N6-methyladenosine (m6A) has emerged as a pivotal regulator of post-transcriptional gene control, yet its contribution to chemotherapy resistance remains insufficiently defined. Here, we describe a previously unrecognized METTL3-ADAM23 epitranscriptomic regulatory relationship associated with platinum (Pt) resistance in ovarian cancer (OC). We show that cisplatin treatment increases global m6A levels and METTL3 expression, linking Pt exposure to activation of the m6A machinery. Functional perturbation studies demonstrate that METTL3 overexpression enhances cisplatin resistance, whereas METTL3 knockdown or pharmacologic inhibition with the selective METTL3 inhibitor STM2457 sensitizes OC cells to Pt treatment in vitro and improves Pt response in vivo. Transcriptomic profiling identifies ADAM23, a cell-adhesion-related tumor suppressor, as a METTL3-dependent, m6A-associated transcript, with altered mRNA expression observed across multiple experimental systems and several high-confidence predicted m6A sites within its transcript. Cisplatin-associated METTL3 upregulation correlates with reduced ADAM23 expression, suggesting a potential regulatory relationship that may contribute to chemoresistance. Together, these findings support a model in which METTL3-associated increases in m6A methylation are linked to Pt resistance, in part through modulation of ADAM23 expression, and highlight METTL3 as a potential therapeutic target in OC.

Ovarian cancer has the highest mortality rate among gynecological cancers. Relapse of the disease accompanied by chemoresistance is common; however, the biological causes are still unclear. Although initial chemotherapy succeeds in debulking the burden of OC, it promotes emergence of cancer stem cells (CSCs), which are responsible for driving tumor relapse and developing chemo-resistant tumors in OC. Myeloid Ecotropic Insertion Site 2 (MEIS), is a homeobox transcription factor (TF), deregulated in various cancers, which plays critical roles in regulating stem cell identity and cell fate decision. Here, we examined the role of MEIS2 in regulating stemness features that promote chemoresistance of OC, and the impact of a MEIS2 blockade on OC initiation and progression. ​ Paired isogenic OC cell lines (OVCAR4/5 and COV362 platinum sensitive (Pt-S) and resistant (Pt-R)) were used. MEIS2 expression level in these cells were assessed by q-RT-PCR and Western Blotting. The effects of MEIS2 inhibition (by using a pharmacological inhibitor MEIS2i and biological knockdown (KD) on OC cell survival were assessed using colony formation and cell proliferation assays, and on stemness features by flow cytometry for analysis of ALDH+ cancer stem cell (CSC) population and measurement of stemness-related gene expression. ​MEIS2 direct binding targets in OC cells, were examined using ChIP-seq. The anti-tumor effects of MEIS2 inhibition on Pt-R OC cells were tested in vivo. MEIS2 expression levels were increased in Pt-R vs. Pt-S OC cells at mRNA and protein levels. Treatment with MEIS2i (500nM-1μM) reduced the number of colonies in OC cells by at least 2-fold change (p<0.001); however, proliferation of cells was not diminished. Treatment with MEIS2i (250nM-1μM, 48 hours) decreased ALDH+ CSCs population by at least ∼2-fold (p<0.05), and inhibited the expression of stemness associated genes ALDH1A1, ALDH1A3, and Sox2 (p<0.05), compared to vehicle control. Knock down of MEIS2 decreased the CSC population from 29.4% to 14.3% (p<0.05) and inhibited stemness gene expression (p<0.05). ​MEIS2 knockdown inhibited tumor initiating capacity in vivo. OC xenografts derived from MEIS2 knockdown OVCAR5 cells were more responsive to carboplatin in vivo (p<0.05) compared with control xenografts. By integrating ChIP seq and RNA seq, we identified stemness-associated genes, ALDH1A2 and CD44 as direct MEIS2 targets contributing to maintaining stemness and chemo-resistance. Overexpression of MEIS2 contributes to stemness in OC. MEIS2 blockade inhibited stemness traits that contribute to chemotherapy resistance and tumor relapse. Our results put forward MEIS2 as a potential new target. Yinu Wang, Natalia Obrochta, Junzui Li, Daniela Matei. Targeting MEIS2 in platinum resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4410.

