in the invasive cancer at 8-month-old, compared to the pre-invasive stage. Interestingly, significant infiltration of novel M1-like tumor-associated macrophages (TAMs) correlated with the histologic tumor progression from HGPIN to invasive cancer. Targeting TAMs with anti-CSF1R antibody delayed prostate cancer progression and invasion, which appears to be independent of CD8 T-cells. Mechanistically, TAMs expressed a high level of IL-1b, which stimulated Hi-Myc prostate cell proliferation in an organoid co-culture system. More importantly, depleting IL-1b with a neutralizing antibody slowed prostate cancer progression in Hi-Myc mice. Lastly, we found that increased macrophage gene expression was associated with higher Gleason scores and worse disease-free survival in a TCGA clinical patient cohort. In summary, we have identified a molecular mechanism involving pro-inflammatory TAMs and IL-1b in driving early prostate cancer progression in a MYC-driven prostate cancer model, opening a potential therapeutic avenue to target TAMs and pro-inflammatory cytokine in preventing early prostate cancer progression. Citation Format: Jimmy L. Zhao, Joseph Chan, Max Land, Perianne Smith, Anuradha Gopalan, Michael Haffner, Dana Pe'er, Charles Sawyers. Single cell RNA-seq analysis reveals a role of pro-inflammatory tumor-associated macrophages in driving prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1728.

MEDB-73. Lipid metabolism as a therapeutic vulnerability in BET inhibitor-resistant medulloblastoma

MYC-driven medulloblastomas are a particularly devastating group of pediatric brain tumors that exhibit resistance and continued progression despite standard of care treatments. Our preclinical work identified BET-bromodomain inhibitors as a potentially promising new class of drugs for children with medulloblastoma and other MYC-driven cancers, providing rationale to evaluate these agents in clinical trials. However, treatment with BET inhibitor (BETi) alone is unlikely to be sufficient to cure, with most tumors evolving to acquire resistance to single-agent targeted therapies. We applied an integrative genomics approach to identify genes and pathways mediating BETi response in medulloblastoma. These studies revealed that MYC-driven medulloblastoma cells with acquired resistance to BETi reinstate transcription of essential genes suppressed by drug and exhibit changes in cell state and new vulnerabilities not present in drug-sensitive cells. We now have a growing body of evidence showing that BET inhibition downregulates the expression of key lipid metabolism genes and metabolism-related signaling pathways, and that medulloblastoma cells with adaptive resistance to drug differentially express and exhibit preferential dependency on specific lipid metabolic genes and transcriptional regulators. Our studies explore the possibility of exploiting these metabolic vulnerabilities to overcome BETi resistance and provide a more efficacious upfront therapy.

MEDB-85. Transcriptional complexes as resistance drivers to BET inhibition

BET-bromodomain inhibition (BETi) is a promising therapeutic strategy to target MYC-driven cancers, including Group 3 medulloblastoma, a deadly childhood brain tumor. We have shown that BET inhibitors exhibit preclinical efficacy against MYC¬-amplified medulloblastoma, providing motivation to evaluate this drug class in early phase clinical trials. However, our work has also found that MYC-amplified medulloblastoma cells can acquire resistance to BETi, suggesting that curative responses for this disease will require combination therapy. To guide the development of such combination therapies, we have focused our efforts on elucidating the mechanisms through which medulloblastoma cells acquire resistance to BETi. We found that medulloblastoma cells can develop tolerance to BETi by reinstating the expression of cell-essential “rescue genes,” which include bHLH transcription factors, cell-cycle regulators, and anti-apoptosis genes. This transition to the resistant cell state is mediated through changes in chromatin structure including the upregulation of H3K4me3 promoters. Our preliminary results suggest that BETi-resistant cells maintain mRNA transcription and protein translation of important mediators of resistance. Importantly, we observe that BETi-resistant medulloblastoma cells are more dependent on specific protein complexes involved in transcriptional regulation. This project explores the mechanisms through which these transcriptional regulators help maintain transcription of rescue genes that drive BETi resistance and evaluates the potential of targeting these drivers of BETi resistance. These results will help guide the development of combination approaches to improve the efficacy of BETi for the treatment of MYC-driven medulloblastoma.

Small Molecule 20S Proteasome Enhancer Regulates MYC Protein Stability and Exhibits Antitumor Activity in Multiple Myeloma.

