Dysregulated RPB1 Is A Targetable Master Driver Of Overgrowth And Drug Resistance In Acute Myeloid Leukemia

Background:Acute myeloid leukemia(AML)is a group of highly aggressive malignancies with cytogenetic and molecular abnormalities,as characterized by a blockade in differentiation of hematopoietic stem cells and a clonal expansion of myeloid blasts in the bone marrow and peripheral blood.Although a number of therapies have been approved for AML over the past decades,such as the standard "7+3" induction therapy,which combines a nucleoside analogue such as cytarabine for 7 days with an anthracycline for 3 days(DA regimen),and are effective in killing leukemia cells in AML,the vast majority of AML patients suffer from lethal disease relapse within 3 years.The 5-year overall survival(OS)is below 40%.One of biggest challenges in treating AML is the inability of conventional therapies to kill rapidly proliferating tumor cells with defective apoptosis.Thus,elucidation of the molecular mechanisms by which cancer cells maintain rapidly proliferating ability and drug resistance is expected to provide a basis for developing more potent and safer therapies for curing AML.Transcription is a "vital process" required for all living cells.However,cancer cells need high levels of transcription for maintaining rapid proliferation and survival,and dysregulated transcription is universal in cancer.For instance,RNA polymerase ?(RNAPI)transcriptional overactivity was thought to be essential for the survival of hematological tumor cells and can be therapeutically targeted in vivo.RNAP? activity is increased in tumor cells compared with normal cells.In contrast to the higher expression of RNAPI and RNAP ? in cancer,heterozygous loss of POLR2A,which encodes the largest subunit of RNAPII(RPB1),is frequently observed in human colorectal,breast cancer and prostate cancers,although it is thought to be required for transcribing oncogenic genes and anti-apoptotic factors maintaining rapid growth and apoptosis resistance of tumor cells.In addition,POLR2A mutation was also reported in a distinct subset of meningiomas.However,the expression of RPB1 has not previously been linked to human AML and other cancer.Objective:1.To analyze the expression of RPB1 and its coding gene POLR2A in AML patients,cell lines and control samples,and to explore the association between the expression of RPB1 with clinical features in acute myeloid leukemia samples.2.To analyze the molecular mechanism of RPB1 in accelerating the development of acute myeloid leukemia.Methods:1.We analyzed the RPB1 expression in AML cell lines,primary AML patients,cord blood and peripheral blood samples.The expression and location of RPB1 was analyzed in AML cell lines,primary AML patients,cord blood and normal hematopoietic stem cells.We analyzed the expression of phosphorylated and total RPB1 in AML cell lines,CD34+AML cells,quiescent or mobilized human hematopoietic stem/progenitor cells.POLR2A mRNA expression was retrospectively analyzed with AML cell lines,newly diagnosed AML samples and healthy PB samples as a control.Meanwhile,the mRNA expression levels of POLR2A in AML and 30 other tumor samples were analyzed by the Cancer Genome Atlas dataset.2.Correlation analysis was used to explore the correlation between the expression of RPB1 with tumor burden and survival time in untreated AML samples.We analyzed the POLR2A expression in different AML subtypes,ALL and normal hematopoietic cells from BloodSpot analysis.3.First,we constructed inducible POLR2A knockout AML cells.And detected the effect of RPB1 silencing on cell survival,proliferation and cell cycle through cell growth curve,soft agar clone formation,cell apoptosis and cell cycle assay.4.Overexpressed RPB1 AML cells were constructed,the effect of RPB1 expression on cell growth and proliferation was detected by cell growth curve and soft agar clone formation assay.5.Using RNA-Seq approach to identify the differentially expressed genes between RPB1 silencing AML cells with the controlled cells after knocking out RPB1,and classifing the differentially expressed genes.6.Using orthotopic AML models established with RPB1 silencing and control AML cells to investigate the effect of RPB1 protein level on the anti-leukemia activity of triptolide.Results:1.RPB1 is aberrantly activated in AML cell lines and primary AML patients compared to normal hematopoietic cells.Moreover,we analyzed the POLR2A mRNA expression data from TCGA dataset and found the expression of POLR2A in AML was higher than normal blood cell samples.2.RPB1 levels positively correlated with the malignant proliferative potential of leukemia cells in AML patients.And the AML patients with high level of RPB1 had a significantly shorter overall survival than patients with low level of RPB1.There is no lineage differences in POLR2A expression among AML subtypes and ALL.3.We constructed an inducible CRISPR/Cas9 system to silence RPB1.RPB1 protein levels in POLR2A-KO cells was reduced to 6.81%compared to control cells at the fourth day after Dox induction and RPB1 silence dramatically decreased viability of AML cells.In addition,the MOLM-13 cells displayed potent apoptosis and cell-cycle is arrested in S phase.4.RPB1 overexpression enhances proliferation and colony formation of AML cells.5.RNA-Seq technology was used to identify differentially expressed genes in RPB1-silencing and control AML cells.A total of 1836 differentially expressed genes in the basis of at least>1.5-fold were screened,of which 995 genes were down-regulated and 10%down-regulated genes were associated with cancer.6.Consistent with the in vitro results,a significant reduction in tumor signal was observed with RPB1 silencing compared with control at 7 day after POLR2A-KO initiation in AML orthotopic model.The mouse with low expression of RPB1 is more sensitive to Triptolide mediated anti-leukemia activity.Conclusion:1.Dysregulated RPB1 is a critical oncogenic hub that drives overgrowth and drug resistance by hijacking an array of oncogenic and anti-apoptosis factors.2.Targeting silencing RPB1 leads to potent regression of human refractory AML in mouse models and enhance anti-leukemia activity of Triptolide.