Deficient Tumors Research Articles

Abstract 6930: Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion

Abstract Colorectal cancer (CRC) persists as a leading cause of cancer-related deaths in the United States. Intratumoral heterogeneity stems from a multi-ancestral origin or clonal divergence and contributes to treatment resistance. Unfortunately, tumors in existing mouse models are largely homogenous, rendering predictions of drug efficacy inaccurate. To investigate whether signaling between distinct neoplastic clones in a tumor and cells in the surrounding microenvironment impacts CRC biology, we performed spatial transcriptomic analysis on samples collected from a transgenic mouse model previously established in the Halberg laboratory. This model (referred to as B6.FCTG3KM) develops three types of tumors: homotypic Adenomatous Polyposis Coli (APC)-deficient tumors that express red fluorescent protein (RFP), homotypic APC-deficient tumors that express constitutively active phosphoinositide 3-kinase (PI3K) and green fluorescent protein (GFP), and heterotypic tumors. Previous characterization of the model clearly demonstrated that heterotypic tumors are significantly more aggressive than either type of homotypic tumor. Analysis was conducted on four serially sectioned slides containing three tumors and adjacent normal tissue obtained from the colon of a male B6.FCTG3KM mouse using the 10x Genomics Visium CytAssist platform with the mouse whole transcriptome probe set. Collected tumors were staged using traditional histopathology techniques by a board-certified pathologist (Matkowskyj). Immunohistochemistry and immunofluorescence were used to assess β-catenin localization and to re-probe for RFP and GFP expression. Sequencing was performed on the Illumina NovaSeqX platform to an average depth of 255,528,294 reads per sample. Data analysis was performed using the 10x Genomics SpaceRanger and Kendziorski laboratory R/SpatialView packages. A fold-change threshold of 1.5 and an adjusted p-value threshold of 0.05 were used when establishing marker genes for each of the 8 clusters identified. One distinct cluster was identified by marker genes including Notum, Wif1, and Dkk2, factors previously implicated in CRC tumorigenesis, and corresponded to the center of each region of surface-level dysplastic epithelia. The remainder of the surface-level dysplastic epithelia corresponded to a second cluster, with marker genes including those coding for TRIM family proteins (Trim5, Trim10, Trim40, Trim12c), and matrix metalloproteinases (Mmp12, Mmp9, Mmp10, Mmp13). A third cluster corresponded to the desmoplastic stroma underlying each neoplasm. Ongoing analyses are underway to identify clonal interactions that alter gene expression and tumor aggressiveness. Citation Format: Mary M. Stangis, Lingxin Cheng, Dawn Albrecht, Santina Snow, Tessa Wirtz, Kristina A. Matkowskyj, Huy Q. Dinh, Christina Kendziorski, Richard B. Halberg. Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion [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 6930.

Abstract 4527: TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress

Abstract Defective maintenance of genomic integrity is a hallmark of cancer cells that can result from oncogene-induced replication stress and by loss of DNA repair mechanisms. DNA repair deficiencies and elevated replication stress present targetable vulnerabilities for cancer treatment. Notably, BRCA1/2 mutant and homologous recombination deficient (HRD) tumors cannot repair double-strand breaks by homologous recombination and rely on alternative pathways of DNA repair. PARP inhibitors (PARPi), which are a standard of care in many BRCA1/2 mutant tumors, cause synthetic lethality with BRCA1/2 mutation by inhibiting the DNA base excision repair pathway. Despite the clinical benefit of PARPi, they are not effective in every HRD tumor and the acquisition of PARPi resistance limits long-term response. TNG348, a selective allosteric inhibitor of the deubiquitinating enzyme USP1, was specifically designed to target HRD vulnerabilities through an alternative mechanism. We previously showed that the anti-tumor activity of USP1 inhibition results from disruption of the translesion synthesis DNA damage tolerance pathway, a mechanism of action that is functionally distinct from base excision repair targeted by PARPi. Our preclinical studies show that TNG348 is active in HRD models and strongly synergizes with PARP inhibitors to drive strong anti-tumor responses. We have identified replication stress as a predictive biomarker of TNG348 response using cell line profiling and genome-wide CRISPR screens. For example, overexpression of oncogenes known to induce replication stress sensitized to the TNG348 and PARPi combination both in vitro and in vivo. These data indicate that cancer-specific elevated DNA replication stress could contribute to tumor sensitivity to TNG348 and provide additional patient stratification strategies and opportunities for indication expansion. Citation Format: Antoine Simoneau, Hsin-Jung Wu, Charlotte Pratt, Grace Comer, Shangtao Liu, Samuel Meier, Tenzing Khendu, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Douglas Whittington, Sirimas Sudsakorn, Wenhai Zhang, Yi Yu, Yingnan Chen, Brian Haines, Adam Crystal, Jannik N. Andersen, John Maxwell, Scott Throner. TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress [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 4527.

