Cancer Therapeutics Research Articles

Abstract 7154: Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models

Abstract Polyamines are small polycationic alkylamines that are absolutely required for the continual growth and proliferation of cancer cells. As cancer cells maintain elevated polyamine pools through dysregulated polyamine metabolism, its pharmacological modulation is a promising avenue in cancer therapeutics. The polyamine analogue ivospemin has shown efficacy in slowing pancreatic and ovarian tumor progression in vitro and in vivo and has demonstrated encouraging results in pancreatic cancer clinical trials. Considering nearly 75% of late-stage ovarian cancer patients develop resistant to platinum-based chemotherapies, limiting treatment options, the aim of our study is to determine the potential efficacy of ivospemin in combination with doxorubicin, a common chemotherapeutic used in platinum-resistant ovarian tumors. We have previously shown that ivospemin exposure decreases polyamine content in a variety of cancer cell lines through downregulation of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and induction of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT). Here we examine the potential of combining ivospemin with doxorubicin. Ivospemin treatment reduces cell viability in ovarian adenocarcinoma cell lines and increases the toxicity of doxorubicin regardless of cisplatin sensitivity. Cells treated with the combination exhibit a greater decrease in polyamine levels than cells treated with either single agent. This increased polyamine depletion and decreased survival is accomplished through modulation of polyamine metabolism, predominately through an additive induction of SSAT activity. Using the syngeneic VDID8+ ovarian murine model, we further evaluated the ability of ivospemin to improve response to doxorubicin at clinical and sub-clinical dosing. Ascites fluid was used as a marker for tumor burden and evaluated for polyamine content. We found that the combination treatment increases median survival, delays tumor onset, and decreases overall tumor burden compared to either clinical or subclinical doxorubicin dosing schemes. Combination treatment also decreases overall polyamine content in the ascites by 75%. N1-acetylated spermidine is enriched in ascites from combination-treated mice, consistent with an upregulation of SSAT in response to treatment. Recognizing the non-representative mutational status of the VDID8+ model as a limitation, we are currently evaluating the combination of ivospemin and doxorubicin in genetically defined murine models that better recapitulate human high-grade serous ovarian carcinomas. Ongoing studies will determine influences on the tumor microenvironment and will mechanistically evaluate the cooperativity of ivospemin and doxorubicin on pathways outside of polyamine metabolism. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Ashley C. Nwafor, Ting-Ann Liu, Elizabeth Bruckheimer, Jennifer K. Simpson, Tracy Murray Stewart, Robert A. Casero. Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian 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 7154.

Abstract 4979: Genomic profiling of early-onset metastatic colorectal cancer based on tumor sample location

Abstract Background: Here, we describe analyses towards identifying and comparing genetic variations between the primary and metastatic sites in early-onset metastatic colorectal cancer patients. Methods: We selected patients with early-onset metastatic colorectal cancer (under 50 years old) using data from the American Association for Cancer Research Project GENIE (version 14.1). We used the Fisher exact test to compare genetic mutations between the primary tumor and metastatic sites, complemented by the Benjamini-Hochberg adjustment method. Results: In a total of 1,791 patients with early-onset metastatic colorectal cancer, 27.1% had tumor samples from metastatic sites, while the majority, 72.9%, were from the primary tumor location. The most common mutations found were in the TP53 gene (77.6%), followed by APC (76.0%), KRAS (42.4%), and PIK3CA (19.0%). Notably, the metastatic samples had a significantly higher occurrence of SMAD4 mutations compared to their primary site counterparts (20.8% vs. 14.0%, p<0.001, q<0.001). Conversely, the primary tumor samples exhibited higher frequencies of TCF7L2 and ARID1A mutations (16.9% vs. 10.6%, p<0.001, q<0.001, and 13.0% vs. 6.4%, p<0.001, q<0.001, respectively). Conclusion: Determining the specific genes responsible for the formation of metastatic tumors is crucial for a deeper understanding of the disease's underlying biology in early-onset metastatic colorectal cancer. Such genomic details are crucial to pave the way to the discovery of potential biomarkers for diagnosis and the development of new cancer therapeutics. Citation Format: Muhammet Ozer, Suleyman Y. Goksu, Busra Bacik Goksu, Nina N. Sanford, Amy Jones, Nilesh Verma, Syed Kazmi. Genomic profiling of early-onset metastatic colorectal cancer based on tumor sample location [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 4979.

Abstract 2186: Bacterial lung cancer therapeutics from soil bacteria in New York City

Abstract The microbiome's diverse functions in human health have generated increasing interest in using live bacteria for cancer therapy. Due to their natural presence in colon, lung and breast tissue, bacterial therapies have been augmented using synthetic biology in order to treat infections, inflammation, and cancer. One major challenge is finding safe and effective host species and therapeutic payloads for a particular type of cancer. Here we explored the vast diversity of microbial isolates in the environment as a valuable source of novel therapeutic compounds that can be expressed by bacteria. Specifically, we isolated and identified soil bacteria from 25 local parks, and investigated their secreted compounds for anti-cancer activity using monolayer and 3D-spheroid cultures of lung cancer. Through metagenomic analysis, toxicology assays, varied growth conditions, and bacteria-cancer co-culture experiments, we found Bacillus spp. isolates from Manhattan parks' soil that secreted products with high, dose-dependent cytotoxicity against lung cancer models. We also demonstrate that Bacillus subtilis, a model organism for gram-positive bacteria, can colonize lung cancer tumor spheroids, pointing to its use as a viable strain for bacteria cancer therapy. The work presented here underscores the potential of harnessing environmental microbial diversity for the development of gram positive species and their compounds as cancer therapeutics. Citation Format: Dhruba Deb, Tal Danino. Bacterial lung cancer therapeutics from soil bacteria in New York 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 2186.

