Person:

Chai, Li

Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2–4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.

Background The embryonic stem cell (ESC) factor, SALL4, plays an essential role in both development and leukemogenesis. It is a unique gene that is involved in self-renewal in ESC and leukemic stem cell (LSC).Methodology/Principal Findings To understand the mechanism(s) of SALL4 function(s), we sought to identify SALL4-associated proteins by tandem mass spectrometry. Components of a transcription repressor Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex were found in the SALL4-immunocomplexes with histone deacetylase (HDAC) activity in ESCs with endogenous SALL4 expression and 293T cells overexpressing SALL4. The SALL4-mediated transcriptional regulation was tested on two potential target genes: PTEN and SALL1. Both genes were confirmed as SALL4 downstream targets by chromatin-immunoprecipitation, and their expression levels, when tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR), were decreased in 293T cells overexpressing SALL4. Moreover, SALL4 binding sites at the promoter regions of PTEN and SALL1 were co-occupied by NuRD components, suggesting that SALL4 represses the transcriptions of PTEN and SALL1 through its interactions with the Mi-2/NuRD complex. The in vivo repressive effect(s) of SALL4 were evaluated in SALL4 transgenic mice, where decreased expressions of PTEN and SALL1 were associated with myeloid leukemia and cystic kidneys, respectively.Conclusions/Significance In summary, we are the first to demonstrate that stem cell protein SALL4 represses its target genes, PTEN and SALL1, through the epigenetic repressor Mi-2/NuRD complex. Our novel finding provides insight into the mechanism(s) of SALL4 functions in kidney development and leukemogenesis.

Inositol trisphosphate 3-kinase B (InsP3KB) belongs to a family of kinases that convert inositol 1,4,5-trisphosphate (Ins(1,4,5)P3 or IP3) to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). Previous studies have shown that disruption of InsP3KB leads to impaired T cell and B cell development as well as hyperactivation of neutrophils. Here, we demonstrate that InsP3KB is also a physiological modulator of myelopoiesis. The InsP3KB gene is expressed in all hematopoietic stem/progenitor cell populations. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population was expanded, and GMP cells proliferated significantly faster. Consequently, neutrophil production in the bone marrow was enhanced, and the peripheral blood neutrophil count was also substantially elevated in these mice. These effects might be due to enhancement of PtdIns(3,4,5)P3/Akt signaling in the InsP3KB null cells. Phosphorylation of cell cycle-inhibitory protein (p21^{cip1}), one of the downstream targets of Akt, was augmented, which can lead to the suppression of the cell cycle-inhibitory effect of p21.

SALL4 is aberrantly expressed in human myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We have generated a SALL4 transgenic (SALL4B Tg) mouse model with pre-leukemic MDS-like symptoms that transform to AML over time. This makes our mouse model applicable for studying human MDS/AML diseases. Characterization of the leukemic initiation population in this model leads to the discovery that Fancl (Fanconi anemia, complementation group L) is down-regulated in SALL4B Tg leukemic and pre-leukemic cells. Similar to the reported Fanconi anemia (FA) mouse model, chromosomal instability with radial changes that can be detected in pre-leukemic SALL4B Tg bone marrow (BM) cells after DNA damage challenge. Results from additional studies using DNA damage repair reporter assays support a role of SALL4 in inhibiting the homologous recombination pathway. Intriguingly, unlike the FA mouse model, after DNA damage challenge, SALL4B Tg BM cells can survive and generate hematopoietic colonies. We further elucidated that the mechanism by which SALL4 promotes cell survival is through Bcl2 activation. Overall, our studies demonstrate for the first time that SALL4 has a negative impact in DNA damage repair, and support the model of dual functional properties of SALL4 in leukemogenesis through inhibiting DNA damage repair and promoting cell survival.

Hematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-CreLinducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling (Lin^-Sca-1^+c-kit^+) (LSK) cells is increased in (Fak^{-/-}) mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from (Fak^{-/-}) mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs ((Lin^-Sca-1^+c-kit^+CD34^-Flk-2^-) cells) sorted from (Fak^{-/-}) mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of (Fak^{fl/fl}Mx1-Cre^+) bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady- state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.

