Parental Cell Line Research Articles

Abstract 1455: In vitro blood brain barrier model for evaluation of brain metastasis

Abstract Introduction: Brain metastasis significantly reduces the patient's quality of life and survival, but there are only a few choices of treatment for brain metastasis. The research for brain metastasis is difficult because of its complex and unique microenvironment, including blood-brain barrier (BBB). The BBB maintains strong cell adhesion of the vascular endothelial cells (ECs) to protect the brain parenchyma. The BBB is composed of three cell types: vascular endothelial cells, pericytes, and astrocytes. As the role of pericytes in maintaining the BBB function has become more evident, the reconstituted models of the BBB in which ECs, pericytes, and astrocytes are seeded on the front and back of the insert membrane have gotten our attention. In the field of cancer, the role of not only ECs but also wall cells, corresponding to pericytes in the brain, has been focused on. In this study, we aimed to create the in vitro BBB models and elucidate the interaction of the BBB and the metastasizing cancer cells. Methods: We applied a reconstituted model of the BBB consisting of ECs, pericytes, and astrocytes of human immortalized cells (Ito R, et al. Mol Pharm 2019) and created the co-culture system for evaluating brain metastasis. We constructed four co-culture systems and compared the migration ability of MDA231 in those models: EPA model (ECs, pericytes, and astrocytes), E0A model (ECs and astrocytes), EP0 model (ECs and pericytes), and E00 model (ECs only). We also analyzed the migration ability of the pairs of parental and brain metastatic (BrM) cell lines. Results: The BBB reconstituted model (EPA model), in which ECs were co-cultured with pericytes and astrocytes, showed higher trans-epithelial electrical resistance and lower permeability than the other co-culture models. When cancer cells were seeded, the EPA model strongly inhibited metastatic invasion of cancer cells compared to the other models. It suggests that both pericytes and astrocytes are essential for regulating cancer cell invasion. In addition, when the BrM cells of MDA231 and Ex3LL were seeded, the BrM cells invaded the EPA model more easily than the parental cells. It indicates that this model can reproduce in vivo brain vascular functions. Conclusion: We created the BBB reconstituted model which mimics the brain vascular microenvironment. This model reproduced the in vivo BBB function to protect brain from cancer cells and evaluated the brain metastatic ability of the cancer cells. Using this model, we showed that pericytes and astrocytes were essential for the BBB barrier function against the cancer cells. We need further investigation to elucidate the mechanism of brain metastasis using this BBB model. Citation Format: Shoko Noda-Narita, Atsu Aiba. In vitro blood brain barrier model for evaluation of brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1455.

Abstract 2680: Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis

Abstract Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of breast cancer. Lack of known targets, patient-to-patient variations of target antigens and resistance to immune checkpoint inhibitors make TNBC one of the most challenging cancers to treat. TNBC is enriched in tumor-infiltrating neutrophils (TINs). TINs are key contributors to pro-tumorigenic processes linked to “immunologically cold” tumors, in part by preventing recruitment of T-lymphocytes (TILs). Prior studies identified the Enhancer of zeste homolog 2 (EZH2) as a prominent methyltransferase expressed in primary and metastatic TNBC cells. EZH2 is an upstream regulator of the stimulator of interferon genes (STING), a critical innate sensor mediating anti-tumor responses that is inhibited in cold tumors. To date, EZH2/STING signaling in TINs is relatively unknown. Here, we hypothesized that EZH2 inhibition and subsequent STING activation could exert anti-tumor activity by modulating TINs. We derived EZH2-knockout (KO) and EZH2-overexpressed (OX) cell lines from the well-established 4T1 TNBC mouse cell line. We demonstrate a 10-fold decrease in lung metastasis and significant reduction of 4T1 EZH2 KO tumor growth compared to the parent 4T1 cell line. Concomitantly, the TIN:TIL ratio was significantly reduced in 4T1 EZH2 KO vs. control tumors. Overexpression of EZH2 in 4T1 TNBC cells did not affect the primary tumor growth or lung metastasis. A therapeutic study using the repeated injection of the STING agonist MSA-2 after tumor engraftment in vivo also demonstrated a decrease in lung metastasis and tumor growth in MSA-2 vs. vehicle injection with both 4T1 parent and EZH2 KO cell lines. Together, our findings suggest a key role for STING pathway modulation and TIN/TIL poise in primary TNBC progression and lung metastasis. Citation Format: Lenore Monterroza, Maria Parrilla, Sarah Samaranayake, Dormarie Rivera, Brian Dobosh, Periasamy Selvaraj, Rabindra M. Tirouvanziam. Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2680.

