Dose-dependent Tumor Growth Inhibition Research Articles

Abstract PO5-05-02: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of cyclin D-CDK4/6 signaling node. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate due to its potency in vitro, in CDK4/6i resistant models and its effectiveness at inhibiting tumor growth in vivo. Degradation of CDK4/6 by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with BTX-9341 for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC50s of < 1nM. Similar degradation depth and DC50 values were seen in HR+ breast cancer cells including T47D, MCF7 and ZR-751. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. To determine the effect of CDK4/6 degradation on downstream signaling, we examined RB phosphorylation by in-cell western. Twenty-four hours treatment with BTX-9341 blocked RB phosphorylation robustly, with phospho-RB IC50s at < 30nM in HR+ breast cancer cells as well as MDA-MB-231 cells. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with BTX-9341 caused G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) as a readout for inhibition of proliferation by cell cycle arrest. BTX-9341 showed potent inhibition of cell proliferation with CFA IC50s of < 50nM in TNBC cell lines and < 20nM in HR+ cell lines. We demonstrated that BTX-9341 was significantly more potent in vitro than the CDK4/6i, which had CFA values between 50 and 700nM in HR+ cell lines and between 100 and 1000nM in TNBC. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell lines models which exhibit high CDK6 expression, BTX-9341 maintained a low CFA IC50 (< 150nM) while other CDK4/6 inhibitors displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties and oral bioavailability, which allowed for oral dosing in xenograft studies. MCF7 xenograft results with BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher doses and overall greater potency compared to the clinical CDK4/6i. BTX-9341 shows good exposure in the brain and exhibits tumor growth inhibition in an intracranial MCF-7 xenograft model, indicating that BTX-9341 could inhibit the growth of brain metastases. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-02.

Abstract 2601: Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker

Abstract Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2. Citation Format: Yulia Liubomirski, Galia Tiram, Anna Scomparin, Samer Gnaim, Sayantan Das, Sachin Gholap, Liang Ge, Eilam Yeini, Omri Shelef, Arie Zauberman, Nir Berger, Doron Kalimi, Mira Toister-Achituv, Christian Schröter, Stephan Dickgiesser, Jason Tonillo, Min Shan, Carl Deutsch, Stanley Sweeney-Lasch, Doron Shabat, Ronit Satchi-Fainaro. Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2601.

Abstract 6588: Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile

Abstract In response to cellular stress, the tumor suppressor p53 is activated to modulate cell cycle progression, DNA repair, and apoptosis. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 prevents its degradation and can reactivate p53 to elicit an anti-cancer effect. Targeting the p53-MDM2 interaction therefore remains a promising strategy for cancer therapy. However, development of first generation MDM2 antagonists has been challenged by dose-limiting, on-target bone marrow toxicities. Understanding of differential effects of p53 pathway activation in normal hematopoietic versus cancer cells (to be presented in a separate abstract) together with our expertise in structure-based drug design have led to the discovery of ASTX295, a potent MDM2 antagonist with differentiated pharmacokinetic profile aimed at sparing bone marrow toxicities and increasing the therapeutic index. Here, we present the first disclosure of the structure and pre-clinical characterisation of ASTX295. ASTX295 exhibits potent activity (IC50<1 nM) against MDM2 in an ELISA-based in vitro assay and induces significant growth reduction in p53 wild-type, MDM2-amplified SJSA-1 cells (GI50=27 nM). Antiproliferative activity of ASTX295 was further demonstrated in a panel of 219 p53 wild-type cell lines, with 143 cell lines showing GI50 values less than 1 μM and 50 showing values less than 0.1 μM. Effects of ASTX295 are shown in cell lines carrying functional p53 as confirmed in three p53 wild-type and mutant cell line pairs (SJSA1 and SN40R2, A2780 and A2780CP, HCT116 and HCT116 p53−/−). In addition to inhibiting cell cycle progression and cell proliferation, ASTX295 also potently induces apoptosis following 24-48 hour treatment. Further in vitro analyses of ASTX295 demonstrated an increase in the levels of p53 (EC50=10 nM after 2 hours) and its transcriptional targets such as p21 and MDM2. In vivo, ASTX295 shows robust induction of p53 and its target genes at 3 and 6 hours after oral administration together with dose-dependent inhibition of tumour growth in the SJSA-1 xenograft model. Importantly, ASTX295 exhibits optimised pharmacokinetic and pharmacodynamic profiles with relatively short duration of pathway modulation and a desired predicted human half-life of 2-8 hours. Based on our pre-clinical hypothesis on differential time-dependent sensitivities of normal versus cancer cells to p53 activation, achieving such a profile while maintaining potency may increase the therapeutic index. These data highlight the therapeutic potential of ASTX295, which is currently being tested in a Phase 1/2 clinical trial in advanced solid tumors with wild-type p53 (NCT03975387). We plan to present preliminary clinical data in a separate abstract at this meeting. Citation Format: Maria Ahn, Luke Bevan, Ildiko Buck, Celine Cano, Juan Castro, Ben Cons, Jane Endicott, Lynsey Fazal, Nicola Ferrari, Ian Hardcastle, Keisha Hearn, Rhian Holvey, Steven Howard, Chris Johnson, Claire Jennings, Justyna Kucia-Tran, Suzanne Kyle, John Lunec, John Lyons, Duncan Miller, David Rees, Martin Noble, David R. Newell, Judith Reeks, Harpreet Saini, Jeffrey St. Denis, Emiliano Tamanini, Huw Thomas, Neil Thompson, Mladen Vinkovic, George Ward, Nicola Wallis, Hugh Walton, Stephen Wedge, Pamela Williams, Elaine Willmore, Nicola Wilshire, Yan Zhao, Gianni Chessari. Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6588.

Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition

Abstract CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors. Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5705.

Abstract 7264: OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models

Abstract The Hippo pathway regulates critical cellular processes during development. In ~10% of human cancers, Hippo pathway dysregulation results in YAP/TAZ overactivation and increased TEAD-dependent transcription that promotes tumor growth and therapy resistance - often by supporting drug-tolerant persister survival. In 40% of malignant mesotheliomas (MM), neurofibromatosis 2 (NF2) gene mutations inactivate the Merlin protein, an upstream negative regulator of TEAD. Preclinical studies indicate high TEAD dependency in NF2-mutant MM. We have discovered an array of potent covalent and non-covalent small molecule inhibitors that occupy the palmitate-binding pocket of TEAD proteins, with varying paralog selectivity profiles. OPN-9840 is an oral non-covalent pan-TEAD inhibitor displaying dose-dependent and on-target in vitro and in vivo efficacy for NCI-H226, an NF2-mutant MM cell line. OPN-9840 showed equivalent binding in thermal shift assays (10°C delta Tm) for depalmitoylated TEAD1-4 proteins, and blocked TEAD reporter activity (IC50 100nM) and NCI-H226 cell growth (IC50 400nM). In an NCI-H226 xenograft model, OPN-9840 dosed orally for 14 days exhibited dose-dependent tumor growth inhibition (TGI) from 5 to 50 mg/kg PO (88 to >100% TGI) with tumor regression in the 15 (2/8 mice) and 50 mg/kg (4/8 mice) dose groups. qPCR of OPN-9840-treated tumors confirmed 50-70% downregulation of canonical TEAD targets, CTGF and CYR61. OPN-9840 shows a favorable safety profile, including a lack of cytotoxicity (IC50>50µM) in HEK293T and HepG2 cells. Additionally, OPN-9840 caused no body weight loss in the NCI-H226 xenograft model following 14 days of dosing. In vitro evaluation of OPN-9840 in the blood-brain barrier (BBB) specific parallel artificial membrane permeability assay exhibited high BBB penetration potential (-Log Pe = 4.7). High oral exposure was achieved at all dose levels (AUC0-24 > 100,000 h•ng/mL) after 14 days in the NCI-H226 study. We next investigated synergy between TEAD inhibition and targeted therapy in MM. Combined treatment of one of our pan-TEAD covalent compounds, OPN-9652 (from a separate chemical series to OPN-9840), at 50 mg/kg with 1 mg/kg trametinib resulted in NCI-H226 xenograft tumor regression (130% TGI) whereas trametinib alone only led to decreased tumor growth (74% TGI). RNA-Seq of treated tumors revealed that OPN-9652 and trametinib synergistically inhibited TEAD downstream target genes, including ANKRD1 and CTGF. OPN-9652 as a single agent caused significant downregulation of E2F targets, such as MYC and TP53, suggesting decreased proliferation. In summary, OPN-9840 is a potent pan-TEAD inhibitor that shows monotherapy efficacy in MM. As we observed synergy between another pan-TEAD inhibitor, OPN-9652, with trametinib, we are exploring synergy of OPN-9840 with additional targeted therapies in tumor types beyond MM. Citation Format: Pan-Yu Chen, Bernice Matusow, James Tsai, Peipei Li, Hoa Nguyen, Gaston Habets, Gideon Bollag, Somenath Chowdhury. OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7264.

