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STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (–NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated. Cancer Res; 74(8); 2316–27. ©2014 AACR.

PDF file - 163K, Representative Annexin V flow cytomety plots of chemotherapy-resistant primary cell lines.

PDF file - 172K, EPR spectra from organ tissue samples obtained from HO-3867-treated mice.

PDF file - 146K, Methods for HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.

PDF file - 77K, Immunoblot analysis for pSTAT3, and Bcl-2 using 2 different mice tissue.

PDF file - 220K, Representative Annexin V flow cytomety plots of CHO, H9c2 and A2780 cells.

PDF file - 122K, Illustration of the GFP-Histone 2B fusion protein (pBOS H2BGFP) vector.

PDF file - 156K, Effect of H-4073 in both cancer and normal cells. Phosphokinase assay array using CHO cells.

PDF file - 177K, Effect of DAPs on SKOV3 ovarian cancer and hOSE cells.

PDF file - 116K, Viability of SKOV3, hOSE, and H9c2 cells treated with H-4073 or HO-3867.

PDF file - 95K, Results of Tukey's post-hoc test and Molecular weights of the pure DAP compounds.

PDF file - 692K, (A) H-4073 Cell uptake in A2780 and CHO cell lines; (B) Ion chromatogram of pure HO-3867 in A2780 cells; (C) Ion chromatogram of pure HO-3867 in CHO cells; (D) Extracted ion chromatograms - H-4073; (E)Extracted ion chromatograms - H-4073.

Objective Constitutive activation of STAT3 is a hallmark of various human cancers, however an increased pSTAT3 expression in high grade human endometrial cancer has not been reported. In the present study, we examine the expression of STAT family of proteins in endometrial cancer cell lines and the efficacy of HO-3867, a novel STAT3 inhibitor designed in our lab. Methods Expression of STAT family proteins was evaluated via Western blot. The cell viability, post-treatment with HO-3867, was assessed using MTT, cell-cycle profile and Annexin assay. In vivo efficacy of HO-3867 was evaluated using xenograft mice. Results Expression of activated STATs was inconsistent among the cell lines and 18 human endometrial cancer specimens tested. While pSTAT3 Tyr705 was not expressed in any of the cell lines, pSTAT3 Ser727 was highly expressed in endometrial cancer cell lines and tumor specimens. HO-3867 decreased the expression of pSTAT3 Ser727 while total STAT3 remained constant; cell viability decreased by 50–80% and induced G2/M arrest in 55% of Ishikawa cells at the G2/M cell cycle checkpoint. There was an increase in p53, a decrease in Bcl2 and Bcl-xL, and cleavage of caspase-3, caspase-7 and PARP. HO-3867 mediated a dosage-dependent inhibition of the growth of xenografted endometrial tumors. Conclusions HO-3867 treatment decreases the high levels of pSTAT3 Ser727 in endometrial cancer cells by inducing cell cycle arrest and apoptosis. This suggests a specific role of serine-phosphorylated STAT3, independent of tyrosine phosphorylation in the oncogenesis of endometrial cancer. HO-3867 could potentially serve as an adjunctive targeted therapy.

The objective of this study was to evaluate gynecologic oncology provider (GOP) practices regarding weight loss (WL) counseling, and to assess their willingness to initiate weight loss interventions, specifically bariatric surgery (WLS).Members of the Society of Gynecologic Oncology were invited to complete an online survey of 49 items assessing knowledge, attitudes, and behaviors related to WL counseling.A total of 454 participants initiated the survey, yielding a response rate of 30%. The majority of respondents (85%) were practicing GOP or fellows. A majority of responders reported that50% of their patient population is clinically obese (BMI ≥ 30). Only 10% reported having any formal training in WL counseling, most often in medical school or residency. Providers who feel adequate about WL counseling were more likely to offer multiple WL options to their patients (p.05). Over 90% of responders believe that WLS is an effective WL option and is more effective than self-directed diet and medical management of obesity. Providers who were more comfortable with WL counseling were significantly more likely to recommend WLS (p.01). Approximately 75% of respondents expressed interest in clinical trials evaluating WLS in obese cancer survivors.The present study suggests that GOP appreciate the importance of WL counseling, but often fail to provide it. Our results demonstrate the paucity of formal obesity training in oncology. Providers seem willing to recommend WLS as an option to their patients but also in clinical trials examining gynecologic cancer outcomes in women treated with BS.

STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (–NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated. Cancer Res; 74(8); 2316–27. ©2014 AACR.

Importance Poly(adenosine diphosphate-ribose) polymerase inhibitor and anti-programmed death receptor-1 inhibitor monotherapy have shown limited clinical activity in patients with advanced triple-negative breast cancer (TNBC). Objective To evaluate the clinical activity (primary) and safety (secondary) of combination treatment with niraparib and pembrolizumab in patients with advanced or metastatic TNBC. Design, setting, and participants This open-label, single-arm, phase 2 study enrolled 55 eligible patients with advanced or metastatic TNBC irrespective of BRCA mutation status or programmed death-ligand 1 (PD-L1) expression at 34 US sites. Data were collected from January 3, 2017, through October 29, 2018, and analyzed from October 29, 2018, through February 27, 2019. Interventions Patients were administered 200 mg of oral niraparib once daily in combination with 200 mg of intravenous pembrolizumab on day 1 of each 21-day cycle. Main outcomes and measures The primary end point was objective response rate (ORR) per the Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary end points were safety, disease control rate (DCR; complete response plus partial response plus stable disease), duration of response (DOR), progression-free survival (PFS), and overall survival. Results Within the full study population of 55 women (median age, 54 years [range, 32-90 years]), 5 patients had confirmed complete responses, 5 had confirmed partial responses, 13 had stable disease, and 24 had progressive disease. In the efficacy-evaluable population (n = 47), ORR included 10 patients (21%; 90% CI, 12%-33%) and DCR included 23 (49%; 90% CI, 36%-62%). Median DOR was not reached at the time of the data cutoff, with 7 patients still receiving treatment at the time of analysis. In 15 evaluable patients with tumor BRCA mutations, ORR included 7 patients(47%; 90% CI, 24%-70%), DCR included 12 (80%; 90% CI, 56%-94%), and median PFS was 8.3 months (95% CI, 2.1 months to not estimable). In 27 evaluable patients with BRCA wild-type tumors, ORR included 3 patients (11%; 90% CI, 3%-26%), DCR included 9 (33%; 90% CI, 19%-51%), and median PFS was 2.1 months (95% CI, 1.4-2.5 months). The most common treatment-related adverse events of grade 3 or higher were anemia (10 [18%]), thrombocytopenia (8 [15%]), and fatigue (4 [7%]). Immune-related adverse events were reported in 8 patients (15%) and were grade 3 in 2 patients (4%); no new safety signals were detected. Conclusions and relevance Combination niraparib plus pembrolizumab provides promising antitumor activity in patients with advanced or metastatic TNBC, with numerically higher response rates in those with tumor BRCA mutations. The combination therapy was safe with a tolerable safety profile, warranting further investigation. Trial registration ClinicalTrials.gov identifier: NCT02657889.

Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. In this study, we demonstrate the level of pSTAT3 Tyr705 is increased in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens, as determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. In addition, we show that S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. STAT3-target proteins Bcl-xL, cyclin D2 and VEGF showed similar decreases in HO-3867 treated cells. Taken together, these findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors.

