Increased Tumor Cell Death Research Articles

EXTH-69. DEVELOPMENT OF A NOVEL, HUMAN ANTIBODY TO IMPROVE ONCOLYTIC VIRUS THERAPY FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive, malignant, primary brain tumor with abysmal overall survival. GBM is regarded as an “immune cold” tumor due to poor immune cell infiltration and immunosuppressive signaling in the tumor microenvironment (TME). Oncolytic herpes virus (oHSV) therapy has emerged as a possible therapeutic avenue for solid tumors such as GBM. We have observed that oHSV-treated tumor cells release extracellular ATP (eATP) which functions as immunostimulatory signaling molecule. In the TME, eATP is rapidly hydrolyzed into immunosuppressive adenosine by ectoenzymes CD39 and CD73. Using global CD73 knock out (CD73KO) mice or a function-blocking murine antibody, we found that blocking CD73 activity improves oHSV therapy in vivo. Intracranial tumor-bearing CD73KO mice respond significantly better to oHSV therapy than wild type (WT). These long-term survivors also rejected a subsequent tumor challenge, demonstrating development of a memory response. scRNA-seq revealed significantly increased immune cell infiltration in CD73KO mice compared to WT. We evaluated cellular crosstalk using CellChat and uncovered a major CCL-directed signaling pathway in the CD73KO mice. Furthermore, we identified a novel, human CD73 antibody from panning an scFv phage display library derived from human tonsils. This antibody, Ab6, demonstrated functional inhibition of purified CD73 and very high binding affinity for CD73. Co-culture of human GBM cells treated with OV and Ab6 and donor PBMCs resulted in increased tumor cell death. Additionally, human DCs exposed to Ab6 and OV-treated conditioned media were better able to activate T cells. To evaluate in vivo efficacy of this novel antibody, we have generated custom humanized CD73 knock-in mice wherein human CD73 has replaced murine CD73. We are currently evaluating the efficacy of Ab6 and oHSV in these mice against intracranial glioma. These results will support the translation of Ab6 as a potential immune therapeutic to add to the arsenal against brain tumors.

Aptamin C enhances anti-cancer activity NK cells through the activation of STAT3: a comparative study with vitamin C.

Vitamin C is a well-known antioxidant with antiviral, anticancer, and anti-inflammatory properties based on its antioxidative function. Aptamin C, a complex of vitamin C with its specific aptamer, has been reported to maintain or even enhance the efficacy of vitamin C while increasing its stability. To investigate in vivo distribution of Aptamin C, Gulo knockout mice, which, like humans, cannot biosynthesize vitamin C, were administered Aptamin C orally for 2 and 4 weeks. The results showed higher vitamin C accumulation in all tissues when administered Aptamin C, especially in the spleen. Next, the activity of natural killer (NK) cells were conducted. CD69, a marker known for activating for NK cells, which had decreased due to vitamin C deficiency, did not recover with vitamin C treatment but showed an increasing with Aptamin C. Furthermore, the expression of CD107a, a cell surface marker that increases during the killing process of target cells, also did not recover with vitamin C but increased with Aptamin C. Based on these results, when cultured with tumor cells to measure the extent of tumor cell death, an increase in tumor cell death was observed. To investigate the signaling mechanisms and related molecules involved in the proliferation and activation of NK cells by Aptamin C showed that Aptamin C treatment led to an increase in intracellular STAT3 activation. In conclusion, Aptamin C has a higher capability to activate NK cells and induce tumor cell death compared to vitamin C and it is mediated through the activation of STAT3.

Interaction of SMAC with a survivin-derived peptide alters essential cancer hallmarks: Tumor growth, inflammation, and immunosuppression

SMAC/Diablo is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in-vivo study, a survivin/BIRC5-derived peptide was selected due to its overexpression in many cancers, and involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD8+ cells and increased cell-intrinsic PD-1/PD-L1 expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents a innovative therapeutic cancer paradigm.

