Bromodomain Inhibitors Research Articles

Abstract 4747: Novel targets for BRAFV600E and BRAFV600ERAC1P29S drug resistant melanoma

Abstract The most common mutational drivers in malignant melanoma are the oncogene BRAFV600E/K (60%), NRASQ61L/R (30%) and the RHO GTPase RAC1P29S/L (9%). BRAFV600E/K melanoma standard of care comprises the use of checkpoint inhibitors (CPIs) such as anti-PD1 or anti-CTLA-4 in combination with targeted therapies against BRAF and MEK kinases. BRAF mutant melanomas develop intrinsic, acquired, or after treatment resistance to BRAF and MEK inhibitors : around half of targeted therapy combination treatments with anti-PD1 and 70% with anti-CTLA-4 will be unsuccessful due to melanoma intrinsic resistance. Among other causes, intrinsic drug resistance can be associated with additional genome mutations, such as the activating Rac1P29S mutation present in 6% of patients with BRAF positive melanoma. A deeper understanding on the biology, progression, and tumor microenvironment of drug resistant melanomas is needed to aid in the search of novel targets and to increase the effectivity of proposed treatments. We generated 3 sets of isogenic BRAFV600E and BRAFV600E/RAC1P29S cell lines by transfecting 501mel, 451Lu and YUMM1.7 cell lines with Rac1P29S. We then exposed them for 3 months to increasing concentrations of PLX4720 to generate drug resistant cell lines (DR). Rac1 P29S -transfected and PLX4720 resistant cell lines had an increase in the Mek/Erk signaling pathway. A differential analysis using a kinome and an epigenome drug screening library in the 501mel cell lines (501mel, 501mel Rac1P29S, 501mel-DR, 501mel Rac1P29S -DR) showed that BRAFV600E/RAC1P29S and the DR melanoma cell lines growth can be inhibited with mTor, PI3K, cdk9 and BRD4 inhibitors. BRD4 is an epigenetic regulator that recruits P-TEFb to stimulate RNA polymerase II elongation. BRD4 also interacts with SMAD3 and CDK9 to regulate transcription through eIF4G increasing oncogenic c-Myc and increasing Mek/Erk signaling pathways. We tested 501mel, 451Lu and YUMM1.7 isogenic cell line series against Rapamycin to confirm mTORC1 as an effective target. Drugs that target different subunits of the eIF4F complex (CR-1-31-B, Ribavirin and Briciclib) also decrease BRAFV600E/RAC1P29S and the DR melanoma cell lines growth specifically. Furthermore, silencing of eIF4A1 and Raptor genes reduces cell growth in the BRAFV600E/RAC1P29S, and the DR cells when compared to the parental cell lines. Silencing Rictor had no effect on cell growth on any cells. When tested in a syngraft model, CR-1-31-B and rapamycin reduced tumor size when using YUMM1.7, YUMM1.7- RAC1P29S and YUMM1.7 RAC1P29S-DR cell lines. Next, we sought to investigate the effect of inhibiting mTOR and eiF4F in the PD-1/PD-L1 axis in tumor cells and tumor associated dendritic cells and macrophages, and how targeting these modifies the tumor microenvironment. Citation Format: Cristina Uribe-Alvarez, Daniela Araiza-Olivera, Alexa Cannon, Jonathan Chernoff. Novel targets for BRAFV600E and BRAFV600ERAC1P29S drug resistant melanoma [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 4747.

