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The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein–coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ–activated eosinophils facilitated CD4+ and CD8+ T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. Significance: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer.

Background: Breast cancer (BC) is a leading cause of cancer death in Ghana and around the world. Ghanaian women are diagnosed at younger ages with the more aggressive Triple Negative BC (TNBC) subtype where West African ancestry is associated with advanced BC diagnosis and higher mortality rates compared to age-matched women of European ancestry. Genomic comparisons of BC tumors from women of African and European ancestry show differences in frequencies of single nucleotide polymorphisms (SNP) and copy number variations. These differences may contribute to disparities in disease and treatment outcomes observed in women of African ancestry. Chemotherapy plays a major role in treatment of recurrent and metastatic BC. Long-term BC survival remains poor especially in Africa due to multidrug resistance (MDR). MDR has been associated with binding cassette (ABC) protein transporters. ABCB1, ABCC1 and ABCG2 are ABC transporter genes that code for proteins involved in drug efflux. We hypothesize that SNPs in ABC transporter genes may alter their physiological protective role and increase risk of MDR, treatment failure and death among BC patients. Preliminary dataWe explored gene expression profiles of ABCB1, ABCC1 and ABCG2 in an African ancestry-enriched subset of women with TNBC (ICSBCS cohort: Ghanaian n = 6, African American (AA) n = 9, Ethiopian n = 11). Preliminary data showed significantly higher expression of ABCC1 among Ghanaian and AA patients compared to Ethiopians, and a significant positive correlation with African ancestry. ABCB1 and ABCG2 showed lower expression in all three groups (ns).To study the relationship between ABC transporter gene SNPs and MDR, we have collected data over a 3-year period (2019-2021) from the Oncology Department of the Komfo Anokye Teaching Hospital in Ghana. The overall prevalence of BC recurrence was 3.4% (CI = 2.5 - 4.7%), and prevalence of metastatic BC was 47.6% (CI = 44.6 - 50.6%). Methodology: SNPs in ABC transporter genes will be obtained from 150 consented female BC patients who have undergone chemotherapy. We will compare genotype frequencies among patients with disease recurrence and/or metastasis (n = 100) to those with no disease recurrence or metastasis (n = 50). Single-plex genotyping of the ABC gene SNPs will be completed using a real-time PCR allelic discrimination assay. Conclusion: ABCC1 has been established to be associated with African ancestry. Determining the association of ABC gene SNPs and MDR among Ghanaian BC patients will provide further information on allelic variants and their effects on BC treatment outcomes. Citation Format: Gloria Agyekum Boaitey, Rachel Martini, Melissa B. Davis, Lisa Newman, Brian Stonaker, Linda Ahenkorah Fondjo, Christian Obirikorang, Ernest Osei Bonsu, Ernest Adjei, Ishmael Kyei, Mavis Bobie Ansah, Mahteme Bekele, Timothy Chu, Nicolas Robine. Evaluation of multidrug resistant genes among breast cancer patients in Ghana [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2247.

Quadruple-negative breast cancer (QNBCs) is a highly aggressive and metastatic disease and remains clinically challenging breast cancer subtype with worst prognosis. Due to its exceeding heterogeneity, absence of the AR, ER, PR, Her2 receptors and lack of established therapeutic targets, QNBC is hard to treat cancer and recurs rapidly with standard chemo regimen. QNBC demonstrated vulnerability to proteasome inhibition due to their higher metabolic needs. But FDA approved 20S proteasome inhibitors failed to treat solid tumors including QNBC. Hence, developing an effective strategy to overcome the limitations associated with current 20S proteasome inhibitors is mandated to provide alternate treatment options for QNBC patients. Preclinical, Up Therapeutics lead candidate small molecule inhibitor Up284 has shown significant tumor growth inhibition as a single agent in animal models. To explore potential synergy of Up284 with other therapeutic agents, we conducted the in vivo combination experiments. In this work, we present ADRM1 amplification in QNBC tumors, identification of a small molecule selective inhibitor of ADRM1, Up284 (Up Therapeutics), Up284 demonstrated efficacy against panel of BC cell lines and patient derived organoids. In vivo combination results for Up284 with widely used BC standard-of care chemotherapy regimen demonstrated strong in vivo synergy in tumor mouse syngeneic models. Taken together, these data demonstrated combination synergy of a small molecule ADRM1 inhibitor with other agents. These results pave the road for potential clinical evaluation of combination treatment of QNBC in patients. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung-Nien Chang, Rachel Martini, Florencia Madorsky Rowdo, Melissa B. Davis. Up284, A small molecule inhibitor of ADRM1 demonstrates significant anti-tumor efficacy in preclinical models of aggressive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4478.

