Jiayun Lu

Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment

The existence of a haematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that haematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in bone marrow cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where most closely interact with sinusoidal and non-sinusoidal bone marrow microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF- 1α, regardless of localization throughout the bone marrow, adjacency to vascular structures or cell-cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms.

Stem Cell Factor SALL4 Represses the Transcriptions of PTEN and SALL1 through an Epigenetic Repressor Complex

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.

SALL4, a stem cell factor, affects the side population by regulation of the ATP-binding cassette drug transport genes.

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.

Dissecting the role of SALL4, a newly identified stem cell factor, in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematological malignancy that expresses the BCR-ABL fusion oncogene. It has three distinct clinicopathological phases: chronic and accelerated phase and blast crisis. Tyrosine kinase inhibitors, which target the BCR-ABL fusion protein, can induce complete cytogenic remission (CCR) in more than 70% of newly diagnosed chronic CML patients. However, complete molecular response is a rare event in these CML patients with CCR, suggesting that they are not effective in eliminating CML leukemia initiating cells (LIC), although there are differences between the trosine kinase inhibitors. Once CML progresses to blast crisis, it becomes resistant to most treatment approaches, and survival rapidly declines. The exact underlying mechanism of transformation of chronic phase CML to blast crisis is still unclear; however, it is thought to be supported by self-renewing LIC. Therefore, identifying genes or signaling pathways involved in the self-renewal of LIC may promote more effective leukemia treatments. So far, two key regulators in self-renewal of LIC have been linked to CML progression, Wnt/β-catenin and Bmi-1(1–4). Recently, several research groups, including ours, have demonstrated that SALL4 plays an essential role in the maintenance of pluripotent and self-renewal properties of embryonic stem cells (ESC) by interacting with Nanog and Oct4 (5). In addition, our group has shown that constitutive expression of SALL4 contributes to leukemogenesis in adult mice by interacting with two other key regulators in LIC, Wnt/β-catenin and Bmi-1 (6, 7). It appears that SALL4 is a unique gene involved in self-renewal in ESC and LIC. Therefore, we examined SALL4 expression in CML to determine whether it could be involved in the pathogenesis of this disease. Using immunohistochemistry staining, we observed that SALL4 expression was present in blast-crisis CML (9/12, 75%) but not in chronic phase (0/11, 10%). In accelerated phase (1/6, 16.7%), wherein the blast count was 10–19%, immature blasts expressing SALL4 were observed in a background of negative, more mature myeloid cells (Figure 1A). Similar results were observed when we performed FACS analysis on whole bone marrow cells from normal human and CML samples at different disease phases using a conjugated SALL4 antibody (Figure 1B). No SALL4 positive population was detectable in normal whole bone marrow or chronic phase CML samples. A distinct SALL4 positive population was present in accelerated phase and blast crisis CML marrows, which correlated well with the blast count and overlapped with the CD34+ population. The CD34+CD38+ cells had the highest SALL4 RNA expression when tested by qRT-PCR (Figure 1C and Supplemental Table 1). This finding is of particular interest since Granulocyte-Macrophage Progenitors, which are CD34+CD38+, have been proposed as candidate LIC in CML, that transforms to acute myeloid leukemia (AML)(1). Figure 1 SALL4 expression correlates with disease