Virtual International Research Articles

Abstract B065: Enhancing the therapeutic gain of proton radiotherapy in Locally Advanced Rectal Cancer (LARC) through manipulation of WSB1, ATM, and β-Catenin/c-Myc pathway Ubiquitination

Abstract Just 20% of patients with LARC attain a complete response to preoperative chemoradiotherapy which portends an excellent prognosis and opportunity to preserve the rectum through nonoperative surveillance. Moreover, normal tissue radiation toxicity can lead to significant morbidity, and becomes an even more important consideration in non-operative approaches where irradiated tissue is not resected. The geometry of conventional proton beams is already being used clinically to spare patient’s normal tissue, but the biology of entrance (ENT) vs. Bragg Peak (BP) protons has yet to be fully exploited to enhance tumor efficacy. Protons, unlike conventional x-ray radiation, spare normal tissue by lacking an exit dose - but they also exhibit a Bragg Peak (BP), which causes more complex DNA damage and is more biologically effective. ATM is critical in the repair of double strand DNA breaks (DSBs) and our lab has demonstrated that inhibitors of ATM (iATM) preferentially radiosensitize BP protons in CRC (Fig. 1A). Overactivation of the Wnt/β-catenin pathway via loss of function of APC is the most common genetic mutation in colorectal cancer (75-80%) and β-catenin over-expression negatively correlates with prognosis. WSB1 has been discovered by our lab to regulate ATM ubiquitination and degradation, it decreases metastates-free survival in CRC, and also forms a positive feedback loop with β-catenin and c-Myc that drives oncogenic progression. It is unknown if this upregulation of WSB1 promotes ATM degredation in LARC specifically, and a deubiquitinase (DUB) for ATM (which would counter WSB1) has yet to be identified. We preformed a comprehensive database search (UbiBrowser 2.0) for the most likely predicted DUBs for ATM (Fig. 1B), several of which have been identified as regulators of β-catenin/c-Myc, DSB repair pathways, and correlated with poor prognosis in CRC. We have also performed a screen of DUBs predicted to interact with ATM, and have preliminary evidence suggesting USP14 as the worlds first identified DUB of ATM (Fig 1C). USP14 expression is upregulated in CRC compared to surrounding normal tissue, promotes radioresistance, and promotes Wnt/β-catenin signalling. Higher USP14 expression in CRC is associated with liver and lymph node metastases and worse overall survival. USP14 regulates β-catenin by deubiquitinating K63-linked polyubiquitin chains of Dvl, and several small molecule inhibitors of USP14 have been identified. Our lab has preliminary data demonstrating USP14 is preferentially activated after BP proton irradiation (Fig. 1D), suggesting ATM may be necessary for repair of complex DSBs. While more work will be necessary to confirm these findings, many of the other predicted DUBs of ATM have overlapping functions regulating DSB repair and/or β-catenin/c-Myc signalling. We hypothesize that targeting ATM ubiquitination will open a therapeutic window in LARC by disrupting β-catenin/c-Myc signalling and selectively radiosensitizing BP protons. Citation Format: Sonja Dragojevic, Cameron M. Callaghan. Enhancing the therapeutic gain of proton radiotherapy in Locally Advanced Rectal Cancer (LARC) through manipulation of WSB1, ATM, and β-Catenin/c-Myc pathway Ubiquitination [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B065.

Abstract A033: Anti-proliferative effects of lentinan, a beta-glucan from shiitake mushroom (lentinula edodes)

Abstract Cancer is one of the most significant health challenges worldwide. Lentinan, a β-glucan from shiitake mushroom, Lentinula edodes, has been demonstrated to have an inhibitory effect on selected cancer cells, but results are inconclusive. The aim of the present study was to investigate the anti-proliferative effect of lentinan on breast adenocarcinoma cells (MCF-7), prostate carcinoma (DU-145), and ovarian carcinoma (SKOV3) using MTS (3-(4,5-dimethylthiazol-2-yl)-5—(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and explore possible mechanisms of action of this compound. In the MCF-7 cells that were used as a positive control, lentinan without phenol red resulted in a significant inhibition of cell viability by 96% while in the presence of phenol red, the weak estrogen effect attributable to phenol red eliminated the significant inhibitory effect of lentinan. Treatment of DU-145 cells with different doses of lentinan resulted in up to a 42% inhibition in cell growth. In SKOV3 cells, lentinan induced a biphasic response where doses of 800 µg/ml or less increased cell viability by 76% – 148%; however, a dose of 1.6 mg/ml inhibited cell proliferation significantly by 87%. When SKOV3 cells were co-treated with lentinan (800 µg/ml or less) and laminarin (a Dectin-1 receptor antagonist), the proliferation-promoting effect of lentinan was dominant over the proliferation-inhibiting effect of laminarin. However, when lentinan was presented at significant higher levels (1600 µg/ml), the inhibitory effect of the two agents became additive in suppressing SKOV3 cell proliferation. Lentinan induced a biphasic response in human ovarian surface epithelial cells (HOSEpC) similar to SKOV3 cells, where the highest dose (1600 µg/ml) resulted in the inhibition of cell growth by 53%. Western blots after treatment of SKOV3 cells with 1600 µg/ml of lentinan demonstrated caspase-3 activation and a decreased anti-apoptotic protein Bcl-2 levels. Our findings reveal that lentinan can be considered as a potential anti-cancer drug for effective treatment of breast and prostate cancer. Citation Format: Titus Sombuor, Kenneth Shane Broughton, Michael Bergel. Anti-proliferative effects of lentinan, a beta-glucan from shiitake mushroom (lentinula edodes) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A033.

