scholarly journals BCR-ABL and Src Family Kinases Induce YAP Tyrosine Phosphorylation Resulting in Survivin and Cyclin D1 Expression in Chronic Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5366-5366
Author(s):  
Toshiyuki Hori ◽  
Kenta Moriyama
Keyword(s):  
Cell Growth ◽  
Cyclin D1 ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Hippo Pathway ◽  
Src Family Kinases ◽  
Growth And Survival ◽  
Downstream Signaling ◽  
Erythroleukemia Cell

Chronic myeloid leukemia (CML) is caused by the BCR-ABL fusion protein. To date, several downstream signaling pathways of BCR-ABL have been reported to underlie the leukemogenesis of CML such as the JAK/STAT pathway, the PI3K/AKT pathway, and the Grb2/MAPK pathway. Furthermore, the Src family kinases (SFKs), especially Hck, Lyn and Fyn, have been suggested to be involved in BCR-ABL-induced transformation. Although these studies have revealed important aspects of the downstream signals of BCR-ABL, the detailed molecular mechanism of CML has not been thoroughly elucidated. Yes-associated protein (YAP) is a transcriptional cofactor that functions as an effector of the Hippo pathway which regulates cell growth and survival. In the classical Hippo pathway, YAP phosphorylated at serine 127 (S127) by LATS1/2 is bound to 14-3-3 and prevented from nuclear translocation. Apart from this serine/threonine phosphorylation, YAP undergoes phosphorylation at several tyrosine residues by various kinases to be activated. SFKs can phosphorylate and activate YAP, which has been demonstrated in some tumors. Among the several possible phosphorylated tyrosine residues, the phosphorylation at Y357 (p-Y357) has been demonstrated to be the most important for tumorigenesis. Therefore, it is possible that BCR-ABL directly or indirectly phosphorylates YAP through SFKs and thus activated YAP is translocated into the nucleus and together with TEAD induces expression of genes necessary for cell growth and survival. In the present study, we investigated the effects of imatinib and an SFK-specific inhibitor RK-20449 on viable cell number, YAP p-Y357 and expression of Survivin as well as Cyclin D1 in CML-derived cell lines in comparison with AML-derived cell lines. Furthermore, we established BCR-ABL stable transfectants and the control lines derived from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML-derived cell lines. We first checked the phosphorylation status of YAP and found that it was constitutively phosphorylated at tyrosine 357in CML-derived cell lines (TCC-S and K562) but not in AML-derived cell lines (HL-60 and KG-1a). Treatment with imatinib or RK-20449 inhibited cell growth and decreased tyrosine phosphorylation of YAP in both CML lines. Expression of Survivin or Cyclin D1 was decreased at least in TCC-S but not in AML cell lines. Furthermore, we established BCR-ABL stable transfectant and control empty vector transfectant from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML cell lines. YAP was phosphorylated at Y357 constitutively in BCR-ABL stable transfectant but not in control transfectant, and treatment with imatinib or RK-20449 inhibited cell growth, YAP tyrosine phosphorylation, and expression of Cyclin D1 in BCR-ABL stable transfectant. These results suggest that BCR-ABL induces tyrosine phosphorylation of YAP presumably through Src family kinases, which results in expression of Survivin and Cyclin D leading to leukemogenesis in CML cells. We have not determined which SFK is involved in the downstream signaling of BCR-ABL. Overexpression experiments in HEK293T have indicated that Lck, Lyn and Fyn can phosphorylate YAP at Y357 without BCR-ABL. However, it remains to be determined which SFK is activated by BCR-ABL and practically involved in YAP phosphorylation resulting in leukemogenesis. Further studies are required to specify the relevant SFKs and disclose the downstream signaling from activated YAP. Disclosures No relevant conflicts of interest to declare.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3526-3526
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Sophia Adamia ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Genomic Alteration ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Cell Growth And Survival

Abstract Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

Cell Cycle Regulation by Iron: Novel Approaches to Mantle Cell Lymphoma Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2515-2515 ◽  
Author(s):  
Heather Gilbert ◽  
John Cumming ◽  
Josef T. Prchal
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
Lymphoma Cell ◽  
Protein Levels ◽  
Mantle Cell

