BET Inhibitors Research Articles

Genome-Wide Analysis Identifies Nuclear Factor 1C as a Novel Transcription Factor and Potential Therapeutic Target in SCLC

IntroductionRecent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy. MethodsTranscriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared with normal human small airway epithelial cells (SAECs) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitive site sequencing (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to evaluate growth, tumorigenicity, and changes in transcriptomes and glucose metabolism of SCLC cells after NFIC knockdown and to evaluate NFIC expression in SCLC cells after exposure to BET inhibitors. ResultsConsiderable commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DHS-seq to RNA sequencing enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in vitro and in vivo. ChIP-seq analysis identified numerous sites occupied by BRD4 in the NFIC promoter region. Knockdown of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETis) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes down-regulated by BETi treatment were repressed by NFIC knockdown in SCLC, whereas 34% of genes repressed after NFIC knockdown were also down-regulated in SCLC cells after BETi treatment. ConclusionsNFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.

Abstract LB159: Discovery of HP560, a novel BET inhibitor for the treatment of myelofibrosis

Abstract Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by defective clonal hematopoiesis, bone marrow fibrosis, splenomegaly, severe anemia, and the propensity for transformation to acute myeloid leukemia (AML). Inhibitors targeting JAKs (Janus kinases) have revolutionized the treatment of MF, but are limited by modest efficacy or acquired resistance after prolonged treatment. Here, we report a novel inhibitor, HP560 that targets BET (bromodomain and extra-terminal) proteins and shows promising therapeutic activities against MF and AML. Through engaging the bromodomain pocket of BET proteins in a competitive manner with acetylated histones, HP560 causes the disruption of BET proteins from chromatin. HP560 has high binding affinities for all BET proteins and strong anti-proliferative activity as well as synergistic anti-proliferative effect in combination with ruxolitinib, a JAK1/2 inhibitor. In an MF cell line derived tumor xenograft (CDX) mouse model, HP560 prolongs survival and improves splenomegaly and anemia, and these effects are enhanced when combined with ruxolitinib. HP560 also significantly and dose-dependently inhibits tumor growth in an AML CDX mouse model. Together, HP560 is a potent pan-BET inhibitor and can be developed as a monotherapy or in combination with a JAK inhibitor for the treatment of MF. HP560 structure will not be disclosed. Citation Format: Jing Li, Lei Fan, Hongwei Yuan, Yongxu Huo, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP560, a novel BET inhibitor for the treatment of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB159.

Transcriptional elongation control of hypoxic response

The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.

Targeting Metabolic Dependencies Fueling the TCA Cycle to Circumvent Therapy Resistance in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is one of the most prevalent blood cancers, characterized by a dismal survival rate. This poor outcome is largely attributed to AML cells that persist despite treatment and eventually result in relapse. Relapse-initiating cells exhibit diverse resistance mechanisms, encompassing genetic factors and, more recently discovered, nongenetic factors such as metabolic adaptations. Leukemic stem cells (LSC) rely on mitochondrial metabolism for their survival, whereas hematopoietic stem cells primarily depend on glycolysis. Furthermore, following treatments such as cytarabine, a standard in AML treatment for over four decades, drug-persisting leukemic cells exhibit an enhanced reliance on mitochondrial metabolism. In this issue of Cancer Research, two studies investigated dependencies of AML cells on two respiratory substrates, α-ketoglutarate and lactate-derived pyruvate, that support mitochondrial oxidative phosphorylation (OXPHOS) following treatment with the imipridone ONC-213 and the BET inhibitor INCB054329, respectively. Targeting lactate utilization by interfering with monocarboxylate transporter 1 (MCT1 or SLC16A1) or lactate dehydrogenase effectively sensitized cells to BET inhibition in vitro and in vivo. In addition, ONC-213 affected αKGDH, a pivotal NADH-producing enzyme of the TCA cycle, to induce a mitochondrial stress response through ATF4 activation that diminished the expression of the antiapoptotic protein MCL1, consequently promoting apoptosis of AML cells. In summary, targeting these mitochondrial dependencies might be a promising strategy to kill therapy-naïve and treatment-resistant OXPHOS-reliant LSCs and to delay or prevent relapse. See related articles by Monteith et al., p. 1101 and Su et al., p. 1084.

BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney—an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery.

Abstract 5779: Enhancing selectivity and overcoming tissue-specific toxicity in drug discovery: A novel BET inhibitor with targeted delivery for prostate cancer treatment

