Distinct Mechanisms of Mismatch-Repair Deficiency Delineate Two Modes of Response to Anti-PD-1 Immunotherapy in Endometrial Carcinoma.

Mismatch repair-deficient (MMRd) cancers have varied responses to immune-checkpoint blockade (ICB). We conducted a phase II clinical trial of the PD-1 inhibitor pembrolizumab in 24 patients with MMRd endometrial cancer (NCT02899793). Patients with mutational MMRd tumors (6 patients) had higher response rates and longer survival than those with epigenetic MMRd tumors (18 patients). Mutation burden was higher in tumors with mutational MMRd compared with epigenetic MMRd; however, within each category of MMRd, mutation burden was not correlated with ICB response. Pretreatment JAK1 mutations were not associated with primary resistance to pembrolizumab. Longitudinal single-cell RNA-seq of circulating immune cells revealed contrasting modes of antitumor immunity for mutational versus epigenetic MMRd cancers. Whereas effector CD8+ T cells correlated with regression of mutational MMRd tumors, activated CD16+ NK cells were associated with ICB-responsive epigenetic MMRd tumors. These data highlight the interplay between tumor-intrinsic and tumor-extrinsic factors that influence ICB response. SIGNIFICANCE: The molecular mechanism of MMRd is associated with response to anti-PD-1 immunotherapy in endometrial carcinoma. Tumors with epigenetic MMRd or mutational MMRd are correlated with NK cell or CD8+ T cell-driven immunity, respectively. Classifying tumors by the mechanism of MMRd may inform clinical decision-making regarding cancer immunotherapy. This article is highlighted in the In This Issue feature, p. 247.

Synergistic activity of neratinib in combination with olaparib in uterine serous carcinoma overexpressing HER2/neu.

INTRODUCTION: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with a poor prognosis. Approximately 30% of USC overexpress HER2/neu, a recognized target for trastuzumab in advanced/recurrent HER2/neu-positive USC. We evaluated the efficacy of the pan-c-erb inhibitor neratinib and the poly (ADP-ribose)-polymerase (PARP) inhibitor olaparib as single agents and in combination against USC cell lines and xenografts. METHODS: In-vitro cell-viability assays with olaparib, neratinib, and olaparib/neratinib were assessed using flow-cytometry based assays against a panel of USC cell lines with high and low HER2/neu expression. Homologous recombination deficiency (HRD) signatures were evaluated as described by Alexandrov et al. (Nature;2020;578:94-101) while downstream signaling affected by neratinib/olaparib exposure was assessed with immunoblotting. Efficacy of single- versus dual-agent inhibition was evaluated in-vivo using two USC-xenografts with 3+ HER2/neu expression. RESULTS: Neratinib was more potent than olaparib in suppression of in-vitro growth of HER2/neu 3+ cell lines (ARK1: p = 0.0047; ARK2: p = 0.0428) while no difference was noted against HER2/neu 1+ tumors (ARK4). Importantly, the combination of olaparib with neratinib synergistically improved tumor suppression compared to either single-agent in vitro. USC cells exposed to olaparib upregulated HER2/neu expression, while neratinib treatment increased PARP activity (ARK1: p < 0.0001; ARK2: p < 0.0001). Single-agent neratinib transiently inhibited in vivo growth of USC xenografts harboring HER2/neu gene amplification (ARK1: p < 0.05; ARK2: p < 0.05). In contrast, the combination of the two inhibitors caused a stronger and durable growth inhibition in both USC xenografts (ARK1: p < 0.05; ARK2: p < 0.05). CONCLUSION: The combination of olaparib and neratinib is active and synergistic against primary HER2/neu + USC. This combination may represent a novel therapeutic option for USC patients with HER2/neu+, homologous recombination-proficient tumors resistant to chemotherapy.

A Benzenesulfonamide-Based Mitochondrial Uncoupler Induces Endoplasmic Reticulum Stress and Immunogenic Cell Death in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and requires new therapeutic strategies to improve clinical outcomes. EOC metastasizes in the abdominal cavity through dissemination in the peritoneal fluid and ascites, efficiently adapt to the nutrient-deprived microenvironment, and resist current chemotherapeutic agents. Accumulating evidence suggests that mitochondrial oxidative phosphorylation is critical for the adaptation of EOC cells to this otherwise hostile microenvironment. Although chemical mitochondrial uncouplers can impair mitochondrial functions and thereby target multiple, essential pathways for cancer cell proliferation, traditional mitochondria uncouplers often cause toxicity that precludes their clinical application. In this study, we demonstrated that a mitochondrial uncoupler, specifically 2,5-dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide, hereinafter named Y3, was an antineoplastic agent in ovarian cancer models. Y3 treatment activated AMP-activated protein kinase and resulted in the activation of endoplasmic reticulum stress sensors as well as growth inhibition and apoptosis in ovarian cancer cells in vitro Y3 was well tolerated in vivo and effectively suppressed tumor progression in three mouse models of EOC, and Y3 also induced immunogenic cell death of cancer cells that involved the release of damage-associated molecular patterns and the activation of antitumor adaptive immune responses. These findings suggest that mitochondrial uncouplers hold promise in developing new anticancer therapies that delay tumor progression and protect patients with ovarian cancer against relapse.

