HIF-2α Inhibitor Research Articles

Treatment of Refractory Metastatic Renal Cell Carcinoma.

Simple SummaryThe treatment of metastatic renal cell carcinoma continues to rapidly evolve, with various combinations of immune checkpoint inhibitors and targeted tyrosine kinase inhibitors improving outcomes in the first-line setting. However, a significant subset of patients fail to respond to these therapies, and many patients eventually progress, necessitating effective therapeutic options in the treatment-refractory setting. Here, we review the treatment of refractory renal cell carcinoma, including the current standard of care, ongoing trials with the potential to alter current paradigms, and considerations for non-clear cell histologies.First-line treatment for metastatic renal cell carcinoma (mRCC) rapidly shifted in recent years with the advent of combination therapies, including immune checkpoint inhibitor (ICI) doublets and combinations of an ICI with a vascular endothelial growth factor receptor (VEGFR) targeted tyrosine kinase inhibitor (TKI). Despite improvements in overall survival and many durable responses, there exists a significant number of patients who fail to respond to these agents, and many patients eventually progress. Given the rapid changes in the front-line setting, it is essential to understand treatment options in refractory mRCC. Here, we review the evidence behind current options for later-line therapies, often involving additional VEGFR-TKIs alone or in combination with mammalian target of rapamycin (mTOR) targeted agents, as well as situations where consideration of immunotherapy rechallenge may be appropriate. Additionally, we describe ongoing clinical trials examining concurrent ICI and TKI in the refractory setting, as well as those studying novel agents, such as targeted drug–antibody conjugates and hypoxia inducible factor 2α (HIF-2α) inhibitors. Finally, we review considerations for non-clear cell histologies in the refractory setting and mechanisms of resistance in mRCC.

Neurological Applications of Belzutifan in von Hippel Lindau Disease.

Von Hippel Lindau (VHL) disease is a tumor predisposition syndrome caused by mutations in the VHL gene that presents with visceral neoplasms and growths, including clear cell renal cell carcinoma, and central nervous system manifestations, such as hemangioblastomas of the brain and spine. The pathophysiology involves a dysregulation of oxygen sensing caused by inability to degrade HIFα, leading to overactivation of hypoxic pathways. Hemangioblastomas are the most common tumors in patients with VHL and cause significant morbidity. Until recently, there were no systemic therapies available for patients that could effectively reduce the size of these lesions. Belzutifan, the first approved HIF-2α inhibitor, has demonstrated benefit in VHL-associated tumors, with a 30% response rate in hemangioblastomas and ~30-50% reduction in their sizes over the course of treatment. Anemia is the most prominent adverse effect, affecting 76-90% of participants and sometimes requiring dose reduction or transfusion. Other significant adverse events include hypoxia and fatigue. Overall, belzutifan is well tolerated; however, long term data on dosing regimens, safety, and fertility are not yet available. Belzutifan holds promise for treatment of neurological manifestations of VHL and its utility may influence the clinical management paradigms for this patient population.

58 (PB048) - HIF-2α inhibitor AB521 modulates erythropoietin levels in healthy volunteers following a single oral dose

Background: Hypoxia-inducible factor (HIF)-2α is a transcription factor that is an oncogenic driver in clear cell renal cell carcinoma (ccRCC). The posttranslational regulation of the HIF-2α protein is oxygen-dependent and, in hypoxic or pseudohypoxic conditions, results in the stabilization of HIF-2α and downstream transcription of tumor-inducing genes. HIF-2α inhibition has been shown to mitigate tumor growth in ccRCC cases with mutation or dysregulation of the von Hippel-Lindau tumor suppressor gene.

Structural basis for the allosteric inhibition of hypoxia-inducible factor (HIF)-2 by belzutifan.

