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Induction of prolonged early G1 arrest by CDK4/CDK6 inhibition reprograms lymphoma cells for durable PI3K? inhibition through PIK3IP1.

ABSTRACT: Phosphatidylinositol-3-kinase (PI3K) signaling is constitutive in most human cancers. Selective inhibition of PI3K? (p110?) by GS-1101 has emerged as a promising therapy in chronic lymphocytic leukemia and indolent lymphomas. In aggressive non-Hodgkin lymphomas such as mantle cell lymphoma (MCL), however, efficacy has been observed, but the extent and duration of tumor control is modest. To determine if tumor killing by GS-1101 is cell cycle-dependent, we show in primary MCL cells by whole-transcriptome sequencing that, despite aberrant expression and recurrent mutations in Cyclin D1, mutations are rare in coding regions of CDK4, RB1 and other genes that control G1-S cell cycle progression or PI3K/AKT signaling. PI3K? is the predominant PI3K catalytic subunit expressed, and inhibition by GS-1101 transiently inhibits AKT phosphorylation but not proliferation in MCL cells. Induction of prolonged early G1-arrest (pG1) by selective inhibition of CDK4/CDK6 with PD 0332991 amplifies and sustains PI3K? inhibition, which leads to robust apoptosis. Accordingly, inhibition of PI3K? induces apoptosis of primary MCL tumor cells once they have ceased to cycle ex vivo, and this killing is enhanced by PD 0332991 inhibition of CDK4/CDK6. PIK3IP1, a negative PI3K regulator, appears to mediate pG1 sensitization to PI3K inhibition; it is markedly reduced in MCL tumor cells compared with normal peripheral B cells, profoundly induced in pG1 and required for pG1 sensitization to GS-1101. Thus, the magnitude and duration of PI3K inhibition and tumor killing by GS-1101 is pG1-dependent, suggesting induction of pG1 by CDK4/CDK6 inhibition as a strategy to sensitize proliferating lymphoma cells to PI3K inhibition.

SUBMITTER: Chiron D

PROVIDER: S-EPMC3735703 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Induction of prolonged early G1 arrest by CDK4/CDK6 inhibition reprograms lymphoma cells for durable PI3Kδ inhibition through PIK3IP1.

Chiron David D Martin Peter P Di Liberto Maurizio M Huang Xiangao X Ely Scott S Lannutti Brian J BJ Leonard John P JP Mason Christopher E CE Chen-Kiang Selina S

Cell cycle (Georgetown, Tex.) 20130515 12

Phosphatidylinositol-3-kinase (PI3K) signaling is constitutive in most human cancers. Selective inhibition of PI3Kδ (p110δ) by GS-1101 has emerged as a promising therapy in chronic lymphocytic leukemia and indolent lymphomas. In aggressive non-Hodgkin lymphomas such as mantle cell lymphoma (MCL), however, efficacy has been observed, but the extent and duration of tumor control is modest. To determine if tumor killing by GS-1101 is cell cycle-dependent, we show in primary MCL cells by whole-trans ...[more]

PMID: 23676220

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Project description:BACKGROUND:Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. METHODS:Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by ?-galactosidase staining and p16INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. RESULTS:Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. ?-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. CONCLUSIONS:We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

Project description:Background CDK4 and CDK6 are frequently over expressed or hyper activated in human cancers. Due to the importance of CDK4/6 activity in cancer cells, CDK4/6 inhibitors have emerged as promising candidates for cancer treatment. LEE011, a novel inhibitor of CDK4/6, until now the molecular function of LEE011 in acute myeloid leukemia is still unknown. Methods AML cell growth was assessed by CCK-8 and annexin V/propidium iodide staining followed by flow cytometry detected apoptosis in cell culture. AML cell senescence was assessed by β-Galactosidase Staining, p16INK4a expression and HP1α Staining analysis. Gene expressions of LEE011-treated tumor cells were analyzed using an Arraystar Human LncRNA Array. The Multi Experiment View cluster software analyzed the expression data. Differentially expressed genes from the cluster analyses were imported into the GO and KEGG pathway analysis. Results Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and do not induce apoptosis. Hoechst 33342 staining analysis showed that DNA fragmentation and abnormal nuclear cells was observed after LEE011 treatment. Cell cycle analysis showed in these eight acute leukemia cells, LEE011 induce the cell cycle G1 arrest very significantly, except THP-1 cells. Cell senescence β-Galactosidase Staining analysis, p16INK4a expression and HP1α Staining analysis all showed that LEE011 treatment can induce the cell senescence of AML cells. LncRNA microarray was used to identify 2083 differently expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with control group. Molecular function analysis showed that LEE011 induced cellular senescence in leukemia cells partially through down regulation the transcriptional expression of MYBL2. Conclusions In this study, we showed for the first time that LEE011 treatment resulted in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in AML cells. LncRNA microarray was used to identify differentially expressed genes and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induce cellular senescence partially through down regulation the expression of MYBL2. These results may provide new insights into the molecular mechanism of LEE011-induced cellular senescence; however, further research will be required to determine the underlying details. Taken together, our findings firstly suggest that LEE011 may act as new candidate’s drug for AML.

Dataset Information

Induction of prolonged early G1 arrest by CDK4/CDK6 inhibition reprograms lymphoma cells for durable PI3K? inhibition through PIK3IP1.

Publications

Induction of prolonged early G1 arrest by CDK4/CDK6 inhibition reprograms lymphoma cells for durable PI3Kδ inhibition through PIK3IP1.

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