Bim Is Required to Sensitize Mantle Cell Lymphoma Cells for Killing by Bortezomib, but Not Ara-C, by Selective Inhibition of CDK4/CDK6

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1655-1655
Author(s):  
Xiangao Huang ◽  
Maurizio Di Liberto ◽  
Jamieson Bretz ◽  
David Chiron ◽  
Peter Martin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Phase Synchronization ◽  
Selective Inhibition ◽  
S Phase ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Mantle Cell

Abstract Abstract 1655 Mantle cell lymphoma (MCL) is characterized by aberrant cyclin D1 expression due to the t (11: 14) translocation. In conjunction with elevation of CDK4/CDK6, this promotes cell cycle progression through G1 and unrestrained cell proliferation. As MCL remains incurable despite initial response to therapy, mechanism- and genome-based therapies that both control the cell cycle and enhance cytotoxic killing are urgently needed. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), a selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable. We demonstrate that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs; and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog Ara-C (cytarabine), both in vitro and in a mouse model of MCL. These findings implicate a unified mechanism for cell cycle sensitization of cytotoxic killing. To elucidate the underpinning mechanism, we show that sensitization to cytotoxic killing by CDK4/CDK6 inhibition requires an intact Rb, the substrate of CDK4/CDK6, but is independent of p53. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that prolonged inhibition of CDK4/CDK6 with PD halts the gene expression program in early G1 and depletes the expression of genes programmed for other phases of the cell cycle, such as cyclin A (G1/S), thymidine kinase (S), CDK1 and cyclin B (G2/M) and selective metabolic genes. Removal of PD restores the CDK4/CDK6 activities and the expression of scheduled cell cycle genes but leaves many others in the pG1 state. This leads to S phase synchronization with impaired metabolism. Accordingly, the magnitude of bortezomib and Ara-C killing in pG1-S greatly exceeds the enrichment of S phase cells. Selective inhibition of CDK4/CDK6, therefore, sensitizes MCL cells for cytotoxic killing in S phase synchronization through induction of a persistent metabolic imbalance in prior pG1. pG1 alone induces caspase activation moderately in MCL cells, but markedly augments apoptosis induced by either bortezomib or Ara-C in pG1-S. This enhancement of apoptosis is apparently mediated by an alteration of the ratios of pro-apoptotic BH3-only proteins (Bim, Noxa and Puma) to anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-xL), which lowers the threshold for caspase-9 activation. Importantly, Bim is selectively required to sensitize MCL cells for killing by bortezomib, but not Ara-C, at low doses as indicated in studies of Bim-deficient MCL cell lines. Corroborating these findings, loss of one allele of Bim attenuates the enhancement of bortezomib killing in pG1-S in untransformed primary mouse B cells after activation by BCR and CD40 signaling. Thus, the synergistic actions of PD-bortezomib and PD-AraC in MCL therapy are distinguishable by the requirement for Bim. Furthermore, we found that the three Bim isoforms are expressed at variable levels but undetected in 30% of primary MCL tumor cells, consistent with the reported mutations and bi-allelic deletion of Bim (BCL2L11) in MCL. RNA-Seq analysis of samples from patients enrolled in a phase I study of PD in combination with bortezomib in MCL further reveals that the mutation burden in BCL2L11 is ∼3-fold higher in a clinically non-responder compared with a responder. Collectively, our data demonstrate that by halting scheduled gene expression in prolonged early G1 arrest, selective and reversible inhibition of CDK4/CDK6 provides a mechanism-based strategy to sensitize MCL cells for cytotoxic killing by bortezomib, Ara-C, and potentially other emerging agents. By lowering the threshold for caspase activation, Bim is selectively required for sensitization to killing by low dose bortezomib, but not Ara-C, and may serve as a biomarker for genome-based selection of cytotoxic partners in therapeutic targeting of CDK4/CDK6 in MCL. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Mutation Burden in CDKN2C, CDK1 and E2F2 Is Associated with Differential Response to Targeting CDK4/CDK6 in Combination with Bortezomib in Mantle Cell Lymphoma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3738-3738 ◽  
Author(s):  
Christopher E. Mason ◽  
Maurizio Di Liberto ◽  
Xiangao Huang ◽  
David Chiron ◽  
Jamieson Bretz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Clinical Study ◽  
Research Funding ◽  
Cell Lymphoma ◽  
S Phase ◽  
Cytotoxic Agents ◽  
G1 Arrest ◽  
Mutation Burden ◽  
Mantle Cell

