Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas.

Repurposing Clemastine to Target Glioblastoma Cell Stemness.

Brain tumor-initiating cells (BTICs) and tumor cell plasticity promote glioblastoma (GBM) progression. Here, we demonstrate that clemastine, an over-the-counter drug for treating hay fever and allergy symptoms, effectively attenuated the stemness and suppressed the propagation of primary BTIC cultures bearing PDGFRA amplification. These effects on BTICs were accompanied by altered gene expression profiling indicative of their more differentiated states, resonating with the activity of clemastine in promoting the differentiation of normal oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Functional assays for pharmacological targets of clemastine revealed that the Emopamil Binding Protein (EBP), an enzyme in the cholesterol biosynthesis pathway, is essential for BTIC propagation and a target that mediates the suppressive effects of clemastine. Finally, we showed that a neural stem cell-derived mouse glioma model displaying predominantly proneural features was similarly susceptible to clemastine treatment. Collectively, these results identify pathways essential for maintaining the stemness and progenitor features of GBMs, uncover BTIC dependency on EBP, and suggest that non-oncology, low-toxicity drugs with OPC differentiation-promoting activity can be repurposed to target GBM stemness and aid in their treatment.

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX , defining molecular alterations in IDH -mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX knockout glioma models in the presence and absence of the IDH1 R 132 H mutation. ATRX-deficient glioma cells were sensitive to dsRNA-based innate immune agonism and exhibited impaired lethality and increased T-cell infiltration in vivo . However, the presence of IDH1 R 132 H dampened baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1 R132H inhibition. IDH1 R132H co-expression did not interfere with the ATRX KO-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1 R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytoma.

Purine Synthesis Inhibitor L-Alanosine Impairs Mitochondrial Function and Stemness of Brain Tumor Initiating Cells.

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for MTAP-deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.

MTAP loss correlates with an immunosuppressive profile in GBM and its substrate MTA stimulates alternative macrophage polarization.

Glioblastoma (GBM) is a lethal brain cancer known for its potent immunosuppressive effects. Loss of Methylthioadenosine Phosphorylase (MTAP) expression, via gene deletion or epigenetic silencing, is one of the most common alterations in GBM. Here we show that MTAP loss in GBM cells is correlated with differential expression of immune regulatory genes. In silico analysis of gene expression profiles in GBM samples revealed that low MTAP expression is correlated with an increased proportion of M2 macrophages. Using in vitro macrophage models, we found that methylthioadenosine (MTA), the metabolite that accumulates as a result of MTAP loss in GBM cells, promotes the immunosuppressive alternative activation (M2) of macrophages. We show that this effect of MTA on macrophages is independent of IL4/IL3 signaling, is mediated by the adenosine A2B receptor, and can be pharmacologically reversed. This study suggests that MTAP loss in GBM cells may contribute to the immunosuppressive tumor microenvironment, and that MTAP status should be considered for characterizing GBM immune states and devising immunotherapy-based approaches for treating MTAP-null GBM.

Epigenetic Regulation of Fanconi Anemia Genes Implicates PRMT5 Blockage as a Strategy for Tumor Chemosensitization.

Strengthened DNA repair pathways in tumor cells contribute to the development of resistance to DNA-damaging agents. Consequently, targeting proteins in these pathways is a promising strategy for tumor chemosensitization. Here, we show that the expression of a subset of Fanconi anemia (FA) genes is attenuated in glioblastoma tumor cells deficient in methylthioadenosine phosphorylase (MTAP), a common genetic alteration in a variety of cancers. Subsequent experiments in cell line models of different cancer types illustrate that this reduced transcription of FA genes can be recapitulated by blockage of Protein Arginine Methyltransferase 5 (PRMT5), a promising therapeutically targetable epigenetic regulator whose enzymatic activity is compromised in MTAP-deficient cells. Further analyses provide evidence to support that PRMT5 can function as an epigenetic regulator that contributes to the increased expression of FA genes in cancer cells. Most notably and consistent with the essential roles of FA proteins in resolving DNA damage elicited by interstrand crosslinking (ICL) agents, PRMT5 blockage, as well as MTAP loss, sensitizes tumor cells to ICL agents both in vitro and in xenografts. Collectively, these findings reveal a novel epigenetic mechanism underlying the upregulated expression of FA genes in cancer cells and suggest that therapeutically targeting PRMT5 can have an additional benefit of chemosensitizing tumor cells to ICL agents. IMPLICATIONS: PRMT5 positively regulates the expression of FA genes. Inhibition of PRMT5 attenuates FA-dependent DNA repair pathway and sensitizes tumor cells to ICL agents.

A PRMT5-RNF168-SMURF2 Axis Controls H2AX Proteostasis.

H2AX safeguards genomic stability in a dose-dependent manner; however, mechanisms governing its proteostasis are poorly understood. Here, we identify a PRMT5-RNF168-SMURF2 cascade that regulates H2AX proteostasis. We show that PRMT5 sustains the expression of RNF168, an E3 ubiquitin ligase essential for DNA damage response (DDR). Suppression of PRMT5 occurs in methylthioadenosine phosphorylase (MTAP)-deficient glioblastoma cells and attenuates the expression of RNF168, leading to destabilization of H2AX by E3 ubiquitin ligase SMURF2. RNF168 and SMURF2 serve as a stabilizer and destabilizer of H2AX, respectively, via their dynamic interactions with H2AX. In supporting an important role of this signaling cascade in regulating H2AX, MTAP-deficient glioblastoma cells display higher levels of DNA damage spontaneously or in response to genotoxic agents. These findings reveal a regulatory mechanism of H2AX proteostasis and define a signaling cascade that is essential to DDR and that is disrupted by the loss of a metabolic enzyme in tumor cells.