Vitamin D is a steroid hormone that confers anti-proliferative and anti-inflammatory properties in prostate cells. Vitamin D deficiency is associated with advanced prostate cancer (PCa) stage, grade, and increased mortality. Since AA men are more likely to be vitamin D deficient compared to EA men, elucidating the pleiotropic effects of vitamin D may provide additional drug targets to mitigate disparate outcomes for AA men with PCa. Total RNA sequencing (RNAseq) was performed on an AA prostate cell line, RC-77N/E, derived from non-malignant epithelial tissue obtained from a PCa patient. We compared untreated RC-77N/E cells with RC-77N/E cells treated with 10 nM of vitamin D3 metabolite, 1α,25(OH)2D3 at 24h. Bioinformatic analysis, public cohort data mining, and RT-qPCR was performed to filter and validate selected statistically significant differentially expressed genes from RNAseq analysis. Our comparison of 1α,25(OH)2D3 treated versus untreated control RC-77N/E cell replicates revealed, 1601 significantly differentially expressed genes (DEGs) (FDR p < 0.05). Ingenuity Pathway Analysis software was used to conduct pathway enrichment analysis, which predicted repression of signaling pathways involved in actin cytoskeleton organization, epithelial-mesenchymal transitioning, adherens junction pathway, and RhoA pathway signaling with high enrichment z-score and statistically significant p-value. We additionally report predicted repression of PCa cell viability, invasion, cell proliferation and organismal death, for disease and function. We prioritized differentially expressed genes by identifying genes with 1) vitamin D response elements (VDREs) and 2) predicted poor prognosis, by performing overall survival analyses using The Cancer Genome Atlas (TCGA) Prostate Adenocarcinoma (PRAD) Cohort. We identified ANLN (Anillin) and ECT2 (Epithelial Cell Transforming 2), which were further validated using an AA tumor/benign contralateral matched clinical cohort, and a public cohort study using a non-malignant European prostate cell line, RWPE1. Our data suggest 1α,25(OH)2D3 significantly downregulates ANLN and ECT2 (FDR p < 0.05) in the RC-77N/E cell line, where survival analyses in the TCGA PRAD cohort show low expression of both ANLN and ECT2 are associated with significantly better overall survival outcome (p < 0.05). In our clinical cohort validations, both ANLN and ECT2 also show significantly decreased expression in non-malignant compared to tumor (p < 0.05). In conclusion, our study provides more insight of vitamin D3 regulation of PCa biomarkers and their potential roles in AA prostate cancer prevention, adjuvant treatment, as well as improving cancer survivor outcome Citation Format: Jabril R. Johnson, Rachel N. Martini, Yate Yuan-Ching, Leanne Woods-Burnham, Mya Walker, Greisha L. Ortiz-Hernandez, Dorothy Galloway, Melissa B. Davis, Sean K. Kimbro, Adam B. Murphy, Rick A. Kittles. 1,25-dihydroxyvitamin D3-mediated suppression of genes associated with cell cycle regulation and actin organization in a non-malignant African American prostate cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3509.

