Single-cell epigenomic variability reveals functional cancer heterogeneity.

BackgroundCell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally.ResultsWe develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity.ConclusionSingle-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution

Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells

Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma

A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology

The indoleamine-2,3-dioxygenase (IDO) inhibitor 1-methyl-D-tryptophan upregulates IDO1 in human cancer cells.

1-methyl-D-tryptophan (1-D-MT) is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO)-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp) to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn) pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT

Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells

1-D-MT upregulates IDO1 expression and kyn release induced by different concentrations of IFN-γ in diverse cancer cells.

<p>(<b>A</b>) Representative HPLC graphs of kyn production of HeLa cells, which were either untreated, treated with 1 mM 1-D-MT and/or 1000 U IFN-γ for 72 h. Absorption of kyn was measured at 365 nm. (<b>B</b>) In untreated HeLa cells 1 mM 1-D-MT did not induce <i>de novo</i> IDO1 mRNA, but increased IDO1 mRNA after its induction by 1000 U IFN-γ mRNA expression was analyzed by qRT-PCR 24 h after treatment. (<b>C</b>) Representative example of the effect of different IFN-γ concentrations on IDO1 mRNA induction by 1-D-MT, shown in U251 glioma cells. (<b>D</b>) IDO1 mRNA expression of indicated cell lines that were stimulated for 24 h with appropriate concentrations of IFN-γ alone (white bars) or in combination with 1 mM 1-D-MT (black bars). (<b>E</b>) Representative example of IDO1 mRNA induction by 200 µM 1-D-MT in IFN-γ-stimulated T98G glioma cells. (<b>F</b>) Kyn release of indicated cell lines that were stimulated for 72 h with appropriate concentrations of IFN-γ alone (white bars) or in combination with 1 mM 1-D-MT (black bars), measured by HPLC. Experiments were performed in triplicate. Data are mean ± SEM. * (p<0.05).</p

1-D-MT reduces T cell proliferation in cocultures of SKOV-3 cells with mixed leukocyte reactions.

<p>(<b>A</b>) Alloreactive T cell proliferation after addition of 25 µM kyn to mixed leukocyte reactions (MLR). (<b>B</b>) Alloreactive T cell proliferation in the presence of 6000 SKOV-3 cells. (<b>C</b>) T cell proliferation in MLR cocultured with 2000 control SKOV-3 cells (white bar) or 2000 SKOV-3 cells with a knockdown of IDO1 (black bar). (<b>D</b>) T cell proliferation in cocultures of MLR with 2000 SKOV-3 cells after addition of increasing concentrations of 1-L-MT. (<b>E</b>) T cell proliferation in cocultures of MLR with 2000 SKOV-3 cells after addition of increasing concentrations of 1-D-MT. (<b>F</b>) Representative result of MLR/SKOV-3 coculture experiments with PBMC from five different donors and 2000 or 6000 SKOV-3 cells. Cells were treated with or without 1 mM 1-D-MT in combination with or without 250 µM trp. Proliferation was measured by ³[H] methylthymidine uptake. Experiments were performed at least in triplicate. Data are mean ± SEM. * (p<0.05).</p