Abstract 5702: BTX-9341, a bifunctional degrader of CDK4 and CDK6 for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with limited treatment options. GBM has a high frequency of dysregulation of the CDKN2A-cyclin D-CDK4/CDK6 signaling node and should subsequently be sensitive to CDK4/6 inhibition. Despite this, the CDK4/6 inhibitors that are approved for HR+/HER2- breast cancer have shown limited efficacy in GBM due to poor blood brain barrier (BBB) penetration. BTX-9341 is a Cereblon (CRBN) mediated CDK4/6 bifunctional degrader that we have developed for HR+/HER2- breast cancer. This degrader shows good exposure in brain tissues with a high brain to plasma ratio. Given the exposure in the brain, we explored the in vitro and in vivo efficacy of BTX-9341 in GBM cell line and xenograft models. GBM cell lines treated with BTX-9341 showed up to 86% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. We examined RB phosphorylation by immunoblot and cell cycle progression by propidium iodide staining followed by flow cytometry. BTX-9341 was potent in all downstream assays, with phospho-RB IC50s <10nM, and G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation by cell cycle arrest. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 13-100nM in GBM cell lines. BTX-9341 displays excellent pharmacokinetic properties with good oral bioavailability and high brain to plasma ratios which allowed for oral dosing in both subcutaneous and orthotopic xenograft studies. We examined tumor growth inhibition efficacy in two GBM xenograft models: an intracranial U87 model and a subcutaneous U118 model and saw robust tumor growth inhibition in both models with BTX-9341 treatment. The U118 cells have partially unmethylated MGMT, which lead to a loss of efficacy of temozolomide over time, while BTX-9341 maintained inhibition of tumor growth. This indicates that BTX-9341 may be effective in patient populations for which temozolomide is ineffective. The U87 intracranial xenograft model also showed dose-dependent tumor growth inhibition upon treatment with BTX-9341, indicating that BTX-9341 can cross the BBB and reach concentrations high enough to execute cell proliferation inhibition effects. BTX-9341 treated mice also had much better survival than abemaciclib treated mice, the only CDK4/6i which has BBB penetration. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo in GBM models. The xenograft data indicates that BTX-9341 can inhibit growth of MGMT methylated and unmethylated GBM cell lines, and that BTX-9341 can inhibit tumor growth in the brain more effectively than BBB penetrant CDK4/6i. Together this data shows that BTX-9341 may be a promising candidate for treating GBM. Citation Format: Hannah Majeski, Kirti Chahal, Akinori Okano, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. BTX-9341, a bifunctional degrader of CDK4 and CDK6 for glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5702.

Abstract 4540: SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo

Abstract Cancer therapies such as ionizing radiation or topoisomerase II inhibitors (for example, doxorubicin) induce DNA double-strand breaks (DSBs), which can subsequently be repaired by homologous recombination or non-homologous end joining (NHEJ). DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine protein kinase complex, is a pivotal component of the NHEJ process that maintain genome integrity. The critical role of DNA-PK in DNA damage response (DDR) and the dysregulated DNA-PK expression in cancer make it an intriguing therapeutic target for cancer, especially when combined with DSBs agents. Here, we identified a highly potent and selective DNA-PK inhibitor, SY-7021, with an IC50 value of 0.242 nM on DNA-PK kinase and high selectivity on ATM and ATR (greater than 400-fold) in biochemical assays. In a reporter assay for NHEJ repair, SY-7021 was able to dose-dependently reduce cellular NHEJ efficiency. Functionally, SY-7021 inhibited cell proliferation in multiple cancer cell lines alone or combined with doxorubicin. Basically, combination of SY-7021 and doxorubicin induced significant G2/M phase arrest and cell apoptosis, as well as enhanced the phosphorylation on Ser139 of γH2AX, Tyrosine 68 of CHK2 and Serine15 of p53 in MDA-MB-468 cells. In a subcutaneous NCI-H1703 xenograft tumor model, SY-7021 administered orally twice daily achieved dose-dependent tumor growth inhibition in vivo, with a tumor growth inhibition (TGI) of 105.6% at 60 mg/kg and no significant weight loss was observed. In addition, SY-7021 demonstrates good PK properties and acceptable safety profiles in in vivo studies. Collectively, SY-7021, a potent and selective DNA-PK inhibitor, shows significant inhibition on tumor growth in in vitro and in vivo studies, providing a rationale treatment for multiple tumors in monotherapy or in combination with other agents. Citation Format: Kai Zhang, Zhihua Liu, Yuhao Gao, Chang Lu, Shikang Cheng, Xijie Liu, Hong Luo, Yinghui Sun. SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4540.

Abstract 2461: Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy

Abstract Boosting anti-tumor immune responses can be achieved by activating the innate immune system. Despite the ongoing development of numerous STING and TLR agonists for this purpose, none of them have progressed to late-stage clinical evaluation. Alpha protein kinase-1 (ALPK1) is an intracellular innate immune receptor newly identified for its ability to detect ADP-heptose, a metabolite released during the biosynthesis and degradation of lipopolysaccharides (LPS). Since heptose is not naturally present in mammalian cells, the recognition of heptose metabolites by ALPK1 serves as a reliable mechanism to identify bacterial infections. When ADP-heptose binds to ALPK1, it initiates the phosphorylation of TIFA, thereby triggering a cascade of immune responses. Systemic administration with ADP-heptose induces robust ALPK1-dependent cytokine responses in mice. While high doses of ADP-heptose demonstrate anti-tumor effects, its instability and low in vivo potency hinder its clinical development. Through a SBDD medicinal chemistry effort, a novel, stable, and potent ADP-heptose analog, designated PTT-936, has been discovered and is progressing into clinical evaluation in cancer patients. PTT-936 exhibits remarkable nanomolar potencies for ALPK1 activation in various cellular assays. Notably, in human PBMCs, PTT-936 induces both concentration-dependent release of cytokines and activation of NK and T cells. Consistent with in vitro studies, oral administration of PTT-936 triggered significant cytokine responses in mice at microgram dose levels. However, overt toxicities were observed only at milligram dose levels, indicating a wide therapeutic window for this compound. In syngeneic tumor models, oral administration of PTT-936 induced dose-dependent tumor growth inhibition, which correlated with elevations in several cytokine biomarkers. Mechanistic studies show that PTT-936, working exclusively through the ALPK1 pathway, elicited strong innate and adaptive immune responses, as evidenced by robust cytokine induction and immune cell activation and recruitment. In syngeneic mouse models, the anti-tumor effects of PTT-936 were significantly hindered by either CD8+ T cell depletion or neutralization of select cytokines. Besides monotherapy studies, compelling evidence demonstrates strong synergistic anti-tumor effects when combining PTT-936 with immune checkpoint inhibitors. Intriguingly, PTT-936 exhibits favorable tolerability and a wider therapeutic index compared to certain clinical-stage STING and TLR agonists. Furthermore, PTT-936 does not enhance cytokine elevations induced by checkpoint inhibitors, which have been linked to adverse events (including CRS) observed in clinical combination studies. The dosing frequency of PTT-936 in the upcoming first-in-human clinical trial is carefully designed to prioritize both tolerability and patient compliance. Citation Format: Yupeng Wang, Nana Du, Xiaona Dong, Xuebing Sun, Yuan Hong, Xiaolong Yang, Zhijiang Ji, Qianyu Wang, Yun Lv, Zhichen Shi, Yajuan Gao, Xiaofu Hou, Yaxin Zhang, Juan Wang, Rui Mao, Yinxin Liu, Liang Li, Jingjin Ding, Hexiang Wang, Nan Hu, Counde O’Yang, Zhihong Li, Keith Bley, Feng Shao. Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2461.

