Dose-dependent Tumor Growth Inhibition Research Articles

Abstract PR001: KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability

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 synthetic lethality 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, ATX020, inhibits KIF18A ATPase activity with an IC50 of 0.014 µM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 > 10 µM) and EG5 (IC50 5.87 µM). ATX020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 0.053 µM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX020, 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 ATX020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In these sensitive cell lines, ATX020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX020 exhibit fragmented nuclei and malformed mitotic spindles. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX020 treatment, cell division is unaffected, and no induction of Annexin V, phospho-Histone H3, and gamma-H2AX is observed. This selective dependency is also observed in vivo, where administration of ATX020 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 ATX020 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, Laura Ghisolfi, 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. KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR001.

Enhancing the efficacy of anti-PD-L1 therapy by cabozantinib in the treatment of advanced esophageal squamous cell carcinoma (ESCC): Rationales from a preclinical study.

e16036 Background: Anti-PD-1/PD-L1 therapy has changed the therapeutic landscape of ESCC. However, the efficacy of anti-PD-1 therapy alone remains modest. Cabozantinib, a multikinase inhibitor with immune modulatory effect in preclinical studies, in combination with atezolizumab has been investigated in various cancer types. We conducted a preclinical study to investigate the efficacy of cabozantinib plus anti-PD-L1 therapy using immunocompetent mouse ESCC models. Methods: From esophageal tumors developed in C57BL/6 mice following chronic exposure to 4-nitroquinoline 1-oxide, we generated several ESCC clones, which could be successfully propagated by in vitro culture for more than 25 passages. The viability of mouse ESCC clones in in vitro cell culture treated with various concentrations of cabozantinib (Adooq Bioscience LLC, Irvine, CA, US) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. We used a subcutaneous mouse model by implanting 5 x 106 murine ESCC cells (clone 104) in male 5-week-old C57BL/6 mice to evaluate the in vivo therapeutic efficacy of anti-PD-L1 therapy (10F.9G2 clone [a rat anti-mouse PD-L1 antibody], Bio X Cell, Lebanon, NH, US) by intraperitoneal (ip) injection, cabozantinib by oral gavage, or the combination of both agents. Tumor volumes were measured twice per week using bilateral calipers. Following two weeks of treatment and an additional week of observation, all mice were sacrificed for tumor harvest. Tumor weights were determined and compared among different treatment groups. Results: Cabozantinib, up to 30 μM, induced only mild growth suppression (13.8 to 26.6%) of 3 mouse ESCC cell clones (clones 001, 103, and 104) in vitro. In the sc tumor model implanted with mouse ESCC clone 104, the anti-PD-L1 monotherapy (10mg/kg, twice per week) did not affect the tumor growth ( P = 0.2000), suggesting that the sc tumor of clone 104 is an anti-PD-L1 primary resistant model. On the contrary, cabozantinib (10 to 30 mg/kg/day, 5 days per week, for 2 weeks) induced a dose-dependent inhibition of tumor growth (tumor weight decreasing from 22.7 to 84.4%) in the sc tumor model of clone 104. The combination of anti-PD-L1 plus cabozantinib induced a more significant suppression of tumor growth than either single treatment alone, and cabozantinib at the dose of 20 mg/kg exhibited more pronounced enhancing effect than at the dose of 10 mg/kg. Conclusions: This preclinical study demonstrated a synergistic antitumor effect of combining anti-PD-L1 therapy with cabozantinib in immunocompetent murine models of ESCC. A phase II trial is ongoing to investigate the efficacy of cabozantinib plus atezolizumab in advanced ESCC (NCT05007613) (This work was supported by NTUCCS-112-06, NSTC112-2314-B-002-150-, NSTC112-2314-B-002-268-, MOHW111-TDU-B-221-114006, and MOHW112-TDU-B-221-124006).

A phase I/II, first-in-human study of VLS-1488, an oral KIF18A inhibitor, in patients with advanced cancer.

