Disrupting the interaction between tumor-cell surface PD-L1 and T cell membrane PD-1 can elicit durable clinical responses. However, only about 10% of ovarian cancer patients respond to PD-1/PD-L1 blockade. Here, we show that PD-L1 expression in ovarian cancer-patient tumors is predominantly intracellular. Notably, PARP inhibitor treatment highly increased intracellular PD-L1 accumulation in both ovarian cancer-patient tumor samples and cell lines. We investigated whether intracellular PD-L1 might play a critical role in ovarian cancer progression. Mutating the PD-L1 acetylation site in PEO1 and ID8Brca1-/- ovarian cancer cells significantly decreased PD-L1 levels and impaired colony formation, which was accompanied by cell cycle G2/M arrest and apoptosis induction. PEO1 and ID8Brca1-/- tumors with PD-L1 acetylation site mutation also exhibited significantly reduced growth in mice. Furthermore, targeting intracellular PD-L1 with a cell-penetrating antibody effectively decreased ovarian tumor-cell intracellular PD-L1 level and induced tumor-cell growth arrest and apoptosis, as well as enhanced DNA damage and STING activation, both in vitro and in vivo. In conclusion, we have shown the critical role of intracellular PD-L1 in ovarian cancer progression.

The introduction of poly (ADP-ribose) polymerase inhibitors (PARPi) into the management of ovarian cancer has transformed the treatment landscape for patients affected by this malignancy. However, as the use of PARPi expands into both frontline maintenance and recurrence settings, the emergence of drug resistance has become a significant clinical challenge in the treatment of these patients. Although platinum-based chemotherapy (PBC) and PARPi act through different mechanisms—PBC causes DNA damage while PARPi blocks its repair—both depend on the integrity of DNA damage repair (DDR) pathways, leading to overlapping mechanisms of resistance. Here, we review the key resistance mechanisms shared by PARPi and PBC, and then we discuss their clinical implications in the management of patients with ovarian cancer. We also examine clinical rationale supporting the hypothesis that prior PARPi exposure may reduce the efficacy of subsequent PBC in patients experiencing a disease recurrence. Furthermore, we review preliminary clinical data assessing the potential role of PARPi retreatment in patients who have previously progressed on PARPis.

Background Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment.Methods By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG’s catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition.Results Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors.Conclusions We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

Immune checkpoint blockade (ICB) therapy has significantly benefited patients with several types of solid tumors and some lymphomas. However, many of the treated patients do not have a durable clinical response. It has been demonstrated that rescuing exhausted CD8+ T cells is required for ICB-mediated antitumor effects. We recently developed an immunostimulatory strategy based on silencing STAT3 while stimulating immune responses by CpG, a ligand for Toll-like receptor 9 (TLR9). The CpG-small interfering RNA (siRNA) conjugates efficiently enter immune cells, silencing STAT3 and activating innate immunity to enhance T cell-mediated antitumor immune responses. In the present study, we demonstrate that blocking STAT3 through locally delivered CpG-Stat3 siRNA enhances the efficacies of the systemic PD-1 and CTLA4 blockade against mouse A20 B cell lymphoma. In addition, locally delivered CpG-Stat3 siRNA combined with systemic administration of PD-1 antibody significantly augmented both local and systemic antitumor effects against mouse B16 melanoma tumors, with enhanced tumor-associated T cell activation. Furthermore, locally delivered CpG-Stat3 siRNA enhanced CD8+ T cell tumor infiltration and antitumor activity in a xenograft tumor model. Overall, our studies in both B cell lymphoma and melanoma mouse models demonstrate the potential of combinatory immunotherapy with CpG-Stat3 siRNA and checkpoint inhibitors as a therapeutic strategy for B cell lymphoma and melanoma.

Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

IntroductionWhether adaptive immune cells are required for the formation of pre-metastatic niches critical for tumor metastasis remains unknown. Prior research indicates that Signal transducer and activator of transcription 3 (STAT3) contributes to the accumulation and function of innate immune cells in these niches. This study investigates whether CD4+ T cells can directly condition future metastatic sites and if STAT3 is necessary for CD4+ T cell-mediated niche formation.MethodsWe evaluated CD4+ T cell infiltration in non-metastatic lung regions of mice and examined the role of STAT3 signaling using CD4Cre-Stat3Flox transgenic mice lacking functional Stat3 in T cells. Clinical correlations in ovarian cancer patients‘ disease-free lung and liver tissue sites from rapid autopsy tissue collection were also analyzed.ResultsOur findings reveal that CD4+ T cells accumulate in distant tumor-free sites, forming pre-metastatic niches and subsequently promoting tumor metastasis in mice. Moreover, STAT3 activation is necessary for CD4+ T cell-mediated pre-metastatic niche formation. Depleting CD4+ T cells and STAT3 activation prior to primary tumor establishment significantly reduced tumor growth and almost completely blocked spontaneous lung metastasis, as evidenced in CD4-Stat3–/– mice. Importantly, analysis of presumed disease-free lung and liver tissue sites of ovarian cancer patients showed CD4+ T cell accumulation with activated STAT3 in non-tumor regions and surrounding micro-metastases.Conclusion/ImplicationsBlocking the pre-metastatic niche has the potential to prevent tumor cell seeding at distant sites, and our studies now show that targeting STAT3 in CD4+ T cells may be an effective strategy to prevent tumor metastasis.

Reprogrammed metabolism is a hallmark of cancer. However, the metabolic dependency of cancer, from tumour initiation through disease progression and therapy resistance, requires a spectrum of distinct reprogrammed cellular metabolic pathways. These pathways include aerobic glycolysis, oxidative phosphorylation, reactive oxygen species generation, de novo lipid synthesis, fatty acid β-oxidation, amino acid (notably glutamine) metabolism and mitochondrial metabolism. This Review highlights the central roles of signal transducer and activator of transcription (STAT) proteins, notably STAT3, STAT5, STAT6 and STAT1, in orchestrating the highly dynamic metabolism not only of cancer cells but also of immune cells and adipocytes in the tumour microenvironment. STAT proteins are able to shape distinct metabolic processes that regulate tumour progression and therapy resistance by transducing signals from metabolites, cytokines, growth factors and their receptors; defining genetic programmes that regulate a wide range of molecules involved in orchestration of metabolism in cancer and immune cells; and regulating mitochondrial activity at multiple levels, including energy metabolism and lipid-mediated mitochondrial integrity. Given the central role of STAT proteins in regulation of metabolic states, they are potential therapeutic targets for altering metabolic reprogramming in cancer.

Objectives To elucidate an off-target mechanism by which niraparib induces ovarian cancer cell apoptosis regardless of homologous recombination (HR) status through downregulation of the oncogenic SRC/STAT3 axis. Methods A variety of techniques were used to determine the underlying mechanisms by which niraparib regulates the SRC/STAT3 axis including tumor organoid formation, cell viability assays, colony formation assays, real-time PCR, western blot, apoptosis assays, cell transfections, in-cell and in-vitro thermal shift assays, and confocal microscopy. Results Niraparib exhibited more potent antitumor effects than olaparib in both HR deficient and proficient models. In addition to inhibiting PARP catalytic function, niraparib-promoted cell death in ovarian cancer cells was found to be mediated by its inhibitory effects on activated STAT3 (p-STAT3). Niraparib altered the expression of STAT3 downstream target genes, specifically those involved in apoptosis. The anti-apoptotic gene BCL-XL (BCL2L1), usually induced by STAT3 activation, was significantly reduced while the proapoptotic CASP3, CASP8, and CASP9 genes, which are suppressed by STAT3 activity, were markedly upregulated. Niraparib-mediated inhibition of the STAT3 pathway was found to be at least partially attributed to the downregulation of SRC kinase activity as demonstrated in all tested ovarian cancer cell lines and patient tumor-derived organoid models. Conclusions Niraparib inhibits the growth of ovarian cancer cells, regardless of HR status, more effectively than olaparib. Unlike olaparib, which is known to activate STAT3, niraparib inhibits STAT3 activity by interfering with SRC tyrosine kinase. These findings provide a potential off-target mechanism by which niraparib may provide benefit to ovarian cancer patients regardless of HR biomarker status.