Abstract Abstract 3738 Dysregulation of the cell cycle is a hallmark of mantle cell lymphoma (MCL) in which cyclin D1 expression is constitutive due to the t (11:14) translocation and CDK4 levels are elevated. MCL remains incurable despite initial response to therapy. Our goal was to develop a mechanism- and genome-based therapy to both inhibit lymphoma cell proliferation and sensitize them for cytotoxic killing. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), the only known selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable, in combination with cytotoxic agents. We demonstrate, for the first time, that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs, and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant cytotoxic agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog cytarabine, in vitro and in a mouse model of MCL (Huang et al, submitted). In a completed phase I clinical study in MCL, PD potently and preferentially inhibited CDK4/CDK6 in lymphoma cells despite extensive chromosomal abnormalities, with an excellent toxicity profile and promising clinical response (Leonard et al, submitted). To advance targeting CDK4/CDK6 in MCL, we have now combined PD with escalating dose of bortezomib in an ongoing phase I clinical study (PD-B) in MCL. In this proof-of-concept study, PD is administered on days 1–12 of a 21-day cycle; bortezomib is administered first in prolonged G1 arrest concurrent with PD on days 8 and 11, and again after PD withdrawal in pG1-S on days 15 and 18. CD19+ MCL tumor cells were isolated at baseline, on day 8 and day 21 for analysis. To elucidate the mechanisms that underlie the progression of MCL and the differential response to this novel, cell-sensitizing therapy, we preformed 50×50 paired-end RNA-Sequencing on a HiSeq2000, using one lane for each sample of clinically responding and non-responding patients enrolled in this clinical trial. We generated an average of 76 million reads for each sample, then used the Burrow-Wheeler Aligner (BWA) to align the reads to the genome (Build 37), and SAMtools and the Genome Analysis Toolkit (GATK) to call non-reference variants. We focused on examining genes in the cell cycle and apoptotic pathways, and our data show 400 mutations in 16 genes including CDKN2C (p18), CDK1, E2F2, BBC3 (PUMA), BCL2L11(BIM), JUN and TP53, which are specific to each patient and whose expression changes dynamically during treatment. Moreover, we observe that the overall mutation burden is higher in a non-responding patient relative to the responding patient, and that certain genes (CDKN2C, CDK1, E2F2) show a highly significant (p=2.2×10–16) enrichment of mutations at baseline in the non-responder. By inhibiting CDK4/CDK6, p18 (CDKN2C) is essential for homeostatic cell cycle control of B cell activation and plasma cell differentiation in immunity. Conversely, mutations and deletions of CDKN2C are frequent in MCL, suggesting that loss of CDKN2C contributes to cell cycle dysregulation in this disease. Our RNA-Seq data reveal specific mutations in CDKN2C that are associated with compromised clinical response to PD, in line with cooperative inhibition of CDK4/CDK6 by p18 and PD in BCR-activated B cells as we reported previously. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that induction of prolonged G1 arrest by inhibition of CDK4/CDK6 with PD halts gene expression in early G1 and depletes the expression of those programmed for other phases of the cell cycle. This leads to a metabolic imbalance, which is not restored in pG1-S, thereby sensitizing MCL cells to cytotoxic killing. Mutations in E2F2, which promotes G1/S transition, and CDK1, which functions in G2/M, may therefore antagonize cell cycle sensitization to cytotoxic killing by CDK4/CDK6 inhibition. These data provide new mechanistic insight into therapeutic targeting of CDK4/CDK6 in MCL, and suggest novel molecular targets for personalizing and advancing cell cycle-based therapy in MCL. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Leonard:Pfizer, Inc: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Integrative Whole Transcriptome and Exome Sequencing Reveals Reprogramming Of Mantle Cell Lymphoma In Prolonged Early G1 Arrest For Clinical Response To Selective Inhibition Of CDK4 In Combination Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 80-80
Author(s):  
David Chiron ◽  
Peter Martin ◽  
Maurizio Di Liberto ◽  
Xiangao Huang ◽  
Priyanka Vijay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Clinical Response ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Selective Inhibition ◽  
G1 Arrest ◽  
Cyclin D ◽  
Mantle Cell ◽  
Whole Transcriptome