Despite the addition of several new agents to the armamentarium for the treatment of multiple myeloma (MM) in the last decade and improvements in outcomes, the refractory and relapsing disease continues to take a great toll, limiting overall survival. Therefore, additional novel approaches are needed to improve outcomes for MM patients. The oncogenic transcription factor MYC drives cell growth, differentiation and tumor development in many cancers. MYC protein levels are tightly regulated by the proteasome and an increase in MYC protein expression is found in more than 70% of all human cancers, including MM. In addition to the ubiquitin-dependent degradation of MYC by the 26S proteasome, MYC levels are also regulated in a ubiquitin-independent manner through the REGγ activation of the 20S proteasome. Here, we demonstrate that a small molecule activator of the 20S proteasome, TCH-165, decreases MYC protein levels, in a manner that parallels REGγ protein-mediated MYC degradation. TCH-165 enhances MYC degradation and reduces cancer cell growth in vitro and in vivo models of multiple myeloma by enhancing apoptotic signaling, as assessed by targeted gene expression analysis of cancer pathways. Furthermore, 20S proteasome enhancement is well tolerated in mice and dogs. These data support the therapeutic potential of small molecule-driven 20S proteasome activation for the treatments of MYC-driven cancers, especially MM.

A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth

BackgroundChromosome translocations involving mixed lineage leukemia 1 (MLL1) cause acute leukemia in most infants and 5–10% children/adults with dismal clinical outcomes. Most frequent MLL1-fusion partners AF4/AFF4, AF9/ENL and ELL, together with CDK9/cyclin-T1, constitute super elongation complexes (SEC), which promote aberrant gene transcription, oncogenesis and maintenance of MLL1-rearranged (MLL1-r) leukemia. Notably, ENL, but not its paralog AF9, is essential for MLL1-r leukemia (and several other cancers) and therefore a drug target. Moreover, recurrent ENL mutations are found in Wilms tumor, the most common pediatric kidney cancer, and play critical roles in oncogenesis.MethodsProteolysis-Targeting Chimera (PROTAC) molecules were designed and synthesized to degrade ENL. Biological activities of these compounds were characterized in cell and mouse models of MLL1-r leukemia and other cancers.ResultsCompound 1 efficiently degraded ENL with DC50 of 37 nM and almost depleted it at ~ 500 nM in blood and solid tumor cells. AF9 (as well as other proteins in SEC) was not significantly decreased. Compound 1-mediated ENL reduction significantly suppressed malignant gene signatures, selectively inhibited cell proliferation of MLL1-r leukemia and Myc-driven cancer cells with EC50s as low as 320 nM, and induced cell differentiation and apoptosis. It exhibited significant antitumor activity in a mouse model of MLL1-r leukemia. Compound 1 can also degrade a mutant ENL in Wilms tumor and suppress its mediated gene transcription.ConclusionCompound 1 is a novel chemical probe for cellular and in vivo studies of ENL (including its oncogenic mutants) and a lead compound for further anticancer drug development.

Combined Inactivation of CTPS1 and ATR Is Synthetically Lethal to MYC-Overexpressing Cancer Cells.

Inhibition of CTPS in MYC-overexpressing cells blocks pyrimidine synthesis while maintaining ribosome synthesis activity to create an anabolic imbalance that induces replication stress, providing a new approach to selectively target MYC-driven cancer. See related commentary by Chabanon and Postel-Vinay, p. 969.

MiR-32 promotes MYC-driven prostate cancer

miR-32 is an androgen receptor (AR)-regulated microRNA, expression of which is increased in castration-resistant prostate cancer (PC). We have previously shown that overexpression of miR-32 in the prostate of transgenic mice potentiates proliferation in prostate epithelium. Here, we set out to determine whether increased expression of miR-32 influences growth or phenotype in prostate adenocarcinoma in vivo. We studied transgenic mice expressing MYC oncogene (hiMYC mice) to induce tumorigenesis in the mouse prostate and discovered that transgenic overexpression of miR-32 resulted in increased tumor burden as well as a more aggressive tumor phenotype in this model. Elevated expression of miR-32 increased proliferation as assessed by Ki-67 immunohistochemistry, increased nuclear density, and higher mitotic index in the tumors. By gene expression analysis of the tumorous prostate tissue, we confirmed earlier findings that miR-32 expression regulates prostate secretome by modulating expression levels of several PC-related target genes such as Spink1, Spink5, and Msmb. Further, we identified Pdk4 as a tumor-associated miR-32 target in the mouse prostate. Expression analysis of PDK4 in human PC reveals an inverse correlation with miR-32 expression and Gleason score, a decrease in castration-resistant and metastatic tumors compared to untreated primary PC, and an association of low PDK4 expression with a shorter recurrence-free survival of patients. Although decreased PDK4 expression induces the higher metabolic activity of PC cells, induced expression of PDK4 reduces both mitotic respiration and glycolysis rates as well as inhibits cell growth. In conclusion, we show that miR-32 promotes MYC-induced prostate adenocarcinoma and identifies PDK4 as a PC-relevant metabolic target of miR-32-3p.

