Activation of Notch1 Signaling Suppresses Granulocytic Differentiation and Maintains a Part of Myeloid Progenitor Cells At the Immature Stage

Abstract 2375 The Notch signaling pathway is evolutionally conserved and has crucial roles in the control of fate decision and differentiation in numerous cell types. Although Notch1 is continuously expressed in differentiation myeloid cells, the role of Notch1 signaling in regulating differentiation remains controversial. Here, we enhanced and/or suppressed Notch signaling in myeloblasts and then determined the effects of Notch signaling modulation on granulocytic differentiation. Specifically, we first transduced myeloblastic 32D and HL60 cells with retroviruses that express the intracellular domain of Notch1 (ICN1; pMSCV-ICN1-IRES-GFP) to activate Notch1 signaling, or alternatively expressed a dominant-negative form of Mastermind-like 1 (DNMAML1; pMSCV-DNMAML1-GFP) to inhibit Notch signaling. Then the transduced (GFP+) and untransduced (GFP-) cells were induced into granulocytic differentiation using G-CSF for 32D cells and ATRA for HL60 cells. The degree of granulocytic differentiation was then assessed by flow cytometric analysis of surface expression of CD11b, a marker of granulocytic lineage. We found that the percentage of differentiated cells (CD11b high for 32D and CD11b+ for HL60) in the ICN1 expressing (GFP+) group was significantly ( p Next, We further studied the role of Notch1 signaling in granulopoiesis by first activating Notch1 signaling in 32D cells by stable expression of exogenous ICN1 followed by Notch inhibition via DNMAML1 expression within the same cells. The subsequent four sub-populations of 32D cells termed as Vec/GFP (control), Vec/DNMAML1 (cells with endogenous Notch signaling blocked by DNMAML1, ICN1/GFP (cells with activating Notch1), and ICN1/DNMAML1 (cells with activating Notch1 followed by Notch signaling inhibition) were then cultured with G-CSF and evaluated for differentiation by CD11b staining. We found that on days 6 and 8 after the induction of differentiation, the proportion of differentiated (CD11b high ) cells in ICN1/GFP was significantly ( p Our data indicate that Notch1 signaling activation suppresses granulocytic differentiation, and maintains a part of myeloid progenitor cells at the immature stage. Therefore, it suggests that aberrant Notch1 signaling could support the granulocytic transformation and the maintenance of the malignant phenotype. Disclosures: Griffin: Novartis Pharmaceuticals: Consultancy, Research Funding.