Abstract CDK4 and CDK6, which drive cell cycle entry and progression through G1 in the presence of cyclin D, are overexpressed at a high frequency in human cancers. Targeting CDK4 with the first selective and potent CDK4/CDK6 inhibitor, palbociclib (PD 0332991), has recently achieved unprecedented clinical efficacy in both hematologic malignancies and solid tumors. Most notably, palbociclib more than tripled the progression free survival of breast cancer patients treated with letrozole. In mantle cell lymphoma (MCL), CDK4 overexpression and aberrant cyclin D1 expression leads to unrestrained cycling and proliferation that underlies disease progression. In the first phase I single-agent palbociclib clinical trial in recurrent MCL, inhibition of CDK4 by palbociclib alone resulted in a durable clinical response with tumor regression in some MCL patients, including one complete response and two partial response. However, the fundamental mechanism for differential clinical response to selective targeting of CDK4/CDK6 remains obscure. To address this question, we have developed a novel strategy that both inhibits proliferation of cancer cells and reprograms them for cytotoxic killing by reversible inhibition of CDK4/CDK6. We have shown that: 1) inhibition of CDK4/6 with palbociclib leads to early G1 arrest that is dependent on Rb, the substrate for CDK4/CDK6; 2) prolonged early G1 arrest (pG1) reprograms cancer cells for killing by diverse agents; 3) pG1 sensitization is exacerbated in synchronous S phase entry (pG1-S) upon palbociclib withdrawal. Further, pG1 sensitization appears to stem from restricted expression of genes scheduled for early G1 only, which is exacerbated in pG1-S due to incomplete restoration of cell cycle-coupled gene expression. To advance targeting CDK4 in MCL, we have implemented this strategy combining palbociclib with bortezomib at a reduced dose (1.0 mg /m2) in a phase I clinical trial (Pa-Btz) in recurrent MCL. palbociclib was administered to MCL patients on days 1-12 of a 21-day cycle to induce pG1; bortezomib was given on days 8 and 11 in pG1 and on days 15 and 18 in pG1-S. Pa-Btz was well tolerated and appeared to have a palbociclib dose-dependent durable clinical activity, with only one of 6 patients progressed at the optimal dose combination. We investigated the genes that mediate pG1 reprogramming by integrative whole exome sequencing (WES) and whole transcriptome sequencing (WTS). The dynamic changes in cell cycle-coupled gene expression were determined within individual patients in primary MCL tumor cells isolated from serial lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) in conjunction with immunohistochemistry (IHC). palbociclib inhibited CDK4 and induced pG1 in all patients initially, regardless of the clinical response, mutations in p53 or ATM, cyclin D1 3’UTR deletion, or other patient-specific deletions, amplifications and mutations. No mutations were detected in CDK4, which was expressed in primary MCL cells over CDK6. Induction of pG1 maintained the expression of cell cycle genes programmed for early G1 (CDK4, cyclin D and Rb), and prevented the expression of those scheduled for late G1 (cyclin A), S phase (Ki 67, TK) and G2/M (CDK1, cyclin B), and this was completely reversible upon release of the early G1 block. However, induction of pG1 also led to an imbalance in the expression of other cellular genes due to restricted expression of only genes programmed for early G1. Among the 868 genes that were suppressed in pG1 (not programmed for early G1) in MCL tumor of clinically-responding patients (N=4, EdgeR, FDR 0.05), 9 were conversely activated in the non-responding patients (N=4, EdgeR, FDR 0.05). These genes are involved in redox stress, metabolism and cell migration, suggesting a potential role of cell cycle-coupled metabolic imbalance in differential clinical response to targeting CDK4/6. Thus, selective inhibition of CDK4 led to Rb-dependent pG1 in tumor cells of all MCL patients despite a multitude of genomic aberrations. Integrative WES and WTS analysis of serial tumor biopsies revealed that pG1 reprograms MCL cells by inducing an imbalance in gene expression that is associated with the clinical response to the Pa-Btz therapy. Defining the functions of the candidate genes identified in the context of clinical response should shed light on the mechanism for therapeutic targeting of CDK4/CDK6 and advance genome-based patient stratification. Disclosures: Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor. Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau. Leonard:millennium: Consultancy.

