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.

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. vating the metazoan program of apoptosis (also known as cytotoxicity). 2 Apoptosis is a replication-control program that senses cell stress or injury as stimuli to upregulate the master regulator p53 (TP53) and its key co-factor p16 (CDKN2A); these in turn activate gene expression modules that force cell cycle exits as an interlude for repair, then if the stress persists, compel orderly cell self-destructions. Some tissue lineages, mainly testicular, lymphoid and myeloid, can transform and advance to disseminated malignancy without TP53 or CDKN2A deletions and/or mutations (reviewed in Reference 3). The apoptosis master switch is therefore available for activation, enabling cures even in advanced cases. Underscoring the centrality of p53/p16 to these treatment successes, p53/p16 loss-of-function or dominant-negative alterations typify relapsed/refractory testicular, lymphoid and myeloid malignancies. 3 Transformation of most other lineages into advanced malignancy, however, usually does entail attenuation or deep inactivation of the p53/p16-system. Cancers derived from these lineages thus demonstrate (a) anticipatory resistance to cytotoxic therapy (primary refractory), or (b) ready selection by cytotoxic therapy for the most apoptosis-attenuated malignant subclones, that resist subsequent treatments (relapsed-refractory), 3
Thus, durable remissions as pay-offs for short-and long-term toxicities of aggressive chemotherapy and radiation are expected only for the few glioma types containing genetically intact p53/p16-systems (reviewed in Reference 3), and it would seem logical to develop new treatments not reliant on p53/p16/apoptosis.

| REPLICATION: THE HEART OF EVOLUTION, INCLUDING NEOPLASTIC EVOLUTION
All cancers share an essential foundation of relentless replication, the engine that drives evolution. Synchronized duplication of life's materials and machines is inexplicably complex. Neoplastic evolution is unlikely to reinvent such complexity, refined over millennia, and therefore hijacks it from the normal cellular contexts in which it occurs: (a) tissue stem cells, and/or (b) lineage-committed progenitors. 3 In the adult mammalian brain, neuronal stem cells (NSC) reside in neuroglial-genic niches in the dentate gyrus of the hippocampus and in subventricular zones lining the lateral ventricles. 11-13 NSC replicate rarely, for example, once every several months, and these replications can be naturally decoupled from onward lineage-differentiation for self-replications needed to maintain NSC pools through life-span (linear proliferation kinetics over long time-scales). 11,14,15 Instead of self-replication, daughter cells may commit toward neuronal or glial lineage-fates, to produce lineage-committed progenitors. [12][13][14] Lineage-committed progenitors replicate every day for exponential proliferation kinetics over short time scales. 14 Each replication is coupled to the acquisition of neuronal-or glial-lineage programs (lineagematuration), culminating after 4 or more cell divisions in activation of final neuronal or glial lineage-fate programs that terminate proliferation (terminal-differentiation). 11,14,16 3 | WHICH OF THESE NORMAL REPLICATION CONTEXTS DO GLIOMAS HIJACK?
Malignant clonal expansion requires a substantial proportion of replications to be self-replications, a biological and mathematical requirement that can be computationally modeled. 17 Since NSC are the only normal brain cells to self-replicate, an intuitive assumption is that glioma self-replications derive from NSC ( Figure 1A). To examine this assumption it is useful to recap the role of "master transcription factors" (MTFs) in cell fate determination. MTFs are sequence-specific, DNA-binding proteins that cooperate in specific combinations (MTF circuits) to activate the hundreds to thousands of genes that define specific cell fates and functions. The MTF circuit that creates NSC, SOX2/POU5F1/NANOG, has been reproducibly identified (reviewed in Reference 18), as has the MTF circuit NFIA/ATF3/RUNX2 that commits NSC to glial-lineage fates. 20,21 What then is the MTF combination highly expressed in glioma cells?
Gliomas express the MTF circuit SOX2/POU5F1/NANOG, which converts other somatic cells into NSC (and also into embryonic stem cells, ESC) in the same pattern as that observed in normal whole brain ( Figure 1A,B): SOX2 is highly expressed in gliomas, but SOX2 is normally highly expressed through glial-lineage maturation, 20 and accordingly, is also highly expressed in normal brain that consists mainly of terminally-matured cells. The other two MTF in the NSC circuit POU5F1 and NANOG are expressed at barely detectable levels and are not more elevated in aggressive GBMs than less aggressive grade 2/3 oligodendrogliomas or astrocytomas ( Figure 1B). Instead, the MTF circuit that forces NSC commitment into the glial-lineage NFIA/ ATF3/RUNX2 is expressed at log levels higher than POU5F1 or NANOG, with further upregulation in GBM vs grade 2/3 oligodendrogliomas or astrocytomas ( Figure 1B). SELECT TO TRANSFORM IN LINEAGE-SPECIFYING MTF CIRCUIT CONTEXTS? MYC, and its paralogues MYCN and MYCL, are the ancient MTF that regulate hundreds of genes essential for nutrient supply, energy production, provision of cellular building blocks, cell cycle entry and progression (replication). 23 The emergence of multicellularity (metazoa) occasioned MYC subordination to MTF circuits that create diverse lineages and hierarchies 24   5 | CLARIFYING CANCER " STEM" CELL TERMINOLOGY Self-replicating cancer cells are often referred to in the literature as "stem" or "stem-like" (cancer "stem" cells), which is true in so far as they self-replicate, but the terminology obscures that by MTF content, dependency, and many other parameters, these cells phenocopy lineage-committed progenitors. [38][39][40][41][42][43][44][45] Tens of glioma cell lines that indefinitely self-replicate in vitro, as well as other human and murine glioma cells shown to initiate GBM in mice, faithfully recapitulate the high lineage-specifying MTF circuit/early-glial/ MYC-target gene expression configuration observed in bulk glioma samples. 46-52 Self-replicating malignant cells in other tissues are also characterized by high expression and dependency on lineage-specifying MTF circuits that activate and cooperate with MYC for transit-amplification (reviewed in 3 ). Thus, normal self-replication is restricted to tissue stem cells, but malignant self-replication is not (self-replication = replication without lineage-maturation; Figure 1A).

