HIF Inhibitor 32-134D Combined with Anti-PD1 Therapy Blocks Murine Hepatocellular Carcinoma Growth and Eradicates Tumors

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 (HIF) 1a in the diagnostic biopsy 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 as monotherapy. 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. 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 anti-tumor immunity. Taken together, these preclinical findings suggest that combining 32-134D with immune checkpoint blockade may represent a breakthrough therapy for HCC. Overall design: Hep3B cells were seeded into 6-well plates in three biological replicates and exposed to 20% or 1% O2 for 24 hours. Total RNA was isolated using TRIzol and treated with DNase (Qiagen). Library preparation and sequencing using the NovaSeq6000 platform (Illumina) were performed by Johns Hopkins Genetics Resources Core Facility High-Throughput Sequencing Center. The Fastq files were subjected to quality check and analyzed by Genialis (https://www.genialis.com/). Differential expression results with FDR < 0.05 and mRNA fold change > 1.5 were used as a cutoff for further downstream analysis.