Oncogenic Transcription Factor Research Articles

Abstract A015: Exploiting endogenous replication stress with a novel targeted therapy in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a rare and aggressive disease that typically affects the bone of children and young adults. Although there are moderate 5-year survival rates for primary disease, there is a high rate of recurrence and a lack of targeted therapies in this setting. Furthermore, pediatric exposure to chemotherapies leads to an increased risk of long-term complications, including secondary cancers, cardiomyopathy, and infertility. Therefore, there is a desperate need for targeted therapies which improve therapeutic outcomes and reduce exposure to chemotherapy in these patients. Like many pediatric cancers, EwS has a low mutational burden. Instead, it is driven by chromosomal translocation between a FET family member, EWSR1, and an ETS family member, usually FLI1. The EWSR1-FLI1 fusion protein acts as an aberrant and oncogenic transcription factor, disrupting cell cycle control, cell migration, and proliferation. ETS family members in these fusions retain their ability to bind to the SWI/SNF chromatin remodeling complex, further dysregulating DNA methylation and gene expression. Although targeting of the EWSR1 fusion has not yet been successful, these elevated levels of replication stress (RS) present a unique element of vulnerability within the cell. To this end, our lab has previously identified replication stress response (RSR) inhibitors, DDKi and WEE1i, which can be utilized to exploit the characteristic ability of EwS to maintain high levels of replication stress (RS) and push EwS cells into mitotic catastrophe in vitro. Here, we evaluate the efficacy of the replication stress response inhibitors (RSRi) TAK931 (DDKi) and MK1775 (WEE1i) with chemotherapy regimens commonly utilized in the relapsed/refractory setting in vivo. Using cell line derived xenografts in NSG mice, we show that DDKi and WEE1i with irinotecan limits tumor growth significantly more than existing therapy (temozolomide + irinotecan). Dosing schedules are optimized through dosing de-escalation and alternative-day dosing strategies. We further demonstrate the ability of our RSRi combination to effectively limit tumor growth over time in vivo even when irinotecan dosage is decreased. We go on to investigate the RSRi combination in the presence of ultra-low dose irinotecan. Finally, we utilize reduced representation bisulfite sequencing to examine the epigenetic effects of these treatment regimens. Overall, the exploitation of high endogenous replication stress in EwS by targeted RSRi presents a promising potential therapeutic option for this pediatric patient population. Citation Format: Emily Isenhart, Ajay Gupta, Bryan Gillard, Kristopher Attwood, Joyce Ohm. Exploiting endogenous replication stress with a novel targeted therapy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A015.

Effect of RGT-61159 on inhibition of oncogene c-MYB synthesis and tumor growth inhibition in a broad range of ACC PDX models, at well tolerated doses in rodents and non-human primates.

6107 Background: RGT-61159 is a first in-class oral inhibitor of the oncogenic transcription factor c-MYB via selective alteration of its RNA splicing machinery. RGT-61159 selectively induces the inclusion of the cryptic “poison” exon into the c-MYB RNA transcripts, resulting in the robust elimination of c-MYB canonical mRNA transcript and consequently of its protein in cells. Overactivation of the c-MYB oncogene, primarily due to chromosome rearrangements, is a hallmark of adenoid cystic carcinoma. (ACC). ACC is a rare and aggressive type of cancer for which effective treatment does not exist. By inhibiting oncogenic MYB protein production, RGT-61159 has the potential to efficiently and selectively inhibit proliferation or induce cell death of cancer cells overexpressing MYB protein. Methods: RGT-61159 cell killing activity was measured against a broad panel of cancer cell lines in a CellTiter-Glo assay. MYB RNA and MYB protein levels were assessed after drug treatment by RT-qPCR and JESS assay respectively. Anti-tumor activity of RGT-61159 as single agent or in combination was evaluated in a panel of ACC PDX mouse models harboring different molecular alterations in c-MYB, including ACCX6, ACCX11, ACCx5M1 and ST105B2. Results: Here, we showed that RGT-61159 has potent cancer cell killing activity (EC50 – 100nM -200nM) against a large panel of cancer cell lines overexpressing c-MYB, while sparing normal cells. Robust c-MYB RNA and c-MYB protein depletion (>80%) was observed in treated cells confirming RGT-61159 on-target cell killing effect. In addition, RGT-61159 single agent showed a remarkable anti-tumor activity (up to 90% TGI) at tolerated doses in the four ACC PDX models evaluated: ACCX6, ACCx11, ACCx5M1 and STB105B2. Importantly, we confirmed that RGT-61159 anti-tumor activity correlated with c-MYB target modulation. RGT-61159 efficacious doses reduced c-MYB transcript and c-MYB protein levels (by >80%) in all ACC PDx models evaluated. In addition, RNAseq analysis of ACC tumor after treatment showed that RGT-61159 drug treatment reversed the published ACC overexpression gene signature. Notably, the optimal RGT-61159 regimen was well tolerated, as assessed by changes in animal body weight and clinical signs in rodents and non-human primate. Finally, we showed that the combination of RGT-M001, an analog of RGT-61159, with the NOTCH Inhibitor AL-101 resulted in significant tumor regression at well tolerated doses. Conclusions: Altogether, these data demonstrated that RGT-61159 efficiently and safely targets the undruggable oncogenic transcription factor c-MYB. Down-regulation of c-MYB by the RGT-61159 RNA-targeting small molecule is a very promising therapeutic strategy to treat ACC and other cancers driven by c-MYB dysregulation.

Depletion of histone H3K27 demethylase exerts anti-tumor activity as a monotherapy and in combination therapy with ceralasertib in non-small cell lung carcinoma.

e20035 Background: Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide. Non-small cell lung carcinoma (NSCLC) represents 85% of lung cancers and is the most common type with poor prognosis. The lysine-specific demethylase UTX (gene name KDM6A) demethylates H3K27me2/3 at genes and enhancers and have been shown to support oncogenic transcription factors. We hypothesize that UTX is a highly pleiotropic factor in tumorigenesis and may plays a critical role in controlling DNA replication-associated gene activation as well as in replication stress-related DNA damage repair. Methods: To evaluate the role of UTX in tumorigenesis, shRNA-mediated UTX knockdown was employed in NCI-H1975 cells followed by cell cycle and cell proliferation assays. In the UTX-knocked down NCI-H1975 cells, target genes involved in DNA replication and DNA damage repair were discovered by RNA sequencing and further validated with RT-qPCR. Effects of UTX depletion on DNA replication and radiation-induced DNA repair were analyzed by immunofluorescence. Effects of UTX depletion on cell cycle arrest and the ATR–CHK1 checkpoint signaling pathway were analyzed by flow cytometry and immunoblotting. In vivo response to UTX inhibition monotherapy (UTX knockdown) and combination therapy of UTX knockdown with ceralasertib, a selective ATR kinase inhibitor, was evaluated in dox-inducible UTX knockdown NCI-H1975 xenografts. Results: Induction of UTX knockdown significantly reduced the NCI-H1975 tumor volume compared to the control group, showing anti-tumor activity. Furthermore, loss of UTX induced hyperactivation of ATR–CHK1 checkpoint signaling pathway, suggesting the UTX-depleted tumor cells attempt to alleviate replication stress to escape from apoptosis. To inhibit the UTX-depletion induced ATR–CHK1 pathway, ceralasertib (formerly AZD6738), an oral potent selective inhibitor of ATR, was combined with UTX-knockdown for in vitro and in vivo evaluation. Consistent with in vitro cell-based assay, combination therapy of ceralasertib with UTX depletion showed enhanced tumor growth inhibition and regression in the NCI-H1975 xenograft model. Conclusions: Our findings provide new insights into a pro-oncogenic role of UTX in supporting tumor maintenance via engaging DNA replication and repair pathway. Depletion of UTX alone may serve as a new therapeutic target in non-small cell lung carcinoma; and further combination with ceralasertib enhances the anti-tumor activity of UTX knockdown by inhibiting the ATR–CHK1 checkpoint signaling pathway.

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

Molecular Pathways of Genistein Activity in Breast Cancer Cells.

The most common malignancy in women is breast cancer. During the development of cancer, oncogenic transcription factors facilitate the overproduction of inflammatory cytokines and cell adhesion molecules. Antiapoptotic proteins are markedly upregulated in cancer cells, which promotes tumor development, metastasis, and cell survival. Promising findings have been found in studies on the cell cycle-mediated apoptosis pathway for medication development and treatment. Dietary phytoconstituents have been studied in great detail for their potential to prevent cancer by triggering the body's defense mechanisms. The underlying mechanisms of action may be clarified by considering the role of polyphenols in important cancer signaling pathways. Phenolic acids, flavonoids, tannins, coumarins, lignans, lignins, naphthoquinones, anthraquinones, xanthones, and stilbenes are examples of natural chemicals that are being studied for potential anticancer drugs. These substances are also vital for signaling pathways. This review focuses on innovations in the study of polyphenol genistein's effects on breast cancer cells and presents integrated chemical biology methods to harness mechanisms of action for important therapeutic advances.