Deletions/translocations of genes for other activating machinery.
Genes for other key components in the machinery needed to activate  tumor-initiation in vivo. 46 Consistent with a nonapoptosis pathway, there was no significant effect on glioma cell viability, even as sphere morphology (a measure of self-replication) and proliferation were reduced. 46 In H3F3A K27M-mutated gliomas, EZH2 inhibition decreased proliferation in vitro and increased survival in mice. 107 Another study looked specifically for a cytotoxic effect of EZH2 inhibition in the glioma cells to explain the cytoreduction, and did not find any. 108 ATP-dependent chromatin remodelers of the ISWI and CHD families, for example, CHD4, SMARCA5, oppose SWI/SNF ATP-dependent chromatin remodelers. That is, they execute a linchpin epigenetic repression event of re-positioning nucleosomes to obstruct access to genes by basal transcription factor machinery. CHD4 depletion using shRNA promoted astrocyte differentiation in vitro, 109 implying ISWI/ CHD-family members are candidate targets for therapy.
Mitochondrial outputs other than AKG, for example, cytidine triphosphate (CTP), also facilitate lineage-differentiation: small molecules that inhibit de novo pyrimidine synthesis and decrease CTP, including dihydroorotate dehydrogenase (DHODH)-inhibitors (several available) and the cytidine triphosphate synthase 2 (CTPS2)-inhibitor cyclopentenyl cytosine (CPEC), release cancer cells including glioma cells to terminal-differentiation. [110][111][112] Implicating CTP specifically, CTP-restoration with exogenous cytidine prevented terminaldifferentiation induction by the DHODH-inhibitor leflunomide. 113 These results imply that CTP operates as a cofactor in a corepressor complex, and interestingly, key DNA packaging proteins in prokaryotes are CTP-dependent and related to eukaryotic condensins. 114 The deoxycytidine analog decitabine, a clinical pro-drug approved to treat myeloid malignancies, inhibits and depletes DNMT1 from dividing cells. Self-replication of IDH1-mutated glioma cells was terminated by decitabine treatment in vitro, without activation of apoptosis, but with activation of neuronal/glial lineage-differentiation genes, and with morphology changes of terminal-differentiation. 115 The cytidine analog 5-azacytidine also inhibits/depletes DNMT1: long-term administration of 5-azacytidine to mice with IDH1-mutated anaplastic astrocytoma significantly decreased tumor growth; histological examination indicated terminal-differentiation was the pathway of tumor  No improvement in PFS and OS c cytoreduction. 116 5-azacytidine also suppressed IDH-wildtype GBM growth in vitro and in xenografts. 117  objective response rates by Response Assessment in Neuro-Oncology (RANO) criteria ranged from $3% to 29% (Table 1) One caveat with magnetic resonance imaging (MRI) assessment of glioma-response is that advancing glioma can be difficult to distinguish from radiation-induced changes in the normal brain. 132 All authors. Accountable for all aspects of the manuscript: All authors.
The work reported in the paper has been performed by the authors unless clearly specified in the text.

FUNDING INFORMATION
Yogen Saunthararajah: National Heart, Lung and Blood Institute