A novel tumour-suppressor function for the Notch pathway in myeloid leukaemia

Abstract

Notch signalling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase (γSE) complex. Although Notch signalling can play both oncogenic and tumour-suppressor roles in solid tumours, in the haematopoietic system it is exclusively oncogenic, notably in T-cell acute lymphoblastic leukaemia, a disease characterized by Notch1-activating mutations. Here we identify novel somatic-inactivating Notch pathway mutations in a fraction of patients with chronic myelomonocytic leukaemia (CMML). Inactivation of Notch signalling in mouse haematopoietic stem cells (HSCs) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMPs), extramedullary haematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signalling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signalling during early haematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumour-promoting and -suppressive roles within the same tissue.

Figure 1. Ncstn deficiency leads to CMML-like disease and a significant enlargement of the GMP progenitor population

a, Histological analyzis showing accumulation of monocytes and granulocytes in peripheral blood (Wright Giemsa staining), spleen and liver (H&E staining). A magnification of each infiltrant is shown in the lower panel. b, Absolute numbers of each monocytic/granulocytic subset from the spleen of control and Ncstn^f/f Vav-cre⁺ littermate animals (12 weeks of age, mean ±S.D. n=10). c, Detailed FACS analysis of bone marrow and spleen myeloid progenitors (MP: Lin⁻/c-Kit⁺/Sca-1⁻) populations of Ncstn^f/f Vav-cre⁺ and Ncstn^f/fVav-cre⁻ littermates showing a significant enlargement of the GMP (FcγRII/III+, CD34=) subset. d, Absolute numbers of each progenitor subpopulation in the bone marrow (mean ±S.D. n=10).

Figure 2. Notch signaling suppresses an extensive myeloid gene expression program through the induction of the transcriptional repressor Hes1

a, Heat map showing regulation of genes representative of the myeloid-signature from the indicated cell populations and mice. b, Expression data were analyzed for lists of genes positively involved in myelopoiesis using GSEA analysis. Enrichment plots show up-regulation of myeloid-specific genes in Ncstn^f/fMx1-cre⁺ and downregulation in Notch1^IC Mx1-cre⁺ LSK cells (when compared to WT counterparts). c, Purified cKit+ progenitors from WT and Ncstn^f/fMx1-cre⁺ mice were transduced with retroviruses encoding Hes-1 or empty vector, subsequently plated on methylcellulose for 7 days and analyzed for expression of myeloid or megakaryocyte differentiation markers (Gr1, CD41). A representative of four experiments is shown.

Figure 3. Ectopic expression of Notch1-IC is able to prevent CMML-like disease in Ncstn^−/− mice

a, PolyI-polyC-induced Notch1-IC expression in Ncstn^f/flsl-N1-IC Mx1-Cre⁺ animals suppresses myeloid cells in the Spleen. b, Notch1-IC expression suppresses GMP progenitor population in the bone marrow. c, Induction of cell death in wild-type GMP cells cultured in the presence (OP9-DL1-) or absence (OP9) of Notch ligands. Cell death was measure by the combination of 7AAD and Annexin-V staining 48h after coculture initiation. For a–c a representative of more than three experiments is shown.

Figure 4. Novel, loss-of-function Notch pathway mutations in human CMML

a, Sequence traces of identified Notch pathway mutations in tumor of CMML patients but not in normal tissues show somatic origin. b, Comparison of percentage of Notch pathway mutations in CMML, MF and PV patient speciments. The “*” denotes that only verified somatic CMML mutations are included. c, OP9-DL1 co-culture of wild-type LSK cells infected with specified constructs. Analysis of CD11b⁺ population was studied 14 days after the initiation of the culture. d, A similar experiments as in c utilizing LSK Ncstn^−/− progenitors infected with the specified constructs. In all cases, a representative of more than three experiments is shown.