Abstract 4036: Integrating dendrimers and peptide-based biologics to co-target immune checkpoints and oncogenic pathways for HNSCC treatment

Abstract The utilization of peptides as cancer therapeutics has tremendous potential given their nature of mimicking protein functionalities while maintaining a high degree of modularity in their molecular design. However, their translation into the clinic remains a challenge due to their inferior binding kinetics, poor bioavailability, and short plasma half-life. To address these, we employed multivalent dendrimers that improve the binding and stability of the peptide-based biologis. We herein present a translatable, modular nanoparticle platform, utilizing generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers conjugated with engineered peptides (dendrimer-peptide conjugates, DPCs). This approach capitalizes on the multivalent binding effect mediated by dendrimers. We found that DPCs functionalized with peptides targeting programmed death-ligand 1 (PD-L1, DPCPD-L1), a crucial immune checkpoint protein in HNSCC, exhibit significantly lower KD at 163 nM, which was three orders of magnitude stronger than free peptides. Furthermore, DPCPD-L1 prolonged the serum half-life to 10.8 hours, approximately 14 times longer than that observed with PD-L1-binding peptide alone. The enhanced binding kinetics and plasma stability of DPCPD-L1 were translated into the in vivo efficacy, as evidenced by a significant 40% reduction in tumor volume after four injections of DPCPD-L1 at a dosage of 50 mg/kg in mice bearing oral carcinoma (MOC1) tumors. The transcriptomic analysis of DPCPD-L1-treated tumor tissues revealed an increased population of CD4- and CD8-positive lymphocytes while decreasing the regulatory T cells and tumor cell proliferation, which is likely related to the observed efficacy. Expanding on this study, we have employed peptides designed to bind to the epidermal growth factor receptor (EGFR), a key player in the oncogenic pathways of HNSCC. These peptides were conjugated to G7 PAMAM dendrimer, forming DPCEGFR. Similar to DPCPD-L1, DPCEGFR displayed improved binding affinity as proven by biophysical measurements and a series of in vitro assays. All these results highlight the versatility of our DPC platform, suggesting its potential applicability not only for targeting immune checkpoint proteins but also for intervening oncogenic pathways. With its simplicity, modularity, and versatility, this platform indicates the possibility of broadening its scope to incorporate multiple targeting and therapeutic factors, paving the way for its clinical translation, and providing new avenues in HNSCC treatment. Citation Format: DaWon Kim, Piper Rawding, Mari Iida, Kourtney Kostecki, Bridget Mehall, Deric Wheeler, Seungpyo Hong. Integrating dendrimers and peptide-based biologics to co-target immune checkpoints and oncogenic pathways for HNSCC treatment [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 4036.

Abstract 2115: Assessing BCL2 protein complex level in peripheral blood mononuclear cells as a surrogate pharmacodynamic biomarker of BCL2 inhibitors in hematological cancers

Abstract In the last decade, cancer therapeutics have adhered to the practice of administering drugs at the maximum tolerated dose (MTD) selected from limited patient cohorts. The emergence of targeted therapies has disrupted this dose selection paradigm, as optimal efficacy may be achievable at lower dose than MTD. Recently, a regulatory agency initiated "OPTIMUS" to redefine the dose selection process, recommending quantitative assessments of drug efficacy in relation to the target protein throughout development and clinical trials. ABT-199 is a protein interaction inhibitor that specifically disrupts the interaction of BCL2 protein. Consequently, the assessment of changes in the level of BCL2 protein complex is necessary to evaluate the ABT-199 efficacy for optimal dose selection. In this study, we adapted the Single-molecule Protein Interaction Detection platform for evaluating pharmacodynamics of BCL2 inhibitors in clinical samples. We assessed the dissociation of two BCL2-related biomarkers, BCL2-BAX and BCL2-BIM, from a model system under various ABT-199 doses. Following one hour treatment with 300 nM of ABT-199, the BCL2-BAX complex dissociated almost by 40%, while 80% of the BCL2-BIM complex remained. The reduction of the BCL2-BIM complex was not changed when extending the treatment for 3 hours, indicating that the BCL2-BAX complex may serve as a more sensitive biomarker for assessing the target engagement of ABT-199. To determine the assay sensitivity, we evaluated the ABT-199 response across different cell numbers and found that 100,000 cells are sufficient for performing the BCL2-BAX assay. Subsequently, we validated the utility of our method in assessing clinical samples, specifically BMMCs and PBMCs. We collected 4 PBMCs and the corresponding BMMCs from hematological cancer patients and treated them with various doses of ABT-199 ex vivo. With 100,000 BMMCs and 1,000,000 PBMCs, we can generate dose-response curves for the BCL2-BAX complex at doses of ABT-199 ranging from 1 to 1,000 nM. To explore the feasibility of employing the BCL2-BAX complex in PBMCs as a surrogate biomarker for assessing ABT-199 engagement with BCL2 in BMMCs, we compared the averaged dose-response curves from 4 PBMCs with that of BMMCs. Remarkably, the two curves showed a coherent dissociation trend, indicating that the biochemical effect of ABT-199 on the BCL2-BAX complex may be conserved across different sample types. Collectively, we have developed a target engagement assay for BCL2 inhibitors by measuring the BCL2-BAX complex. Notably, we observed a consistent dissociation pattern of the BCL2-BAX complex by ABT-199 treatment in both PBMCs and BMMCs. This molecular information offers an alternative way to assess the pharmacodynamics of BCL2 inhibitors, particularly in the case where collecting a series of BMMCs with sufficient amounts may not be feasible. Citation Format: Hongwon Lee, Byungsan Choi, Saem Hong, Yunseo Lee, Tae-Young Yoon, Janghee Woo. Assessing BCL2 protein complex level in peripheral blood mononuclear cells as a surrogate pharmacodynamic biomarker of BCL2 inhibitors in hematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2115.

