Purpose: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TGs) that ~50% of clear cell renal cell carcinomas (ccRCCs) 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 anti-tumor activity. Orthogonal RNA-seq 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 paralleled those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed obviating the need for phlebotomies, 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.

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Introduction. Translocation Renal Cell Carcinoma (tRCC) is a distinct and aggressive molecular subtype of kidney cancer unusually prevalent in children and adolescents. tRCC has a poor prognosis with no specific therapies and is incurable in the metastatic setting. tRCC is characterized by chromosomal translocations involving three out of the four Microphthalmia (MiT) family transcription factors, TFE3, TFE3, and MITF. The most commonly involved gene is TFE3, which most often fuses with ASPSCR1. However, the molecular mechanisms by which ASPSCR1-TFE3 (and more broadly MiT/TFE fusion proteins) dictates tRCC tumorigenesis remain unclear and advances are hampered by the paucity of animal models. Herein, we sought to model tRCC and performed integrative genomic analyses to dissect molecular mechanisms underpinning tRCC tumorigenesis with the goal of translating this knowledge into therapeutic opportunities. Methods. We conditionally expressed a human ASPSCR1-TFE3 fusion in the Pax8 and Sglt2 lineages and performed phenotypic analyses as well as WES and RNAseq of emerging tRCC tumors. Similar genomic analyses of a human tRCC cohort were performed. The mutational landscape, cooperating pathways, and potential therapeutic targets were identified through comparative genomic and transcriptomic analyses. Emerging hypotheses were tested through in vitro and in vivo assays. Results. We show that human ASPSCR1-TFE3 driven by Pax8-Cre, a credentialled RCC driver, disrupted nephron development causing neonatal death, whilst Sglt2-Cre resulted in tRCC with complete penetrance and short latency. In both models, ASPSCR1-TFE3 induced characteristic morphological changes, loss of epithelial markers, and an EMT program, but only in the Sglt2-Cre model was proliferation observed. Given that Pax8-Cre, but not Sglt2-Cre¸ induce clear cell RCC (ccRCC), these data show that tRCC and ccRCC originate from different cell types. Comparative murine/human genomic analyses show that tumorigenesis by MiT/TFE fusion proteins is broadly cooperative (i.e. p53, ATM/ATR, Fanconi anemia, SWI/SNF, Hippo, and Wnt). Gene expression and functional studies implicate EPHA5 as well as Met/Ret in tRCC tumorigenesis. Antitumor/antiproliferative activity was observed with the therapeutic targeting of these pathways. Conclusion. This study provides insight into the tRCC mutational landscape, the cell of origin, and MiT/TFE-driven transcriptional programs offering several avenues for therapeutic development. Citation Format: Gopinath Prakasam, Akhilesh Mishra, Alana Christie, Jeffrey Miyata, Vanina T. Tcheuyap, Hui Ye, Quyen N. Do, Kevin B. Jones, Mai-Carmen Requena-Komuro, Ivan Pedrosa, Dinesh Rakheja, Payal Kapur, James Brugarolas. Comparative genomic analysis of novel translocation renal cell carcinoma model reveals molecular mechanisms of disease progression and therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR004.

The role of Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens (alone and in combination) in inducing systemic resistance in chickpea exposed to Fusarium oxysporum ciceris was investigated. Chickpea seed (var. Radhey) treatment with Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens alone and in combination on the germination, wilt incidence, plant growth promotion, yield, production of chlorophyll, protein, peroxidase, polyphenol oxidase and total phenol content was recorded. The results of in vitro studies revealed better performances of treatment T6 (Seed treatment with 1% Trichoderma harzianum + 2% Pseudomonas fluorescens + 2% Bacillus subtilis in combination). This treatment significantly reduced wilt incidence and increased germination, root length, shoot length, and yield as compared to untreated control. Treatment T6 also induced 1.4 per cent increase in peroxidase, 1.6 in polyphenol oxidase and 2.3 in total phenol in chickpea during pathogenesis by F. oxysporum ciceris f. sp. Similarly, 1.9% increase in chlorophyll and protein was recorded with the treatment T6 as compared to control. Present investigations will be helpful in formulating novel bioformulations using fungal and bacterial bioagents to control wilt in chickpea.

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