Abstract 233: Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an approximate 10% 5-year survival rate. PDAC patients rarely benefit from treatment with immune checkpoint inhibitors (ICIs) due to a tumor microenvironment (TME) associated with constrained effector T cell infiltration, and the presence of a suppressive myeloid cell population. Objective: To identify a combinatorial therapeutic strategy targeting the myeloid derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs) within the PDAC TME to increase the efficacy of ICIs. Methods: C57BL/6 mice were orthotopically transplanted with syngeneic 7940b PDAC cells derived from transgenic KPC mouse. 7 days post-transplantation mice were treated with InVivoPlus anti-mouse PD-1 (CD279), cabozantinib (cabo, tyrosine kinase inhibitor) or a combination of drugs for a further 7 days. NanoString CosMx™ spatial molecular imaging (CosMx™ SMI) was performed using FFPE sections collected from core biopsies and surgically resected tissues. PDAC patient derived organoids (PDOs) were generated from the core biopsies or surgical biospecimens. Cancer-associated fibroblasts (CAFs) were isolated from the organoid cultures. Autologous immune cells (IMM) were cultured from patient peripheral blood mononuclear cells. PDO/CAF/IMM co-cultures were used to predict the patient response to cabo plus pembrolizumab combinatorial therapy. Results: Mice treated with a combination of anti-PD-1 and cabo exhibited significant EpCAM/PD-L1-expressing cell death that correlated with increased CTL proliferation and decreased PMN-MDSC death. Mice that were depleted of CD8+ CTLs using anti-CD8 antibody in vivo did not exhibit tumor cell death in response to combinatorial therapy and thus supporting the role of CTLs in driving effective immunotherapy in combination with cabozantinib treatment. Unexpectedly, a significant decrease in extracellular matrix protein fibronectin was observed. Human PDOs orthotopically transplanted into NSG mice developed pathology consistent with human PDAC with strong expression of CD44v9, IL6, vimentin and αSMA. Using PDAC PDO/CAFs/IMM co-cultures, pembro significantly induced tumor cell death in the presence CTLs, MDSCs significantly inhibited this response. Depletion of MDSCs using cabo in the PDO/CAFs/IMM co-cultures resulted in a significant increase in tumor cell death with a concomitant increase in CTL proliferation. Compared to core biopsies, surgical biospecimens collected post-standard of-care demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) increases MDSCs, 2) change in CAFs phenotype and 3) increased expression of CD44v9. Conclusions: Cabozantinib depletes the PDAC TME of MDCSs leading to sensitization to ICI and tumor cell death and thus offers a potential combinatorial therapeutic approach. Citation Format: Pritha Adhikary, Jayati Chakrabarti, Meenu Priya Resmi, Jiang Wang, Davendra Sohal, Rachna T. Shroff, Syed A. Ahmad, Yana Zavros. Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab [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 233.

Abstract 626: EGFL6 induced HER2-HER3 signaling controls accurate centrosome deposition of activated ERK and mitotic spindle formation in ovarian cancer cells

Abstract Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer (HGSOC) cells and ovarian tumor vasculature. EGFL6 acts on tumor cells to drive tumor cell proliferation and migration. To better understand EGFL6 signaling and develop therapeutic strategies to target EGFL6 signaling in HGSOC, we evaluated phosphorylation of putative EGFL6 receptors and their downstream signaling. We demonstrated that EGFL6 induced phosphorylation and activation of ERBB/HER family of receptors; EGFL6 treatment of ovarian cancer cells led to rapid and transient phosphorylation of EGFR while inducing prolonged activation of both HER2 and HER3, with subsequent phosphorylation of ERK. Interestingly, we found that in EGFL6 stimulated ovarian cancer cells undergoing mitosis, pERK localized to the centrosome and the contractile ring. EGFL6 neutralizing antibodies reduce ERK phosphorylation and resulted in pERK being aberrantly localized. This resulted in altered mitotic spindle alignment and an increased number of cells undergoing mitotic catastrophe. Furthermore, combination anti-EGFL6 therapy with the pan-EGFR receptor inhibitor neratinib, compared to either therapy alone, led to an increase in aberrant pERK localization and cancer cell death in vitro. Consistent with these findings, dual anti-EGFL6 and neratinib therapy significantly restricted tumor growth in vivo, resulting in increased tumor cell death and a reduction in angiogenesis. Combined our data identify an unexplored role for pERK at the centrosome and suggest that dual targeting of the EGFL6/HER signaling axis maybe an effective therapeutic strategy in ovarian cancer. Citation Format: Shoumei Bai, Navneet Gupta, Qi Jiang, Ronald J. Buckanovich. EGFL6 induced HER2-HER3 signaling controls accurate centrosome deposition of activated ERK and mitotic spindle formation in ovarian cancer cells [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 626.

