Growth Of Cell Lines Research Articles

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [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 138.

Abstract 4441: The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells

Abstract Dysregulation of epigenetic factors is a key component of tumorigenesis. BMI1, a member of the polycomb repressor complex 1 (PRC1), is an oncogenic factor studied in many types of cancer, including pancreatic ductal adenocarcinoma (PDAC). PDAC is the third most common cause of cancer-related death in the United States and investigation of the biology involved in transformation is critical for improving outcomes in this devastating disease. In PDAC, BMI1 is required for initiation of pancreatic cancer in mice and enhances in vitro growth of human and murine tumor cell lines. CRISPR-mediated knock out of BMI1 results in reduced subcutaneous and orthotopic tumor growth in mice and decreased expression of genes related to metabolism, specifically glycolysis, and cell proliferation. BMI1 has also been implicated in regulation of metabolism in other epithelial tumor types, including ovarian cancer. Given these preliminary findings, we hypothesized that loss of BMI1 in multiple human PDAC cell lines would result in altered metabolic activity, reducing the growth and tumorigenic properties of these cells. We demonstrated that BMI1 expression is higher in PDAC cell lines compared to normal pancreatic epithelial cells and normal human donor pancreatic tissue. Following CRISPR-mediated BMI1 knock out in human PDAC cell lines, we investigated the alterations to metabolism using metabolic flux analysis. Loss of BMI1 resulted in lower basal and compensatory rates of glycolysis and increased oxygen consumption rates compared to wild-type cells. Metabolomic analysis demonstrated reduction in all the enzymes involved in the glycolysis pathway with loss of BMI1. Cell growth was reduced by loss of BMI1 in vitro, and orthotopic injection into mice of human cells with BMI1 knock out resulted in decreased tumor size compared to wild-type, recapitulating these findings in vivo. Together, these data identify a role of BMI1 in promotion of tumor growth through regulation of metabolism in pancreatic cancer. Experiments are ongoing to determine the underlying mechanism of this regulation and implications on therapeutic targeting with BMI1/PRC1 complex inhibitors in conjunction with traditional chemotherapy. Citation Format: Jamie N. Mills, Dominik Awad, Heather K. Schofield, Joyce K. Thompson, Hannah Watkoske, Damien Sutton, Nicholas Nedzesky, Donovan Drouillard, Zeribe Nwosu, Carlos Espinoza, Yaqing Zhang, Annachiara Del Vecchio, Christopher J. Halbrook, Marina Pasca di Magliano, Costas A. Lyssiotis, Filip Bednar. The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4441.

Abstract 383: Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment

Abstract Background: Mitophagy is a pivotal cellular process crucial for maintaining homeostasis in hypoxic conditions, selectively eliminating dysfunctional mitochondria through autophagy. This process is implicated in various diseases, including cancer. Non-Small Cell Lung Cancer (NSCLC), known for its prevalence and aggressiveness, often encounters hypoxic microenvironments. Mic19, a core protein of the mitochondrial cristae membrane, plays vital roles in maintaining mitochondrial architecture and function. However, its involvement in mitophagy in hypoxic NSCLC and the underlying molecular mechanisms remain inadequately explored. Methods: NSCLC cells were cultured under conditions with 1% O2 oxygen concentration to simulate a hypoxic microenvironment. Protein expression levels of Mic19 were analyzed using Western Blot and immunofluorescence (IF). Cellular proliferation and metastasis capability were assessed through CCK8 and transwell experiments. Immunoprecipitation experiments were conducted to validate the interaction between Mic19 and key mitophagy regulatory proteins. IF, confocal microscopy, ATP measurement, oxygen consumption rate (OCR), mitochondrial membrane potential, and transmission electron microscopy were employed to assess the formation of mitochondrial cristae and the level of mitophagy. Results: In hypoxic conditions, Mic19 exhibited up-regulation in NSCLC cells. Subsequent in vitro experiments confirmed Mic19's pivotal role in promoting the growth and metastasis of lung cancer cell lines. Through clinical data analysis, Mic19 emerged as an independent prognostic indicator for NSCLC. Inhibition of Mic19 expression exacerbated the disruption of mitochondrial morphology and structure in lung cancer cells, accompanied by the loss of mitochondrial cristae. Notably, Mic19 expression closely correlated with ATP and OCR production in tumor cells. Assessment of mitochondrial membrane potential and transmission electron microscopy revealed Mic19 as a critical downstream target of hypoxia, mediating mitophagy. Overall, these findings not only underscore the significance of Mic19 in regulating mitochondrial dynamics but also highlight its multifaceted role in tumor progression, positioning Mic19 as a promising therapeutic target and prognostic biomarker for NSCLC. Conclusion: This study provides novel insights into the mechanistic understanding of hypoxia-induced mitophagy in NSCLC, emphasizing the regulatory role of Mic19. Mic19 emerges as a potential therapeutic target for future interventions in NSCLC patients. Citation Format: Hua Huang, Chen Ding, Zhanrui Zhang, Di Wu, Chen Chen, Yongwen Li, Hongyu Liu, Jun Chen. Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment [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 383.

