Regression In Xenograft Models Research Articles

Abstract PO-010: Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma

Abstract Background: Non-Hodgkin lymphoma (NHL) represents a biologically and clinically diverse group of hematologic malignancies originating from B cells, T cells, and/or natural killer cells, with those of B-cell origin constituting ∼80–85% of all NHL cases. The BCL6 transcription factor is a key oncogenic driver of B-cell lymphomagenesis, and deregulated BCL6 expression is a common feature of diffuse large B-cell lymphoma (DLBCL), the most common type of NHL. BCL6 translocations have been observed in follicular lymphomas, the second most prevalent NHL, with studies suggesting that these translocations may portend a higher risk of transformation into aggressive lymphoma. BCL6 is also a lineage-defining transcription factor of T follicular helper cells, the cell of origin for angioimmunoblastic T-cell lymphoma (AITL). Moreover, BCL6 expression has been identified in biopsies of patients with AITL, and its continued expression was required for tumor growth in a mouse model of AITL, supporting BCL6 as a cancer driver in this type of NHL as well. ARV-393, an orally administered PROteolysis TArgeting Chimera (PROTAC) BCL6 degrader, is a bifunctional small molecule consisting of a BCL6-binding domain attached by a linker to a cereblon E3 ubiquitin ligase–binding domain. ARV-393 induces ubiquitination and subsequent degradation of BCL6 by the proteasome. In preclinical studies, ARV-393 induced rapid and robust degradation (>90%) of BCL6 in NHL cell lines and demonstrated dose-responsive tumor growth inhibition leading to tumor stasis or regression in xenograft models, supporting further investigation in patients with NHL. This multicenter, first-in-human, phase 1 study will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of ARV-393 in patients with advanced B-cell NHL or AITL. Methods: Eligible patients (aged ≥18 years) have relapsed/refractory mature B-cell NHL and ≥2 prior systemic therapies, or histologically confirmed AITL that has recurred or progressed following institutional standard-of-care therapy. Patients must also have ≥1 measurable lesion at study entry, Eastern Cooperative Oncology Group performance status of 0 or 1, freshly biopsied or archival tumor tissue available, adequate organ function, no active central nervous system involvement, and no prior allogeneic stem cell transplant or solid organ transplantation. Autologous stem cell transplant must not have occurred ≤100 days and previous CAR T-cell therapy ≤60 days prior to cycle 1 day 1 of ARV-393 treatment. ARV-393 will be administered orally in 28-day cycles with dose escalation according to a Bayesian optimal interval design. The primary objectives are to evaluate the safety and tolerability of ARV-393, determine maximum tolerated dose if necessary, and identify the recommended phase 2 dose(s) and dosing schedule. Secondary objectives are to characterize the pharmacokinetic profile of ARV-393 and evaluate its preliminary anti-tumor activity. Enrollment for this study will open in 2024. Citation Format: Paolo Caimi, Scott Huntington, Sean Landrette, Sheryl M Gough, Vivian Dai, Bryan Jackson, Juan Chavez, Sarah Tannenbaum-Dvir, Matthew Matasar. Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-010.

Raludotatug Deruxtecan, a CDH6-Targeting Antibody-Drug Conjugate with a DNA Topoisomerase I Inhibitor DXd, Is Efficacious in Human Ovarian and Kidney Cancer Models.

Cadherin-6 (CDH6) is expressed in several cancer types, but no CDH6-targeted therapy is currently clinically available. Here, we generated raludotatug deruxtecan (R-DXd; DS-6000), a novel CDH6-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and evaluated its properties, pharmacologic activities, and safety profile. In vitro pharmacologic activities and the mechanisms of action of R-DXd were assessed in serous-type ovarian cancer and renal cell carcinoma cell lines. In vivo pharmacologic activities were evaluated with several human cancer cell lines and patient-derived xenograft mouse models. The safety profile in cynomolgus monkeys was also assessed. R-DXd exhibited CDH6 expression-dependent cell growth-inhibitory activity and induced tumor regression in xenograft models. In this process, R-DXd specifically bound to CDH6, was internalized into cancer cells, and then translocated to the lysosome. The DXd released from R-DXd induced the phosphorylation of Chk1, a DNA damage marker, and cleaved caspase-3, an apoptosis marker, in cancer cells. It was also confirmed that the DXd payload had a bystander effect, passing through the cell membrane and impacting surrounding cells. The safety profile of R-DXd was favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys. R-DXd demonstrated potent antitumor activity against CDH6-expressing tumors in mice and an acceptable safety profile in monkeys. These findings indicate the potential of R-DXd as a new treatment option for patients with CDH6-expressing serous-type ovarian cancer and renal cell carcinoma in a clinical setting.

