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extrinsic factors recapitulating the BM microenvironment. Since HIF-1α critically regulates the interactions of CLL cells with the BM stroma,17 the cytotoxic effects exerted by BAY87-2243 in culture systems mimicking the tumor niche could, in part, be the result of a perturbation of molecular circuits triggered by microenvironmental stim- uli that are implicated in cell survival and drug resistance. Data showing that hypoxia and SC induce HIF-1α overex- pression support the hypothesis that, within the tumor niche, leukemic cells become highly dependent on its pro-
survival effect. In line with this assumption, we found that treatment with BAY87-2243 induced a marked reduction in leukemic infiltration and a parallel increase in the pro- portion of apoptotic leukemic cells in the BM of a CLL transplantable model derived from the Eμ-TCL1 trans- genic mice. Of note, the inhibition of HIF-1α may have beneficial effects also on the non-leukemic milieu. Recent data have shown that infiltration by CLL cells into BM could result in tissue-site hypoxia, causing: i) increased expression of HIF-1α in hematopoietic progenitors, which
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Figure 7. BAY87-2243 plus ibrutinib combinations exerts synergistic cytotoxic effects and is effective in impairing chronic lymphocytic leukemia (CLL) cell viability in the presence of stromal cells (SC). (A) Normalized 48-hour (h) viability of TP53dis and TP53wt CLL cells exposed to 1 μM BAY87-2243 (BAY) and 10 μM ibrutinib, used as single agents or in combination (striped pattern). Box plots represent median value and 25-75% percentiles; whiskers represent minimum and maximum values for each group, together with all points. (B) Heatmaps showing normalized viability after 48-h treatment with BAY87-2243 + ibrutinib as single agents or in combination, used at different concentrations. Asterisks indicate the combinations which determined a significant reduction in cell viability compared to each single agent, at the corresponding concentration. (C) Normalized 48-h viability of TP53dis and TP53wt CLL cells exposed to 1 μM BAY87-2243 and 10 μM ibrutinib, used as single agents or in combination (striped pattern), in co-culture with M2-10B4 SC. Box plots represent median value and 25-75% percentiles; whiskers represent min- imum and maximum values for each group, together with all points. *P<0.05; **P<0.01; ***P<0.001.
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