Abstract C175: The potential effect of KRAS inhibitors on tumor microenvironments

Abstract KRAS is one of the most common driver oncogenes among various cancer types. Researches aiming to develop KRAS inhibitors has been surging recently and the first clinical KRAS inhibitor sotorasib was approved for non-small cell lung cancer in the US and Japan. As KRAS activity has been reported to have certain effects on tumor microenvironments, KRAS inhibitor could potentially induce collateral outcomes on tumor microenvironment. Therefore, I have analyzed the potential influences of resistance to KRAS inhibitors on the tumor microenvironment. At first, I have established pancreatic cancer cell line MIA-Paca2 that acquired resistance to sotorasib, MIA/AMG-R, by continuously exposing them to sotorasib (1 µmol/L). An RNA sequencing analysis were performed with MIA/AMG-R and revealed that a gene set related to hypoxia were upregulated. When non-small cell lung cancer cell line NCI-H358 cells were exposed to sotorasib (1 µmol/L) for 4 hrs, the protein levels of HIF-1A decreased while that in MIA Paca-2 cells were unaffected. To evaluate at which steps of protein synthesis from gene transcription to through protein degradation of HIF-1A, NCI-H358 cells were treated with transcription inhibitor actinomycin D, translation inhibitor cycloheximide or proteasome inhibitor MG-132. The treatment of transcription inhibitor actinomycin D on NCI-H358 cells for 6 hrs did not lead to the decrease of HIF-1A protein level however, translation inhibitor cycloheximide induced drastic decrease in HIF-1A protein. Although exposure of NCI-H358 cells to MG-132 increased the expression level of HIF-1A, the combinational treatment of sotorasib and MG-132 still led to slight decrease in HIF-1A protein level. Also, MG-132 failed to recover the protein expression level decreased by cycloheximide treatment. In total, the cellular expression level of HIF-1A is possibly dependent on translation activity and KRAS inhibitor potentially affect the HIF-1A expression through the regulation of its translation. Conclusively, KRAS inhibitors could potentially be used to reprogram the hypoxic tumor microenvironments besides its primary effect against the downstream signaling of KRAS. Citation Format: Noritaka Tanaka, Takeharu Sakamoto. The potential effect of KRAS inhibitors on tumor microenvironments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C175.

Abstract C032: NUC-7738 in combination with pembrolizumab in patients with metastatic melanoma: Phase 2 results from the NuTide:701 study

Abstract Background: 3’-deoxyadenosine (3’-dA) is a natural nucleoside analogue that demonstrated potent anti-cancer activity in vitro but was not successfully developed in the clinic due to rapid breakdown and limited activation to the anti-cancer metabolite (3’-dATP). NUC-7738 is a phosphoramidate modification of 3’-dA designed to overcome these shortcomings and generate higher levels of 3’-dATP. Primarily, 3’-dATP disrupts RNA polyadenylation and can cause metabolic stress, blockade of cell division and apoptosis. Secreted forms of PD-L1, associated with resistance to PD-1 inhibitors, are partially controlled by alternative polyadenylation and were found to be reduced by NUC-7738 in vitro and in some patient (pt) samples. NuTide:701 is a two-part, Phase 1/2 clinical study designed to establish the RP2D of NUC-7738 monotherapy and to investigate NUC-7738 at the RP2D in combination with pembrolizumab. NUC-7738 monotherapy has shown a favourable safety profile and anti-tumour activity (prolonged SD and tumour reductions). Here we focus on the Phase 2 NUC-7738 + pembrolizumab part of the study. Methods: Pts with advanced/metastatic cutaneous melanoma who had progressed on 1- 2 prior lines, one of which may have contained immunotherapy, are included in the ongoing NUC-7738 + pembrolizumab cohort and receive NUC-7738 1125 mg/m2 Q1W (with option to escalate to 1350 mg/m2 beyond C2D8) and pembrolizumab 200 mg/m2 Q3W until progression. In pts with accessible disease, biopsies were taken prior to and 28 days after initiation of treatment to assess tumour levels of NUC-7738 and its metabolites and evaluate changes in the tumour microenvironment. Results: At the time of data cut-off, 4 pts with cutaneous melanoma have received NUC-7738 + pembrolizumab. To date, 1 pt experienced reversable Grade 4 transaminitis, related to pembrolizumab, and 2 pts experienced Grade 3 abdominal pain, diarrhoea and fatigue. At the time of writing, all 4 remain on treatment, 1 pt has SD and 8 additional pts are being recruited. Conclusions: NUC-7738 has a favourable toxicity profile as monotherapy and has been well-tolerated to date in combination with pembrolizumab. Available clinical and translational data for all pts treated with NUC-7738 + pembrolizumab will be presented. Clinical trial information: NCT03428958 Citation Format: Sarah P Blagden, Stefan N Symeonides, Aglaia Skolariki, Noor Md Haris, Zhuang Boh, In Hwa Um, Mustafa Elshani, Alison L Dickson, Ying Zhang, David J Harrison, Fiona G McKissock, Elisabeth Oelmann, Jeffrey D Bloss, Natalie Cook, TR Jeffry Evans, E. Ruth Plummer. NUC-7738 in combination with pembrolizumab in patients with metastatic melanoma: Phase 2 results from the NuTide:701 study [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C032.

Abstract LB_A02: Predicting breast cancer patients’ response to neoadjuvant chemotherapy from the deconvolved transcriptome of different cell types in the tumor microenvironment

Abstract Introduction: The tumor microenvironment (TME) constitutes a multifaceted ecosystem that influences tumor growth and clinical response via dynamic interactions between diverse cell types. Considerable attention has been devoted in recent years in exploring the TME’s impact on clinical response following immunotherapy, warranting further investigation into its impact in the context of chemotherapy. To address this, we present a novel computational framework called DECODEM (DEcoupling Cell-type-specific Outcomes using DEconvolution and Machine learning). Through DECODEM, we investigate the association of cell-type-specific transcriptomic profiles within the TME with response to neoadjuvant chemotherapy (NAC) in breast cancer (BC). Methods: DECODEM leverages cellular deconvolution of bulk transcriptomics data to extract cell-type-specific transcriptomics profiles for different cell types present in the TME, and further utilizes machine learning to build cell-type-specific predictors of clinical response. Using DECODEM, we analyzed the bulk transcriptomics data across three publicly available cohorts of HER2-negative BC patients who underwent NAC. We compared the predictive powers of these cell types, both individually and in concert with each other, against a state-of-the-art predictor employing clinical and transcriptomic features. We then validated our findings in a single-cell chemotherapy cohort of triple-negative breast cancer. Furthermore, we extended DECODEM to DECODEMi that can incorporate the cell-cell interactions (CCIs) among the prominent cell types to predict clinical response, hinting at the underlying mechanism at play. Results: Our analysis revealed that the diverse cell types within the TME significantly contribute to the prediction of chemotherapy response in HER2-negative BC, thus extending beyond the impact of malignant tumor cells alone. Notably, our key findings are: 1. We demonstrated the superior predictive powers of immune cells (myeloid, plasmablasts, B-cells) and stromal cells (endothelial, normal epithelial, cancer-associated fibroblasts), as compared to the bulk mixture and the state-of-the-art predictor. 2. We illustrated the complementary impacts of the prominent cell types via building highly accurate multi-cell-ensembles, where, remarkably, a three-cell-ensemble of endothelial, myeloid, and plasmablasts displayed the best predictive capability across cohorts. 3. We displayed that DECODEMi can capture the regulatory roles of CCIs and identified new CCIs likely to mediate chemotherapy response in HER2-negative BC. Conclusion: We provide an unprecedented computational approach to objectively delineate the cell-type-specific impacts of the TME in clinical response. Our analysis provides comprehensive insights into the intricate interplay of the cells in TME and their associations with chemotherapy response in HER2-negative BC, contributing to a deeper understanding of BC treatment efficacy. Citation Format: Saugato Rahman Dhruba, Sahil Sahni, Binbin Wang, Di Wu, Yael Schmidt, Eldad Shulman, Sanju Sinha, Stephen-John Sammut, Carlos Caldas, Kun Wang, Eytan Ruppin. Predicting breast cancer patients’ response to neoadjuvant chemotherapy from the deconvolved transcriptome of different cell types in the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A02.

