Clinical Application And Mechanism Research Of Stem Cell Factor SALL4 In MDS Initiation And Progression

BackgroundMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) disorders characterized by refractory cytopenias with dysplasia as a result of ineffective hematopoiesis. Approximately 30 percent of MDS cases progress to acute myeloid leukemia (AML). Because of its heterogeneity and lack of molecular markers that effectively monitor disease progression, clinical management of MDS patients is challenging.SALL4 is the human homologue of the Drosophila spalt homeotic gene, which is mapped to chromosome 20q13.13-13.2. Recently it has been reported that SALL4 played essential role for the maintenance of pluripotent and self-renew of embryonic stem cell (ESC) and was also involved in normal hematopoiesis and leukemogenesis. We have previously reported that SALL4 was constitutively expressed in human AML and SALL4 transgenic mice developed MDS-like features and subsequently AML. However, the role of SALL4 in human MDS has not been extensively investigated. In this study, we evaluate the diagnostic/prognostic value of SALL4 in MDS by examining its expression levels in a cohort of MDS patients. Furthermore, we explore the mechanism of SALL4 in MDS and leukemogenesis by using SALL4B transgenic mouse model and SALL4 conditional knockout mouse model in both gain-of-function and loss-of-function.Materials and methodsThis study is divided into three parts. In the first part, Fifty-five newly diagnosed MDS, twenty MDS-AML, and sixteen post-treatment MDS patients were selected for our study along with ten healthy donors. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blot were used to analysis the expression of SALL4 in both mRNA and protein level. Furthermore, this cohort of MDS patients was followed up for about two years to evaluate the prognosis value of SALL4 in MDS.In the second part, homologous recombination (HR) and non-homologous DNA end joining (NHEJ) reporter assay were used to study the role of SALL4 in DNA damage repair in SALL4B transgenic mouse model and further explore the mechanism of SALL4 in the progression of MDS to AML.In the third part, SALL4 conditional knockout mouse model was used to investigate the essential role of SALL4 in Hoxa9/Meis1 mediate leukemogenesis by in-vivo serial bone marrow transplant and in-vitro re-plating colony forming assay.ResultsIn the first part, we demonstrated that SALL4 was over-expressed in MDS patients and proportionally increased in MDS patients with high grade/IPSS scores. This expression pattern was similar to that of Bmi-1, an important marker in predicting MDS/AML progression. In addition, the level of SALL4 was positively correlated with increased blast counts, high-risk keryotypes and increased significantly in MDS-AML transformation. Furthermore, higher level of SALL4 expression was associated with worse survival rates and SALL4 level decreased following effective therapy.In the second part, our studies indicate that SALL4 can negatively affect the HR DNA damage repair process, at least in part, through repressing Fancl. In addition, SALL4 can promote cell survival by activating the anti-apoptosis gene Bcl2. All together, it is possible that the SALL4 transgenic mice are more prone to accumulate secondary mutations, which may lead to their progression from MDS into the AML stage.In the third part, we use SALL4 conditional knockout mouse model to showed evidence that the proliferative potential of leukemic stem and progenitor cells lacking SALL4 is compromised since SALL4 KO-Hoxa9/Meis1 leukemic cells eventually undergo proliferation arrest and show signs of differentiation, leading to transplant failure of leukemia in recipient mice after second serial bone marrow transplant.ConclusionTo the best of our knowledge, this is the largest series and the first to report the expression pattern of SALL4 in detail in various subtypes of MDS in comparison to that of Bmi-1. We conclude that SALL4 is a potential molecular marker in predicting the prognosis of MDS.Our studies have shown for the first time that SALL4 has dual functional roles in inhibiting DNA damage repair and promoting cell survival under challenge. Our findings have provided new insight on the pathogenesis of SALL4 in MDS/AML disease progression.We are the first to use conditional SALL4 knockout mouse model to show that SALL4 have essential role in the maintenance of leukemogenesis. Targeting SALL4 could be an innovative approach in treating MDS and leukemia with high SALL4 expression.