Abstract 541: Establishment of Inotuzumab ozogamicin resistant leukemia cell lines; Focusing on p-gp and DNA damage repair for overcoming drug resistance

Abstract Introduction: Inotuzumab Ozogamicin (IO) is a humanized anti-CD22 monoclonal antibody conjugated with chalicheamicin, which causes single- and double-strand DNA breaks. Although IO shows a high CR/CRi rate in relapsed/refractory CD22-positive B-cell acute lymphoblastic leukemia (ALL), the duration of remission is short due to early acquisition of resistance. Therefore, the present study attempted to enhance the cytotoxicity of IO by focusing on polyadenosine diphosphate-ribose polymerase (PARP) inhibition. Furthermore, IO-resistant cell lines were newly established to investigate the mechanism of IO resistance. Methods: Reh cell line derived from Ph-negative B-ALL cells was used. To establish IO-resistant cell lines, Reh cells were long-term exposed to IO, followed by limiting dilution. FACS analysis evaluated the CD22 positivity and the induction of apoptosis. WTS assay determined the cell growth inhibition effects. The Comet assay quantitatively assessed DNA damage. Gene expression profiles were determined by using DNA microarray. Results: The 50%-inhibitory concentration (IC50) of IO was 2.1 ng/mL in Reh cells. Reh cells were synergistically sensitized to IO by the addition of non-toxic concentrations of PARP inhibitors, olaparib or talazoparib, with the Combination Index of 0.19 and 0.42, respectively. Moreover, these combinations augmented the induction of apoptosis (23.5% in IO alone, 51.7% in IO + olaparib, 66.1% in IO + talazoparib). The Comet assay demonstrated that DNA strand breaks observed 1h after IO administration were repaired 6h later. In the combination of IO with olaparib or talazoparib, DNA damage remained even at 6h, suggesting the inhibition of DNA repair by PARP inhibitors. Next, six IO-resistant cell lines were established with IC50 more than 60-fold that of parental cells. All these cell lines kept CD22 expression. These cell lines were also resistant to apoptosis induced by IO. In addition of olaparib or talazoparib to IO, the IC50 of IO decreased by 40% to 70% in 6 IO-resistant cells. These combinations also augmented the induction of apoptosis (24.2% in IO alone, 45.3% in IO+olaparib, and 56.8% in IO+talazoparib). DNA microarray was performed to compare gene expression profiles between parental and IO-resistant cell lines. ABCB1 (p-gp), a drug efflux pump that causes multi-drug resistance, was overexpressed in resistant cell lines. Moreover, the addition of p-gp inhibitor, PSC-833, to IO enhanced the cell growth inhibition and induction of apoptosis due to more DNA strand break. Conclusion: The present study demonstrated that the combination of IO with PARP inhibitors exerted synergistic cytotoxicity via the inhibition of DNA repair. In addition, it was suggested that overexpression of p-gp caused IO resistance, which was partially reversed with the combination of PARP inhibitors or a p-gp inhibitor. Citation Format: Naoko Ida, Miyuki Okura, Naoko Hosono, Takahiro Yamauchi. Establishment of Inotuzumab ozogamicin resistant leukemia cell lines; Focusing on p-gp and DNA damage repair for overcoming 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 541.

Abstract 1931: Targeting YAP1/TEAD signaling re-sensitizes MAPK/ERK pathway inhibitors in KRAS- driven cancer cells

Abstract Many TEAD small-molecule inhibitors (SMIs) have recently reported initial clinical evaluations on Hippo-mutated cancer types. Several studies have demonstrated that activation of the YAP1/TEAD transcriptional complex via a Hippo-independent manner can drive resistance to MAPK/ERK pathway inhibitors. Here, we elucidated the potential mechanism of TEAD inhibition overcoming MAPK/ERK pathway resistance with a TEAD SMI (hereafter abbreviated as TEADi). Firstly, the MoA of TEADi was validated in Hippo-mutated cells. TEADi inhibited cell growth of Hippo-mutated cancer cell lines NCI-H226 and NCI-H2052 but not that of MKN45, a YAP1-deletion cell line. Co-IP and qRT-PCR results demonstrated that TEADi disrupted the interaction between YAP1 and TEAD, and thus markedly repressed the expression of CTGF and CYR61, two downstream targets of YAP1/TEAD, in NCI-H226 cells. Secondly, we hypothesized that hyper-activation of TEAD confers resistance to KRAS mutant cancer types. To test this hypothesis, we generated two resistant cell lines. One is KARS G12C inhibitor Sotorasib-resistant NCI-H358 (NCI-H358-R). The other is MEK inhibitor Trametinib-resistant HCT116 (HCT116-R). Immunofluoresence assay showed that YAP1 nucleus translocation was enhanced in both resistant cells (NCI-H358-R and HCT116-R), but not in their parental counterparts (NCI-H358-P and HCT116-P). The enhanced YAP1 nucleus translocation resulted in increased transcription activities of TEAD in both resistant cells, as illustrated by luciferase reporter assay. Accordingly, CTGF and CYR61 were observed upregulated in both resistant cells. The above data indicate that YAP1/TEAD mediated-transactivation plays a role in MAPK pathway resistance. Indeed, TEADi treatment alone displayed substantial difference in cell growth inhibition between parental and resistant cell lines in both NCI-H358 and HCT116, further confirming that the resistant cells are more dependent on YAP1/TEAD signaling. Finally, we evaluated the combinational efficacies of TEADi and MAPK/ERK pathway inhibitors in the resistant cells. It was observed that addition of TEADi could significantly restore the response of NCI-H358-R and HCT116-R to Sotorasib and Trametinib, respectively. Mechanistically, TEADi efficiently suppressed TEAD transcriptional activities and subsequently the expression of CTGF and CYR61 in both resistant cells. Moreover, TEADi had almost no impact on ERK phosphorylation in either of the resistant cells, suggesting that re-sensitization of MAPK/ERK pathway inhibitors by TEADi is independent of primary onco-genetic signaling pathway. Taken together, our study demonstrates that inhibition of YAP1/TEAD signaling would be an efficient approach to overcome resistance to MAPK/ERK pathway inhibitors in the patients carrying KRAS mutations, and provides the scientific basis for development of combination therapy strategies. Citation Format: Xianwen Yang, Guiping Li, Peiling Ren, Xiaoyun Shi, Ying Yu, Baoyu Hao, Pan Wang, Min Cheng, Guangxiu Dai. Targeting YAP1/TEAD signaling re-sensitizes MAPK/ERK pathway inhibitors in KRAS- driven 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 1931.