Abstract C079: Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy

Abstract Immuno-oncology has revolutionized cancer treatment by harnessing the immune system to target and eliminate tumor cells. In this study, we employed a combination of fluorescence-activated cell sorting (FACS) and Cytek full spectrum flow cytometry to investigate the impact of anti-PD1 (programmed cell death protein 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) treatments on the 4T1 breast cancer model. We comprehensively analyzed 25 surface markers on immune cells to gain insights into the dynamic changes induced by immunotherapy. The 4T1 murine breast cancer model is known for its aggressive and metastatic nature, resembling triple-negative breast cancer in humans. We established a syngeneic orthotopic tumor graft of 4T1 cells in immunocompetent mice and treated them individually with anti-PD1 or anti-CTLA4 antibodies and compared the results to the respective isotype controls. Using FACS, we characterized immune cell populations within the tumor microenvironment and spleenocytes at multiple time points (before treatment, day 5, and day 17) during the treatment course. The Cytek full spectrum flow cytometry platform allowed simultaneous measurement of 25 surface markers, enabling a detailed analysis of immune cell phenotypes and functional states in a single experiment. Our findings revealed significant alterations in the immune landscape following anti-PD1 or anti-CTLA4 treatments. We observed an increase in tumor-infiltrating lymphocytes (TILs) and specifically CD4+ as well as CD8+ T cell infiltration within the tumor, accompanied by upregulation of T cell activation markers, such as CD69, CD38, CD44, PD1 and CD25. The immune cell composition within 4T1 tumors included CD4+ and CD8+ TILs, as well as increased populations of monocytes and macrophages. Additionally, a decrease in regulatory T cells (TRegs) was observed, indicating a potential restoration of immune balance. Moreover, F4/80+ CD206+ tumor-associated macrophages (TAMs) were identified in the 4T1 tumors vs. the spleen. This was associated with increased granzyme B production by CD8+ T cells and a shift towards an effector memory phenotype, suggesting improved cytotoxic function. To gain insights into spatial infiltration patterns of immune cells, we performed Immunohistochemistry (IHC) stainings of various immune cell markers in the 4T1 tumors. In line with the FACS results, we observed increased immune cell infiltration in tumors treated with anti-PD1 and anti-CTLA4 antibodies. Our comprehensive immunophenotypic analysis, utilizing Cytek full spectrum flow cytometry and IHC sheds light on the dynamic interplay between immune cells and the 4T1 breast cancer model during anti-PD1 and anti-CTLA4 immunotherapy. These findings provide valuable insights into the mechanisms underlying the therapeutic efficacy of immune checkpoint blockade in this aggressive cancer model. Citation Format: Christos Nikolaou, Simon Heller, Stefan Kaulfuss, Carlo Stresemann, Alexandra Eichten, Oliver von Ahsen, Martin Lange. Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C079.

Abstract B028: Phosphodiesterase 3A modulators sensitize tumor cells to Bcl-2/Bcl-xL inhibitors

Abstract Phosphodiesterase 3A (PDE3A) modulators are promising anticancer agents for tumors that coexpress PDE3A and Schlafen 12 (SLFN12), including sarcomas. The cytotoxic effect of these modulators is based on their ability to form a complex between PDE3A and SLFN12, and correlates with the expression levels of the two proteins. In cases where the two proteins are only moderately expressed, PDE3A modulator efficacy may remain poor and additional therapeutic options are needed. In this study, we investigated the combinatorial effects of a PDE3A modulator, anagrelide, with 526 other oncological compounds in two PDE3A and SLFN12 expressing liposarcoma cell lines (SA4 and GOT3). Both cell lines were responsive to PDE3A modulators, as expected. Interestingly, we found synergy with seven different drug classes that can be grouped based on their protein and protein family targets: Bcl-2l/Bcl-xL, HDAC, integrin α2 (ITGA2), mTOR, NEDD-activating enzyme E1 regulatory subunit proteins, and PAK. Additional detailed drug combination synergy studies in PDE3A modulator –sensitive liposarcoma, gastrointestinal stromal tumor, melanoma, and astrocytoma cell lines indicated a sensitizing effect of PDE3A modulators to Bcl-2/Bcl-xL inhibitors. These cell lines, excluding GOT3, showed no response to Bcl-2/Bcl-xL inhibitors otherwise. Furthermore, we discovered that the sensitization effect depends on the coexpression of PDE3A and SLFN12, and that cotreatment resulted in stronger activation of programmed cell death. Our findings suggest additional combinatorial opportunities between PDE3A modulators and inhibitors of key cancer pathways that could be explored for their therapeutic value. Citation Format: Kirsi L. J. Toivanen, Astrid Murumägi, Mariliina A. Arjama, Nanna J. Merikoski, Sami I. Salmikangas, Olli P. Kallioniemi, Tom O. Böhling, Harri J. Sihto. Phosphodiesterase 3A modulators sensitize tumor cells to Bcl-2/Bcl-xL inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B028.

