Related Deaths Research Articles

Abstract 944: Pre-clinical activity of the Wnt pathway inhibitor RXC004 in combination with MAPK pathway inhibitors in GI cancer models

Abstract Background: Gastrointestinal (GI) cancers cause more cancer deaths than any other body system, with 3.4 million related deaths in 2018. Wnt pathway activation is common in GI cancers, with the pathogenic upstream Wnt pathway variant sub-population (RNF43 loss of function/RSPO gain of function) showing a combined prevalence of 4.7% in GI cancer patients; pre-clinically this sub-population shows exquisite sensitivity to RXC004, a small molecule Porcupine inhibitor. A striking co-occurrence of upstream Wnt pathway variants with MAPK pathway driver mutations (KRAS, BRAF, NRAS) was detected in 77% of Microsatellite stable (MSS) GI cancers, suggesting co-inhibiting these pathways could enhance clinical activity in these patients. Methods: In vitro proliferation assays were performed in pre-clinical models of upstream Wnt pathway mutated MSS GI cancer (SNU-1411, HPAF-II and AsPC-1) using Wnt (RXC004) or MAPK (Trametinib) pathway inhibitors at multiple concentrations as single agents or in combination. Control cells with downstream Wnt pathway mutations (WiDr and HCT116) were also tested. Synergistic combination effects were detected by BLISS scores. RXC004 (1.5mg/kg QD) and Trametinib (0.3mg/kg QD) were evaluated in an RSPO-fusion xenograft model (SNU-1411) as single agents or in combination. Efficacy was measured by tumour volume and weight; PD markers were assessed in end of study tumour samples. Relative AXIN2 and DUSP6 gene expression changes were determined upon inhibitor treatment by quantitative Polymerase Chain Reaction (qPCR). Results: RXC004 in combination with Trametinib demonstrated strong anti-proliferative synergy in SNU-1411 cells and moderate synergy/additivity in HPAF-II and AsPC-1 cells; no synergy was observed in control cells. In vivo, RXC004 and Trametinib monotherapy led to significant moderate tumour growth control (p<0.05) at the doses tested, whereas their combination led to significantly enhanced efficacy (p<0.05). Gene expression analysis demonstrated a reciprocal Wnt/MAPK signaling mechanism; RXC004 strongly suppressed Wnt signaling (AXIN2) but increased MAPK signaling (DUSP6), whilst Trametinib strongly suppressed MAPK signaling (DUSP6) but increased Wnt signaling (AXIN2). Combination of RXC004 and Trametinib sustained inhibition of both Wnt and MAPK signalling. Similar effects were observed with inhibitors of other MAPK pathway components. Conclusion: Wnt and MAPK pathways are frequently co-activated in GI cancers, particularly in the upstream Wnt pathway mutated sub-population. Pre-clinically we show that Wnt and MAPK pathways act as potential reciprocal resistance mechanisms following single agent inhibition of either pathway; co-inhibition of these pathways leads to synergistic effects in vitro and enhanced efficacy in vivo. This provides a clear rationale to assess this combination approach in the clinic. Citation Format: Simon A. Woodcock, Dorottya Keppel, Catherine Eagle, James Kelly, Eimear Flanagan, Emma Bishop, Inder Bhamra, Clifford D. Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical activity of the Wnt pathway inhibitor RXC004 in combination with MAPK pathway inhibitors in GI cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 944.

Abstract 1851: Comprehensive multi-level proteomics characterization of gastric cancer

Abstract Gastric cancer is the leading cause of cancer related deaths worldwide, accounts for over 700,000 deaths each year. There is an urgent need for advancing therapeutic strategies for patients with gastric cancer. Addressing this need, the Clinical Proteomics Tumor Analysis Consortium (CPTAC) has conducted a comprehensive characterization of gastric cancer, aiming to elucidate the complex molecular mechanisms driving this cancer type, and to discover underlying cancer biology that will form the basis for developing new approaches for precision medicine. This comprehensive study includes an integrated multi-level proteomics analysis of 165 surgically resected gastric cancer and 41 normal adjacent tissues. We employed a multi-faceted approach, analyzing the proteome, phosphoproteome and glycoproteome, along with other protein modifications including ubiquitination, acetylation, and tyrosine phosphorylation. Furthermore, we extended our research to include 74 pairs of early-onset gastric cancer and normal adjacent tissues, analyzed using a proteomic approach. We can totally identify more than 12,000 protein groups with median identification of 9750 protein groups per sample. This expansive dataset helps constructing a comprehensive multi-omics atlas of gastric cancer, offering a deep understanding into the disease's molecular landscape. The depth of this study was enhanced by incorporating multi-level proteomics data across 6 tiers, facilitating an unprecedented multi-level proteomics analysis. Our integrated approach allows us to illustrate the changes in protein expression, protein modifications, signaling pathways, and protein networks during tumorigeneis. This analysis uncovers potential therapeutic targets for personalized treatments. Overall, our study provides a multi-omics atlas for gastric cancer, a profound insight into gastric cancer biology, contributing significantly to the understanding of its pathogenesis. It could pave the way for precise treatments for gastric cancer, underscoring the potential of multi-level proteomics analyses in advancing personalized medicine. Citation Format: Yuefan Wang, Clinical Proteomic Tumor Analysis Consortium (CPTAC). Comprehensive multi-level proteomics characterization of gastric 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 1851.