Abstract Abstract 2515 Poster Board II-492 Mantle cell lymphoma is a well defined subtype of B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13 (which encodes cyclin D1) next to the immunoglobulin heavy chain gene promoter on chromosome 14q32. The result is constitutive overexpression of cyclin D1 (CD1) resulting in deregulation of the cell cycle and activation of cell survival mechanisms. There are no “standard” treatments for MCL. Despite response rates to many chemotherapy regimens of 50% to 70%, the disease typically progresses after treatment, with a median survival time of approximately 3-4 years. Mantle cell lymphoma represents a small portion of malignant lymphomas, but it accounts for a disproportionately large percentage of lymphoma-related mortality. Novel therapeutic approaches are needed. In 2007, Nurtjaha-Tjendraputra described how iron chelation causes post-translational degradation of cyclin D1 via von Hippel Lindau protein-independent ubiquitinization and subsequent proteasomal degradation (1). Nurtjaha-Tjendraputra demonstrated that iron chelation inhibits cell cycle progression and induces apoptosis via proteosomal degradation of cyclin D1 in various cell lines, including breast cancer, renal carcinoma, neuroepithelioma and melanoma. Our preliminary data show similar findings in mantle cell lymphoma. To establish whether iron chelation can selectively inhibit and promote apoptosis in mantle cell derived cell lines, the human MCL cell lines Jeko-1, Mino, Granta and Hb-12; the Diffuse Large B cell lymphoma line SUDHL-6; and the Burkitt's Lymphoma lines BL-41 and DG75 were grown with media only, with two different iron chelators (deferoxamine (DFO) and deferasirox) at various concentrations (10, 20, 40, 100 and 250 μM), and with DMSO as an appropriate vehicle control. Cells were harvested at 24, 48 and 72 hours. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V antibody and PI (BD Pharmingen, San Diego, CA). Cell growth was analyzed using the Promega MTS cytotoxicity assay. CD1 protein levels were assessed using standard Western blot techniques. At 24, 48 and 72 hours of incubation with iron chelators, the mantle cell lymphoma cell lines showed significantly increased rates of apoptosis compared to the non-mantle cell lymphoma cell lines (p<0.0001 for all time points). DFO and deferasirox inhibted cell growth with an IC50 of 18 and 12 μM respectively. All of the mantle cell lines had measurable cyclin D1 levels at baseline. None of the non-mantle cell lines expressed baseline measurable cyclin D1. In the mantle cell lines, cyclin D1 protein levels were no longer apparent on western blot after 24 hours of incubation with chelation. We then added ferrous ammonium sulfate (FAS) to DFO in a 1:1 molarity ratio and to deferasirox in a 2:1 ratio, and then treated the same lymphoma cell lines with the FAS/chelator mixture and with FAS alone for 72 hours. Adding iron to the chelators completely negated all the pro-apoptotic effects that were seen with iron chelation treatment. Treating with FAS alone had no effect on cell growth or apoptosis. Iron chelation therapy with both DFO and deferasirox results in decreased cell growth, increased cellular apoptosis, and decreased cyclin D1 protein levels in vitro in mantle cell lymphoma. The cytotoxic effects are prevented by coincubation with ferrous ammonium citrate, confirming that the effects are due to iron depletion. Proposed future research includes further defining the molecular basis of iron chelation effects; studying these therapies in combination with other cancer treatments both in vitro and in vivo; and studying iron chelation therapy in mantle cell lymphoma patients. 1. Nurtjahja-Tjendraputra, E., D. Fu, et al. (2007). “Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression.” Blood109(9): 4045–54. Disclosures: No relevant conflicts of interest to declare.

