Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells

Abstract

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to "induced" TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies. PAPERCLIP:

Figure 1. Epigenetic landscapes distinguish functionally distinct GBM models

(A) GBM cells (MGG8) grown as gliomaspheres in serum-free conditions propagate tumor in vivo while serum-differentiated cells fail to do so. (B) Flow cytometry of MGG8 TPCs shows positivity for the GBM stemlike markers SSEA-1 and CD133, while serum-differentiated cells do not. (C) Cells grow in serum as adherent monolayers and express the differentiation markers GFAP (astroglial), beta III tubulin (neuronal), MAP-2 (neuronal) and GalC (oligodendroglial). (D) Xenografted tumors from MGG8 TPCs (left) are invasive, crossing the corpus callosum (boxed region), infiltrating along white matter tracks (arrowhead). At high magnification, the cells are atypical and mitotic figures are evident (arrow). Xenografted tumors from MGG4 TPCs (right) are more circumscribed but also infiltrate adjacent parenchyma (boxed region, arrowhead). At high magnification areas of necrosis (*) and mitotic figures (arrow) are readily identified. LV: lateral ventricle. (E) TPC-specific, DGC-specific and shared regulatory elements. Shared elements tend to be located proximal to promoters, while the vast majority of TPC- and DGC-specific elements are distal. Motif analyses predict binding sites for TF families within each set of sites. See also Supplemental FigureS1.

Figure 2. Candidate regulators for the specification of alternate epigenetic states in GBM

(A) A set of 19 TPC-specific TFs is identified based on RNA-Seq expression and promoter H3K27ac signals in TPCs and DGCs. TF family is indicated at right. (B) Western blots confirm exclusive protein expression in TPCs for selected TFs. Lower panel indicates tubulin loading control. (C) ChIP-Seq tracks show H3K27ac signals for loci encoding TPC-specific TFs OLIG1, OLIG2 and SOX2, or (D) the differentiation factor BMP4 in the respective GBM models. TPC-specific TF loci are enriched for TPC-specific regulatory elements.

Figure 3. A core TF network for tumor-propagating GBM cells

(A) Data points indicate percentage of single-cell DGCs capable of forming spheres in serum-free conditions. Each of the 19 TFs in Figure 2A was tested alone (first column, ‘single TF’), in combination with POU3F2 (second column) or in combination with POU3F2 and SOX2 (third column). HLH family TFs were also tested in combination with POU3F2, SOX2 and SALL2 (fourth column), based on an enrichment of HLH motifs in regulatory elements that failed to activate in 3TF-induced DGCs. TF combinations that enhanced in vitro spherogenicity (blue) were selected for in vivo testing. (B) Flow cytometry profiles show expression of the stem cell marker CD133 for DGCs induced by the single, double, triple and quadruple TF combinations with the highest in vitro sphere-forming potential. (C) For TF combinations with in vitro spherogenic potential (blue in panel 3A), 100,000 cells were injected in the brain parenchyma (n=4 mice per TF combination). Survival curve is shown for this in vivo tumor-propagation assay. Only the quadruple TF combination POU3F2+SOX2+SALL2+OLIG2 initiated tumors in mice. (D) Tumor histopathology shows characteristic features of glioblastoma, including necrotic areas (*) and crossing of corpus callosum (boxed area). At high magnification cells show atypical features and mitotic figures are evident (arrows). LV: lateral ventricle. (E) Secondary TPC sphere cultures (“iTPC”) derived from xenotransplant tumors express the stem-cell marker CD133. (F) Contrast field image of iTPC spheres. (G) Left: bar graph shows iTPC and TPC proliferation rates measure by BrdU incorporation. Right: data points indicate percentage of single cells capable of serial sphere formation in three consecutive passages in serum-free conditions. Self-renewal properties and proliferation of iTPCs are comparable to corresponding TPCs. (H) Orthotopic serial xenotransplantation in limiting dilution shows that as few as 50 MGG8 iTPC are sufficient to initiate tumors. (I) Data points indicate in vitro sphere formation of MGG4 TPCs infected with lentivirus containing shRNA for POU3F2, OLIG2 or SALL2, compared to control (two hairpins per TF). (J) Survival curve depicts in vivo tumor propagating potential of MGG4 TPCs infected with POU3F2 shRNA, SALL2 shRNA, OLIG2 shRNA or control shRNA. See also Supplemental FiguresS2–S4.

Figure 4. Core TFs reprogram the epigenetic landscape of DGCs

(A) Left: heatmap depicts H3K27ac signals for TPC-specific, DGC-specific or shared regulatory elements defined in Figure 1E. Relative to control vector infected DGCs, iTPCs gain H3K27ac over TPC-specific elements and lose H3K27ac over DGC-specific elements, consistent with genome-wide reprogramming of the epigenetic landscape. Right: pie charts show fraction of regulatory elements (dark cyan) in each set with H3K27ac in iTPC. (B) RNA-Seq expression and promoter H3K27ac levels at promoter are shown for TPC-specific TFs defined in Figure 2A (NES: Nestin). (C) Hierarchical clustering of MGG8 DGCs, TPCs and replicate iTPCs (iTPC1/2) by H3K27ac ChIP-Seq signal. (D) RNA-Seq tracks show that core TF mRNAs in iTPCs include 3′UTRs (shaded in gray). This indicates the endogenous loci are reactivated in iTPCs as the exogenous vectors lack 3′UTRs. (E) H3K27ac signal tracks for loci encoding core TFs show that endogenous regulatory elements (highlighted with grey shading) are reactivated in iTPCs. (F) Serum-induced differentiation leads iTPCs to convert to an adherent phenotype, up-regulate differentiation markers GFAP, beta III tubulin, MAP-2, GalC and (G) to lose CD133 expression. (H) Western blots confirm serum-induced differentiation of iTPCs leads to down-regulation of core TFs. Lower panels: tubulin loading control. These data indicate that the core TFs can reprogram DGCs into stem-like GBM cells, which have an epigenetic landscape similar to TPCs that is sustained by endogenous regulatory programs. See also Supplemental Figures S2.

Figure 5. All four core TFs are coordinately expressed in a subset of primary GBM cells with stem-like markers

(A) Quadruple immunofluorescence for core TFs in three human GBM samples shows co-expression in a subset of cells; shown at right are the fractions of SOX2+ cells that express each other individual TF or all four TFs in each tumor. (B) Flow cytometry analysis from acutely resected GBM tumors. A majority of cells positive for the four core TFs express the stem-cell marker CD133. Enrichment is significantly greater than for SOX2-expressing cells. (C) Heatmap shows H3K27ac signal from three freshly resected GBM tumors for regulatory elements defined in Figure 1E. Right: pie-charts show fraction of regulatory elements (dark cyan) in each set with H3K27ac. TPC-specific elements show significant enrichment, consistent with a TPC-like regulatory program in a subset of cells.

Figure 6. TF network reconstruction and targeting

(A) ChIP-Seq signal for core TFs profiled in TPCs (MGG8) shows preferential binding at TPC-specific regulatory elements. (B) Pie charts indicate proportion of TF binding sites that coincide with the indicated sets of regulatory elements. (C) Sequence motifs identified in TF ChIP-Seq peaks. With the exception of SALL2 (see text and Figure S5), motifs correspond to the expected class of TFs, further validating ChIP-Seq experiments. (D) Model for core TF regulatory interactions reconstructed from binding profiles and expression data (see text and methods). Other TFs defined in figure 2A (green) and chromatin regulators (red) are highlighted. (E) Signal tracks depict core TF binding over TPC-specific regulatory elements within loci containing the corresponding TF genes. See also Supplemental Figure S5.

Figure 7. The LSD1-RCOR2 chromatin complex is essential for GBM TPCs

(A) Plots depict LSD1 and RCOR2 RNA-Seq expression values for TPCs and DGCs. (B) Western blot for RCOR2 (MGG8 TPC and DGC lysates) confirms exclusive expression in TPC. (C) Western blot for LSD1 on RCOR2 immunoprecipitate indicates co-association between the two proteins in TPCs. (D) Signal tracks depict TF binding and H3K27ac enrichment in the RCOR2 locus. OLIG2 binds a TPC-specific regulatory element in the locus. (E) Survival curve of mice injected with DGCs induced with the combination of POU3F2+SOX2+SALL2+RCOR2 indicates that RCOR2 can substitute for OLIG2 in the cocktail. (F) Coronal section of a xenografted GBM tumor (dashed line) established from iTPCs reprogrammed with the POU3F2+SOX2+SALL2+RCOR2 combination. (G) Representative images of TPCs and DGCs infected with LSD1 shRNA show reduced viability specifically in the TPCs. (H) Bar graphs depict percent viability for MGG4 TPCs or DGCs infected with control shRNA or two different LSD1 shRNAs. LSD1-depletion causes decreased viability in TPCs and has effect on DGCs. (I) Data points indicate in vitro sphere formation of MGG4 TPCs infected with lentivirus shRNA for LSD1 (two hairpins), compared to control in three serial passages. (J) Graph depicts percent viability for TPCs and DGCs (MGG4 and MGG8) and primary astrocytes (NHA) exposed to increasing doses of the synthetic LSD1 inhibitor S2101. A representative image of TPCs exposed to 20uM S2101 for 96 hours is shown below. (K) Survival curve depicts in vivo tumor propagating potential of MGG4 TPCs infected with LSD1 shRNA (two hairpins) or control shRNA. These data suggest that the RCOR2/LSD1 complex is essential for stem-like TPCs, and thus represents a candidate therapeutic target for eliminating this aggressive GBM sub-population. See also Supplemental Figure S4.