Ovarian cancer is lethal because of near-universal development of resistance to platinum-based chemotherapy. Metabolic adaptations can play a pivotal role in therapy resistance. In this study, we aimed to identify key metabolic pathways that regulate platinum response and represent potential therapeutic targets. Transcriptomic and metabolomic analyses in cisplatin-sensitive and -resistant ovarian cancer cells identified enrichment of pyrimidine metabolism related to upregulated de novo pyrimidine synthesis. The 15N-glutamine flux analysis confirmed increased de novo pyrimidine synthesis in cisplatin-resistant cells. Targeting this pathway using brequinar (BRQ), an inhibitor of the key enzyme dihydroorotate dehydrogenase, decreased cell viability, delayed G2/M cell-cycle progression, and altered expression of genes related to mitochondrial electron transport in resistant cells. Under basal conditions, cisplatin-resistant cells had a lower oxygen consumption rate and spare respiratory capacity than cisplatin-sensitive cells. BRQ suppressed the oxygen consumption rate in both sensitive and resistant cells but only inhibited spare respiratory capacity in resistant cells. In cell line-derived and patient-derived xenograft models, BRQ attenuated the growth of cisplatin-resistant ovarian tumors and enhanced the inhibitory effects of carboplatin. Together, these results identify metabolic reprogramming in cisplatin-resistant ovarian cancer that induces an acquired dependency on de novo pyrimidine synthesis, which can be targeted to sensitize tumors to chemotherapy.Significance: De novo pyrimidine synthesis supports platinum resistance in ovarian cancer and can be targeted with DHODH inhibitors to suppress tumor growth, pointing to potential metabolic therapies for treating recurrent ovarian cancer.

5596 Background: Abemaciclib inhibits cyclin dependent kinases (CDK) 4 and 6 and, in synergy with anti-estrogens, is an FDA approved treatment of ER+ breast cancer. Endometrioid endometrial cancer frequently express the estrogen receptor and harbors PTEN, PI3K, KRAS and CTNNB1 mutations which induce estrogen-mediated upregulation of cyclin D1 and cancer cell proliferation. We hypothesized that the combination of letrozole and abemaciclib will inhibit cellular proliferation, as measured by Ki-67, in patients with endometrial cancer. Methods: This is a single-arm prospective multicenter surgical window of opportunity clinical trial for patients with endometrial cancer planned to undergo definitive surgical management with hysterectomy. Eligibility criteria included endometrioid histology, no prior treatment for endometrial cancer, and a window of greater than 15 days prior to scheduled hysterectomy. Patients received 14 days of abemaciclib 150mg twice daily and letrozole 2.5mg once daily. The primary endpoint was change in Ki-67 (%) from pretreatment biopsy to posttreatment hysterectomy specimen. Secondary endpoints included biological tumor characteristics (MMR, PTEN, PI3K, cyclin D1, pRB) associated with Ki-67 changes. The planned sample size was n=21 evaluable patients which would achieve 80% power with two-sided α = 0.05 to detect a 12.9% decrease in Ki67 expression based on a paired t-test. Results: A total of 27 women were enrolled, 2 patients withdrew consent leaving 25 evaluable patients. The median age was 62 (IQR: 54 - 66) and 19 patients (76%) were menopausal. Most patients were White (68%), 24% identified as Black; 20% were of Hispanic ethnicity. Median BMI was 37 (IQR: 30-40). All cancers were endometrioid with the majority being FIGO grade 1 (76%) and 24% grade 2. Nine (33%) patients had mismatch repair deficient tumors. Twenty-two (88%) patients were stage I, two (8%) stage II, and 1 (4%) stage IV. Mean baseline Ki-67 was 34% (SD 22%). After 2 weeks of treatment with abemaciclib and letrozole mean Ki67 was 14% (SD 16%). The mean Ki-67 reduction was 19% (SD 23%, p<0.001) meeting the prespecified endpoint. We found no significant difference in the magnitude of Ki-67 reduction based upon present or absent staining for pRb, PTEN, cyclin D1, p16, or MMR proteins. Post-menopausal patients had a decrease in Ki-67 of 26% compared to 15% for those who were premenopausal, but this did not reach statistical significance (p=0.5). There were no serious adverse events recorded. The most common study related adverse events were grade 1-2 diarrhea (68%) and grade 1-2 nausea (36%). Two patients had grade 3 hematological events (anemia and neutropenia), which resolved. Conclusions: Abemaciclib and letrozole induced a reduction in cellular proliferation in endometrioid endometrial cancer over a relatively short two week time period prior to surgery, suggesting clinical efficacy. Clinical trial information: NCT04049227 .

