High-Grade Endometrial Cancer: Molecular Subtypes, Current Challenges, and Treatment Options.

Although many recent advancements have been made in women's health, perhaps one of the most neglected areas of research is the diagnosis and treatment of high-grade endometrial cancer (EnCa). The molecular classification of EnCa in concert with histology was a major step forward. The integration of profiling for mismatch repair deficiency and Human Epidermal Growth Factor 2 (HER2) overexpression, can further inform treatment options, especially for drug resistant recurrent disease. Recent early phase trials suggest that regardless of subtype, combination therapy with agents that have distinct mechanisms of action is a fruitful approach to the treatment of high-grade EnCa. Unfortunately, although the importance of diagnosis and treatment of high-grade EnCa is well recognized, it is understudied compared to other gynecologic and breast cancers. There remains a tremendous need to couple molecular profiling and biomarker development with promising treatment options to inform new treatment strategies with higher efficacy and safety for all who suffer from high-grade recurrent EnCa.

Phase I/II Study of Combined BCL-xL and MEK Inhibition with Navitoclax and Trametinib in KRAS or NRAS Mutant Advanced Solid Tumors.

PURPOSE: MEK inhibitors (MEKi) lack monotherapy efficacy in most RAS-mutant cancers. BCL-xL is an anti-apoptotic protein identified by a synthetic lethal shRNA screen as a key suppressor of apoptotic response to MEKi. PATIENTS AND METHODS: We conducted a dose escalation study (NCT02079740) of the BCL-xL inhibitor navitoclax and MEKi trametinib in patients with RAS-mutant tumors with expansion cohorts for: pancreatic, gynecologic (GYN), non-small cell lung cancer (NSCLC), and other cancers harboring KRAS/NRAS mutations. Paired pretreatment and day 15 tumor biopsies and serial cell-free (cf)DNA were analyzed. RESULTS: A total of 91 patients initiated treatment, with 38 in dose escalation. Fifty-eight percent had ≥3 prior therapies. A total of 15 patients (17%) had colorectal cancer, 19 (11%) pancreatic, 15 (17%) NSCLC, and 32 (35%) GYN cancers. The recommended phase II dose (RP2D) was established as trametinib 2 mg daily days 1 to 14 and navitoclax 250 mg daily days 1 to 28 of each cycle. Most common adverse events included diarrhea, thrombocytopenia, increased AST/ALT, and acneiform rash. At RP2D, 8 of 49 (16%) evaluable patients achieved partial response (PR). Disease-specific differences in efficacy were noted. In patients with GYN at the RP2D, 7 of 21 (33%) achieved a PR and median duration of response 8.2 months. No PRs occurred in patients with colorectal cancer, NSCLC, or pancreatic cancer. MAPK pathway inhibition was observed in on-treatment tumor biopsies. Reductions in KRAS/NRAS mutation levels in cfDNA correlated with clinical benefit. CONCLUSIONS: Navitoclax in combination with trametinib was tolerable. Durable clinical responses were observed in patients with RAS-mutant GYN cancers, warranting further evaluation in this population.

Targeting Galectin 3 illuminates its contributions to the pathology of uterine serous carcinoma.

BACKGROUND: Uterine serous cancer (USC) comprises around 10% of all uterine cancers. However, USC accounts for approximately 40% of uterine cancer deaths, which is attributed to tumor aggressiveness and limited effective treatment. Galectin 3 (Gal3) has been implicated in promoting aggressive features in some malignancies. However, Gal3's role in promoting USC pathology is lacking. METHODS: We explored the relationship between LGALS3 levels and prognosis in USC patients using TCGA database, and examined the association between Gal3 levels in primary USC tumors and clinical-pathological features. CRISPR/Cas9-mediated Gal3-knockout (KO) and GB1107, inhibitor of Gal3, were employed to evaluate Gal3's impact on cell function. RESULTS: TCGA analysis revealed a worse prognosis for USC patients with high LGALS3. Patients with no-to-low Gal3 expression in primary tumors exhibited reduced clinical-pathological tumor progression. Gal3-KO and GB1107 reduced cell proliferation, stemness, adhesion, migration, and or invasion properties of USC lines. Furthermore, Gal3-positive conditioned media (CM) stimulated vascular tubal formation and branching and transition of fibroblast to cancer-associated fibroblast compared to Gal3-negative CM. Xenograft models emphasized the significance of Gal3 loss with fewer and smaller tumors compared to controls. Moreover, GB1107 impeded the growth of USC patient-derived organoids. CONCLUSION: These findings suggest inhibiting Gal3 may benefit USC patients.

Structural basis for antibody recognition of the proximal MUC16 ectodomain.