Abstract 2024: Deep and durable responses to ALK-targeted therapy require CD8+ T cells

Abstract Despite major advances in therapies for lung cancer, it remains the leading cause of cancer related deaths in the United States. Tyrosine kinase inhibitors (TKI) designed to inhibit oncogenic anaplastic lymphoma kinase (ALK) fusions have transformed the treatment of patients with ALK+ NSCLC and many survive for several years on TKIs. While most patients experience a decrease in tumor burden on initial alectinib therapy, for many, tumor shrinkage is incomplete and persistent, residual disease remains. Critically, the degree of tumor shrinkage and amount of residual disease that persists despite TKI associates with overall survival. Multiple cancer-cell intrinsic mechanisms of persistence despite TKI therapy have been reported, but they do not fully account for the heterogeneity of this initial tumor response, suggesting other determinants are present beyond inhibiting the primary oncogene. However, extrinsic mediators of persistent disease, such as immune cells within the tumor microenvironment (TME), have not been rigorously studied in ALK+ NSCLC as they are considered immunologically “cold” tumors. Using EA-1 and EA-2, two distinct murine ALK+ adenocarcinoma cells lines derived from immunocompetent C57BL/6J mice, we have demonstrated that ALK+ NSCLC response to alectinib requires the adaptive immune system. C57BL/6J mice bearing orthotopic EA-1 tumors experience tumor shrinkage but have persistent, residual disease on CT imaging; those bearing EA-2 tumors have no residual disease. In this model system, when alectinib treatment stops, no EA-2 tumors recur but all animals bearing EA-1 tumors experience tumor outgrowth. TME interrogations by multispectral tissue imaging demonstrated more CD8+ T cells in the EA-2 TME compared to EA-1 and no difference in CD4+ T cells. Flow cytometry revealed a significant increase in activated CD8+ T cells and CD86+ CD103+ dendritic cells within the TME of EA-2 tumors compared to EA-1. When implanted into nude mice, EA-1 and EA-2 both experience initial disease shrinkage on alectinib, but EA-2 tumors now exhibit residual disease on imaging. Notably for both cell lines, tumor outgrowth occurs in the nude mice despite continued TKI therapy. Using knock out animal models, we established that CD8+ T cells, but not CD4+ T cells, were required for response to alectinib. In animals lacking CD8+ T cells, EA-2 tumors shrink with alectinib therapy but are not eliminated as in the wild type controls. Notably, despite continued alectinib therapy, tumors grew out in the CD8+ knock out animals. In the EA-2 model, CD4+ T cells we not required for response to alectinib, and CD4+ knock out animals did not experience tumor outgrowth when alectinib was held. Taken together, these data suggest that the adaptive immune system, specifically CD8+ T cells, are required for a deep and durable response to molecularly targeted therapy for ALK+ NSCLC. Citation Format: Greg Reis, Stevie Phelabaum, Andre Navarro, Emily Kleczko, Lynn Heasley, Raphael Nemenoff, Erin L. Schenk. Deep and durable responses to ALK-targeted therapy require CD8+ T 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 2024.

Abstract 7056: The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. In PCa the expression of anoctamin 7 (ANO7) has been shown to diminish as the cancer progresses. We have previously linked single-nucleotide polymorphisms in the ANO7 gene to the risk of aggressive prostate cancer and shown that in homozygous carriers of rs77559646, the variant leads to a total loss of ANO7 protein. ANO7 is a prostate-specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. To uncover the cellular functions of ANO7 has proven challenging because ANO7 is not expressed in commercially available cell lines. To study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and gene set enrichment analysis. Interestingly, we found enrichment and downregulation of mitochondrial genes participating in oxidative phosphorylation in ANO7 cells. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the basal and maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells. Furthermore, the glycolysis stress assay showed that glycolytic capacity is increased in ANO7 overexpressing cells. In addition, a targeted metabolite screening revealed that aspartate is decreased in ANO7 cells, which again points towards perturbed mitochondrial function as aspartate is produced from a tricarboxylic acid cycle intermediate. By inhibiting the uptake of extracellular aspartate, we measured slower proliferation of ANO7 expressing cells, while inhibiting aspartate had no effect on control cells. Interestingly, preliminary results also suggest ANO7's involvement in regulation of phospholipid acyl chain length and saturation, offering insights into altered signaling observed in ANO7 cells. Taken together, this study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism, which could explain why it is beneficial for cancer cells to lose ANO7 expression. Citation Format: Christoffer Löf, Nasrin Sultana, Neha Goel, Gudrun Wahlström, Johanna Schleutker. The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism [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 7056.

Abstract 4266: Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation

Abstract Lung cancer is the leading cause of cancer related deaths worldwide, with the most prevalent subtype being non-small cell lung cancer (NSCLC). As only a subset of patients exhibits durable clinical responses to current treatments, including targeted therapy and immunotherapy, new treatment options are needed. Integrins (ITGs) are a diverse class of heterodimeric, transmembrane receptors that bind extracellularly to the extracellular matrix (ECM) and intracellularly to the cytoskeleton to integrate signals bi-directionally between the extracellular environment and cell interior. One of 18 α subunits is paired with one of 6 β subunits to create the 24 known αβ ITGs in mammals. ITGs are expressed at low levels in normal tissue and can be upregulated in cancer cells in response to stress signals in the tumor microenvironment (TME). Altered expression of ITGs is associated with enhanced cell proliferation, metastasis, and immune evasion. Targeting of the diverse ITG family members by inhibitors not specific to individual isoforms has obscured understanding of potentially distinct functions of each heterodimer. Through data mining of a genome wide CRISPR screening publicly available at the DepMap portal, we have identified a novel cancer dependency to ITG αvβ5 in approximately 40% of NSCLC cell lines. Using small molecule ITG αv inhibitors, we have mirrored sensitivity of NSCLC cell lines to their ITG αvβ5 CRISPR scores, suggesting that these inhibitors function specifically through ITG αvβ5 in cell lines. RNA sequencing analysis following αvβ5 inhibition in human NSCLC cell lines revealed downregulation of YAP and E2F1 target genes with alterations in gene ontologies associated with cell cycling and cell death in sensitive, but not resistant, lines. This is consistent with the known function of YAP driving gene transcription promoting cell proliferation and survival, and of E2F1 being implicated in regulating the proliferation and self-renewal of cancer stem cell (CSC). Additional changes to immune processes, specifically perturbations to interferon (IFN) pathways, were identified in response to ITG αvβ5 inhibition, suggesting that ITG αvβ5 may play a role in host anti-tumor immune responses. Pharmacological inhibition of ITG αvβ5 also led to a reduction of tumor growth in NSCLC murine models accompanied with increased CD8 T cell activation. These findings support ITG αvβ5 as a unique target of cancer dependency in NSCLC whose inhibition promotes anti-tumor responses through both tumor intrinsic and host immune regulatory mechanisms. Citation Format: Bitta P. Kahangi, William P. Crosson, Ji-Ann Lee, Camelia Dumitras, Tianhao Zhang, Ramin Salehi-Rad, Linh M. Tran, Michael Palazzolo, Dylan Conklin, Steven M. Dubinett, Bin Liu. Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation [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 4266.