Abstract 3919: Targeting tissue-resident macrophage progenitors by anti-IL34 restores anti-tumor immunity and suppresses tumor growth

Abstract Macrophages play important roles in tumor microenvironment, where they can promote immune suppression, tumor growth and resistance to checkpoint inhibitor therapy and are the focus of efforts to develop novel cancer therapeutics. Strategies to inhibit macrophage accumulation to suppress tumor growth by blocking their recruitment from circulation have been only partially effective, leading to the identification of roles in tumor progression for tissue resident macrophages that are seeded during embryonic development. We examined the origins of lung tumor associated macrophages using lineage tracing, flow cytometry, and single cell sequencing. By comparing gene expression profiles of resident F4/80hi Lyve1+ Ly6C- macrophages and Ly6C+ F4/80lo CCR2+ recruited macrophages in subcutaneous, lung, central nervous and renal normal and tumor tissues, we identified generic tissue resident macrophage and recruited macrophage transcriptomic signatures, revealing that resident macrophages are characterized by signatures of Myc driven proliferation, fatty acid and cholesterol metabolism and immune suppression, while recruited macrophages are characterized by signature of inflammation and immune stimulation. Resident macrophages were associated with worse outcomes in cancer patients than recruited macrophages. Single cell sequencing and flow cytometry of genetically engineered mouse models of CC10-Cre KrasG12D;p53-/- and SPC-Cre KrasG12D lung cancer revealed that lung tumor progression was characterized by extensive expansion of immune suppressive, Ki67+ resident alveolar macrophages and loss of T cells in the lung. Single cell transcriptomics identified IL-34 expressed exclusively by tumor cells as a possible driver of alveolar macrophage proliferation and IGF1 expressed by tumor associated alveolar macrophages as a possible regulator of tumor cell proliferation. Accordingly, blockade of IL-34 profoundly reduced tissue resident macrophage proliferation, stimulated recruitment of activated T cells, and suppressed growth of KrasG12D driven tumors. Analysis of TCGA data sets revealed that expression of IGF1 or IL34 was associated with reduced survival in lung, kidney, liver, head and neck and ovarian cancer. These studies identify key roles for IL-34 and IGF1 in solid tumor progression and suggest new therapeutic targets for the development of therapeutic strategies to stimulate anti-cancer immunity. Citation Format: Hui Chen, Jaroslav Zak, Mark Paradise, Guangfang Wang, John Teijaro, Mark Onaitis, Judith Varner. Targeting tissue-resident macrophage progenitors by anti-IL34 restores anti-tumor immunity and suppresses tumor growth [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 3919.

Abstract 4236: PDX-derived organoids (PDXO) are valuable tools to unveil the shortcomings of new anti-cancer drug candidates

Abstract Introduction: Tumor organoids are increasingly being used as predictive preclinical cancer models, as they reflect the original tumor features and patient drug responses. However, systematic comparisons between organoids and traditional 2D cell lines or 3D spheroids are lacking. Method: We established over 30 PDX-derived organoid (PDXO) models with KRAS mutations and tested three inhibitors targeting the KRAS pathway: VS-6766(RAF/MEK inhibitor), Trametinib (MEK inhibitor), and MRTX1133 (a Phase II drug targeting KRASG12D) against these models. Results: IC50 results indicated that most models responded to treatment. For some cell lines we detected differential drug responses in 2D vs. 3D testing with 3Dspheroids being more sensitive to inhibitors than 2D cell lines (on average ~10 fold). PDXO models exhibited greater individual differences in drug response, with a subset of models demonstrating no sensitivity to MRTX1133. This is consistent with published results showing that some KRASG12D-mutant PDX models were less sensitive to MRTX11331. Similarly, the clinical trial data for the KRASG12C inhibitor Adagrasib revealed distinctions in the response of patients2. Further analysis with WES (whole exome sequencing) and RNA-sequencing identified potential genotypic mutations and differential expressions of signaling pathways related to drug resistance. Subsequent drug combination testing showed that targeting these pathways could enhance the MRTX1133 efficacy in the resistant PDXO models. Summary: Our research confirms that drug testing on organoids predicts responses in mouse models and clinical trials. In combination with multi-omics and bioinformatics analysis, the platform can be used for evaluating drug resistances, identifying new targets, developing new drug combination therapies, and ultimately improving the creation of new cancer therapeutics. Citation Format: Yuhui Wang, Lin Feng, Han Liu, Junwen Zhang, Kaiqiang Hu, Pengwei Pan, Fang He. PDX-derived organoids (PDXO) are valuable tools to unveil the shortcomings of new anti-cancer drug candidates [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 4236.

Abstract 356: Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca® PLX Image Cytometer

Abstract Apoptosis is a critical pathway for programmed cell death that cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, allowing for the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase 3/7, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer. First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, and were monitored at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using the Cellaca® PLX. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early to late-stage apoptosis, and ultimately necrosis. A clear trend was observed during the first 2 h showing differences of up to 25% between the single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis amongst apoptotic populations, Annexin V+ only populations were higher than Caspase-3+ only populations by up to 30% between 0-2 hours. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image-based detection method using the Cellaca® PLX may provide an effective and efficient tool for rapid and reliable simultaneous detection of early, late-stage apoptosis, and necrosis. This may allow researchers to better characterize and screen potential cancer therapeutic drug candidates in a high-throughput manner to determine their treatment efficacy. Citation Format: Joydeep Mukherjee, Mackenzie Pierce, Yongyang Huang, Allen Lin, Leo Li-Ying Chan. Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca® PLX Image Cytometer [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 356.

