Activity In Preclinical Models Research Articles

Abstract PO5-05-05: Clinical outcomes of CDK4/6 inhibitors in patients with bone only metastatic breast cancer

Abstract Background: CDK4/6 inhibitors (CDK 4/6i) have significantly advanced the treatment of hormone receptor-positive (HR+) metastatic breast cancer in the last decade. Several clinical trials including PALOMA, MONALEESA, and MONARCH have shown improved progression-free survival (PFS) rates when CDK4/6i were combined with endocrine therapy (ET). These results led to recommending their use in combination with ET as the first line of therapy in the metastatic settings. Bone metastasis is the most common site of disease in patients with HR+ metastatic breast cancer, about 40% of whom have bone as the only site of metastasis (bone-only disease [BoD]). While previous studies have shown that BoD can have a unique clinical course and a more favorable prognosis compared to non-BoD metastasis, most of the aforementioned CDK 4/6i clinical trials have not shown significant improvements in PFS in BoD subset of patients. Here we aimed to investigate the survival outcomes of BoD patients at a single institution center and evaluate CDK 4/6i activity in preclinical models of bone metastasis in breast cancer. Patients and methods: Patients with HR+, HER2-negative metastatic breast cancer from a retrospective CDK4/6i study at Washington University School of Medicine were included in this analysis. We selected all the patients who received CDK4/6i in the first- or second-line settings from June 2014 to March 2020. Outcomes were collected through June 10, 2023. The Kaplan-Meier method with log rank test and multivariable cox model were used for survival analysis. Results: 114 female patients (n=75 first line, n=39 second line, n=20 African American, n=94 Caucasian) were included in this study. All patients received Palbociclib as the first exposure to CDK4/6 inhibitor in combination with ET for at least two months. Median age was 60 years (range, 28-87) and forty-seven patients (41%) were in the BoD group. Survival outcomes for BoD group were compared to the rest of patients who had either bone and visceral or visceral only metastatic lesions (BV group). Our results demonstrated a significantly reduced median PFS in BoD group compared to BV group (13.1 months [95%CI:8-18.2] vs 19.6 months [95%CI:6.8-32.4], p=0.01) respectively. There was no significant difference in overall survival between BoD and BV groups (36.6 months [95%CI:32.2-41] vs 41.7 months [95%CI 36.1-47.2] p=0.74) respectively. In multivariate analysis regarding the associated risk factors with PFS, after adjusting for age and race, BoD had a HR of 1.62 (95%CI:1.04-2.54) for PFS. Among the 75 patients who received CDK4/6i as their first line of therapy, BoD (n=32 patients [42.7%]) also showed an inferior (p=0.05) median PFS (20 months [95%CI 12.9-27]) compared to BV group (n=43 patients [57.3%], median PFS=28.4 months [95%CI: 16.3-40.4]). To investigate potential treatment resistance mechanisms in bone metastases, we used the triple negative 4T1 and the ER+ luminal B PyMT Bo1 breast tumor cell line derived mammary gland models in immunocompetent mice. Single agent CDK4/6i, ribociclib, decreased primary mammary gland tumor burden. This effect was abrogated when CD8+ T-cells were depleted. In the bone colonization model using these cell lines, ribociclib had little effect on bone tumor burden. Arg1+ immune suppressive macrophages were significantly decreased in the primary mammary tumor but not in the bone tumor after ribociclib administration, suggesting a relative treatment resistance and immune suppression in the bone tumor microenvironment compared to the primary mammary gland. Conclusion: CDK4/6i play a significant role in the treatment of patients with HR+ metastatic breast cancer. Our single institution, retrospective study and preclinical data suggests that CDK4/6i may not be as effective for the treatment of tumors residing in the bone microenvironment, warranting future studies to understand the mechanisms of CDK4/6 inhibitors action in bone versus visceral metastases. Citation Format: Mahta Mardani, Suleyman Noordeen, Katherine Clifton, Cynthia Ma, Jingqin Luo, Jing Xi, Nusayba Bagegni, Foluso Ademuyiwa, Rama Suresh, Ashley Frith, Andrew Davis, Ron Bose, Lindsay Peterson, Shana Thomas, Yu Tao, Takayuki Kobayashi, Jingyu Xiang, Yalin Xu, Xinming Su, Katherine Weilbaecher. Clinical outcomes of CDK4/6 inhibitors in patients with bone only metastatic 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 PO5-05-05.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract Targeted Protein Degradation (TPD) is a novel therapeutic modality that uses heterobifunctional small molecules to redirect the cellular machinery responsible for protein degradation and cellular homeostasis, the ubiquitin-proteasome system (UPS), toward specific disease-causing proteins through spatial proximity with the use of affinity rather than occupancy-based catalysis, delivering a potentially transformative new way to treat disease. To realize on the promise of this disease-agnostic technology, we have taken a unique approach to target selection, where we focus on targets that are either undrugged or inadequately drugged within key signaling pathways with clear clinical validation and validation through human genetics/causal biology, and where TPD is the best or the only solution - defining a clear path to early differentiation from existing technologies and agents. Additionally, our comprehensive drug discovery engine utilizes computational tools, fit-for-purpose technologies, and quantitative translational models to design potent and selective degraders and drive consistent fidelity of translation of safety, pharmacokinetics (PK), pharmacodynamics (PD), and early efficacy from preclinical models to patients, as observed across all our clinical programs. Within oncology, TPD has the potential to redefine treatment paradigms given the existence of numerous clinically validated targets coupled with the limitations of existing cancer therapies in key areas such as druggability, selectivity, resistance, potency, and durability. Here, we share vignettes that highlight the advantage of TPD over other technologies and strong preclinical to clinical translation from our first-in-class oncology programs. KT-333 is a potent, highly selective, heterobifunctional small molecule protein degrader of signal transducer and activator of transcription 3 (STAT3), a traditionally undrugged transcription factor recognized as a key component of the Janus Kinase (JAK)-STAT signaling pathway with both tumor cell intrinsic and tumor cell extrinsic effects on the tumor microenvironment. Although multiple drugs have been approved that target upstream effectors signaling through STAT3, no known drugs selectively block STAT3 broadly across all relevant cell types or address both phosphorylation-dependent and -independent functions of STAT3. For these reasons, we believe that STAT3 degraders may provide a solution to the development of targeted and selective drugs to address multiple STAT3 dependent pathologies. In preclinical studies, KT-333 has shown durable single agent antitumor activity across multiple liquid and solid tumor models. Additionally, degradation of STAT3 has both antiproliferative and proapoptotic effects on tumor cells as well as immunomodulatory effects on tumor cells and the tumor microenvironment. This leads to robust anti-tumor activity in preclinical models of STAT3-dependent T-cell lymphomas and syngeneic models of solid tumors combined with immunotherapies such as checkpoint blockade - which only work in a small percentage of patients. Interim data from the Phase 1a clinical trial demonstrated early signs of antitumor activity at doses that were generally well-tolerated and associated with substantial STAT3 knockdown in blood and tumor. Preclinical to clinical translation showed strong responses in both cutaneous T-cell lymphoma (CTCL) and in Hodgkin's lymphoma as well as induction of an Interferon (IFN)-ƴ response in tumor and blood that preclinically was shown to enhance the response of solid tumors to anti-PD-1 drugs. We believe this supports KT-333’s potential to address both hematological malignancies as a single agent and solid tumors as a potential novel combination therapy with anti-PD-1 or other targeted therapies. KT-253 is a highly potent heterobifunctional degrader of the murine double minute 2 (MDM2) oncoprotein, a key E3 ligase that modulates the most common tumor suppressor, p53. p53 is a transcription factor that regulates cellular responses to stress and guides cell fate decisions such as cell cycle arrest, DNA repair, senescence, and apoptosis. Loss of p53 function leads to inability of cells to respond to cellular stressors such as DNA damage and leads to genetic instability, a hallmark of cancer. Notably, p53 remains intact in close to 50% of cancers. While small molecule inhibitors have been developed to stabilize and upregulate p53 expression, they have been found to induce a feedback loop that increases MDM2 protein levels, which can repress p53 and therefore limit their efficacy. In preclinical studies, KT-253, unlike small molecule inhibitors of MDM2, has shown the ability to overcome the MDM2 feedback loop and thereby robustly activate the p53 pathway, even with brief exposures. KT-253 more effectively upregulates and activates p53 in tumors in vivo compared to small molecule MDM2 inhibitors, and this translates into antitumor responses in p53 wild-type acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and Merkel cell carcinoma (MCC) models with just single doses of KT-253. These results support an intermittent dosing strategy for KT-253 that enables maximum p53 pathway activation for a limited period of time in tumor cells leading to rapid apoptosis while mitigating the impact of target engagement in normal cells in order to improve the therapeutic index relative to MDM2 small molecule inhibitors. Interim data from the Phase 1a clinical trial show evidence of target engagement and p53 pathway activation, along with initial signs of antitumor activity without dose limiting toxicities (DLTs), including typical hematological toxicity, and are supportive of our therapeutic hypothesis for MDM2 degraders and the potential to improve the therapeutic index compared to MDM2 small molecule inhibitors. We demonstrated proof of mechanism in the first 2 dose levels (DLs) with exposure-dependent upregulation of plasma GDF-15 levels. GDF-15 is a transcriptional target of p53, and as such it serves as a downstream biomarker of p53 upregulation following MDM2 degradation. This was consistent with the pattern of p53 activation in preclinical models associated with KT-253 antitumor activity. One patient with a partial response had MCC metastatic to abdominal lymph nodes and skin who had previously been treated with chemotherapy as well as multiple different immune checkpoint inhibitors. Lymph node metastases were responding after the first 2 cycles of treatment, as was the skin metastasis, and after 4 cycles there was an approximately 60% reduction in nodal tumor burden and resolution of the skin metastasis. In summary, these preclinical and early clinical findings support a clear degrader advantage by eliminating oncogenic proteins in key signaling pathways and validate our differentiated target selection and translational strategies to advance a new generation of medicines. Citation Format: Nello Mainolfi. Targeting validated but un-drugged oncogenes with small molecule protein degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY12-01.