Abstract 6068: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of the cyclin D-CDK4/6 signaling node. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate. Breast cancer cell lines treated with BTX-9341 showed up to 85% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. We examined RB phosphorylation by in-cell western, E2F target gene expression by qPCR and cell cycle progression by propidium iodide staining followed by flow cytometry. BTX-9341 was potent in all downstream assays, with phospho-RB IC50s <30nM, E2F target gene downregulation and G0/G1 cell cycle arrest at concentrations as low as 10nM. These downstream effects were sustained throughout 72 hours with BTX-9341 treatment but recovered more rapidly with Palbociclib treatment. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation by cell cycle arrest. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 20-50nM while CDK4/6i had CFA IC50s of 50-1000nM. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell line models BTX-9341 maintained a low CFA IC50 (<150nM) while CDK4/6i displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties which allowed for oral dosing in xenograft studies. In several breast cancer xenograft models, BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher dose levels, and was effective with multiple alternate dosing regimens. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6068.

Abstract 3124: MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most challenging cancers with high unmet medical need. Glypican-3 (GPC3) is an oncofetal glycoprotein anchored to the cell surface via GPI (glycosylphosphatidylinositol) and frequently over-expressed in HCC with limited expression in normal adult tissues, which makes it an appealing antibody-drug conjugate (ADC) target for HCC. MRG006A is a potentially first-in-class GPC3-targeting ADC composed of a novel humanized IgG1 antibody conjugated to a proprietary linker-payload platform utilizing a potent topoisomerase 1 inhibitor via a peptide-based cleavable linker. Materials and Methods: As a GPC3-targeting ADC intended for clinical therapy of GPC3-expressing cancers, the mechanism of action and therapeutic potential of MRG006A were investigated in a series of preclinical studies, including (1) MRG006A and its antibody moiety binding to human GPC3 expressed on cells assessed by flow cytometry; (2) binding affinity to human and cynomolgus monkey GPC3 measured by SPR (surface plasmon resonance); (3) internalization in Huh7 cells assessed by pHAb Reactive Dye and confocal microscopy; (4) in vitro cytotoxicity against a panel of HCC cell lines (Huh7, HepG2, Hep3B and GPC3-negative SK-HEP-1); (5) anti-tumor activity of MRG006A evaluated in Huh7 and HepG2 cell line-derived xenograft (CDX) as well as a panel of HCC patient-derived xenograft (PDX) mouse models with a different expression level of GPC3; (6) plasma stability of MRG006A evaluated in human, cynomolgus monkey, rat and mouse plasma; and (7) an exploratory pharmacokinetic (PK) and toxicology study of MRG006A in cynomolgus monkey. Results: The GPC3-targeting ADC, MRG006A, and its antibody moiety exhibited superior binding activity to GPC3-expressing cancer cell lines than a clinical antibody benchmark and similar nanomolar binding affinity to both human and cynomolgus monkey GPC3, which makes cynomolgus monkey suitable for toxicological studies. Rapid internalization of MRG006A antibody was observed in GPC3-expressing Huh7 cells, and MRG006A maintained comparable internalization capability to the naked antibody. More importantly, MRG006A exhibited potent GPC3-dependent cytotoxic activity in a panel of HCC cells expressing varying levels of GPC3. Administrations of MRG006A resulted in a robust and dose-dependent tumor growth inhibition of multiple CDX models and HCC PDX models. Remarkable stability of MRG006A was shown in the plasma of various species tested. MRG006A demonstrated a favorable PK and safety profile with good tolerability in the exploratory toxicology study. Conclusion: Overall, the preclinical study results suggest that MRG006A is a feasible ADC drug candidate for treating GPC3-expressing cancers in clinical studies. MRG006A was aimed to submit IND in China and the U.S in 2024. Citation Format: Yanchun Wang, Hongfeng Li, Hao Shen, Wenchao Liu, Shoujia Liu, Kequan Yin, Haili Xu, Xueyuan Cui, Wei Li, Wei Liu, Xiangyu Wu, Liu Yang, Tian Ma, Zhongrun Zhao, Jun Wang, Feifei Cui, Lei Fang, Minmin Qin, Chaohong Hu. MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3124.