Oncogene EVI1 drives acute myeloid leukemia via a targetable interaction with CTBP2.

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

MYC Inhibition Potentiates CD8+ T Cells Against Multiple Myeloma and Overcomes Immunomodulatory Drug Resistance.

Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, are a cornerstone of multiple myeloma (MM) therapies, yet the disease inevitably becomes refractory. IMiDs exert cytotoxicity by inducing cereblon-dependent proteasomal degradation of IKZF1 and IKZF3, resulting in downregulation of the oncogenic transcription factors IRF4 and MYC. To date, clinical IMiD resistance independent of cereblon or IKZF1/3 has not been well explored. Here, we investigated the roles of IRF4 and MYC in this context. Using bone marrow aspirates from patients with IMiD-naïve or refractory MM, we examined IKZF1/3 protein levels and IRF4/MYC gene expression following ex vivo pomalidomide treatment via flow cytometry and qPCR. We also assessed exvivo sensitivity to the MYC inhibitor MYCi975 using flow cytometry. We discovered that although pomalidomide frequently led to IKZF1/3 degradation in MM cells, it did not affect MYC gene expression in most IMiD-refractory samples. We subsequently demonstrated that MYCi975 exerted strong anti-MM effects in both IMiD-naïve and -refractory samples. Unexpectedly, we identified a cluster of differentiation 8+ (CD8+ T) cells from patients with MM as crucial effectors of MYCi975-induced cytotoxicity in primary MM samples, and we discovered that MYCi975 enhanced the cytotoxic functions of memory CD8+ T cells. We lastly observed synergy between MYCi975 and pomalidomide in IMiD-refractory samples, suggesting that restoring MYC downregulation can re-sensitize refractory MM to IMiDs. Our study supports the concept that MYC represents an Achilles' heel in MM across disease states and that MYCi975 may be a promising therapeutic for patients with MM, particularly in combination with IMiDs.

Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2’s biochemical properties, providing valuable insights into its serine protease domain.

SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both invitro and invivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both invitro and invivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

BackgroundGenetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models.ResultsWe treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells.ConclusionsNB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.

Hold the MYCrophone: MYC Invades Enhancers to Control Cancer-Type Gene Programs.

MYC is an oncogenic transcription factor that binds gene promoters to facilitate oncogenic gene expression. When overexpressed, as is the case in most human cancers, MYC also invades active enhancers-cis-regulatory elements that are critical for regulating gene expression. In previous studies, the regulatory significance of MYC enhancer invasion in cancer cells has been debated. In their study published in Nature Genetics, Jakobsen and colleagues establish a new role for MYC in enhancer regions: regulating cancer type-specific gene programs. Their work reveals a mechanism in which MYC cooperates with other oncogenic transcription factors to recruit epigenetic regulators to enhancers, resulting in an epigenetic "switch" that promotes enhancer activation through BRD4 and RNA polymerase II. This activity was highly cancer-type specific, highlighting gene expression programs that predicted clinical outcome in a subtype-specific manner in patients with breast cancer.

Disrupting prostate cancer research: Challenge accepted; report from the 2023 Coffey-Holden Prostate Cancer Academy Meeting.

The 2023 Coffey-Holden Prostate Cancer Academy (CHPCA) Meeting, themed "Disrupting Prostate Cancer Research: Challenge Accepted," was convened at the University of California, Los Angeles, Luskin Conference Center, in Los Angeles, CA, from June 22 to 25, 2023. The 2023 marked the 10th Annual CHPCA Meeting, a discussion-oriented scientific think-tank conference convened annually by the Prostate Cancer Foundation, which centers on innovative and emerging research topics deemed pivotal for advancing critical unmet needs in prostate cancer research and clinical care. The 2023 CHPCA Meeting was attended by 81 academic investigators and included 40 talks across 8 sessions. The central topic areas covered at the meeting included: targeting transcription factor neo-enhancesomes in cancer, AR as a pro-differentiation and oncogenic transcription factor, why few are cured with androgen deprivation therapy and how to change dogma to cure metastatic prostate cancer without castration, reducing prostate cancer morbidity and mortality with genetics, opportunities for radiation to enhance therapeutic benefit in oligometastatic prostate cancer, novel immunotherapeutic approaches, and the new era of artificial intelligence-driven precision medicine. This article provides an overview of the scientific presentations delivered at the 2023 CHPCA Meeting, such that this knowledge can help in facilitating the advancement of prostate cancer research worldwide.