Abstract B159: A first-in-human study of CDK9 inhibitor KB-0742 demonstrates evidence of tolerability and clinical activity

Abstract Background: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator that mediates expression and downstream activity of oncogenic transcription factors (TFs) including MYC and chimeric TFs. Inhibiting CDK9 presents a promising approach to treat transcriptionally addicted cancers. KB-0742 is a potent, selective, orally bioavailable inhibitor of CDK9 that is currently being studied in a phase 1/2 dose escalation in solid tumors and NHL and cohort expansion for patients with transcription-dependent tumors (NCT04718675). Methods: KB-0742 is administered orally once daily for 3 consecutive days with 4 days off, weekly, in 28-day cycles until toxicity or disease progression. Eligibility criteria include age > 18 years, relapsed or refractory solid tumors or NHL, acceptable organ function and ECOG PS < 2. Study objectives include evaluation of safety, tolerability, PK, PD, and identification of KB-0742 MTD and RP2D using a modified Continuous Reassessment Method (mCRM). PK was measured from patient plasma and PD was assessed in peripheral blood mononuclear cells (PBMCs). PD measurements included analysis of phosphorylation of the CDK9 substrate serine 2 of the RNA Polymerase II C-terminal domain (pSER2), and changes in gene expression of prospectively defined CDK9 responsive genes. Results: As of 1 June 2023, 28 patients enrolled in dose escalation up to 60 mg. Patients had received a median of 4 (2-11) prior lines of therapy and remained on KB-0742 for a median of 86 days (10-311+). Twelve of 28 patients received at least 4 cycles of KB-0742. The most common tumor types enrolled were colorectal (5), chordoma (4), sarcoma (4), and breast (3). Treatment-emergent adverse events (TEAEs) occurring in >20% of patients included nausea, vomiting, anemia, fatigue, nervous system disorders, and peripheral edema. The most common reasons for treatment discontinuation were progressive disease, TEAEs, and withdrawal of consent. Across 4 dose levels, AUC and Cmax of KB-0742 increased linearly with a terminal half-life of 24 hours. At 60 mg, evidence of target engagement was observed by pSER2 reduction and proportional changes to CDK9 responsive genes. In the escalation phase 60 mg cohort, three patients, (2 colorectal and 1 PTCL) had MYC over-expressing tumors and achieved SD. Of the two patients with myxoid liposarcoma, both exhibited radiographic regression of their target lesions. One patient achieved 26% reduction in the sum of partial diameters lasting > 5 mos. The second patient achieved a RECIST 1.1 partial response and remains on treatment at > 12 mos. Consistent with the therapeutic hypothesis, both patients had TF fusions. Conclusions: KB-0742 treatment was well tolerated with manageable toxicity and no evidence of neutropenia. CDK9 inhibition was observed at the 60 mg dose level, and expansion cohorts in tumor types with a high prevalence of MYC amplification or overexpression and other transcriptionally addicted tumors are accruing. The MTD has not been reached and further dose escalation is continuing. Citation Format: Miguel Villalona-Calero, Monica Mita, Alain Mita, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Glenn J Hanna, Gregory M Cote, Richard E Cutler, Pavan Kumar, Crystal MacKenzie, Charles Lin, Jorge F DiMartino, Elizabeth A Olek, Brian Van Tine. A first-in-human study of CDK9 inhibitor KB-0742 demonstrates evidence of tolerability and clinical activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B159.

Abstract LB_C10: An orally available small molecule inhibitor for synthetic lethal targeting of MYC expressing tumors

Abstract The potential for synthetic lethality occurs when the same oncogenic events that promote carcinogenesis, also create vulnerabilities for cancer cells. Targeting cancers with these vulnerabilities, but not resident normal tissues, is the promise of synthetic lethal therapies. The compound LC30 is a potent and orally bioavailable compound that is synthetic lethal with deregulated MYC. No off-target liabilities have been demonstrated by kinome and safety profiling and long-term treatment is well tolerated by rodents and canines. LC30 is active against a wide spectrum of cell line xenografts derived from both liquid and solid cancers, including B and T cell malignancies, lung, colon, gastric, mammary and liver cancer cell lines, with efficacy that correlates with levels of MYC. Experiments aimed at unlocking the key to LC30’s synthetic lethality have revealed that LC30 is active in both resting cells and during cell division, so it is not simply an anti-mitotic compound. In resting cells, LC30 disrupts golgi structure, a phenotype that possibly relates to MYC control of centrosomal gene expression. Anti-angiogenic effects are observed in xenografts and have been confirmed using in vitro assays of endothelial cell migration and tubule formation. We speculate that the golgi phenotype and resulting faulty trafficking of secretory vesicles containing pro-angiogenic factors may underly the vascular phenotype. In dividing cells, LC30 potently deregulates the positioning of the chromosomal passenger protein complex (CPPC), a mitotic complex comprised of a catalytic subunit, Aurora B kinase (AURKB), the scaffolding protein inner centromere protein (INCENP) as well as survivin and borealin. The CPPC is dynamically relocated during mitotic progression so that AURKB activity can be localized to specific locations where activity is required. The phenotypic effects of LC30 treatment are consistent with this compound functioning as an inhibitor of the localization, but not catalytic activity of the CPPC. This includes the induction of multipolar spindles, micronuclei and polyploidy, leading to mitotic arrest and both mitotic and post-mitotic apoptosis. This unique mode-of-action, to disrupt CPPC passage in the mitotic cell, is a key component of the MYC synthetic lethality as cells without MYC overexpression do not develop multipolar spindles, lethal polyploidy and apoptose with treatment. LC30 represents a new class of anticancer compound that is a non-kinase, synthetic lethal inhibitor that unlocks MYC-induced vulnerabilities in cancer cells. Citation Format: Qiong Shi, Ting Zhang, Julia Kalashova, Jinhua Li, Chenglu Yang, Hongmei Li, Xiaohu Zhou, Yan Long, Yidan Xia Abuliezi, Gang Lv, Duo Yu, Shenqiu Zhang, Jing Zhang, Thaddeus D Allen, Hong Liu, Dun Yang. An orally available small molecule inhibitor for synthetic lethal targeting of MYC expressing tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C10.