Abstract 6767: Multivariate analysis of ex vivo kidney 3D immune-microtumors for functional precision medicine

Abstract Immuno-oncology (IO) represents a significant paradigm shift in cancer treatment, focused on leveraging and mobilizing a patient’s own immune response and anti-tumor capabilities. Despite accounting for the majority of available treatment options, there are currently no diagnostic tests capable of predicting combination immunotherapy response in kidney cancer. We have developed a fully human 3D IO model for clinical drug efficacy prediction through multivariate analysis of the tumor-immune microenvironment (TiME) combining functional assays, artificial intelligence and multi-omics. These immune-microtumors recapitulate a patient’s unique pathophysiology and allow 3D spatio-temporal analyses of functional metrics at both bulk and single-cell resolutions. Renal cell carcinoma (RCC) resections and matched blood were collected and processed for single cell isolation of tumor and PBMCs (N=20, ISRCTN10001405). Flow cytometry (FC) and immunofluorescence (IF) were used to characterize TiME cell subpopulations (cancer, immune, stromal etc.) in pellets and 3D cultures, and to deconvolute bulk RNAseq data. Target (tumor) and effector cells (PBMCs and CD8+) were stained with fluorescent probes, activated using ɑCD3 and encapsulated in TiME-mimicking 3D matrices. 3D immune-microtumors were treated with FDA-approved regimens including immunotherapies (ipilimumab, nivolumab, pembrolizumab) and receptor-tyrosine kinase inhibitors (TKIs)(cabozantinib, lenvatinib, axitinib) as monotherapies or combination therapies. Differential drug efficacies were functionally quantified over 4 days using 3D confocal microscopy and computer vision pipelines to extract cell behavior metrics such as immune cell infiltration, immune/tumor cell migration speed, and tumor killing/viability. We devised a specialized RCC hydrogel formulation with superior viability compared to MatrigelTM alone (N=7). FC/IF/RNAseq were used to quantify relative cell subpopulations after isolation and show protein/transcriptional signature retention after 3D culture (N=7). The platform was conducive to the testing of various drugs with varying MoAs including immunotherapies, TKIs, targeted therapies and cell therapies. Treatment with pembrolizumab led to 26% higher PBMC infiltration, 14% higher CD8+ infiltration and 15% increased tumor cell death compared to control. When in combination, pembrolizumab reduced cell viability by 10% and 19% with lenvatinib and axitinib respectively (N=1). Population analyses revealed intra-patient response heterogeneity (N=7). In conclusion, we showed MoA-agnostic, patient-dependent responses to FDA-approved clinical treatment regimens via molecular and functional spatio-temporal analyses. Clinical outcome correlation is currently underway in our advanced RCC study PEAR-TREE2 as well as in breast NCT05435352 and brain NCT06038760 tumors. Citation Format: Eleonora Peerani, Keqian Nan, Demi A. Wiskerke, Thomas D. Richardson, Polina Maximchick, Jay Kearney, George R. de Fraine, Kerrie L. Loughrey, Jonathan Ient, Jessica A. Hudson, Andreas Kaffa, Aston M. Crawley, Nourdine K. Bah, Edgar G. Lopez Molina, Elli Tham, Francesco Iori, Matthew H. Williams, Maxine G. Tran, Duleek N. Ranatunga. Multivariate analysis of ex vivo kidney 3D immune-microtumors for functional precision medicine [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 6767.

Abstract 19: Patient derived tumor-on-a-chip platform potentiates adaptive immune response against primary tumor cells

