Search

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

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 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

Individual mouse data for in vivo experiments.

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

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

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

Prognostic significance of SUVmax for local (cervix) recurrence (A) and any pelvic recurrence (B).

Oleic acid and radiation modulates PPARγ regulated gene expression

Differential effect of polyunsaturated fatty acid, Arachidonic acid (AA) in cervical cancer cells.

Number of patients in each of the patient cohorts used, and overlap between the cohorts.

Unsaturated fatty acids radio-sensitize cervical cancer cells through a mechanism that includes upregulation in pp53 and p53 and induction of apoptosis.

Supplemental Material and Methods

Tissue microarray staining for CD163, a marker for M2 polarized macrophages.

Representative image of an auto-contoured metabolic tumor volume on simulation CT scan fused with a diagnostic PET scan.

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.

Tumor growth kinetics of TC-1 orthograft tumors

Bootstrap multivariate analysis results.

Oleic acid and linoleic acid supplementation does not affect radiation sensitivity in TP53

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 thatSERPINB3was positively correlated withCXCL1/8, S100A8/A9and myeloid cell infiltration. Induction of SERPINB3in vitroresulted in increased CXCL1/8 and S100A8/A9 production, and supernatants from SERPINB3-expressing cultures attracted monocytes and MDSCs. In murine tumors, the orthologuemSerpinB3apromoted 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.

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 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

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.

Accurate HPV genotyping is crucial in facilitating epidemiology studies, vaccine trials, and HPV-related cancer research. Contemporary HPV genotyping assays only detect

Background: Identifying pathways that can be targeted to reduce breast density and breast cancer incidence is an unmet need, especially in premenopausal women. Receptor activator of nuclear factor-κB ligand (RANKL) signaling mediates the major proliferative response of mammary epithelium to progesterone and is positively associated with breast density but there are no clinical trial data on the impact of RANKL inhibition on breast tissue markers in women with dense breasts. We, therefore, determined the impact of RANKL inhibition on breast tissue gene expression in high-risk premenopausal women with dense breasts. Study design: Pilot phase I clinical trial of 9 healthy high-risk pre-menopausal women (≥35 years of age) with dense breasts performed at Washington University School of Medicine, St. Louis, MO from July 2018-December 2018. Fifty-five percent of participants had a positive family history of breast cancer in a first degree relative. Intervention: Participants were given a single dose of subcutaneous RANKL antibody, denosumab (60mg), at baseline (Day 1). On the same day, prior to the denosumab injection, participants had an ultrasound guided core needle biopsy and a blood draw. All study participants returned for repeat core needle biopsy and blood draw after 60 days (Day 60). Outcome Measures: Changes in breast tissue gene expression between Day 1 and Day 60 with a focus on immune/inflammatory and hormone markers. Gene expression was profiled using NanoString nCounter platform. We performed pathway enrichment analysis and used Cell Maker Enrichment library from EnrichR to identify differentially expressed genes. Genes with a p-value Results: Macrophage markers, including CCR5, CD86, CD300A, and CD84 and targets downstream of IRF8: (p-value =4.73e-7), neutrophil degranulation/neutrophil activation in the immune response (p-value=6.0e-21), and the secretory granule membrane pathways (p-value=2.4e-14) were downregulated in breast tissues on Day 60. Pathways involved in progesterone metabolism (p-value=0.0003), estrogen response (p-value=0.0014) and fatty acid metabolism, (p-value=0.0001) were significantly upregulated on Day 60. Specifically, genes involved in steroid hormone metabolism such as DHRS2 (p-value=3.13e-05), and AKR1B15: p-value=4.12e-05) were upregulated on Day 60. Conclusions: A single 60 mg dose of subcutaneous denosumab injection was associated with alterations in pathways involved in hormone, immune and inflammatory signaling in the breast tissues of healthy premenopausal women with dense breasts. Citation Format: Michael Waters, Kay Jayachandran, Debbie Bennet, Jin Zhang, Katherine Weilbaecher, Ian S. Hagemann, Graham A. Colditz, Catherine M. Appleton, Adetunji T. Toriola. Pilot phase I clinical trial of RANKL inhibition and breast tissue gene expression in high-risk premenopausal women with dense breasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2206.

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.

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 (18)F-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.

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 (SUVAn SUVSUVSUV

Purpose/Objective(s) Medulloblastoma has been categorized into four subgroups based on genetic and epigenetic profiling. However, molecular pathways determining radiotherapy response in this tumor remain unknown. Here, we investigated the role of the EZH2-dependent histone H3K27 tri-methylation in radiation response in medulloblastoma. Materials/Methods We analyzed the H3K27me3 expression medulloblastoma tumor samples and determined the correlation between the H3K27me3 status and PFS and OS in 36 non-WNT/SHH medulloblastoma patients. We used an ex vivo mouse model of group 3 medulloblastoma to elucidate the molecular mechanism of radiation resistance in H3K27me3-deficient tumors and develop an approach to target radiotherapy resistance. Results We demonstrate that 47.2% of group 3 and 4 medulloblastoma patients have H3K27me3-deficient tumors. Loss of H3K27me3 was associated with a radioresistant phenotype, high relapse rates and poor overall survival. We show that an epigenetic switch from H3K27me3 to H3K27ac occurs at certain genomic loci in H3K27me3-deficient medulloblastoma cells altering the transcriptional profile. The resulting up-regulation of EPHA2 (ephrin type-A receptor 2) stimulates an over-activation of the pro-survival AKT signaling pathway leading to radiotherapy resistance. We show that BET inhibitors target radiation resistance in H3K27me3-deficient medulloblastoma by suppressing H3K27ac levels, blunting EPHA2 overexpression and mitigating the excessive AKT signaling. Additionally, BET inhibition sensitizes medulloblastoma cells to radiation by enhancing apoptotic response through suppression of Bcl-XL and up-regulation of Bim expression. Conclusion Our work demonstrates a novel mechanism of radiation resistance in medulloblastoma and identifies an epigenetic marker predictive of radiotherapy response. Based on these findings we propose an epigenetically guided personalized treatment approach to target radiotherapy resistance in medulloblastoma patients.