The MYCN 5′ UTR as a therapeutic target in neuroblastoma

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

FOXA1 regulates ribosomal RNA transcription in prostate cancer.

Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation. Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays. In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis. Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.

Cell Context Is the Third Axis of Synergy for the Combination of ATR Inhibition and Cisplatin in Ewing Sarcoma.

The importance of cellular context to the synergy of DNA damage response (DDR)-targeted agents is important for tumors with mutations in DDR pathways, but less well-established for tumors driven by oncogenic transcription factors. In this study, we exploit the widespread transcriptional dysregulation of the EWS-FLI1 transcription factor to identify an effective DDR-targeted combination therapy for Ewing sarcoma. We used matrix drug screening to evaluate synergy between a DNA-PK inhibitor (M9831) or an ATR inhibitor (berzosertib) and chemotherapy. The combination of berzosertib and cisplatin was selected for broad synergy, mechanistically evaluated for Ewing sarcoma selectivity, and optimized for in vivo schedule. Berzosertib combined with cisplatin demonstrates profound synergy in multiple Ewing sarcoma cell lines at clinically achievable concentrations. The synergy is due to loss of expression of the ATR downstream target CHEK1, loss of cell-cycle check-points, and mitotic catastrophe. Consistent with the goals of the project, EWS-FLI1 drives the expression of CHEK1 and five other ATR pathway members. The loss of CHEK1 expression is not due to transcriptional repression and instead caused by degradation coupled with suppression of protein translation. The profound synergy is realized in vivo with a novel optimized schedule of this combination in subsets of Ewing sarcoma models, leading to durable complete responses in 50% of animals bearing two different Ewing sarcoma xenografts. These data exploit EWS-FLI1 driven alterations in cell context to broaden the therapeutic window of berzosertib and cisplatin to establish a promising combination therapy and a novel in vivo schedule. See related commentary by Ohmura and Grünewald, p. 3358.

Abstract LB037: E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333

Abstract To elucidate the underlying mechanisms of STAT3 degradation and the suitability of VHL as an E3 ligase partner for targeting STAT3 in cancer using our clinical degrader, KT-333. Signal transducer and activator of transcription 3 (STAT3) is an undrugged oncogenic transcription factor and its role as cancer driver and tumor microenvironment modulator has been validated in a multitude of studies. Based on it’s potential as a target for cancer therapeutics and limitations of prior approaches, we developed KT-333, a first-in-class, potent, highly selective, heterobifunctional STAT3 degrader currently in Phase 1 clinical trials. Here, based on STAT3 degradation by multiple E3s based degraders, structure of STAT3-KT333-VHL and a lysine site-resolved target ubiquitination model, we provide evidence for VHL as the ideal partner E3 for targeting STAT3 in cancer. We successfully identified potent and selective STAT3 degraders using either CRBN or VHL as the E3 ligase, but ultimately preferred VHL based degraders for their increased potency, and consistent STAT3 degradation across multiple cancer lines. KT-333, our VHL based clinical STAT3 degrader, induces a strong ternary complex between STAT3 and VHL. We present a high resolution cryo-electron microscopy based ternary complex structure of STAT3-KT333-VHL which provides mechanistic insights further validating VHL as the E3 of choice for deep, selective and fast STAT3 degradation. Specifically, KT-333 enables favorable protein-protein interactions between STAT3 and VHL, burying a large protein interface in the ternary complex to the extent typically observed only in native protein complexes. Additionally, the residues forming the STAT3-VHL interface are not conserved in other STAT family members corroborating with degradation selectivity observed with KT-333. Next, we leveraged this structure and generated a ubiquitination super-complex model by deploying a combination of biochemical and proteomics techniques and reveal that KT-333-induced ubiquitination occurs by precise targeting of specific lysine resides on STAT3. Furthermore, to decode the mechanism for KT-333 activity against SUDHL1 cell line both in vitro and in a mouse xenograft model, we measured temporal changes at protein level by discovery proteomics. Reduced expression of canonical STAT3 targets and down-regulation of cytokine-mediated signaling and cell cycle signature genes indicated that cell cycle arrest and subsequent apoptosis are the main drivers of efficacy in vitro and in vivo. In summary, we show that our clinical STAT3 degrader, KT-333, is designed and optimally paired with VHL resulting in a very stable ternary complex exhibiting properties of native protein complexes. Our data provides precise structural and molecular mechanisms behind potent, selective, consistent, and fast degradation of STAT3, and downstream mechanism of action observed in vitro and in vivo. Citation Format: Kirti Sharma, Xue Fei, Yatao Shi, Christopher Browne, Dirk Walther, Caroline Daigle, Anand Ramanathan, Richard Miller, Karen Yuan, Kiran Mahasenan, Sean Zhu, Xin Huang, Bin Yang. E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333 [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 LB037.

EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells

The androgen receptor (AR) is a primary target for treating prostate cancer (PCa), forming the bedrock of its clinical management. Despite their efficacy, resistance often hampers AR-targeted therapies, necessitating new strategies against therapy-resistant PCa. These resistances involve various mechanisms, including AR splice variant overexpression and altered activities of transcription factors like the glucocorticoid receptor (GR) and FOXA1. These factors rely on common coregulators, such as EP300/CREBBP, suggesting a rationale for coregulator-targeted therapies. Our study explores EP300/CREBBP acetyltransferase inhibition’s impact on steroid receptor and FOXA1 signaling in PCa cells using genome-wide techniques. Results reveal that EP300/CREBBP inhibition significantly disrupts the AR-regulated transcriptome and receptor chromatin binding by reducing the AR-gene expression. Similarly, GR’s regulated transcriptome and receptor binding were hindered, not linked to reduced GR expression but to diminished FOXA1 chromatin binding, restricting GR signaling. Overall, our findings highlight how EP300/CREBBP inhibition distinctively curtails oncogenic transcription factors’ signaling, suggesting the potential of coregulatory-targeted therapies in PCa.

Transcription Factors in Prostate Cancer: Insights for Disease Development and Diagnostic and Therapeutic Approaches.

Transcription factors (TFs) are proteins essential for the regulation of gene expression, and they regulate the genes involved in different cellular processes, such as proliferation, differentiation, survival, and apoptosis. Although their expression is essential in normal physiological conditions, abnormal regulation of TFs plays critical role in several diseases, including cancer. In prostate cancer, the most common malignancy in men, TFs are known to play crucial roles in the initiation, progression, and resistance to therapy of the disease. Understanding the interplay between these TFs and their downstream targets provides insights into the molecular basis of prostate cancer pathogenesis. In this review, we discuss the involvement of key TFs, including the E26 Transformation-Specific (ETS) Family (ERG and SPDEF), NF-κB, Activating Protein-1 (AP-1), MYC, and androgen receptor (AR), in prostate cancer while focusing on the molecular mechanisms involved in prostate cancer development. We also discuss emerging diagnostic strategies, early detection, and risk stratification using TFs. Furthermore, we explore the development of therapeutic interventions targeting TF pathways, including the use of small molecule inhibitors, gene therapies, and immunotherapies, aimed at disrupting oncogenic TF signaling and improving patient outcomes. Understanding the complex regulation of TFs in prostate cancer provides valuable insights into disease biology, which ultimately may lead to advancing precision approaches for patients.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Oncogenic STAT Transcription Factors as Targets for Cancer Therapy: Innovative Strategies and Clinical Translation.

Despite advances in our understanding of molecular aspects of oncogenesis, cancer remains a leading cause of death. The malignant behavior of a cancer cell is driven by the inappropriate activation of transcription factors. In particular, signal transducers and activators of transcription (STATs), which regulate many critical cellular processes such as proliferation, apoptosis, and differentiation, are frequently activated inappropriately in a wide spectrum of human cancers. Multiple signaling pathways converge on the STATs, highlighting their importance in the development and progression of oncogenic diseases. STAT3 and STAT5 are two members of the STAT protein family that are the most frequently activated in cancers and can drive cancer pathogenesis directly. The development of inhibitors targeting STAT3 and STAT5 has been the subject of intense investigations in the last decade, although effective treatment options remain limited. In this review, we investigate the specific roles of STAT3 and STAT5 in normal physiology and cancer biology, discuss the opportunities and challenges in pharmacologically targeting STAT proteins and their upstream activators, and offer insights into novel therapeutic strategies to identify STAT inhibitors as cancer therapeutics.