Abstract 5170: Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors

Abstract Accurate patient stratification is crucial for the successful application of targeted anti-cancer therapeutic approaches. It has been shown that PARP inhibitors are synthetic lethal with defective homologous recombination (HR) repair, with genes such as BRCA1 and BRCA2 identified as conferring therapy resistance or sensitivity, depending on their mutational status. Moreover, there is evidence of patient benefit of carboplatin-based therapy in HR deficient (HRD) tumors. With a growing emphasis on HRD in cancer treatment, efforts have been made to define the HRD phenotype. Nonetheless, the current approaches employed in the clinic, such as Next Generation Sequencing (NGS), HRD score or RAD51 assays have significant limitations and their interpretation can be unclear. To answer the need for a functional HR-specific biomarker, we have recently developed and introduced two new assays, called collectively dSTRIDE-HR. The assays are based on the STRIDE platform technology that enables direct and sensitive detection of single- or double-strand DNA breaks in situ in fixed cells. Novel dSTRIDE-HR explicitly detect double-strand DNA breaks localized in close proximity to RPA (dSTRIDE-RPA) or RAD51 (dSTRIDE-RAD51) proteins. The assays have been shown to be reliable reporters of HR activation in cellular models after exogenous DNA damage induction, yet their potential to predict HR status in an in vivo setting has not been tested so far. To assess the suitability of the dSTRIDE-HR assays for determining the HR status of patients’ tumor cells, we applied these assays in FFPE sections from biopsies derived from breast cancer patients with a different mutational status of BRCA1 and BRCA2 genes. The assays showed low dSTRIDE-RPA and dSTRIDE-RAD51 readouts in BRCA1/2 mutated tumors in comparison to wild type tumors, as well as a great concordance of readouts within a given genetic background. Additionally, the cell cycle analysis proved nearly twofold increase in foci numbers of dSTRIDE-RPA and dSTRIDE-RAD51 in G2/S phase. Our study unveils a potential of dSTRIDE-HR assays to become predictive biomarkers reporting on the status of HR repair in breast tumors, although more patient samples need to be tested to confirm initial findings. Citation Format: Monika Jarosz, Zsombor Prucsi, Franek Sierpowski, Karolina Stepien, Sophie Ostrowska-Paton, Szymon Koman, Nadeem Riaz, Simon Powell, Fresia Pareja, Edaise da Silva, Magdalena Kordon-Kiszala, Kamil Solarczyk. Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5170.

Abstract 5492: Spatial organization and cellular composition of immunity hubs in human colorectal cancer

Abstract Background: Among colorectal cancer (CRC) tumors, mismatch repair deficient (MMRd) tumors have more immunogenic neo-antigens, hence more cytotoxic T-cell mediated anti-tumor immunity than mismatch repair proficient (MMRp) tumors. Previously1, we transcriptionally profiled over 300,000 cells from 62 MMRd and MMRp CRC tumors and found an MMRd-unique network of immunologically active transcriptional states enriched in interferon-stimulated genes (ISGs). With 4-color imaging, we showed that this program arises from spatially organized hubs of at least 3 cell types: IFNã-secreting T and interferon-responsive CXCL9/10/11+ myeloid and tumor cells. To characterize the breadth of cells involved in tumor immunity hubs, we must simultaneously measure the expression of 100s of genes in situ. Methods: To describe the cellular composition, structural organization and intercellular spatial interactions of these immunity hubs, we profiled primary untreated resection specimens from 17 CRC donors (n=12 MMRd, 5 MMRp) using multiplexed error-robust FISH (MERFISH) mRNA microscopy. Efforts were made to sample both the tumor body and the invasive border of the tumor. We designed a panel of 479 genes to capture key transcriptional programs from our single-cell atlas1. We wrote analytical pipelines to segment transcripts into cells, select high-quality cells, and organize cells into distinct zones in tissue. To leverage the rich information in the scRNA-seq atlas1, we designed a robust label transfer analysis to assign cell state labels to MERFISH cells by comparison to scRNAseq cells. Results: We profiled 7.5 million high quality MERFISH cells across 21 CRC tissue sections and labeled the cells into 7 major lineages: 63.19% epithelial, 22.52% stromal, 7.75% myeloid, 3.72% T/NK/ILCs, 1.45% plasma, 1.2% B, and 0.17% mast cells. We visualized the spatial location of these cell types. T cells aggregated into 3 distinct regions of tissue: tertiary lymphoid structures (TLS) outside the tumor, and smaller clusters near the invasive border and in the tumor center. We identified immunity hubs defined by colocalized CXCL9/10/11 expressing cells and T cells near the invasive border and in the center of MMRd tumors. As expected, we found enrichment of CXCL13, IFNG, and ISGs such as CD74 and MHCII genes at these locations. In contrast, we found evidence of CCL17+CCL19+LAMP3+ activated dendritic cells and B cells enriched in the TLS. These results support a distinct composition and localization of immunity hubs in tumors, in contrast to TLS outside tumors. Conclusions: Our study defines the composition, spatial organization, and intercellular interactions of immunity hubs, shedding light on the anti-tumor immune response in CRC. Ethics Approval: Approved by the DF/HCC Institutional Review Board (protocol #02-240).(1) Pelka, K. et al. Spatially organized multicellular immune hubs in human colorectal cancer. Cell 184, 4734-4752.e20 (2021) Citation Format: Mukta G. Palshikar, Maxwell Spurrell, Jack Demaray, Han Chen, Scott Engels, Roopa Madhu, Milan Parikh, Izabella Gazmora, Arnav Mehta, Nicolas Fernandez, Colles Price, Jiang He, Sonia Cohen, Karin Pelka, Nir Hacohen, Jonathan Chen, Ilya Korsunsky. Spatial organization and cellular composition of immunity hubs in human colorectal 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 5492.