Abstract LB063: Anti-tumor activity in preclinical models utilizing a conditional bispecific redirected activation molecule (COBRA) targeting Her2

Abstract Among strategies to recruit cytotoxic T-cells into the tumor micro-environment (TME) are the use of T-cell engager molecules. One such therapeutic modality are molecules that recognize specific tumor associated antigens (TAA) but are activated preferentially in the TME. Conditional Bispecific Redirected Activation molecules (COBRA) are designed to be administered as a prodrug, and cleaved via matrix metalloproteinase to engage with both a tumor TAA and CD3 (Panachi, et.al. MAbs 2020 12(1) 1792130). In this study, we sought to determine the preclinical efficacy of a COBRA molecule targeting the Her2 receptor. Sub-cutaneous xenograft models (SCID mice using adoptive T-cell transfer) were utilized representing various levels of the TAA (HT-55, SNU-16, JIMT-1, and HCC827) to measure both efficacy and mechanism of action (T-cell extravasation). The COBRA molecule was dosed at 0.03, 0.1, 0.3, and 1 mg/kg using a Q3dx7 schedule. A non-cleavable COBRA molecule with the Her2 TAA was included as a control at 1 mg/kg. Efficacy was determined by caliper measurements of sub-cutaneous tumor size twice per week after dosing, and T-cell extravasation into the TME was measured by immunohistochemistry (IHC) at days 10, 19, and termination. IHC data was quantified using HALO imaging software. In our study, we measured dose-dependent tumor regression and/or stasis in all models tested. No anti-tumor activity was measured after dosing with the non-cleavable COBRA molecule. T-cell extravasation as measured by IHC and Halo analysis followed a dose and time dependent course. Comparison of Day 19 T-cell extravasation (one day after the last dose) showed a linear relationship to TAA expression. Our data indicate that treatment with a COBRA molecule in preclinical models with a Her2 TAA results in anti-tumor activity as measured by tumor stasis/regression, and exhibit the expected mechanism of action as evaluated by T-cell extravasation into the TME. Citation Format: Bradley Stringer, Johara Chouitar, Jacqualine Cerbone, Sarah Fortin, Alina Ainbinder, Cassie Kysilovsky, Brittany Scott, Antara Banerjee. Anti-tumor activity in preclinical models utilizing a conditional bispecific redirected activation molecule (COBRA) targeting Her2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB063.

