Discovery,Leukemogenic Mechanism And Treatment Of A Novel Recurrent CPSF6-RARG Fusion

Objectives1.To discover novel recurrent CPSF6-RARG fusions in atypical acute promyelocytic leukemia.2.To explore the functions of CPSF6-RARG fusions in hematopoietic system.3.To explore whether CPSF6-RARG fusions can drive leukemia development or aggressiveness.4.To explore the function mechanism of CPSF6-RARG fusions.5.To explore whether CPSF6-RARG fusions are resistant to retinoic acid.Methods1.We retrospectively analyzed the clinical characters of suspected APL patients from January 2003 to December 2016 in our center.2.To explore the functions of CPSF6-RARG fusions,we transfected human cord blood CD34+stem/progenitor cells by CPSF6-RARGs to perform clone formation,longterm liquid culture and xenograft mice experience.3.We generated CPSF6-RARGs knock-in mice to study whether CPSF6-RARG fusions could drive leukemia development or aggressiveness.We detected the immunophenotype of hematopoietic cells from CPSF6-RARGs KI mice.Furthermore,we transduced CPSF6-RARGs KI murine primary hematopoietic stem/progenitor cells with NRAS-G12D mutation to explore leukemia development.4.To compare with PML-RARA,we performed cellular localization,protein-protein interaction and luciferase assay.Meanwhile,the pathway affected by CPSF6-RARG fusions was studied by RNA sequencing,ChIP sequencing and RIP sequencing.5.We transduced U937 cells or human cord blood CD34+cells by CPSF6-RARGs.And then we detected the cell viability,proliferation,apoptosis,differentiation and protein degradation to study the response to ATRA.Results1.We collected a total of 1,401 patients with suspected APL from January 2003 to December 2016 in our center.In total,19 patients with alternative RARA or RARG fusions were identified.We first found a novel recurrent CPSF6-RARG fusion in 2 patients.The 3’region of RARG(from exon 1 or exon 4 to exon 9)was reversed and fused in-frame with the 5’-region of CPSF6 gene(from exon 1 to exon 4).The longer transcription was named CPSF6-RARG-L,the other one was named CPSF6-RARG-S.2.CPSF6-RARG fusions increased self-renewal ability of human cord blood CD34+cells(clone replating capacity:CPSF6-RARGs vs.Vector=5 times vs.3 times).CPSF6RARG fusions increased myeloblast cells(CPSF6-RARGs vs.Vector:51.0±1.7%/41.4±9.6%vs.15.5±4.8%,P<0.001)and hematopoietic stem/progenitor cells(HSPCs)(CPSF6RARGs vs.Vector:18.8±3.8%/21.0±6.2%vs.12.3±1.5%,P<0.001).CPSF6-RARG fusions inhibited lymphoid reconstitution and blocked myeloid maturation(CD 19+:CPSF6RARGs vs.Vector:40.9±16.3%/35.0±14.8%vs.73.6±6.9%,P<0.001;CD33+CD66b+:CPSF6-RARGs vs.Vector:0.683±0.006%/0.589±0.013%vs.29.8±8.1%,P<0.001).3.CPSF6-RARGs knock in mouse(CPSF6-RARGs KI)increased myeloid development and inhibited lymphoid and erythrocyte development(myeloblast:CPSF6RARGs KI vs.KI-:8.6±0.6%/6.9±0.4%vs.3.8± 1.0%,P<0.001).CPSF6-RARGs KI mouse increased stem and progenitor populations(MPP:CPSF6-RARGs KI vs.KI-:0.11±0.03%/0.13±0.007%vs.0.05±0.007%,P<0.001).CPSF6-RARGs KI mouse cells owned more self-renewal ability(GM colonies:CPSF6-RARG-L KI vs.KI-:29.9±8.1%vs.11.3±2.8%,P=0.003;CPSF6-RARG-S KI vs.KI-:27.6±1.5%vs.11.3±2.8%,P<0.001).With a follow-up of 592 days,CPSF6-RARGs alone could not induce leukemia.4.CPSF6-RARGs fusions increased NRAS-G12D cells self-renew.CPSF6-RARGs fusions combined with NRAS-G12D mutation accelerated the occurrence of leukemia.With a median survival time of 19/20 days,the phenotype of mice showed amplification of myeloblast cells(CPSF6-RARG-L-NRAS vs.WT-Vector:78.9±9.1%vs.6.4±1.5%,P<0.001;CPSF6-RARG-S-NRAS vs.WT-Vector:80.5±5.1%vs.6.4±1.5%,P<0.001).5.CPSF6-RARG fusions formed a homodimer,recruited corepressors and repressed transcription as PML-RARA.CPSF6-RARG fusions formed Cleavage factor complex I with CPSF5 or CPSF7.CPSF6-RARG fusions influenced the pathway in pluripotency of stem cells and transcription in cancers.6.CPSF6-RARG fusions could not be degraded with ATRA or ATO.CPSF6-RARGs cells were induced to apoptosis(CPSF6-RARGs vs.Vector:70%vs.12%,P<0.001)after ATRA treatment.The rest live cells could not be induced to differentiation.Ratio of myeloblast cells was 32±2%.ATRA partially released transcription repression.Conclusions1.All harbor the first four exons of CPSF6 including a RNA recognition motif(RRM)fuses in frame to either 5’ UTR or exon 4 of RARG.None of the patients do not show any ATRA sensitivity.2.CPSF6-RARG fusions are critical for cell differentiation resulting in both a differentiation block and an expansion of myeloid progenitors.3.CPSF6-RARG fusions provide a pre-leukemia status and combine with NRASG12D mutation can accelerate the occurrence of leukemia.4.CPSF6-RARG fusions form a homodimer,recruit corepressors and repress transcription.CPSF6-RARG fusions can influence the pathway in pluripotency of stem cells and transcription in cancers.5.CPSF6-RARG fusions are partial resistant to ATRA.