Abstract 3337: Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors

Abstract KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX-21020, inhibits KIF18A ATPase activity with an IC50 of 14.5 nM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 > 10 µM) and EG5 (IC50 5.87 µM). ATX-21020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 53.3 nM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX-21020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX-21020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX-21020 treatment, and Annexin V, phospho-Histone H3, and gamma-H2AX are not induced. In sensitive cell lines, ATX-21020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX-21020 exhibit fragmented nuclei and malformed mitotic spindles, in contrast to A2780 cells where cell division is unaffected, suggesting that KIF18A is dispensable in CIN negative cells. This selective dependency is also observed in vivo, where administration of ATX-21020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX-21020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3337.

Abstract 7146: HSK39775: A USP1 inhibitor for the treatment of cancers with homologous recombination deficiencies

Abstract Tumors with BRCA1/2 mutations or homologous repair deficiency (HRD) are vulnerable to agents that target DNA repair pathways, including platinum-containing chemotherapeutics and poly (ADP-ribose) polymerase-1 (PARP1) inhibitors. Despite the significant therapeutic benefits in patients with BRCAm or HRD, innate and acquired resistance to PARPi is still a challenge in the clinic. To address this clinical challenge, we developed HSK39775, a potent, highly selective inhibitor of ubiquitin carboxyl-terminal hydrolase 1 (USP1), which acts as an oncogenic and PARP inhibitor resistance driver involved in DNA damage repair pathway through the deubiquitylation of proteins in Translesion Synthesis pathway and Fanconi Anemia pathway, paired with BRCA as a synthetic lethality target. HSK39775 was active in enzymatic level to inhibit USP1/UAF1 complex. Enzymatic panel screening containing 50 deubiquitinase demonstrated high selectivity of HSK39775. Ubiquitinated PCNA was accumulated treated by HSK39775, indicating target engagement. HSK39775 also demonstrated potent anti-proliferation activity against cancer cell lines with BRCA mutation and excellent selectivity between BRCA WT vs. BRCAm. Combining HSK39775 with PARP inhibitors indicated synergy in cell lines with partial or insensitivity to each agent alone. Triple-negative BRCA mutant breast cancer xenograft model demonstrated strong dose-dependent tumor growth inhibition of HSK39775 as a single agent with Tumor Growth Inhibition (TGI) of 63.2% at 30 mg/kg QD, and ubiquitinated PCNA was also accumulated in HSK39775 treated tumor tissue. Meanwhile, combination with PARP inhibitors significantly enhanced the therapeutic efficacy of HSK39775 in xenograft model and led to tumor regressions and durable tumor control even at low dose (TGI:89% (HSK39775, 5 mg/kg + Olaparib, 50 mg/kg)). Interestingly, HSK39775 inhibited the tumor growth of a HRP and BRCA wild-type lung cancer xenograft as a single agent with TGI of 94% at 30 mg/kg QD, suggesting the therapeutic effect of USP1 inhibitors beyond canonical BRCA mutant or HRD tumors. In summary, these results indicate HSK39775 as a potent and selective USP1 inhibitor and support further development. Citation Format: Jun Xu, Yangguang Li, Ju Wang, Pingming Tang, Jianmin Wang, Chen Zhang, Pangke Yan. HSK39775: A USP1 inhibitor for the treatment of cancers with homologous recombination deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7146.

Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Identification of a novel potent CDK inhibitor degrading cyclinK with a superb activity to reverse trastuzumab-resistance in HER2-positive breast cancer in vivo

CDK12 is overexpressed in HER2-positive breast cancers and promotes tumorigenesis and trastuzumab resistance. Thus CDK12 is a good therapeutic target for the HER2-positive breast tumors resistant to trastuzumab. We previously reported a novel purine-based CDK inhibitor with an ability to degrade cyclinK. Herein, we further explored and synthesized new derivatives, and identified a new potent pan-CDK inhibitor degrading cyclinK (32e). Compound 32e potently inhibited CDK12/cyclinK with IC50 = 3 nM, and suppressed the growth of the both trastuzumab-sensitive and trastuzumab-resistant HER2-positive breast cancer cell lines (GI50's = 9–21 nM), which is superior to a potent, clinical pan-CDK inhibitor dinaciclib. Moreover, 32e (10, 20 mg/kg, ip, twice a week) showed a dose-dependent inhibition of tumor growth and a more dramatic anti-cancer effect than dinaciclib in mouse in vivo orthotopic breast cancer model of trastuzumab-resistant HCC1954 cells. Kinome-wide inhibition profiling revealed that 32e at 1 μM exhibits a decent selectivity toward CDK-family kinases including CDK12 over other wildtype protein kinases. Quantitative global proteomic analysis of 32e-treated HCC1954 cells demonstrated that 32e also showed a decent selectivity in degrading cyclinK over other cyclins. Compound 32e could be developed as a drug for intractable trastuzumab-resistant HER2-positive breast cancers. Our current study would provide a useful insight in designing potent cyclinK degraders.

