Prolonged halting of gene expression in early G1 arrest by inhibition of CDK4/CDK6 reprograms myeloma cells for cytotoxic killing
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ABSTRACT: By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. Induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell cycle phases. S-phase synchronization upon removal of the early-G1 block (pG1-S) fails to completely restore scheduled gene expression. Consequently, coordinate loss of IRF4 and gain of Bim and Noxa expression sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 and more profoundly in pG1-S in vitro. Induction of pG1 and pG1-S by CDK4/CDK6 inhibition augments tumor-specific bortezomib killing in myeloma xenografts. Inhibition of CDK4/CDK6 in combination therapy thus represents a novel mechanism-based cancer therapy. PD 0332991 (PD) is the only known specific and reversible CDK4/CDK6 inhibitor. Gene expression was measured in myeloma MM1.S cells treated with PD (0.25 uM) in triplicate for 12, 24 or 36 h, or in cells released from G1, induced by 24hPD, for 4 or 18 h.
Project description:By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. Induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell cycle phases. S-phase synchronization upon removal of the early-G1 block (pG1-S) fails to completely restore scheduled gene expression. Consequently, coordinate loss of IRF4 and gain of Bim and Noxa expression sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 and more profoundly in pG1-S in vitro. Induction of pG1 and pG1-S by CDK4/CDK6 inhibition augments tumor-specific bortezomib killing in myeloma xenografts. Inhibition of CDK4/CDK6 in combination therapy thus represents a novel mechanism-based cancer therapy.
Project description:Cdk4 and Cdk6 are two related kinases that bind D-type cyclins and regulate cell cycle progression. Due to their relevance in cancer, Cdk4/6 inhibitors are currently in advanced clinical trials in multiple tumor types. Cdk4/6 are inhibited by INK4 proteins that exert tumor suppressing functions. To test the significance of this inhibitory mechanism we have generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6R/R mice display altered development of the hematopoietic system without resulting in enhanced tumor susceptibility, either in the presence or absence of p53. The presence of the Cdk6 R31C allele results in defective potential of hematopoietic progenitors in adoptive transfer assays or after induced damage. These defects are rescued after complete insensitivity to INK4 inhibitors in Cdk4R/R; Cdk6R/R double mutant mice, and INK4-resistant mice display increased susceptibility to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, the presence of the Cdk6 R31C allele results in increased binding of p16INK4a to wild-type Cdk4, whereas the double mutant is fully insensitive to INK4 inhibitors resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in tumor development and suggest a role for Cdk6 in buffering INK4 protein levels thus contributing to the development of hematopoietic tumors. The presence of the Cdk4 R24C and Cdk6 R31C alleles results in relevant changes in the expression profiles of cancer cells including deregulation of apoptosis and other processes. p185BCR-ABL1 was used to transform wild-type or double knock-in Cdk4 R24C; Cdk6 R31C fetal livers. Cell lines were isolated as spontaneous immortal and transformed clones after transduction of fetal liver with a p185 BCR-ABL1-transgene. RNA was isolated from asynchronous cultures. Two-condition experiment, Cdk4R-Cdk6R cells versus wild-type cells. Biological replicates: 3 control replicates, 3 transfected replicates.
Project description:PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and arrayCGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort ovarian cancer patients. Concentration-dependent anti-proliferative effects of PD-0332991were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, Rb, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration and time dependent manner, and enhanced the effects of chemotherapy. Rb proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and associated with adverse clinical outcome (progression free survival, adjusted relative risk 1.49, 95%CI 0.99-2.22, p =0.054). PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Gene expression of 40 individual ovarian cell lines relative to an ovarian cell line reference mix containing equal amounts of 41 ovarian cell lines (including OCC-1 which was later identified as originating from mouse). The expression data was correllated with cell line growth response to CDK 4/6 inhibitor PD-0332991 to identify genes associated with drug sensitivity and resistance.
Project description:PD-0332991 is a small molecule inhibitor for Cdk4 and Cdk6. It exerted growth inhibitory effects on PDAC cell lines (AsPC-1 and COLO-357). Microarray analysis was used to characterize the changes in gene expression profiles of AsPC-1 and COLO-357 upon PD-0332991 incubation AsPC-1 and COLO-357 cells were treated in the absence or presence of 5 µM PD-0332991 for 24 h and 72 h. Each expreimental condition had biological triplicates. Twenty-four samples were analyzed in total.
Project description:CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer. Previous work identified CDK6 overexpression as one critical determinant of acquired resistance to CDK4/6 inhibition. This dataset interrogates CDK6 interactors/recruiters to determine their role in transcriptional regulation of CDK4/6i resistance mechanisms and will provide novel therapeutic strategies to overcome resistance.
Project description:We report a novel resistance mechanism to CDK4/6 inhibition in Hedgehog-associated medulloblastoma where cell models and mouse models demonstrate that prolonged inhibition of CDK4/6 inhibits ribosome biogenesis, activates the unfolded protein response, and increases the amount of Smoothened-activating lipids. This RNA-Sequencing dataset represents genomically-engineered mouse medulloblastoma models that either have wild-type Cdk6 or genomic knockout of Cdk6. We find that tumors that grew despite genetic loss of Cdk6 have suppresed ribosome biogenesis.
Project description:CDK4/6 inhibitors (CDK4/6i) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function, and are exquisitely sensitive to CDK4/6i. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6i and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90/CDC37 complex. In contrast, CDK4/6i and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6i and CDK4/6i-derived degraders and the need for novel inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics.
Project description:The aim of this study is to estimate gene expression variations involved in glucose meatbolism in HepG2 cell line after cell cycle(G1 to S phase) inhibition by using drug(PD-0332991) or CDK4-6-knock down. We hypothesized that transition of glycolytic gene expression might indicates the effect of PD-0332991 drug treatment in Hepatocellular carcinoma cells.