Hypoxia-Inducible Factor-1 Alpha (HIF-1α) and HIF-2α Are Independent Prognostic Factors for Normal Karyotype Acute Myeloid Leukemia (NK AML)

Abstract Emerging data has shown that severe hypoxia in vitro selects for highly immature or therapy-resistant leukemia clones and may be a crucial component of leukemia stem cell niches. One mechanism utilized by normal cells to survive hypoxia is upregulation of hypoxia inducible factor-1α (HIF-1α), a master transcription factor that directly transactivates genes important for cellular responses to hypoxia including angiogenesis and anaerobic metabolic pathways. HIF-1α is rapidly degraded under normoxia but is stabilized and synthesized under hypoxia or following malignant transformation. Overexpression of HIF-1α protein is associated with increased patient (pt) mortality in multiple solid cancer types, and with worse clinical outcome in pediatric acute lymphoblastic leukemia; however, its role in acute myeloid leukemia (AML) is unknown. First, we examined the response of human AML cells in vitro to hypoxic stress. We found that in vitro exposure of human AML cell lines (HEL, HL60/VCR) with low baseline HIF-1α levels to hypoxia (≤1% O2, 5%CO2) resulted in significantly increased HIF-1α mRNA levels after 4 hours followed by increased HIF-1α protein and elevated VEGF-A and VEGFR-1 mRNA levels peaking at 8 hours. We then examined expression of HIF-1α and a related hypoxia factor, HIF-2α, in diagnostic marrow samples from 91 consecutive AML pts (46% male, 54% female) treated at our institute from 1995–2005. As karyotype is the most important prognostic factor in AML, we examined only normal karotype AML samples associated with intermediate prognosis. Median patient age was 66 years (range 21–87). Less than half (n=46, 49%) achieved complete remission (CR) following induction chemotherapy. Median overall survival (OS) was 9.6 months. HIF-1α and HIF-2α levels were measured by Q-PCR and expressed relative to normal bone marrow controls (level of mRNA expression=1). HIF-1α protein expression was also evaluated by immunohistochemistry (IHC) and qualified as nuclear vs. cytoplasmic. We found that HIF-1α mRNA levels were consistently higher in AML cells than normal bone marrow controls (median fold change 2.78; range 0.48–22.89), although HIF-1α protein levels was increased in only a minority of samples. In contrast, HIF-2α mRNA levels were consistently lower in AML samples than normal bone marrow (median 0.14; range 0.7–0.42). Univariate analysis demonstrated that age, CR, and nuclear HIF-1α protein expression by IHC (p=0.0081) impacted OS. Significant factors for event free survival (EFS) were age, CR, and cytoplasmic HIF-1α IHC expression (p=0.0422). Multivariate analysis demonstrated that age, CR, cytoplasmic HIF-1α IHC expression (p=0.0056; HR=0.22; 95% CI=0.07–0.64) and HIF-2α mRNA expression (p=0.0101; HR=0.16; 95% CI=0.04–0.65) were independent predictors for OS. Similarly, age, CR, cytoplasmic HIF-1α IHC expression (p=0.0302; HR=0.26; 95% CI=0.08–0.88), and HIF-2α mRNA levels (p=0.0016, HR 0.08, 95% CI= 0.02–0.39) were also independent factors for EFS. Conclusions: Our results are the first to demonstrate that overexpression of HIF-1α and HIF-2α are independent prognostic factors in normal karyotype AML (constituting 40–49% of all adult AML diagnoses). Given the fact that HIF expression can be upregulated by oncogenes and tumor suppressors, additional studies examining potential correlations between HIF-1/2α expression and FLT-3/NPM-1 gene mutations in NK-AML; and in other prognostic AML subgroups (i.e. AML with recurrent or complex cytogenetics) are warranted. Based on these data, inhibition of HIF-1/2α mediated pathways with targeted agents may represent a future means to improve clinical outcome for subsets of AML patients.