Methylome changes in peripheral blood mononuclear cell (PBMCs) from small cell lung cancer (SCLC) patients treated with an epigenetic therapy revealed global hypomethylation and altered cancer signaling processes associated with tumor progression, immune response, therapy resistance and significant change in the proportion of immune cells. Integrating blood-based methylation biomarkers into clinical trials of epigenetic therapy and methylomic analysis of PBMCs provides direct monitoring of treatment effects in cancer patients, which may improve patient selection and enable real-time response assessment in patients receiving hypomethylating agents. Background:Small-cell lung cancer (SCLC) represents 15% of lung cancers and with a 5-year survival rate under 7% remains one of the deadliest malignancies. Although initially responsive to chemotherapy, rapid recurrence and resistance are common. Epigenetic modifications, particularly DNA methylation, contribute to tumor progression and therapy resistance. Guadecitabine, a hypomethylating agent (HMA), has shown promising clinical activity when combined with carboplatin in preclinical models. We evaluated the combination of guadecitabine with carboplatin as a second-line treatment for extensive-stage SCLC (NCT03913455). Here we report methylome changes in peripheral blood mononuclear cell (PBMCs) collected at baseline and during treatment from patients on the trial. Results:PMBC DNA was analyzed using Infinium HumanMethylationEPIC v1.0 bead chips. Data were processed and differentially methylated positions (DMPs) were identified and analyzed for pathway enrichment using bioinformatic approaches and immune deconvolution analyses were conducted to investigate the impact on immune cell composition. Direct comparison of PBMCs between cycle 2 day 5 (C2D5; post-treatment) vs cycle 1 day 1 (C1D1; pre-treatment) revealed a greater number of hypomethylated DMPs (380 DMPs in C2D5 vs C1D1 PBMCs; p < 0.05, |β| > 20%). Moreover, when first compared with normal PBMCs from cancer-free controls, the number of hypomethylated DMPs was even greater in C2D5 than in C1D1 (1,771 vs 237 DMPs, respectively; p < 0.05, |β| > 20%). Long interspersed nucleotide elements-1 (LINE-1) were also significantly hypomethylated in PBMCs after HMA treatment (C2D5), compared to C1D1. Pathway analysis of hypomethylated DMPs revealed significant alterations in key signaling pathways including NF-κB, Rho GTPase, pulmonary fibrosis, and p75 NTR in C1D1 vs C2D5. When normal PBMCs were compared to C1D1 PBMCs, changes in IL-3 signaling, Fcγ receptor-mediated phagocytosis, and molecular mechanisms of cancer were observed. Deconvolution analysis revealed a significantly higher percentage of monocytes in C1D1 PBMCs vs normal PBMCs. However, after HMA treatment, percentages of monocytes and B cells decreased, while eosinophil percentage increased in C1D1 compared to C2D5 PBMCs. Conclusion:In the first study on the global impact of HMA treatment on PBMC methylomes in SCLC patients, DNA methylation changes associated with biological pathways related to PBMC function reveal shifts in distinct immune cell populations.

Background/Objectives: Epigenetic modifications, particularly DNA methylation, contribute to tumor progression and therapy resistance. Guadecitabine, a hypomethylating agent (HMA), has shown promising clinical activity when combined with carboplatin in preclinical models. We evaluated the combination of guadecitabine with carboplatin as a second-line treatment for extensive-stage small-cell lung cancer (SCLC; NCT03913455), one of the deadliest malignancies. Here, we report methylome changes in peripheral blood mononuclear cells (PBMCs) collected at baseline and during treatment from patients on the trial. Methods: PMBC DNA was analyzed using Infinium HumanMethylationEPIC v1.0 bead chips. Data were processed, and differentially methylated positions (DMPs) were identified and analyzed for pathway enrichment using bioinformatic approaches, and immune deconvolution analyses were conducted to investigate the impact on immune cell composition. Results: Direct comparison of PBMCs between cycle 2 day 5 (C2D5; post-treatment) vs. cycle 1 day 1 (C1D1; pre-treatment) revealed a greater number of hypomethylated DMPs (380 DMPs in C2D5 vs. C1D1 PBMCs; p < 0.05, |β| > 20%). Moreover, when first compared with normal PBMCs from cancer-free controls, the number of hypomethylated DMPs was even greater in C2D5 than in C1D1 (1771 vs. 237 DMPs, respectively; p < 0.05, |β| > 20%). Long interspersed nucleotide elements-1 (LINE-1) were significantly hypomethylated in PBMCs after HMA treatment (C2D5 vs. C1D1). Pathway analysis of hypomethylated DMPs revealed significant alterations in key signaling pathways, including NF-κB, Rho GTPase, and pulmonary fibrosis in C1D1 vs. C2D5. Normal PBMCs to C1D1 PBMCs revealed changes in IL-3 signaling, Fcγ receptor-mediated phagocytosis, and molecular mechanisms of cancer. Deconvolution analysis revealed a greater percentage of monocytes in C1D1 vs. normal PBMCs; after HMA treatment, percentages of monocytes and B cells decreased, while the eosinophil percentage increased in C1D1 vs. C2D5. Conclusions: HMA treatment has a global impact on PBMC methylomes in cancer patients. DNA methylation changes were associated with biological pathways related to PBMC function, and shifts in distinct immune cell populations were observed.