Mutations in the TP53 gene can be found in more than 50% of tumors. We recently showed in 2D breast cancer cell lines that mutant p53 (mtp53) proteins tightly associate with replicating DNA, chromatin and Poly (ADP-ribose) polymerase (PARP) protein. In addition, missense mtp53 R273H causes an increase in the chromatin association of replication proteins including PARP, and mini-chromosome maintenance complex 2-7 (MCM2-7). The expression of mtp53 R273H enhances overall MCM2 levels, promotes cell proliferation, and improves the synergistic cytotoxicity of treatment with the alkylating agent temozolomide in combination with the PARP inhibitor (PARPi) talazoparib. Currently, PARPis are indicated for patients that present BRCA1/2 mutations, but there might be patients with other alterations associated with DNA damage repair that could benefit from PARPi treatment; this includes expression of mtp53. Patient-derived Tumor Organoids (PDTO) are 3D culture models that retain cell-cell and cell-matrix interactions and are shown to reproduce drug responses observed in patients. Here, we evaluated the sensitivity of wild-type BRCA1/2 breast and lung PDTO with either mtp53 or wild-type p53 (wtp53) to the combination of DNA damaging agent temozolomide plus the PARPi talazoparib. First, we tested the sensitivity of the organoids to talazoparib and temozolomide individually. To determine if there was synergy between the two treatments, the organoids were treated with an inhibitor matrix to evaluate multiple combinatorial concentrations. Three breast cancer and two lung cancer PDTO with mtp53 presented synergistic cytotoxicity of the combination treatment. Two breast organoid lines with wtp53 showed no synergistic interaction between the two drugs, and the same was observed in a wtp53 lung organoid line. We analyzed wtp53 and mtp53 PDTO cell extracts by western blot to assess the activation of downstream p53 effectors and DNA damage markers after treatment with temozolomide and/or talazoparib. Combination of talazoparib and temozolomide induced higher DNA double-strand breaks in mtp53 organoids as shown by increased gamma-H2AX expression. The results obtained demonstrated that in mtp53 PDTO synergism is achieved with combined talazoparib-temozolomide treatment supporting the idea that tumors expressing mtp53 may be potential candidates for this combination PARPi treatment. Citation Format: Florencia P. Madorsky Rowdo, Gu Xiao, Melissa B. Davis, Laura Martin, Olivier Elemento, Jill Bargonetti. Patient-derived tumor organoids with p53 mutations, and not wild-type p53, demonstrate synergistic sensitivity to treatment with temozolomide in combination with talazoparib PARP inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6186.

African American (AA) men are much more likely to be diagnosed with prostate cancer (PCa) and die from the disease than any other race/ethnicity in the United States. The pursuit of therapeutic strategies that incorporate tailored medicine for AA men remains a paramount goal. Metastatic PCa initially responds to suppression of androgen signaling through standard-of-care androgen deprivation therapy. However, relapse is inevitable, and treatment options are limited. For this reason, inhibiting androgen-independent signaling pathways that promote metastasis —such as human epidermal growth factor receptor 2 (HER2) signaling— is a promising area of investigation. HER2 overexpression in PCa tumors correlates with worse prognosis and treatment-resistance, but HER2 overexpression has not been evaluated specifically in AA men. We hypothesize that HER2 overexpression correlates with West African genetic ancestry (WAA) in AA PCa patients and worsens clinical features, treatment response, and survival outcomes. In this study, we report that HER2/ERBB2 is upregulated in the majority of PCa tumors in the Oncomine database. We quantified HER2/ERBB2 with RNAseq analysis of prostate tissue samples collected from AA patients (n=36) and observed a moderate correlation with WAA determined by genotyping using a validated set of Ancestry Informative Markers. Using immunohistochemistry, we also observed detectable HER2 protein expression in primary prostate tumors of AA patients (n=10). Our observation that 30% of AA primary tumor tissue samples were HER2-positive is remarkable, especially as previous studies have reported that HER2 overexpression is directly associated with metastasis as opposed to early, localized tumors. Our findings may underscore a basal/increased