Abstract 5516: Aberrant activation and perpetuation of lung wound response facilitates osteosarcoma metastasis

Abstract Introduction: The mechanisms that allow tumor cells to integrate with unfamiliar parenchymal cells within hostile distant tissues during metastatic colonization remain poorly defined. Nowhere are these mechanisms more important than in osteosarcoma-a pediatric bone tumor where metastases cause nearly all osteosarcoma related deaths and occur almost exclusively in the lung. The purpose of this study was to define the cellular and molecular events that facilitate osteosarcoma lung colonization to devise novel therapeutic strategies that prevent and treat metastasis. Experimental Approach: We generated experimental lung metastases using a panel of immunocompetent murine and human xenograft osteosarcoma models. A combination of immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics were used to define the metastatic niche. Relevant findings were validated in human primary tumor, lung metastasis, and normal lung tissue specimens. Results: Disseminated osteosarcoma cells physically associate with alveolar epithelial cells and trigger changes in surrounding epithelial cells that are characteristic of the aberrant, non-resolving wound response seen in non-malignant lung diseases such as idiopathic pulmonary fibrosis (IPF). This process drives the accumulation of fibrotic epithelial cells and scar-associated macrophages confined within a dense fibronectin-based matrix. Anti-fibrotic tyrosine kinase inhibitors used to treat patients with IPF (nintedanib) prevent the observed fibrogenic changes and inhibit metastatic colonization. Epithelial-osteosarcoma paracrine signaling is dominated by an inflammatory cytokine milieu that facilitates this transition towards fibrosis. Blocking one of the dominant paracrine signals with a recombinant IL1 receptor antagonist (anakinra) has similar effects on both fibrosis and metastatic colonization. Conclusions: Our study demonstrates that tumor interactions with host stromal cells are critical for metastasis. Metastatic colonization requires transformation of the local lung microenvironment into a fibrotic, non-resolving, cytokine-rich, wound-like state that is both provoked and sustained by tumor-epithelial interactions. These insights have identified several potential therapeutic approaches to prevent metastatic colonization and treat established metastases. Citation Format: James Brandon Reinecke, Amy C. Gross, Maren Cam, Leyre Jimenez Garcia, Matthew V. Cannon, Ryan D. Roberts. Aberrant activation and perpetuation of lung wound response facilitates osteosarcoma metastasis [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 5516.

Abstract 702: Development of a radioresistant PDX prostate cancer model to evaluate potential radiosensitizing compounds

Abstract Prostate cancer is the second most common cancer diagnosis worldwide and the fifth leading cause of cancer related death among men. Several treatment strategies are used including the FDA approved 177Lu-vipivotide tetraxetan (177Lu-PSMA-617) for the treatment of prostate-specific membrane antigen (PSMA)-positive metastatic castration-resistant prostate cancer. However, not all patients respond to treatment due to tumor intrinsic resistance mechanisms. Therefore, the combination of targeted radionuclide therapy with inhibitors against e.g. DNA damage response, cell cycle progression or cell survival pathways are investigated to enhance the radiosensitivity of the tumor. Current models to study radioresistance in prostate cancer are based on cancer cell lines or cell-line derived xenograft models, but these models do not account for the heterogenous and complex biology of the disease. To overcome this issue, we have developed a radioresistant patient-derived xenograft (PDX) prostate cancer model (ST1273, XenoSTART), that is positive for PSMA. Resistance to radiation was initially established by sequential irradiation of the tumors with external beam radiation. Moreover, the model has proven to be resistant towards treatment with 30 MBq 177Lu-PSMA-617. Utilizing this radioresistant model we are now able to investigate the treatment response of 177Lu-PSMA-617 in combination with different drug inhibitor classes to evaluate their potential radiosensitizing effect. The PARP inhibitor, Olaparib, was administered at 50 mg/kg QDx48 alone or in combination with a single dose of 177Lu-PSMA-617 in radioresistant ST1273 tumor bearing NMRI nude mice. First results showed a prolonging treatment effect of Olaparib given in combination with 177Lu-PSMA-617 compared to 177Lu-PSMA-617 alone. Tumor relapse was observed 29 days after treatment with 177Lu-PSMA-617 alone, whereas it was observed 49 days after combination treatment. Olaparib given alone did not influence tumor growth. To further investigate the mechanism of resistance we are currently investigating the radiosensitizing potential of additional compounds and evaluating the tumors by RNA- and whole-exome- sequencing. Altogether, we successfully established a PSMA positive prostate cancer model resistant to PSMA targeted radionuclide therapy. With this model we are able to investigate the radiosensitizing effect of different drug classes. Citation Format: Maria Zeiler Alfsen, Maria Thaysen, Nikoline Nielsen, Michael Wick, Luke Piggott, Sebastian Gnosa, Carsten Haagen Nielsen. Development of a radioresistant PDX prostate cancer model to evaluate potential radiosensitizing compounds [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 702.

