The Research On The Mechanism Of PRMT1-RBM15 Axis Regulatory Pathway In Normal Hematopoietic Regulation And Malignant Hematopoietic Diseases

BackgroundProtein Arginine Methlytransferase 1(PRMT1)is the predominant protein arginine methyltransferase in mammalian cells.It plays important regulatory roles in many biological processes through its arginine methyltransferase activity,such as alternative splicing,nuclei export and signal transduction.PRMT1 stimulate granulocyte differentiation through the arginine methylation of RUNX1,a critical transcription factor for definitive hematopoiesis,myeloid differentiation,and lymphocyte development.The PRMT1 protein interacts with and directely methylates AML1-ETO fusion protein in acute myeloid leukemia(AML).Knockdown of PRMT1 reduces the transcriptional activation of target genes that are activated by AML1–ETO and thus inhibits proliferation and self-renewal of AML1-ETO expressing leukemia cells.In addition,PRMT1 is significantly upregulated in the samples of newly diagnosed patients with pediatric ALL(acute lymphoblastic leukemia).RNA Binding Motif Protein 15(RBM15)is encoded by the RBM15 / OTT gene,which is located on chromosome 1.RBM15 recognizes RTE(RNA transport element)directly and specifically then stimulates the RNA export and expression of RTE-containing m RNAs;RBM15 cooperates with NXF1 during RTE-mediated m RNA export and expression.c-Myc is a target of RBM15 in adult hematopoietic stem cell and in the course of megakaryocytic development.To further understand the role of PRMT1 in normal and abnormal hematopoiesis,we found that RBM15 is methylated by PRMT1 at residue R578,leading to its degradation via ubiquitylation mediated by E3 ligase CNOT4.The study of conditional deletion of RBM15 in adult mice demonstrated the requirement for RBM15 in pre-B development and its essential role in myeloid progenitors and megakaryocytes.RBM15 binds to the intronic regions of certain pre-m RNAs that are encoded by genes crucial for megakaryopoiesis,such as GATA1,RUNX1,TAL1,and c-MPL.Furthermore,the preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for mediating alternative RNA splicing.Thus,the PRMT1-RBM15 axis regulates alternative RNA splicing via arginine methylation.These data imply that dysregulation of PRMT1-RBM15 axis may contribute to the leukemiagenesis of AML initiated by AML1-ETO as well as RBM15-MKL1 fusions.However,the detailed mechanism of PRMT1-RBM15 axis in the differentiation from stem cells to megakaryocytes and myeloid cells remains unclear.Many hematological malignancies have mutation genes and RNA alternative splicing.Myelodysplastic syndrome(MDS)is a group of heterogeneous blood disorders,it is believed that MDS is a clonal disease;MDS is characterized by ineffective bone marrow hematopoiesis,peripheral blood cytopenia and a risk of progression to acute myeloid leukemia.Approximately 80% of patients with MDS carry mutations of genes,such as epigenetic regulators(TET2,ASXL1,DNMT3 A,IDH1,IDH2,EZH2),transcription factors(ETV6,RUNX1,TP53),signal transduction proteins(CBL,JAK2,KRAS,NRAS),and genes related to the RNA splicing machinery(SF3B1,SRSF2,U2AF1,ZRSR2).Mutations on SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast(RARS),a subtype of MDS.SF3B1K700 E is the most frequently mutated site among mutations on SF3B1.Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear.Previously we demonstrated that RBM15 is responsible for recruiting SF3B1 and TAL1 to certain RNA intronic elements.TAL1 encodes a helix-loop-helix transcription factor,which is required for early hematopoiesis and for the generation of erythrocytes and megakaryocytes in adult hematopoiesis.TAL1 is one of the nine transcription factors critical for the formulation of the transcriptional regulatory network for hematopoiesis.TAL1 encodes full length and a short form TAL1 proteins missing N terminal region.Overexpression of the shortest form TAL1(TAL1s),which does not have N-terminal