HIF-2α Inhibition Research Articles

56 Clinical Pharmacokinetic/Pharmacodynamic (PK/PD) Relationship Confirms Best-in-class Potential of Casdatifan (AB521), a Small Molecule Inhibitor of HIF-2α Being Developed in Renal Cancer

Abstract Background Casdatifan, an orally bioavailable small molecule inhibitor of HIF-2α, potently inhibits transcription of HIF-2α-dependent genes in cell lines and preclinical species. The objective of this analysis was to develop an understanding of the relationship between clinical dose, casdatifan PK, erythropoietin (EPO), a PD biomarker for peripheral (non-tumor) HIF-2α inhibition, and hemoglobin and to use this information to guide dose selection in future clinical trials. Methods Casdatifan plasma concentrations, serum EPO concentration, and hemoglobin data were obtained from 79 healthy participants in two Phase 1 studies, ARC-14 (NCT05117554) and ARC-28 (NCT05999513), and from 71 patients with clear cell renal cell carcinoma (ccRCC) and other solid tumors in an ongoing Phase 1 study, ARC-20 (NCT05536141). The available PK and PD data were collected following single oral doses of casdatifan ranging from 3 mg to 100 mg in healthy participants, and multiple oral doses of casdatifan ranging from 15 mg to 150 mg once daily (QD) in healthy participants and cancer patients. Serial PK, EPO, and Hb data were gathered in all study participants pre-dose till end of treatment. The population PKPD model was developed using mixed effects methodology with NONMEM software to relate dose, PK, and PD (EPO and Hb) data. Results Casdatifan showed dose-proportional increases in plasma exposure over the 3-150 mg dose range after single and multiple doses. Casdatifan PK was also invariant over time in patients with an approximately 2.0-fold accumulation at steady-state compared to first dose. The mean terminal half-life of casdatifan was approximately 24 h. The PK was similar in healthy participants and cancer patients. Dose-dependent reduction in EPO was observed after single and multiple doses in healthy participants and patients. A two-compartment model with first-order absorption adequately described the casdatifan plasma PK across the dose range tested. The effect of casdatifan plasma concentrations on EPO production rate was modeled using an inhibitory function. Analysis of the casdatifan dose-PD (EPO suppression) relationship indicated that casdatifan 20 mg once daily provided a similar level of EPO suppression in patients as belzutifan 120 mg daily (benchmark peripheral PD). Furthermore, due to the dose-proportional PK of casdatifan, the selected dose (100 mg daily) for further development results in plasma levels approximately 5 times higher than those associated with the benchmark peripheral PD. Conclusions Casdatifan exhibits dose-linear and time-invariant PK in the dose range 15-150 mg. Dose-dependent reduction in EPO levels, consistent with the mechanism of action of HIF-2α inhibition, was seen after casdatifan administration. A PKPD analysis of available data indicated that 20 mg daily dose of casdatifan would result in similar EPO effect as the registered dose of belzutifan. Available PKPD data and analyses indicated best-in-class properties of casdatifan.

HIF-2α inhibition disrupts leukemia stem cell metabolism and impairs vascular microenvironment to enhance chronic myeloid leukemia treatment

Leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) contribute to treatment resistance and disease recurrence. Metabolism regulates LSCs, but the mechanisms remain elusive. Here, we show that hypoxia-inducible factor 2α (HIF-2α) is highly expressed in LSCs in mouse and human CML and increases after tyrosine kinase inhibitor (TKI) treatment. Deletion of HIF-2α suppresses disease progression, reduces LSC numbers, and enhances the efficacy of TKI treatment in BCL-ABL-induced CML mice. Mechanistically, HIF-2α deletion reshapes the metabolic profile of LSCs, leading to increased production of reactive oxygen species (ROS) and apoptosis in CML. Moreover, HIF-2α deletion decreases vascular endothelial growth factor (VEGF) expression, thereby suppressing neovascularization in the bone marrow of CML mice. Furthermore, pharmaceutical inhibition of HIF-2α by PT2399 attenuates disease progression and improves the efficacy of TKI treatment in both mouse and human CML. Overall, our findings highlight the role of HIF-2α in controlling the metabolic state and vascular niche remodeling in CML, suggesting it is a potential therapeutic target to enhance TKI therapy.

