Changing paradigms in oncology: Toward noncytotoxic treatments for advanced gliomas

Abstract

Glial-lineage malignancies (gliomas) recurrently mutate and/or delete the master regulators of apoptosis p53 and/or p16/CDKN2A, undermining apoptosis-intending (cytotoxic) treatments. By contrast to disrupted p53/p16, glioma cells are live-wired with the master transcription factor circuits that specify and drive glial lineage fates: these transcription factors activate early-glial and replication programs as expected, but fail in their other usual function of forcing onward glial lineage-maturation-late-glial genes have constitutively "closed" chromatin requiring chromatin-remodeling for activation-glioma-genesis disrupts several epigenetic components needed to perform this work, and simultaneously amplifies repressing epigenetic machinery instead. Pharmacologic inhibition of repressing epigenetic enzymes thus allows activation of late-glial genes and terminates glioma self-replication (self-replication = replication without lineage-maturation), independent of p53/p16/apoptosis. Lineage-specifying master transcription factors therefore contrast with p53/p16 in being enriched in self-replicating glioma cells, reveal a cause-effect relationship between aberrant epigenetic repression of late-lineage programs and malignant self-replication, and point to specific epigenetic targets for noncytotoxic glioma-therapy.

Keywords: cancer epigenetics; epigenetic; glioma; glioma therapy; neurooncology.

FIGURE 1

(A) Normal self‐replication (replication without onward lineage‐differentiation) is restricted to tissue stem cells, but malignant self‐replication is not. (B) Of the master transcription factor (MTF) circuit SOX2/POU5F1/NANOG, that produces neural stem cells (NSC), only SOX2 that is stably expressed through glial lineage‐maturation is also highly expressed in gliomas, however, the MTF circuit NFIA/ATF3/RUNX2 that compels NSC commitment into glioma lineage‐precursors is highly expressed. Normal brain (n = 5); Oligo‐2/3 = oligodendroglioma, IDH‐mutant and 1p/19q‐codeleted, WHO grade 2 or 3 (n = 176); Astro‐2/3 = astrocytoma, IDH‐mutant, WHO grade 2 or 3 (n = 241); Astro‐4 = astrocytoma, IDH‐mutant, WHO grade 4 (n = 38); GBM = glioblastoma, IDH wild‐type, WHO grade 4 (n = 196). TCGA RNA‐seq public data, RSEM values (counts normalized by RNA‐Seq by Expectation‐Maximization). Mann‐Whitney two‐sided test ***P < .0001, **P < .005. (C) Consistent with lineage MTF circuit wiring, gliomas significantly upregulate 539 genes that characterize astroglial lineage‐commitment/early maturation (early glial) (67 known MYC‐target genes excluded), and 337 MYC‐target genes identified by chromatin‐immunoprecipitation, but there is anomalous suppression of 310 astroglial late lineage‐differentiation (late glial) genes. Average expression of each gene in glioma samples of each glioma sub‐type (samples as per panel B). (D) More aggressive gliomas demonstrate deeper suppression of late‐glial, and more upregulation of early‐glial, genes. Average expression per sample of all genes in a category. Lines = median ± IQR, ***P < .0001 Mann‐Whitney two‐sided test (samples as per panel B). (E) Early‐glial and MYC‐target gene expression positively correlate (67 known MYC‐target genes were excluded from early glial genes analyzed); Late‐glial and MYC‐target gene expression negatively correlate [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Less onward maturation of cells committed into the glial lineage (higher expression of commitment/early‐glial genes, lower expression of late‐glial genes) independently predicts and stratifies for worse overall survival within well‐established EANO/WHO glioma subtypes. Cases within pathologic subgroups were stratified around the median average expression of late oligodendroglial lineage genes (as identified in Reference 27) for Oligo‐2/3, or early astroglial lineage genes (as identified in Reference 20) for Astro‐2/3, Astro‐4 and GBM. Overall survival data TCGA. (A) Oligo‐2/3. (B) Astro‐2/3. (C) Astro‐4. (D) GBM [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Glioma‐genesis exploits differences in epigenetic landscape: MYC‐target and early‐glial genes have a constitutively accessible epigenetic configuration but late‐glial genes do not. (A) me‐CpG at early‐glial, late‐glial and MYC‐target genes in embryonic stem cells (ESC) (gene groups as per Figure 1) (A) Public data GSE31848116. Median ± interquartile range (IQR). ESC (n = 19). me‐CpG measured by Illumina 450K array. (B) me‐CpG at early‐glial, late‐glial and MYC‐target genes in normal cerebral cortex vs clinicopathologic types of glioma. me‐CpG measured by Illumina 450K array, TCGA public data as per Figure 1. P‐value Mann‐Whitney test 2‐sided. (C) H3K27me3 and H3K27ac distributions at early‐glial, late‐glial and MYC‐target genes in ESC, normal brain cortex and gliomas without and with histone 3 gene (H3F3A) mutations. Public ChIP‐seq data (FastQ files processed by UseGalaxy suite of tools): ESC H3K27me3—GSM428295 (Encode); Normal cerebral cortex H3K27me3—GSM772833 (Encode); ESC H3K27ac—GSM466732 (Encode); Normal cerebral cortex H3K27ac—GSM1112812 (Encode); GBM (SF9402), H3K27M glioma (SF7761) and H3G34V glioma (KNS42) H3K27me3 and H3K27ac GSE162976117. Plots using EASEQ. (D) Pediatric gliomas recapitulate the glial lineage‐specifying MTF (NFIA, ATF3, RUNX2) wiring observed in adult gliomas (Figure 1). Of NSC‐specifying MTF, only SOX2 is highly expressed, again as also seen in adult gliomas, and as expected from stable SOX2 expression through normal glial lineage‐maturation. Oligo‐glioma = oligodendroglioma (n = 2); Glioma‐2 = glioma grade 2 (n = 236); Glioma‐HG = glioma high‐grade (n = 53); Glioma‐K27M = glioma containing H3F3A K27M mutation (n = 22). Pediatric Brain Tumor Atlas public data, RSEM values (counts normalized by RNA‐Seq by Expectation‐Maximization). (E) More aggressive pediatric gliomas display deeper late‐glial gene suppression, accompanied by more upregulation of early‐glial and MYC‐target genes. Heat map collapsed on average expression per gene in all the samples in each subtype (samples as per panel D). (F) Average expression of all early‐glial, late‐glial and MYC‐target genes in each pediatric glioma sample (samples as per panel D). P values Mann‐Whitney test two‐sided [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Gliomas contain recurrent genetic alterations expected to preserve or increase H3K27me3 and me‐CpG, and simultaneously decrease H3K27ac and other chromatin remodeling needed to activate late‐glial genes. TCGA public data (n = 651). (A) Significant correlation between gene copy number and expression of the chromatin remodelers. The GISTIC2 method produced segmented copy number variant data mapped to genes to produce gene‐level estimates. Gene‐level transcript estimates by RNA‐Sequencing were analyzed as log₂(x + 1) transformed RSEM normalized counts. Pearson correlation coefficients, P‐value two‐sided. (B) Glioma‐genesis alters several classes of chromatin remodelers, at frequencies that increase with the aggression of disease. Percentage of cases in each WHO/EANO glioma sub‐group with the indicated gene copy number or mutation changes. Gene‐level copy number estimates generated by the GISTIC2 method were thresholded to estimated values −2, −1, 0, 1, 2 representing homozygous or single‐copy deletion (del), diploid normal copy or low‐level or high‐level copy number amplification (amp). (C) The H3K27 demethylase KDM6A is significantly less expressed in gliomas in males vs females. ***P < .0001, Mann‐Whitney test, two‐sided [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Summary. Glioma‐genesis selects to impede chromatin‐remodeling needed to activate late‐glial lineage genes, thus converting the exponential replications of glial‐lineage committed progenitors into self‐replications (glioma “stem” cells). Inhibiting repressing epigenetic enzymes enables glial‐lineage transcription factors, already highly expressed in glioma stem cells, to activate late‐glial genes and hence terminate malignant self‐replications, without a need for an intact apoptosis program [Color figure can be viewed at wileyonlinelibrary.com]