Tumor Expression Research Articles

Abstract 5288: Soy isoflavone genistein reboosts targeted therapy sensitivity in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is a very aggressive type of breast cancer that does not expression important proteins called estrogen, progesterone and HER2. This makes TNBC hard to treat because it does not respond to common targeted treatments. However, recent treatments using the immune system to target key immune proteins such as PD-1 and PD-L1 have been successful in many types of cancer. This form of immunotherapy works by blocking PD-1/PD-L1 pathway and leads to stimulating the body's own immune system to recognize and attack cancer cells, thus providing a potential new treatment option for TNBC patients. Nevertheless, only approximately 30% of TNBC patients may respond to these drugs, suggesting the majority of TNBC patients would not benefit from these innovative immunotherapies. It is believed that low levels of immune cells and PD-L1 expression in tumors are the primary reasons for the lack of response or resistance to anti-PD-1/PD-L1 treatments. The use of nontoxic and healthy food and nutrition to increase treatment response is an emerging field of cancer research. Our studies showed that adding genistein (GE), a bioactive dietary component found in soybean products such as soy milk, soy protein and tofu, could significantly enhance the efficacy of anti-PD-1 immunotherapy in a TNBC mouse model that was previously not responsive to the treatment. Our research also found that soybean GE can increase tumor immune response by significantly recruiting active immune cells into the tumor and increasing PD-L1 expression. Our studies will help to establish a novel nutrition-based intervention targeting TNBC patients who might not qualify for this treatment initially. Eventually, we will develop a novel strategy of administering a tailored dietary plan to resensitize these patients to emerging anti-PD-1/PD-L1 immunotherapies. Citation Format: Taiwo Bankole, Zhenhai Li, Yuanyuan Li. Soy isoflavone genistein reboosts targeted therapy sensitivity 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 5288.

Abstract 673: Mechanisms of drugs in ovarian cancer and prevention of invasion and metastasis

Abstract Introduction: Ovarian cancer patients are typically diagnosed late stage after the cancer has left the primary site, this is the major factor contributing to the 70% 5-year mortality rate. Standard of care is Platinum therapies or debulking surgery followed by maintenance therapies to keep cancer from coming back. The limitation to these therapies is overall toxicity to the patient forcing them to stop treatments or eventual resistance to the therapy. The objective of this study is to evaluate the mechanisms in which the novel drug, SHetA2, alone compares to the competitor, Olaparib. Additionally, discover the genetic differences between attached tumor and floating tumor in ascites.Objective: Our objective was to evaluate the mechanisms in which the novel drug, SHetA2, alone compares to the front-line competitor, Olaparib, in affecting Filamin A and cyclin d1 levels in ovarian cancer. Methods: Individual and combination drug treatments were preformed on two ovarian cancer cell lines and patient samples for cell viability assays. Immunocytochemistry was performed to visualize colocalization of proteins. RNA isolation was performed for Nanostring analysis. Results: Ovarian cancer cells treated with SHetA2 or olaparib demonstrate a decrease of cyclin D1 on the membrane colocalized with filamin A, a structural protein that facilitates movement by acting as a signaling molecule for downstream effects. Nanostring analysis of attached tumor and spheroids form ascites shows significant differential gene expression in tumor versus ascites. Conclusions: These results suggest the decrease of cyclin D1 with filamin A upon treatment with SHetA2 or olaparib could be one of the primary factors leading to each drug’s efficacy and additivity with each other. The differential gene expression of tumor versus ascites begins to elucidate the fundamental differences between attached and floating tumors and spheroids. Citation Format: Laura F. Mortan, Doris Benbrook. Mechanisms of drugs in ovarian cancer and prevention of 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 673.

Abstract 2587: Evaluating the potential of radiomics features to predict outcome to an antibody-drug conjugate therapy in non-small cell lung cancer patients

Abstract This study evaluated radiomics, a non-invasive biomarker, for assessment of tumor heterogeneity in non-small cell lung cancer (NSCLC) patients treated with an AXL-targeting antibody-drug conjugate (ADC), enapotamab vedotin (EnaV), through analyzing baseline and post-treatment radiomic features from CT images for predicting outcome. Clinical development of EnaV was discontinued in 2020 due to low response rates unimproved by dose optimization and/or predictive biomarkers. Study data included CT images, target expression and clinical data from 77 advanced NSCLC patients treated at 1.0 mg/kg (3Q4W), 1.8 mg/kg (Q3W), or 2.2 mg/kg EnaV (1Q3W) in the first in human phase I/II study assessing safety and preliminary efficacy of EnaV (NCT02988817). Radiologist-segmented tumors were examined using the cancer imaging phenomics tooklit CaPTk1, deriving 225 radiomic features from 77 baseline and 50 Cycle 2 post-treatment CT images. Batch effects were balanced using nested ComBat2, and radiomic feature dimensionality was reduced in two stages: clustering features with ≥80% similarity followed by principal component analysis (PCA). Radiomic PC1 was utilized in statistical models to assess overall and progression-free survival (OS, PFS), overall response rate (ORR), and disease control rate (DCR). Both baseline and delta radiomic PC1 were evaluated for correlation with biomarker and clinical activity, independently and with soluble or tumoral AXL expression. Baseline radiomic PC1 significantly predicted DCR: confirmed (p=0.03), and unconfirmed (p=0.03). Baseline tumor membrane AXL predicted ORR for this NSCLC subset: confirmed (p=0.03) or unconfirmed (p=0.02). Delta analysis showed delta PC1 predicted unconfirmed (p=0.03) and confirmed (p=0.03) DCR and remained predictive for DCR with soluble AXL (p=0.04). No association was found between baseline and delta radiomics with OS or PFS. Baseline and delta radiomic signatures showed potential in predicting NSCLC response. Preliminary findings suggest that radiomics combined with target expression predicted DCR, an early clinical activity indicator, although they did not predict OS or PFS. These outcomes pertain to a NSCLC subset and might not apply to other tumor types. Tumoral AXL expression did not linearly correlate with EnaV activity in a patient-derived soft tissue sarcoma model3, possibly due to AXL expression in both tumor cells and myeloid-derived suppressor cells, or the presence of tumor-intrinsic MMAE resistance4, implying the AXL expression and EnaV activity relationship is contingent on the tumor microenvironment5. Further validation in larger cohorts is planned. 1 Davatzikos C, et al. J Med. Imaging 2018 2 Horng H, et al. Sci. Reports 2022 3 Van Renterghem B, et al. Int J Mol Sci. 2022 4 Holtzhausen A, et al. Cancer Immunol Res 2019 5 Boshuizen J, et al. Cancer Res. 2021 Citation Format: Andrew W. Chen, Lauren K. Brady, Eric A. Cohen, Walter C. Mankowski, Leonid Roshkovan, Mohammed Qutaish, Sharyn Katz, Patricia G. Castro, Nora Pencheva, Maria Jure-Kunkel, Brandon W. Higgs, Despina Kontos. Evaluating the potential of radiomics features to predict outcome to an antibody-drug conjugate therapy in non-small cell lung cancer 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 2587.