region upstream of the DNA binding domain,promotes erythroid differentiation of mouse bone marrow cells.Therefore,we hypothesized that the change of interaction among PRMT1-RBM15,SF3B1 and TAL1 may play a key role in the pathogenesis of MDS.In the second part of this thesis,we focused on the function of mutant SF3B1 in MDS via dysregulation of TAL1 alternative RNA splicing.One type of Acute megakaryoblastic leukemia(AMKL)features with the t(1;22)(p13;q13)chromosomal translocation,resulting in expression of the one twenty-two megakaryocytic acute leukemia(OTT-MAL)(also known as RBM15-MKL1)fusion protein.Rbm15-MKL1 knock-in mouse and knock-out Rbm15 mouse both display impaired terminal megakaryocytic differentiation.Rbm15-MKL1 knock-in mice develop AMKL with low penetrant.Introduction of MPLW515L(a constitutively active mutant of thrombopoietin receptor(TPOR/MPL)mutant)into Rbm15-MKL1 knock-in bone marrow cells induces AMKL with 100% penetrance.6133 cells,which are isolated from Rbm15-MKL1 knock-in mice,cause AMKL when transplanted.Using mouse Rbm15-MKL1 leukemia cell line 6133,we demonstrated that overexpression of MPLW515 L activates PRMT1 expression when MPLW515 L was introduced into 6133 cells.Furthermore,6133 cells expressing MPLW515 L grow in a cytokine independent fashion.PRMT1 expression level is higher in AMKL patient samples compared to those of other types of AML.Treatment of normal CD34+ cells with DB75(PRMT1 inhibitor)promoted megakaryocyte differentiation.DB75 has been demonstrated be a PRMT1 inhibitor.It is permeable and well tolerated in animal study.DB75(20?M)treatment was able to significantly inhibit the growth of various kinds of leukemia cell lines in culture.In this thesis,we investigated RBM15-MKL1 leukemia also has high PRMT1 expression and if inhibition of PRMT1 activity rescues the abnormal proliferation of AMKLIn summary,this thesis contributes the knowledge on mechanism of PRMT1-RBM15 axis in normal hematopoietic regulation and leukemogenesis in three parts.In part 1,we will address the mechanism of PRMT1-RBM15 axis in normal erythroid and megakaryocytic differentiation.The second part addresses the mechanism of PRMT1-RBM15 axis in SF3B1K700 E mutant-related abnormal erythroid hematopoiesis.The third part addresses the dysregulation of PRMT1-RBM15 axis in RBM15-MKL1-related AMKL.Base on the results on my thesis,we argue that PRMT1 inhibition may be a valid approach for curative therapy of AML.Part 1: The regulatory mechanism of PRMT1-RBM15 axis in normal erythroid and megakaryocytic differentiationObjectivesTo study the role of PRMT1 and RBM15 in the differentiation from normal hematopoietic stem cells to megakaryocytes and erythrocytes using stem progenitor isolated from human umbilical cord blood.MethodsCD34+ stem progenitor cells were isolated from human umbilical cord blood by using the anti-CD34 antibody magnetic beads and separator.PRMT1,RBM15,sh PRMT1,sh RBM15 # 1,sh RBM15 # 2 lentiviruses were packaged from transfected 293 T cells,using calcium phosphate transfection and then concentrated to 1/100 of the original volume by addition of PEG6000.CD34+ cells were infected with these viruses by standard spinfection method for suspension cells.48 hours after the last round of infection,infected cells were selected by flow cytometry or with puromycin.Cells were further cultured in medium supplemented with either 2IU /ml Erythropoietin(EPO)or 50 ng/ml Thrombopoietin(TPO)respectively.Flow cytometry analysis for CD71 or CD42/CD41 cells was performed after seven days of culture.Results1.PRMT1-RBM15 axis regulates the differentiation to mature megakaryocytes.The number of mature megakaryocytes increases in CD34+ cord blood stem progenitor cells overexpressing RBM15 or treated with PRMT1 inhibitor DB75;while overexpressing PRMT1 or using sh RNA method to knock down RBM15 lead to reduced megakaryocytic differentiation.2.PRMT1-RBM15 axis regulates the differentiation of erythroid.Erythroid