Abstract 7135: Preclinical effects of Tumor Treating Fields (TTFields) with PARP inhibitors in ovarian cancer

Abstract Purpose: Among gynecological malignancies, ovarian cancer is the leading cause of mortality. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent standard-of-care treatment for selected cases of advanced epithelial ovarian cancer. PARPi exhibit sensitivity in homologues recombination deficient (HRD) tumors, in which there are mutations in the Fanconi Anemia (FA)-BRCA pathway, while about 75% of ovarian cancer patients are homologues recombination proficient (HRP). TTFields are non-invasive electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Recent research showed that TTFields induce an HRD-like state (also known as BRCAness state) in various cancer types. The current study aims to investigate the impact of TTFields applied together with PARPi in ovarian cancer preclinical models. Methods: In vitro, A2780 (HRP), OVCAR3 (HRD), and A2780cis (platinum-resistant) ovarian cancer cells were treated with TTFields (1 V/cm RMS, 200 kHz, 72 h), alone or with addition of PARPi (olaparib or niraparib, at various concentrations). Efficacy was measured by analyzing cell count, colony formation, and apoptosis induction. The overall effect was calculated by multiplying cell count with colony formation. TTFields-treated cells were evaluated for the expression of FA-BRCA pathway proteins and for formation of γH2AX, a DNA damage marker, using Western blot and fluorescent microscopy, respectively. In vivo, ID8-fLuc (HRP) cells were inoculated intraperitoneally to 6-8-week-old C57BL/6 mice. Seven days post inoculation, mice were treated with sham-vehicle, TTFields, olaparib (50 mg/kg), or both over a period of four weeks. Tumor growth was analyzed using bioluminescent imaging at treatment cessation, and survival analysis was performed. Results: TTFields application resulted in reduced cell count, increased overall effect, and increased apoptosis in vitro. When TTFields were applied together with PARPi, a high and moderate synergistic interaction was shown respectively the BRCA wild-type A2780 cells and the platinum-resistant A2780cis cells. However, in the BRCA mutant OVCAR-3 cells the effect was additive. A downregulation of FA-BRCA protein expression was confirmed after TTFields treatment of the ovarian cells, with consequent DNA damage. In the tumor-bearing mice, TTFields and olaparib co-treatment resulted in reduced tumor volume and a survival benefit relative to the monotherapies and to control-treated mice. Conclusions: The data suggest potential advantages for TTFields concomitant with PARPi in ovarian cancer, even without underlying BRCA mutations - consistent with the BRCAness state induced by TTFields - and also suggests benefits in platinum-resistant ovarian cancer. Citation Format: Yani Berckmans, Hila Ene, Kerem Ben-Meir, Antonia Martinez-Conde, Roni Monin, Bieke Van den Ende, Sara Van Mechelen, Roni Frechtel-Gerzi, Hila Gabay, Rotem Engelman, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti, Ignace Vergote, An Coosemans. Preclinical effects of Tumor Treating Fields (TTFields) with PARP inhibitors in 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 7135.

Abstract 1040: Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype

Abstract Introduction: MSI-H and mismatch repair deficient (dMMR) tumors are associated with favorable immune checkpoint inhibitor (ICI) responses (PMID: 3326454). However, up to 95% of CRC tumors are MSS/proficient MMR (pMMR) leading to poorer prognosis and treatment outcomes than MSI-H/dMMR patients (PMID: 12867608, 26028255). In MSS/pMMR metastatic CRC, multiple combination therapies are being investigated despite a lack of biomarkers to guide therapy. Therefore, we developed an RNA-based MSS-PRS with a primary objective to identify MSS tumors that have MSI-H/dMMR molecular features indicative of ICI response. Experimental Procedures: The MSS-PRS was developed using the TCGA CRC cohort (COAD; n=268). Training labels were assigned using the MSI Mantis score and mutation status from 8 genes with reported MSI association. Samples called “activated” had both a gene mutation and an MSI Mantis score > 0.4 (i.e., MSI-H), and samples called “non-activated” were wild type for all genes and had MSI Mantis score < 0.4. All other samples were designated “ambiguous” and excluded from training. Two thirds of the non-ambiguous samples were assigned to the classifier training set and all remaining samples were assigned to the test set. Using ClaNC software (PMID: 16269418) and cross-validation in the training set, a nearest centroid classifier was developed from a set of high mean, high variance candidate genes to select an optimal gene set to separate activated and non-activated groups. The classifier performance was evaluated in the test set. Results: The MSS-PRS contained 112-genes enriched for mismatched DNA binding, DNA damage repair, PD-L1, innate and adaptive immune response, cellular immunity, and cytokines. In the test set, the classifier called 44 of 46 MSI-H samples activated; of the remaining 77 samples that were MSS tumors, 38 were called activated and 39 were called not activated. Visual inspection of heat maps of tumor by MSI/MSS-PRS status and their association with TMB and CRC-related mutations suggest greater differences between MSS tumors called activated and MSI-H tumors than between MSS tumors called activated and MSS tumors called not activated. In contrast, immune marker expression profiles in MSS tumors called activated were markedly more similar to MSI-H tumors compared to MSS tumors called non-activated. Summary and Conclusions: Herein we described the development of a novel MSS-PRS that captures an MSI-H/dMMR-like molecular phenotype in a subset of patients with MSS tumors despite their lack of high TMB or overt MSI defects. Based upon these initial findings, further development of the MSS-PRS and its clinical validation as a tool to select patients with MSS tumors who may benefit from ICI-containing treatment regimens is warranted. Citation Format: Kirk Pappan, Gregory Mayhew, Jonathan Shepherd, Yuelong Guo, Kirk Beebe, Joel Eisner, Michael Milburn. Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype [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 1040.

