Ronald J. Buckanovich

Quiescent Ovarian Cancer Cells Secrete Follistatin to Induce Chemotherapy Resistance in Surrounding Cells in Response to Chemotherapy

Abstract Purpose: We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance. Experimental Design: We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy. Results: We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2–dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post–progression-free, and overall survival. Conclusions: FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Phase I trial of ribociclib with platinum chemotherapy in ovarian cancer

BACKGROUNDNew therapeutic combinations to improve outcomes of patients with ovarian cancer are clearly needed. Preclinical studies with ribociclib (LEE-011), a CDK4/6 cell cycle checkpoint inhibitor, demonstrate a synergistic effect with platinum chemotherapy and efficacy as a maintenance therapy after chemotherapy. We tested the safety and initial efficacy of ribociclib in combination with platinum-based chemotherapy in recurrent ovarian cancer.METHODSThis phase I trial combined weekly carboplatin and paclitaxel chemotherapy with ribociclib, followed by ribociclib maintenance in patients with recurrent platinum-sensitive ovarian cancer. Primary objectives were safety and maximum tolerated dose (MTD) of ribociclib when given with platinum and taxane chemotherapy. Secondary endpoints were response rate (RR) and progression-free survival (PFS).RESULTSThirty-five patients were enrolled. Patients had a mean of 2.5 prior lines of chemotherapy, and 51% received prior maintenance therapy with poly(ADP-ribose) polymerase inhibitors and/or bevacizumab. The MTD was 400 mg. The most common adverse events included anemia (82.9%), neutropenia (82.9%), fatigue (82.9%), and nausea (77.1%). The overall RR was 79.3%, with a stable disease rate of 18%, resulting in a clinical benefit rate of 96.6%. Median PFS was 11.4 months. RR and PFS did not differ based on the number of lines of prior chemotherapy or prior maintenance therapy.CONCLUSIONThis work demonstrates that the combination of ribociclib with chemotherapy in ovarian cancer is feasible and safe. With a clinical benefit rate of 97%, this work provides encouraging evidence of clinical efficacy in patients with recurrent platinum-sensitive disease.TRIAL REGISTRATIONClinicalTrials.gov NCT03056833.FUNDINGThis investigator-initiated trial was supported by Novartis, which provided drugs and funds for trial execution.

Targeting Therapeutic Resistance and Multinucleate Giant Cells in CCNE1-Amplified HR-Proficient Ovarian Cancer

Abstract Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.

EGFL6 is a novel HER3 ligand, inducing HER3/integrin heterodimers to induce pERK centrosomal deposition and therapeutic resistance

EGF-like domain multiple-6 (EGFL6) is a secreted tumor growth/migration factor linked with poor outcomes in many tumor types. While EGFL6 is known to signal, in part, via its integrin-binding RGD domain, little else is known about EGFL6 receptors. We evaluated putative EGFL6 receptors and found that EGFL6 treatment of ovarian cancer cells leads to both transient phosphorylation of EGFR and prolonged phosphorylation of HER2 and HER3 and subsequent phosphorylation of ERK (pERK). We found that EGFL6 directly binds HER3. However, EGFL6-driven prolonged activation of HER3 is dependent on an intact EGFL6 integrin-binding RGD domain. Immunoprecipitation and proximity ligation assays confirmed that EGFL6 treatment of cancer cells induces HER2/3-integrin-β3 heterocomplexes. Suggesting EGFL6 could play a role in resistance to HER targeting therapies, EGFL6 is upregulated in EGFR/HER receptor inhibitor-resistant cells, and EGFL6 treatment increases resistance to EGFR/HER inhibitors in vitro. Interestingly, we found that, in EGFL6-treated ovarian cancer cells undergoing mitosis, pERK localizes to the centrosome. Both EGFL6-neutralizing antibodies and HER protein-targeted inhibitors resulted in aberrant pERK centrosomal localization with associated altered mitotic spindle alignment and mitotic catastrophe. Furthermore, combination anti-EGFL6 therapy with the pan-EGFR receptor inhibitor neratinib, compared to either therapy alone, led to an increase in aberrant pERK localization and cancer cell death in vitro and significant restricted tumor growth in vivo. Combined, our data suggests that EGFL6 is a new ligand for HER3 and that dual targeting of the EGFL6/HER signaling axis, via altered pERK localization, may be an effective therapeutic strategy in ovarian cancer. SIGNIFICANCE: This work reveals that EGFL6 is a previously unrecognized ligand for HER3 which can increase resistance to HER family-targeted therapy. We also reveal a novel function of pERK downstream of pHER3 at the centrosome in mitosis. Importantly, we show that EGFL6 is an important therapeutic target to enhance the efficacy of EGFR/HER-targeted therapy.

Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) play a critical role in resistance to immunotherapy. In this study, we identified epidermal growth factor-like 6 (Egfl6) as a regulator of myeloid cell functions. Our analyses indicated that Egfl6, via binding with β3 integrins and activation of p38 and SYK signaling, acts as a chemotactic factor for myeloid cell migration and promotes their differentiation toward an immunosuppressive state. In syngeneic mouse models of ovarian cancer (OvCa), tumor expression of Egfl6 increased the intratumoral accumulation of polymorphonuclear (PMN) MDSCs and TAMs and their expression of immunosuppressive factors, including CXCL2, IL-10, and PD-L1. Consistent with this, in an immune 'hot' tumor model, Egfl6 expression eliminated response to anti-PD-L1 therapy, while Egfl6 neutralizing antibody decreased the accumulation of tumor-infiltrating CD206+ TAMs and PMN-MDSCs and restored the efficacy of anti-PD-L1 therapy. Supporting a role in human tumors, in human OvCa tissue samples, areas of high EGFL6 expression colocalized with myeloid cell infiltration. scRNA-Seq analyses revealed a correlation between EGFL6 and immune cell expression of immunosuppressive factors. Our data provide mechanistic insights into the oncoimmunologic functions of EGFL6 in mediating tumor immune suppression and identified EGFL6 as a potential therapeutic target to enhance immunotherapy in patients with OvCa.

Phase I and Randomized Phase II Study of Ruxolitinib With Frontline Neoadjuvant Therapy in Advanced Ovarian Cancer: An NRG Oncology Group Study

PURPOSE The interleukin-6/Janus kinase (JAK)/signal transducers and activators of transcription 3 axis is a reported driver of chemotherapy resistance. We hypothesized that adding the JAK1/2 inhibitor ruxolitinib to standard chemotherapy would be tolerable and improve progression-free survival (PFS) in patients with ovarian cancer in the upfront setting. MATERIALS AND METHODS Patients with ovarian/fallopian tube/primary peritoneal carcinoma recommended for neoadjuvant chemotherapy were eligible. In phase I, treatment was initiated with dose-dense paclitaxel (P) 70 mg/m2 once daily on days 1, 8, and 15; carboplatin AUC 5 intravenously day 1; and ruxolitinib 15 mg orally (PO) twice a day, every 21 days (dose level 1). Interval debulking surgery (IDS) was required after cycle 3. Patients then received three additional cycles of chemotherapy/ruxolitinib, followed by maintenance ruxolitinib. In the randomized phase II, patients were randomly assigned to paclitaxel/carboplatin with or without ruxolitinib at 15 mg PO twice a day for three cycles, IDS, followed by another three cycles of chemotherapy/ruxolitinib, without further maintenance ruxolitinib. The primary phase II end point was PFS. RESULTS Seventeen patients were enrolled in phase I. The maximum tolerated dose and recommended phase II dose were established to be dose level 1. One hundred thirty patients were enrolled in phase II with a median follow-up of 24 months. The regimen was well tolerated, with a trend toward higher grade 3 to 4 anemia (64% v 27%), grade 3 to 4 neutropenia (53% v 37%), and thromboembolic events (12.6% v 2.4%) in the experimental arm. In the randomized phase II, the median PFS in the reference arm was 11.6 versus 14.6 in the experimental, hazard ratio (HR) for PFS was 0.702 (log-rank P = .059). The overall survival HR was 0.785 ( P = .24). CONCLUSION Ruxolitinib 15 mg PO twice a day was well tolerated with acceptable toxicity in combination with paclitaxel/carboplatin chemotherapy. The primary end point of prolongation of PFS was achieved in the experimental arm, warranting further investigation.

Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

Abstract The fundamental steps in high-grade serous ovarian cancer (HGSOC) initiation are unclear, presenting critical barriers to the prevention and early detection of this deadly disease. Current models propose that fallopian tube epithelial (FTE) cells transform into serous tubal intraepithelial carcinoma (STIC) precursor lesions and subsequently into HGSOC. In this study, we report that an epigenetically altered mesenchymal stem cell niche, termed high-risk mesenchymal stromal/stem cell (hrMSC), exists prior to STIC lesion formation. hrMSCs are enriched in STIC stroma and contribute to a stromal “field effect” extending beyond the borders of the STIC lesion. hrMSCs promote DNA damage in FTE cells while also fostering FTE cell survival. hrMSCs induce malignant transformation of the FTE, resulting in metastatic cancer in vivo, indicating that hrMSCs promote cancer initiation. hrMSCs are significantly enriched in BRCA1/2 mutation carriers and increase with age. Combined, these findings indicate that hrMSCs can incite ovarian cancer initiation and have important implications for ovarian cancer detection and prevention. Significance: This work demonstrates a critical role of fallopian tube stromal cells in HGSOC initiation with implications for the pathophysiology of HGSOC formation and the development of prevention and early detection strategies critically needed in this disease. Additionally, the identification of stromal-mediated epithelial transformation has broad implications for understanding pan-cancer initiation. See related commentary by Recouvreux and Orsulic, p. 1093

Identification of the MRTFA/SRF pathway as a critical regulator of quiescence and chemotherapy resistance in cancer

Chemoresistance is a major cause of cancer deaths. One understudied mechanism of chemoresistance is quiescence. We used single-cell culture to identify and isolate patient-derived proliferating and quiescent ovarian cancer cells (qOvCa). RNA-seq analysis indicated that hundreds of genes that are differentially expressed in qOvCa cells are transcriptional targets of the Myocardin-Related Transcription Factor-A/Serum Response Factor (MRTFA/SRF) pathway, and both genetic disruption and pharmacologic inhibition of MRTFA/SRF interaction (with the inhibitor CCG257081) induced quiescence across multiple cancer types. MRTFA/SRF inhibition-mediated quiescence is p27/Kip1 dependent and associated with a downregulation of cell cycle regulators, NCL, MYH9, and alterations in the proteasome. We show that the MRTFA/SRF axis plays a dual role in chemotherapy resistance, with both pathway inhibition and activation contributing to chemotherapy resistance in vitro and in patient samples. CCG081 treatment results in a proteasome-dependent downregulation of the stem-cell marker CD133. Suggesting a critical role for the proteasome in quiescent cells, CCG081 therapy sensitized OvCa cells to proteasome inhibitors. In vivo, we found that CCG257081 therapy could be used to induce tumor growth-arrest and delay disease growth to improve overall survival. Moreover, we found that dual therapy with CCG081 and proteasome inhibition further improved outcomes, leading to undetectable tumors in ∼20% of mice. Together, these data suggest that the MRTFA/SRF pathway is a critical regulator of quiescence in cancer and a potential therapeutic target.

Ribociclib (Ribociclib (LEE-011)) With Platinum-based Chemotherapy in Recurrent Platinum Sensitive Ovarian Cancer

Investigators hypothesize that concurrent ribociclib treatment and chemotherapy will enhance the response to platinum-based therapy and maintenance therapy will slow ovarian cancer tumor growth leading to prolongation in progression free survival.

Ruxolitinib Phosphate, Paclitaxel, and Carboplatin in Treating Patients With Stage III-IV Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

This phase I/II trial studies the side effects and the best dose of ruxolitinib phosphate when given together with paclitaxel and carboplatin and to see how well they work in treating patients with stage III-IV epithelial ovarian, fallopian tube, or primary peritoneal cancer. Ruxolitinib phosphate may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Drugs used in chemotherapy, such as paclitaxel and carboplatin, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Giving ruxolitinib phosphate together with paclitaxel and carboplatin may be a better treatment for epithelial ovarian, fallopian tube, or primary peritoneal cancer compared to paclitaxel and carboplatin alone.