Abstract 7496: The piritramide-based perioperative analgesia can affect the cancer dissemination in colorectal cancer patients

Abstract Introduction: Colorectal cancer (CRC) stands as a prominent contributor to global cancer-related mortality. Post-surgical management of CRC patients often involves the administration of opioid analgesics. These analgesics operate through opioid and cannabinoid receptors, pathways implicated in tumor progression and metastasis, potentially impacting patient survival adversely. This investigation delves into the influence of piritramide and morphine on cancer dissemination, encompassing in vitro and in vivo assessments, cytotoxicity analyses, and elucidation of their mode of action. Methods: The presence of circulating tumor cells (CTCs) in the blood of 131 CRC patients was quantitatively assessed using the real-time polymerase chain reaction (qPCR) method. Cytotoxic effects of morphine and piritramide were evaluated via colony-forming assays (CFA) utilizing HCT116 and HT29 cell lines. The impact of piritramide and morphine on opioid receptor activity was gauged through transfected reporter cell lines and fluorescence imaging plater reader (FLIPR) calcium assays. In vivo models involving subcutaneous and intrasplenic administration in SCID and BALB mice, respectively, utilizing HCT116 and HT29 cell lines, were conducted. Morphological profiling, employing the cell painting assay, facilitated high-content image-based analysis on both cell lines. Results: Perioperative analgesia with piritramide exhibited a tendency towards longer time to recurrence (TTR) survival in CRC patients (HR=1.8, p=0.104). Conversely, morphine-treated patients demonstrated a twofold increase in TTR events (HR=4.7, p=0.011). Piritramide, but not morphine, significantly inhibited colony growth in HCT116 and HT-29 cell lines in the CFA, consistent with cytotoxicity test results. Functional assays revealed agonistic effects of both piritramide and morphine on opioid receptor kappa 1 (OPRK1) and mu 1 (OPRM1), with piritramide exhibiting greater potency as an OPRK1 agonist. In the SCID mice model, subcutaneous application of HT29 cell lines resulted in significantly reduced tumor growth in piritramide-treated mice. In the intrasplenic model utilizing BALB mice, mice treated with piritramide exhibited diminished tumor growth and a reduction in both the number and size of metastases. Comparative analysis with reference compounds revealed a distinct phenotypic profile of piritramide affecting the endoplasmic reticulum. Conclusion: Piritramide, as opposed to morphine, demonstrated cytotoxic effects on both in vitro and in vivo CRC cell lines, resulting in reduced tumor growth and dissemination. Perioperative analgesia with piritramide exhibited potential in improving time to recurrence survival in CRC patients. Acknowledgement: This study received support from the European Union - Next Generation EU (LX22NPO5102) and Palacky University Olomouc (LF 2023_006). Citation Format: Josef Srovnal, Emil Berta, Monika Vidlarova, Alona Rehulkova, Katerina Jecmenova, Miroslav Popper, Alzbeta Srovnalova, Petr Prasil, Pavel Skalicky, Tomas Gabrhelik, Jan Maca, Pavel Michalek, Marian Hajduch. The piritramide-based perioperative analgesia can affect the cancer dissemination in colorectal 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 7496.

Abstract 4221: Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models

Abstract The monocarboxylic acid transporter 4 (Mct-4), a downstream biomarker of hypoxia inducing factor (HIF)-1α, is involved in the cellular response to hypoxia, as indicated by the hypoxic response element in its promoter region. Using a tumorsphere assay as an in vitro 3-dimensional (3D) model generated using 384-well ultra-low attachment (ULA) plates for cell proliferation analysis using a plate-based image cytometer, we identify a hypoxic response in the tumorsphere model that is distinct from that of cells grown under 2-dimensional (2D) normoxic conditions and demonstrate a key role for Mct-4 in enabling 3D growth. The tumorsphere model yields evidence of an essential role for Mct-4 in multiple cell lines which were genetically modified to underexpress and overexpress Mct-4, evidence not apparent in a standard 2D model of growth in the same cell lines. In addition, we identify the effects of overexpressing Mct-4 in cancer cell migration using a transwell chamber assay. We also show that the response to hypoxia may be circumvented by transfection with a CMV promoter driven Mct-4, which confers constitutive 3D growth, wherein tumorsphere growth inhibition by small molecule HIF-1α inhibitors is mitigated. Finally, we demonstrate quantifiable gene/protein expression differences between 2D and 3D cancer models based on the normoxic and hypoxic conditions. Therefore, the tumorsphere 3D model generated using 384-well ULA plates in combination with high-throughput image cytometer is demonstrated to provide a convenient, robust, and reproducible tool and method for the elucidation of mechanisms of action underlying tumor growth and migration in the hypoxic tumor microenvironment. Citation Format: James McDonald, Leo Li-Ying Chan, Sarah Kessel, Bo Lin, Paul Weingarten. Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4221.

