The oncogenic transcription factor RUNX1/ETO corrupts the cell cycle to drive leukaemic transformation

RUNX1/ETO is required for leukaemic clonogenicity and proliferation and is traditionally thought of as a transcriptional repressor. Our RNAi experiment identifies target genes that are transcriptionally upregulated.

RNAi screens were performed both in vitro and in vivo, directed at genes found bound by RUNX1/ETO and differentially expressed upon RUNX1/ETO knockdown. For the RNAi screens, we used a doxycycline (dox)-inducible lentiviral RNAi library covering each gene with 3 shRNAs. We transduced two t(8;21)-positive AML cell lines with the library and performed parallel screens employing colony formation and long-term suspension culture assays in the in vitro arm, and intrafemoral xenotransplantation of immunodeficient NSG mice for the in vivo arm. Comparison of Dox and no dox treated groups identified Cyclin D2 (CCND2) as a top hit.

CCND2 knockdown downregulates both cell proliferation and colony formation in t(8;21) positive cells by causing G1 phase cell cycle arrest via a reduction in Rb phosphorylation, which is a phenotype copied by our RUNX1/ETO knockdown. Inhibition of CDK4/6-CCND2 by palbociclib (PD-0332991) in t(8;21) positive cells similarly reduces cell proliferation and colony formation via a G1 cell cycle arrest.

We conclude CCND2 is essential for RUNX1/ETO function and demonstrate that inhibition of G1 CCND-CDK complexes is a promising therapeutic strategy for RUNX1/ETO-driven AML.