Hypoxia-inducible factor (HIF)-2α and its obligate heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT), are both members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family. Previous studies have identified HIF-2α as a key oncogenic driver in clear cell renal cell carcinoma (ccRCC), rendering it a promising drug target for this type of kidney cancer. Belzutifan is the first HIF-2α inhibitor approved for treating ccRCC and other cancers associated with the von Hippel-Lindau (VHL) disease. However, the detailed inhibitory mechanism of belzutifan at molecular level is still unclear. Here we obtained the crystal structure of HIF-2α-ARNT heterodimer in complex with belzutifan at 2.75 Å resolution. The complex structure shows that belzutifan binds into the PAS-B pocket of HIF-2α, and it destabilizes the dimerization of HIF-2α and ARNT through allosteric effects mainly mediated by the key residue M252 of HIF-2α near the dimer interface. We further explored the inhibitory effects of belzutifan using biochemical and functional assays. The time-resolved fluorescence energy transfer (TR-FRET)-based binding assay showed that belzutifan disrupts the dimerization of HIF-2α and ARNT with a Ki value of 20 nM. The luciferase reporter assay indicated that belzutifan can efficiently inhibit the transcriptional activity of HIF-2α with an IC50 value of 17 nM. Besides, the real-time PCR assay illustrated that belzutifan can reduce the expression of HIF-2α downstream genes in 786-O kidney cancer cells in a dose-dependent manner. Our work reveals the molecular mechanism by which belzutifan allosterically inhibits HIF-2α and provides valuable information for the subsequent drug development targeting HIF-2α. Significance Statement The bHLH-PAS family of transcription factors are an emerging group of small-molecule drug targets. Belzutifan, originally developed by Peloton Therapeutics, is the first FDA-approved drug directly binding to a bHLH-PAS protein, the hypoxia-inducible factor (HIF)-2α. Based on the protein-drug complex structure, biochemical binding assays, and functional profiling of downstream gene expression, this study reveals the regulatory mechanism of how belzutifan allosterically destabilizes HIF-2α's heterodimerization with its obligate partner protein, thus reducing their transcriptional activity that links to tumor progression.

1453P Phase II study of belzutifan plus cabozantinib for previously treated advanced renal cell carcinoma (RCC): Update from cohort 2 of LITESPARK-003

First-line treatment with immune checkpoint inhibitors (ICIs) is standard of care for advanced RCC. However, most patients will experience disease progression. Belzutifan, a first-in-class HIF-2α inhibitor, has shown durable responses in heavily pretreated RCC. Cabozantinib, a VEGF, AXL, and MET inhibitor, is approved for advanced RCC. The ongoing, open-label, phase 2 LITESPARK-003 study (NCT03634540) investigated belzutifan + cabozantinib in pts with no prior treatment (cohort 1) and pts with prior ICI therapy (cohort 2).

1447O Phase II study of belzutifan plus cabozantinib as first-line treatment of advanced renal cell carcinoma (RCC): Cohort 1 of LITESPARK-003

HIF-2α is constitutively activated in RCC and is a transcription factor of multiple oncogenic pathways including TGF-α, GLUT1, cyclin D1, CXCR4, and VEGF. VEGF, and its receptor, are key regulators of angiogenesis and are upregulated in RCC. Belzutifan, a first-in-class HIF-2α inhibitor, has shown antitumor activity and favorable safety in heavily pretreated advanced RCC. Cabozantinib, a multikinase inhibitor that targets VEGF, is approved for advanced RCC. We present the first data of the use of belzutifan plus cabozantinib for treatment-naive pts with advanced RCC (cohort 1) enrolled in the ongoing, open-label, phase 2 LITESPARK-003 study (NCT03634540).

LBA69 Belzutifan, a HIF-2α Inhibitor, for von Hippel-Lindau (VHL) disease-associated neoplasms: 36 months of follow-up of the phase II LITESPARK-004 study

Transcription factor HIF-2α is an established oncogenic driver, and its activation in VHL disease is caused by germline alterations in the VHL gene. Belzutifan is a first-in-class HIF-2α inhibitor approved for patients (pts) with VHL disease who require therapy for associated renal cell carcinoma (RCC), CNS hemangioblastomas, or pancreatic neuroendocrine tumors (pNET) not requiring immediate surgery. Results with more than 3 years of follow-up from the phase 2 LITESPARK-004 study (NCT03401788) are presented.

Angiogenesis in NENs, with a focus on gastroenteropancreatic NENs: from biology to current and future therapeutic implications.