Abstract 5075: Transcriptomic landscape of tumor microenvironment in gastric cancer in treatment with different types of cancer drugs

Abstract Gastric cancer (GC) in the fifth-leading types of cancer and the third-leading cause of death from cancer. Characterization of tumor microenvironment (TME) in GC is important not only to comprehensively understand tumor biology in GC but also to develop a novel cancer therapeutics targeting TME. In this study, we conducted multi-omics analyses of single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (bulk RNA-seq), and whole exome sequencing (WES) using biopsied tumor samples through endoscopy from 23 GC patients: 3 treatment-naïve patients, 7 patients in treatment with chemotherapy (Chemo), 5 patients in combination treatment with chemotherapy and immune checkpoint blockade (Chemo+CPI), 3 patients in combination treatment with chemotherapy and Her2-antibody targeted therapy (Chemo+Her2i), and 5 patients in third-line or later treatment. We initially classified the GC samples into 4 molecular subtypes (CIN, GS, MSI, and EBV) using datasets of WES, bulk RNA-seq and scRNA-seq, showing that our samples were mainly composed of CIN (n=7) and GS (n=16) types. In scRNA-seq, we revealed that drug treatments profoundly modulated not only proportions of TME components but also their molecular characteristics. For instance, Chemo+CPI treated patients showed higher proportion of CD8+ cytotoxic T cells with activation and/or exhaustion markers. Combination treatments with Chemo+Her2i, in addition, resulted in higher Trail and Estrogen signaling pathways in epithelial cells of TME components. These findings suggest that interventions with certain cancer therapeutic drugs could substantially convert transcriptome landscape and reconstitute GC TME components. High-resolution of GC TME analyses should provide a clue for developing a next generation of cancer therapeutic drugs. Citation Format: Hiroki Yamashita, Jason Guoqiang Zhang, Chiaki Mashima, Ryo Kamata, Tomoko Yamamori, Izuma Nakayama, Akihito Kawazoe, Kohei Shitara, Tomonori Yano, Qunli Xu, Akihiro Ohashi, Takeshi Saito. Transcriptomic landscape of tumor microenvironment in gastric cancer in treatment with different types of cancer drugs [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 5075.

Abstract 4582: HDAC6 inhibition reshapes the epigenome of cancer via P300 stabilization

Abstract Introduction: Histone deacetylases (HDACs)are crucial in regulating gene expression, DNA synthesis, and metabolic processes, including cancer, in various cellular environments. The unique structural and functional attributes of class IIb HDAC6 position it as a key player in influencing protein stability and chromatin dynamics. While its role in cancer progression and immunomodulation is well-recognized, the in-depth mechanisms following HDAC6 inactivation, particularly its influence on chromatin remodeling and gene regulation, are not thoroughly understood. This knowledge gap represents a critical area of investigation, given the potential of HDAC6 as a therapeutic target in oncology. This study aims to elucidate HDAC6 inactivation-dependent epigenetic effects of, exploring the possibility to exploit these latter for therapeutic purposes in cancer. This aim includes an examination of the stabilization of the lysine acetyltransferase P300 and its implications for chromatin structure and function. Methods & Results: In this study, ITF3756 and CRISPR/Cas9 were employed to inactivate HDAC6 across a variety of cancer cell lines (Jurkat, MDA 231, HCC1806, 4T1, and B16F10 melanoma). ATAC-seq and H3K27Ac ChIP-seq were used to investigate changes in chromatin accessibility, revealing significant alterations, particularly in introns and distal intergenic regions. Gene Ontology (GO) analysis further supported this alteration in the chromatin landscape, which indicated a differential expression of genes involved in critical cellular processes like cell proliferation, adhesion, migration, and apoptosis. A notable outcome of this study was the stabilization of P300 following ITF3756 treatment, an effect that was paralleled by changes in the expression of critical genes, including AKT1, ITGB3, GAS6, SOX9, NF1, TGFB2, and CASP7. These genes are integral to cellular survival mechanisms, and their modulation emphasizes the role of HDAC6 in cancer cell physiology. Conclusions: This research shows that the chemical or genetic inactivation of HDAC6 impacts cancer cell proliferation and chromatin architecture, underscoring the potential of HDAC6 as a target in cancer therapeutics.. A critical finding of this study is the stabilization of P300, which offers new perspectives on HDAC6's functionality and its role as an epigenetic modifier in cancer cells. This study contributes to the field by suggesting therapeutic strategies focusing on HDAC/HAT pharmacological targeting, thereby advancing our understanding of the role of HDAC6 in regulating cancer cell behavior and epigenetic landscape. Citation Format: Michela Gottardi Zamperla, Barbara Illi, Veronica Barbi, Chiara Cencioni, Stella Gagliardi, Maria Garofalo, Gabriele Antonio Zingale, Irene Pandino, Diego Sbardella, Lina Cipolla, Simone Sabbioneda, Antonella Farsetti, Chiara Ripamonti, Gianluca Fossati, Christian Steinkühler, Sandra Atlante, Carlo Gaetano. HDAC6 inhibition reshapes the epigenome of cancer via P300 stabilization [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 4582.