Abstract CT162: Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor

Abstract Background ZL-1218 is an anti-chemokine receptor 8 (CCR8) humanized IgG1 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. CCR8 demonstrates a preferential expression in tumor-associated Tregs over peripheral Tregs. Thus, targeting CCR8 may allow for specific depletion of intratumoral Tregs without impacting peripheral Tregs/other immune cells. ZL-1218 is intended to decrease the intratumoral Tregs by binding to both high- and low-expressing CCR8+ cells and eliciting potent ADCC activity. ZL-1218 blocked CCR8 ligand CCL1 from binding to CCR8, and reduced Treg recruitment by blocking chemotaxis of CCR8+ cells (unpublished data). Additionally, ZL-1218 demonstrated enhanced antitumor activity in preclinical models in combination with anti-PD-1.  Methods This is a Phase I study of ZL-1218 as a single agent and in combination with pembrolizumab (P) to evaluate the safety, tolerability, pharmacokinetics/pharmacodynamics, and preliminary antitumor activity in subjects with advanced solid tumor malignancies. The study consists of Part 1 Dose Escalation and Part 2 Cohort Expansion. In Part 1, subjects will be treated with escalating doses of ZL-1218 alone and in combination with a fixed dose of P to identify the maximum tolerated dose (MTD) and/or the recommended expansion dose (RD).  The Bayesian optimal interval (BOIN) design is used and adopted with overdose control. Upon progression in the single agent group, a subject may enroll into combination therapy of ZL-1218 with P. Subjects will start at their respective single agent dose or the cleared combination dose level, whichever is lower. Part 2 will explore the combination with pembrolizumab in subjects with selected tumor types, with or without prior CPI therapy.   Major inclusion criteria include: (1) Subjects must have histologically confirmed and documented diagnosis of locally advanced unresectable/metastatic advanced solid tumor that is refractory/intolerant to standard treatment, or for which no standard treatment exists, and no greater than 3 prior lines of therapy in the relapse/refractory setting (2). Subjects must have at least one target lesion as defined by RECIST v1.1 on CT, PET/CT, or MRI scan. (3) Subjects must have a site of disease which is not previously irradiated and is safe and amenable to biopsy. Major exclusion criteria include: (1) Prior exposure to CCR8 inhibitor, or severe hypersensitivity (≥ Grade 3) to P and/or any of its excipients. (2) Subjects with symptomatic or uncontrolled brain metastasis requiring concurrent treatment, impaired major organ functions, active autoimmune disease, or active infections.  Part 1 Dose Escalation is ongoing in the US and EU.  Citation Format: Elena Garralda, Valentina Boni, David Vincente Baz, Arvind Chaudhry, Ignacio Gil Bazo, Martin Gutierrez, Jyoti Malhotra, Casal Guzman Alonso, Valentina Gambardella, Michael Chisamore, Renke Zhou, Maria Tea, Deborah Doroshow. Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT162.

Characterization of a novel anti-PVRIG antibody with Fc-competent function that exerts strong antitumor effects via NK activation in preclinical models

Poliovirus receptor-related immunoglobulin domain-containing protein, or PVRIG, is a newly discovered immune checkpoint that has emerged as a promising target for cancer immunotherapy. It is primarily expressed on activated T and natural killer (NK) cells, and once engaged with its ligand, PVRL2, it induces inhibitory signaling in T cells, thereby promoting the functional exhaustion of tumor-infiltrating lymphocytes (TILs). Here, we characterized IBI352g4a, a novel humanized anti-PVRIG antibody with Fc-competent function, explored the mechanism of its antitumor activity in preclinical models, and systemically evaluated the contribution of FcrR engagement to PVRIG blockade-induced antitumor activity. IBI352g4a binds to the extracellular domain of human PVRIG with high affinity (Kd = 0.53 nM) and specificity, and fully blocks the interaction between PVRIG and its ligand PVRL2. Unlike other immune checkpoints, IBI352g4a significantly induced NK cell activation and degranulation, but had a minimal effect on T-cell activation in in vitro functional assays. IBI352g4a induced strong antitumor effect in several preclinic models, through in vivo mechanism analysis we found that both NK and T cells contribute to the antitumor effect, but NK cells play predominant roles. Specifically, a single dose of IBI352g4a induced significant NK cell activation in TILs, but T-cell activation was observed only after the second dose. Moreover, the Fc effector function is critical for both NK cell activation and treatment efficacy in vitro and in vivo. Our study, for the first time, demonstrates that both NK activation and FcrR engagement are required for antitumor efficacy induced by PVRIG blockade.

Abstract 1911: Dato-DXd mediates anti-tumor activity in preclinical TROP2-expressing intracranial tumor model

Abstract Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 monoclonal antibody linked to a potent DNA topoisomerase I (TOP1) inhibitor payload via a plasma-stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd is currently under clinical investigation for the treatment of patients with solid tumors including non-small cell lung cancer (NSCLC), hormone receptor positive (HR+ BC) and triple negative breast cancer (TNBC). Pharmacotherapy of brain tumors can be limiting due to restricted drug delivery across blood brain and blood tumor barrier. Enhertu®, that uses the same DXd ADC technology, has reported clinical activity in patients with brain metastases from HER2+ breast cancer, but very little information is available on what drives ADC biodistribution and activity in CNS-involved cancers. Here, we investigated whether systemically administered ADCs can penetrate the brain microenvironment and mediate anti-tumor activity in a preclinical model. Luciferase-tagged H1373 (TROP2-expressing NSCLC) tumor cells were intracranially implanted into NSG mice. Tumor-bearing mice were dosed with Dato-DXd or matched isotype Control IgG-ADC (DAR4) at 10mpk 7 days or 14 days post intracranial tumor implant. Dato-DXd inhibited intracranial tumor growth better than Control ADC (Day 7: 105% TGI vs 38% TGI; Day 14: 65% TGI vs <10% TGI, respectively, compared to Vehicle). Immunohistochemistry analysis of brain tissue validated localization of Dato-DXd in the tumor, suggesting Dato-DXd can distribute into the local tumor microenvironment in this preclinical tumor model. Additionally, treatment with Dato-DXd provided a significant survival benefit over Control ADC (Median survival of 63 days vs 43 days, P=0.0002). This preclinical study supports the inclusion of Dato-DXd in treatment of patients with CNS-involved tumors. Understanding the pharmacological determinants of Dato-DXd activity in the CNS will help outline strategies to implement Dato-DXd-based treatment of patients with CNS-involved tumors. Citation Format: Kristen Jones Jones, Montira Suksomboon, SaraAnn rosenthal, Jacob Gordon, Corinne Reimer, Matthew Sung, Chetan Rane. Dato-DXd mediates anti-tumor activity in preclinical TROP2-expressing intracranial tumor model [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 1911.

