Differentiation Of Acute Leukemia Cells Research Articles

Differentiation of Acute Leukemia Cells Including Cells with MLL-AF4 Rearrangements Induced by Jiyuan Oridonin A.

Chromosomal rearrangements involving the Mixed lineage leukemia (MLL) gene are observed in acute leukemia (AL) patients, which have poor prognosis, especially in infants. Hence, there is still a challenge to develop other effective agents to treat AL with MLL rearrangements (MLLr). MLL has been shown to rearrange with partner genes, of which the most frequently observed are AF4 and AF9. Moreover, AL is characterized by a differentiation blockage resulting in the accumulation of immature cells. An ent-kaurene diterpenoid compound, Jiyuan Oridonin A (JOA), has been shown to reduce the viability of AML cells by differentiation. We aimed to evaluate the effect of JOA on the growth and differentiation of AL cells (SEM, JURKAT and MV4-11) including cells with MLLr-AF4 by cell proliferation assay, colony formation assay, cell cycle analysis, cell apoptosis analysis, measurement of cell surface antigens, cell morphology, mRNA-sequencing analysis, quantitative Real-time PCR and Western blotting analysis. Our findings demonstrated that the proliferation of AL cells including cells with MLLr-AF4 was significantly suppressed by JOA, which induced cell differentiation followed by G0/G1 cell cycle withdrawal. Moreover, JOA-mediated cell differentiation was likely due to activation of G-CSFR in MV4-11 cells. Our results suggest that JOA may be considered a promising anti-leukemia compound to develop to surmount the differentiation block in AL patients.

The Histone Deacetylase Inhibitor I1 Induces Differentiation of Acute Leukemia Cells With MLL Gene Rearrangements via Epigenetic Modification.

Acute leukemia (AL) is characterized by excessive proliferation and impaired differentiation of leukemic cells. AL includes acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Previous studies have demonstrated that about 10% of AML and 22% of ALL are mixed lineage leukemia gene rearrangements (MLLr) leukemia. The prognosis of MLLr leukemia is poor and new therapeutics are urgently needed. Differentiation therapy with all-trans-retinoic acid (ATRA) has prolonged the 5-years disease-free survival rate in acute promyelocytic leukemia (APL), a subtype of AML. However, the differentiation therapy has not been effective in other acute leukemia. Here, we aim to explore the cell differentiation effect of the potent HDACs inhibitor, I1, and the possible mechanism on the MLLr-AML and MLLr-ALL cells (MOLM-13, THP-1, MV4-11 and SEM). It is shown that I1 can significantly inhibit the proliferation and the colony-forming ability of MOLM-13, THP-1, MV4-11 and SEM cells by promoting cell differentiation coupled with cell cycle block at G0/G1 phase. We show that the anti-proliferative effect of I1 attributed to cell differentiation is most likely associated with the HDAC inhibition activity, as assessed by the acetylation of histone H3 and H4, which may dictates the activation of hematopoietic cell lineage pathway in both MOLM-13 and THP-1 cell lines. Moreover, the activity of HDAC inhibition of I1 is stronger than that of SAHA in MOLM-13 and THP-1 cells. Our findings suggest that I1, as a chromatin-remodeling agent, could be a potent epigenetic drug to overcome differentiation block in MLLr-AL patients and would be promising for the treatment of AL.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

MicroRNA-16 Regulates Myeloblastosis Oncogene Expression to Affect Differentiation of Acute Leukemia Cells.

This study was designed to evaluate the effects of micro-RNA-16 (miR-16)-regulated expression of myeloblastosis oncogene (MYB) on the differentiation of acute leukemia cells, the expressions of miR-16 and MYB mRNA, and protein in differently differentiated leukemia cells were detected by real-time PCR and western blot. 1,25-Dihydroxyvitamin D3 (1,25 D3) induced monocytic differentiation of HL60 cells, and the resulting changes in miR-16 and MYB expressions were detected. Morphology of the cells induced by 1,25 D3, after being transfection with miR-16 mimics, was observed by Wright-Giemsa staining. The expression of mononuclear cell surface marker CD14 was detected by flow cytometry. Minimum miR-16 was expressed in early-differentiation KG-1a cells, while late-differentiation U937 and THP-1 cells had higher expressions (p < 0.01). The expressions of MYB changed oppositely. During the monocytic differentiation of HL60 cells, miR-16 expression showed a time-dependent increase, but MYB expression gradually decreased. Overexpression of miR-16 in HL60 cells promoted 1,25 D3-induced morphological changes and CD14 expression (p < 0.05). MR-16 facilitated the monocytic differentiation of leukemia HL60 cells by negatively regulating MYB expression.

Effect of Erbin on proliferation and differentiation of acute myeloid leukemia cells

Objective To elucidate the impact of Erbin on the cell cycle, proliferation, apoptosis and differentiation of acute myeloid leukemia (AML) cells. Methods The expression levels of Erbin in acute myeloid 1eukemia cell lines (HL60, THP-1, SHI-1, U937 and NB4) were analyzed by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. We exogenously increased the level of Erbin in the leukemia cell line with low-Erbin-expression and exogenously reduced the level of Erbin in the leukemia cell line with high-Erbin-expression. The proliferation capacity was assayed by cell counting kit-8 (CCK-8), cell cycle by Hoechst/PY, and apoptosis by Annexin V and differentiation by flow cytometry. Results U937 was selected as cell line with high-Erbin-expression and HL60 was selected as cell line with low-Erbin-expression. We found that proliferation (0.65±0.02 vs. 0.78±0.02) and G2/M cell cycle [(26.00±0.44)% vs. (33.24±1.29)%] were inhabited, early apoptosis [(9.51±0.81)% vs. (2.47±1.13)%] and differentiation were promoted [CD11b: (53.02±4.51)% vs. (38.59±4.62)%] in HL60 with exogenously elevated level of Erbin. Theaccordant results were also observed in U937 with exogenously decreased level of Erbin. The proliferation (0.68±0.04 vs. 0.56±0.05) and G2/M cell cycle [(20.30±0.83)% vs. (14.76±0.86)%] were promoted, differentiation [CD11b: (30.52±3.57)% vs. (51.26±6.21)%] was inhabited. Besides, the expression of Erbin was positively correlated with the expression of p21Waf1/CIP1 and p27Kip1. Conclusion Erbin may act as a cancer suppressor gene in AML cells, and may be a novel target for AML. Key words: Acute myeloid leukemia; Erbin gene; Cell proliferation; Apoptosis; Differentiation

MicroRNA-150 Expression Induces Myeloid Differentiation of Human Acute Leukemia Cells and Normal Hematopoietic Progenitors

In acute myeloid leukemia (AML) and blast crisis (BC) chronic myeloid leukemia (CML) normal differentiation is impaired. Differentiation of immature stem/progenitor cells is critical for normal blood cell function. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Aberrant miRNA expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. We have identified miRNAs differentially expressed in AML and BC CML and identified a new role for miR-150 in myeloid differentiation. Expression of miR-150 is low or absent in BC CML and AML patient samples and cell lines. We have found that expression of miR-150 in AML cell lines, CD34+ progenitor cells from healthy individuals, and primary BC CML and AML patient samples at levels similar to miR-150 expression in normal bone marrow promotes myeloid differentiation of these cells. MYB is a direct target of miR-150, and we have identified that the observed phenotype is partially mediated by MYB. In AML cell lines, differentiation of miR-150 expressing cells occurs independently of retinoic acid receptor α (RARA) signaling. High-throughput gene expression profiling (GEP) studies of the AML cell lines HL60, PL21, and THP-1 suggest that activation of CEPBA, CEBPE, and cytokines associated with myeloid differentiation in miR-150 expressing cells as compared to control cells contributes to myeloid differentiation. These data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role for this miRNA, and that absent or low miR-150 expression contributes to blocked myeloid differentiation in acute leukemia cells.

TRIADs: a new class of proteins with a novel cysteine-rich signature.

Triad1 was recently identified as a nuclear RING finger protein, which is up-regulated during retinoic acid induced granulocytic differentiation of acute leukemia cells. Here we show that a cysteine-rich domain (C6HC), present in Triad1, is conserved in at least 24 proteins encoded by various eukaryotes. The C6HC consensus pattern C-x(4)-C-x(14-30)-C-x(1-4)-C-x(4)-C-x(2)-C-x(4)-H-x(4)-C defines this structure as the fourth family member of the zinc-binding RING, LIM, and LAP/PHD fingers. Strikingly, in 22 of 24 proteins the C6HC domain is flanked by two RING finger structures. We have termed the novel C6HC motif DRIL (double RING finger linked). The strong conservation of the larger tripartite TRIAD (two RING fingers and DRIL) structure indicates that the three subdomains are functionally linked and identifies a novel class of proteins.