Abstract 2990: Differential micro-RNA expression in circulating exosomes of pancreatic ductal adenocarcinoma patients

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is a highly lethal solid tumor with 5-year survival rate of 5-8%. Extracellular vesicles like exosomes circulating in the blood are crucial mediators of intracellular communication, immunomodulation and tumor progression. The microRNAs being the major cargo of exosomes, are the primary carriers of intracellular information. The role of exosomal-miRNA (exoMIR’s) in regulating the pathogenesis of tumors is well-established in oncology but its potential is still unexplored. To the best of our knowledge no study has yet explored the contrasting exoMIR expression in cancer patients before and after their surgery. In this study, differential exoMIR’s profile of three pancreatic ductal adenocarcinoma patients at three stages: pre-surgery, post-surgery, and follow-up was established using small RNA sequencing on the Illumina platform. In blood samples taken before and after surgery total of 181, 201 and 201 microRNAs were dysregulated in circulating exosomes of three different patients viz. P_1, P_9 & P_11 (Patient identifiers) respectively. Out of the total dysregulated microRNAs in post-op samples 76 were upregulated and 105 downregulated in P_1, 95 upregulated and 106 downregulated in P_9 & 69 upregulated and 132 downregulated in P_11 as compared to their Pre surgery samples. Further, when exoMIR’s of post-op samples were compared to that of follow-up samples after 6 months of surgery their expression profile changed notably such that a total of 184, 157 & 190 microRNAs dysregulated in follow-up of P_1, P_9 and P_11 respectively. Out of the total dysregulated microRNAs in follow up samples 93 were upregulated and 91 downregulated in P_1, 69 upregulated and 88 downregulated in P_9 & 96 upregulated and 94 downregulated in P_11 as compared to their post-op samples. On Venn diagram analysis 27 microRNAs were found to be commonly dysregulated in all 3 patients before surgery and 22 common micro RNAs were again dysregulated even after 6 months of surgery. Bioinformatic analysis of these differentially expressed miRNAs and their respective protein targets revealed that their role in oncogenic molecular pathways is very prominent, thus highlighting their potential use in cancer therapeutics, diagnostics as well as prognosis. Citation Format: Rupinder Kaur, Sandeep Bhoriwal, Promod Kumar Gautam. Differential micro-RNA expression in circulating exosomes of pancreatic ductal adenocarcinoma patients [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 2990.

Abstract 5115: Designing targeted approaches for recurrent medulloblastoma

Abstract Designing targeted therapies for recurrent medulloblastomaPediatric brain tumors are the leading cause of cancer-related death in children, with medulloblastoma (MB) being the most common type. Despite significant progress in the field, approximately 30% of children with MB will relapse. Recurrent MB is commonly metastatic and virtually none of these patients will live beyond the one-year hallmark. These sobering statistics highlight the significant unmet need for effective therapies for recurrent MB. The likely underlying cause of the constant failure of our current clinical approaches, which are based on data obtained from disease models recapitulating primary tumors, is the disparity in genomic and transcriptomic data between primary and relapsed disease. Because of these disparities, we hypothesize that by conducting mechanistic studies and drug screens in relapse disease models, we can improve the outcomes for children with recurrent MB. Our data, obtained through a bioinformatics mining approach to predict tumor response, enabled us to identify a clinically relevant candidate drug for the treatment of recurrent MB: Minnelide. This compound acted on MYC to reduce tumor growth and the metastatic dissemination of tumors with the genetic background often found in relapsed MB patients. Additionally, to further support the translational relevance of our data, Minnelide increased the efficacy of adjuvant therapeutics used in the clinical management of MB. Our findings not only highlight the potential for repurposing Minnelide to treat children with recurrent MB, but also the potential of our research approaches aimed at identifying novel therapeutics for relapsed cancers. Citation Format: Jezabel Rodriguez Blanco. Designing targeted approaches for recurrent medulloblastoma [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 5115.

Abstract 5082: Novel TSHR CAR T therapy for targeting oncocytic carcinoma

Abstract Oncocytic carcinoma (Hurthle cell carcinoma) has the highest incidence of metastasis among the differentiated thyroid cancers. Metastatic disease is reported at the initial diagnosis in 10-20% of patients and 34% overall. Thyroid-stimulating hormone receptor (TSHR) expression is limited to the thyroid gland. It is abundantly expressed on thyroid tumor cells, making TSHR a compelling target for advanced thyroid cancer diagnostics and therapeutics. We developed a potent anticancer strategy for treating oncocytic carcinoma using a novel thyroid hormone stimulating receptor (TSHR)- targeted (chimeric antigen receptor) CAR T cell therapy. TSHR CAR T was generated by transducing T cells derived from healthy donors. In vitro effector functions (cytotoxicity, proliferation, degranulation) were tested in TSHR+ tumor models, and subsequent in vivo studies using NSG mice were performed to assess anti-tumor activity efficacy. We observed strong anti-tumor activity against metastatic oncocytic carcinoma cell line XCT.UC1 using the TSHR CAR T therapy in vivo. Re-challenging treated mice with XCT.UC1 cells reactivated TSHR CART cells with enhanced proliferation and complete suppression of tumor growth of the newly injected tumor cells. Our work represents a promising strategy to improve treatment outcomes of patients with metastatic oncocytic carcinoma that may provide a durable response. Citation Format: Justyna J. Gleba, Aylin Alasonyalilar-Demirer, Matthew Pawlush, Claudia Manriquez-Roman, Elizabeth L. Siegler, Robert C. Smallridge, Abba Zubair, Han W. Tun, Saad S. Kenderian, John A. Copland. Novel TSHR CAR T therapy for targeting oncocytic carcinoma [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 5082.

