As ex pected, there was a significant amuvatinib dose dependent apoptosis induction in the U266 cells after 48 h treatment. In contrast there was only a minor apoptosis induction in the RPMI 8226/S cells which was not statistically signifi cant. These results suggest that the apoptosis induction is due to targeting MET kinase which U266 cells are addicted to while RPMI 8226/S cells are not. Tumoricidal Effects of Amuvatinib in Myeloma Cells Grown in a Protective Stromal Environment Bone marrow stroma provides a protective environment for MM cells . thus, it is important to assess the effi cacy of therapeutic agents in the context of a stromal environment. To assess this, we treated U266 cells co 72 h, respectively. This apoptotic induction was blocked by a pan caspase inhibitor, ZVAD, suggesting a role for caspases in amuvatinib mediated cell death.

Consistent with the annexin V/PI staining results was our finding that amuvatinib induced poly ADP ribose polymerase cleavage in these cells in a dose dependent manner. Under full serum conditions an induction of PARP cultured with and without stromal cells with amuvatinib for 48 h and measured viability by using flow cytometry analysis of annexin V/PI staining. Under these condi tions, the U266 do not attach to the stromal cells, but are protected by them through both cell to cell contact and by various soluble factors produced by the stromal cells. Amuvatinib induced 50% cell killing during this time period and co culture with the stromal cells provided no protection from this effect.

In contrast, these stromal cells were able to protect U266 cells from bortezomib treatment as they reduced the amount of bortezomib induced apoptosis from 75% to 40%. To determine whether amuvatinib had an effect on the survival of stro mal cells, stromal cells cultured alone were treated with amuvatinib, harvested by trypsinization, and Cilengitide similarly assessed for viability. Interestingly, amuvatinib had a very minimal effect on the survival of this population of cells, though they express MET. These results indicate that the tumor icidal action of amuvatinib was largely restricted to the U266 myeloma cells, whereas the stromal cells, which are not addicted to MET, are not affected by this inhibitor. Furthermore, the stromal cells were not able to protect the U266 cells from amuvatinibs tumoricidal activity.

Since amuvatinib also inhibits PDGFR and KIT, we vali dated MET kinase inhibition as the primary cause of cell death by using imatinib as a negative control. In addition to ABL, imatinib is known to also inhibit PDGFR and KIT but not MET. In contrast to amuvatinib, 25 uM ima tinib did not induce significant cell death indicating that amuvatinib mediated cell death is not due to its effects on PDGFR and KIT.