Abstract 6579: DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models

Abstract Background: Tumor-associated mucin-1 (TA-MUC1) is a tumor-specific transmembrane glycoprotein with aberrant glycosylation. It is highly expressed in various human epithelial cancers, making it an attractive target for cancer therapy. DS-3939a is a TA-MUC1 targeting antibody-drug conjugate (ADC) structurally composed of a humanized anti-TA-MUC1 antibody, an enzymatically cleavable tetrapeptide-based linker, and a DNA topoisomerase I inhibitor (DXd) with a high drug-to-antibody ratio (DAR). In this study, the pharmacological activity of DS-3939a was evaluated using preclinical in vitro and in vivo models. Methods: TA-MUC1 expression was assessed by immunohistochemistry and flow cytometry. Binding specificity of DS-3939a was confirmed by enzyme-linked immunosorbent assay. Induction of DNA damage and apoptosis to cancer cells by DS-3939a treatment were detected by Simple Western System. In vitro cell growth inhibitory and in vivo antitumor activities of DS-3939a were evaluated using various cancer cell lines and patient-derived xenograft (PDX) models. Results: Many specimens across multiple cancer types showed positive staining for TA-MUC1, with a particularly high positive rate observed in bladder, lung, and breast cancers. DS-3939a specifically bound to aberrantly glycosylated MUC1 peptides by recognizing both the glycan and backbone peptide moiety of MUC1. In vitro, DS-3939a inhibited the growth of TA-MUC1-positive cancer cells but not of TA-MUC1-negative cancer cells. Neither a parental anti-TA-MUC1 antibody nor isotype control ADC inhibited the growth of these cells. In addition, treatment with DS-3939a and DXd alone induced DNA damage and apoptosis in cancer cells. These results demonstrate that DS-3939a exhibits TA-MUC1-dependent cell growth inhibition through DXd-mediated cytotoxic effect. Moreover, DS-3939a exhibited significant antitumor effects in the several TA-MUC1-positive cancer cell line-derived xenograft models and various PDX models. DS-3939a also induced strong tumor regression even after treatment of other cytotoxic ADCs in several xenograft models. Conclusion: Based on these preclinical results, DS-3939a could provide valuable therapy with potential benefits for patients with TA-MUC1-expressing tumors. A first-in-human phase 1/2 study in patients with advanced solid tumors is currently in progress (NCT05875168). Citation Format: Mayuko Yukiura, Kohei Takano, Kazuki Takahashi, Akiko Zembutsu, Michiko Kitamura, Yoshinobu Shiose, Kokichi Honda, Kazunori Oyama, Wataru Obuchi, Makiko Yamada, Riki Goto, Ken Sakurai, Kazuyoshi Kumagai, Takashi Kagari, Yuki Abe, Toshinori Agatsuma. DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical 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 6579.

Abstract 665: MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib

Abstract Myelofibrosis (MF) is a pathological chronic bone marrow (BM) condition characterized by abnormal growth of erythroid and megakaryocytic precursors and deposition of fibrotic tissue. The JAK/STAT pathway plays an important role in MF (Cardoso 2015), and multiple JAK1/2 inhibitors (JAKis) are approved for MF treatment. Despite initially limiting disease symptoms, JAKis often fail due to persistent JAK2 activation and survival of MF-driving cells, hence new treatment strategies are highly needed. PIM kinases also contribute to MF pathogenesis. PIM1 is highly overexpressed in granulocytes, peripheral blood mononuclear cells (PBMCs), and BM of patients with MF. PIM1/2 expression is dependent upon JAK2 signaling in myeloproliferative neoplasms (MPNs). Hence, PIM inhibitors represent potential therapeutic targets for MPNs and have been evaluated in several preclinical and clinical studies (Rampal 2021). MEN1703/SEL24 (MEN) is an oral, first-in-class, dual PIM/FLT3 kinase inhibitor in development for hematologic malignancies. This study aims to investigate the efficacy of MEN alone and in combination with the JAKi ruxolitinib (RUX) in preclinical MF models and to elucidate the underlying signaling pathways. MF cell lines (JAK2V617F and JAK2 wild type) were used in MTS assays to assess the in vitro cytotoxicity of MEN alone and in combination with RUX, compared with the PIM inhibitor TP-3654 (Dutta 2022). Cell cycle distribution and signaling pathways were analyzed via propidium iodide/FACS analysis and immunocapillary electrophoresis, respectively, upon treatment with the single agents and MEN + RUX. In vivo efficacy was assessed in a murine xenograft model treated with MEN +/- RUX. In vitro studies showed single agent MEN activity with an IC50 ranging from 0.3 to 2.3 µM. Moreover, a synergistic drug interaction was found with MEN + RUX in all tested cell lines (Combination Index [CI] range 0.41-0.80). MEN presented cytotoxic and cytostatic capacity, inducing apoptosis and arresting a proportion of cells in G2/M phase. This effect was maintained in combination with RUX. Signaling pathway analysis showed that MEN inhibits phosphorylation of STAT proteins and decreases Mcl1 protein expression. These effects were enhanced with MEN + RUX. Compared with TP-3654, MEN was more effective both as a single agent (IC50 2-3 fold lower than TP-3654) and in combination with RUX (mean CI of 0.77-0.80 compared to 0.79-0.98 with TP-3654). MEN showed anti-tumoral efficacy in MF preclinical models, moderately higher than TP-3654, exhibiting in vitro activity at clinically relevant concentrations. Importantly, MEN combined with the standard of care RUX was synergistic, and molecular analyses confirmed the role of PIM downstream target inhibition. Our results support the therapeutic potential and relevance of MEN in MF treatment strategies. Citation Format: Maria Luisa Iannitto, Diego Bisignano, Sonia Zicari, Francesco Belli, Alessio Fiascarelli, Simone Talucci, Giuseppe Merlino, Vittoria Nicolis di Robilant, Clelia Irrissuto, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Monica Binaschi. MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib [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 665.