Abstract 2444: Plasma levels of matrix metalloproteinases 8 are significantly increased in patients with colorectal cancer (CRC) and mRNA levels are increased in 71% of tumors vs normal tissue

Abstract Introduction: Matrix metalloproteinases 8 (MMP-8) is a member of the MMP protease family mainly involved in degrading extracellular matrix. A variety of cells express MMP-8 and during pathological inflammation neutrophils release IL-1, IL-8, TNF-α, and GM-CSF which triggers MMP-8 secretion. MMP-8 aids tumors in evading immune detection during the development of CRC and also regulates the role of endothelial cell in angiogenesis. Increased MMP-8 tumor expression and serum levels has been noted in hepatocellular cancer and melanoma patients and are associated with adverse outcomes. There is limited data regarding blood levels of MMP-8 in CRC patients. This study compared preoperative (PreOp) plasma MMP-8 levels in patients with CRC and benign colonic pathology (BCP) undergoing bowel resection. Method: Patients with CRC or BCP who had elective colorectal resection who were enrolled in an IRB approved tissue and data bank for whom preop plasma samples were available were eligible. Data analyzed included demographic, clinical, operative, and final pathology reports. Blood specimens were quickly processed and plasma stored at -80°C. MMP-8 levels in Preop plasma samples were measured via ELISA, in duplicate, and reported as median +95% CI (ng/ml). MMP8 expression was examined in paired CRC and normal mucosal tissue samples from a subset of the CRC patients (n=20) via QRT-PCR. Immunohistochemistry (IHC) was conducted on formalin-preserved tissue sections (tumor/normal pairs) following standard protocols. The concept of MMP8 as CRC diagnostic marker was evaluated by assessing the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC). The analysis was done with the Mann-Whitney test (significance, p<0.05). Results: 96 CRC patients (74% colon and 26% rectal cancer) and 96 BCP patients (32% adenoma, 64% diverticulitis, 4% other) met entry criteria. The CRC stage distribution was: Stage 1, 26%; Stage 2, 26% Stage 3, 40%; and stage 4, 8%. Median plasma MMP-8 levels were higher in CRC (8.21, CI: 6.92, 10.81) compared to BCP patients (5.59, CI: 4.68, 7.30; p<0.001). A non-significant increase in median MMP-8 levels were noted in STG IV vs STG I and STG II and STG III was found (38-89%). IHC validated the expression of MMP-8 in CRC tumors relative to normal colon. Increased expression of MMP-8 was seen in 71% of the tested CRC samples (12/17). The AUC value for the ROC curve was 0.653 (sensitivity 35%, specificity 0.95%). Conclusion: The median MMP-8 level in CRC patients was found to be 47% higher compared to BCP patients and 89% higher in stage IV vs stage II patients (p=0.06). The tumor is the most likely the cause of elevated plasma levels. Increased tumor expression of MMP-8 mRNA was noted in 71% of tumors. The low sensitivity of the ROC curve suggests MMP8 alone has no value as diagnostic factor. Larger studies are justified. Citation Format: Chandana S. K. Herath Mudiyanselage, Anuj R. Sharma, Neil Mitra, Aashutosh Sah, Xiaohong Yan, Pablo Palacios, Vesna Cekic, Richard L. Whelan. Plasma levels of matrix metalloproteinases 8 are significantly increased in patients with colorectal cancer (CRC) and mRNA levels are increased in 71% of tumors vs normal tissue [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 2444.

Abstract 6254: Casein kinase II (CK2) expression in pediatric solid tumors

Abstract Despite recent advances, high-risk neuroblastoma (NB) and sarcomas account for 15% and 5% of all pediatric cancer deaths, respectively. Many patients attain remission, but approximately half of patients relapse. Genomic alterations such as MYCN amplification and ALK mutations have been shown to be important in prognosis and treatment. Despite improvements, approximately 20% of patients have refractory or recurrent disease after therapy, underscoring the need for further genomic understanding and new approaches to treatment.CK2 is a serine/threonine kinase that regulates numerous cellular processes such as cell growth, proliferation, and survival. Overexpression of CK2 is associated with a poor prognosis in several adult solid tumors, making it a potential target of interest in pediatric solid tumors. This study aims to investigate CK2 expression among patients with NB, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) and its correlation with patient survival. MethodsPatients were enrolled in NMTRC008/009: “Feasibility Trial Using Molecular Guided Therapy for Patients with Relapsed/Refractory Childhood Cancer” and BCC-001. WES and RNA-Seq were performed at the Translational Genomics Research Institute and Sema4 or Caris respectively. RNA expression levels were compared to a normal whole-body reference and a pediatric cancer reference. Differential expression data were interpreted in the context of systems biology.ResultsTumor WES was performed on 297 patients (212 NB, 50 EWS, 35 RMS) and RNA-Seq on 284 patients (210 NB, 39 EWS, 35 RMS). The most common mutations or copy number alterations were seen in MYCN, ATRX, ALK, CDKN2A, and chr17q+ in NB; EWSR1-FLI1 fusion in EWS; PAX3-FOXO1 fusion in RMS. RNA expression analysis was performed to understand MYCN-driver status by covariance and interactions and to identify subsets of patients with high/low expression of CK2 in different tumors. CK2 bimodal expression is seen across various cancers and normal tissues, implying distinct sub-groups exist in the data. In NB, overexpression of the CK2 gene with statistical significance was found in MYCN-amplified and chr17q+ tumors. A positive and negative correlation of the CK2 gene is seen with several target genes like MYCN, TRIM71, LIN28B, and HMGA2, JAK2, AR, STAT3, EGFR, LEF1, and NFKB2, respectively. In EWS and RMS, subgroups are identified with high and low expression of the CK2 gene.ConclusionGenomic sequencing of pediatric solid tumors showed a low mutation burden. RNA analysis resulted in the identification of potential novel therapeutic targets involving CK2 pathways. High expression of CK2 correlates with MYCN status. Further analysis of patient molecular subgroups is ongoing. Citation Format: Abhinav Beeravally Nagulapally, Chandrika Behura, Divya Gandra, Giselle Saulnier Sholler. Casein kinase II (CK2) expression in pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6254.

