Oncofetal Protein SALL4 Is Highly Expressed in Myelodysplastic Syndrome Alongside with NAT10 and P53
Authors: Tatetsu Hiro , Watanabe Miho , Mason Emily F , Lovitch Scott B , Tokunaga Kenji , Iwanaga Eisaku , Matsuoka Masao , Tenen Daniel G. , Chai Li 
Published: November 2020

Introduction: Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases characterized by cytologic dysplasia and refractory cytopenias as a result of ineffective hematopoiesis. We have reported that oncofetal protein SALL4 can be used as a prognostic biomarker in MDS disease. SALL4 is a transcription factor that is important for development and embryonic stem cell properties. While SALL4 expression is down-regulated or absent in most adult tissues, SALL4 is re-expressed in various cancers. We have previously reported that aberrant expression of SALL4 can be detected in MDS and AML patients. Studies in murine models, as well as in human samples, have demonstrated that SALL4 is essential for leukemic cell survival and that SALL4 transgenic mice develop an MDS-like phenotype prior to transformation to AML, suggesting that SALL4 can be a driver of MDS/AML pathogenesis.

However, SALL4 expression status and related pathway in bone marrow (BM) cells and relationship with somatic gene mutations in MDS patients has not been explored. In this study, we evaluated the expression of SALL4 and related factors using single-cell mass cytometry (CyTOF) and NanoString technology in MDS patients alongside with target sequence for MDS-related mutations.

Materials and methods: We evaluated the expression of SALL4 using single-cell mass cytometry (CyTOF) utilizing 28 antibodies including surface lineage markers and intracellular proteins, such as p53, ki67, c-myc and pAKT to identify SALL4 expressed cells and related pathway in bone marrow (BM) for 10 MDS patients. SALL4 expression was also analyzed in formalin-fixed paraffin-embedded (FFPE) 14 patient samples utilizing two SALL4 probes designed for NanoString technology alongside with target sequence for MDS-related mutations. One probe only detects expression of SALL4A, which is full length SALL4, while the other probe can detect both SALL4A as well as its splicing variant SALL4B. We examined the expression of a panel of 17 known SALL4 downstream target genes.

Results: First, we assessed an expression level of SALL4 using CyTOF in 10 MDS patients as compared with normal BM samples (N=5). We observed that Lin-CD34+CD38+ progenitor cells in 8 out of the 10 MDS patients had higher SALL4 expression levels, while the progenitor cells of normal BM cells did not express SALL4. Similar results were observed in Lin-CD34+CD38- hematopoietic stem cells, CD11b+ mature myeloid cells, CD19+ B cells, CD235+ erythroid cells. Conversely only 3 MDS patient had weak SALL4 expression in CD3+ T cells. Subsequently, we investigated the correlation with p53, ki67, c-myc and pAKT. SALL4 is only correlated with p53 (P=0.005), while others were not.

Next, we also assessed an expression level of SALL4A and SALL4A&B in 14 MDS patient bone marrow (BM) samples using Nanostring nCounter. We observed that 12 out of 14 MDS patients had higher SALL4 expression levels as compared with control normal BM samples (N=3). Next, we evaluated whether MDS patients expressed SALL4A, SALL4B, or both. Of the 14 MDS patients, 8 predominantly expressed SALL4A Of these 8 MDS patients, 5 harbored RNA splicing-related mutations, such as SF3B1 (N=4), U2AF1 (N=1). Only 1 out of 7 patients who predominantly expressed SALL4B demonstrated a splicing related mutation. Subsequently, we investigated the correlation between SALL4 and its known downstream targets. An expression of SALL4A is moderately correlated with CBLb (γ=0.53), CDH1 (γ=0.42) and NAT10 (γ=0.41). The expression of SALLA&B is moderately correlated with NAT10 (γ=0.47) and RUNX1 (γ=0.46). NAT10 (N-acetyltransferase 10) has been reported to promote transcription of RNA polymerase I and is a critical regulator of p53 homeostasis. It was identified to be a potential SALL4 downstream target in our early Chip-seq studies. In the isogenic K562 cell lines with SALL4 overexpression, higher levels of NAT10 were observed, suggesting that NAT10 is one of the downstream targets of SALL4.

Conclusions: Our study has demonstrated for the first time that 1) SALL4 was expressed in various MDS BM cells confirmed by CyTOF 2) there are SALL4 splicing variants in MDS patients, particularly with SF3B1 mutations 3) a novel SALL4/NAT10/p53 link has been identified in these MDS patients. Future studies on mechanism(s) and biological role(s) of SALL4 splicing variants in MDS are needed.

Disclosures

No relevant conflicts of interest to declare.