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.

Abstract C145: ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer

Abstract Mesenchymal epithelial transition (MET) proto-oncogene receptor tyrosine kinase (RTK) is a cell surface receptor selective for hepatocyte growth factor (HGF), and is involved in embryogenesis regulation, wound healing, organ regeneration, angiogenesis, and immunomodulation. Aberrant MET oncogenic alterations include: MET exon 14 skipping (MET∆ex14) mutations; activating mutations in the kinase domain; MET gene amplification; MET fusions; and MET protein overexpression. These oncogenic alterations occur in a wide range of human solid cancers. Small-molecule type I c-Met inhibitors bind to the active (Asp-Phe-Gly “DFG-in” state) of c-Met within the adenosine triphosphate (ATP) binding pocket via the Y1230 residue, resulting in down-regulation of multiple pro-oncogenic downstream factors. Presently, type I c-Met inhibitors such as Capmatinib are used as monotherapies in patients with locally advanced or metastatic NSCLC with MET∆ex14 mutations. However, development of post-treatment resistance to type I c-Met inhibitors occurs clinically, including through acquired mutations in codons D1228 and Y1230. No drugs have been approved globally for MET alteration indications other than MET∆ex14 mutations. Next generation MET inhibitors are thus needed to treat patients harboring various MET oncogenic alterations beyond MET∆ex14, including post-treatment acquired mutations. ANS014004 is a newly developed small-molecule type II inhibitor of c-Met, binding to the inactive state of c-Met (“DFG-out”) in the ATP pocket. ANS014004 showed greater kinase inhibition activity against c-Met mutant kinases Y1230H and D1228A in vitro (IC50’s = 2.596 and 2.971 nM, respectively), when compared with Capmatinib, Cabozantinib, and Merestinib (IC50 values = >5000, ~10, and ~5 nM respectively). Further, ANS014004 showed a stronger anti-proliferative effect in MET mutant Ba/F3 cells TPR-Met D1228A and TPR-Met-Y1230H in vitro (IC50’s = 49.34 nM and 30.32 nM, respectively), when compared with Capmatinib (IC50 values>10000nM) and Merestinib (IC50 values: 37~100 nM). ANS014004 also showed potent antitumor effects in vitro in the human cancer tumor cell lines harboring various pathogenetic MET alterations: Hs-746T (gastric); MKN-45 (gastric); EBC-1 (NSCLC); NCI-H1993 (NSCLC); with IC50 ranging from 6.38~62.32 nM. In several subcutaneous xenograft tumor model studies using these same cell lines implanted in BALB/c nude mice, orally administered ANS014004 produced significant dose-dependent tumor growth inhibition, and the corresponding exposures of ANS014004 in plasma, tumor and brain samples increased with increasing dose. ANS0104001 was also observed to induce tumor regression in a MET-amplified intracranial xenograft model. Overall, the non-clinical studies of ANS014004 demonstrated it is a potent, orally-bioavailable type II c-Met inhibitor with activity against various pathogenetic MET alterations and with favorable absorption, distribution, pharmacokinetic, efficacy, and tolerability profiles in vivo. The molecule will enter a Phase 1 clinical study in the US soon. Citation Format: Gong Li, Hepeng Shi, Peilong Zhang, Xiangqiu Li, Wenli Lan, Linna Li, David Chung, Kevin Schaab. ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer [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 C145.

Abstract PR011: Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers

Abstract Somatic gain of function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor or loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1) E3 ligase, which regulates NRF2 protein levels, are frequently identified in many solid cancers such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), and head and neck squamous cell carcinoma (HNSCC). These genomic alterations drive the activation of cytoprotective NRF2 transcriptional programs. In addition to mutational activation, NRF2 pathway activation has been documented with high frequency in patients without mutations in the pathway. Since tumor cells with hyperactivated NRF2 have been demonstrated to be dependent on this pathway for survival, targeting NRF2 represents an attractive therapeutic approach in tumors with aberrant NRF2 activation. Here, we report the identification of VVD-065, a first-in-class NRF2 inhibitor that acts via an unprecedented mechanism of action. Covalent modification of sensor cysteines on KEAP1 during electrophilic or oxidative stress is known to inhibit KEAP1 mediated NRF2 degradation. In complete contrast, covalent modification of a KEAP1 sensor cysteine by VVD-065 dramatically enhances NRF2 degradation. At low nM concentrations, VVD-065 covalently targets KEAP1 E3 ligase, and allosterically increases the affinity between KEAP1 and CUL3, thereby promoting the formation of active KEAP1-CUL3 E3 ligase complexes and increasing the rate of NRF2 degradation. VVD-065 profoundly reduces NRF2 protein levels in WT KEAP1/NRF2 settings, as well as various KEAP1 and NRF2 mutant settings. Consequently, VVD-065 substantially inhibits NRF2-dependent gene expression and proliferation of NRF2-dependent cell lines. In vivo, oral administration of VVD-065 results in robust degradation of NRF2 and decreased expression of NRF2 target genes. Tumor growth inhibition studies with cell-line derived, and patient derived NSCLC/ESCC xenograft (CDX/PDX) models revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of any overt toxicity. Since constitutive NRF2 activation often contributes to chemotherapeutic resistance, we also conducted TGI studies with 100+ PDX models to assess combination opportunities with chemotherapeutic agents. A marked combination response was achieved with cisplatin and nab-paclitaxel in various solid tumor types such as NSCLC, ESCC, HNSCC, and uterine cancers. Combination with chemotherapy was safe and well-tolerated in these mouse studies. In summary, we have identified VVD-065, a potent and selective KEAP1 activator that promotes the degradation of NRF2. Degradation of NRF2 led to robust monotherapy response in NRF2-activated cancers and also chemo-sensitized chemo-refractory tumors. A related molecule, acting through the same mechanism of action has now entered into Phase I clinical trials. Citation Format: Nil Roy, Tine Wyseure, I-Chung Lo, Jordon Inloes, Aaron Snead, Steffen Bernard, Justine Metzger, Jonathan Pollock, Stephanie Grabow, Christie Eissler, Melaminah Williams, Sarah Jacinto, Gabe Simon, Todd Kinsella, David Weinstein, Matt Patricelli. Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers [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 PR011.