Abstract 431: Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer

Abstract Background & Hypothesis: Treatments for triple-negative breast cancer (TNBC) are limited to chemotherapy, PARP inhibitors and immunotherapies. Tumor metabolism seeks to identify metabolic pathways preferentially utilized by cancer. Malic enzyme 2 (ME2) has a role in the malate-aspartate shuttle and pyruvate and NADPH production, contributing to the redox balance and metabolite synthesis. We demonstrated that ME2 knockdown inhibits growth of TNBC cells with upregulated serine biosynthesis pathway. We hypothesized that ME2 knock-down (kd) would be growth inhibitory in vivo. Methods: We used a non-tumorigenic control (MCF10a) and TNBC lines (BT20, MDA-MB-468, HCC1806, HCC70, Hs578T, SUM149). Nude mice received mammary fat pad xenografts of MDA-MB-468, to assess the impact of ME2-shRNA on growth. ME2 relevant metabolite profiling was achieved through the Promega Metabolite-Glo assay, measuring NAD, NADH, NADP, NADPH, Malate, and Glutamate. Cellular proliferation was quantified via the BioTek BioSpa 8 system and Promega CellTiter Glo. Comprehensive profiling of metabolic genes was achieved with the Nanostring nCounter across all cell lines. The potential therapeutic efficacy of NPD-389, MDSA, disodium embonate and doxorubicin was evaluated, alone or in combination, through targeted drug assays. Metabolic flux analyses, specifically mitochondrial and glycolytic function, were conducted using the Seahorse XF96, contrasting ME2-shRNA modified and control cells. Results: ME2 kd resulted in reduced proliferation in several TNBC cell lines to varying degrees, in contrast to control MCF10a, and to Hs578T. In response to the loss of ME2, several cell lines upregulated serine biosynthesis genes, including PSAT1. MDA-MB-468 ME2 kd cells developed significantly smaller tumors in a nude mouse xenograft model. Increased malate levels, but reduced NADH and NADPH, were detected in TNBC cells with inhibited growth post-ME2 kd, but not in MCF10a or Hs578T cells. Among the tested ME2 inhibitors, NPD-389 had superior efficacy vs. MDSA and disodium embonate. Metabolic flux analysis showed compromised mitochondrial and glycolytic processes in the TNBC cell lines exhibiting growth inhibition upon ME2 kd. Conclusions: ME2 knockdown selectively impairs cell proliferation and tumor growth in TNBC cell lines, validating its potential as a therapeutic target. The differential metabolic response, including altered malate and NADH/NADPH levels, confirms ME2's importance in TNBC metabolism. NPD-389 effectively reduced growth in ME2-sensitive lines, meriting further investigation. These findings support targeting ME2 in as a novel treatment strategy. Citation Format: Mark D. Slayton, Zackariah A. Farah, Jin Heon Jeon, Abhinav Achreja, Ben Krinkel, Brisilda Nilaj, Yi-Hsien Eu, Justin Sung, Alyssa Rosenfeld, Alisa Lui, Mason Collard, Liwei Bao, Xu Cheng, Celina Kleer, Kerry Loomes, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent 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 431.

Abstract 634: Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD) is a multifaceted and genetically diverse cancer, shaped by a myriad of oncogenic and tumor suppressive events. Through recent clinical and in vitro investigations, we have discovered that the TBX2 subfamily of genes, encompassing TBX2, TBX3, TBX4, and TBX5, are linked to LUAD progression. Specifically, we have observed marked downregulation of their expression in clinical LUAD specimens, and reduced growth in LUAD cell lines when the subfamily is re-activated- hinting at a potential tumor suppressive function. Paradoxically, the role of TBX2 subfamily genes in other cancer types remains enigmatic, with varying reports of both oncogenic and tumor suppressive effects. In this study, we harnessed the power of Tumor Barcoding with Ultradeep Barcode Sequencing technology (Tuba-seq) and CRISPR/Cas9-mediated genome editing to systematically investigate the effects of knocking-out Tbx genes and associated partners EGR1, CHD2, A20 and ATF3 in LUAD preclinical models. We investigated their effects in both normal lung epithelium and Ras-driven lung tumors via in situ gene editing and assessed tumor burden via histology and targeted DNA sequencing of tumor barcodes. TBX2 subfamily gene losses appear to have a profound effect on tumor initiation and early growth, which somewhat attenuates with progression. In Ras-driven tumors, we found that these genes are moderately tumor suppressive when measuring mean growth rate, although these effects (sans Egr1 loss) are more pronounced at 6 weeks of growth than at 20 weeks. Strikingly, however, we observed an unprecedented increase in total tumor burden when deleting these genes in genomically-normal (Kras w.t.) cells, including 4-20+ fold increase in Tbx2, Tbx3, Tbx4, and Tnfaip3-deficient cell lineages 20-week following transduction. Increases in total lineage burden of this magnitude are typically only seen for frequently-deleted hallmark tumor suppressor genes such as Tp53, Stk11, or Pten. This increased burden, however, seems to primarily affect early-stage progression, as no appreciable increases in mean lineage size relative to w.t. lineages are observed after 40 weeks of growth. Our study reveals the context-specific functions of the TBX2 subfamily while introducing the first in vivo model for TBX2-driven LUADs. This model facilitates the development of targeted therapeutic strategies and encourages accurate tumor modeling across various progression stages and diverse genetic backgrounds. Citation Format: Athar Khalil, Jaff Maltas, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher McFarland. Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma [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 634.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [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 4250.