scholarly journals Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

1993 ◽  
Vol 13 (6) ◽  
pp. 3577-3587 ◽  
Author(s):  
E A Musgrove ◽  
J A Hamilton ◽  
C S Lee ◽  
K J Sweeney ◽  
C K Watts ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cell Cycle Progression ◽  
S Phase ◽  
G1 Phase ◽  
Cycle Progression

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

scholarly journals Restoration of miR-193a-5p and miR-146 a-5p Expression Induces G1 Arrest in Colorectal Cancer through Targeting of MDM2/p53

2019 ◽  
Vol 10 (1) ◽  
pp. 130-134 ◽  
Author(s):  
Saeed Noorolyai ◽  
Elham Baghbani ◽  
Leili Aghebati Maleki ◽  
Amir Baghbanzadeh Kojabad ◽  
Dariush Shanehbansdi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cell Cycle Progression ◽  
Cyclin B ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Ht 29

Purpose: Colorectal cancer (CRC) remains a universal and lethal cancer owing to metastatic and relapsing disease. Currently, the role of microRNAs has been checked in tumorigeneses. Numerous studies have revealed that between the tumor suppressor miRNAs, the reduced expression of miR-146a-5p and -193a-5p in several cancers including CRC tissues are related with tumor progression and poor prognosis of patients. The purpose of this study is to examine the role of miR-146 a-5p and -193 a-5p in CRC cell cycle progression. Methods: The miR-193a-5p and -146 a-5p mimics were transfected into HT-29 CRC cells via jetPEI transfection reagent and their impact was assessed on p53, cyclin B, and NF-kB gene expression. The inhibitory effect of these miRNAs on cell cycle was assessed by flow cytometry. The consequence of miR-193a-5p and miR-146 a-5p on the protein expression level of Murine double minute 2 (MDM2) was assessed by western blotting. Results: miR193a-5p and -146a-5p regulated the expression of MDM2 protein and p53, cyclin B, and NF-kB gene expression in CRC cells. Treatment of HT-29 cells with miRNA-146a-5p and -193a-5p induced G1 cell cycle arrest. Conclusion: The findings of our study suggest that miR146a-5p and -193a-5p may act as a potential tumor suppressor by their influence on cell cycle progression in CRC cells. Thus, miRNA-146a-5p and -193a-5p restoration may be recommended as a potential therapeutic goal in the treatment of CRC patients.

Selective Inhibition of CDK4 and CDK6 Primes Chemoresistant MCL Cells for Killing by Proteasome Inhibitor Bortezomib by Activating Cell Cycle-Coupled Apoptosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3624-3624
Author(s):  
Maurizio Di Li berto ◽  
Xiangao Huang ◽  
Amy Chadburn ◽  
Peter Martin ◽  
Ruben Niesvizky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
Selective Inhibition ◽  
S Phase ◽  
Rational Approach ◽  
Effective Control ◽  
Cycle Progression ◽  
Selective Targeting ◽  
Phase Entry