ObjectiveThe optimal role of bevacizumab (Bev) in the treatment of ovarian cancer has not yet been established. Furthermore, it is unclear whether there is a benefit of Bev after progression on a Bev-containing regimen in ovarian cancer. The objective of this study was to compare response rates, progression-free survival (PFS), and overall survival between patients who were treated with chemotherapy and Bev after progression on Bev (BAB) versus patients who were treated with chemotherapy without Bev (CWOB).MethodsWe conducted a retrospective chart review of all patients who received treatment with Bev (with or without cytotoxic chemotherapy) for recurrent ovarian cancer at a single institution. Patients who received additional therapy after progression while on Bev were included.ResultsForty-six patients were included (16 CWOB group and 30 BAB). The median number of previous chemotherapy regimens was 2.5 for CWOB compared with 4 for BAB (P= 0.11). Fifty-two percent of patients had an objective response to the first Bev regimen before progressing on Bev. Response rates for the regimen after progression on Bev were 19% (3/16) in the CWOB group and 23% (7/30) in the BAB group (P= 1). Twenty-five percent of the patients who responded to the first Bev regimen and 18% of those who did not respond to the first Bev regimen responded to the second Bev regimen (P= 0.72). The median PFS for patients in the CWOB group was 2.6 months (95% confidence interval [CI], 1.3–5 months), compared with 5.0 months (95% CI, 3.5–7.3 months) for patients in the BAB group (P= 0.01). Overall survival was similar, 9.4 months (95% CI, 5.0–12.0 months) for CWOB versus 8.6 months (95% CI, 5.8–15.5 months) for BAB (P= 0.19). One patient in the BAB group died of a bowel perforation.ConclusionsIn patients previously treated with Bev for recurrent ovarian cancer, the subsequent addition of Bev to cytotoxic chemotherapy increased the PFS compared with patients not receiving a second course of Bev, but did so without an impact on overall survival. The response to the first Bev regimen did not predict whether a patient would respond again to the next Bev regimen. Randomized, larger studies will have to be performed to confirm this observation.

Background: PARP inhibitor (PARPi) monotherapy has previously demonstrated clinical activity only in patients with a germline BRCA mutation (BRCAmut), while single-agent anti-programmed cell death protein 1 (PD-1) therapy has achieved response rates of only 5–20% in advanced triple negative breast cancer (TNBC). In preclinical studies, PARP inhibition enhanced anti-tumor immunity, increased infiltration of proliferating CD8+ T cells, and synergized with anti-PD-1 agents in BRCA wildtype (wt) tumors. TOPACIO is a fully enrolled, phase I/II trial of niraparib + pembrolizumab (pembro) in advanced TNBC. This combination achieved 28% objective response rate (ORR) and 50% disease control rate (DCR) in evaluable patients. Although activity was highest in patients with BRCAmut (ORR=60%; DCR=80%), durable clinical benefit was also observed in patients without BRCA mutations. In this study, we conducted exploratory biomarker analyses to evaluate their potential correlation with durable clinical benefit (any complete response [CR] or partial response [PR] regardless of duration or stable disease [SD] for ≥16 weeks) beyond BRCA mutations. Method: ORR was assessed by RECIST v1.1. Duration of disease control (DDC) was defined as time from first dose of study treatment to radiologic disease progression or death. Tumor mutational status of homologous recombination repair (HRR) and other DNA damage repair (DDR) pathway genes was determined using an NGS panel. Immunoprofiling was conducted using NanoString IO360 panel complimented with 30 DNA repair spike-ins. Tumor immune micro-environment was characterized using multiplex immuno-fluorescence (CycIF). PD-L1 status was determined using the Agilent/DAKO 22C3 IHC clinical trial assay. Results: Of 46 evaluable patients, 20 achieved durable clinical benefit (any CR/PR or SD≥16 weeks) with niraparib + pembro combination, of which 8 were tumor BRCA wild type (wt), and 1 was BRCA unknown. Of the 9 BRCAwt/unknown patients, 5 had deleterious mutations in HRR/DDR pathway genes, whereas the remaining 4 had no mutations (HRR/DDR wt). Mutations that were associated with response include CHEK1 (CR; DDC=10.3 mos), ATR (CR; DDC=6.4 mos), PALB2 (PR; DDC=3.5 mos), BLM (SD; DDC=8.1 mos), and NBN/RAD51C (SD; DDC=3.7 mos). Of 4 patients that had no identified mutations (HRR/DDR wt), 1 patient had CR; DDC=10.3 mos, and the remaining 3 patients had SD with DDC ranging from ∼4-8 mos. Of note, all 4 HRR/DDR wt patients were also PD-L1-negative. Table:Durable clinical benefit in BRCAwt/unknown patientsHRR/DRR MutationsPD-L1 StatusBest ResponseDDC (Months)CHEK1+CR10.3†ATR+CR6.4PALB2*UnknownPR3.5BLM-SD8.1NBN/RAD51C+SD3.7None-CR10.3†None-SD8.2None-SD4.2None-SD3.9*BRCA status unknown; †Treatment is ongoing. Conclusion: Patients with mutations beyond BRCA achieved durable clinical benefit with niraparib + pembro treatment; five of these patients had DDC >6 mos. Mutations in genes that are associated with the HRR/DDR pathway appear to confer sensitivity to niraparib + anti-PD1. Additional translational analyses, including immunoprofiling and CycIF, will be presented. Funding: TESARO, Inc., Waltham, MA, USA sponsored the study. Citation Format: Vinayak S, Tolaney SM, Schwartzberg L, Mita M, McCann G, Tan AR, Wahner Hendrickson A, Forero A, Anders C, Wulf G, Dillon P, Lynce F, Zarwan C, Erban J, Färkkilä A, Zhou Y, Buerstatte N, Graham JR, Arora S, Dezube B, Telli ML. Durability of clinical benefit with niraparib + pembrolizumab in patients with advanced triple-negative breast cancer beyond BRCA: (TOPACIO/Keynote-162) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-02.

