The HDAC inhibitor LBH589 enhances the anti-myeloma effect of the IGF-1 RTK inhibitor picropodophyllin

Our previous studies have shown that inhibition of IGF-1R pathway by picropodophyllin (PPP) can be achieved with favorable therapeutic window in multiple myeloma (MM) models in vivo and in vitro. Considering the acquirement of compensatory genetic lesions during MM progression approaches targeting single receptors are however not likely to be curative for MM. In line with this notion remaining MM cells in the in vivo 5TMM model eventually leads to relapse and mortality. To counteract this, we combined PPP in a combinatorial drug screen (HTS). In this approach we focused on the use of one of these candidates, the HDAC inhibitor LBH589, in combination with PPP in MM models in vivo and in vitro. The studies show that PPP and LBH589 used at suboptimal concentrations has synergistic effects in a combinatorial regimen both in vitro and in vivo. The contribution from the single drugs and the combination were monitored for apoptosis, cell cycle distribution, and the impact on downstream gene and protein expression in human and mouse MM models in vitro. In the RPMI8226 human MM cell line, simultaneous treatment with both compounds for 48h caused a 5-fold increase of apoptotic and late apoptotic/necrotic cells as compared to controls, while treatment with either compound alone only induced a 3-fold increase. After 24h cleavage of apoptotic proteins caspase -9, -8 and -3 could be found in RPMI8226 cells treated with both drugs individually, but in the combination we observed an additive effect on the cleavage of the active forms of caspase 8 as compared to single drug treatments. The combination of LBH589 and PPP could be monitored as an accumulation of cells in the G2/M phase, and subsequent down-regulation of cell cycle regulated proteins. The effect of both compounds on the expression of cyclin B1, -E and -D2 was additive, as demonstrated by western blot. Similar in vitro experiments using cells of the 5T33MM murine model confirm these data. At lower combinatorial concentrations, a significant additive reduction of VEGF secretion (56%) when compared to the single treatments (45% and 13% for LBH589 and PPP respectively) of 5T33MM cells was observed. Finally, we examined the in vivo effect of the combinatorial treatment using sub-optimal concentrations of LBH589 (2.5 mg/kg/day i.p. injection) and PPP (1,5mg/day orally) and assessed the overall survive rate using Kaplan-Meier analysis. Combined treatment resulted in a significant (P