Reversing Metabolic and Epigenetic Cellular Alterations to Overcome Chemo-Resistance in Aggressive B Cell Lymphomas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1305-1305
Author(s):  
Kejie Zhang ◽  
Lan V Pham ◽  
Archito T. Tamayo ◽  
John Lee ◽  
Jerry Bryant ◽  
...  
Keyword(s):  
Cell Growth ◽  
Glucose Metabolism ◽  
Tumor Cell ◽  
Cell Lines ◽  
Histone Methylation ◽  
Ros Production ◽  
Tumor Cell Growth ◽  
Growth And Survival ◽  
Methylation Inhibitor ◽  
Cell Growth And Survival

Abstract Abstract 1305 Cancer cells exist in a stressed environment, mainly due to lack of nutrients and oxygen, particularly during chemotherapy, and rely on metabolic homeostatic regulatory mechanisms for protection against these lethal challenges. Increasing glucose metabolism and continuous reactive oxygen species (ROS) production is one strategy of metabolic adaptation utilized by tumor cells to relieve this stress. Thioredoxin interacting protein (TXNIP) is a negative regulator for both redox thioredoxin (ROS production) and cellular glucose uptake, not well understood but found to be repressed in various cancers, including diffuse large B-cell lymphomas (DLBCL), the most common form of non-Hodgkin lymphoma that continues increasing in incidence and remains incurable in many cases, primarily due to development of chemo-resistance. The molecular mechanisms by which TXNIP expression is down-regulated during cancer progression and chemo-resistance development have not been completely elucidated. Since key gene silencing events have now been identified in the pathogenesis of DLBCL, recent therapeutic interest has focused on dysregulated histone modifications as potentially important therapeutic targets, for developing strategies that can reactivate silenced tumor suppressor genes. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), is a highly conserved histone methyltransferase that targets lysine-27 of histone H3 (H3K27). Studies in human tumors show that EZH2 is frequently over-expressed in a wide variety of tumors, including lymphomas. More importantly, recent studies using whole-genome sequencing in primary DLBCL, identified frequent mutations in the EZH2 gene that leads to critical gene silencing in DLBCL pathophysiology. Our study showed that EZH2 is either over-expressed or mutated in representative DLBCL cell lines and primary DLBCL cells, and that down-regulation of EZH2 with siRNA leads to the reactivation of TXNIP, with subsequent inhibition of tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL. We also found that histone deacetylation (HDAC) is also involved in EZH2-mediated silencing of TXNIP in DLBCL. Pharmacologic agents aimed at reactivating TXNIP genes include histone methylation inhibitor 3-Deazaneplanocin A (DZNep) that targets EZH2, as well as HDAC inhibitor Vorinostat. DZNep is currently the only histone methylation inhibitor that is commercially available. Our data indicated that DZNep is highly effective in inhibiting cell growth in various DLBCL cell lines, particularly in chemo-resistant DLBCL cell lines. Vorinostat, on the other hand, has been a good drug and is currently in clinical trial for relapsed DLBCL and has been FDA approved for treating cutaneous T-cell lymphoma patients. Our data showed synergistic activity of DZNep and Vorinostat in reactivating TXNIP gene expression and inhibiting DLBCL cell growth and survival. We also discovered that EZH2 controls constitutive NF-κB activity through both, the canonical and alternative NF-κB pathways in DLBCL. This function of EZH2 is independent of its histone methyltransferase activity. These findings reveal that EZH2 and NF-κB, the two oncogenic factors display functional crosstalk in DLBCL cells. Our findings have indicated that deregulated EZH2 leads to constitutive NF-kB activation and to epigenetic silencing of TXNIP, resulting in uncontrolled tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL, and that targeting this pathway represents a novel, rational, and effective therapeutic approach to selectively reverse chemoresistance in DLBCL patients, particularly relapsed/refractory patients. Disclosures: No relevant conflicts of interest to declare.

Diffuse Large B-Cell Lymphomas Utilize Endogenous and Exogenous Fatty Acids for Cell Growth and Survival

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1581-1581
Author(s):  
Frederick Lansigan ◽  
Wilson L Davis ◽  
Nancy Kuemmerle ◽  
Leslie E Lupien ◽  
Valeriya Posternak ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fatty Acid ◽  
Cell Growth ◽  
B Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Functional Significance ◽  
De Novo ◽  
Growth And Survival ◽  
Large B Cell