MYC overexpression leads to increased chromatin interactions at super-enhancers and MYC binding sites.

The MYC oncogene encodes for the MYC protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. MYC overexpression leads to MYC binding at active enhancers, resulting in a global transcriptional amplification of active genes. Because super-enhancers are frequently dysregulated in cancer, we hypothesized that MYC preferentially invades into super-enhancers and alters the cancer genome organization. To that end, we performed ChIP-seq, RNA-seq, circular chromosome conformation capture (4C-seq), and Spike-in Quantitative Hi-C (SIQHiC) on the U2OS osteosarcoma cell line with tetracycline-inducible MYC. MYC overexpression in U2OS cells modulated histone acetylation and increased MYC binding at super-enhancers. SIQHiC analysis revealed increased global chromatin contact frequency, particularly at chromatin interactions connecting MYC binding sites at promoters and enhancers. Immunofluorescence staining showed that MYC molecules formed punctate foci at these transcriptionally active domains after MYC overexpression. These results demonstrate the accumulation of overexpressed MYC at promoter–enhancer hubs and suggest that MYC invades into enhancers through spatial proximity. At the same time, the increased protein–protein interactions may strengthen these chromatin interactions to increase chromatin contact frequency. CTCF siRNA knockdown in MYC-overexpressed U2OS cells demonstrated that removal of architectural proteins can disperse MYC and abrogate the increase in chromatin contacts. By elucidating the chromatin landscape of MYC-driven cancers, we can potentially target MYC-associated chromatin interactions for cancer therapy.

Abstract LBA004: Identification of GSPT1-directed molecular glue degrader (MGD) for the treatment of Myc-driven breast cancer

Abstract The Myc family of transcription factors is a well-established driver of human cancers. However, despite being amongst the most frequently mutated, translocated and overexpressed oncogenes, no therapy directly targeting the Myc family members has been developed to date. Abnormal activation of Myc results in uncontrolled cell growth that is associated with high translational output and ramp up of the protein translational machinery. This creates a dependency to protein translation and in turn represents a potential therapeutic vulnerability for Myc-driven tumors. Based on these considerations, we hypothesized that targeting the translational termination factor GSPT1, a key player of protein synthesis, may constitute a vulnerability for Myc-driven tumors. Using our proprietary Quantitative and Engineered Elimination of Neosubstrates (QuEENTM) platform we characterized and explored the known G-loop degron in GSPT1 that renders it amenable to cereblon-induced degradation by molecular glue degraders (MGDs). We rationally designed and subsequently screened a proprietary library of cereblon-binding small molecules, including GSPT1-directed MGDs, in human mammary epithelial cells (HMECs) expressing doxycycline-inducible c-Myc. Doxycycline treatment led to sustained c-Myc expression and as a consequence to the induction of key biomarkers of enhanced protein translation, such as phospho 4EBP1 (p4EBP1). We identified MRT-048 as a potent and highly selective GSPT1 degrader and demonstrated its ability to induce cell death in Myc-driven HMEC cells whilst sparing control cells (EC50 0.64 μM vs 30 μM respectively). This confirmed the selective vulnerability of Myc-driven cell growth to GSPT1 degradation. In follow-up studies, we confirmed the correlation between p4EBP1 as biomarker of Myc-activation and sensitivity to MRT-048 in a large panel of breast cancer cell lines. Moreover, MRT-048 treatment of animals xenografted with breast cancer cells induced tumor regression and was associated with complete GSPT1 degradation. Mechanistically, we observed that GSPT1 degradation induced by MRT-048 led to inhibition of genes regulated by Myc and ribosomal stalling at stop codons of several mRNAs. Additionally, polysome profiling of cancer cells treated with MRT-048 was associated with a global reduction of the intensities of the polysome peaks and concomitant increase in the monosome peaks as previously observed in GSPT1 knockdown experiments, suggesting that GSPT1 degradation by our MGD molecules affects both the termination and initiation stages of protein translation. We believe these data support the therapeutic potential of GSPT1-directed MGDs in Myc-driven tumors dependent on protein translation machinery. Citation Format: Gerald Gavory, Bernhard Fasching, Debora Bonenfant, Amine Sadok, Ambika Singh, Martin Schillo, Vittoria Massafra, Anne-Cecile d’Alessandro, John Castle, Mahmoud Ghandi, Agustin Chicas, Frederic Delobel, Alexander Flohr, Giorgio Ottaviani, Thomas Ryckmans, Anne-Laure Laine, Oliv Eidam, Hannah Wang, Ilona Bernett, Laura Chan, Chiara Gorrini, Theo Roumiliotis, Jyoti Choudhary, Yann-Vai LeBihan, Marc Cabry, Mark Stubbs, Rosemary Burke, Rob Van Montfort, John Caldwell, Rajesh Chopra, Ian Collins, Silvia Buonamici. Identification of GSPT1-directed molecular glue degrader (MGD) for the treatment of Myc-driven breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA004.

Abstract P178: RBM39 degradation leads to exceptional responses in high-risk neuroblastoma by targeting the spliceosome

Abstract Genome-wide studies of thousands of human tumors have revealed dysregulated pre-mRNA splicing as a critical pathway in tumorigenesis, which may consequently create vulnerabilities for cancer cells. Indeed, C-MYC-dependent cancers are specifically susceptible to perturbation of spliceosome function. The MYC oncogenes (C-MYC, MYCN, and MYCL1), which are known transcriptional drivers of large numbers of genes, are among the most common genetic abnormalities in human cancer. We found that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires splicing factor RBM39 for survival. Indisulam, a “molecular glue” that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to exceptional responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy. In summary, our study has validated the mechanism by which “molecular glues” targeting RNA binding proteins can be exploited for drug development in cancer, and has a broad impact on understanding of the splicing dependency of MYC-driven cancers and the identification of novel therapeutic approaches against them. Citation Format: Jun Yang. RBM39 degradation leads to exceptional responses in high-risk neuroblastoma by targeting the spliceosome [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P178.

Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models.

Aberrant alternative pre-mRNA splicing plays a critical role in MYC-driven cancers and therefore may represent a therapeutic vulnerability. Here, we show that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires the splicing factor RBM39 for survival. Indisulam, a “molecular glue” that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to significant responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy.

Abstract PO-027: Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of only 10%. A major feature of PDAC tumor microenvironment (TME) is desmoplasia, which are dense fibrotic and inflammatory stromal regions constituting the PDAC TME. This desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a significant deficiency in unsaturated fatty acids (UFAs) because the de novo synthesis of UFAs requires molecular oxygen. Our data indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger a subsequent unfolded protein response (UPR) under tumor-like stress, which can be mitigated by UFAs. The UPR regulates an intricate signaling network mediated by three major stress sensors: Inositol-requiring enzyme 1a (IRE1a), PKR-like ER kinase (PERK) and activating transcription factor 6a (ATF6a). UPR output can be either cytoprotective or cytotoxic depending on tumor types and the severity of stress. Our data suggest that B-I09, a selective IRE1 RNase inhibitor, impairs PDAC cell viability under stress conditions, indicating a pro-survival role for IRE1a. Previous studies showed that MYC-driven cancer cells are highly reliant on UPR pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” may have distinct dependencies on UPR pathways, with the Basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Overall, we hypothesize that IRE1a, PERK and/or ATF6a play(s) an oncogenic role in PDAC tumorigenesis due to lipid saturation abnormalities, and the Basal-like PDAC subtype is more sensitive to inhibition of UPR pathway(s). We are evaluating the role of IRE1a, PERK and ATF6a using pharmacologic inhibition and genetic knockdown in vitro and in vivo under UFA deprived conditions. We will also evaluate PDAC intracellular and interstitial fluid lipidomics using LC-MS. In addition, we will assess whether hypoxic PDAC cells experience ER stress in vivo due to UFA deprivation using Stimulated Raman scattering (SRS) microscopy. To explore the subtype-specific dependency on the UPR pathways, we are querying a UPR gene signature in different tumor cell clusters using single cell RNA-seq datasets of patient samples. In addition, we will employ an isogenic system for PDAC subtypes and examine subtype-specific responses to UPR inhibition in vitro and in vivo. Our goal is to better understand UPR pathways in PDAC cells as a survival mechanism in response to the “lipotoxicity” induced by ER membrane saturation, with a consideration of intratumoral heterogeneity of PDAC cell subtype. Citation Format: Yanqing (Christine) Jiang, Xu Han, Michelle Burrows, Carson Poltorack, Celeste Simon, Brian Keith. Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-027.

