Carboplatin Resistance Research Articles

189 (PB177): Downregulation of microRNA-214-3p by long non-coding RNA DNAJC3-As1 promotes carboplatin resistance in esophageal cancer cells through the overexpression of the anti-apoptotic protein c-IAP1

189 (PB177): Downregulation of microRNA-214-3p by long non-coding RNA DNAJC3-As1 promotes carboplatin resistance in esophageal cancer cells through the overexpression of the anti-apoptotic protein c-IAP1

Methionine Restriction Reduces Lung Cancer Progression and Increases Chemotherapy Response.

Targeting tumor metabolism through dietary interventions is an area of growing interest, and may help to improve the significant mortality of aggressive cancers, including non-small cell lung cancer (NSCLC). Here we show that the restriction of methionine in the aggressive KRAS /Lkb1- mutant NSCLC autochthonous mouse model drives decreased tumor progression and increased carboplatin treatment efficacy. Importantly, methionine restriction during early stages of tumorigenesis prevents the lineage switching known to occur in the model, and alters the tumor immune microenvironment (TIME) to have fewer tumor-infiltrating neutrophils. Mechanistically, mutations in LKB1 are linked to anti-oxidant production through changes to cystathionine-β-synthase (CBS) expression. Human cell lines with rescued LKB1 show increased CBS levels and resistance to carboplatin, which can be partially rescued by methionine restriction. Furthermore, LKB1 rescued cells, but not mutant cells, show less G2- M arrest and apoptosis in high methionine conditions. Knock-down of CBS sensitized both LKB1 mutant and non-mutated lines to carboplatin, again rescuing the carboplatin resistance of the LKB1 rescued lines. Given that immunotherapy is commonly combined with chemotherapy for NSCLC, we next wanted to understand if T cells are impaired by MR. Therefore, we examined the ability of T cells from MR and control tumor bearing mice to proliferate in culture and found that T cells from MR treated mice had no defects in proliferation, even though we continued the MR conditions ex vivo . We also identified that CBS is most highly correlated with smoking, adenocarcinomas with alveolar and bronchiolar features, and adenosquamous cell carcinomas, implicating its roles in oxidative stress response and lineage fate in human tumors. Taken together, we have shown the importance of MR as a dietary intervention to slow tumor growth and improve treatment outcomes for NSCLC.

Extracellular vesicles of Bifidobacterium longum reverse the acquired carboplatin resistance in ovarian cancer cells via p53 phosphorylation on Ser15.

We previously found that the relative abundance of Bifidobacterium was increased after chemotherapy; however, the role of Bifidobacterium longum in chemotherapeutic drug resistance in ovarian cancer (OVC) remains unclear. This study aimed to understand the potential effects and mechanism of B. longum extracellular vesicles (B. longum-EVs) on carboplatin (CBP) resistance in OVC. Eight normal and 11 ovarian tissues were collected and the expression of B. longum genomic DNA and its association with acquired CBP resistance in OVC patients was determined. After isolating EVs by ultracentrifugation from B. longum (ATCC 15707), CBP-resistant A2780 cells were treated with PBS, CBP, B. longum-EVs, or CBP + B. longum-EVs, and subsequently analyzed by CCK-8, Edu staining, Annexin V/PI double staining, wound healing, and Transwell assays to detect cell viability, proliferation, apoptosis, migration, and invasion, respectively. MRP1, ATP7A, ATP7B, and p53 expression as well as p53 phosphorylation were measured by western blot analysis. S15A mutation of p53 was assessed to examine the potential role of p53 Ser15 phosphorylation in CBP-resistant OVC. B. longum levels were elevated and positively associated with CBP resistance in OVC patients. Only high concentrations of B. longum-EVs attenuated A2780 cell proliferation, apoptosis, migration, and invasion. B. longum-EVs exposure significantly enhanced the sensitivity of CBP-resistant A2780 cells to CBP and decreased the expression of drug resistance-related proteins. The effect of B. longum-EVs on reversing CBP resistance was completely inhibited by S15A mutation of p53. B. longum-EVs enhanced the sensitivity of OVC cells to CBP through p53 phosphorylation on Ser15.

Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

Comparison of Photodynamic Priming Efficacy, with BPD or ALA-PpIX, to Overcome PFAS-Induced Platinum Resistance in Ovarian Cancer

Ovarian cancer is the most lethal gynecologic cancer, largely due to the development of platinum-based chemotherapy resistance. Understanding sources of chemoresistance and the development of mechanism-based treatments are critical. In a recent study, we reported that perfluoroalkyl substances (PFAS), endocrine disruptors that pollute drinking water supplies worldwide, induce carboplatin resistance in ovarian cancer cells, potentially by enhancing mitochondrial function. The ability of photodynamic priming (PDP) to overcome PFAS-induced resistance using photosensitizers that, in part, localize to mitochondria, specifically benzoporphyrin derivative (BPD) or aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX)), will be presented. Ovarian cancer cells were exposed to PFAS, then treated with either BPD- or ALA-PpIX-PDP followed by carboplatin. Under PFAS exposure conditions that previously induced chemoresistance, both BPD- and ALA-PpIX-PDP sensitized ovarian cancer cells to carboplatin. A concomitant decrease in mitochondrial membrane potential was observed.

High-dimensional deconstruction of ovarian cancer at single-cell precision reveals HEBP2 that reshape the TIME and drive carboplatin resistance

BackgroundSingle-cell sequencing was employed to analyze the tumor immune microenvironment in ovarian cancer (OC) patients, exploring the evolutionary roles of various macrophage subgroups in OC progression and their correlation with fatty acid metabolism-related genes in contributing to drug resistance. MethodsThis study aimed to decipher the mechanisms underlying OC chemoresistance (OC-CR) and carboplatin resistance by integrating and analyzing multiple single-cell RNA sequencing datasets from OC patients. The tumor immune microenvironment in OC-CR patients exhibited notable alterations in cellular interactions and the proportions of different immune cell populations, in contrast to the cohort sensitive to OC chemotherapy. ResultsThe study demonstrates that the fatty acid-associated gene HEBP2 not only accelerates OC progression but also modifies the immune landscape of OC, driving the polarization from M0_TAM to M2_TAM. This shift results in a diminished efficacy of chemotherapy in OC. Furthermore, both in vitro and in vivo experiments underscored HEBP2′s role in boosting the proliferation of OC-resistant cell lines and suppressing apoptosis, thereby facilitating carboplatin resistance. ConclusionIn conclusion, the immune microenvironments of OC-CR significantly differ from those sensitive to chemotherapy, underscoring HEBP2′s role in fostering OC resistance. This establishes HEBP2 as a promising prognostic marker and a novel target for therapeutic strategies against OC resistance.

Abstract 1986: Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts

Abstract Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts [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 1986.

Abstract 575: Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer

Abstract This study aimed to understand the molecular mechanism of platinum chemoresistance in recurrent ovarian tumors with a focus on the SORL1-regulated pathways. SORL1 is an intracellular sorting receptor that binds to HER2 in breast cancer; this promotes HER2 recycling to the cell surface, which ultimately impacts the progression and treatment of breast cancer. The role of SORL1 in ovarian cancer has not been extensively studied. Method: RNA was extracted from 15 pairs of matched primary and recurrent high grade serous ovarian tumors collected from patients. Human transcriptomic microarray was used to identify the differentially expressed genes. The identified genes were validated by RT-QPCR and western blot in five ovarian cancer cell lines that were treated or untreated with carboplatin. To study the effects of identified target gene SORL1, overexpression and knockdown in ovarian cancer cell lines were established using SORL1 plasmid and shRNA. Proliferation and response to carboplatin were analyzed using celltiter Glo and caspase-3/7 activity assays respectively. EGFR and FGFR4 were identified as the interacting proteins of SORL1 in ovarian cancer cells through FGF1 and EGF1 treatment and co-immunoprecipitation. A proximity ligation assay was performed to validate their interactions. Finally, a xenograft mouse model was used to compare the tumor forming abilities and the response to carboplatin of SORL1-knockdown ovarian cancer cells and the control cancer cells in vivo. Protein expression of SORL1, EGFR, and FGFR4 in the collected tumors was determined via western blot. Result: Transcriptomic analysis of matched human primary and recurrent ovarian tumors identified SORL1 as an upregulated gene in recurrent tumors compared to primary samples. SORL1 expression was also upregulated in ovarian cancer cells surviving carboplatin treatment. Proliferation assays demonstrated that SORL1-overexpressing and knockdown cell lines had increased and inhibited proliferative capabilities respectively. Caspase-3 activity assays showed that SORL1-overexpressing and knockdown cell lines had reduced and increased sensitivity to carboplatin treatment respectively. SORL1 was found to bind to and promote the expression of EGFR and FGFR4. When injected subcutaneously into nude mice, SORL1-knockdown cells had reduced tumor forming capabilities and increased sensitivity to carboplatin. SORL1-knockdown caused the tumors to express lower levels of SORL1, EGFR, and FGFR4 proteins in comparison with the control tumors. Conclusion: This study demonstrates that SORL1 regulates the EGFR and FGF4 pathways to promote the growth and carboplatin resistance of ovarian cancer cells. Targeting this pathway has potential to aid in the treatment of platinum-resistant ovarian cancer. Citation Format: Miranda Mansolf, Ziyan Jian, Fangfang Bi, Tobias M. Hartwich, Yang Yang-Hartwich. Identification of SORL1 as a key regulator of chemoresistance in ovarian 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 575.