BACKGROUND:Platinum resistant ovarian cancer (PROC) is associated with poor survival. This clinical trial sought to determine whether the bispecific CD47 inhibitor and CD40 agonist, SL-172154, could be combined safely and improve the efficacy of mirvetuximab (MIRV) or pegylated liposomal doxorubicin (PLD) through enhanced tumor cell phagocytosis and antigen presentation to T cells. METHODS:Patients with PROC received MIRV (21-day cycle) or PLD (28-day cycle) on day 1 and SL-172154 (3 mg/kg) on days 8 and 15 of each cycle. Objectives included safety, anti-tumor activity, PK, and immunogenicity. RESULTS:65 patients (60% folate receptor alpha (FRα) high and 40% medium/low) were enrolled in the MIRV cohort and 21 patients in the PLD cohort. Most common TEAEs ( > 40%) in the MIRV cohort were blurred vision, nausea, infusion related reaction, transaminase increase, diarrhea, and in the PLD cohort, nausea, constipation, neutropenia and fatigue. The objective response rate in the MIRV cohort was 33% (95% CI, 19%, 50%) for FRα high subgroup, and 15% (95% CI, 4%, 35%) for FRα medium/low subgroup, and in the PLD cohort was 20% (95% CI, 6%, 44%). CONCLUSIONS:Combination of SL172154 with MIRV did not improve upon the previously reported ORR for MIRV, while the combination with PLD resulted in a higher ORR than reported for PLD, albeit in a small number of patients. Limited tumor penetration of SL-172154, lack of durable CD47 blockade, inability to overcome T cell exhaustion and other mechanisms of resistance may explain these findings. Trial register number: NCT05483933.

Background Ovarian cancer (OC) depends on lipids as fuel for metastasis and growth. We previously showed that cisplatin resistant (Pt-R) OC cells uptake higher amounts of fatty acids (FAs) compared to sensitive (Pt-S) cells, a process which facilitates cancer cell survival under cisplatin-induced oxidative stress. Methods Isogenic pairs of Pt-S and Pt-R OC cell lines were cultured in low serum conditions supplemented with either 50 μM oleic acid (OA, unsaturated) or 50 μM palmitic acid (PA, saturated) and used for viability assays, RNA-Sequencing, and cell cycle analysis. The effects of an OA enriched diet were assessed in intraperitoneal ovarian xenografts. The FABP inhibitor BMS-309403 was used to block lipid import in vitro and in vivo. Results Pt-R cells were less viable than Pt-S cells under serum depletion and OA rescued starvation induced inhibition of cell proliferation, with more significant effects in Pt-R compared to Pt-S cells. RNA-sequencing showed that OA promoted upregulation of cell cycle-related pathways, including G2/M checkpoints, driven by the transcription factor E2F1. Supplementation with OA increased S- and G2/M phase cell populations in both Pt-S and Pt-R cells (p<0.05) and E2F1 inhibition reduced OA-induced cell proliferation. An OA enriched diet promoted the growth and peritoneal dissemination of Pt-R ovarian xenografts. When co-cultured with adipocytes, Pt-R cells expressed higher levels of FA transporter proteins FABP4 and CD36 compared to sensitive cells and FABP4 expression was upregulated in paired metastatic and recurrent vs. primary human ovarian tumors (p<0.05). An FABP inhibitor sensitized OC cells to cisplatin and suppressed the in vivo growth of Pt-R xenografts and patient derived xenografts. Conclusions Pt-R OC cells harbor heightened dependence on unsaturated FAs compared to Pt-S cells and upregulate key transporters to increase FAs uptake. OA supports the proliferation of Pt-R cells in vitro and in vivo and a combination of carboplatin and FABP4 inhibitor reduces OC growth in vivo. These findings suggest that lipid composition may influence therapeutic response and raise important considerations for dietary guidance in patients with cancer.