Objectives: STAT3 is known to be hyperactivated in most human cancers and is associated with poor prognoses. The STAT3 activation pathway within human papillomavirus (HPV)-positive cervical cancer cells is known to be essential for cervical cancer cell proliferation and survival. However, whether STAT3 activity is elevated in cervical cancer patient tumors remains to be determined. We sought to assess whether activated STAT3 (p-STAT3) was more highly expressed in cervical squamous cell carcinoma tissue microarrays compared to benign adjacent cervix tissue. We also assessed the ability of a small molecule inhibitor of STAT3 that has been used in the clinic to attenuate the STAT3 activation pathway and induce tumor cell death of HPV-associated human squamous cell carcinoma cells in vitro. Methods: Tumor microarrays from 12 patients with cervical squamous cell carcinoma with adjacent, benign cervix tissue were first probed with an anti-p-STAT3 antibody and detected with a fluorophore-conjugated secondary antibody for p-STAT3. Confocal imaging of immunofluorescence was performed, and fluorescence quantification was performed by ZEN 2.3 lite software and plotted in GraphPad Prism 8 software. The CaSki HPV16 infected cervical squamous cell carcinoma cell line was used for in vitro experiments. CaSki cells were treated with BBI608 (a small molecule inhibitor of STAT3) and assessed for changes in the STAT3 activation axis using Western Blot. Cell viability was analyzed using CellTiter-Glo® Luminescent Cell Viability Assay. Results: Quantification of p-STAT3 immunofluorescence from confocal imaging showed significantly higher expression of p-STAT3 in cervical squamous cell carcinoma tissue microarrays compared to adjacent, benign cervix tissue with mean expression levels of 37.7 relative units (RU) vs 29.1 RU, respectively (p=0.006). On Western Blot, BBI608 (3uM and 4uM) was found to attenuate the STAT3 activation axis in a dose-dependent fashion, including a significant decrease in p-JAK1 and p-STAT3 protein expressions. Furthermore, the protein expression of HIF1A, a known downstream STAT3 target gene and associated with poor survival and treatment resistance in cervical cancer, was also decreased by BBI608 (3uM and 4uM) in a similar fashion. On analysis of cell viability, BBI608 was found capable of inducing cell death in CaSki cells with an IC50 of 2.6uM after a 48-hour incubation period. Objectives: STAT3 is known to be hyperactivated in most human cancers and is associated with poor prognoses. The STAT3 activation pathway within human papillomavirus (HPV)-positive cervical cancer cells is known to be essential for cervical cancer cell proliferation and survival. However, whether STAT3 activity is elevated in cervical cancer patient tumors remains to be determined. We sought to assess whether activated STAT3 (p-STAT3) was more highly expressed in cervical squamous cell carcinoma tissue microarrays compared to benign adjacent cervix tissue. We also assessed the ability of a small molecule inhibitor of STAT3 that has been used in the clinic to attenuate the STAT3 activation pathway and induce tumor cell death of HPV-associated human squamous cell carcinoma cells in vitro. Methods: Tumor microarrays from 12 patients with cervical squamous cell carcinoma with adjacent, benign cervix tissue were first probed with an anti-p-STAT3 antibody and detected with a fluorophore-conjugated secondary antibody for p-STAT3. Confocal imaging of immunofluorescence was performed, and fluorescence quantification was performed by ZEN 2.3 lite software and plotted in GraphPad Prism 8 software. The CaSki HPV16 infected cervical squamous cell carcinoma cell line was used for in vitro experiments. CaSki cells were treated with BBI608 (a small molecule inhibitor of STAT3) and assessed for changes in the STAT3 activation axis using Western Blot. Cell viability was analyzed using CellTiter-Glo® Luminescent Cell Viability Assay. Results: Quantification of p-STAT3 immunofluorescence from confocal imaging showed significantly higher expression of p-STAT3 in cervical squamous cell carcinoma tissue microarrays compared to adjacent, benign cervix tissue with mean expression levels of 37.7 relative units (RU) vs 29.1 RU, respectively (p=0.006). On Western Blot, BBI608 (3uM and 4uM) was found to attenuate the STAT3 activation axis in a dose-dependent fashion, including a significant decrease in p-JAK1 and p-STAT3 protein expressions. Furthermore, the protein expression of HIF1A, a known downstream STAT3 target gene and associated with poor survival and treatment resistance in cervical cancer, was also decreased by BBI608 (3uM and 4uM) in a similar fashion. On analysis of cell viability, BBI608 was found capable of inducing cell death in CaSki cells with an IC50 of 2.6uM after a 48-hour incubation period.