predisposition for HER2 overexpression in the AA population. We also detected HER2/ERBB2 mRNA expression using qPCR in a racially diverse panel of commonly used PCa cell lines: AA (MDA-PCa-2b and RC-77T/E) and non-Hispanic white (PC3, DU145, 22Rv1). To determine the effect of HER2 blockade on cell viability, we treated cells with 20 nM of anti-HER2 drug Trastuzumab for 72 hours and performed CellTiter-Glo™ assays. The only cell lines with significantly reduced viability were the AA cell lines. Furthermore, the RC-77T/E cell line which had the highest expression of HER2/ERBB2 also had the most significant reduction in cell viability. This finding suggests that AAs may be more responsive to HER2-targeting drugs, and that this responsiveness may be increased in PCa patients with higher levels of HER2 overexpression. It is plausible that HER2 overexpression may occur at earlier disease stages in AA men than previously reported and could be utilized to characterize aggressive PCa tumors eligible for HER2-targeting inhibitors, thus providing an early and effective therapeutic intervention that may improve outcomes in this population and reduce this appreciable health disparity. Citation Format: Nicole Ursula Mavingire, Mya Walker, Jabril R. Johnson, Isaiah J. Sailors, Greisha L. Ortiz-Hernandez, Rachel N. Martini, Melissa B. Davis, Justin Tran, Frank A. Myers, Sean Kimbro, Zhirong Yin, Tanya B. Dorff, Rick A. Kittles, Leanne Woods-Burnham. HER2 expression in African American men with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1955.

Malignant pleural effusions (MPE) are a common complication of advanced cancers, particularly those adjacent to the pleura such as lung and breast cancer and are a frequent complication in metastatic disease. The pathophysiology of MPE formation in advanced breast cancer remains poorly understood, and their composition and biology are understudied. To characterise the phenotypic diversity of malignant pleural effusion, we performed single-cell RNA sequencing on 10 MPEs from 7 metastatic breast cancer patients with diverse molecular subtypes: two triple negative (TNBC) patients, three luminal B patients including one with a rare inflammatory subtype, and two luminal A patients. For all patients, we sequenced cells from the entire MPE, without performing any enrichment or selection, in order to ascertain the cellular composition and molecular phenotypes in an unbiased manner. We identified pronounced differences in the relative proportions of malignant, mesothelial and immune cell populations: both TNBC and two Luminal B patients had extensive malignant cell populations, while the other three patients had few or no detectible malignant cells but extensive immune populations. We also observed heterogeneity in the expression of subtype-specific gene signatures and in copy number aberration patterns that captured variability across malignant cell populations both within and between patients. We observed that most malignant cells retained the molecular subtype diagnosed in the primary tumour, however sub-populations of malignant cells could be identified that mapped to different molecular subtypes, indicative of complexity in the molecular phenotypes of cancer cells disseminated to or metastasising in the pleural cavity. We distinguished mesothelial cell populations from malignant cells using key markers, expression of the PAM50 signatures, and copy number aberration patterns. We found that pleural mesothelial cells expressed a cancer associated fibroblast-like transcriptomic program that may support cancer growth within the pleural cavity through the secretion of growth factors that target malignant cells. Our dataset presents the first unbiased and unselected assessment of breast cancer associated MPEs at single cell resolution, providing the community with a vital resource for the study of MPEs. Our work highlights the molecular and cellular diversity captured in MPEs and advances the use of these clinically relevant biopsies both in monitoring disease progression and in the development of targeted therapeutics for patients with advanced breast cancer. Citation Format: Holly J. Whitfield, Jean Berthelet, Stefano Mangiola, Caroline Bell, Robin L. Anderson, Bhupinder Pal, Anthony T. Papenfuss, Belinda Yeo, Delphine Merino, Melissa J. Davis. Defining the cellular composition and associated molecular phenotypes of malignant and non-malignant cells in breast cancer pleural effusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6232.