Abstract Background: Adoptive cellular therapy (ACT) is a promising antitumor immunotherapy showing success in select trials/patients. However, major challenges limit ACT in all tumors including insufficient cell numbers and failure to target heterogeneous tumors. To improve ACT, we developed a novel biomimetic device where patient-derived tumor organoids, enriched with antigen presenting cells, are cocultured with autologous peripheral blood mononuclear cells (PBMCs) in a microfluidic tumor-on-a-chip system (TOC). As PBMCs circulate through the device, they are exposed to dispersed tumor polyclonal neoantigens, leading to formation of patient- and tumor-specific organoid interacting lymphocytes (OILs). Objectives: Demonstrate that a 3D tumor-on-a-chip (TOC) platform generates organoid interacting lymphocytes (OILs) with enhanced antitumor reactivity. Methods: Specimens were collected from 15 surgical patients: melanoma (2), mesothelioma (6), and metastatic appendiceal adenocarcinoma (9). Tumor cells were combined with autologous antigen presenting cells (healthy spleen/lymph node) in a 3D matrix to generate immune-enhanced tumor organoids in the TOC system. Autologous PBMCs circulated through the system to produce OILs (7 days). Uncirculated PBMCs and tumor infiltrating lymphocytes (TILs) were cultured as controls. Resultant CD8+ controls and OILs were examined by single cell proteomics to compare cytokine secretion polyfunctionality. To assess anti-tumor reactivity, OILs and controls were reintroduced to tumor cells via coculture. Secreted bulk cytokine profiles were compared by Isoplexis CodePlex and confirmed by ELISA. NanoString Geomx spatial proteomic analysis of coculture PTOs identified key lymphocyte activity and phenotypes during anti-tumor reactivity. Coculture cytotoxic lymphocyte activation and tumor cell death was confirmed by flow cytometry. Data were analyzed using student t-test and differential expression from linear mixed-effect model. Results: Compared to controls, CD8+ OILs showed increased polyfunctionality within single cells immediately after training, particularly in effector T-cell associated cytokine profiles (INF-γ, Granzyme B, Perforin). Upon re-introduction to PTOs, OILs excreted significantly increased inflammatory-associated proteins IFN-γ, MCP-1, and Granzyme A (p<0.05). Furthermore, OIL re-exposure to tumor led to cytotoxic lymphocyte activation (CD8+CD69+) and significantly increased tumor cell death compared to control (p<0.05). Conclusion: Compared to controls, OILs produced in the TOC system show enhanced cytotoxic lymphocyte polyfunctionality and induce tumor cytotoxicity, independent of tumor type. With further validation of the OILs’ increased tumor-targeting efficacy, we propose a new potential ACT modality to target a broad array of tumors previously untreatable by current therapies. Citation Format: Cecilia R. Schaaf, Tiefu Liu, Damian Hutchins, Mitra Kooshki, Calvin Wagner, Nicholas Edenhoffer, Steven Forsythe, Robyn Greissinger, Lance Miller, Pierre Triozzi, Edward Levine, Shay Soker, Adam R. Hall, Konstantinos I. Votanopoulos. Patient derived tumor-on-a-chip platform potentiates adaptive immune response against primary tumor cells [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 19.

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract C014: CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma

Abstract Introduction Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The CXC chemokine receptor type 4 (CXCR4)/chemokine (CXC motif) ligand 12 (CXCL12) axis is one key immunosuppressive pathway. CXCL12 (released by cancer-associated fibroblasts (CAF)) binds to CXCR4 on CD8+ T cells (CTL) and decreases its infiltration into the tumor. Encouraging preclinical studies from murine PDAC models and human PDAC tumor sections have demonstrated infiltration of T cells with tumors after co-inhibition of CXCR4 and PD1/PDL1, however with limited tumor regressions in mouse model. We established an innovative ex vivo autologous patient-derived immune/organoid (PDIO) co-culture system that allows studying the interactions between distinct immune cells and tumor cells. Methods Patient-derived tumor organoids (PDTOs) generated from tumors resected from patients diagnosed with pancreas cancer were grown to confluence in Matrigel domes. We conducted ex vivo migration assays with autologous peripheral blood mononuclear cells (PBMCs) which were placed in the surrounding liquid phase of the matrigels. We separately treated PBMCs and PDTOs with AMD3100, an inhibitor of CXCR4, and tested the role of CXCR4 inhibition on PBMCs and PDTOs in T cell exclusion and immune cell activation. Results Pre-treatment of PBMCs with AMD3100 increased CD8+ T cell mobilization towards autologous PDTOs in matrigel dome migration assays by up to 11-fold. After co-culture in the liquid interphase of PDTOs matrigel domes, AMD3100 pre-treated PBMCs exhibit a more robust proliferative response compared to untreated PBMCs and a significant increase in the number of reactive T cells migrated inside the PDTO matrigel dome and expressing activation markers (p=0.02). Nearly 50% of T cells in proximity of tumor cells produce the cytotoxic factor Granzyme B (p=0.002), with AMD3100 treatment of PBMC increasing it by up to 3-fold compared to untreated PBMC. CXCR4 inhibition of PDTOs resulted in modulation of immune checkpoint ligands involved in the tumor immune response and tumor cell immunogenicity. Combination treatment of PBMCs and PDTOs with AMD3100 and anti-PD1 antibody ultimately resulted in increased tumor cell death with up to 45% of PDTOs exhibiting apoptosis markers after coculture with AMD3100-treated PBMC, implicating a role of this combination in the clinic. Conclusions We demonstrate that inhibition of CXCR4, not only increases migration of autologous T cells towards PDTOs, but also results in proliferation of PBMCs and activation of T cells. Furthermore, CXCR4 inhibition of PDTOs increases antigen presentation and tumor cell death. Inhibition of CXCR4 is an attractive therapeutic target as it enhances anti-tumor immune response via dual targeting of immune and neoplastic compartments. We are testing this combination in a multicenter investigator-initiated clinical trial in treatment-naïve metastatic PDAC patients. Citation Format: Ilenia Pellicciotta. CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C014.