Background:High grade serous ovarian cancer (HGSC) is initially a responsive tumor to platinum (Pt)-based therapy. Pt resistance in HGSC is associated with epigenetic modifications and hypomethylating agents (HMAs) have been studied as carboplatin resensitizing agents. As DNA methylation is detectable in cancer cells and in blood, here we aimed to develop a blood-based methylation signature associated with cancer and cancer recurrence in HGSC. Results:We evaluated genome-wide DNA methylation in de-identified peripheral blood mononuclear cells (PBMCs) from women 1) without cancer (controls, n=20); 2) newly diagnosed HGSC (prior to treatment, Pt-naive, n=60) 3) Pt-resistant recurrent HGSC before and after treatment with the novel HMA/DNA methyltransferase inhibitor (DNMTI) guadecitabine (Pt-resistant, n=30). The Pt-resistant patients were enrolled in NCT02901899 clinical trial testing guadecitabine and the PD-1 inhibitor pembrolizumab. DNA extracted from PBMCs was analyzed by using Infinium MethylationEPIC BeadChips. There were 30,369 differentially methylated loci (DMLs) in Pt-naïve patients vs. controls (adj. p < 0.05, β >10%), with most loci being demethylated. Enriched pathways in PBMCs from cancer patients included mechanisms of cancer, neutrophil degranulation, and cancer-related signaling pathways (PI3K/AKT, STAT3, HGF, interleukins). The number of DMLs was greater (880 DMLs; adj. p<0.05, β>10%) in Pt-resistant vs. Pt-naïve patients, and top enriched pathways associated with Pt-resistant HGSC included pathways in cancer, metabolic pathways, platelet activation, ABC transporters and signaling pathways (calcium, PI3K/AKT, MAPK, Ras, ErbB, Hippo, Wnt). Massive genome-wide hypomethylation 5 days after treatment with guadecitabine was observed (13,742 DMLs; adj. p<0.05, β>10%), which persisted 30 days after discontinuation of treatment. Pathways enriched by hypomethylated genes in PBMCs following guadecitabine treatment interestingly included pathways related to neuronal signaling, such as glutaminergic receptor signaling, axonal guidance signaling, synaptic long-term depression, synaptogenesis signaling and serotonin receptor signaling. Deconvolution analysis of the methylome data of PBMCs from Pt-resistant recurrent HGSC before versus after HMA treatment predicted increased naïve B cells, memory and naïve CD+ T cells, naïve CD4+ T cells, and neutrophils and decreased monocytes. Conclusions:We propose new DMLs associated with Pt-naive versus Pt-resistant HGSC. These findings can lead to new biomarkers for HGSC.

Sarcopenia, defined as a progressive loss of skeletal muscle mass, strength, and function, is a leading contributor to disability, dependence, and reduced quality of life (HRQoL) in older adults. This study aimed to evaluate the impact of a personalized six-month rehabilitation program, centered on tailored kinetic therapy, on physical performance and HRQoL in older women with primary sarcopenia. Methods: This prospective controlled study included 80 women aged ≥65 years, allocated into a Study Group (SG, n = 40), who followed a supervised personalized kinetic program, and a control group (CG, n = 40), who received general advice regarding physical activity and nutrition. Physical performance was measured using the short physical performance battery (SPPB), while HRQoL was assessed with the disease-specific SarQoL questionnaire. Evaluations were conducted at baseline and after six months. Results: At baseline, both groups had comparable scores (SPPB: SG = 5.75 ± 0.86 vs. CG = 5.8 ± 0.88, p = 0.798; SarQoL: SG = 54.42 ± 8.76 vs. CG = 55.59 ± 4.61, p = 0.457). After six months, the SG showed significant improvements (SPPB = 8.05 ± 0.90, p < 0.001; SarQoL = 62.55 ± 7.00, p < 0.001). Significant gains were observed in domains related to physical and mental health, locomotion, functionality, and leisure activities (p < 0.05). In contrast, the CG showed only minor, non-significant changes (SPPB = 6.17 ± 0.78; SarQoL = 56.51 ± 5.51). Conclusions: A structured, personalized kinetic program significantly improves physical performance and HRQoL in older women with primary sarcopenia. These results support the need for individualized, supervised rehabilitation programs in optimizing functional recovery and enhancing patient-centered outcomes in sarcopenia management.