Abstract 5649: PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumors

Abstract Background: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. Methods: PRRX1 expression in human Malignant peripheral nerve sheath tumors (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro, and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA seq, and structural prediction were used to identify proteins interacting with PRRX1. Results: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumor malignancy-related gene sets including mTORC1, KRAS, and SRC signaling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. Conclusion: Targeting the PRRX1-TOP2A interaction in malignant tumors with high PRRX1 expression might provide a novel tumor-selective therapeutic strategy. Significance: MPNST is a malignant tumor with a poor prognosis and still lacks definitive treatment. Our results could hold promise as an alternative therapeutic strategy to inhibit the interaction between PRRX1 and TOP2A, and may improve the prognosis for malignant tumor patients with high expression of PRRX1 and TOP2A such as MPNSTs. Citation Format: Shota Takihira, Eiji Nakata, Daisuke Yamada, Tatsunori Osone, Tomoka Takao, Takeshi Takarada, Akira Hirasawa, Takuto Itano, Tomohiro Fujiwara, Toshiyuki Kunisada, Toshifumi Ozaki. PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5649.

Abstract 2681: Blockade of SIRPα in glioblastoma cells inhibits mitochondrial fatty acid β-oxidation and increase immune cell mediated cytotoxicity

Abstract Glioblastoma (GBM) is the most malignant brain tumor with limited immunotherapeutic options, likely due to significant GBM-associated systemic and intra-tumoral immunosuppression. The binding of SIRPα on myeloid cells to CD47 on cancer cells restricts immuno-phagocytosis against cancer cells. However, less attention has been given to SIRPα is also expressed in cancer cells, and the role of SIRPα on cancer cells is still unclear. Immunohistochemistry (IHC) staining was used to examine the expression of SIRPα in normal brain tissues and GBM. A single-cell database on the Broad Institute Portal was utilized to analyze the SIRPα expression in different cell types. SIRPα antisense morpholino was applied for gene knockdown. Established stable SIRPα overexpressed GBM cell lines were used to examine the mitochondrial function through the Seahorse Mito Stress test. In vitro, immune cell-mediated cytotoxicity was measured by Agilent xCELLigence Real-Time Cell Analysis. Patient biopsies show a 2-fold increase in SIRPα expression in GBM tumors compared to the normal adjacent (n=14-21; p < 0.01). Single-cell RNA-seq data on GBM tumors shows that SIRPα is expressed not only in the myeloid compartment but also in malignant cells. Studies have shown that mitochondrial function and fatty acid β-oxidation (FAO) play a critical role in GBM progression. Respirometry data revealed that blocking SIRPα in murine GBM cell line (CT2A) decreases mitochondrial ATP production. Seahorse Mito Stress test combined with fuel inhibitors showed that SIRPα expression mainly affects the usage of FAO (approximately 50%). In addition, stably, the GFP-tagged SIRPα overexpressed GBM cell line showed an increase of mitochondrial respiration with enhanced FAO ratio compared to the GFP control cell line. These suggested that SIRPα regulates mitochondrial FAO. Studies have shown that tumor-intrinsic FAO confers cell resistance to immune cell-mediated cytotoxicity. Interestingly, by coculturing with microglia, the innate immune population in the brain, our data showed that SIRPα blockade on GBM enhanced microglia-mediated cytotoxicity. Our data showed that targeting SIRPα on GBM may be a prospective therapeutic option by inhibiting FAO in mitochondria and increasing immune cell-mediated cytotoxicity. Citation Format: Yu-Ting Tsai, Glenn J. Lesser, David R. Soto-Pantoja. Blockade of SIRPα in glioblastoma cells inhibits mitochondrial fatty acid β-oxidation and increase immune cell mediated cytotoxicity [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 2681.

Abstract 6715: CLDN6: Biological insight into therapeutic potency of SAIL66, a next generation of T-cell engager targeting CLDN6

Abstract Background: Because of high recurrence rate, ovarian cancer continues to demand new treatments. Recently, we have developed a next-generation T-cell engager SAIL66, which is a tri-specific monoclonal antibody designed to bind to CLDN6 on cancer cells with one Fab arm and to both CD3 and CD137 with the other Fab arm. By boosting T cell activation through both CD3 and CD137 binding, SAIL66 shows potent anti tumor efficacy against CLDN6-positive cancers and are currently being evaluated in patients with CLDN6-positive solid cancers including ovarian cancer (NCT05735366). CLDN6 is upregulated in many solid tumors but shows minimal or no expression in healthy adult tissues. Due to its cancer specific expression profile, CLDN6 has been utilized as a target for cancer immune therapies. Here, we demonstrate that CLDN6 is an attractive therapeutic target from its biological perspective in ovarian cancer. Methods and Results: In a HuNOG tumor-bearing mouse model transplanted with CLDN6-positive ovarian cancer cells (OV90, OVCAR3), SAIL66 showed potent antitumor activity as a single agent. Surprisingly, CLDN6 expression in transplanted tumors was significantly lower compared to in vitro cultured cells, suggesting that CLDN6 expression is highly variable. In vitro, CLDN6 expression reversibly fluctuated depending on cell density, and its expression was consistent with the expression of epithelial-mesenchymal transition (EMT) and stemness-related genes. Furthermore, FACS and qPCR revealed that chemotherapeutic agents increased CLDN6-positive cells, and this fluctuation was accompanied by the expression of EMT and stemness-related genes. Based on this biology, we tested the combination effect of SAIL66 and chemotherapies in a non-clinical tumor-bearing mouse model. As a result, significant tumor regression was observed in mice treated with SAIL66 in combination with carboplatin. Conclusion and Discussion: Ovarian cancer is characterized by significant tumor heterogeneity. The presence of cancer stem/stem-like cells or EMT has been proposed to elucidate the underlying mechanisms of the heterogeneity. Our study potentially could offer valuable insights into the biology of CLDN6-positive cells and the causes of ovarian cancer heterogeneity. Furthermore, it could help in patient selection, the development of combination strategies, and future clinical validation of SAIL66. Citation Format: Naoki Kimura, Kenji Taniguchi, Takayuki Kamikawa, Moe Yoshimoto, Shinya Ishii, Masaru Muraoka, Mei Shimada, Mika Kamata-Sakurai, Takehisa Kitazawa, Tomoyuki Igawa. CLDN6: Biological insight into therapeutic potency of SAIL66, a next generation of T-cell engager targeting CLDN6 [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 6715.

Abstract 874: Expanded detection of BAP1 alterations in cancer and comparison of tumor type-specific gene expression scores

Abstract BAP1 is a tumor suppressor gene that was originally studied in uveal melanoma (UVM), kidney renal cell clear cell carcinoma (KIRC), and malignant mesothelioma (MESO). Early analyses focused on single-nucleotide variants, but other alteration types such as larger indels and gene-level copy number (CN) loss can also lead to loss of BAP1 expression. By incorporating these, we can improve our assessment of cancer type-specific consequences of BAP1 loss and broaden our findings beyond the initial small subset of cancer types which were studied in the context of mutations alone. We performed integrated multi-omic analyses using data from The Cancer Genome Atlas (TCGA), for 33 cancer types and more than 10,000 individuals. By combining and manually reviewing existing variant calls and new calls derived from a de novo local realignment pipeline across multiple independent variant callers including indel callers, we increased detection of high-quality somatic variant calls by 30% from 85 to 111, including 14 indels ≥5bp. Including CN loss alterations, we found a total of 1561 samples from 32 cancer types to be BAP1-altered, with alterations being predominantly CN-driven. We confirmed that expression of BAP1 was lower in altered versus unaltered tumors (p<0.001). Alteration frequencies across cancer types were highly variable, ranging from 0% in thyroid cancer to >30% in KIRC, cholangiocarcinoma (CHOL), UVM, MESO, lung squamous carcinoma (LUSC), and head-neck squamous cell carcinoma (HNSC). Tumor types such as CHOL, UVM, MESO, and liver hepatocellular carcinoma (LIHC) were relatively more mutation-driven whereas other tumor types like KIRC, LUSC, and HNSC were almost entirely CN-driven. To better understand the tissue and context-specific consequences of BAP1 alteration, we performed differential expression analyses across BAP1-altered and -unaltered samples. For each cancer type, we separately computed per-sample BAP1 alteration signature z-scores summarizing expression of the set of differentially expressed genes. In UVM, we observed distinct patterns of RNA expression, consistent with established literature on BAP1 loss and serving as a baseline for comparison for the biological effects of BAP1 loss in other less-studied cancer types. The BAP1 altered scores for 66% of cancer types were positively correlated with the UVM BAP1 altered signature, suggesting shared biological responses and affected pathways including immune responses, epithelial-mesenchymal transition, oxidative phosphorylation, and proliferation – all known roles of BAP1. In LIHC, we noted enrichment of bile duct gene expression in BAP1-altered tumors, suggesting a possible role for BAP1 in maintaining cell identity distinguishing hepatocytes and cholangiocytes. Our findings broadly emphasize the improvements that are gained by using new computational approaches in large cancer-genome studies such as TCGA. Citation Format: Ian R. Sturgill, Jesse R. Raab, Katherine A. Hoadley. Expanded detection of BAP1 alterations in cancer and comparison of tumor type-specific gene expression scores [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 874.

Abstract 2079: Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [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 2079.

Abstract 1149: Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor

Abstract Recent advance of treatment strategy for triple negative breast cancer (TNBC) including immune checkpoint inhibitor improved survival outcome. Especially, adding pembrolizumab on neoadjuvant chemotherapy (NAC) increased pathologic complete response (pCR) rate at the time of curative surgery. Previous studies suggested that PD-L1 expression in both tumor and immune cells was associated with the response of pembrolizumab in TNBC, but PD-L1 expression state were not associated with pembrolizumab response in NAC setting. In addition, neoadjuvant pembrolizumab increased event-free survival (DFS) rate after NAC even though they had not achieved pCR. However, there were no biomarker for EFS increasement of using pembrolizumab. We designed prospective translational research for evaluating tumor microenvironment (TME) of residual TNBC which had treated with NAC followed by curative surgery. We collected formalin fixed paraffin embedded (FFPE) surgical specimens after NAC with or without pembrolizumab. TNBC samples were used for spatial transcriptomics (ST) analysis, with H&E staining and inspection by an experienced pathologist. Libraries were prepared using Visium Spatial Gene Expression Reagent Kit protocol for FFPE (10x Genomics). Sequencing was conducted on the NovaSeq 6000 System (Illumina). From February 2023 to October 2023, 23 TNBC specimens from 22 patients were collected. Twenty specimens were breast tissues and three were lymph nodes, 18 TNBCs had been treated with NAC with pembrolizumab and four with only NAC, and five had residual cancer burden(RCB) class I, 11 of RCB class II and one of RCB class III. We collected BC and LN samples from BC with class III. Of 23 samples, we performed spatial transcriptomic analysis in 12 samples. In intrinsic subtyping, eight specimens were categorized into normal like, two of basal like and two of luminal A BC subtypes. In TNBC subtyping, mesenchymal stem cell like (MSL) were in six specimens, three of stem cell like (SL), two of luminal androgen receptor (LAR) and one of immunomodulatory (IM). We also performed cell type annotation with unsupervised method and categorized into 11 cell types; three cancer cell types, two macrophages, B and T cells, adipocytes, normal epithelium and perivascular-like and cancer associated fibroblasts. Among these 11 cell types, increasement of immune cells including B cells, T cells and macrophage proportions was associated with pembrolizumab treatment in NAC setting. In our study, we evaluated TME state of TNBC after NAC with or without pembrolizumab. We suggested that MSL or SL TNBC subtypes were associated with residual disease after NAC regardless of pembrolizumab use and pembrolizumab may recruit more immune cells in TME of TNBC. For concreating our suggestion, further analyses would be warranted. Citation Format: Ji-Yeon Kim, Kyunghee Park, Yoon La Choi, Jeong Eon Lee, Byung Joo Chae, Jonghan Yu, Seok Won Kim, Seok Jin Nam, Jin Seok Ahn, Woong-Yang Park, Yeon Hee Park. Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor [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 1149.

Abstract 5148: Analysis of HER2 prevalence by RNA expression across solid tumors

Abstract Background: The frequency of HER2 expression in solid tumors other than breast (BC) and gastric cancer (GC) is not well defined. This study describes the frequency of HER2 expression by correlating HER2 IHC/ISH and ERBB2 mRNA levels by NGS in tumor samples of patients with locally advanced/metastatic (LA/m) BC or GC and applying those findings to predict HER2 expression in other solid tumors. Methods: De-identified records from NGS-tested patients with LA/m BC, GC, HNSCC, NSCLC, endometrial, and ovarian cancers in the Tempus database from 2015 - 2021 were analyzed. Patients with BC and GC who underwent HER2 IHC/ISH testing were categorized as: RNA-zero (IHC0), -low (IHC1+, IHC2+/ISH-), and -positive (HER2+; IHC2+/ISH+, IHC3+). To identify thresholds separating HER2 subgroups, a logistic regression model was fit to NGS and IHC status data in BC and GC. These thresholds were applied to other solid tumors to predict the distribution of HER2 expression. HER2 amplification and mutation rates were evaluated in the Tempus NGS data. Results: Logistic regression modeling used 3,898 samples. HER2+ samples had the highest IHC-to-RNA agreement at 80% (BC) and 81% (GC) compared to HER2 low and zero samples which ranged ~56-75% agreement. When applying thresholds identifying RNA-low and RNA-positive sample to other tumors (N = 4,726), the models predicted up to 33% (BC thresholds) or 56% (GC thresholds) of tumors express HER2 at levels that may be detectable (Table 1). Conclusions: A notable amount of solid tumors apart from BC or GC have RNA expression that may correspond to HER2 IHC ≥1+. This opens the possibility for novel agents such as HER2-directed antibody drug conjugates to impact patient care in several tumor types. Further IHC testing in these populations are needed to corroborate our results. Table 1. ERBB2 (HER2) Categories in Solid Tumors HER2 Status HNSCC (N = 630) NSCLC (N = 2945) OC (N = 855) EC (N = 295) NGS BC thresholds ERBB2 Log2(TPM+1) RNA-Zero 589 (93.5%) 1,974 (67.0%) 626 (73.2%) 205 (69.5%) RNA-Low 33 (5.2%) 853 (29.0%) 214 (25.0%) 64 (21.7%) RNA-Positive 8 (1.3%) 118 (4.0%) 15 (1.8%) 26 (8.8%) RNA-Low + RNA-positive 41 (6.5%) 971 (33.0%) 229 (26.8%) 90 (30.5%) NGS GC thresholds ERBB2 Log2(TPM+1) RNA-zero 492 (78.1%) 1,345 (45.7%) 395 (46.2%) 130 (44.1%) RNA-Low 124 (19.7%) 1,190 (40.4%) 384 (44.9%) 127 (43.1%) RNA-Positive 14 (2.2%) 410 (13.9%) 76 (8.9%) 38 (12.9%) RNA-Low + RNA-positive 138 (21.9%) 1,600 (54.3%) 460 (53.8%) 165 (55.9%) DNA Amplification Unamplified 622 (98.7%) 2,911 (98.8%) 843 (98.6%) 277 (93.9%) Amplified 8 (1.3%) 34 (1.2%) 12 (1.4%) 18 (6.1%) DNA mutation Wild-Type 623 (98.9%) 2,916 (99.0%) 850 (99.4%) 287 (97.3%) Mutated 7 (1.1%) 29 (1.0%) 5 (0.6%) 8 (2.7%) NGS: next-generation sequencing; HNSCC: head and neck squamous cell carcinoma; NSCLC: non-small cell lung cancer; OC: ovarian cancer; EC: endometrial cancer; TPM: transcripts per million Citation Format: Katherine Moxley, Emilie Scherrer, Martin Bontrager, Samantha Whitman, Naomi R. Schwartz, Chithra Sangli, Seung Won Hyun, Halla Nimeiri, Jonathan Ball, Charu Aggarwal. Analysis of HER2 prevalence by RNA expression across solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5148.

Abstract 7645: Differentially expressed genes as prognostic markers in racial disparities of prostate cancer

Abstract Racial disparities in prostate cancer are prevalent, with African American (AA) men experiencing 60% greater incidence and 2 to 3 times higher mortality than Caucasian American (CA) men. Consequently, there is a pressing need for improved means of interrogating race/ethnicity-based prostate cancer biology in order to identify biomarkers to help stratify men at high-risk for prostate cancer-specific progression and death and offer treatment recommendation that would mostly benefit AA men undergoing early aggressive therapies. Current predictive models rely on clinical variables but do not account for these disparities. The overall goal is to construct a race-specific prostate cancer prognostic prediction model for risk assessment. Here we performed comprehensive bioinformatics analysis using the data from 17 pairs of gene expression array from AA and CA cohorts and identified 63 differentially expressed genes (DEGs) by race. Among them, chemokine receptor 4 (CXCR4), viz. p-Akt (Ser473), fatty acid synthase (FASN), interleukin-6 (IL-6) and matrix metallopeptidase 9 (MMP-9) gene expressions were significantly altered in AA groups. Immunohistochemistry was performed on 163 prostate cancer patient specimens to validate DEG expression and immunoreactive scores (IRS) were calculated. CXCR4 exhibited significantly higher expression in AA tumors, with > 85% of AA prostate tumors showing weak to strong CXCR4 expression, compared to < 5% in CA specimens. While immunostaining for IL-6, FASN, MMP9 and p-Akt did not provide a significant distinction between CA and AA cases. IRS analysis confirmed higher expression of CXCR4 in AA cancers, compared to CA prostate cancer (p < 0.001). MMP9 and IL6 expression were moderately higher in AA cancer compared to CA prostate cancer (p < 0.05). In contrast, FASN expression was significantly higher in CA cancer, compared to AA prostate cancer (p < 0.001). We further investigated the impact of these genes on biochemical recurrence-free survival (BCRFS) using Kaplan-Meier analysis to examine the differences between low-risk versus high-risk groups. BCRFS analysis demonstrated that CXCR4 is a determinant factor of recurrence risk in AA prostate cancer patients. The activation of the PI3K-Akt signaling pathway, particularly in AA patients, suggests its role in prostate cancer aggressiveness. Taken together, this study demonstrates the importance of differential expression of biomarkers which constitute the bulk tumors in driving cancer. The distinct gene expression patterns, especially the upregulation of CXCR4, in AA prostate cancer highlight its potential as a prognostic biomarker and a target for personalized treatment strategies. These findings encourage further study into the role of population-specific difference in adding CXCR4 in risk model construction, especially in machine learning models. Citation Format: Amel A. Ahmed, Shiv Verma, Ibrahim M. Atawia, Daniel L. Shen, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Differentially expressed genes as prognostic markers in racial disparities of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7645.

Abstract 2855: ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3

Abstract Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [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 2855.

Abstract 2508: EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies

Abstract Background: Glioblastoma (GBM) is characterized as the most aggressive and lethal primary brain tumor. One of the hallmarks of GBM tumor growth and progression lies within the proangiogenic tumor microenvironment. Current anti-angiogenic drugs such as the anti-VEGF antibody take effect by inducing tumor cell starvation, leading to a functional normalization of the tumor vasculature. However, the survival benefits of anti-angiogenic drugs have been very limited. To this end, we hypothesized that the tetraspan Epithelial Membrane Protein-2 (EMP2) may help mediate resistance. Objective: We hypothesize that Epithelial Membrane Protein-2 may serve as a targetable biomarker for anti-VEGF resistance. We further explored the effectiveness of combination therapy in SB28 and GL261 GBM mouse models using anti-VEGF drug, Aflibercept, with a novel anti-EMP2 monoclonal antibody (mAb). Results: Tumor tissue was collected prospectively from patients undergoing surgery for suspected glioblastoma and stained for EMP2 and HIF2a using standard immunohistochemistry. Patients had banked tumor tissue both prior to and after bevacizumab treatment with 3 months of clinical and radiologic follow-up available. Bevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. Similarly, HIF-2a showed increased expression post-bevacizumab treatment and showed expression in both tumor and stromal cells. Limited animal models exist for anti-VEGF therapy for GBM. To establish syngeneic models, 10 week-old mice were subcutaneously implanted with murine SB28 or GL261 GBM cells. Treatment with either polyclonal sera to VEGF or Aflibercept failed to reduce tumor load or provide a therapeutic benefit. To next investigate if this model could recapitulate the response obtained patients tumors, formalin fixed paraffin embedded tissue was analyzed for both EMP2 and HIF-2a expression. Both markers were significantly upregulated in Aflibercept treated samples compared to the control.To next investigate if combination treatment could produce a significant reduction in tumor load, animals were treated with a combination treatment with Aflibercept and anti-EMP2 mAb. The combination therapy significantly reduced tumor volume and weight in comparison to control-treated tumors. Combination treatment in SB28 tumors resulted in a two fold reduction in tumor load and 50% reduction in tumor weight. Moreover, tumors showed reduced Ki67 expression. Similarly, treated GL261 tumors produced a 2.3 fold reduction in tumor size and ~2.7 fold reduction in tumor weight. Our results suggest that EMP2 can be a promising new target therapeutic target that can be combined with anti-VEGF treatment to potentially increase overall survival in GBM patients. Citation Format: Brian Aguirre, Mahlet Mekonnen, Riddhi Mehta, Meera Suresh, Anubhav Chandla, Karam Han, Isaac Yang, Madhuri Wadehra. EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies [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 2508.

