ABSTRACT
Acute myeloid leukaemia (AML) with chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukaemia (MLL) is an aggressive subtype with low overall survival. Bortezomib (Bort) is first applied in multiple myeloma. However, whether bort possesses anti-self-renewal and leukemogenesis of leukaemia stem cell (LSC) in AML with MLL rearrangements is still unclear. Here, we found that bort suppressed cell proliferation and decreased colony formation in human and murine leukaemic blasts. Besides, bort reduced the frequency and function of LSC, inhibited the progression, and extended the overall survival in MLL-AF9 (MF9) -transformed leukaemic mice. Furthermore, bort decreased the percentage of human LSC (CD34+ CD38- ) cells and extended the overall survival in AML blasts-xenografted NOD/SCID-IL2Rγ (NSG) mice. Mechanistically, cyclin dependent kinase 6 (CDK6) was identified as a bort target by RNA sequencing. Bort reduced the expressions of CDK6 by inhibiting NF ĸB recruitment to the promoter of CDK6, leading to the abolishment of NF ĸB DNA-binding activity for CDK6 promoter. Overexpression of CDK6 partially rescued bort-induced anti-leukemogenesis. Most importantly, bort had little side-effect against the normal haematological stem and progenitor cell (HSPC) and did not affect CDK6 expression in normal HSPC. In conclusion, our results suggest that bort selectively targets LSC in MLL rearrangements. Bort might be a prospective drug for AML patients bearing MLL rearrangements.
Subject(s)
Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Cell Self Renewal/drug effects , Cyclin-Dependent Kinase 6/metabolism , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , NF-kappa B/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Animals , Cell Line, Tumor , Computational Biology , Cyclin-Dependent Kinase 6/genetics , Disease Models, Animal , Dose-Response Relationship, Drug , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Gene Order , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Prognosis , Xenograft Model Antitumor AssaysABSTRACT
[This corrects the article DOI: 10.7150/thno.42795.].
ABSTRACT
YAP1 is a key mediator of the Hippo pathway capable of exerting a profound effect on organ size as well as tumorigenesis. Alternative mRNA splicing of human YAP1 results in at least 8 protein isoforms that differ within the 2nd WW motif and the transcriptional activation domain. Methods: To investigate the isoform-specific differences in their mRNA expression, transcriptional activity and tumor-promoting function, we cloned cDNA encoding all of the eight YAP1 protein isoforms. Then, we examined their mRNA expression, subcellular localization, transcriptional regulation properties, interactions with key regulatory partners, and protein stability in response to changes in cell density, as well as their effects on pancreatic cancer cell malignancy both in vitro and in vivo. Results: Multiple YAP1 mRNA isoforms are expressed in commonly used pancreatic cancer lines as well as human pancreatic cancer PDX lines. Based on the analysis of heterologous reporter and endogenous target genes, all YAP1 isoforms are capable of activating transcription, albeit to a different extent. Importantly, we unveiled a marked discrepancy between the mRNA and protein expression levels of the YAP1-1 and YAP1-2 isoforms. We further discovered that the YAP1-2 isoform, which contains two tandem WW motifs, is less stable at the protein level, particularly at high cell densities. Mechanistically, we found that the presence of the 2nd WW motif in YAP1-2 facilitates the de novo formation of the YAP1-2/AMOT/LATS1 complex and contributes to a stronger binding of YAP1-2 to LATS1 and subsequently increased YAP1-2 ubiquitination and degradation by ß-TRCP. Conclusion: Our data reveals a potent effect of YAP1-1 on pancreatic cancer malignancy in vitro and in vivo and provides novel mechanistic insight into isoform-specific and cell density-dependent regulation of YAP1 stability, as well as its impact on cancer malignancy.
