Mutation profiles in circulating cell-free DNA predict acquired resistance to olaparib in high-grade serous ovarian carcinoma.

Although resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) has gradually become a major challenge in the maintenance therapy for high-grade serous ovarian carcinoma (HGSOC), there are no universal indicators for resistance monitoring in patients. A key resistance mechanism to PARPi is the restoration of homologous recombination repair (HRR), including BRCA reversion mutations and changes in DNA damage repair proteins. To explore mutation profiles associated with PARPi resistance, we undertook targeted 42-gene deep sequencing of circulating cell-free DNA (cfDNA) extracted from HGSOC patients pre- and post-treatment with olaparib maintenance therapy. We found that pathogenic germline mutations in the HRR pathway, including BRCA1/2, were strongly associated with improved clinical outcomes, and newly acquired MRE11A mutations significantly shortened the progression-free survival (PFS) of patients. Furthermore, dynamic fluctuations of somatic mutation sites in CHEK2:p.K373E and CHEK2:p.R406H can be used for evaluating the therapeutic efficacy of patients. MRE11A:p.K464R might be a vital driving factor of olaparib resistance, as patients with newly acquired MRE11A:p.K464R in post-treatment cfDNA had significantly shorter PFS than those without it. These findings provide potential noninvasive biomarkers for efficacy evaluation and resistance monitoring of olaparib treatment, and lay the foundation for developing combination treatment after olaparib resistance.

The Diversity of Vaginal Microbiota Predicts Neoadjuvant Chemotherapy Responsiveness in Locally Advanced Cervical Cancer.

The vaginal microbiota is closely related to HPV infection and cervical cancer (CC), but its relationship with platinum-based chemotherapy responsiveness is unknown. The study aimed to investigate the vaginal microbiota diversity of women with locally advanced cervical cancer (LACC) and compare the differences between responders and nonresponders. We characterized the 16S rRNA gene sequencing of vaginal microbiome of 66 vaginal samples, including 26 LACC patients before neoadjuvant chemotherapy and 40 healthy controls. Compared with the healthy controls, alpha diversity was significantly increased in CC patients (p <0.05) with more unconventionality bacterial colonization. Beta diversity also significantly differed between cervical cancer patients and controls (p <0.01). Within the CC patients, alpha diversity in vaginal samples was significantly higher in the nonresponders versus the responders (p <0.01), and the Ace index and Chao index were negatively correlated with mass reduction (p <0.001). Moreover, the Bacteroides genus enriched in the nonresponders had a ROC-plot AUC value reaching 0.84. The study suggests the vaginal microbiota in LACC patients is associated with platinum-based chemotherapy responsiveness. Alpha diversity and Bacteroides abundance have the potential of identifying platinum-resistant patients at an early time. These findings provide a basis for further research on the relationship between vaginal microbiome and chemotherapy effect in LACC.

WEE1 inhibitor and ataxia telangiectasia and RAD3-related inhibitor trigger stimulator of interferon gene-dependent immune response and enhance tumor treatment efficacy through programmed death-ligand 1 blockade.

WEE1 plays an important role in the regulation of cell cycle G2/M checkpoints and DNA damage response (DDR). Inhibition of WEE1 can increase the instability of the genome and have anti-tumor effects in some solid tumors. However, it has certain limitations for multiple cancer cells from different lineages. Therefore, we consider the use of synthetic lethal interactions to enhance the therapeutic effect. Our experiments proved that WEE1 inhibitor (WEE1i) can activate the ataxia telangiectasia and RAD3-related (ATR) pathway and that blockage of ATR dramatically sensitized the WEE1i-induced cell death. The tumor-selective synthetic lethality between bioavailable WEE1 and ATR inhibitors led to tumor remission in vivo. Mechanistically, the combination promoted the accumulation of cytosolic double-strand DNA, which subsequently activated the stimulator of the interferon gene (STING) pathway and induced the production of type I interferon and CD8+ T cells, thereby inducing anti-tumor immunity. Furthermore, our study found that immune checkpoint programmed death-ligand 1 is upregulated by the combination therapy, and blocking PD-L1 further enhances the effect of the combination therapy. In summary, as an immunomodulator, the combination of WEE1i with ATR inhibitor (ATRi) and immune checkpoint blockers provides a potential new approach for cancer treatment.

KMT2E Haploinsufficiency Manifests Autism-Like Behaviors and Amygdala Neuronal Development Dysfunction in Mice.

