bernda ireenah sigmund
Ceumar Pee
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The devastating transcriptional programs influenced by H3-alterations in DMG are fundamentally controlled by posttranslational modification (PTMs) of the 59 amino acid long N-terminal tail of H3, in both histone 3 isoform 1 (H3.1) and histone 3 isoform 3 (H3.3). These PTMs include acetylation, methylation, phosphorylation and ubiquitination and dictate protein structure, stability and accessibility, hence promotes or represses the activation of the transcriptional machinery complexes [10]. H3.1 variants are encoded by a cluster of intronless genes (HIST1 cluster), expressed in a replication dependent manner during the S-phase of the cell cycle [11]. Comparatively, H3.3 variants are independently encoded by two, intron-possessing genes (H3F3A and H3F3B) and are expressed throughout the cell cycle, however, are enriched at various stages of differentiation [12]. Of the genes encoding H3 histone variants, HIST1H3B (H3.1) and H3F3A (H3.3) harbor recurring mutations in DMG leading to the establishment of the molecular subtypes [9]. Between these two histone H3 genes, there are significant structural differences in both the presence of exon and introns, and nucleotide length (Fig. 1A, B), with only 3.4% genomic homogeny. However, the resultant histone proteins show 98.5% homogeny (pairwise sequence alignment using the EMBOSS needle alignment tool [13]). Key lysine residues such as K27 and K36 are conserved and play pivotal roles in the epigenetic regulation of transcription.
As the dominant molecular feature, treatment strategies targeting H3K27M are a priority, however, it has remained undruggable to-date. Given the associated loss of trimethylation and hence increased H3K27 acetylation (H3K27ac), it is of some surprise that most research has focused on the use of histone deacetylase (HDAC) inhibitors (HDACis), such as panobinostat, rather than inhibitors of histone acetyltransferases (HATs). Nevertheless, HDACis show low nanomolar cytotoxicity against DMG cell cultures and are effective in DMG patient-derived xenograft (PDX) mouse models [21, 22]. The HDACis, valproic acid, panobinostat, quisinostat and romidepsin, induce a dose-dependent global increase in H3K27ac and H3K27me3 [21,22,23], suggestive of a partial rescue of global H3K27 hypomethylation. This is consistent with findings that polyacetylation at residues both proximal and distal to K27M can greatly diminish PRC2 inhibition. Unfortunately, HDACi-induced partial rescue of global hypomethylation is transient [24], encouraging exploration into chemicals that synergize with HDACi. One promising candidate that is in preclinical development is the proteasome inhibitor marizomib which invokes acute toxicity through uncoupling of respiration and inhibition of glycolysis leading to metabolic catastrophe in DMG cells [25].
We are gaining a greater understanding of the somatic heterogeneity of DMG [1], however, what is less understood is the impact of distinct genomic subclonal populations [6], potentially underpinning the lack of effective treatments. While recurring genetic H3-alterations are hallmark features of DMG, multiple co-segregating mutations are patient-specific, conferring their own varying level of poor prognoses and midline localization (Table 1, Fig. 2). The genomic landscapes of DMG have been comprehensively characterized [1], and in most cases, highlight the alterations that harbor potential for therapeutic targeting [29]. Even though we are yet to translate these discoveries into improved outcomes, these sophisticated real-time studies are providing us with increased knowledge of the co-occurring somatic events that underpin the genomic heterogeneity of DMG, critical information for the design of effective combination treatment strategies. To aid in the development of such combination strategies, in the following sections we summarize recurring somatic mutations linked with each DMG a subtype, list the recurrent midline localizations of these driver gene alterations (Fig. 2), and highlight potential therapies and research priorities that we hope will help to increase the durability and effectiveness of strategies targeting these mutations.
Given that the intracellular activity of SMADs is reliant on their phosphorylation, numerous protein phosphatases are known to downregulate their activity and may serve as targets for therapies to enhance ALK2 inhibition [47]. The serine/threonine protein phosphatase, PP2A is a SMAD-associated phosphatase frequently showing reduced activity in cancer, with activity increased using fingolimod (FTY720), an FDA approved drug used in the treatment of multiple sclerosis, and hence has excellent brain penetration [48]. This encourages preclinical assessment of FTY720 in combination with therapies targeting ALK2 for ACVR1 mutant DMG.