All Roads Lead to Rome: YAP/TAZ Activity Influences Efficacy of KRASG12C Inhibitors

AbstractThe development of direct inhibitors of KRASG12C represents a monumental step forward in the field of oncology. Nevertheless, there is considerable opportunity to enhance response rates to KRASG12C inhibitors. In this issue of Cancer Research, three investigative teams explore the modulation of KRASG12C inhibitor activity in lung, colorectal, and pancreatic cancers using CRISPR-based knockout screens. While each group identified and validated a variety of genes and pathways conferring resistance to KRASG12C inhibition, all three groups converged upon activation of YAP/TAZ as a common means of resistance. While coinhibition of KRASG12C and YAP/TAZ did not cause complete tumor regression in xenograft models, combining YAP/TAZ inhibition was capable of significantly extending the response of tumors to KRASG12C inhibition.See related articles by Mukhopadhyay et al., p. 4095, Edwards et al., p. 4112, and Prahallad et al., p. 4130

EXTH-63. MTA-COOPERATIVE PRMT5 INHIBITORS ARE EFFICACIOUS IN MTAP-DELETED MALIGNANT PERIPHERAL NERVE SHEATH TUMOR MODELS

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with limited treatment options and poor survival rates, necessitating the development of novel therapeutics. Approximately 25-50% of MPNST harbor loss of the enzyme methylthioadenosine phosphorylase (MTAP) due to a passenger deletion driven by loss of the proximal tumor suppressor gene, CDKN2A. PRMT5 was identified as a selective dependence in MTAP-deleted cells due to the accumulation of the substrate methylthioadenosine (MTA), which is itself an endogenous PRMT5 inhibitor. TNG908 and TNG462 are clinical stage MTA-cooperative PRMT5 inhibitors that demonstrate selectivity for MTAP-deleted cells over MTAP-intact cells of 15X and 45X, respectively. Previous reports show both molecules drive durable tumor regressions in xenograft models of various MTAP-deleted cancer histologies. Here, our objectives are to examine the activity of TNG908 and TNG462 in preclinical MPNST models. METHODS The proliferation effects of MTA-cooperative PRMT5 inhibitors, TNG908 or TNG462, and a SAM-cooperative PRMT5 inhibitor, GSK3326595, on MTAP-deleted and MTAP-intact MPNST cell lines were determined using CellTiter-Glo (CTG) assays. TNG908 and TNG462 were further profiled in two MTAP-deleted MPNST patient-derived xenograft (PDX) models, WU-356 and WU-386. RESULTS Incubation with the MTA-cooperative PRMT5 inhibitors, TNG908 and TNG462, selectively decreased the proliferation of MTAP-deleted MPNST cell lines relative to MTAP-intact MPNST cell lines. TNG908 and TNG462 drove dose-dependent antitumor activity including tumor regressions in the MTAP-deleted MPNST PDX models, WU-356 and WU-386, at well-tolerated doses. CONCLUSIONS The clinical stage MTA-cooperative PRMT5 inhibitors TNG908 (NCT05275478) and TNG462 (NCT05732831) are efficacious in MPNST models in vitro and in vivo and are therefore promising therapeutic agents for patients with MTAP-deleted MPNST.

Abstract 1560: Orally bioavailable macrocycles that target cyclins A and B RxL motifs cause tumor regression in xenograft models and in vitro show activity across multiple cancer types

Abstract RB and E2F genes are frequently altered in cancer. Cyclin/Cdk (cyclin-dependent kinase) complexes regulate the activity of RB and E2F to drive cell cycle progression and ensure fidelity of DNA replication. A critical subset of their substrates (Rb, E2F, Cdc6, etc.) requires a short linear motif (RxL) that interacts with the hydrophobic patch on cyclins. Peptides that disrupt the cyclin/RxL interaction were found to be synthetically lethal in cancer cell lines displaying Rb-E2F dysregulation (Chen, 1999). Using structure-guided design, cell-permeable macrocycles were synthesized that selectively inhibit RxL-mediated binding to cyclins A and B. Preclinical studies with an orally bioavailable cyclin A/B RxL inhibitor show activity in cell lines with Rb-E2F pathway dysregulation, and inhibition of tumor growth and regression in xenograft models of small cell lung carcinoma (SCLC). RB-null mutations occur with high frequency in SCLC tumors (Febres-Aldana 2022). Previously we reported (AACR April 2022 Abs.#5379) sensitivity of a large proportion of SCLC cell lines to cyclin RxL inhibitors. Further studies have shown that such compounds have growth inhibitory activity in cell lines representing multiple types of solid tumors and hematological malignancies. Sensitivity of the cell lines to these macrocyclic inhibitors is associated with increased expression of genes involved in the E2F pathway, consistent with the proposed Rb/cyclin/E2F pathway synthetic lethality mechanism. Disruption of RxL binding to both cyclin A and B is required for sensitivity. Cyclin A/B RxL inhibitors disrupt the interactions of E2F and Cdc6 with cyclin A/Cdk complexes in co-immunoprecipitation studies and have EC50 values consistent with biochemical and cellular activity. Cyclin A/B RxL inhibition results in accumulation of cells in the G2/M phase of the cell cycle which leads to apoptosis both in vitro and in vivo. A separate study (Singh et al. AACR 2023) utilizing a CRISPR screen identified that genes of the spindle assembly checkpoint (SAC) are required for the synthetic lethality induced by the cyclin A/B RxL inhibitors. The inhibitors assessed in vitro for off-target activity showed negligible activity in a panel of 468 kinases and in safety panels that include GPCRs, ion channels, and transporters. In a 14-day tolerability study, neither neutropenia nor significant effects in other blood cell counts were noted. Cyclin A/B RxL inhibitors cause significant tumor regression via oral administration in SCLC xenograft models. Given their compelling characteristics, we are progressing the development of these orally bioavailable macrocyclic cyclin A/B RxL inhibitors to the clinic. Citation Format: Catherine E. Gleason, Pablo D. Garcia, Ranya Odeh, Frances Hamkins-Indik, Daphne He, Meisam Nosrati, Gavin Situ, Roberta Sala, Bernard Levin, Li-Fen Liu, Evelyn W. Wang, Siegfried Leung, Breena Fraga, Andrew T. Bockus, Justin Shapiro, Nathan Dupper, Chinmay Bhatt, Kai Yang, Megan DeMart, Sammy Metobo, James Aggen, Peadar Cremin, Ramesh Bambal, Constantine Kreatsoulas, David J. Earp, Rajinder Singh. Orally bioavailable macrocycles that target cyclins A and B RxL motifs cause tumor regression in xenograft models and in vitro show activity across multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1560.