Abstract Abstract 1581 Background It is well-recognized that de novo long chain fatty acid (FA) synthesis, driven by the key enzyme fatty acid synthase (FASN), is crucial for the growth and survival of many types of cancer cells. We and others have observed FASN protein expression in diffuse large B-cell lymphoma (DLBCL) tumors. Furthermore, we have shown that higher levels of FASN in DLBCL tumors strongly predicted inferior survival, which was independent from the international prognostic index. We also recently demonstrated that, in addition to FA synthesis, various cancer cells can acquire FA from circulating lipoproteins, using the secreted enzyme lipoprotein lipase (LPL), and that this promotes cell growth. DLBCL, however, has never been examined in this regard. In this study, we investigated the functional significance of both de novo FA synthesis via FASN and exogenous FA uptake via LPL in DLBCL. Methods Levels of FASN and LPL mRNAs in DLBCL cell lines (SUDHL4, SUDHL10, OCI-LY3, OCI-LY19) were studied using reverse transcriptase polymerase chain reaction. We determined FASN and LPL protein expression by flow cytometry using a novel anti-LPL antibody that we developed. DLBCL cell lines were cultured +/− Cerulenin (an inhibitor of FASN), Orlistat (an inhibitor of FASN and LPL), or in lipoprotein-depleted serum +/− supplementation with very low density lipoprotein (VLDL) particles. The MTT assay was used to assess cell proliferation. Results DLBCL cell lines exhibited >10-fold variation in levels of FASN mRNA. Cerulenin and Orlistat each caused dose-dependent inhibition of proliferation of each cell line. The cells were partially rescued by the addition of palmitic acid, the FA product of FASN. Surprisingly, flow cytometry revealed that SUDHL4 and OCI-LY3 cells, which did not secrete LPL or show detectable LPL activity, displayed the enzyme on the cell surface. Moreover, in stark contrast to several other cancer cell lines, DLBCL cells were exquisitely sensitive to withdrawal of lipoproteins from the culture media. Indeed, 75–95% of the cells underwent apoptosis after only 24 hours in lipoprotein-depleted serum. In complete serum, the provision of VLDL particles did not rescue DLBCL cells from FA synthesis inhibition using Cerulenin, suggesting that the serum contains sufficient lipoproteins to saturate the FA uptake system. This prediction was validated in experiments utilizing lipoprotein-depleted serum, in which add-back of VLDL particles completely rescued the cells from Cerulenin-induced demise in a dose-related manner, with full restoration at approximately 100–200mcg/ml of VLDL. Conclusions Our data demonstrate that DLBCL cells employ both de novo FA synthesis via FASN and exogenous FA uptake using LPL to satisfy their strict requirement for FA. Interference with either pathway, using FASN inhibitors or lipoprotein-depleted serum, is cytotoxic indicating that neither alone is sufficient to support proliferation. Further, DLBCL cells show a striking dependency on exogenous FA of dietary origin compared with all other cancer cells we have examined. The observation that the cell lines can be rescued by provision of VLDL particles strongly supports the functional significance of the exogenous FA uptake pathway for DLBCL. Our data thus demonstrate that the extracellular lipase LPL is critical for the growth and survival of DLBCL cells. Surprisingly, the cells deploy LPL to their surface, and we speculate that this promotes efficient FA acquisition from circulating lipoproteins. Recognition that DLBCL relies on both synthesis and uptake of FA will provide guidance for drug development and dietary modifications to effectively target the metabolic requirements of this tumor. Disclosures: No relevant conflicts of interest to declare.

IRF2-INPP4B axis participates in the development of acute myeloid leukemia by regulating cell growth and survival

Gene ◽  
2017 ◽  
Vol 627 ◽  
pp. 9-14 ◽  
Author(s):  
Feng Zhang ◽  
Junfeng Zhu ◽  
Jiajia Li ◽  
Fangbing Zhu ◽  
Pingping Zhang
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Myeloid Leukemia ◽  
Growth And Survival ◽  
Acute Myeloid ◽  
Cell Growth And Survival

scholarly journals Phosphorylation of p27Kip1 Regulates Assembly and Activation of Cyclin D1-Cdk4

2008 ◽  
Vol 28 (20) ◽  
pp. 6462-6472 ◽  
Author(s):  
Michelle D. Larrea ◽  
Jiyong Liang ◽  
Thiago Da Silva ◽  
Feng Hong ◽  
Shan H. Shao ◽  
...  
Keyword(s):  
Complex Formation ◽  
Cyclin D1 ◽  
Tyrosine Phosphorylation ◽  
Combined Therapy ◽  
Src Family Kinases ◽  
Kinase Activation ◽  
Constitutive Activation ◽  
Catalytic Activation ◽  
Inhibitory Function

ABSTRACT p27 mediates Cdk2 inhibition and is also found in cyclin D1-Cdk4 complexes. The present data support a role for p27 in the assembly of D-type cyclin-Cdk complexes and indicate that both cyclin D1-Cdk4-p27 assembly and kinase activation are regulated by p27 phosphorylation. Prior work showed that p27 can be phosphorylated by protein kinase B/Akt (PKB/Akt) at T157 and T198. Here we show that PKB activation and the appearance of p27pT157 and p27pT198 precede p27-cyclin D1-Cdk4 assembly in early G1. PI3K/PKB inhibition rapidly reduced p27pT157 and p27pT198 and dissociated cellular p27-cyclin D1-Cdk4. Mutant p27 allele products lacking phosphorylation at T157 and T198 bound poorly to cellular cyclin D1 and Cdk4. Cellular p27pT157 and p27pT198 coprecipitated with Cdk4 but were not detected in Cdk2 complexes. The addition of p27 to recombinant cyclin D1 and Cdk4 led to cyclin D1-Cdk4-p27 complex formation in vitro. p27 phosphorylation by PKB increased p27-cyclin D1-Cdk4 assembly in vitro but yielded inactive Cdk4. In contrast, Src pretreatment of p27 did not affect p27-cyclin D1-Cdk4 complex formation. However, Src treatment led to tyrosine phosphorylation of p27 and catalytic activation of assembled cyclin D1-Cdk4-p27 complexes. Thus, while PKB-dependent p27 phosphorylation appears to increase cyclin D1-Cdk4-p27 assembly or stabilize these complexes in vitro, cyclin D1-Cdk4-p27 activation requires the tyrosine phosphorylation of p27. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process.

Phosphoproteomic Analysis of Primary Acute Myeloid Leukemia Cells Reveals Redundant Roles for Src Family Kinases in AML Survival

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2650-2650
Author(s):  
Jamil K. Dierov ◽  
Tae Kon Kim ◽  
Xiaowei Yang ◽  
Alexander Perl ◽  
Beth A Burke ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Src Family Kinases ◽  
Variable Degree ◽  
Acute Myeloid Leukemia Cells ◽  
Lck Kinase ◽  
Sirna Knockdown ◽  
Acute Myeloid

Abstract Recent results have demonstrated that multiple signal transduction pathways are activated in acute myeloid leukemia (AML) cells, however, the tyrosine kinase(s) that phosphorylates these signaling proteins is not identified. We have analyzed AML cells using a phosphoproteomics screen and demonstrate that the Src family kinases, Lyn, Lck and Fgr, are phosphorylated on their activation sites in AML samples. Expression and activation of Lyn has been previously confirmed. Evaluation of Lck demonstrated that Lck is expressed to a variable degree but consistently in AML samples (n=20). Lck kinase assays show activation of Lck in 17/20 samples tested at levels above the level of activation detectable in normal CD34+ progenitor cells. Lyn and Lck both contribute to AML cell growth as siRNA depletion of either kinase leads to decreased leukemia colony forming activity. Interestingly, both Lyn and Lck contribute to phosphorylation of STAT5 as STAT5 phosphorylation is decreased but not abrogated by siRNA modification of either kinase alone. Consistent with the necessity for this signaling pathway for optimal AML cell growth, siRNA knockdown of STAT5 leads to decreased expression of both STAT5A and STAT5B, decreased expression of the STAT5 target protein, Bclxl and decreased AML colony forming ability. Based on this data, we have studied the FDA approved compound, Dasatinib, and demonstrate that Dasatinib decreases AML colony formation in 4 out of 5 samples tested. Overall, these results demonstrate that SFK’s act redundantly to regulate STAT5 phosphorylation and AML cell growth in primary cells and that phosphoprotein analysis is a robust approach to identify new targets for therapy of malignancy. Src family kinase inhibitors may be valuable in the therapy of AML.

Sign in / Sign up

Export Citation Format

Share Document