Abstract 7122: A novel USP1 inhibitor coupled with a novel proteomic response biomarker for precision oncology therapy

Abstract Ubiquitin-specific protease 1 (USP1) is a well-characterized deubiquitinating enzyme (DUB) that plays a critical role in DNA damage repair (DDR). It was shown to regulate various key repair processes, most notably proliferating cell nuclear antigen (PCNA) in the Translesion Repair (TLS) pathway and FANCD2/FANCI in the Fanconi Anemia (FA) repair pathway. Research indicates that USP1 deficiency leads to a decrease in cell survival and disruption of genomic stability, suggesting that USP1 inhibitors (USP1i) may hold promise in treating DDR deficient tumors. Through the integration of computational and medicinal chemistry approaches, we discovered a novel, potent inhibitor of USP1, showing anti-USP1 activity in both cell-free enzymatic assay and in measurement of PCNA-ubiquitination change upon USP1 inhibition in cells. We demonstrate anti tumor activity in DDR-related and other indications. By utilizing our platform to analyze the basal proteomics of resistant and sensitive cell lines treated with USP1i, we revealed a distinctive proteomic biomarker signature for USP1i response. This data demonstrates the potential of our novel USP1 inhibitor to serve as a potent therapeutic agent for DDR-deficient and other biomarker-positive cancer patients. Citation Format: Iris Alchanati, Andrew Morley, Avital Hay-Koren, Fabrizio Fierro, Galina Otonin, Shay Herman, Alon Shtrikman, Yonatan Katzenelenbogen, Gali Arad, Barr Tivon, Kirill Pevzner, Eran Seger. A novel USP1 inhibitor coupled with a novel proteomic response biomarker for precision oncology therapy [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 7122.

Abstract 5801: Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody

Abstract The formulation of novel chemotherapeutics capable of precisely targeting and damaging tumor cells, while preserving the integrity of healthy tissues, represents a principal challenge and is recognized as one of the biggest achievements in cancer treatment in the last few decades. Antibody-drug conjugates (ADCs) hold significant potential as an effective cancer treatment, by targeting and delivering cytotoxic drugs exclusively to tumor cells that express distinct antigens associated with malignancy. Due to the necessity for specific surface antigens, therapies with ADCs are limited to specific tumor subtypes. In this study, we show the potential of a lupus-derived, cell-penetrating, anti-DNA antibody for delivering of cytotoxic payload to tumors in vivo. We utilized a fully humanized variant, named as V66, which was chemically linked to the Topoisomerase I inhibitor, Exatecan. The primary mechanism involved in V66 cellular penetration is mediated by the Equilibrative Nucleoside Transporter ENT2. The latter shows elevated expression in a substantial subset of cancer types in comparison to healthy tissues, broadening the range of applications for ADCs in tumor therapy. Using labeled V66, we demonstrated specific tumor delivery in vivo, with minimal impact on healthy tissues. In vitro experiments validated the nuclear localization of V66-Exatecan, where it triggers the activation of the DNA damage repair pathway (DDR). As a consequence of the profound DNA damage, exposure to V66-Exatecan leads to cell death, exhibiting low nanomolar EC50 values across a diverse range of cell lines. Furthermore, V66-Exatecan treatment presents synthetic lethality with BRCA2 deficiency, evidenced by a substantial increase in sensitivity. In vivo efficacy studies conducted in BRCA2 deficient tumors revealed a remarkable effectiveness of V66-Exatecan, leading to almost complete tumor regression and substantial improvement in the probability of survival. In addition to the lack of significant weight changes observed throughout the treatment, further toxicological studies conducted in serum and tissues demonstrated the absence of short- and long-term toxicity, with optimal liver and kidney function and the absence of damage to other tissues. Likewise, bone marrow cellularity remained unaffected. Collectively, these studies indicate that V66-Exatecan achieves targeted treatment of tumor cells, where it induces DNA damage leading to cell death. The in vivo application of V66-Exatecan leads to tumor regression without triggering general toxicity that is otherwise associated with Topoisomerase I inhibitors. This work provides the basis to advance a novel nuclear-penetrating ADC for delivering cytotoxic payloads. Citation Format: Zaira Ianniello, Elias Quijano, Peter M. Glazer. Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody [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 5801.