Abstract Mantle Cell Lymphoma (MCL) remains generally incurable, suggesting that more effective control of unrestrained tumor growth is essential. Loss of cell cycle control is a hallmark of cancer, in particular of MCL where cell cycle progression through G1 is accelerated due to elevation of cyclin-dependent kinase 4 (CDK4) and constitutive cyclin D1 expression. Thus, one rational approach to improve MCL therapy is to target CDK4/6 in combination with cytotoxic killing. Although success in targeting the cell cycle in cancer with broad-spectrum CDK inhibitors has been modest, PD 0332991, the only known CDK4/6-specific inhibitor with oral bioavailability, has been shown to selectively and potently inhibit CDK4/6 in MCL cells ex vivo. Additionally, in a proof-of-mechanism study in patients with recurrent MCL, we have found that PD 0332991 is well tolerated, and effective in inhibiting CDK4 and CDK6 and suppressing tumor growth in vivo. Of note, 50% of the patients (8/16) have achieved a stable disease for greater then 40 weeks (Leonard et al, abstract submitted to ASH 2008). These findings suggest that selective targeting of CDK4 and CDK6 with PD 0332991 is a promising therapy for MCL. To advance targeting of the cell cycle in cancer, we have developed two novel approaches to both inhibit tumor cell proliferation and activate cell cycle-coupled apoptosis in MCL. We show in primary MCL tumor cells and MCL cell lines by BrdU pulse labeling and DNA content analysis that selective inhibition of CDK4/6 with PD 0332991 leads to a complete G1 arrest, despite high level of c-Myc expression and extensive chromosomal abnormality. As PD 0332991 acts reversibly, removal of PD 0332991 immediately releases the G1 block and induces synchronous (>90%) G1-S cell cycle progression and S phase entry. This sensitizes chemoresistant MCL cells to killing by suboptimal doses of cytotoxic agents such as bortezomib, through activating cell cycle-coupled apoptosis during S phase entry. Synergistic killing of MCL cells by induction of cell cycle synchronization with PD 0332991 in combination with bortezomib is mediated by induction of mitochondrial membrane depolarization and activation of caspase-9. In a complementary study, we have demonstrated that selective targeting of CDK4 and CDK6 by PD 0332991 similarly primes chemoresistant primary myeloma cells for cytotoxic killing by activating cell cycle-coupled apoptosis, and induces synergistic tumor suppression in animal models. Selective targeting of CDK4 and CDK6 by PD 0332991 in combination with cytotoxic killing, therefore, represents a promising new strategy for cell cycle-based therapy for MCL and other hematopoietic malignancies.

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression

1993 ◽  
Vol 13 (6) ◽  
pp. 3577-3587
Author(s):  
E A Musgrove ◽  
J A Hamilton ◽  
C S Lee ◽  
K J Sweeney ◽  
C K Watts ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cell Cycle Progression ◽  
S Phase ◽  
G1 Phase ◽  
Cycle Progression

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Induction of Metabolic Impairment In Prolonged Early G1 Arrest Induced by CDK4/CDK6 Inhibition Sensitizes Myeloma Cells for Proteasome Inhibitor Killing During Subsequent S Phase Synchronization

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2989-2989 ◽  
Author(s):  
Maurizio Di Liberto ◽  
Xiangao Huang ◽  
Jamieson Bretz ◽  
Scott A Ely ◽  
Rediet Zewdu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Combination Therapy ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Phase Synchronization ◽  
Phase 1 ◽  
S Phase ◽  
G1 Arrest ◽  
Tumor Killing ◽  
Sequential Combination