Ovarian cancer (OC) depends on fat as fuel for metastasis and growth. We have previously demonstrated that cisplatin resistant (Pt-R) OC cells uptake higher amounts of fatty acids (FAs) compared to sensitive (Pt-S) cells. Here, we determined the mechanism through which FAs support cancer survival. We used two isogenic OC cell lines pairs, sensitive and resistant to cisplatin: OVCAR5 Pt-S (IC50 = 2.881mM) vs Pt-R (IC50 = 6.831 mM) and PEO1 (IC50 = 4.901 mM) vs PEO4 (IC50 = 8.285 mM). Cells were cultured under low serum conditions, supplemented with 50 µM oleic (OA) or 50 µM palmitic acid (PA), and used for viability assays, RNA-Seq, cell cycle, and cell death analysis. Quantitative RT-PCR measured expression of key FAs transporters in Pt-S and Pt-R cells. Immunohistochemistry (IHC) assessed expression of FABP4 in primary and recurrent human tumors. The FABP inhibitor BMS-309403 was used to pharmacologically inhibit the FA transporters in vitro and in vivo. Pt-R cells rely more on unsaturated FAs than Pt-S cells, upregulating transporters for increased FA uptake. Cell viability assays showed Pt-R cells were less viable under serum depletion, with oleic acid (OA, unsaturated) promoting proliferation of both OC cell lines. OA rescued Pt-R cells significantly more than Pt-S cells: 268.4% vs 91.6% for OVCAR-5 and 38.1% vs 12.1% for PEO4 vs PEO1. OA also reduced apoptosis, as shown by Annexin V assays, and decreased Caspase-3 and -8 cleavage. RNA-Seq analysis linked OA-induced proliferation to upregulation of cell cycle-related pathways, including G2/M checkpoints, driven by E2F1. OA increased S- and G2/M phase populations (e.g., 6.95% to 11.6%; p<0.05), while E2F1 inhibition reduced OA-induced proliferation. Pt-R cells also expressed higher levels of transported proteins FABP4 and CD36. Inhibiting FABPs reduced Pt IC50 values (e.g., from 11.893 μM to 5.0415 μM in OVCAR5) and blocked ovarian cancer progression in vivo. IHC showed FABP4 overexpression in metastatic and recurrent tumors (n=21 paired tumors, p<0.05). Pt-R OC cells are more dependent on unsaturated FAs compared to Pt-S cells and upregulate key transporters to allow increased uptake of FAs. Inhibiting intracellular FA transport reduces OC progression in vivo. OA supports proliferation of Pt-R cells by rescuing them from apoptosis and by promoting cell cycle progression from G1 to S- and G2/M phases. Ana Maria Isac, Andres Valdivia, Guangyuan Zhao, Yinu Wang, Jian-Jun Wei, Sandra Orsulic, Daniela Matei. Unsaturated fatty acids promote cell cycle progression and proliferation of platinum resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1536.

Ovarian cancer (OC) is the 8th most common cancer in women. Most patients with high-grade serous OC present with intraperitoneal disseminated disease when diagnosed, due to mild symptoms in earlier disease stages. They respond efficiently to cisplatin or taxol-based compounds in the front-line treatment; however, resistance emerges in the majority of cases, which allows tumors to progress. There is an urgent need for improved treatment to increase patients’ survival. Tissue transglutaminase (TG2) is a protein overexpressed in many solid tumors, including ovarian; it interacts with fibronectin (FN) in the extracellular matrix and promotes OC intraperitoneal dissemination. This makes TG2 a promising target for treatment. We previously identified MT4 as a promising small molecule inhibitor (SMI) able to disrupt TG2-FN protein-protein interaction, prevent cancer cell adhesion and spheroid formation, and sensitize OC cells to chemotherapeutic drugs, such as paclitaxel (Sima LE et al., Mol Cancer Ther., 2019). We recently investigated the effect of 5 new MT4 analogues on OC cells behavior. We found that compound #3011 completely prevented spheroids formation, similar to Tgm2 gene excision, while #3002 decreased cell adhesion onto FN. We used phosphoproteomics to analyze the adaptive signaling of OC cells treated with MT4 that escape treatment. New promising molecular TG2 co-targets for OC treatment have been identified as effectors of MT4 escape pathways: sirtuins and p21-activated kinases (PAKs). Combination of signaling inhibitors and TG2-directed SMIs induce OC cell death in both 2D and 3D conditions, with potential for future therapeutic use. Our multiplex IHC data shows that in OC tumors TG2 is expressed by cancer cells, as well as by α-SMA+ CAF subset. Therefore, we next sought to test the effect of TG2 drug targeting on OC cells-fibroblasts 3D co-cultures. Our results showed that MT4 prevents heterospheroid self-assembly in vitro, which indicates potential disruption in tumor-stroma communication using TG2-directed therapies. In parallel, we developed new tools to test drug combinations, as well as radiation therapy regimens for their potential to inhibit cancer spreading. We produced a new glass microfluidic chip and adapted a miniaturized mesothelial clearance assay for live cell imaging. We obtained new SKOV3 and SKOV3-TG2KO LifeAct+ stable cell lines to allow real time imaging measurements of OC cell behavior. Subpopulations were sorted based on actin-GFP expression level and characterized by cell motility quantification. Cell lines with behavior similar with parental cells were selected for further use. We currently use these tools to investigate the potential of TG2 targeting to enhance response of cancer cells to new FLASH radiation sources (Orobeti S et al, Sci. Rep., 2024), such as very high energy electrons generated with a high-intensity laser plasma accelerator system. Monica Tudor, Cristian Munteanu, Gabriela Chiritoiu, Stefana Orobeti, Alexandra Bran, Florin Jipa, Felix Sima, Gary E. Schiltz, Daniela Matei, Livia Elena Sima. Small molecule inhibitors targeting TG2 in ovarian cancer and identification of drug escape pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3867.