Abstract 1594: Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response

Abstract Background: While T-cell-based immunotherapy emerged as a promising tool for cancer management, clinical trials and translational studies revealed that cancer cells develop resistance by modulating tumor microenvironment and cell-intrinsic mechanisms. Pancreatic cancer (PC), consisting of dense stroma, immunosuppressive environment, and aberrant mucin expression, has a dismal survival rate and responds poorly to immunotherapies. MUC4, a member of the mucin family, has been reported to block lymphokine-activated killer cells and induce apoptosis of cytotoxic T-cells, suggesting its possible role in immune modulation. However, how the T-cell secretome influences MUC4 expression in PC and its role in modulating the T-cell response is poorly investigated. Method: The scRNA-seq data analysis and primary T-cell conditioned media (CM) were utilized to investigate the MUC4 and T-cell crosstalk. T-cell CM-treated murine PC cell line (KCT-3266) was analyzed by RNA-seq analysis followed by MUC4 silencing studies to investigate its role in evading T-cell response. The subcutaneous murine model was utilized to investigate the influence of MUC4 KO on immune cells and cytotoxic cell infiltration. The RNA isolated from tumor tissues from KrasG12D/+, Trp53R127H/+, Pdx-1-Cre (KPC), and Muc4 knockout (KPMC) murine models were used for PanCancer immune profiling. Results: The scRNA-seq analysis suggests that intratumoral T-cells positively correlate with MUC4 expression in PC patients. The activated T-cell CM consisting of predominantly IL-2, IFN-γ, and TNF-α cytokines, significantly induce the expression of MUC4 transcriptionally and translationally in both human (SW1990 and COLO357) and murine (KCT-3248 and KCT3266) PC cell lines. RNA-seq analysis from T-cell CM-treated cancer cells revealed that T-cell secretome induced the pathways related to cancer cell death. The CRISPR knockout of MUC4 in PC cells showed increased expression of cleaved caspase-3 after T-cell CM treatment, indicating that MUC4 plays a protective role against the T-cell CM-mediated killing. Subcutaneous implantation of MUC4 proficient and deficient PC cells on the flanks of C57BL/6 immunocompetent mice demonstrated a significantly higher infiltration of CD3 positive and cytotoxic T-cell infiltration in the MUC4 deficient tumors, resulting in significantly lower tumor weight. The PanCancer immune profiling also showed that depletion of Muc4 increases the T-cells and cytotoxic T-cell signaling scores in the KPMC tumor tissues compared to KPC. Conclusion: T-cell secretome induces MUC4 expression in pancreatic cancer cells, and the increased MUC4 expression reduces T-cell infiltration and protects PC cells against T-cell-mediated killing. Citation Format: Xiaoqi Li, Imran Khan, Rachel Kehrberg, Zahraa Wajih Alsafwani, Rakesh Bhatia, Sushil Kumar, Surinder K. Batra. Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response [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 1594.

Abstract 1416: Resolvins enhance immunotherapy to induce Fanconi anemia tumor regression via inflammation resolution

Abstract Fanconi anemia (FA) is a disorder characterized by early-onset solid tumors unresponsive to chemotherapy and radiation. Unresolved inflammation is a hallmark of FA, the mechanisms underlying the initiation and resolution of inflammation in FA are poorly characterized. We hypothesized that FA tumor progression is controlled by dysregulated resolution metabolomes and a systemic pro-inflammatory eicosanoid storm of inflammation-initiating mediators. Using a novel transplantable murine FA tumor model, we show that Fancc-/- HNSCC (head and neck squamous cell carcinoma) progression disrupts the resolution of inflammation via an imbalance between specialized pro-resolving lipid mediators (SPMs) and eicosanoids. Fancc-/- tumor-bearing mice exhibited an increase in eicosanoids, including leukotriene B4 (LTB4) and thromboxane B2 (TXB2) in the spleen and a 113% increase in prostaglandin E metabolite (PGEM), a marker of PGE2 production, in the plasma compared to non-tumor-bearing (NT) mice. Fancc-/- tumor-bearing mice exhibited a loss in the SPM/LTB4 ratio including RvD1/LTB4, RvD2/LTB4, and MaR1/LTB4, compared to NT mice. Given the activity of immune checkpoint blockade in mismatch repair deficient tumors including head and neck cancers, immunotherapy may be relevant to FA-induced cancers. Importantly, the number of absolute T cells and CD4+ T cell function is preserved in individuals with FA. We combined stimulation of the resolution of inflammation (i.e., treatment with RvDs) with immunotherapy in a new transplantable murine FA tumor model. While treatment with RvD4, RvD5, or anti-PD1 immunotherapy alone delayed tumor growth compared to control at treatment day 20, the tumors did escape monotherapy by treatment day 30. Remarkably, RvD4 or RvD5 combined with anti-PD1 immunotherapy induced sustained Fancc-/- tumor regression. Moreover, RvD4 and/or anti-PD1 immunotherapy inhibited orthotopic Fancc-/- tumor growth compared to control. Resolvins and immune checkpoint blockade synergistically induced Fancc-/- tumor regression via macrophage phagocytosis of apoptotic debris, counter-regulation of pro-angiogenic cytokines, and inhibition of angiogenesis. Triggering Receptor Expressed on Myeloid cells-2 (TREM2)-antagonism combined with resolvins and immunotherapy restored SPM/eicosanoid ratios in Fancc−/- tumor-bearing mice to pre-cancer levels. SPMs and eicosanoids provide potential early biomarkers and biological targets in FA-induced cancer progression. Stimulating the resolution of inflammation via pro-resolution lipid mediators to enhance immunotherapy is a novel host-centric therapeutic approach to prevent FA-induced cancer progression via debris clearance and cytokine suppression. These results provide evidence for targeting the resolution of inflammation via resolvins to enhance immunotherapy to prevent and/or reverse FA-induced cancer progression. Citation Format: Katherine Quinlivan, Haixia Yang, Diane R. Bielenberg, Susanne I. Wells, Charles N. Serhan, Dipak Panigrahy. Resolvins enhance immunotherapy to induce Fanconi anemia tumor regression via inflammation resolution [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 1416.