Abstract Improving selectivity and circumventing tissue-specific toxicity remain significant challenges in the realm of contemporary drug discovery. Despite the advanced development of highly selective inhibitors, their systemic distribution often stimulates off-tissue unwanted side-toxicity. To address this, we engineered a highly selective inhibitor that specifically targets and releases a small molecule drug payload to a desired cell type. As a proof of concept, we apply this method to epigenetic inhibitors of BET proteins, which are crucial regulators of gene transcription implicated in the progression and survival of cancer. We engineered a novel BET-selective chemotype conjugated to a Prostate-Cancer specific targeting component in order to overcome the off-tissue effects of state-of-the-art BET inhibitors. To achieve delivery of the compound, we modified the benzodiazepine (BzD) scaffold of the well-established BET inhibitor I-BET762, conjugating the tertiary amine with a self-immolating motif, allowing for intracellular compound release upon an enzymatic trigger. This new chemotype, RT53, maintains the biochemical properties of I-BET762, showing potent and selective binding to BET bromodomains in a range of established biophysical assays. In cells, RT53 phenocopied I-BET762 on cancer cell lines, including prostate cancer. We validated the established strong anti-proliferative effect following BET inhibition using clonogenic assays, flow cytometry, and monitored the suppression of MYC expression. Moreover, we established the transcriptional rationale for loss of cellular fitness using RNA sequencing, discovering strong down-regulation of solute carriers, depriving cells of nutrient flux and disrupting normal processes. Furthermore, RT53 exhibits antitumor activity in vivo, in localized models of prostate cancer established in male athymic nude mice. The targeted delivery of RT53 demonstrated superior efficacy in vivo ameliorating on-target, off-tissue toxicity, as low-dose treatment led to almost complete elimination of grafted tumor sites, significant extension of survival, and no signs of toxicity on non-targeted tissues. Taken together, our data establish a novel class of BET-specific inhibitors offering a handle for selective delivery in vivo, enhancing the therapeutic index while avoiding on-target off-tissue toxicity. Citation Format: Cong Tang, Rui Traquete, Elizabeth Henderson, João Conde, Sara Picaud, Panagis Filippakopoulos, Gonçalo Bernardes. Enhancing selectivity and overcoming tissue-specific toxicity in drug discovery: A novel BET inhibitor with targeted delivery for prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5779.

BET inhibitors drive Natural Killer activation in non-small cell lung cancer via BRD4 and SMAD3

Non-small-cell lung carcinoma (NSCLC) is the most common lung cancer and one of the pioneer tumors in which immunotherapy has radically changed patients’ outcomes. However, several issues are emerging and their implementation is required to optimize immunotherapy-based protocols. In this work, we investigate the ability of the Bromodomain and Extra-Terminal protein inhibitors (BETi) to stimulate a proficient anti-tumor immune response toward NSCLC. By using in vitro, ex-vivo, and in vivo models, we demonstrate that these epigenetic drugs specifically enhance Natural Killer (NK) cell cytotoxicity. BETi down-regulate a large set of NK inhibitory receptors, including several immune checkpoints (ICs), that are direct targets of the transcriptional cooperation between the BET protein BRD4 and the transcription factor SMAD3. Overall, BETi orchestrate an epigenetic reprogramming that leads to increased recognition of tumor cells and the killing ability of NK cells. Our results unveil the opportunity to exploit and repurpose these drugs in combination with immunotherapy.

Abstract 7544: Bromodomain and extra-terminal protein inhibitors deplete nuclear factor 1C and reduce stemness and growth of human small cell lung cancer cells in vitro and in vivo

Abstract Objectives: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to examine complex epigenetic mechanisms underpinning the aggressiveness of SCLC, with a focus on understanding those mediating stemness in these highly lethal cancers. Methods: Normal small airway epithelial cells (SAECs) were reprogrammed into iPSCs, validated by various assays, such as spectral karyotyping and teratoma formation. Genome-wide chromatin landscapes of lung iPSCs (Lu-iPSC) and SAECs, as well as NSCLC and SCLC lines were characterized using RNA-seq, DNA methylome, and DNase-hypersensitivity sequencing (DHS). Gene knock-downs were established using siRNA/shRNA techniques, along with qRT-PCR, immunoblotting, proliferation, and tumorigenicity assays. Glycolysis was analyzed using Seahorse assays. In-silico analysis of the NFIC promoter was performed using TFmapper. NFIC and related gene expression in human SCLC cells following NFIC or Bromodomain-containing protein (BRD) knockdown or exposure to BET inhibitors (BETi) were evaluated. Stemness scores were calculated using established RNA-seq criteria, and gene set enrichment analyses were conducted. Proliferation assays and xenograft models were used for in-vitro and in-vivo SCLC growth assessment. Results: RNA-seq,DNA methylome and DHS analyses revealed shared characteristics among SAEC, Lu-iPSC, SCLC, and NSCLC, whereas SOXs and POUs showed strong activity in Lu-iPSC but not SCLC. NFIC, with elevated chromatin occupancy in SCLC, exhibited increased expression compared to SAEC. NFIC KD in SCLC significantly curtailed cell growth in-vitro and in-vivo, affecting multiple pathways, including glycolysis and stem cell signaling etc, confirmed by Seahorse assays. Furthermore, we identified occupancy sites for BRD4 in the NFIC promoter, and knockdown of BRD4, but not BRD2 or BRD3, reduced NFIC expression in SCLC cells. BET inhibitor (BETi) treatment dose-dependently decreased NFIC expression in SCLC lines and human SCLC PDXs, irrespective of subtype. BETi treatment also reduced SCLC stemness and showed a significant overlap in gene regulation with NFIC knockdown, affecting pathways related to gluconeogenesis, neuronal differentiation, and embryonic organ morphogenesis. Importantly, BETi treatment effectively reduced SCLC cell growth in-vitro and in-vivo, suggesting the potential therapeutic value of targeting NFIC via BET inhibition in SCLC. Conclusions: NFIC, a unique transcription factor in SCLC, emerges as a key downstream target of BRD4. These findings underscore the potential druggability of NFIC in clinical settings, provide compelling rationale for evaluating BETi for SCLC therapy. Citation Format: Haitao Wang, Ruihong Wang, Frank Villa-Hernandez, Vivek Shukla, Lyuba Varticovski, Songjoon Baek, Frank Echtenkamp, Sudheer Kumar Gara, Mary R. Zhang, Stephanie Shiffka, Razi Raziuddin, Leonard M. Neckers, W. Marston Linehan, Gordon L. Hager, Shamus R. Carr, Chuong D. Hoang, Haobin Chen, David S. Schrump. Bromodomain and extra-terminal protein inhibitors deplete nuclear factor 1C and reduce stemness and growth of human small cell lung cancer cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7544.