TMIC-61. INVESTIGATING REPROGRAMING OF THE GBM’S TUMOR MICROENVIRONMENT AS A RESULT OF CO-TRANSFER OF HEMATOPOIETIC STEM CELLS (HSCS) WITH ADOPTIVE CELLULAR THERAPY

Abstract Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and the powerful regulatory mechanisms within the tumor microenvironment (TME) that hamper immune activation have proved to be formidable challenges to the development of effective therapies. Fortunately, immunotherapy has emerged as a potentially powerful approach to achieve of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition Our previous work has demonstrated that the survival benefit of adoptive cellular therapy (ACT) is significantly enhanced by concomitant transfer of bone marrow derived hematopoietic stem cells (HSC) with tumor-reactive T cells in preclinical models. Using a novel organotypic brain slice culture (BSC) model study using glioma-bearing C57BL/6J mice, we interrogate tumor microenvironment and directly demonstrate that the presence of HSC-derived cells in the microenvironment increases T cell migration to tumor, and increased tumor cell death. In addition, we can investigate reprogramming of the TME as a result of treatment with this ACT+HSC platform. These studies are unique in that through this model system, we are capable of studying the cell-cell interactions occurring within the tumor microenvironment TME longitudinally while accounting for the complexity and the spatial orientation of the components within the TME that promote immunosuppression and tumor escape.

IMMU-28. TREM2 INHIBITION REPROGRAMS MYELOID CELLS TO STIMULATE ANTI-TUMOR IMMUNITY THROUGH BOTH DIRECT AND INDIRECT MECHANISMS

Abstract The prevailing view is that myeloid cells—macrophages and microglia—in the tumor microenvironment (TME) are immunosuppressive and promote glioblastoma (GBM) progression. However, myeloid cells have the functional plasticity to restrict or support tumor cell growth. TREM2 plays important roles in brain microglial function in neurodegenerative diseases, but the functional role of TREM2 in the GBM TME and whether TREM2 represents a meaningful therapeutic target remain largely unknown. To address these questions, we investigated the immune and tumor cell consequences of TREM2 inhibition by multiple orthogonal approaches—transgenic mice, antisense oligonucleotides, and blocking antibodies—in immunocompetent preclinical models (SB28, NPA C54B) and human GBM samples through bulk and single cell RNA-sequencing, flow cytometry, biochemistry, and functional assays. We found TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors, and that high TREM2 expression correlates with poor prognosis in GBM patients. TREM2 loss of function in human macrophages and mouse myeloid cells increased IFNγ-induced immunoactivation and proinflammatory polarization. Conditioned media from TREM2 deficient human macrophages directly killed patient-derived GBM tumor cells, and biochemical analysis identified TNF and other putative tumoricidal proteins in this conditioned media. In mouse GBM models, mice with chronic and acute Trem2 loss of function demonstrated increased tumor cell death and increased animal survival. Trem2 inhibition reprogrammed myeloid cell phenotypes and increased PD-1+CD8+ T cells in the TME. Remarkably, Trem2 deficiency enhanced the effectiveness of anti-PD-1 treatment in vivo. Trem2 inhibition suppresses tumor cell growth through direct and indirect immune cell mechanisms--by reshaping myeloid subset and T cell functions. Thus, Trem2 inhibitory strategies, in combination with immune checkpoint blockade, may represent an attractive new approach for GBM therapy.

Pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, sensitizes cancer cells to VSVΔ51 oncolytic virotherapy

The clinical efficacy of VSVΔ51 oncolytic virotherapy has been limited by tumor resistance to viral infection, so strategies to transiently repress antiviral defenses are warranted. Pevonedistat is a first-in-class NEDD8-activating enzyme (NAE) inhibitor currently being tested in clinical trials for its antitumor potential. In this study, we demonstrate that pevonedistat sensitizes human and murine cancer cells to increase oncolytic VSVΔ51 infection, increase tumor cell death, and improve therapeutic outcomes in resistant syngeneic murine cancer models. Increased VSVΔ51 infectivity was also observed in clinical human tumor samples. We further identify the mechanism of this effect to operate via blockade of the type 1 interferon (IFN-1) response through neddylation-dependent interferon-stimulated growth factor 3 (ISGF3) repression and neddylation-independent inhibition of NF-κB nuclear translocation. Together, our results identify a role for neddylation in regulating the innate immune response and demonstrate that pevonedistat can improve the therapeutic outcomes of strategies using oncolytic virotherapy.