Abstract 5362: Targeting IRE1α alleviates the immunosuppressive tumor microenvironment in prostate cancer

Abstract Unfolded protein response (UPR) is a central stress response pathway in normal cells that is hijacked by tumor cells for their survival. However, how activation of UPR in cancer cells shapes the tumor microenvironment (TME) remain largely unexplored. Here, we investigated the role of IRE1α-XBP1s pathway on modulation of TME dynamics in prostate cancer (PCa). We found that IRE1α is increased in PCa patients and genetic or pharmacological inhibition of IRE1α signaling in the immunocompetent syngeneic mouse PCa model dramatically reduced tumor growth. Multiomics analysis of tumor samples upon IRE1α deletion in cancer cells showed significantly potentiated interferon (IFN) response and activation of immune system related pathways in the TME. Single-cell RNA sequencing (scRNA-seq) analysis revealed that the abundance of immunosuppressive cells, such as tumor-associated macrophages (TAMs) and T regulatory cells were markedly reduced in the IRE1α deficient tumors. Analysis of differentially expressed genes in the annotated cell types in the TME demonstrated that expression signatures associated with IFN responses were significantly enriched in TAMs, cancer cells, and dendritic cells. In addition, a novel scRNA-seq derived TAM gene signature is strongly associated with poor PCa survival. Furthermore, IRE1α inhibition by the small molecule MKC8866 (ORIN1001) that is in clinical trials significantly enhanced anti-PD-1 checkpoint inhibitor therapy in mice. Our findings indicate that IRE1α not only promotes cancer cell growth and survival, but it also strongly inhibits anti-tumor immunity in the PCa TME. Citation Format: Bilal Unal, Omer Faruk Kuzu, Yang Jin, Daniel Camilo Hurtado, Marieke Lydia Kuijjer, Mads Daugaard, Htoo Zarni Oo, John B. Patterson, Fahri Saatcioglu. Targeting IRE1α alleviates the immunosuppressive tumor microenvironment in prostate 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 5362.

Abstract 4539: ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics

Abstract Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway, and its dysfunction leads to cell genome instability and can result in tumorigenesis. In the situation of HR deficiency, DNA polymerase theta (Polθ) mediated microhomology-mediated end joining (MMEJ) is up-regulated to serve as a backup pathway for DSB repair. Several studies have proved that the inhibition of Polθ causes synthetic lethality with HR deficiency, and Polθ emerges as a potential DNA damage repair (DDR) target for the treatment of HR deficient tumors. In addition, there is evidence showing that the depletion of Polθ improves the sensitivity of some other DDR-related targeted therapies, chemotherapy, and radiotherapy. Here we identify a novel small molecular Polθ inhibitor, ZM-2311, which inhibits Polθ activity with an IC50 of 24 nM, and strongly inhibits cellular MMEJ pathway with a single nanomolar IC50. ZM-2311 illustrates a robust potency against tumor growth in a MDA-MB-436 SHLD2-/- xenograft model, suggesting potential monotherapy application in BRCA1 mutation patients with TP53BP1/Shieldin complex deficiency. Besides monotherapy, ZM-2311 elicits strong synergetic anti-proliferation activities in combination with PARP inhibitors on BRCA2-/- DLD-1 and endogenous HR deficient tumor cells in vitro and in vivo. Tumor regression is observed even at a low dosage, indicating a high potency of ZM-2311. As reversion mutation of HR genes such as BRCA1/2 mediated by the MMEJ pathway is reported to be a mechanism of PARP inhibitor resistance, ZM-2311 is evaluated and demonstrates its potential to overcome the resistance to PARP inhibitors. Furthermore, the combination of ZM-2311 with other DDR agents, such as ATR inhibitors, induces fatal DNA damage to HR deficient cells. Considering a low risk of hematotoxicity of Polθ inhibition, ZM-2311 may provide a new combination option for DDR agents. Besides, ZM-2311 was assessed in combination with different therapies respectively which functionally induce DNA damage, such as radiotherapy and chemotherapy. ZM-2311 renders tumor cells more sensitive to radiotherapy in both HR deficient cells and proficient cells in vitro, accompanying with the upregulation of Polθ expression after radiotherapy and an enhanced phosphorylation level of H2AX in the combination treatment. Similarly, ZM-2311 also achieves a robust synergetic effect in combination with chemotherapy in cell lines with different HR statuses. These results indicate a potential application expansion of ZM-2311 to HR-proficient tumors, which is expected to bring better efficacy and reduced hematotoxicity. Taken together, ZM-2311 has proved to be a highly potent Polθ inhibitor and has promising combination potential with multiple therapy strategies. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Shuo Qian, Liting Xue, Mengying Li, Zhengtao Li, Zhen Li, Jian Li, Renhong Tang. ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics [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 4539.