Abstract C085: OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains an urgent unmet medical need despite existing therapies that target androgen receptor (AR) signaling. Disease progression is commonly a result of acquired resistance to existing anti-androgen therapies. Biological models have revealed potential resistance mechanisms of CRPC through the reactivation of the AR pathway. Transcriptional coactivators CREB-binding protein (CBP) and E1A binding protein (EP300) are high value targets in mCRPC as they mediate androgen-dependent and -independent activity of the AR pathway. EP300 and CBP proteins harbor both histone acetyltransferase (HAT)- and bromo-domains, and the HAT activity of EP300 regulates AR function. EP300 bromodomain inhibition partially blocks its HAT activity, reducing AR-mediated gene expression and tumor growth. OPN-6602 and OPN-6742 are potent, selective, and orally active small molecule dual EP300/CBP bromodomain inhibitors which abrogate persistent AR signaling in CRPC by modulating H3K27 acetylation and the recruitment of EP300, AR full length (AR-FL), and AR variant 7 (AR-v7) to AR response elements. OPN-6602 and OPN-6742 obstruct the EP300 bromodomain leading to downregulation of the AR pathway and consequent anti-tumor effects in AR-dependent CRPC. OPN-6602 and OPN-6742 are highly selective (>100-fold) against other bromodomains based on a screening panel of 40 bromodomains. Biochemical screens using AlphaScreen technology show these compounds to be equipotent against EP300 and CBP. OPN-6602 is particularly potent by Surface Plasmon Resonance analysis, displaying a KD of 0.87 nM, with a fast association rate (1.5 E+06/Ms) and a slow dissociation rate (1.3 E-03/s). Consequently, OPN-6602 displays a noteworthy residence time of 13 minutes. Both compounds show an AR-selective mode of action with anti-proliferative activity in AR-dependent prostate cancer cell lines (e.g. VCaP, LNCaP, and 22Rv-1) but are inactive against AR-negative prostate cancer cell lines (e.g. DU145). VCaP and mCRPC patient-derived xenograft (PDX) RNA profiling provides evidence of a marked, dose-dependent, pharmacodynamic response indicative of EP300/CBP inhibition. In the VCaP xenograft model, OPN-6602 and OPN-6742 show dose-dependent tumor growth inhibition and corresponding pharmacodynamic responses with prostate-specific antigen level decreases and decreased H3K27 acetylation. In the QR (AR-v7+) PDX model of CRPC (post-androgen deprivation therapy), a synergistic effect was observed between OPN-6602 and enzalutamide. This combination further modulates the expression of AR-driven transcripts such as MYC compared to single agent treatments. The pharmacokinetic profiles of OPN-6602 and OPN-6742 are favorable for oral single daily dose administration due to a high Cmax and short half-life. Due to its higher potency, OPN-6602 has been chosen as clinical candidate and a first-in-human study will start in 2024. Citation Format: Bernice Matusow, Wayne Spevak, Chao Zhang, Yan Ma, Rafe Shellooe, Peipei Li, James Tsai, Pan- Yu Chen, Parmveer Singh, Jackie Walling, Christine Nichols, Jason Halladay, Kerry Inokuchi, Gaston Habets, Gideon Bollag. OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC [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 C085.

Discovery of Novel, First-in-Class Allosteric Modulators of MALT1 Scaffolding Function with Differentiated Pharmacology for NFκB-Driven Malignancies

Constitutive activation of NFκB is an oncogenic driver for many lymphoid and selected solid tumor malignancies. BTK inhibitors, which affect the upstream NFκB pathway, have been successfully used in some diseases, but their utility is limited. The majority of DLBCL patients, for instance, are insensitive to BTK inhibition, supporting the need for alternative mechanisms to block NFκB-driven tumor progression. Knockdown of MALT1, a key component of the CBMT complex (CARD-BCL10-MALT1-TRAF6) involved in direct signal transduction of NFκB, is able to block NFκB-dependent DLBCL tumor growth in BTKi sensitive or resistant tumors, but pharmacologic modulation has been challenged by the dual-functionality of MALT1 in a complexed signalosome. MALT1 functions as the scaffolding component of the CBMT complex. Activated MALT1 recruits multiple proteins and directly activates NFκB canonical signaling. MALT1 also has protease activity that fine-tunes various signaling pathways including NFκB via the cleavage of many substrates. The scaffolding activity of MALT1 is critical for cancer cell survival. Most MALT1 inhibitors discovered so far only block protease activity, which results in incomplete NFκB inhibition, suboptimal anti-tumor activity, and the induction of severe autoimmunity due to protease inhibition. Using our Smart Allostery platform, we elucidated and then drugged a natural hotspot on MALT1 that selectively impacts scaffolding but not protease function. The series of scaffolding modulators demonstrate potent inhibition of MALT1 scaffolding activity with cellular IC 50 values ranging from 15 to 250 nM, while exerting minimal influence on MALT1 protease function. Unlike protease inhibitors, which only partially affect NFκB activity, scaffolding modulators completely inhibit the NFκB downstream signaling manifested by the suppression of NFκB activity with cellular IC 50 values ranging from 2 to 90 nM. Consequently, scaffolding modulators exhibit a superior and broader anti-proliferation profile compared to MALT1 protease inhibitors and BTK inhibitors in a panel of lymphoma cell lines. Pathway and genetic analyses indicate that B-cell lymphoma lines driven by NFκB are especially sensitive to scaffolding modulators. In vivo studies demonstrate a robust, dose-dependent tumor growth inhibition (TGI) by one representative scaffolding modulator in several B-cell lymphoma xenograft models. In a protease inhibitor sensitive ABC-DLBCL model, OCI-LY10, our scaffolding modulator achieved a maximal TGI of 94%, while a clinical MALT1 protease inhibitor showed 62% maximal TGI (p<0.001). In protease resistant NFκB driven DLBCL models, MALT1 scaffolding modulation resulted in tumor regression or stasis. These findings highlight the differentiated pharmacology of our scaffolding modulators. Distinct from protease inhibitors that suppress T cell activation and Treg population in vivo, scaffolding modulators also did not deplete Treg cells nor suppress T cell activation in a 14-day study. This potentially mitigates the risk of development of autoimmune disease caused by chronic MALT1 protease inhibition. In summary, we have discovered novel, first-in-class allosteric modulators of MALT1 scaffolding activities. These scaffolding modulators drive enhanced and broader anti-tumor effect in models resistant to MALT1 protease or BTK inhibitors and do so without immune dysregulation. Scaffolding modulation represents a potential best-in-class therapeutic opportunity to modulate MALT1 in patients with NFκB-driven malignancies.

EP102: Pharmacological Inhibition of METTL3 Elicits Tumor Growth Inhibition In Vivo and Demonstrates Synergy with Venetoclax in Various AML Models

METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of acute myeloid leukemia (AML) 1. EPICS has discovered and optimized a small molecule inhibitor of METTL3 (“M3i”). M3i was shown to inhibit the enzymatic activity of METTL3 (IC 50 = 1.8 nM, SPA assay). The anti-proliferative effects of M3i was demonstrated in various AML cell lines (Kasumi, IC 50 = 63 nM; MV-411, IC 50 = 506 nM; KG1a, IC 50 = 99 nM). Biomarkers were evaluated following M3i treatment of Kasumi and MV-411 cells with consistent results demonstrating depletion of oncoproteins relevant to apoptotic (BCL-2, MCL1) and proliferative (MYC, BRD4, SP1) pathways. In addition, combined treatment with M3i and venetoclax showed clear synergistic effects 2 to inhibit proliferation on both cell lines consistent with the depletion of BCL-2 and MCL1, known targets for venetoclax activity and resistance mechanisms. M3i has been optimized for oral dosing with oral exposure confirmed in mouse, rat and minipig (oral bioavailability >80%, rat; >30%, minipig). In vivo efficacy was evaluated in a disseminated xenograft model using established systemic MV-411-Luc-mCh-Puro 3 in female NSG mice where M3i (10, 30 mg/kg, oral, QDx31 days) demonstrated dose-dependent tumor growth inhibition (p<0.01, relative to vehicle control) of cancer progression as measured by in-life imaging and confirmed by flow cytometry demonstrating significant, dose-dependent pharmacological knockdown of hCD45+ cells in bone marrow, blood and spleen (p<0.01, in all cases). These results confirm that the pharmacological inhibition of METTL3 arrests proliferation of AML and further provide a mechanistic basis for the synergistic activity of M3i in combination with venetoclax as is a viable strategy for the treatment of disease. 1Yankova E et al. (2021), Nature 593:597-601; 2Ianevski A et al. (2022), Nucl Acids Res 50:W739-W743; 3Trachet E (2016), LabCorp Drug Development website: model spotlight MV (4;11)