Autism spectrum disorders (ASD) are highly heterogeneous neurodevelopmental disorders characterized by impaired social interaction skills. Whole exome sequencing has identified loss-of-function mutations in lysine methyltransferase 2E (KMT2E, also named MLL5) in ASD patients and it is listed as an ASD high-risk gene in humans. However, experimental evidence of KMT2E in association with ASD-like manifestations or neuronal function is still missing. Relying on KMT2E+/- mice, through animal behavior analyses, positron emission tomography (PET) imaging, and neuronal morphological analyses, we explored the role of KMT2E haploinsufficiency in ASD-like symptoms. Behavioral results revealed that KMT2E haploinsufficiency was sufficient to produce social deficit, accompanied by anxiety in mice. Whole-brain 18F-FDG-PET analysis identified that relative amygdala glycometabolism was selectively decreased in KMT2E+/- mice compared to wild-type mice. The numbers and soma sizes of amygdala neurons in KMT2E+/- mice were prominently increased. Additionally, KMT2E mRNA levels in human amygdala were significantly decreased after birth during brain development. Our findings support a causative role of KMT2E in ASD development and suggest that amygdala neuronal development abnormality is likely a major underlying mechanism.

Codelivery of adavosertib and olaparib by tumor-targeting nanoparticles for augmented efficacy and reduced toxicity.

Ovarian cancer (OC) ranks first among gynecologic malignancies in terms of mortality. The benefits of poly (ADP-ribose) polymerase (PARP) inhibitors appear to be limited to OC with BRCA mutations. Concurrent administration of WEE1 inhibitors (eg, adavosertib (Ada)) and PARP inhibitors (eg, olaparib (Ola)) effectively suppress ovarian tumor growth regardless of BRCA mutation status, but is poorly tolerated. Henceforth, we aimed to seek a strategy to reduce the toxic effects of this combination by taking advantage of the mesoporous polydopamine (MPDA) nanoparticles with good biocompatibility and high drug loading capacity. In this work, we designed a tumor-targeting peptide TMTP1 modified MPDA-based nano-drug delivery system (TPNPs) for targeted co-delivery of Ada and Ola to treat OC. Ada and Ola could be effectively loaded into MPDA nanoplatform and showed tumor microenvironment triggered release behavior. The nanoparticles induced more apoptosis in OC cells, and significantly enhanced the synergy of combination therapy with Ada plus Ola in murine OC models. Moreover, the precise drug delivery of TPNPs towards tumor cells significantly diminished the toxic side effects caused by concurrent administration of Ada and Ola. Co-delivery of WEE1 inhibitors and PARP inhibitors via TPNPs represents a promising approach for the treatment of OC. STATEMENT OF SIGNIFICANCE: Combination therapy of WEE1 inhibitors (eg, Ada) with PARP inhibitors (eg, Ola) effectively suppress ovarian tumor growth regardless of BRCA mutation status. However, poor tolerability limits its clinical application. To address this issue, we construct a tumor-targeting nano-drug delivery system (TPNP) for co-delivery of Ada and Ola. The nanoparticles specifically target ovarian cancer and effectively enhance the antitumor effect while minimizing undesired toxic side effects. As the first nanomedicine co-loaded with a WEE1 inhibitor and a PARP inhibitor, TPNP-Ada-Ola may provide a promising and generally applicable therapeutic strategy for ovarian cancer patients.

MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

BACKGROUND: Although the clinical application of PARP inhibitors has brought hope to ovarian cancer, the problem of its resistance has become increasingly prominent. Therefore, clinical experts have been focused on finding specific indicators and therapeutic targets that can be used for resistance monitoring of PARP inhibitors. RESULTS: By cfDNA detecting during Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer, we found the presence of MRE11:p.K464R mutation was strongly associated with acquired Olaparib resistance. Structural analysis revealed that the MRE11:p.K464R mutation is situated at a critical site where the MRE11 protein interacts with other biomolecules, leading to potential structural and functional abnormalities of MRE11 protein. Functionally, MRE11:p.K464R mutation enhanced the tolerance of Olaparib by reducing the DNA damage. Mechanistically, MRE11:p.K464R mutation improved the efficiency of DNA damage repair and induce Olaparib resistance by enhancing its binding activity with the interacting proteins (including RAD50 and RPS3). Among them, the enhanced binding of MRE11:p.K464R mutation to RAD50/RPS3 facilitated non-homologous end joining (NHEJ) repair in tumor cells, thereby expanding the scope of research into acquired resistance to PARP inhibitors. CONCLUSIONS: Our findings provide a theoretical basis for MRE11:p.K464R mutation as a specific indicator of resistance monitoring in Olaparib treatment, and the exploration of its resistance mechanism provides a novel insights for the formulation of combination ther therapies after Olaparib resistance.

