Targeting of the Hypoxia-Induced Acid Microenvironment of Multiple Myeloma Cells Increases Hypoxia-Mediated Apoptosis

Multiple myeloma (MM) is an incurable disease in which malignant plasma cells engraft within the bone marrow (BM). It is postulated that components of the BM microenvironment provide pro-survival and pro-growth signals to MM cells thereby facilitating their survival and proliferation. Because the BM is known to be hypoxic, we hypothesized that low pO₂ activates an adaptive response mediated by hypoxia inducible transcriptional factors (HIFs) that protect MM cells from hypoxia-mediated apoptosis. We recently showed that targeting HIF activity with a polyamide compound (HIF-PA) that targets the hypoxic response element and blocks the ability of HIF to bind to its cognate DNA sequence both inhibits HIF-mediated gene expression and makes MM cells in culture MM xenografts engrafted in the BM of NOG mice sensitive to hypoxia-mediated killing in vitro and. In other tumors, hypoxia leads to acidification of the microenvironment that is a critical factor in their survival and spread. To examine if this is also the case for MM, we exposed a panel of cell lines to severe hypoxia (down to 0.2%) for 48 hrs. This produced a marked acidification of the microenvironment (decrease in pH from ~7.4 to ~6.4) as well as increase in the expression of carbonic anhydrase IX (CA9), a HIF-dependent enzyme important to pH regulation. Moreover, while MM cells are usually resistant to hypoxia-mediated apoptosis, exposure of 8226 MM cells to a specific CA9 inhibitor (acetazolamide) or Na⁺/H⁺ transporter inhibitor (amiloride) increased hypoxia-mediated apoptosis by 3-4 fold. Tumor cells are able to sense changes in pO₂ levels through the activity of the Pryol-Hydroxylase (PHD1-3) sensing pathways. In particular, PHD3 is a known tumor suppressor gene that is frequently silenced in MM cells (including 8226), and confers resistance to hypoxia by constitutively activating HIF2a. We exogenously re-expressed PHD3 in MM cells and this concomitantly downregulated HIF2a expression in an oxygen-dependent manner and sensitized these cells to hypoxia-mediated apoptosis. Apoptosis was further increased with an acid pH. In addition, HIF-PA treatment of cells cultured under hypoxic and acidic pH (6.8) conditions showed a synergistic increase in apoptosis compared to control cells which was dependent on the PHD3 expression. Re-expression of PHD3 also resulted in an increased sensitivity to both acetazolamide and amiloride, suggesting that inhibiting HIF2a expression is critical for sensitizing MM cells to a low pH. Finally, exposure of MM cells to a low pO₂ and acidic pH (6.8) significantly enhanced MM migration as detected with the transwell migration assay; whereas the movement of the MM cells was almost totally inhibited (>80%) by treatment with low concentrations of acetazolamide. The results of these studies support our hypothesis that hypoxic conditions within the BM results in MM-mediated acidification of the tumor microenvironment that facilitates the survival and migration of malignant myeloma cells. Targeting the pathways that lead to acidification of the microenvironment might be a novel and effective primary or ancillary strategy for the treatment of MM.