Compared to European American men (EAM), African American men (AAM) have 2-3 times higher prostate cancer (PCa) mortality rates. AAM diagnose with more aggressive PCa at an earlier age & clinically advanced disease compared to EAM. Studies suggested that tumor biology & cellular heterogeneity/gene profile, have a potential contribution to racial differences, which remain even when controlled for access to care and stage at presentation. Moreover, men of African ancestry from the Caribbean & South America experience incidence & mortality rates similar to AAM, suggesting a possible ancestral basis for some of these expected outcomes. Additionally, the association of the genomic findings with patient ancestry and with other characteristics such as tumor biology & transcriptomic alterations remains poorly understood. Here we hypothesize that African ancestry drives aggressive prostate cancer and leads to genetic alterations with upregulation of unique Immune-inflammatory signature in AAM of African descent. To assess our hypothesis, we performed genome-wide sequencing (WES & RNA Seq), for a total of (n= 72) patients obtained from treatment-naive PCa who self-reported their race. Out of those 72 samples, 47 samples RNA Seq match with WES. To verify the self-reported race, we used ADMIXTURE to generate a quantitative estimate of each individual ancestral composition. Most of our cohorts who self-reported as AAM, their ancestry assigned to African Ancestry (Ancestry proportion > 70%) with either Bantu subpopulation in Sub-Saharan area and/or Yoruba (Nigeria) subpopulation. Descriptive statistical analyses of the study population were conducted and stratified by race & pathology stage. Our results showed that AA men are diagnosed with PCa at a younger age and higher pathology stage. We ran differential gene expression (DGEs) using EdgR/DESeq2 and enriched pathways based on the patient's ancestry/race using GSEA & EdgR gene ontology function. Our DGEs analyses revealed that gene sets such as activation of the innate immune system and immune-inflammation pathway, as well as interleukin signaling, are positively enriched (p-value 0.05), while gene sets such as RNA Polymerase II transcription and metabolisms signaling are negatively enriched (p-value 0.05) in AAM. Moreover, our WES analysis showed that patients with high African Ancestry associated with AR-related mutation such as FOXA1, PTEN, TP53, EPHB2 with SPOP mutation (18% in AAM vs 8% in EAM) on top of these mutations. Interestingly, when we stratified patients based on PD-L1 expression, AAM with high PD-L1 (Immunogenic) has higher CD8+ T cells/PD-1+ expression than AAM patients with low PD-L1 (Non-Immunogenic). WES analysis showed that AAM with high PD-L1 express SPOP mutation. Our study's findings could lead to new therapeutic strategies using anti-inflammatory drugs and immune modulators to decrease the disease burden among men at high risk for PCa. Citation Format: Isra A. Elhussin, Jason White, Tiffany Dorsey, Moray J. Campbell, Melissa B. Davis, Stefan Ambs, Isaac Kim, Clayton Yates. Genomic comparison between African American men & European American men with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1167.

Therapeutic strategies that improve survival outcomes for advanced-stage breast cancers have proven a major clinical challenge. Here, we define an androgen receptor (AR) signalling network that governs the maintenance and de novo formation of cancer stem cells in triple-negative breast cancer. In response to chemotherapy, AR activation switches cells into a cancer stem cell state, while AR antagonism suppresses cancer stem cell formation and function. In vivo, we validate that the AR antagonist, seviteronel, significantly improves chemotherapy-mediated inhibition of primary and metastatic tumour growth. Analysing three independent triple-negative breast cancer patient cohorts we identify that cytoplasmic AR expression prognosticates poor survival in treatment-naïve patients and predicts poor response to chemotherapy. Additionally, Phase II clinical trial data demonstrates that seviteronel treatment followed by chemotherapy improved survival outcomes in triple-negative breast cancer patients expressing high cytoplasmic AR levels. Seviteronel combined with chemotherapy represents a promising therapeutic strategy for triple-negative breast cancer patients. Citation Format: Beatriz P. San Juan, Soroor Hediyah-Zadeh, Laura Rangel, Vanina Rodriguez, Heloisa H. Milioli, Felix Kohane, Carley A. Purcell, Therese E. Hickey, Leonard D. Goldstein, John G. Lock, Melissa J. Davis, Christine L. Chaffer. The anti-androgen seviteronel sensitizes triple-negative breast cancer to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1029.

KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1130.

The development of androgen resistance is a major limitation to androgen deprivation treatment in prostate cancer (PCa). We have developed an in vitro model of androgen-resistance using the androgen sensitive cell line LNCaP to characterize the phenotypic and transcriptomic changes occurring as androgen resistance develops. Our aim is to understand biological network profiles of transcriptomic changes during the transition to androgen-resistance and to validate these changes between our in vitro model and previously published clinical datasets (paired samples before and after androgen-deprivation therapy of patients with advanced PCa) (1). Methods: PCa cells, LNCaP, expressing mutated AR which are androgen-dependent (2,3), are used in the development of an androgen-resistant subline. Subline cells were established by prolonged cultures in media + 10% CS-FBS to mimic the clinical course of PCa. Cell proliferation, cell motility and invasion, morphology, AR expression were examined. RNA-sequencing was performed using the parental LNCaP cells and an androgen-resistant subline (LNCaP-AI) established by chronic exposure to the androgen-deprivation. Reads from cells and clinical samples (1), pre- vs. post-treatment, were processed through the same standard pipeline and quality control. Data outputs were analysed as differential expression [DEG] (EdgeR) (4) and top scoring protein-protein interaction (PPI) networks [PINA2 (5) and BioNet (6)]. Data outputs from the cell line model and clinical samples were compared. Results: LNCaP cells initially showed poor growth after prolonged exposure to androgen-deprived conditions but later adapted and started to grow well. After 24 weeks of androgen-deprivation, LNCaP-AI's growth was no longer responsive to addition of androgen [0.1 - 10 nM]. AR expression was not different in LNCaP and LNCaP-AI (P>0.05). LNCaP-AI cells had increased proliferation and cell invasion compared to LNCaP. We identified key genes that overlap between our cell line and clinical RNAseq (1) datasets and analyzed the overlapping PPI network that showed the same pattern of behavior in both datasets. The network revealed several potential mechanisms and gene interactions that warrant further investigation, including cooperative behaviors of other nuclear receptors, TP63 mediated signalling pathway and Aryl hydrocarbon receptor transcriptional pathway. Conclusion: Cell line model of androgen-resistance will be used for further longitudinal study of the mechanism of castrate resistant prostate cancer (CRPC). Our approach allows for better characterization of biological processes of CRPC. Knowledge of the genetic profiles during transition to androgen resistance will improve our understanding of this common clinical scenario and may lead to biomarker discovery. References: 1. Rajan P, Sudbery IM, Villasevil ME, Mui E, Fleming J, Davis M, et al. Next-generation sequencing of advanced prostate cancer treated with androgen-deprivation therapy. Eur Urol 2013;66(1):32-9. 2. Marques RB, van Weerden WM, Erkens-Schulze S, de Ridder CM, Bangma CH, Trapman J, et al. The human PC346 xenograft and cell line panel: A model system for prostate cancer progression. Eur Urol 2006;49(2):245-57. 3. Pienta KJ, Bradley D. Mechanisms underlying the development of androgen-independent prostate cancer. Clin Cancer Res 2006;12(6):1665-71. 4. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010;26(1):139-40. 5. Cowley MJ, Pinese M, Kassahn KS, Waddell N, Pearson JV, Grimmond SM, et al. PINA v2.0: mining interactome modules. Nucleic Acids Res 2012;40(Database issue):D862-5. 6. Beisser D, Klau GW, Dandekar T, Müller T, Dittrich M. BioNet: an R-Package for the functional analysis of biological networks. Bioinformatics 2010;26(8):1129-30. Citation Format: Sujitra Detchokul, Aparna Elangovan, Melissa J. Davis, Geoff Macintyre, Edmund J. Crampin, Albert G. Frauman. Biological network analysis using an in vitro model of androgen-resistance in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-40.