Abstract 1541: NextGen conditionally active biologic (CAB) anti-Nectin-4-ADC with improved stability and safety

Abstract In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents. Today, development of a new ADC faces two major challenges: on-target, off-tumor toxicity based on target expression on healthy tissue and off-target, off-tumor toxicity caused by extracellular linker vulnerability and/or premature payload release. On-target, off-tumor toxicity can be effectively eliminated by using a Conditionally Active Biologics (CAB) antibody to specifically target tumor cells. CAB technology is a proprietary platform used to engineer antibodies that target antigens in the context of diseased tissues, but not normal tissues by exploiting the unique extracellular acidic conditions found on the surface of cancer cells (Chang et al. 2021). This platform has allowed us to generate CAB antibody drug conjugates (ADCs) specific to tumor associated antigens that are often expressed on healthy tissues. CAB selectively reduces on-target toxicity, as well as off-target toxicity by eliminating target mediated drug disposition. Conventional ADCs can induce unacceptable toxicities caused by binding to antigen expressed on normal cells, resulting in the undesired release of the payload. This can severely limit the therapeutic window of the drug candidate. Two CAB-ADCs (Mecbotamab Vedotin and Ozuriftamab Vedotin) using the well-established vedotin linker with MMAE as payload are currently in phase 2 clinical trials and show promising results with increased safety profiles, with currently no signs of on-target, off-tumor toxicity. BA3361, a CAB-ADC targeting Nectin-4, showed good in vitro and in vivo efficacy data demonstrating cytotoxicity against human Nectin-4-expressing tumor cell lines and tumor regression in xenograft models using the vedotin linker (AACR 2020, Poster #4560). To eliminate vedotin linker-related off-target toxicities, we developed a NextGen Nectin-4 CAB ADC with a novel cleavable linker that is highly stable in human serum and has increased solubility. In vivo efficacy data from CDX models as well as data from non-GLP tox studies in rats and cynomolgus monkeys will be presented. In conclusion, our data indicates that the CAB-Nectin-4-ADC with its optimized linker payload, has superior serum stability while maintaining the CAB target selectivity. Reference: 1. Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ, Short JM. Proc Natl Acad Sci U S A. 2021 Mar 2;118(9):e2020606118. 2. Jing Wang, Charles Xing, Haizhen Liu, Ana Paula Cugnetti, Christina Wheeler, Mathew Lucas, Gerhard Frey, Cathy Chang, William J. Boyle, Jay M. Short. Novel conditionally active ADCs (CAB ADCs): A novel class of molecules targeting solid tumors. AACR Annual Meeting 2020, Abstract #7890, Poster #4560 Citation Format: Gerhard Frey, Jing Wang, Kyrie Johnson, Haizhen Liu, Christina Wheeler, Charles Xing, Ana Paula Cugnetti, Patricia McNeeley, Solmarie Joyner, Cathy Chang, William J. Boyle, Jay M. Short. NextGen conditionally active biologic (CAB) anti-Nectin-4-ADC with improved stability and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1541.