Recently, poly(ADP-ribosyl)ation polymerase inhibitors (PARPis), which induce synthetic lethality of tumor cells with DNA damage repair defects, have emerged as a promising therapy for ovarian, breast, and pancreatic cancer. Although the PARPi Olaparib is limited to treating cancer patients with DNA repair deficiencies, the PARPi Niraparib is FDA approved to treat ovarian cancer patients regardless of their status in DNA repair pathways. Despite differences in the affinity to PARP enzymes, the rationale behind the clinical use of Niraparib in patients without DNA repair deficiencies is still lacking. Moreover, only Olaparib has been approved for pancreatic ductal adenocarcinoma (PDAC) patients with BRCA mutations, accounting for only 5-7% of total PDACs. It remains unclear whether Niraparib could be beneficial to PDACs without BRCA mutations. We found that Niraparib inhibits ovarian and PDAC tumor cell growth, regardless of BRCA mutational status, more effectively than Olaparib. Unlike Olaparib, which is known to activate STAT3, Niraparib inhibits STAT3 activity in ovarian and PDAC cancer cell lines and patient tumors. Moreover, Niraparib regulates the expression of several STAT3 downstream genes involved in apoptosis. Overexpression of a constitutively activated STAT3 mutant rescues Niraparib-induced cancer cell apoptosis. Our results suggest that Niraparib inhibits pSTAT3 by interfering with SRC tyrosine kinase. Collectively, our studies provide a mechanism underlying Niraparib’s ability to induce tumor cell apoptosis without BRCA mutations, suggesting the potential use of Niraparib for treating PDAC patients regardless of BRCA status.

ObjectivePreclinical evidence and early clinical trials have demonstrated the activity of SPL-108, a targeted agent that inhibits CD44 mediated induction of multidrug resistance specifically to paclitaxel and platinum agents. We conducted a phase I, open label, dose escalation study of the safety and tolerability of the combination of SPL-108 with weekly paclitaxel in patients with platinum resistant CD44+ ovarian, primary peritoneal, or fallopian tube cancer.MethodsPatients with platinum resistant histologically proven epithelial ovarian, primary peritoneal, or fallopian tube cancers and measurable disease according to RECIST (Response Evaluation Criteria in Solid Tumours) version 1.1 were selected. Tumors were tested for CD44 expression for eligibility, defined as strong (+++) or moderate (++) staining in ≥20% of the tumor tissue or diffuse + staining. Patients were treated with daily and then twice daily SPL-108 subcutaneous injections and weekly intravenous paclitaxel on days 1, 8, and 15 of a 28 day cycle. Endpoints included safety, determination of maximum tolerated dose, and efficacy. Tumors underwent comprehensive genomic profiling, and cell lines and western blotting were used to study markers of response.ResultsWe screened 16 patients, and 14 were enrolled based on CD44+ expression. A total of 86% of patients had high grade serous tumors and all had received multiple prior therapies. There were no grade 4–5 toxicities. One patient had grade 3 peripheral sensory neuropathy attributed to paclitaxel and one patient developed presumed colonic perforation attributed to the study drug. No dose reductions or treatment discontinuations were required. All patients tolerated the maximum planned dose; no maximum tolerated dose was reached. Overall response rate was 36%; 5 (36%) patients had partial response and 5 (36%) patients had stable disease.ConclusionsThe combination of SPL-108 with weekly paclitaxel was safe and well tolerated. Encouraging antitumor activity was observed, with 72% of patients deriving a clinical benefit.Trial registrationNCT03078400.