Direct visualization of metabolic conversions within living systems is essential for understanding metabolic activities yet challenging due to the absence of reaction-specific reporters and the limited sensitivity of current imaging modalities. Herein, we report an approach to monitor fatty acids (FAs) desaturation, primarily catalyzed by stearoyl-CoA desaturase, in cancer cells using deuterium (D)-labeled palmitic acid (PA-d31) as the reaction-specific reporter and mid-infrared photothermal (MIP) microscopy as the bond-selective imaging modality. The desaturation of PA-d31 produced a peak at 2246 cm-1 in the cell-silent region, corresponding to the stretching vibration of unsaturated C-D bonds (D-C=C-D) in unsaturated fatty acids. Penalized least squares fitting was employed to remove water background for enhancing the visibility of this peak. Our study revealed heterogeneous spatial distributions of both saturated FAs and their desaturated metabolites within lipid droplet pools in cancer cells. Furthermore, we observed an increase in fatty acid unsaturation level in OVCAR5 cells under cisplatin-induced stress. By directly visualizing fatty acid desaturation, this study offers new insights into fatty acid metabolism and opens avenues for evaluating new therapeutic strategies targeting fatty acid metabolism.

Ovarian cancer (OC) is the fifth leading cause of cancer death in women. OC inevitably acquires resistance to platinum-based chemotherapy, leading to treatment failure and disease progression. There is an unmet need to overcome platinum OC resistance. Arsenic trioxide (ATO) is an approved drug for treating acute promyelocytic leukemia, but not in solid tumors. Arsenoplatins (APs), a family of novel anticancer dual-pharmacophore agents containing ATO in the form of arsenous acid attached to a platinum center, can deliver ATO into the tumor cells. APs have previously shown in vitro potency in leukemia, breast, and ovarian cell lines in NCI-60 screens. It was hypothesized that APs (AP-1 and AP-7) could have therapeutic effects in cisplatin (CDDP) resistant OC due to the unique nature of the drug delivery. Three paired CDDP-sensitive and resistant OC cell lines (A2780, SKOV3, and OVCAR5) were used to test the potency of AP-1 and AP-7 compared to CDDP, ATO, or a combination of both. In addition, a single dose of maximum tolerated dose (MTD) was determined in mice, and the efficacy of AP-1 and AP-7 was evaluated in clinically translational platinum-resistant OC PDX tumor models. RNA-seq analysis of tumor tissue and in silico molecular docking was further studied to explore the mechanism of action to identify potential biomarkers for AP-7 therapy. AP-1 and AP-7 demonstrated great potency and showed similar cell growth inhibition in all tested CDDP-resistant cell lines. AUC, a parameter of combined measurement of both Emax and IC50, was used to rank the effectiveness of APs in these cell lines, with ATO being the most potent, followed by a combination of ATO and CDDP, then AP-1, AP-7, and CDDP was the least potent. Compared to the MTD for CDDP and ATO in mice, APs have much lower toxicity(higher MTD). AP-7, but not AP-1, demonstrated efficacy in the platinum-resistant OC PDX tumor study. AP-7 showed significant tumor volume reduction starting from week 3 (P<0.01); at the end of 5 weeks, there was a significant reduction of 60.78% (P<0.001) in tumor volume and 42.80% (P<0.01) in tumor weight over control. No body weight change was observed throughout the study. Molecular docking of AP-7 with GLI1 revealed that AP-7 binds to a pocket in proximity to the Zn-binding site of GLI1, potentially disrupting Zn coordination by interacting with key cysteine and histidine residues. The RNA-seq sequencing from AP-7 treated PDX tumor tissues was completed, and data analysis is ongoing for differential gene expressions and alternative splicing. Our preliminary results indicate that AP-7 is a potential therapeutic agent for overcoming platinum resistance in OC by delivering arsenous acid to tumor cells with greater efficacy and less toxicity. However, the prediction of potential target GLI1 warrants further validation. Wenan Qiang, Denana Miodragovic, Yi Yang, Bong Jin Hong, Chi-Hao Luan, David Fang, Daniela E. Matei, Thomas V. O’Halloran. Arsenoplatin-7, a family member of arsenoplatins, as a novel therapeutic strategy to overcome platinum-resistance in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1763.

