Hypoxia Inducible Factors' Transcriptional Activity in Polycythemia Vera

Polycythemia vera (PV), a myeloproliferative neoplasms (MPN), is characterized by high red cell mass due to excessive erythrocytosis and by clonal proliferation of myeloid cells. The major PV complication, increased risk of thrombosis, is also seen in Chuvash erythrocytosis (CE), a disorder of upregulated hypoxia sensing. Hypoxia inducible factor (HIF)-1 and HIF-2 are the principle regulators of the hypoxic response by regulating the transcription of hypoxia responsive genes. We previously showed that MPN patients have upregulation of some HIF target genes in granulocytes and platelets along with increased glucose uptake (Sana, et al., Blood 2013 122:1604). Further, stem cell factor and thrombopoietin signaling increase HIF-1 activity (Kirito et al, Blood 2005); however, the role of HIFs in PV is unknown.

We evaluated HIF transcriptional activity in PV by unbiased RNA sequencing of JAK2^V617F positive PV granulocytes (22 patients and 10 controls) and platelets (24 patients and 4 controls). Differential gene expression was determined by DEseq2 and data stratified by p<0.05 and Log2fold change>1. BaseSpace Correlation Engine (Illumina Inc.) was used to identify genes regulated by HIF.

Fifty-seven HIF target genes were dysregulated in PV granulocytes. There were 44 upregulated genes involved in apoptosis, metabolism, cellular proliferation, intracellular signal transduction, and ion transport. Thirteen genes were downregulated in PV, all associated with Ras and Rap1 signaling. Thirty-five genes were correlated with JAK2^V617F allele burden; 27 genes were positively correlated while 8 genes were inversely correlated. SOCS2, FLT1, PDLIM1, ALDH1A2, NPR3, and BATF3 were regulated by both STAT5 and HIF suggesting that HIFs and the JAK-STAT pathway may cross-talk. CAV1, PLOD2, NRIP3, NGFRAP1, and UBXN10 inversely correlated with the white blood cell counts.

Eighty-seven HIF target genes were differentially expressed in PV platelets. Seventy-two genes were upregulated and 15 genes were downregulated. Upregulated genes were associated with apoptosis, proliferation, and metabolism. Unlike in PV granulocytes, some of these upregulated genes were associated with immune responses including the TNF and NF-kB signaling pathways; NF-kB and HIF-1 co-stimulate each other. Twenty genes positively correlated with JAK2^V617F allele burden but BCL2 and PNOC inversely correlated. There were 13 genes regulated by both STAT5 and HIF. DOT1L, GYG1, HOXA1, PRELID1, and RPIA transcript levels inversely correlated with the platelet counts.

In both granulocytes and platelets, 17 genes were dysregulated. CAV1, CHAD, and SOCS2 are associated with negative regulation of receptor signaling via JAK-STAT. CHAD was downregulated while CAV1 and SOCS2 were upregulated in both granulocytes and platelets. ALDH1A2, CAV1, KCNMA1, and MMP9 genes were upregulated and associated with apoptosis.

We also observed an increased transcript of inflammatory genes in PV. PV granulocytes had 82 upregulated and 11 downregulated inflammatory genes. Among these upregulated genes, MGLL, PROK2, and MAP2K6 were also HIF target genes. In platelets, 34 genes were dysregulated (31 genes were upregulated and 3 genes were downregulated).

It is of interest that 2 prothrombotic genes are reported to be regulated by HIFs, tissue factor encoded by F3 (Giannarelli, et al., Circulation 2014) and protein S (encoded by PROS1). We found F3 transcript to be upregulated also in PV and CE granulocytes. PROS1 was reported to be negatively regulated by HIF-1 as detected by decreased protein S in mouse plasma and by decreased PROS1mouse liver transcript and human cell line HepG2 (Pilli, et al., Blood 2018). We found PROS1 to be also expressed in human granulocytes and platelets; however, it was overexpressed in PV and CP granulocytes but not in platelets.

We demonstrate here high HIF transcriptional activity in granulocytes and platelets of PV; some of these HIF-regulated genes are also associated with immune response genes. Dysregulated HIF target genes were involved in apoptosis and cell proliferation pathways. Most HIF target gene transcription correlated with JAK2^V617F allele burden, platelet counts, and white blood cell counts. These results suggest that HIF might be a therapeutic target of PV. Of drugs in clinical use, digoxin is a potent HIF-1 inhibitor (Zhang, et al., PNAS 2008); its potential role in PV and CE is being explored.