Abstract 3327: Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors

Abstract The non-receptor protein tyrosine phosphatase SHP2 (PTPN11) plays an important role in the regulation of RAS/MAPK signal transduction downstream of growth factor receptor activation. Loss of SHP2 activity suppresses tumor cell growth, making SHP2 a potential target for cancer therapy. Here we report the discovery of GDC-1971 (formerly RLY-1971), a highly potent, selective, and orally bioavailable small-molecule SHP2 inhibitor that stabilizes SHP2 in a closed, auto-inhibited conformation. GDC-1971 potently inhibits both wild-type SHP2 (IC50 <1nM) and the E76K activating mutant (IC50 <250nM) in biochemical assays. In standard 2-dimensional and anchorage-independent growth conditions, GDC-1971 inhibits cellular proliferation in models harboring receptor tyrosine kinases (RTKs), SHP2, NF1, KRAS, or BRAF mutations in a dose-dependent manner. GDC-1971 potently inhibits the proliferation of cellular models harboring KRAS G12C or G12A mutations (median IC50 <80 nM) compared to models harboring other KRAS G12, G13 or Q61 mutations (median IC50 >1 uM), indicating a link between KRAS GTP hydrolysis and SHP2 dependency. Despite this trend, some non-KRAS G12C or G12A cell lines harboring other KRAS mutations responded to GDC-1971 in vitro, suggesting some heterogeneity in RTK/SHP2 signaling dependence in subsets of other KRAS mutants. In vivo, GDC-1971 demonstrates dose-dependent RAS/MAPK pathway inhibition and induces significant tumor-growth inhibition in human xenograft models harboring EGFR and KRAS alterations at continuous daily doses that are well tolerated. Given the reported role of SHP2 as a critical mediator of resistance to targeted therapies, we assessed the activity of GDC-1971 combinations in multiple contexts. We observed increased suppression of the MAPK signaling cascade and anti-proliferation synergy when combining GDC-1971 with EGFR, ALK, and KRAS G12C inhibitors in vitro. The observed in vitro synergy translated to dramatic anti-tumor growth effects in vivo. GDC-1971 in combination with the KRAS G12C covalent inhibitor GDC-6036 resulted in significant regressions at doses well below those required for single agent activity in a KRAS G12C-mutant NSCLC xenograft model. In rodent and dog toxicology studies, GDC-1971 is well tolerated at exposures above those required to induce regression in xenograft models. The biochemical and cellular potency and favorable pharmaceutical properties of GDC-1971 support the further clinical development in RTK/MAPK pathway altered tumors using continuous daily dosing alone and in combination with other targeted agents, including the KRAS-G12C inhibitor GDC-6036 (clinical trial NCT04449874). Citation Format: Bret Williams, Alexander Taylor, Olivia Orozco, Christopher Owen, Elizabeth Kelley, Andre Lescarbeau, Kelley Shortsleeves, Randy Kipp, Vy Nguyen, Erin Brophy, Jeremy Wilbur, Yong Tang, David Lanzetta, Nigel Waters, Sherri Smith, Fabrizio Giordanetto, Paul Maragakis, Jack Greismann, Lindsay Willmore, Eric Therrien, Yang Xiao, Marie Evangelista, Luca Gerosa, Eva Lin, Mark Merchant, Alfonso Arrazate, Emily Chan, Pablo Sáenz-López Larrocha, Stefan Chun, Thomas Hunsaker, Gauri Deshmukh, Christine M. Bowman, David E. Shaw, Mark Murcko, Mahesh Padval, W Patrick Walters, James Watters, Donald A. Bergstrom. Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3327.

Abstract LB197: Tucatinib has selective activity in HER2-positive cancers and significant combined activity with approved and novel breast cancer targeted therapies

Abstract Pharmacologically targeting the HER2 oncoprotein with therapeutics such as the monoclonal antibody (mAb), trastuzumab, provides clinical benefit for patients with HER2-positive (HER2+) cancers. However, a significant number of patients eventually progress on these therapies. Efforts to overcome therapeutic resistance through combination therapy with small molecule inhibitors of HER2 have been limited by toxicities associated with off-target activity and/or limited efficacy. In this preclinical study, we explore single-agent and combined activity of tucatinib, a novel HER2-selective small molecule inhibitor. Tucatinib demonstrated potent, selective activity in a panel of 456 human cancer cell lines, with activity restricted to cell lines (breast and non-breast) with HER2-amplification, including models of acquired resistance to trastuzumab. Within the HER2+ population, tucatinib response tracked strongly with HER2-driven signaling. Single agent tucatinib induced tumor regressions in xenograft models of HER2+ breast cancer and combination with trastuzumab induced a complete and sustained blockade of HER2/PI3K/AKT-signaling. Efficacy of the tucatinib/trastuzumab combination matched that induced by current standard of care (SOC) trastuzumab/pertuzumab/docetaxel combination, with the exception that the chemotherapy-sparing tucatinib/trastuzumab combination did not require a dosing holiday to achieve the same efficacy. In xenograft models of HER2+ breast cancer that also express estrogen receptor (ER) (HER2+/ER+), tucatinib showed combined efficacy with inhibitors of CDK4/6 and ER, indicating potential novel therapeutic strategies for difficult-to-treat subtypes of HER2+ breast cancer. These data support expanded clinical investigations of tucatinib as a combination partner for other novel and approved targeted therapies for HER2-driven malignancies. Citation Format: Neil A. O'Brien, Holly K. Huang, Martina S. McDermott, Athena Madrid, Tong Luo, Raul Ayala, Shawnt Issakhanian, Ke Wei Gong, Ming Lu, Jun Zhang, Dennis J. Slamon. Tucatinib has selective activity in HER2-positive cancers and significant combined activity with approved and novel breast cancer targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB197.

Tucatinib has Selective Activity in HER2-Positive Cancers and Significant Combined Activity with Approved and Novel Breast Cancer-Targeted Therapies.

