Targeting hypoxia-mediated gene transcription with a novel polyamide drug designed to disrupt the HIF1α/β heterodimer binding overcomes resistance to hypoxia in myeloma cell lines

Abstract

Despite recent advancements in therapy, multiple myeloma (MM) remains incurable. While the reasons for this are unclear, it is felt that the bone marrow (BM) microenvironment may confer protective advantages to MM. The BM is known to be hypoxic compared to other tissues, and while oxygen stress can kill tumor cells, it is also known that hypoxia promotes tumor progression, metastasis, angiogenesis, and resistance to chemo- and radiation therapy. This suggests that hypoxic conditions in the BM may contribute to MM resistance, and therefore targeting the hypoxia response may be of therapeutic benefit. The adaptive response to hypoxia is mediated by a constitutively expressed β-subunit (HIF-1β), which forms a heterodimer with the inducible α-subunit (HIF-1α), although HIF-1α is frequently found to be constitutively expressed in some MM cell lines. In order to target the hypoxic response in MM, we blocked HIF-mediated gene transcription using a novel class of DNA-binding pyrrole-imidazole (Py-Im) polyamide (PA) drugs that are designed to target and disrupt the HIF1α/β heterodimer from binding to its cognate DNA sequence. Using a hypoxia chamber, we cultured MM cell lines (OPM-2, 8226, U266, MM1.S) under normoxia (22% O2, 5% CO2) or hypoxia (0.1% O2, 5% CO2) for 72 hours and assayed for apoptosis. We found that 8226 and U266 cell lines were relatively resistant to hypoxia-mediated apoptosis (∼15-20% apoptosis), but that OPM-2 was much more sensitive (>50% apoptosis), whilst MM1.S was intermediate in sensitivity (∼25% apoptosis). Using immunoblots, we found that cell lines with constitutive expression of HIF-1α were resistance to hypoxia, suggesting that constitutive HIF activity predicts MM resistance to hypoxia. We then tested whether treatment with our novel PA drug could overcome resistance to hypoxia-induced apoptosis. After confirming the ability of the PA inhibitor to specifically block HIF-mediated transcription using MM cells with a HIF-response element-reporter construct, as well as inhibition of VEGF mRNA expression, we next assayed the effect of PA (0.1-10 μM) treatment in MM cells as described above. PA was only mildly cytotoxic to MM cells under normoxia, but we observed a significant synergistic induction of apoptosis under hypoxic conditions. These effects were further potentiated when PA treatment was combined with rapamycin (0.1-100 nM). Our data suggests that the HIF-mediated hypoxic response plays an important role in MM sensitivity to hypoxia, likely through the induction of specific survival genes. In conclusion, our preliminary data shows that targeting the hypoxic response in MM with a specific polyamide drug that targets the HIF-response, alone or in combination with mTOR inhibitors, may provide an effective and novel treatment in the hypoxic BM microenvironment.

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