Highlights • We describe bilateral groin reconstruction with a single anterolateral thigh flap. • Anatomy and surgical technique of the anterolateral thigh flap are discussed.

ObjectivesThe objective of this study is to determine (1) if there is a relationship between increasing body mass index (BMI) and postoperative complications in patients undergoing robotic hysterectomy for endometrial cancer and (2) if there are additional patient characteristics, specifically preoperative comorbidities, which increase the risk of postoperative complicationMethodsA retrospective chart review was conducted on women who underwent a robotic staging surgery for endometrial cancer from 2006 to 2012. Basic demographics and preoperative and postoperative complications were extracted from the medical records. Obesity was divided into 4 categories, and complication rates were compared across these subgroups. Patients were also divided by the number of comorbidities and compared.ResultsThe cohort included 543 patients. The BMI ranged from 17.3 to 69.5 kg/m2. Three hundred eighty patients (70%) were obese (BMI >30 kg.m2). One hundred ninety patients (35%) had no comorbidities other than obesity, and 180 patients (33%) had only 1 comorbidity other than obesity (Table 1).Postoperative complications occurred in 102 (18.7%) of the patients. Severe postoperative complications, including intensive care unit admission, reintubation, reoperation, and perioperative death, occurred in 14 patients (2.6%). Of the nonobese patients, 27 (16.5%) had postoperative complications; of the obese patients, 75 (19.7%) had a complication (P = 0.38). In patients with no comorbidities, 16.3% had a complication; 18% of patients with 1 to 2 comorbidities had a complication, and 28% of patients with 3 or more comorbidities had a complication (P = 0.08).ConclusionsThe postoperative complication rate based on BMI or number of comorbidities was not statistically significant, but patients with greater number of comorbidities had an increased rate of postoperative complications. Patients with certain comorbidities, cardiac and renal specifically, had the highest rates of postoperative complications.

Highlights • The VRAM flap is commonly used for perineal and vaginal reconstruction at the time of pelvic exenteration. • Prolapse of the VRAM flap may be under reported. • We have shown successful repair of VRAM flap prolapse via an obliterative technique and sacral suspension.

