Unconventional mechanism of action and resistance to rapalogs in renal cancer.

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.

Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ferroptosis.

Lipids are essential for tumours because of their structural, energetic, and signaling roles. While many cancer cells upregulate lipid synthesis, growing evidence suggests that tumours simultaneously intensify the uptake of circulating lipids carried by lipoproteins. Which mechanisms promote the uptake of extracellular lipids, and how this pool of lipids contributes to cancer progression, are poorly understood. Here, using functional genetic screens, we find that lipoprotein uptake confers resistance to lipid peroxidation and ferroptotic cell death. Lipoprotein supplementation robustly inhibits ferroptosis across numerous cancer types. Mechanistically, cancer cells take up lipoproteins through a pathway dependent on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. Tumour GAGs are a major determinant of the uptake of both low and high density lipoproteins. Impairment of glycosaminoglycan synthesis or acute degradation of surface GAGs decreases the uptake of lipoproteins, sensitizes cells to ferroptosis and reduces tumour growth in mice. We also find that human clear cell renal cell carcinomas, a distinctively lipid-rich tumour type, display elevated levels of lipoprotein-derived antioxidants and the GAG chondroitin sulfate than non-malignant human kidney. Altogether, our work identifies lipoprotein uptake as an essential anti-ferroptotic mechanism for cancer cells to overcome lipid oxidative stress in vivo, and reveals GAG biosynthesis as an unexpected mediator of this process.

HIF2 Inactivation and Tumor Suppression with a Tumor-Directed RNA-Silencing Drug in Mice and Humans.

PURPOSE: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TG) that ∼50% of clear cell renal cell carcinomas (ccRCC) are HIF2α dependent. However, prolonged treatment induced resistance mutations, which we also identified in humans. Here, we evaluated a tumor-directed, systemically delivered, siRNA drug (siHIF2) active against wild-type and resistant-mutant HIF2α. EXPERIMENTAL DESIGN: Using our credentialed TG platform, we performed pharmacokinetic and pharmacodynamic analyses evaluating uptake, HIF2α silencing, target gene inactivation, and antitumor activity. Orthogonal RNA-sequencing studies of siHIF2 and PT2399 were pursued to define the HIF2 transcriptome. Analyses were extended to a TG line generated from a study biopsy of a siHIF2 phase I clinical trial (NCT04169711) participant and the corresponding patient, an extensively pretreated individual with rapidly progressive ccRCC and paraneoplastic polycythemia likely evidencing a HIF2 dependency. RESULTS: siHIF2 was taken up by ccRCC TGs, effectively depleted HIF2α, deactivated orthogonally defined effector pathways (including Myc and novel E2F pathways), downregulated cell cycle genes, and inhibited tumor growth. Effects on the study subject TG mimicked those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed, and a partial response was induced. CONCLUSIONS: To our knowledge, this is the first example of functional inactivation of an oncoprotein and tumor suppression with a systemic, tumor-directed, RNA-silencing drug. These studies provide a proof-of-principle of HIF2α inhibition by RNA-targeting drugs in ccRCC and establish a paradigm for tumor-directed RNA-based therapeutics in cancer.

Spectrum of diverse genomic alterations define non-clear cell renal carcinoma subtypes.

To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.