Abstract 3218: Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML

Abstract Background: Acute myeloid leukemia (AML) with KMT2A-rearrangements have a poor prognosis and so there is an urgent need for novel therapies. Menin inhibitors have shown promising anti-leukemic effects in KMT2A-rearranged and NPM1-mutated AML. However, the efficacy of menin inhibitors as single agents may be limited by the development of resistance mutations. Combining menin inhibitors with other epigenetic drugs may present a strategy to enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic drug library in KMT2A-rearranged (MV4;11) and NPM1-mutant (OCI-AML3) AML cell lines to identify synergistic therapies that can be used in conjunction with menin inhibitors. Methods: During assay optimization, MV4;11 and OCI-AML3 cells were treated with the menin inhibitor DSP-5336, and their viability was assessed. Quality control metrics were applied with Z’ values consistently greater than 0.5 for all assay plates and then the cells were subjected to a small molecule compound library screen containing a set of 932 epigenetic drugs. The screening process included a systematic evaluation of each drug's effect on cell viability in combination with the DSP-5336. Results: The combination drug screen demonstrated robust performance in both MV4;11 and OCI-AML3 cell lines, with reliable quality control metrics. Among the epigenetic drugs tested, five compounds were identified as potential hits that synergistically enhanced the efficacy of DSP-5336 across both cell lines.The five identified compounds exhibited enhanced anti-leukemic effects when combined with the menin inhibitor in either one of the cell lines. The targets of these drugs varied, including histone deacetylases, histone acetyltransferases and BRD4 inhibition, suggesting many alternate mechanisms of action involving epigenetic modulation. Remarkably, one of the 5 hits C301-3161, a KAT2A inhibitor, showed limited activity in either cell line alone but increased cell killing with DSP-5336 in both cell lines, highlighting its potential as a specific therapeutic for combining with DSP-5336 in AML patients. Conclusions: Our findings suggest combining menin inhibitors with other epigenetic drugs may improve AML treatment outcomes. The combination drug screen successfully identified four novel compounds that synergistically enhance menin inhibitor efficacy in both KMT2A-rearranged and NPM1-mutant AML cell lines. These compounds hold significant promise as potential therapeutic candidates for further investigation. Future studies should focus on elucidating the underlying mechanisms of action for each hit and evaluating their effects in preclinical animal models. Ultimately, this research may pave the way for the development of innovative combination therapeutic strategies for AML patients with KMT2A-rearragned and NPM1-mutant AML. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Maurizio Affer, Anya K. Sondhi, Alexandra Chirino, Claude-Henry Volmar, Shaun Brothers, Justin Watts, Justin Taylor. Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML [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 3218.

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 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 5852: Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia

Abstract Background: Venetoclax (VEN), a B-cell lymphoma-2 (BCL-2) inhibitor, is an important drug in acute myeloid leukemia (AML) treatment regimens. However, resistance to VEN-based therapy is a common challenge in AML management. Some factors contributing to resistance include dysregulation of BCL-2 family proteins, mutations in the TP53 gene, activating kinase mutations, and oxidative phosphorylation activity. Despite these insights, other mechanisms of resistance remain elusive. We aim to analyze tumor gene expression patterns associated with treatment response in patients who received VEN-based therapy. Methods: Molecular and clinical data of the patients were obtained as part of the Oncology Research Information Exchange Network (ORIEN) Avatar dataset. RNA-Seq data from diagnostic Bone Marrow were processed to remove batch effects, and differential expression analysis was performed using DESeq2, while correcting for age at sample collection and sex of patients. Results: We included 16 individuals treated with VEN-based therapy. Median age at the time of specimen collection was 65 years (interquartile range, 58-69), 9 (56%) male, 11 (69%) had active disease despite treatment with VEN. VEN-based therapy was used as 1st line, 2nd line, 3rd line, 4th line or more in 3, 4, 4, 2, and 3 patients, respectively. We found a total of 332 genes differentially expressed (fdr<0.05 and abs(log2FoldChange)>2.0) among patients having no evidence of active disease and those with no active disease at the time of last contact. Among those genes, we found HOXA7 and PRAME of particular interest. HOXA7 has been previously implicated in Bromodomain and Extraterminal Domain Inhibitors (BETi) inhibitor resistance in AML. This makes HOXA7 a potentially universal mechanism of chemotherapy resistance in AML. PRAME has been associated with leukemia cell survival, and its knockdown has been shown to induce apoptosis in K562 cells, which is only expressed in AML cells but not in normal hematopoietic cells. Pathway analysis found that the top two enriched pathways for the differentially expressed genes were MET activates PTK2 signaling (R-HSA-8874081) and Metabolism of Angiotensinogen to Angiotensins(R-HSA-2022377). Conclusions: This study highlights some molecular mechanisms that give rise to AML resistance to VEN. Our results indicate that HOXA7 may potentially be a universal mechanism of chemotherapy resistance not limited to VEN-based therapy. Similarly, pathways enriched in resistant cells could be targeted to prevent or treat resistance to VEN-based therapy. These and other findings may help us identify resistance mechanisms in all AML cases vs those specific to VEN-based therapy, allowing the development of treatment strategies based on treatment mechanism. Also, analysis of these mechanisms can aid therapeutic decision-making and the development of effective treatment strategies to improve outcomes in AML. Citation Format: Juan Antonio Raygoza Garay, Seongseok Yun, Afaf Osman, Reshma Ramlal, David Feith, Michelle Churchman, Prajwal Dhakal. Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia [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 5852.