Abstract 6985: The oncogenic functions and action mechanism of UTP14A, a subunit required for 18S rRNA synthesis, in head and neck cancer

Abstract Head and neck cancer (HNC) is the sixth common malignancy in the world and > 90% of HNC cases are squamous cell carcinoma in origin. Oral cancer is one major HNC subtype. Despite the recent advancements in cancer diagnostics and therapeutics, the 5-yr survival rate of advanced HNC patients remains <50%. Ribosomes are the cellular factories responsible for making proteins. The hyper-activation of ribosome biogenesis initiated by oncogene gain or tumor suppressor gene loss plays a critical role in cancer initiation and progression. Ribosome biogenesis is a complex and energy intensive process that involves hundreds of factors. UTP14A gene encodes a member of the uridine triphosphate 14 family and is an essential component of a large ribonucleoprotein complex bound to the U3 small nucleolar RNA. Previous studies have demonstrated that UTP14A plays a key role in the synthesis of ribosomes and 18S rRNA, and is overexpressed in several malignancies. However, the biological functions and underlying mechanisms of UTP14A in HNC remain elusive. We found not only the increase of UTP14A mRNA expression in TCGA-HNC database but also its negative impact on patient clinical outcome. UTP14A depletion significantly impaired HNC cell proliferation, migration and invasion in vitro. Ectopic overexpression had the opposite effect, suggesting an oncogenic role for UTP14A in HNC. Through gene set enrichment analysis of pairwise RNA sequencing results, several genes involved in the process of epithelial mesenchymal transition (EMT) were potentially associated with manipulated UTP14A expression. We also detected a concordant change of lipid droplet numbers and its surface marker expression in UTP14A-manipulated cells, respectively, by BODIPY staining and immunhistochemical staining. Western blot analysis validated a positive relationship of EMT markers and COX-2, a potential mediator of lipid metabolism and EMT, with altered UTP14A expression. The involvement of COX2 in UTP14A mediation was confirmed by using both pharmacological inhibition and genetic manipulation. We also identified p38 as a central downstream effector but acting upstream from COX-2 in UTP14A mediation in oral cancer cells. We believe that our study of UTP14A-mediated oncogenic signaling axis can potentially be harnessed as a novel therapeutic target against HNC although more mechanistic studies may be needed. Citation Format: Li-Wha Wu, Chen-Ni Chang, Pulak K. Biswas, Sen-Tien Tsai. The oncogenic functions and action mechanism of UTP14A, a subunit required for 18S rRNA synthesis, in head and neck 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 6985.

Abstract 6460: Incidence of pathogenic germline variants and presumed germline pathogenic variants in Japanese lung cancer patients using comprehensive genomic profiling tests

Abstract Introduction: Pathogenic germline variants (PGVs) and presumed germline pathogenic variants (PGPVs) are often detected with comprehensive genomic profiling (CGP) tests. Using the Center for Advanced Cancer Genomics and Therapeutics (C-CAT) database, we investigated the incidence of PGV and PGPV detected by CGP tests in Japanese lung cancer patients. Methods: Among 3,240 lung cancer cases registered in C-CAT between June 2019 and August 2023, 321 were tumor/normal paired NCC Oncopanel (NOP), 773 were FoundationOne Liquid CDx (F1L), and 2,145 were tumor-only FoundationOne CDx (F1CDx). We analyzed cancer susceptibility genes in accordance with the national guideline. In F1L and F1CDx, PGPV was determined based on variant allele frequency (VAF) and medical history. Differences between groups in the detection of PGV and PGPV were tested using chi-squared test, and differences in age were tested using t-test. Results: The incidence of PGV in NOP and PGPV in F1L and F1CDx were 4/321 (1.2%), 36/773 (4.7%), and 141/2145 (6.6%), respectively (NOP vs F1L vs F1CDx; p = 0.001 NOP vs F1L; p = 0.006, NOP vs F1CDx; p < 0.001, F1L vs F1CDx;p=0.190). The 4 PGVs detected by NOP were in the ATM, BRCA2, MSH6, and TP53 genes. Notably, two out of four patients were in their 30s while the other two were in their 70s. In the F1L dataset, 36 patients (4.7%) had PGPV: 8 patients (1.0%) had PALB2, 6 patients (0.8%) had BRCA2, and 4 patients (0.5%) had CHEK2 variants. In the F1CDx dataset, 141 had PGPV: 19 patients (0.9%) each had BRCA1 or BRCA2 variants and 18 patients (0.8%) had PALB2 variant. There was no significant age difference between PGPV(-) and PGPV(+) patients (F1L:63.3 vs. 62.4 years; p = 0.938, F1CDx: 65.5 vs 65.0 years; p = 0.749). Most cases with PGV or PGPV were non-small cell lung cancer: 4/4 in NOP, 34/36 in F1L, and 110/141 in F1CDx. Discussion: The incidence of PGV in lung cancer patients was at least 1% and found in both younger and older patients. A circulating tumor DNA panel and a tumor-only tissue panel detected PGPVs at a significantly higher rate than detection of true PGVs by tumor/normal paired tissue panel, suggesting the use of tumor/normal paired panels may mitigate the burden of both patients and clinicians with concerns from false positive PGPV. The majority of detected PGV/PGPV were in homologous recombination repair genes. Citation Format: Michiko Ueki, Kousuke Watanabe, Momoko Morishita, Koki Fujii, Hiroki Ikushima, Hideaki Isago, Katsutoshi Oda, Hidenori Kage. Incidence of pathogenic germline variants and presumed germline pathogenic variants in Japanese lung cancer patients using comprehensive genomic profiling tests [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 6460.