A dysfunctional bone marrow (BM) microenvironment is thought to contribute to the development of hematologic diseases. However, functional replacement of pathologic BM microenvironment through BM transplantation has not been possible. Furthermore, the study of hematopoietic inductive BM microenvironment is hampered by the lack of a functional nonhematopoietic reconstitution system. Here, we show that a deficiency of SH2-containing inositol-5'-phosphatase-1 (SHIP) in a nonhematopoietic host microenvironment enables its functional reconstitution by wild-type donor cells. This microenvironment reconstitution normalizes hematopoiesis in peripheral blood and BM and alleviates pathology of spleen and lung in the SHIP-deficient recipients. SHIP-deficient BM contains a significantly smaller population of multipotent stromal cells with distinct properties, which may contribute to the reconstitution by wild-type cells. We further demonstrate that it is the nonhematopoietic donor cells that are responsible for the reconstitution. Thus, we have established a nonhematopoietic BM microenvironment reconstitution system to functionally study specific cell types in hematopoietic niches.

An exciting recent approach to targeting transcription factors in cancer is to block formation of oncogenic complexes. We investigated whether interfering with the interaction of the transcription factor SALL4, which is critical for leukemic cell survival, and its epigenetic partner complex represents a novel therapeutic approach. The mechanism of SALL4 in promoting leukemogenesis is at least in part mediated by its repression of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through its interaction with a histone deacetylase (HDAC) complex. In this study, we demonstrate that a peptide can compete with SALL4 in interacting with the HDAC complex and reverse its effect on PTEN repression. Treating SALL4-expressing malignant cells with this peptide leads to cell death that can be rescued by a PTEN inhibitor. The antileukemic effect of this peptide can be confirmed on primary human leukemia cells in culture and in vivo, and is identical to that of down-regulation of SALL4 in these cells using an RNAi approach. In summary, our results demonstrate a novel peptide that can block the specific interaction between SALL4 and its epigenetic HDAC complex in regulating its target gene, PTEN. Furthermore, targeting SALL4 with this approach could be an innovative approach in treating leukemia.

Background: Myelodysplastic syndromes (MDS) are a group of heterogeneous diseases with variable clinical course. Predicting disease progression is difficult due to lack of specific molecular marker(s). SALL4 plays important roles in normal hematopoiesis and leukemogenesis. SALL4 transgenic mice develop MDS prior to acute myeloid leukemia (AML) transformation. 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. Methods: Fifty-five newly diagnosed MDS, twenty MDS-AML, and sixteen post-treatment MDS patients were selected for our study along with ten healthy donors. Results: 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. Conclusions: To 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.

Aggressive cancers and embryonic stem (ES) cells share a common gene expression signature. Identifying the key factors/pathway(s) within this ES signature responsible for the aggressiveness of cancers can lead to a potential cure. In this study, we find that SALL4, a gene involved in the maintenance of ES cell self-renewal, is aberrantly expressed in 47.7% of primary human endometrial cancer samples. It is not expressed in normal or hyperplastic endometrial. More importantly, SALL4 expression is positively correlated with worse patient survival and aggressive features such as metastasis in endometrial carcinoma. Further functional studies have shown that loss of SALL4 inhibits endometrial cancer cell growth in vitro and tumorigenicity in vivo, as a result of inhibition of cell proliferation and increased apoptosis. In addition, down-regulation of SALL4 significantly impedes the migration and invasion properties of endometrial cancer cells in vitro and their metastatic potential in vivo. Furthermore, manipulation of SALL4 expression can affect drug sensitivity of endometrial cancer cells to carboplatin. Moreover, we show that SALL4 specifically binds to the c-Myc promoter region in endometrial cancer cells. While down-regulation of SALL4 leads to a decreased expression of c-Myc at both protein and mRNA levels, ectopic SALL4 overexpression causes increased c-Myc protein and mRNA expression, indicating that c-Myc is one of the SALL4 downstream targets in endometrial tumorigenesis. In summary, we are the first to demonstrate that SALL4 plays functional role(s) in metastasis and drug resistance in aggressive endometrial cancer. As a consequence of its functional roles in cancer cell and absence in normal tissue, SALL4 is a potential novel therapeutic target for the high risk endometrial cancer patient population.