Abstract B51: Dynamic remodeling of CEBPα complexes in myeloid differentiation
Authors: Rositsa I. Koleva, Scott Ficarro, Manor Askenazi, Jignesh Parikh, Jarrod A. Marto
Journal: Cancer Research (2009)

Abstract Acute myeloid leukemia (AML) remains a highly lethal malignancy with limited therapeutic options. An integral component of the AML phenotype is an inability of hematopoietic progenitors to differentiate into mature myeloid cells. Mounting evidence indicates that the basic leucine zipper transcription factor C/EBPα plays a critical role in development of granulocytes, and that impaired function of C/EBPα may disrupt myeloid differentiation and ultimately lead to blast crisis in AML. Recent studies demonstrated that oncogenic Flt3 signaling leads to phosphorylation of C/EBPα on serine 21, with concomitant impairment of myeloid differentiation in cell line models and primary hematopoietic cells. While these data provide compelling evidence for the central role of C/EBPα in normal hematopoiesis and leukemia, the mechanism by which pS21 disrupts differentiation remains unresolved. Given the general thesis that cellular control of biological processes is orchestrated through a delicate balance of protein-protein interactions and post-translational modifications, we speculated that C/EBPα phosphorylation catalyzes the assembly of a transcriptionally inactive protein complex. To test this hypothesis we established affinity tagged C/EBPα under control of a tet-inducible promoter in myeloid progenitors with constitutive Flt3 activity. Treatment of these cells with Flt3 inhibitors modulated pS21 in a dose-dependent manner. Next advanced quantitative proteomics methodology, including true nanoflow LC coupled with multidimensional fractionation and on-column iTRAQ stable isotope labeling, was used to monitor remodeling of C/EBPα protein complexes as a function of pS21. Our data represent by far the largest catalog C/EBPα interactors involved in chromatin organization, transcriptional modulation, and cell cycle regulation. Furthermore, our quantitative proteomics data demonstrate that 1) C/EBPα interacts with proteins genetically linked to leukemia (DEK, SET and other); and 2) many of these interact with C/EBPα in a phosphorylation-dependent manner. Our functional validation studies show that DEK and SET bind to C/EBPα-target promoters in C/EBPα-dependent manner to enhance C/EBPα attachment to its DNA binding site. Knock-down of newly-identified, leukemia-associated interactors, DEK and SET, reduce the ability of C/EBPα to drive expression of granulocytic target genes. Furthermore the depletion of DEK and SET reduce capacity of myeloid cells to undergo terminal granulocytic differentiation. Our data demonstrate that phosphorylation on serine 21 modulates association of C/EBPα with protein partners that are functionally relevant for myeloid differentiation. Our ability to quantitatively monitor multiple leukemia-related gene products in the context of C/EBPα protein complexes provides valuable insight into the mechanisms by which oncogenic kinase activity disrupts transcription and leads to leukemogenesis. Citation Information: Cancer Res 2009;69(23 Suppl):B51.