Abstract Abstract 2989 Sequential drug combination is a rational approach to maximize tumor killing and minimize side effects in cancer therapy. However, this is rarely achieved because the mechanism of drug action is often incompletely understood and the cell cycle specificity of individual drugs unknown. Dysregulation of cyclin-dependent kinase (CDK)4 and CDK6 is common in human cancer and precedes unrestrained proliferation of tumor cells in multiple myeloma (MM) patients, especially during refractory relapse. This highlights the critical importance of targeting CDK4/CDK6 in MM. We have now developed, for the first time, a novel therapeutic strategy to selectively inhibit CDK4/CDK6 in sequential combination with clinically relevant cytotoxic drugs for maximal tumor killing at reduced doses in MM. CDK4 and CDK6 promote reentry and progression of the cell cycle through G1. PD 0332991, the only known CDK4/CDK6-specific inhibitor, is potent, reversible and bioavailable. We showed that inhibition of CDK4/CDK6 with PD 0332991 induces early G1 arrest and upon release from the G1 block, synchronous progression to S phase and G2/M with exceptional precision and efficiency in MM cells in vitro and in animal models. This provides a unique means to determine the cell cycle targeting specificity of individual compounds for optimal combination. Simultaneous analysis of BrdU pulse-labeling (30 minutes) and DNA content per cell reveals that bortezomib, a reversible proteasome inhibitor; carfilzomib (PR-171), an irreversible selective inhibitor of the proteasom; and its oral analog ONX-0912 (PR-047) all preferentially target MM cells synchronized into S phase over those arrested in G1, but not cells in G2/M. On this basis, killing of myeloma cells by proteasome inhibitors is markedly enhanced in prolonged G1 arrest induced by PD 0332991 and further augmented during synchronous entry into and progression through S phase upon release from the G1 block, in vitro and in vivo in the native bone marrow niches. Induction of early G1 arrest by PD 0332991 requires Rb, the substrate of CDK4 and CDK6, but not p53. Importantly, the increase in carfilzomib, ONX-0912 or bortezomib mediated killing after S phase synchronization significantly surpasses the enrichment of S phase cells. It is in fact proportional to the time of prior G1 arrest. Kinetics analyses of global gene expression patterns, specific RNA and protein levels and functional shRNA interference show that prolonging early G1 arrest leads to time-dependent uncoupling of gene expression from the cell cycle. PD 0332991 withdraw rapidly restores the CDK4 and CDK6 catalytic activity and scheduled expression of cell cycle genes, hence synchronous progression to S phase and mitosis. This includes upregulation of cyclin A synthesis and Skp2 mediated ubiquitin-proteasome degradation of p27 for S phase entry, mini chromosome maintenance(MCM)s and thymidine kinase for DNA replication, and genes critical for G2/M checkpoint control and mitosis. However, it fails to fully reverse the metabolic impairment (altered glucose, nucleotide and ATP metabolism) induced in prolonged early G1 arrest. This culminates in the loss of IRF-4 required for myeloma survival and selective gain of pro-apoptotic Bim function in G1 arrest and Noxa in S phase in synergy with carfilzomib and bortezomib, which lowers the threshold for activation of the intrinsic apoptosis pathway. Selective inhibition of CDK4/CDK6 in sequential combination therapy thus not only halts tumor cell proliferation but also potently induces synergistic tumor killing. This sequential combination therapy has been implemented in a multi-center phase 1/2 clinical trial targeting CDK4/6 with PD 0332991 in combination with bortezomib and dexamethasone in relapsed refractory MM. Phase 1 data indicate that PD 0332991 is well tolerated, and directly and completely inhibits CDK4/CDK6 and the cell cycle in tumor cells in MM patients with promising clinical efficacy. Evidence from phase 2 trials of carfilzomib indicates that it is also well tolerated. The peripheral neuropathy commonly observed with bortezomib appears to be less severe and less frequent. Selective combination with carfilzomib or the oral agent ONX-0912 thus represents a promising alternative to refine targeting CDK4/6 in sequential combination therapy for multiple myeloma and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is a cell cycle CDK4/CDK6 inhibitor Carfilzomib is a proteasome inhibitor. Kirk:Onyx: Employment, Equity Ownership. Randolph:Pfizer: Employment, Equity Ownership. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau.

scholarly journals Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

2020 ◽  
Author(s):  
Lina Sui ◽  
Yurong Xin ◽  
Daniela Georgieva ◽  
Giacomo Diedenhofen ◽  
Leena Haataja ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Endocrine Cells ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Growth Potential ◽  
Proliferative Potential ◽  
G1 Arrest ◽  
Cycle Progression

AbstractLimitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown. Understanding proliferation potential is important for the safty of cell replacement therapy with cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells both in vitro and after grafting in vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.

