Gene expression analysis suggests immunosuppressive roles of endolysosomes in glioblastoma.

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.

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.

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.

Lack of urea transporters, UT-A1 and UT-A3, increases nitric oxide accumulation to dampen medullary sodium reabsorption through ENaC.

Although the role of urea in urine concentration is known, the effect of urea handling by the urea transporters (UTs), UT-A1 and UT-A3, on sodium balance remains elusive. Serum and urinary sodium concentration is similar between wild-type mice (WT) and UT-A3 null (UT-A3 KO) mice; however, mice lacking both UT-A1 and UT-A3 (UT-A1/A3 KO) have significantly lower serum sodium and higher urinary sodium. Protein expression of renal sodium transporters is unchanged among all three genotypes. WT, UT-A3 KO, and UT-A1/A3 KO acutely respond to hydrochlorothiazide and furosemide; however, UT-A1/A3 KO fail to show a diuretic or natriuretic response following amiloride administration, indicating that baseline epithelial Na+ channel (ENaC) activity is impaired. UT-A1/A3 KO have more ENaC at the apical membrane than WT mice, and single-channel analysis of ENaC in split-open inner medullary collecting duct (IMCD) isolated in saline shows that ENaC channel density and open probability is higher in UT-A1/A3 KO than WT. UT-A1/A3 KO excrete more urinary nitric oxide (NO), a paracrine inhibitor of ENaC, and inner medullary nitric oxide synthase 1 mRNA expression is ~40-fold higher than WT. Because endogenous NO is unstable, ENaC activity was reassessed in split-open IMCD with the NO donor PAPA NONOate [1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine)], and ENaC activity was almost abolished in UT-A1/A3 KO. In summary, loss of both UT-A1 and UT-A3 (but not UT-A3 alone) causes elevated medullary NO production and salt wasting. NO inhibition of ENaC, despite elevated apical accumulation of ENaC in UT-A1/A3 KO IMCD, appears to be the main contributor to natriuresis in UT-A1/A3 KO mice.

Chronic lithium treatment induces novel patterns of pendrin localization and expression.

Prolonged lithium treatment is associated with various renal side effects and is known to induce inner medullary collecting duct (IMCD) remodeling. In animals treated with lithium, the fraction of intercalated cells (ICs), which are responsible for acid-base homeostasis, increases compared with renal principal cells (PCs). To investigate the intricacies of lithium-induced IMCD remodeling, male Sprague-Dawley rats were fed a lithium-enriched diet for 0,1, 2, 3, 6, 9, or 12 wk. Urine osmolality was decreased at 1 wk, and from 2 to 12 wk, animals were severely polyuric. After 6 wk of lithium treatment, approximately one-quarter of the cells in the initial IMCD expressed vacuolar H+-ATPase, an IC marker. These cells were localized in portions of the inner medulla, where ICs are not normally found. Pendrin, a Cl-/[Formula: see text] exchanger, is normally expressed only in two IC subtypes found in the convoluted tubule, the cortical collecting duct, and the connecting tubule. At 6 wk of lithium treatment, we observed various patterns of pendrin localization and expression in the rat IMCD, including a novel phenotype wherein pendrin was coexpressed with aquaporin-4. These observations collectively suggest that renal IMCD cell plasticity may play an important role in lithium-induced IMCD remodeling.