Parental Cell Line Research Articles

Abstract PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

Abstract P09: AN EX-VIVO COMBINATION DRUG SENSITIVITY PLATFORM FOR PREDICTING SENSITIVITY TO FLT3 INHIBITOR-BASED COMBINATION IN ACUTE MYELOID LEUKEMIA

Abstract FMS-like tyrosine kinase-3 (FLT3) mutations are often linked with poor prognosis in Acute myeloid leukemia (AML). Despite increasing availability of targeted therapy, such as FLT3 inhibitors (FLT3i), remission is often short-term and followed by relapse and drug resistance. With varying response rates to standard treatments, the need to understand the molecular mechanisms driving drug resistance and accurately predict responses to treatment is crucial. Here, we present the ability of Quadratic phenotypic optimization platform (QPOP), in accurately identifying potential responders to possible combination therapies in AML. AML cell lines and AML patient samples were treated with a set panel of drugs including FLT3i. QPOP drug combination scores and ranking were derived from cell viability response following treatment. 40 patients were recruited for the study, of which 20 patients were evaluated for their clinical concordance. RNA sequencing and molecular analysis was further conducted to determine the accuracy of QPOP predicted response and to determine mechanism of resistance to FLT3i. QPOP predicted response was concordant to patient treatment outcome with 80% sensitivity and specificity. QPOP analysis on AML cell lines accurately identified responders to FLT3i including the differential response between a FLT3- ITD isogenic parental and FLT3i resistant cell line. Serial QPOP analysis in a FLT3 mutant patient sample indicated a decrease in FLT3i sensitivity concordant with an increasing FLT3 allelic burden and the development of resistance. The development of resistance to FLT3i based combinations was correlated to an increase in phosphorylated AKT expression. Activation of AKT is associated with the upregulation of Forkhead box M1 (FoxM1) in the resistant cell line. Co-treatment with an AKT inhibitor restored sensitivity to FLT3i treatment in the resistant cells. We demonstrate that QPOP analysis accurately predicts sensitivity to drug-combinations and can be used as a tool to identify sensitivity to FLT3i-based combination therapy in AML. Citation Format: Noor Rashidha Bte Meera Sahib, Jen Wei Ying, Melissa Ooi, Yen Lin Chee, Jayalakshmi, Jameelah Bte Sheik Mohamed, Masturah Rashid, Edward KH Chow. AN EX-VIVO COMBINATION DRUG SENSITIVITY PLATFORM FOR PREDICTING SENSITIVITY TO FLT3 INHIBITOR-BASED COMBINATION IN ACUTE MYELOID LEUKEMIA [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P09.

PARP1 promotes EGFR-TKI drug-resistance via PI3K/AKT pathway in non-small-cell lung cancer.

Tyrosine kinase inhibitor (TKI) resistance is the main type of drug resistance in lung cancer patients with epidermal growth factor receptor (EGFR) mutations, but its underlying mechanism remains unclear. The purpose of this work was to investigate the mechanism by which PARP1 regulates EGFR-TKI resistance to identify potential targets for combating drug resistance. The GEO databases, TCGA databases, western blot and qPCR studies were used to investigate the expression of PARP1 in lung cancer cells and tissues and its correlation with the prognosis of lung cancer. The expression of PARP1 in lung cancer TKI resistant cell PC9-ER and TKI sensitive cell PC9 was analyzed by qPCR and western blot. After knocking down of PARP1, CCK-8 assays, colony formation, flow cytometry were used to investigate its impact on erlotinib sensitivity, cell survival, cell cycle, and apoptosis. RNA-seq was used to investigate the mechanism by which PARP1 participates in EGFR-TKI resistance, and the results were validated in vitro and in vivo studies. PARP1 was highly expressed in both lung cancer tissues and cells. Subsequently, increased PARP1 expression was observed in PC9-ER compared with its parental cell line. Knockdown of PARP1 increased erlotinib sensitivity, promoted cell apoptosis, and suppressed cell growth. RNA-seq and previous studies have shown that the PI3K/AKT/mTOR/P70S6K pathway is involved in PARP1-mediated TKI resistance, and these results were confirmed by Western blot in vitro and in vivo. PARP1 may serve as a potential therapeutic target for reversing EGFR-TKI resistance in NSCLC via the PI3K/AKT/mTOR/P70S6K pathway.

Development and in-depth characterization of BRAFi-resistant melanoma cell lines in vitro and in vivo

Regardless of the clinical response and improved patient survival observed following treatment with BRAFi like Vemurafenib (Vem), rapid development of resistance still remains as a major obstacle in melanoma therapy. In this context, we developed and characterized two acquired Vem-resistant melanoma cell lines, A375V and SK-MEL-28V, and an intrinsically Vem-resistant cell line, RPMI-7951. Altered morphology and growth rate of the resistant cell lines displayed spindle-shaped cells with filopodia formation and enhanced proliferation rate as compared to parental cells. Further in vitro characterization in 2D models confirmed the emergence of a resistant phenotype in melanoma cells. To mimic the in vivo tumor microenvironment, spheroids were developed for both parental and resistant cell lines to recognize materialization of invadopodia structures demonstrating elevated invasiveness and proliferation of resistant cells-based spheroids, especially A375V. Importantly, we validated A375V cell line in vivo to prove its tumorigenicity and drug resistance in tumor xenograft model. Taken together, our established clinically relevant Vem-resistant tumor model could be beneficial to elucidate drug resistance mechanisms, screen and identify novel anticancer therapies to overcome BRAFi resistance in melanoma.

Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

Abstract LB210: Identification of histone deacetylase 10 as a repressor of cancer cell migration

Abstract Histone deacetylases (HDACs) are a family of 18 enzymes with the potential to be epigenetic modifiers. Of the 4 classes of HDACs, HDAC6 and HDAC10 belong to the class IIb subgroup. Unlike HDAC6, HDAC10 contains two deacetylase-like domains, with one active deacetylase domain and one inactive leucine-rich domain, and a preference for a specific acetylated polyamine over other substrates. Aberrant expression of HDAC10 has been implicated in cancer and other diseases. However, the deacetylation targets and biological functions of HDAC10 have not been well characterized due to a lack of specific and potent HDAC10 inhibitors with cellular activity. We previously established CRISPR/Cas9-mediated HDAC10 knockout (KO) cell lines in HCT116 and HeLa S3 cells. In the current study, we used the HCT116 wildtype and KO pair for bulk RNA sequencing, differential gene expression, and gene set enrichment analyses (GSEA), which revealed an upregulation of gene markers and pathways potentially associated with metastasis and invasion in the HDAC10-KO cells, including S100A14, LAMA4, and TGF-beta. As the role of HDAC10 expression on cell migration and invasion has not been previously reported, we performed cell migration assays using our HDAC10 parental and knockout cell line pairs. In both HCT116 and HeLa systems, HDAC10 KO cells exhibited dramatically higher rates of cell migration in Boyden chamber and scratch assays. These same assays were performed in HDAC10-expressing cells in the presence of the HDAC10-specific inhibitor DKFZ-728, which similarly caused a dose-dependent increase in migration, strongly supporting a catalytic role for HDAC10 in repressing migration. In addition to these results, we present data investigating the role of HDAC10 in cell migration/invasion in representative prostate cancer cell lines (LNCAP and LAPC4), as the prostate tumor microenvironment is particularly rich in polyamines, including N 8-acetylated spermidine, the substrate of HDAC10. Finally, using our panel of HDAC10-KO cell lines and inhibitors, we analyze the contribution of commonly perturbed genes to the migratory phenotype with the goal of defining the precise mechanism of action. Overall, our studies provide functional evidence that loss of HDAC10 can increase cell migration, and thus, may lead to increased metastatic potential. These results emphasize the need for isoform-specific inhibitors and may have important clinical implications, as commonly utilized HDAC inhibitors, including vorinostat (SAHA), tubastatin A, and trichostatin A (TSA), also inhibit HDAC10. Our ongoing studies include incorporating HDAC10 proteolysis targeting chimeras (PROTACs) into our functional assays to examine differences between HDAC10 inhibition and PROTAC-mediated degradation, providing further rationale for modulating HDAC10 activity as an antineoplastic strategy. Citation Format: Ting-Ann Liu, Ashley Nwafor, Rajendra Kumar, Laura Sena, Raphael R. Steimbach, Aubry K. Miller, Robert A. Casero, Tracy Murray Stewart. Identification of histone deacetylase 10 as a repressor of cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB210.

Cisplatin-Resistant Urothelial Bladder Cancer Cells Undergo Metabolic Reprogramming beyond the Warburg Effect.

Advanced urothelial bladder cancer (UBC) patients are tagged by a dismal prognosis and high mortality rates, mostly due to their poor response to standard-of-care platinum-based therapy. Mediators of chemoresistance are not fully elucidated. This work aimed to study the metabolic profile of advanced UBC, in the context of cisplatin resistance. Three isogenic pairs of parental cell lines (T24, HT1376 and KU1919) and the matching cisplatin-resistant (R) sublines were used. A set of functional assays was used to perform a metabolic screening on the cells. In comparison to the parental sublines, a tendency was observed towards an exacerbated glycolytic metabolism in the cisplatin-resistant T24 and HT1376 cells; this glycolytic phenotype was particularly evident for the HT1376/HT1376R pair, for which the cisplatin resistance ratio was higher. HT1376R cells showed decreased basal respiration and oxygen consumption associated with ATP production; in accordance, the extracellular acidification rate was also higher in the resistant subline. Glycolytic rate assay confirmed that these cells presented higher basal glycolysis, with an increase in proton efflux. While the results of real-time metabolomics seem to substantiate the manifestation of the Warburg phenotype in HT1376R cells, a shift towards distinct metabolic pathways involving lactate uptake, lipid biosynthesis and glutamate metabolism occurred with time. On the other hand, KU1919R cells seem to engage in a metabolic rewiring, recovering their preference for oxidative phosphorylation. In conclusion, cisplatin-resistant UBC cells seem to display deep metabolic alterations surpassing the Warburg effect, which likely depend on the molecular signature of each cell line.

