DON inhibits FGAMS, a de novo purine biosynthesis enzyme, with the lowest Ki.

Inhibiting MTAP sensitizes tumor cells to DRP-104 treatments.

Prostate cancer cells develop adaptation to purine shortage inducer, such as DRP-104, and employ microtubules to assemble purinosome to enhance purine biosynthesis efficiency.

Treating tumor-bearing mice with DRP-104 in combination with MTDIA does not affect animal’s body weight but alters the tumor’s immune microenvironment.

Gene expression and functional assays implicate purine biosynthesis as a driver of prostate cancer pathogenesis.

The MTAP-dependent purine salvage is a source of purine supply for cancer cells.

A graphical abstract summarizing tumor cells’ strategies for adapting to and their vulnerabilities under purine shortage stress.

PURPOSE:Purine metabolism is a promising therapeutic target in cancer; however, how cancer cells respond to purine shortage, particularly their adaptation and vulnerabilities, remains unclear. EXPERIMENTAL DESIGN:Using the recently developed purine shortage-inducing prodrug DRP-104 and genetic approaches, we investigated the responses in prostate, lung, and glioma cancer models. RESULTS:We demonstrate that when de novo purine biosynthesis is compromised, cancer cells employ microtubules to assemble purinosomes, multiprotein complexes of de novo purine biosynthesis enzymes that enhance purine biosynthesis efficiency. Although this process enables tumor cells to adapt to purine shortage stress, it also renders them more susceptible to the microtubule-stabilizing chemotherapeutic drug docetaxel. Furthermore, we show that although cancer cells primarily rely on de novo purine biosynthesis, they also exploit methylthioadenosine phosphorylase (MTAP)-mediated purine salvage as a crucial alternative source of purine supply, especially under purine shortage stress. In support of this finding, combining DRP-104 with an MTAP inhibitor significantly enhances tumor suppression in prostate cancer models in vivo. Finally, despite the resilience of the purine supply machinery, purine shortage-stressed tumor cells exhibit increased DNA damage and activation of the cGAS-STING pathway, which may contribute to impaired immunoevasion and provide a molecular basis of the previously observed DRP-104-induced antitumor immunity. CONCLUSIONS:Together, these findings reveal purinosome assembly and purine salvage as key mechanisms of cancer cell adaptation and resilience to purine shortage while identifying microtubules, MTAP, and immunoevasion deficits as therapeutic vulnerabilities.

Targeting endolysosomes is a strategy extensively pursued for treating cancers, including glioblastomas (GBMs), on the basis that the intact function of these subcellular organelles is key to tumor cell autophagy and survival. Through gene expression analyses and cell type abundance estimation in GBMs, we showed that genes associated with the endolysosomal machinery are more prominently featured in non-tumor cells in GBMs than in tumor cells, and that tumor-associated macrophages represent the primary immune cell type that contributes to this trend. Further analyses found an enrichment of endolysosomal pathway genes in immunosuppressive (pro-tumorigenic) macrophages, such as M2-like macrophages or those associated with worse prognosis in glioma patients, but not in those linked to inflammation (anti-tumorigenic). Specifically, genes critical to the hydrolysis function of endolysosomes, including progranulin and cathepsins, were among the most positively correlated with immunosuppressive macrophages, and elevated expression of these genes is associated with worse patient survival in GBMs. Together, these results implicate the hydrolysis function of endolysosomes in shaping the immunosuppressive microenvironment of GBM. We propose that targeting endolysosomes, in addition to its detrimental effects on tumor cells, can be leveraged for modulating immunosuppression to render GBMs more amenable to immunotherapies.

Supplementary Data from Epigenetic Regulation of Fanconi Anemia Genes Implicates PRMT5 Blockage as a Strategy for Tumor Chemosensitization

Cancer cells depend on nicotinamide adenine dinucleotide phosphate (NADPH) to combat oxidative stress and support reductive biosynthesis. One major NAPDH production route is the oxidative pentose phosphate pathway (committed step: glucose-6-phosphate dehydrogenase, G6PD). Alternatives exist and can compensate in some tumors. Here, using genetically-engineered lung cancer model, we show that ablation of G6PD significantly suppresses KrasG12D/+;Lkb1-/-(KL) but not KrasG12D/+;p53-/- (KP) lung tumorigenesis. In vivo isotope tracing and metabolomics revealed that G6PD ablation significantly impaired NADPH generation, redox balance and de novo lipogenesis in KL but not KP lung tumors. Mechanistically, in KL tumors, G6PD ablation caused p53 activation that suppressed tumor growth. As tumor progressed, G6PD-deficient KL tumors increased an alternative NADPH source, serine-driven one carbon metabolism, rendering associated tumor-derived cell lines sensitive to serine/glycine depletion. Thus, oncogenic driver mutations determine lung cancer dependence on G6PD, whose targeting is a potential therapeutic strategy for tumors harboring KRAS and LKB1 co-mutations.

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.

Glioblastoma stem cells (GSCs), also known as brain tumor initiating cells (BTICs), drive tumor progression, heterogeneity, and resistance to treatments, posing formidable challenges to advancing effective treatments against glioblastoma (GBM). We postulated that inducing BTIC differentiation can serve as a solution to diminishing their stem-like features. Here, we report that clemastine, an over-the-counter oral medication for allergy relief, attenuates stemness and proliferation of BTICs. These effects of clemastine were accompanied by altered transcriptional programs suggestive of a shift from maintaining stem cell identity to differentiation, resonating with the ability of clemastine to promote oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes. Genetic perturbation and small-molecule inhibition of putative pharmacological targets of clemastine revealed that Emopamil-binding protein (EBP), an enzyme in the sterol biosynthesis pathway, played a pivotal role in mediating the differentiating and anti-tumor effects of clemastine. Notably, loss-of-function assays showed that EBP expression was indispensable for BTIC propagation. In contrast, overexpression of EBP stimulated BTIC proliferation, thus uncovering a previously unknown role of sterol metabolism in BTIC maintenance. Finally, we demonstrated that a mouse neural stem cell-derived glioma model was similarly susceptible to clemastine treatment. Taken together, our study identifies pathways essential for the perpetuation of stemness in GBM, and implicates a non-oncology drug with a well-established safety profile that can be repurposed to mitigate the stem-like properties of GBM.

Abstract 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.