Abstract 3351: Identifying novel therapies from unbiased screens in AML with MECOM re-arrangement

Abstract EVI1 is a transcriptional regulator encoded from the MECOM locus (3q26.2). Aberrant EVI1 expression is observed in ~10% of de novo AML, of which about half are due to inv3(q21;q26.2) or t(3;3)(q21;q26.2) chromosomal rearrangements [3q26.2-r]. 3q26.2-r AMLs generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Therefore, there is an unmet need to develop novel targeted therapies with improved efficacy in repressing EVI1 and its targets, thereby improving outcomes in AML with EVI1 overexpression. We conducted an unbiased high-throughput drug screen, utilizing a library of mechanistically annotated drugs (NCATS Mechanism Interrogation Plates [MIPE 5.0]), against seven AML cell lines (3q26.2-r: UCSD-AML1, HNT-34, AML191, AML194; non-3q26.2-r AML: SET-2, MV4-11, OCI-AML3). This exploited the mechanistic redundancy in the library to identify druggable, target-level dependencies in 3q26.2-r AML, exhibiting absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. An unbiased epigenetic modifier CRISPR screen in UCSD-AML further confirmed BRD4 as a dependency, confirming previous reports of BET inhibition (BETi) efficacy in 3q26.2-r AML. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, IAP family inhibitors/SMAC mimetics induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines, which was associated with reduction of IAP proteins, p-ERK, MCL1 and Bcl-xL, but increase in levels of cleaved caspase-3 and PARP. BETi (mivebresib) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, IAP proteins, and CDK4/6, while increasing levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment of mivebresib with SMAC mimetic birinapant or IAP inhibitor LCL161 was synergistically lethal against 3q26.2-r cells. Co-targeting the other identified druggable vulnerabilities from the drug screen with BETi (mTOR/PI3K with BGT-226 and dactolisib, Bcl-xL with navitoclax and A1155463, as well as CBP/p300 (with GNE781, identified through the CRISPR screen), exerted synergistic lethality in 3q26.2-r AML cells. Finally, in a xenograft model of 3q26.2-r AML in immune-depleted mice, while monotherapy with LCL161 or dactolisib significantly reduced the AML burden with minimal toxicity, co-treatment with mivebresib and LCL161 or dactolisib was superior in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of BETi and IAP protein or mTOR/PI3K antagonists in the cellular models of AML with EVI1 overexpression. This supports the rationale to determine in vivo efficacy of these BETi-based combinations against this therapy-resistant AML sub-type. Citation Format: Christine E. Birdwell, Warren C. Fiskus, Christopher P. Mill, Tapan M. Kadia, Naval Daver, Courtney D. DiNardo, Koji Sasaki, John A. Davis, Kaberi Das, Hanxi Hou, Michele Cerebelli, Kapil N. Bhalla. Identifying novel therapies from unbiased screens in AML with MECOM re-arrangement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3351.