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 5719: Targeting CDK4/6 and BET proteins for the treatment of RB+ and RB- osteosarcoma

Abstract Osteosarcoma (OS) is the most common malignant bone cancer characterized by heterogeneity and genomic instability. Treatment and survival of patients have not improved in several decades, making it urgent to develop safe and effective therapies. Dysregulation of cell cycle regulators CDKN2A, cyclins, and CDK4/6 represent high-risk signatures in aggressive OS and can be targeted via CDK4/6 inhibitors (CDK4/6i). However, monotherapy often fails due to resistance from speculated RB loss as well as hyperactivation of compensatory signaling pathways in RB proficient cancers. In addition, >70% of OS patients have RB loss which is viewed as a biomarker of therapeutic response that negates the use of CDK4/6i. However, more research is needed in RB-deficient preclinical models to clearly define and validate the utility of RB as a biomarker for patient stratification. Based on our screening data, we hypothesize that combining CDK4/6i with bromodomain and extraterminal domain inhibitors (BETi) will potentiate anti-tumorigenic effects regardless of RB status in OS. BETi creates imbalances between transcription and replication which worsens oncogenic stress. In OS cell lines, combination index and Bliss analysis indicated additive-to-synergistic growth inhibition using CDK4/6i (abemaciclib or palbociclib) and BETi (AZD5153). Dual therapy also resulted in increased apoptosis at clinically relevant concentrations in an OS xenoline (TT2) compared to monotherapy. To determine potential for emergence of therapeutic resistance, an in-vitro longitudinal growth delay assay was established. While single agent therapies resulted in transient decreases in cell numbers over time, combination CDK4/6i+BETi resulted in cell death (p<0.05). To determine the effect of RB loss on therapeutic response to CDK4/6i, CRISPR mediated RB-knockout (KO) clones were developed in 143B human and K7M2 murine OS cell lines. RB KO cells exhibited sensitivity to CDK4/6i irrespective of RB status. However, levels of CDK4/6i required for 50% growth inhibition in RB KO were 1-3-fold higher using palbociclib, with negligible differences observed with abemaciclib in RB KO vs WT clones. To determine if therapeutic response is achievable in-vivo, a single-agent dosing study was conducted using palbociclib (40mg/kg, SID) and AZD5153 (1mg/kg, BID) in patient-derived xenograft (PDX) model derived from a metastatic lesion in the pelvis (TT2 PDX) harboring a CDK4/6 hyperactivation signature and monoallelic RB loss. Tumor growth reduction and survival was significantly higher in monotherapy groups compared to vehicle. However, one week after dosing was stopped, survival rates based on tumor volume endpoint were 40% and 100% for palbociclib and AZD5153 respectively. In-vivo efficacy of dual CDK46i+BETi is underway. This data provides rationale for further study of novel therapeutic options which may expand the clinical utility of CDK4/6i regardless of RB status. Citation Format: Rada Malko, Pankita H. Pandya, Harlan E. Shannon, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Farinaz Barghi, Nikan Riyahi, Ryli Justice, Melissa A. Trowbridge, Kathryn L. Coy, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, Micheal J. Ferguson, M. Reza Saadatzadeh, Karen E. Pollok. Targeting CDK4/6 and BET proteins for the treatment of RB+ and RB- osteosarcoma [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 5719.

Abstract 1548: Olfactomedin 4 deficiency promote de novo progression of prostate cancer in TRAMP mouse model