Pharmacologically targeting the HER2 oncoprotein with therapeutics such as the mAb, trastuzumab, provides clinical benefit for patients with HER2-positive (HER2+) cancers. However, a significant number of patients eventually progress on these therapies. Efforts to overcome therapeutic resistance through combination therapy with small-molecule inhibitors of HER2 have been limited by toxicities associated with off-target activity and/or limited efficacy. In this preclinical study, we explore single-agent and combined activity of tucatinib, a novel HER2-selective small-molecule inhibitor. Tucatinib demonstrated potent, selective activity in a panel of 456 human cancer cell lines, with activity restricted to cell lines (breast and non-breast) with HER2-amplification, including models of acquired resistance to trastuzumab. Within the HER2+ population, tucatinib response tracked strongly with HER2-driven signaling. Single-agent tucatinib induced tumor regressions in xenograft models of HER2+ breast cancer and combination with trastuzumab induced a complete and sustained blockade of HER2/PI3K/AKT signaling. Efficacy of the tucatinib/trastuzumab combination matched that induced by current standard-of-care trastuzumab/pertuzumab/docetaxel combination, with the exception that the chemotherapy-sparing tucatinib/trastuzumab combination did not require a dosing holiday to achieve the same efficacy. In xenograft models of HER2+ breast cancer that also express estrogen receptor (ER; HER2+/ER+), tucatinib showed combined efficacy with inhibitors of CDK4/6 and ER, indicating potential novel therapeutic strategies for difficult-to-treat subtypes of HER2+ breast cancer. These data support expanded clinical investigations of tucatinib as a combination partner for other novel and approved targeted therapies for HER2-driven malignancies.

RING-finger protein 6 promotes colorectal tumorigenesis by transcriptionally activating SF3B2

RNF6 is a RING finger protein with oncogenic potential. In this study, we established colon-specific RNF6 transgenic (tg) mice, and demonstrated that RNF6 overexpression accelerated colorectal carcinogenesis compared to wild-type littermates in a chemically induced colorectal cancer (CRC) model. To understand whether transcriptional activity of RNF6 underlies its oncogenic effect, we performed integrated chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing analysis to identify splicing factor 3b subunit 2 (SF3B2) as a potential downstream target of RNF6. RNF6 binds to the SF3B2 promoter and the overexpression of RNF6 activates SF3B2 expression in CRC cells, primary CRC organoids, and RNF6 tg mice. SF3B2 knockout abrogated the tumor promoting effect of RNF6 overexpression, whereas the reexpression of SF3B2 recused cell growth and migration/invasion in RNF6 knockout cells, indicating that SF3B2 is a functional downstream target of RNF6 in CRC. Targeting of RNF6-SF3B2 axis with SF3B2 inhibitor with pladienolide B suppressed the growth of CRC cells with RNF6 overexpression in vitro and in vivo. Moreover, the combination of 5-fluorouracil (5-FU) plus pladienolide B exerted synergistic effects in CRC with high RNF6 expression, leading to tumor regression in xenograft models. These findings indicate that tumor promoting effect of RNF6 is achieved mainly via transcriptional upregulation of SF3B2, and that RNF6-SF3B2 axis is a promising target for CRC therapy.

Abstract 1276: Identification of S65487/VOB560 as a potent and selective intravenous 2nd-generation BCL-2 inhibitor active in wild-type and clinical mutants resistant to Venetoclax

Abstract The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Venetoclax (Venclexta™), a selective BCL-2 inhibitor, is the first member of a new class of anti-cancer drugs, called BH3 mimetics, to be approved for CLL and AML. Here, we describe the identification of a novel potent and selective BCL-2 inhibitor named S65487/VOB560 that has a different binding mode on BCL-2 compared to Venetoclax. This inhibitor binds to the BH3 hydrophobic groove of BCL-2. Its selectivity profile demonstrates lack of significant binding to MCL-1, BFL-1 and poor affinity for BCL-XL. S65487/VOB560 induces apoptosis in a panel of hematological cancer cell lines and inhibits cell proliferation with IC50s in the low nM range. S65487/VOB560 induces complete regression in BCL-2-dependent RS4;11 tumors in vivo after a single IV (intravenous) administration. Strong and persistent tumor regression in xenograft models of lymphoid malignancies in mouse and rat were observed at well tolerated doses following weekly IV administration of S65487 in combination with the MCL-1-specific inhibitor, S64315/MIK665. These positive findings were further confirmed in a panel of AML PDX tumor models. Recently, acquired BCL-2 mutations (such as G101V and D103Y) were identified in patients with Chronic Lymphocytic Leukemia becoming resistant to Venetoclax. Interestingly, S65487/VOB560 is active on such BCL-2 mutants and induces apoptosis in preclinical resistance models. Altogether, these data demonstrate that S65487/VOB560 has significant therapeutic potential against human lymphoid and myeloid malignancies as well as in patients with Venetoclax resistant leukemias. Clinical studies are currently ongoing with S65487/VOB560 (NCT03755154). Citation Format: Arnaud Le Tiran, Audrey Claperon, James Davidson, Jérôme-Benoit Starck, Thierry Le Diguarher, Maïa Chanrion, Prakash Mistry, Youzhen Wang, Elodie Monceau, Fabienne Bernhardt, Francesca Rocchetti, Gaelle Lysiak-Auvity, Ijen Chen, Zoe Daniels, Chris Pedder, Mandy Fallowfield, Jean-Michel Henlin, Imre Fejes, Janos Tatai, Miklos Nyerges, Didier Durand, Marion Zarka, Sneha Sanghavi, Anne-Marie Girard, Marie Schoumacher, Laurence Kraus-Berthier, Rick Newcombe, Ensar Halilovic, Sébastien Banquet, Alain Rupin, Heiko Maacke, James Murray, Erick Morris, Francesco Hofmann, Frédéric Colland, Olivier Geneste. Identification of S65487/VOB560 as a potent and selective intravenous 2nd-generation BCL-2 inhibitor active in wild-type and clinical mutants resistant to Venetoclax [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1276.