differentiation was enhanced in CD34+ cord blood stem cells expressing PRMT1 or sh RBM15 respectively;while overexpressing RBM15 or DB75 treatment lead to repressed red blood cell formation.Part II: SF3B1K700 E dysregulation of PRMT1-RBM15 axis in abnormal erythropoiesisObjectivesTo investigate the mechanism of PRMT1-RBM15 axis in SF3B1K700E-initiated myelodysplastic syndrome.Methods1.To use Real-time PCR assays and Western blot analyze how PRMT1-RBM15 axis regulate the alternative RNA splicing of TAL12.We constructed lentiviral plasmids expressing-TAL1 s and Flag-TAL1 fl and used them to package viruses for infections of K562 cells.Fourty-eight hours after infection,positive cells were sorted by flow cytometry.After RNA extraction and c DNA synthesis,the expression level of endogenous TAL1 s,TAL1fl,ALAS2,?-globin and ?-globin were measured by Real-time PCR assays.Benzidine dye staining was used to identify hemoglobin-expressing cells.The number of mature red blood cells was counted by microscope.We constructed lentiviral plasmids expressing TAL1 s and TAL1 fl.We packaged lentiviruses in 293 T cells and used PEG600 to concentrate the volume of viral solution of 1/100.Human umbilical cord blood CD34+ cells were spinfected with the concentrated viruses.After 48 hours,virus-positive cells were sorted by flow cytometry.Positive cells were further cultured in medium containing 2IU/ ml EPO and the percentage of CD71+ red blood cell calculated by FACS analysis.3.Plasmids expressing Flag-TAL1 s,Flag-TAL1 fl,Flag-TAL1 s + ETO2,Flag-TAL1 fl + ETO2 were transfected into 293 T cells respectively.Whole cell extract was collected after 48 hours and Flag-tagged protein was purified by immunoprecipitation using anti-Flag beads.We used Western blot to detect the binding of two different isoforms of TAL1 to ETO2.Whole cell extract of sorted cells was made for immunoprecipitation and the binding of two different isforms of TAL1 to ETO2 was determinded by Western blot after IP.4.We obtained wild-type Flag-SF3B1 and mutant SF3B1K700 E lentiviral constructs from collaborators at Memorial Sloan-Ketterin Cancer Center.After confirming the plasmids by Sanger sequencing.48 hours after infection,positive cells were collected by flow cytometry and the total protein was collected and purified via immunoprecipitation.The effect of RBM15 on wild-type SF3B1 and mutant SF3B1K700 E was analyzed by Western blotting.Results1.PRMT1-RBM15 axis regulates TAL1 aternative splicing.Inhibition of PRMT1 enzymatic activity by DB75 enhances the production of TAL1 fl.PRMT1 overexpression or RBM15 knockdown increase the ratio of TAL1s/TAL1,while PRMT1 knockdown or RBM15 overexpression decreases the ratio of TAL1s/TAL1.These real-time PCR and Western blot results validate that RBM15 regulates TAL1 alternative splicing.2.When Flag-TAL1 s and Flag-TAL1 fl were overexpressed in K562 cells,cell pellets turned red,especially TAL1s-overexpressing K562 cells.Increased expression of TAL1 s did not increase the m RNA level of endogenous TAL1 s or AL1 fl,but the increased expression of TAL1 fl increased the endogenous TAL1 fl while endogenous TAL1 s was untouched.Increased expression of TAL1 s by ectopic expression promoted K562 cells to produce more ?-hemoglobin m RNA;also both TAL1 fl and TAL1 s activated the transcription of hemoglobin-producing gene ALAS2.TAL1s-expressing K562 cells contained higher percentage of Bezidine-positive cells,compare to the cells with TAL1 fl overexpression.The expression of both TAL1 s and TAL1 fl in umbilical cord blood stem cells could increase the number of mature red blood cells in culture,TAL1 s is more efficient to produce red blood cells.In this vein,overexpression of PRMT1 in cord blood stem cells lead to elevated production of CD71+ mature red blood cells.3.TAL1 fl binds to ETO2 in both 293 T cells or in K562 cells,while TAL1 s does not,which implies that the N-terminal of TAL1 fl is required for ETO2 binding.4.SF3B1K700 E binds to RBM15 