BRD4 inhibition impairs DNA mismatch repair, induces mismatch repair mutation signatures and creates therapeutic vulnerability to immune checkpoint blockade in MMR-proficient tumors.

BACKGROUND: Mismatch repair deficiency (dMMR) is a well-recognized biomarker for response to immune checkpoint blockade (ICB). Strategies to convert MMR-proficient (pMMR) to dMMR phenotype with the goal of sensitizing tumors to ICB are highly sought. The combination of bromodomain containing 4 (BRD4) inhibition and ICB provides a promising antitumor effect. However, the mechanisms underlying remain unknown. Here, we identify that BRD4 inhibition induces a persistent dMMR phenotype in cancers. METHODS: We confirmed the correlation between BRD4 and mismatch repair (MMR) by the bioinformatic analysis on The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium data, and the statistical analysis on immunohistochemistry (IHC) scores of ovarian cancer specimens. The MMR genes (MLH1,MSH2,MSH6,PMS2) were measured by quantitative reverse transcription PCR, western blot, and IHC. The MMR status was confirmed by whole exome sequencing, RNA sequencing, MMR assay and hypoxanthine-guanine phosphoribosyl transferase gene mutation assay. The BRD4i AZD5153 resistant models were induced both in vitro and in vivo. The transcriptional effects of BRD4 on MMR genes were investigated by chromatin immunoprecipitation among cell lines and data from the Cistrome Data Browser. The therapeutic response to ICB was testified in vivo. The tumor immune microenvironment markers, such as CD4, CD8, TIM-3, FOXP3, were measured by flow cytometry. RESULTS: We identified the positive correlation between BRD4 and MMR genes in transcriptional and translational aspects. Also, the inhibition of BRD4 transcriptionally reduced MMR genes expression, resulting in dMMR status and elevated mutation loads. Furthermore, prolonged exposure to AZD5153 promoted a persistent dMMR signature both in vitro and in vivo, enhancing tumor immunogenicity, and increased sensitivity to α-programmed death ligand-1 therapy despite the acquired drug resistance. CONCLUSIONS: We demonstrated that BRD4 inhibition suppressed expression of genes critical to MMR, dampened MMR, and increased dMMR mutation signatures both in vitro and in vivo, sensitizing pMMR tumors to ICB. Importantly, even in BRD4 inhibitors (BRD4i)-resistant tumor models, the effects of BRD4i on MMR function were maintained rendering tumors sensitive to ICB. Together, these data identified a strategy to induce dMMR in pMMR tumors and further, indicated that BRD4i sensitive and resistant tumors could benefit from immunotherapy.

Multi-omics characterization of silent and productive HPV integration in cervical cancer.

Cervical cancer (CC) that is caused by high-risk human papillomavirus (HPV) remains a significant public health problem worldwide. HPV integration sites can be silent or actively transcribed, leading to the production of viral-host fusion transcripts. Herein, we demonstrate that only productive HPV integration sites were nonrandomly distributed across both viral and host genomes, suggesting that productive integration sites are under selection and likely to contribute to CC pathophysiology. Furthermore, using large-scale, multi-omics (clinical, genomic, transcriptional, proteomic, phosphoproteomic, and single-cell) data, we demonstrate that tumors with productive HPV integration are associated with higher E6/E7 proteins and enhanced tumor aggressiveness and immunoevasion. Importantly, productive HPV integration increases from carcinoma in situ to advanced disease. This study improves our understanding of the functional consequences of HPV fusion transcripts on the biology and pathophysiology of HPV-driven CCs, suggesting that productive HPV integration should be evaluated as an indicator of high risk for progression to aggressive cancers.

Akkermansia supplementation reverses the tumor-promoting effect of the fecal microbiota transplantation in ovarian cancer.

Ovarian cancer (OC) remains a clinical challenge for its difficulty in early diagnosis and insensitivity to treatments. Gut microbiota modulate multiple carcinoma progression through immunoregulation. The relationship between OC and gut microbiota has not been fully characterized. We find that the feces of patients with OC demonstrate different characteristics from benign controls. After fecal microbiota transplantation (FMT) from patients with OC into OC-bearing mice, the tumor development accelerates. Further, an Akkermansia supplementation with FMT significantly suppresses OC progression in mice. RNA sequencing of tumors shows that T cell activation pathways are upregulated after Akkermansia supplementation with FMT. Moreover, acetate accumulation accompanies Akkermansia abundance elevation, which is associated with enhanced interferon γ (IFNγ) secretion of CD8+ T cells and also its tumor-killing property. This work highlights the importance of protective gut microbiome in immune surveillance of OC, which connects accumulation of acetate and the cytotoxic function of CD8+ T cells by increasing IFNγ secretion.