Neuroendocrine neoplasms (NENs) are highly vascularized malignancies arising from cells of the diffuse neuroendocrine system. An intricated cross-talk exists between NEN cells and the tumor microenvironment, and three main molecular circuits (VEGF/VEGFR pathway, FGF-dependent signaling and PDGF/PDGFR axis) have been shown to regulate angiogenesis in these neoplasms. Multiple randomized trials have investigated antiangiogenic agents over the past two decades, and sunitinib is currently approved for the treatment of advanced, progressive, G1/G2 pancreatic NENs. In recent years, two phase III clinical trials have demonstrated the efficacy and safety of surufatinib, a multi-tyrosine kinase angioimmune inhibitor, in patients with well-differentiated pancreatic and extrapancreatic NENs, and two studies of this agent are currently underway in Europe and US. The HIF-2α inhibitor belzutifan has recently received regulatory approval for the treatment of tumors arising in the context of Von-Hippel Lindau syndrome including pancreatic NENs, and a study of this drug in patients with sporadic tumors is presently ongoing. Combinations of antiangiogenic agents with chemotherapeutics and targeted drugs have been tested, with accumulating toxicities being a matter of concern. The potential of antiangiogenic agents in fine-tuning the immune microenvironment of NENs to enhance the activity of immune checkpoint inhibitors has been only partially elucidated, and further research should be carried out at this regard. Here, we review the current understanding of the biology of angiogenesis in NENs and provide a summary of the latest clinical investigations on antiangiogenic drugs in this malignancy.

HIF-2α Inhibits Ameloblast Differentiation via Hey2 in Tooth Development

Enamel is the highly mineralized outer layer of teeth; the cells responsible for enamel formation are ameloblasts. Local hypoxia and hypoxia inducible factor (HIF) in embryonic tissues are important to promote normal organogenesis. However, hypoxic state in tooth germs and the roles of HIF in ameloblast differentiation have not been understood. The aim of this study is to clarify the role of HIF in ameloblast differentiation during tooth germ development. We found that tooth germs were under hypoxia and HIF-1α and HIF-2α were expressed in tooth germs in embryonic mice. Then, we used HIF inhibitors to evaluate the function of HIF during tooth germ development. The HIF-2α inhibitor significantly decreased the size of tooth germs in organ culture, while the HIF-1α inhibitor did not apparently affect the size of tooth germs. The HIF-2α inhibitor enhanced the expression of amelogenin, a marker of ameloblast differentiation, in the tooth germs in organ culture and rat dental epithelial SF2 cells. Moreover, we found that the HIF-2α inhibitor-stimulating amelogenin expression was regulated by hes-related family basic helix-loop-helix transcription factor with YRPW motif 2(Hey2) in SF2 cells. These findings suggest that the HIF-2α–Hey2 axis plays an important role in ameloblast differentiation during tooth germ development.

First clinical experience with belzutifan in von Hippel-Lindau diseaseassociated CNS hemangioblastoma.

We present two cases of von Hippel–Lindau (VHL) disease-associated hemangioblastomas in the CNS treated with the newly approved HIF-2α inhibitor, belzutifan. The first case is a 31-year-old female with confirmed pathogenic germline VHL mutation who presented with multiple hemangioblastomas. The patient was started on belzutifan, and a brisk reduction in perilesional edema was observed after 2 months of treatment. The second patient is a 30-year-old male with familial VHL disease. Imaging revealed multiple cerebellar hemangioblastomas, and follow-up imaging after three cycles of belzutifan revealed a reduction in perilesional edema. Both patients tolerated belzutifan well, with only anemia and fatigue. We highlight our initial experience and early imaging findings associated with belzutifan in VHL disease-associated CNS hemangioblastomas.

SGLT2 inhibitor dapagliflozin attenuates cardiac fibrosis and inflammation by reverting the HIF-2α signaling pathway in arrhythmogenic cardiomyopathy.

Excessive cardiac fibrosis and inflammation aberrantly contribute to the progressive pathogenesis of arrhythmogenic cardiomyopathy (ACM). Whether sodium-glucose cotransporter-2 inhibitor (SGLT2i), as a new hypoglycemic drug, benefits ACM remains unclear. Cardiomyocyte-specific Dsg2 exon-11 knockout and wild-type (WT) littermate mice were used as the animal model of ACM and controls, respectively. Mice were administered by gavage with either SGLT2i dapagliflozin (DAPA, 1mg/kg/day) or vehicle alone for 8 weeks. HL-1 cells were treated with DAPA to identify the molecular mechanism in vitro. All mice presented normal glucose homeostasis. DAPA not only significantly ameliorated cardiac dysfunction, adverse remodeling, and ventricular dilation in ACM but also attenuated ACM-associated cardiac fibrofatty replacement, as demonstrated by the echocardiography and histopathological examination. The protein expressions of HIF-2α and HIF-1α were decreased and increased respectively in cardiac tissue of ACM, which were compromised after DAPA treatment. Additionally, NF-κB P65 and IκB phosphorylation, as well as fibrosis indicators (including TGF-β, α-SMA, Collagen I, and Collagen III) were increased in ACM. However, these trends were markedly suppressed by DAPA treatment. Consistent with these results in vitro, DAPA alleviated the IκB phosphorylation and NF-κB p65 transcriptional activity in DSG2-knockdown HL-1 cells. Interestingly, the elective HIF-2α inhibitor PT2399 almost completely blunted the DAPA-mediated downregulation of indicators concerning cardiac fibrosis and inflammation. SGLT2i attenuated the ACM-associated cardiac dysfunction and adverse remodeling in a glucose-independent manner by suppressing cardiac fibrosis and inflammation via reverting the HIF-2α signaling pathway, suggesting that SGLT2i is a novel and available therapy for ACM.

