Littérature scientifique sur le sujet « CCND-CDK4 »

Abstract Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival. Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor. Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib. Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. Disclosures Iriyama: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

524 Background: Hormone receptor positive breast cancer is a therapeutic challenge. Despite optimal anti-endocrine therapies, most breast cancer deaths follow a diagnosis of early luminal cancer. To understand the impact of multiple aberrations in the context of current therapy, we assessed the prognostic ability of genomic signatures as a putative stratification tool to targeted therapies. Methods: This a priori study is based on molecular pathways which might predict response to targeted therapies. DNA from 420 patients from the phase III TEAM pathology cohort were used. Patients with a distant recurrence within 5 years were matched by clinical variables to those disease-free at follow up. Copy number analysis was performed using the Affymetrix Oncoscan Assay. Targeted sequencing was performed in a subset of samples for genes based on signaling cassettes mined from the ICGC. Pathways were identified as aberrant if there were copy number variations (CNVs) and/or mutations in any of the pre-determined pathway genes: 1) CCND1/CCND2/CCND3/CDK4/CDK6; 2) FGFR1/FGFR2/FGFR2/FGFR4; and 3) AKT1/AKT2/PIK3CA/PTEN. Kaplan-Meier and log-rank analyses were used for DFS between groups. Hazard ratios were calculated using the Cox proportional hazard models adjusted for age, tumour size, grade, lymph node and HER2 status. Results: 390/420 samples passed informatics QC filters. For the CCND/CDK pathway, patients with no CNV changes experienced a better DFS (HR = 1.7, 95% CI 1.3-2.3, p < 0.001). For the FGFR/FGF pathway, a similar outcome is seen among patients without CNVs (HR = 1.5, 95% CI 1.1-2.0; p = 0.005). For AKT/PIK3CA, a decrease in DFS was seen in those with aberrations (HR = 1.4, 95% CI 1.0-1.8, p = 0.03). Conclusions: We demonstrated that CNVs of genes within CDK4/CCND, PIK3CA/AKT and FGFR pathways are independently linked to high risk of relapse following endocrine treatment, with implications for identifying those patients who are at high-risk for recurrence despite optimal anti-endocrine therapy and linking molecular features driving these cancers to targeted therapies.

PURPOSE Hormone receptor–positive breast cancer remains an ongoing therapeutic challenge, despite optimal anti-endocrine therapies. In this study, we assessed the prognostic ability of genomic signatures to identify patients at risk for recurrence after endocrine therapy. Analysis was performed on the basis of an a priori hypothesis related to molecular pathways, which might predict response to existing targeted therapies. PATIENTS AND METHODS A subset of patients from the Tamoxifen Versus Exemestane Adjuvant Multinational trial ( ClinicalTrials.gov identifiers: NCT00279448 and NCT00032136, and NCT00036270) pathology cohort were analyzed to determine the prognostic ability of mutational and copy number aberration biomarkers that represent the cyclin D/cyclin-dependent kinase (CCND/CDK), fibroblast growth factor receptor/fibroblast growth factor (FGFR/FGF), and phosphatidylinositol 3-kinase/protein kinase B (PI3K/ATK) pathways to inform the potential choice of additional therapies to standard endocrine treatment. Copy number analysis and targeted sequencing was performed. Pathways were identified as aberrant if there were copy number aberrations and/or mutations in any of the predetermined pathway genes: CCND1/CCND2/CCND3/CDK4/CDK6, FGFR1/FGFR2/FGFR2/FGFR4, and AKT1/AKT2/PIK3CA/PTEN. RESULTS The 390 of 420 samples that passed quality control were analyzed for distant metastasis–free survival between groups. Patients with no changes in the CCND/CDK pathway experienced a better distant metastasis–free survival (hazard ratio, 1.94; 95% CI, 1.45 to 2.61; P < .001) than those who possessed aberrations. In the FGFR/FGF and PI3K/AKT pathways, a similar outcome was observed (hazard ratio, 1.43 [95% CI, 1.07 to 1.92; P = .017] and 1.34 [95% CI, 1.00 to 1.81; P = .053], respectively). CONCLUSION We show that aberrations of genes in these pathways are independently linked to a higher risk of relapse after endocrine treatment. Improvement of the clinical management of early breast cancers could be made by identifying those for whom current endocrine therapies are sufficient, thus reducing unnecessary treatment, and secondly, by identifying those who are at high risk for recurrence and linking molecular features that drive these cancers to treatment with targeted therapies.

