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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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 2

Effect of 32-134D on Hep3B tumor xenograft growth and vascularization.

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Effect of 32-134D on Hep3B tumor xenograft growth and vascularization. (...
(A) Female nude mice received a subcutaneous injection of 5 × 106 Hep3B cells. When tumors reached a volume of 150 mm3 (designated treatment day 1), the mice were randomized to receive a daily intraperitoneal injection of 32-134D at a dose of 0 (blue), 20 (black), 40 (red), or 80 (green) mg/kg. Data are presented as mean tumor volume (± SEM; n = 4 each). **P < 0.01, ***P < 0.001 (ANOVA with Bonferroni’s post hoc test). (B) Gross pathology of tumors harvested from vehicle-treated (top panel) and 32-134D–treated (bottom panel) mice. (C) Nuclear extracts prepared from tumors were assayed by immunoblotting using antibodies against the indicated proteins. (D) Total RNA was isolated from tumor tissue and analyzed by RT-qPCR using primers specific for the indicated mRNAs and results (mean ± SEM, n = 4) were normalized to the mean value for tumors from vehicle-treated mice. (E) ELISA for the indicated proteins was performed using aliquots of tumor lysates (mean ± SEM; n = 3–4 tumors each). *P < 0.05 (Mann-Whitney test). (F) Formalin-fixed and paraffin-embedded tumor sections were analyzed by immunohistochemistry using an antibody against CD31 to identify vascular endothelial cells. Scale bar: 100 μm. The total CD31+ vessel area per field was quantified using ImageJ (mean ± SEM; n = 4 tumors with 5 sections per tumor). *P < 0.05 (Student’s t test).