Haploinsufficiency Of SAMD9L, An Endosome Fusion Facilitator, In Mice Induces The Development Of Myeloid Malignancies Mimicking Human Diseases With Monosomy 7

Monosomy 7 is a common chromosomal abnormality found frequently in MDS and AML. We previously identified a common microdeletion cluster in 7q21.3 in juvenile myelomonocytic leukemia patients. This cluster contains three poorly characterized genes: sterile alpha motif (SAM) domain-9 (SAMD9) which is absent in mice, samd9-like (SAMD9L) and Miki (LOC253012). Although mutations have rarely been seen in these genes, the genes reside proximal to the 7q22 band that is deleted in single copy in nearly 25% of AML and MDS patients. We recently reported that Miki, a centrosomal protein that promotes alignment of chromosomes at metaphase, is a candidate gene responsible for mitotic/nuclear abnormalities observed in MDS patients (Mol Cell 2012).

In this study, we established and characterized SAMD9L-deficient mice, along with the analysis of molecular function of SAMD9L protein. Among SAMD9L^-/- (n=15) and SAMD9L^+/- (n=15) littermates, 13 mice developed myeloid dysplasia, 2 mice developed myeloid leukemia and one mouse developed myeloproliferative disease after the age of 18 months, while all but one SAMD9L^+/+ mice (n=23) maintained normal hematopoiesis throughout the 24-month observation period. Infection of MOL4070A retrovirus into newborn mice developed myeloid leukemia within 15 months preferentially in SAMD9L-deficient genetic background with Evi1 and Fbxl10 (encoding a H3K36 demethylase) genes as common virus integration sites.

While bone marrow (BM) cells from SAMD9L^+/+ mice (12 weeks old) formed fewer colonies by the third replating, cells from SAMD9L-deficient mice continued to form similar numbers and sizes of well-differentiated colonies beyond the 7th plating. The excess number of colonies formed was reduced by retrovirus-mediated forced expression of Samd9L. These data suggested enhanced self-renewal and/or delays in differentiation of SAMD9L-deficient stem cells. In addition, enhanced reconstitution ability of SAMD9L-deficient stem cells was demonstrated by competitive repopulation assay using the Ly5 congenic mouse system, where irradiated Ly5.1 mice were transplanted with long term-LSK cells from SAMD9L^+/+ or SAMD9L-deficient Ly5.2 mice (10 weeks old) together with BM cells from SAMD9L^+/+Ly5.1 mice. This was confirmed by limiting-dilution transplants, results of which showed a higher frequency of multi-lineage repopulating cells at 8 weeks in SAMD9L^-/- donor BM.

Moreover, growth advantage in the presence of cytokines was evident in liquid cultures of SAMD9L-deficient BM progenitor cells. Hypersensitivity of SAMD9L-deficient BM progenitors to cytokines was also shown in in vivo experiments, in which SAMD9L-deficient mice injected with cyclophosphamide (day 0) and G-CSF (days 1-4) showed significantly higher WBC counts than SAMD9L^+/+ mice at the nadir (day 3). These findings suggested that SAMD9L-deficiency sensitizes hematopoietic progenitors to cytokines. Immunostaining using SAMD9L antibody showed a vesicular pattern of SAMD9L localization in approximately 15% of BM progenitor cells that overlapped with the localization of EEA1, an early endosomal protein. In SAMD9L^-/- fibroblasts, while rapid endocytosis of PDGF-receptor (PDGFR) by PDGF stimulation occurred in a time-course similar to that in SAMD9L^+/+ cells, homotypic fusion of endosomes containing PDGFR delayed. Inhibition of endosome fusion in SAMD9L^-/- cells lead to the accumulation of PDGFR that were remained to be phosphorylated in early endosome, resulting in the prolonged activation of cytokine signals. Accumulation of cytokine receptors in early endosome and persistent cytokine signals were also found in BM progenitors obtained from SAMD9L-deficient mice. These observations suggest that SAMD9L is a crucial component of a protein complex that facilitates the degradation of cytokine receptors through the homotypic fusion of endosomes. Collectively, our study suggests the contribution of haploinsufficiency of SAMD9L to the pathogenesis of myeloid diseases harboring -7/7q- through the prolonged activation of cytokine signals that results in the enhancement of stem cell self-renewal and/or delay in differentiation of early progenitors. Deletion of other haploinsufficient tumor-suppressor genes that reside in 7q would collaborate with the deficiency of SAMD9L for myeloid leukemogenesis.