TPS3181 Background: Chromosomal instability (CIN) is a hallmark of cancer characterized by high rates of chromosomal segregation errors during mitosis. This creates structural and numerical changes to chromosomes, driving genetic heterogeneity and cancer evolution. KIF18A is a mitotic kinesin protein shown to be important for successful division of cancer cells with CIN but not required for mitosis in normal diploid cells. Pre-clinical studies demonstrated that treatment of chromosomally unstable cancer cells in vitro and in xenograft models (HCC15 and OVCAR-3) with a KIF18A inhibitor resulted in failure of chromosomal congression, mitotic arrest, cell death and dose-dependent inhibition of tumor growth. VLS-1488 is an oral small molecule inhibitor of KIF18A that is being examined in an open-label, phase I/II, first-in-human study evaluating the safety, tolerability, pharmacokinetics (PK) and preliminary anti-tumor activity in advanced solid tumors with high prevalence of CIN. Methods: Key eligibility criteria include specific histologically confirmed metastatic or advanced tumor types with at least one site of measurable disease per RECIST 1.1, adequate organ function, and mismatch repair proficient/microsatellite stable.Eligible tumor types include high grade serous ovarian/fallopian tube/primary peritoneal cancer, squamous non-small cell lung cancer, triple negative breast cancer, gastric adenocarcinoma, gastroesophageal cancer, esophageal cancer, colorectal adenocarcinoma, transitional cell carcinoma of bladder, head and neck squamous cell carcinoma, ovarian or uterine carcinosarcoma, and uterine serous carcinoma. Subjects will receive VLS-1488 monotherapy continuously for 28-day cycles once daily. The study consists of two parts, Dose Escalation and Dose Expansion. Utilizing a Bayesian Optimal Interval (BOIN) design, Dose Escalation will examine the safety and tolerability of VLS-1488 at various dose levels (DLs) to identify the Maximum Tolerated Dose (MTD) and to select DLs for Dose Expansion. Dose Expansion may commence after the MTD or DLs of interest are identified. Dose Expansion will examine the safety, tolerability, preliminary efficacy and preliminary drug-drug interaction (DDI) and food effect risk of VLS-1488 in selected tumor types. Subjects enrolled to specific tumor type cohorts may be randomized to DLs of interest. Enrollment to Dose Escalation began in September 2023 and is ongoing. Clinical trial information: NCT05902988 .

Efficacy of GCN2 inhibition by a novel small molecule AP030 in acute leukemia.

6532 Background: GCN2 is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR) that is activated in response to amino acid scarcity. Active GCN2 phosphorylates translation initiation factor eIF2α resulting in the attenuation of global protein synthesis. ISR signaling primarily promotes cell survival but may trigger cell death, dependent on the cellular context. In hematological tumors GCN2 promotes tumor cell survival under conditions of nutrient scarcity. Several GCN2 inhibitors are in clinical development for treatment of both solid and hematological tumors. Here, we describe the preclinical results of a novel, selective ATP-competitive inhibitor of GCN2. Methods: GCN2 inhibition by AP030 was determined using a biochemical Lanthascreen assay and a cell based HTRF assay measuring eIF2a phosphorylation following stimulation with Borrelidin. Kinase selectivity was determined using KinomeScan, and bespoke biochemical and cell-based assays. Interaction of AP030 with GCN2 was determined using X-ray crystallography. AP030 activity in disease-relevant tumor cell lines was determined using cell viability, caspase activation, protein expression and qPCR gene expression endpoints. Inhibition of hematological tumor growth and induction of cell death was investigated in patient samples, and ALL and AML in vivo animal models. Results: Potent inhibition of GCN2 in the Lanthascreen assay was demonstrated with AP030 (Ki of 4.4nM). Following stimulation with Borrelidin, AP030 inhibited eIF2a phosphorylation with an IC50 of 50.8nM and inhibited downstream targets CHAC1 and DDIT3 measured by qPCR. X-ray crystallography confirmed that AP030 binds to the ATP-binding site of GCN2. The KinomeScan confirmed that AP030 was highly selective against the human kinome. AP030 led to partial or complete reduction of AML cell line viability as a single agent and acted synergistically with asparaginase in ALL cell lines. Caspase 3/7 induction was observed in the AML and ALL cell lines evaluated and AP030 increased the proportion of cells undergoing apoptosis in AML patient primary samples. In the CCRF-CEM ALL systemic in vivo tumor model AP030 inhibited tumor growth in combination with asparaginase (TGI 79.33%). Treatment with 0.5-5mg/kg (q.d.) of AP030 resulted in dose-dependent tumor growth inhibition and regression of the MOLM-16 AML in vivo model (TGI 97.37% 5mg/kg). RNA sequencing of residual tumor cells revealed disruption of amino acid and protein metabolism, consistent with GCN2 inhibition. Conclusions: Targeting hematological tumor reliance on the GCN2 arm of the ISR during nutrient scarcity is a novel approach to preventing tumor cell survival. AP030 a novel, potent, selective, ATP-competitive kinase inhibitor has been shown to lead to impressive efficacy in acute leukemia. Based on these preclinical results, a phase 1/2 study has been initiated in AML.

