Hypoxia-Inducible Factor (HIF)-1alpha Inhibitionin Myeloma Cells Significantly Increases the Anti-Myeloma Effect of Lenalidomide in Vivo
Authors: Storti Paola , Marchica Valentina , Toscani Denise , Airoldi Irma , Maiga Sophie , Bolzoni Marina , Campanini Nicoletta , Martella Eugenia , Mancini Cristina , Ferri Valentina , Donofrio Gaetano , Amiot Martine , Aversa Franco , Giuliani Nicola 
Published: October 2019

Abstract
The immunomodulatory drugs (IMiDs®) exert an anti-myeloma effect by cereblon-dependent destruction of IKZF proteinseither through a direct action on multiple myeloma (MM) cells or through indirect immunomodulatory and anti-angiogenic effects.Previous data indicated that MM cells overexpress hypoxia inducible factor (HIF)-1α and that HIF-1α suppression significantly blocks MM-induced angiogenesis and reduces in vivo tumoral burden in MM mouse models. Interestingly, it has been recently reported that HIF-1α knock-down in MM cells potentiates the in vitro effect of Lenalidomide (LEN) on cell proliferation without changing cell viability and that LEN is not able to suppress HIF-1α expression in MM cells. These evidences give the rationale design to investigate the in vivo effect of HIF-1α stable suppression in MM cells on LEN sensitivity. Thus, in this study, we assessed the effect of LEN in vivo in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous mouse model using JJN3, a cell line known to be resistant to the cytotoxic effect of LEN.
Different groups of animals were injected with JJN3-pLKO.1 (empty vector) or JJN3-anti-HIF1α, obtained by anti-HIF1α lentiviral shRNA pool. When tumors became palpable, mice were treated with LEN (5mg/kg), using the intraperitoneal route. After three weeks, we evaluated tumor volume and weight. Moreover, by immunohistochemistry on ex vivo plasmacytomas, we evaluated the expression of p27 and the microvascular density (MVD), checked by CD34 immunostaining. In addition, the expression of a p27 inhibitor, the S-phase kinase-associated protein 2 (SKP2), the expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and the levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasmacytomas lysates by western blot. We found that LEN treatment induced a significant weight and volume reduction of the tumor burden in mice injected with JJN3 anti-HIF1α as compared to JJN3-pLKO.1. The p27 nuclear expression was significantly increased by LEN treatment in JJN3-anti-HIF1α as compared to JJN3 pLKO.1 mice and compared to JJN3-anti-HIF1α mice treated with vehicle. In addition, we demonstrated that LEN in combination with HIF-1α inhibition significantly reduced in vivo the expression of p-ERK1/2, total Casp-3, HK2 and the p27 inhibitor, SKP2.
Because it is known that LEN exerts its anti-MM effect targeting the IKZF proteins, we further checked in vitro whether the effect of HIF-1α suppression and LEN treatment combination could be mediated by IKZF proteins modulation. Interestingly, after LEN (2-10µM) treatment we found that both IKZF1 and IKZF3 were not differentially expressed, whereas IRF4 was down regulated, in JJN3-anti-HIF1α as compared to JJN3 pLKO.1. Finally, regarding a possible combinatory effect on the in vivo angiogenesis, we found that both the number of CD34 positive vessels and the MVD were reduced in mice colonized by JJN3-anti-HIF1α as compared to JJN3-pLKO.1. On the other hand, LEN treatment did not further significantly reduce the number of CD34 positive vessels and the MVD. Accordingly, we did not find any significant inhibitory effect by LEN treatment on the main pro-angiogenic molecules in JJN3 anti-HIF1α as compared to JJN3 pLKO.1 even after 72 hours.
Overall, our data indicate that HIF-1α suppression in MM cells significantly increases the anti-MM effect of LEN in vivo, mainly through the inhibition of proliferation signaling including the modulation of p27 pathway and the IKZF target protein IRF4, rather than to an anti-angiogenic effect. These data suggest that the combination of LEN and HIF-1α inhibition has a therapeutic rationale in MM.

Disclosures
No relevant conflicts of interest to declare.