Abstract 167: Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy

Abstract Introduction: Statins, known as inhibitors of HMG-CoA, not only reduce cholesterol but also exhibit pleiotropic effects which include anti-tumor activities by targeting tumor microenvironments. They inhibit the synthesis of FPP and GGPP, which is important for prenylation of signaling proteins. Although PD-1/PD-L1 inhibitors has been approved for treatment for TNBC patients with PD-L1 positive, the response remains unsatisfactory. Also, it has been reported that statins affect PD-L1 expression in other tumors, but it is still uncertain in TNBC. Therefore, we aimed to identify the effect of statins on the expression of PD-L1 in TNBC and to verify the efficacy of combination therapy with statins and immune checkpoint inhibitors. Methods: Human and mouse TNBC cell lines, mouse macrophage cell line (RAW 264.7), and clinically approved simvastatin were used. Flow cytometry, western blot, proliferation assay, qRT-PCR, immune phenotyping and immunohistochemistry were conducted. A co-culture of macrophages and cancer cells was performed. An orthotopic syngeneic mouse model by injection of EMT6 cells into mammary gland fat pad were produced. Mice were treated with simvastatin(20mg/kg) by oral gavage daily and anti-PD-1(200μg) by intraperitoneal injection two times per week. Results: Among thirteen human TNBC cell lines, we confirmed that TNBC cell lines showed various PD-L1 expression, and especially MDA-MB-231 and HCC38 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative effects in MDA-MB-231, HCC38, and EMT6 in a dose- and time-dependent manner. Phosphorylation of AKT and STAT3 was reduced when simvastatin was treated. Mice with combination therapy of anti-PD-1 and simvastatin had less effective inhibitory effect on tumor growth than mice with anti-PD-1 monotherapy, which showed the highest tumor suppression effect. In immune phenotyping, tumor-infiltrated T cells did not have significant differences among groups. However, M1:M2 macrophage ratio was highest in anti-PD-1 group but decreased in combination therapy group with simvastatin. To determine the effect of simvastatin on macrophages, RAW264.7 cells were stimulated with MDA-MB-231 conditioned medium, revealing an upregulation of PD-1 expression. Conclusions: Our findings show that simvastatin has an anti-tumor effect, which reduces Pd-L1 expression and kills breast cancer cells, reducing phosphorylation of STAT3 and AKT. However, when combined with PD-1 checkpoint inhibitor, it reduces the anti-tumor effect by increasing PD-1 expression in macrophages and promoting M1 to M2 polarization. Further study is needed to demonstrate the mechanism by which simvastatin regulate polarization of macrophages and to confirm the polarization of macrophages in samples of TNBC patients who had been taking simvastatin. Citation Format: Sangeun Lee, Ju Hee Kim, A Young Park, Jihong Baik, Ji-Jung Jung, Han-Byoel Lee, Wonshik Han. Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy [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 167.

Abstract 3500: Refining diagnosis of renal cell carcinoma subtypes through single-cell resolution transcriptomic signatures

Abstract A remaining challenge in the field of renal cell carcinoma (RCC) is diagnosis of rare or mixed subtypes, which hampers the delivery of the most effective healthcare to patients. While a new classification scheme, integrating molecular characteristics of tumors, is emerging, it requires a deep understanding of the tumor heterogeneity and etiopathogenesis of each subtype. We have generated and analyzed single-cell transcriptome profiles of 38 human RCCs and normal kidneys 16 RCCs with diverse histological subtypes and established gene signatures specific to each cell type. We have applied these gene signatures to bulk RNA-sequencing data of 1000 RCC tumors with different histological subtypes and normal kidneys from The Cancer Genome Atlas (TCGA) to characterize different cancer types and cellular origins. Unsupervised clustering analysis of TCGA data shows diagnostic discrepancies for 9.3% cases and subclassifies each major subtype (clear cell, papillary and chromophobe) into clusters with differential clinical outcomes.[KM1]. The pathological marker expression in misdiagnosed tumors is consistent with clustering-based classification, not their original diagnosis. Notably, using unique and subtype-specific gene signature patterns, we identified clear cell papillary renal cell tumor (ccpRCT), which is a newly classified benign entity and the main tumor type representing the mis-diagnosed tumors. These observations have further been confirmed by reviewing histological images of the tumors by an experienced genitourinary pathologist. Interestingly, our gene signatures reveal that ccpRCT does not originate from proximal tubule cells unlike clear cell and papillary RCCs. Taken together, our approach suggests a potential new classification method connected to clinical behaviors and drug responses based on overall transcriptomic differences. Citation Format: Minjun Kim, Ariel Madrigal, Zohreh Mehrjoo, Kate I. Glennon, Madeleine Arseneault, Morag Park, Fadi Brimo, Simon Tanguay, Hamed S. Najafabadi, Yasser Riazalhosseini. Refining diagnosis of renal cell carcinoma subtypes through single-cell resolution transcriptomic signatures [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 3500.

Abstract 464: Chromatin associated long non-coding RNAs involved in dormancy for late recurrence of ER-positive breast cancer

Abstract Late recurrence is one of the major problems for hormone receptor-positive breast cancer patients. It is suggested that the risk of recurrence may be maintained by cancer cells that have been already disseminated in the body at surgery. They enter long-term dormancy, thus evade immune surveillance and become resistant to therapies. The detailed molecular mechanism for this process is largely unknown.Nuclear long noncoding RNAs, which are not translated to proteins, often function as epigenetic regulators and determine the phenotype of cells and individuals. We found that a cluster of long noncoding RNAs, ELEANORS, is specifically expressed in ER (estrogen receptor)-positive recurrent breast cancer cells, forms a molecular condensate (RNA cloud) in the nucleus, and activates the ESR1 gene that encodes ER. ELEANORS define a large chromatin domain, the 0.7 Mb topologically associated domain (TAD), which contains the ESR1 and three other breast-cancer-associated genes. ELEANORS also regulates the three-dimensional genome architecture. They mediate a long-range chromatin interaction of ESR1 and FOXO3 genes, which are 42.9 Mb apart, and contribute to their cooperatively activate transcription, equilibrating cell proliferation (ESR1) and apoptosis (FOXO3). Upon ELEANOR knockdown, the ELEANOR clouds are disappeared, and ESR1 gene transcription is repressed. Further, the two genes are dissociated and only the FOXO3 gene remains transcriptionally active, resulting in the induction of apoptosis.Further analysis of clinical specimens revealed that ELEANOR expression in primary tumors correlates with recurrences over 5 years after surgeries, suggesting that ELEANORs are involved in late recurrence. We also found that ELEANOR activates transcription of CD44, a breast cancer stem cell marker gene. Cancer stem cell maintenance and equilibration of proliferation and apoptosis by ELEANOR may be involved in long-term tumor dormancy in late recurrence. This study suggests the possibility of ELEANORS as a biomarker to predict late recurrence, as well as a therapeutic target for ER-positive breast cancer. (References)Abdalla, MOA. et al., Nat. Commun, 10, 3778 (2019) Tomita, S. et al., Nat. Commun, 6, 6966 (2015) Citation Format: Noriko Saitoh, Maierdan Palihati, Hiroaki Tachiwana. Chromatin associated long non-coding RNAs involved in dormancy for late recurrence of ER-positive 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 464.