more tightly to dysregulate alternative RNA splicing of TAL1.Compared with wild type SF3B1;mutant SF3B1 has increased RBM15 binding affinity.Mutant SF3B1 increases the m RNA level of TAL1 fl and decreased the m RNA ratio of TAL1 s / TAL1 fl.SF3B1 mutant has no effects on the m RNA levels of ALAS2 and hemoglobin genes.Part 3: The role of PRMT1-RBM15 axis in pathogenesis of RBM15-MKL1 fusion gene-initiated AMKLObjectivesTo study the mechanism of PRMT1-RBM15 signaling pathway in acute megakaryocytic leukemia initiated by RBM15-MKL1 fusion gene.Methods1.CD34+ stem progenitor cells were isolated from umbilical cord blood by using the anti CD34 magnetic beads.lentiviruses of RBM15-MKL1 and MPLW515 L were packaged,concentrated to 100 x then used for spinfection of CD34+ cells.Cells were infected with RBM15-MKL1,RBM15-MKL1 + MPLW515 L and MPLW515 L,respectively.After infection of 48 hours,single or double positive cells were sorted by flow cytometry.Positively Sorted positive cells were cultured in CD34+ cell growth medium and the number of cells was counted daily.At the same time,according to the procedure of the Canadian Stem Cell cloning assay kit,serial colony formation analysis was performed to quantify colony-forming units in each population.PRMT1 m RNA level was analyzed by Real-time PCR and PRMT1 protein was analyzed by flow cytometry.FACS sorted cells were cultured in megakaryocyte medium and the expression of CD41 CD42 was analyzed by flow cytometry on day 7.2.PRMT1 inhibitor(DB75)was added to the double positive cells overexpressing RBM15-MKL1 and MPLW515 L,and the effect of methylation inhibitor on cells was measured by cell viability assays and serial colony formation assays.Double-positive cells of RBM15-MKL1 + MPLW515 L sorted in 1 were cultured in megakaryocyte culture medium containing PRMT1 inhibitor(DB75)and then CD41/ CD42 were analyzed by flow cytometry after 7 days of culture.Results1.RBM15-MKL1 fusion protein supports long-term proliferation in cooperation of MPLW515 L active mutant.CD34+ umbilical cord blood stem cells co-expressing RBM15-MKL1 and MPLW515 L are capable of long-term proliferation in hematopoietic stem cell liquid culture medium and as well as on semi-solid medium a containing CD34+ grown cytokine mixtures for maintaining stem cells.PRMT1 was elevated in CD34+ cord blood stem progenitor cells co-expressing RBM15-MKL1 and MPLW515 L at both m RNA and protein level.Cells expressing by RBM15-MKL1 + MPLW515 L did not express CD41,after cultured for 7 days in megakaryocyte culture medium.2.PRMT1 inhibitor DB75 promotes the differentiation of transformed CD34+cells.When we added DB75 to cells co-expressing RBM15-MKL1 + MPLW515 L,cells stopped growing and died within three days.DB75 suppressed the colony formation of RBM15-MKL1 + MPLW515 L cells on methylcellulose medium.PRMT1 inhibitor(DB75)was able to reverse the megakaryocyte maturation disorder in the leukemia model caused by RBM15-MKL1 and MPL,resulting in an increase in the number of mature megakaryocytes.Conclusions1.PRMT1-RBM15 axis plays an important role in the regulation of megakaryocytes and erythrocytes in normal hematopoietic stem cells.2.RBM15 and SF3B1K700 E have crucial roles in SF3B1K700 E mutant myelodysplastic syndrome,leading to the reduction of TAL1 s and erythrocyte maturation.The increase of TAL1 s induced by PRMT1 elevation results in higher expression of ALAS2 and ?-globin,which are related to erythrocyte differentiation,leads to the increase of mature red blood cells.TAL1 s promotes red blood cell maturation differentiation due to the fact that it has lost the binding site for ETO2.PRMT1 might be a potential therapeutic target for improving the symptoms of anemia.3.PRMT1 promotes the transformative effect of RBM15-MKL1 during hematopoietic stem cell differentiation.PRMT1 inhibitor(DB75)was able to reverse this transformation.PRMT1 inhibitors may be novel targeting drugs for the treatment of RBK15-MKL1-induced AMKL.