Abstract #1511: Dynamic remodeling of CEBP\#945; complexes in myeloid differentiation and Leukemogenesis
Authors: Rositsa Koleva, Scott Ficarro, Manor Askenazi, Jignesh Parikh, Shaojuan Li, Chance Luckey, Jarrod Marto
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

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/EBP\#945; plays a critical role in development of granulocytes, and that impaired function of C/EBP\#945; may disrupt myeloid differentiation and ultimately lead to blast crisis in AML. Recent studies demonstrated that oncogenic Flt3 signaling leads to phosphorylation of C/EBP\#945; 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/EBP\#945; 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/EBP\#945; phosphorylation catalyzes the assembly of a transcriptionally inactive protein complex. To test this hypothesis we established affinity tagged C/EBP\#945; 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/EBP\#945; protein complexes as a function of pS21. Our data represent by far the largest catalog C/EBP\#945; interactors, including more than 100 proteins involved in chromatin organization, transcriptional modulation, and cell cycle regulation. Furthermore, our quantitative proteomics data demonstrate that 1) C/EBP\#945; interacts with proteins genetically linked to leukemia; and 2) many of these interact with C/EBP\#945; in a phosphorylation-dependent manner. Knock-down of newly-identified, leukemia-associated interactors reduced the ability of C/EBP\#945; to drive expression of granulocytic target genes. Furthermore enforced expression of the newly identified genes enhances transactivation capacity of C/EBP\#945; and promotes myeloid differentiation. Our data demonstrate that phosphorylation on serine 21 modulates association of C/EBP\#945; 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/EBP\#945; protein complexes provides valuable insight into the mechanisms by which oncogenic kinase activity disrupts transcription and leads to leukemogenesis. 

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1511.