Enhancing immunogenic responses through CDK4/6 and HIF2α inhibition in Merkel cell carcinoma

Approximately 50% of Merkel cell carcinoma (MCC) patients facing this highly aggressive skin cancer initially respond positively to PD-1-based immunotherapy. Nevertheless, the recurrence of MCC post-immunotherapy emphasizes the pressing need for more effective treatments. Recent research has highlighted Cyclin-dependent kinases 4 and 6 (CDK4/6) as pivotal cell cycle regulators gaining prominence in cancer studies. This study reveals that the CDK4/6 inhibitor, palbociclib can enhance PD-L1 gene transcription and surface expression in MCC cells by activating HIF2α. Inhibiting HIF2α with TC-S7009 effectively counteracts palbociclib-induced PD-L1 transcription and significantly intensifies cell death in MCC. Simultaneously, co-targeting CDK4/6 and HIF2α boosts ROS levels while suppressing SLC7A11, a key regulator of cellular redox balance, promoting ferroptosis- a form of immunogenic cell death linked to iron. Considering the rising importance of immunogenic cell death in immunotherapy, this strategy holds promise for improving future MCC treatments, markedly increasing immunogenic cell death across various MCC cell lines, thus advancing cancer immunotherapy.

The transcription factor HIF2α partakes in the differentiation block of acute myeloid leukemia

One of the defining features of acute myeloid leukemia (AML) is an arrest of myeloid differentiation whose molecular determinants are still poorly defined. Pharmacological removal of the differentiation block contributes to the cure of acute promyelocytic leukemia (APL) in the absence of cytotoxic chemotherapy, but this approach has not yet been translated to non‐APL AMLs. Here, by investigating the function of hypoxia‐inducible transcription factors HIF1α and HIF2α, we found that both genes exert oncogenic functions in AML and that HIF2α is a novel regulator of the AML differentiation block. Mechanistically, we found that HIF2α promotes the expression of transcriptional repressors that have been implicated in suppressing AML myeloid differentiation programs. Importantly, we positioned HIF2α under direct transcriptional control by the prodifferentiation agent all‐trans retinoic acid (ATRA) and demonstrated that HIF2α blockade cooperates with ATRA to trigger AML cell differentiation. In conclusion, we propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and leukemia debulking.

LITESPARK-024: A randomized phase 1/2 study of belzutifan with or without palbociclib for previously treated advanced renal cell carcinoma

Abstract Background Immunotherapy is a standard-of-care first-line treatment for advanced clear cell renal cell carcinoma (ccRCC). However, many patients will develop resistance to first-line therapy, and effective second- and later-line treatment options are therefore needed. The von Hippel-Lindau (VHL) gene is inactivated in approximately 90% of patients with RCC and results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. Belzutifan, a first-in-class HIF-2α inhibitor, has demonstrated antitumor activity with manageable safety in previously treated patients with advanced ccRCC. The cyclin-dependent kinase (CDK) pathway is also associated with poor clinical outcomes in ccRCC. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. CDK 4/6 inhibition has shown synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α–dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods LITESPARK-024 is a 2-part, open-label, multicenter, phase 1/2 randomized study (NCT05468697). Part 1 is intended to establish the recommended phase 2 dose (RP2D) of belzutifan plus palbociclib using a modified toxicity probability interval design. After the RP2D is established, part 2 will directly compare the safety and efficacy of belzutifan monotherapy with belzutifan + palbociclib in patients with advanced ccRCC. In both parts, patients with measurable disease per RECIST v1.1, a Karnofsky Performance Status score of ≥70%, and histologically confirmed unresectable stage IV RCC with a clear cell component and disease progressing on or after receiving at least 2 systemic treatments (both an anti–PD-1/L1 monoclonal antibody and a VEGF receptor–targeted tyrosine kinase inhibitor, in sequence or in combination) will be enrolled. Up to 30 patients will be enrolled into 3 dose groups in part 1 and will receive belzutifan 120 mg once daily plus palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 patients will be randomly assigned 2:1 to receive belzutifan 120 mg once daily plus palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Patients will be stratified by International metastatic RCC Database Consortium risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events and to determine the RP2D of belzutifan plus palbociclib. The primary end point for part 2 is objective response rate per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are clinical benefit rate, duration of response, and progression-free survival per RECIST v1.1 by investigator assessment; overall survival, and safety and tolerability. Enrollment began in July 2022. ©2023 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2023 ASCO Genitourinary Cancers Symposium. All rights reserved. CDMRP DOD Funding: no