Abstract 2819: Novel drug resistant CDX models for anti-cancer drugs discovery

Abstract Oncogene BRAF is located on chromosome 7q34, consisting of 766 amino acids and 22 exons, encoding a serine/threonine protein kinase belonging to the RAF family and plays a critical role in the MAPK signaling pathway. The mutation of BRAF was documented in nearly 8% of all human cancers including melanoma (60%), thyroid (60%), and lung adenocarcinoma (10%). The most common mutation of BRAF is V600E (Class I), which was found in more than 70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because of primary or acquired drug resistance. The construction of BRAF drug-resistant cell lines plays a pivotal role in cancer research. It serves as a crucial tool for discovery of the mechanisms of drug resistance. In this study, we have developed a systematic approach for constructing drug-resistant cell lines, inducing resistance through the gradual escalation of drug concentrations. The validation of drug resistance involves morphological recordings of parent and drug-resistant cell lines, drug resistance index in vitro through IC50 measurements and in vivo through the inhibition of tumor growth (TGI) which compare to those in the parental cell line. We successfully established Vemurafenib and Dabrafenib resistant A375 cell lines (A375 Vemurafenib R, A375 Dabrafenib R) with 21 and 31.5 folds of resistance index, respectively. The in vivo drug resistance efficacy was also tested by implanting Vemurafenib resistant cells subcutaneously into NOD-SCID mice and showed that A375 Vemurafenib R exhibited robust tolerance to 50 mg/kg Vemurafenib treatment (P.O.21 days, 50mpk, TGI=17.4%), whereas the parental cells were nearly completely inhibited (P.O.21 days, 50mpk, TGI=92.3%). In addition, single-cell sequencing of A375 Vemurafenib R revealed significant gene expression differences in the MAPK and EGFR signaling pathways, aiding in the identification of new targets, and signaling pathways associated with drug resistance, thereby providing potential targets for new drug development. Citation Format: Hai-Ting Dai, Tie-Jun Bing, Zhi-Da An, Jie Yang, Tian-Yu Pang, Jia-Wei Gao, Yun-Yi Bo, Wen-Jen Yu. Novel drug resistant CDX models for anti-cancer drugs discovery [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 2819.

Abstract 4877: Morphological detection of chemotherapy resistance in cancer cell lines using AI-based analysis

Abstract Chemoresistance can develop during the course of treatment of many cancers, which can limit the utility of standard therapeutics and lead to poor outcomes. The mechanism by which resistance arises is not well understood, and developing models to study resistance can be challenging. Resistance can spontaneously arise in cell lines, however, it is difficult to separate resistant from sensitive cells using traditional biomarkers, hampering their utility for studying mechanisms. Morphology presents another modality to examine resistance, independently of protein markers, and may solve a previously intractable problem of separating resistant from sensitive cells for further study. Here we sought to study the mechanism of chemoresistance in cell lines using multi-parameter morphology in combination with gene expression and other standard molecular approaches. We induced resistance to specific chemotherapeutic drugs in parent cancer cell lines from lung, ovarian, and melanoma cancers. IC-50s of each drug increased by ≥1 order of magnitude in the resistant cells. Whole exome sequencing and bulk RNAseq were performed to examine differentially expressed genes and pathways between the parent and resistant cell lines. Each cell line was analyzed using multi-dimensional morphology using the Deepcell platform, which extracts >100 morphological features from images of unlabeled single cells using artificial intelligence (AI), advanced imaging, and microfluidics. The morphology features represent both Deep Learning (DL)-derived features and human-interpretable morphometric features, such as size and shape. Unique morphotypes were observed differentiating each combination of parent and resistant cell lines, with notable changes in granularity distinguishing them. A subset of images was used to train a random forest classifier to predict the resistant state of each cell image, which performed with up to 85% accuracy on independent cell images. Strikingly, the combination of DL plus morphometric features outperformed the use of morphometric features alone, demonstrating the increased utility of combined analysis. The top gene pathways and top morphological features were examined to infer potential mechanisms underlying chemoresistance. This work demonstrates that morphology alone can be used to distinguish drug resistant vs. parent populations without the use of markers. Morphology analysis can be applied to understanding complex phenotypes, and future platform improvements will enable sorting of these resistant populations to better identify molecular pathways in both cell lines and in primary tumor samples. Citation Format: Andreja Jovic, Manisha Ray, Ryan Carelli, Kiran Saini, Tiffine Pham, Christian Corona, Stephane C. Boutet, Chassidy Johnson, Mahyar Salek, Maddison Masaeli, Matt Barnes, Cyril Y. Ramathal. Morphological detection of chemotherapy resistance in cancer cell lines using AI-based analysis [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 4877.

Abstract 1930: Combination of KRASG12C and FGFR1 inhibitors as a resistance-overcoming therapeutic strategy for maximizing therapeutic impact of KRASG12C inhibitors

Abstract INTRODUCTION: Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the most frequent mutated driver gen in lung adenocarcinoma (LUAD). KRASG12C inhibitors (G12Ci) have revealed promising results in the clinic, having being approved two G12Ci for the treatment of LUAD patients, which marks the first approved targeted therapy for KRAS-mutant tumors. Nevertheless, these therapies face the same limitation as other targeted therapies, the therapeutic potential of these inhibitors can be impaired by resistance mechanisms. Deciphering resistance mechanisms to G12Ci is of prime relevance to predict which patients may benefit from these therapies and to propose resistance-overcoming therapeutic strategies for maximizing therapeutic impact of these inhibitors. MATERIAL AND METHODS: We generated 10 acquired resistant LUAD cell lines to G12Ci (Sotorasib and Adagrasib), exposing sensitive cells to increasing concentrations of drugs. All the acquired resistant models were characterized at the proteomic (phospho-Array and Western Blot) and transcriptomic (WTS) levels to find signatures that define the resistance.The efficacy of G12Ci was tested in vitro in a panel of 8 cell lines and 7 PDX-derived organoids (PDXDO) models and in vivo in 7 patient-derived xenografts (PDX) models to classify them as resistant, sensitive or partially sensitive. The efficacy of the combination of FGFR1 and G12Ci was tested in vitro in the panel of 10 acquired resistant cell lines but also in the parental cell lines and PDXDOs (sensitive and intrinsic resistant) and in vivo in the intrinsic resistant PDXs. RESULTS AND DISCUSSION: The characterization of 10 acquired resistant cell lines at the proteomic and transcriptomic levels showed differences in the expression and/or activation of Fibroblast Growth Factor Receptors (FGFRs) in more than 50% of them. The combination of KRASG12C and FGFR1 inhibitors in all acquired resistant cell lines showed efficacy, regardless of whether acquired resistance was mediated or not by FGFR1 overexpression/overactivation.In addition, the combination of KRASG12C and FGFR1 inhibitors showed more efficacy than the monotherapies in most intrinsically resistant cell lines, PDXDOs and PDXs models. Finally, we tested the combination with FGFR1i in sensitive or partially responsive models, observing an improvement in efficacy compared to monotherapy with G12Ci. CONCLUSIONS: The activation or overexpression of FGFR1 acts as a mechanism of acquired resistance to KRASG12C inhibitors.-The combination of FGFR and KRASG12C inhibitors is effective as an acquired/intrinsic resistance-overcoming therapeutic strategy in cell lines, PDXDOs and PDXs models.-The combination of FGFR and KRASG12C inhibitors is also synergistic for sensitive models, which could maximize therapeutic impact of KRASG12C inhibitors. Citation Format: Alba Santos, Patricia Plaza, Marta Jimenez, David Gómez-Sánchez, Luis Paz-Ares, Irene Ferrer. Combination of KRASG12C and FGFR1 inhibitors as a resistance-overcoming therapeutic strategy for maximizing therapeutic impact of KRASG12C inhibitors [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 1930.