Abstract 1656: ISM6331, a novel and potent pan-TEAD inhibitor, exhibits strong anti-tumor activity in preclinical models of Hippo pathway-dysregulated cancers

Abstract The Hippo-YAP/TAZ-TEAD signaling pathway plays critical roles in tumorigenesis and tumor progression. Genetic alterations of Hippo pathway regulators, such as NF2 or LATS1/2, have been shown to induce YAP/TAZ-TEAD transcriptional activity, promoting initiation and progression of many solid tumors. In addition, many studies have shown that YAP/TAZ-TEAD activation mediates resistance to multiple targeted therapies, particularly EGFR-KRas-MAPK pathway inhibitors. The TEAD family of transcription factors (TEAD1/2/3/4) share structural similarity and have overlapping functions in tumor development and TEAD auto-palmitoylation is critical for its interaction with YAP/TAZ and its transcriptional function. Therefore, blocking TEAD palmitoylation and allosterically disrupting YAP/TAZ-TEAD-mediated transcriptional regulation, is emerging as a promising approach for the treatment of tumors with Hippo pathway dysregulation. In this work, ISM6331, a novel and potent TEAD inhibitor with excellent inhibition against TEAD1/2/3/4 was identified leveraging an AI generative model. ISM6331 reversibly binds to the TEAD palmitoylation site and significantly suppresses TEAD transcriptional activity. ISM6331 potently and selectively inhibited the growth of many Hippo pathway-dysregulated tumor cells and dose-dependently suppressed the expression of TEAD target genes in vitro. In NF2-deficient or LATS1/2-mutant in vivo mesothelioma models, ISM6331 exhibited promising anti-tumor efficacy as monotherapy in a dose-dependent manner from 3 to 30mg/kg. ISM6331 also showed synergism with EGFR or KRasG12C inhibitors in inhibiting tumor cell growth and overcame drug resistance in in vitro and in vivo models. In addition, ISM6331 showed excellent pharmacokinetic properties in multiple preclinical species and was well-tolerated in GLP-toxicity studies. In summary, ISM6331 has promising potential to treat Hippo pathway-dysregulated tumors as monotherapy as well as in combination with targeted therapies. Citation Format: Qi Li, Jainfei Wan, Jinxin Liu, Jing Shang, Jiaojiao Yu, Wei Zhu, Celia X.-J. Chen, Junwen Qiao, Ling Wang, Man Zhang, Xiao Ding, Supriya Bavadekar, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM6331, a novel and potent pan-TEAD inhibitor, exhibits strong anti-tumor activity in preclinical models of Hippo pathway-dysregulated cancers [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 1656.

Abstract 20: KSQ-001EX: An engineered TIL therapy manufactured from a clinical-scale, feeder-free process for the treatment of solid tumor indications

Abstract Tumor Infiltrating Lymphocyte (TIL) therapy is an autologous adoptive cell therapy involving the isolation, ex vivo expansion and infusion of tumor infiltrating lymphocytes (TIL) into late-stage cancer patients for therapeutic benefit. While clinical responses to TIL therapy have been observed in metastatic melanoma, efficacy in other tumor types including non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC) [SM1] is limited by the intrinsic functionality and persistence of transferred T cells and the immunosuppressive tumor microenvironment. To improve the clinical outcome of TIL therapy, we have developed KSQ-001EX, an engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene, for the treatment of solid tumors. SOCS1 is a negative regulator of cytokine signaling in T cells that we previously identified as a top target restraining T cell anti-tumor function and persistence using genome-wide in vivo CRISPR screens. To streamline and shorten TIL manufacture, we established a next-generation manufacturing process with resected tumors or core biopsies as starting materials that eliminates the use of feeder cells and REP, shortening the length of manufacture to 22 days. We demonstrated robust manufacture of cryopreserved KSQ-001EX at clinical scale from NSCLC, HNSCC, checkpoint refractory melanoma, and heavily pre-treated colorectal carcinoma (CRC). KSQ-001EX manufactured from clinical-scale runs were highly viable (>90%) following cryopreservation and thaw, with > 90% SOCS1 on-target editing and complete knockdown of SOCS1 at the protein level. Importantly, KSQ-001EX across all indications assessed showed high frequency of CD8 (median ~80%), an attribute associated with TIL clinical responses. KSQ-001EX also retained high diversity of TCR repertoire. Consistent with the biology of SOCS1 editing, KSQ-001EX exhibited greater cytotoxicity and higher IFNγ production against tumor spheroids in comparison to TIL and enhanced induction of pSTAT4 upon IL-12 stimulation, demonstrating heightened sensitivity of KSQ-001EX to cytokines. In conclusion, KSQ-001EX, an engineered TIL therapy with CRISPR/Cas9 mediated inactivation of SOCS1, can be manufactured at clinical doses from surgical resections or core biopsies within 22 days using a simplified feeder-free manufacturing process from multiple indications. KSQ-001EX exhibits heightened anti-tumor activity in preclinical models, with therapeutic benefit to be evaluated in a planned clinical trial in patients with treatment-refractory melanoma, NSCLC, and HNSCC. [SM1]Spell out these at first instance? Citation Format: Sharon Lin, Gustavo Martinez, Marie-Andrée Forget, Katrina Adlerz, Mitali Ghose, Leila Williams, Paul Dunbar, Frank Thompson, Fiona Sharp, Hugh Gannon, Conor Calnan, Ashish Yeri, Dipen Sangurdekar, Donald Sakellariou-Thompson, Samantha Fix, Tamara Griffith, Ellie Jafari, Jane Zulovich, Priya Balasubramanian, Erin Willert, MIcah Benson, Chantale Bernatchez, Karrie Ka Wai Wong. KSQ-001EX: An engineered TIL therapy manufactured from a clinical-scale, feeder-free process for the treatment of solid tumor indications [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 20.

