Hypoxia-Inducible Factor (HIF)-1a Is A Therapeutic Target in Myeloma-Induced Angiogenesis and Bone Destruction in Vivo.

Abstract 2947

It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that hypoxia inducible factor (HIF)-1α is overexpressed by MM cells. However, the potential role of HIF-1α as a therapeutic target in MM is not known and is currently under investigation.

In this study we explored the effect of persistent HIF-1α inhibition by expression of a lentivirus shRNA pool in MM cells on proliferation, survival and transcriptional and pro-angiogenic profiles of MM cells either in vitro or in vivo in mouse models. A HIF-1α Lentivirus shRNA pool was used for HIF-1α stable knock-down in human myeloma cell lines (HMCL)s and the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs were infected and then selected with puromycin. Selected clones were screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. The transcriptional profiles were evaluated in the HMCL JJN3 cells transduced with shRNA forHIF-1α (JJN3-anti-HIF-1α) and on those infected with the control vector pKLO.1 (JJN3-pKLO.1) by U133 Plus2.0 Arrays (Affymetrix®) either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR and by ELISA assays for protein levels. Finally the effect of HIF-1α inhibition in MM cells was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models. Together with tumor volume and weight, microvascular density was evaluated by CD34 immunostaining. Cortical bone thickness was determined by microQcT in the intratibial mouse model.

Among the genes significantly modulated by HIF-1α inhibition (327 and 361 genes in hypoxic and normoxic condition, respectively), we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated by HIF-1α inhibition. Interestingly some pro-osteoclastogenic cytokines were also inhibited including IL-7 and CCL3/MIP-1α. In the in vivo mouse models, we found that mice, injected either subcutaneously or intratibially with JJN3-anti-HIF-1α, showed a dramatic reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-pKLO.1. A significant reduction in the number of vessels X field and VEGF immunostaining were observed in both mouse models. Moreover in the intratibial experiments HIF-1α inhibition significantly blocked MM-induced bone destruction.

Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenesis and reduces the MM tumor burden and bone destruction in vivo suggesting that HIF-1α is a potential therapeutic target in MM.