Abstract 4675: Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2

Abstract Lung cancer will account for 21% of US cancer deaths in 2023 with 238,340 cases and 127,070 deaths. Treatment with EGFR-TKI (epidermal growth factor receptor tyrosine kinase) inhibitors initially decrease tumor growth, but often leads to TKI resistance. According to studies, non-small cell lung cancer (NSCLC) tumors express more SphK2 (Sphingosine kinase 2) than normal lung tissue and patients with overexpression have worse survival chances. However, SphK2's role in TKI resistance is unclear. We hypothesize that inhibiting/downregulating SphK2 could reduce tumorigenicity and overcome ER (Erlotinib) and OR (Osimertinib) resistance. We studied the expression of SphK2 in both drug-resistant (OR/ER) and drug sensitive (parental) H358, H2170, H3255, and PC9 NSCLC cell lines using Western blotting, qRT-PCR, and immunofluorescence. H3255OR cells were also bioprinted with GelMA hydrogel using an R-Gen 200 bioprinter to mimic the physiological tumor environment and treated with SphK2 siRNA or ABC294640 (SphK2i). Immunoblotting was done to analyze changes in the expression of SphK2 in H358 (WT-EGFR) and H3255 (mutant EGFR- L858R) parental and OR-resistant cell lines. Results indicated a 1.5-1.8-fold upregulation of SphK2. Increased SphK2 gene expression was observed using qPCR in TKI-resistant H3255OR (4.3-fold), PC9ER/OR with mutant EGFR (E746-A750del) (1.8-3.3-fold), and H2170ER/OR with WT EGFR (1.3-1.5-fold) compared to parental cells. Immunofluorescence studies showed increased fluorescence of SphK2 in H3255OR (2.6-fold), H358 ER (2.4-fold) and OR cells (2.3-fold) compared to parental cells. Spheroids formed from single cancer cells can "re-create" the growth of a tumor. The spheroid count of H3255OR was reduced by 73.98% and 56%, respectively, when SphK2i was added to the 3D-cell culture with Matrigel, compared to the diluent and OR alone (p≤0.01). We used 3D bioprinting to precisely stack cells on biological scaffolds to recreate lung tumors from patients and boost the therapeutic efficacy of these findings. When bioprinted spheroids of H3255OR were treated with SphK2i in combination with OR, the number of spheroids per microscopic field were reduced by 87.3% compared to OR alone. Bioprinted H3255OR spheroids treated with SphK2 siRNA and OR revealed a 75.8% and 84% reduction of spheroid count, respectively, compared to treatments with siRNA alone or OR alone (p≤0.05). This data supports the hypothesis that SphK2 suppression or downregulation can assist in mitigating oncogenesis. We also used H3255OR transwell migration assays to examine how SphK2 inhibition affects cell migration. Combining OR with SphK2i resulted in a 94.7% and 91.4% decrease in cell migration compared to diluent and OR alone (p≤0.01). In H358OR-resistant cells, combining OR with SphK2i resulted in a 94.97% inhibition compared to diluent (p≤0.01). These results suggest that targeting SphK2 could be a valuable approach for treating lung cancer. Citation Format: Ayesha Khan, Bamby Dieng, Namrata Dube, Mohammad Fazle Alam, Neelu Puri. Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2 [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 4675.

Abstract 5897: Understanding and targeting fatty acid CoA ligase ACSL4 in human prostate cancer

Abstract Prostate cancer (PCa) is the most common non-cutaneous malignancy among men. Although localized prostate cancer is curable, recurrence of metastatic castration-resistant prostate cancer (mCRPC) is almost inevitable and is the major cause of death in human PCa. Recent findings from our lab and others demonstrate that RB1 loss drives lineage plasticity, metastasis, and lethality of prostate tumors initiated by PTEN loss. A downstream target upregulated by RB1 loss is acyl CoA synthetase long chain family member 4 (ACSL4), a fatty acid ligase with an essential role in utilization of long chain fatty acids. ACSL4 has been reported to be upregulated in cancers of various histological origins, including prostate cancer, and its high expression is correlated with aggressiveness of cancer. We hypothesize that ACSL4 is a critical downstream effector of RB1 loss-driven prostate tumorigenesis and pharmacological inhibition of ACSL4 could represent a novel therapeutic approach to the treatment of lethal RB1 deficient or ACSL4 overexpressing prostate cancer. Here we aim to develop novel ACSL4 inhibitors for preclinical studies. Through a structure based virtual screening that targeted ATP binding domains of ACSL4, we successfully identified 15 potential ACSL4 inhibitors. To validate the result, we performed an in vitro screening using various parental and isogenic human PCa cell lines, and found that 4 out of 15 compounds effectively inhibited PCa cell proliferation in a dose-response manner and their inhibitory effects positively correlated with cellular levels of ACSL4. Next, we will perform isotope-labeling enzyme activity assays to determine the specificity of the 4 candidate drugs, and PDPK analyses followed by characterizing their in vivo efficacies in preclinical studies, with a final goal to develop a well-validated ACSL4 inhibitor. Successful completion of this work will provide great insights into contributions of ACSL4 to prostate tumorigenesis as well as preclinical data of ACSL4 inhibitors to treat lethal prostate cancer. Citation Format: Jinjin Wu, Mu-En Wang, Ming Chen, Pyeong Hwa Jeong, Jiyong Hong, Lixin Wan. Understanding and targeting fatty acid CoA ligase ACSL4 in human 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 5897.

Abstract 5512: Cerebral endothelial FAK paradoxically inhibits brain metastasis in vivo

Abstract Introduction: In cancer metastasis in the trunk, the secreted proteins from tumors, such as VEGFs and TNF-α, activate focal adhesion kinase (FAK) in vascular endothelial cells (VECs). The activated FAK promotes VE-cadherin phosphorylation and the expression of cell adhesion molecules, and it facilitates the extravasation of cancer cells. In the cerebral blood vessels, the VECs maintain very strong cell adhesion supported by the surrounding pericytes and astrocytes. The strong barrier of cerebral VECs is called the blood-brain barrier (BBB) and is regulated differently from trunk VECs. Although the role of FAK in trunk metastasis is shown in many studies, its importance in brain metastasis is not verified. In this study, we focused on the role of the cerebral endothelial FAK and compared the role of FAK in brain metastasis with that in trunk metastasis. Methods: We generated tamoxifen-inducible VEC-specific FAK knockout (FAK-cKO) mice (VEcad-CreERT2; Ptk2-flox/flox) and brain metastatic (BrM) cell lines of Ex3LL (murine lung cancer cell line). We intracardially injected BrM cells of Ex3LL into the FAK-cKO mice and analyzed the tumor development using in vivo bioluminescence imaging and a CUBIC tissue clearing system. We also analyzed the intracranially injected mice of the parental and BrM cell lines to evaluate the tumor angiogenesis, which is reported to be promoted by the endothelial FAK. Results: Although FAK-cKO in VECs has been reported to inhibit lung metastasis, brain metastasis was accelerated in FAK-cKO mice compared with control mice. The number of brain metastatic sites increased in FAK-cKO mice, but there was no difference in the individual tumor size. It suggests that the brain metastasis was promoted by FAK-cKO, but that the proliferation of brain metastasis was not promoted by FAK-cKO. Blood vessel density was decreased with irregular shape in the brain tumors of FAK-cKO mice, suggesting that the angiogenesis was inhibited by FAK-cKO. It indicates that the angiogenic function of endothelial FAK in the brain vasculature was not different from that in the trunk. Conclusion: FAK-cKO in VECs exacerbated the brain metastasis of the BrM cells although it has been reported to inhibit the metastasis in the trunk. FAK-cKO in VECs decreased tumor angiogenesis both in the brain and the trunk. It suggests that the regulatory mechanism of cell adhesion and the FAK function in the brain vasculature is partly different from that in the other organs in the body trunk. We need further investigation to elucidate the function of endothelial FAK in brain metastasis. Citation Format: Shoko Noda-Narita, Atsu Aiba. Cerebral endothelial FAK paradoxically inhibits brain metastasis in vivo [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 5512.