Pembrolizumab in patients from China with microsatellite instability-high/mismatch repair deficient tumors: KEYNOTE-158.

Aim: To evaluate pembrolizumab in patients of Chinese descent with microsatellite instability-high (MSI-H)/deficient mismatch repair (dMMR) tumors enrolled in KEYNOTE-158 (Cohort L). Methods: Patients with MSI-H/dMMR advanced tumors received pembrolizumab 200 mg IV Q3W. Primary endpoint was overall response rate (ORR). Secondary end points were duration of response (DOR), progression-free survival (PFS) and overall survival (OS). Results: 24patients were enrolled (20 were evaluable for efficacy). With median follow-up of 12.4months, the ORR was 70%. DOR, PFS and OS were all not reached. A total of 19 (79%) patients had a treatment-related adverse event (AE; grade ≥3 in 4 [17%]), and 8 (33%) had an immune-mediated AE (grade ≥3 in (4 [17%]). Conclusion: Pembrolizumab provided meaningful and durable responses with manageable safety. These results are consistent with those reported for the global trial.

Long-lasting mRNA-encoded interleukin-2 restores CD8+ T cell neoantigen immunity in MHC class I-deficient cancers

Major histocompatibility complex (MHC) class I antigen presentation deficiency is a common cancer immune escape mechanism, but the mechanistic implications and potential strategies to address this challenge remain poorly understood. Studying β2-microglobulin (B2M) deficient mouse tumor models, we find that MHC class I loss leads to a substantial immune desertification of the tumor microenvironment (TME) and broad resistance to immune-, chemo-, and radiotherapy. We show that treatment with long-lasting mRNA-encoded interleukin-2 (IL-2) restores an immune cell infiltrated, IFNγ-promoted, highly proinflammatory TME signature, and when combined with a tumor-targeting monoclonal antibody (mAB), can overcome therapeutic resistance. Unexpectedly, the effectiveness of this treatment is driven by IFNγ-releasing CD8+ Tcells that recognize neoantigens cross-presented by TME-resident activated macrophages. These macrophages acquire augmented antigen presentation proficiency and other M1-phenotype-associated features under IL-2 treatment. Our findings highlight the importance of restoring neoantigen-specific immune responses in the treatment of cancers with MHC class I deficiencies.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Cost effectiveness of pembrolizumab plus lenvatinib compared with chemotherapy for treating previously treated advanced endometrial cancer in Sweden

Objective Pembrolizumab plus lenvatinib was recently approved for the treatment of advanced or recurrent endometrial carcinoma in women with disease progression on or following prior treatment with a platinum‑containing therapy in any setting, and who are not candidates for curative surgery or radiation (KEYNOTE-775/Study-309; NCT03517449). The objective was to assess the cost effectiveness of pembrolizumab plus lenvatinib compared with chemotherapy from a Swedish healthcare perspective. Materials and methods A lifetime partitioned-survival model with three health states (progression free, progressed disease, death) was constructed. Chemotherapy was represented by paclitaxel or doxorubicin. Overall survival, progression-free survival, time on treatment, and utility data were obtained from KEYNOTE-775 (database lock: March 1, 2022). Costs (in 2020 Swedish Krona [SEK]) included drug acquisition and administration, health state, end of life, adverse event management, subsequent treatment, and societal (scenario analysis). Outcomes were calculated as quality-adjusted life-years (QALY) and life-years. Model results were presented as incremental cost-effectiveness ratios for all-comers, patients with proficient mismatch repair tumors, and deficient mismatch repair tumors. Deterministic and probabilistic sensitivity analyses were conducted. Results Pembrolizumab plus lenvatinib is a cost-effective treatment when compared with chemotherapy, with estimated deterministic and probabilistic incremental cost-effectiveness ratios of SEK 795,712 and 819,757 per QALY gained. Pembrolizumab plus lenvatinib was associated with a large incremental QALY and life-year gain per person versus chemotherapy over the model time horizon (1.49 and 1.76). Limitations Time-to-event data were incomplete and semiparametric and parametric curves were utilized for lifetime extrapolation. Willingness-to-pay thresholds, costs, and utility weights vary by country, which would vary the treatment’s cost effectiveness in different countries. Conclusions This partitioned survival analysis suggests that pembrolizumab plus lenvatinib is cost effective compared with chemotherapy in Sweden for women with advanced or recurrent endometrial carcinoma following previous systemic therapy. Results were robust to mismatch repair status and to changes in parameters/assumptions.