Abstract 5988: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified a ubiquitin complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines. USP7 inhibition induced apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70, stabilizing Ku70 at the basal level, leading to stabilization of Ku70/c-FLIPL/Bax complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, due to the inhibition of Ku70 and c-FLIPL proteins degradation leading to the blockade of caspase-8 activation and the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, CARLA SAMPAIO, PETER E. ZAGE. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma [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 5988.

Abstract 1661: Preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor

Abstract Background: Although therapeutic agents targeting alterations in the MAPK pathway have achieved great clinical success, the overall benefit is still suboptimal. Several studies have shown that reactivation of MAPK signaling is the main basis to compromise the efficacy. Thus, co-inhibition of ERK, the terminal master kinase of MAPK pathway, and the upstream targets may effectively shut down the MAPK signaling cascade and induce deeper and more durable anti-tumor activities. Herein, we present the preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor, discovered and being currently developed in phase I clinical trial (NCT04908046) by HUTCHMED. Methods: The inhibition on ERK 1/2 kinase was determined using Z-LYTE™ kinase assay. The selectivity of HMPL-295 was evaluated against 394 kinases (KinaseProfilerTM) and 86 safety-related proteins (SafetyScreen87TM) by Eurofins. Cellular target inhibition on phosphorylation of RSK (p-RSK) was detected by ELISA. In vitro anti-proliferation activity of HMPL-295 as a single agent was measured by CCK-8 or CellTiter-Glo luminescent assay. Colony formation assay was used to evaluate the combination effect of HMPL-295 with targeted therapy. Multiple tumor models with RAS/RAF/MAPK pathway activation were applied in immune-deficient mice to investigate the anti-tumor activity of HMPL-295 as a single agent or in combination with either chemotherapy or targeted therapy. Results: HMPL-295 strongly inhibited ERK1 and ERK2 kinase activities with equal IC50 at 4 nM. In human colon cancer cell line COLO 205 harboring BRAFV600E mutation, HMPL-295 inhibited p-RSK, a direct substrate of ERK1/2, with an average IC50 of 180 nM from 3 independent experiments. With the inhibition of ERK and its downstream substrates, HMPL-295 treatment induced tumor cells arresting at G0/G1 phase via down-regulation of cyclin D1/p-RB signaling and consequently attenuated cell growth in a panel of MAPK pathway dysregulated tumor cell lines, but barely affected the growth of normal cells. Furthermore, HMPL-295 demonstrated favorable selectivity in the kinase and safety pharmacology profiling, indicating a low off-target risk. For in vivo studies, oral administration of HMPL-295 induced time- and dose-dependent inhibition of p-RSK and resulted in dose-dependent anti-tumor activity in several tumor models harboring KRAS or BRAF mutations. Moreover, HMPL-295 significantly improved the anti-tumor activity of standard-of-care chemotherapy as well as targeted agents in KRAS- or BRAF-mutant tumor models both in vitro and in vivo. Conclusion: HMPL-295 is a potent and selective ERK1/2 inhibitor which demonstrates strong anti-tumor activity in preclinical models, supporting further clinical evaluation. Citation Format: Jia Hu, Jun Ni, Yuanzhi Lv, Wenjun Li, Hui Zhang, Xin Li, Ying Yu, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor [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 1661.

Abstract 1351: Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic

Abstract Introduction: Blockade of the CTLA-4 pathway with ipilimumab (IPI), alone and in combination with nivolumab (anti-programmed death-1 antibody), has shown clinical benefit in multiple tumor types. However, not all tumors respond to IPI ± nivolumab, and peripheral effects can lead to immune-related adverse events. To enhance the therapeutic index of CTLA-4-directed therapy, a proprietary Probody® therapeutics technology platform was used to develop a modified version of IPI. BMS-986288 is an anti-CTLA-4 antibody that combines a Probody® therapeutic masking peptide and protease-cleavable linker, designed to localize anti-CTLA-4 activity to the tumor microenvironment, with an NF Fc region hypothesized to enhance antigen-presenting cell (APC)-mediated T-cell priming and regulatory T cell (Treg) modulation via increased binding to the FcγRIIIA (CD16) receptor. Here, we present the characterization of the mechanism of action and pharmacodynamic (PD) response of BMS-986288 in preclinical models. Methods: APC-mediated T-cell priming and antigen-specific responses were evaluated in a superantigen model using peripheral blood mononuclear cells (PBMCs) and human CTLA-4 knock-in (KI) mice. Antitumor activity and immune cell population changes were assessed in an MC38 tumor model implanted in human CTLA-4 KI mice. Intratumoral Treg depletion was further investigated using patient-derived dissociated tumor samples as a physiologically relevant model. Peripheral PD effects and tolerability of BMS-986288 were evaluated in a non-human primate (NHP) Ad5 vaccine model. Results: Protease-cleaved BMS-986288 demonstrated enhanced APC-mediated T-cell priming compared with IPI in superantigen-stimulated PBMCs. Similarly, administration of superantigen staphylococcal enterotoxin B (SEB) peptide elicited an increased antigen-specific T-cell receptor (Vβ8+) T-cell response after treatment with cleaved BMS-986288 vs IPI in human CTLA-4 KI mice. In the MC38 tumor-bearing mouse model, BMS-986288 showed enhanced antitumor activity as compared to IPI, with tumor clearance in all mice. Although BMS-986288 enhanced intratumoral CTLA-4+ Treg depletion in the MC38 model, limited Treg depletion was observed in the patient-derived dissociated tumor model with an NF anti-CTLA-4 antibody without the masking peptide, potentially reflecting differences in CTLA-4 expression on human vs mouse Tregs. BMS-986288 reduced PD effects in the peripheral blood compartment and increased tolerability in NHP relative to IPI. Conclusion: BMS-986288 leverages unique characteristics to differentiate it from IPI, enhancing APC-mediated T-cell priming and anti-tumor activity while also reducing peripheral activity in preclinical models. These data support an ongoing phase 1/2 clinical trial of BMS-986288 (NCT03994601) in patients with advanced solid tumors. Citation Format: Amy Jhatakia, Mohammed Nasser, Anandaroop Mukhopadhyay, Kyeongah Kang, Courtni Newsome, Neha Gupta, Remie Gail Z. Mandawe, Felix Findeisen, Jack A. Lohre, Leslie Leung, Yun Wei, Joshua Dobroff, Karen Price, John Engelhardt, Mark Selby, Alan J. Korman, Nicholas Wilson. Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic [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 1351.