Background: Sarcopenic obesity, characterized by reduced skeletal muscle mass and excess adiposity, is strongly associated with chronic pain and functional decline in older adults. Objective: This prospective controlled trial without randomization investigated the effects of a structured, three-phase rehabilitation program on physical performance, pain, and sarcopenia-specific quality of life in elderly patients with sarcopenic obesity and chronic pain. Methods: In this study, 82 participants were enrolled and allocated to a study group (SG, n = 40), receiving supervised rehabilitation, nutritional counseling, and supplementation, or to a control group (CG, n = 42), which did not receive rehabilitation. The final analysis included 35 patients in SG and 36 in CG. Outcomes were assessed at baseline and six months using the Sarcopenia Quality of Life Questionnaire (SarQoL), Short Physical Performance Battery (SPPB), Numeric Rating Scale (NRS), and Pressure Pain Threshold (PPT). Results: The SG showed significant improvements in all outcomes: SarQoL increased from 57.02 to 63.98, SPPB increased from 7.14 to 8.4, PPT increased from 69.31 to 78.05, and NRS decreased from 6.94 to 4.65 (all p < 0.001). The CG showed no significant changes. Conclusions: The implementation of a structured, three-phase rehabilitation program resulted in clinically and statistically significant improvements in physical performance, pain perception, and sarcopenia-related quality of life in older adults with sarcopenic obesity and chronic pain.

High grade serous ovarian cancer (OC) is initially a responsive tumor to platinum (Pt)-based therapy. Tumor suppressor genes (TSGs) are implicated in OC initiation and progression and can be inactivated through genetic or epigenetic events. DNA methylation has been shown to silence TSGs and other cancer-related genes and is detectable in cancer cells and in blood. Here we aimed to develop a blood-based methylation assay associated with cancer and cancer recurrence in OC. We evaluated genome-wide DNA methylation in de-identified peripheral blood mononuclear cells (PBMCs) from women: without cancer (controls, n=20), newly diagnosed OC (prior to treatment, Pt-sensitive, n=50), and platinum-resistant recurrent HGSOC before and after treatment with a hypomethylating agent (HMA, Pt-resistant, n=31). The Pt-resistant patients were enrolled on NCT02901899 clinical trial testing guadecitabine and pembrolizumab. DNA extracted from PBMCs was analyzed by using Infinium MethylationEPIC BeadChips. DNA methylation analysis used R package SeSAMe to derive beta (β) values and identify differential methylated loci (DMLs) and differential methylated regions (DMRs) between groups. Bioinformatics analysis was conducted to identify biological pathways and processes, determined using Ingenuity Pathway Analysis (IPA) and KEGG, GO:BP, REAC databases. Deconvolution analyses used the HEpiDISH algorithm to determine the cellular make-up of the samples. Comparing controls vs. Pt-sensitive groups, there were 30, 369 DMLs (adj. p<0.05, β>10%), with most loci being demethylated. Enriched pathways related to presence of cancer included: mechanisms of cancer, neutrophil degranulation, and cancer-related signaling pathways (PI3K/AKT, STAT3, HGF, interleukins). The number of DMLs was greater (880, adj. p<0.05, β>10%) in Pt-resistant vs Pt-sensitive and enriched pathways associated with Pt-resistant OC included: pathways in cancer, metabolic pathways, platelet activation, ABC transporters and signaling pathways (calcium, PI3K/AKT, MAPK, Ras, ErbB, Hippo, Wnt). Massive genome-wide hypomethylation 5 days after treatment with the HMA was seen (13, 742, adj. p<0.05, β>10%), consistent with observed LINE1 hypomethylation in PBMCs, and genome-wide hypomethylation persisted 30 days after end of HMA treatment. Enriched pathways among hypomethylated genes included: Th17 cell differentiation, virus infection, focal adhesion, signaling (chemokine, cGMP-PKG, Rap1) and hormone (thyroid, parathyroid, oxytocin, growth hormone) pathways. Deconvolution analysis identified decreased B and NK cells, monocytes, and eosinophils and a trend towards decreased CD4+ and CD8+ cells in Pt-sensitive OC vs controls. In contrast, neutrophils were increased in cancer vs control patients. We propose new DMLs associated with Pt-sensitive vs. Pt-resistant OC. These findings can lead to new biomarkers for HGSOC. Daniela Matei, Horacio Cardenas, Elnaz Abbasi Farid, Zhen Fu, Collin M. Coon, Adam Vieth, Kenneth P. Nephew. Blood based biomarkers of DNA methylation associated with platinum resistance in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4101.