C-Rel-dependent Chk2 signaling regulates the DNA damage response limiting hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC. Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone. Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma

BackgroundMultiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies.MethodsWe performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt’s and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment’ synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy.ResultsWe identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects.ConclusionsThe present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Abstract A014: Inhibition of KrasG12D with a novel small molecule inhibitor alters the pancreatic cancer microenvironment

Abstract Approximately 90% of pancreatic ductal adenocarcinoma (PDAC) patients harbor an oncogenic KRAS mutation, with Gly-to-Asp mutations (KrasG12D) being most common. PDAC is characterized by a robust, immunosuppressive tumor microenvironment that is rich in myeloid-derived suppressor cells (MDSCs) and has a paucity of T cells. The majority of therapeutic interventions in PDAC are ineffective, in part, due to this immunosuppressive TME. We aimed to examine the efficacy of a small molecule KrasG12D inhibitor, MRTX1133, in implantable and autochthonous PDAC models with an intact immune system. In vitro studies validated the specificity and potency of MRTX1133. In vivo, MRTX1133 prompted deep tumor regressions in all models tested, including complete or near-complete remissions after 14d. MRTX1133 treatment additionally reduced metastatic burden after 7d of treatment. An increase in tumor cell death was identified after only 36h of treatment, with concomitant loss of tumor cell proliferation. Inhibition of KrasG12D led to marked shifts in the TME composition. MRTX1133-treated tumors had an increase in fibroblasts, collagen deposition, and blood vessel density. There was a robust increase in M1-like macrophages after MRTX1133 treatment. Further, there was a loss of MDSCs, along with an increase in cytotoxic T cells. Proteome profiling on tumor cells treated with MRTX1133 identified a loss of tumor cell-derived GM-CSF (a MDSC recruiter), as well as an increase in CCL2 (a macrophage recruiter), providing evidence that MRTX1133 alters the TME through tumor secreted factors. T cells were necessary for MRTX1133’s full anti-tumor effect, and T cell depletion accelerated tumor regrowth after therapy. These results validate the specificity, potency, and efficacy of MRTX1133 in immunocompetent KrasG12D-mutant PDAC models, providing a rationale for clinical testing and a platform for further investigation of combination therapies. Citation Format: Samantha B. Kemp, Noah Cheng, Nune Markosyan, Rina Sor, Il-Kyu Kim, Jill Hallin, Jason Shoush, Liz Quinones, Natalie Brown, Jared B. Bassett, Nikhil Joshi, Salina Yuan, Molly Smith, William P. Vostrejs, Kia Z. Perez-Vale, Benjamin Kahn, Feiyan Mo, Timothy R. Donahue, Caius G. Radu, Cynthia Clendenin, James G. Christensen, Robert H. Vonderheide, Ben Z. Stanger. Inhibition of KrasG12D with a novel small molecule inhibitor alters the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A014.

Abstract LB232: Targeting MEK activity in pancreatic tumors resistant to the combination of 5FU, irinotecan, and oxaliplatin promotes response to immunotherapy

Abstract FOLFIRINOX, a chemotherapy regimen consisting of 5FU, Leucovorin, Irinotecan, and Oxaliplatin, has been a first-line standard of care for patients with pancreatic adenocarcinoma (PDAC) for the last decade. However, low patient survival rates following treatment highlight frequent occurrence of resistance. Hence there is increasing interest in developing appropriate models of FOLFIRINOX resistance to identify subsequent line therapies. We have generated PDAC cell lines that exhibit acquired resistance to a combination of 5FU (F), Irinotecan (I), and Oxaliplatin (O) (FIO) in vitro. In mice, they form tumors that resemble FOLFIRINOX neoadjuvant-treated PDAC patients in regard to immune cell infiltration and a lack of response to single-agent anti-PD-1 therapy. We also show that similar to human PDAC tumors treated with neoadjuvant FOLFIRINOX, FIO-resistant tumors exhibit increased ERK1/2 phosphorylation. We demonstrate that the MEK inhibitor Trametinib reprograms the FIO-resistant tumors, but not the parental tumors, towards a less immunosuppressive tumor immune microenvironment. Importantly, Trametinib sensitizes FIO-resistant tumors to anti-PD-1 therapy, with the combination treatment significantly enhancing the cytolytic activity of the infiltrating CD8 + T cells and increasing tumor cell death. Taken together, our findings identify enhanced MEK/ERK signaling as a therapeutic target in FOLFIRINOX-resistant PDAC tumors, and suggest evaluating the combination of Trametinib and anti-PD-1 therapy in patients who progress on FOLFIRINOX. Citation Format: Thao D. Pham, Anastasia E. Metropulos, Nida Mubin, Dhavan N. Shah, Christina Spaulding, Mario Shields, David J. Bentrem, Hidayatullah G. Munshi. Targeting MEK activity in pancreatic tumors resistant to the combination of 5FU, irinotecan, and oxaliplatin promotes response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB232.

