AML1-ETO induced leukemia stem cell self-renewal depends on Plcg1

Leukemia stem cells are characterized by aberrant self-renewal capacity. Targeting oncogenic fusions that mediate this aberrant self-renewal capacity remains a therapeutic challenge. The t(8;21) translocation, resulting in the oncogenic fusion AML1-ETO (AE, RUNX1-RUNXT1) is among the most common chromosomal rearrangements found in acute myeloid leukemia (AML). By conducting high-resolution proteomic analysis on myeloid leukemia stem cells we identified Phospholipase C- and Ca++-signaling pathways to be differentially regulated in AE/t(8;21) AML. Phospholipase C gamma 1 (Plcg1) was specifically, and highly expressed in t(8;21) AML and could be identified as a direct target of the AML1(RUNX1)-ETO fusion. Genetic inactivation of Plcg1 resulted in abrogation of disease initiation by AE, reduction of intracellular Ca++ release and loss of AE-driven self-renewal programs. Plcg1 deletion after onset of AE-induced leukemia significantly reduced disease penetrance, number of leukemia stem cells and resulted in abrogation of leukemia development in secondary recipients. Inactivation of Plcg1 in human AE-positive AML cells by RNAi reduced colony formation and AML development in vivo. In contrast, Plcg1 was dispensable for steady state hematopoiesis and maintenance of murine and human hematopoietic stem cells (HSC). Translationally, we used pharmacologic inhibition of Ca++ signaling downstream of Plcg1 in AE-driven AML and this resulted in impaired proliferation and self-renewal capacity. The Plcg1 pathway represents a novel, specific vulnerability of AE-driven leukemia and represents an important new therapeutic target. mRNA profiles of Plcg1 -/- in AML1-ETO9a stem cells (LCSs) were generated using SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara) and Nextera XT Kit (Illumina) followed by sequencing with 50 bp single-end reads on HiSeq 2000.