Inhibition of HIF-1a By PX-478 Normalizes Blood Vessels, Improves Drug Delivery and Suppresses Progression and Dissemination in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy localized in the bone marrow (BM) characterized by the continuous metastasis. Despite the introduction of novel therapies, MM patients relapse due to the development of drug resistance that is, at least in part, promoted by hypoxia (insufficient oxygen). MM cells develop a hypoxic phenotype, leading to cellular adaptations that cause metastasis, angiogenesis, stemness and resistance to drugs, such as carfilzomib, promoted by a hypoxia-inducible factor-1α (HIF-1α) transcription factor. Herein, we explored the mechanisms underlying HIF pathway inhibition using for the first time in MM a HIF-1α selective small molecule inhibitor, PX-478, both in vitro and in vivo.

Methods: In vitro, to test the effect of PX-478 (0 - 50 µM) in combination with carfilzomib on MM cell survival exposed to normoxia (21% O₂) or hypoxia (1% O₂) was assessed using MTT assay. Cell adhesion to endothelial cells (HUVECs), and cell migration to stromal cells of prelabeled MM cells treated with PX-478 was measured by fluorescent spectrophotometer and flow cytometry, respectively. Tube-like formation of HUVECs as well as survival was tested in the presence of PX-478.

For in vivo study, MM.1S-Luc-GFP cells were injected intravenously (i.v.) into 40 SCID mice; 3 weeks post injection the mice were divided into 4 groups and treated with vehicle (PBS), carfilzomib, PX-478, and a combination of PX-478 and carfilzomib. Tumor progression and weight was monitored weekly by bioluminescent imaging, and survival was monitored daily. At day 28, 3 mice from each group were randomly taken: (i) to test the number of circulating tumor cells (MM-GFP+) in the peripheral blood counted by flow cytometry; (ii) to test the MM apoptosis in the femurs by TUNEL staining; and (iii) to test extramedullar metastasis of MM in the kidney, spleen and the liver using immunohistochemistry. Additionally, tumor vasculature was demonstrated in the skull using photoacoustic imaging as well as tumor involvement using fluorescent microscopy. Moreover, we tested the drug delivery by injecting fluorescent large molecule (Dextran-AF405 Mw=70,000) i.v. in MM-bearing mice treated with and without PX-478. Lastly, we tested the effect of PX-478 on prelabeled MM cell retention in the blood and homing to the BM one hour post-MM injection in naïve mice.

Results: We found that PX-478 reversed the hypoxia-induced resistance of MM cells to carfilzomib, inhibited metastasis-related cell processes such as adhesion and migration, and reduced MM-mediated tube-like formation of HUVECs in vitro. In vivo, in MM-bearing mice PX-478 decreased the number of MM circulating cells, suppressed tumor metastasis, improved vascularization of the tumor thus delivery of chemotherapy, and as a result re-sensitized MM cells to carfilzomib by increasing tumor apoptosis thus completely abrogating tumor growth and significantly extending mice survival.

Conclusions: This is the first study to show the efficacy of PX-478 in MM demonstrating that PX-478 is acting as a pleiotropic molecule in which it inhibited many different hypoxia-induced biological processes - migration, angiogenesis and drug resistance. By overcoming these cancer adaptations, PX-478 has a clear advantage over using agents that carry an effect against one of these processes. This data provides a preclinical basis for future clinical trials testing efficacy of PX-478 in MM.