A diagnosis of advanced ovarian cancer is the beginning of a long and arduous journey for a patient. Worldwide, approximately half of the individuals undergoing therapy for advanced cancer will succumb to the disease, or consequences of treatment. Well-known and widely-used chemotherapeutic agents such as cisplatin, paclitaxel, 5-fluorouracil, and doxorubicin are toxic to both cancer and non-cancerous cells, and have debilitating side effects Therefore, development of new targeted anticancer therapies that can selectively kill cancer cells while sparing the surrounding healthy tissues is essential to develop more effective therapies. We have developed a new class of synthetic curcumin analogs, diarylidenyl-piperidones (DAPs), which have higher anticancer activity and enhanced bio-absorption than curcumin. The DAP backbone structure exhibits cytotoxic (anticancer) activity, whereas the N-hydroxypyrroline (-NOH) moiety found on some variants functions as a cellular- or tissue-specific modulator (antioxidant) of cytotoxicity. The anticancer activity of the DAPs has been evaluated using a number of ovarian cancer cell lines, and the safety has been evaluated in a number of non-cancerous cell lines. Both variations of the DAP compounds showed similar levels of cell death in ovarian cancer cells, however the compounds with the -NOH modification were less toxic to non-cancerous cells. The selective cytotoxicity of the DAP–NOH compounds suggests that they will be useful as safe and effective anticancer agents. This article reviews some of the key findings of our work with the DAP compounds, and compares this to some of the targeted therapies currently used in ovarian cancer therapy.

While it is known that positive surgical margins increase the risk of cervical cancer recurrence, little is known about the effect of close surgical margins (CSM). Therefore, we set out to determine the impact of margin status on recurrence and survival in patients with early-stage cervical cancer.A retrospective review was conducted of patients undergoing radical hysterectomy from 2000 to 2010 with Stage IA2-IIA cervical cancer. CSM were defined as ≤5mm; association with other clinicopathologic factors as well as recurrence and survival was evaluated.Of the 119 patients, 75 (63%) with CSM had a recurrence rate of 24% compared to 9% without CSM. Though not independently associated with recurrence, CSM were significantly associated with positive lymph nodes (44% vs. 18%), positive parametria (33.3% vs. 2.3%), larger tumors (3.5 vs. 2.5cm), greater depth of stromal invasion (DOI) (84% vs. 33%), and lymphovascular space invasion (LVSI) (61.3% vs. 34.1%). We failed to find an association between adjuvant therapy and recurrence in those with CSM. Exploratory analysis revealed that a surgical margin of ≤2mm was significantly associated with an increased risk of overall recurrence (36% vs. 9%, p=0.009) as well as loco-regional recurrence (22% vs. 4%, p=0.0034).Surgical margins of ≤5mm on radical hysterectomy specimens are often associated with other high or intermediate risk factors for recurrence. While not a proven independent risk factor, the distance to surgical margin may warrant further investigation as an intermediate risk factor along with tumor size, DOI and LVSI.

BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, tend to have better response to platinum-based chemotherapy as compared with sporadic cases of epithelial ovarian cancer (EOC). In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Our novel agent, HO-3867 and a commercial STAT3 inhibitor, STATTIC, significantly inhibited BRCA-mutated ovarian cancer cells in vitro in a dose- and time-dependent manner. BRCA-mutated ovarian cancer cells treated with HO-3867 exhibited a significant degree of apoptosis with elevated levels of cleaved caspase-3, caspase-7 and PARP. HO-3867 treatment induced more reactive oxygen species (ROS) in BRCA-mutated cells compared with wild-type cells, however, there was no increased ROS when benign ovarian surface epithelial cells were treated with HO-3867. BRCA1-mutated cancer cells had higher expression of Tyrosine-phosphorylated STAT3 (pTyr705) as compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and its downstream targets cyclin D1, Bcl-2 and survivin. In addition, overexpression of STAT3 cDNA provided resistance to HO-3867-induced apoptosis. Our results show that HO-3867, a potent STAT3 inhibitor, may have a role as a biologically targeted agent for BRCA1-mutated cancers either as an adjunct to cytotoxic chemotherapy or as a single agent.