The identification of a novel orally available ferroptosis inducer for the treatment of clear cell renal carcinoma

Abstract Background Background: Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene resulting in constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. HIF-2α is a central driver of kidney cancer progression and is a well validated therapeutic target for ccRCC. Additionally, VHL loss and HIF activation promote lipid accumulation that increases the sensitivity of ccRCC cells to ferroptosis, a form of cell death characterized by iron dependent lipid peroxidation. However, classical inducers of ferroptosis, which induce ferroptosis in vitro, show limited activity in vivo due to possible redundancies in the extracellular space, suggesting a need for novel molecular targets for the induction of ferroptosis in vivo for therapeutic benefit. Methods Method: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α and performed additional validation and structure activity relationship studies to identify compounds with improved potency of HIF-2α inhibition. The molecular target of these inhibitors was determined by mass spectrometry using the Drug Affinity Responsive Target Stability (DARTS) assay, and target validation was confirmed using siRNA knockdown and NMR. In vivo anti-tumor efficacy was verified using syngeneic RENCA cells injected into Balb/C mice. Exposure to KD061 was verified using LC-MS/MS detection of KD061 in the plasma and tumors of treated mice. Results Results: We identified a novel molecule, KD061, which decreases levels of HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. Cell death induced by KD061 was decreased by re-expression of VHL, suggesting synthetic lethality in the context of VHL loss. ISCA2 inhibition also decreased HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreased HIF-1α translation through unknown mechanisms. Treatment of tumor-bearing RENCA-Balb/C mice with KD061 significantly reduced tumor growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Treatment with KD061 was well tolerated at the therapeutic dose with no detectable effects on blood chemistry and blood counts. Conclusions Conclusions: Here, we describe the first orally available inducer of ferroptosis with anti-tumor efficacy in vivo. The targeting of ISCA2 using KD061 is a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells. CDMRP DOD Funding: yes

Abstract A002: Targeting hypoxia inducible factor (HIF)-2α with AB521, a novel and potent small molecule HIF-2α inhibitor, for the treatment of clear cell renal cell carcinoma