Abstract LB_A23: The role of SMARCA1 in Rhabdomyosarcoma and skeletal muscle differentiation

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue of pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically and histologically, is divided into two subtypes: PAX-FOXO1 fusion positive (alveolar) RMS (aRMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative (embryonal) RMS (eRMS), which is marked by mutations in RAS isoforms and some genes such as TP53, PIK3CA, CTNNB1 and FGFR4. Chromatin was one of the earliest identified targets for cancer therapy. Several chromatin remodeling proteins are associated with cancer progression processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Components of chromatin remodeling complexes, such as the imitation switch (ISWI) complex, have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 (also known as SNF2L), has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact in myogenesis is not well understood. In this study, we will characterize the function of SMARCA1 in RMS cells and skeletal muscle. We hypothesize that SMARCA1 acts to modulate chromatin accessibility, and drive RMS tumorigenic growth. Methods: We will use RNA-seq, ATAC-seq, and CUT&RUN to study the impact of SMARCA1 deletion in RMS cells. Furthermore, phenotypic experiments will be performed to determine the influence of SMARCA1 on differentiation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Furthermore, our findings show that SMARCA1 is modestly upregulated during the differentiation in normal myogenesis. Curiously, eRMS cell lines show no change in the protein level of SMARCA1 when treated with a MEK inhibitor, trametinib. Analysis of gene clusters in RMS transcriptomic data of indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells as determined by co-immunoprecipitation experiments. Conclusions: This study will deepen our understanding of how SMARCA1 impacts RMS differentiation and proliferation and may credential SMARCA1 as a novel therapeutic target in rhabdomyosarcoma. Citation Format: Ashwaq K Aljabri, Katie E Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Alex Emmons, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Peter Fitzgerald, Bao Tran, Matthew Geisler, Judith K Davie, Marielle E Yohe. The role of SMARCA1 in Rhabdomyosarcoma and skeletal muscle differentiation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A23.

Abstract B086: Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models

Abstract Background: Triple negative breast cancer subtype is associated with poor prognosis and is mostly treated with chemotherapy, including the DNA crosslinking agent Carboplatin. Carboplatin treatment leads to DNA damage and a cell cycle inhibition allowing DNA damage repair. However, many patients are resistant or develop resistance to Carboplatin. The identification of novel treatments against chemoresistant TNBC is a major unmet clinical need. Method: TNBC patient-derived cell lines (PDCs) from patient-derived xenografts (PDXs) generated from chemotherapy resistant tumors were used for shRNA high throughput screens to identify genetic vulnerabilities that could resensitize these cells to Carboplatin. Validation of this target was performed genetically by shRNA silencing and pharmacologically with commercially available target inhibitors both in vitro and in vivo, using Alamar Blue viability assays and calculating synergy using the Chou Talalay method. DNA damage was assessed using yH2AX expression, and apoptosis using c-PARP and Annexin V, by confocal microscopy, flow cytometry and/or western blotting. Results: We identified ataxia telangiectasia and Rad3-related protein (ATR) as a target in one of the cell lines screened and validated it with an ATR shRNA. We tested the combination of the ATR inhibitor (ATRi), Elimusertib, with Carboplatin and found the combination to be synergistic (Average CI=0.30). DNA damage was markedly increased with combination treatment and a pan-nuclear yH2AX staining together with increased pHH3 expression were observed, suggestive of mitotic catastrophe. Cells treated with Carboplatin accumulated in S phase and a shift toward G2/M when Elimusertib was added. Apoptosis was significantly increased with combination treatment. PKMYT1 and WEE1 are two kinases regulating cell cycle entry by inhibiting CDK1, which controls entry into mitosis. Carboplatin treatment resulted in an increase in PKMYT1 expression. PKMYT1 inhibition with RP6306 re-sensitized the cells to Carboplatin more than WEE1 inhibition with Adavosertib, and this synergy was further increased with low doses of Elimusertib. Gene silencing of WEE1 and PKMYT1 both re-sensitized the cells to Carboplatin, but only WEE1 silencing alone significantly prevent proliferation, suggesting the requirement of PKMYT1 activity for resistance but not for cell proliferation without Carboplatin. In vivo validation using the PDXs from which the cell lines have been derived, demonstrated a significant delayed in tumor growth and prolonged survival when combining Carboplatin with ATRi compared to Carboplatin alone in two models. Conclusion: ATR inhibition resensitizes drug resistant TNBC cells to Carboplatin both in vitro and in vivo. The combination of Carboplatin with ATRi led to DNA damage and mitotic catastrophe. PKMYT1 plays a role in Carboplatin-resistant TNBC, collaborating with ATR in managing the cell cycle response to DNA damage, and PKMYT1 inhibition also re-sensitizes cells to Carboplatin, an effect increased with the triple combination of adding ATR inhibition. Citation Format: Juliet Guay, Catherine Chabot, Cédric Darini, Marguerite Buchanan, Adriana Aguilar-Mahecha, Tim Kong, Connie Yang, Mark Basik, Sidong Huang. Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B086.

Abstract B088: VT3989, the first-in-class and first-in-human TEAD auto-palmitoylation inhibitor, enhances the efficacy and durability of multiple targeted therapies of the MAPK and P13K/AKT/mTOR pathways

Abstract TEAD transcription factors are the major effectors of the Hippo-YAP/TAZ-TEAD pathway involved in the regulation of cell proliferation, survival, and cell migration. TEAD auto-palmitoylation has been shown to be required for TEAD interaction with coactivator YAP/TAZ and hence activation of transcriptional activity. There are multiple reports that YAP/TEAD activation provides the essential survival signal in drug-tolerant persister/dormant cells in cancer in response to treatment with targeted therapies, leading to drug resistance and cancer relapse in BRAF-mutant, KRAS-mutant, EGFR-mutant, and ALK-rearranged non–small cell lung cancers (NSCLC). The TEAD inhibitor VT3989, currently in clinical testing, was evaluated for effect on emerging drug tolerant persister/dominant cancer cells and drug resistance. VT3989, with similar biological activity in vitro and in vivo as the potent small molecule TEAD auto-palmitoylation inhibitors reported (Tang et al, 2021, Mol Cancer Ther. 20(6):986-998), disrupts YAP/TAZ-TEAD protein interaction, suppresses TEAD transcriptional activity, and selectively blocks proliferation of NF2-deficient mesothelioma in vitro and inhibit NF2 mutant xenograft tumor growth in vivo. In in vitro assays as well as in vivo studies using cell line derived (CDX) and patient derived (PDX) xenograft models, VT3989 enhanced the efficacy and durability of clinically approved EGFR inhibitors (osimertinib, lazertinib), MET inhibitors (savolitinib, capmatinib), EGFR-MET bispecific antibody (amivantinib), KRAS-G12C inhibitors (sotorasib, adagrasib), BRAF inhibitors (encorafenib, dabrafenib), MEK inhibitors (trametinib, cobimetinib), and mTORC inhibitor (everolimus). With the addition of VT3989, combination anti-tumor effect was observed and/or tumor regrowth was delayed compared to monotherapy alone. Therefore, in addition to Hippo-YAP/TAZ-TEAD pathway driven cancers, such as mesothelioma, where VT3989 can show monotherapy efficacy, future VT3989 clinical studies will include combination therapy with approved targeted therapies to enhance the efficacy and durability of treatment. Citation Format: Tracy T Tang, Leonard Post. VT3989, the first-in-class and first-in-human TEAD auto-palmitoylation inhibitor, enhances the efficacy and durability of multiple targeted therapies of the MAPK and P13K/AKT/mTOR pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B088.