Abstract 3978: B-cell activating factor (BAFF) enhances antitumor immunity

Abstract Immunotherapies that modulate T cell function have been firmly established in cancer immunotherapy, whereas the potential for B cells in the antitumor immune response is not as well understood. B cell-activating factor (BAFF) is a cytokine belonging to the TNF ligand family that activates B cells and is linked to autoimmunity. Our preliminary research has identified BAFF as a target for enhancing antitumor immunity through enhancing presentation of tumor neoantigens and inducing tertiary lymphoid structures which can orchestrate tumor-specific T cell immune responses and correlate with improved clinical response. Since broad activation of B cell response could be detrimental and possibly induce autoimmune side effects, identification of specific B cell receptors that coordinate anti-tumor immunity is prudent. BAFF has three known receptors (BAFF-R, TACI and BCMA), and the effects of these specific BAFF ligand-receptor interactions on antitumor immunity are not known. Here we first generated BAFF-overexpressing cell lines using genomic editing [KPC-BAFF (pancreas), B16F10-BAFF (melanoma), and PancO2-BAFF (pancreas)] and examined their tumor growth in syngeneic immunocompetent host. Secondly, we investigated the effects of BAFF-overexpression on tumor response when individual receptors are knocked out using BAFF-R-KO, TACI-KO and BCMA-KO mice. Our results show significantly hindered tumor growth when the BAFF expressing stable cell lines are introduced to immunocompetent host, compared to parental cell lines. Tumor response experiments in receptor knockout mice is currently in progress. Successful identification of the BAFF receptor candidate that drives anti-tumor response is an exciting prospect for clinical translation. Development of agonists for the specific receptor may allow selective modulation of this particular B cell function in context of cancer therapy without perturbation of broad systemic humoral immunity. Citation Format: Khaled Aziz, Kabeer Munjal, Kathryn Howe, Mark Yarchoan. B-cell activating factor (BAFF) enhances antitumor immunity [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 3978.

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 1571: Upregulation of phosphoglyceric acid mutase (PGAM)-1 is related to lymph node metastasis in gastric cancer

Abstract Gastric cancer (GC) is the most common malignancy of the digestive system, causing over 750,000 deaths worldwide. Gastric cancer mortality is largely due to its high level of invasiveness and metastasis to distal organs. Thus, it is an urgent priority to identify mechanisms underlying GC metastasis to improve cancer diagnosis develop of effective biological targeting agents. In this study, we demonstrate that phosphoglyceric acid mutase-1 (PGAM1) was preferentially over-expressed in lymph node metastasis (LNM) tissues of GC patients and correlated with poor prognosis. PGAM1 knock down in two human GC cell lines resulted in impaired cell proliferation and increased cell cycle arrest. Further, PGAM knockdown inhibited migration of both GC cell lines, which correlated with decreased expression of MMP2, MMP9 and ICAM-1. In a murine xenograft model, GC cells with a PGAM1 knockdown displayed decreased growth capacity in vivo. Western blot analysis showed that PGAM1 knockdown in SGC7901 and BGC823 decreased the phosphorylation of Src, FAK and Paxillin relative to the parental cell lines. These results indicate that PGAM1 may play an important role in promoting GC lymph node metastasis by driving cell proliferation and activating cytoskeletal reorganization and cell migration through the regulating Src/FAK/Paxillin pathway. Citation Format: Helei Wang, Zhicheng Liu, Lei Wang, Zitian Wang, Christina Jamieson, Nicholas A. Cacalano. Upregulation of phosphoglyceric acid mutase (PGAM)-1 is related to lymph node metastasis in gastric 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 1571.

Abstract 7407: Melanoma clonal subline analysis reveals genetic factors driving intra-tumor heterogeneity

Abstract Melanoma is the most invasive skin cancer caused by the malignant melanocytes. The use of immune checkpoint blockade (ICB) improved the survival rate in advanced melanoma. Yet, the response rate to ICB varies across patients due to the highly heterogeneous nature of melanoma. Recent studies reported genomic and epigenetic factors contributing to the therapeutic response. Identifying these factors involved in clonal evolution in melanoma is a key to better understanding the tumor progression and divergence in the therapy response. To study melanoma heterogeneity, we generated twenty-four clonal sublines, each derived from a single cell isolated from a parental cell line derived from the M4 mouse melanoma model. DNA prepared from each subline and a spleen from a healthy mouse were subjected to long-read sequencing. Importantly, long-read sequencing allows direct detection of methylation states, small variants, and structural variants in the same run. The results thus provide excellent means to study genetic and epigenetic factors in clonal evolution. Each of these sublines was also implanted in distinct mice for survival and tumor growth analysis and further functional evaluation. We developed Severus, a structural variation (SV) caller for long reads, which works with single (germline), paired (tumor-normal), and multiple samples (e.g., multi-site or time series). Severus takes advantage of improved phasing in long-read sequencing, attributes somatic variants to germline haplotype, and builds a haplotype-specific breakpoint graph that is used to cluster multi-break rearrangements and represent the derived chromosomal structure. We benchmarked Severus using multiplatform validated COLO829 truthset and CHM1/CHM13 haploid genomes, and Severus outperformed all other methods in terms of recall and precision. The structural variation (SV) analysis using Severus with all the sublines showed enrichment of possibly clonal SVs in chr4, chr11, and mainly in chr13. Further investigation revealed SVs in chr13 were not distributed across the chromosome but clustered in certain regions. Analysis of somatic SNVs in chr13 corroborates the localized hypermutation profile, which indicates a possible kataegis. Interestingly, a loss of the chr13 copy affected by kataegis in one of the sublines coincides with a better survival rate, as compared to other sublines, in an allograft melanoma mouse model, which further supports the association between the kataegis and tumor progression. We also identified other clonal and subclonal events, including the homozygous deletion of Cdkn2a in most of the sublines, associated with their aggressiveness in vivo. Our analysis allowed us to identify clonal and subclonal genetic and epigenetic factors involved in tumor progression and evolution in a melanoma model, which can potentially translate to human disease. Citation Format: Ayse Keskus, Anton Goretsky, Yuelin Liu, Xiwen Cui, Tanveer Ahmad, Eva Perez Guijarro, Asher Bryant, Erin Malloy, Salem Malikic, Glenn Merlino, Chi-Ping Day, Cenk Sahinalp, Mikhail Kolmogorov. Melanoma clonal subline analysis reveals genetic factors driving intra-tumor heterogeneity [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 7407.