Abstract B100: Perfluoroalkyl substances (PFAS) induce platinum resistance in ovarian cancer through altering mitochondrial function

Abstract Platinum resistance is a major barrier to the effective treatment of advanced-stage ovarian cancer; however, factors leading to the development of resistance are not well understood. In fact, the role of environmental endocrine disrupting chemicals, such as perfluoroalkyl substances (PFAS), have seldom been explored. This is important because select PFAS have been linked to adverse reproductive outcomes in females including endometriosis, oocyte apoptosis, infertility, and increased even ovarian cancer risk at very high exposure levels. The relationship between PFAS and adverse female reproductive outcomes, as well as reports of other endocrine disruptors leading to therapy resistance, led, in the current study, to the evaluation of the effect of PFAS exposure on response to carboplatin in ovarian cancer cells. We recently showed that select PFAS, at human-relevant concentrations, induced carboplatin resistance in OVCAR-3 and Caov-3 cells; however, the mechanism underlying the onset of resistance remains largely unknown. Since platinum-resistant ovarian cancer is characterized, in part, by increased mitochondrial networks and enhanced bioenergetic capacities, the central hypothesis of this study is that PFAS induce alterations to mitochondrial biology. Mitochondrial endpoints of interest included redox ratio and superoxide production. To measure redox ratio, nicotinamide dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are inherently fluorescent, were measured using a fluorescence plate reader. Since NADH and FAD are metabolites produced via oxidative phosphorylation and glycolysis, evaluating redox ratio can provide an understanding of which pathway is preferred in the presence of environmental stimuli like PFAS. In OVCAR-3 and Caov-3 cells, baseline NADH:FAD ratios were 3.3 and 3.5, respectively. After 48 hours of PFAS exposure, NADH:FAD ratios of OVCAR-3 and Caov-3 cells ranged from 8-10 or 6-10, respectively. This increase after PFAS exposure could suggest enhanced energy production capabilities that warrant further investigation. As an additional measure, superoxide production was measured using flow cytometry. OVCAR-3 and Caov-3 cells were exposed to PFAS for 6 days prior to isolating cell pellets and incubating them with MitoSOX (mitochondrial) and SYTOX Blue (nuclear) dyes. After 15–30-minute incubations, samples were run on the flow cytometer. Compared to controls, superoxide production in PFAS-exposed ovarian cancer cells increased by up to 200%. Often, cancer cells have elevated levels of reactive oxygen species due to enhanced energy production and signaling pathways, thus this finding further suggests that PFAS affect mitochondrial energy pathways. Altogether, these data show that PFAS exposure, which can induce carboplatin resistance in OVCAR-3 and Caov-3 cells, increases redox ratios and superoxide production in ovarian cancer cells. Therefore, targeting alterations in mitochondrial biology arising from PFAS exposure using precision medicine could be effective in overcoming platinum resistance in ovarian cancer. Citation Format: Brittany P. Rickard, Marta Overchuk, Vesna A. Chappell, Carl D. Bortner, Victoria L. Bae-Jump, Suzanne E. Fenton, Imran Rizvi. Perfluoroalkyl substances (PFAS) induce platinum resistance in ovarian cancer through altering mitochondrial function [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 B100.