Abstract 457: Persister cell state-associated lncRNA signatures in non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, largely due to late-stage diagnosis and a 5-year survival rate of 9%. Targeted therapies for oncogene-dependent tumors like tyrosine kinase inhibitors (TKIs) improve outcomes yet their clinical efficacy is often limited by occult drug-tolerant persister cells (DTPCs), causing recurrence. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides and typically non-protein coding. They regulate cellular functions by interacting with substrates through their complex secondary structures. Their deregulation is linked to oncogenesis and therapy resistance. While their role in lung cancer has been documented, their involvement in the DTPC state remains unexplored. Our study aims to investigate the role of lncRNAs in mechanisms that drive failure to targeted therapy in NSCLC. We comprehensively analyzed single-cell RNA-seq data from 49 NSCLC biopsies, pre- and during TKI therapy (residual disease, RD). We identified several lncRNAs that were significantly, differentially expressed in RD when compared to treatment naïve (TN) tumors. We orthogonally validated these findings in a minimum of 2 lung cancer cell lines in TN or DTPCs (9-15 days upon TKI exposure in culture) [p<0.05, absolute log2 fold change > 0.3]. We found that MALAT1, HCG11, and EPB41L4A-AS1 (lncRNA-UP signature) displayed significantly increased expression in RD relative to TN tumors and parental cell lines. We also found significant downregulation of LUCAT1 and LINC00152 (lncRNA-DOWN signature) in RD samples. We further performed Kaplan-Meier survival analysis in an independent set of RD samples stratified by differential expression of our lncRNA signatures (n=18/group). The analysis showed improved overall survival in patients whose DTPCs expressed a high level of the lncRNA-UP signature (hazard ratio, HR=0.56; log-rank test p=0.0331). We did not observe significant differences in survival for the lncRNA-DOWN signature. We additionally performed survival analysis using the GEPIA database for lung adenocarcinoma (primarily TN, n= 239/group). Consistently, the analysis showed improved overall survival for high levels of the lncRNA-UP signature (HR=0.71; p=0.029), whereas high levels of the lncRNA-DOWN signature were indicative of inferior outcomes (HR=1.4, p=0.046). We systematically identified DTPC-associated lncRNA signatures from our unique DTPC cohort, indicating that deregulation of lncRNA expression may play a role in acquired drug resistance in lung cancer. DTPC-associated lncRNA expression patterns are linked to differential overall survival, suggesting a possible tumor-suppressive or promoting role and highlighting their potential as predictive biomarkers and therapeutic targets. Further investigation into the functional roles of these lncRNAs and their interplay with coding molecules is warranted. Citation Format: Jovanka Gencel-Augusto, Wei Wu, Trever G. Bivona. Persister cell state-associated lncRNA signatures in non-small cell lung 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 457.

Abstract 414: Accelerating DDR related drug discovery through a customized cell panel

Abstract DNA damage is a major threat to cell survival. Not properly repaired DNA damage can lead to cell senescence, apoptosis or tumor development. DDR (DNA Damage Repair) is a collection of bioprocesses which cells utilize in order to identify and correct the DNA damage of the genome. The usage of various compounds for targeting DNA damage responses as well as attenuating DNA repair is also an approach for cancer therapy that has been widely studied in recent years. Despite the success of drug discovery of DDR related inhibitor, drug resistance has become an emerging issue of the field. Here we have constructed multiple cancer cell lines which are either DDR gene, such as XRCC1 and FANCD2, knock-out or resistant to certain DDR inhibitors. Together with the corresponding parental cell lines, we have generated a DDR cell panel which covers 11 different cancer types. We have first validated those DDR inhibitor sensitive or resistant cell lines with functional cell proliferation assays. Additionally, RNAseq has been performed for all constructed resistant cell lines. In combination with a thorough bioinformatic analysis using our in-house generated algorithms, we can provide the genetic background information of the resistant cell lines and top features that potentially contribute to the resistance mechanism. The representative data in this study has shown that LINC02709, BDKRB1, and PRKCQ are the top featured genes in A375 Vemurafenib resistant cell. Meanwhile, cell cycle, ECM-receptor interaction, and DNA replication are the most enriched KEGG terms. Lastly, we have validated this cell panel against multiple inhibitors targeting different DDR vital proteins such as ATM, PARP, POLQ etc. The result has shown that our unique DDR cell panel is capable of providing fast and comprehensive evaluation of DDR related inhibitors, thus facilitate faster and more efficient discovery of DDR inhibitors in the cancer therapy field. Citation Format: Cong Huang, Xiaoqing Li, Zhengtai Li, Yan Tan, Huawei Pan, Lizhen Zhou, Kejun Mao, Yanyan Qin, Zhida An, Wanxiao Song, Yifan Li, Shuyu Jin, Tiejun Bing. Accelerating DDR related drug discovery through a customized cell panel [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 414.

Abstract 306: The role of exosomal UBE2R2 in colorectal cancer drug resistance

Abstract Background: Ctherapy resistance has been a major barrier against successful theratment. Exosome (EV), an important intercellular communication mediator, participates in a highly and complex network that drug resistance of cancer cells. EV has been reported to be able to transfer drug resistance from drug-resistant tumor cells to adjacent drug-sensitive cells. We identified a novel target, UBE2R2, based on our previous exosome proteomics analysis of resistant colorectal cancer cell lines. The main goal of this study is to validate the underlying mechanisms leading to chemotherapy resistance. Methods: We isolated EV from drug resistant cancer cell lines including coloractal cancer cell lines (HCT116-OXAR, DLD-1 OXA-R, DLD-1-OXA-R/SN38) its parental cell lines (HCT116, DLD-1) using the ExoLutE isolation kits based on a combination of multiple isolation techniques. Based on the existing data (proteomic analysis), we conducted experiments to elucidate the functions and mechanisms involved in cancer resistance of the identified UBE2R2. Cell viability assay and invasion asaay were performed to assess the effects of EVs drived from drug resistance colorectal cells on oxaliplatin and irinotecan resistance. In addition, cells transfected with siControl and siUBE2R2 were treated with oxaliplatin to access the relative UBE2R2 drug resistance effect. To explore the related mechanisms, we performed real time PCR and western blot to identify the UBE2R2-SMYD3-P53 pathway, and FACS analysis to confirm apoptosis. Additionally, TMA samples were utilized to confirm resistance according to patient stage, and UBE2R2 expression was validated by immunohistological methods. Results: UBE2R2, which is involved in ubiquitination, was most prominently expressed in resistant cell lines compared to controls in our previous proteomic analysis. Based on these results, we found that cell lines treated with exosomes which were derived from resistant cell lines showed lower sensitivity to the drug. Notably, treatment of resistant cell lines with siUBE2R2 resulted in increased sensitivity, confirming the involvement of UBE2R2 in resistance. As a possible mechanism in which UBE2R2 may be involved, western analysis targeting the SMYD-P53 mechanism showed that the combination of UBE2R2-SMYD3-p53 promoted the ubiquitination and degradation of p53. Analyzed using TMA patient samples, we found that the expression of UBE2R2 increased towards more advenced stage. Conclusion: Our results suggest a novel mechanism of chemotherapy resistance may be associated with UBE2R2. Exosome (including UBE2R2) which was drived from drug resistance cells may induce oxaliplatin and irinotecan resistance in parental colorectal cells via regulation of SMYD3-UBE2R2-P53 signaling pathway, which will be useful for investigating a potential clinical target in prediction drug resistance. Citation Format: Chi Hoon Maeng, Minji Choi. The role of exosomal UBE2R2 in colorectal cancer drug resistance [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 306.

Abstract 5868: Upregulation of HER3 and MET in non-small cell lung cancer cells resistant to EGFR tyrosine kinase inhibitor

Abstract Introduction: Prior research demonstrates the importance of studying epidermal growth factor receptor (EGFR) activating mutations for better understanding the biology of non-small cell lung cancer (NSCLC), which comprises the majority (~80%) of lung cancers. EGFR-targeted tyrosine kinase inhibitors (TKIs) have been used in the clinic to treat NSCLC with EGFR activating mutations. First and second-generation EGFR-TKIs are effective in NSCLC patients with the most common EGFR mutations (exon 19 deletion (Del19) and exon 21-point mutation (L858R)). A third generation EGFR-TKI, osimertinib (AZD9291), has been developed to target the T790M resistance mutation, but patients are starting to show resistance to this line of therapy as well. Our study aims to elucidate the underlying mechanism(s) of EGFR-TKI resistance in NSCLC cells. Methods: NSCLC cell lines, including PC-9 and HCC827, which have the Del19 EGFR activating mutation, were utilized for experiments. From the parental cell lines, several cell lines resistant to the EGFR-TKIs were previously established. Cell proliferation (MTS) assays were used to confirm the resistance phenotype of the established cell lines. Western blot assays and RT-qPCR were utilized to determine changes in the expression level of several membrane receptors and their downstream effectors. Results: Our preliminary analysis showed that both human epidermal growth factor 3 (HER3) and mesenchymal-epithelial transition factor (MET) were upregulated in the EGFR-TKI-resistant cell lines as compared to their parental counterparts. Further studies revealed an increased level of phosphorylated STAT3 (p-STAT3) and ERK1/2 (p-ERK1/2) in the resistant cell lines when compared to the parental cell lines, suggesting the possible involvement of HER3 and/or MET in the activation of downstream signaling pathways. Upregulation of HER3 and MET was found at both the protein and mRNA level. Conclusion: Based on our initial findings, membrane receptors, including HER3 and MET, could be working together to continue activating downstream signaling pathways to evade the effects of osimertinib, resulting in resistance. Additional work is underway to define the role of HER3 and MET in the NSCLC cells resistant to EGFR-TKIs. Citation Format: Margaret Larsen, Hui Lyu, CongCong Tan, Yakun Wu, Bolin Liu. Upregulation of HER3 and MET in non-small cell lung cancer cells resistant to EGFR tyrosine kinase 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 5868.

