Role of Hypoxia and Hypoxia-Inducible-Factor 1α (HIF1A) In the BM Microenvironment-Mediated Protection of Leukemic Cells.

Abstract 2586

Resistance to chemotherapy can be mediated by genetic, epigenetic and microenvironmental causes. Only recently the connection between leukemia growth and survival and the hypoxic state of the BM microenvironment has been appreciated, by work conducted by us and others (Fiegl M et.al. Blood 2009; 113: 1504–1512; Harrison JS et. al., Blood 2002; 99). In extension of this concept we investigated the role of Hypoxia-Inducible-Factor 1α (HIF1A), the master regulator of hypoxia induced responses, in the microenvironment and its relevance for leukemia progression. Here we focused on the role of hypoxia and HIF transcription factors in cells contributing to the BM microenvironment, the mesenchymal stromal cells (MSC). Co-culture of lymphoid (NALM6) and myeloid (OCI-AML3) leukemic cell lines with BM-derived MSC under hypoxic conditions (1% O₂) stimulated the secretion of a number of pro-survival cytokines and chemokines (including IL-6, VEGF, Beta-NGF and SDF-1α) that were quantified in co-culture supernatants by Luminex flow cytometry (Table 1). These findings suggest that hypoxia, and possibly its main mediator, the transcription factor HIF1A, may be responsible for the increased production of these factors. Since the chemokine stromal cell-derived factor-1α (SDF-1α) is involved in the attraction of leukemic cells towards cells of the BM microenvironment, we next investigated the role of HIF1A expression in MSC and its effect on SDF-1 secretion and migration of leukemic cells under hypoxic conditions. To this end, we generated primary human BM MSC stably transduced with lentiviral-encoded shRNA against HIF1A. SDF-1α transcription levels measured by qRT-PCR were diminished (∼30%, p<0.01) in HIF1A-silenced MSCs compared to control MSCs expressing non-silencing shRNA. This correlated with significantly reduced transwell migration of OCI-AML3 cells towards HIF1A-silenced MSCs compared with control (non-silencing) MSCs (∼35%, p<0.05) under hypoxic conditions. We next examined the contribution of hypoxia and HIF1A in the protective role of the BM microenvironment against standard chemotherapy with AraC and Doxorubicin. To this end, we performed in vitro experiments co culturing OCI-AML3 cells with either HIF1A-silenced MSCs or control MSCs under hypoxic conditions. After 48h of drug treatment a significant decrease in chemotherapy-induced apoptosis in leukemic cells co-cultured with control MSCs compared to leukemic cells cultured alone was observed. In turn, chemoresistance was reduced in OCI-AML3 co-cultured with HIF1A-silenced MSC, suggesting that hypoxia mediates chemoresistance largely through its effects on cells of the BM microenvironment. It has been shown that leukemic cells seem to exhibit increased dependency on glycolysis for ATP generation, which is frequently associated with resistance to therapeutic agents. Therefore, we measured the production of lactic acid (LA) in leukemic cells co-cultured with MSC in hypoxia compared to normoxia. In agreement with previous observations, we found that REH and primary ALL cells produced more LA when they were co-cultured with MSC under hypoxia compared to normoxia (∼1.8 fold, p<0.05). When REH cells were co-cultured with HIF1A-silenced MSCs in hypoxic conditions the lactic acid production was slightly but significantly reduced (∼20%, p<0.05) compared with the values observed in REH-control MSCs co-culture supernatants. Altogether, these findings strongly point to hypoxia and HIF1A as pivotal components in the protection from chemotherapy mediated by the BM microenvironment. We propose that targeting HIF1A and hypoxia in the protective cells of the bone marrow niches may represent a new approach to increase chemosensitivity of leukemic cells and hopefully improve the existing therapeutic strategies.