Differential HIF2α Protein Expression in Human Carotid Body and Adrenal Medulla under Physiologic and Tumorigenic Conditions.

Simple SummaryThoraco-abdominal paraganglioma and pheochromocytoma (PPGL) are pathogenically linked to mutations in SDH genes. Loss-of-function of SDH has been associated with stabilization of HIF2α, which otherwise would be degraded via oxygen-sensitive mechanisms. SDH dysfunction also predispose to the development of paragangliomas arising at the carotid body or other head and neck paraganglia (HNPGL). Although PPGL and HNPGL share similar features, they have certain clinical and genetic peculiarities. By comparison of HIF2α expression in HNPGL and PPGL, we found that functional HIF2α is overexpressed in 80% of PPGLs, including those with SDH mutations as compared with non-tumor tissue. However, HIF2α is already highly expressed in the carotid body under physiologic conditions, and it is not overexpressed in HNPGL. These data suggest that selective HIF2α inhibitors already in clinical trials may benefit a wide spectrum of PPGL.Hypoxia-inducible factors (HIF) 2α and 1α are the major oxygen-sensing molecules in eukaryotic cells. HIF2α has been pathogenically linked to paraganglioma and pheochromocytoma (PPGL) arising in sympathetic paraganglia or the adrenal medulla (AM), respectively. However, its involvement in the pathogenesis of paraganglioma arising in the carotid body (CB) or other parasympathetic ganglia in the head and neck (HNPGL) remains to be defined. Here, we retrospectively analyzed HIF2α by immunohistochemistry in 62 PPGL/HNPGL and human CB and AM, and comprehensively evaluated the HIF-related transcriptome of 202 published PPGL/HNPGL. We report that HIF2α is barely detected in the AM, but accumulates at high levels in PPGL, mostly (but not exclusively) in those with loss-of-function mutations in VHL and genes encoding components of the succinate dehydrogenase (SDH) complex. This is associated with upregulation of EPAS1 and the HIF2α-regulated genes COX4I2 and ADORA2A. In contrast, HIF2α and HIF2α-regulated genes are highly expressed in CB and HNPGL, irrespective of VHL and SDH dysfunctions. We also found that HIF2α and HIF1α protein expressions are not correlated in PPGL nor HNPGL. In addition, HIF1α-target genes are almost exclusively overexpressed in VHL-mutated HNPGL/PPGL. Collectively, the data suggest that involvement of HIF2α in the physiology and tumor pathology of human paraganglia is organ-of-origin-dependent and HIF1α-independent.

Epithelial HIF2α expression induces intestinal barrier dysfunction and exacerbation of arthritis

ObjectiveTo investigate how the mucosal barrier in the intestine influences the development of arthritis, considering that metabolic changes in the intestinal epithelium influence its barrier function.MethodsIntestinal hypoxia inducible factor (HIF)-2α...

Abstract 6330: NKT2152: A highly potent HIF2α inhibitor and its therapeutic potential in solid tumors beyond ccRCC

