HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy
Shaima Salman, … , Michelle A. Rudek, Gregg L. Semenza
Shaima Salman, … , Michelle A. Rudek, Gregg L. Semenza
Published May 2, 2022
Citation Information: J Clin Invest. 2022;132(9):e156774. https://doi.org/10.1172/JCI156774.
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Research Article Oncology Therapeutics

HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy

Abstract

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide and available therapies, including immunotherapies, are ineffective for many patients. HCC is characterized by intratumoral hypoxia, and increased expression of hypoxia-inducible factor 1α (HIF-1α) in diagnostic biopsies is associated with patient mortality. Here we report the development of 32-134D, a low-molecular-weight compound that effectively inhibits gene expression mediated by HIF-1 and HIF-2 in HCC cells, and blocks human and mouse HCC tumor growth. In immunocompetent mice bearing Hepa1-6 HCC tumors, addition of 32-134D to anti-PD1 therapy increased the rate of tumor eradication from 25% to 67%. Treated mice showed no changes in appearance, behavior, body weight, hemoglobin, or hematocrit. Compound 32-134D altered the expression of a large battery of genes encoding proteins that mediate angiogenesis, glycolytic metabolism, and responses to innate and adaptive immunity. This altered gene expression led to significant changes in the tumor immune microenvironment, including a decreased percentage of tumor-associated macrophages and myeloid-derived suppressor cells, which mediate immune evasion, and an increased percentage of CD8+ T cells and natural killer cells, which mediate antitumor immunity. Taken together, these preclinical findings suggest that combining 32-134D with immune checkpoint blockade may represent a breakthrough therapy for HCC.

Authors

Shaima Salman, David J. Meyers, Elizabeth E. Wicks, Sophia N. Lee, Emmanuel Datan, Aline M. Thomas, Nicole M. Anders, Yousang Hwang, Yajing Lyu, Yongkang Yang, Walter Jackson III, Dominic Dordai, Michelle A. Rudek, Gregg L. Semenza

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Figure 1

Identification of HIF inhibitors.

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Identification of HIF inhibitors. (A) Hep3B-c1 cells were stably transfe...
(A) Hep3B-c1 cells were stably transfected with firefly luciferase (FLuc) reporter p2.1, which contains a hypoxia-response element (HRE), and Renilla luciferase (RLuc) reporter pSVR. (B) Hep3B-c1 cells were incubated with vehicle (Veh; 0.1% DMSO; blue bars) or 10 μM 32-134D (red bars) at 20% O2 (n = 6) or 1% O2 (n = 12) for 24 hours. Cell lysates were assayed for Fluc/Rluc activity (mean ± SEM); *P < 0.05 versus vehicle (χ2 test). (C) Chemical structures and IC50 values for HIF inhibitors. (D and E) Hep3B cells were exposed to 20% O2 with vehicle, or 1% O2 with vehicle (blue bar), 32-134D (red bars), 33-063 (green bars), or PT2385 (brown bars) for 24 hours and CA9 (D) and EPO (E) mRNAs were quantified by RT-qPCR. Data are presented as mean ± SEM (n = 3). *P < 0.05 versus 20% O2-vehicle; **P < 0.01 versus 1% O2-vehicle (ANOVA with Bonferroni’s post hoc test); NS, not significantly different from 1% O2-vehicle. (F) Hep3B cells were exposed to 20% or 1% O2 in the presence of vehicle or 1% O2 in the presence of 5 μM 32-134D (n = 3 each) for 24 hours. RNA sequencing identified genes with hypoxia-induced expression (blue circle) and genes that were inhibited by 32-134D (orange circle), based on FDR < 0.05 and fold change > 1.5. (G) Hep3B cells were exposed to 20% or 1% O2 for 24 hours in the presence of vehicle or 5 μM 32-134D, nuclear extracts were prepared, and immunoblot assays were performed. (H) Hep3B cells were exposed to 20% or 1% O2 for 24 hours with vehicle, 5 μM 32-134D, or 5 μM MG132 (during last 8 hours of exposure), nuclear extracts were prepared, and immunoblot assays were performed.