Abstract 2171: A multivariate biomarker predicts sacituzumab govitecan response in solid tumors

Abstract Despite the recent clinical success of antibody drug conjugates (ADC) in oncology, predictive biomarkers are lacking, potentially limiting their impact. Herein, we evaluated the ability of candidate biomarkers alone and in combination to predict objective response rates observed in solid tumor patients treated with the TROP2-targeted ADC, sacituzumab govitecan (SG), as determined in the IMMU-12-01 basket trial. We leveraged available next generation sequencing (NGS)-based molecular profiling data from an independent advanced solid tumor cohort (n = 23,968) and developed a multivariate biomarker algorithm that produced biomarker positive rates correlating with the objective response rates (ORR) observed in IMMU-12-01. Candidate biomarkers evaluated included TROP2 gene expression, proliferation (by gene expression) and tumor cellularity. Notably, while TROP2 gene expression was highly correlated with protein expression across 45 tumor types (r = 0.93), TROP2 gene expression alone did not significantly predict ORR across 9 tumor types (r = 0.40, p = 0.29). In contrast, a biomarker algorithm combining TROP2 and proliferation by gene expression with tumor cellularity strongly predicted response both when using tumor type-specific biomarker rates in a discovery cohort (r = 0.83, p = 0.006) and in an independent validation cohort (r = 0.82, p = 0.007). These results indicate that the multivariate biomarker accounts for 67% of the variability observed in response rates and may thus identify patients likely to benefit from SG. Among tumor types with objective responses in IMMU-12-01, biomarker positive rates ranged from 9.9% in colorectal cancer to 57.4% in bladder cancer. Additional tumor types with biomarker positive rates >30% included cancers of the head and neck, cervix, salivary gland, skin (non-melanoma) and ovary, all with positive biomarker rates >30%. Interestingly, most tumor types had biomarker positive rates >5%, suggesting the potential for a tumor type-agnostic approach to patient selection. Considering SG and other ADC’s mechanism of action, a plausible model for response is that (1) higher target expression increases ADC drug delivery, (2) higher tumor cellularity increases ADC bystander effect and (3) higher tumor cell proliferation increases tumor cell death. In summary, we uncovered a novel biomarker algorithm capable of predicting SG response across solid tumors that may be generalizable to ADCs as a class, with the potential to further optimize use and maximize benefit. Citation Format: Nickolay A. Khazanov, Laura E. Lamb, Daniel H. Hovelson, Kat Kwiatkowski, D. Bryan Johnson, Daniel R. Rhodes, Scott A. Tomlins. A multivariate biomarker predicts sacituzumab govitecan response in solid tumors [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 2171.