Characterization of BTX-10908, a first-in-class, orally bioavailable SOS1 bifunctional degrader for the treatment of KRAS- and RTK-driven cancers.

e15192 Background: Despite early clinical responses of targeted therapeutics against specific nodes in the RTK-RAS-MAPK pathway, limitations in the depth and duration of efficacy occur due to the emergence of adaptive and acquired resistance mechanisms. Resistance can result from the removal of feedback inhibition to reactivate RTKs, on-target amplification and new mutations. The efficacy of these oncogene-targeted agents in the clinic could be improved by co-treatment with additional therapies that address either type of resistance mechanism. The RAS guanine nucleotide exchange factor, Son of Sevenless 1 (SOS1), represents an ideal target to enhance clinical potency of other pathway inhibitors for two reasons. In addition to mitigating inputs from upstream RTKs to downstream RAS proteins, SOS1’s activity is also regulated by ERK- and RSK-mediated negative feedback inhibition with upstream and downstream inhibitors alleviating this inhibition to reengage pathway activation. Methods: To address this need, we utilized our PRODEGY platform to generate orally bioavailable, Cereblon (CRBN)-based SOS1 bifunctional degrader, BTX-10908, for the treatment of KRAS- and RTK-driven tumors. Results: BTX-10908 demonstrated rapid and potent SOS1 degradation with <10nM DC50 and maximum degradation > 90%. Consistent with BTX-10908’s mechanism of action, SOS1 degradation required CRBN and the proteasome. Not only did it reduce active RAS levels, but BTX-10908 also inhibited downstream signaling proteins (pERK, pRSK, pS6) and lowered ERK-mediated transcripts in KRAS- and EGFR-mutant as well as MET amplified cell lines under anchorage-independent (3D) conditions. Additionally, drug washout experiments demonstrated prolonged suppression of pERK and ERK-regulated transcripts with SOS1 degradation whereas rebound occurred quicker with SOS1 inhibitors. BTX-10908 displayed CRBN- and SOS1-dependent antiproliferative activity with IC50 values ranging from 0.5-20nM. Notably, BTX-10908 was significantly more potent than the clinical SOS1 inhibitor at reducing pERK levels and cell viability in multiple KRAS- and RTK-driven cell lines. Oral administration of BTX-10908 resulted in dose-dependent SOS1 degradation and tumor growth inhibition in H358 (KRAS G12C) and H441 (KRAS G12V) xenografts. The combination of BTX-10908 with KRAS G12C inhibitors (Sotorasib, Adagrasib) and various RTK inhibitors (EGFR, Met, BCR-ABL, and FLT3) prevented pathway reactivation driven by these inhibitors resulting in synergistic effects in in vitro proliferation assays and enhanced tumor growth inhibition in KRAS-mutant xenograft models. Conclusions: Our developmental candidate, BTX-10908, displayed robust in vitro and in vivo activity supporting its further development for monotherapy and combination therapies of KRAS and RTK-driven cancers.

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.

CLX-155: A Novel, Oral 5-FU Prodrug Displaying Antitumor Activity in Human Colon Cancer Xenograft Model in Nude Mice

Introduction: Capecitabine is an oral prodrug of 5-FU, which interpatient pharmacokinetic (PK) variability related to liver function and severe adverse events (e.g., hand-foot syndrome, myelosuppression, and neurotoxicity) limits. CLX-155 is a novel oral 5’-DFCR prodrug involving 5’-DFCR as an intermediate for generating 5-FU, unlike capecitabine, which the liver does not metabolize. This study addresses the following research question: what is the activity of CLX-155 in a human colon cancer xenograft model in nude mice? Methods: This study involved 50 Foxn1 athymic nude female mice implanted with the human colon cancer cell line HCT116 (5 million cells per site). Investigators randomized animals into five treatment groups (N = 10): vehicle control, CLX-155 at doses of 125, 250, and 500 mg/kg/day, or capecitabine 1000 mg/kg/day. Animals received oral treatment once daily for five days a week with two days off for a total of three consecutive weeks. Investigators evaluated treatment toxicity based on body weight loss. Calculations for tumor growth inhibition involved comparing changes in tumor volume on a given day to tumor volumes on Day 1. Results: CLX-155 demonstrated statistically significant, dose-dependent tumor growth inhibition at all doses compared to vehicle control (p<0.0001). Tumor growth inhibition at Day 15 for CLX-155 treatment groups of 125, 250, and 500 mg/kg/day was 57.8%, 70.4%, and 90.6% respectively. Two animals in the CLX-155 500 mg/kg/day treatment group experienced complete tumor regression, and all animals in the CLX-155 treatment groups survived. Two animals in the CLX-155 250 and 500 mg/kg/day dosing groups experienced a decrease in body weight. In contrast, two mice in the capecitabine group exhibited clinical signs of hunchback and scaly skin, progressive weight loss, and eventual death. Conclusion: CLX-155 demonstrated comparable tumor growth inhibition to capecitabine but at a lower dose, suggesting increased potency. In addition, CLX-155 exhibited improved tolerability and fewer adverse effects. These promising results support further investigation in Phase 1 clinical trials for managing colon cancer.

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.