Abstract Abstract 3971 Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomid, Bortezomib and Lenalidomide improve response rate and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations including high levels of cyclin D. In addition, MM displays a high genomic instability. The most common translocations in MM lead to dysregulation of cyclin D (CCND), a regulatory protein that governs the activation of a key cell cycle regulator – cyclin dependent kinase (CDK). Cell-proliferation is tightly regulated by activity of CDKs at checkpoints located at the boundaries of cell cycle stages. The association of CDK4/6 with CCND is critical for G1 phase progression, whereas CDK2-cycllin E is necessary for S phase initiation. Deviation from the orderly control may lead to malignant transformation. Furthermore, genomic instability was reported to be affected by over expression of cyclin E. Cyclin E deregulation induces genomic instability as an early event in tumorgenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore, sought to investigate the role of cyclin E in MM. We first examined the expression profile of CCNE1 in various MM cell lines (hMMCLs) including ArP-1, ARH77, CAG, NCIH929, RPMI8226 and U266 by immunoblotting utilizing CCNE1 Abs and by qRT-PCR. CCNE1 expression was found to be heterogeneous being high in U266 and NCIH292, intermediate in ARH77, CAG and PRMI8226 and low in ArP-1 MM cell lines, respectively. A direct correlation between the level of protein and the transcript was observed. We then evaluated the effect of Seliciclib (Roscovitine, CYC202), a selective inhibitor of CDK's on hMMCLs viability, using the MTT assay as hMMCLs have been reported to be sensitive to CDK inhibition. The various hMMCLs were incubated with increasing concentration of Seliciclib (0-100 μM) for 3 days. Seliciclib inhibition of CDK resulted in dose-dependent cytotoxicity with IC50 of 20–80 μM with PRMI8226 being the most sensitive, while ARH77 hMMCL the most resistant. Cell cycle study demonstrated increased apoptosis (based on sub G1 and annexin V analysis) in the sensitive hMMCLs, with no effect on the resistant cell lines. Annexin V binding was observed within 5h and reached maximal level 12h flowing Seliciclib (50 μM) exposure. We next assessed the effect Seliciclib on MCL1, an anti-apoptotic BCL2 family member that is consistitutively expressed in MM cells. Seliciclib significantly down-regulated MCL1 expression in a time and dose dependant manner. Assessing the effect of Seliciclib on cell cycle regulators including CCND, CCNE and P27kip1 indicated a very significant reduction in CCND2 and P27 expression levels in most of the hMMCLs in a dose and time dependant manner, while CCNE1 expression levels increased. Transfection of the low expressing hMMCL RPMI8226 with CCNE1, inducing over expression of cyclin E, resulted in reduced sensitivity of the MM tumor cells to the Seliciclib induced cell death in comparison to the paternal cell line. Lastly, we investigated the effect of Seliciclib on MM cell adhesion to fibronectin (FN), as cell-adhesion-mediated-drug-resistance (CAM-DR) is a major importance protecting MM tumor cells from cytotoxic drugs induced apoptosis. hMMCLs were pre-incubated with increasing concentration of Seliciclib for 1h, plated onto 40 μg/ml FN coated plates for 1h, washed and stained by crystal violet. Seliciclib resulted in a direct dose dependant inhibition of adhesion. The adhesive protective property was evalued by CAM-DR assay, whereas FN-adhesion protected the MM tumor cells against doxorubicin mediated cytotoxicity. There was no difference between the cytotoxic effects of Seliciclib on FN-MM adherent vs. suspended tumor cells. In summary incubation of MM tumor cells with Seliciclib, a selective inhibitor of CDK's, results in apoptosis which is mediated by down regulation of MCL1 and is accompanied by reduced adhesion to FN. Its ability to prevent adhesion – mediated – drug resistance of MM tumor cells may turn Seliciclib as essential component of modern anti MM drug combination therapy. Further experiments should test its synergism with anti MM novel agents. Disclosures: No relevant conflicts of interest to declare.