BACKGROUND:Poly (ADP-ribose) polymerase (PARP) inhibition (PARPi) has demonstrated potent therapeutic efficacy in patients with BRCA-mutant ovarian cancer. However, acquired resistance to PARPi remains a major challenge in the clinic. METHODS:PARPi-resistant ovarian cancer mouse models were generated by long-term treatment of olaparib in syngeneic Brca1-deficient ovarian tumors. Signal transducer and activator of transcription 3 (STAT3)-mediated immunosuppression was investigated in vitro by co-culture experiments and in vivo by analysis of immune cells in the tumor microenvironment (TME) of human and mouse PARPi-resistant tumors. Whole genome transcriptome analysis was performed to assess the antitumor immunomodulatory effect of STING (stimulator of interferon genes) agonists on myeloid cells in the TME of PARPi-resistant ovarian tumors. A STING agonist was used to overcome STAT3-mediated immunosuppression and acquired PARPi resistance in syngeneic and patient-derived xenografts models of ovarian cancer. RESULTS:In this study, we uncover an adaptive resistance mechanism to PARP inhibition mediated by tumor-associated macrophages (TAMs) in the TME. Markedly increased populations of protumor macrophages are found in BRCA-deficient ovarian tumors that rendered resistance to PARPi in both murine models and patients. Mechanistically, PARP inhibition elevates the STAT3 signaling pathway in tumor cells, which in turn promotes protumor polarization of TAMs. STAT3 ablation in tumor cells mitigates polarization of protumor macrophages and increases tumor-infiltrating T cells on PARP inhibition. These findings are corroborated in patient-derived, PARPi-resistant BRCA1-mutant ovarian tumors. Importantly, STING agonists reshape the immunosuppressive TME by reprogramming myeloid cells and overcome the TME-dependent adaptive resistance to PARPi in ovarian cancer. This effect is further enhanced by addition of the programmed cell death protein-1 blockade. CONCLUSIONS:We elucidate an adaptive immunosuppression mechanism rendering resistance to PARPi in BRCA1-mutant ovarian tumors. This is mediated by enrichment of protumor TAMs propelled by PARPi-induced STAT3 activation in tumor cells. We also provide a new strategy to reshape the immunosuppressive TME with STING agonists and overcome PARPi resistance in ovarian cancer.

Despite the promising activity of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) in many cancer types with defects in the DNA damage response the majority of the treated patients acquire PARPi resistance and succumb to their diseases. Consequently, there is an urgent need to identify the mechanisms of PARPi resistance. Here, we show that PARPi treatment promotes STAT3 activation in ovarian cancer cells, tumor-associated immune cells and fibroblasts, resulting in PARPi resistance and immunosuppression. Comparison of ovarian cancer patient-matched tumor biopsies before and after PARPi therapy revealed that STAT3 activity was significantly higher in tumor cells and tumor-associated immune cells and fibroblasts post PARPi treatment. Moreover, one-time PARPi treatment activated STAT3 both in tumor cells as well as diverse immune subsets and fibroblasts. PARPi-treated immune cells exhibited decreased expression of immunostimulatory interferon (IFN)-γ and Granzyme B while increasing immunosuppressive cytokine IL-10. Finally, we demonstrate that the acquisition of PARPi resistance in ovarian cancer cells was accompanied by increased STAT3 activity. Ablating STAT3 inhibited PARPi-resistant ovarian tumor cell growth and/or restored PARPi sensitivity. Therefore, our study has identified a critical mechanism intrinsic to PARPi that promotes resistance to PARPi and induces immunosuppression during PARPi treatment by activating STAT3 in tumor cells and tumor-associated immune cells/fibroblasts.