Abstract 6311: From bench to bedside: GMP manufacturing and testing of an LGR5-targeting CAR-T against colorectal cancer

Abstract Colorectal cancer is the third most diagnosed cancer worldwide, and is responsible for the second largest number of cancer related deaths, warranting the need for new treatments. The successful use of chimeric antigen receptor T (CAR-T) cells against B-cell malignancies has inspired the search for CAR-T cell therapies against solid tumours such as colorectal cancer. Cancer stem cells (CSC) are a small population of cells within a tumour with the capacity to self-renew, differentiate, and initiate new tumours. CSC are resistant to chemotherapy and radiotherapy and are enriched in residual disease. Targeting CSC is a strategy to reduce the tumours’ ability to generate new cells. Leucine-rich G protein-coupled receptor 5 (LGR5) is a marker of CSC and LGR5+ cancer cells are implicated in tumour progression and metastasis in colorectal cancer. We designed and tested a chimeric antigen receptor (CAR) targeting the extracellular domain of LGR5. LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity and IFNγ release against colorectal cancer (CRC) cell lines in vitro, and in vivo efficacy in a CRC xenograft mouse model. We confirmed the specificity and safety of the LGR5 targeting CAR-T cells by testing against normal human tissues. A GMP ready protocol for clinical scale manufacture of LGR5-targeting CAR-T cells have been developed using CD3+ T-cells from healthy donors. We have successfully tested this protocol using CRC patient-derived CD3+ T-cells and were able to generate clinically relevant cell numbers (80-fold expansion) with high CAR expression. Cells manufactured using this GMP protocol expressed markers of self-renewing naïve-like and central memory phenotype and showed very low percentage of cells that were triple-positive for PD1, TIM3 and LAG3. This protocol has been successfully tested and validated in a GMP manufacturing facility for the production of LGR5 targeting CAR-T cells, in preparation for an FDA approved first in man clinical trial against metastatic colorectal cancer, starting in 2023 (NCT05759728). Citation Format: Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Jade Foeng, Dylan McPeake, Timona Tylis, Emma Thompson, Stuart Mills, Allison Cowin, Claudine Bonder, Timothy Sadlon, Shaun McColl, Simon Barry. From bench to bedside: GMP manufacturing and testing of an LGR5-targeting CAR-T against 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 6311.

Abstract 7028: Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers

Abstract Prostate cancer is the second leading cause of cancer related deaths among men in the United States. Health disparities in men diagnosed with prostate cancer are observed between patients of African and those of European ancestry. To elucidate molecular mechanisms behind prostate tumorigenesis and racial disparities, researchers have identified genetic alterations such as gene fusions, deletions, and mutations that occur at varying frequencies among different ethnic groups. However, results are inconsistent across studies, suggesting that racial disparities may be multifactorial. Our approach to further understand the mechanisms underlying racial disparities is to compare DNA methylation profiles between cancers from different ethnic groups using formalin-fixed paraffin-embedded (FFPE) prostate cancer tissue samples. For this study, FFPE prostate cancer specimens from 32 Black men and 30 age-matched White men were obtained in the form of tissue sections on glass slides. To profile DNA methylation in these samples, tumor and normal tissue regions (in a subset of cases) were outlined on a hematoxylin & eosin (H&E) reference slide and the respective regions were macro-dissected off the corresponding unstained slides from the same block. The areas of macro-dissected regions were measured using ImageJ, and the number of slides were taken to normalize DNA yield. Next, we compared magnetic bead and column based DNA extraction methods. We evaluated them on their relative DNA yield per tissue size, and identified that the bead-based extraction method had both higher and more consistent DNA yield. In total, we used 95 unique tissue regions to profile global DNA methylation levels using Illumina Epic array. We found that a subset of samples harbored an aberrant DNA methylation pattern. Therefore, we extracted DNA from additional FFPE tissue slides using the bead-based method and repeated the assay with a larger amount of DNA, successfully rescuing the majority of the aberrant samples. Next, we will evaluate associations between DNA methylation levels in different ethnic groups, clinicopathological features, and follow-up data to help identify potential DNA methylation biomarkers and therapeutic targets linked to prostate cancer from different ethnicities. Citation Format: Colton Duran Stensrud, Leonardo Gonzalez-Smith, Claire Stevens, Huan Cao, Jenaye Mack, Olumide Arigbede, Daniel Weisenberger, Sarah G. Buxbaum, Sara M. Falzarano, Suhn Rhie. Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers [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 7028.

Abstract 4278: Role of WNK1 in extracellular matrix modification in breast cancer

Abstract Introduction: Breast cancer (BCa) is the most common cancer and the second leading cause of cancer related deaths in women in the United States. Metastatic dissemination of cancer cells remains the leading cause of mortality in BCa. Therefore, determination of the mechanisms that promote metastasis is important for development of new therapeutic strategies. The extracellular matrix (ECM), which is the main non-cellular component of the tumor microenvironment (TME), plays an important role in BCa progression and metastasis. The cancer-associated fibroblasts (CAFs) are the principal cell types that together with BCa cells drive the modification of ECM during cancer development. Lysine deficient receptor protein kinase 1 (WNK1), a widely expressed serine threonine kinase that regulates blood pressure and electrolyte balance in the body, and is highly overexpressed in BCa microenvironment. The aim of the present study was to investigate the role of WNK1 in modification of ECM in BCa. Methods: The clinical relevance of WNK1 overexpression in BCa was determined by immunohistochemically staining human BCa sections, analyzing data from publicly available databases and creating orthotopic mouse models of BCa using 4T1 mouse BCa cells followed by treatment of the tumor bearing mice with WNK1 inhibitors, WNK-IN-11 and WNK463 (10 mg/kg , oral for 10 days). In vitro assays were conducted using 4T1 cells and mouse embryonic fibroblasts (MEFs) to determine the regulatory role of WNK1 on cancer cells and fibroblasts. Results and Conclusion: Our results indicated that WNK1 is strongly expressed in BCa cells and fibroblasts, the two most abundant cell types in the BCa microenvironment. Furthermore, WNK1 expression strongly correlated with expressions of matrix regulatory and cross-linking genes. Our results demonstrated that WNK1 inhibitors significantly reduced orthotopic 4T1 growth and metastasis. WNK1 inhibition also significantly inhibited collagen deposition in tumor tissues, which is a negative prognostic indicator in BCa. In vitro proliferation and migration assays using 4T1 cells and MEFs identified that WNK1 inhibitors (1-100nM) can significantly inhibit the proliferation and migration of 4T1 cells and MEFs. Results from co-culture studies with 4T1 cells and MEFs further indicated that WNK1 inhibition in MEFs could significantly reduce the invasive properties of 4T1 cells. Taken together, our results suggest that blocking the actions of WNK1 in BCa may be a promising therapeutic approach. Citation Format: Prabhat Suman, Sooraj Kakkat, Suhas S. Patil, Veronica Ramirez, Elba A. Turbat-Herrera, Seema Singh, Chandrani Sarkar, Debanjan Chakroborty. Role of WNK1 in extracellular matrix modification in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4278.