Abstract 5815: Next-generation RNAi therapeutics: siRNA-loaded exosomes targeting KRAS G12C in non-small cell lung cancer (NSCLC)

Abstract Background: NSCLC represents the predominant subtype of lung cancer, boasting an overall 5-year survival rate of 26%, a figure that fluctuates based on the disease stage. Within NSCLC, certain cancer gene mutations, notably KRAS G12C, are currently classified as 'undruggable targets,' creating hurdles in achieving effective treatment. The development of innovative anticancer drugs aimed at these challenging targets is expected to present a viable solution to these difficulties. Methods: Exosomes have garnered attention as an inventive drug delivery system for in vivo gene delivery through systemic injection. We used a new post-loading approach by actively loading KRAS G12C siRNA into exosomes using the shock waves (SWEET: shock wave exosome engineering technology ) in this study. The effect of in vitro and in vivo were tested in NCI-H358 (KRAS G12C mt NSCLC) cells and its xenograft mouse model. The tumor size and weight were measured twice a week. Results: We developed an exosome-based therapeutic using siRNA to effectively inhibit KRAS G12C, targeting NSCLC. The SWEET platform's post-loading method showed a high efficiency in loading KRAS G12C siRNA into exosomes. The KRAS G12C siRNA-loaded exosomes effectively silenced oncogenic KRAS G12C expression in cancer cells; they inhibited tumor growth when administered intravenously in a NSCLC xenograft mouse model. Remarkably, the anticancer effect of KRAS G12C-loaded exosome was similar to that of Sotorasib, a KRAS G12C mutation targeting anti-cancer drug. Conclusion: Our study showed KRAS G12C siRNA loaded exosomes were effectively delivered to NSCLC tumor tissue via intravenous (IV) administration. And its anti-cancer effects were very similar to sotorasib, showing potential as a treatment for KRAS G12C mutation-expressing cancer. The effective delivery of siRNA to tumor tissues through exosomes highlights the potential of this technology as a promising approach for developing new RNAi therapeutics for cancer. Citation Format: Hyo Kyeong Kim, Yujeong Choi, Kyoung Hwa Kim, Yeongju Byun, Tae Hee Kim, Jae Hwan Kim, Shung Hyun An, DaeHo Bae, MyeongKwan Choi, Jihwa Chung, Kihwan Kwon. Next-generation RNAi therapeutics: siRNA-loaded exosomes targeting KRAS G12C in non-small cell lung cancer (NSCLC) [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 5815.

Abstract 1219: In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables ATAD3A gene depletion to enhance RAS-targeted therapy

Abstract Targeting mitochondrial oncoproteins presents a new concept in developing effective cancer therapeutics. ATAD3A is a nuclear-encoded mitochondrial enzyme that contributes to mitochondrial dynamics, cholesterol metabolism, and signal transduction. Our previous study has demonstrated elevated expression of ATAD3A in head and neck squamous cell carcinoma (HNSCC) primary tissues and cultured cell lines. Loss of function suppresses HNSCC cell growth and elicits tumor regression in orthotopic tumor-bearing mice. Mechanistically, ATAD3A interacts with ERK1/2 in the mitochondria of HNSCC cells in the presence of VDAC1, and the ATAD3A-ERK1/2 signaling axis drives HNSCC development in a RAS-independent fashion. CRISPR-Cas9 has revolutionized genome engineering and has been intensively used to modify cancer-associated genes. However, challenges remain as CRISPR editing components must be transported into cells to exert their function. There is a need to explore effective in vivo delivery systems to accelerate the clinical use of CRISPR-Cas9. As no drug is available to target ATAD3A directly, we developed CRISPR-based ATAD3A-targeting nanodrugs by loading Cas9 mRNA and anti-ATAD3A sgRNAs into lipid nanoparticles (LNPs) that have become clinically acceptable delivery system due to low toxicity and high delivery efficacy and biocompatibility. As expected, treatment tongue tumors established in NSG mice with LNP-sgATAD3A remarkably repressed tumor growth. Most interestingly, the addition of LNP-sgATAD3A to the RAS inhibitor salirasib potentiated anticancer activity for HNSCC compared with the efficacy resulting from single-arm treatment. These novel and significant findings demonstrate a novel approach to inhibiting ATAD3A in tumor cells and provide a possible therapeutic strategy to enhance the success of multimodality therapy in cancer patients. Citation Format: Fanghui Chen, Liwei Lang, Yamin Li, Wei Zheng, Yong Teng. In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables ATAD3A gene depletion to enhance RAS-targeted 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 1219.

Abstract 1886: BRY812, an anti-LIV-1 antibody drug conjugate with novel conjugation method for cancer therapeutics

Abstract LIV-1, also known as SLC39A6 or ZIP6, belongs to the zinc transporter family and was initially identified as an estrogen-inducible gene in breast cancer. Immunohistochemical (IHC) analysis have revealed escalated LIV-1 expression in estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites) as well as ER-/PR-/Her2- (triple-negative) breast cancers. Notably, healthy human tissues exhibit limited LIV-1 expression, primarily in hormone regulated organs (prostate, uterus, and breast). The escalated expression of LIV-1 in breast and prostate cancer, as well as melanoma and other tumors, positions LIV-1 as an excellent target for ADC therapeutics. BRY812 is a monomethyl auristatin E (MMAE)-conjugated humanized IgG1 ADC against LIV-1. With Bioray’s irreversible conjugation method CysLinkTM, BRY812 enables superior in vivo stability and eliminates off-target payload migration. The PK studies in rat and monkey demonstrated that the free MMAE released from ADC is 1 to 2 logs lower compared with approved ADCs prepared by the standard conjugation method. BRY812 is well tolerated in cynomolgus monkeys with repeated doses. With best-in-class anti-tumor efficacy in various models, BRY812 demonstrated a wider therapeutic window for cancer treatment. BRY812 is currently in Phase Ia dose escalation stage clinical trial to explore the maximum tolerate dosage (MTD) from 0.25 to 3.6 mg/kg Q3W in patients with advanced malignancies. Citation Format: Xiaobei Zhao, Jie Zhu, Yaqiong Zhou, Jing Li, Lei Nie, Haibin Wang, Gang Chen. BRY812, an anti-LIV-1 antibody drug conjugate with novel conjugation method for cancer 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 1886.