Delivery of hereditary cancer genetics services to patients newly diagnosed with ovarian and endometrial cancers at three gynecologic oncology clinics in the USA, Brazil, and Mexico

ObjectiveThree gynecologic oncology clinics located in the USA, Brazil, and Mexico collaborated to evaluate their delivery of hereditary cancer genetics services. This descriptive retrospective review study aimed to establish baseline...

Abstract PR-012: Proteogenomic analysis of enriched tumor epithelium identifies prognostic signatures and an increased dependency of homologous recombination proficient cells on bmi1 in high grade serous ovarian cancer

Abstract Background: Proteogenomic characterization efforts of high-grade serous ovarian cancer (HGSOC) have largely assessed tumors with high tumor cellularity (“purity”) to enhance detection of cancer-related biomarkers. Comprehensive analyses of cancers independent of purity are needed to reflect a “real-world” spectrum of patient phenotypes. To improve identification of clinically relevant molecular alterations associated with HGSOC independent of tumor purity, we applied cellular enrichment techniques coupled with comprehensive multi-omic analyses in HGSOC patient tumors spanning a broad spectrum of purity. Methods: Seventy HGSOC patient tumors were selected exhibiting low to high tumor purity (20-90%) balanced by clinical outcomes. Whole genome sequencing (WGS), mRNA-seq, quantitative global proteomics, methylation array and reverse phase protein array analyses of Bulk Tumor and laser microdissected (LMD) Enriched Tumor (ET) cells separately for each case. Prognostic and homologous recombination deficient (HRD) expression signatures were validated in two independent cohorts. Results: Analysis of WGS in ET compared to WT resulted in significant increases in sensitivity to identify somatic SNV, indel, structural variants, and neoepitopes (SNV p = 4.8E−3; indel p = 7.7E−5; SV, p = 1.1E−8, neoepitopes, p= 1.0E−10). Following LMD, 63% of cases characterized as mesenchymal subtype (C4) in WT samples were reclassified to other molecular subtypes (1.0E−3) suggesting that historical HGSOC expression subtypes strongly reflect tumor purity. Analysis of paired primary and metastatic tumors demonstrated that WT proteomic profiles largely cluster by anatomic location while ET proteomic profiles co-cluster in a patient centric manner. Hierarchical cluster analysis identified patients with longer progression free survival associated with increased immune signatures and validated proteins correlating with tumor infiltrating lymphocytes (TILs) in 65 tumors collected from an independent cohort of 12 HGSOC patients, as well as with overall survival in an additional cohort of 126 HGSOC patients. We identified that homologous recombination deficient (HRD) tumors express transcriptomic and proteomic pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts, including 69 HRD-positive HGSOC tumors. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells exhibit increased sensitivity to BMI-1 inhibition. Conclusion: Proteogenomic alterations in HGSOC tumors uncovered using enrichment techniques highlight the importance of specimen preparation in the identification of tumor alterations. Our efforts provide insights into low purity HGSOC correlating TILs with improved disease prognosis, expression alterations associated with HRD status, and increased sensitivity of HRP tumor cells to BMI-1 inhibitors. Citation Format: George Larry Maxwell, Nicholas Bateman, Tamara Abulez, Anthony Soltis, Andrew McPherson, Seongmin Choi, Dale Garsed, Chunqiao Tian, Brian Hood, Kelly Conrads, Pang-ning Teng, Julie Oliver, Glenn Gist, Dave Mitchell, Tracy Litzi, Christopher Tarney, Clifton Dalgard, Matthew Wilkerson, Mariaelena Pierobon, Emmanuel Petricoin, Chunhua Yan, Daoud Meerzaman, Clara Bodelon, Nicolas Wentzensen, Jerry S. H. Lee, David Huntsman, Sohrab Shah, Craig Shriver, Neil Phippen, Kathleen Darcy, David Bowtell, Thomas Conrads. Proteogenomic analysis of enriched tumor epithelium identifies prognostic signatures and an increased dependency of homologous recombination proficient cells on bmi1 in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-012.

Abstract P23: RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma

Abstract P23: RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma

Abstract B024: Maintenance therapy inhibition of ptk2 yields decreased disease in preclinical HRP/HRD models of recurrent HGSOC

Abstract B024: Maintenance therapy inhibition of ptk2 yields decreased disease in preclinical HRP/HRD models of recurrent HGSOC