Abstract Prostate cancer is the most diagnosed solid tumor and the second leading cause of cancer-related death in American men. Olfactomedin (OLFM4) plays tumor suppressor roles and is associated with the initiation, growth, and clinical outcome of human prostate cancer. Olfm4-null mouse sporadically developed prostate epithelial neoplasia through up-regulated hedgehog signaling pathways. In this study, we have generated Olfm4+/+/GFP/TRAMP and Olfm4−/−/TRAMP mouse models to investigate Olfm4 gene biological functions underlying prostate cancer de novo progression. We have observed that Olfm4−/−/TRAMP mice developed more advanced prostate cancer at 47.6% (20/42) compared to Olfm4+/+/TRAMP at 11.9 % (5/42) and had poor survival compared to Olfm4+/+/TRAMP mice (P<0.01). Histopathological and immunofluorescent staining revealed that tumor tissues containing more heterogeneous cells such as Ar+/Ck8+ epithelial or Syn+ neuroendocrinal cells or Vim+/Ck8+ epithelium mesenchymal transition cells in the primary tumors obtained from Olfm4−/−/TRAMP mouse. We found more tumor angiogenesis and metastases into lymph nodes, liver, lung, kidney, and soft tissues in Olfm4−/−/TRAMP mice. The accumulations of the dendritic cell population in primary tumor and lung metastasis tumors were detected. The primary tumor was sensitive to androgen deprivation therapy by using castration with both Olfm4+/+/GFP/TRAMP and Olfm4−/−/TRAMP mice. However, castration-resisted EMT tumor cells or expressing neuroendocrine marker cells regrew after castration 7-8 months in both Olfm4+/+/GFP/TRAMP and Olfm4−/−/TRAMP mice. Furthermore, we tested JQ1, a BET bromodomain inhibitor, as an epigenetic targeted therapy reagent and found that JQ1 significantly inhibited tumor cell growth of Olfm4−/−/TRAMP mouse tumor in the organoids culture. Taken together, we have provided novel evidence that Olfm4 plays a tumor-suppressing role during prostate cancer de novo progression. Citation Format: Hongzhen Li, Wenli Liu, Jianqiong Zhu, Kyung Chin, Griffin P. Rodgers. Olfactomedin 4 deficiency promote de novo progression of prostate cancer in TRAMP mouse model [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 1548.

Abstract 7553: Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast cancer

Abstract Epigenetics plays a central role in the initiation, establishment, and progression of cancer. In this capacity, epigenetics regulates several abnormal nuclear processes in the cancer cell including DNA replication, DNA damage repair and most prominently transcription. Because of their reversibility, epigenetic modifications have been aggressively pursued pharmacologically to correct abnormal cancer biology in the hopes of reversing cancer phenotypes. To develop novel epigenetic based therapeutic strategies for triple negative breast cancer (TNBC) we combined the DNA methyltransferase inhibitor (DNMTi) guadecitabine (Gua) with a genetic CRISPR/Cas9 mediated knockout (KO) of the chromatin remolding protein Bromodomain PHD-finger Transcription Factor (BPTF). Using the E0771 and 4T1 mouse TNBC models we show that BPTF depleted tumor cells are more susceptible to the antitumor effects of Gua. These antitumor effects are not observed in culture, or in immune compromised NSG mice, suggesting that their effects on cancer cell growth are not cancer cell intrinsic. RNA-Seq, ATAC-Seq and MBD-Seq analysis from FACS purified cancer cells shows that the combination of BPTF inhibition and Gua alters immune regulatory cytokine expression (BMP4 and CSF3) in the cancer cell, and it is theorized that these changes alter the tumor microenvironment to become less immune suppressive and permissive to the antitumor effects of immune cells. This hypothesis is supported by Cytek phenotyping of tumor resident and peripheral immune cells from treated mice showing reduced levels of immune suppressive myeloid derived suppressor cells (MDSC). Inhibiting BPTF pharmacologically using the bromodomain inhibitor AU1 does not recapitulate the effects of the genetic inhibition suggesting that the benefits of BPTF depletion to sensitization to Gua are not captured by inhibition of its bromodomain. To model the effects of pharmacologic BPTF inhibition we created a BPTF mouse KO genetically modified mouse model (GEMM). We show using this GEMM that BPTF KO in the adult mouse (a tamoxifen inducible Cre) is not lethal and has beneficial effects to the control of E0771 tumors, possibly through depleting tumor resident naïve T cells, and that transplanting BPTF KO E0771 tumors into BPTF KO adult mice further improves tumor growth control. These benefits in tumor growth control are prevented with CD8 depletion. These results in total suggests that a combination therapy regimen including BPTF inhibition (a PROTAC degradation approach) with a DNMTi could provide clinical benefits to patients with TNBC through both tumor cell intrinsic and extrinsic mechanisms. Citation Format: Joseph W. Landry, Jubin Kang, Christiane Morecock, Harry Bear, Rebecca Martin. Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast 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 7553.