progression of human CML To explore the role of SALL4 in CML, we first tested whether overexpression of SALL4B could block myeloid differentiation and cooperate with BCR-ABL in, consequently, promoting blastic transformation of chronic phase CML. For induction of CML-like leukemia, we harvested bone marrow cells from SALL4B transgenic and wild-type donor mice 4 days post intravenous administration of 200 mg per kg (body weight) 5-fluorouracil (5-FU), transduced with BCR-ABL-GFP retrovirus as described (8), and injected 4×105 cells intravenously into sublethally irradiated (750cGy) C57BL/6 recipients. Slightly sublethal irradiation in this mouse model has been shown not to inhibit induction of CML-like disease. Both, recipients of BCR-ABL induced SALL4B transgenic and wild-type bone marrow, succumbed to fatal CML-like leukemia within 3–6 weeks with increased WBC counts (Supplemental Table 2). Flow cytometry analysis demonstrated that 80% of BCR-ABL-positive bone marrow cells from both groups were Mac1+and Gr-1+ neutrophils (Supplemental Figure 1A). In addition, the percentage of CD34+ or c-Kit BCR-ABL-positive cells from bone marrow and spleen increased about 2- and 3-fold in leukemic SALL4B transgenic recipients compared to wild-type counterparts (Supplemental Figure 1B and 1C and data not shown). This suggests that BCR-ABL- transduced SALL4B leukemic cells were more immature and less differentiated. We next investigated the functional role of SALL4 in CML progression using a loss-of-function approach by knocking down SALL4 expression in the human CML cell line KBM5. Two shRNA retroviral constructs targeting different regions of the SALL4 mRNA as we previously reported (6), were shown by qRT-PCR to reduce SALL4 and Bmi-1 mRNA level in KBM5 cells (Figure. 2A). Figure 2 SALL4 expression is essential for CML cell survival The KBM5 cells expressing reduced levels of SALL4 grew slowly. To better explain this phenomenon, we measured the level of caspase-3, which is a marker for the apoptosis signaling pathway. In KBM5 cells that retained 50% of the wild–type (WT) levels of SALL4, there was a 3-fold increase of caspase-3 activity from 27.9 % in WT cells to 93.6% in cells with reduced SALL4 levels as measured by FACS (Figure 2B). We have previously shown that Bmi-1 is a major downstream target of SALL4 in leukemic cells (6). To determine if overexpression of Bmi-1 could rescue SALL4-induced apoptosis, we transfected SALL4 shRNA treated KBM5 cells with an expression vector containing Bmi-1. As shown in figure 2B, SALL4-induced caspase-3 activity (Fig. 2B-ii) was restored to a near normal level by overexpression of Bmi-1 (Fig. 2B-iii). To further study the role of SALL4 in cell growth in a leukemic cell line, we monitored cell-cycle changes and cellular DNA synthesis in SALL4-reduced and WT KBM5 cells using the BrdU incorporation assay by FACS. A 50% reduction of SALL4 expression level in KBM5 cells resulted in G0/G1 phase (37.8%) and G2 phase (21.7%) arrests (Fig 2C-ii). A more than two-fold decrease in S phase cells was also observed, paralleling the drop in DNA synthesis measured by the level of BrdU incorporation. In contrast, no significant change in the cell-cycle profile was observed when WT- KBM5 cells were infected with control viruses (Fig. 2C-i). Reintroduction of Bmi-1 in SALL4-reduced KBM5 cells can rescue this phenotype (Fig. 2C-iii). Our data suggest that the stem cell factor SALL4 plays an important role in the proliferation and survival of CML cells, and its expression is associated with an advanced stage of CML disease. The mechanism of SALL4-mediated proliferation and survival of CML cells, at least in part, is mediated by the Bmi-1 pathway. In support of this concept, downregulation of SALL4 leads to apoptosis and cell cycle arrest in CML cells, which is rescued by restoring its downstream target gene Bmi-1. This finding suggests that targeting SALL4 may provide a novel therapeutic modality for advanced-stage CML disease.