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [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 2305.

Abstract 5661: Identifying and intervening rare resistant subclones to BRAF/MEK inhibitors in metastatic melanoma

Abstract Acquired drug resistance poses a significant challenge in the treatment of cancer. This study focuses on melanoma as a model system to understand and address pre-existing subclonal resistance. The use of BRAF and MEK inhibitors (BRAFi/MEKi) in patients with metastatic melanoma harboring BRAF activating mutations has resulted in remarkable responses in the majority of cases. However, most patients have disease relapse after one year, and there are limited subsequent treatment options. One possible cause of relapse is the existence of a subpopulation of cells resistant to BRAFi/MEKi prior to treatment, raising the possibility that commencing counter-resistance treatment earlier may delay or abolish resistance to BRAFi/MEKi. We aim to enhance the detection and treatment of low-frequency resistant mutations, addressing the current oversight by existing technologies. This research is crucial due to a lack of consensus on the clinical management of these mutations. By using quantitative blocker displacement amplification (qBDA) technology and integrating patient samples with cell line models, our project seeks to determine 1) the prevalence of low-frequency resistant subclones by measuring the Variant Allele Frequency (VAF) of known resistance mutations along the MAPK/PI3K pathways in FFPE biopsies from 149 metastatic melanoma patients and 2) determine the functional impact of different VAF levels on the outgrowth of resistant subclones. We assessed 14 loci in 6 known resistance genes: NRAS, KRAS, PIK3CA, AKT1, MAP2K1, and MAP2K2, as well as BRAF itself as a positive control. As expected, all 55 patients analyzed harbored BRAF activating mutations, while 17 patients had at least one resistant subclone above our preliminary cutoff of 60% VAF. To simulate the behavior of low-VAF resistance mutations, we conducted in vitro experiments by spiking in labeled BRAF/MEK inhibitor-resistant melanoma cells into their isogenic sensitive parental cell line. Our initial data demonstrated that, at least in this model system, a 10% VAF spike-in leads to the eventual outgrowth of resistant cells, but not in a 1% VAF spike-in, suggesting that either the density or the absolute number of the pre-existing resistant cells can dictate resistance evolution. In future experiments, we will assess multiple VAFs in an independent cell line as well as in vivo to identify the nature of its correlation with resistance outcomes and expand to additional models. In summary, our data suggest that a substantial proportion of metastatic melanoma patients with BRAF mutations have pre-existing resistant cell subclones, and that accurately assessing its VAF could provide clinically-relevant information. Ultimately, results from this work would be the basis for future personalized counter-resistance therapies, potentially extending treatment effectiveness for patients with pre-existing resistant mutations. Citation Format: Han Wang, Michael Nakazawa, Omid Veiseh, Lawrence Kwong, David Zhang. Identifying and intervening rare resistant subclones to BRAF/MEK inhibitors in metastatic 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 5661.

Abstract 4205: Investigating micro-environmental changes in a syngeneic radio-recurrent prostate cancer model