Abstract 2770: A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis

Abstract Metastasis of cancer cells to vital organs remains the leading cause of cancer related deaths, emphasizing a strong need for actionable targets in advanced stage cancer. To address this, we study novel dysregulated mitochondrial signaling mechanisms that cells utilize to metastasize. Here, we focus on how outer mitochondrial membrane protein—Mitochondrial Rho GTPase 2 (MIRO2)—promotes tumor cell invasion and metastasis. Our previous work identified higher MIRO2 mRNA expression in cancer vs. normal patient samples in a multitude of cancer types, which correlated with worse patient outcomes. Furthermore, we demonstrated that MIRO2 was critical for prostate cancer cell growth and survival in vitro and in vivo. However, it remains unknown if MIRO2 only affects primary tumor growth or if this protein is important throughout tumor progression. Using siRNA mediated knockdown (KD) of MIRO2 we find MIRO2 KD ubiquitously reduces tumor cell invasion in breast, melanoma, pancreatic, and prostate cancer cells. Utilizing metastatic prostate and breast cancer models, we demonstrate that mice injected with MIRO2 shRNA cells have significantly lower metastatic burden compared to mice injected with control shRNA cells. To determine the mechanism that MIRO2 controls invasion and metastasis, we looked at which of the top protein-binding partners for MIRO2 may regulate tumor cell invasion. Of our top hits, we found that depletion of atypical myosin IXB (MYO9B) by siRNA most significantly reduced tumor cell invasion and phenocopied MIRO2 depletion. MYO9B has a well-defined role in controlling cell motility via inactivation of RhoA. Excitingly, we have found 1) MIRO2 KD results in increased active RhoA, phenocopying MYO9B KD, 2) dual ablation of MIRO2 and RhoA fully rescues tumor cell invasion, and 3) MIRO2 is required for MYO9B driven invasion. Taken together, we propose a novel signaling mechanism by which MIRO2 broadly promotes invasion and metastasis through MYO9B dependent inactivation of RhoA. Citation Format: Dillon P. Boulton, Connor J. Hughes, Valentina Vaira, Ajita Jindal, Masoom Raza, Stephen Connor Purdy, Shilpa Sant, Heide L. Ford, M. Cecilia Caino. A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis [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 2770.

Abstract 4317: The role of autophagy in triple negative breast cancer

Abstract Breast cancer (BC) remains a global health concern, being the most common cancer amongst women in the world, and the second-leading cause of cancer related death. Triple-negative breast cancer (TNBC) represents a challenging frontier in oncology, characterized by its aggressiveness and its unique molecular profile devoid of the known receptors. Its elusiveness lies in the inherent heterogeneity within its subtypes, contributing to diverse clinical behaviours and treatment responses. TNBC has also shown elevated basal autophagy level as a pro-survival mechanism to make up for the increased nutrients demand mandated by the high K-RAS signalling. To tackle such tumour, we chose an autophagy blocker in addition to multi-kinase inhibitor. The combination index calculated for the proposed drugs has shown promising results in reduction of cell viability more than each drug separately. Selectivity index proved the safety of our doses on normal fibroblast cells in contrast to two breast cancer cell lines (MDA-MB 231 & MCF7). To further complement the findings, cell cycle arrest was witnessed at S phase which lead to apoptosis seen by flowcytometry and other expression analysis studies. Finally, Imaging studies and expression analysis on protein level have documented the apoptotic effects rendered by autophagy inhibition.Research endeavours continue to unravel the intricate landscape of TNBC, seeking novel avenues for targeted treatments, immunotherapies, and personalized medicine, aiming to transform the paradigm of care for those affected by this enigmatic form of breast cancer. As evident from the results, our combination has also managed to reduce the cells migration effectively which reflects that it can be initiated with aggressive metastatic tumours like TNBC. Citation Format: Sameh Gamal, Nefertiti A. El-Nikhely, Hesham Nematallah, Hesham M. Saeed. The role of autophagy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4317.

Abstract 1387: SETD2 loss promotes macrophage infiltration and metastasis in RCC

Abstract Across human cancer, tumors that are high grade, poorly differentiated, and have undergone epithelial-to-mesenchymal transition (EMT) carry a worse prognosis with a high likelihood of metastasizing to distant organs. Despite significant research efforts, metastatic cancers and relapse remain the primary cause of cancer related deaths. In renal cell carcinoma (RCC), metastasis is present in one-third of patients at the time of diagnosis, and one-third of patients who initially present with locally advanced disease eventually develop local or distant recurrence following resection of the primary tumor, highlighting the need for new RCC therapies and further insight into the metastatic process. Using RCC patient biopsies, we found that patients with more epithelial tumors have a significantly lower risk of recurrence. Additionally, tumors that had a mesenchymal protein signature had significantly increased macrophage infiltration, which was significantly associated with a higher stage at diagnosis and worse overall survival, suggesting that macrophages may play an important role in EMT and metastasis in ccRCC. Whole exome sequencing of these patient samples revealed that mutational loss of SETD2, a histone methyltransferase that is lost in about 20% of RCC patients, correlated strongly with increased macrophage infiltration within the primary tumor and was necessary for the correlation between macrophages and EMT. SETD2 loss in syngeneic kidney cancer mouse models confirmed that SETD2 loss within tumor cells resulted in increased EMT, macrophages within the primary tumor microenvironment (TME), and spontaneous metastasis, further supporting the role of macrophages in promoting metastasis in the absence of SETD2. Further characterization of the gene signatures and immune cell infiltrate in RCC will lead to a better understanding of metastasis in an oncogene- and tissue-specific manner and thus lead to more efficacious therapies for metastatic patients. Citation Format: Emily N. Arner, Melissa M. Wolf, Logan Vlach, Jeffrey C. Rathmell, W. Kimryn Rathmell. SETD2 loss promotes macrophage infiltration and metastasis in RCC [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 1387.

