Identification Of Genes/Signaling Pathways That Predispose To Therapy Related MDS (t-MDS)

Myelodysplastic syndrome (MDS) constitutes a complex set of hematopoietic stem cell diseases that involves defective differentiation into either one, some or all of the hematopoietic lineages. MDS is caused due to the accumulation of mutations in hematopoietic stem/progenitor cells (HSPCs). Therapy related MDS (t-MDS) is a secondary complication associated with prior chemo-radiation therapy for either cancer or non-cancer related diseases and accounts about 20% of the total MDS cases. Genes predisposing t-MDS in response to chemo-radiation therapy remain enigmatic. We combined a retroviral insertional mutagenesis approach with a radiation treatment regimen in the mouse in an attempt to identify novel t-MDS predisposing genes and signaling pathways. To this end, C57BL/6 low-density bone marrow (LDBM) cells were transduced with the SFβ91 EGFP+ retrovirus, which results in a very high level of insertional mutagenesis (dysregulation of genes in the vicinity of the integration site). Transduced LDBM cells were transplanted into BoyJ recipient mice. After confirming successful transplantation of donor cells into recipients, animals were treated 3-4 weeks post transplant with either 0, 0.5 or 2Gy radiation once a day for 3 days. Mice were monitored for aberrations in any of the hematopoietic lineages in both peripheral blood (PB) and bone marrow (BM, intra-femural aspirates) at frequent intervals thereafter. Mice irradiated with 2 Gy exhibited cytotoxic effects on different hematopoietic lineages 1-week post radiation. Currently at 35 weeks post-radiation, 4 mice treated with 0.5 Gy and 1 mouse treated with 2 Gy developed a tMDS/AML-like disease. In these animals, BM was saturated with transduced donor cells (measured by GFP^pos cells, ranging from 82-97% in BM of different mice) and mice exhibited anemia, thrombocytopenia, splenomegaly and myeloid blasts in BM at different time points. Additionally, 1 mouse treated with 2 Gy developed T-cell leukemia with 95.5% GFP^pos T cells in PB and lymphoid blasts in BM and 1 mouse with 98.7% GFP^neg cells in BM, with 75% of contribution from Mac1/Gr1. Mice developing t-MDS/AML like disease were sacrificed and bone marrow (BM) cells were collected for analysis of retroviral integration sites. We performed ligation-mediated PCR (LM-PCR) using retroviral long terminal repeats (LTR) sequence based primers and amplifying into the mouse genomic region to identify the unique insertion sites. We observed unique band patterns for each of the four t-MDS/AML mice indicating that the clones driving the disease are probably unique, which were confirmed by sequencing of LM-PCR products to identify the genome integration sites. Further gene expression analysis will be performed on the genes located around the integration site to identify individual genes and/or signaling pathways that are altered in these mice. Validation of such genes or pathways in mice might facilitate the identification of novel genes that predispose to t-MDS/AML.