The BH3 Mimetic, ABT-737, Overcomes Stromal-Mediated Pro-Survival Signals and Synergizes with PHA-767491, a Dual Cdc7/CDK9 Inhibitor, In Acute Myeloid Leukaemia

Abstract 1841

ABT-737 is a small molecule inhibitor of Bcl-2 and Bcl-xL with reported activity in pre-clinical in-vitro and in-vivo studies of acute myeloid leukaemia(AML) but to date no data has been reported on its activity in an AML co-culture model. To address this, we examined the effects of co-culture of AML cell lines (MOLM-13, ML-1, KG-1, OCI-AML2) with HS5 cells, a human stromal cell line, on sensitivity to Ara-C and ABT-737. All cell lines cultured in the presence of HS-5 stroma demonstrated a significant reduction in Ara-C-induced apoptosis (% relative reduction - OCI-AML2:80%; ML1:65%; MOLM-13:53%; KG-1:55%) as compared to cells cultured in suspension in normal complete media, with the effect on expression of Bcl-2 family members being currently under evaluation. In contrast, in the presence of ABT-737, HS-5 co-culture did not provide any protective effect whatsoever to AML cells, with IC₅₀ ranging from 0.1 to 0.3μM in the cell lines noted above, regardless of the presence of stroma. OCI-AML3, an AML cell line known to express high levels of Mcl-1 was resistant to ABT-737 in both normal suspension cultures and co-culture. Indeed Mcl-1, an important pro-survival protein in haematopoietic cells is thought to be a key factor promoting resistance to ABT-737 and it has recently been reported that transcriptional upregulation of Mcl-1 may follow exposure to ABT-737. Thus, the combination of ABT-737 with strategies to deplete Mcl-1 is particularly attractive. Cdk9 inhibition is such a strategy. Since Cdk9 phosphorylates RNA polymerase II affecting the rate of transcription, inhibition leads to a depletion of proteins with short half-lives, such as Mcl-1. Here we report that resistance of OCI-AML3 cells to ABT-737-induced apoptosis can be overcome by combination with PHA-767491, a novel dual Cdc7/CDK9 inhibitor. OCI-AML3 cells were treated with increasing concentrations of ABT-737, PHA-767491 or both. Co-administration resulted in a strong synergistic apoptosis-inducing effect as assessed by AnnexinV staining, with combination indices, as calculated by Chou et Talalay, for a range of doses of both drugs of <1 (range 0.3–0.9). Importantly, the sensitising effect of PHA-767491 was seen only at concentrations (≥ 2μM) that resulted in significant downregulation of Mcl-1 protein expression, implicating Mcl-1 downregulation as a possible cause of synergy. We are currently investigating the precise role of Mcl-1 in this regard. In conclusion, taken together, these studies support that ABT-737, possibly in combination with agents to deplete Mcl-1, represents a promising therapeutic strategy for AML and warrants further evaluation.