5-Fluorouracil Enhances the Antitumor Activity of the Glutaminase Inhibitor CB-839 against PIK3CA-Mutant Colorectal Cancers

PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. SIGNIFICANCE: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.

Abstract B26: The SHOC2 phosphatase complex as a therapeutic target for ERK-pathway inhibition in RAS-driven tumors

Abstract Mutual exclusivity of RAS and BRAF mutations in multiple cancer types illustrates the key role of the RAF-MEK-ERK signaling pathway in mediating RAS oncogenesis. Although MEK inhibitors in particular are potent and specific, their on-target toxicity has severely limited their clinical efficacy against RAS-driven tumors and strategies to enhance their therapeutic index are sorely needed. RAF activation is a complex process that involves multiple regulatory steps in addition to RAS binding. Key among them is the dephosphorylation of a conserved inhibitory site (S259-RAF1, S365-BRAF). We have shown that a ternary complex comprising SHOC2, MRAS and PP1 functions as a specific “S259” RAF phosphatase holoenzyme. Gain-of-function mutations at this site in RAF, and indeed SHOC2 itself, are found in the RASopathy Noonan syndrome. We have used CRISPR to knock out SHOC2 in a panel of NSCLC cell lines and show that SHOC2 inhibition strongly sensitizes RAS-mutant cells to MEK inhibitors. SHOC2 inhibition lowers the concentration of MEK inhibitor required for cytotoxicity and drives tumor regressions in xenograft models. Biochemically, we show that SHOC2 inhibition impairs the dimerization of RAF upon signaling rebound after MEK inhibitor treatment, and thereby potentiates the duration of ERK-pathway inhibition. In vivo, conditional SHOC2 ablation in the adult mouse is relatively well tolerated, and strikingly SHOC2 inactivation in the lung suppresses tumor initiation in KRAS-driven models to prolong overall survival. Taken together, our results suggest the SHOC2 complex may provide an attractive target for therapeutic intervention—both as a monotherapy and by significantly enhancing the therapeutic index of clinically available MEK inhibitors. Citation Format: Greg G. Jones, Isabel Boned Del Rio, Sibel Sari, Aysen Sekerim, Lucy C. Young, Nicole Hartig, Mona A. El-Bahrawy, Miriam Molina-Arcas, Julian Downward, Pablo Rodriguez-Viciana. The SHOC2 phosphatase complex as a therapeutic target for ERK-pathway inhibition in RAS-driven tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B26.

ERK Inhibitor LY3214996 Targets ERK Pathway-Driven Cancers: A Therapeutic Approach Toward Precision Medicine.

The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

An Open-Label Phase I/IIa Study to Evaluate the Safety and Efficacy of CCS1477, a First in Clinic Inhibitor of the p300/CPB Bromodomains, As Monotherapy in Patients with Advanced Haematological Malignancies

An Open-Label Phase I/IIa Study to Evaluate the Safety and Efficacy of CCS1477, a First in Clinic Inhibitor of the p300/CPB Bromodomains, As Monotherapy in Patients with Advanced Haematological Malignancies