Background: Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized that the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. Methods: The increase of pSTAT3 Tyr705 in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens was determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies of pSTAT3 Tyr705 were used to demonstrate the necessity for cell survival and proliferation under hypoxic conditions. In addition, S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). This association was evaluated with knock down of S1PR1 in HOCCs. HOCCs were treated with our novel STAT3 inhibitor, HO-3867. Results: pSTAT3 Tyr705 is increased in the hypoxic regions of EOC specimens. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. There was decreased expression of the STAT3-target proteins Bcl-xL, cyclin D2, and VEGF in all HO-3867 treated cells. Conclusions: These findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Therefore, targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors. Citation Format: Georgia A McCann, MD, Adam ElNaggar, MD, Shan Naidu, MS; Kellie S Rath, MD; Brent J Tierney, MD; Adrian Suarez, MD; David E Cohn, MD; Karuppaiyah Selvendiran, PhD. Targeting constitutively-activated STAT3 in hypoxic ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1413.

We have developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group which selectively inhibits STAT3 activation, translocation, and DNA binding activity, without affecting the expression of other pSTATs. This new STAT3 inhibitor offers advantages when compared to other STAT3-targeting molecules currently available, under development or undergoing preclinical trials. Most notably, these compounds exhibit minimal toxicity toward normal cells and tissues while inducing apoptosis in ovarian cancer cells, and exhibit good oral bioavailability. Results show that selective cytotoxicity of the lead compound, HO-3867, towards cancer cells is multi-faceted. AutoDock molecular modeling indicates that the DAP compounds interact with STAT3 at the DNA-binding domain. Liquid chromatography / Mass spectroscopy analysis demonstrates greater bioabsorption and bioavailability of active (cytotoxic) metabolites in cancer cells when compared to normal cells. Differential activation of the Akt pathway was demonstrated using Akt siRNA in normal versus cancer cells. Knockdown of STAT3 expression using two different shRNAs shows the requirement of STAT3 for HO-3867-mediated apoptosis. In vivo testing shows that HO-3867 inhibits xenograft tumor growth without toxic side effects. In addition, HO-3867 significantly inhibited cell survival, migration/invasion and induced cell death in primary ovarian cancer cell populations isolated from ascites of patients with recurrent and advanced ovarian cancer. This study provides evidence of a safe, targeted STAT3 inhibitor (HO-3867) with demonstrated efficacy in treating multiple ovarian cancer models. This suggests significant translational potential of these compounds for the treatment of ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A253. Citation Format: Karuppaiyah Selvendiran, Kellie S. Rath, Shan Naidu, Pushpa Latha, Bid Hemant, Georgia A. McCann, Veronica Bravo, Peter Houghton, Gustavo Leone, Kálmán Hideg, Periannan Kuppusamy, David E. Cohn. HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A253.

Background: BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation, and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, patients have shown improved responses with platinum-based chemotherapy versus sporadic cases. In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer cell models. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Methods: Expression of STAT family proteins, cell cycle, apoptotic and DNA repair proteins were evaluated via western blot on BRCA- mutated and non-mutated ovarian cancer cell lines. Cell viability and proliferation were analyzed with MTT and clonogenic assays. Cell cycle analysis and apoptosis assays were performed using flow cytometry and Annexin V staining. Reactive oxygen species (ROS) staining was performed using immunofluoresence. Finally, STAT3 overexpression or suppression experiments were performed using transfected wild-type STAT3 cDNA and STAT3 siRNA, respectively. Results: Our novel agent, HO-3867, and a commercial STAT3 inhibitor, STATIC, significantly inhibited BRCA-mutated cancer cells in vitro, in a dose-dependent manner. HO-3867 induced G2/M arrest with increased expression of p53 and p21 and with decreased cdk5 and cyclin D1 expression. BRCA-mutated cells treated with HO-3867 exhibited an increased induction of apoptosis with elevated levels of cleaved caspase-3, caspase-7, and PARP. Tyrosine-phosphorylated STAT3 (pTyr705) exhibited higher expression levels in BRCA-mutated cancer cells compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and other DNA repair proteins. In addition, HO-3867 treatment induced more ROS in BRCA-mutated cells compared with wild-type cells, with no increased induction in benign ovarian surface epithelial cells. Furthermore, we confirmed that the over-expression of STAT3 cDNA provides resistance to HO-3867 induction of apoptosis. Conclusion: Our results show that HO-3867, a potential STAT3 inhibitor, may be effective in the treatment of BRCA-mutated ovarian cancer, although continued studies are needed to elucidate the exact mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4723. doi:1538-7445.AM2012-4723

