The Role Of C/EBPα Abnormal Expression And Mutation In Drug Resistance And Immune Evasion Of Acute Myeloid Leukemia

Chapter 1Impact of C/EBPα abnormal expression on the differentiation block and ATRA resistance of APL cellsIntroduction CCAAT enhancer binding protein alpha (C/EBPa) is an important transcription factor in myeloid differentiation, which determines the destiny of hematopoietic cells in early hematopoiesis. Supression of C/EBPα was observed in many AML subtypes including M3 (APL), AML1-ETO positive M2 and CBFB-MYH11 positive M4. We found that C/EBPa was expressed differently in all-trans-retinoic acid (ATRA) sensitive APL cell line NB4 cells and ATRA resistance APL cell line NB4-R1 cells. Impact of C/EBPa abnormal expression on the sensitivity of APL cells to ATRA has not been researched yet. Restoring of C/EBPa was surposed to be a potential method to enhance ATRA sensitivity of APL cells.Aim This part intended to reveal the reasons of C/EBPa abnormal expression in ATRA resistant APL cells, then to explore the impact of abnormal C/EBPa expression on the differentiation block and ATRA resistance of APL cells. This research will provide theoretic support to the prophylaxis and treatment of APL relapse.Methods We compared C/EBPa expression in ATRA sensitive and resistant APL cells(NB4 and NB4-R1 respectively) through western blot analysis. To establish 42KDa or 30KDa C/EBPα over-expressed cell lines, we employed lentivirus to infect NB4-R1 cells and selected positive cells by blasticidin. Then we performed FCM (flow cytometry) and PCR (polymerase chain reaction) to measure the expression of differentiation clusters and differentiation related genes. We then treated NB4-R1 cells with ATRA and evaluated cell differentiation and apoptosis by FCM. Morphological changes was observed by giemsa staining. Finally we treated NB4-R1 cells with histone deacetylaze inhibitor TSA and measured the change of C/EBPα expression by western blot analysis. Synergistic effect of TSA and ATRA on promoting cell differentiation and inhibiting cell growth was tested by FCM and MTT (methyl thiazolyl tetrazolium) assay.Results C/EBPα expression in NB4-R1 cells was significantly lower than that in NB4 cells.42KDa C/EBPα almost disappeared in NB4-R1 cells. Reduced PI3K/Akt/mTOR pathway activation and increased eIF2α activation were found in NB4-R1 cells. Inhibiting PI3K/Akt/mTOR pathway by LY294002 and RAD001 reduced C/EBPα expression in NB4 cells. Restoring of 42KDa but not 30KDa C/EBPα expression in NB4-R1 cells increased the expression of differentiation clusters CD11b and CD14 and differentiation related genes G-CSFR and GM-CSFR. Over-expressing of 42KDa C/EBPα also suppressed transcription of PU.1 encoding gene SPI1. This surpression effect might favor granulocyte over macrophage differentiation. FLT3 gene also suppressed by over-expressing of 42KDa C/EBPa in NB4-R1 cells. Restoring 42KDa C/EBPα expression also enhanced cell differentiation induced by ATRA. Expression of CD11b obviously up-regulated when 42KDa C/EBPα over-expressing NB4-R1 cells was treated with 1μM ATRA. TSA treatment up regulated C/EBPα expression in NB4-R1 cells and showed a synergistic effect with ATRA in differentiation promoting and growth arrest.Conclusions Abnormal expression of C/EBPα induced by PI3K/Akt/mTOR inhibition and increased eIF2a activation was an important reason of ATRA resistance in APL cells. Restoring 42KDa but not 30KDa C/EBPa expression in NB4-R1 cells could promote cell differentiation and enhance the sensitivity to ATRA treatment. Up-regulation of C/EBPa expression was also the mechanism of synergistic effect of TSA with ATRA in promoting NB4-R1 cell differentiation and inhibiting cell growth.Chapter 2The role of CEBPA mutation in immune evasion of acute myeloid leukemia cells from NKG2D induced cytotoxicityIntroduction Nature killing (NK) cells are one kind of the most important cytotoxic cells in anti-leukemia immune surveillance. Recognition and binding of NKG2D receptor with its ligands are essential for the cytotoxic effect of NK cells. Reduced expression of NKG2D ligands were often found in leukemia cells and were main reason of immune evasion and relapse. Therefor the mechanisms involved in the regulation of NKG2D ligands expression urgently need to be research. Mutations of CCAAT enhancer binding protein alpha (C/EBPa) encoding gene CEBPA were reported responsible for leukemia onset and relapse. In our early study we found that CEBPA gene and its different types of mutations regulated the expression of NKG2D ligand UL-16 binding proteins (ULBPs). These regulatory effects might provide targets for anti-leukemia immunotherapy.Aim This part intended to confirm the regulatory effect of C/EBPa on the expression of NKG2D ligands, and to research the impact of different kinds of CEBPA mutation on the regulation ability of C/EBPa. The final aim of this study was to reveal the role of CEBPA mutation in the immune evasion of leukemia cells and to provide target for anti-leukemia immunotherapy.Methods Wide type C/EBPa, truncated 30KDa C/EBPaand different kinds of mutations coding sequence were over-expressed in leukemia cell lines through lentivirus infection system. Mutation types were as follow:frameshift mutation in first transcription activation domain of N’terminal(NMl), duplication in second transcription activation domain of N’terminal(NM2) and duplication in basic leucine zipper domain of C’terminal(CM). C/EBPα expression was tested through western blot analysis.NKG2D ligands expression in infected leukemia cell lines was measured using flow cytometry (FCM). Infected cells were incubated with NK92MI cells to test cytotoxic lysis of leukemia through PI/CFSE staining and test degranulation of NK92MI cells through CD 107a staining. Binding information of C/EBPaand promoter sequence of ULBP1 gene was predicted using information from NCBI and SABiosciences database.Results Wide type C/EBPa up-regulated ULBPs expression in leukemia cell line NB4 cells, whose innate ULBPs were rarely expressed. Sensitivity of NB4 cells were also increased when over-expressed wide type C/EBPa. NM2 mutation kept the regulatory ability of C/EBPa to ULBPs expression. ULBPs and cytotoxic lysis were also up-regulated when NB4 cells were over expressed NM2 mutated C/EBPa. However, NM1 mutation, CM mutation and P30 lost the regulatory ability of C/EBPa to ULBPs expression. These isotypes of C/EBPa could not increase ULBPs expression and cytotoxic lysis induced by NK92MI cells. Even worse NM1 mutation, CM mutation and P30 decreased innate ULBP2/5/6 expression in K562 cells. Granzyme pathway was involved in cytotoxic effect of NK92MI cells. Degranulation rate of NK92MI cells was higher when co-incubated with wide type or NM2 C/EBPaover expressed NB4 cells but not in NM1 mutation or CM mutation or P30 C/EBPaover expressed NB4 cells.Conclusions C/EBPa up-regulated expressions of ULBPs as a transcription factor. Over expression of C/EBPa in leukemia cell NB4 could increase cytotoxic lysis induced by granzyme releasing of NK92MI cells. Integrity of TAD1 and bZIP domain was necessary for regulatory ability of C/EBPα to ULBPs expression. Leukemia cells with disrupted C/EBPα at TAD1 or bZIP domain could escape from surveillance of NK cells and act as origin of leukemia relapse.