A Novel TWIST2-ID2Axis That Modulates The Growth And Imatinib Sensitivity Of CML CD34+Cells

Objective: This study aims to elucidate the role of TWIST2-ID2instem/progenitor cells from healthy donors and chronic myeloid leukemia patients.Methods:(1) We first collected bone marrow from CML patients and healthydonors and use Immunomagnetic beads to purify CD34+cells, then used Q-RT-PCR(Quantitative Real-time PCR) and IF (Immunofluorescence) to detect the mRNAand protein levels of TWIST2expression in NBM (normal bone marrow) and CMLcells;(2) We used lentiviral vectors to construct shTWIST2, then transduced shNCand shTWIST2viruses to NBM-CD34+cells to detect the effect of shTWIST2oncell proliferation;(3) To obtain molecular insight of the function of TWIST2, weused lentiviral vectors to construct TWIST2and its several mutants, including theN-terminal truncation (ΔN), C-terminal truncation (ΔC), b-(deficiency in basicregion for DNA binding) and F86P (deficient to form hetero or homo dimmer), thenwe used western blot to identify the expression of them;(4) Lentiviral vectors wereconstructed to overexpress TWIST2and its mutants and then transduced intoMEG-01and K562CML cells, then we detected the effects of TWIST2on cellproliferation, the colony formation and nude mice tumorigenicity experiment;(5)We used Q-RT-PCR to test ID2mRNA levels and We prepared the luciferasereporter vector with ID2promoter and its mutant (the only E-box sequence in thepromoter was mutated), then detected the transactivation activity of the wide typepromoter in both K562and MEG-01cells;(6) Wild type ID2was amplified withPCR, and then we used lentiviral vector to constructed ID2to test its effect on CMLcell proliferation, CFC production and the response of Imatinib;(7) We usedlentiviral vectors to construct shID2, then transduced shNC and shTWIST2virusesto K562and NBM-CD34+cells to detecte the effect of shID2on cell proliferation;(8) NBM-CD34+cells were forced to express BCR-ABL oncogene, and then wetested its effect on TWIST2expresssion and the colony formation of NBM-CD34+. Results: Suppression the expression of TWIST2could produce more CFCscompared with that of control vectors. ChIP assay suggested that TWIST2coulddirectly bind ID2promoter in normal bone morrow CD34+cells, TWIST2silencecould inhibit the expression of ID2. We found that the expression of both TWIST2and ID2were lower in CD34+cells from chronic myeloid leukemia (CML) patients(n=14) than those from healthy donors (n=6), the protein expression of TWIST2andID2was lower in CD34+leukemic cells than normal controls as well. BCR-ABLsuppresses the expression of TWIST2in NBM-CD34+cells and TWIST2expressionwas enhanced when K562cells were treated with Imatinib mesylate. BCR-ABL andTWIST2silence cooperatively could promote the CFC production in normal CD34+cells. The forced expression of TWIST2was capable to inhibit the growth and theCFC production of CML cells including CML patients. TWIST2could also reducethe tumorigenic ability of MEG-01cells subcutaneously injected into nude mice(0/8for TWIST2group,7/8for control group). In addition, TWIST2could sensitizethe imatinib response of K562cells and CD34+cells from CML patients withimatinib resistance in chronic phase or in blast crisis. The dimmer formation wascritical for the function of TWIST2. ChIP assay was used to identify that TWIST2could directly regulate ID2in K562and MEG-01cells，forced expression of ID2could inhibit the growth of CML cells，and ID2silence could enhance the growth ofCML cells. We found that the suppressed CML cell growth, CFC production andsensitized Imatinib response by TWIST2was partially rescued by ID2knockdown.Conclusion:(1) TWIST2-ID2axis plays a novel regulatory role in normalhematopoietic cells；(2) BCR-ABL impairs TWIST2-ID2axis in CML cells；(3)TWIST2-ID2axis modulates the growth, IM sensitivity of CML cells；(4) TWIST2is a novel tumor suppressor of CML.