Abstract B111: Mechanism-informed photochemical strategies to overcome fluid shear stress-induced platinum resistance in ovarian cancer

Abstract Background: Over 75% of patients with disseminated ovarian cancer develop resistance to platinum-based chemotherapies (carboplatin or cisplatin). Patients with a high volume of malignant ascites or excessive fluid buildup in the peritoneum are more likely to present with primary platinum-resistant disease. It is hypothesized that the physical forces generated by ascites may contribute to the dissemination and progression of ovarian cancer. Our research group has shown that fluid shear stress (FSS) induces carboplatin resistance and a motile and aggressive phenotype in monolayer cultures and 3D models for adherent ovarian cancer. In this study, the effects of FSS on treatment response to cisplatin and doxorubicin were examined. The latter is used to manage recurrent platinum-resistant disease. Changes in mitochondrial function under FSS as they relate to platinum resistance were also characterized. Finally, the use of photodynamic therapy (PDT), which utilizes 690 nm light and a clinical photosensitizer benzoporphyrin derivative (BPD) to locally generate cytotoxic reactive molecular species as a means of overcoming FSS-induced platinum resistance, was explored. Methods: Human epithelial ovarian adenocarcinoma cells OVCAR-3 and Caov-3 (ATCC) were cultured in tissue culture plates (static culture) or chamber slides (ibidi) connected to a perfusion pump for 48 h (flow rate: 0.11 mL/min, shear stress: 0.41 dyn/cm2). Cells in static and flow cultures were exposed to cisplatin, doxorubicin, BPD-PDT or combinations thereof and survival fractions were measured 72 h post-treatment. Mitochondrial membrane potential was measured using JC-1 fluorescence assay, and mitochondrial DNA (mtDNA) copy number was quantified using qPCR. ATP content per cell was measured with a CellTiterGlo luminescence assay. Results: OVCAR-3 and Caov-3 cells grown under FSS developed significant resistance to cisplatin. Specifically, 3.13 µM cisplatin induced a 50% decrease in the survival fraction in OVCAR-3 static cultures but failed to decrease the survival fraction under flow. In contrast to these findings with platins, no resistance to doxorubicin was observed under flow. Evaluation of the mitochondrial function revealed increased ATP content and mtDNA copy number in flow cultures, suggesting that increased mitochondrial activity may contribute to platinum resistance. Finally, low-dose BPD-PDT (IC10, 0.15 J/cm2) enhanced the efficacy of cisplatin in OVCAR-3 cells under flow, indicating that PDT is a viable strategy to sensitize tumors exposed to FSS to platinum-based chemotherapy. Conclusions: Our data suggest that ovarian cancer cells that acquired resistance to platinum due to FSS remain sensitive to doxorubicin. Changes in mitochondrial function, including increased ATP content per cell and mtDNA copy number, have been identified as potential contributing factors to platinum resistance under FSS. Finally, low-dose BPD-PDT can be used to re-sensitize cells to cisplatin and overcome platinum resistance. Citation Format: Marta Overchuk, Brittany P. Rickard, Justin Tulino, Frances S. Ligler, Imran Rizvi. Mechanism-informed photochemical strategies to overcome fluid shear stress-induced platinum resistance in ovarian cancer [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 B111.

CircRAD23B promotes proliferation and carboplatin resistance in ovarian cancer cell lines and organoids

BackgroundCircular RNAs (circRNAs) are involved in the regulation of progression and drug resistance in ovarian cancer (OC). In the present study, we aimed to explore the role of circRAD23B, a newly identified circRNA, in the regulation of carboplatin-resistant OC.MethodsCircRAD23B expression levels were measured using qRT-PCR. The biological roles of circRAD23B were analysed using CCK-8, colony formation, EDU, flow cytometry, and cell viability assays. RNA pull-down and luciferase assays were used to investigate the interactions of circRAD23B with mRNAs and miRNAs.ResultsCircRAD23B was significantly increased in carboplatin-resistant OC tissues. CircRAD23B promoted proliferation and reduced sensitivity to carboplatin in cell lines and patient-derived organoids (PDOs), consistent with in vivo findings. Mechanistically, circRAD23B acted as a molecular sponge, abrogating its inhibitory effect on Y-box binding protein 1 (YBX1) by adsorbing miR-1287-5p. Rescue experiments confirmed that the pro-proliferation and carboplatin resistance mediated by circRAD23B was partially reversed by the upregulation of miR-1287-5p.ConclusionsOur results demonstrated, for the first time, the role of the circRAD23B/miR-1287-5p/YBX1 axis in OC progression and carboplatin resistance in cell lines, PDOs, and animal models, providing a basis for the development of targeted therapies for patients with OC.