The ability of breast cancer cells to switch between epithelial and mesenchymal phenotypes may be key to their survival in new environments, resistance to therapies and ability to form metastases. Epithelial mesenchymal plasticity (EMP) is instrumental in embryological development and has been implicated in stemness, therapy resistance and metastasis of breast cancer. EMP markers are enriched in basal-like, triple negative breast cancer, which is a type of breast cancer associated with early recurrence and poor prognosis, and established as a common phenotype in women with BRCA1 mutations. The EMPathy Breast Cancer Network (BCN) is a national collaborative effort including scientists, surgeons, medical oncologists and a consumer advocate investigating the role of EMP in breast cancer recurrence. The 7 thematic research projects of EMPathy BCN, including the 9 program-funded ‘Satellite’ projects, are aligned with the Cooperative Research Centre for Cancer Therapeutics (CTx) (www.cancercrc.com/index), so that any potential drug targets identified may progress into the CTx drug development program. Multiple parallel approaches in the Target Discovery theme were used to identify candidate regulators and effectors of EMP. A total of 10 functional or gene expression experiments provided 7,950 significant events in any one system, which were cross referenced against 10 public breast cancer datasets relevant to EMP and/or breast cancer stem cells. A series of criteria were used to select a panel of 127 candidates that were combined with 123 ad hoc candidates (mainly hits close to the cut-off and breast cancer context genes) to give a total of 250 candidates to be analysed in breast cancer tissues using Nanostring technology. The 2,301 ‘significant events’ in any functional screen were further cross-referenced to 10 public functional datasets relevant to EMP in any system and a series of criteria were used to select a panel of 320 candidates that are to be analysed in an siRNA ‘functional screen' of multiple breast cancer cell lines, to support the choice of Candidate Targets for drug development. Ongoing studies will address the biology behind selected Candidates. This work and the EMPathy BCN is supported by a NBCF National Collaborative Research Program Grant. Citation Format: Tony Blick, Gayle Phillip, Eva Tomaskovic-Crook, Annet Hammacher, Nicholas Wong, Izhak Haviv, The EMPathy Breast Cancer Network, Greg Goodall, Melissa Davis, Erik W. Thompson. Integrated target discovery in the EMPathy Breast Cancer Network - Multidimensional analysis of epithelial mesenchymal plasticity (EMP) in experimental systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1060. doi:10.1158/1538-7445.AM2014-1060

Prostate cancer (PCa) is one of the leading causes of cancer death in men. CD151 is a member of the tetraspanin family and is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. Previous studies from our laboratory revealed that expression of CD151 differs across histological grades of PCa and high levels of expression are linked to shorter survival, independent of Gleason score1. In vitro motility assays of human PCa cell lines suggest CD151 is a motility promoter2. We have sought to develop a number of CD151 inhibitors and examine their ability to modulate prostate cancer motility and metastasis. The human PCa cell line, LNCaP (low endogenous level of CD151), transfected with CD151 shows increased motility and invasion compared to control LNCaP, whilst CD151 siRNA knock-down (KD) of PC-3 cells (high endogenous level of CD151), reduces motility compared to control PC-3. We have conducted in silico screening with compounds predicted to bind a model of the large extracellular domain (EC2) of CD151 and found that a number of these small molecules possess bioactivity in vitro and in vivo in inhibiting prostate cancer motility and progression. LNCaP growth and migration was confirmed by stimulating with 1 and 10 nM DHT at 24 hrs in charcoal stripped FBS. It was found that cell proliferation and motility of LNCaP cells was significantly increased after stimulation by 10 nM DHT at 24 hrs (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 469. doi:1538-7445.AM2012-469