Abstract 7116: Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair

Abstract Prostate cancer (PCa) is the 2nd leading cause of cancer related deaths in US men. PCa is an androgen dependent disease driven by the androgen receptor (AR). Currently, androgen-deprivation therapy (ADT) is the standard of care first-line therapy for metastatic PCa. Resistance to ADT uniformly leads to lethal disease, termed castration-resistant prostate cancer (CRPC). Thus, there is an unmet clinical need to identify and develop novel strategies to treat CRPC. CBP/p300 are potent co-activators for AR. High expression of CBP/p300 is associated with locally advanced disease and castration resistant function of AR, resulting in poor patient outcomes and further highlighting the need to discern the role of CBP/p300 to potentially develop therapeutic targets for precision medicine. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. In this study, CBP/p300 mediated bromodomain activity is targeted by CCS1477 (inobrodib), a first-in-class bromodomain inhibitor developed by Cell Centric. CCS1477 treatment demonstrated effective inhibition in growth and clonogenicity assays. Inhibition of the CBP/p300 bromodomain with CCS1477 resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression in addition to inhibition of associated factors such as c-MYC and its downstream targets in PCa cell lines as well as patient derived xenograft (PDX) models. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC patient samples. The clinical significance of CBP/p300 expression in PCa has been investigated via elucidation of CBP/p300 transcriptional reprogramming and its role in DNA damage response (DDR) pathways. Specifically, findings revealed that CBP/p300 bromodomain suppression sensitizes to AR-dependent DNA-repair. Transcriptional mapping identified CBP/p300 as regulators of cell proliferation and DNA repair processes, which were functionally confirmed across several PCa model systems. To assess relevance, exogenous challenge with radiation revealed that CBP/p300 are required for AR-mediated DNA repair, and CBP/p300 expression is linked to DNA repair capacity in the clinical setting. Molecular analyses revealed that CBP/p300 facilitate double-strand break (DSB) repair efficiency via homologous recombination (HR) mediated DDR. Congruently, CBP/p300 strongly correlated with HR gene expression in PCa patient tissue. These collective findings reveal that CBP/p300 govern repair of DNA DSBs by regulating HR, thus modulating genome integrity and promoting CRPC growth. These studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati N. Chand, Wei Yuan, Denisa Bogdan, Jonathan Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair [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 7116.

Abstract 363: Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy of B lineage progenitors. It remains a leading cause of death in childhood, while outcomes in adults are dismal. There is therefore a need to better understand drivers of high-risk B-ALL and to develop novel therapeutic approaches targeting these challenging patient cohorts. Loss-of-function mutations affecting CREBBP are recurrent second-hit mutations across multiple genetic subtypes of B-ALL and are associated with adverse features, including high-risk genetic subtypes and persistent measurable residual disease. In addition, they have been mechanistically associated with chemoresistance and are more frequently found in relapse. We sought to identify novel treatment options for CREBBP-mutated high-risk B-ALL. CREBBP-mutated isogenic human B-ALL cell lines were genome-engineered to provide a platform for synthetic lethal drug screening. We subjected these cell lines to a targeted drug screen focused on clinically-actionable drugs in classes that have either been implicated or hypothesized to show differential sensitivity patterns in published models of B cell lymphoma and other CREBBP-mutated malignancies. Unexpectedly, CREBBP-mutated cells were not differentially sensitive to traditional cytotoxic chemotherapy, and paradoxically showed a degree of sensitization to the glucocorticoid Dexamethasone, used in current ALL induction regimens. As anticipated, and validating our screen design, inhibitors of the CREBBP paralogue EP300 (the CREBBP/EP300-specific bromodomain inhibitor Inobrodib and the CREBBP/EP300 acetylase inhibitor A485) exhibited synthetic lethality, consistent with previous reports in B-cell lymphoma. The most potent hit was the BCL2 inhibitor Venetoclax, which we show acts through a non-canonical, but BCL2-dependent mechanism resulting in ferroptotic programmed cell death. CREBBP-mutated cell lines were transcriptionally and functionally characterized, revealing underlying differences in cell-cycle, metabolism and response to oxidative stress. Acquisition of resistance to Venetoclax further dysregulated these pathways and resulted in a transcriptionally-convergent state. Lastly, we demonstrate that small-molecule inhibition of CREBBP sensitizes B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in vitro and in vivo, providing a potential novel drug combination for broader clinical translation in B-ALL. In summary, we have identified a number of actionable compounds that specifically target CREBBP-mutated high-risk B-ALL, demonstrate a novel mechanism-of-action for the BCL2 inhibitor Venetoclax in B-ALL and propose CREBBP-inhibitors and Venetoclax as a novel treatment combination for B-ALL across genotypes. Citation Format: Alicia Garcia Gimenez, Jonathan Ditcham, Dhoyazan M. Azazi, Eshwar Meduri, Ryan Asby, Nathalie Sakakini, Cecile K. Lopez, Nisha Narayan, Jaana Bagri, Tumas Beinortas, Shuchi Agrawal Singh, George Giotopoulos, Michael P. Murphy, Sarah J. Horton, Brian J. Huntly, Simon E. Richardson. Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition [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 363.