Abstract Background: Signal transducer and activator of transcription 3 (STAT3) activation promotes tumor cell survival, immune evasion, metastasis, and drug resistance in PARP inhibitors and platinum-based chemotherapy. Objective: Based on our in-vitro findings of the tumoricidal potential of STAT3 inhibition in BRCAwild-type and PARP inhibitor-resistant tumor cells, we sought to evaluate whether ovarian and uterine cancer patients with somatic mutations in the STAT3 activation pathway had improved overall survival (OS) compared to those with intact STAT3 activation pathway-related genes. Method: We induced Olaparib resistance in BRCAwild-type ovarian cancer cell lines (OVCAR8 and A2780) and treated both parental and PARP inhibitor-resistant cell lines with various STAT3 inhibitors. We then attempted to establish clinical correlations of our in-vitro studies and queried the Cancer Genome Atlas (TCGA) Pan-Cancer Atlas for uterine and ovarian cancers. OS in patients with somatic mutations in the STAT3 gene and/or associated STAT3 activation genes (IL6ST, GNAS, JAK1, JAK2) was compared to OS in patients with intact STAT3 pathway-related genes. Kaplan-Meier survival curves were compared using the log-rank test. Results: In-vitro, STAT3 inhibition demonstrated potent, tumoricidal propensity in all cell lines (EC50: 0.34uM-0.46uM) including A2780parental, A2780parpi-resistant, OVCAR8parental, and OVCAR8parpi-resistant. Clinically, 1,154 total patients were identified in TCGA, including 571 high-grade serous ovarian carcinoma, 397 uterine endometrioid adenocarcinomas, 109 uterine serous carcinoma/uterine papillary serous carcinomas, 56 uterine carcinosarcomas/uterine malignant mixed Mullerian tumors, and 21 uterine mixed endometrial carcinomas. When pooling all uterine and ovarian cancer patients, median survival (MS) was 52 months (0-185) in patients with intact STAT3 (n=1,007) compared to not reached (NR, 0-225) in the STAT3 mutated group (n=147) with a Hazard Ratio (HR) of 0.25 and Confidence Interval (CI) 0.20-0.32 (p<0.001). In ovarian cancer patients, MS was 45 months (0-180) in patients with intact STAT3 (n=551) compared to 95 months (0-145) in the STAT3 mutated group (n=20) with a HR of 0.48 and CI 0.29-0.80 (p=0.038). In uterine cancer patients, MS was 112 months (0-185) in patients with intact STAT3 (n=456) compared to NR (0-225) in the STAT3 mutated group (n=127) with a HR of 0.40 and CI 0.25-0.64 (p=0.003). Conclusion: Pharmacologic and genetic (siRNA) inhibition of the STAT3 activation pathway killed PARP inhibitor-resistant ovarian cancer cells in-vitro. Clinically, somatic mutations in the STAT3 activation pathway were associated with improved OS in both ovarian and uterine cancer. These findings provide a rationale for the therapeutic targeting of this pathway in gynecologic malignancies. Citation Format: Adrian Kohut, Antons Martincuks, Thanh Dellinger, Hua Yu, Lorna Rodriguez-Rodriguez. Somatic mutations in the STAT3 activation pathway are associated with improved survival in gynecologic malignancies and provide a molecular rationale for therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2432.

