Hypoxia-Induced Chemoresistance to Doxorubicin in Acute Myeloid Leukemia (AML) Cells Occurs Via HIF-1α Independent Mechanism.

Abstract 2749

Poster Board II-725

Hypoxia-inducible factors (HIFs) are master transcription factors, which directly activate genes promoting proliferation, angiogenesis, and anti-apoptotic effects in normal tissues under low oxygenation conditions. Constitutive upregulation of HIF-1/2α in solid tumors is correlated with therapy resistance, invasion, and increased risk of metastases. We previously showed that high HIF-1α and HIF-2α levels in primary acute myeloid leukemia (AML) cells independently predicted for poor clinical outcome (Demock K et al, ASH 2008). These results imply a potential role for HIFs in promoting leukemogenesis and chemoresistance in AML cells. To confirm these findings, we asked: (1) what are the roles of HIF-1 and HIF-2α in mediating hypoxic responses in AML cells and (2) can hypoxia and/or changes in HIF-1/2α expression alter chemosensitivity of AML cells to standard cytotoxic agents (cytarabine, doxorubicin). Two human AML cell lines (HEL, HL60) were utilized for these studies. AML cells were placed under normoxia and hypoxia (0.5% O2) for 1–72 hours (h) and harvested at different time points for evaluation of HIF-1α, HIF-2α, and VEGF-A (a key downstream mediator of HIFs) expression by Q-PCR and Western blot analysis. We found that HIF-1α gene expression was induced within 1h of hypoxia in AML cells with subsequent upregulation and maintenance of HIF-1α protein expression for up to 48h. In contrast, HIF-2α gene expression peaked at 1h and again at 8h, with HIF-2α protein expression maintained for at least 72h. VEGF-A protein levels rose within 8h of hypoxia and continued to increase up to 72h in a HIF-1α dependent manner. Next, we examined the effects of normoxia or hypoxia on apoptosis induced by doxorubicin (Dox) treatment from 20–100 nM for 1–72h. While normoxic HL60 cells treated with Dox underwent caspase 3-dependent apoptosis and VEGF-A induction in a time- and concentration-dependent manner (as demonstrated by PARP cleavage and flow cytometry), the same cells cultured under chronic hypoxia (48–72h) demonstrated significantly less apoptosis and decreased VEGF-A production following Dox. These effects were not agent-specific, as similar results were also noted following cytarabine treatment. To determine whether these changes were related to HIF-1α upregulation, we repeated these experiments using mock and HIF-1α shRNA knockdown (KD) HL60 clones and found no change in apoptotic response or VEGF-A induction following Dox treatment in control versus HIF-1α KD cells. As compared to mock-transfected cells, HIF-1α KD AML cells also displayed no difference in overall in vitro proliferation or colony-formation capacity under normoxic or hypoxic conditions and in fact grew faster when engrafted as in vivo tumor xenografts in immunodeficient mouse models. Of note, hypoxic treatment of HIF-1α KD cells was also associated with a relative increase in HIF-2α protein expression and upregulation of microRNAs (mir-92A and mir-210) associated with angiogenic and HIF-1α regulation, respectively, in other cell systems. In conclusion, these data support distinct roles for HIF-1α during acute hypoxia and HIF-2α during chronic hypoxia in AML. Attenuation of chemotherapy-induced apoptosis appears to occur following prolonged (48–72 hours) hypoxia of AML cells via HIF-1α independent mechanisms. The role of HIF-2α in mediating these hypoxia-induced effects is under active investigation. These findings imply that AML chemotherapy regimens, which eradicate marrow vasculature as well as hematopoietic cells, could also paradoxically promote survival of some AML cells by enhancing marrow hypoxia and chemoresistance.