Endogenous Retrovirus RNA Expression Differences between Race, Stage and HPV Status Offer Improved Prognostication among Women with Cervical Cancer.

Endogenous human retroviruses (ERVs) are remnants of exogenous retroviruses that have integrated into the human genome. Using publicly available RNA-seq data from 63 cervical cancer patients, we investigated the expression of ERVs in cervical cancers. Four aspects of cervical cancer were investigated: patient ancestral background, tumor HPV type, tumor stage and patient survival. Between the racial subgroups, 74 ERVs were significantly differentially expressed, with Black Americans having 30 upregulated and 44 downregulated (including MER21C, HERV9-int, and HERVH-int) ERVs when compared to White Americans. We found that 3313 ERVs were differentially expressed between HPV subgroups, including MER41A, HERVH-int and HERVK9. There were 28 downregulated (including MLT1D and HERVH-int) and 61 upregulated (including MER41A) ERVs in locally advanced-stage compared to early-stage samples. Tissue microarrays of cervical cancer patients were used to investigate the protein expression of ERVs with protein coding potential (i.e., HERVK and ERV3). Significant differences in protein expression of ERV3 (p = 0.000905) were observed between early-stage and locally advanced-stage tumors. No significant differential expression at the protein level was found for HERVK7 (p = 0.243). We also investigated a prognostic model, supplementing a baseline prediction model using FIGO stage, age and HPV positivity with ERVs data. The expression levels of all ERVs in the HERVd were input into a Lasso-Cox proportional hazards model, developing a predictive 67-ERV panel. When ERVs expression levels were supplemented with the clinical data, a significant increase in prognostic power (p = 9.433 × 10-15) relative to that obtained with the clinical parameters alone (p = 0.06027) was observed. In summary, ERV RNA expression in cervical cancer tumors is significantly different among racial cohorts, HPV subgroups and disease stages. The combination of the expression of certain ERVs in cervical cancers with clinical factors significantly improved prognostication compared to clinical factors alone; therefore, ERVs may serve as future prognostic biomarkers and therapeutic targets. Novelty and Impact: When endogenous retroviral (ERV) expression signatures were combined with currently employed clinical prognosticators of relapse of cervical cancer, the combination outperformed prediction models based on clinical prognosticators alone. ERV expression signatures in tumor biopsies may therefore be useful to help identify patients at greater risk of recurrence. The novel ERV expression signatures or adjacent genes possibly impacted by ERV expression described here may also be targets for the development of future therapeutic interventions.

GAiN: An integrative tool utilizing generative adversarial neural networks for augmented gene expression analysis.

Big genomic data and artificial intelligence (AI) are ushering in an era of precision medicine, providing opportunities to study previously under-represented subtypes and rare diseases rather than categorize them as variances. However, clinical researchers face challenges in accessing such novel technologies as well as reliable methods to study small datasets or subcohorts with unique phenotypes. To address this need, we developed an integrative approach, GAiN, to capture patterns of gene expression from small datasets on the basis of an ensemble of generative adversarial networks (GANs) while leveraging big population data. Where conventional biostatistical methods fail, GAiN reliably discovers differentially expressed genes (DEGs) and enriched pathways between two cohorts with limited numbers of samples (n = 10) when benchmarked against a gold standard. GAiN is freely available at GitHub. Thus, GAiN may serve as a crucial tool for gene expression analysis in scenarios with limited samples, as in the context of rare diseases, under-represented populations, or limited investigator resources.

SCCA1/SERPINB3 promotes suppressive immune environment via STAT-dependent chemokine production, blunting the therapy-induced T cell responses.

Radiotherapy is a commonly used cancer treatment; however, patients with high serum squamous cell carcinoma antigen (SCCA1/SERPINB3) are associated with resistance and poor prognosis. Despite being a strong clinical biomarker, the modulation of SERPINB3 in tumor immunity is poorly understood. We investigated the microenvironment of SERPINB3 high tumors through RNAseq of primary cervix tumors and found that SERPINB3 was positively correlated with CXCL1/8, S100A8/A9 and myeloid cell infiltration. Induction of SERPINB3 in vitro resulted in increased CXCL1/8 and S100A8/A9 production, and supernatants from SERPINB3-expressing cultures attracted monocytes and MDSCs. In murine tumors, the orthologue mSerpinB3a promoted MDSC, TAM, and M2 macrophage infiltration contributing to an immunosuppressive phenotype, which was further augmented upon radiation. Radiation-enhanced T cell response was muted in SERPINB3 tumors, whereas Treg expansion was observed. A STAT-dependent mechanism was implicated, whereby inhibiting STAT signaling with ruxolitinib abrogated suppressive chemokine production. Patients with elevated pre-treatment serum SCCA and high pSTAT3 had increased intratumoral CD11b+ myeloid cell compared to patients with low SCCA and pSTAT3 cohort that had overall improved cancer specific survival after radiotherapy. These findings provide a preclinical rationale for targeting STAT signaling in tumors with high SERPINB3 to counteract the immunosuppressive microenvironment and improve response to radiation.