Abstract 4747: Novel targets for BRAFV600E and BRAFV600ERAC1P29S drug resistant melanoma

Abstract The most common mutational drivers in malignant melanoma are the oncogene BRAFV600E/K (60%), NRASQ61L/R (30%) and the RHO GTPase RAC1P29S/L (9%). BRAFV600E/K melanoma standard of care comprises the use of checkpoint inhibitors (CPIs) such as anti-PD1 or anti-CTLA-4 in combination with targeted therapies against BRAF and MEK kinases. BRAF mutant melanomas develop intrinsic, acquired, or after treatment resistance to BRAF and MEK inhibitors : around half of targeted therapy combination treatments with anti-PD1 and 70% with anti-CTLA-4 will be unsuccessful due to melanoma intrinsic resistance. Among other causes, intrinsic drug resistance can be associated with additional genome mutations, such as the activating Rac1P29S mutation present in 6% of patients with BRAF positive melanoma. A deeper understanding on the biology, progression, and tumor microenvironment of drug resistant melanomas is needed to aid in the search of novel targets and to increase the effectivity of proposed treatments. We generated 3 sets of isogenic BRAFV600E and BRAFV600E/RAC1P29S cell lines by transfecting 501mel, 451Lu and YUMM1.7 cell lines with Rac1P29S. We then exposed them for 3 months to increasing concentrations of PLX4720 to generate drug resistant cell lines (DR). Rac1 P29S -transfected and PLX4720 resistant cell lines had an increase in the Mek/Erk signaling pathway. A differential analysis using a kinome and an epigenome drug screening library in the 501mel cell lines (501mel, 501mel Rac1P29S, 501mel-DR, 501mel Rac1P29S -DR) showed that BRAFV600E/RAC1P29S and the DR melanoma cell lines growth can be inhibited with mTor, PI3K, cdk9 and BRD4 inhibitors. BRD4 is an epigenetic regulator that recruits P-TEFb to stimulate RNA polymerase II elongation. BRD4 also interacts with SMAD3 and CDK9 to regulate transcription through eIF4G increasing oncogenic c-Myc and increasing Mek/Erk signaling pathways. We tested 501mel, 451Lu and YUMM1.7 isogenic cell line series against Rapamycin to confirm mTORC1 as an effective target. Drugs that target different subunits of the eIF4F complex (CR-1-31-B, Ribavirin and Briciclib) also decrease BRAFV600E/RAC1P29S and the DR melanoma cell lines growth specifically. Furthermore, silencing of eIF4A1 and Raptor genes reduces cell growth in the BRAFV600E/RAC1P29S, and the DR cells when compared to the parental cell lines. Silencing Rictor had no effect on cell growth on any cells. When tested in a syngraft model, CR-1-31-B and rapamycin reduced tumor size when using YUMM1.7, YUMM1.7- RAC1P29S and YUMM1.7 RAC1P29S-DR cell lines. Next, we sought to investigate the effect of inhibiting mTOR and eiF4F in the PD-1/PD-L1 axis in tumor cells and tumor associated dendritic cells and macrophages, and how targeting these modifies the tumor microenvironment. Citation Format: Cristina Uribe-Alvarez, Daniela Araiza-Olivera, Alexa Cannon, Jonathan Chernoff. Novel targets for BRAFV600E and BRAFV600ERAC1P29S drug resistant 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 4747.

Abstract 1224: 3D models of chemotherapy- and PARPi-resistant ovarian cancer indicate essential roles for JAK/STAT signaling in mediating drug resistance

Abstract Ovarian cancer is the deadliest gynecological cancer with a low 5-year overall survival rate that affects over 300,000 women globally each year. Given lack of routine screening procedures, ovarian cancer patients are frequently diagnosed with late-stage disease that rapidly progress despite initial responses to front line therapy. PARPi and VEGFi targeted therapies have improved progression free survival in small subsets of patients but have largely failed to improve overall survival. Since the robust uptake of PARPis in the front line setting, resistance to this class of drugs is a growing problem. The literature is filled with many novel treatment approaches, but the vast majority fail to gain traction in pre-clinical models and early phase trials. A contributing factor to these disappointments relates to the prevalent use of 2D monoculture model systems that do not recapitulate the tumor microenvironment. To address this issue, we utilized the Rastrum 3D cell printer to grow ovarian cancer cells and PDX models in bio-functional matrices which provide optimal stiffness and extracellular matrix components specific to the ovarian tumor microenvironment. We found that ovarian cancer cells grown under these conditions exhibit dramatic up-regulation of the JAK/STAT signaling pathway, including autocrine/paracrine signaling factors, which correlated with increased sensitivity to JAK/STAT inhibitors. This affect was lost when STAT1 and STAT3 knockout cell lines were grown in this 3D environment. Ovarian tumors are known to be rich in cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). We incorporated primary CAFs into this 3D culture system and found they could promote ovarian cancer cell growth only in cancer cells with intact JAK/STAT signaling further indicating the essential role of this pathway in mediating tumor progression. Co-culture with M2 macrophages was also found to induce ovarian cancer cell growth, particularly in resistant models. Conversely, co-culture with M1 macrophages inhibited treatment naïve ovarian cancer cell growth but not resistant cell line growth. Single-cell RNAseq of the complex 3D cultures elucidated profound differences in the cross-talk between these cell types that is dependent on cancer cell sensitivity to standard-of-care agents. Finally, the addition of JAK/STAT inhibitors was found to substantially alter this cross-talk between cell types and inhibit cancer cell viability. These data further implicate the importance of JAK/STAT signaling, specifically in clinically relevant 3D models, in supporting ovarian cancer progression and response to chemotherapy and PARPi treatment. Ongoing studies are aimed at confirming these findings in pre-clinical mouse models and to elucidate the most effective JAK/STAT inhibitors in vivo with the goal of informing the design of future clinical trials. Citation Format: Esther Rodman, Xiyin Wang, Hannah Smith, Xiaonan Hou, Scott Kaufmann, John Weroha, John Hawse. 3D models of chemotherapy- and PARPi-resistant ovarian cancer indicate essential roles for JAK/STAT signaling in mediating 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 1224.

Abstract 5960: Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS

Abstract Alveolar rhabdomyosarcoma (aRMS), characterized by poor overall survival and limited advancements in therapy over the past four decades, poses a great challenge in childhood cancer treatment. More than 60% of high-risk aRMS is driven by the oncofusion protein PAX3-FOXO1. The direct targeting of transcription factor fusions, however, is quite difficult. Given that PAX3-FOXO1 is reported to drive disease by controlling transcriptional programs to maintain an undifferentiated myoblastic state, an alternative approach would be to target the transcriptional machinery recruited by the fusion. To identify potential novel targets in aRMS, we utilized the Broad Institute’s Cancer Dependency Map dataset, a collection of genome-wide CRISPR-Cas9 screens performed in over 1000 cancer cell line models. We focused on druggable dependencies that are selective to fusion-positive aRMS and intersected this data with PAX3-FOXO1-associated transcriptional complexes. Among these candidates, we identified CDK8 as a top selective dependency. Importantly, other components of the CDK8 kinase module in the mediator complex, such as CCNC, also scored as a strong selective dependency in fusion-positive aRMS. CDK8 expression is notably elevated in aRMS tumors compared to other pediatric tumors, and aRMS cell lines exhibit significantly higher CDK8 protein levels than non-transformed primary skeletal muscle cells. We next validated these in silico results through the application of shRNA knockdown, CRISPR knockout, and the use of CDK8 kinase inhibitory small molecules, including BI-1347, SEL-120-34A, and JH-XII-178, which demonstrated on-target activity as assessed by STAT1Ser727 phosphorylation. Both genetic loss of CDK8 function and kinase inhibition impaired aRMS cell line growth in vitro. In vivo studies demonstrated a significant inhibition of xenograft tumor growth with both doxycycline inducible CDK8-directed shRNAs and with pharmacologic inhibition of CDK8 with SEL120-34A treatment. To understand the mechanisms by which CDK8 inhibition impairs aRMS growth, we evaluated the transcriptional consequences of CDK8 inhibition by RNA-sequencing in aRMS, as well as the chromatin accessibility by ATAC-sequencing. We discovered that loss of CDK8 led to gain of chromatin accessibility and the upregulation of hallmark genes involved in myogenesis, such as MYH3, MYOG, MEF2C, and MYBPH, as well as alteration of PAX3-FOXO1 gene expression programs. We next validated this differentiation phenotype both in vitro through detection of increased myogenin staining by immunofluorescence and in vivo by visualization of myofibrils in the xenograft tumors and increased myoglobin quantified by qRT-PCR after CDK8 inhibition. In conclusion, this work identifies CDK8 as a novel and readily translatable therapeutic target in fusion-positive aRMS laying a robust foundation for further mechanistic and preclinical studies. Citation Format: Susu Zhang, Clare Malone, Brian Guedes, Kathleen Engel, Marissa Just, Kenneth Ross, Assil Fahs, Giulia Digiovanni, Diyana Granum, Rex Bentley, Leen Barbar, Christian Cerda-Smith, Elizabeth Mendes, Ozgun Erdogan, Alexander Kovach, Kristianne Oristian, Seth Zimmerman, Jack F. Shern, Kris Wood, Chris Counter, Corinne M. Linardic, Kimberly Stegmaier. Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS [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 5960.

Abstract 4724: Optimizing 500 pooled PRISM cell lines for a 10-day assay to elucidate viability effects and mechanism of action for slower-acting therapeutics

Abstract PRISM (Profiling Relative Inhibition Simultaneously in Mixtures), using DNA-barcoded cell lines cultured as ~25 cell line pools, has been an effective drug discovery screening platform to rapidly test large numbers of potential therapeutics on over 900 different cancer cell lines. However, the conventional 5-day assay may be too short to offer the resolution needed to identify the full cellular response for slower-acting therapeutics such as those directed against epigenetic pathways. Here, we describe development of the PRISM 10-day extended assay with ~500 adherent solid tumor cancer cell lines to overcome this challenge. We found that the ranges of cell line growth rates within the standard PRISM pools were too large and not amenable for a long-term assay beyond 6 days—after 10 days of growth, slower cell lines dropped below the limit of barcode detection as faster cell lines overtook the pools. We therefore regenerated pools to reduce the range of growth rates within a pool. We show that this re-pooling, coupled with seeding density optimization, enables recovery of > 98% of cell lines at day 10 in vehicle-treated wells, with acceptable data variability. The average estimated number of cell doublings across our cell lines, 7, is significantly greater than the number of doublings in the PRISM standard 5-day assay (~3 doublings). Importantly, we observe greater drug effects on day 10 versus day 6 when cells are treated with slow-acting DNA methyltransferase inhibitors such as decitabine; this also results in increased sensitivity to identify cellular biomarkers underlying differential response. In summary, we have successfully engineered a PRISM 10-day assay in ~500 adherent cell lines, where the extended duration can be beneficial in elucidating the effects and mechanism of action for epigenetic drug candidates. Citation Format: Melissa Ronan, Blanche C. Ip, Ellen Nguyen, Ursula Widocki, Tenzin Sangpo, Alvin Kalathungal, Nashielli Diaz-Gallegos, Anthony Fazio, Aydin Golabi, Maha Naim, Ashish Bino George, YuhJong Liu, Mustafa Kocak, Rishi Puram, Matthew G. Rees, Jennifer Roth. Optimizing 500 pooled PRISM cell lines for a 10-day assay to elucidate viability effects and mechanism of action for slower-acting therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4724.

Abstract 4658: Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with a dismal 5-year survival rate, highlighting the urgent need to improve drug response and overcome primary or acquired (secondary) resistance to conventional chemotherapy. Glycogen synthase kinase 3β (GSK-3β) is a target in PDAC and the GSK-3 inhibitor, elraglusib, has been shown to improve chemosensitivity of PDAC and survival in preclinical cancer models. In addition, data from phase II clinical trials in patients with previously untreated metastatic pancreatic cancer is demonstrating preliminary evidence of clinical benefit in patients receiving a combination of gemcitabine/nab-paclitaxel. While this early evidence is highly encouraging, the biological determinants of elraglusib response are unknown. Using genome wide CRISPR/Cas9 library screening, we identify aurora kinase A (AURKA), an oncogenic kinase with major roles in mitosis, as a critical regulator for elraglusib sensitivity. Elraglusib treatment activates AURKA by inducing a G2/M cell cycle arrest. We show that knocking out AURKA reduces the phosphorylation of polo like kinase 1 (PLK1), arresting cells in mitosis, and leading to mitotic catastrophe and significant apoptosis after elraglusib treatment. Strikingly, the AURKA inhibitor, alisertib, works synergistically with elraglusib to inhibit PDAC cell line growth and induce apoptosis. In summary, our results demonstrate that targeting AURKA is an effective approach to sensitize PDAC tumors to elraglusib. Citation Format: Li Ding, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Daniel Schmitt, Andrew Mazar, Hu Li, Daniel Billadeau. Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4658.

Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

Melanoma is a type of skin cancer that originates in melanin-producing melanocytes. It is considered a multifactorial disease caused by both genetic and environmental factors, such as UV radiation. Dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) phosphorylates many substrates involved in signaling pathways, cell survival, cell cycle control, differentiation, and neuronal development. However, little is known about the cellular function of DYRK3, one of the five members of the DYRK family. Interestingly, it was observed that the expression of DYRK3, as well as p62 (a multifunctional signaling protein), is highly enhanced in most melanoma cell lines. This study aimed to investigate whether DYRK3 interacts with p62, and how this affects melanoma progression, particularly in melanoma cell lines. We found that DYRK3 directly phosphorylates p62 at the Ser-207 and Thr-269 residue. Phosphorylation at Thr-269 of p62 by DYRK3 increased the interaction of p62 with TRAF6, an already known activator of mTORC1 in the mTOR-involved signaling pathways. Moreover, the phosphorylation of p62 at Thr-269 promoted the activation of mTORC1. We also found that DYRK3-mediated phosphorylation of p62 at Thr-269 enhanced the growth of melanoma cell lines and melanoma progression. Conversely, DYRK3 knockdown or blockade of p62-T269 phosphorylation inhibited melanoma growth, colony formation, and cell migration. In conclusion, we demonstrated that DYRK3 phosphorylates p62, positively modulating the p62-TRAF6-mTORC1 pathway in melanoma cells. This finding suggests that DYRK3 suppression may be a novel therapy for preventing melanoma progression by regulating the mTORC1 pathway.

Novel 2-Aminobenzothiazole Derivatives: Docking, Synthesis, and Biological Evaluation as Anticancer Agents.

Sixteen novel 2-aminobenzothiazole compounds with different amines or substituted piperazine moieties were designed, synthesized, and tested using various methods. Potential interactions were assessed by docking new compounds in the adenosine triphosphate (ATP) binding domain of the PI3Kγ enzyme (PDB code: 7JWE) by nucleophilic substitution or solvent-free/neat fusion for docked compound synthesis. Final 2-aminobenzothiazole compounds were characterized by direct probe gas chromatography-mass spectrometry (GC-MS), proton (1H-NMR), carbon-13 (13C-NMR), and attenuated total reflectance-infrared Fourier transform infrared (ATR FT-IR). The synthesized compounds were investigated for anticancer activities on lung cancer (A549) and breast cancer (MCF-7) cell lines. The compounds' PI3Kγ inhibition was evaluated at a 100 μM concentration. 4-Nitroaniline and piperazine-4-nitroaniline combination in OMS5 and OMS14 reduced lung and breast cancer cell line growth. IC50 values for OMS5 and OMS14, the strongest compounds, ranged from 22.13 to 61.03 μM. OMS1 and OMS2 inhibited PI3Kγ at the highest rates (47 and 48%, respectively) at a 100 μM concentration. Results show that the PI3Kγ enzyme suppression is not the main mechanism behind these OMS5 and OMS14 anticancer effects. CDK2, Akt, mTOR, and p42/44 MAPK are affected. EGF receptor suppression matters. AKT1, AKT3, CDK1/cyclin B, PDK1 direct, PIK3CA E542 K/PIK3R1 (p110 α/p85 α), PIK3CD/PIK3R1 (p110 δ/p85 α), and PKN inhibition were measured to evaluate the possible mechanism of compound OMS14. PIK3CD/PIK3R1 (p110 δ/p85 α) is the most, with 65% inhibition, suggesting a possible mechanism of anticancer properties. Furthermore, the NCI 60-cell line inhibition demonstrates promising broad anticancer inhibition against numerous cancer cell lines of OMS5 and OMS14, which could be good lead compounds for future development.

Enoxacin fluoroquinolone antibacterial agent complexes with Pt(IV), Ru(III) and Ir(III) ions: Synthesis, spectroscopic, physicochemical characterizations and biological studies

The synthesis, physicochemical characterizations, and biological investigations on the chelation of the antibiotic medication enoxacin (EXN) with certain vital metal ions, such as platinum(IV), ruthenium(III), and iridium(III) are discussed in this study. These compounds structures were interpreted based on elemental analysis and spectral measurements (FTIR, 1H-NMR, UV-Vis electronic, and XRD). The findings show that there is same coordination pathway for the chelation behavior of enoxacin complexes: a monodentate manner. The Pt(IV), Ru(III), and Ir(III) complexes are coordinated through the N atom of the piperazine ring, which is an unusual mode of coordination for this class of compounds. The dissolved complexes in DMSO were found to have non-electrolyte nature based on their molar conductance measurements. The formula for these complexes is [Pt(EXN)3(H2O)2Cl] (1), [Ru(EXN)3(H2O)3] (2), and [Ir(EXN)3(H2O)3] (3). Using scanning electron microscope (SEM) analysis and X-ray powder diffraction (XRD), the nano-scale range of the Pt(IV), Ru(III), and Ir(III) complexes has been determined. Results of in vitro cytotoxicity were examined using MCF-7 cells (human breast cancer cell line) and HepG-2 cells (human hepatocellular carcinoma) that had been grown. The cytotoxic activity of the metal complexes demonstrated a cytotoxic effect against the growth of human breast and liver cancer cell lines.
 KEY WORDS: Enoxacin, Platinum(IV), Complexity, Spectral analyses, Biological assessments
 Bull. Chem. Soc. Ethiop. 2024, 38(3), 685-699. 
 DOI: https://dx.doi.org/10.4314/bcse.v38i3.11

Computational and in vitro analyses on synergistic effects of paclitaxel and thymoquinone in suppressing invasive breast cancer cells.

In the present experiment, we evaluated the impact of thymoquinone (TQ) and paclitaxel (PTX) treatment on MDA-MB-231 cell line growth inhibition via controlling apoptosis/autophagy. MDA-MB-231cells were exposed to PTX (0, 25, 50, 75, and 100 nM), TQ (0, 25, 50, 75, and 100 µM), and combinations for 48h. After the MTT assessment, dose-response curves and IC50 values were calculated, and the combination synergism was evaluated using the Compusyn software. Following the treatment with PTX, TQ, and combinations at IC50 doses, the expression of apoptosis and autophagy genes was assessed in cells. The GraphPad Prism program was used to analyze the data, and Tukey's test at p < 0.05 was then run. PTX, TQ, and their combinations inhibited MDA-MB-231cell proliferation and viability dose-dependently. TQ reduced the effective concentration (IC50) of PTX in co-treatment groups. PTX and TQ showed antagonistic effects when cell proliferation declined above 70%. Antagonistic effects shifted into additive and synergistic effects upon increasing PTX concentration, indicated by diminished cell proliferation below 70%. PTX-TQ co-treatment significantly enhanced P53 and BAX expression while reducing Bcl-2 expression. Also, their combination increased Beclin-1, ATG-5, and ATG-7 expression in treated cells. Effective concentrations of TQ and PTX had synergic effects and inhibited breast cancer cells via prompting apoptosis and autophagy in vitro.