OBJECTIVE: Signal transducer and activator of transcription 3 (STAT3) proteins have important roles in cancer cell survival and proliferation. Recent studies show constitutive activation of STAT3 is associated with tumor growth and oncogenic transformation in several human cancers; however, little is known about STAT3 expression in endometrial cancer. The objectives of this study were to examine the expression of the STAT family of proteins in endometrial cancer cell lines and to evaluate the efficacy of HO-3867, a novel STAT3 inhibitor. METHODS: Expression of STAT family proteins, cell cycle and apoptotic proteins were evaluated via western blot on Ishikawa, AN3-CA, RL95-2, HEC1B, HEC-1A, and SK-UT1B human endometrial cancer cell lines. Cell viability and proliferation of these cell lines were analyzed with MTT and nucleo counter assay after treatment with increasing concentrations of HO-3867. Cell cycle analysis and apoptosis were performed using flow cytometry and annexin V staining. An ubiquitination assay was used to examine the inhibition of CDK5 degradation. Finally, STAT3 over-expression or suppression experiments were performed using wild-type STAT3 cDNA and STAT3 siRNA. RESULTS: Expression of activated STAT1, 2, 5, and 6 was inconsistent among cell lines tested, while pSTAT3 Ser727 had consistently high expression. HO-3867 decreased cell viability through induced G2/M arrest and apoptosis within 24h of treatment. HO-3867 inhibited pSTAT3 Ser727 with no change in total STAT3. This was associated with an increase in expression of cell cycle related proteins p53 and p21, a decrease in anti-apoptotic proteins Bcl2 and Bcl-XL, as well as increased cleavage of caspases. After 24h of treatment with HO-3867, 55-88% of cells were apoptotic. The downregulation of Ser727 was, at least in part, due to an accelerated inhibition of ubiquitin-dependent CDK5 degradation, as a positive regulator of STAT3 Ser727. Suppression of STAT3 expression with siRNA significantly reduced cell survival. In addition, we confirmed that over-expression of STAT3 cDNA provides resistance to HO-3867. CONCLUSION: Endometrial cancer cells express high levels of pSTAT3 Ser727. HO-3867 induces cell cycle arrest and apoptosis by targeting pSTAT3 Ser727. Our findings suggest a specific role of serine-phosphorylated STAT3 that is independent of tyrosine phosphorylation. HO-3867 may be a potential adjunct to chemotherapy and radiation in the treatment of endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2802. doi:1538-7445.AM2012-2802