Ovarian BDNF promotes survival, migration, and attachment of tumor precursors originated from p53 mutant fallopian tube epithelial cells.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. New evidence supports a hypothesis that HGSOC can originate from fallopian tube epithelium (FTE). It is unclear how genetic alterations and pathophysiological processes drive the progression of FTE tumor precursors into widespread HGSOCs. In this study, we uncovered that brain-derived neurotrophic factor (BDNF) in the follicular fluid stimulates the tropomyosin receptor kinase B (TrkB)-expressing FTE cells to promote their survival, migration, and attachment. Using in vitro and in vivo models, we further identified that the acquisition of common TP53 gain-of-function (GOF) mutations in FTE cells led to enhanced BDNF/TrkB signaling compared to that of FTE cells with TP53 loss-of-function (LOF) mutations. Different mutant p53 proteins can either increase TrkB transcription or enhance TrkB endocytic recycling. Our findings have demonstrated possible interplays between genetic alterations in FTE tumor precursors (i.e., p53 GOF mutations) and pathophysiological processes (i.e., the release of follicular fluid upon ovulation) during the initiation of HGSOC from the fallopian tube. Our data revealed molecular events underlying the link between HGSOC tumorigenesis and ovulation, a physiological process that has been associated with risk factors of HGSOC.

Inhibition of Heat Shock Protein 90 suppresses TWIST1 Transcription.

Molecular chaperone heat shock protein 90 (HSP90) is involved in oncogenic signaling pathways including epithelial-mesenchymal transition (EMT), a key process in tumor initiation, progression, metastasis, and chemoresistance. The molecular mechanisms underlying the involvement of HSP90 in EMT are still under investigation. In this study, we identified a previously unrecognized role of HSP90 in cooperating with signal transducer and activator of transcription 3 (STAT3) to regulate TWIST1 transcription in cancer cells. The HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin suppressed TWIST1 mRNA expression and promoter activity in epithelial ovarian cancer, renal clear cell cancer, and nasopharyngeal cancer cell lines. The interactions between HSP90 and transcription factors were visualized in cancer cell lines and tumor tissues using proximity ligation assays. Our findings reveal that HSP90 promotes the binding of STAT3 to the TWIST1 promoter, leading to the transcription of TWIST1. The inhibition of HSP90 downregulates STAT3 activity and TWIST1 transcription, thereby suppressing EMT and potentially inhibiting tumor progression, metastasis, and chemoresistance in different types of cancers. SIGNIFICANCE STATEMENT: Our study provides new evidence that HSP90 promotes EMT through enhancing TWIST1 transcription, which can be suppressed by HSP90 inhibitors. The HSP90 inhibitor inhibits EMT, thus potentially slowing down tumor growth, invasion, dissemination, metastasis, and drug resistance. These findings will hopefully pave the way for new therapeutic opportunities to target EMT and metastasis using HSP90 inhibitors.

Determination of two-photon photoactivation rates of fluorescent proteins.

The application of two-photon activation of photoactivatable fluorescent proteins is limited by a lack of information about two-photon activation rates. Here we present rates for the commonly used photoactivatable proteins PAmCherry, PAmKate and PA-GFP at different wavelengths using a novel method that allows us to determine the two-photon activation rates directly, independent of any reference data, with microscopic sample volumes. We show that PAmCherry features the highest rates of the tested proteins at 700 nm activation wavelength followed by PAmKate. Towards longer wavelengths, two-photon activation rates decrease for all three proteins. For PAmCherry, our data contradicts an activation model relying solely on two-photon activation and suggests additional activation pathways requiring at least two absorption steps. Our method is readily expandable to other photoactivatable fluorescent molecules. The presented results allow optimization of experimental conditions in spectroscopic and imaging techniques such as super-resolution fluorescence microscopy.

Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms.

Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.