SCCA1/SERPINB3 suppresses antitumor immunity and blunts therapy-induced T cell responses via STAT-dependent chemokine production.

Patients with cancer who have high serum levels of squamous cell carcinoma antigen 1 (SCCA1, now referred to as SERPINB3) commonly experience treatment resistance and have a poor prognosis. Despite being a clinical biomarker, the modulation of SERPINB3 in tumor immunity is poorly understood. We found positive correlations of SERPINB3 with CXCL1, CXCL8 (CXCL8/9), S100A8, and S100A9 (S100A8/A9) myeloid cell infiltration through RNA-Seq analysis of human primary cervical tumors. Induction of SERPINB3 resulted in increased CXCL1/8 and S100A8/A9 expression, which promoted monocyte and myeloid-derived suppressor cell (MDSC) migration in vitro. In mouse models, Serpinb3a tumors showed increased MDSC and tumor-associated macrophage (TAM) infiltration, contributing to T cell inhibition, and this was further augmented upon radiation. Intratumoral knockdown (KD) of Serpinb3a resulted in tumor growth inhibition and reduced CXCL1 and S100A8/A expression and MDSC and M2 macrophage infiltration. These changes led to enhanced cytotoxic T cell function and sensitized tumors to radiotherapy (RT). We further revealed that SERPINB3 promoted STAT-dependent expression of chemokines, whereby inhibition of STAT activation by ruxolitinib or siRNA abrogated CXCL1/8 and S100A8/ A9 expression in SERPINB3 cells. Patients with elevated pretreatment SCCA levels and high phosphorylated STAT3 (p-STAT3) had increased intratumoral CD11b+ myeloid cells compared with patients with low SCCA levels and p-STAT3, who had improved overall survival after RT. These findings provide a preclinical rationale for targeting SERPINB3 in tumors to counteract immunosuppression and improve the response to RT.

Loss of H3K27 Trimethylation Promotes Radiotherapy Resistance in Medulloblastoma and Induces an Actionable Vulnerability to BET Inhibition.

Medulloblastoma has been categorized into four subgroups based on genetic, epigenetic, and transcriptional profiling. Radiation is used for treating medulloblastoma regardless of the subgroup. A better understanding of the molecular pathways determining radiotherapy response could help improve medulloblastoma treatment. Here, we investigated the role of the EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit)-dependent histone H3K27 trimethylation in radiotherapy response in medulloblastoma. The tumors in 47.2% of patients with group 3 and 4 medulloblastoma displayed H3K27me3 deficiency. Loss of H3K27me3 was associated with a radioresistant phenotype, high relapse rates, and poor overall survival. In H3K27me3-deficient medulloblastoma cells, an epigenetic switch from H3K27me3 to H3K27ac occurred at specific genomic loci, altering the transcriptional profile. The resulting upregulation of EPHA2 stimulated excessive activation of the prosurvival AKT signaling pathway, leading to radiotherapy resistance. Bromodomain and extraterminal motif (BET) inhibition overcame radiation resistance in H3K27me3-deficient medulloblastoma cells by suppressing H3K27ac levels, blunting EPHA2 overexpression, and mitigating excessive AKT signaling. In addition, BET inhibition sensitized medulloblastoma cells to radiation by enhancing the apoptotic response through suppression of Bcl-xL and upregulation of Bim. This work demonstrates a novel mechanism of radiation resistance in medulloblastoma and identifies an epigenetic marker predictive of radiotherapy response. On the basis of these findings, we propose an epigenetically guided treatment approach targeting radiotherapy resistance in patients with medulloblastoma. SIGNIFICANCE: This study demonstrates a novel epigenetic mechanism of radiation resistance in medulloblastoma and identifies a therapeutic approach to improve outcomes in these patients.

DANSR: A Tool for the Detection of Annotated and Novel Small RNAs.

Existing small noncoding RNA analysis tools are optimized for processing short sequencing reads (17-35 nucleotides) to monitor microRNA expression. However, these strategies under-represent many biologically relevant classes of small noncoding RNAs in the 36-200 nucleotides length range (tRNAs, snoRNAs, etc.). To address this, we developed DANSR, a tool for the detection of annotated and novel small RNAs using sequencing reads with variable lengths (ranging from 17-200 nt). While DANSR is broadly applicable to any small RNA dataset, we applied it to a cohort of matched normal, primary, and distant metastatic colorectal cancer specimens to demonstrate its ability to quantify annotated small RNAs, discover novel genes, and calculate differential expression. DANSR is available as an open source tool.

Monounsaturated and Diunsaturated Fatty Acids Sensitize Cervical Cancer to Radiation Therapy.

Obesity induces numerous physiological changes that can impact cancer risk and patient response to therapy. Obese patients with cervical cancer have been reported to have superior outcomes following chemoradiotherapy, suggesting that free fatty acids (FFA) might enhance response to radiotherapy. Here, using preclinical models, we show that monounsaturated and diunsaturated FFAs (uFFA) radiosensitize cervical cancer through a novel p53-dependent mechanism. UFFAs signaled through PPARγ and p53 to promote lipid uptake, storage, and metabolism after radiotherapy. Stable isotope labeling confirmed that cervical cancer cells increase both catabolic and anabolic oleate metabolism in response to radiotherapy, with associated increases in dependence on mitochondrial fatty acid oxidation for survival. In vivo, supplementation with exogenous oleate suppressed tumor growth in xenografts after radiotherapy, an effect that could be partially mimicked in tumors from high fat diet-induced obese mice. These results suggest that supplementation with uFFAs may improve tumor responses to radiotherapy, particularly in p53 wild-type tumors. SIGNIFICANCE: Metabolism of monounsaturated and diunsaturated fatty acids improves the efficacy of radiotherapy in cancer through modulation of p53 activity. See related commentary by Jungles and Green, p. 4513.

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation.

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

Integrating imaging and RNA-seq improves outcome prediction in cervical cancer.

Approaches using a single type of data have been applied to classify human tumors. Here we integrate imaging features and transcriptomic data using a prospectively collected tumor bank. We demonstrate that increased maximum standardized uptake value on pretreatment 18F-fluorodeoxyglucose-positron emission tomography correlates with epithelial-to-mesenchymal transition (EMT) gene expression. We derived and validated 3 major molecular groups, namely squamous epithelial, squamous mesenchymal, and adenocarcinoma, using prospectively collected institutional (n = 67) and publicly available (n = 304) data sets. Patients with tumors of the squamous mesenchymal subtype showed inferior survival outcomes compared with the other 2 molecular groups. High mesenchymal gene expression in cervical cancer cells positively correlated with the capacity to form spheroids and with resistance to radiation. CaSki organoids were radiation-resistant but sensitive to the glycolysis inhibitor, 2-DG. These experiments provide a strategy for response prediction by integrating large data sets, and highlight the potential for metabolic therapy to influence EMT phenotypes in cervical cancer.

Standardized Uptake Value for 18F-Fluorodeoxyglucose Is a Marker of Inflammatory State and Immune Infiltrate in Cervical Cancer.

PURPOSE: Chemoradiotherapy for locally advanced cervical cancer fails in over a third of patients. Biomarkers with therapeutic implications are therefore needed. We investigated the relationship between an established prognostic marker, maximum standardized uptake value (SUVmax) on 18F-fluorodeoxyglucose positron emission tomography, and the inflammatory and immune state of cervical cancers. EXPERIMENTAL DESIGN: An SUVmax most prognostic for freedom from progression (FFP) was identified and compared with known prognostic clinical variables in a cohort of 318 patients treated with definitive radiation with prospectively collected clinical data. Gene set enrichment analysis (GSEA) and CIBERSORT of whole-transcriptome data from 68 patients were used to identify biological pathways and immune cell subpopulations associated with high SUVmax. IHC using a tissue microarray (TMA, N = 82) was used to validate the CIBERSORT findings. The impact of macrophages on cervical cancer glucose metabolism was investigated in coculture experiments. RESULTS: SUVmax <11.4 was most prognostic for FFP (P = 0.001). The GSEA showed that high SUVmax is associated with increased gene expression of inflammatory pathways, including JAK/STAT3 signaling. CIBERSORT and CD68 staining of the TMA showed high SUVmax tumors are characterized by a monocyte-predominant immune infiltrate. Coculture of cervical cancer cells with macrophages or macrophage-conditioned media altered glucose uptake, and IL6 and JAK/STAT3 signaling contribute to this effect. CONCLUSIONS: SUVmax is a prognostic marker in cervical cancer that is associated with activation of inflammatory pathways and tumor infiltration of myeloid-derived immune cells, particularly macrophages. Macrophages contribute to changes in cervical cancer glucose metabolism.See related commentary by Williamson et al., p. 4136.

HPV-EM: an accurate HPV detection and genotyping EM algorithm.

Accurate HPV genotyping is crucial in facilitating epidemiology studies, vaccine trials, and HPV-related cancer research. Contemporary HPV genotyping assays only detect < 25% of all known HPV genotypes and are not accurate for low-risk or mixed HPV genotypes. Current genomic HPV genotyping algorithms use a simple read-alignment and filtering strategy that has difficulty handling repeats and homology sequences. Therefore, we have developed an optimized expectation-maximization algorithm, designated HPV-EM, to address the ambiguities caused by repetitive sequencing reads. HPV-EM achieved 97-100% accuracy when benchmarked using cell line data and TCGA cervical cancer data. We also validated HPV-EM using DNA tiling data on an institutional cervical cancer cohort (96.5% accuracy). Using HPV-EM, we demonstrated HPV genotypic differences in recurrence and patient outcomes in cervical and head and neck cancers.

Glutaminase Inhibitors Induce Thiol-Mediated Oxidative Stress and Radiosensitization in Treatment-Resistant Cervical Cancers.

The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.