The BH3-only protein Bim plays a critical role in leukemia cell death triggered by concomitant inhibition of the PI3K/Akt and MEK/ERK1/2 pathways.

Mechanisms underlying apoptosis induced by concomitant interruption of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways were investigated in human leukemia cells. Inhibition of these pathways using the MEK inhibitor PD184352 or U0126 and the PI3K/Akt inhibitor perifosine strikingly induced apoptosis in multiple malignant human hematopoietic cells, and substantially reduced the colony-forming capacity of primary acute myeloblastic leukemia, but not normal CD34+ cells. These events were associated with pronounced Bim up-regulation, Mcl-1 down-regulation, marked Bak/Bax conformational change accompanied by Bax membrane translocation, and a pronounced increase in Bax/Bak association. Molecular studies using tet-inducible Akt, constitutively active MEK1, dominant-negative Akt, and MEK1 small interfering RNA revealed that inhibition of both MEK/ERK1/2 and Akt pathways plays a critical functional role in perifosine/PD184352-mediated lethality. Ectopic Mcl-1 expression potently inhibited perifosine/PD184352-induced apoptosis, as did Bak or Bax knockdown. Notably, knockdown of Bim, but not Bad, blocked Bak and Bax conformational change, inhibited Bax membrane translocation, diminished Bax/Bak binding, and sharply attenuated perifosine/PD184352-induced apoptosis. Finally, enforced expression of Bim significantly enhanced apoptosis induced by PI3K/Akt inhibitors, analogous to the effects of MEK1/2 inhibitors. Collectively, these findings suggest that Bim, and Mcl-1, but not Bad, integrate death signaling triggered by concomitant disruption of the PI3K/Akt and MEK1/2/ERK1/2 pathways in human leukemia cells.

[1]  R. Bataille,et al.  Endogenous association of Bim BH3‐only protein with Mcl‐1, Bcl‐xL and Bcl‐2 on mitochondria in human B cells , 2005, European journal of immunology.

[2]  D. Green,et al.  Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. , 2006, Molecular cell.

[3]  Peter Kraft,et al.  PIK3CA mutation is associated with poor prognosis among patients with curatively resected colon cancer. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[4]  R. Craig,et al.  MCL1 is phosphorylated in the PEST region and stabilized upon ERK activation in viable cells, and at additional sites with cytotoxic okadaic acid or taxol , 2004, Oncogene.

[5]  John Calvin Reed,et al.  Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. , 2006, Cancer cell.

[6]  H. Aburatani,et al.  The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas , 2009, British Journal of Cancer.

[7]  P. Dent,et al.  The Kinase Inhibitor Sorafenib Induces Cell Death through a Process Involving Induction of Endoplasmic Reticulum Stress , 2007, Molecular and Cellular Biology.

[8]  S. Grant,et al.  The Histone Deacetylase Inhibitor Sodium Butyrate Interacts Synergistically with Phorbol Myristate Acetate (PMA) to Induce Mitochondrial Damage and Apoptosis in Human Myeloid Leukemia Cells through a Tumor Necrosis Factor-α-Mediated Process , 2002 .

[9]  Dan Xu,et al.  Specific Cleavage of Mcl-1 by Caspase-3 in Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis in Jurkat Leukemia T Cells* , 2005, Journal of Biological Chemistry.

[10]  Xiao-Ming Yin,et al.  Degradation of Mcl-1 by Granzyme B , 2004, Journal of Biological Chemistry.

[11]  T. Kuwana,et al.  BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. , 2005, Molecular cell.

[12]  Brian J. Smith,et al.  Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. , 2005, Molecular cell.

[13]  E. Oliva,et al.  Pathogenetic Pathways in Ovarian Endometrioid Adenocarcinoma: A Molecular Study of 29 Cases , 2009, The American journal of surgical pathology.

[14]  N. Munshi,et al.  Targeting MEK induces myeloma-cell cytotoxicity and inhibits osteoclastogenesis. , 2007, Blood.

[15]  Xiao-Fan Wang,et al.  Transcriptional Activation of p 21 waf 1 / cip 1 by Alkylphospholipids : Role of the Mitogen-Activated Protein Kinase Pathway in the Transactivation of the Human p 21 waf 1 / cip 1 Promoter by Sp 1 , 2004 .

[16]  P. Pandolfi,et al.  Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. , 2008, The Journal of clinical investigation.

[17]  E. Vellenga,et al.  Constitutive NF-κB DNA-binding activity in AML is frequently mediated by a Ras/PI3-K/PKB-dependent pathway , 2004, Leukemia.

[18]  E. Sausville,et al.  Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation. , 2003, Molecular cancer therapeutics.

[19]  P. Dent,et al.  Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells. , 2001, Cancer research.

[20]  R. Bataille,et al.  Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells. , 2002, Blood.

[21]  Ceri M. Wiggins,et al.  ERK1/2‐dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl‐1 and Bcl‐xL , 2007, The EMBO journal.