Abstract 5877: Disulfiram and its metal complexes reversed hypoxia induced stemness and synergized with first line drugs in primary patient derived pancreatic ductal adenocarcinoma cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate (12%) and is predicted to become the second leading cause of cancer related deaths by 2030. The main treatment option for advanced PDAC, gemcitabine (GEM) and nab-paclitaxel (PAC) chemotherapy, has limited efficacy due to adverse toxicities and chemoresistance. Extensive desmoplasia in the PDAC microenvironment induces hypoxia that drives epithelial to mesenchymal transition (EMT) and promotes cancer stem cells (CSCs) leading to metastasis and acquired resistance. Drugs that target CSCs and EMT to overcome chemoresistance could improve patient outcome. Disulfiram (DSF), an FDA approved anti alcoholism drug, has excellent anticancer activity on chelation with Cu2+/Zn2+ ions to form the anticancer metabolites, Copper or Zinc Diethyldithiocarbamate (CuDDC or ZnDDC). The anticancer application of the current oral version of DSF is prevented by its short plasma half-life (< 4 minutes) and separate administration of oral Cu/Zn supplement leading to bioavailability issues. A stable, long circulating, injectable formulation of DSF, CuDDC or ZnDDC may overcome these pharmacokinetic issues. In this study albumin was used to develop soluble, stable and homogenous CuDDC and ZnDDC nanoparticles (Alb-CuDDC, Alb-ZnDDC) and their efficacy was compared with free DSF-Cu in a panel of normoxic, hypoxic and spheroid cultures of patient PDAC cells derived from primary, circulating and metastatic sites. Hypoxia and spheroid cultures of PDAC cells were highly resistant to GEM and PAC and had increased expression of CSC markers such as ALDH, CD133, ABCG2 and CA9. Alb-CuDDC and Alb-ZnDDC were cytotoxic to all PDAC cells (IC50s ~300 nM and ~7000 nM, respectively) in normoxia and hypoxia and synergized with GEM and PAC. While DSF-Cu and Alb-CuDDC significantly reduced the expression of CSC markers and inhibited sphere reformation and migration of PDAC cells, Alb-ZnDDC was less effective in inhibiting these features. Therefore, the ability of PDAC cells to acquire resistance to Alb-ZnDDC was investigated. After long term exposure (> 4 months) to increasing doses of each drug (DSF-Cu, Alb-CuDDC and Alb-ZnDDC), PANC1 and BXPC3 PDAC cells acquired resistance only to Alb-ZnDDC. PANC1ZR6 and BXPC3ZR5 resistant cells showed increased expression of CSC markers and significant cross-resistance to PAC and GEM suggesting multidrug resistance. Importantly, no cross-resistance to Alb-CuDDC or DSF-Cu was observed, suggesting alternative mechanisms for Alb-ZnDDC. RNA sequencing is underway to identify potential mechanisms involved. To conclude, albumin formulation improved the solubility and stability of DSF-metal complexes. Alb-CuDDC targets CSCs and overcomes resistance in PDAC, similar to free DSF-Cu. Alb-ZnDDC may have alternative mechanisms which are yet to be elucidated. Citation Format: Benjamin J. Small, Ogechi Nkeonye, Vinodh Kannappan, Shalini Kumarasamy, Kate Butcher, Karim Azar, Gowtham Rajendran, Yoshitha Lakshmanan Lakshmanan, Mark Morris, Christopher Heeschen, Yaohe Wang, Weiguang Wang. Disulfiram and its metal complexes reversed hypoxia induced stemness and synergized with first line drugs in primary patient derived pancreatic ductal adenocarcinoma 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 5877.

Abstract 1592: Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer related death in the US. This mortality is strongly linked to the complex PDA tumor microenvironment that drives resistance to chemotherapy and immune suppression. Most patients succumb to metastatic disease, with liver being the most frequently colonized organ. Despite this, most pre-clinical studies have focused on primary PDA models to study the disease. Accordingly, the extent that the liver metastatic niche promotes immune evasion and resistance to treatment remains unclear.To directly compare primary vs. metastatic tumors in PDA tumors, we have optimized a syngeneic experimental model to contrast primary vs. metastatic lesions. To define the similarities and differences between cell types present in these tumors, we employed a single-cell transcriptomics approach. Our analysis has identified shared populations of malignant epithelial, stromal, and immune cells between lesions, allowing for a direct comparison of programming. Here, we have observed that the dominant populations of PDA cells in pancreas vs. liver tumors engage in distinct metabolic programs that suggest different actionable vulnerabilities.Beyond these, we also observed fibroblasts and myeloid sub-populations that are exclusive to liver tumors. These data further support our hypothesis that alternative mechanisms of metabolic, immune, and stromal interactions take place in the metastatic tumor microenvironment. To define these crosstalk networks, we employed the interactome tool CellChat, which has uncovered unique signaling interactions between the CAF and cancer cell populations in pancreatic or liver tumors. Overall, these data have the potential to lead to new avenues to directly target cancer cells or engage an effective immune response that can be designed to treat metastatic pancreatic cancer- an urgent clinical need. Citation Format: Rima Singh, Christopher Halbrook. Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic 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 1592.