Mutations in the components of the Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling axis are recognized drivers of gliomagenesis in DMG [43]. Mutations in the PI3K catalytic subunit alpha (PIK3CA) are seen in 12% of DMG (Fig. 2), while PI3K regulatory subunit 1 (PIK3R1) mutations are present in 18%, most commonly in H3.3K27M and EZHIP subtypes [43, 49] (Table 1, Figs. 2B, C). PIK3CA mutations lead to constitutive lipid kinase activity thereby driving cellular transformation [50], while PIK3R1 mutations activate wildtype PIK3CA or PI3K signaling [51]. Phosphatase and tensin homolog (PTEN) is the well-established negative regulator of this signaling cascade, and an important tyrosine kinase tumor suppressor [52]. PTEN is mutated in 4% of H3.1K27M [53], 6% of H3.3K27M [49], and 6% of EZHIP DMG [27] (Table 1, Fig. 2). Loss of PTEN can occur through a chromosomal deletion of 10q and is determined to be an early event in DMG development [52].
Paxalisib is an FDA approved, PI3K/Akt inhibitor developed to penetrate the brain and decrease activity of signaling cascades [54]. Recently, paxalisib was shown to reduce growth and proliferation in PI3K-mutant and wildtype DMG cell lines [54] and has recently entered combination clinical trials for DMG (NCT05009992). Fimepinostat (CUDC-907), is a dual PI3K/HDAC inhibitor (Table 2), that inhibits radiation-induced DNA repair pathways including homologous recombination and nonhomologous end-joining, leading to G1 cell-cycle arrest and apoptosis [55] and is also in DMG clinical trials (NCT03893487). Despite PI3K alterations being some of the common events in DMG and other cancers, pharmacological inhibition of PI3K has resulted in variable clinical responses. This raises the possibility of an inherent mechanism of resistance. Indeed, mouse tumor models show insulin feedback is induced by PI3K inhibitors, reactivating PI3K signaling, thus compromising their efficacy [56]. Insulin-feedback is effectively controlled using anti-glycemic approaches, which greatly enhances the therapeutic effectiveness of PI3K inhibitors, an approach that warrants rigorous testing in DMG.
Hyperacetylation of H3K27 in DMG is driven by the activity of acetyl-binding, bromodomain and extraterminal (BET) proteins, particularly bromodomain-containing protein 4 (BRD4) which are implicated in tumor progression and aggressiveness [59]. MYC is highly occupied by H3.3K27M and H3K27ac super-enhancers and hence highly expressed in DMG. This is slightly at odds with what has been previously shown, where MYC/MYCN amplification was only observed in EZHIP DMG [7]. Panobinostat decreased oncogenic MYC target gene expression causing cell death in DMG preclinical models [21].
Targeting BRD4-driven MYC activity, using the BBB-penetrant bromodomain inhibitor, JQ1, is effective as a monotherapy in EZHIP DMG models when used at high-doses, however, H3K27M DMG are less sensitive [23]. Combinations of JQ1 and MRK003, a gamma-secretase inhibitor that reduces NOTCH1 expression, reduced H3K27M DMG growth and survival [60], highlighting this as a potential strategy for DMG.
Given the importance of cyclin proteins in mitosis, several CDK4/6 inhibitors have been developed and are under clinical evaluation in DMG [63] (Table 2). Palbociclib has completed dose escalation studies, while two other trials are testing ribociclib. Abemaciclib is suggested to be the most effective CCND2-CDK4/6 inhibitor due to higher CDK4/6 affinity/potency compared to palbociclib and ribociclib [63]. While these CDK4/6 inhibitors have shown some efficacy in tumors with deregulated cell-cycle control, resistance to these therapies is common. Phosphoproteomic profiling of cancer models resistant to CDK4/6 inhibitors revealed enhanced Mitogen-activated protein kinase (MAPK) signaling, therapeutically exposed using the FDA approved MEK inhibitor, trametinib [64] (Table 2). Targeting MEK is a treatment paradigm that has recently come into clinical thinking particularly for HGGs and DIPGs harboring germline or somatic mutations in Neurofibromatosis type 1 (NF1) [65, 66]. Furthermore, the combination of MEK1/2 and CDK4/6 inhibition showed therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models [67], a treatment paradigm that warrants exploration in DMG.
The somatic alterations summarized above offer some biological insight into the roles these recurring mutations play in DMG and present us with a suite of therapeutic vulnerabilities. Despite the number of currently used targeted therapies developed to combat the various co-occurring mutations in DMG, most are still undergoing early-stage clinical trials to establish toxicity profiles. More recent studies are now using them in combination, however, the maximum tolerated doses for these combinations are yet to be determined, and efficacy signals are unknown (Table 2).
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