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.

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.

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

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.

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