Squamous cell carcinoma antigen (SERPINB3) is a widely used biomarker for epithelial cancers including uterine cervix, head and neck, and esophagus. We have previously shown that SERPINB3 contributes to radiation resistance, mainly due to its predominant function as a protease inhibitor that interfered with lysosomal cell death. In addition, elevated SERPINB3 is also observed in various inflammatory conditions related to increased cytokine expression such as, atopic dermatitis, psoriasis, asthma and chronic obstructive pulmonary disease, indicative of potential immunoregulatory function of SERPINB3. We hypothesized that elevated SERPINB3 may also contribute to altered tumor immunity, leading to reduced sensitivity and therapeutic effects of radiation treatment. Thus, we sought evidence of SERPINB3 expression in relation to immune regulation using RNA sequencing analysis of cervical tumor biopsies from 66 patients, and identified a positive correlation of SERPINB3 with chemokines CXCL1, CXCL8, S100A8, and S100A9. These chemokines are known to induce myeloid cell accumulation in the tumor microenvironment, and the presence of myeloid cells have been shown to reduce the efficacy of radiotherapy in cervical cancer. Indeed we found enrichment of myeloid cell subsets on immune cell prediction analysis with xCell in SERPINB3-high tumors. We then examined expression and secretion of myeloid-attractant chemokines via quantitative PCR and ELISA in SERPINB3-overexpressing cervical cancer cells, and confirmed the upregulation of CXCL1/8 and S100A8/A9. Supernatants collected from SERPINB3-overexpressing cells contained chemoattractant to myeloid cells as evidenced by transwell assay using human peripheral blood mononuclear cells from patients with cervical cancer. In the mouse models, tumors formed by SERPINB3-expressing cells induced an immunosuppressive microenvironment with the increased infiltration of M2 macrophages and MDSCs in tumors and spleens. Furthermore, we observed the elevated phosphorylation of STAT1/3/5 transcription factors, known to regulate inflammatory chemokine expression, in SERPINB3-high cells. In our patient cohort, RNA-seq analysis of biopsies collected from 20 patients both before and 3 weeks into chemoradiation therapy (CRT) were analyzed for changes of SERPINB3 in comparison to chemokine expression. We found that patients with decreased SERPINB3 expression in the mid-CRT biopsy (n=7) showed downregulated S100A8/A9 levels in tumors while patients with increased SERPINB3 (n=13) had upregulated S100A8/A9 expression. Our findings suggest that SERPINB3 promotes chemokine secretion involved in myeloid cell-mediated immune suppression which may contribute to negative impacts on radiation treatment in patients with cervical cancer. Citation Format: Liyun Chen, Songyan Wang, Victoria Shi, Fiona Ruiz, Kay Jayachandran, Jin Zhang, Pippa Cosper, Perry Grigsby, Julie Schwarz, Stephanie Markovina. SERPINB3 promotes myeloid cell chemotaxis and associated chemokine production in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2680.

Medulloblastoma has been categorized into four subgroups based on genetic, epigenetic and transcriptional profiling. However, molecular pathways determining radiotherapy response in this tumor remain elusive. Here, we investigated the role of the EZH2-dependent histone H3K27 tri-methylation in radiotherapy response in medulloblastoma. We demonstrate that 47.2% of group 3 and 4 medulloblastoma patients have H3K27me3-deficient tumors. Loss of H3K27me3 was associated with a radioresistant phenotype, high relapse rates and poor overall survival. We show that an epigenetic switch from H3K27me3 to H3K27ac occurs at specific genomic loci in H3K27me3-deficient medulloblastoma cells altering the transcriptional profile. The resulting up-regulation of EPHA2 (ephrin type-A receptor 2) stimulates an excessive activation of the pro-survival AKT signaling pathway leading to radiotherapy resistance. We show that BET inhibition targets radiation resistance in H3K27me3-deficient medulloblastoma by suppressing H3K27ac levels, blunting EPHA2 overexpression and mitigating the excessive AKT signaling. Additionally, BET inhibition sensitizes medulloblastoma cells to radiation by enhancing apoptotic response through suppression of Bcl-XL and up-regulation of Bim expression. Our work demonstrates a novel mechanism of radiation resistance in medulloblastoma and identifies an epigenetic marker predictive of radiotherapy response. Based on these findings we propose an epigenetically guided treatment approach targeting radiotherapy resistance in medulloblastoma patients.