Background: The hypoxic microenvironment plays a key role in tumorigenesis and the development of therapeutic resistance in epithelial ovarian cancer (EOC). The goal of this study was to examine the expression of STAT3 in hypoxic EOC, to understand its mechanism of activation, and to explore the therapeutic efficacy of a novel compound HO-3867. Methods: Expression of STAT family proteins in cell lines and human tumor specimens was analyzed via western blot and IHC. MTT was used to assess cell viability and apoptotic analysis was performed using flow cytometry. Over-expression or suppression experiments were performed using S1PR1 cDNA and shRNA. Tumor xenografts were made with cell lines exposed to hypoxic preconditioning. Results: pSTAT3 Tyr705 is highly expressed in ovarian cancer cell lines exposed to hypoxia as well as in hypoxic regions of advanced human EOC tumor specimens. Accelerated xenograft tumor growth was observed when implanted cells were pre-conditioned in a hypoxic microenvironment. Similar to our in vitro findings, the tumor xenografts of hypoxic ovarian cancer cells (HOCCs) had high expression of pSTAT3 and its target proteins. We found an increased expression of sphingosine-1-phosphate receptor 1(S1PR1) in HOCCs, a known positive feedback regulator of STAT3 transcription. Suppression of S1PR1 expression with shRNA significantly reduced the expression of pSTAT3 Tyr705 through the inhibition of the JAK kinases, resulting in decreased cell survival. Conversely, overexpression of S1PR1 with cDNA increased pSTAT3 Tyr705 and promoted cell proliferation under hypoxic conditions. Finally, treatment of HOCCs with the novel synthetic compound HO-3867 resulted in decreased expression of pSTAT3 Tyr705, as well as STAT3-regulated proteins VEGF, Bcl-xL, Bcl-2, and cyclin D1. The rapid downregulation of pSTAT3 Tyr705 was associated with accelerated ubiquitin-dependent degradation. An induction of apoptosis contributed to inhibition of cell proliferation as HO-3867-exposed cells exhibited elevated levels of cleaved caspases and PARP. Conclusions: The hypoxic microenvironment results in STAT3 activation as evidenced in our in vitro and in vivo models. S1PR1 is involved in STAT3 Tyr705 activation in hypoxic ovarian cancer via the JAK signaling pathway. The specific targeting of hypoxia-induced tyrosine phosphorylation of STAT3 with HO-3867 is a novel therapeutic strategy in the treatment of hypoxia-mediated chemotherapeutic resistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3940. doi:1538-7445.AM2012-3940

Objectives: Hypoxia plays a role in tumorigenesis and the development of resistance to radio- and chemotherapy. We have shown that human xenograft tumors in mice are severely hypoxic and that hypoxic exposure increases the phosphorylation of STAT3 at the Tyr705 residue in A2780 cell lines and is associated with chemotherapeutic resistance. HO-3867, a novel curcumin analogue designed with our institution, induces cell cycle arrest and apoptosis via inhibition of the STAT3 signaling pathway. The goal of this study was to understand the mechanistic pathways involved. Methods: The study was performed using A2780 and SKOV3 human ovarian cancer cell lines grown under normoxic (21% O2) and hypoxic (1% O2) conditions for 24 hours. Hypoxia-exposed cells were subsequently treated with 10 or 20 uM of HO-3867 for 2 and 24 hours. The expression levels of pSTAT3, associated proteins, and apoptotic markers were evaluated by Western blot and RT-PCR. Quantification of apoptosis was performed by staining with Annexin V. We also examined cell viability and involvement of ubiquitin-dependent degradation of pSTAT3. Results: Treatment with HO-3867 under hypoxic conditions resulted in decreased protein expression of pSTAT3 Tyr705 and Ser727. Rapid downregulation of STAT3 correlated with accelerated ubiquitin-dependent degradation of Tyr705-phosphorylated STAT3. Treatment was associated with decreased levels of STAT3-targeting proteins VEGF, Bcl-2, Bcl-xL, and cyclin D1. RT-PCR revealed decreased BCL-2 mRNA expression with treatment. There was no change in expression of proteins upstream of STAT3 including pJAK1, JAK1, pTYK2, TYK2, SOCS1 and SOCS2. An induction in apoptosis contributed to inhibition of cell proliferation as HO-3867-exposed cells exhibited elevated levels of cleaved caspase-3, caspase-7, and PARP as well as an increase in apoptotic cell count measured by flow cytometry. Conclusion: Under hypoxic growth conditions, HO-3867 demonstrates cytotoxicity via inhibition of STAT3. The use of HO-3867 may be a potential adjunct in the treatment of hypoxia-mediated chemotherapy-resistant ovarian cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2011-2059