Abstract LB-272: KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition

Abstract Purpose: This work assessed the antitumor activity of selective small molecule IRAK4 degraders in human ABC DLBCL cell lines in vitro and in tumor xenograft models in vivo, alone and in combination with BTK inhibition. Introduction: ABC DLBCL comprises approximately 45% of DLBCL and has a worse outcome with R-CHOP chemotherapy compared to GCB DLBCL. Activating mutations in MYD88 occur in 30-40% of ABC DLBCL; L265P, the most prevalent MYD88 mutation, causes constitutive assembly and activation of the Myddosome. IRAK4 kinase and scaffolding functions are essential for full signaling through the Myddosome to NFκB and MAPK pathways. Kymera Therapeutics is using a chemical knockdown strategy to develop heterobifunctional small molecule IRAK4 degraders, exemplified by KYM-001, for the treatment of MYD88-driven lymphomas. Methods: IRAK4 in human PBMC, ABC DLBCL cell lines and xenografts was quantified by immunoassays or targeted MS/MS. Myddosome signaling was monitored by mRNA and phosphoprotein endpoints. Cell viability and cell cycle were monitored by flow cytometry. Tumor xenograft studies were conducted by implanting human ABC DLBCL lines into immunocompromised mouse strains and assessing tumor volume. Key data: KYM-001 led to potent E3 ligase-dependent degradation of IRAK4. Notably, KYM-001 more effectively inhibited TLR-activated Myddosome signaling compared to IRAK4 kinase inhibitors in human PBMC. Degradation was highly selective for IRAK4 vs >10,000 other detected proteins in the MYD88 L265P mutant ABC DLBCL line OCI-LY10. IRAK4 degradation by KYM-001 resulted in cell cycle inhibition and apoptosis within 48-72 h in ABC DLBCL, with preferential activity in MYD88-mutant vs MYD88-WT cell lines. Oral dosing of KYM-001 showed dose-dependent antitumor activity in several mouse xenograft models of human MYD88-mutant ABC DLBCL at tolerated doses and schedules. In the OCI-LY10 model, tumor regression was associated with >80% degradation of IRAK4, establishing the pharmacodynamic effect required for maximal efficacy. Since alterations in BCR signaling and MYD88 frequently co-occur in B-cell malignancies, we investigated the potential for combined activity of IRAK4 degradation and BTK inhibition. In the OCI-LY10 xenograft model, which has activating mutations in both CD79B and MYD88, BTK inhibition with ibrutinib had an additive effect on KYM-001 antitumor activity. Conclusions: KYM-001 is a first-in-class, potent, selective and orally active IRAK4 degrader that causes tumor regression in ABC-DLBCL models. Degradation of IRAK4 removes both the kinase and scaffolding functions of IRAK4, and may be superior to kinase inhibition alone. These data support IRAK4 degraders as a promising new therapeutic opportunity for MYD88-driven lymphoma, both alone and in combination with other targeted approaches such as BTK inhibition. Citation Format: Joseph F. Kelleher, Veronica Campbell, Jesse Chen, Jared Gollob, Nan Ji, Hari Kamadurai, Christine Klaus, Henry Li, Christine Loh, Alice McDonald, Haojing Rong, Scott Rusin, Kirti Sharma, Dominico Vigil, Duncan Walker, Matt Weiss, Karen Yuan, Yi Zhang, Laurent Audoly, Nello Mainolfi. KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-272.

Abstract 3512: AZD7648, a potent and selective inhibitor of DNA-PK, potentiates the activity of ionising radiation and doxorubicin in vitro and causes tumour regression in xenograft models

Abstract DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex that is a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of DNA double strand breaks (DSB). Cancer therapies such as ionising radiation (IR) or topoisomerase II inhibitors (doxorubicin) generate DSB which can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). It can therefore be hypothesised a DNA-PK inhibitor would potentiate the activity of these agents. We have developed a highly potent and selective inhibitor of DNA-PK, AZD7648, which inhibits IR-induced DNA-PK S2056 auto phosphoryalation with an IC50 = 92 nM in A549 non-small cell lung cancer (NSCLC) cells. AZD7648 is a potent radiosensitiser where treatment in combination with IR led to a concentration-dependent reduction of the colony survival capacity of A549 and H1299 NSCLC cells (DEF37 at 100 nM = 1.7 and 2.5, respectively). In A549 cells, AZD7648 (≥1 µM) in combination with 2Gy IR for 48 hours led to a significant accumulation of cells arrested in the G2/M of the cell cycle, a 4-fold increase in micronuclei formation, and 3-fold induction of γH2AX, pATM S1981 and 53BP1 foci formation compared with IR alone. AZD7648 was also found to combine synergistically with doxorubicin in a panel of ovarian and triple negative breast cancer (TNBC) cell lines in cell growth inhibition assays when applying the Loewe additivity model (synergy scores 4 - 35). In vivo the combination of AZD7648 with IR (5x 2Gy) induced tumour regression in H1299 and A549 NSCLC xenografts in a dose-dependent manner (84 and 11% regression respectively), while monotherapy treatment only achieved tumour growth inhibition. In these two models the increased activation by IR of three primary DNA-PK pharmacodynamic markers, pDNAPK (S2056), pRPA32 (S4/8) and γH2AX, was inhibited by AZD7648 treatment (70-90% inhibition 2 h after IR + AZD7648). Similarly, liposomal doxorubicin (2.5 mg/kg weekly) in combination with AZD7648 (37.5 mg/kg bid) induced tumour regressions in the BT474c ER+ breast cancer xenograft model and in a TNBC PDX model (63% and 33% regression respectively), while monotherapy treatments only achieved tumour growth inhibition. These data confirm that DNA-PK inhibition with AZD7648 enhances the efficacy of a range of DSB inducing agents in vitro and in vivo, providing a clear rationale for its clinical investigation. Citation Format: Jacqueline H. Fok, Antonio Ramos-Montoya, Neil James, Mercedes Vazquez-Chantada, Valeria Follia, Ankur Karmokar, Anna Staniszewska, Lenka Oplustil O’Connor, Emma Dean, Simon J. Hollingsworth, Barry R. Davies, Elaine B. Cadogan. AZD7648, a potent and selective inhibitor of DNA-PK, potentiates the activity of ionising radiation and doxorubicin in vitro and causes tumour regression in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3512.