Abstract Cells in the solid tumor microenvironment are frequently exposed to hypoxic or pseudohypoxic conditions, necessitating molecular adaptations such as induction of transcriptional programs for survival. HIF-2α is a transcriptional regulator of hypoxia-induced genes that is regulated post-translationally in an oxygen-dependent manner via hydroxylation by prolyl hydroxylase enzymes - a modification required for recognition by pVHL for subsequent degradation. Therefore, under hypoxic conditions or in the case of pVHL inactivation (pseudohypoxia), HIF-2α is stabilized and translocates to the nucleus where it forms a heterodimer with ARNT to initiate transcription of its target genes. HIF-2α has been shown to be an oncogenic driver in clear cell renal cell carcinoma (ccRCC) where VHL mutations occur frequently, resulting in a pseudohypoxic state and stable HIF-2α expression. There is both preclinical and clinical evidence in ccRCC demonstrating that inhibiting HIF-2α is a valid therapeutic approach in blocking tumor progression. AB521 is a clinical-stage novel and potent small-molecule HIF-2α inhibitor. In vitro, AB521 effectively inhibits transcription of pro-tumorigenic HIF-2α target genes in multiple human cell types relevant to the tumor microenvironment, including cancer cells, primary endothelial cells, and pro-tumorigenic M2-polarized macrophages. AB521 also blocks HIF-2α-mediated gene transcription in vivo, as detected using both intratumoral and peripheral markers, an activity that is accompanied by a marked reduction in tumor burden in two VHL-mutant ccRCC xenograft models. Supporting a potential combinatorial treatment approach for HIF-2α inhibition in ccRCC, AB521 combines favorably with standard-of-care tyrosine kinase inhibitor cabozantinib in mouse models, further enhancing tumor efficacy in VHL-mutant ccRCC compared to either single agent alone. In healthy human volunteers, AB521 exhibits favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties including a half-life of 18-24 hours, supportive of once-daily dosing, dose-dependent reductions in serum erythropoietin (EPO) following a single dose of 10 to 100 mg, and sustained EPO reduction after multiple daily dosing. In summary, AB521 is a potent and selective HIF-2α inhibitor that has been thoroughly characterized in a suite of in vitro assays, reduces tumor burden in ccRCC xenografts both as a single agent and in combination with cabozantinib, and exhibits favorable PK/PD properties in human healthy volunteers. Phase I clinical evaluation of AB521 in subjects with ccRCC and other solid tumors has been initiated and is currently ongoing (NCT05536141). Citation Format: Dana Piovesan, Soonweng Cho, Kenneth V. Lawson, Kai H. Liao, Paul G. Foster, Tzuling Cheng, Matthew J. Walters, Kelsey E. Sivick Gauthier. Targeting hypoxia inducible factor (HIF)-2α with AB521, a novel and potent small molecule HIF-2α inhibitor, for the treatment of clear cell renal cell carcinoma [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 A002.

Abstract B033: Targeting acquired resistance to HIF2α inhibition in kidney cancer

Abstract Recently, a HIF-2α inhibitor MK-6482 was developed and approved by FDA to treat certain patients with VHL disease-associated RCC. However, in preclinical studies, both intrinsic and adaptive resistance to this drug was reported, which highlights the necessity of uncovering the mechanisms underlying drug resistance and developing alternative treatments for HIF-2α inhibitor-resistant patients. To this end, we established several 786-O cell lines that are resistant to PT2399 (analog of MK-6482) treatment. To characterize the differences between sensitive and resistant cell lines, we performed RNA sequencing, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and CUT&RUN sequencing experiments. Through integrative analyses of all sequencing data, we identified a series of candidate genes which might contribute to the acquirement of drug resistance for further validation. We identified 2'-5'-oligoadenylate synthetase 2 (OAS2) as one of the candidate genes exhibiting both significant upregulation in RNA level and dramatically increased chromatin accessibility in the promoter region in resistant lines comparing to sensitive lines. We further validated the upregulation of OAS2 mRNA level as well as protein level in all resistant clones by RT-qPCR and western blot, respectively. OAS2 belongs to 2-5A synthetase family, which is involved in the activation of latent RNase L and degradation of viral RNA during innate immune response to viral infection. Interestingly, the other 3 members in this protein family, OAS1, OAS3 and OASL, are also significantly upregulated at RNA level in resistant cell lines, although the upregulation of chromatin accessibility is modest. Furthermore, OAS2, along with the other 3 family members, all show increased expression levels in ccRCC tumor tissues comparing to normal tissues. Therefore, we hypothesize that OAS2 protein promotes ccRCC cell proliferation under HIF-2α inhibitor treatment, thus contributing to the adaptive resistance to HIF-2α inhibitor. We will test this hypothesis through a series of loss-of-function and gain-of-function experiments, while continuing to investigate other candidate genes. Citation Format: Fangzhou Zhao, Jeremy Simon, Qing Zhang. Targeting acquired resistance to HIF2α inhibition in kidney cancer [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 B033.

Discovery of Cycloalkyl[c]thiophenes as Novel Scaffolds for Hypoxia-Inducible Factor-2α Inhibitors.

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy-aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology.

LITESPARK-024: Randomized phase 1/2 study of belzutifan with or without palbociclib for treatment of advanced renal cell carcinoma (RCC).

