EOMES and IL-10 regulate antitumor activity of T regulatory type 1 CD4+ T cells in chronic lymphocytic leukemia.

The transcription factor eomesodermin (EOMES) promotes interleukin (IL)-10 expression in CD4+ T cells, which has been linked to immunosuppressive and cytotoxic activities. We detected cytotoxic, programmed cell death protein-1 (PD-1) and EOMES co-expressing CD4+ T cells in lymph nodes (LNs) of patients with chronic lymphocytic leukemia (CLL) or diffuse large B-cell lymphoma. Transcriptome and flow cytometry analyses revealed that EOMES does not only drive IL-10 expression, but rather controls a unique transcriptional signature in CD4+ T cells, that is enriched in genes typical for T regulatory type 1 (TR1) cells. The TR1 cell identity of these CD4+ T cells was supported by their expression of interferon gamma and IL-10, as well as inhibitory receptors including PD-1. TR1 cells with cytotoxic capacity accumulate also in Eµ-TCL1 mice that develop CLL-like disease. Whereas wild-type CD4+ T cells control TCL1 leukemia development after adoptive transfer in leukopenic Rag2-/- mice, EOMES-deficient CD4+ T cells failed to do so. We further show that TR1 cell-mediated control of TCL1 leukemia requires IL-10 receptor (IL-10R) signaling, as Il10rb-deficient CD4+ T cells showed impaired antileukemia activity. Altogether, our data demonstrate that EOMES is indispensable for the development of IL-10-expressing, cytotoxic TR1 cells, which accumulate in LNs of CLL patients and control TCL1 leukemia in mice in an IL-10R-dependent manner.

Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype.

Glioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural-mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.

Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate macrophage phenotype.

Elevated amino acid catabolism is common to many cancers. Here, we show that glioblastoma are excreting large amounts of branched-chain ketoacids (BCKAs), metabolites of branched-chain amino acid (BCAA) catabolism. We show that efflux of BCKAs, as well as pyruvate, is mediated by the monocarboxylate transporter 1 (MCT1) in glioblastoma. MCT1 locates in close proximity to BCKA-generating branched-chain amino acid transaminase 1, suggesting possible functional interaction of the proteins. Using in vitro models, we demonstrate that tumor-excreted BCKAs can be taken up and re-aminated to BCAAs by tumor-associated macrophages. Furthermore, exposure to BCKAs reduced the phagocytic activity of macrophages. This study provides further evidence for the eminent role of BCAA catabolism in glioblastoma by demonstrating that tumor-excreted BCKAs might have a direct role in tumor immune suppression. Our data further suggest that the anti-proliferative effects of MCT1 knockdown observed by others might be related to the blocked excretion of BCKAs.

Gcn5 Modulates the Cellular Response to Oxidative Stress and Histone Deacetylase Inhibition.

To identify chemical genetic interactions underlying the mechanism of action of histone deacetylase inhibitors (HDACi) a yeast deletion library was screened for hypersensitive deletion mutants that confer increased sensitivity to the HDACi, CG-1521. The screen demonstrated that loss of GCN5 or deletion of components of the Gcn5 histone acetyltransferase (HAT) complex, SAGA, sensitizes yeast to CG-1521-induced cell death. Expression profiling after CG-1521 treatment reveals increased expression of genes involved in metabolism and oxidative stress response, and oxidative stress response mutants are hypersensitive to CG-1521 treatment. Accumulation of reactive oxygen species and increased cell death are enhanced in the gcn5Δ deletion mutant, and are abrogated by anti-oxidants, indicating a central role of oxidative stress in CG-1521-induced cell death. In human cell lines, siRNA mediated knockdown of GCN5 or PCAF, or chemical inhibition of GCN5 enzymatic activity, increases the sensitivity to CG-1521 and SAHA. These data suggest that the combination of HDAC and GCN5/PCAF inhibitors can be used for cancer treatment.

High throughput screening identifies modulators of histone deacetylase inhibitors.

BACKGROUND: Previous studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521. RESULTS: Screening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5Δ strains show a G1 delay after CG-1521 treatment, however the gcn5Δ strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5Δ strain. CONCLUSION: Genome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.

Tumor progression in the LPB-Tag transgenic model of prostate cancer is altered by vitamin D receptor and serum testosterone status.

Previous studies have suggested that 1,25 dihydroxyvitamin D(3) (1,25(OH)2D3) induces cell cycle arrest and/or apoptosis in prostate cancer cells in vitro, suggesting that vitamin D may be a useful adjuvant therapy for prostate cancer and a chemopreventive agent. Most epidemiological data however shows a weak link between serum 25(OH)D3 and risk of prostate cancer. To explore this dichotomy we have compared tumor progression in the LPB-Tag model of prostate in VDR knock out (VDRKO) and wild type (VDRWT) mice. On the C57BL/6 background LPB-Tag tumors progress significantly more rapidly in the VDRKO mice. VDRKO tumors show significantly higher levels of cell proliferation than VDRWT tumors. In mice supplemented with testosterone to restore the serum levels to the normal range, these differences in tumor progression, and proliferation are abrogated, suggesting that there is considerable cross-talk between the androgen receptor (AR) and the vitamin D axis which is reflected in significant changes in steady state mRNA levels of the AR, PCNA, cdk2 survivin and IGFR1 and 2 genes. These alterations may explain the differences between the in vitro data and the epidemiological studies.