PURPOSE Soft tissue and bone sarcomas are rare malignancies that exhibit significant pathologic and molecular heterogeneity. Deregulation of the CDKN2A-CCND-CDK4/6-retinoblastoma 1 (Rb) pathway is frequently observed in about 25% of unselected sarcomas and is pathognomonic for specific sarcoma subtypes. This genomic specificity has fueled the clinical evaluation of selective CDK4/6 inhibitors in sarcomas. Here, we highlight successes, opportunities, and future challenges for using CDK4/6 inhibitors to treat sarcoma. MATERIALS AND METHODS This review summarizes the current evidence for the use of CDK4/6 inhibitors in sarcoma while identifying molecular rationale and predictive biomarkers that provide the foundation for targeting the CDK4/6 pathway in sarcoma. A systematic review was performed of articles indexed in the PubMed database and the National Institutes of Health Clinical Trials Registry (ClinicalTrials.gov). For each sarcoma subtype, we discuss the preclinical rationale, case reports, and available clinical trials data. RESULTS Despite promising clinical outcomes in a subset of sarcomas, resistance to CDK4/6 inhibitors results in highly heterogeneous clinical outcomes. Current clinical data support the use of CDK4/6 inhibitors in subsets of sarcoma primarily driven by CDK4/6 deregulation. When dysregulation of the Rb pathway is a secondary driver of sarcoma, combination therapy with CDK4/6 inhibition may be an option. Developing strategies to identify responders and the mechanisms that drive resistance is important to maximize the clinical utility of these drugs in patients with sarcoma. Potential biomarkers that indicate CDK4/6 inhibitor sensitivity in sarcoma include CDK4, CCND, CCNE, RB1, E2F1, and CDKN2A. CONCLUSION CDK4/6 inhibitors represent a major breakthrough for targeted cancer treatment. CDK4/6 inhibitor use in sarcoma has led to limited, but significant, early clinical success. Targeted future clinical research will be key to unlocking the potential of CDK4/6 inhibition in sarcoma.

Abstract Purpose: Cyclin D/CDK4/6 is critical in controlling the G1 to S checkpoint. CCND, the gene encoding cyclin D, is known to be amplified in a variety of solid tumors. Palbociclib is an oral CDK4/6 inhibitor, approved in advanced breast cancer in combination with endocrine therapy. We explored the efficacy of palbociclib in patients with nonbreast solid tumors containing an amplification in CCND1, 2, or 3. Patients and Methods: Patients with tumors containing a CCND1, 2, or 3 amplification and expression of the retinoblastoma protein were assigned to subprotocol Z1B and received palbociclib 125 mg once daily for 21 days of a 28-day cycle. Tumor response was assessed every two cycles. Results: Forty patients were assigned to subprotocol Z1B; 4 patients had outside assays identifying the CCND1, 2, or 3 amplification and were not confirmed centrally; 3 were ineligible and 2 were not treated (1 untreated patient was also ineligible), leaving 32 evaluable patients for this analysis. There were no partial responses; 12 patients (37.5%) had stable disease as best response. There were seven deaths on study, all during cycle 1 and attributable to disease progression. Median progression-free survival was 1.8 months. The most common toxicities were leukopenia (n = 21, 55%) and neutropenia (n = 19, 50%); neutropenia was the most common grade 3/4 event (n = 12, 32%). Conclusions: Palbociclib was not effective at treating nonbreast solid tumors with a CCND1, 2, or 3 amplification in this cohort. These data do not support further investigation of single-agent palbociclib in tumors with CCND1, 2, or 3 amplification.

Introduction: Activation of cardiac cell cycle re-entry is considered the primary therapeutic strategy for cardiomyocyte (CM) regeneration. However, the role of cardiac cell-cycle control in cardiomyogenesis remains elusive. Here, we combined RNA interference and stem cell modeling to investigate the role of Retinoblastoma (RB) in human cardiomyogenesis. Hypothesis: RB regulates proliferation and differentiation of cardiac progenitors (CPCs) but not CM replication. Methods: H9 human embryonic stem cells (hESCs) stably expressing tetracycline (tet)-inducible shRNAs against RB (hESCshRB) or hemagglutinin-tagged RB (hESCHA-RB) were tet-induced at selected time-points during or after CM differentiation. Results: Analysis of ser-608 illustrated stage-specific differences in the degree of RB inactivation during normal hESCs-cardiogenesis. Transient shRB knockdown in hESCshRB-derived embryoid bodies (EBs) during the CPC-stage (EB-days 5-8), significantly upregulated GATA4, ISL1, CTNNI, and cKit transcription (p<0.05), while increasing the yield of beating EBs by 2.4-fold (n=6/group, p<0.0001 vs. vehicle). Gene-expression arrays of 22 RB-related genes, illustrated that shRB-knockdown upregulated CCND1, CCND2, CCND3, and CDK4, CDK6 (p<0.05), followed by a 3.6-fold increase in E2F3 (p<0.05) expression. Moreover, expression of p107 and p130, p27, p57, ARF and CDKN3 were also significantly increased (p<0.05), whereas TP53 and MDM2 remained unchanged. Ectopic HA-RB in CPCs did not significantly affect cardiogenesis (n=18). Conversely, shRB knockdown in EB-day 60-derived CMs (n=15) did not stimulate cell cycle re-entry, as assessed by analysis of EdU incorporation and Aurora-B kinase (AurB). Remarkably, co-culture of hESCHA-RB-derived CMs with adult cardiac (CSCs) and/or mesenchymal (MSCs) stem cells (n=15/group), increased cell-cycle re-entry ~2.8-fold, assessed by ser-10 Histone H3 (p=0.0002) and AurB (p<0.0001). Conclusions: These findings suggest that RB regulates proliferation and differentiation of human CPCs in a cell-autonomous manner, via a CCND-CDK4/6-E2F3 mechanism. Conversely, CM replication may be enhanced via cell-cell interactions with MSCs and/or CSCs, but not cell-autonomously via RB inactivation.

Approximately 10-15% of breast carcinomas are classified as triple receptor-negative breast cancer (TNBC) subtype because of the lack of expression of hormone receptors. Despite the advent of new therapeutic strategies, tumor relapses remain the major challenge in TNBC management. Several studies show that treatment failure and cancer recurrence are primarily due to drug resistance acquisition and self-renewal that are specific properties of cancer stem cells (CSCs). Here I show that a fasting mimicking diet (FMD) reduces the percentage of the staminal population in mouse models of TNBC, increasing cancer free survival, and that the mechanism through which it affects CSCs is glucose dependent and mediated, at least in part, by the down-regulation of the protein kinase A (PKA) pathway. Moreover, the use of RNA-seq analysis on TNBC tumor masses, after FMD, allowed the identification of druggable escape pathways, in particular PI3K/AKT, mTOR and CCND/CDK4-6 axis, activated selectively by differentiated cells. My results show that addition of FMD to inhibitors of these pathways promotes TNBC regression, leading to complete tumor shrinkage. Notably, FMD protects also from hyperglycemia induced by PI3K pathway inhibitors, preventing side effects associated with it. Taken together, these data indicate that FMD has wide but differential effects reaching normal as well as differentiated cancer cells and CSCs, thus representing a promising strategy for the treatment of TNBC, which can be hopefully translated into the clinic.