Abstract Clear cell Renal Cell Carcinoma (ccRCC) is characterized in most cases (>80%) by the inactivation of von Hippel-Lindau (VHL) tumor suppressor. VHL loss leads to HIF2α accumulation and constitutive activation of its downstream genes important in carcinogenesis. Preclinical and clinical evidence demonstrate that HIF2α inhibition by the first-in-class belzutifan (MK-6482) offers an effective treatment for ccRCC. Here we discovered a novel, potent, selective, and orally available small molecule HIF2α inhibitor (NKT2152) through rational drug design. Cellular thermal shift assay demonstrated NKT2152 engaged HIF2α and stabilized it against heat-induced denaturation in ccRCC 786-O cells. NKT2152 disrupted HIF2α/HIF1β complex in a dose-dependent manner, resulting in increased cytosol localization and reduced nucleus localization of HIF2α in 786-O cells. In vitro, NKT2152 specifically suppressed the expression of HIF2α target genes including vascular endothelial growth factor A, cyclin D1, and glucose transporter 1 in 786-O cells. Pharmacodynamic analysis of NKT2152 in the 786-O xenograft tumor model confirmed that NKT2152 potently inhibited HIF2α as demonstrated by the repression of HIF2α-regulated genes at both mRNA and protein levels. Moreover, NKT2152 treatment, administrated twice daily by oral gavage, led to a dose-dependent tumor growth inhibition or tumor regression in both ccRCC cell line-derived (A498 and 786-O) and patient-derived xenograft tumor models. More pronounced tumor growth inhibition was observed when NKT2152 was combined with a VEGFR inhibitor or a CDK4/6 inhibitor in the 786-O xenograft tumor model. In addition to directly promoting tumor progression by regulating gene expression in tumor cells, HIF2α has also been proposed to play an integral role in shaping the tumor microenvironment under hypoxia. Given hypoxia is a hallmark of most solid tumors and is associated with poor survival in a wide range of cancer types, we tested the hypothesis that HIF2α inhibition by NKT2152 may exert broader anti-tumor activity in other solid tumors that lack a VHL gene deficiency. Indeed, we demonstrated here that NKT2152 caused significant tumor growth inhibition in multiple solid tumor xenograft models, including but not limited to hepatocellular carcinoma. NKT2152 had excellent oral bioavailability and achieved high exposures in mouse, rat and dog. These data not only support clinical development of NKT2152 in ccRCC, but also provide exciting opportunities for expanding to other solid tumor indications with tremendous unmet medical needs. Clinical evaluation of NKT2152 for the treatment of ccRCC is ongoing. Citation Format: Jing Lu, Hairong Wei, Wenfeng Sun, Jianlin Geng, Ke Liu, Jessica Liu, Zhihong Liu, Jiping Fu, Yigang He, Keshi Wang, Yan Lou, Zhenhai Gao. NKT2152: A highly potent HIF2α inhibitor and its therapeutic potential in solid tumors beyond ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6330.

Abstract 122: Hypoxia-reoxygenation couples enzyme and cofactor upregulation to quicken prostate cancer androgen biosynthesis and hormone therapy resistance

Abstract Androgen deprivation therapy depletes circulating testosterone and is the mainstay treatment for advanced prostate cancer by suppressing tumor androgen receptor signaling. Despite initial treatment response, castration-resistant prostate cancer nearly always develops and remains driven primarily by the androgen axis. We investigated how changes in oxygenation affect androgen synthesis. Our study indicates that when chronic hypoxia is coupled to reoxygenation, prostate cancer cells efficiently metabolize androgen precursors to produce androgens and activate AR. Hypoxia induces 3βHSD1, the rate-limiting androgen synthesis regulator, and reoxygenation replenishes necessary cofactors. Therefore, hypoxia and reoxygenation are both essential for potent androgen synthesis. The EGLN1/VHL/HIF2α pathway promoted transcription of HSD3B1 by directly binding the 5’ regulatory region of HSD3B1. HIF2α overexpression facilitated prostate cancer progression, which largely depends on 3βHSD1. PT2399, a specific HIF2α inhibitor, prevented prostate cancer cell proliferation. Our study indicates HIF2α is a potential therapeutic target in prostate cancer. Citation Format: Liang Qin, Yoon-Mi Chung, Michael Berk, Bryan Naelitz, Eric Klein, Abhishek A Chakraborty, Nima Sharifi. Hypoxia-reoxygenation couples enzyme and cofactor upregulation to quicken prostate cancer androgen biosynthesis and hormone therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 122.

Phase 2 open-label study of pembrolizumab plus lenvatinib and belzutifan in patients with advanced solid tumors.