Metagenomic analysis of the microbiome of the upper reproductive tract: combating ovarian cancer through predictive, preventive, and personalized medicine.

Purpose: We investigated whether ovarian cancer could alter the genital microbiota in a specific way with clinical values. Furthermore, we proposed how such changes could be envisioned in a paradigm of predictive, preventive, and personalized medicine (PPPM). Methods: The samples were collected using cotton swabs from the cervical, uterine cavity, fallopian tubes, and ovaries of patients subjected to the surgical procedures for the malignant/benign lesions. All samples were then analyzed by metagenomic shotgun sequencing. The distribution patterns and characteristics of the microbiota in the reproductive tract of subjects were analyzed and were interpreted in relation to the clinical outcomes of the subjects. Results: While the ovarian cancer was able to alter the genital microbiota, the bacteria were the dominant microorganisms in all samples across all cohorts in the study (median 99%). The microbiota of the upper female reproductive tract were mainly from the cervical, identified by low bacterial biomass and high bacterial diversity. Ovarian cancer had a distinct microbiota signature. The tubal ligation affects its microbial distribution. There were no different species on the surface of platinum-sensitive ovarian tissues compared to samples from platinum-resistant patients. Conclusion: The ovarian cancer-induced changes in microbiota magnify the potential of microbiota as a biotherapeutic modality in the treatment of ovarian cancer in this study and very likely for several malignancies and other conditions. Our findings demonstrated, for the first time, that microbiota could be dissected and applied in more specific fashion based on a predictive, preventive, and personalized medicine (PPPM) model in the treatment of ovarian cancer. Utilizing microbiota portfolio in a PPPM system in ovarian cancer would provide a unique opportunity to a clinically intelligent and novel approach in the treatment of ovarian cancer as well as several other conditions and malignancies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-022-00286-1.

AZD5153 reverses palbociclib resistance in ovarian cancer by inhibiting cell cycle-related proteins and the MAPK/PI3K-AKT pathway.

The CDK4/6 inhibitor, palbociclib has recently entered clinic-trial stage for breast cancer treatment. However, translating its efficacy to other solid tumors has been challenging, especially for aggressive solid tumors. We found that the effect of palbociclib as a single agent was limited due to primary and acquired resistance in multiple ovarian cancer (OC) models. Among these, patient-derived organoid and xenograft models are two most representative models of drug responsiveness in patients with OC. In preclinical models, this study demonstrated that activated MAPK/PI3K-AKT pathway and cell cycle-related proteins induced the resistance to palbociclib, which was overcome by the addition of the bromodomain protein 4 (BRD4) inhibitor AZD5153. Moreover, this study revealed that AZD5153 and palbociclib had a synergistic lethal effect on inducing the cell cycle arrest and increasing apoptosis, even in RB-deficient cell lines. Based on these results, it is anticipated that this class of drugs, including AZD5153, which inhibit the cell cycle-related protein and MAPK/PI3K-AKT pathway, will exhibit synergistic effects with palbociclib in OC.

Spatial heterogeneity of infiltrating T cells in high-grade serous ovarian cancer revealed by multi-omics analysis.

Tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, represent a favorable prognostic factor in high-grade serous ovarian cancer (HGSOC) and other tumor lineages. Here, we analyze the spatial heterogeneity of different TIL subtypes in HGSOC. We integrated RNA sequencing, whole-genome sequencing, bulk T cell receptor (TCR) sequencing, as well as single-cell RNA/TCR sequencing to investigate the characteristics and differential composition of TILs across different HGSOC sites. Two immune "cold" patterns in ovarian cancer are identified: (1) ovarian lesions with low infiltration of mainly dysfunctional T cells and immunosuppressive Treg cells and (2) omental lesions infiltrated with non-tumor-specific bystander cells. Exhausted CD8 T cells that are preferentially enriched in ovarian tumors exhibit evidence for expansion and cytotoxic activity. Inherent tumor immune microenvironment characteristics appear to be the main contributor to the spatial differences in TIL status. The landscape of spatial heterogeneity of TILs may inform potential strategies for therapeutic manipulation in HGSOC.