Abstract B062: Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells

Abstract Understanding chemoresistance mechanisms in BRCA-deficient cells will allow for identification of biomarkers for predicting tumor response to therapy, as well as the design of novel therapeutic approaches targeting this chemoresistance. Here, we show that the protein MED12, a component of the Mediator transcription regulation complex, plays an unexpected role in regulating chemosensitivity in BRCA-deficient cells. We found that loss of MED12 confers resistance to cisplatin and PARP inhibitors in both BRCA1- and BRCA2-deficient cells, which is associated with restoration of both homologous recombination and replication fork stability. Surprisingly, MED12-controlled chemosensitivity does not involve a function of the Mediator complex, but instead reflects a distinct role of MED12 in suppression of the TGFβ pathway. Importantly, we show that ectopic activation of the TGFβ pathway is enough to overcome the fork protection and DNA repair defects of BRCA-mutant cells, resulting in chemoresistance. Our work identifies the MED12-TGFβ module as an important regulator of genomic stability and chemosensitivity in BRCA-deficient cells. Citation Format: Lindsey M Pale, Ashna Dhoonmoon, Anastasia Hale, Kady A Dennis, Emily M Schleicher, Claudia M Nicolae, George-Lucian Moldovan. Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B062.

Abstract A063: Epigenetic regulation of Neuregulin 1 promotes breast cancer progression associated to hyperglycemia

Abstract Hyperglycemia is a risk factor for breast cancer-related morbidity and mortality. Hyperglycemia induces Neuregulin 1 (Nrg1) overexpression in breast cancer, which subsequently promotes tumor progression. However, molecular mechanisms underlying hyperglycemia-induced Nrg1 overexpression remain poorly understood. Here, we show that hyperglycemia causes active histone modifications at the Nrg1 enhancer, forming enhanceosome complexes where recombination signal binding protein for immunoglobulin kappa J region (RBPJ), E1A binding protein p300 (P300), and SET domain containing 1A (SETD1A) are recruited to upregulate Nrg1 expression. Deletions in RBPJ-binding sites causes hyperglycemia-controlled Nrg1 levels to be downregulated, resulting in decreased tumor growth in vitro and in vivo. Mice with modest-temporary hyperglycemia, induced by low-dose short-exposure streptozotocin, display accelerated tumor growth and lapatinib resistance, whereas combining lapatinib with N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S42 phenylglycine t-butyl ester (DAPT) ameliorates tumor growth under these modest hyperglycemic conditions by inhibiting NOTCH and EGFR superfamilies. NOTCH activity is correlated with NRG1 levels, and high NRG1 levels predicts poor outcomes, particularly in HER2-positive breast cancer patients. Our findings highlight the hyperglycemia-linked epigenetic modulation of NRG1 as a potential therapeutic strategy for treating breast cancer patients with diabetes. Citation Format: Changhu Lee, Min Kim, Chanho Park, Woobeen Jo, Jiyoung Park. Epigenetic regulation of Neuregulin 1 promotes breast cancer progression associated to hyperglycemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A063.

Abstract A101: Novel KRAS inhibitors suppress MAPK pathway signalling and display potent anti-proliferative activity across a broad range of KRAS mutant cell lines

Abstract KRAS is the most frequently mutated oncogene across different cancer types. Redx Pharma are developing novel, orally bioavailable, small molecule inhibitors of KRAS. These compounds display activity against a broad spectrum of the most frequently occurring KRAS mutations whilst maintaining selectivity over NRAS and HRAS protein isoforms. Isoform selective multi-KRAS inhibitors were identified using biochemical SOS1-independent nucleotide exchange assays (NEA) and validated by surface plasmon resonance (SPR) spectroscopy. Compounds of interest were the further triaged using relevant cancer cell lines for inhibition of phospho-ERK (pERK) and anti-proliferative effects both in 2 and 3-dimensional assay formats. KRAS selective inhibitors retaining activity across a spectrum of mutations were identified, these compounds exhibit picomolar affinity for KRAS, translating into low nanomolar antiproliferative activity. As further confirmation of MAPK pathway inhibition, treatment of relevant cancer cell lines with Redx compounds resulted in suppression of DUSP6 and SPTY4 gene expression. Cellular washout experiments indicated sustained inhibition of pERK post washout of compounds consistent with high affinity compounds with extended residency time at KRAS. Optimisation towards KRAS inhibitors suitable for clinical evaluation is ongoing. Citation Format: Inder Bhamra, Helen Mason, Simon Woodcock, Katie Anderson, James Kelly, Grace Haydon, Manuela La Montagna, Megan Brierley, Emma Bishop, Ross Keeler, James Ryan, Paula Jackson, Helen Aylott, Kelvin Ho, Daniel Leng, Clifford Jones, Caroline Phillips. Novel KRAS inhibitors suppress MAPK pathway signalling and display potent anti-proliferative activity across a broad range of KRAS mutant cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A101.