Abstract 4328: Epigenetic modulation of SSTR2 expression provides the potential to broaden PEN-221 treatment population

Abstract The expression of somatostatin receptor subtype 2 (SSTR2) is increased in up to 80% of neuroendocrine tumors (NETs), 20-40% of small cell lung cancers (SCLC) and a number of other tumor types. As a cell surface receptor, SSTR2 is a logical target for a drug conjugate. However, marginal, or loss of, expression of SSTR2 in tumor tissues has potential to limit the benefit from such conjugates. Recent published preclinical studies have demonstrated that epigenetic modulators, such as HDAC inhibitors, can result in increased SSTR2 expression with treatment. Given that the expression of SSTR2 can be variable from patient to patient and in some cases low to absent, we hypothesized that the combination of epigenetic modulators with an SSTR2 target therapy may provide a benefit for patients who are not eligible for these targeted approaches on their own. PEN-221 is a miniature drug conjugate of an SSTR2 targeting peptide attached to the potent cytotoxic DM1 through a cleavable disulfide linker. PEN-221, currently in Phase 1/2a (NCT02936323), is designed as a potent and selective anticancer agent to treat patients whose tumors express SSTR2. The targeting peptide of PEN-221 selectively binds to SSTR2, triggers receptor internalization leading to the accumulation of the DM1 to provide antitumor activity by disrupting microtubule networks causing apoptosis and mitotic catastrophe. In preclinical studies, single agent PEN-221 treatment leads to complete and sustained tumor regression in xenograft models that over-express SSTR2. We hypothesize that increased expression of SSTR2 as a result of epigenetic modulation from HDAC or other epigenetic modulators, may allow for the broadening of PEN-221 treatable tumors. To evaluate these hypotheses, we have combined PEN-221 and epigenetic modulators in preclinical models of cancer. Efficacy studies were carried out in models expressing various degrees of SSTR2, and combinations resulted in greater efficacy than that of the single agent. In addition, a pharmacodynamic assessment of epigenetic modulator treatment on SSTR2 expression levels was performed. These data demonstrate that combination of PEN-221 and epigenetic modulators provide greater efficacy than single agent activity alone and support the evaluation of such combinations in the clinical setting. Citation Format: Samantha Perino, James Quinn, Jessica Freda, Brian White, Scott Bailey, Viren Bhatia, Beata Sweryda-Krawiec, Mark Bilodeau, Jeffrey D. Bloss, Kerry Whalen, Richard Wooster. Epigenetic modulation of SSTR2 expression provides the potential to broaden PEN-221 treatment population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4328.

Highlights of This Issue

The complex, highly interdependent nature of intracellular processes often hinders drug discovery. Hoang and colleagues report on a counter example by coupling the cytotoxicity of pancreatic-type ribonucleases (ptRNases) with extant cancer therapies. They discover that the ERK pathway is responsible for phosphorylating a cytosolic ribonuclease inhibitor protein and, thereby, confounding ptRNase cytotoxicity. ERK-pathway inhibitors undo this effect. The authors demonstrate this surprising synergy between seemingly unrelated processes by co-administering a clinical ptRNase (QBI-139) and KRAS/BRAF/MEK inhibitors to human lung cancer and melanoma cells. Such a combination could enhance the efficacy of kinase inhibitors and minimize drug resistance.The high incidence of KRAS and CDKN2A mutations in pancreatic cancer supports dual targeting of MEK and CDK4/6 to treat this disease. Maust and colleagues show that the combination of trametinib and palbociclib elicits synergy and is highly active against two adenosquamous pancreatic cancer models. Sensitive tumors showed strong downregulation of cyclooxygenase-2 (COX-2) in response to co-targeting of MEK and CDK4/6. Furthermore, genetic manipulation of COX-2 expression blunted effectiveness of combination treatment. These results suggest a prognostic role for COX-2 in the identification of patients who might derive the greatest therapeutic benefit from this combination strategy.Targeting androgen receptor (AR) is an attractive therapeutic strategy for breast cancer (BC) and PARP inhibitors are effective for treating BC patients with germline BRCA 1/2 mutation. Min and colleagues explored the potential therapeutic effects of AR inhibitor AZD3514 and PARP inhibitor olaparib in BC cells. AZD3514 and olaparib co-treatment showed antitumor effect caused by compromised DNA damage repair activity. AR inhibition attenuated ATM-chk2 axis activity via downregulation of NKX3.1 mediated by TOPORS, subsequently led to enhance sensitivity to olaparib. The combination therapy of AR inhibitor with PARP inhibitor could expand the clinical usage of PARP inhibitor by inducing HRD-phenotype for AR-positive BC patients.Antibody-drug conjugate (ADC) technology is limited by the diversity of payloads that are efficacious and tolerated in this delivery format. NAMPT is a critical enzyme in NAD biosynthesis, and inhibitors are potently cytotoxic due to disruption to energy metabolism. Neumann and colleagues describe the development of ADCs with NAMPT inhibitor payloads that deplete NAD in cells and induce tumor regression in xenograft models, while improving the tolerability of the drug class in rodent toxicology models. These findings introduce a new mechanism of action to the ADC field with promise to translate into efficacious and tolerated clinical agents.

RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.