Leukemia (2008) 22, 686–707 & 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00 www.nature.com/leu REVIEW

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

LS Steelman1, SL Abrams1, J Whelan1, FE Bertrand1, DE Ludwig2,JBa¨secke3, M Libra4, F Stivala4, M Milella5, A Tafuri6, P Lunghi7, A Bonati7,8, AM Martelli9,10 and JA McCubrey1

1Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA; 2ImClone Systems, New York, NY, USA; 3Division of Hematology and Oncology, Department of Medicine, Georg-August University, Go¨ttingen, Germany; 4Department of Biomedical Sciences, University of Catania, Catania, Italy; 5Regina Elena Cancer Center, Rome, Italy; 6Department of Cellular Biotechnology and Hematology, University La Sapienza of Rome, Rome, Italy; 7Department of Clinical Sciences, University of Parma, Parma, Italy; 8Unit of Hematology and Bone-Marrow Transplantation, University Hospital of Parma, Parma, Italy; 9Department of Human Anatomical Sciences, University of Bologna, Bologna, Italy and 10IGM-CNR, c/o IOR, Bologna, Italy

Mutations and chromosomal translocations occur in leukemic and preventing apoptosis in hematopoietic cells.1–5 An over- cells that result in elevated expression or constitutive activation view of the effects of the Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/ of various growth factor receptors and downstream . The Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT path- mTOR and Jak/STAT pathways on downstream signaling ways are often activated by mutations in upstream . The pathways leading to growth and the prevention of apoptosis Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways are regu- is presented in Figure 1. After receptor ligation, Shc, a Src lated by upstream Ras that is frequently mutated in human homology (SH) 2 domain-containing protein, becomes asso- cancer. Recently, it has been observed that the FLT-3 and Jak ciated with the C terminus of the growth factor receptor.6–8 Shc kinases and the phosphatase and tensin homologue deleted on recruits the GTP-exchange complex growth factor receptor 10 (PTEN) phosphatase are also frequently mutated or their expression is altered in certain hematopoietic bound-2 (Grb2)/son of sevenless exchange (Sos) proteins . Many of the events elicited by the Raf/MEK/ERK, (Grb2/Sos) resulting in the loading of membrane-bound 9,10 PI3K/PTEN/Akt/mTOR and Jak/STAT pathways have direct Ras with GTP. Ras:GTP then recruits Raf to the membrane effects on survival pathways. Aberrant regulation of the where it becomes activated, likely via a Src family tyrosine survival pathways can contribute to uncontrolled cell growth (Y) .11–13 and lead to leukemia. In this review, we describe the Raf/MEK/ Raf is a multigene family that consists of RAF1, ARAF and ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT signaling cascades and summarize recent data regarding the regulation and BRAF, which encode proteins of 74, 68 and 94 kDa, respec- mutation status of these pathways and their involvement in tively. The Raf proteins have three distinct functional domains: leukemia. CR1, CR2 and CR3. The CR1 domain is necessary for Ras Leukemia (2008) 22, 686–707; doi:10.1038/leu.2008.26; binding and subsequent activation. The CR2 domain is a published online 13 March 2008 regulatory domain. CR3 is the kinase domain. Deletion of the Keywords: Raf; PI3K; Akt; signal transduction; inhibitors; CR1 and CR2 domains produces a constitutively active Raf chemotherapeutic drugs protein due to, in part, the removal of phosphorylation sites that serve to negatively regulate the kinase in the CR2 domain.14 Introduction Raf-1 is fairly ubiquitously expressed. B-Raf was originally thought to be expressed primarily in neuronal and hematopoie- In this first review, we summarize the Raf/MEK/ERK, PI3K/PTEN/ tic tissues, but has since been shown to be expressed in more Akt/mTOR and Jak/STAT pathways with regard to how these diverse tissues including melanocytes and thyroid cells that are cascades are regulated. We also discuss how these pathways hormonal responsive cell types. B-Raf mutations play significant can interact and cross-regulate each other. Furthermore, we roles in malignant transformation in melanocytes and thyroid discuss the effects of key mutations at critical components in cells as the hormones stimulate proliferation of B-Raf-dependent these pathways and how they influence the leukemogenic signaling pathways. A-Raf has a more limited tissue expression process. In the accompanying review in this issue of Leukemia, and is expressed in urogenital and intestine cells. A-Raf is we summarize how specific targeting of these pathways may expressed predominately in urogenital tissues, including the 15 enhance leukemia therapy. kidney. The different Raf genes have been knocked out in mice to examine some of the global roles of the Raf genes on development. ARAF deletion results in postnatal lethality Overview of the Ras/Raf/MEK/ERK pathway attributed to neurological and gastrointestinal defects. BRAF deletion results in intrauterine death between days 10.5 and The Ras/Raf/MEK/ERK pathway is activated by many growth 12.5 and the mouse embryos display enlarged blood vessels and factors and cytokines that are important in driving proliferation increased apoptosis of endothelial cells. Many of the functions of Raf-1 in RAF1 knockout mice are believed to be still present Correspondence: Dr JA McCubrey, Department of Microbiology and due to the presence of functional BRAF genes, and B-Raf can Immunology, Brody School of Medicine at East Carolina University, fulfill many of the functions of Raf-1. 600 Moye Boulevard, 5th Floor Brody Building 5N98C, Greenville, Raf kinases are required for phosphorylation of the mitogen- NC 27858, USA. 16–18 E-mail: [email protected] associated/extracellular regulated kinase-1 (MEK1). MEK1 Received 28 November 2007; revised 22 January 2008; accepted 23 phosphorylates extracellular regulated kinases 1 and 2 (ERKs 1 January 2008; published online 13 March 2008 and 2) on specific threonine (T) and Y residues.16–18 Activated Signaling and apoptotic pathways and leukemia LS Steelman et al 687 Overview of Raf/MEK/ERK, into an inactive complex. This complex of Raf-1:MST-2 is PI3K/PTEN/Akt/mTOR and Jak/STAT Pathways independent of MEK and downstream ERK. Raf-1 can also interact with the apoptotic signal kinase (ASK1) to inhibit apoptosis.49 ASK1 is a general mediator of apoptosis and it is Growth Factor induced in response to a variety of cytotoxic stresses including β tumor necrosis factor (TNF), Fas and reactive oxygen species α (ROS). ASK1 appears to be involved in the activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs). These are examples of interactions of Raf-1 with kinases and antiapoptotic molecules that are independent of MEK and ERK. Raf-1 can also interact with Bcl-2 at the to influence its activity (Figure 1).

Overview of the PI3K/PTEN/Akt/mTOR pathway

Growth factor/cytokine receptor ligation also leads to rapid activation of phosphatidylinositol 3-kinase (PI3K).51,52 PI3K consists of an 85-kDa regulatory subunit, which contains SH2 Regulation of 51,52 protein and SH3 domains, and a 110-kDa catalytic subunit. translation and Cytokine stimulation often creates a PI3K- on the cell size and growth cytokine receptor. The p85 subunit SH2 domain associates with AAA AAA this site.51,52 The p85 subunit is then phosphorylated which Regulation of leads to activation of the p110 catalytic subunit. transcription Class IA PI3Ks are heterodimeric proteins that consist of Figure 1 Overview of Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/ 85-kDa regulatory and 110-kDa catalytic subunits. An overview STAT Pathways. The Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/ of the PI3K/PTEN/Akt/mTOR pathway is presented in Figure 1. STAT pathways are regulated by upstream growth factor receptors as The p85 regulatory subunit contains a SH2 domain, that well as various kinases. Many kinases serve to phosphorylate serine/ recognizes phosphorylated Y residues, an inter SH2 domain (a threonine (S/T) and tyrosine (Y) residues on various proteins in this rigid tether for p110), a conserved domain related to sequences cascade. Some of these phosphorylation events serve to enhance activity (shown by a black P in a white circle) whereas others serve to present in the breakpoint cluster region (BCR) gene and a SH3 inhibit activity (shown by a white P in a black circle. Phosphatases domain that are found in proteins that interact with other such as phosphatase and tensin homologue deleted on chromosome proteins and mediate assembly of specific protein complexes via 10 (PTEN) that inhibit the function of proteins are indicated by a black binding to proline-rich motifs.53,54 The 110-kDa PI3K class 1A octagon with white lettering. The downstream transcription factors catalytic subunit contains a p85-binding domain, a Ras-binding regulated by these pathways are indicated in diamond-shaped domain, a kinase domain and a helical domain.14,52–55 The p85 outlines. subunit SH2 domain associates with this site.50,51,55 The p85 subunit is then phosphorylated that leads to activation of the p110 catalytic subunit. This often occurs at the inner leaflet of ERK1 and ERK2 serine (S)/T kinases phosphorylate and activate a the cytoplasmic membrane, although there are other important variety of substrates.19–25 90 kDa ribosomal six kinase 1 roles of PI3K in nuclear membranes that have been reviewed (p90Rsk1) is one such substrate. p90Rsk1 can activate the cyclic- recently.56–58 A diagram illustrating some of the important roles AMP response element-binding protein (CREB) transcription of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in the factor.22 Moreover, ERK can translocate to the nucleus and cytoplasm and nucleus is presented in Figure 2. It is important to phosphorylate additional transcription factors such as Elk1 and note that these two cascades, as well as the Jak/STAT pathway, Fos that bind promoters of many genes, including growth factor also have critical features in the nucleus, although their and cytokine genes important in stimulating the growth and initial activation often occurs by ligation of cytokine and other survival of hematopoietic cells.26–37 The Raf/MEK/ERK pathway types of receptors expressed on the plasma membrane can also modulate the activity of many proteins involved in hematopoietic cells. apoptosis including: B cell leukemia-2 (Bcl-2), Bad, Bim, PI3K is a multigene family. There are at least eight distinct myeloid cell leukemia-1 (Mcl-1), caspase 9 and survivin.38–47 isoforms of the PI3K catalytic subunit and at least seven Raf-1 has many roles that are independent of MEK and ERK. It regulatory subunits.58 These genes encode proteins with is important to discuss these interactions as they serve to different functions and there are probably many unknown illustrate the concept that targeting Raf, which will be covered in functions that may play critical roles in activation/differentiation the accompanying review, will have additional effects than just of hematopoietic cells which remain to be elucidated. PI3K lipid inhibition of downstream MEK/ERK. Many of these non-MEK/ kinases have been grouped into three classes (I–III) according to ERK functions are involved in the prevention of apoptosis.4 Raf- their substrate preference and .59–62 The 1 interacts with mammalian sterile 20-like kinase (MST-2) and class I PI3K catalytic subunits have been grouped into two prevents its dimerization and activation.48–50 MST-2 is a kinase, families, class IA and class IB. Class 1A consists of p110a which is activated by proapoptotic agents such as staurosporine (PIK3CA), p110b (PIK3CB) and p110d (PIK3CD). Class IA p110 and Fas ligand. Raf-1, but not B-Raf, binds MST-2. Depletion of are activated by tyrosine kinase receptors and Ras. Class IA p110 MST-2 from Raf-1À/À cells abrogated sensitivity to apoptosis. PI3Ks bind one of the three regulatory subunits p85a or the Overexpression of MST-2 increased sensitivity to apoptosis. It splice variants p55a or p50a that are encoded by PIK3R, p85b was proposed that Raf-1 might control MST-2 by sequestering it encoded by PIK3R2 or p55g encoded by PIK3R3.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 688 Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Have Cytoplasmic as Well as Nuclear Roles

PTEN PTEN PTEN PTEN regulates regulates apoptosis Fak, Shc cell growth by protein PTEN by controlling dephosphorylation Akt activation (controversial)

Cytoplasm

PTEN PTEN Mitochondrion regulates apoptosis in Nucleus cytoplasm by Regulation of controlling protein Akt activation translation and cell size and growth

Cell Cycle Gene Regulation PTEN Nuclear PTEN regulates ERK, CyclinD1, cell cycle progression centromeres and Gene Transcription chromosome stability

Growth factors, p27Kip-1, Puma, Noxa anti-apoptotic Cell Cycle Apoptotic Gene gene expression Progression Expression

Figure 2 Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR have cytoplasmic as well as nuclear roles. Although the cytoplasmic membrane localized roles of these pathways are more frequently discussed, these pathways often have nuclear roles that are only beginning to be understood. Many of the nuclear functions of these pathways may regulate gene transcription, cell cycle progression as well as chromosome stability and replication. The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase is illustrated by a stop sign octagon as it has been shown to be an important tumor suppressor and have roles regulating apoptosis in the cytoplasm and cell cycle progression in the nucleus.