Abstract B086: Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models

Abstract Background: Triple negative breast cancer subtype is associated with poor prognosis and is mostly treated with chemotherapy, including the DNA crosslinking agent Carboplatin. Carboplatin treatment leads to DNA damage and a cell cycle inhibition allowing DNA damage repair. However, many patients are resistant or develop resistance to Carboplatin. The identification of novel treatments against chemoresistant TNBC is a major unmet clinical need. Method: TNBC patient-derived cell lines (PDCs) from patient-derived xenografts (PDXs) generated from chemotherapy resistant tumors were used for shRNA high throughput screens to identify genetic vulnerabilities that could resensitize these cells to Carboplatin. Validation of this target was performed genetically by shRNA silencing and pharmacologically with commercially available target inhibitors both in vitro and in vivo, using Alamar Blue viability assays and calculating synergy using the Chou Talalay method. DNA damage was assessed using yH2AX expression, and apoptosis using c-PARP and Annexin V, by confocal microscopy, flow cytometry and/or western blotting. Results: We identified ataxia telangiectasia and Rad3-related protein (ATR) as a target in one of the cell lines screened and validated it with an ATR shRNA. We tested the combination of the ATR inhibitor (ATRi), Elimusertib, with Carboplatin and found the combination to be synergistic (Average CI=0.30). DNA damage was markedly increased with combination treatment and a pan-nuclear yH2AX staining together with increased pHH3 expression were observed, suggestive of mitotic catastrophe. Cells treated with Carboplatin accumulated in S phase and a shift toward G2/M when Elimusertib was added. Apoptosis was significantly increased with combination treatment. PKMYT1 and WEE1 are two kinases regulating cell cycle entry by inhibiting CDK1, which controls entry into mitosis. Carboplatin treatment resulted in an increase in PKMYT1 expression. PKMYT1 inhibition with RP6306 re-sensitized the cells to Carboplatin more than WEE1 inhibition with Adavosertib, and this synergy was further increased with low doses of Elimusertib. Gene silencing of WEE1 and PKMYT1 both re-sensitized the cells to Carboplatin, but only WEE1 silencing alone significantly prevent proliferation, suggesting the requirement of PKMYT1 activity for resistance but not for cell proliferation without Carboplatin. In vivo validation using the PDXs from which the cell lines have been derived, demonstrated a significant delayed in tumor growth and prolonged survival when combining Carboplatin with ATRi compared to Carboplatin alone in two models. Conclusion: ATR inhibition resensitizes drug resistant TNBC cells to Carboplatin both in vitro and in vivo. The combination of Carboplatin with ATRi led to DNA damage and mitotic catastrophe. PKMYT1 plays a role in Carboplatin-resistant TNBC, collaborating with ATR in managing the cell cycle response to DNA damage, and PKMYT1 inhibition also re-sensitizes cells to Carboplatin, an effect increased with the triple combination of adding ATR inhibition. Citation Format: Juliet Guay, Catherine Chabot, Cédric Darini, Marguerite Buchanan, Adriana Aguilar-Mahecha, Tim Kong, Connie Yang, Mark Basik, Sidong Huang. Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B086.