TPS4603 Background: Immunotherapy is a standard-of-care first-line treatment for advanced clear cell RCC (ccRCC). However, many patients (pts) will develop resistance to first-line therapy, and effective second- and later-line treatment options are therefore needed. The von Hippel-Lindau ( VHL) gene is inactivated in approximately 90% of ccRCC cases and results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. Belzutifan, a first-in-class HIF-2α inhibitor, has demonstrated antitumor activity with manageable safety in previously treated pts with advanced ccRCC. The cyclin-dependent kinase (CDK) pathway is also associated with poor clinical outcomes in ccRCC. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. CDK 4/6 inhibition has shown synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α–dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: LITESPARK-024 is a 2-part, open-label, multicenter, phase 1/2 randomized study (NCT05468697). Part 1 is intended to establish the recommended phase 2 dose (RP2D) of belzutifan + palbociclib using a modified toxicity probability interval design. After the RP2D is established, part 2 will directly compare the safety and efficacy of belzutifan monotherapy with belzutifan + palbociclib in pts with advanced ccRCC. In both parts, pts with measurable disease per RECIST v1.1, a KPS score of ≥70%, and histologically confirmed unresectable stage IV RCC with a clear cell component with disease progressing on or after receiving at least 2 systemic treatments (both an anti–PD-1/L1 monoclonal antibody and a VEGF receptor–targeted tyrosine kinase inhibitor, in sequence or in combination) will be enrolled. Up to 30 pts will be enrolled into 3 dose groups in part 1 and will receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events and to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are CBR, DOR, and PFS per RECIST v1.1 by investigator assessment, OS, and safety and tolerability. Enrollment began in July 2022. Clinical trial information: NCT05468697 .

Abstract CT120: The randomized phase 1/2 LITESPARK-024 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma (RCC)

Abstract Background: First-line treatment with immunotherapy alone or in combination with antiangiogenic agents is a standard of care for advanced RCC. Many patients (pts) develop resistance to first-line treatment and effective second-line and beyond options are needed. The von Hippel-Lindau (VHL) gene is inactivated in approximately 90% of RCC cases, which results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. The first-in-class HIF-2α inhibitor belzutifan has demonstrated promising antitumor activity with manageable safety in previously treated pts with advanced RCC. The cyclin-dependent kinase (CDK) pathway has also been implicated in RCC and is associated with poor clinical outcomes. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. The addition of CDK 4/6 inhibition had synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α-dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: The two-part, open-label, multicenter, phase 1/2 randomized LITESPARK-024 study (NCT05468697) is intended to establish the recommended phase 2 dose (RP2D) of belzutifan + palbociclib in combination with a modified toxicity probability interval design (Part 1), followed by a direct comparison of belzutifan monotherapy to the combination with respect to safety and efficacy in pts with advanced RCC (Part 2). Pts with histologically confirmed unresectable stage IV RCC with a clear cell component, whose disease progressed on or after having received at least 2 systemic treatments (both an anti-PD-1/PD-L1 monoclonal antibody and a VEGF receptor-targeted tyrosine kinase inhibitor, in sequence or in combination), have measurable disease per RECIST v1.1 by blinded independent central review, and have KPS score of ≥70% will be enrolled. Up to 30 pts will be enrolled into 3 dose groups in Part 1 and will receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are CBR, DOR, PFS, OS, and safety and tolerability. This abstract was accepted and previously presented at the 2023 ASCO Genitourinary Cancers Symposium. All rights reserved. Citation Format: David F. McDermott, Avivit Peer, Neeraj Agarwal, Michael B. Atkins, Jerry Cornell, Rodolfo F. Perini, Kenneth F. Grossmann, Howard Gurney. The randomized phase 1/2 LITESPARK-024 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT120.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

LITESPARK-024: A randomized phase 1/2 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma.

