Modeling Bone Marrow Failure and MDS in Shwachman Diamond Syndrome Using Induced Pluripotent Stem Cells

Myelodysplastic syndrome (MDS) caused by monosomy 7 or del(7q) is a frequent clonal abnormality that arises in the context of inherited bone marrow failure syndromes, such as Shwachman Diamond Syndrome (SDS). Monosomy 7/del(7q) also develops in a subset of patients with acquired aplastic anemia or de novo MDS in the general population. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation, most frequently to acute myelogenous leukemia (AML). Bone marrow failure and clonal evolution to MDS and AML remain major causes of morbidity and mortality for individuals with SDS. Currently, the only curative therapy for these hematological complications is a hematopoietic stem cell transplant. Prognosis is extremely poor once SDS patients develop leukemia. The basis for this propensity to develop monosomy 7 clones remains unclear. The longterm aim of this study is to understand the molecular mechanisms underlying leukemia predisposition and develop more effective treatments.

Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in bone marrow failure remains a critical question. The lack of synteny between murine versus human chromosome 7 has posed a major barrier to the development of mouse models of monosomy 7/del(7q). To study the biological and molecular consequences of monosomy 7/del(7q) in SDS, induced pluripotent stem cells (iPSCs) were generated from bone marrow mononuclear cells of two patients with SDS. Each patient harbored homozygous c.258+2 T>C mutations in the canonical splice donor site of intron 2 in the SBDS gene. The SDS-iPSCs retained the pathogenic homozygous IVS2+2 T>C SBDS mutations, expressed stem cell markers, formed teratomas, and expressed reduced levels of SBDS protein similar to levels noted in the primary patient samples. Proliferation of 4 distinct SDS-iPSC clones derived from two different patients was reduced relative to wild type controls without an increase in cell death. SDS-iPSC formed smaller embryoid bodies with reduced production of CD34+ hematopoietic stem/progenitor cells. Hematopoietic differentiation from CD34+ to CD45+ cells was also impaired. Preliminary data suggest that SDS-iPSCs retain the capacity to give rise to hematopoietic stem/progenitor cells and early myeloid progenitor cells in vitro. These populations were also observed in primary SDS patient-derived bone marrow samples. Because the number of CD34+ cells derived from SDS-iPSCs are limiting, a previously reported 5 transcrition factor re-specification system was used to expand multilineage hematopietic progenitors for further characterization. SDS iPSCs were able to differentiate into an expandable CD34+ population in vitro. Further studies to characterize the hematopoietic impairment in SDS iPSC and primary marrow samples are ongoing.

To model del(7q) in SDS iPSCs, a deletion of the MDS-associated long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q at locus (11.2) was confirmed by karyotyping and by qPCR across chromosome 7. The SDS (del7q) iPSCs retained the SBDS pathogenic mutations, expressed stem cell markers, and formed teratomas. Proliferation of the SDS del(7q) iPSC was markedly impaired compared to isogenic SDS iPSCs. No increase in cell death was observed in the SDS del7q iPSCs. Studies are in progress to determine the effects of del7q on hematopoiesis. Investigation is ongoing to determine the molecular consequences of deleting 7q. These isogenic SDS+/- del(7q) iPS models provide a platform to study the role of 7q loss in clonal evolution from bone marrow failure and to screen for novel therapeutic compounds or pathways to treat bone marrow failure and MDS.