Abstract 2028: The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer

Abstract The NRF2 cytoprotective pathway, a drug target for many inflammation-related diseases, regulates immune cell function. While the anti-inflammatory nature of NRF2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs. Up to 30% of human lung adenocarcinomas acquire mutations in either NFE2L2 or its negative regulator KEAP1 which result in constitutive activation of the NRF2 pathway. However, despite our knowledge of the important immunomodulatory effects of NRF2, these effects are not well characterized in the context of cancer. With NRF2 activators now approved for clinical use, it is critical to understand the impact of these drugs in cancer. Triterpenoids including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl) are potent pharmacological NRF2 activators with demonstrated anti-cancer activity in preclinical models. In an early-stage preclinical model of lung cancer, CDDO-Me significantly decreased tumor burden in a dose- and Nrf2-dependent manner and improved immune signatures within the tumor microenvironment. However, most human lung cancers are not diagnosed until more advanced stages. To test CDDO-Me in an established tumor intervention model, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 knockout (KO) mice. Tumors were allowed to develop for 8 weeks post initiation, after which mice were fed either a vehicle control diet or CDDO-Me (50-100 mg/kg of diet) for an additional 8-12 weeks, alone or in combination with carboplatin and paclitaxel (C/P) at 50 mg/kg and 15 mg/kg, respectively, by IP injection every other week. CDDO-Me significantly (p < 0.05) decreased surface lung tumor counts 35-71% in a Nrf2-dependent manner, and C/P significantly (p < 0.001) reduced tumor burden 53-59% regardless of Nrf2 status. The combination of CDDO-Me + C/P reduced tumor burden in WT lungs by 84% (p < 0.05), more than either agent alone. Nrf2 KO mice had an approximately two-fold increase (p < 0.001) of surface tumors compared to WT mice in both studies, regardless of treatment. C/P reduced tumor burden similarly in Nrf2 WT and Nrf2 KO mice. Interestingly, the combination of CDDO-Me + C/P increased T cell infiltration into the lungs of WT mice, but this change was not observed in groups treated with either agent alone. Importantly, CDDO-Me did not decrease the efficacy of C/P but protected WT mice from mortality and weight loss and lowered white and red blood cell counts. These studies suggest that NRF2 activation in advanced lung cancers can still decrease tumor burden by favorably modulating the immune microenvironment and, importantly, complements the anti-tumor efficacy of conventional chemotherapy while decreasing drug toxicity. Citation Format: Jessica Ann Moerland, Karen T. Liby. The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung 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 2028.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p<0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p<0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p<0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p<0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p<0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian 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 5909.

Abstract 2902: CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Background: Although recent advances in immunotherapy for metastatic melanoma have transformed the treatment paradigm for a disease that once carried a grim prognosis, nearly 50% of patients fail to reach a complete response to immune checkpoint blockade (ICB) due to multiple mechanisms of tumor-induced immune resistance. These challenges are amplified in the CNS tumor microenvironment (TME) where metastatic brain melanoma (MBM) demonstrates diminished clinical benefit to ICB treatment. A deeper understanding of these mechanisms are required to improve therapeutic response to ICB for these patients. Our group has previously identified that aberrant activation of the myddosomal cascade through downstream interleukin (IL)-1 receptor-associated kinase (IRAK-4) signaling results in chronic stimulation of MAPK and NF-κB in MBM. Further investigation reveals myddosome signaling emanates in large part from the TME, resulting in reflexive immune suppression. CA-4948 (emavusertib), a small molecule inhibitor of IRAK-4, is capable of reaching therapeutic dose levels in the brain where it demonstrates on-target inhibition of this pathway. These data highlight a unique therapeutic opportunity where selective inhibition of immune-suppressive signaling via targeted IRAK-4 blockade may sensitize MBM to anti-PD-1 ICB. Methods Multispectral imaging of patient MBM samples were analyzed for myddosome expression in the TME. Melanoma preclinical models, including both primary site (cutaneous) and metastatic brain disease, were evaluated for tumor burden and survival in response to CA-4948 treatment alone and in combination with anti-PD-1 ICB. Immunological responses of tumor to treatment were evaluated in two transgenic reporter models, CCR2RFPCX3CR1GFP and IFNγIRES-EYFP, to determine both the distribution and activation of infiltrating immune cells. Tumor-associated myeloid cells were assessed for phenotypic markers by multiparameter flow cytometry. Results Myddosome signaling was found to be elevated in tumor-associated immune cells in patient MBM specimens. Treatment of preclinical melanoma with CA-4948 combined with anti-PD-1 ICB resulted in significantly reduced tumor growth and improved survival in three independent models. CA-4948 treatment was associated with reduced tumor recruitment of peripherally derived tumor-associated macrophages, as well as increased lymphocyte infiltration and improved pro-inflammatory signaling measured by increased IFNγ expression in the combinatorial setting. Conclusion Targeted inhibition of IRAK-4 with CA-4948 improves immune surveillance and activation in preclinical models when combined with anti-PD-1 ICB, resulting in reduced tumor growth and increased survival. Thus, CA-4948 may be an effective adjuvant treatment strategy alongside anti-PD-1 ICB for the treatment of metastatic melanoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Amanda M. Acevedo, Jeet A. Patel, Savannah L. Carpenter, Alisha Bhatia, Reinhard W. von Roemeling, Jamison Hoffman, Maureen Lane, Duane A. Mitchell. CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [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 2902.