Abstract 4739: Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. Fusion-positive (FP-)RMS expressing the oncogenic chimeric transcription factor (TF) PAX3-FOXO1 (P3F) is at high risk of recurrence. FP-RMS cells show survival dependency on P3F. Despite P3F, as a TF, is considered undruggable, we and others demonstrated that P3F levels/functions can be epigenetically modulated. The BET protein and epigenetic reader BRD4 binds super-enhancers (SEs) to foster oncogenic transcription in cancer. We showed that P3F rewires the RMS enhancer landscape by recruiting BRD4 on oncogenic SEs. Accordingly, JQ1, a BET inhibitor (BETi) shuts down P3F functions halting tumor growth in vitro and in vivo. Therefore, BRD4 inhibition in FP-RMS results in a tumor subtype-specific vulnerability. Nonetheless, as for other targeted therapies, cancer cells can acquire resistance to BETi, thus, we investigated whether FP-RMS cells could develop BETi resistance. Therefore, we established resistant cells from two sensitive FP-RMS cell lines chronically exposed to escalating doses of JQ1, as a model of naturally acquired resistance. Our data show that the two established FP-RMS cell lines, acquired resistance by increasing JQ1 IC50 over the sensitive cells both in 2D and 3D settings. Compared to the sensitive ones, both the resistant cell lines show cross-resistance to other monovalent and bivalent BETi and degraders. Resistant cells also maintain the ability to form colonies in a clonogenic assay and to invade in a 3D Matrigel assay in the presence of JQ1. Moreover, conversely to sensitive cells, JQ1 treatment is unable to down-regulate P3F, MYCN and BCL2 (P3F target) levels in resistant cells. Furthermore, integration of transcriptomic and proteomic data reveals differentially modulated subset of genes/proteins among which MYCN and P3F targets, suggesting a restored activity of the TFs circuitry in resistant cells. Accordingly, MYCN silencing completely halts growth of resistant cells. In line, MYCN protein levels are reduced by JQ1 in a dose-dependent manner in sensitive but not in resistant cells and its half-life is 3 folds longer in resistant than in sensitive cells. Being MYCN protein stability affected by specific post-translational modifications among which the phosphorylation at Ser62 by ERKs, we evaluated the expression of phosphorylated MYCN and the activated form of ERK (pERK) and found both of them increased in resistant cells. In agreement, resistant cells are more sensitive to MEK-ERKs pathway inhibition, showing a lower IC50 of Trametinib (MEKi) compared to sensitive cells. Moreover, Trametinib and JQ1 co-treatment downregulates MYCN protein levels in resistant but not in sensitive cells, suggesting that ERKs overactivation could participate to BETi acquired resistance by stabilizing MYCN, thus representing an acquired vulnerability. The study has received funding from Ministero della Salute to SP. Citation Format: Matteo Cassandri, Erika Ferraro, Lucrezia D'Archivio, Francesca Aiello, Hsien-Chao Chou, Young K. Song, Berkley E. Gryder, Jun S. Wei, Simone Sidoli, Robert G. Hawley, Rossella Rota, Franco Locatelli, Javed Khan, Silvia Pomella. Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4739.

Abstract 7600: Synergy of ZEN3694 and VIP152 for dual transcriptional targeting of BET and CDK9 in patient derived organoid models of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer related mortality in the United States, largely due to ineffective systemic therapy. While novel KRAS target strategies are in development, few agents have been investigated to target the transcriptional machinery for global inhibition of expression. Recent small molecule inhibitors targeting transcription (i.e., CDK7) have shown preliminary evidence of disease control; however, combination therapies have lagged, likely due to uncertainty in clinical toxicity. Text mining analysis suggests combination BET and CDK9 inhibition may be promising against PDAC. Here, we present preclinical evidence of synergistic activity for the combination of bromodomain and extraterminal (BET) inhibitor (ZEN3694) and CDK9 inhibitor (VIP152) using patient derived cancer organoids (PCOs). Methods: Dose response curves were generated for ZEN3694 and VIP152 to assess their single agent activity against PCOs using a low volume format. ZEN3694 was treated continuously while VIP152 was removed after 24h to mimic pharmacokinetics with assay endpoint at 144h. Synergy assays were designed using a combination dose titration grid. Viability was determined in all assays using 3D CellTiter Glo (CTG, 50% v/v). Synergy scores were produced using SynergyFinder 3.0 including Zero Interaction Potency (ZIP), Bliss Independence (Bliss), Highest Single Agent (HSA), and Loewe Additivity (Loewe). Western blot analysis was performed against global markers of active RNA polymerase II (phosphorylation at serine 2, 5, 7) and antiapoptotic protein, BCL xL, and MCL 1. Results: ZEN3694 was observed to have a single agent IC50 in the single uM range across 3 independent PDAC PCO lines (PDAC1 3.7uM, PDAC2 1.1uM, PDAC3 2.9uM). VIP152 showed significant potency in the same 3 PDAC PCO lines (PDAC1 140.2nM, PDAC2 30.5nM, PDAC3 43.4nM). The combination of ZEN3694 and VIP152 was found to have increased therapeutic activity with double digit synergy scores across all reference models: PDAC1 (Loewe 17.3, HSA 19.8, ZIP 20.2, and Bliss 17.2) and PDAC2 (Loewe 34.7, HSA 28.3, ZIP 19.7, and Bliss 19.6). Western blots of protein collected from organoids treated for 48h showed on target activity, including decrease in the phosphorylation of Rbp1 (Ser 2, Ser 5, and Ser 7) compared to both control and single agents and decreased expression in antiapoptotic proteins (MCL 1 and BCL xL). Conclusion: Here, we show the activity of dual transcription targeting with BET and CDK9 inhibition in patient derived pancreatic cancer organoid models. The combination of ZEN3694 and VIP152 has in vitro synergy in PDAC PCO models with on target activity. Ongoing work is validating this combination in animal models to assess toxicity, in vivo activity, and RNA expression profiling to understand transcriptional pathway dependency and therapeutic resistance. Citation Format: Lucas J. Koeppel, Austin Stram, Robert J. Millikin, Ellie Riedl, Md Shahadat Hossan, Ethan Samuel Lin, Ron Stewart, Jeremy D. Kratz. Synergy of ZEN3694 and VIP152 for dual transcriptional targeting of BET and CDK9 in patient derived organoid models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7600.