TPS4173 Background: There is an unmet medical need in patients (pts) with advanced/metastatic hepatocellular carcinoma (HCC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and biliary tract cancer (BTC), that progresses after standard therapy. The combination of pembrolizumab (pembro; anti–PD-1 antibody) and lenvatinib (len; anti-angiogenic multikinase inhibitor) demonstrated antitumor activity and manageable toxicity in many tumor types. Hypoxia-inducible factor-2 alpha (HIF-2α) acts on multiple pathways including cell survival and proliferation, angiogenesis, genomic instability and treatment resistance. Monotherapy with the HIF-2α inhibitor belzutifan (MK-6482) impairs hypoxic signaling in cancer cells and has antitumor activity in clear cell renal cell carcinoma (RCC), von Hippel-Lindau (VHL) disease associated non-RCC tumors such as pancreatic neuroendocrine tumors. Based on the potential role of hypoxia signaling and HIF-2α in tumorigenesis and tumor progression, the combination of belzutifan with pembro and len may improve antitumor activity further in the selected tumor types. Methods: This phase 2, single-arm, open-label, multicenter study (NCT04976634) is evaluating pembro 400 mg IV Q6W + oral len 20 mg QD (8 or 12 mg QD for body weight < 60 or ≥60 kg, respectively, for pts with HCC) + oral belzutifan 120 mg QD. Eligible pts: ≥18 y old with histologically confirmed unresectable HCC (Child-Pugh A; 1L); previously treated unresectable, metastatic non–MSI-H/mismatch repair deficient CRC (3L+); metastatic PDAC (2L/3L); or locally advanced/metastatic BTC (includes intra/extrahepatic cholangiocarcinoma and gall bladder cancer; 2L+) with progressive disease (PD); measurable disease per RECIST v1.1 (verified by blinded independent central review [BICR]); ECOG PS ≤1 and archival/new tumor sample for biomarker analysis. All pts should be naïve to prior immune checkpoint inhibitor therapy, len or any HIF-2α inhibitor. The safety lead-in phase is based on the modified toxicity probability interval design with a target dose-limiting toxicity (DLT) rate of 30% and DLT-evaluable period of 21 days (3 wk/1 cycle) in ≤10 HCC pts and ≤15 pts with CRC, PDAC and BTC pooled for each dose level. The study will enroll 120 pts (30/tumor type) to receive triplet therapy for up to 2 y (for pembro) or until PD or discontinuation criteria are met. Enrollment may be expanded by an additional 70 pts/tumor type at the chosen dose level after safety and efficacy review with ≥6 mo follow-up. Primary endpoints are safety (DLTs [safety lead-in], AEs, and treatment discontinuation due to AEs) and ORR per RECIST v1.1 by BICR. Secondary endpoints include DOR, disease control rate and PFS per RECIST v1.1 (and mRECIST for HCC) by BICR, and OS. Efficacy analyses will report binomial proportion or Kaplan-Meier estimates with 95% CIs. Safety analyses will be descriptive. Enrollment began August 2021. Clinical trial information: NCT04976634.

LITESPARK-022: A phase 3 study of pembrolizumab + belzutifan as adjuvant treatment of clear cell renal cell carcinoma (ccRCC).

TPS4602 Background: Patients with locally advanced renal cell carcinoma (RCC) may experience recurrence after surgery. In the phase 3 KEYNOTE-564 (NCT03142334) trial, adjuvant pembrolizumab demonstrated significant improvement in disease-free survival (DFS) in ccRCC, leading to FDA approval of pembrolizumab (November 17, 2021) for adjuvant treatment of patients with RCC at intermediate-high or high risk for recurrence following nephrectomy or following nephrectomy and resection of metastatic lesions. Despite benefit of pembrolizumab, there is an unmet need for more effective adjuvant treatment for patients at risk for recurrence after definitive surgery. The HIF-2α inhibitor belzutifan (MK-6482) has shown activity and good tolerability in patients with advanced ccRCC and von Hippel-Lindau (VHL) disease–associated RCC. Combining belzutifan with pembrolizumab may be a therapeutic option as adjuvant treatment of ccRCC. This global, multicenter, double-blind, randomized, phase 3 study LITESPARK-022 (NCT05239728) is designed to compare the efficacy and safety of belzutifan + pembrolizumab with that of placebo + pembrolizumab as adjuvant treatment of ccRCC after nephrectomy. Methods: Approximately 1600 patients with histologically or cytologically confirmed RCC (intermediate-high [pT2, grade 4 or sarcomatoid, N0, M0 or pT3, any grade, N0, M0], high [pT4, any grade, N0, M0 or pT, any stage/grade, N+, M0] or M1 NED [patients who present with the primary kidney tumor and solid, isolated, soft tissue metastases that can be resected at the time of nephrectomy or ≤2 years from nephrectomy) with a clear cell component and had not previously received systemic therapy will be enrolled. Patients must have undergone nephrectomy and/or metastasectomy ≤12 weeks before randomization and be tumor free, confirmed by CT/MRI. Stratification factors are tumor grade (1 or 2 vs 3 or 4) and risk type (intermediate-high vs high vs M1 NED). Patients will be randomly assigned 1:1 to receive belzutifan 120 mg orally once daily + pembrolizumab 400 mg IV every 6 weeks (Q6W) or oral placebo + pembrolizumab 400 mg IV Q6W. Pembrolizumab treatment will be administered for up to 9 doses (̃1 year); belzutifan and placebo may be continued for a maximum of 54 weeks. Disease recurrence will be evaluated radiologically Q12W from randomization through year 2, Q16W in years 3-5, and Q24W in years 6 and beyond. Adverse events (AEs) will be monitored by NCI CTCAE v5.0 through 30 days (90 days for serious AEs) after cessation of study drug. The primary end point is DFS, as assessed by investigator, defined as time from randomization to first documented event of local recurrence, occurrence of distant kidney cancer metastasis, or death from any cause, whichever occurs first. The key secondary end point is overall survival. Other secondary end points are safety, disease recurrence–specific survival and patient-reported outcomes. Clinical trial information: NCT05239728.