TPS747 Background: The combination of immunotherapy with antiangiogenic agents is a well-established first-line treatment option for patients (pts) with advanced renal cell carcinoma (RCC), but many pts develop resistance, and effective second- or subsequent-line options are needed. The von Hippel-Lindau ( VHL) gene is inactivated in approximately 90% of RCC cases, which results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α) signaling. HIF-2α is involved in angiogenesis, tumor growth, proliferation, and metastasis, and is a key oncogenic driver in RCC. The HIF-2α inhibitor belzutifan has demonstrated promising antitumor activity with manageable safety in pts with heavily pretreated RCC. The cyclin-dependent kinase (CDK) pathway is altered in several cancer types, including RCC, and is associated with poor clinical outcomes. The CDK 4/6 inhibitor palbociclib inhibited cell growth in RCC cell lines, and the antiproliferative effects of CDK 4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α–dependent VHL -/- clear cell RCC cell lines. We hypothesized palbociclib could potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: LITESPARK-024 (NCT05468697) is an open-label, multicenter, phase 1/2 randomized study of belzutifan + palbociclib versus belzutifan monotherapy in pts with advanced RCC. Pts must have histologically confirmed unresectable stage IV RCC with a clear cell component, received at least 2 prior systemic regimens (both an anti–PD-1/PD-L1 monoclonal antibody and a VEGF receptor–targeted TKI, in sequence or in combination), have measurable disease per RECIST v1.1 by BICR, have KPS score of ≥70%, and have radiographic disease progression on or after the most recent regimen per investigator. Part 1 will evaluate the safety of belzutifan + palbociclib and determine the recommended phase 2 dose (RP2D) for the combination using a modified toxicity probability interval design. Part 2 will evaluate the safety and efficacy of belzutifan + palbociclib versus belzutifan alone. In part 1, ≤30 pts will be enrolled into 3 dose groups and receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are clinical benefit rate, DOR, PFS, OS, and safety and tolerability. Clinical trial information: NCT05468697 .

Hypoxia-Inducible Factor 2 Alpha (HIF2α) Inhibitors: Targeting Genetically Driven Tumor Hypoxia.

Tumors driven by deficiency of the VHL gene product, which is involved in degradation of the hypoxia-inducible factor subunit 2 alpha (HIF2α), are natural candidates for targeted inhibition of this pathway. Belzutifan, a highly specific and well-tolerated HIF2α inhibitor, recently received FDA approval for the treatment of nonmetastatic renal cell carcinomas, pancreatic neuroendocrine tumors, and central nervous system hemangioblastomas from patients with von Hippel-Lindau disease, who carry VHL germline mutations. Such approval is a milestone in oncology; however, the full potential, and limitations, of HIF2α inhibition in the clinic are just starting to be explored. Here we briefly recapitulate the molecular rationale for HIF2α blockade in tumors and review available preclinical and clinical data, elaborating on mutations that might be particularly sensitive to this approach. We also outline some emerging mechanisms of intrinsic and acquired resistance to HIF2α inhibitors, including acquired mutations of the gatekeeper pocket of HIF2α and its interacting partner ARNT. Lastly, we propose that the high efficacy of belzutifan observed in tumors with genetically driven hypoxia caused by VHL mutations suggests that a focus on other mutations that similarly lead to HIF2α stabilization, such as those occurring in neuroendocrine tumors with disruptions in the tricarboxylic acid cycle (SDHA/B/C/D, FH, MDH2, IDH2), HIF hydroxylases (EGLN/PHDs), and the HIF2α-encoding gene, EPAS1, are warranted.

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.

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

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. 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. 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. 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.

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.

Hypoxia-Inducible Factor-2 Alpha Regulates the Migration of Fibroblast-like Synoviocytes via Oxidative Stress-Induced CD70 Expression in Patients with Rheumatoid Arthritis.