[22]  M. Inouye,et al.  NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. , 2007, Genes & development.

[23]  Lin Chen,et al.  Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. , 2005, Genes & development.

[24]  P. Dent,et al.  Apoptosis Induced by the Kinase Inhibitor BAY 43-9006 in Human Leukemia Cells Involves Down-regulation of Mcl-1 through Inhibition of Translation* , 2005, Journal of Biological Chemistry.

[25]  Paul Dent,et al.  The Multikinase Inhibitor Sorafenib Induces Apoptosis in Highly Imatinib Mesylate-Resistant Bcr/Abl+ Human Leukemia Cells in Association with Signal Transducer and Activator of Transcription 5 Inhibition and Myeloid Cell Leukemia-1 Down-Regulation , 2007, Molecular Pharmacology.

[26]  Yi-Te Hsu,et al.  Movement of Bax from the Cytosol to Mitochondria during Apoptosis , 1997, The Journal of cell biology.

[27]  Audy G. Whitman,et al.  Targeting the PI3K and MAPK pathways to treat Kaposi’s sarcoma-associated herpes virus infection and pathogenesis , 2007, Expert opinion on therapeutic targets.

[28]  P. Dent,et al.  Inhibition of PI-3 kinase sensitizes human leukemic cells to histone deacetylase inhibitor-mediated apoptosis through p44/42 MAP kinase inactivation and abrogation of p21CIP1/WAF1 induction rather than AKT inhibition , 2003, Oncogene.

[29]  Erinna F. Lee,et al.  Apoptosis Initiated When BH3 Ligands Engage Multiple Bcl-2 Homologs, Not Bax or Bak , 2007, Science.

[30]  S. Cory,et al.  The Bcl-2 apoptotic switch in cancer development and therapy , 2007, Oncogene.

[31]  J. LoPiccolo,et al.  Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. , 2008, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[32]  F. Khuri,et al.  14-3-3 Integrates prosurvival signals mediated by the AKT and MAPK pathways in ZNF198-FGFR1-transformed hematopoietic cells. , 2007, Blood.

[33]  M. Carroll,et al.  Survival of acute myeloid leukemia cells requires PI3 kinase activation. , 2003, Blood.

[34]  Neal Rosen,et al.  The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells. , 2005, Cancer cell.

[35]  M. Loda,et al.  Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. , 2005, The Journal of molecular diagnostics : JMD.

[36]  S. Korsmeyer,et al.  Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Keith L. Ligon,et al.  Coactivation of Receptor Tyrosine Kinases Affects the Response of Tumor Cells to Targeted Therapies , 2007, Science.

[38]  Michael E. Greenberg,et al.  Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis , 1995, Science.

[39]  S. Payne,et al.  Coadministration of histone deacetylase inhibitors and perifosine synergistically induces apoptosis in human leukemia cells through Akt and ERK1/2 inactivation and the generation of ceramide and reactive oxygen species. , 2005, Cancer research.

[40]  J. Engel,et al.  D-21266, a new heterocyclic alkylphospholipid with antitumour activity. , 1997, European journal of cancer.

[41]  Dennis C. Sgroi,et al.  Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis , 2003, Nature Cell Biology.

[42]  J. Lammers,et al.  Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1 , 2000, Current Biology.

[43]  Wenhua Gao,et al.  Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. , 2003, Genes & development.

[44]  Suzanne Schubbert,et al.  Hyperactive Ras in developmental disorders and cancer , 2007, Nature Reviews Cancer.

[45]  J. Hsieh,et al.  Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies , 2006, Nature Cell Biology.

[46]  S. Willis,et al.  Life in the balance: how BH3-only proteins induce apoptosis. , 2005, Current opinion in cell biology.

[47]  E. Wagner,et al.  Activation of ERK1/2 by ΔRaf-1 : ER* represses Bim expression independently of the JNK or PI3K pathways , 2003, Oncogene.

[48]  Stephen L. Abrams,et al.  Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. , 2006, Advances in enzyme regulation.

[49]  G. Wildey,et al.  Evidence That Ser87 of BimEL Is Phosphorylated by Akt and Regulates BimEL Apoptotic Function* , 2006, Journal of Biological Chemistry.

[50]  Xiaojie Li,et al.  Rapid and selective death of leukemia stem and progenitor cells induced by the compound 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (TDZD-8). , 2007, Blood.

[51]  T. Chou,et al.  Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. , 1984, Advances in enzyme regulation.

[52]  Bernd Giese,et al.  Targeting phosphoinositide 3-kinase: moving towards therapy. , 2008, Biochimica et biophysica acta.

[53]  K. Murphy,et al.  Bcl-2 Inhibits a Fas-induced Conformational Change in the Bax N Terminus and Bax Mitochondrial Translocation* , 2000, The Journal of Biological Chemistry.

[54]  Hiroshi Yasui,et al.  Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells. , 2005, Blood.