Phase 1 LITESPARK-001 (MK-6482-001) study of belzutifan in advanced solid tumors: Update of the clear cell renal cell carcinoma (ccRCC) cohort with more than 3 years of total follow-up.

4509 Background: Hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in RCC. Antitumor activity of the HIF-2α inhibitor belzutifan has been observed in RCC and is approved for treatment in patients (pts) with VHL disease who require therapy for associated RCC, CNS hemangioblastomas, or pNETs not requiring immediate surgery. Previous data from the phase 1 LITESPARK-001 trial (NCT02974738) designed to evaluate belzutifan in heavily pretreated RCC showed durable antitumor activity and an acceptable safety profile. After more than 3 years of follow-up for pts with ccRCC still receiving treatment, updated data are presented. Methods: Pts enrolled in the ccRCC cohort were previously treated with ≥1 therapy, had RECIST-measurable disease, ECOG PS score of 0 or 1, adequate organ function, and life expectancy of ≥6 months. Pts received oral belzutifan 120 mg once daily. The primary end point was safety. Secondary end points were ORR, DCR (CR + PR + SD), PFS, and DOR per RECIST v1.1 by investigator. The data cutoff date was July 15, 2021. Results: Of 55 pts enrolled in the ccRCC cohort, 9 (16%) remain on treatment as of the data cutoff date of July 15, 2021; the primary reason for discontinuation was progressive disease (n = 34; 62%). Pts received a median of 3 prior therapies (range, 1-9); 39 (71%) received prior VEGF and immunotherapy. Pts were followed while on treatment and for 30 days after the last dose for a median of 41.2 months (range, 38.2-47.7). Twenty-two pts (40%) experienced grade 3 TRAEs. The most common (≥10%) grade 3 TRAEs were anemia (n = 13; 24%) and hypoxia (n = 7; 13%). There were no grade 4 or 5 TRAEs. ORR was 25%, with 1 confirmed CR (2%) and 13 PRs (24%); DCR was 80%. Median DOR was not reached (range, 3.1+ to 37.9+ months); 8 of 14 responding pts (57%) remain in response as of the data cutoff date. Per IMDC risk, 4 of 13 pts with favorable risk achieved response (ORR = 31%; all PRs) and 10 of 42 pts with intermediate/poor risk achieved response (ORR = 24%; 1 CR, 9 PRs). DCR was 92% for pts with favorable risk and 76% for pts with intermediate/poor risk. For pts who received prior VEGF and immunotherapy, 8 of 39 pts achieved response (ORR = 21%; 1 CR; 7 PR); DCR was 74%. For the 16 pts who did not receive prior VEGF/immunotherapy, 6 achieved response (ORR = 38%; all PRs); DCR was 94%. Median PFS for the total cohort was 14.5 months (95% CI, 7.3-22.1); PFS rate at 156 weeks (̃36 months) was 34%. Conclusions: As seen after a median follow-up of > 3 years for pts still receiving treatment, belzutifan monotherapy continued to show a high rate of disease control and durable responses in previously treated pts with advanced ccRCC. Belzutifan exhibited a favorable safety profile, and no new safety signals were observed. In several phase 3 studies, belzutifan is being evaluated as monotherapy and combined therapy for ccRCC. Clinical trial information: NCT02974738.

