Aberrant differentiation is a hallmark of myeloid leukemia, reflecting a disruption of the intrinsic balance between proliferation and differentiation. Our published and preliminary data indicate that subverted RARα- CAK signaling is a pivotal feature of myeloid leukemogenesis and challenges the current paradigm that RA induces cell differentiation solely by transactivating target genes. We propose to determine how RARα-CAK signaling couples CAK-dependent G1 exit to transcriptional control of granulocytic differentiation in normal and leukemic myeloid cells. The long-term goal of our research is to provide fundamental basis for development of differentiation therapy that can force the terminal differentiation of myeloid leukemia cells. Normal cellular differentiation requires timely exit from the cell cycle. CAK, whose activity is determined by its targeting subunit MAT1, regulates cell cycle G1 exit when cells commonly commit to proliferation or to differentiation. The mechanisms by which transcription factor RARαmediates the effect of RA to coordinate the granulocytic proliferation/differentiation (P/D) transition during granulopoiesis are unknown. We discovered that, in myeloid leukemic cells, the lack of RA-induced ubiquitination-proteolysis of MAT1 inhibits CAK-dependent G1 exit and sustains CAK hyperphosphorylation of RARαto prevent granulocytic differentiation. This contrasts with the P/D transition in normal hematopoietic cells, where intrinsic MAT1 degradation proceeds together with progressive granulocytic differentiation, and the loss of RARα phosphorylation by CAK induces RA-target gene expression and granulocytic differentiation. Using in vitro cellular models and NOD/SCID mice, the proposed experiments seek to: a) evaluate the role and mechanism of MAT1 expression and degradation in normal granulopoiesis;b) evaluate the mechanisms of RA-induced ubiquitination-proteolysis of MAT1, c) determine the role and mechanism of RARα hypophosphorylation in transcriptional control of granulocytic differentiation;and d) define the role of ALDH1A1 and 1B1 in initiating RARα-CAK signaling during granulopoiesis. The proposed specific aims will examine, for the first time, the novel mechanisms by which CAK-RARα signaling coordinates CAK-dependent post-translational regulation of G1 exit to RARα-dependent transcriptional control of granulocytic differentiation. This project has the potential to generate valuable insights into the molecular regulation of granulopoiesis and therefore has clear relevance to the design of effective differentiation therapies against myeloid leukemia.
Deregulation of the normal differentiation program with restricted cell division is a key event in myeloid leukemogenesis. An improved differentiation therapy for myeloid leukemia has been delayed due to the lack of fundamental knowledge on how restricted proliferation is coupled with differentiation during granulopoiesis. We propose to close this gap by determining how RARα-CAK signaling coordinates proliferation/differentiation transition in normal and leukemic myeloid cells and, ultimately, devise strategies to force the myeloid leukemic cells into a terminal stage of development.