Induction of sequential G1 arrest and synchronous S phase entry by reversible CDK4/CDK6 inhibition sensitizes myeloma cells for cytotoxic killing through loss of IRF-4.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 299-299
Author(s):  
Xiangao Huang ◽  
Maurizio Di Liberto ◽  
Scott Ely ◽  
David S Jayabalan ◽  
Isan Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Specific Inhibitor ◽  
S Phase ◽  
G1 Arrest ◽  
Myeloma Cells ◽  
Cycle Dependent ◽  
Phase Entry

Abstract Abstract 299 Dysregulation of the cell cycle is a hallmark of cancer. However, targeting the cell cycle in cancer therapy has only been modestly successful since broad-spectrum cyclindependent kinase (CDK) inhibitors lack specificity and are highly toxic. The critical importance of controlling CDK4/CDK6 in cancer treatment is further exemplified by recent evidence of prominent CDK4/CDK6 dysregulation in human cancers, including breast cancer, metastatic lung adenocarcinoma, glioblastoma, mantle cell lymphoma and multiple myeloma (MM). To advance mechanism-based targeting of the cell cycle in cancer, we have developed a novel strategy that both inhibits cell cycle progression and enhances cytotoxic killing in tumor cells using PD 0332991(PD), the only known CDK4/CDK6-specific inhibitor that is also reversible, potent and orally bioavailable. We demonstrated by BrdU pulse-labeling that inhibition of CDK4/CDK6 with PD in primary bone marrow (BM) myeloma cells and human myeloma cell lines (HMCL) (IC50 60nM) leads to a complete early G1 arrest in the absence of apoptosis and upon release of the G1 block, synchronous cell cycle progression to S phase. Furthermore, prolonged early G1 arrest enhances cytotoxic killing of MM cells by diverse clinically relevant drugs at low dose, including bortezomib, carfilzomib (PR-171) and dexamethasone, and this is dramatically augmented during synchronous S phase entry. The enhancement of cytotoxic killing in either G1 arrest or synchronous S phase entry is sustained in the presence of BM stromal cells. This killing is caspase-dependent and triggered by the loss of mitochondrial outer membrane potential and activation of the intrinsic apoptosis pathway. Time course studies of cell cycle-specific gene expression by expression profiling, quantitative real time RT-PCR and immunoblotting further revealed that the expression of IRF-4, essential for normal plasma cell differentiation and myeloma cell survival, is strictly cell cycle-dependent: elevated in G1 and markedly declined in S phase. The IRF-4 protein is also markedly reduced (50%) by bortezomib treatment, resulting in a combined 5-fold reduction in S phase. This suggests that differential enhancement of cytotoxic killing in G1 arrest and S phase is mediated by cell cycle-dependent IRF-4 expression. Indeed, shRNA interference confirms that by antagonizing mitochondrial depolarization, IRF-4 is required to protect myeloma cells from cell cycle-dependent enhancement of bortezomib killing. By timely administration and discontinuation of PD treatment, we have further demonstrated in a human MM 1.S. xenograft myeloma model that it is feasible to induce sequential G1 arrest and synchronous S phase in vivo. This leads to synergistic tumor suppression through amplification of bortezomib killing of myeloma cells, but not normal BM cells. As PD is orally bio-available, specific and low in toxicity, our novel strategy has been implemented in the first phase I/II multi-center clinical trial targeting CDK4/CDK6 with PD in combination with bortezomib and dexamethasone in MM. Preliminary bone marrow immunohistochemistry demonstrates PD preferentially and completely inhibits CDK4/CDK6-specific phosphorylaton of Rb and DNA replication in tumor cells, but not other bone marrow cells in all patients. One patient achieved VGPR (12.5%) while 1 patient each achieved MR and SD respectively for an ORR 25% (Niesvizky et al, submitted). Collectively, our preclinical and clinical data indicate, for the fist time, that selective targeting of CDK4/CDK6 in combination therapy is a promising mechanism-based therapy for MM and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor.. Chen:Pfizer, Inc.: Employment, Equity Ownership. Wilner:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee. Chen-Kiang:Pfizer Inc.: Research Funding.

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