Abstract B095: Sacituzumab govitecan-based drug combinations overcome platinum/PARP inhibitor resistance in ovarian cancer models

Abstract Background: Ovarian cancer causes more deaths than any other gynaecological cancer per year in the USA. Sacituzumab govitecan (SG) is a TROP2-targeted antibody-drug conjugate, approved for the treatment of triple-negative and hormone receptor-positive breast cancers. TROP2 is commonly overexpressed in ovarian cancer and represents an attractive, potential therapeutic target in ovarian cancer. This study investigated the in vitro activity of SG alone and in combination with targeted therapies/chemotherapies in ovarian cancer cell line models. Methods: This study utilized the PEO1 and PEO4 cell lines, which were derived from the same patient, with PEO4 acquiring a secondary restorative BRCA2 mutation to confer platinum resistance. Olaparib-resistant PEO1 (PEO1-Ola) cells were generated through continuous exposure to olaparib, to a maximum 13 μM, for four months. The anti-proliferative effects of SG, talazoparib, olaparib, carboplatin, and paclitaxel were tested in PEO1, PEO1-Ola, and PEO4 cell lines. Synergism was assessed using Combenefit software with the Loewe additivity model. Induction of caspase 3/7 activation, a marker of apoptosis, was tested with single agents and combinations using kinetic, fluorescent microscopy with the Incucyte S3 imaging system. Results: Olaparib and carboplatin resistance did not result in their cross-resistance. PEO1-Ola showed similar sensitivity to carboplatin as PEO1 parental cells, while carboplatin-resistant PEO4 cells displayed clinically achievable sensitivity to olaparib and talazoparib. SG was highly effective in all models tested. SG in combination with carboplatin or talazoparib was synergistic across all cell lines and resulted in induction of apoptosis. Conclusions: This pre-clinical study showed that TROP2-targeting ADC-based drug combinations warrant further investigation for the treatment of ovarian cancer. Citation Format: Neil T Conlon, Elena Möhrle, Luna Stockmann, John Crown. Sacituzumab govitecan-based drug combinations overcome platinum/PARP inhibitor resistance in ovarian cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B095.

Carboplatin-induced upregulation of pan β-tubulin and class III β-tubulin is implicated in acquired resistance and cross-resistance of ovarian cancer.

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III β-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other β-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking β-tubulin isotype compensation visible at the level of total β-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

HLF promotes ovarian cancer progression and chemoresistance via regulating Hippo signaling pathway

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, the role of HLF in the regulation of ovarian cancer (OC) remains unknown. Herein, we reported that HLF expression was upregulated in OC tissues and ovarian cancer stem cells (CSCs). Functional studies have revealed that HLF regulates OC cell stemness, proliferation, and metastasis. Mechanistically, HLF transcriptionally activated Yes-associated protein 1 (YAP1) expression and subsequently modulated the Hippo signaling pathway. Moreover, we found that miR-520e directly targeted HLF 3′-UTR in OC cells. miR-520e expression was negatively correlated with HLF and YAP1 expression in OC tissues. The combined immunohistochemical (IHC) panels exhibited a better prognostic value for OC patients than any of these components alone. Importantly, the HLF/YAP1 axis determines the response of OC cells to carboplatin treatment and HLF depletion or the YAP1 inhibitor verteporfin abrogated carboplatin resistance. Analysis of patient-derived xenografts (PDXs) further suggested that HLF might predict carboplatin benefits in OC patients. In conclusion, these findings suggest a crucial role of the miR-520e/HLF/YAP1 axis in OC progression and chemoresistance, suggesting potential therapeutic targets for OC.

The PRMT5 inhibitor C9 mitigates hypoxia-induced carboplatin resistance in lung cancer by inducing autophagy.

Hypoxia, a common feature of solid tumors, can promote chemoresistance in cancer cells. PRMT5 mediates various cellular processes involved in cancer development and progression. However, the role of PRMT5 in hypoxia-induced chemoresistance is unclear. In this study, hypoxia upregulated PRMT5 expression in lung cancer cells. Additionally, PRMT5 overexpression promoted cancer cell resistance to carboplatin. In carboplatin-resistant cancer cells, PRMT5 overexpression promoted the methylation of ULK1, a critical regulator of autophagy. ULK1 hypermethylation leads to the upregulation of autophagy, which can improve the survival of cancer cells under hypoxic conditions. Furthermore, this study demonstrated that the PRMT5 inhibitor C9 significantly enhanced the sensitivity of lung cancer cells to carboplatin. These findings suggest that targeting PRMT5-mediated autophagy with C9 can overcome hypoxia-induced carboplatin resistance and improve the efficacy of chemotherapy in patients with cancer.