The p85a and p85b proteins display a wide tissue distribution, PDK1 then phosphorylates Akt on a T regulatory residue. while the p55g protein is more restricted and is highly expressed Akt is also a member of a multigene family (Akt-1, Akt-2 and in brain and testis. The class IA regulatory subunits recruit the Akt-3) and is also called (PKB) encoded by p110 catalytic subunit to phosphotyrosine (pY) residues in AKT1, AKT2 and AKT3, respectively. Depending on the Akt receptors, adapter proteins and other molecules. This localizes isoform, PDK1 can phosphorylate Akt on T308/309/305. A the class IA p110 subunits in the membranes where their lipid second kinase phosphorylates Akt on an S regulatory residue substrates reside. The adapter/regulatory subunits act to localize (S473/474/472).63–67 The identity of the second kinase (often PI3K to the plasma membrane by the interaction of their SH2 referred to as PDK2) that phosphorylates Akt has remained domains with pY residues in activated receptors. They also serve elusive. Integrin linked kinase (ILK), PDK1, Rictor-mTOR to stabilize p110 and to limit its activity. complex (see below) and Akt autophosphorylation have all Class Ib p110 (p110g) is encoded by PIK3CG and is activated been suggested to be responsible for phosphorylation of by G-protein-coupled receptors (GPCR) and Ras and binds the Akt at the second S phosphorylation site.68 The PH domain p101 regulatory molecule encoded by PIK3R5. The biological leucine-rich repeat protein phosphatase (PHLPP) dephosphor- functions of the class IA PI3K regulatory and catalytic subunits ylates S473 on Akt that induces apoptosis and inhibits tumor are better described than the class Ib and classes 2 and 3 PI3K growth.69 proteins. The rest of this review focuses on the class Ia PI3Ks and Activated Akt is present both in the cytosol and the nucleus. In their downstream signaling partners. Although it should be the cytosol, Akt-1 is functional when it is phosphorylated at realized that undoubtedly some of these other classes of PI3K T308 and S473 and translocated to the membrane via the PH proteins will be shown to have important roles in hematopoiesis domain and PtdIns(3,4,5)P3. Similar events are required for and leukemia. activation of Akt-2 and Akt-3. Nuclear Akt may play important The class I PI3K-preferred substrate in vivo is phosphatidyli- antiapoptotic roles.56,70 The differential biochemical contribu- nositol 4,5 bisphosphate (PtdIns(4,5)P2) that is phosphorylated to tions of Akt in the cytosol and the nucleus remain to be yield phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). elucidated. Akt-1 and Akt-2 are fairly ubiquitously expressed. PtdIns(3,4,5)P3 serves as an anchor for pleckstrin homology (PH) Akt-3 has a more limited tissue distribution and is expressed in domain-containing proteins, such as Akt or phosphoinositide- the heart, kidney, brain, testes, lung and skeletal muscle.68 Akt 71,72 dependent protein kinase-1 (PDK1). PtdIns(3,4,5)P3 is required has been postulated to phosphorylate over 9000 proteins. for the membrane localization of Akt and PDK1. Thus, Akt is clearly a critical growth regulatory switch.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 689 Akt can transduce an antiapoptotic signal by phosphorylating proliferation 1 (MTCP1). TCL1, TCL1b and MTCP1 were downstream target proteins involved in the regulation of cell identified as translocated genes to the T-cell receptor (TcR) loci growth (for example, glycogen synthase kinase-3b (GSK-3b), in chromosomal translocations present in human T-cell prolym- ASK1, Bim, Bad, murine double minute-2 (MDM-2), p21Cip1 phocytic and mature leukemia.118–120 These proteins contain X-linked inhibitor of apoptosis (XIAP) and the Foxo3a 114, 106 and 128 amino acids, respectively. TCL1 is aberrantly transcription factor.63,73–83 Phosphorylated Foxo3a loses its expressed in many human diseases including Epstein–Barr Virus ability to induce Fas, p27Kip1 Bim, Noxa and Puma gene (EBV) transformed B-cell lymphoma, ataxia-telangiectasia, transcription.84,85 seminoma, dysgerminoma and AIDS-related lymphomas.68 Akt also phosphorylates I-kB kinase (I-kK), which subse- TCL1 functions as an Akt coactivator and enhances its activity quently phosphorylates inhibitory subunit (I-kB) that binds and nuclear translocation.121 TCL1 functions as a homodimer nuclear transcription factor k light chain in B cells (NF-kB) that is required for TCL1 to enhance Akt activation.122–124 transcription factor. When I-kB is phosphorylated it is ubiqui- tinated and subsequently degraded in proteosomes.86–93 Disassociation of I-kB from NF-kB enables NF-kB to translocate into the nucleus to promote gene expression. The PI3K pathway Effects of Akt on protein translation can also phosphorylate and activate CREB that regulates transcription of antiapoptotic genes including Mcl-1, Bcl-2 and Downstream of Akt are a complicated set of proteins critical for c-Jun.94–96 The PI3K pathway also results in activation of the the regulation of cell growth that may also serve as targets for mammalian target of rapamycin (mTOR) and ribosomal protein leukemia therapy. An overview of these downstream pathways kinases such as p70 ribosomal six protein kinase (p70S6K)97–104 and how they are regulated by both the Raf/MEK/ERK and PI3K/ these later two proteins play key roles in growth and size PTEN/Akt/mTOR pathways is presented in Figure 3. One key control. It is worth noting that Akt can cause the activation of downstream protein of Akt is mTOR. mTOR is a 289-kDa S/T specific substrates (for example, IkKa, CREB and MDM-2) or kinase. It regulates translation in response to nutrients/growth factors by phosphorylating components of the protein synthesis may mediate the inactivation of other proteins (for example, S6K Raf-1, B-Raf (by the Akt-related kinase, serum glucocorticoid machinery, including p70 and eukaryotic initiation factor kinase (SGK)), p21CipÀ1, Bim, Bad, procaspase-9, Foxo3a and (eIF)-4E-binding protein (4EBP-1), the latter resulting in release GSK-3b). This concept is important to remember as targeting of the translation initiation factor eIF-4E, allowing eIF-4E to participate in the assembly of a translational initiation com- Akt, which will be discussed in the accompanying review, may 125 S6K actually turn on (derepress) certain pathways (for example, Raf/ plex. p70 , which can also be directly activated by PDK1, MEK/ERK) which have pro-proliferative effects. phosphorylates the 40S ribosomal protein, S6, leading to active translation of mRNAs.126 Integration of a variety of signals (mitogens, growth factors, hormones) by mTOR assures cell Phosphatase regulation of the PI3K/PTEN/Akt/mTOR cycle entry only if nutrients and energy are sufficient for cell pathway Interactions Between Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR The PI3K pathway is negatively regulated by phosphatases. Pathways in Protein Translational Control Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is primarily a lipid phosphatase that removes the 3- phosphate from the PI3K lipid product PtdIns(3,4,5)P3 to 14,55,105–108 produce PtdIns(4,5)P2 that prevents Akt activation. PTEN is also a protein phosphatase.109–111 The ability of PTEN to function as a protein phosphatase remains controversial. Rapamycin Insensitive PTEN has been proposed to dephosphorylate Fak and Shc that Energy 14 may alter cell motility. PTEN also has roles in both the nucleus Depletion Activation and the cytoplasm. Some of these roles are illustrate in Figure 2. by other pathways In the cytoplasm, PTEN is thought to have roles of suppressing and stress apoptosis and regulating cell growth. In the nucleus PTEN has been postulated to function in regulating cell cycle progression, Rapamycin but it is also thought to have roles regulating cell growth by Sensitive controlling p70S6K. Two other phosphatases, SH2 domain-containing inositol 50phosphatase 1 and 2 (SHIP-1 and -2), remove the 5-phosphate 112–116 from PtdIns(3,4,5)P3 to produce PtdIns(3,4)P2. Mutations in these phosphatases as well as PTEN, which eliminate their mRNA Translation activity, can lead to tumor progression. Consequently, the genes Growth encoding these phosphatases are referred to as antioncogenes or Cell Size tumor suppressor genes. Figure 3 Interactions between Raf/MEK/ERK and PI3K/PTEN/Akt/ mTOR pathways in protein translational control. The Raf/MEK/Erk and PI3K/PTEN/Akt/mTOR pathways both serve to regulate the activity of Interactions of Akt with activator proteins mTORC1 a protein complex consisting of MLST8, raptor and most importantly mammalian target of rapamycin (mTOR). This rapamycin- In a yeast two-hybrid search for proteins that interact with Akt sensitive complex has both positive effects on mRNA translation growth, and cell size via regulating the activity of p70S6K and 4E-BP1. and have roles in leukemia, the T-cell leukemia protein-1 (TCL1) Furthermore, this complex can negatively regulate Akt activity. In 117 was determined to bind Akt. TCL1 is a member of a contrast, the rapamycin-insensitive mTORC2 complex consisting of multigene family that includes TCL1b and mature T-cell MLST8, SIN1, Raptor and mTOR phosphorylates and activates Akt.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 690 duplication.127,128 Therefore, mTOR controls multiple steps resistance in AML cells.142,143 b-Catenin expression in AML involved in protein synthesis, but importantly enhances produc- cells predicts enhanced clonogenic capacities and is associated tion of key molecules such as c-Myc, cyclin D1, p27Kip1 and with a poor prognosis.143 Thus, GSK-3b also plays key roles in retinoblastoma protein (pRb).129 mTOR also controls the regulating proliferative loops involved in malignant transforma- translation of hypoxia-inducible transcription factor-1a tion of hematopoietic cells. (HIF-1a) mRNA. HIF-1a upregulation leads to increased A recently discovered proliferative loop in melanoma is expression of angiogenic factors such as vascular endothelial controlled by c-Jun activity regulated by the CREB transcription growth factor (VEGF) and platelet-derived growth factor factor that is activated by ERK and Akt.144 c-Jun can be (PDGF).130 Moreover, HIF-1a regulates the glycolytic pathway inactivated by GSK-3b. Elevated ERK leads to GSK-3b phos- by controlling the expression of glucose-sensing molecules phorylation and inactivation and results in a feed-forward loop including glucose transporter (Glut) 1 and Glut3.131 which results in receptors for activated C kinase (RACK) By regulating protein synthesis, p70S6K and 4E-BP1 also transcription that acts in concert with (PKC) control cell growth and hypertrophy, which are important and MKK4/7 to regulate JNK and c-Jun phosphorylation and processes for neoplastic progression resulting in leukemia. Akt- stability.144 When c-Jun is active it can induce the transcription mediated regulation of mTOR activity is a complex multistep of cyclin D1 that can affect hematopoietic cell proliferation and phenomenon. Akt inhibits tuberous sclerosis 2 (TSC2 or leukemia. The roles of this ERK-mediated inactivation of GSK-3b hamartin) function through direct phosphorylation.132 TSC2 is in leukemia are not currently known, however, since activated a GTPase-activating protein (GAP) that functions in association ERK is detected frequently at elevated levels leukemia, there is a with the putative tuberous sclerosis 1 (TSC1 or tuberin) to potential for this abnormal regulatory circuit playing a critical inactivate the small G protein Rheb (Ras homologue enriched in function in leukemogenic transformation. brain).133 TSC2 phosphorylation by Akt represses GAP activity of the TSC1/TSC2 complex, allowing Rheb to accumulate in a GTP-bound state. Rheb-GTP then activates, through a mechan- Overview of Jak/STAT pathway ism not yet elucidated, the protein kinase activity of mTOR when complexed with the regulatory associated protein of The Jak/STAT pathway is another key signaling pathway mTOR (Raptor) adapter protein and mLST8, a member of the activated after receptor ligation.1,145 The Jak/STAT pathway lethal-with-sec-thirteen gene family, first identified in yeast.72 consists of three families of genes: the JAK, or Janus family of The mTOR/Raptor/mLST8 complex (mTORC1) is sensitive to tyrosine kinases, the signal transducers and activators of rapamycin and, importantly, inhibits Akt via a negative feedback transcription (STAT) family and the suppressors of cytokine loop that involves, at least in part, p70S6K, insulin receptor signaling/cytokine-induced SH2-containing (SOCS/CIS) family, substrate-1 (IRS-1) and PI3K72 (Figure 3). The relationship which serves to downregulate the activity of the Jak/STAT between Akt and mTOR is further complicated by the existence pathway.1,145 The Jak/STAT pathway involves signaling from the of the mTOR/Rictor (rapamycin-insensitive companion of cytokine receptor to the nucleus. Jaks are stimulated by mTOR/mLST8 complex (mTORC2), which displays rapamycin- activation of a cytokine receptor. Stimulation of Jaks results in insensitive activity. The mTORC2 complex has been found to STAT transcription factor activity. directly phosphorylate Akt on S473 in vitro and to facilitate Jaks are a family of large tyrosine kinases, having molecular T308 phosphorylation. Thus, the mTORC2 complex might be weights in the range of 120–140 kDa (1130–1142aa).145 Four the elusive PDK2 that phosphorylates Akt on S473 in response to Jaks (JAK1, JAK2, JAK3 and TYK2) have been identified in growth factor stimulation.134 Akt and mTOR are linked to each mammals. Jak proteins consist of seven different conserved other via ill-defined positive and negative regulatory circuits, domains (JH1–7). The JH1 constitutes a kinase domain, while which restrain their simultaneous hyperactivation through a JH2 is a pseudokinase domain. Many possible roles have been mechanism involving p70S6K and PI3K. Assuming that equili- proposed for the different domains of the Jak proteins: (1) JH2 brium exists between these two complexes, when the mTORC1 inhibits JH1, (2) JH2 promotes STAT binding, (3) JH2 is required complex is formed, it could antagonize the formation of the for kinase activity of JH1 and (4) JH6 and JH7 are necessary for mTORC2 complex and reduce Akt activity.134 Thus, at least in association of Jaks with cytokine receptors.145 principle, inhibition of the mTORC1 complex could result in Akt Loss of Jak1 produces prenatal lethality due to neurological hyperactivation. This is one complication with rapamycin disorders,146 while Jak2À/À results in embryonic lethality due to treatment (see below) and the accompanying review. defects in erythropoiesis.147,148 Jak3 expression is limited to Akt directly phosphorylates mTOR on S2448 that results in its hematopoietic cells, and Jak3 knockout mice have develop- activation.135 mTOR was found to be phosphorylated in acute mental defects in lymphoid cells.147–160 myeloid leukemia (AML) blasts, along with its two downstream Aggregation of cytokine receptors following activation allows substrates, p70S6K and 4E-BB1, in a PI3K/Akt-dependent formation of receptor homodimers and heterodimers. Receptor fashion.136,137 Nevertheless, others failed to detect any relation- aggregation allows transphosphorylation of receptors and ship between PI3K/Akt signaling upregulation and p70S6K activation of the associated Jaks and STATs. Transphosphoryla- phosphorylation in AML primary cells.138 This might occur via tion occurs when one receptor kinase complex, in the absence the Raf/MEK/ERK pathway activating mTOR via ERK 1/2 of the ligand for the second receptor complex, phosphorylates phosphorylation.139 The Raf/MEK/ERK pathway is frequently and activates the second receptor complex. This may occur by activated in AML.140 Consistently, in some AML cases, interaction of functionally similar domains present on the two treatment of blast cells with pharmacological inhibitors of ERK different signaling molecules. The best evidence of transpho- 1/2 signaling (U0126) suppressed p70S6K phosphorylation.137 sphorylation is Jak1 mutant cell lines, which cannot activate GSK-3b can negatively regulate mTOR by phosphorylating Tyk2 after stimulation with interferon a/b.145 Another example and activating TSC-2.141 These results further suggest GSK-3b of this transphosphorylation is that interleukin-2 (IL-2) cannot may be involved in the regulation of cell growth and malignant activate Jak1 in the absence of Jak3.146,161 Together these data transformation. GSK-3b activity seems also important for indicate that receptor aggregation and transphosphorylation are adhesion and Wnt-pathway b-catenin expression and drug important in activation of the Jaks.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 691 The STAT gene family consists of seven proteins (STAT1, The Jak/STAT pathway is negatively regulated by the SOCS STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6) ranging in and CIS family of proteins.145 The more accepted name of this molecular weights from 73 to 95 kDa (748–851aa).145 The family is SOCS. This consists of SOCS1–SOCS5 structure of the STAT family proteins consists of an and CIS1.172–182 This family of genes has a conserved SH2 N-terminal oligomerization domain, a DNA-binding domain domain and SOCS box.172–182 The SOCS box, the C-terminal 40 in the central part of the protein, an SH2 domain and a amino acids, is implicated in stability and degradation of the transactivation domain near the C terminus. The transactivation protein by targeting it to the proteosome.175–177 CIS inhibits domain is the most divergent in size and sequence STAT5 activation by competing for its receptor-binding and is responsible for activation of transcription. The oligomer- site.178,179 SOCS2 directly binds and inhibits the kinase domain ization domain contains a tyrosine that is rapidly phosphory- (JH1) of Jak2.179–182 Gene ablation studies have indicated that lated by Jaks, allowing the pY product to interact with the SOCS proteins have important roles in regulating the effects the SH2 domains of other STAT proteins. Formation of STAT of interferon-g, growth hormone and erythropoietin. SOCS2 and dimers promotes movement to the nucleus, DNA binding SOCS3 knockout mice are lethal, whereas SOCS2 knockout and activation of transcription, as well as increased mice are 30% larger than their wild-type counterparts.182–185 protein stability. Threonine phosphorylation has been proposed There are other mechanisms to downregulate Jak/STAT to play a further role in the regulation of STAT activity.162,163 signaling. Protein phosphatases, including CD45 and protein This may be mediated by ERK,163 indicating a point of tyrosine phosphatase-e C (PTPeC), are also implicated in the interaction between the Raf/MEK/ERK and Jak/STAT pathways negative regulation of Jak/STAT signaling.186,187 (Figure 1). The roles of the STAT and Jak proteins in hematopoietic growth have been investigated by the creation of knockout Interactions Between PI3K/PTEN/Akt/mTOR, Raf/MEK/ERK strains of mice.163–166 STAT3À/À mice have severe develop- and Jak/STAT pathways that regulate apoptosis mental problems resulting in fetal death. Cytokine signaling abnormalities are associated with other STAT knockout models, Now that we have examined the basic mechanism of regulation but all mice are viable. of these pathways, we now discuss how these cascades interact Constitutive STAT activity is associated with viral infections. to regulate apoptosis. When apoptosis is deregulated, which can STAT3 is known to have oncogenic properties.167–171 v-Abl occur by aberrant expression of these pathways, leukemia may and BCR-ABL induce constitutive STAT activity.167,168 STAT arise. Akt can phosphorylate Raf-1 and B-Raf and leads to their transcription factors can induce antiapoptotic gene expression inactivation.188–191 Akt can also activate Raf-1 through a Ras- including Bcl-XL. independent but PKC-dependent mechanism that results in the