Abstract 4374: PP2A-B56α in nutrient scavenging: Tipping the balance from macropinocytosis to cell death in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths in the US, with the lowest five-year survival rate of all major cancers. Nutrients in the PDAC microenvironment are commonly depleted, with the vital amino acid glutamine among the most deficient metabolites. To circumvent this deprivation, PDAC cells initiate KRAS dependent macropinocytosis, an actin-driven nutrient scavenging pathway. Macropinosomes fuse with lysosomes in a process mediated by the kinase PIKfyve. It allows cells to replenish the nutrients required for survival. As glutamine is essential for PDAC cell survival, therapeutic inhibition of macropinocytosis represents a novel strategy to suppress nutrient acquisition and drive cell death. Protein phosphatase 2A (PP2A) is a heterotrimeric Serine/Threonine phosphatase known to inhibit downstream targets of the KRAS signaling cascade and is implicated in macropinocytosis regulation. PP2A holoenzyme comprises of A, B and C subunits, where the regulatory B subunit provides substrate specificity to the enzyme. Using high molecular weight TMR-Dextran, we demonstrate that overexpression of PP2A-B56α subunit or pharmacological activation by DT061 promotes macropinosome accumulation in cells. Macropinosome accumulation was caused by the inability of macropinosomes to fuse with lysosomes, leading to cell death. The cell death caused by DT061 was significantly rescued when treated with a Rac family inhibitor. To understand if B56α regulates the process of macropinosome-lysosome fusion, we performed co-immunoprecipitation and established the interaction of B56α with PIKfyve. Further, pharmacological PIKfyve inhibition with Apilimod phenocopies DT061 induced vesicle accumulation in cells, suggesting that B56α likely inhibits PIKfyve to promote aberrant macropinocytosis. Prevention of lysosomal fusion will impact nutrient uptake through macropinocytosis. Our metabolomics study confirmed the deprivation of glutamine with activation of PP2A-B56α. This regulation can be therapeutically leveraged to metabolically stress cells with a combination of DT061 and glutamine transport inhibitor V-9302. Combination drug treatment was found to be synergistic in a panel of PDAC cells. Together, these findings indicate that activation of PP2A in late stage PDAC promotes aberrant macropinocytosis by preventing lysosomal fusion through PIKfyve regulation and establishes a novel role of PP2A-B56α in nutrient scavenging and cell death. These pathways can be further exploited to identify potential combination therapeutics in PDAC. Citation Format: Garima Baral, Claire M. Pfeffer, Indiraa Doraivel, Sara N. Filippelli, Brittany L. Allen-Petersen. PP2A-B56α in nutrient scavenging: Tipping the balance from macropinocytosis to cell death in pancreatic 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 4374.

Abstract 2156: Exploring barriers to colorectal cancer screening among adult male immigrants in Salt Lake City

Abstract Background: The United States (U.S) foreign-born population aged 65+ increased substantially between 1990 and 2010 with migrants from Africa and the Middle East being a part of these new refugee admissions. According to 2010 data, socioeconomic disparities and reduced access to health care is common in this population with 16 percent of foreign-born elderly living below the poverty threshold in comparison with 8 percent of U.S.-born elderly. Colorectal Cancer (CRC) is the second leading cause of cancer related deaths in the U.S despite being easily preventable and treatable. Studies among male African Americans reveal a CRC incidence >20% higher than what obtains among whites. Since, genetic factors are believed to contribute as much as 35% to the overall risk of CRC, male adult immigrants aged 45 years and above from Africa and the Middle East may be at increased risk of developing the disease. Determining CRC screening uptake in this population will be critical in planning interventions which may ultimately reduce disparities in CRC-related morbidity and mortality in the future. Methods: We conducted a mixed methods study comprising administration of questionnaires and organizing four Focus Group Discussion (FGD) sessions among adult males in Salt Lake City who identified as refugees from Africa or the Middle East. The sessions were audio-taped, transcribed and coded using the Dedoose software. Results: We had thirty-one participants who were between 45 and 64 years of age. Twenty-five of them (83%) reported awareness of the existence of CRC screening tests. Ten participants (33%) had undergone a CRC test in the past. However, only nine participants (30%) indicated that they will be willing to participate (WTP) in CRC screening if offered for free. Increased WTP was associated with younger age (OR=1.45, 95% CI: 1.23-1.81), length of stay in the United States (OR=1.41, 95% CI: 1.13-1.78) and higher education (OR= 1.26, 95% CI 1.13-1.37). Three major themes identified in the FGDs include: (1) Mistrust - distrust of the US health system (2) Fear - fear of cancer and nature of test and (3) Fatalism - the belief that screening cannot lead to cancer cure. Conclusion: The low level of WTP in CRC screening tests among the respondents may be associated with past experiences. There may be a need to consider the correlates of increased WTP and explore culture-specific CRC prevention measures in planning interventions designed to improve screening uptake in this population. Citation Format: Olanrewaju Olusola Onigbogi, Kebba Kah, Kolawole Okuyemi. Exploring barriers to colorectal cancer screening among adult male immigrants in Salt Lake City [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 2156.

Abstract 6220: TRIP13 augments pancreatic cancer progression- An integrated systems biology study

Abstract Background- Pancreatic cancer (PanCa) is one of the most aggressive forms of cancer and its incidence rate is continuously increasing every year. It is expected that by 2030, PanCa will become the 2nd leading cause of cancer related deaths in the United States due to the lack of understanding of the complexity of disease, early diagnostic methods, and extremely poor survival. Despite great advancements in biomedical research, there are very limited modalities available for the early detection of PanCa and its treatment. Thus, understanding of disease biology and identification of newer diagnostic and therapeutic modalities are high priority research in the field. Our group has identified Thyroid Receptor Interacting Protein -13 (TRIP13) as an oncogenic protein which contributes to early events of PanCa. Methodology- High dimensional omics and structural elucidation of TRIP13 was conducted using publicly available databases like Consurf, Phosphosite, GTEx and GEPIA2. Molecular biology techniques were applied to cross validate all our bioinformatics data. qPCR, immunoblotting and IHC analysis was performed in progressive PDAC cell lines and human tissues to decipher the expression level of TRIP13 in various pathological staging including functional enrichment analysis using Linkdomics. Results- The structural elucidation analysis revealed that most of the functionally exposed residues, phosphorylation and ubiquitylation sites situated at AAA domain of TRIP13. As compared to normal, TRIP13 is significantly over expressed in pancreatic tumor samples. Pathological staging 2 and 1 (early stages) showed relatively higher expression of TRIP13 as compared advanced stage. Of the total 7 isoforms of TRIP13, only one isoform (TRIP13-001) has shown the over expression in pancreatic tumor samples and shown association with poor survival. In correlation studies, TRIP13 has shown positive association with other pancreatic cancer biomarkers (CEACAM5, S100A4, MUC1, MSLN, CA125). The functional enrichment analyses suggest that TRIP13 is involved in important patho-physiological pathways like DNA repair, viral carcinogenesis, cellular senescence, and cell cycle. Our wet lab experiments also support our computational biology observation. Conclusion- This integrated computational biology study suggests a potential role of TRIP13 in pancreatic cancer progression via modulation of important oncological pathways. Wet lab experimentations also validated the involvement of TRIP13 in pancreatic cancer progression. TRIP13 expression may be associated with patient prognosis. Thus, its inhibitory small molecules can be useful for boosting pancreatic cancer therapies in clinical settings. Key words- TRIP13, Pancreatic cancer, Early events of cancer, Integrative Biology, Transcriptomics, Computational biology Citation Format: Swati Dhasmana, Anupam Dhasmana, Stella Rios, Iris A. Perez, Sheema Khan, Farrukh Afaq, Upender Manne, Murali M. Yallapu, Subhash C. Chauhan. TRIP13 augments pancreatic cancer progression- An integrated systems biology study [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 6220.

Abstract 6872: A spatio-temporal approach to mapping the dynamics of cutaneous squamous cell carcinoma progression and immunotherapy response: A journey through TiME

Abstract Background: Cutaneous squamous cell carcinoma (cSCC), arising from proliferation of malignant epidermal keratinocytes, is the second most common non-melanoma skin cancer. cSCC accounts for 75% of all skin cancer related deaths excluding melanoma, and metastatic and immunotherapy-resistant cases pose an emerging global threat. Tumor development is a gradual process characterized by high mutational burden and an immunosuppressive tumor immune microenvironment (TiME); therefore, identifying the underlying changes across the spatial and temporal landscape of these tumors will be crucial to understanding the key cellular and molecular drivers of disease progression and response. Methods: In this longitudinal study, we used a single-cell, whole-slide approach to mapping key proteins of immune lineage, tissue architecture, cellular metabolism, and stress to study the spatial landscape of cSCC at baseline (pre-treatment) and multiple time points over the course of immunotherapy with the aim to develop a temporal atlas of the tumor. An ultra-high plex antibody panel encompassing over 50 major determinants in the tumor microenvironment was employed on the PhenoCycler®-Fusion spatial biology platform. Deep bioinformatic analyses was performed to identify cellular phenotypes, spatial neighborhoods, heterogenous functional states and cellular interactions by spatial proximity determinations. Results: Previous studies in our laboratory have identified unique phenotypes correlating with response and resistance to immunotherapies in pre-treatment cSCC biopsies. Our results expand on these findings with temporal data tracking the dynamic changes in the TiME over the course of immune checkpoint inhibitor therapy. Apart from differences in immune cell composition and spatial localization patterns, we also saw differences in macrophage polarization and expression of metabolic markers in the responder and non-responder cohorts. Conclusions: Overall, ultrahigh-plex spatio-temporal monitoring of cSCC revealed distinct signatures of response and resistance and identified key features in the TiME that reveal deeper insights into the pathobiology of the tumor. Our discovery-based approach identifies novel biomarkers for patient stratification and therapeutic modulation. Citation Format: Niyati Jhaveri, Bassem Ben Cheikh, Dmytro Klymyshyn, Ning Ma, Aditya Pratapa, James Monkman, Nadine Nelson, Arutha Kulasinghe. A spatio-temporal approach to mapping the dynamics of cutaneous squamous cell carcinoma progression and immunotherapy response: A journey through TiME [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 6872.

Abstract 7116: Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair

Abstract Prostate cancer (PCa) is the 2nd leading cause of cancer related deaths in US men. PCa is an androgen dependent disease driven by the androgen receptor (AR). Currently, androgen-deprivation therapy (ADT) is the standard of care first-line therapy for metastatic PCa. Resistance to ADT uniformly leads to lethal disease, termed castration-resistant prostate cancer (CRPC). Thus, there is an unmet clinical need to identify and develop novel strategies to treat CRPC. CBP/p300 are potent co-activators for AR. High expression of CBP/p300 is associated with locally advanced disease and castration resistant function of AR, resulting in poor patient outcomes and further highlighting the need to discern the role of CBP/p300 to potentially develop therapeutic targets for precision medicine. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. In this study, CBP/p300 mediated bromodomain activity is targeted by CCS1477 (inobrodib), a first-in-class bromodomain inhibitor developed by Cell Centric. CCS1477 treatment demonstrated effective inhibition in growth and clonogenicity assays. Inhibition of the CBP/p300 bromodomain with CCS1477 resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression in addition to inhibition of associated factors such as c-MYC and its downstream targets in PCa cell lines as well as patient derived xenograft (PDX) models. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC patient samples. The clinical significance of CBP/p300 expression in PCa has been investigated via elucidation of CBP/p300 transcriptional reprogramming and its role in DNA damage response (DDR) pathways. Specifically, findings revealed that CBP/p300 bromodomain suppression sensitizes to AR-dependent DNA-repair. Transcriptional mapping identified CBP/p300 as regulators of cell proliferation and DNA repair processes, which were functionally confirmed across several PCa model systems. To assess relevance, exogenous challenge with radiation revealed that CBP/p300 are required for AR-mediated DNA repair, and CBP/p300 expression is linked to DNA repair capacity in the clinical setting. Molecular analyses revealed that CBP/p300 facilitate double-strand break (DSB) repair efficiency via homologous recombination (HR) mediated DDR. Congruently, CBP/p300 strongly correlated with HR gene expression in PCa patient tissue. These collective findings reveal that CBP/p300 govern repair of DNA DSBs by regulating HR, thus modulating genome integrity and promoting CRPC growth. These studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati N. Chand, Wei Yuan, Denisa Bogdan, Jonathan Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair [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 7116.