Human acute myeloid cell lines undergo genetic re-programming in response to differentiation inducers. The M3 AML cell line, HL-60, has served as a model system for studying phorbol ester-induced myeloid differentiation into macrophage-like cells. Macrophage differentiation is preceded by cell cycle arrest and followed by apoptosis. Therefore, the system elucidates the changes in the leukemia cell gene expression program necessary to override the genetic alterations driving continuous cell division, preventing myeloid differentiation, and blocking programmed cell death. We hypothesize that defining the evolving transcriptome as AML cells reverse their proliferative, undifferentiated, and pro-survival phenotype will reveal genes regulating these processes and possibly identify new targets for AML treatment. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. Among these genes are members of the BCL-2 family that encode both pro- and anti-apoptotic proteins (BBC3, PMAIP1, BID and BCL2, MCL1, BCL2A1, respectively). Myeloid cell leukemia 1 (MCL1) is a BCL-2 family protein defined as an anti-apoptotic protein, however MCL1 increases in expression following phorbol ester treatment in HL-60 cells, as well as other AML lines including THP-1, Kasumi-1, and KG-1. The MCL1 gene can be alternatively spliced to generate a transcript encoding a BH3-only variant (MCL1S) known to exhibit pro-apoptotic function. Western blotting demonstrates the accumulation of MCL1S protein prior to the initiation of apoptosis in phorbol ester treated cells. Here we present evidence for splice variant switching and explore the possibility that reduction in the pro-survival form, MCL1L, and accumulation of MCL1S mediates the p53-independent initiation of apoptosis. Currently, we are evaluating the effects on the leukemic phenotype and apoptotic response to phorbol ester in HL-60 cell lines overexpressing MCL1L, MCL1S, and PMAIP1, as well as HL-60 lines containing CRISPR-Cas9 mediated knockouts of MCL1 and PMAIP1.

Citation Format: Cassandra Holbert, Jeffrey Forrester, Michael Roberts. Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3561.