Abstract 3909: PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer

Abstract Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair in conjunction with PARP1 by removal of poly (ADP-ribose) (PAR). Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting it is a potential therapeutic target for cancer therapy. Indeed, PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with BRCA1/2-mutation. In this study, we investigated PARG inhibitor (PARGi)-PDD00017273 activity in preclinical models of ovarian cancer cell lines and primary patient-derived organoids. PARGi exhibited sensitivity in a subset of ovarian cancer cell lines that was distinct from PARP inhibitor sensitivity. Initially, we evaluated a PARGi-sensitive cell line, RMUGS. By gradually increasing the concentration of PARGi, we generated drug resistant cells. PARGi treatment resulted in increased S phase specific PAR, DNA-PARylation, DNA damage, and replication stress in the sensitive cells but not in cells with de novo or acquired resistance. In the sensitive cells, PARGi exposure also increased accumulation of PAR at the replication fork resulting from unligated Okazaki fragment processing. More importantly, PARGi treatment or PARG loss caused accumulation of single strand DNA (ssDNA) gaps in sensitive cells, as detected by the DNA fiber assays combined with S1 nuclease treatment. Regardless of the BRCA-status, the induction of ssDNA gaps in ovarian cancer cells correlated with PARGi sensitivity. Consistent with an increase in ssDNA gaps, PARGi exposure increased p-RPA, a ssDNA surrogate, at the replication fork in sensitive cells. p-RPA sites also colocalized with PAR suggesting that accumulation of PAR occurs at the ssDNA sites, perhaps, leading to the ssDNA gaps. Collectively, these results suggested that toxic accumulation of PAR at the replication fork leads to accumulation of ssDNA gaps due to Okazaki fragment processing defects and ovarian cancer cell killing after PARG inhibition. We further assessed PARG inhibition in patient-derived organoids (PDOs) and patient-derived xenograft (PDX)-derived organoids (PDXOs) as model systems of ovarian cancer. PARGi exhibited monotherapy activity in specific HR-deficient PDOs and PDXOs with BRCA1 or RAD51C mutation and in a HR-proficient PDXO model. Importantly, PARGi exposure resulted in accumulation of ssDNA gaps in organoids of ovarian cancer and this phenotype directly correlated with drug sensitivity. PDOs can therefore be a useful model system for testing PARGi sensitivity. The detection of ssDNA gaps in PDOs may provide a functional biomarker to predict the response to PARG inhibitors in clinical trials. Citation Format: Ramya Ravindranathan, Ozge Somuncu, Yuqing Jiao, Alexandre André B A da Costa, Benjamin P Lamarre, David B Martignetti, Gabriella A Zambrano, Sirisha Mukkavalli, Joyce Liu, Bose Kochupurakkal, Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I Shapiro, Alan D. D'Andrea. PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian 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 3909.

Abstract 6585: Combination RASG12C(ON) and RASMULTI(ON) inhibition overcomes resistance and prolongs durability in preclinical models of mutant KRASG12C-driven cancers

Abstract Inhibitors of the GDP-bound state of mutant KRASG12C protein (KRASG12C(OFF)), e.g., adagrasib and sotorasib, have shown clinical activity in patients with KRASG12C-driven NSCLC, CRC and PDAC. However, most patients exhibit progression on monotherapy treatment in months, underlining the need for combination therapeutic approaches that improve the durability of the anti-tumor activity seen with KRASG12C inhibitors. RMC-6291 is a mutant-selective covalent inhibitor of the GTP-bound RAS(ON) form of RASG12C, while RMC-6236 is a non-covalent RASMULTI(ON) inhibitor of both mutant and wild-type variants of canonical RAS isoforms. In models harboring diverse alterations driving resistance mechanisms to KRASG12C(OFF) inhibitors – including reactivation of KRASG12C and wild-type RAS by increased RTK signaling, acquired secondary oncogenic RAS mutations, and KRAS amplification – both RMC-6291 and RMC-6236 exhibited superior anti-cancer activity as single agents when compared to KRASG12C(OFF) inhibitors. Thus, we hypothesized that a combination of RMC-6291 and RMC-6236 could address a broad spectrum of observed clinically relevant resistance mechanisms to KRASG12C(OFF) inhibitors that reactivate RAS signaling via mutant and wild-type RAS isoforms and compromise the depth and durability of anti-tumor response. In a mouse clinical trial with KRASG12C xenograft models, daily oral administration of RMC-6291 induced objective responses in 18 out of 25 NSCLC PDX and CDX models as assessed by mRECIST. In a subpanel of 7 NSCLC models that are relatively refractory to KRASG12C(OFF) inhibitor monotherapy, RMC-6236 alone somewhat prolonged the durability of response, while the combination of RMC-6291 with RMC-6236 improved the depth of response and significantly extended the durability thereof. In contrast to NSCLC, in models of KRASG12C CRC tumors a more heterogeneous response to RMC-6291 monotherapy was observed with objective responses in 4 out of 15 CRC models. The addition of RMC-6236 to RMC-6291 substantially improved the depth and duration of anti-tumor response in the resistant models to KRASG12C inhibitor alone. The combination of RMC-6291 with RMC-6236 at preclinical target doses did not affect the systemic or tumor exposures of each compound and was well tolerated in tumor-bearing immune-deficient animals. Both investigational agents RMC-6236 and RMC-6291 have shown promising anti-tumor activity as monotherapies in preclinical models and, more recently, showed preliminary clinical activity in patients with RAS-driven tumors at exposures that were well tolerated. The addition of RMC-6236 to RMC-6291 shows combinatorial activity in preclinical models that are relatively less responsive to either inhibitor alone. A clinical trial combining RMC-6291 with RMC-6236 in patients with mutant KRASG12C tumors is currently enrolling. Citation Format: Xing Wei, Matthew Holderfield, Yu Chi Yang, Kyle Seamon, Jay Dinglasan, Nilufar Montazar, Bianca Lee, Shurui Cai, Lick Lai, Ida Aronchik, Stephanie Chang, Zhican Wang, Pete Wildes, Jacqueline A. Smith, Jingjing Jiang, Mallika Singh. Combination RASG12C(ON) and RASMULTI(ON) inhibition overcomes resistance and prolongs durability in preclinical models of mutant KRASG12C-driven cancers [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 6585.

Anti-CD47 and Anti-PDL1 Immunotherapy Synergize for Anti-tumor Activity in Preclinical Models of Head and Neck Cancer

Anti-CD47 and Anti-PDL1 Immunotherapy Synergize for Anti-tumor Activity in Preclinical Models of Head and Neck Cancer

Inhibitors of NAD+ Production in Cancer Treatment: State of the Art and Perspectives.

The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called "Preiss-Handler (PH) pathway", which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery.