scholarly journals Combination of Palbociclib and Erlotinib Exhibits Synergistic Antitumor Effect in Colorectal Cancer Patient-Derived Xenograft (PDX) Models

2021 ◽  
Author(s):  
Xiaohui Zhou ◽  
Yeqing Gong ◽  
Xiaorong Liu ◽  
Jiaqi Mao ◽  
Zhen Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Lines ◽  
Colorectal Cancer Patient ◽  
Single Agent ◽  
Cycle Arrest ◽  
Ros Accumulation ◽  
Er Positive ◽  
Rb Phosphorylation ◽  
Pdx Models

Abstract Background: The heterogenetic nature of colorectal cancer (CRC) constitutes a major challenge for drug development. Simultaneous targeting multiple molecules by combination therapy provides a promising strategy, but it requires identification of more potentially useful targeted agents. Palbociclib, a selective CDK4/6 inhibitor approved for the treatment of HR/ER-positive and HER2-negative breast cancer, exhibited anti-cancer versatility in several types of cancer. In this study, we evaluated its usefulness in the treatment of CRC either by single-agent or combined with a small molecule EGFR inhibitor erlotinib. Methods: The impacts of palbociclib, erlotinib, and their combination on cell proliferation, colony formation, cell cycle, apoptosis, senescence, and ROS accumulation in CRC cells were assessed. Their efficacies were evaluated in CRC patient-derived organoids (PDO) and xenograft (PDX) models.Results: Single-agent palbociclib efficiently inhibited proliferation, suppressed the RB phosphorylation, and caused G1-phase arrest in KRAS/BRAF mutated CRC cell lines. IC50 of all cell lines were below 1 µM. Moreover, it induced ROS accumulation and consequently caused apoptosis and senescence of CRC cells. The addition of erlotinib further aggravated palbociclib-induced anti-proliferation, cell cycle arrest, ROS accumulation, apoptosis, and senescence via blocking multiple critical effectors on RB/PI3K/RAS pathways and such interaction between two agents are synergistic. Finally, both palbociclib and erlotinib demonstrated anti-CRC activities, but only their combination caused statistically meaningful inhibition of tumor growth and prolonged survival with tolerable toxicity in KRAS wildtype/mutated PDX models. Conclusion: Our work demonstrated that the palbociclib and erlotinib combination treatment is a promising therapy for CRC and worthy of further clinical evaluation.

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

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

scholarly journals Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Yuxuan Che ◽  
Yang Liu ◽  
Lingzhi Li ◽  
Holly Hill ◽  
Joseph McIntosh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Therapeutic Resistance ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Ic50 Values

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

scholarly journals Curcumin Reverses NNMT-Induced 5-Fluorouracil Resistance via Increasing ROS and Cell Cycle Arrest in Colorectal Cancer Cells

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1295
Author(s):  
Guoli Li ◽  
Sining Fang ◽  
Xiao Shao ◽  
Yejia Li ◽  
Qingchao Tong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Induced Resistance ◽  
Phase Cell ◽  
Ros Generation ◽  
Antitumor Effects ◽  
Cycle Arrest

Nicotinamide N-methyltransferase (NNMT) plays multiple roles in improving the aggressiveness of colorectal cancer (CRC) and enhancing resistance to 5-Fluorouracil (5-FU), making it an attractive therapeutic target. Curcumin (Cur) is a promising natural compound, exhibiting multiple antitumor effects and potentiating the effect of 5-FU. The aim of the present study is to explore the effect of Cur on attenuating NNMT-induced resistance to 5-FU in CRC. A panel of CRC cell lines with different NNMT expressions are used to characterize the effect of Cur. Herein, it is observed that Cur can depress the expression of NNMT and p-STAT3 in CRC cells. Furthermore, Cur can induce inhibition of cell proliferation, G2/M phase cell cycle arrest, and reactive oxygen species (ROS) generation, especially in high-NNMT-expression CRC cell lines. Cur can also re-sensitize high-NNMT-expression CRC cells to 5-FU both in vitro and in vivo. In summary, it is proposed that Cur can reverse NNMT-induced cell proliferation and 5-FU resistance through ROS generation and cell cycle arrest. Given that Cur has long been used, we suppose that Cur is a promising anticancer drug candidate with minimal side effects for human CRC therapy and can attenuate NNMT-induced resistance to 5-FU.

Identification of transcriptomic determinants to predict the efficacy of anti-EGFR treatment of colorectal cancer patients.

2021 ◽  
Vol 39 (3_suppl) ◽  
pp. 130-130
Author(s):  
Saikat Chowdhury ◽  
John Paul Y.C. Shen
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Single Agent ◽  
Gene Set Enrichment Analysis ◽  
P Value ◽  
Intrinsic Resistance ◽  
Chi Square ◽  
Pathway Gene ◽  
Chi Square Test ◽  
Pdx Models

130 Background: It is now well known that in colorectal cancer (CRC) activating mutations in KRAS, NRAS, and BRAF confer resistance to anti-EGFR therapy. However, only ~ 40% of patients with RASWT (KRASWT/NRASWT/BRAFWT) tumors respond to therapy. We have used transcriptomic data in addition to somatic mutations to decipher intrinsic resistance mechanisms and identify novel biomarkers of anti-EGFR response in CRC. Methods: The transcriptomic profiles of metastatic CRC patients treated with the anti-EGFR drug cetuximab plus chemotherapy (n = 193) or as a single agent (n = 80) were retrospectively obtained from two independent clinical cohorts (Okita et al., 2018; & Khambata-Ford et al., 2007). Tumor samples were further divided into RASWT and RASMut groups and subsequently in four consensus molecular subtypes (CMS) based on their transcriptomic profile. The transcriptomic profiles of cetuximab treated CRC cell lines (n = 148, Medico et al., 2015) and PDX models (n = 230, Bertotti et al., 2015) were used for validation. Results: In RASWT population of cetuximab plus chemotherapy cohort, the disease control rates (DCR) observed for cetuximab in the second line were 92% (33/36) for CMS2 and 83% (15/18) for CMS4 tumors relative to 50% (2/4) and 59% (13/22) for CMS1 and CMS3 respectively (Chi-square test p-value = 0.037). Similarly, in cetuximab single agent cohort, the DCR were 0% (0/4), 68% (15/22), and 29% (5/17) for CMS1, CMS2, and CMS4, respectively (Chi-square test p-value = 0.029). In the preclinical study of 148 CRC cell lines, 60% (12/20) of CMS2 cell lines with RASWT genes were sensitive to cetuximab, versus no sensitive cell lines from other CMS (CMS1 = 0/4; CMS3 = 0/5; CMS4 = 0/12). In the PDX study, 84% (11/13) of CMS2 tumor showed clinical benefit, followed by CMS4 with 75% (6/8) in RASWT group (CMS1 = 2/5; CMS3 = 2/4). Gene Set Enrichment Analysis (GSEA) of tumor tissue samples identified that Myc (NES = 1.32, FDR = 0.3), E2F (NES = 2.24, FDR = 0.02), and mTOR (NES = 1.54, FDR = 0.1) pathways were active in the cetuximab refractory RASWT CMS2 patients relative to cetuximab sensitive tumors. Similarly, these pathways were also activated (FDR ≤ 0.05) in cetuximab resistant RASWT cell lines and PDX models, suggesting that preclinical models are appropriate for testing of potential therapeutic combinations to overcome resistance to anti-EGFR therapy. Conclusions: Resistance to anti-EGFR therapy in extended RASWT CRC tumors is mediated in part by transcriptional activation and can be predicted by consensus molecular subtype (CMS) and quantifying transcriptional states of Myc, E2F, and mTOR pathway gene sets.

scholarly journals Scorpion (Androctonus bicolor) venom exhibits cytotoxicity and induces cell cycle arrest and apoptosis in breast and colorectal cancer cell lines

2016 ◽  
Vol 48 (5) ◽  
pp. 537 ◽  
Author(s):  
AbdulrahmanK Al-Asmari ◽  
Anvarbatcha Riyasdeen ◽  
Rajamohamed Abbasmanthiri ◽  
Mohammed Arshaduddin ◽  
FahadAli Al-Harthi
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Colorectal Cancer Cell ◽  
Cycle Arrest ◽  
Colorectal Cancer Cell Lines

scholarly journals Synthesis of 2,6-Diamino-Substituted Purine Derivatives and Evaluation of Cell Cycle Arrest in Breast and Colorectal Cancer Cells

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1996 ◽  
Author(s):  
Bartolomeo Bosco ◽  
Andrea Defant ◽  
Andrea Messina ◽  
Tania Incitti ◽  
Denise Sighel ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Density Functional ◽  
Cell Cycle Progression ◽  
Cycle Arrest ◽  
M Phase

Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe three reversine-related molecules, designed by docking calculation, that present structural modifications in the diamino units at positions 2 and 6. We investigated the conformations of the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas phase, water and chloroform, the last solvent considered to give insights into the detection of broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the HN(7) form, but the most stable conformations changed in different solvents. Molecules 1–3 were evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy. Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell lines used, similar concentrations of 1–3 were effective only in cells where p53 was deleted or down-regulated. Therefore, our findings support a potential selective role of these structurally simplified, reversine-related molecules in p53-defective cancer cells.

The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2732-2732
Author(s):  
Enrico Derenzini ◽  
Ilaria Iacobucci ◽  
Elisa Brighenti ◽  
Federica Cattina ◽  
Richard Eric Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cycle Arrest ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 2732 The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 2075-2083 ◽  
Author(s):  
Lisheng Wang ◽  
Jie Wang ◽  
Bradley W. Blaser ◽  
Anne-Marie Duchemin ◽  
Donna F. Kusewitt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Single Agent ◽  
Acquired Resistance ◽  
Myelogenous Leukemia ◽  
Cyclin D2 ◽  
Down Regulation ◽  
Cycle Arrest ◽  
Pharmacologic Inhibition

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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