Abstract 5943: Development of a SMART theranostic agent for precision pancreatic cancer therapeutics

Abstract Pancreatic cancer is predicted to emerge as the second leading cause of cancer-related deaths in the United States by 2030. Pancreatic ductal adenocarcinoma (PDAC) - the most common form of pancreatic cancer - is often diagnosed as metastatic disease and has demonstrated the least improvement in overall survival over the last three decades with incidence rising by 0.5% to 1.0% per year due in part to lack of effective and tumor-selective treatment strategies. One approach to reduce host toxicity is the development of novel molecules and innovative therapeutic approaches that are nontoxic to normal cells but can be activated only under tumor-specific conditions for lethality, which we coined as SMART (Selective Method of Activation for Response in Tumors) therapeutic agents. We utilized explicit properties and biomarkers present in PDAC tumors but not in normal tissues to develop a SMART DNA-damaging anti-cancer agent with both therapeutic and diagnostic (theranostic) activities. We evaluated the tumoricidal effect of our SMART agent using long-term survival assays in PDAC cell lines and 3D co-culture spheroids, as well as in tumor xenograft model studies in mice. We also tested the ability of our SMART agent to treat, detect/diagnose (via click chemistry), and identify critical resistance mechanisms (via proteomics) as a single agent or as a chemosensitizer with predictive tumor biomarkers for tumor-selective therapeutic response. We found that our SMART small molecule induces tumor-selective lethal effects as a monotherapy or in combination with DNA repair inhibitors or small molecules known to generate hydrogen peroxide (H2O2) in a tumor-selective manner for activation. Using “click” chemistry, we detected and quantified the incorporation of our SMART small molecule as a DNA-damaging agent via flow cytometry that correlated with therapeutic response, which is a feature that could facilitate the stratification, treatment monitoring, and management of patients with pancreatic cancers. We also found that our SMART agent is less toxic and more effective as a monotherapy than cisplatin in PDAC cell line-derived xenograft studies in mice. Overall, our novel SMART theranostic agent could provide a significant advancement as they are preferentially activated under tumor-specific conditions to induce tumor-selective therapeutic response. Furthermore, the diagnostic component our SMART agent could provide oncologists with essential information to make rapid clinical decisions on treatment strategies. Our SMART agent is also remarkably versatile as it can be utilized as a chemosensitizer or as a chemical probe to identify targetable vulnerabilities to potentiate its therapeutic effects at lower doses. Citation Format: Edward A. Motea, Jarrett J. Smith, Oluwasijibomi Ketiku. Development of a SMART theranostic agent for precision pancreatic cancer 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 5943.

Abstract 7041: Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder cancer

Abstract Background: Cisplatin-based chemotherapy followed by radical cystectomy remains the standard-of-care treatment for patient with localized muscle-invasive bladder cancer (MIBC). Neoadjuvant clinical trials studying the combination of cisplatin-based chemotherapy and immune checkpoint blockade (ICB) in MIBC reported encouraging pathologic complete response rates. However, the molecular mechanisms and cellular programs involved in response and resistance to cisplatin and ICB in MIBC remain incompletely characterized. Our goal is to define cellular transcriptional programs and chromatin accessible regions in cancer cells and infiltrating immune cells associated with response and resistance to neoadjuvant systemic therapy. Methods: We performed single-cell Multiome ATAC and Gene expression sequencing (simultaneous identification of gene expression and open chromatin regions within the same cell) of tumors that had a complete response – “responders” – and tumors that had no response or progressed – “non-responders” – through neoadjuvant cisplatin-based chemotherapy (n=15). Single cell suspensions were prepared following the 10x Genomics protocol. Cell Ranger ARC was used for sequence alignment, peak calling, and generation of count matrices. Downstream single-cell gene expression analysis was performed with Scanpy and ATAC-seq with Seurat. Results: We identified two distinct urothelial cell populations in "responders" vs "non-responders" based on single cell gene expression and genome wide chromatin accessibility regions. Cancer cells in “non-responders” preferentially express basal cell gene expression signatures. “Non-responders” were significantly enriched in suppressive tumor-associated macrophages (TAMs). Interestingly, SPP1+ TAMs known to have a pro-tumoral role and promote resistance to ICB were exclusively identified in “non-responders”. Conclusion: Single cell and genome wide chromatin accessibility regions identify heterogenous urothelial cell populations expressing basal cell markers in tumors resistant to neoadjuvant cisplatin-based chemotherapy. “Non-responders” are significantly enriched with TAMs implicated in resistance to ICB. Future studies will better define the implications of these findings in cancer therapeutics for patients with MIBC. Citation Format: Filipe La Fuente De Carvalho, Jihyun Lee, Kevin Bi, Breanna Titchen, Konrad Stawiski, Jihye Park, Amanda Garza, Daniel Michaud, Jennifer Guerriero, Elizabeth Mittendorf, Kent Mouw, Eliezer Van Allen. Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder 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 7041.