FAK silencing inhibits leukemogenesis in BCR/ABL-transformed hematopoietic cells†

Focal adhesion kinase (FAK) is constitutively activated and tyrosine phosphorylated in BCR/ABL‐transformed hematopoietic cells, but the role it plays during leukemogenesis remains unclear. Here, we examined the effects of RNA interference‐mediated FAK silencing on leukemogenesis induced by a BCR/ABL‐transformed cell line. Transduction of BCR/ABL‐BaF3 cells with FAK shRNA inhibited FAK expression and reduced STAT5 phosphorylation, but induced caspase‐3 activation. In vitro studies showed that treatment with FAK shRNA resulted in impaired cell proliferation and colony formation, while increasing cell apoptosis. Mice that received transplants of BCR/ABL‐BaF3 cells with FAK shRNA displayed significantly prolonged survival time and diminished leukemia progression. In addition, FAK silencing enhanced in vitro and in vivo efficacy of ABL tyrosine kinase inhibitor imatinib in BCR/ABL‐BaF3 cells. Our results suggest that FAK is critical for leukemogenesis and might be a potential target for leukemia therapy. Am. J. Hematol. 2009.

A SALL4/MLL/HOXA9 pathway in murine and human myeloid leukemogenesis

The embryonic self-renewal factor SALL4 has been implicated in the development of human acute myeloid leukemia (AML). Transgenic mice expressing the human SALL4B allele develop AML, which indicates that this molecule contributes to leukemia development and maintenance. However, the underlying mechanism of SALL4-dependent AML progression is unknown. Using SALL4B transgenic mice, we observed that HoxA9 was significantly upregulated in SALL4B leukemic cells compared with wild-type controls. Downregulation of HoxA9 in SALL4B leukemic cells led to decreased replating capacity in vitro and delayed AML development in recipient mice. In primary human AML cells, downregulation of SALL4 led to decreased HOXA9 expression and enhanced apoptosis. We found that SALL4 bound a specific region of the HOXA9 promoter in leukemic cells. SALL4 overexpression led to enhanced binding of histone activation markers at the HOXA9 promoter region, as well as increased HOXA9 expression in these cells. Furthermore, we observed that SALL4 interacted with mixed-lineage leukemia (MLL) and co-occupied the HOXA9 promoter region with MLL in AML leukemic cells, which suggests that a SALL4/MLL pathway may control HOXA9 expression. In summary, our findings revealed a molecular mechanism for SALL4 function in leukemogenesis and suggest that targeting of the SALL4/MLL/HOXA9 pathway would be an innovative approach in treating AML.

Fak depletion in both hematopoietic and nonhematopoietic niche cells leads to hematopoietic stem cell expansion

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-Cre-inducible 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.

Increased miR-155-5p and reduced miR-148a-3p contribute to the suppression of osteosarcoma cell death

Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer-related death in young adults. Currently, 5-year survival rates have plateaued at ~70% for patients with localized disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS may yield new approaches to improve outcomes for OS patients. To this end, we applied murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNAs) in OS. miRNA expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross-species comparison with human osteoblasts and OS cultures. We identified miR-155-5p and miR-148a-3p as deregulated in OS. miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, we used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. Although we could not resolve the mechanism through which miR-148a-3p impacts OS, we identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine–threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, small interfering RNA against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively, we show that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNAs was specifically toxic to tumor cells but not normal osteoblasts, raising the possibility that these may be tractable targets for miRNA-based therapies for OS.

Aberrant expression of SALL4 in acute B cell lymphoblastic leukemia: mechanism, function, and implication for a potential novel therapeutic target.

Treatment for high-risk pediatric and adult acute B cell lymphoblastic leukemia (B-ALL) remains challenging. Exploring novel pathways in B-ALL could lead to new therapy. Our previous study has shown that stem cell factor SALL4 is aberrantly expressed in B-ALL, but its functional roles and the mechanism that accounts for its upregulation in B-ALL remain unexplored. To address this question, we first surveyed the existing B-ALL cell lines and primary patient samples for SALL4 expression. We then selected the B-ALL cell lines with the highest SALL4 expression for functional studies. RNA interference was used to downregulate SALL4 expression in these cell lines. When compared with control cells, SALL4 knockdown cells exhibited decreased cell proliferation, increased apoptosis in vitro, and decreased engraftment in a xenotransplant model in vivo. Gene expression analysis showed that in SALL4 knockdown B-ALL cells, multiple caspase members involved in cell apoptosis pathway were upregulated. Next, we explored the mechanisms of aberrant SALL4 expression in B-ALL. We found that hypomethylation of the SALL4 CpG islands was correlated with its high expression. Furthermore, treatment of low SALL4-expressing B-ALL cell lines with DNA methylation inhibitor led to demethylation of the SALL4 CpG and increased SALL4 expression. In summary, to our knowledge, we are the first to show that the aberrant expression of SALL4 in B-ALL is associated with hypomethylation, and that SALL4 plays a key role in B-ALL cell survival and could be a potential novel target in B-ALL treatment.

Leukemic survival factor SALL4 contributes to defective DNA damage repair

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 downregulated in SALL4B Tg leukemic and pre-leukemic cells. Similar to the reported Fanconi anemia (FA) mouse model, chromosomal instability with radial changes 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.