Abstract
Abstract 126
Oxygen tension regulates the function of hematopoietic stem cells (HSCs). The hypoxic microenvironment in bone marrow, known as the hypoxic niche, supports a quiescent state of HSCs and maintains long-term repopulating activity. In addition to normal HSCs, hypoxia also affects the fate of leukemic stem cells. Giuntoli et al. reported that hypoxia selects for cells with high-repopulating activity in BCR-ABL-positive leukemia cell lines that have reduced sensitivity to imatinib. The V617F-activating mutation in JAK2 plays a major role in BCR-ABL-negative myeloproliferative neoplasms (MPN). JAK2V617F activates a variety of signal transduction molecules and induces cytokine-independent growth while suppressing apoptosis in HSCs.In this study, we investigated the effects of hypoxia on JAK2V617F-positive cells. To this end, we used JAK2V617F-harboring human leukemia cell lines, HEL and SET-2. We found that culturing under hypoxic conditions (1% O2) significantly suppressed growth of these cells. Interestingly, we found that hypoxia reduced the autophosphorylation of JAK2V617F. Phosphorylation of STAT5, a major downstream target of JAK2V617F, was also highly suppressed in the hypoxic condition. Furthermore, expression of several target genes of STAT5, including cyclin D and Bcl-xL, was drastically decreased after exposure to hypoxia. For further study, after obtaining informed consent, we performed an endogenous erythroid colony (EEC) formation assay on peripheral blood mononuclear cells from JAK2V617F-positive polycythemia vera patients (n=7). We found that hypoxia suppressed EEC formation (Normoxia: 43.8±10.0; Hypoxia: 25.9±4.7, P < 0.05) and suppressed phosphorylation of JAK2 in these cells. Next, we investigated the molecular mechanisms of how hypoxia suppressed activity of JAK2V617F. To elucidate these mechanisms, we initially determined whether hypoxia induced the expression of several negative regulators for JAK2. Hypoxia did not change protein levels of SOCS1 or SOCS3, suppressors of the cytokine signaling (SOCS) family of proteins. We also confirmed that the expression of the tyrosine phosphatases PTP1B and CD45, which could dephosphorylate JAK2, were not altered in hypoxia-treated cells. In contrast, the expression of SHP-2, an SH-2 domain-containing protein tyrosine phosphatase, was drastically diminished after exposure to hypoxia in both HEL and SET2 cells. Importantly, hypoxia also suppressed the expression of SHP-2 proteins in peripheral blood mononuclear cells derived from polycythemia vera patients. To confirm that SHP-2 is required for activation of JAK2V617F, we treated HEL and SET-2 cells with an inhibitor of SHP-2. The inhibitor clearly diminished auto-phosphorylation of JAK2V617F in both cell lines. Our observations show that hypoxia suppressed activity of JAK2V617F and induced growth arrest in JAK2V617F-positive MPN cells. SHP-2 plays important roles in these processes. Interestingly, several studies have reported that SHP-2 is required for the activation of JAK2 by cytokines. In addition, recent studies have revealed that the activity of SHP-2 is increased in platelets from MPN patients. In conclusion, a hypoxic environment may modulate the fate of JAK2-positive MPN cells through suppression of SHP-2 levels and through subsequent suppression of JAK2V617F activity.
Disclosures:
No relevant conflicts of interest to declare.
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