Abstract 7548: Role of bromodomain extra terminal inhibitor (BETi) in combination with radiotherapy for the treatment of NF1-associated malignant peripheral nerve sheath tumor

Abstract Introduction: Neurofibromatosis Type 1 (NF-1) associated malignant peripheral nerve sheath tumors (MPNST) harbor mutations in the polycomb repressive complex 2 (PRC2) in 70% of tumors. PRC2 loss results in epigenetic changes, which increase histone acetylation and make the DNA vulnerable to binding by bromodomain proteins. This interaction could be targeted to uncouple histone acetylation from transcription using bromodomain extra terminal inhibitors (BETi). As radiation therapy (RT) is a cornerstone of MPNST treatment, we analyzed the effects of BETi with and without RT. Methods: Mice were implanted with patient-derived xenografts (PDX) of PRC2-wildtype model developed in the Torres laboratory, MPNST007. Mice were segregated into vehicle and I-BET 762 treatment groups and treated with either vehicle or drug for one week before the RT was initiated. Half of the mice in each treatment group (n = 10) received radiation (2 Gy per fraction for 5 days for a total of 10 Gy). The study endpoints were (1) tumor growth rates, (2) tumor size and weight at study termination, (3) immunohistochemical (IHC) staining for biomarkers of proliferation (Ki67), apoptosis (cleaved caspase 3, CC3), and DNA damage (γH2AX), and (4) RNA sequencing of PDXs from each experimental arm. Results: Significant tumor regression was observed in PRC2-mutant PDX tumors treated with I-BET 762 based on tumor growth kinetics over 45 days of treatment (p < 0.001). Tumor volume was significantly reduced by I-BET 762 compared to vehicle (p < 0.01). Combining I-BET 762 and RT resulted in significant tumor volume reduction (p <0.001). RNA sequencing and intersection of differentially expressed genes related to “DNA damage repair” and those repressed by I-BET 762 was performed for the MPNST007 PDXs. The expression of 259 genes related to DNA damage repair decreased after I-BET 762 treatment, including XRCC1, ERCC4, RAD50, RAD51D, and LIG4 (data not shown). IHC analysis prompted by the RNA sequencing results of the I-BET 762-treated PDXs revealed higher levels of γH2AX. An increase in necrotic and fibrotic tissue with increased CC3 in the BETi plus radiation group was observed. Conclusions: Genetic mutations in NF-1-associated MPNST tumors result in susceptibility to BETi treatment. In a PDX model of PRC2 wild-type tumors, BETi treatment resulted in tumor regression and sensitized MPNST xenografts to RT. Combining BETi and RT enhanced tumor death and increased expression of genes associated with DNA damage and apoptosis. Citation Format: Alcia A. Gingrich, Sharon Landers, Angela Bhalla, Cristian Rodriguez-Aguayo, Rossana Lazcano, Suresh Satpati, Alines Lebron-Torres, Emily Z. Keung, Christopher P. Scally, Christina L. Roland, Kelly K. Hunt, Heather Lyu, Heather Lillemoe, Ashleigh Guadagnolo, Alexander Lazar, John Slopis, Ian McCutcheon, John A. Livingston, Ahsan Farooqi, Kunal Rai, Keila E. Torres. Role of bromodomain extra terminal inhibitor (BETi) in combination with radiotherapy for the treatment of NF1-associated malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7548.

Abstract 3953: BET inhibitor increases DNA damage, modulates Wnt signaling, and suppresses osteosarcoma growth in naïve and metastatic disease models

Abstract Osteosarcoma (OS) is an aggressive bone cancer in pediatric adolescent and young adult patients. Survival rate for metastatic and relapsed OS patients remains dismal at <30%. Additionally, no effective standardized salvage therapy currently exists for these patients, in part, due to genomic complexities arising from moderate levels of replication stress (RS). Bromodomain and extra-terminal domain protein inhibitors (BETi) are an underexplored option to target RS for BETi creates an imbalance between transcription-replication kinetics resulting exacerbation of oncogenic RS and cell death. BET proteins are epigenetic readers that play a role in regulating gene expression networks as well as DNA replication and repair. We tested the hypothesis that BET inhibition leads to decreased OS cell growth and potentiates the efficacy of salvage therapy via gene dysregulation and increased DNA damage and RS. Combination index and Bliss independence analyses of bivalent BET inhibitor (BETi) AZD5153 or PROTAC ARV825 in combination with salvage agents demonstrated additive-to-synergistic cell growth inhibition in OS lines. Increased apoptotic-mediated cell death was observed following AZD5153+topotecan. In addition, γ-H2AX levels and comet assays demonstrated that BETi+topotecan induces its effect, in part through increased RS in-vitro. AZD5153 monotherapy significantly suppressed OS tumor growth in patient-derived xenografts (PDXs) derived from both naïve (PDX96) and metastatic (TT2) OS compared to vehicle (p<0.05). Moreover, AZD5153 reduced lung metastatic lesions and increased survival in the 143B OS lung colonization model. In-vivo mechanisms of drug-induced tumor response were evaluated. Anti-tumor effect correlated with increased γ-H2AX following AZD5153 exposure in PDX96, indicative of increased RS. RNA-seq analysis and protein validation from vehicle versus BETi-treated PDX96 highlighted dysregulation of several key genes involved in DNA damage response including elevated TXNIP, a tumor suppressor which can induce DNA damage and apoptosis. Downregulation of TCF7, a downstream effector of the Wnt pathway, was also observed following BETi. Global kinome profiling and validation experiments revealed increased activity and expression of EphA2 and EphA4 receptors both implicated in development of drug resistance. Furthermore, in BETi-treated PDX96 tumors, Wnt/β-catenin pathway activation emerged as tumors rebounded once BETi treatment was stopped. TT2 PDX was resistant to commonly used salvage agents ifosfamide and irinotecan; however, combination BETi+ topotecan increased the probability of survival compared to each agent alone (p<0.05) and was well tolerated. These data collectively suggest that BET inhibition alone or in combination with low-dose salvage therapy holds promise as novel treatment strategies in aggressive OS. Citation Format: Niknam Riyahi, Pankita H. Pandya, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Harlan E. Shannon, Farinaz Barghi, Rada Malko, Khadijeh Bijangi-Vishehsaraei, Melissa A. Trowbridge, Kathy Coy, Felicia M. Kennedy, Anthony L. Sinn, Steve Angus, Michael J. Ferguson, M. Reza Saadatzadeh, Karen E. Pollok. BET inhibitor increases DNA damage, modulates Wnt signaling, and suppresses osteosarcoma growth in naïve and metastatic disease models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3953.

Abstract 1691: p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma

Abstract Multiple myeloma (MM), an incurable plasma cell malignancy, is addicted to oncogenic transcription factor (TF) IRF4 signaling. Lineage TFs like IRF4 are difficult to drug but represent high value oncology targets owing to the depth and context selectivity of their genetic dependency. Using a transcription regulatory network (TRN) based approach to integrate a high dimensional multi-platform dataset, we defined the key regulatory activity of IRF4 and its druggable co-factors. This unbiased analysis identified p300 lysine acetyl transferase (KAT) function as the nearest selective neighbor for therapeutic intervention, with more potent and selective effects than historically observed with p300/CBP bromodomain inhibitors. To further probe this biology, we developed a highly selective inhibitor of the KAT domain of p300 and its close homolog CBP. p300 KAT inhibition led to selective downregulation of IRF4 at the mRNA and protein level as well as reduction in acetylation at highly specific sites co-localized with IRF4, in contrast to more general agents like BET inhibitors. Gene expression reduction was most pronounced at IRF4/p300 co-bound genes, in particular IRF4 itself and MYC. IRF4 and p300 also directly bind to each other and other key MM nodes regulating them at the chromatin, mRNA, and protein levels. p300 KAT inhibition exhibits strong antiproliferative activity across in vitro MM models regardless of cytogenetics or resistance to other therapies. Moreover, this relationship is mediated by the KAT domain and not its acetyl-lysine recognizing bromodomain. Comparative pharmacology studies found that a p300 KAT inhibitor downregulated the IRF4 TRN and induced apoptosis in MM cells more effectively than existing standard of care agents, including IMiDs and glucocorticoid receptor agonists, or p300/CBP bromodomain inhibitors. Combinations of a p300 KAT inhibitor with clinically active molecules such as IMiDs and glucocorticoid agonists showed synergistic impacts on reducing key MM TFs and cell line viability. Ex vivo treatment of primary patient samples demonstrated broad anti-proliferative activity with loss of IRF4 and induction of apoptosis in relapsed-refractory MM patient samples, independent of prior lines of therapy. Importantly, the induction of MM cell apoptosis occurs in the milieu of CD138 negative bone marrow stromal cells while also sparing them from toxicity, supporting a potential therapeutic index. Finally, p300 KAT inhibition demonstrated activity in in vivo MM models with robust tumor growth inhibition at tolerated doses and exposures correlated to reduced p300-dependent substrate acetylation. IRF4 dependency is a hallmark of MM that has been challenging to target with existing therapies, motivating further development of p300 KAT targeting therapeutics. Citation Format: Michael R. McKeown, Giulia Giorgetti, Walter F. Lenoir, Marek J. Kobylarz, Tamara D. Hopkins, Wayne L. Glore, Michelle G. Shum, Yare Calderon, Kameron R. Mori, Hua Gao, Luis A. Carvajal, Nikolaus D. Obholzer, Benjamin W. Trotter, Christopher D. Dinsmore, Nikhil C. Munshi, Peter B. Rahl, Mariateresa Fulciniti, Charles Y. Lin. p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1691.

Abstract 3826: Predictive and prognostic factors in NUT carcinoma: A systematic review and individual patient data meta-analysis

Abstract Background: NUT carcinoma (NC) is a rare cancer affecting midline structures and involving the lung, bones, and lymph nodes. Data on NC are limited mostly to case reports and case series. Methods: PubMed, Web of Science, and Scopus were systematically searched for original articles describing confirmed NC patients. A quantitative individual patient data meta-analysis (IPD-MA) analyzing overall survival and risk factors for progression was performed for all NC patients with a clear age at diagnosis. Relevant patient characteristics and clinical parameters were compared in a multivariate Cox regression model. Findings: Out of 4,701 studies screened, 175 studies met the inclusion criteria for the systematic review. 117 (66.9%) were case reports. 284 patients were included in the IPD-MA. 139 (48.9%) patients were male, 145 (51.1%) underwent surgery, 156 (54.9%) received radiotherapy, and 173 (60.9%) developed metastasis. The mean age at diagnosis was 35.82±18.33 and the median OS was 9 months. 77 (27.1%) patients received platinum-based agents, 39 (13.7%) received taxanes, and 15 (5.3%) received BET inhibitors as part of their first-line regiment. Age at diagnosis, gender, and the aforementioned drugs did not significantly influence OS. Presence of metastasis was an independent predictor of poor survival, while radiotherapy and surgery were significantly associated with better survival (table 1). Interpretation: NC is associated with poor survival. Surgery and radiotherapy appear to be more effective treatment strategies, while medical modalities may not play a major role in the treatment of NC. Predictors of overall survival in NUT carcinoma patients. Parameter Hazard ratio 95% confidence interval P-value Age at diagnosis 0.99 0.99-1 0.35 BET inhibitors 0.61 0.28-1.15 0.16 Platinum-based agents 0.73 0.47-1.13 0.16 Taxanes 1.29 0.78-2.1 0.31 Surgery 0.41 0.29-0.58 <0.0001 Female gender 1.12 0.83-1.51 0.45 Metastasis 1.76 1.26-2.49 0.0011 Radiotherapy 0.48 0.36-0.67 <0.0001 Citation Format: Sameeha K. Abdulwali, Abduljalil M. El-Sibai, Saad S. Ali, Abdulaziz M. Alaklabi, Hytham M. Abdalla, Ahmad N. Sabbah, Areez Shafqat, Nader A. Fawzy, Belal N. Sabbah, Ahmed Badran, Tarek Z. Arabi. Predictive and prognostic factors in NUT carcinoma: A systematic review and individual patient data meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3826.

Abstract 4737: The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer

Abstract Efforts to develop targeted therapies for drug resistant tumors have been relatively unsuccessful. The 5-year survival for patients with advanced pancreatic cancer is ~3%. Gemcitabine is usually combined with nab-paclitaxel as a standard of care for treatment of locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, no chemotherapeutic regimens thus far evaluated have been curative. We developed the first PDAC patient-derived xenograft (PDX) models in which resistance against gemcitabine was acquired in vivo. We used RNA-seq and ingenuity pathway analysis to compare expression profiles of paired parent vs gemcitabine resistant PDX models. This comparison suggested a previously unreported mechanism for gemcitabine resistance, involving aberrations of cholesterol biosynthesis and lipid metabolism pathways. We also made the unexpected observation that gemcitabine-resistant PDAC models derived from primary tumors have high levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential to cholesterol biosynthesis and rate limiting in ketogenesis. Notably, the BET bromodomain inhibitor JQ1 decreases HMGCS2 levels; and BET inhibitors + gemcitabine exert synergistic cytotoxicity in gemcitabine-resistant PDAC cells. We determined that the BET bromodomain inhibitor JQ1 + gemcitabine induces regressions of two in vivo gemcitabine-resistant tumor models which we developed directly from primary human tumor tissue. These mechanism-based studies suggest that inhibiting HMGCS2 represents a novel approach to therapy for PDAC. We propose that combining gemcitabine with agents that decrease levels of HMGCS2 comprises effective treatment for patients with drug resistant PDAC. Citation Format: Aubrey L. Miller, Samuel C. Fehling, Eric Josh Brown, Eric O. Heard, Tracy L. Gamblin, Rebecca B. Vance, Karina J. Yoon. The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4737.

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 534.

Abstract 6042: CBP-8008: A first-in-class targeted pan-Bet protein degradation therapy using bi-specific XDC (Bi-XDC) technology for TNBC and mCRPC

Abstract Bi-XDC is a breakthrough cellular targeting technology using multi-targeting ligands to effectively deliver the payload to and into specific cells. Several anticancer Bi-ligand drug conjugates including CBP-1008, CBP-1018, and CBP-1019 have been brought into various clinical stages in China and US. Targeted protein degradation molecules can degrade previously “undruggable” target proteins through their catalytic mechanism of action. The field target protein degradation has evolved since 2001, and more than 20 PROTACs are under clinical trials at different stages. To increase the efficacy vs. tolerability window, we combine Bi-XDC technology and the catalytic approach of targeted protein degradation, to form C-PROTAC, by taking advantage of our privileged Bi-XDC technology and PROTAC’s degradation property. Further medicinal chemistry optimization, spacer optimization, and evaluation of many linker payloads led to the identification of our preclinical candidate CBP-8008, a highly potent pan-Bet protein degrader conjugated to bi-ligand via a cleavable linker. A small panel of cell lines was screened to identify which tumors were sensitive to the pan-Bet protein degradation. All the tested breast cancer and prostate cell lines were the most sensitive to the payload. CBP-8008 had a high affinity to the Prostate Specific Membrane Antigen (PSMA) and the folate receptor alpha (FRα). Much more CBP-8008 were binding the 293T-FP which overexpress both FR-α and PSMA than the cell lines which only express one target (293T-F or 293T-P) or negative cell line (293T). CBP-8008 can quickly enter cells through endocytosis after treatment for 15 min, and degradation of BRD2, BRD3, and BRD4 was observed in MDA-MB-231 after 6 h, which was similar to its payload. The compound was very stable in mouse and human plasma in vitro, and limited free payload was detected which potentially avoids hematotoxicity in clinical. In the tumor-beaning mice DMPK study, fewer free payload was detected in 6 hours while much more free payload was detected in the tumor than in the blood and the time was extended to 72 hours after a single dose treatment. We evaluated CBP-8008 in TNBC and mCRPC xenograft model and robust efficacy was observed as lower as 3 MPK, significantly better than BMS-986158, a potent Bet inhibitor in clinical. In conclusion: CBP-8008 is currently in preclinical development as a potential first-in-class pan-Bet protein degradation therapy using Bi-XDC technology for TNBC and mCRPC Citation Format: Tao Wang, Zhiguang Zhang, Guitao Wang, Fan Pan, Robert Huang. CBP-8008: A first-in-class targeted pan-Bet protein degradation therapy using bi-specific XDC (Bi-XDC) technology for TNBC and mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6042.

Abstract 150: Transcriptional rewiring of BET inhibitor treated Ewing sarcoma cells augments their dependency on focal adhesion kinase

Abstract Outcomes for patients with recurrent and metastatic Ewing sarcoma (EwS) are dismal and novel therapies are needed. As EwS are initiated and maintained by the EWS::FLI1 transcription factor, agents that target aberrant gene transcription are of interest and bromodomain inhibitors (BETi) have shown promise in preclinical models. Nevertheless, despite initial efficacy, BETi tolerance emerges across tumor types, often via transcriptional rewiring. We reasoned that the therapeutic potential of BETi in EwS could be augmented by identifying and targeting mechanisms of drug resistance. To investigate this, we performed transcriptional and phenotypic profiling of BETi-naïve, responsive, and drug-tolerant (DT) EwS cells and used these data to nominate biologically-informed drug combinations. EwS cells (A673, CHLA10, TC32) were exposed for 72 hrs or 20 days to BMS-986158, a clinical grade BETi being tested in a phase I pediatric clinical trial (NCT0393465). A profound cytostatic effect was observed at 72 hrs but in all cell lines proliferation was restored by 20 days. RNA-seq confirmed reversion of the EWS::FLI1-dependent gene signature at 72 hrs and this was maintained at 20 days, demonstrating continued on-target effects despite restored growth of DT cells. Interrogation of differentially expressed genes between drug naïve, cytostatic, and DT cells indicated that that DT cells had been transcriptionally rewired to more mesenchymal states, with notable upregulation of genes involved in integrin signaling. In support of this, DT populations displayed more fibroblastic morphologies, increased F-actin filaments, and enhanced invasion in collagen-rich 3D culture. In parallel, an unbiased kinase inhibitor screen that exploits mathematical modeling revealed that, while BETi-treated cells were overall more resistant than parent cells to kinase inhibitors, their viability depended on focal adhesion kinase (FAK) activity. Western blot confirmed increased phospho-FAK in DT cells. We next tested BMS-986158 and the FAK-inhibitor Defactinib singly or in combination in vitro and in vivo. Defactinib induced cell death in 2D cultures and blocked invasion of 3D spheroids in collagen. Conversely, BMS-986158 enhanced invasive potential in 3D. The combination of Defactinib and BMS-986158 in naïve and DT populations was highly synergistic in 2D assays. In 3D collagen assays, viability and invasion of DT tumor spheroids were significantly reduced by the combination. Finally, ongoing studies show that the combination of FAK and BET inhibition prolongs survival of mice with EwS tumor xenografts. Thus, our studies reveal that exposure of EwS cells to BETi induces transcriptional rewiring that activates integrin and FAK signaling, restoring proliferation. This work supports further investigation of FAKi as agents that could prevent or reverse the BETi tolerant state in EwS. Citation Format: Shireen S. Ganapathi, Emma Wrenn, Nicolas Garcia, Neerja Katiyar, Aya Miyaki, Yuqi Kang, Marina Chan, Taran S. Gujral, Elizabeth R. Lawlor. Transcriptional rewiring of BET inhibitor treated Ewing sarcoma cells augments their dependency on focal adhesion kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 150.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.