Abstract 2978: The utilization of a human MCL1 knockin mouse suggests that reductions in B-cells and monocytes may serve as clinically relevant pharmacodynamic markers of MCL1 inhibition

The development of small-molecule inhibitors targeting pro-survival BCL-2 family members, such as MCL1 and BCL-2, is an attractive approach for the treatment of cancer. AMG 176 is a potent and selective first-in-class MCL1 inhibitor currently in phase I clinical trials in multiple myeloma and AML. AMG 176 and its analogue AM-8621 exhibit picomolar affinity for human MCL1. However, binding affinity for murine MCL1 is reduced by ~200-fold. Examination of the AMG 176 binding pocket in human MCL1 suggests Leu248 contributes to the high affinity interaction with AMG 176. In mouse MCL1, the corresponding amino acid is a phenylalanine, reducing the affinity of AMG 176 for the mouse protein. This reduced affinity makes it challenging to assess the pharmacodynamic effect of AMG 176 on normal hematopoietic cell types at efficacious doses in human tumor xenograft models. To overcome this challenge we generated a human MCL1 knockin mouse in which the mouse genomic Mcl1 locus was replaced with its human counterpart. Human MCL1 knockin (KI) mice were created by targeting embryonic stem (ES) cells with a vector containing the human MCL1 genomic locus flanked by the homologous mouse sequences up- and downstream of mouse Mcl1. Targeted ES cells were confirmed by Southern blot and used to generate chimeric mice. Germline transmission of the human MCL1 KI allele and the removal of selectable markers was accomplished by breeding with cre-expressing transgenic mice. Homozygous human MCL1 KI mice were created by breeding heterozygous human MCL1 KI mice lacking the Cre transgene and selectable markers. We used a splenocyte viability assay to confirm the functional replacement of murine Mcl1 with its human ortholog. The in vitro treatment of splenocytes with AM-8621 for 6 hours resulted in dose-dependent Caspase 3 activation and reduced viability in B and T cells derived from human MCL1 knockin mice. Oral administration of AMG 176 at doses of 30 and 60 mg/kg resulted in the induction of apoptosis and the subsequent inhibition and regression of OPM-2 multiple myeloma tumor xenografts (54% tumor growth inhibition and 21% regression, respectively). The human MCL1 knockin mice were used to test the effect of AMG 176 at an equivalent dose and schedule on B cells, T cells and monocytes derived from peripheral blood, spleen and bone marrow. Samples treated with 30 and 60 mg/kg of AMG 176 were harvested 24 hours post-second dose of the first and second cycles. We observed a dose-dependen