Abstract 564: Therapeutic targeting of JAK/STAT signaling and histone acetyltransferase activity in post-myeloproliferative neoplasm secondary AML

Abstract Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling was identified in 50-90% of MPNs - underscoring its role in driving MPN pathogenesis and transformation to post-MPN sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. The trial revealed that ruxolitinib improved the quality of life in patients but was not disease-modifying. Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML. Genome-wide CRISPR loss-of-function screens in JAK2-mutated HEL cells determined that knockout of CBP sensitizes leukemic cells to JAK2 inhibitors. Consistently, functional validation through CRISPR/Cas9 demonstrated that genetic deletion of CBP but not of p300 renders HEL cells sensitive to ruxolitinib treatment. Pharmacologic inhibition of JAK/STAT signaling and HAT activity through ruxolitinib and CBP30 treatment in post-MPN sAML lines HEL and SET2 decreases proliferation and induces apoptosis and cell cycle arrest. Mechanistically, the synergism between ruxolitinib and CBP30 elicits a unique transcriptome profile in HEL with significant downregulation of E2F and MYC targets. In addition, epigenetic profiling revealed that the synergism induces global changes in H3K27ac signals with downregulation and upregulation of H3K27ac signals in predicted STAT5/BCL3 and GATA-bounded regions, respectively. Remarkably, combination treatment of ruxolitinib and CBP30 in HEL luciferase NSG mouse xenografts showed significant leukemia regression and decrease in spleen size compared to mice treated with vehicle or single agents. Overall, these results substantiate the potential of targeting JAK/STAT signaling and histone acetyltransferase activity in the treatment of post-MPN sAML. Citation Format: Kalay Bertulfo, Koen Debackere, Hannah Miller, Cindy Ma, Ryan Najac, Adolfo Ferrando, Teresa Palomero. Therapeutic targeting of JAK/STAT signaling and histone acetyltransferase activity in post-myeloproliferative neoplasm secondary AML [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 564.

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.

An LC-MS/MS method for determination of the bromodomain inhibitor ZEN-3694 and its metabolite ZEN-3791 in human plasma.

We have developed and validated a novel LC-MS/MS method for the simultaneous quantification of ZEN-3694 and its active metabolite ZEN-3791 in human plasma afterprotein precipitation. Stable isotope-labeled versions were used as internal standards. Chromatographic separation was achieved on a Kinetex C18 column using 0.1% formic acid in H2O and 0.1% formic acid in MeOH as mobile phases. Detection was performed via positive electrospray ionization mode with multiple reaction monitoring. The assay exhibited linearity in the concentration range of 5-5000ng/ml for both analytes. Intra- and inter-assay precision and accuracy were within ±11%. ZEN-3694 and ZEN-3791 recoveries were between 93 and 105%. This LC-MS/MS assay is an essential tool to study ZEN-3694 in an ongoing clinical trial (NCT04840589).

Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction

BackgroundInjury to contractile organs such as the heart, vasculature, urinary bladder and gut can stimulate a pathological response that results in loss of normal contractility. PDGF and TGFβ are among the most well studied initiators of the injury response and have been shown to induce aberrant contraction in mechanically active cells of hollow organs including smooth muscle cells (SMC) and fibroblasts. However, the mechanisms driving contractile alterations downstream of PDGF and TGFβ in SMC and fibroblasts are incompletely understood, limiting therapeutic interventions.MethodsTo identify potential molecular targets, we have leveraged the analysis of publicly available data, comparing transcriptomic changes in mechanically active cells stimulated with PDGF and TGFβ. Additional Analysis of publicly available data sets were performed on SMC and fibroblasts treated in the presence or absence of the MYC inhibitor JQ1. Validation of in silico findings were performed with qPCR, immunoblots, and collagen gel contraction assays measure the effect of JQ1 on cytoskeleton associated genes, proteins and contractility in mechanically active cells. Likelihood ratio test and FDR adjusted p-values were used to determine significant differentially expressed genes. Student ttest were used to calculate statistical significance of qPCR and contractility analyses.ResultsComparing PDGF and TGFβ stimulated SMC and fibroblasts identified a shared molecular profile regulated by MYC and members of the AP-1 transcription factor complex. Additional in silico analysis revealed a unique set of cytoskeleton-associated genes that were sensitive to MYC inhibition with JQ1. In vitro validation demonstrated JQ1 was also able to attenuate TGFβ and PDGF induced changes to the cytoskeleton and contraction of smooth muscle cells and fibroblasts in vitro.ConclusionsThese findings identify MYC as a key driver of aberrant cytoskeletal and contractile changes in fibroblasts and SMC, and suggest that JQ1 could be used to restore normal contractile function in hollow organs.

Novel BRD4-p53 Inhibitor SDU-071 Suppresses Proliferation and Migration of MDA-MB-231 Triple-Negative Breast Cancer Cells.

A promising alternative for cancer treatment involves targeted inhibition of the epigenetic regulator bromodomain-containing protein 4 (BRD4); however, available BRD4 inhibitors are constrained by their potency, oral bioavailability, and cytotoxicity. Herein, to overcome the drawback of the translational BRD4 inhibitors, we describe a novel BRD4-p53 inhibitor, SDU-071, which suppresses BRD4 interaction with the p53 tumor suppressor and its biological activity in MDA-MB-231 triple-negative breast cancer (TNBC) cells in vitro and in vivo. This novel small-molecule BRD4-p53 inhibitor suppresses cell proliferation, migration, and invasion by downregulating the expression of BRD4-targeted genes, such as c-Myc and Mucin 5AC, and inducing cell cycle arrest and apoptosis, as demonstrated in cultured MDA-MB-231 TNBC cells. Its antitumor activity is illustrated in an orthotopic mouse xenograft mammary tumor model. Overall, our results show that SDU-071 is a viable option for potentially treating TNBC as a new BRD4-p53 inhibitor.

Therapeutic Potential of Bromodomain and Extra-Terminal Domain Inhibitors for Synovial Sarcoma Cells.

Synovial sarcoma (SS), a rare subtype of soft-tissue sarcoma distinguished by expression of the fusion gene SS18-SSX, predominantly affects the extremities of young patients. Existing anticancer drugs have limited efficacy against this malignancy, necessitating the development of innovative therapeutic approaches. Given the established role of SS18-SSX in epigenetic regulation, we focused on bromodomain and extra-terminal domain protein (BET) inhibitors and epigenetic agents. Our investigation of the BET inhibitor ABBV-075 revealed its pronounced antitumor effects, inducing G1-phase cell-cycle arrest and apoptosis, in four SS cell lines. Notably, BET inhibitors exhibited regulatory control over crucial cell-cycle regulators, such as MYC, p21, CDK4, and CDK6. Additionally, RNA sequencing findings across the four cell lines revealed the significance of fluctuating BCL2 family protein expression during apoptotic induction. Notably, variations in the expression ratio of the anti-apoptotic factor BCLxL and the pro-apoptotic factor BIM may underlie susceptibility to ABBV-075. Additionally, knockdown of SS18-SSX, which upregulates BCL2, reduced the sensitivity to ABBV-075. These findings suggest the potential utility of BET inhibitors targeting the SS18-SSX-regulated intrinsic apoptotic pathway as a promising therapeutic strategy for SS.