Abstract Approximately 1 in 8 men in Canada will be diagnosed with prostate cancer (PCa) in their lifetime, with ~20% presenting with high-risk disease. A standard treatment is radiotherapy (RT) and while many patients respond well to RT, up to 40% of men with high-risk disease can recur, often within the prostate. It can be challenging to salvage prostate relapses due to the morbidity of re-irradiation and the high rate of subsequent progression. The tumor micro-environment (TME) is altered after radiotherapy with increased immune-suppressor and immune-stimulatory effects occurring concurrently. Therapies capitalizing on these changes in the TME might provide an advantage to tackling radiation-recurrent cancer. Identifying TME changes in radio-recurrent PCa is the first step; however, existing PCa animal models do not accurately model the TME. Our collaborator’s lab has created the DVL3 mouse model with a clinically relevant TME. It forms tumors with distinct glandular morphology and displays micro-environmental responses to RT like what is seen in high-risk PCa patients, making this model an innovative tool for investigating radio-recurrence. The objective of this study is to create a radio-recurrent DVL3 PCa cell line to explore changes in the TME both in vitro and in vivo via orthotopic injection. To generate the radio-recurrent model, the DVL3 Parental cells (DVL3-Par) underwent a conventionally fractionated RT schedule (78Gy/39fx), herein referred to as DVL3-CF cells. The first part of the project involves in vitro characterization. Radiation resistance was evaluated via clonogenic assay which demonstrated a significant increase in clonogenic survival in the DVL3-CF cells relative to DVL3-Par. Characterization of DVL3-CF cells demonstrates that they are more proliferative at baseline, they recover quicker following G2/M blocks, they have decreased senescence following high dose radiation, and they are more invasive in vitro. The second part of the project evaluates the radio-recurrent model in vivo using an orthotopic mouse model as this best emulates the TME and clinical tumor progression. Parental and CF cells will be injected orthotopically into the prostate of C57/BL-6 mice and monitored for growth by MRI which will provide information on tumor volume as well as vasculature. At the time of sacrifice, the prostate and draining lymph nodes will be cryo-preserved and formalin fixed for downstream histological analysis. Transcriptional analyses, MRI data, and histological analysis of the tumors and draining lymph nodes will be compared across the radio-recurrent model and its parental cell line to evaluate environmental changes and potentially implicated mechanisms. This study will elucidate some of the complex interactions that are occurring within the TME of radio-recurrent PCa, increasing our understanding of mechanisms of radio-recurrence. Citation Format: Stephanie D. White, Xiaoyong Huang, Ian Mills, Stanley K. Liu. Investigating micro-environmental changes in a syngeneic radio-recurrent prostate cancer model [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 4205.

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [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 540.

Abstract 5845: Cytarabine resistance leads to a unique mutational signature

Abstract The prognosis of recurrent T-cell acute lymphoblastic leukemia (T-ALL) is poor, mainly due to chemotherapy resistance. The mechanism(s) leading to chemoresistance at relapse are incompletely understood. We previously characterized a mouse strain that is hypomorphic for the DNA replication factor Mcm2. Mcm2hypo mice develop T-ALL due to acquired DNA copy number variations (CNV) involving tumor suppressor or oncogenes. We hypothesize that treatment of Mcm2hypo cells with specific chemotherapy agents may reveal CNV that lead to chemoresistance. We treated two Mcm2hypo T-ALL cell lines (designated 2883 and 2869) and an Mcm2 WT T-ALL cell line (designated 7298) with cytarabine (ARAC), an active agent often incorporated in T-ALL treatment regimens. After one year of treatment with gradually increasing ARAC concentrations, we obtained ARAC resistant cell lines (designated with the suffix CR) that tolerate drug concentrations up to 10,000X that of parental cell lines. These samples were characterized by sparse WGS (for CNV assessment), RNA-Seq and WES. Both the 2883CR and 2869CR cell lines had bi-allelic mutations involving Dck, which is the rate-limiting enzyme in the cytidine salvage pathway, and is thought to be required for metabolism of ARAC. In both cases, one allele was deleted while the second allele had a splice site/region mutation leading to partial intron retention and resultant frameshift. The 7298CR cell line had homozygous deletion of Dck. Western blotting showed absence of WT Dck protein in all three cell lines. Further analysis of WES data revealed thousands of acquired single nucleotide variants (SNV) in both Dck mutant Mcm2hypo cell lines. These mutations occurred in a specific trinucleotide context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, and C>G in a GCC context) generating a mutational signature that is not present in the COSMIC database. In addition, the parental 2883 and 2869 cell lines showed fewer mutations (ie, hundreds vs thousands) in a similar context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, but not the C>G in GCC context). A GEMINI (Genotoxic Mutational Signature Identified After Clonal Expansion In Vitro) assay of untreated 2883CR and 2869CR cells demonstrated that ongoing ARAC exposure was not required for the mutational signature. We hypothesize that the basic signature (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context) can be produced by Mcm2 deficiency, and this signature is amplified and modified (to include a C>G in GCC context) by Dck inactivation. In summary, we identified three mechanisms responsible for Dck inactivation associated with ARAC resistance: copy number loss, splice site mutations, and splice region mutations. Moreover, Dck inactivation leads to a specific mutation signature. These findings add to emerging data that exposure to specific chemotherapy agents can lead to specific mutational signatures. Citation Format: Dengchao Cao, Mianmian Yin, Zhenhua Zhang, Taimour Baslan, Jack Zhu, Ryan Bertoli, Paul Meltzer, Peter Aplan. Cytarabine resistance leads to a unique mutational signature [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 5845.

