HIF 1 Alpha: A Suitable Target for Multiple Myeloma

Abstract 2901

Hypoxia-inducible factor-1 alpha (HIF1 α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1 α, in response to hypoxia or activation of growth factor pathways, is associated with tumor proliferation, metastasis, and drug resistance and correlated with poor prognosis. In contrast to solid tumors, the role of HIF1 α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF1 α has been suggested to be constitutively expressed and HIF1 α knockdown cell lines have shown higher sensitivity to standard chemotherapy, suggesting a role in the pathophysiology of MM.

In the present study, we explored the effect of EZN2968, an antisense oligonucleotide against HIF1 α, as a molecular target in MM.

We showed, using real time PCR, and Western blotting analysis, that the expression of HIF1 α in several MM cell lines (MM1S, U266, OPM2, RPMI8226) is detectable under conditions of normoxia or hypoxia and is increased in the presence of growth stimuli (IL-6 and stroma cells). The immunofluorescence analysis suggested that the protein is ubiquitously present in both the cytosol and nucleus.

To evaluate the specificity of the oligonucleotide for the target, we tested whether EZN2968 was able to induce a selective and stable down-modulation of HIF1 α mRNA and protein expression. We confirmed that the downmodulation was lasting in a long term culture experiment (up to 96 hours) either in normoxic or hypoxic conditions, and did not affect the expression of other family members of hypoxia inducible transcription factors (HIF2 α).

We next explored the effects of EZN-2968 on the growth and survival of MM cells. Using an MTT colorimetric survival assay, we showed that, after 48 hours of culture in the presence of the HIF1 α inhibitor (20μM), MM1.S and U266 cell lines exhibited a reduction of 30% of viability compared to untreated cells, while RPMI8226 of 15%.

AnnexinV/PI staining revealed that EZN-2968 (20μM) increased, after 48 hours of culture, the percentage of PI⁺ cells compared to the control, suggesting a disruption on membrane permeability. In addition, immunoblotting revealed PARP cleavage as early as 24 hours.

Evaluation of cell cycle profile, by flow cytometric analysis, showed an increase of the sub-G0/G1 population from 3.5% to 30 %, after 48 hour of exposure to EZN-2968.

To evaluate if the impact on cell viability was irreversible, we performed a cell death commitment assays. MM1S cells were incubated with EZN2968 (20 μM) for 24 to 96 hours, following incubation in drug-free medium for additional 24 to 72 hours. MTT colorimetric survival assay showed that EZN-2968 treatment for as early as 24h resulted in commitment to death in all cell lines tested.

To evaluate the effect of microenvironment, MM cells treated with EZN2968 were exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame the proliferative effect induced by cytokines.

We next evaluated the impact of EZN-2968 on purified CD138⁺ cells from MM patients with advanced MM. MTT colorimetric survival assay showed a reduction of cells viability of 30% after 24 hours of incubation. In addition we observed a low sensitivity of PBMCs and CD34⁺cells, derived from healthy donors, to EZN-2968 treatment suggesting that EZN-2968 has selective in vitro activity against MM cells. Evaluation of gene expression profiling modulation induced by EZN 2968 is on going.

In summary, our results suggests that the inhibition of HIF1 α activity can be used as an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitors.