The Mechanisms Of MAT1 Protein Fragmentation And Downstream RARα Hypophosphorylation In Inhibiting Myeloid Leukemia Proliferation

Section 1 The mechanism study of MAT1 expression/fragmentation in normal and malignant hematopoietic cells’proliferation/differentiationObjective:Myeloid leukemia is a malignant hematopoietic disease, which pathogenesis remains incompletely understood and patient survival rate is low. MAT1, a protein involved in regulating cell cycle and gene transcription, is cleaved during normal granulocyte differentiation of hematopoietic stem cells, but is highly expressed in its full-length form in ATRA-resistant myeloid leukemic cells. This suggests that the expression/fragmentation of MAT 1 is closely related to the proliferation/differentiation of normal and malignant hematopoietic cells, though the mechanism remains elusive. This section will discuss the effect and mechanism of MAT 1 expression/fragmentation on normal and malignant hematopoietic cells’proliferation/differentiation in vitro and in vivo.Methods:Human hematopoietic stem cells CD34+ cells, primary myeloid leukemia cells and myeloid leukemia cell lines were used in this study. (1) Trypan blue staining and hemocytometer counting were used to calculate cell survival, cell death and doubling time; (2) Molecular cloning techniques were applied to construct MAT1 and pM9 plasmid, and to produce lentivirus for the overexpression of MAT1 and pM9 protein in cells; (3) Western Blotting assay was used to detect the expression and phosphorylation levels of proteins; (4) Flow cytometry was performed to analyze the percentage of GFP-expressing cells and cell surface antigen CD11b, CD66, CD34 and CD19; (6) Wright-Giemsa staining was used for the detection of nuclear morphology; (5) qRT-PCR assay was used to analyze mRNA levels; (7) Immunofluorescence was used to detect MAT1 and p21Cip/Kip protein expression in CD34+ cells; (8) Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine the amino acid sequence of MAT 1 fragments; (9) Immunoprecipitation was used for the detection of protein binding; (10) HE staining and immunohistochemistry staining were used to analyze protein expression in tissue sections.Results:(1) MAT1 overexpression sustains CD34+cell expansion and suppresses granulocytic differentiationThe overexpression of MAT1 in CD34+ cells not only inhibited endogenous MAT1 fragmentation, but also promoted CD34+ cells expansion in vitro. Furthermore, flow cytometry analysis found that MAT1 overexpression inhibited the expression of CD66 and CD11b, the maturation markers of granulocytic differentiation. Wright-Giemsa staining further confirmed that MAT1 overexpression inhibited nuclear segmentation, which demonstrated that MAT1 expression sustains the hematopoietic expansion and inhibits the granulocytic differentiation of CD34+ cells in vitro. Next, a humanized mouse microenvironment by coengrafting CD34+ cells together with human mesenchymal stem cells into NSG mice was established for studying MAT 1-modulated granulopoiesis. Flow cytometry and qRT-PCR analysis showed that MAT1 overexpression promoted bone marrow reconstitution of hematopoietic precursors, enhanced expansion of CD34+ cells in peripheral blood, inhibited CD11b and CD66 marker transcription and expression. The above results repeat that MAT1 maintains hematopoietic expansion while inhibiting granulopoiesis of hematopoietic precursors in vivo. As the increased level of MAT1 protein was associated with decreased p21Cip/Kip expression, these findings suggest that down-regulation of p21Cip/Kip expression is one of the mechanisms by which MAT1 inhibits granulopoiesis and promotes the expansion of primitive hematopoietic precursors.(2) MAT1 fragment pM9 inhibits the proliferation of myeloid leukemia cells by decreasing CAK and TFIIH activityLC-MS/MS sequencing analysis of MAT1 and its cleavage fragment M30 showed that MAT1 is cleaved at C-terminus to generate M30 and pM9. After transfecting lentiviral Flag-pM9 in myeloid leukemic HL60, HL60R and Jurl-MKl cell lines, we found that pM9 competed with endogenous MAT1 to consume cyclin H and CDK7 to form ΔCAK, while inhibited endogenous MAT1 expression. Owing to the lack of domain that interacts with the XPD and XPB subunits of TFIIH-core, ΔCAK cannot assemble TFIIH kinase, thus pM9 could significantly inhibit the endogenous formation of CAK and TFIIH. Further study revealed that pM9 inhibited the in vitro proliferation of myeloid leukemia cells and promoted the granulocytic differentiation of primary leukemic cells, which may result from the reduction of CDK7 phosphorylation, CAK’s hypophosphorylation of downstream targets CDK1 and RARa, and interference with TFIIH-mediated gene transcription. However, pM9 did not affect the expansion of normal hematopoietic CD34+ precursor cells, but specifically inhibited leukemic growth, which likely resulted from the different phosphorylation levels of RNA Pol Ⅱ. In vivo data also demonstrated that pM9 can overcome the resistance of MAT1 fragmentation in ATRA-resistant cells to inhibit proliferation and metastasis.Conclusion:MAT1 expression promotes the expansion of hematopoietic precursors and inhibits granulocyte differentiation, whereas pM9 shares a mechanistic resemblance with MAT1 fragmentation in specifically suppressing myeloid leukemogenesis by inversely coordinating CAK and TFIIH activities.Section 2 Decrease in RARaS77 phosphorylation overcomes ATRA-resistance to induce transcription response inhibiting t(8;21) AML cell proliferationObjective:Impaired CAK-RARa signaling pathway is one of the mechanisms contributing to non-APL AML cells’ATRA resistance. The first section has confirmed that MAT1’s fragment pM9 can form ΔCAK, thereby inhibiting RARa phosphorylation and suppressing the proliferation and metastasis of myeloid leukemia cells. In this section, we will attempt to clarify the effect of RARa phosphorylation on gene transcription and cell proliferation in ATRA-resistance t(8;21) AML, and reveal the correlation between RARa phosphorylation and ATRA-resistance, which may provide insight for the design of novel anti-leukemia drugs targeting RARa phosphorylation.Methods:Primary t(8;21) AML cells and t(8;21) AML cell lines were used in this study. (1) Molecular cloning techniques were applied to construct Flag-tagged RARa and RARaS77A plasmid, and to produce lentivirus for the overexpression of RARa and RARaS77A protein in cells; (2) Trypan blue staining and hemocytometer counting were used to calculate cell survival and cell death; (3) DNA agarose gel electrophoresis was used to detect nucleosome fragments during apoptosis; (4) Western Blotting assay was used to detect the expression and phosphorylation levels of proteins; (5) qRT-PCR assay was used to analyze mRNA levels; (6) Flow cytometry was performed to analyze cell cycle and the percentage of GFP-expressing cells; (7) Immunoprecipitation was used for the detection of protein binding; (8) HE staining and immunohistochemistry staining were used to analyze protein expression in tissue sections; (9) Chromatin immunoprecipitation and qPCR analysis were used to detect the effect of specific protein on target gene transcription.Result:By overexpressing hypophosphorylated RARaS77 mutant RARaS77A in many myeloid leukemia cells, we found that RARaS77A can promote the transcription of RA-target genes regulating granulocytic differentiation and apoptosis, thereby inhibiting cell proliferation and induce apoptosis of ATRA-resistant t(8;21) AML cells in vitro. The RARaS77A-induced gene transcription can be further enhanced by ATRA combination. Depletion of retinoid or blockade of RARa-LBD by a selective RARa antagonist Ro 41-5253 did not abolish RARaS77A-mediated suppression of proliferation and induction of RA-target genes transcription, which suggested that the effect of RARaS77A is independent of both ligand stimulation and RARa-LBD activation. In NSG mice xenografted with ATRA-resistant SKNO-1 cells expressing RARaS77A and treated with or without ATRA, flow cytometry analysis found that compared to vector group, RARaS77A or RARaS77A+ATRA can significantly reduce the percentage of donor cells in peripheral blood and bone marrow, while preventing lymph node metastasis in vivo. To elucidate how hypophosphorylated RARaS77 induces transcription of RA-target genes to suppress proliferation of t(8;21) AML cells, we investigated RARaS77A, RARa, RNA Pol Ⅱ and ETO-mediated genetic interaction with the RARE loci of RA-target genes by performing a series of ChIP analyses. The results showed that RARaS77A enhanced its binding to RARE loci, allowed its interaction with TFIIIH p89 and recruitment of RNA Pol Ⅱ, but prevented formation of complexes with AML1/ETO, HDAC1 and DNMT3a, which suggested that RARaS77A could induce gene transcription via remodelling the repressive genetic and epigenetic chromatin surrounding the promoter of RA-target genes.Conclusion:Hypophosphorylation of RARaS77 can circumvent ATRA-dependent activation to induce transcription of RA-target genes regulating differentiation and apoptosis by remodeling genetic and epigenetic chromatins, thereby inhibiting the proliferation of ATRA-resistant t(8;21) AML cells.