BACKGROUND: Mucin 16 (MUC16) overexpression is linked with cancer progression, metastasis, and therapy resistance in high grade serous ovarian cancer and other malignancies. The cleavage of MUC16 forms independent bimodular fragments, the shed tandem repeat sequence which circulates as a protein bearing the ovarian cancer biomarker (CA125) and a proximal membrane-bound component which is critical in MUC16 oncogenic behavior. A humanized, high affinity antibody targeting the proximal ectodomain represents a potential therapeutic agent against MUC16 with lower antigenic potential and restricted human tissue expression. RESULTS: Here, we demonstrate the potential therapeutic versatility of the humanized antibody as a monoclonal antibody, antibody drug conjugate, and chimeric antigen receptor. We report the crystal structures of 4H11-scFv, derived from an antibody specifically targeting the MUC16 C-terminal region, alone and in complex with a 26-amino acid MUC16 segment resolved at 2.36 Å and 2.47 Å resolution, respectively. The scFv forms a robust interaction with an epitope consisting of two consecutive ß-turns and a ß-hairpin stabilized by 2 hydrogen bonds. The VH-VL interface within the 4H11-scFv is stabilized through an intricate network of 11 hydrogen bonds and a cation-π interaction. CONCLUSIONS: Together, our studies offer insight into antibody-MUC16 ectodomain interaction and advance our ability to design agents with potentially improved therapeutic properties over anti-CA125 moiety antibodies.

Phase IIa Study of PLX2853 in Gynecologic Cancers With Known ARID1A Mutation and Phase Ib/IIa Study of PLX2853/Carboplatin in Platinum-Resistant Epithelial Ovarian Cancer.

PURPOSE: The Bromodomain and Extra-Terminal (BET) domain proteins facilitate the development of many human cancers via epigenetic regulation. BET inhibitors may be effective in reversing platinum resistance in ovarian cancer (OC) and may generate synthetic lethality with ARID1A loss. PLX2853 is an orally active, small-molecule inhibitor of BET bromodomain-mediated interactions that exhibits low nanomolar potency in blocking all four BET family members. METHODS: We conducted a multicenter and open-label study with two parallel arms: a phase IIa study of PLX2853 monotherapy in patients with advanced gynecologic malignancies with an ARID1A mutation and a phase Ib/IIa combination study of PLX2853 plus carboplatin in women with platinum-resistant OC. The primary objectives were safety and tolerability for phase Ib and efficacy for both phase IIa portions. Thirty-four of 37 enrolled patients completed at least one post-baseline response assessment. RESULTS: Of the 14 evaluable patients on the monotherapy arm, 1 (7.1%) achieved a best overall response of partial response (PR), 5 (35.7%) had stable disease (SD), and 8 (57.1%) had progressive disease (PD). Of the 20 evaluable patients on the combination arm, 1 (5.0%) had PR, 9 (45.0%) had SD, and 10 (50%) had PD. CONCLUSION: This study confirmed the safety profile of PLX2853 and demonstrated the feasibility of combination with carboplatin. Although these results did not meet the prespecified response criteria, evidence of clinical activity highlights the rationale for further exploration of BET inhibitors in patients with ARID1A-mutated gynecologic malignancies, possibly in combination with agents targeting potential feedback mechanisms such as the PI3K pathway.

Intraperitoneal Therapy for Ovarian Cancer - Some Answers, More Questions, and Missed Opportunities.

Intraperitoneal (i.p.) therapy set a new treatment standard for patients with advanced-stage ovarian cancer in 2006 based on data showing improved overall survival in the trial by Armstrong et al.1 This trial showed a statistically significant and clinically meaningful improvement in median overall survival of almost 16 months in favor of patients treated with i.p. chemotherapy compared with an intravenous approach. Since then, several clinical trials have aimed to better understand what population of patients are most likely to benefit from this therapy. Will patients with earlier-stage disease or suboptimal cytoreduction after surgery benefit? Does tumor histology matter?

Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents.

Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure-activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non-receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Effect of Mismatch Repair Status on Outcome of Early-Stage Grade 1 to 2 Endometrial Cancer Treated With Vaginal Brachytherapy.

OBJECTIVES: The objective of this study was to determine if deficiency of mismatch repair (dMMR) proteins in patients with early-stage favorable endometrial cancer treated with vaginal brachytherapy (VB) is associated with increased recurrence. MATERIALS AND METHODS: A multi-institutional retrospective cohort study of 141 patients with stage I to II grade 1 and 2 endometrioid adenocarcinoma treated with surgery and adjuvant VB was performed to compare recurrence risk in dMMR (n=41) versus MMR-preserved (pMMR) (n=100). Additional clinical and pathologic risk factors were also collected. Univariate analysis and multivariable analysis Cox regression analysis was performed to identify factors associated with any recurrence. Kaplan-Meier method and log rank test were used to compare recurrence free survival and overall survival (OS). RESULTS: Median follow up was 42 months. Forty-one patients (29%) were dMMR. There were 7 recurrences (17%) in dMMR versus 4 recurrences (4%) in pMMR (P=0.009). On univariate analysis of any recurrence, both dMMR (hazard ratio: 5.3, P=0.008) and stage (hazard ratio: 3.8, P=0.05) were statistically significantly associated with time to first recurrence. The 5-year recurrence free survival was 90% (95% CI: 73%-96%) in pMMR versus 61.0% (95% CI: 19%-86%) in dMMR (P=0.003). Five-year OS was 96% (95% CI: 76%-99%) in pMMR versus 86% (95% CI: 62%-95%) in dMMR (P=0.03). CONCLUSIONS: MMR deficiency in stage I to II grade 1 to 2 endometrial cancer patients treated with adjuvant VB alone was associated with statistically significant increased risk for any recurrence and worse OS. MMR status may be an important prognosticator in this cohort of patients warranting adjuvant treatment intensification in the clinical trial setting.