Abstract P33: Leveraging probabilistic causal disease models to understand molecular pathways and target resistance mechanisms in Multiple Myeloma with the CREBBP and EP300 bromodomain inhibitor, pocenbrodib

Abstract To explore and devise innovative approaches for the treatment of Multiple Myeloma (MM) – a disease often deemed incurable due to the development of resistance mechanisms against available therapies – we adopted an integrative data approach, constructing causal AI models of MM that integrate co-expression relationships, DNA variation, longitudinal clinical data, and expansive knowledge graphs. Diverging from traditional methods, our approach centers on constructing probabilistic causal models to elucidate the regulatory mechanisms of disease and capture existing known causal relationships published in the scientific literature or curated in pathway databases. We employ generative AI and other advanced machine learning approaches to stratify patients into mechanistically-informed subtypes that we can directly associate with clinical outcomes. These models, and the ability to map driver mechanisms to outcomes, serve as a foundation for analyzing our real-world data, a dataset made up of more than 200,000 cancer patients with multimodal molecular data generated on tumor tissues and matched to longitudinal clinical data. Finally, we can validate and refine therapeutically relevant hypotheses using a scalable high-content organoid and cell line screening platform to characterize drug and/or targeted genetic perturbation signatures.We analyzed our models in MM to reveal sub-populations of relapsed refractory patients that could be susceptible to specific pathway inhibition. This analysis revealed a focused causal subnetwork that predicts poor overall survival in standard MM treatment protocols. We demonstrate that not only does this subnetwork show promise in predicting response to a novel CREBBP and EP300 bromodomain inhibitor, pocenbrodib, but it sheds light on the molecular underpinnings of MM, beyond the currently known IRF4, MYC, and immune cell regulation mechanisms that the inhibition of CREBBP/EP300 is known to target. Our network models reveal that this novel subnetwork is intricately linked to a range of driver mechanisms that strongly predict disease progression and are associated with known mechanisms of resistance. Notably, the signature persists across various MM stages, from initial diagnosis to relapsed/refractory states, and intensifies with each additional line of therapy. Further, signature scores are higher in patients with known high-risk cytogenetic abnormalitiesOur findings suggest that stratifying patients based on this gene signature could be instrumental in identifying individuals at high risk of MM progression. Such stratification could transform the selection process for pocenbrodib treatment. By leveraging an integrated, AI-driven approach, we advance the understanding of MM and pave the way for more targeted and effective therapeutic strategies. Citation Format: Allison E Roder, Alice M Walsh, Taylor K Krebs, Veronica Calvo, Bonnie V Dougherty, Preston Smith, Jens Renstrup, Eric E Schadt. Leveraging probabilistic causal disease models to understand molecular pathways and target resistance mechanisms in Multiple Myeloma with the CREBBP and EP300 bromodomain inhibitor, pocenbrodib [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P33.

Inhibition of spinal BRD4 alleviates pyroptosis and M1 microglia polarization via STING-IRF3 pathway in morphine-tolerant rats

BackgroundMorphine tolerance has been a challenging medical issue. Neuroinflammation is considered as a critical mechanism for the development of morphine tolerance. Bromodomain-containing protein 4 (BRD4), a key regulator in cell damage and inflammation, participates in the development of chronic pain. However, whether BRD4 is involved in morphine tolerance and the underlying mechanisms remain unknown. MethodsThe morphine-tolerant rat model was established by intrathecal administration of morphine twice daily for 7 days. Behavior test was assessed by a tail-flick latency test. The roles of BRD4, pyroptosis, microglia polarization and related signaling pathways in morphine tolerance were elucidated by Western blot, real-time quantitative polymerase chain reaction, and immunofluorescence. ResultsRepeated morphine administration upregulated BRD4 level, induced pyroptosis, and promoted microglia M1-polarization in spinal cord, accompanied by the release of proinflammatory cytokines, such as TNF-α and IL-1β. JQ-1, a BRD4 antagonist, alleviated the development of morphine tolerance, diminished pyroptosis and induced the switch of microglia from M1 to M2 phenotype. Mechanistically, stimulator of interferon gene (STING)- interferon regulatory factor 3 (IRF3) pathway was activated and the protective effect of JQ-1 against morphine tolerance was at least partially mediated by inhibition of STING-IRF3 pathway. ConclusionThis study demonstrated for the first time that spinal BRD4 contributes to pyroptosis and switch of microglia polarization via STING-IRF3 signaling pathway during the development of morphine tolerance, which extend the understanding of the neuroinflammation mechanism of morphine tolerance and provide an alternative strategy for the precaution against of this medical condition.