Prognostic value of immune-related genes and immune cell infiltration analysis in the tumor microenvironment of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the few malignant tumors that respond well to immunotherapy. We aimed to investigate the immune-related genes and immune cell infiltration of HNSCC and construct a predictive model for its prognosis. METHODS: We calculated the stromal/immune scores of patients with HNSCC from The Cancer Genome Atlas using the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data algorithm and investigated the relationship between the scores and patients' prognosis. Three machine learning algorithms (LASSO, Random Forest, and Rbsurv) were performed to screen key immune-related genes and constructed a predictive model. The immune cell infiltrating was calculated by the Tumor Immune Estimation Resource algorithm. RESULTS: The stromal and immune scores significantly correlated with prognosis. A 6-gene signature was selected and displayed a robust predictive effect. The expressions of key genes were associated with immune infiltrating. GSE65858 validated the results. CONCLUSION: Our study comprehensively analyzed the tumor microenvironment of HNSCC and constructed a robust predictive model, providing a basis for further investigation of therapy.

Clinical and molecular characteristics of COVID-19 patients with persistent SARS-CoV-2 infection.

The characteristics of COVID-19 patients with persistent SARS-CoV-2 infection are not yet well described. Here, we compare the clinical and molecular features of patients with long duration of viral shedding (LDs) with those from patients with short duration patients (SDs), and healthy donors (HDs). We find that several cytokines and chemokines, such as interleukin (IL)-2, tumor necrosis factor (TNF) and lymphotoxin α (LT-α) are present at lower levels in LDs than SDs. Single-cell RNA sequencing shows that natural killer (NK) cells and CD14+ monocytes are reduced, while regulatory T cells are increased in LDs; moreover, T and NK cells in LDs are less activated than in SDs. Importantly, most cells in LDs show reduced expression of ribosomal protein (RP) genes and related pathways, with this inversed correlation between RP levels and infection duration further validated in 103 independent patients. Our results thus indicate that immunosuppression and low RP expression may be related to the persistence of the viral infection in COVID-19 patients.

Impact of chemotherapy and immunotherapy on the composition and function of immune cells in COVID-19 convalescent with gynecological tumors.

Ongoing pandemic and potential resurgence of Coronavirus disease 2019 (COVID-19) has prompted urgent efforts to investigate the immunological memory of convalescent patients, especially in patients with active cancers. Here we performed single-cell RNA sequencing in peripheral blood samples of 3 healthy donors (HDs), 4 COVID-19 patients (Covs) and 4 COVID-19 patients with active gynecological tumor (TCs) pre- and post- anti-tumor treatment. All Covs patients had recovered from their acute infection. Interestingly, the molecular features of PBMCs in TCs are similar to that in Covs, suggesting that convalescent COVID-19 with gynecologic tumors do not have major immunological changes and may be protected against reinfection similar to COVID-19 patients without tumors. Moreover, the chemotherapy given to these patients mainly caused neutropenia, while having little effect on the proportion and functional phenotype of T and B cells, and T cell clonal expansion. Notably, anti-PD-L1 treatment massively increased cytotoxic scores of NK cells, and T cells, and facilitated clonal expansion of T cells in these patients. It is likely that T cells could protect patients from SARS-CoV-2 virus reinfection and anti-PD-L1 treatment can enhance the anti-viral activity of the T cells.

The Immunological Role of CDK4/6 and Potential Mechanism Exploration in Ovarian Cancer.

Background: Ovarian cancer (OC) is one of the most lethal gynecologic cancers. Growing evidence has proven that CDK4/6 plays a key role in tumor immunity and the prognosis of many cancers. However, the expression and function of CDK4/6 in OC remain unclear. Therefore, we aimed to explore the influence of CDK4/6 in OC, especially on immunity. Methods: We analyzed CDK4/6 expression and prognosis using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Genotype Tissue Expression (GTEx) data. Subsequently, we used the cytoHubba plug-in of Cytoscape software and starBase to identify the noncoding RNAs (ncRNAs) regulating CDK4/6. Finally, we verified the effect of CDK4/6 on immunity in OC cell lines and animal models. Results: CDK4/6 expression was higher in OC tissues than in normal ovarian tissues, and the high expression levels of CDK4/6 contributed to the immunosuppressive state of OC and were thus related to the poor prognosis of OC patients. This was also in general agreement with the results of OC cell line and animal experiments. Mechanistically, the CDK4/6 inhibitor palbociclib increased the secretion of interferon (IFN)-γ and the interferon-stimulated gene (ISG) response, thereby upregulating the expression of antigen-presenting molecules; this effect was partly dependent on the STING pathway and thus activated immunity in OC. Additionally, according to public data, the LRRC75A-AS1-hsa-miR-330-5p axis could inhibit the immune response of OC patients by upregulating CDK4/6, leading to a poor prognosis. Conclusion: CDK4/6 affects the immune microenvironment of OC and correlates with the prognosis of OC patients.