Abstract 495: Targeting undruggable transcription factor HOXB13 in metastatic prostate cancer by CRISPR/Cas13d-based nanoparticle therapy

Abstract Background: The oncogenic transcription factor HOXB13, which is highly expressed in castration-resistant prostate cancer (CRPC), has been shown to promote CRPC growth and metastasis. However, HOXB13 is considered untargetable by traditional small-molecule-based drug design. Gene therapy is critical alternative strategy with potential to directly target such traditionally undruggable genes. Methods: Selective cell in organ targeting (SCORT) nanoparticles for precise delivery of nanoparticles to metastatic cancer cells in an organ were constructed and characterized by the NanoAssemblr Spark, Zetasizer Nano ZS, and transmission electron microscopy (TEM); Cas13d mRNA with pseudouridine modification was synthesized using in vitro transcription; Hemi-spleen injection of CRPC cells was performed to build CRPC liver metastatic models; RNA-seq was employed to evaluate off-target effects of Cas13d targeting. Results: By incorporating a prostate cancer-specific E3 aptamer to functionalized lipid-like nanoparticles (FTT5 LNP-E3), we have successfully constructed SCORT nanoparticles that enable preferential delivery of mRNAs to metastatic CRPC cells in the liver, as opposed to various normal cells in the surrounding liver tissue. We demonstrated that Cas13d-pre-gHOXB13 mediates highly effective and specific HOXB13 mRNA knockdown in CRPC cells. Significantly, systemic treatment of SCORT nanoparticles carrying Cas13d-pre-gHOXB13 mRNA decreased HOXB13 expression in the metastatic tumors, inhibited metastasis, and prolonged survival of mice bearing androgen receptor (AR)-positive (AR+) or negative (AR-) tumors. Notably, long term (6 and a half weeks) administration of SCORT nanoparticle-Cas13d-pre-gHOXB13 did not significant alter body weight, hepatic and renal function, chemokines and cytokines, and other factors, effectively highlighting its safety. Conclusions: This study is the first to demonstrate that undruggable oncogenic transcription factors can be targeted using nanoparticle-delivered gene therapy based on CRISPR/Cas13 RNA-targeting. The SCORT-CRISPR/Cas13d system is a highly flexible technology that would impact the larger field of translational science by allowing the design of Cas13d-based, cancer cell-specific delivered nanoparticles targeting other previously-undruggable oncogenic transcription factors in metastatic prostate cancer and other solid tumors. Citation Format: Zhifen Cui, Furong Huang, Kun Fang, Jingyue Yan, Yufan Zhou, Jeffrey Everitt, Yue Zhao, William Hankey, Zhong Chen, Hongyan Wang, Victor X. Jin, Yizhou Dong, Qianben Wang. Targeting undruggable transcription factor HOXB13 in metastatic prostate cancer by CRISPR/Cas13d-based nanoparticle 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 495.

Abstract 2051: Innovations in ADC technology platform with legumain-cleavable KSP-inhibitor payloads adaptable to various aspects of cancer biology

Abstract Despite recent advances in the antibody-drug conjugate (ADC) field it remains challenging to achieve a sufficient therapeutic window. To address key issues of current ADCs such as aggregate formation and payload-related toxicities, we have developed a platform technology with tailored solutions for ADCs and small molecule-drug conjugates (SMDCs). Key features of the ADC technology comprise of kinesin spindle protein inhibitors (KSPi) as a novel payload class, a unique linker selectively cleaved by the tumor-associated protease legumain, and a CellTrapper® modification of the KSPi payload to reduce membrane permeability and provide accumulation in tumor cells beneficial for efficacy and safety1,2. Our advanced ADC VIP943 is targeting CD123 for the treatment of AML and MDS and is currently in Phase 1 clinical trial (NCT06034275). Here we show the extension and applicability of the platform in combination with various antibodies. Two case studies demonstrate how the payload-linker optimization increases the selectivity for tumor over healthy cells and potentially overcomes transporter-mediated drug resistance. Tuning the cleavable linker of cetuximab-ADCs for a higher challenge of legumain-mediated payload release resulted in a 52-fold improved selectivity of cytotoxicity against tumor cells versus HaCaT skin cells. A trastuzumab-ADC using a KSPi payload modified with the CellTrapper, shows low nanomolar potency against JIMT-1 cells resistant to trastuzumab-deruxtecan and thus may overcome resistance. An increased potency was not observed with an ADC releasing a permeable KSPi without CellTrapper. To address heterogenous target expression in solid tumors, novel antibody-2-drug conjugates (A2DCs) were synthesized to combine the advantages of intracellular accumulation of non-permeable payloads with the release of a second, membrane permeable KSPi payload. In vitro studies comparing the A2DCs with their respective mono-drug ADCs on 4 different targets shows the highest cytotoxicity achieved with A2DCs independent of the efficacy driver in mono ADCs. Lastly, synthesis of homogenous KSPi ADCs with a drug to antibody ratio of ~8 is compatible with different antibodies without significant aggregate formation. Furthermore, an easy and efficient process for ring-opening of the thiosuccinimide ring after conjugation was developed and shown to successfully avoid ADC de-conjugation via retro-Michael reaction. Selected examples of each of these subclasses were coupled to trastuzumab; all conjugates were highly efficacious and resulted in tumor regressions in the SKOV-3 model similar to trastuzumab-deruxtecan. Conclusion: Our technology platform offers options for efficient and safe cancer treatments and allows for versatile adaptations to the various aspects of cancer biology. 1. Kirchhoff et al, Cancers (2020). 2. Lerchen et al, Bioconj Chem. (2020). Citation Format: Hans-Georg Lerchen, Anne-Sophie Rebstock, Mareike Wiedmann, Beatrix Stelte-Ludwig, Amy J. Johnson, Raquel Izumi, Ahmed Hamdy. Innovations in ADC technology platform with legumain-cleavable KSP-inhibitor payloads adaptable to various aspects of cancer biology [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 2051.