Despite significant progress in cancer therapy over the last decades, ovarian cancer remains the most lethal gynecologic malignancy worldwide with the five-year overall survival rate less than 30% due to frequent disease recurrence and chemoresistance. CD44 is a non-kinase transmembrane receptor that has been linked to cancer metastatic progression, cancer stem cell maintenance, and chemoresistance development via multiple mechanisms across many cancers, including ovarian, and represents a promising therapeutic target for ovarian cancer treatment. Moreover, CD44-mediated signaling interacts with other well-known pro-tumorigenic pathways and oncogenes during cancer development, such as signal transducer and activator of transcription 3 (STAT3). Given that both CD44 and STAT3 are strongly implicated in the metastatic progression and chemoresistance of ovarian tumors, this review summarizes currently available evidence about functional crosstalk between CD44 and STAT3 in human malignancies with an emphasis on ovarian cancer. In addition to the role of tumor cell-intrinsic CD44 and STAT3 interaction in driving cancer progression and metastasis, we discuss how CD44 and STAT3 support the pro-tumorigenic tumor microenvironment and promote tumor angiogenesis, immunosuppression, and cancer metabolic reprogramming in favor of cancer progression. Finally, we review the current state of therapeutic CD44 targeting and propose superior treatment possibilities for ovarian cancer.

Life-threatening cardiomyopathy is a severe, but common, complication associated with severe trauma or sepsis. Several signaling pathways involved in apoptosis and necroptosis are linked to trauma- or sepsis-associated cardiomyopathy. However, the underling causative factors are still debatable. Heparan sulfate (HS) fragments belong to the class of danger/damage-associated molecular patterns liberated from endothelial-bound proteoglycans by heparanase during tissue injury associated with trauma or sepsis. We hypothesized that HS induces apoptosis or necroptosis in murine cardiomyocytes. By using a novel Medical-In silico approach that combines conventional cell culture experiments with machine learning algorithms, we aimed to reduce a significant part of the expensive and time-consuming cell culture experiments and data generation by using computational intelligence (refinement and replacement). Cardiomyocytes exposed to HS showed an activation of the intrinsic apoptosis signal pathway via cytochrome C and the activation of caspase 3 (both p < 0.001). Notably, the exposure of HS resulted in the induction of necroptosis by tumor necrosis factor α and receptor interaction protein 3 (p < 0.05; p < 0.01) and, hence, an increased level of necrotic cardiomyocytes. In conclusion, using this novel Medical-In silico approach, our data suggest (i) that HS induces necroptosis in cardiomyocytes by phosphorylation (activation) of receptor-interacting protein 3, (ii) that HS is a therapeutic target in trauma- or sepsis-associated cardiomyopathy, and (iii) indicate that this proof-of-concept is a first step toward simulating the extent of activated components in the pro-apoptotic pathway induced by HS with only a small data set gained from the in vitro experiments by using machine learning algorithms.

NF-κB and STAT3 are essential transcription factors in immunity and act at the interface of the transition from chronic inflammation to cancer. Different functional crosstalks between NF-κB and STAT3 have been recently described arguing for a direct interaction of both proteins. During a systematic analysis of NF-κB/STAT3 crosstalk we observed that appearance of the subcellular distribution of NF-κB and STAT3 in immunofluorescence heavily depends on the fixation procedure. Therefore, we established an optimized fixation protocol for the reliable simultaneous analysis of the subcellular distributions of both transcription factors. Using this protocol we found that cytokine-induced nuclear accumulation of NF-κB or STAT3 did not alter the subcellular distribution of the other transcription factor. Both knockout and overexpression of STAT3 does not have any major effect on canonical TNFα-NF-κB signalling in MEF or HeLa cells. Similarly, knockout of p65 did not alter nuclear accumulation of STAT3 in response to IL-6. However, p65 expression correlates with elevated total cellular levels of STAT3 and STAT1 and supports activation of these transcription factors. Our findings in MEF cells argue against a direct physical interaction of free cellular NF-κB and STAT3 but point to more intricate functional interactions.