Abstract 1790: Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma

Abstract Antigenic heterogeneity and limited immune cell infiltration has historically hindered immunotherapy approaches to treat the pediatric brain tumor medulloblastoma (MB). Therefore, modern MB treatment modalities rely on broadly cytotoxic chemotherapy and/or radiation, leaving survivors with side effects that diminish quality of life, stressing an urgent need for novel approaches. Gamma delta (γδ) T cells represent an emerging class of cellular immunotherapy with preclinical potency against the CNS tumor glioblastoma and pediatric solid tumors. The ability of γδ T cells to recognize tumor stress antigens, alongside their MHC independence, supports γδ T cell usage as an off-the-shelf allogeneic cellular immunotherapy for MB and other immunologically cold cancers. Our team recently identified protein tyrosine kinase 7 (PTK7) as a novel immunotherapy target highly expressed in neuroblastoma, with low pediatric healthy tissue expression. Alpha beta T cells engineered with CARs targeting PTK7 demonstrated impressive antitumor efficacy against PTK7+ neuroblastoma xenografts. In that study, we also noted MB and other pediatric solid tumors express elevated levels of PTK7 mRNA. This work therefore serves to test the susceptibility of MB to γδ T cell immunotherapy and validate if targeting PTK7 can further enhance γδ T cell potency. Six MB cell lines representing three out of the four MB molecular subgroups were chosen: DAOY, ONS-76, UW228, D341, D425 and D283. Western blot analysis showed five of six MB cell lines express PTK7 protein, and flow cytometry confirmed PTK7 membrane localization. PTK7high (DAOY) and PTK7low (ONS-76) cells were chosen to test for the antigen-specific killing potential of PTK7-targeted CAR γδ T cells against MB. To generate PTK7-targeted γδ T cells, previously cryopreserved γδ T cells were electroporated with mRNA encoding a PTK7-targeted CAR construct containing the CD28 co-stimulatory domain. After a four hour co-culture, both ONS-76 and DAOY showed moderate rates of apoptosis from exposure to mock electroporated or naïve γδ T cells at 1:1, 2:1 and 5:1 effector:target (E:T) ratios, with approximately 30-40% cell death at 5:1. There was no change in susceptibility of ONS-76 cells to PTK7-CAR γδ T cells when compared to mock electroporated or naïve γδ T cell controls. However, co-culture of DAOY cells with PTK7-CAR γδ T cells markedly increased tumor cell death by as much as 40% compared to the mock controls at E:T ratios as low as 1:1. In summary, naïve expanded γδ T cells induce pronounced MB tumor cell death. Cytotoxicity is further increased against PTK7high MB cells following the introduction of an anti-PTK7 CAR. These results provide a strong rationale for additional preclinical studies regarding the feasibility of γδ T cell-based immunotherapy against MB. Citation Format: Benjamin S. Lowry, Hunter C. Jonus, Jasmine Lee, Trent H. Spencer, Anna M. Kenney, Kelly Goldsmith. Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma [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 1790.

Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma via the combination therapy with FOXM1 inhibitor RCM1.

Rhabdomyosarcoma (RMS) is a highly metastatic soft-tissue sarcoma that often develops resistance to current therapies, including vincristine. Since the existing treatments have not significantly improved survival, there is a critical need for new therapeutic approaches for RMS patients. FOXM1, a known oncogene, is highly expressed in RMS, and is associated with the worst prognosis in RMS patients. In the present study, we found that the combination treatment with specific FOXM1 inhibitor RCM1 and low doses of vincristine is more effective in increasing apoptosis and decreasing RMS cell proliferation in vitro compared to single drugs alone. Since RCM1 is highly hydrophobic, we developed innovative nanoparticle delivery system containing poly-beta-amino-esters and folic acid (NPFA), which efficiently delivers RCM1 to mouse RMS tumors in vivo. The combination of low doses of vincristine together with intravenous administration of NPFA nanoparticles containing RCM1 effectively reduced RMS tumor volumes, increased tumor cell death and decreased tumor cell proliferation in RMS tumors compared to RCM1 or vincristine alone. The combination therapy was non-toxic as demonstrated by liver metabolic panels using peripheral blood serum. Using RNA-seq of dissected RMS tumors, we identified Chac1 as a uniquely downregulated gene after the combination treatment. Knockdown of Chac1 in RMS cells in vitro recapitulated the effects of the combination therapy. Altogether, combination treatment with low doses of vincristine and nanoparticle delivery of FOXM1 inhibitor RCM1 in a pre-clinical model of RMS has superior anti-tumor effects and decreases CHAC1 while reducing vincristine toxicity.

Lethal effects of mitochondria via microfluidics.

Tumor cells can respond to therapeutic agents by morphologic alternations including formation of tunneling nanotubes. Using tomographic microscope, which can detect the internal structure of cells, we found that mitochondria within breast tumor cells migrate to an adjacent tumor cell through a tunneling nanotube. To investigate the relationship between mitochondria and tunneling nanotubes, mitochondria were passed through a microfluidic device that mimick tunneling nanotubes. Mitochondria, through the microfluidic device, released endonuclease G (Endo G) into adjacent tumor cells, which we referred to herein as unsealed mitochondria. Although unsealed mitochondria did not induce cell death by themselves, they induced apoptosis of tumor cells in response to caspase-3. Importantly, Endo G-depleted mitochondria were ineffective as lethal agents. Moreover, unsealed mitochondria had synergistic apoptotic effects with doxorubicin in further increasing tumor cell death. Thus, we show that the mitochondria of microfluidics can provide novel strategies toward tumor cell death.