Abstract A019: eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor

Abstract Despite recent advances in immunotherapy and adjuvant-therapy, resistance to cancer therapeutics is common and requires novel approaches. Moreover, highly aggressive triple-negative breast cancers lack effective targeted treatments. Deregulation of translation factors is associated with the increased proliferation of TNBC cells, heightened CSC activity, and poor patient outcomes. Thus targeting translation regulatory factors upregulated in TNBC is a promising approach to developing novel treatments. Pateamine A (PatA), a natural marine macrodiolide product, inhibits RNA cap-dependent translation by binding and sequestering the eukaryotic translation initiation factor eIF4A. eIF4A promotes the translation of polypurine-rich mRNA sequences, which are enriched within breast cancer oncogenes. Here we report the anticancer efficacy of novel PatA analogs we have synthesized against breast cancer cell lines. The PatA analogs exhibited selective cytotoxicity to breast and colorectal cancers compared to non-cancerous cells measured by Cell Titer-Blue viability studies. Novel analogs show greater antiproliferative potency against models of triple-negative than against human mammary epithelial cells (HMLE). To further elucidate eIF4A as a molecular target, we conducted analyses of patient tissue samples in the Cancer Genome Atlas (TCGA). Our analyses reveal the overexpression of eIF4A in tumor tissues compared to normal tissues and gene correlation studies reveal the upregulation of key co-expressed tumor drivers including beta-catenin, fibronectin, and epithelial-to-mesenchymal transition (EMT) markers such as SNAIL and SLUG. Further analyses show a downregulation of tumor suppressor protein PDCD4, an endogenous inhibitor of eIF4A, in cancer tissues. We have generated eIF4A shRNA knockdown and PDCD4 shRNA knockdown cell lines to uncover the eIF4A-PDCD4 regulatory axis across breast adenocarcinoma and colorectal adenocarcinoma cell types. We show that the loss of eIF4A attenuates the mesenchymal and stem-like phenotype of breast cancer stem cells revealed by phenotypic assays. Our results suggest that targeting translation initiator eIF4A is an effective approach against highly proliferative and metastatic breast cancer types. The presented findings underscore the potential of eIF4A as a promising molecular target and supports translational control as a viable approach against aggressive cancers. Citation Format: Alagu Subramanian, Haoran Xue, Kenneth G Hull, Daniel Romo, Joseph Taube. eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A019.

Abstract A122: Deciphering macrophage targeting cancer immunotherapies using a novel in vitro assay modelling the tumor microenvironment

Abstract Background: Macrophages are one of the major infiltrating immune cells in the tumor microenvironment and have been proposed as a primary driver of epithelial to mesenchymal transition (EMT) through interactions with tumour cells. These interactions play a key role in tumor progression, metastasis and fibrosis, processes which are common to all types of cancer. Understandably therefore, targeting macrophages is an attractive approach for treating wide range of tumors including those resistant to checkpoint inhibitors. Evaluation of cancer therapeutics is hampered by a lack of suitable in vitro models which closely recapitulate the complexity of tumor microenvironment. We addressed this challenge by developing a model system with potential to faithfully represent the key molecular interactions occurring between tumor associated macrophages and cancer cells, driving the latter to become invasive and metastatic. In order to demonstrate suitability of the assay, galunisertib, a TGFbRI/ALK5 inhibitor was used to block one of pathways that has been evaluated clinically in cancer patients. Methods: Human macrophages were differentiated using key growth factors prior to polarisation with various cytokines to generate different putative macrophage phenotypes as evidenced by differences in cytokine secretion, gene transcription and protein expression. Epithelial cells were stimulated with macrophage conditioned medium in a scratch wound assay (Incucyte) and analysed by immunocytochemistry. Results: Macrophages derived supernatants were shown to drive EMT, both in migratory capacity and EMT associated protein expression. Our results demonstrate that whilst galunisertib could potently inhibit TGF-b driven EMT it was only partly capable of reversing the effects associated with addition of macrophage conditioned media. Conclusions: The data show that macrophages can directly drive the EMT process in vitro via multiple mechanisms. One such mechanism is through the secretion of TGF-b. Indeed, blockade of TGF-b signalling appears to be a tangible approach in cancer immunotherapy, but to date this approach has delivered mixed results for clinical efficacy as a monotherapy. By way of example, development of galunisertib has recently been halted. As shown herein, macrophages effects on cancer migration and metastasis span beyond TGF-b only effects. We offer a potential explanation for why single therapies such as galunisertib alone are ineffective in cancer treatment. The described model provides a powerful tool to researchers to better understand and replicate in vitro the complex interactions occurring in the tumor microenvironment. Thus, enabling the identification of molecular targets and combination therapies with potential to block cancer cell migration and metastasis and to evaluate candidate drug efficacy. Citation Format: Darryl Turner, Ross Dobie, Justyna Rzepecka, Hayley Gooding, Matilda Bingham. Deciphering macrophage targeting cancer immunotherapies using a novel in vitro assay modelling the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A122.

Abstract B014: Characterization of Nectin-4 protein expression in non-small cell lung cancer patients

Abstract Nectin-4, a cell adhesion molecule from the Nectin-like family, is a validated tumor target overexpressed on the surface of cells in several cancers. Using Bicycles, a novel class of fully synthetic, short, constrained peptides, we are developing Bicycle Toxin Conjugates® (BTCs) and Bicycle Tumor-Targeted Immune Cell Agonists (Bicycle TICAs) that target Nectin-4 expressing tumors. BT8009 is a BTC™ which consists of a bicyclic peptide targeting Nectin-4 linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker. BT7480 is a Bicycle TICA™ that binds Nectin-4 on tumor cells and CD137 on immune cells to induce CD137 mediated immune agonism. Both BT8009 and BT7480 are currently under investigation in clinical trials, BT8009-100 (NCT04561362) and BT7480-100 (NCT05163041), respectively. To identify tumors expressing Nectin-4, we previously developed a proprietary Nectin-4 monoclonal antibody to measure its protein expression in an immunohistochemistry (IHC) assay. In collaboration with The Christie Hospital & The Manchester Cancer Research Centre Biobank (MCRC), we performed Nectin-4 IHC on tissue microarrays (TMAs) consisting of tumor biopsies from 59 non-small cell lung cancer (NSCLC) patients, including 4 longitudinal pairs, and 10 synchronous primaries with an average of 4 cores per tissue. A pathologist manually scored the TMAs for Nectin-4, evaluating both H-score and tumor proportion score (TPS) independently in the tumor cell membrane and tumor cytoplasm. Given the high degree of heterogeneity observed across multiple TMA cores from a single sample, Nectin-4 positivity was defined as a tumor proportion score (TPS) > 1 in either the tumor membrane or tumor cytoplasm, in at least 1 core. In samples collected around diagnosis, 69.5% of patients were positive for Nectin-4. In the 4 longitudinal pairs, all samples were Nectin-4 positive at the later timepoint. No statistically significant differences were observed in Nectin-4 positivity when analyzed by grade (grade 1 66.6% (n=6), grade 2 88.9% (n=9), grade 3 69.7% (n=33)), stage (stage I 75% (n=24), stage II 75% (n=16), stage III 64.3% (n=14), stage IV 40% (n=5)) or morphology (adenocarcinoma 65.2% (n=23), squamous cell carcinoma 77.4% (n=31)) when compared using Fisher’s exact test. A similarly high percent of Nectin-4 positivity was observed when comparing samples across demographic and mutational data. The high percent of Nectin-4 positivity observed in NSCLC supports the inclusion of this indication in Bicycle's BT8009-100 and BT7480-100 studies. Citation Format: Sean M Santos, Emma Darlington, Laura Woodhouse, Niamh Peters, Erin Peat, Assunta De Rienzo, Rob Smale, Shawn Watson, Stephen Blakemore, Louise Carter, Carly Campbell. Characterization of Nectin-4 protein expression in non-small cell lung cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B014.

Abstract C127: Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas

Abstract MALT1 is a key regulator of antigen receptor signaling in B and T cells. MALT1 partners with BCL10 and CARMA1 to form the CBM complex in which protease activity of MALT1 cleaves the negative regulators (A20, CYLD, RelB) leading to activation of canonical NF-kB pathway. Constitutive activation of NF-kB signaling is a key driver in B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). Therapeutic targeting of MALT1 protease activity can therefore be a useful approach for the treatment of B-cell lymphomas with dysregulated NF-kB signaling. We sought to discover and develop MALT1 inhibitors with the “best-in-class” profile with an emphasis on lowering plasma protein binding (PPB) to achieve improved pharmacological outcome. After extensive SAR efforts utilizing various computational methods, AUR-112 has been identified as a potential development candidate. The allosteric inhibitor AUR-112 exhibited potent and selective inhibition of MALT1 protease activity in both biochemical and cellular assays. Inhibition of MALT1 correlated with potent antiproliferative activity in an ABC-DLBCL cell line. The mechanistic activity of AUR-112 is supported by the inhibition of IL-10 in a cell line and IL-2 in human whole blood. Consistent with our objective, AUR112 showed low plasma protein binding across multiple species, which translated to greater potency in whole blood compared to published MALT1 inhibitors, positioning it as a best-in-class contender. AUR-112 has excellent oral bioavailability across species and exhibited desirable selectivity profile in kinase, protease and CEREP safety screen panels. Oral administration of AUR-112 led to dose dependent anti-tumor activity including durable tumor regression in cell line and patient derived xenograft models. Treatment with AUR-112 led to potent pharmacodynamic modulation in xenograft models as measured by significant and sustained reduction in serum IL-10 levels that correlated with the compelling efficacy. AUR-112 also demonstrated dose dependent inhibition of T-reg cells in vitro and in vivo supporting the immune modulatory role of MALT1 inhibition. AUR-112 is identified as a development candidate with potentially best-in-class physico-chemical properties resulting in a high free fraction for treatment of ABC-DLBCL patients. IND enabling studies are currently ongoing to support the clinical development of AUR-112. Citation Format: Wesley Roy Balasubramanian, Sammeta Srinivasa Raju, Vasantha K.Y, Archana Bhumi Reddy, Janith Mary Maben, Sandeep Sadashiv Patil, Raghavendra N.R, Kiran Aithal B, Charamanna K.B, Dinesh Chikkanna, Kavitha Nellore, Samiulla D.S, Girish Daginakatte, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C127.

Abstract A090: Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations

Abstract Alterations in the RAS-MAPK pathway, such as mutations in NF1, RAS, and BRAF, often lead to oncogenic signaling and result in aberrant cell proliferation and tumor growth. Oncogenic BRAF mutations may be activated as either monomers or dimers, while oncogenic RAS mutations or loss-of-function mutations in NF1, a negative regulator of RAS, drive activation of RAF dimers to transduce MAPK signaling. Currently approved BRAF inhibitors are selective against monomeric BRAF mutations and largely inactive against dimeric RAF mutants. Additionally, these agents may also promote deleterious paradoxical activation of RAF in select contexts. Moreover, FDA-approved KRAS G12C mutant-selective inhibitors are ineffective for cancer patients who harbor other (non-G12C) KRAS, NRAS, and NF1 mutations which all lead to active RAF dimer. There remains a high unmet clinical need for a highly CNS penetrant RAF inhibitor that targets a broad spectrum of oncogenic RAF conformations in the context of RAF, RAS, and other upstream driver mutations. BDTX-4933 is a next generation MasterKey RAF inhibitor designed to address the shortcomings of previous generation RAF inhibitors. BDTX-4933 is an orthosteric inhibitor that potently targets the oncogenic RAF conformation shared by groups of RAF driver mutations and induces an inactive conformation of RAF. As a MasterKey inhibitor, BDTX-4933 potently inhibits a broad spectrum of BRAF mutations and active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as KRAS, NRAS, and NF1 mutations. In vivo, once daily oral dosing of BDTX-4933 resulted in robust and sustained target engagement, inhibiting ERK phosphorylation without inducing paradoxical activation. In a mouse breadth of efficacy study with patient derived xenografts (PDX), BDTX-4933 demonstrated tumor growth inhibition and regression as a single agent across models expressing a variety of targeted BRAF or MAPK pathway oncogenic mutations. Additionally, BDTX-4933 as a single agent and in combination with a MEK inhibitor, demonstrates anti-tumor efficacy in KRAS mutant lung PDX models. In a PDX model of BRAF V600E positive tumor that progressed following BRAF and MEK combination therapy, BDTX-4933 achieved robust tumor growth regression. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class RAF inhibitor profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. BDTX-4933 is currently in a Phase I clinical study in patients with solid tumor harboring sensitive BRAF and RAS alterations (NCT05786924). For more information on this active Phase 1, please contact: ClinicalTrials@bdtx.com. Citation Format: Yoon-Chi Han, Pui Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Darlene Romashko, Tai-An Lin, Elizabeth Buck. Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A090.