This study aimed to examine the role of CD70, which is highly expressed on fibroblast-like synoviocytes (FLS), in rheumatoid arthritis (RA) patients. FLS isolated from RA (n = 14) and osteoarthritis (OA, n = 4) patients were stimulated with recombinant interleukin-17 (IL-17; 5 ng/mL) and tumor necrosis factor alpha (TNF-α; 5 ng/mL) for 24 h. Expression of CD70, CD27/soluble CD27 (sCD27), and hypoxia-inducible factor-2 alpha (HIF-2α) was analyzed by RT-qPCR, flow cytometry, and ELISA assays, respectively. Reactive oxygen species (ROS) expression and cell migration were also examined. The HIF-2α inhibitor PT-2385 and CD70 inhibitor BU69 were used to specifically suppress these pathways. Stimulation with IL-17 and TNF-α significantly induced CD70 expression in RA FLS. Although the synovial fluids from patients with RA contained high levels of sCD27, surface expression of CD27, a ligand of CD70, was rarely detected in RA FLS. Cytokine-induced CD70 expression was significantly decreased following antioxidant treatment. Following HIF-2α inhibition, RA FLS had decreased expression of CD70 and ROS levels. Migration of RA FLS was also inhibited by inhibition of CD70 or HIF-2α. The surface expression of CD70 is regulated by HIF-2α and ROS levels and is a key contributor to cytokine-enhanced migration in RA FLS.

Therapeutic Effects of Inhibition of Sphingosine-1-Phosphate Signaling in HIF-2α Inhibitor-Resistant Clear Cell Renal Cell Carcinoma.

Simple SummaryClear cell renal cell carcinoma is a common malignancy that represents 80% of all kidney tumors. Most tumors harbor an inactivation of the VHL gene, leading to the accumulation of HIF-1α and HIF-2α. Promising clinical results of specific HIF-2α inhibitors will soon lead to new treatment options for advanced cancer patients, although primary and acquired resistance to these agents are common. We here show that Acriflavine, which inhibits both HIF-1α and HIF-2α, and Fingolimod (FTY720), which inhibits sphingosine-1-phosphate signaling, show therapeutic activities in several experimental ccRCC models that are resistant to HIF-2α-inhibitor treatment. Additionally, we show that specific HIF-2α-inhibition suppresses the tumor immune microenvironment, which will be important to consider for future combination studies with immune checkpoint inhibitors.Specific inhibitors of HIF-2α have recently been approved for the treatment of ccRCC in VHL disease patients and have shown encouraging results in clinical trials for metastatic sporadic ccRCC. However, not all patients respond to therapy and pre-clinical and clinical studies indicate that intrinsic as well as acquired resistance mechanisms to HIF-2α inhibitors are likely to represent upcoming clinical challenges. It would be desirable to have additional therapeutic options for the treatment of HIF-2α inhibitor resistant ccRCCs. Here we investigated the effects on tumor growth and on the tumor microenvironment of three different direct and indirect HIF-α inhibitors, namely the HIF-2α-specific inhibitor PT2399, the dual HIF-1α/HIF-2α inhibitor Acriflavine, and the S1P signaling pathway inhibitor FTY720, in the autochthonous Vhl/Trp53/Rb1 mutant ccRCC mouse model and validated these findings in human ccRCC cell culture models. We show that FTY720 and Acriflavine exhibit therapeutic activity in several different settings of HIF-2α inhibitor resistance. We also identify that HIF-2α inhibition strongly suppresses T cell activation in ccRCC. These findings suggest prioritization of sphingosine pathway inhibitors for clinical testing in ccRCC patients and also suggest that HIF-2α inhibitors may inhibit anti-tumor immunity and might therefore be contraindicated for combination therapies with immune checkpoint inhibitors.

717TiP Randomized, open-label, 3-arm phase III study comparing MK-1308A + lenvatinib and pembrolizumab (pembro) + belzutifan + lenvatinib versus pembro + lenvatinib as first-line (1L) treatment for advanced clear cell renal cell carcinoma (ccRCC)

Combination therapy with the PD-1 inhibitor pembro and the VEGF inhibitor lenvatinib showed activity in patients (pts) with advanced ccRCC in the phase III KEYNOTE-581/CLEAR trial. The HIF-2α inhibitor belzutifan (MK-6482) and MK-1308A, a coformulation of pembro and the CTLA-4 inhibitor quavonlimab, have each shown antitumor activity in phase I/II trials. HIF-2α or CTLA-4 inhibition with PD-1 and VEGF inhibition backbone combination may provide additional benefit as 1L treatment in ccRCC.