Abstract B107: Elucidating the influence of long-chain fatty acids on ovarian cancer progression

Abstract Objective: High-fat diets have been associated with cancer progression. Yet, studies indicate that the effects can differ based on the type of fat involved, such as saturated versus unsaturated fatty acids. Uncovering the role of fatty acids may help guide nutrition and provide dietary therapy for cancer patients. In this study, we investigated the effect of common long-chain fatty acids, stearic acid (SA) and oleic acid (OA), on ovarian cancer (OC). Methods: We evaluated the cytotoxicity of SA and the effects of OA using IC50, a 72-hour proliferation assay, flow cytometry, and western blot. The concentration of fatty acids within the tumor was measured using liquid chromatography-mass spectrometry (LC/MS). CAY10566 was used to inhibit SCD1, the enzyme that converts SA to OA. In vivo, tumor growth was analyzed in human OC cell lines and patient-derived xenografts (PDX) using a mouse model fed with a normal diet (ND), an SA-rich diet, or an OA-rich diet combined with CAY10566. The SA-rich diet, which we specially created, had a stearic acid concentration of 33%. Results: SA displayed prominent cytotoxicity compared to other fatty acids. SA dose-dependently increased unfolded protein response (UPR) marker expression. Adding 1μM CAY10566 increased intracellular SA, decreased OA, and reduced IC50 to 20μM (p&amp;lt;0.05). CAY10566 significantly enhanced the cytotoxicity of SA, but the addition of exogenous OA canceled the increase in cell toxicity and UPR marker expression induced by SA (p&amp;lt;0.05). In vivo, tumor growth of OC cell lines and PDXs was significantly suppressed in the SA-rich with CAY10566 group compared to the ND group (p&amp;lt;0.05), while this suppression was nullified in the OA-rich with CAY10566 group. As confirmed by LC/MS, the stearic acid concentration significantly increased in the SA-rich group. Immunohistochemistry demonstrated that CHOP, gH2AX, and caspase-3 expression significantly increased in the SA-rich with CAY10566 group and dramatically decreased in the OA-rich with CAY10566 group (p&amp;lt;0.01). Conclusion: An ectopic supply of SA inhibits OC growth via UPR, while OA counteracts this effect. Creating a Stearate-rich/Oleate-limited environment through diet might affect tumor growth in vivo. Dietary content may greatly contribute to altering function in cancer. Citation Format: Jumpei Ogura, Koji Yamanoi, Mana Taki, Ryusuke Murakami, Ken Yamaguchi, Junzo Hamanishi, Masaki Mandai. Elucidating the influence of long-chain fatty acids on ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B107.

Anticancer Activities of Zinc Oxide Nanoparticles (ZnONPs) Synthesized from Mentha longifolia L. Leaf Extract

Recently, there has been a remarkable increase in cancer and cancer-related deaths. In this study, the impacts of zinc oxide nanoparticles (ZnONPs) produced from the aqueous leaf extract of Mentha longifolia L. (ML) on ovary adenocarcinoma (OVCAR-3), colorectal carcinoma (HCT-116), and healthy retinal pigment epithelial cell (RPE-1) lines were investigated. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed to discover its antiproliferative properties. As a result of the application of ML-ZnONPs on RPE-1, OVCAR-3, and HCT-116 cell lines at doses (µg/mL) of 250, 500, and 1000 for 24 hours, the viability rates (%) in the cell lines were 18.73-30.56, 21.98-28.76, and 27.27-40.93, respectively. In the 48-hour application, the viability rates (%) of the same cells were between 29.51-46.83, 32.49-40.81, and 46.82-44.37, respectively. The MTT test revealed that ML-ZnONPs strongly suppressed the growth of RPE-1, OVCAR-3, and HCT-116 cell lines. The test showed that the effect of dose increase and time on the viability of both cancer cell lines was negative.

Renin-Angiotensin System Inhibitors Suppress the Growth of Leukemia Cells.

The renin-angiotensin system (RAS) regulates blood pressure. The RAS is also related to cell growth, and its activation has been reported in various cancer cells. Therefore, we investigated the effects of RAS inhibitors on the in vitro growth of leukemia cell lines. THP-1, MV4-11, and TMD7 cells derived from acute myeloid leukemia, K-562 cells from chronic myeloid leukemia, and Jurkat and KOPT-K1 cells from T-lymphoblastic leukemia (T-ALL) with NOTCH1 mutations were used. We used four RAS inhibitors: the renin inhibitor aliskiren, angiotensin-converting enzyme 1 inhibitor captopril, angiotensin II type 1 receptor antagonist azilsartan, and angiotensin II type 2 receptor antagonist PD123319. Cells were cultured with the inhibitors and cell growth was assessed using a colorimetric assay. The expression of signaling proteins was assessed using immunoblotting. Treatment with aliskiren, azilsartan, or PD123319 suppressed the growth of all cell lines. Captopril treatment suppressed the growth of K-562, KOPT-K1, and MV4-11 cells. Flow cytometric analysis revealed that the growth suppression was due to the induction of apoptosis. Their suppressive effects on normal lymphocytes were milder than those on leukemia cells. Treatment with these inhibitors decreased MYC expression, induced caspase3 and PARP cleavage, and suppressed mTOR signaling. The treatment also suppressed NOTCH1 signaling in T-ALL cells. RAS inhibitors can be repurposed as molecular-targeted drugs for leukemia. However, the concentrations of the inhibitors were much higher than those in the plasma of patients with hypertension. Therefore, further investigation is required for their clinical use.