Abstract 2603: Targeting chemo-resistant ovarian cancer with a first-in-class, first-in-human immune-oncology agents containing granzyme B

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer-related mortality in women. The current standard of care consists of debulking surgery with platinum-taxane maintenance chemotherapy. However, OC will recur in most patients, resulting in a five-year survival rate of 45%. In the past decades, there have been several developments in front-line maintenance therapy including bevacizumab or PARP inhibitors. While these therapies have shown effectiveness in prolonging progression free survival, this has not translated into improvements in overall survival. It is, therefore, essential to develop more effective maintenance therapies and most of the current efforts are focused on targeted approaches and immune therapeutics. Though immunotherapeutic strategies have induced durable responses in patients with melanoma, lung cancer, and other malignancies, the OC response to immunotherapy is limited as there is a significant subset of patients who do not respond. Our laboratory has developed several fully human granzyme B (GrB) based fusion proteins against key OC targets including folate receptor 1 (GrB-Fc-mov018) and fibroblast growth factor-inducible 14 (GrB-Fc-IT4) which retain high specificity of the targeting agent, combined with the GrB specific activity, a pro-apoptotic mechanism of action, and a robust in vivo efficacy. We assessed the in vitro cytotoxicity of GrB-based constructs against a panel of OC cell lines sensitive (OVCAR8, SKOV3, A2780, HeyA8) or resistant (SKOV3-TX, A2780/Cl-16, HeyA8-MDR) to common chemotherapeutic agents. SKOV3-TX cells were >10 fold more resistant to Paclitaxel than the parental SKOV3 cells while A2780/Cl-16 were 11-fold more resistant to Cisplatin than the parental A2780 cells. In contrast, none of the chemo-resistant cell lines showed resistance to the GrB-based constructs and the IC50 values observed were similar to those of the parental cell lines in the low nanomolar range (<50 nM). Moreover, target expression analysis did not show significant differences between parental and chemo-resistant cell lines. A pilot in vivo efficacy study of GrB-Fc-IT4 and GrB-Fc-mov018 against a subcutaneous, established ovarian tumor (OVCAR8) xenograft showed promising growth inhibition of established tumors compared to vehicle controls. In vivo studies evaluating the response to different treatments of mice bearing chemo-resistant ovarian tumors are ongoing. Our results suggest that the mechanism of resistance developed to conventionally used chemotherapeutic agents such as Paclitaxel, Doxorubicin, Cisplatin or Mitomycin do not confer resistance to GrB-based constructs. Hence, we speculate that GrB-based constructs could be an effective therapy in OC patients with immune-excluded tumors that have developed resistance to conventional treatment. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Michael G. Rosenblum. Targeting chemo-resistant ovarian cancer with a first-in-class, first-in-human immune-oncology agents containing granzyme B [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 2603.

Abstract 1758: Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in uveal melanoma

Abstract Uveal Melanoma (UM) is a rare tumor characterized by mutations in GNAQ or GNA11, followed by a mutation in BAP1, SF3B1 or EIF1AX. Notably, a large subset of BAP1 mutant patients present with a unique chromosome 3 copy-number (CN) loss and higher likelihood of metastases. The biology underlying the causes and consequences of Monosomy 3 (M3), and its role in metastases is largely unexplored. This can be partially attributed to the relatively few numbers of preclinical models of UM, and the inability to delineate M3 from BAP1 loss. To address this, we derived a preclinical isogenic model of chromosome 3 CN loss through CRISPR-based centromere targeting in a well characterized UM cell line lacking a BAP1 mutation. Clones with and without chromosome 3 loss were derived from these efforts. Recent studies have evaluated genomic events in primary UM and described five CN aberration-based subtypes expanding beyond M3, with the fifth subtype being called the “ultra-high-risk UM” based on poor survival and extremely high metastatic rate. Whole genome sequencing across our clones confirmed chromosome 3 CN loss, and in addition to this, recapitulated all of the core genomic features characteristic of the ultra-high-risk UM group. In comparison, our models without chromosome 3 CN loss maintained a genomic profile more characteristic of lower risk UM subtypes. The genomic landscape of our isogenic low risk (LR) and high risk (HR) clones suggest that chromosome 3 CN loss alone in the absence of BAP1 mutations can be the driver for acquisition of downstream genomic events seen in aggressive UM. Further, using single cell DNA sequencing on the parental model, LR and HR clones, we confirmed that chromosome 3 CN loss in our clones was engineered, and not a pre-existing subclone in the parental cell line. Bulk RNAseq on our LR and HR clones demonstrate concordance with genomic regions of losses and gains, while also highlighting upregulation of pathways such as cell cycle and DNA damage response in the HR clones. Next, we developed patient derived xenograft (PDX) models demonstrating differential behavior in paired in vivo settings, where HR clones had higher fitness advantage and metastatic potential, specifically to the mouse liver. This modeling has enabled us to employ spatial transcriptomics in a PDX setting, adapt existing methodology to infer chromosomal copy number events in our system and inform transcriptional and genomic phenotypes akin to the aggressive HR clones. Thus, using our engineered preclinical models, we demonstrate that chromosome 3 loss, in the absence of BAP1 mutations is capable of driving aggressive disease in UM, characterized by genomic evolution, transcriptional reprogramming and in vivo invasiveness. Citation Format: Sanjana Srinivasan, Johnathon Rose, Rosalba Minelli, Christopher Terranova, Parth Shah, Joseph R. Daniele, Michael Peoples, Meng He, Chiu-Yi Liu, Jason Gay, Melinda Soeung, Sanjay Soni, Fangyang Wang, Christopher Bristow, Khalida Wani, Luigi Perelli, Dmitry Loza, Ningping Feng, Maxime Tarabichi, Peter Van Loo, Andrew Futreal, Scott Woodman, Alexander Lazar, Christopher P. Vellano, Joseph Marszalek, Giulio F. Draetta, Timothy Heffernan, Alessandro Carugo, Giannicola Genovese, Virginia Giuliani. Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in uveal melanoma [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 1758.

Abstract 3284: Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer which accounts for more than 90% of occurrences of liver cancer cases. Surgical therapies such as liver transplantation and hepatectomy may help cure hepatocellular carcinoma diagnosed in its early stages. Advanced stages of the disease have a poor prognosis and a high refractoriness because of drug resistance. Sorafenib is a tyrosine kinase inhibitor that has been approved for the systemic treatment of patients with advanced HCC. However, the efficacy of this medication has shown a limited prolongation of the overall survival in patients with advanced HCC. The mechanism of resistance to sorafenib in advanced HCC is poorly understood. The TCGA analysis of the HCC patient cohort shows an elevated expression of YBX1 in tumors as compared to normal. The expression is also correlated with progression, metastasis, and poor survival of HCC patients. Transcription factor YBX1 is involved in the regulation of drug resistance in different cancers, especially breast and colorectal cancers. Our lab is interested in understanding if YBX1 is involved in sorafenib resistance in HCC. Different HCC cell lines procured from ATCC SK-Hep1, Huh-7luc, and C3A, were analyzed for their IC50 values for sorafenib using MTT assays, at different time points. SK-Hep1 showed 8.780µM and 8.174µM, HepG2luc 4.659µM and 3.596µM at 48 and 72 hrs respectively. Overexpression of YBX1 in the SK-Hep1 cell line increased the cell survival and IC50 value of sorafenib at 48 and 72 hrs at 13.19µM and 10.64µM respectively. This supports the hypothesis which demonstrates the role of YBX1 in mediating resistance to sorafenib in advanced HCC. YBX1 could be a major therapeutic target and by inhibiting its action could improve the sensitivity of advanced HCC to sorafenib. We have developed YBX1 overexpressing and knockdown (GFP expressing) cell lines, FACS sorted and Puromycin selected for further mechanistic studies. We are also further developing the sorafenib-resistant HCC cell lines to understand the mechanism underlying sorafenib resistance in HCC. Protein and RNA analyses revealed a relative increase in YBX1 overexpression in sorafenib-resistant cell lines as compared to its parental cell lines. Understanding the mechanism of YBX1-induced drug resistance to sorafenib and its associated signaling pathways could help improve the sensitivity of advanced hepatocellular carcinoma to treatment by sorafenib and thus improve the livelihood of its patients. Citation Format: Dennis Kwabiah, Ana AyalaPazzi, Kyle Doxtater, Sophia Leslie, Manish K. Tripathi. Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines [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 3284.

Abstract 3757: The development of MAGMAS as a potential therapeutic target in temozolomide-resistant glioma

Abstract Glioblastoma (GBM) is one of the most aggressive types of brain cancer and remains a significant challenge for drug development in oncology today. Temozolomide (TMZ) is the standard of care for GBM, and resistance to this drug is modulated partly by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Clinical studies demonstrated that low MGMT expression in GBM enhances sensitivity to TMZ and prolongs survival for patients. However, recent clinical studies indicate MGMT is a discordant biomarker since the predictive value of MGMT methylation is relatively low and there is a lack of modalities to inhibit MGMT expression. Furthermore, glioma stem cells (GSC) are another contributing factor for resistance due to tumor heterogeneity, self-renewal capacity and activation of DNA repair responses to overcome the effect of radiation and TMZ treatment. These resistance mechanisms contribute to tumor recurrence and present major impediments for the development of effective treatments. MAGMAS, 13.8 kDa mitochondria protein, is a translocase of the inner mitochondrial membrane and regulates oxidative phosphorylation. Specifically, MAGMAS regulates the ATP stimulatory activity of DNAJC19 on mtHSP70 that drives the movement of peptides through the TIM23 complex into the mitochondrial matrix. Recent evidence suggests that MAGMAS can regulate oxidative phosphorylation via interactions with the respiratory complexes present in the inner mitochondrial membrane. Previously, our lab has shown MAGMAS overexpression in malignant glioma tissues of patients and murine intracranial xenografts. Additionally, pharmacological inhibition of MAGMAS with the small molecule BT9 promoted cytotoxicity and impaired respiratory functions in malignant glioma cells demonstrating that MAGMAS is a promising potential target for GBM patients. However, further studies are needed to investigate the role of MAGMAS across the spectrum from initial tumorigenesis to disease progression and to further determine how MAGMAS is implicated during the development of treatment resistance. This study is designed to investigate the role of MAGMAS in GSCs and their contribution to TMZ resistance. We observed temozolomide-resistant (TR) U251-TR and D54 MG-TR GBM cell lines expressed significantly higher levels of MAGMAS and several stem cell markers than their drug-sensitive (S) parental cell lines. Therefore, we hypothesized that silencing MAGMAS re-sensitizes TR GBM cell lines to TMZ. We found that MAGMAS knockdown in D54-MG and U251 S/TR cells in the presence of escalating TMZ concentrations, caused enhanced sensitivity to TMZ. Consistent with these results, inhibition of MAGMAS with the novel BT9 inhibitor enhances the TMZ sensitivity in the TMZ-resistant cell lines. Additional studies of patient-derived cell lines are being utilized further to validate MAGMAS as it is an attractive target in TMZ-resistant GBM patients. Citation Format: Jennifer D. Tran, Javier J. Lepe, Maria C. Kenney, Bhaskar C. Das, Daniela A. Bota. The development of MAGMAS as a potential therapeutic target in temozolomide-resistant glioma [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 3757.

Abstract 6916: InnoPanelTMand InnoPediaTM: resources of engineered cell lines and xenografts for drug discovery

Abstract The Development of precision medicine relies on well-characterized experimental models that carry naive, induced, or engineered genomic and epigenetic features targetable by certain treatments. Patient-derived xenografts (PDX) and cell lines can serve as such in vivo and in vitro models by harboring abnormalities such as somatic and driver mutations, dysregulated gene expression and silencing, microsatellite instability, gene fusions, copy number amplification and loss of heterozygosity. In addition, cell lines can also be engineered to introduce sometimes rare-occurring abnormalities that the engineered cell line shares an identical genetic background with its parental cell line, thus providing a clearly contrasting pair to evaluate treatment specificity. With the advancement of disease research and drug discovery, existing cell lines can no longer meet the needs of various types of drug discovery. To evaluate drug efficacy more quickly and accurately, Kyinno has constructed and identified more than 4,000 engineered cell lines, including overexpressed cell lines constructed through transposon systems or lentivirus infection, gene-edited cell lines constructed through CRISPR-Cas9 technology, and reporter cell lines targeting multiple signaling pathways and membrane receptors. Besides, InnoPanelTM, a panel of over 1000 engineered cell lines was established and characterized to cover a wide range of heavily targeted genes such as CLDN18.1, CLDN18.2, LAG3, OX40, PD-L1, PD-L2, and TIM3. Cell line families were also developed for genes with multiple known driver mutations. All cell lines are annotated with background information, engineering details, functional characterization, and efficacy studies. Furthermore, we developed xenografts for several engineered cell lines and showed that the combination of cell lines and xenografts can accelerate drug development. Additionally, all of this information can be accessed in our free database, InnoPediaTM. The database also offers browsing, searching, and analytical capabilities for over 600 commonly used tumor cell lines and thousands of engineered cell lines with essential genomic and pharmacological data, cell line efficacy data for nearly 2000 common anti-tumor drugs, and comprehensive details for over 100 standard antibodies. Our engineered cell lines are now registered in the online cell line database Cellosaurus, which is available on the ExPASy server. Citation Format: Siyu Li, Chang Liu, Na Wang, Yuqing Hu, Jinying Ning, Feng Hao. InnoPanelTMand InnoPediaTM: resources of engineered cell lines and xenografts for drug discovery [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 6916.

Abstract 4301: Increasing the therapeutic vulnerability of heterogenous cell phenotypes within prostate cancer

Abstract Phenotype heterogeneity is a hallmark of prostate adenocarcinoma and is a major source of recurrent disease. We tested pre-clinical therapies to eradicate phenotypically distinct cell populations derived from the parental DU145WT prostate cancer cell line. The derived cell lines are called DU145AA, DU145J7, and DU145G6. The DU145AA variant is a non-aggressive cell population while the DU145J7 and DU145G6 are aggressive phenotypes that invade through muscle and metastasize to bone. Flow cytometry analysis documented the phenotypic plasticity of the DU145 cell variants since DU145 aggressive lines expressed 1.3-fold increases in matrix metalloprotease 14 surface expression and a 2-fold increase in tri-methyl Histone H3 at lysine 27 as compared to the DU145WT parent line. The DU145AA non-aggressive line expressed an epithelial cell-cell lineage phenotype with up to 2-fold increases in E-cadherin, claudin 4, claudin 7, and a unique cytokeratin 6A expression and a 2-fold loss of Zinc finger E-box-binding homeobox 1, EVL, and Kindlin 2. A collaboration with the National Center for Advancing Translational Science (NCATS) resulted in the identification of pharmacological agents active in eradicating DU145J7 and DU145G6 cells as compared to the DU145WT population. The agents were selected from a quantitative high-throughput screen of 10,677 combined investigative and approved anti-cancer agents using the Cell Titer-Glo Luminescent Cell Viability assay. We independently validated 31 compounds from the assay that have 2-fold, or greater, differences in concentration to inhibit 50% cell growth (IC50) between the wild-type and aggressive variants of DU145 cells. The top 10 hits were tested for the efficacy of different combinations to eradicate defined admixtures of aggressive and non-aggressive phenotypes, including the DU145AA cells, to model tumor heterogeneity. Tested compounds with differential sensitivity between aggressive and non-aggressive cells were those inhibiting histone deacetylase (vorinostat, fimepinostat, and pracinostat), proteasomes (bortezomib, delanzomib, ixazomib), topoisomerases (gimatecan and daunorubicin), and other compounds identified independently by us targeting nicotinamide phosphoribosyl transferase (FK866) and DNA (bleomycin). Using admixtures of tumor cell phenotypes, we determined optimal combinations of compounds that would selectively eradicate the sub-populations. The results suggest that determination of phenotypes within tumor populations may eliminate the heterogenous tumor and prevent recurrent disease. Funding was provided by the University of Arizona Cancer Center (NCI-P30 CA23074 and NCI-R01 CA159406) and by the Partnership in Native American Cancer Prevention at the University of Arizona (U54CA143924) and Northern Arizona University (U54CA143925). Collaborators at NCATS were supported by the intramural research program. Citation Format: Allan I. Paxson, Loren H. Chang, Kendra D. Marr, Jaime M. Gard, Colin S. Nelson, Abhijeet Kapoor, William L. Harryman, Juan J. Marugan, Mark J. Henderson, Tino W. Sanchez, Anne E. Cress. Increasing the therapeutic vulnerability of heterogenous cell phenotypes within 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 4301.