TAMI-75. LIPID METABOLISM AS A THERAPEUTIC VULNERABILITY IN BET INHIBITOR-RESISTANT MEDULLOBLASTOMA

Abstract MYC-driven medulloblastomas are a particularly devastating group of pediatric brain tumors that exhibit resistance and continued progression despite standard of care treatments. Our preclinical work identified BET-bromodomain inhibitors as a potentially promising new class of drugs for children with medulloblastoma and other MYC-driven cancers, providing rationale to evaluate these agents in clinical trials. However, treatment with BET inhibitor (BETi) alone is unlikely to be sufficient for a cure, with most tumors evolving to acquire resistance to single-agent targeted therapies. We applied an integrative genomics approach to identify genes and pathways mediating BETi response in medulloblastoma. These studies revealed that MYC-driven medulloblastoma cells with acquired resistance to BETi reinstate transcription of essential genes suppressed by drug, and exhibit changes in cell state and new vulnerabilities not present in drug-sensitive cells. We now have a growing body of evidence showing that BET inhibition downregulates the expression of key lipid metabolism genes and metabolism-related signaling pathways, and that medulloblastoma cells with adaptive resistance to drug differentially express and exhibit preferential dependency on specific lipid metabolic genes and transcriptional regulators. Our studies explore the possibility of exploiting these novel metabolic vulnerabilities in order to overcome BETi resistance and provide a more efficacious upfront therapy.

TMOD-28. MYC GENERATES AGGRESSIVE MEDULLOBLASTOMA BY HSP90 PATHWAY ACTIVATION AND ARF SILENCING

Abstract Medulloblastoma, the most common malignant pediatric brain tumor, often shows amplification or overexpression of the MYC transcription factor and arises in the presence of a functional p53 tumor suppressor protein. To elucidate the mechanism behind this inexplicable tumor development we generated an inducible, immunocompetent transgenic mouse model of MYC-expressing medulloblastoma. Aggressive tumors developed clonally in the presence of an unaltered p53 gene that molecularly resembled Group 3 medulloblastoma. Compared to MYCN-expressing medulloblastoma driven from the same promoter, we instead discovered pronounced and MIZ1-independent silencing of the ARF suppressor, which was also suppressed in MYC-amplified as compared to MYCN-amplified human medulloblastoma. While MYCN-driven tumor malignancy was more sensitive to ARF depletion, it dramatically increased metastatic spread of MYC-driven tumors. DNMT inhibition could restore ARF levels in MYC-expressing tumors but did not show any therapeutic advantage in tumors in vivo. Bioinformatics analysis further showed a strong correlation of the HSP90 pathway with MYC in human Group 3 MB and in the MYC-driven mouse model. The HSP90 inhibitor Onalespib showed significant selectivity for targeting MYC-driven as compared to MYCN-driven tumors. The drug promoted ARF restoration and increased the survival in our animal model which suggests that it could be potentially used in the treatment of MYC-driven ARF-silenced brain cancer patients.

Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Identifies Genomic and Molecular Markers for Decitabine (DAC) Plus Valproic-Acid (VPA) Treatment Response in Patients with Myelodysplastic Syndromes (MDS)

Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Identifies Genomic and Molecular Markers for Decitabine (DAC) Plus Valproic-Acid (VPA) Treatment Response in Patients with Myelodysplastic Syndromes (MDS)

Targeting Myc Interacting Proteins as a Winding Path in Cancer Therapy.

MYC, as a well-known oncogene, plays essential roles in promoting tumor occurrence, development, invasion and metastasis in many kinds of solid tumors and hematologic neoplasms. In tumors, the low expression and the short half-life of Myc are reversed, cause tumorigenesis. And proteins that directly interact with different Myc domains have exerted a significant impact in the process of Myc-driven carcinogenesis. Apart from affecting the transcription of Myc target genes, Myc interaction proteins also regulate the stability of Myc through acetylation, methylation, phosphorylation and other post-translational modifications, as well as competitive combination with Myc. In this review, we summarize a series of Myc interacting proteins and recent advances in the related inhibitors, hoping that can provide new opportunities for Myc-driven cancer treatment.