Abstract 7200: Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy

Abstract Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, most men succumb to Metastatic Castration Resistant Prostate Cancer (mCRPC) due to treatment resistance. MAGMAS, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. MAGMAS is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, MAGMAS plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of MAGMAS in treatment-resistant PCa cells. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of MAGMAS compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX-resistant metastatic PCa cell lines and re-sensitizes them to DTX. MAGMAS knockdown in DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of MAGMAS with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using cell migration/invasion assays and tumor-sphere formation assays are underway to further establish MAGMAS as a potential therapeutic target to attenuate the aggressive properties of chemo-resistant PCa cells. Lastly, we will investigate the diagnostic potential of MAGMAS as a biomarker for advanced PCa and BT#9 as an imaging/therapeutic-specific agent for treatment-resistant tumors. Citation Format: Alfonso Duran, Kristen Whitley, Krystal Santiago, Adil Mohammed, Carlos Casiano, Bhaskar Das, Frankis Almaguel. Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7200.

Abstract 2013: Extracellular vesicles transfer miR-181a to confer PARP inhibitor resistance and induce STING degradation in BRCA-mutated triple-negative breast and ovarian cancers cells

Abstract Background: Patients with BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC), and ovarian cancer (OvCa) are treated with poly (ADP-ribose) polymerase inhibitors (PARPi). BC and OvCa tumors develop similar mechanisms of resistance to platinum-based agents and PARPi. This study aimed to identify microRNAs (miRs) inducing PARPi resistance in cancer cells that can be transferred by extracellular vesicles (EV). Methods: Olaparib-resistant (OlaR) cell lines were analyzed for 2,083 miRs using HTG miRNA Whole Transcriptomic Analysis (WTA) and NGS. Functional assays were performed in BRCA1-mutated TNBC and OvCa cell lines. In-silico analysis were performed using TCGA, GTEx, CCLE, GDSC, and GEO databases. Results: The miR-181 family (including miR-181a, p=0.001) was significantly upregulated in OlaR TNBC cell lines. High miR-181a levels occurred in tumor tissues of TNBC patients (p=0.001). miR-181a binds to the stimulator of interferon genes (STING) mRNA to promote STING mRNA degradation in OvCa. We hypothesized that miR-181a downregulates STING and downstream proinflammatory cytokines, to mediate PARPi resistance. miR-181a-overexpression induced olaparib resistance and STING downregulation in BRCA1-mutated TNBC cell lines. EV isolated from OlaR TNBC cell lines horizontally transferred miR-181a to non-resistant parental cell lines, which conferred PARPi-resistance and decreased STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p=0.01). Low-IFNG response scores in HER2-negative BC patients showed a worse response to neoadjuvant treatment (p=0.001). Consistently, OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to cisplatin showed resistance to olaparib; but miR-181a knockout significantly improved olaparib sensitivity (p=0.001). Conclusions: High levels of miR-181a decrease STING pathway, correlate with IFNG scores, drive PARPi and platinum-based drug resistance. Horizontal miR-181a EVs transfer can facilitate drug resistance in cells. The miR-181a-STING axis can be used for predicting PARPi responses in TNBC and OvCa tumors. Citation Format: Matias A. Bustos, Takamichi Yokoe, Yoshiaki Shoji, Yuta Kobayashi, Shodai Mizuno, Tomohiro Murakami, Sreeja C. Sekhar, Matthew Knarr, Steven A. Vasilev, Analisa DiFeo, Ronny Drapkin, Dave S. Hoon. Extracellular vesicles transfer miR-181a to confer PARP inhibitor resistance and induce STING degradation in BRCA-mutated triple-negative breast and ovarian cancers 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 2013.

Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.

Transcriptome analysis of an AKT inhibitor-resistant endometrial cancer cell line.

Drug resistance in endometrial cancer (EC) is a serious problem and a barrier to improving prognosis. The PI3K/AKT/mTOR pathway is highly activated in EC and can serve as a potential therapeutic target. Inhibitors against AKT have been developed, but resistance to these inhibitors is a concern. This study aimed to establish AKT inhibitor resistant cell lines and identify differentially expressed genes (DEGs) between parental and AKT inhibitor resistant cell lines to understand the mechanism of drug resistance to AKT inhibitors in EC. The sensitivity of eight EC cell lines to AKT inhibitor was analyzed. One of them was used to establish a drug-resistant cell line. DEGs were examined using RNA sequencing (RNA-seq). Furthermore, DEGs were comprehensively analyzed to identify hub genes. Hub genes were evaluated using quantitative real-time polymerase chain reaction. RNA-seq identified 617 DEGs. Hub genes were selected using bioinformatics analysis. The top 10 hub genes were TNF, CDH1, CCND1, COL1A1, CDH2, ICAM1, CAV1, THBS1, NCAM1, and CDKN2A. Relative mRNA expression was significantly upregulated for TNF, CDH1, CCND1, THBS1, p16INK4a, and p14ARF and significantly downregulated for CDH2, ICAM1, and NCAM1 in borussertib-resistant EC cell line. Drug resistance to AKT inhibitors may depend on genes related to cell adhesion-mediated resistance and transforming growth factor β signaling.