Abstract B018: Targeting Notch4 and HIF2α signaling reduces the ability of cells to survive chemotherapy by undergoing the polyaneuploid transition

Abstract Giant cancer cells have been ignored for 200 years, but recently coupled to a role in metastasis and therapeutic resistance. Some cancer cells appear to survive environmental stress, such as chemotherapy or radiotherapy, via a polyaneuploid transition. This results in a PolyAneuploid Cancer Cell (PACC) state with up to 10-fold larger cell sizes. It is proposed that PACCs utilize a conserved evolutionarily or developmental program for polyploidization, which is commonly observed in other organisms and tissues. After a stress-induced formation, the PACC state first undergoes a period of quiescence and eventually undergoes reductive cell division to give rise to proliferative progeny. We hypothesize that PACCs play a role in tumor recurrence and therapy resistance by surviving the initial treatment and subsequently reseeding the tumor via pseudohypoxic HIF signaling. Dosing with LD50 cisplatin for 72h results in the induction of the PACC state in cancer cell lines from breast (HCC1806), renal (786-0), colon (HCT116), and lung (U1690) tumors. Cells in the PACC state had 3-fold larger nuclei after 3 days of treatment and 6-fold larger nuclei after 10 days. Transcriptomic data showed an overall decrease in gene expression across all 4 cell lines. However, there was an increased expression of HIF2 (2.7-fold), Notch4 (2-fold), Plekhs1 (2.3-fold) in PACCs relative to untreated control. Moreover, there was an increase in the expression of replication dependent histones. Overall, ATACseq indicated a reduction in open chromatin in PACCs, as measured by reduced levels of histone 4 and histone 4 modifications (H4k5ac, h4k16ac, H4R3me2). However, H3k27me3 was increased 20% and AP1 binding sites were enriched in open chromatin regions. HIF2 and AP1 cooperate to regulate gene expression and confer therapy resistance. We hypothesize that AP1 is activated in response to treatment and stabilizes HIF2 under oxygenated conditions. HIFs regulate regulators of the cell cycle to induce polyploidy and downregulate β-catenin, a known inducer of cytokinesis. Small molecule inhibition of Notch and HIF-2α reduced PACC formation by 50% and 65% respectively at day 3 and 60% and 75% at day 10. The surviving PACCs were smaller than those untreated by Notch and HIF-2α inhibitors. Large metabolic changes must be undertaken to generate and sustain the larger cellular mass accompanied by polyploidy. HIF signaling metabolically rewires cells to a glycolytic phenotype that provides a carbon source for increased de novo generation of nucleotides, lipids, and proteins. Indicative of this rewiring is that PACCs have increased expression of Hexokinase (2.5-fold), CPT1 (2.3-fold), and GP6D (2-fold). In conclusion, tumor cells that survive chemotherapy are polyaneuploid, larger, and in an altered metabolic state. This process appears driven by Notch4 and HIF-2α signaling. Combining conventional chemotherapy with Notch or HIF2α inhibitors presents an opportunity to reduce tumor recurrence by inhibiting a survival mechanism. Citation Format: Christopher P. Carroll, Emma Hammarlund, Kenneth J. Pienta, Sarah R. Amend. Targeting Notch4 and HIF2α signaling reduces the ability of cells to survive chemotherapy by undergoing the polyaneuploid transition [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B018.

HIF-2α-targeted interventional chemoembolization multifunctional microspheres for effective elimination of hepatocellular carcinoma

Transcatheter arterial chemoembolization (TACE) is widely used for the treatment of advanced hepatocellular carcinoma (HCC). However, the long-term hypoxic microenvironment caused by TACE seriously affects the therapeutic effect of TACE. HIF-2α plays a crucial role on the chronic hypoxia process, which might be an ideal target for TACE therapy. Herein, a multifunctional polyvinyl alcohol (PVA)/hyaluronic acid (HA)-based microsphere (PT/DOX-MS) co-loaded with doxorubicin (DOX) and PT-2385, an effective HIF-2α inhibitor, was developed for enhanced TACE treatment efficacy. In vitro and in vivo studies revealed that PT/DOX-MS had a superior ability to treat HCC by blocking the tumor cells in G2/M phase, prompting cell apoptosis, and inhibiting tumor angiogenesis. The antitumor mechanisms of PT/DOX-MS were possibly due to that the introduction of PT-2385 could effectively inhibit the expression level of HIF-2α in hypoxic HCC cells, thereby down-regulating the expression levels of Cyclin D1, VEGF and TGF-α. In addition, the combination of DOX and PT-2385 could jointly inhibit VEGF expression, which was another reason accounting for the combined anti-cancer effect of PT/DOX-MS. Overall, our study demonstrated that PT/DOX-MS is a promising embolic agent for enhanced HCC treatment via the combined effect of hypoxia microenvironment improvement, chemotherapy, and embolization.