Pharmacologic inhibition of CDK4/6: mechanistic evidence for selective activity or acquired resistance in acute myeloid leukemia

Abstract Entry into the cell cycle is mediated by cyclin-dependent kinase 4/6 (CDK4/6) activation, followed by CDK2 activation. We found that pharmacologic inhibition of the Flt3 internal tandem duplication (ITD), a mutated receptor tyrosine kinase commonly found in patients with acute myelogenous leukemia (AML), led to the down-regulation of cyclin D2 and D3 followed by retinoblastoma protein (pRb) dephosphorylation and G1 cell-cycle arrest. This implicated the D-cyclin-CDK4/6 complex as a downstream effector of Flt3 ITD signaling. Indeed, single-agent PD0332991, a selective CDK4/6 inhibitor, caused sustained cell-cycle arrest in Flt3 ITD AML cell lines and prolonged survival in an in vivo model of Flt3 ITD AML. PD0332991 caused an initial cell-cycle arrest in well-established Flt3 wild-type (wt) AML cell lines, but this was overcome by down-regulation of p27Kip and reactivation of CDK2. This acquired resistance was not observed in a Flt3 ITD and a Flt3 wt sample from a patient with primary AML. In summary, the mechanism of cell-cycle arrest after treatment of Flt3 ITD AML with a Flt3 inhibitor involves down-regulation of cyclin D2 and D3. As such, CDK4/6 can be a therapeutic target in Flt3 ITD AML but also in primary Flt3 wt AML. Finally, acquired resistance to CDK4/6 inhibition can arise through activation CDK2.

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A precision medicine drug discovery pipeline to identify dual CDK2/9 inhibition as a novel treatment for colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e16056 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e16056-e16056

Author(s):

Roham Salman Roghani ◽

Ali Sanjari moghaddam ◽

Gabrielle Rupprecht ◽

Erdem Altunel ◽

So Young Kim ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Cycle ◽

Precision Medicine ◽

Cell Cycle Arrest ◽

Cell Lines ◽

High Throughput ◽

Single Agent ◽

Cdk Inhibitor ◽

Cycle Arrest

e16056 Background: Colorectal cancer (CRC) is the 3rdmost common form of cancer in the US, responsible for over 50,000 death each year. Therapeutic options for advanced colorectal cancer are limited, and there remains an unmet clinical need to identify new therapies to treat this deadly disease. To address this need, we have developed a precision medicine pipeline that integrates high throughput chemical screens with matched patient-derived cell lines and patient-derived xenografts (PDXs) to identify new treatments for CRC. Methods: We used high-throughput chemical screens of 2,100 compounds across five low-passage, patient-derived CRC cell lines. These results were validated using dose-response IC50curves for CDK1, CDK2, CDK9 or CDK1/2/9 inhibitors and by siRNA-mediated knockdown of CDK9 with or without CDK2 inhibition. Cell cycle arrest analysis was performed by flow cytometry and anaphase catastrophe was analyzed by immunofluorescence staining. For in vivo studies, matched PDXs were treated with either CDK2, CDK9 or dual CDK2/9 inhibitors. Results: We identified the CDK inhibitor drug class as among the most effective cytotoxic compounds across all five CRC lines. Further analysis of the CDK inhibitor class revealed that combined targeting of CDK1, 2, and 9 was the most effective, with IC50 in the range of 110 nM to 1.2 μM. We further validated the efficacy of combined CDK2/9 inhibition using siRNA-mediated knockdown of CDK9 in the presence of a CDK2 inhibitor(CVT-313), and showed that CDK9 knockdown acted synergistically with CDK2 inhibition. Dual CDK2/9 inhibition led to significant G2/M cell cycle arrest and anaphase catastrophe. Finally, combined CDK2/9 inhibition in vivo synergistically inhibited PDX tumor growth as compared to single-agent CDK inhibitors. Conclusions: Our precision medicine pipeline revealed CDK2/9 dual inhibition as a combinatorial therapy to treat CRC and can also be used to identify new and novel therapies

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Induction of cell cycle arrest and apoptosis in human colon adenocarcinoma cell lines by beta-carotene through down-regulation of cyclin A and Bcl-2 family proteins

Carcinogenesis ◽

10.1093/carcin/23.1.11 ◽

2002 ◽

Vol 23 (1) ◽

pp. 11-18 ◽

Cited By ~ 77

Author(s):

P. Palozza

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Colon Adenocarcinoma ◽

Beta Carotene ◽

Human Colon ◽

Down Regulation ◽

Adenocarcinoma Cell ◽

Cycle Arrest ◽

Human Colon Adenocarcinoma

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PI3K Signaling Pathway Activation Predicts Class I PI3K Inhibitor GDC-0941 Sensitivity in AML.

Blood ◽

10.1182/blood.v114.22.1057.1057 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1057-1057

Author(s):

Xiaoju Max Ma ◽

Changchun Du ◽

Laura Sun ◽

Xiaoyan Shi ◽

Lori Friedman ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Signaling Pathway ◽

Cell Lines ◽

Akt Signaling ◽

Down Regulation ◽

Employment Equity ◽

Cycle Arrest ◽

Pathway Activation ◽

Equity Ownership

Abstract Abstract 1057 Poster Board I-79 The PI3K-Akt signal transduction pathway plays a key role in the pathogenesis of many human cancers. In AML malignancy, deregulation of upstream receptor tyrosine kinases such as FLT3, c-Kit, and c-FMS or mutations in K-Ras, N-Ras, B-Raf and CEBPA genes lead to activation of PI3K-Akt signaling to promote cell survival and cell growth. A highly selective Class I PI3K inhibitor, GDC-0941, provides exciting therapeutic opportunities for targeting this pathway in AML. Here we show that GDC-0941 significantly inhibits the viability of the majority of a large panel of AML cell lines tested in vitro (80% or 19/24) at a concentration of < 1 uM. Because not all AML cell lines responded to GDC-0941, we show that PI3K-Akt pathway activation, evidenced by basal pAkt level, can serve as a potential predictive biomarker for GDC-0941 in AML in that sensitive cell lines displayed higher level of pAkt relative to resistant cell lines. Consistently, GDC-0941 treatment leads to decreased pAkt, and therefore the down-regulation of this important pro-survival signaling. Our further analysis shows that GDC-0941 treatment can induce apoptosis and/or cell cycle arrest. We also obtained fresh AML tumor samples to test whether GDC-0941 can similarly induces apoptosis in blast cells and showed that GDC-0941 treatment results in a down-regulation of pAkt level and increased apoptosis. Other PD biomarkers such as phospho-BAD level and Bim expression are both consistent with the observed apoptotic responses. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin, synergizes with GDC-0941 to produce an increased amount of apoptosis in several AML cell lines tested. This is likely due to the fact that long-term treatment with rapamycin induces the sensitivity of the PI3K –Akt signaling pathway by releasing the negative feedback loop of mTORC1-S6K-IRS1/2 module. Importantly, in some AML cell lines we observe synergies between GDC-0941 and AraC. Interestingly, while AraC alone does not induce apoptosis in AML cell lines with PTEN loss or mutation, the synergy between AraC and GDC-0941 comes from increased apoptotic response, suggesting that GDC-0941 can synergize with chemo agents that induces S/G2 cell cycle arrest. Together, our preclinical data suggest that GDC-0941 may be used as a targeted therapy in AML patients as a single agent or in combination with other chemotherapies in clinic. Disclosures: Ma: Genentech Inc.: Employment. Du:Genentech, Inc.: Employment, Equity Ownership. Sun:Genentech Inc.: Employment. Shi:Genentech, Inc.: Employment, Equity Ownership. Friedman:Genentech Inc.: Employment. Dornan:Genentech, Inc.: Employment, Equity Ownership. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

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MLN4924 An Investigational Inhibitor Of NEDD8 Activating Enzyme (NAE) Is Active In Pre-Clinical Models Of Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.4409.4409 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4409-4409

Author(s):

Sarah Belliotti ◽

Juan Gu ◽

Cory Mavis ◽

Myron S. Czuczman ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lines ◽

G1 Phase ◽

B Cell Lymphomas ◽

Lymphoma Cells ◽

Down Regulation ◽

Cycle Arrest ◽

Large B Cell

The results of the Collaborative trial in relapsed aggressive lymphoma (CORAL) study suggest that diffuse large B-cell lymphomas (DLBCL) that relapse or fail to respond to rituximab-chemotherapy in the front-line possess a more resistant disease and represent an emerging challenge for clinicians treating aggressive B-cell lymphomas. It also stresses the need to further study and define at the molecular level the mechanisms by which DLBCL are developing resistance to chemo-immunotherapy. We previously demonstrated that the ubiquitin-proteasome system (UPS) plays an important role in the development of rituximab-chemotherapy resistance. Targeting the UPS has become an important therapeutic strategy in relapsed/refractory DLBCL. MLN4924, a NAE inhibitor selectively blocks the UPS up-stream by preventing the activation of a subset of ubiquitin ligases known as cullin-ring ligases. We evaluated the activity of MLN4924 in a panel of rituximab-chemotherapy (RSCL) sensitive and resistant (RRCL) germinal center B-cell (GCB) and ABC-DLBCL cell lines. RSCL and RRCL were exposed to MLN4924 (0.5μM and 1.0μM) for 24-72 hrs. Changes in cell viability, cell cycle and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. MLN4924 induced cell death in ABC-DLBCL cell lines (both RSCL and RRCL) and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. We also observed down regulation of NEDD8 protein across all treated cell lines. Bcl-XL down-regulation appears to be regulated at the transcriptional level. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR), perhaps due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. Our data suggests that MLN4924 is active in ABC-DLBCL by inducing cell cycle arrest in G1 phase and rendering lymphoma cells more susceptible to apoptosis. MLN4924 lowers the apoptotic threshold of lymphoma cells by negatively regulating Bcl-XL levels at the transcription level. Selective inhibition of the NFkB transcription factor most likely play a role in the down-regulation of Bcl-XL observed in all cell lines tested. Ongoing studies aimed to further define the molecular mechanisms of action of MLN4924 can potentially assist scientists and clinicians in the optimal design of clinical trials incorporating this agent in relapsed/refractory DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

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Olean-12-Eno[2,3-c] [1,2,5]Oxadiazol-28-Oic Acid (OEOA) Induces G1 Cell Cycle Arrest and Differentiation in Human Leukemia Cell Lines

PLoS ONE ◽

10.1371/journal.pone.0063580 ◽

2013 ◽

Vol 8 (5) ◽

pp. e63580 ◽

Cited By ~ 9

Author(s):

Yu Pong Ng ◽

Yuewen Chen ◽

Yueqing Hu ◽

Fanny C. F. Ip ◽

Nancy Y. Ip

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Leukemia Cell ◽

Human Leukemia ◽

Human Leukemia Cell ◽

Cycle Arrest ◽

Leukemia Cell Lines ◽

G1 Cell Cycle Arrest

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DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab024.025 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. i6-i7

Author(s):

Alişan Kayabölen ◽

Gizem Nur Sahin ◽

Fidan Seker ◽

Ahmet Cingöz ◽

Bekir Isik ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Mutant Cell ◽

Glioma Cells ◽

Epigenetic Therapy ◽

Low Grade ◽

Orthotopic Model ◽

Cycle Arrest ◽

Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

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