Cytokine Mediated Signal Transduction Pathways and Prevention of Apoptosis Akt phosphorylates Activated β CREB and Gsk-3 Cytokine STAT5 inducing activating Mcl-1 Receptor Bcl-XL transcription transcription and inhibiting Gsk-3β from phosphorylating Bcl-XL induced Mcl-1 thus both PI3K Raf Jak latent Bax enhancing monomers and stabilizing STAT5 PDK MEK in cytoplasm Mcl-1 leveks prevents apoptosis Akt ERK and Akt ERK Bcl-XL phosphorylation P Elevated ERK may of Mcl-1 results P CREB phosphorylate and in Mcl-1 stabilization. P in activate GSK-3β Mcl-1 Bax GSK-3β leading to c-Jun JNK or GSK-3β P expression and orylation of Mcl-1 P phosph cell cycle roteosomal induces p progression degradation, apoptosis JNK

Apoptosis

Figure 4 Cytokines-mediated signal transduction pathways and prevention of apoptosis. Cytokines can induce multiple signal transduction pathways that can affect the expression of apoptotic molecules by transcriptional and posttranscriptional mechanisms.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 692 prevention of apoptosis.192 Suppression of apoptosis in some Both the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways cells by Raf and MEK requires PI3K-dependent signals.193–198 An can phosphorylate the BH3-only domain protein Bim.42,79 overview of the effects of these pathways on the prevention of When Bim is phosphorylated by ERK and Akt, it is targeted for apoptosis is presented in Figure 4. ubiquitination and degradation in the proteosome.46 Mcl-1 can The PI3K/PTEN/Akt/mTOR, Raf/MEK/ERK and Jak/STAT path- bind Bim that prevents the activation and mitochondrial ways contribute to the transcriptional regulation of Bcl-2 family translocation of Bax.46,47 In contrast, JNK can phosphorylate members. Akt and Erk can regulate CREB phosphorylation. Bim at S65 that enhances its ability to induce Bax activation and CREB binds the Mcl-1 and Bcl-2 promoter regions.96,198–200 hence stimulates apoptosis.208 Mcl-1 can also bind proapoptotic 207 STAT can regulate Bcl-XL transcription. Moreover, the PI3K and Bak. The Mcl-1:Bak interaction can be disrupted by the Raf pathways phosphorylate proapoptotic Bcl2 homology-3 binding of the BH3-only domain Noxa protein that results in (BH3)-only domain protein Bad that prevents its apoptotic Mcl-1 being ubiquitinated and degraded in the proteosome.209 effects and it becomes cytoplasmically localized.201–204 Another Bak can then form active dimers and induce apoptosis. Unlike MAPK, JNK phosphorylates 14-3-3 proteins that results in their Bcl-2 and Bcl-XL, the half-life of the Mcl-1 protein is short due to disassociation from cytoplasmically localized Bad. Bad then the N-terminal peptide sequence that is rich in proline (P), translocates to the mitochondrion.205 When Bad associates with glutamic acid (E), serine (S) and threonine (T) (PEST sequence). Bcl-2 or Bcl-XL, it promotes apoptosis by preventing Bcl-2 or Proteins containing PEST sequences are frequently targeted for 206–212 Bcl-XL from interacting with Bax. Bad is phosphorylated proteosomal or calpain degradation. in most AML specimens suggesting that inhibition of Bad The expression of Mcl-1 is regulated by both transcriptional phosphorylation may be therapeutically important in AML.213 In and posttranslational mechanisms.214 Certain chemotherapeutic contrast, the antiapoptotic Mcl-1 protein is not reported to drugs such as taxol will induce Mcl-1 phosphorylation at interact with Bad.207 An overview of the effects of the PI3K/ different sites than those phosphorylated by ERK (T163).46 PTEN/Akt/mTOR and Raf/MEK/ERK pathways on Bad phosphor- Oxidative stress can activate JNK that induces the phosphoryla- ylation and the prevention of apoptosis is presented in Figure 5. tion of Mcl-1 on S121 and T163.215 Cytokine deprivation of

Overview of Interactions between Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR, and Apoptotic Pathways

Chemotherapy Growth and Factor Radiotherapy

Generic Phosphorylation of MDM-2 Receptor by Akt increases p53 P Shc Ras-GTP Fak Dephosphorylated Mcl-1 targeting to proteosomes Grb2 targeted for ubiquitination P and degradation ATM/ATR P and proteosomal degradation SOS PTEN negatively effects Src Family PDK and Akt by Kinase P P dephosphorylation of PIP3, Cyto C Decrease in PTEN also interacts MDM-2 p53 Release Mitochondrial with p53, FAK and Shc P Membrane Cyto C ? Potential Raf PI3K Apaf-1 PTEN Caspase 9

Caspase 3 K Drug Induced Cell Death

Mcl-1 Apoptosis t Mitochondrion Drug Resistance Caspase 3 P P P Caspase 9 P ERK CREB NOXA Apaf-1 Preservation of Cyto C Phosphorylation of Mcl-1 Mitochondrial Release Nucleus Bad, Bim, (NOXA, PUMA??) Membrane By ERK and in some Potential cases Akt, cytosolic Disassociation of Bax sequestration of some Mcl-1 Transcription homodimers, Mcl-1, proteins (e.g., Bad) Transcription Bcl-XL able to bind PTEN, numerous effects in the and neutralize Bax nucleus, on genomic instability, Phosphorylated Bim and centromere breakage, regulation Bad targeted for ubiquitination of Rad51, induction of and proteosomal degradation double strand DNA breaks, chromosomal translocation and cell cycle progression

Figure 5 Overview of interactions between Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR, p53 and apoptotic pathways. All of these pathways interact to regulate the induction of apoptosis. The Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways normally serve to suppress apoptosis while p53, which is induced by certain chemotherapeutic drugs, will result in increases in proapoptotic family members and in some cases, growth factors such as hbEGF that may stimulate growth. Furthermore, p53 activity can be altered by phosphorylation by ERK as well as murine double minute-2 (MDM-2) levels, whose activity is in turn previously regulated by Akt. Hence these pathways are interconnected and serve to regulate each other. Not included in this diagram is the effect of c-Jun N-terminal kinase (JNK) that often is associated with proapoptotic effects and often serves to counterbalance the effects of extracellular regulated kinase (ERK) and Akt.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 693 certain hematopoietic cells induces GSK-3b that in turn Sites of Mutation which can Result in promotes the phosphorylation of Mcl-1 at S159 which results Activation of Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR, and Jak/STAT Cascades in Leukemia in its ubiquitination and degradation.216 Akt phosphorylates Cytokine Receptor GSK-3b suppressing its ability to phosphorylate Mcl-1. Altering FLT-3, Mutations the levels and phosphorylation state of Mcl-1 play important Kit, Fms, Ras & Rafmutatedin roles in the regulation of apoptosis. G-CSF some leukemias BCR-ABL The expression of BH3-only domain Puma and Noxa proteins PI3K, Akt not frequently 217 and other mutated in leukemia but are under the control of the PI3K/Akt pathway. Noxa interacts Ras may be activated 207 translocations specifically with Mcl-1 but not with Bcl-2 or Bcl-XL. Bak PTEN, SHIP may be associates with Mcl-1 and Bcl-XL but not Bcl-2. Upon induction in activated/deleted Raf PI3K in leukemia of Puma and Noxa by p53 after genotoxic stress, Puma and PTEN, SHIP Noxa displace Mcl-1 from Bak and Bak is able to oligomerize TEL-JAK Jak2 mutated Jak in myelo- MEK Akt ERK and Akt and induce apoptosis. This may lead to Mcl-1 degradation and proliferative activated in many apoptosis. The Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR path- diseases leukemias ERK mTOR TSC1/2 ways increase Mcl-1 protein levels and stability. This may lead STAT to an increase in Mcl-1 associated with Noxa and Puma and a p70S6K decrease in free Bak levels and less apoptosis. A diagram depicting the effects of signaling and p53 pathways on Noxa and Deregulation of Proliferation Puma and regulation of apoptosis is present in Figure 5. Figure 6 Sites of mutation that can result in activation of the Raf/ Human caspase 9 was originally thought to be phosphory- MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways in hemato- lated by Akt, but the murine caspase 9 lacks the Akt consensus poietic cells. Mutations have been detected in FLT-3, KIT, G-CSF, RAS phosphorylation site.18 Caspase 9 is phosphorylated by the Raf/ and JAK. The BCR–ABL chromosomal translocation is present in MEK/ERK pathway at T125 that inhibits activation of the caspase virtually all chronic myeloid leukemias (CMLs) and some acute 19 lymphocytic leukemias (ALLs). Many of these mutations and chromo- cascade. Mcl-1 is a substrate for activated caspase 3, thus somal translocations result in activation of the Raf/MEK/ERK and PI3K/ decreased caspase 9 activation by ERK phosphorylation will PTEN/Akt/mTOR cascades. Although PI3KCA and BRAF are frequently reduce caspase 3 activation and Mcl-1 cleavage and apoptosis mutated in certain solid tumors, it has not been documented to be will be suppressed. frequently mutated in leukemia. The frequently mutated genes are Many cytokines and growth factors can also induce the Jak/ indicated by a jagged symbol. 210 STAT pathway that regulates the transcription of Bcl-XL. Bcl- 212 XL can prevent the formation of Bax:Bax homodimers. Hence the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR, Jak/STAT and JNK frequency may change as more and diverse tumors are pathways regulate many molecules involved in prevention of examined for BRAF mutation. Recent studies indicated that apoptosis. Dysregulation of these pathways may contribute to mutated alleles of RAF1 and BRAF are present in therapy- leukemia. induced acute myelogenous leukemia (t-AML).221 These leuke- mias arose after chemotherapeutic treatment of breast cancer patients. The mutated RAF1 genes detected were transmitted in Roles of the Ras/Raf/MEK/ERK pathway in leukemia the germ line, thus they are not spontaneous mutations in the leukemia but may be associated with the susceptibility to Now we discuss the roles that this cascade may play in induction of t-AML in these Austrian cancer patients. Mutations leukemia. We also discuss some relevant examples of where of various genes in the receptor tyrosine kinase (RTK)/RAS-BRAF altered expression of this pathway is important in the malignant signal transduction pathway have been detected in therapy- transformation of other types of cancer (for example, solid related myelodysplasia and AML.222 In this study, the BRAF tumors). It is important to discuss these other cancers as they mutations were always associated to the translocation provide us with information as to how aberrant expression of t(9;11)(p22;q23), involving the mixed lineage leukemia (MLL) these pathways can cause cancer and alter the sensitivity of gene. targeted therapy. The Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and For many years, the RAF oncogenes were not thought to be Jak/STAT pathways can be activated by mutations/amplifications frequently mutated in human cancer and more attention to of upstream growth factor receptors. The FLT-3 kinase/growth abnormal activation of this pathway was dedicated to RAS factor receptor is mutated in greater than 25% of AMLs. The mutations that can regulate both the Raf/MEK/ERK and PI3K/ BCR-ABL chromosomal translocation is present in495% of PTENAkt/mTOR pathways. However, it was shown recently that chronic myeloid leukemias (CMLs) and some acute lymphocytic BRAF is frequently mutated in melanoma (27–70%), papillary leukemia (ALL) and can result in activation of these pathways. thyroid cancer (36–53%), colorectal cancer (5–22%) and Other chromosomal translocations involving diverse genes are ovarian cancer (30%).220,223,224 The reasons for mutation at frequently present in AMLs. An illustration of some of the BRAF and not RAF1 or ARAF in melanoma patients are not receptors, kinases and phosphatases mutated/amplified in entirely clear. On the basis of mechanism of activation of BRAF, leukemia that can result in activation of these pathways is it may be easier to select for BRAF than either RAF1 or ARAF presented in Figure 6. mutations. Due to the amino acids present in certain key Mutations that lead to the expression of constitutively active regulatory sites in the different Raf isoforms, activation of B-Raf Ras proteins have been observed in approximately 30% of would require one genetic mutation whereas activation of either human cancers.218,219 These are often point mutations that alter Raf-1 or A-Raf needs two genetic events. Furthermore, B-Raf key residues which affect Ras activity, although amplification of may be activated in the cytoplasm by non-farnesylated Ras, Ras is also detected in some tumors. Mutations that result in while Raf-1 requires farnesylated Ras for translocation to the cell increased Ras activity also perturb the Raf/MEK/ERK and PI3K/ membrane.224 PTEN/Akt/mTOR cascades. BRAF has been reported to be It was proposed recently that the structure of B-Raf, Raf-1 and mutated in approximately 7% of all cancers.220 However, this A-Raf may dictate the ability of activating mutations to occur at

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 694 these molecules, which can permit the selection of oncogenic sensitive to MEK inhibitors while cells lacking these B-Raf forms.221,224,225 These predictions have arisen from determining mutations are resistant.239 We have shown that introduction of the crystal structure of B-Raf.225 Like many , B-Raf is activated EGFR mutants into hematopoietic cells renders them proposed to have small and large lobes, which are separated, by sensitive to EGFR inhibitors.233,240,241 Furthermore, introduction a catalytic cleft. The structural and catalytic domains of B-Raf of activated Ras, Raf, MEK genes into hematopoietic cells makes and the importance of the size and positioning of the small lobe them sensitive to MEK inhibitors.198,242–245 BCR-ABL-trans- may be critical in its ability to be stabilized by certain activating formed hematopoietic cells are usually highly sensitive to mutations. In contrast, the precise substitutions in A-RAF and inhibitors such as imatinib, providing that the particular BCR- RAF-1 are not predicted to result in small lobe stabilization thus ABL gene present in the cells does not have a mutation that preventing the selection of mutations at ARAF and RAF1, which eliminates sensitivity to the inhibitor. would result in activated oncogenes.225 Raf-1 has been known for years to interact with heat shock protein 90 (Hsp90). Hsp90 may stabilize activated Raf-1, B-Raf and A-Raf. The role that Roles of the PI3K/PTEN/Akt/mTOR Pathway in leukemia Hsp90 plays in selection of activated RAF mutations is highly speculative yet very intriguing. Some Ras mutations can result in PI3K/PTEN/Akt/mTOR The most common BRAF mutation is a change at nucleotide activation.246–251 Mutations at the p85 subunit of PI3K have 600 that converts a valine to a glutamic acid (V600E) (V, valine; been detected in Hodgkin’s lymphoma cells.252 The p110 E, glutamic acid).220 This BRAF mutation accounts for over 90% subunit of PI3K is frequently mutated (B25%) in breast and of the BRAF mutations found in melanoma and thyroid cancer. It some other cancers but it not believed to be frequently mutated has been proposed that BRAF mutations may occur in certain in leukemia.60,253 Mutations and hemizygous deletions of PTEN cells, which express high levels of B-Raf due to hormonal have been frequently detected in AML and non-Hodgkin’s stimulation. Certain hormonal signaling events will elevate Lymphoma (NHL).254,255 PTEN inactivation is believed to occur intracellular cAMP levels, which result in B-Raf activation, frequently in certain hematopoietic neoplasms. Many different leading to proliferation. Melanocytes and thyrocytes are two genetic mechanisms can result in functional inactivation or such cell types, which have elevated B-Raf expression as they silencing of PTEN (see below). are often stimulated by the appropriate hormones.226 Moreover, A recent study observed that decreased PTEN phosphorylation it is now thought that B-Raf is the most important kinase in the was present in approximately 75% of AML patients.256 PTEN Raf/MEK/ERK cascade.220 In some models wild-type and mutant phosphorylation often results in inactivation of PTEN activity.256 B-Raf activate Raf-1, which in turn activates MEK and PTEN phosphorylation was significantly associated with Akt ERK.220,227,228 phosphorylation and with shorter overall survival.256 Phosphor- In some cells, BRAF mutations are believed to be initiating ylation at the C-terminal regulatory domain of PTEN stabilizes events but not sufficient for full-blown neoplastic transforma- the molecule, but renders it less active toward its substrate, 229,230 257 tion. Moreover, there appear to be cases where certain PtdIns(3,4,5)P3. Moreover, PTEN expression has been shown BRAF mutations (V600E) and RAS mutations are not permitted in to be low or absent in some AML patients,258 although the level the transformation process as they might result in hyperactiva- of PTEN expression did not always correlate with the degree of tion of Raf/MEK/ERK signaling and expression, which may lead Akt phosphorylation. However, a subsequent study failed to to cell cycle arrest.222 In contrast, there are other situations, demonstrate that AML blasts have a decreased expression of which depend on the particular BRAF mutation and require both PTEN.259 Another study of 62 AML patients, demonstrated that B-Raf and Ras mutations for transformation. The BRAF mutations 15 of them had aberrant PTEN mRNA transcripts. However, all in these cases are thought to result in lower levels of B-Raf the samples with abnormal transcripts also displayed normal activity.222,230 full-length transcripts, suggesting that the aberrant transcripts Different BRAF mutations have been mapped to various could have resulted from altered RNA splicing. Moreover, no regions of the B-Raf protein. Some of the other BRAF mutations loss of heterozygosity (LOH) or other types of genetic mutations are believed to result in B-Raf molecules with impaired B-Raf were observed.260 PTEN-inactivating mutations do not appear to activity, which must signal through Raf-1.220,228 Heterodimer- occur very frequently in AML.261,262 Therefore, the importance, ization between B-Raf and Raf-1 may allow the impaired B-Raf if any, of PTEN in causing Akt activation in AML blast cells to activate Raf-1. Other mutations, such as D593V, (D, aspartic remains unclear. Nevertheless, we feel that reinvestigation of acid) may activate alternative signal transduction pathways.220 the PTEN role in AML pathogenesis is necessary, since recent It has been reported that a high frequency of AML and ALL studies in mice demonstrated that bone marrow stem cells (450%) displays constitutive activation of the Raf/MEK/ERK without functional PTEN multiplied rapidly, displayed dimin- pathway in absence of any obvious genetic mutation.140,231 ished self-renewal capacity, migrated out of the bone marrow, While there may be some unidentified mutation at one colonized distant organs and initiated a leukemic-like dis- component of the pathway or a chromosomal translocation that ease.263,264 Importantly, these effects were mostly mediated by feeds into the pathway or a phosphatase that regulates the mTOR, as rapamycin not only depleted leukemia-initiating activity of the pathway, the genetic nature of constitutive cells, but also restored normal hematopoietic stem cell activation of the Raf/MEK/ERK pathway is unknown. Elevated function.264 expression of ERK in AMLs and ALLs is associated with a poor The NOTCH1 receptor, which is activated by mutations in at prognosis.232 Raf and potentially more effective MEK inhibitors least 50% of T-cell acute lymphocyte leukemia (T-ALL), inhibits may prove useful in the treatment of a large percentage of AMLs PTEN expression through the HES-1 transcription factor.265,266 and ALLs. This will be addressed in an accompanying review in This in turn leads to Akt activation, and resistance to Leukemia. glucocorticoids. At some point during disease progression, the Recently, it was demonstrated that there can be a genetic PTEN gene is either lost or inactivated through other genetic basis for the sensitivity of non-small cell lung cancers (NSCLC) mechanisms (for example, gene hypermethylation), as even to epidermal growth factor receptor (EGFR) inhibitors.233–238 In inhibition of NOTCH1 did not restore PTEN expression. addition, some melanoma cells carrying B-Raf mutations are Importantly, PTEN-null T-ALLs are resistant to NOTCH1

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 695 inhibitors (g-secretase inhibitors), however they are very PH domain mutation confers many different properties to the sensitive to Akt inhibitors.256 AKT1 gene. Namely, the mutant AKT1 gene (1) has an altered More research has been done on PTEN mutations and gene PH domain conformation, (2) is constitutively active, (3) has an silencing in solid tumors as opposed to hematopoietic cancers. altered cellular distribution as it is constitutively associated with Hence, we discuss what is known about PTEN mutation and the cell membrane, (4) morphologically transforms Rat-1 tissue regulation in breast cancer as some of these mechanisms that culture cells and (5) interacts with c-Myc to induce leukemia in serve to silence this important tumor suppressor gene are likely Em-Myc mice (Em, enhancer of immunoglobulin M (m) gene; to be present in hematopoietic neoplasias where PTEN is Myc, Myc oncogene originally isolated in avian myelocytoma- suppressed. tosis virus).281 This PH domain-mutated AKT1 gene does not Germ-line PTEN mutation is present in approximately 80% of alter its sensitivity to ATP-competitive inhibitors, but does alter patients with Cowden syndrome (CS).53,267 This disease, which its sensitivity to allosteric kinase inhibitors.281 These results is also known as multiple hamartoma syndrome, is a familial demonstrate that targeting the kinase domain of Akt may not be syndrome that includes many different types of cancer condi- sufficient to suppress the activity of various AKT genes that have tions including early onset breast cancer. Mutations have been mutations in the PH domain.281 In a relatively small cohort of reported to occur at PTEN in breast cancer in varying AML patients, such a mutation was not found.282 It will be frequencies (5–21%).268,269 LOH of PTEN is probably more important to determine if this AKT1 mutation (E17K) is also common (30%) than deletion of both PTEN alleles. PTEN present in leukemias. promoter methylation leads to low PTEN expression. In one In summary, mutations do occur at key components of the study, 26% of primary breast cancers had low PTEN levels.269 PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways in var- Low PTEN levels have been correlated with lymph node ious cancers and targeting of these pathways may be important metastases and poor prognoses.270 Mutations at certain residues therapeutic approaches, either by themselves as monotherapy or of PTEN, which are associated with CS, affect the ubiquitination by combination therapies with various chemo, hormonal and of PTEN and prevent nuclear translocation. These mutations antibody treatments (see accompanying review). leave the phosphatase activity intact.271 Inhibition of PTEN The relationship between dysregulated PI3K activity and the activity leads to centromere breakage and chromosome onset of cancer is well documented. The PI3K is the instability.272 PTEN expression may also be silenced in the predominant growth factor-activated pathway in LNCaP human absence of obvious genetic mutations. Disruption of PTEN prostate carcinoma cells.283,284 Other reports directly implicate activity by various genetic mechanisms could have vast effects PI3K activity in a variety of human tumors including breast on different processes affecting the sensitivity of leukemia to cancer,285 lung cancer,286 melanomas287 and leukemia288 diverse therapeutic approaches. Thus, there are many possible among others. Activated Akt can affect the expression and mechanisms that can lead to elevated Akt levels which regulation of the responses of hormone receptors and hence contribute to both leukemogenesis and lead to drug resistance. leads to ineffectiveness of hormone ablation therapies.288–291 PTEN mutation/deletion/inactivation is present in many ALL Activated Akt has been reported to be present in over 50% of lines. SHIP mutations are also detected in AML.273,274 In primary AML samples and detection of activated Akt is summary, the PTEN and SHIP phosphatases play critical roles associated with a poor prognosis.231 Furthermore, the Akt in leukemogenesis. We are only beginning to understand how pathway has been shown to be involved in regulation of these proteins function to regulate growth in normal hemato- multidrug resistance protein-1 and drug resistance in AML and poietic stem cells and as we learn more about their pleiotropic ALL.291–294 Taken together, these data endorse the substantial effects, we may be able to understand their contributions to role that PI3K signaling plays in oncogenesis and drug leukemic stem cells and be able to counteraffect the con- resistance. Moreover, targeted inhibition of the central compo- sequences of mutations that inactivate these proteins. nents of this pathway appears to be an excellent choice for Increased Akt expression is linked with tumor progression and future therapeutic approaches. It has been observed that drug/hormonal resistance.275–279 Although PI3K and Akt have overexpression of both the Raf/MEK/ERK and PI3K/Akt pathways not been observed to be frequently mutated in the leukemia in AML is associated with a worse prognosis than over- samples examined so far, we should be aware of studies that expression of a single pathway.231 Thus, the development of have been performed in solid tumors as this pathway is inhibitors that target both pathways or the formulation of frequently activated in leukemia and in some cases associated combinations of inhibitors may prove effective in leukemic with a poor prognosis. In a recent survey of 40 breast cancer therapy. lines, many were mutated at components of either the PI3K/ PTEN/Akt/mTOR or Raf/MEK/ERK pathways;275 36% were mutated at PIK3CA (PI3K p110 subunit gene), 21% at PTEN Roles of the Jak/STAT pathway in neoplasia (with either PTEN mutation or no protein present), 13% at KRAS, 5% at HRAS,3%atNRAS and 10% at BRAF. In other studies it A chromosomal translocation forming the TEL–Jak2 fusion has been shown that the PIK3CA is mutated in approximately protein that results in constitutive kinase activity has been 25% of breast, 32% of colorectal, 27% of brain, 25% of gastric observed in a limited number of patients with ALL and CML.295 and 4% of lung cancers.279–281 These mutations frequently result This chimeric protein abrogates the cytokine dependence of in activation of its kinase activity. A recent report indicated that certain hematopoietic cell lines. On the other hand, the partner AKT1 is mutated in 8% of breast, 6% of colorectal and 2% of TEL gene (translocated ETS in leukemia) is often rearranged in ovarian cancers examined.281 This mutation results in a lysine human leukemia. TEL rearrangement partners include Abl, (K) substitution for E at amino-acid 17 (E17K) in the PH domain. platelet-derived growth factor receptor (PDGF-R) and Jak, all Cells with this AKT1 mutation have not been observed to have of which encode tyrosine kinases.296 The fusion products mutations at PI3K; a similar scenario is also frequently observed encode constitutively active tyrosine kinases involved in human with RAS and BRAF mutations.282 The AKT1 mutation alters the leukemia. Activation of Jak in TEL–Jak is due to the oligomer- electrostatic interactions of AKT1 that allows it to form new ization domain provided by the TEL transcription factor that hydrogen bonds with the natural phosphoinositol ligand.281 The results in the constitutive, ligand-independent activation of the

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 696 Table 1 List of abbreviations Table 1 (Continued ) Abbreviations Definitions Abbreviations Definitions RTK Receptor tyrosine kinase 4EBP-1 4E binding protein SGK Serum glucocorticoid kinase ALL Acute lymphocytic leukemia Shc A Src homology (SH)-2 (SH2) –domain- ASK1 Apoptotic signal kinase containing protein, becomes associated with BCR Breakpoint cluster region gene the C terminus of the growth factor receptor BH3 Bcl2 homology-3 SHIP-1 and SHIP-2 SH2 domain-containing inositol CML Chronic myeloid leukemias 5’phosphatase 1 and 2 CREB Cyclic-AMP response element-binding SOCS/CIS Suppressors of cytokine signalling/cytokine- protein transcription factor induced SH2-containing CS or multiple Cowden syndrome Sos Son of sevenless exchange proteins hamartoma syndrome STAT Signal transducers and activators of D593V D, aspartic acid transcription Em-Myc mice Em, enhancer of immunoglobulin M (m) gene, t-AML Therapy-induced acute myelogenous Myc, Myc oncogene originally isolated in leukemia avian myelocytomatosis virus TCL1 T-cell leukemia protein-1 EBV Epstein–Barr virus TcR T-cell receptor EGFR Epidermal growth factor receptor TEL gene Translocated ETS in leukemia EIF Eukaryotic initiation factor TNF Tumor necrosis factor ERKs 1 and 2 Extracellular regulated kinases 1 and 2 TSC2 or hamartin Tuberous sclerosis 2 GAP GTPase-activating protein V600E V, valine; E, glutamic acid Glut Glucose transporter VEGF Vascular endothelial growth factor GPCR G-protein-coupled receptors XIAP X-linked inhibitor of apoptosis Grb2 GTP-exchange complex growth factor receptor bound-2 GSK-3b Glycogen synthase kinase-3b Hsp90 Heat shock protein 90 I-kKI-kB kinase Jak kinase domain. Activated Jak proteins have been made from IL-2 Interleukin-2 the TEL–Jak rearranged chromosomal translocation. A summary ILK Integrin-linked kinase of common chromosomal translocations in leukemia and some IRS-1 Insulin receptor substrate-1 of the known signaling pathways activated by these transloca- JNK c-Jun N-terminal kinase tions is presented in Tables 1 and 2.297–335 Recently, it has been KSR Kinase suppressor of Ras LOH Loss of heterozygosity discovered that the Jak2 kinase is frequently mutated in 336–372 Mcl-1 Myeloid cell leukemia-1 myeloproliferative diseases. MDM-2 Murine double minute-2 STAT overexpression is frequently observed in human MEK1 Mitogen-associated/extracellular-regulated cancers. Increased STAT activation may contribute to the kinase-1 myeloproliferative diseases that harbor the Jak2 mutation. The MKK4/7 MAPK kinase 4/7 STAT3 protein can function as an oncogene and other STAT MLL Mixed lineage leukemia gene mLST8 Member of the lethal-with-sec-thirteen gene proteins may function in oncogenic transformation. The STAT family molecules provide novel therapeutic targets for oncogenic MP-1 MEK partner-1 transformation. Activating mutants of STAT5a have been made, MST-2 Mammalian sterile 20-like kinase which will abrogate the cytokine dependence of hematopoietic MTCP1 Mature T-cell proliferation 1 cells. mTOR Mammalian target of rapamycin mTORC2 mTOR/Rictor (rapamycin-insensitive companion of mTOR/mLST8 complex) NF-kB Nuclear transcription factor-k light chain in B cells Conclusions NHL Non-Hodgkin’s lymphoma NSCLC Non-small cell lung cancers Over the past 25 years, there has been much progress in p70S6K p70 ribosomal six protein kinase elucidating the involvement of the Ras/Raf/MEK/ERK, PI3K/ p90Rsk1 90 kDa ribosomal six kinase 1 PDGF Platelet-derived growth factor PTEN/Akt and Jak/STAT cascades in promoting normal cell PDGF-R Platelet-derived growth factor receptor growth, regulating apoptosis as well as the etiology of human PDK-1 Phosphoinositide-dependent protein kinase- neoplasia and the induction of chemotherapeutic drug resis- 1 tance. From initial seminal studies that elucidated the onco- PHLPP PH domain leucine-rich repeat protein genes present in avian and murine oncogenes, we learned that phosphatase ERBB, RAS, SRC, ABL, RAF, PI3K, AKT, JUN, FOS, ETS and PI3K Phosphatidylinositol 3-kinase PKB Protein kinase B NF-kB (Rel) were originally cellular genes which were captured PKC Protein kinase C by retroviruses. Biochemical studies continue to elucidate the PKC Protein kinase C roles that these cellular and viral oncogenes have in cellular pRb Retinoblastoma protein transformation. We have learned that many of these oncogenes PTEN Phosphatase and tensin homologue deleted are connected to the Ras/Raf/MEK/ERK, PI3K/PTEN/PDK/Akt and on chromosome 10 Jak/STAT pathways and either feed into this pathway (for PTPeC Protein tyrosine phosphatase-e C RACK Receptors for activated C kinase example, BCR–ABL, ERBB) or are downstream targets, which Raptor Regulatory associated protein of mTOR regulate gene expression (for example, JUN, FOS, ETS and NF- Rheb Ras homologue-enriched in brain kB). Furthermore, many of these ‘oncogenes’ are also present in RKIP Raf kinase inhibitory protein chromosomal translocations that play key roles in leukemia ROS Reactive oxygen species (BCR–ABL, TEL–JAK, TEL–PDGFR).

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 697 Table 2 Common chromosomal aberrations in AML and ALL

Aberration Frequency Mechanism Associated pathways References (%)

AML FLT3-ITD dup(13) 33 Ligand-independent receptor PI3K/Akt, Raf/MEK/ 297–299 activation ERK, STAT5 AML1/ETO t(8;21) 12 Deregulation of AML1 target genes Unknown 300,301 CBFB/MYH11 inv(16) 10 Dominant-negative function Unknown 302,303 MLL/AF9 t(9;11) 7 Increased Hox gene expression Unknown 304,305 RARa fusions t(15;17),t(11;17),t(5;17) 7 Deregulation of retinoid-inducible Unknown 306–308 genes EVI1 t(3;var) 3 EVI1 overexpression PI3K Akt, inhibits JNK 309,310 NPM/MLF1 t(3;5) 1 Deregulation of target genes Unknown 311–312 DEK/CAN t(6;9) 1 Increased transcriptional activity Unknown 313,314 FUS/ERG t(16;21) 1 Increased transcriptional activity Increased cytokine 315,316 receptor expression NUP98/HOXA9 t(7;11) 1 Increased transcriptional activity Unknown 317–319

ALL TEL/AML1 t(12;21) 20 Deregulation of AML1 target genes Unknown 319,320 MLL fusions e.g., t(4;11),t(1;11), 6 Increased Hox gene expression Unknown 321–323 t(11;19), over 30 others E2A/PBX1 t(1;19) 5 Increased transcriptional activity/ Unknown 324,325 gain of function BCR/ABL t(9;22) 4 Increased kinase activity PI3K/Akt RAF/MEK/Raf 326,327 LYL1, TAL1/2, a(14q11)TCRad 4 Transcription factor overexpression Unknown 328–330 HOX11, MO1/2, MYC LYL1, TAL1/2, a(7q35)TCRb 3 Transcription factor overexpression Unknown 331,332 HOX11, LMO1/2, MYC MYC t(8;14),t(2;8),t(8;22) 2 Increased transcriptional activity Stabilized by ERK PI3K 333,334 Jak/STAT TEL/JAK t(9;12) o1 Constitutive kinase activity Jak/STAT 1,335 aInappropriate V(D)J rearrangements of the T-cell receptor loci result in chromosome translocations and ectopic expression of transcriptional regulations.

The Ras/Raf/MEK/ERK pathway has what often appears to be Activation of the Raf proteins is very complex as there are conflicting roles in cellular proliferation, differentiation and the many phosphorylation sites on Raf. Phosphorylation at different prevention of apoptosis. Classical studies have indicated that sites can lead to either activation or inactivation. It is important Ras/Raf/MEK/ERK can promote proliferation and malignant for the clinician and basic scientist to have a general under- transformation in part due to the stimulation of cell growth standing of the complexity of protein phosphorylation. Targeting and at the same time results in the prevention of apoptosis. a kinase will not necessarily be a simple thing. Inhibition of one Furthermore, an often overlooked aspect of Raf/MEK/ERK kinase might result in activation of another kinase cascade that cascade is its effects on cytokine and growth factor gene may have pro-proliferative effects. Clearly, there are many transcription that can stimulate proliferation. The latest ‘hot’ kinases and phosphatases that regulate Raf activity and the area of the Raf/MEK/ERK pathway is the discovery of BRAF gene phosphorylation will determine whether Raf is active or mutations in human cancer, which can promote proliferation inactive. While the kinases involved in regulation in Raf/MEK/ and transformation.218 However, it should be remembered that ERK have been extensively studied, there is only very limited only a few years ago, hyperactivation of B-Raf and Raf-1 was knowledge of the specific phosphatases involved in these proposed to promote cell cycle arrest.4 Thus, it is probably fine- regulatory events. tuning of these mutations, which dictates whether there is cell Raf-1 has many roles, which are apparently independent of cycle arrest or malignant transformation. downstream MEK/ERK. Some of these functions occur at the Initially it was thought that Raf-1 was the most important Raf mitochondria and are intimately associated with the prevention isoform. RAF1 was the earliest identified RAF isoform and of apoptosis. Raf-1 may function as a scaffolding molecule to homologous genes are present in both murine and avian- inhibit the activity of kinases that promote apoptosis. transforming retroviruses. Originally it was shown that Raf-1 was The Raf/MEK/ERK pathway is both positively (Hsp90, kinase ubiquitously expressed, indicating a more general and important suppressor of Ras (KSR), MEK partner-1 (MP-1)) and negatively role while B-Raf and A-Raf had more limited patterns of (Raf kinase inhibitory protein, RKIP, 14-3-3) regulated by expression. However, it is now believed that B-Raf is the more association with scaffolding proteins. The expression of some important activator of the Raf/MEK/ERK cascade and in some of the scaffolding proteins is altered in human cancer (for cases, activation of Raf-1 may require B-Raf. However, Raf-1 example, RKIP) in some cases. Some of these scaffolding rears its head again in the cancer field by the recent discovery proteins (for example, Hsp90) are being evaluated as potential that there are mutant RAF-1 alleles in certain therapy-induced therapeutic targets (Hsp90 is a target of geldanamycin, modified t-AMLs that are transmitted in a Mendelian fashion.221 The role geldanamycins are in clinical trials). Potential roles of Hsp90 in of A-Raf remains poorly defined yet it is an interesting isoform. stabilizing activated forms of Raf are intriguing and may allow It is the weakest Raf kinase, yet it can stimulate cell cycle the evolution of activated mutant forms of Raf. progression and proliferation without having the negative effects The Raf/MEK/ERK pathway is intimately linked with the PI3K/ on cell proliferation that B-Raf and Raf-1 can exert.243 PTEN/Akt/mTOR pathway and they interact to regulate cell

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 698 growth, apoptosis and malignant transformation. Ras can cell cycle progression and leukemogenesis. Leukemia 2004; 18: regulate both pathways. Furthermore, in some cell types, Raf 189–218. 2 Lee Jr JT, McCubrey JA. Targeting the Raf kinase cascade in activity is negatively regulated by Akt indicating a cross talk F between the two pathways. Both pathways may result in the cancer therapy novel molecular targets and therapeutic strate- gies. Expert Opin Ther Targets 2002; 6: 659–678. phosphorylation of many downstream targets and impose a role 3 Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, in the regulation of cell survival and proliferation. These Wang XY, Algate PA et al. Signal transduction, cell cycle pathways phosphorylate many key proteins involved in apop- regulatory, and anti-apoptotic pathways regulated by IL-3 in tosis (Bad, Bim, Mcl-1, caspase 9, Ask-1 and others), which hematopoietic cells: possible sites for intervention with anti- serve to alter their activities and subcellular localization. The neoplastic drugs. Leukemia 1999; 13: 1109–1166. phosphorylation events mediated by Raf/MEK/ERK and PI3K/ 4 McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW, Chang F et al. Roles of the Raf/MEK/ERK pathway in cell growth, PTEN/Akt/mTOR pathways are associated with the prevention of malignant transformation and drug resistance. Biochem Biophys apoptosis. In contrast, another MAPK, JNK also phosphorylates Acta 2007; 177: 1263–1284. many of these molecules, and these phosphorylation events 5 Kim D, Dan HC, Park S, Yang L, Liu Q, Kaneko S et al. AKT/PKB often have opposite effects as those elicited by the Raf/MEK/ERK signaling mechanisms in cancer and chemoresistance. Front and PI3K/PTEN/Akt/mTOR pathways. Interestingly, Ras and Raf Biosci 2005; 10: 975–987. mutations may not always result in similar outcomes. For 6 Matsuguchi T, Salgia R, Hallek M, Eder M, Druker B, Ernst TJ et al. Shc phosphorylation in myeloid cells is regulated by granulocyte example a Ras mutation would be predicted to activate both the macrophage colony-stimulating factor, interleukin-3, and steel Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. Activation factor and is constitutively increased by p210BCR/ABL. J Biol of PI3K/PTEN/Akt/mTOR could result in the suppression of Raf/ Chem 1994; 269: 5016–5021. MEK/ERK. However, mutation at either B-Raf or Raf-1 would 7 Inhorn RC, Carlesso N, Durstin M, Frank DA, Griffin JD. result in only activation of Raf/MEK/ERK. Identification of a viability domain in the granulocyte/macro- Although we often think of phosphorylation of these phage colony-stimulating factor receptor beta-chain involving molecules as being associated with the prevention of apoptosis tyrosine-750. Proc Natl Acad Sci USA 1995; 92: 8665–8669. 8 Okuda K, Foster R, Griffin JD. Signaling domains of the beta c and the induction of gene transcription, this view is over- chain of the GM-CSF/IL-3/IL-5 receptor. Ann NY Acad Sci 1999; simplified. For example, in certain situations such as those 872: 305–313. leukemias that have a deleted/silenced PTEN gene, the Raf/MEK/ 9 Tauchi T, Boswell HS, Leibowitz D, Broxmeyer HE. Coupling ERK pathway may be inhibited; hence the phosphorylation of between p210bcr-abl and Shc and Grb2 adaptor proteins in Bad and CREB normally mediated by the Raf/MEK/ERK cascade, hematopoietic cells permits growth factor receptor-independent which is associated with the prevention of apoptosis, will be link to ras activation pathway. J Exp Med 1994; 179: 167–175. 10 Lanfrancone L, Pelicci G, Brizzi MF, Aronica MG, Casciari C, inhibited. Likewise it is important to remember that phosphor- Giuli S et al. Overexpression of Shc proteins potentiates the ylation at certain protein residues may result in enhanced proliferative response to the granulocyte-macrophage colony- activity whereas phosphorylation at different residues could stimulating factor and recruitment of Grb2/Sos and Grb2/p140 result in decreased activity. For example, phosphorylation of complexes to the beta receptor subunit. Oncogene 1995; 10: Bim by JNK is linked with the promotion of apoptosis while 907–917. phosphorylation of Bim by Raf/MEK/ERK or PI3K/Akt pathways 11 Minden A, Lin A, McMahon M, Lange-Carter C, De´rijard B, is associated with the prevention of apoptosis. Davis RJ et al. Differential activation of ERK and JNK mitogen- activated protein kinases by Raf-1 and MEKK. Science 1994; 266: Although it has been known for many years that the Raf/MEK/ 1719–1723. ERK pathway can effect cell cycle arrest, differentiation and 12 Lange-Carter CA, Johnson GL. Ras-dependent growth factor senescence, these are probably some of the least studied regulation of MEK kinase in PC12 cells. Science 1994; 265: research areas in the field due to the often cell lineage-specific 1458–1461. effects that must be evaluated in each cell type. An intriguing 13 Marais R, Light Y, Paterson HF, Marshall CJ. Ras recruits Raf-1 to aspect of leukemia therapy is that in some cases stimulation of the plasma membrane for activation by tyrosine phosphorylation. EMBO J 1995; 14: 3136–3145. the Raf/MEK/ERK pathway may be desired to promote terminal 14 Steelman LS, Bertrand FE, McCubrey JA. The complexity of PTEN: differentiation, while in other types of malignant cancer cells mutation, marker and potential target for therapeutic intervention. that proliferate in response to Raf/MEK/ERK activity, inhibition of Expert Opin Ther Targets 2004; 8: 537–550. the Raf/MEK/ERK pathway may be desired to suppress prolifera- 15 Storm SM, Cleveland JL, Rapp UR. Expression of raf family tion. Thus, we must be flexible in dealing with the Raf/MEK/ERK proto-oncogenes in normal mouse tissues. Oncogene 1990; 5: pathway. As we learn more, our conceptions continue to 345–351. change. 16 Marais R, Light Y, Paterson HF, Mason CS, Marshall CJ. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. J Biol Chem 1997; 272: 4378–4383. 17 Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ, Acknowledgements Marais R. Serine and tyrosine phosphorylations cooperate in Raf- 1, but not B-Raf activation. EMBO J 1999; 18: 2137–2148. JAM and LSS have been supported in part by a grant from the NIH 18 Xu S, Robbins D, Frost J, Dang A, Lange-Carter C, Cobb MH. (R01098195). JB was supported in part by the Deutsche MEKK1 phosphorylates MEK1 and MEK2 but does not cause Krebshilfe. AB, PL and AT have been supported in part from activation of mitogen-activated protein kinase. Proc Nat Acad Sci grants from Associazione Italiana Ricerca sul Cancro (AIRC). USA 1995; 92: 6808–6812. 19 Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, AMM has been supported in part by grants from the CARISBO Stanbridge E et al. Regulation of cell death protease caspase-9 by Foundation and the Progetti Strategici Universita` di Bologna phosphorylation. Science 1998; 282: 1318–1321. EF2006. 20 Allan LA, Morrice N, Brady S, Magee G, Pathak S, Clarke PR. Inhibition of caspase-9 by phosphorylation at Thr125 by ERK MAP kinase. Nat Cell Biol 2003; 5: 647–654. References 21 De´rijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ et al. Independent human MAP kinase signal transduction 1 Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE, pathways defined by MEK and MKK isoforms. Science 1995; McCubrey JA. Jak/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in 267: 682–685.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 699 22 Xing J, Ginty DD, Greenberg ME. Coupling of the Ras–MAPK 44 Ley R, Balmanno K, Hadfield K, Weston C, Cook SJ. Activation of pathway to gene activation by Rsk2, a growth factor regulated the ERK1/2 signaling pathway promotes phosphorylation and CREB kinase. Science 1996; 273: 959–963. proteasome-dependent degradation of the BH3-only protein, 23 Coutant A, Rescan C, Gilot D, Loyer P, Guguen-Guillouzo C, Bim. J Biol Chem 2003; 278: 18811–18816. Baffet G. PI3K-FRAP/mTOR pathway is critical for hepatocyte 45 Weston CR, Balmanno K, Chalmers C, Hadfield K, Molton SA, proliferation whereas MEK/ERK supports both proliferation and Ley R et al. Activation of ERK1/2 by deltaRaf-1:ER* represses Bim survival. Hepatology 2002; 36: 1079–1088. expression independently of the JNK or PI3K pathways. Onco- 24 Iijima Y, Laser M, Shiraishi H, Willey CD, Sundaravadivel B, Xu L gene 2003; 22: 1281–1293. et al. c-Raf/MEK/ERK pathway controls protein kinase C-mediated 46 Domina AM, Vrana J, Gregory MA, Hann SR, Craig RW. MCL1 is p70S6K activation in adult cardiac muscle cells. J Biol Chem 2002; phosphorylated in the PEST region and stabilized upon ERK 277: 23065–23075. activation in viable cells, and at additional sites with cytotoxic 25 Blalock WL, Navolanic PM, Steelman LS, Shelton JG, okadaic acid or taxol. Oncogene 2004; 23: 5301–5315. Moye PW, Lee JT et al. Requirement for the PI3K/Akt pathway 47 Ge¨linas C, White E. BH3-only proteins in control: specificity in MEK1-mediated growth and prevention of apoptosis: identifi- regulates MCL-1 and BAK-mediated apoptosis. Genes Dev 2006; cation of an Achilles heel in leukemia. Leukemia 2003; 17: 19: 1263–1268. 1058–1067. 48 O’Neill E, Kolch W. Taming the Hippo: Raf-1 controls apoptosis 26 Deng T, Karin M. c-Fos transcriptional activity stimulated by by suppressing MST2/Hippo. Cell Cycle 2005; 4: 365–367. H-Ras-activated protein kinase distinct from JNK and ERK. 49 O’Neill E, Rushworth L, Baccarini M, Kolch W. Role of the kinase Nature 1994; 371: 171–175. MST2 in suppression of apoptosis by the proto-oncogene Raf. 27 Davis RJ. Transcriptional regulation by MAP kinases. Mol Reprod Science 2004; 306: 2267–2270. Dev 1995; 4: 459–467. 50 O’Neill EE, Matallanas D, Kolch W. Mammalian sterile 20-like 28 Robinson MJ, Stippec SA, Goldsmith E, White MA, Cobb MH. kinases in tumor suppression: an emerging pathway. Cancer Res A constitutively active and nuclear form of the MAP kinase ERK2 2005; 65: 5485–5487. is sufficient for neurite outgrowth and cell transformation. Curr 51 Drexler HG. Expression of the FLT-3 receptor and response to Biol 1998; 21: 1141–1150. FLT3 ligand by leukemic cells. Leukemia 1996; 10: 588–599. 29 Aplin AE, Stewart SA, Assoian RK, Juliano RL. Integrin-mediated 52 Rao P, Mufson RA. A membrane proximal domain of the human adhesion regulates ERK nuclear translocation and phosphoryla- interleukin-3 receptor beta c subunit that signals DNA synthesis tion of Elk-1. J Cell Biol 2001; 153: 273–282. in NIH 3T3 cells 1995 specifically binds a complex of Src and 30 McCubrey JA, May WS, Duronio V, Mufson A. Serine/threonine Janus family tyrosine kinases and phosphatidylinositol 3-kinase. phosphorylation in cytokine signal transduction. Leukemia 2000; J Biol Chem 1995; 270: 6886–6893. 14: 9–21. 53 Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock 31 Tresini M, Lorenzini A, Frisoni L, Allen RG, Cristofalo VJ. Lack of WL et al. Involvement of PI3K/Akt pathway in cell cycle Elk-1 phosphorylation and dysregulation of the extracellular progression, apoptosis, and neoplastic transformation: a target regulated kinase signaling pathway in senescent human fibro- for cancer chemotherapy. Leukemia 2003; 17: 590–603. blast. Exp Cell Res 2001; 269: 287–300. 54 Yu J, Wjasow C, Backer JM. Regulation of the p85/p110alpha 32 Eblen ST, Catling AD, Assanah MC, Weber MJ. Biochemical and phosphatidylinositol 30-kinase. Distinct roles for the N-terminal biological functions of the N-terminal, noncatalytic domain of and C-terminal SH2 domains. Biol Chem 1996; 273: extracellular signal-regulated kinase 2. Mol Cell Biol 2001; 21: 30199–30203. 249–259. 55 Fu Z, Aronoff-Spencer E, Wu H, Gerfen GJ, Backer JM. The iSH2 33 Adachi T, Kar S, Wang M, Carr BI. Transient and sustained ERK domain of PI 3-kinase is a rigid tether for p110 and phosphorylation and nuclear translocation in growth control. not a conformational switch. Arch Biochem Biophys 2004; 432: J Cell Physiol 2002; 192: 151–159. 244–251. 34 Wang CY, Bassuk AG, Boise LH, Thompson CB, Bravo R, Leiden 56 Martelli AM, Faenza I, Billi AM, Manzoli L, Evangelisti C, Fala F JM. Activation of the granulocyte-macrophage colony-stimulating et al. Intranuclear 30-phosphoinositide metabolism and Akt factor promoter in T cells requires cooperative binding of signaling: new mechanisms for tumorigenesis and protection Elf-1 and AP-1 transcription factors. Mol Cell Biol 1994; 14: against apoptosis? Cell Signal 2006; 18: 1101–1107. 1153–1159. 57 Evangelisti C, Bortul R, Fala` F, Tabellini G, Goto K, Martelli AM. 35 Thomas RS, Tymms MJ, McKinlay LH, Shannon MF, Seth A, Kola Nuclear diacylglycerol kinases: emerging downstream regulators I. ETS1, NFkappaB and AP1 synergistically transactivate the in cell signaling networks. Histol Histopathol 2007; 22: 573–579. human GM-CSF promoter. Oncogene 1997; 23: 2845–2855. 58 Cocco L, Follo MY, Faenza I, Bavelloni A, Billi AM, Martelli AM 36 Ponti C, Gibellini D, Boin F, Melloni E, Manzoli FA, Cocco L et al. Nuclear inositide signaling: an appraisal of phospholipase C et al. Role of CREB transcription factor in c-fos activation in beta1 behavior in myeloblastic and leukemia cells. Adv natural killer cells. Eur J Immunol 2002; 32: 3358–3365. Regul 2007; 47: 2–9. 37 Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood 59 Martelli AM, Tazzari PL, Evangelisti C, Chiarini F, Blalock WL, 2002; 99: 3892–3904. Billi AM et al. Targeting the phosphatidylinositol 3-kinase/Akt/ 38 Deng X, Kornblau SM, Ruvolo PP, May Jr WS. Regulation of Bcl2 mammalian target of rapamycin module for acute myelogenous phosphorylation and potential significance for leukemic cell leukemia therapy: from bench to bedside. Curr Med Chem 2007, chemoresistance. J Natl Cancer Inst Monogr 2001; 28: 30–37. 14: 2009–2023. 39 Carter BZ, Milella M, Tsao T, McQueen T, Schober WD, Hu W 60 Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr et al. Regulation and targeting of antiapoptotic XIAP in acute Opin Oncol 2006; 18: 77–82. myeloid leukemia. Leukemia 2003; 17: 2081–2089. 61 Engleman JA, Luo J, Canley LC. The evolution phosphatidylino- 40 Jia W, Yu C, Rahmani M, Krystal G, Sausville EA, Dent P et al. sitol 3-kinases as regulators of growth and metabolism. Nat Rev Synergistic antileukemic interactions between 17-AAG and UCN- Genet 2006; 7: 606–619. 01 involve interruption of RAF/MEK- and AKT-related pathways. 62 Stephens L, Williams R, Hawkins P. Phosphoinositide 3-kinases Blood 2003; 102: 1824–1832. as drug targets in cancer. Curr Opin Pharmacol 2005; 5: 41 Troppmair J, Rapp UR. Raf and the road to cell survival: a tale of 357–365. bad spells, ring bearers and detours. Biochem Pharmacol 2003; 63 Songyang Z, Baltimore D, Cantley LC, Kaplan DR, Franke TF. 66: 1341–1345. Interleukin 3-dependent survival by the Akt protein kinase. Proc 42 Harada H, Quearry B, Ruiz-Vela A, Korsmeyer SJ. Survival factor- Natl Acad Sci USA 1997; 94: 11345–11350. induced extracellular signal-regulated kinase phosphorylates 64 Troussard AA, Mawji NM, Ong C, Mui A, St-Arnaud R, Dedhar S. BIM, inhibiting its association with BAX and proapoptotic Conditional knock-out of integrin-linked kinase demonstrates an activity. Proc Natl Acad Sci USA 2004; 101: 15313–15317. essential role in protein kinase B/Akt activation. J Biol Chem 43 Marani M, Hancock D, Lopes R, Tenev T, Downward J, Lemoine 2003; 278: 22374–22378. NR. Role of Bim in the survival pathway induced by Raf in 65 Xu Z, Ma DZ, Wang LY, Su JM, Zha XL. Transforming growth epithelial cells. Oncogene 2004; 23: 2431–2441. factor-beta1 stimulated protein kinase B serine-473 and focal

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 700 adhesion kinase tyrosine phosphorylation dependent on cell lin-dependent kinases in hydrogen peroxide-induced IkappaB adhesion in human hepatocellular carcinoma SMMC-7721 cells. phosphorylation in human T lymphocytes. J Biol Chem 2002; Biochim Biophys Res Commun 2003; 312: 388–396. 277: 30469–30476. 66 Persad S, Dedhar S. The role of integrin-linked kinase (ILK) in 90 Howe CJ, LaHair MM, McCubrey JA, Franklin RA. Redox cancer progression. Cancer Metastasis Rev 2003; 22: 375–384. regulation of the CaM-kinases. J Biol Chem 2004; 279: 67 Kumar AS, Naruszewicz I, Wang P, Leung-Hagesteijn C, Hannigan 44573–44581. GE. ILKAP regulates ILK signaling and inhibits anchorage- 91 Hu MC, Lee DF, Xia W, Golfman LS, Ou-Yang F, Yang JY et al. independent growth. Oncogene 2004; 23: 3454–3461. IkappaB kinase promotes tumorigenesis through inhibition of 68 Noguchi M, Ropars V, Roumestand C, Suizu F. Proto-oncogene forkhead FOXO3a. Cell 2004; 117: 225–237. TCL1: more than just a coactivator for Akt. FASEB J 2007; 21: 92 Mayo MW, Baldwin AS. The transcription factor NF-kappaB: 1–12. control of oncogenesis and cancer therapy resistance. Biochim 69 Gao T, Furnari F, Newton AC. PHLPP: a phosphatase that directly Biophys Acta 2000; 1470: M55–M62. dephosphorylates Akt, promotes apoptosis, and suppresses tumor 93 Shishodia S, Aggarwal BB. Nuclear factor-kappaB activation growth. Mol Cell 2005; 18: 13–24. mediates cellular transformation, proliferation, invasion angio- 70 Ye K. PIKE/nuclear PI 3-kinase signaling in preventing pro- genesis and metastasis of cancer. Cancer Treat Res 2004; 119: grammed cell death. J Cell Biochem 2005; 96: 463–472. 139–173. 71 Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell 94 Du K, Montminy M. CREB is a regulatory target for the protein proliferation, survival and insulin responses? J Cell Sci 2001; kinase Akt/PKB. J Biol Chem 1998; 273: 32377–32379. 114: 2903–2910. 95 Arcinas M, Heckman CA, Mehew JW, Boxer LM. Molecular 72 Hay N. The Akt-mTOR tango and its relevance to cancer. Cancer mechanisms of transcriptional control of bcl-2 and c-myc in Cell 2005; 8: 179–183. follicular and transformed lymphoma. Cancer Res 2001; 61: 73 Scheid MP, Duronio V. Dissociation of cytokine-induced 5202–5206. phosphorylation of Bad and activation of PKB/akt: involvement 96 Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ, Yang-Yen HF. The of MEK upstream of Bad phosphorylation. Proc Natl Acad Sci antiapoptotic gene Mcl-1 is upregulated by the phosphatidylino- USA 1998; 95: 7439–7444. sitol 3-kinase/Akt signaling pathway through a transcription factor 74 del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. complex containing CREB. Mol Cell Biol 1999; 19: 6195–6206. Interleukin-3-induced phosphorylation of BAD through the 97 Mahalingam M, Templeton DJ. Constitutive activation of S6 protein kinase Akt. Science 1997; 278: 687–689. kinase by deletion of amino-terminal autoinhibitory and rapa- 75 Nakae J, Park BC, Accili D. Insulin stimulates phosphorylation mycin sensitivity domains. Mol Cell Biol 1996; 16: 405–413. of the forkhead transcription factor FKHR on serine 253 through 98 Dufner A, Anjelkovic M, Burgering BMT, Hemmings B, Thomas a Wortmannin-sensitive pathway. J Biol Chem 1999; 274: G. Protein kinase B localization and activation and eukaryotic 15982–15985. translational initiation factor 4E-binding protein phosphorylation. 76 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. Akt Mol Cell Biol 1999; 19: 4525–4534. promotes cell survival by phosphorylating and inhibiting a 99 Romanelli A, Martin KA, Toker A, Bleinis J. p70 S6 kinase is forkhead transcription factor. Cell 1999; 96: 857–868. regulated by protein kinase Cz and participates in a phosphoi- 77 Medema RH, Kops GJ, Bos JL, Burgering BM. Forkhead nositide 3-kinase-regulated signaling complex. Mol Cell Biol transcription factors mediate cell-cycle regulation by Ras and 1999; 19: 2921–2928. PKB through p27kip1. Nature 2000; 404: 782–787. 100 Harada H, Andersen JS, Mann M, Terada N, Korsmeyer SJ. p70S6 78 Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW kinase signals cell survival as well as growth, inactivating the pro- et al. Forkhead transcription factor FKHR-L1 modulates cytokine- apoptotic molecule Bad. Proc Natl Acad Sci USA 2001; 98: dependent transcriptional regulation of p27Kip1. Mol Cell Biol 9666–9670. 2000; 20: 9138–9148. 101 Edinger AL, Thompson CB. An activated mTOR mutant supports 79 Qi XJ, Wildey GM, Howe PH. Evidence that Ser87 of BimEL is growth factor-independent, nutrient-dependent cell survival. phosphorylated by Akt and regulates BimEL apoptotic function. Oncogene 2004; 23: 5654–5663. J Biol Chem 2006; 281: 813–823. 102 Panwalkar A, Verstovsek S, Giles FJ. Mammalian target of 80 Mayo LD, Donner DB. A phosphatidylinositol 3-kinase/Akt rapamycin inhibition as therapy for hematologic malignancies. pathway promotes translocation of Mdm2 from the cytoplasm Cancer 2004; 100: 657–666. to the nucleus. Proc Natl Acad Sci USA 2001; 98: 10983–10985. 103 Jonassen AK, Mjos OD, Sack MN. p70S6 kinase is a functional 81 Gottlieb TM, Leal JF, Seger R, Taya Y, Oren M. Cross-talk target of insulin activated Akt cell-survival. Biochem Biophys Res between Akt, p53 and Mdm2: possible implications for the Commun 2004; 315: 160–165. regulation of apoptosis. Oncogene 2002; 21: 1299–1303. 104 Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. 82 Zhou BP, Hung MC. Novel targets of Akt, p21(Cip1.WAF1), and Phosphorylation and functional inactivation of TSC2 by Erk MDM2. Semin Oncol 2002; 29: 62–70. implications for tuberous sclerosis and cancer pathogenesis. Cell 83 Dan HC, Sun M, Kaneko S, Feldman RI, Nicosia SV, Wang HG 2005; 121: 179–193. et al. Akt phosphorylation and stabilization of X-linked inhibitor 105 Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signaling controls of apoptosis protein (XIAP). J Biol Chem 2004; 279: 5405–5412. tumour cell growth. Nature 2006; 441: 424–430. 84 You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, 106 Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH Erlacher M et al. FOXO3a-dependent regulation of Puma in et al. Identification of a candidate tumour suppressor gene, response to cytokine/growth factor withdrawal. J Exp Med 2006; MMAC1, at chromosome 10q23.3 that is mutated in multiple 203: 1657–1663. advanced cancers. Nat Genet 1997; 15: 356–362. 85 Obexer P, Geiger K, Ambros PF, Meister B, Ausseriechner MJ. 107 Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor FKHRL1-mediated expression of Noxa and Bim induces apoptosis locus, is a novel protein tyrosine phosphatase regulated via the mitochondria in neuroblastoma cells. Cell Death Differ by transforming growth factor beta. Cancer Res 1997; 57: 2007; 14: 534–547. 2124–2129. 86 Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LLM, Donner 108 Li J, Yen C, Liaw D, Podsypanina K, Bolse S, Wang SI et al. PTEN, DB. NF-kappaB activation by tumor necrosis factor requires the a putative protein tyrosine phosphatase gene mutated in Akt serine-threonine kinase. Nature 1999; 401: 82–85. human brain, breast, and prostate cancer. Science 1997; 275: 87 Romashkova JA, Makarov SS. NF-kappaB is a target of Akt in anti- 1943–1947. apoptotic PDGF signaling. Nature 1999; 401: 86–90. 109 Chang F, Steelman LS, Shelton JG, Lee JT, Navolanic PN, Blalock 88 Madrid LV, Wang CY, Guttridge DC, Schottelius AJ, Baldwin AS, WL et al. Regulation of cell cycle progression and apoptosis by Mayo MW. Akt suppresses apoptosis by stimulating the transacti- the Ras/Raf/MEK/ERK pathway. Int J Oncol 2003; 22: 469–480. vation potential of the Rel A/p65 subunit of NF-kappaB. Mol Cell 110 Mahimainathan L, Choudhury GG. Inactivation of platelet- Biol 2000; 20: 1626–1638. derived growth factor receptor by the tumor suppressor PTEN 89 Howe CJ, LaHair MM, Maxwell JA, Lee JT, Robinson PJ, provides a novel mechanism of action of the phosphatase. J Biol Rodriguez-Mora O et al. Participation of the calcium/calmodu- Chem 2004; 279: 15258–15268.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 701 111 Raftopoulou M, Etienne-Manneville S, Self A, Nicholls S, Hall A. 132 Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Regulation of cell migration by the C2 domain of the tumor Identification of the tuberous sclerosis complex-2 tumor suppres- suppressor PTEN. Science 2004; 303: 1179–1181. sor gene product tuberin as a target of the phosphoinositide 112 Damen JE, Liu L, Rosten P, Humphries RK, Jefferson AB, Majerus 3-kinase/akt pathway. Mol Cell 2002; 10: 151–162. PW et al. The 145-kDa protein induced to associate with Shc by 133 Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D. Rheb is a multiple cytokines is an inositol tetraphosphate and phosphati- direct target of the tuberous sclerosis tumour suppressor proteins. dylinositol 3,4,5-triposphate 5-phosphatase. Proc Natl Acad Sci Nat Cell Biol 2003; 5: 578–581. USA 1996; 93: 689–1693. 134 Hresko RC, Mueckler M. mTOR.RICTOR is the Ser473 kinase for 113 Kavanaugh WM, Pot DA, Chin SM, Deuter-Reinhard M, Jefferson Akt/protein kinase B in 3T3-L1 adipocytes. J Biol Chem 2005; AF, Norris FA et al. Multiple forms of an inositol polyphosphate 280: 40406–40416. 5-phosphatase from signaling complexes with Shc and Grb2. 