Bispecific T-Cell Engaging Antibodies Against MUC16 Demonstrate Efficacy Against Ovarian Cancer in Monotherapy and in Combination With PD-1 and VEGF Inhibition.

Immunotherapy for ovarian cancer is an area of intense investigation since the majority of women with relapsed disease develop resistance to conventional cytotoxic therapy. The paucity of safe and validated target antigens has limited the development of clinically relevant antibody-based immunotherapeutics for this disease. Although MUC16 expression is almost universal in High Grade Serous Ovarian Cancers, engagement of the shed circulating MUC16 antigen (CA-125) presents a theoretical risk of systemic activation and toxicity. We designed and evaluated a series of bispecific tandem single-chain variable fragments specific to the retained portion of human MUC16 ectodomain (MUC16ecto) and human CD3. These MUC16ecto- BiTEDs retain binding in the presence of soluble MUC16 (CA-125) and show cytotoxicity against a panel of ovarian cancer cells in vitro. MUC16ecto- BiTEDs delay tumor progression in vivo and significantly prolong survival in a xenograft model of ovarian peritoneal carcinomatosis. This effect was significantly enhanced by antiangiogenic (anti-VEGF) therapy and immune checkpoint inhibition (anti-PD1). However, the combination of BiTEDs with anti-VEGF was superior to combination with anti-PD1, based on findings of decreased peritoneal tumor burden and ascites with the former. This study shows the feasibility and efficacy of MUC16ecto- specific BiTEDs and provides a basis for the combination with anti-VEGF therapy for ovarian cancer.

Tumor derived UBR5 promotes ovarian cancer growth and metastasis through inducing immunosuppressive macrophages.

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic ß-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness.

Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo.

The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment.

Chimeric antigen receptor (CAR) T cell therapy has shown limited efficacy for the management of solid tumor malignancies. In ovarian cancer, this is in part due to an immunosuppressive cytokine and cellular tumor microenvironment which suppresses adoptively transferred T cells. We engineered an armored CAR T cell capable of constitutive secretion of IL-12, and delineate the mechanisms via which these CAR T cells overcome a hostile tumor microenvironment. In this report, we demonstrate enhanced proliferation, decreased apoptosis and increased cytotoxicity in the presence of immunosuppressive ascites. In vivo, we show enhanced expansion and CAR T cell antitumor efficacy, culminating in improvement in survival in a syngeneic model of ovarian peritoneal carcinomatosis. Armored CAR T cells mediated depletion of tumor associated macrophages and resisted endogenous PD-L1-induced inhibition. These findings highlight the role of the inhibitory microenvironment and how CAR T cells can be further engineered to maintain efficacy.

Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy.

Chimaeric antigen receptor (CAR) T-cells are T-cells that have been genetically modified to express an artificial construct consisting of a synthetic T-cell receptor (TCR) targeted to a predetermined antigen expressed on a tumour. Coupling the T-cell receptor to a CD3ζ signalling domain paved the way for first generation CAR T-cells that were efficacious against cluster of differentiation (CD)19-expressing B-cell malignancies. Optimization with additional signalling domains such as CD28 or 4-1BB in addition to CD3ζ provided T-cell activation signal 2 and further improved the efficacy and persistence of these second generation CAR T-cells. Third generation CAR T-cells which utilize two tandem costimulatory domains have also been reported. In this review, we discuss a different approach to optimization of CAR T-cells. Through additional genetic modifications, these resultant armored CAR T-cells are typically modified second generation CAR T-cells that have been further optimized to inducibly or constitutively secrete active cytokines or express ligands that further armor CAR T-cells to improve efficacy and persistence. The choice of the 'armor' agent is based on knowledge of the tumour microenvironment and the roles of other elements of the innate and adaptive immune system. Although there are several variants of armored CAR T-cells under investigation, here we focus on three unique approaches using interleukin-12 (IL-12), CD40L and 4-1BBL. These agents have been shown to further enhance CAR T-cell efficacy and persistence in the face of a hostile tumour microenvironment via different mechanisms.