The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells.

Abstract : The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells We investigated the effect of stable suppression of hypoxia inducible factor (HIF)-1α in myeloma cells on sensitivity to lenalidomide (LEN) in vivo. We found that the in vivo anti-tumoral effect of LEN is enhanced by HIF-1α suppression in myeloma cells. It has been reported that HIF-1α is over-expressed by myeloma cells 1-4 and that HIF-1α suppression significantly blocks myeloma-induced angiogenesis, and reduces both tumoral burden and bone destruction in vivo in multiple myeloma (MM) mouse models. 3 The potential effects of HIF-1α modulation on drug sensitivity in MM cells are not known and are currently under investigation. The immunomodulatory drugs (IMiDs®), including LEN, are a class of drugs derived from thalidomide 4 able to exert anti-myeloma effects by the selective Cereblon-dependent destruction of IKZF proteins, 5,6 either through a direct action on MM cell proliferation and survival, 7 or through indirect immunomodulatory and anti-angiogenic effects. 7 Previous data indicated that HIF-1α inhibition did not increase the anti-proliferative in vitro effect of bortezomib on MM cells; 2,3 this drug induces a strong downregula-tion of HIF-1α in MM cells. 2 We recently reported that HIF-1α knockdown in the human myeloma cell line (HMCL) JJN3 potentiated the in vitro effect of LEN treatment (48-72 h) on cell proliferation through a significant upregulation of p27 without changing cell viability. 3 It has been consistently reported that LEN only slightly down-regulated HIF-1α expression in MM cells. 2 Such evidence has provided the rationale to investigate the effect of stable suppression of HIF-1α in myeloma cells on LEN sensitivity in vivo. Therefore, in the present study, we first inhibited HIF-1α in three HMCLs (JJN3, OPM2 and H929) using an anti-HIF1α lentiviral shRNA pool, as previously described. 3 H929 showed a very high mortality rate after anti-HIF1α lentiviral infection (data not shown) and were not used for the subsequent in vivo experiments. We assessed the effect of LEN in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous in vivo mouse model. 3 Different groups of animals (5 animals in each group) of two sets of independent experiments were injected with JJN3 pLKO.1 (empty vector) or JJN3 anti-HIF1α. When tumors became palpable (approx. 7-10 days after injection) mice were treated with LEN 5 mg/kg (Selleckchem, Houston, TX, USA) or vehicle (DMSO) using the intraperitoneal route. The same mouse model, grouping strategy and treatment conditions were used for OPM2 pLKO.1 or OPM2 anti-HIF1α. After three weeks, we evaluated tumor volume and weight, as previously described, 3 and immunohisto-chemistry was used to evaluate the microvascular density (MVD) and checked by CD34 immunostaining (Santa Cruz, Dallas, TX, USA). 3 In addition, in the first set of mice experiments, the expression of p27 (Abcam, Cambridge, UK) was evaluated by immunohistochem-istry. The expression of S-phase kinase-associated protein 2 (SKP2), a p27 inhibitor, expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasma-cytoma lysates by western blot using the following anti-bodies: SKP2, Casp-3 (Santa Cruz, Dallas, TX, USA), HK2, pERK 1/2, (Cell Signaling, Danvers, MA, USA). b-actin was used as internal control (Millipore,
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Haematologica, Ferrata Storti Foundation, 2016, 101 (3), pp.e107-10. 〈10.3324/haematol.2015〉
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Paola Storti, Denise Toscani, Irma Airoldi, Valentina Marchica, Sophie Maiga, et al.. The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells.. Haematologica, Ferrata Storti Foundation, 2016, 101 (3), pp.e107-10. 〈10.3324/haematol.2015〉. 〈inserm-01285193〉

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