Abstract 3895: New potential mechanism of mnx1-as1 in the regulation of the carboplatin chemoresistant phenotype in ovarian cancer cell lines

Abstract Introduction: Ovarian cancer (OC) is one of the most lethal diseases in the female population worldwide, being considered by 2020 the eighth most common type of cancer and the eighth cause of cancer death in that population. The most widely used chemotherapy treatment is based on the application of platinum based drugs, carboplatin (CBDCA) being one of the most common, in combination with taxanes and cisplatin. Unfortunately, a high percentage of the population relapses shortly after chemotherapy treatment, presenting a drug resistant phenotype. This phenotype reduces overall survival in patients with OC. In this context, various mechanisms have been investigated to understand and study these chemoresistant phenotypes. In the last decade, lncRNAs have been identified in multiple biological processes, including the development of drug-resistant phenotypes in malignant tumors, however, little is known about their role in carboplatin resistance in OC. This study seeks to characterize the effect of lncRNA expression on the chemoresistant phenotype and its potential molecular mechanism involved. Materials and Methods: Ovarian cancer cell lines corresponding to: A2780-R-CBDCA, A2780-P, UCI-101, OVCAR3 and SKOV3 were used. Drug susceptibility assays were performed using MTT. Relative expression was performed via RTqPCR. RNAseq was performed on a CBDCA resistance model in vitro using the Illumina HiSeq 4000 platform. Gene Ontology (GO) was analyzed using ClueGO. Gene set enrichment analysis (GSEA) was performed to define enriched biological pathways. Triplexator was used to predict the triplex forming oligonucleotides (TFO) and triplex target sites (TTS) that may be involved in the formation of RNA-DNA triplexes. Results: RNAseq analysis showed 156 differentially expressed protein coding genes (DEGs). The most enriched and deregulated signaling pathway was Wnt/β-catenin. In this context, several regulators of this pathway were found to be altered, including sFRP1, sFRP4, PCDHB6, CTNNA2, DACT1, PCDHB6, WNT3A, and CER1. In addition, 17 lncRNAs involved in CDBCA resistance were found to be deregulated. In this sense, MNX1-AS1 showed high levels of expression in CBDCA resistant cell lines. Interestingly, it has been shown that MNX1-AS1 has the ability to form a triplex with the PCDHB6 gene, which is a tumor suppressor gene and whose expression was found to be decreased in CBDCA resistant cell lines. Conclusions: Our results indicate that MNX1-AS1 expression could contribute to the modulation of platinum drug resistance through regulation of PCDHB6 expression via triplexes formation in OC. This finding could explain a new mechanism in CBDCA resistance in OC. Citation Format: Tamara Alejandra Viscarra Alvarez, Kurt Leopoldo Buchegger Mena, Daniela Inés León Garrido, Carmen Gloria Ili Gangas, Jorge Sapunar Zenteno, Marcela Berrios Flores, Ramón Silva Pezoa, Sindy Paola Cabarca Barreto, Priscilla Brebi Mieville. New potential mechanism of mnx1-as1 in the regulation of the carboplatin chemoresistant phenotype in ovarian cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3895.

Rewiring of the 3D genome during acquisition of carboplatin resistance in a triple-negative breast cancer patient-derived xenograft

Changes in the three-dimensional (3D) structure of the genome are an emerging hallmark of cancer. Cancer-associated copy number variants and single nucleotide polymorphisms promote rewiring of chromatin loops, disruption of topologically associating domains (TADs), active/inactive chromatin state switching, leading to oncogene expression and silencing of tumor suppressors. However, little is known about 3D changes during cancer progression to a chemotherapy-resistant state. We integrated chromatin conformation capture (Hi-C), RNA-seq, and whole-genome sequencing obtained from triple-negative breast cancer patient-derived xenograft primary tumors (UCD52) and carboplatin-resistant samples and found increased short-range (< 2 Mb) interactions, chromatin looping, formation of TAD, chromatin state switching into a more active state, and amplification of ATP-binding cassette transporters. Transcriptome changes suggested the role of long-noncoding RNAs in carboplatin resistance. Rewiring of the 3D genome was associated with TP53, TP63, BATF, FOS-JUN family of transcription factors and led to activation of aggressiveness-, metastasis- and other cancer-related pathways. Integrative analysis highlighted increased ribosome biogenesis and oxidative phosphorylation, suggesting the role of mitochondrial energy metabolism. Our results suggest that 3D genome remodeling may be a key mechanism underlying carboplatin resistance.

Abstract P1-13-23: Carboplatin resistance-associated changes in the 3D chromatin landscape of a triple-negative breast cancer Patient-Derived Xenograft

Abstract Changes in the three-dimensional (3D) structure of the genome are an emerging hallmark of cancer. Cancer-associated copy number variants and single nucleotide polymorphisms promote rewiring of chromatin loops, disruption of topologically associating domains (TADs), active/inactive chromatin state switching, leading to oncogene expression and silencing of tumor suppressors. However, little is known about 3D changes during cancer progression to a chemotherapy-resistant state. We integrated chromatin conformation capture (Hi-C), RNA-seq, and whole-genome sequencing obtained from triple-negative breast cancer patient-derived xenograft primary tumors (UCD52) and carboplatin-resistant samples and found increased short-range (&amp;lt; 2Mb) interactions, chromatin looping, formation of topologically associating domains (TAD), chromatin state switching into a more active state, and amplification of ATP-binding cassette (ABC) transporters. Transcriptome changes suggested the role of long-noncoding RNAs in carboplatin resistance. Rewiring of the 3D genome was associated with TP53, TP63, BATF, FOS-JUN family of transcription factors and led to activation of aggressiveness-, metastasis- and other cancer-related pathways. Integrative analysis highlighted increased ribosome biogenesis and oxidative phosphorylation, suggesting the role of mitochondrial energy metabolism. Our results suggest that 3D genome remodeling may be a key mechanism underlying carboplatin resistance. Citation Format: Mikhail Dozmorov, Maggie Marshall, Narmeen Rashid, Jacqueline Grible, Aaron D. Valentine, Amy Olex, Kavita Murthy, Abhijit Chakraborty, Joaquin Reyna, Daniela Salgado Figueroa, Da-Inn Lee, Brittany Baur, Sushmita Roy, Ferhat Ay, Chuck Harrell. Carboplatin resistance-associated changes in the 3D chromatin landscape of a triple-negative breast cancer Patient-Derived Xenograft [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-23.

Upregulation of programmed death ligand-1 in tumor-associated macrophages affects chemotherapeutic response in ovarian cancer cells.

To better understand the mechanism of chemoresistance in ovarian cancer cells, we aimed to investigate the influence of macrophages on the tumor cell response to carboplatin and identify the genes associated with chemoresistance. We mimicked the tumor microenvironment (TME) using a co-culture technique and compared the proliferation of ovarian cells with and without macrophages. We also examined M1 and M2 marker expression and the expression of key TME genes. Post the co-culture, we treated ovarian cancer cells with carboplatin and elucidated the function of programmed death-ligand 1 (PD-L1) in carboplatin chemoresistance. We investigated CD68 and PD-L1 expression in normal and cancerous ovarian tissues using immunohistochemistry (IHC). Finally, we analyzed the association between CD68 or PD-L1 expression and survival outcomes. Inducible nitric oxide synthase (iNOS) was downregulated, while the gene expression of M2 macrophage markers was increased in ovarian cancer cells. PD-L1, vascular endothelial growth factor (VEGF), Interleukin (IL)-6, IL-10, IL-12, signal transducer and activator of transcription 3 (STAT3), B-cell lymphoma 2 (BCL2), multidrug resistance 1 (MDR1), and colony stimulating factor 1 (CSF-1) were upregulated. Notably, PD-L1 was upregulated in both the ovarian cancer cells and macrophages. Ovarian cancer cells co-cultured with macrophages exhibited statistically significant carboplatin resistance compared to single-cultured ovarian cancer cells. PD-L1 silencing induced chemosensitivity in both types of co-cultured ovarian cancer cells. However, IHC results revealed no correlation between PD-L1 expression and patient survival or cancer stage. CD68 expression was significantly increased in cancer cells compared to normal or benign ovarian tumor cells, but it was not associated with the survival outcomes of ovarian cancer patients. Our study demonstrated that ovarian cancer cells interact with macrophages to induce the M2 phenotype. We also established that PD-L1 upregulation in both ovarian cancer cells and macrophages is a key factor for carboplatin chemoresistance.