135 Granville CA, Memmott RM, Gills JJ, Dennis PA. Handicapping Curr Bio 1996; 6: 438–445. the race to develop inhibitors of the phosphoinositide 3-kinase/ 114 Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold A, Akt/mammalian target of rapamycin pathway. Clin Cancer Res Rohrschneider LR. P150Ship, a signal transduction molecule 2006; 12: 679–689. with inositol polyphosphate-5-phosphatase activity. Genes Devel 136 Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute 1996; 10: 1084–1095. myeloid leukemia cells requires PI3 kinase activation. Blood 115 Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM 2003; 102: 972–980. et al. 50Phospholipid phosphatase SHIP-2 causes protein kinase B 137 Chow S, Minden MD, Hedley DW. Constitutive phosphorylation inactivation and cell cycle arrest in glioblastoma cells. Mol Cell of the S6 ribosomal protein via mTOR and ERK signaling in the Biol 2000; 20: 6860–6871. peripheral blasts of acute leukemia patients. Exp Hematol 2006; 116 Muraille E, Pesesse X, Kuntz C, Erneux C. Distribution of the src- 34: 1183–1191. homology-2-domain-containing inositol 5-phosphatase SHIP-2 in 138 Grandage VL, Gale RE, Linch DC, Khwaja A. PI3-kinase/Akt is both non-haemopoietic and haemopoietic cells and possible constitutively active in primary acute myeloid leukaemia involvement in SHIP-2 in negative signaling of B-cells. Biochem cells and regulates survival and chemoresistance via J 1999; 342: 697–705. NF-kappaB, Map kinase and p53 pathways. Leukemia. 2005; 117 Laine J, Kunstle G, Obata T, Sha M, Noguchi M. The 19: 586–594. protooncogene TCL1 is an Akt coactivator. Mol Cell 2000; 6: 139 Chambard JC, Lefloch R, Pouyssegur J, Lenormand P. ERK 395–407. implication in cell cycle regulation. Biochim Biophys Acta 2007; 118 Virgilio L, Narducci MG, Isobe M, Billips LG, Cooper MD, Croce 1773: 1299–1311. CM et al. Identification of the TCL1 gene involved in T-cell 140 Ricciardi MR, McQueen T, Chism D, Milella M, Estey E, Kaldjian malignancies. Proc Natl Acad Sci USA 1994; 91: 12530–12534. E et al. Quantitative single cell determination of ERK phosphor- 119 Stern MH, Soulier J, Rosenwajg M, Nakahara K, Canki-Klain N, ylation and regulation in relapsed and refractory primary acute Aurias A et al. MTCP-1: a novel gene on the human chromosome myeloid leukemia. Leukemia 2005; 19: 1543–1549. Xq28 translocated to the T cell receptor alpha/delta locus in 141 Inoki K, Ouyang H, Zhu T, Lindvall C, Wangy Y, Zhang X et al. mature T cell proliferations. Oncogene 1993; 8: 2475–2483. TSC2 integrates Wnt and energy signals via a coordinated 120 Pekarsky Y, Hallas C, Isobe M, Russo G, Croce CM. Abnormal- phosphorylation by AMPK and GSK3 to regulate cell growth. ities at 14q32.1 in T cell malignancies involve two oncogenes. Cell 2006; 126: 955–969. Proc Natl Acad Sci USA 1999; 96: 2949–2951. 142 De Toni F, Racaud-Sultan C, Chicanne G, Mas VM, 121 Pekarsky Y, Koval A, Hallas C, Bichi R, Tresini M, Malstrom S Cariven C, Mesange F et al. A crosstalk between the Wnt and et al. Tcl1 enhances Akt kinase activity and mediates its nuclear the adhesion-dependent signalling pathways governs the chemo- translocation. Proc Natl Acad Sci USA 2000; 97: 3028–3033. sensitivity of acute myeloid leukemia. Oncogene 2006; 25: 122 Auguin D, Barthe P, Royer C, Stern MH, Noguchi M, Arold ST 3113–3122. et al. Structural basis for the co-activation of protein kinase B by 143 Ysebaert L, Chicanne G, Demur C, De Toni F, Prade-Houdellier T-cell leukemia-1 (TCL1) family proto-oncoproteins. J Biol Chem N, Ruidavets JB et al. Expression of beta-catenin by acute myeloid 2004; 279: 35890–35902. leukemia cells predicts enhanced clonogenic capacities and poor 123 Kunstle G, Laine J, Pierron G, Kagami S, Nakajima H, Hoh F et al. prognosis. Leukemia 2006; 20: 1211–1216. Identification of Akt association and oligomerization domains 144 Lopez-Bergami P, Huang C, Goydos JS, Yip D, Bar-Eli M, Herlyn of the Akt kinase coactivator TCL1. Mol Cell Biol 2002; 22: M et al. Rewired ERK–JNK signaling pathways in melanoma. 1513–1525. Cancer 2007; 11: 447–460. 124 French SW, Shen RR, Koh PJ, Malon CS, Mallick P, Teitell MA. A 145 Krebs DL, Hilton DJ. SOCS proteins: negative regulators of modeled hydrophobic domain on the TCL1 oncoprotein mediates cytokine signaling. Stem Cells 2001; 19: 378–387. association with Akt at the cytoplasmic membrane. Biochemistry 146 Rodig SJ, Meraz MA, White JM, Lampe PA, Riley JK, Arthur CD 2002; 41: 6376–6382. et al. Disruption of the Jak1 gene demonstrates obligatory and 125 Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan nonredundant roles of the Jaks in cytokine-induced biologic S et al. Survival signalling by Akt and eIF4E in oncogenesis and responses. Cell 1998; 93: 373–383. cancer therapy. Nature 2004; 428: 332–337. 147 Neubauer H, Cumano A, Muller M, Wu H, Huffstadt U, Pfeffer K. 126 Giles FJ, Albitar M. Mammalian target of rapamycin as a Jak2 deficiency defines an essential development checkpoint in therapeutic target in leukemia. Curr Mol Med 2005; 5: 653–661. definitive hematopoiesis. Cell 1998; 93: 397–409. 127 Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of 148 Parganas E, Wang D, Stravopodis D, Topham DJ, Marine JC, nutrient and growth factor signals and coordinator of cell growth Teglund S et al. Jak2 is essential for signaling through a variety of and cell cycle progression. Oncogene 2004; 23: 3151–3171. cytokine receptors. Cell 1998; 93: 385–395. 128 Tokunaga C, Yoshino K, Yonezawa K. mTOR integrates amino 149 Saharinen P, Silvennoinen O. The pseudokinase domain is acid- and energy-sensing pathways. Biochem Biophys Res required for suppression of the basal activity of Jak2 and Jak3 Commun 2004; 313: 443–446. tyrosine kinase and for cytokine-inducible activation of signal 129 Martin DE, Soulard A, Hall MN. TOR regulates ribosomal protein transduction. J Biol Chem 2002; 277: 47954–47963. gene expression via PKA and the Forkhead transcription factor 150 Gu J, Wang Y, Gu X. Evolutionary analysis for functional FHL1. Cell 2004; 119: 969–979. divergence of Jak protein kinase domain and tissue-specific 130 Witzig TE, Kaufmann SH. Inhibition of the phosphatidylinositol genes. J Mol Evol 2002; 54: 725–733. 3-kinase/mammalian target of rapamycin pathway in hematolo- 151 Cools J, Peeters P, Voet T, Aventin A, Mecucci C, Grandchamp B gic malignancies. Curr Treat Options Oncol 2006; 7: 285–294. et al. Genomic organization of human JAK2 and mutation 131 Mobasheri A, Richardson S, Mobasheri R, Shakibaei M, Hoyland analysis of its JH2-domain in leukemia. Cytogenet Cell Genet JA. Hypoxia inducible factor-1 and facilitative glucose transpor- 1999; 85: 260–266. ters GLUT1 and GLUT3: putative molecular components of the 152 Ho JM, Beattie BK, Squire JA, Frank DA, Barber DL. Fusion of the oxygen and glucose sensing apparatus in articular chondrocytes. ets transcription factor TEL to Jak2 results in constitutive Jak–Stat Histol Histopathol 2005; 20: 1327–1338. signaling. Blood 1999; 93: 4354–4364.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 702 153 Barahmand-Pour F, Meinke A, Groner B, Decker T. Jak2–Stat5 members of STAT induced STAT inhibitor (SSI) family: SSI-2 and interactions analyzed in yeast. J Biol Chem 1998; 273: SSI-3. Biochem Biophys Res Commun 1997; 237: 79–83. 12567–12575. 175 Zhang JG, Farley A, Nicholson SE, Willson TA, Zugaro LM, 154 Luo H, Rose P, Barber D, Hanratty WP, Lee S, Roberts TM et al. Simpson RJ et al. The conserved SOCS box motif in suppressors of Mutation in the Jak kinase JH2 domain hyperactivates Drosophila cytokine signaling binds to elongins B and C and may couple and mammalian Jak–Stat pathways. Mol Cell Biol 1997; 17: bound proteins to proteasomal degradation. Proc Natl Acad Sci 1562–1571. USA 1999; 96: 2071–2076. 155 Fujitani Y, Hibi M, Fukada T, Takahashi-Tezuka M, Yoshida H, 176 Kamizono S, Hanada T, Yasukawa H, Minoguchi S, Kato R, Yamaguchi T et al. An alternative pathway for STAT activation Minoguchi M et al. The SOCS box of SOCS-1 accelerates that is mediated by the direct interaction between JAK and STAT. ubiquitin-dependent proteolysis of TEL-JAK2. J Biol Chem 2001; Oncogene 1997; 14: 751–761. 276: 12530–12538. 156 Riedy MC, Dutra AS, Blake TB, Modi W, Lal BK, Davis J et al. 177 Kamura T, Sato S, Haque D, Liu L, Kaelin Jr WG, Conaway RC Genomic sequence, organization, and chromosomal localization et al. The elongin BC complex interacts with the conserved of human JAK3. Genomics 1996; 37: 57–61. SOCS-box motif present in members of the SOCS, ras, WD-40 157 Park SY, Saijo K, Takahashi T, Osawa M, Arase H, Hirayama N repeat, and ankyrin repeat families. Genes Dev 1998; 12: et al. Developmental defects of lymphoid cells in Jak3 kinase- 3872–3881. deficient mice. Immunity 1995; 3: 771–782. 178 Matsumoto A, Seki Y, Kubo M, Ohtsuka S, Suzuki A, Hayashi I 158 Thomas DC, Berg LJ. The role of Jak3 in lymphoid development, et al. Suppression of STAT5 functions in liver, mammary glands, activation and signaling. Curr Opin Immunol 1997; 9: 541–547. and T cells in cytokine-inducible SH2-containing protein 1 159 Nosaka T, van Deursen JM, Tripp RA, Thierfelder WE, Witthuhn transgenic mice. Mol Cell Biol 1999; 19: 6396–6407. BA, McMickle AP et al. Defective lymphoid development in mice 179 Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, lacking Jak3. Science 1995; 270: 800–802. Mitsui K et al. A new protein containing an SH2 domain that 160 Velazquez L, Mogensen KE, Barbieri G, Fellous M, Uze G, inhibits JAK kinases. Nature 1997; 387: 921–924. Pellegrini S. Distinct domains of the protein tyrosine kinase tyk2 180 Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono required for binding of interferon-alpha/beta and for signal A et al. Structure and function of a new STAT-induced STAT transduction. J Biol Chem 1995; 270: 3327–3334. inhibitor. Nature 1997; 387: 924–929. 161 Oakes SA, Candotti F, Johnston JA, Chen YQ, Ryan JJ, Taylor N 181 Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ et al. Signaling via IL-2 and IL-4 in JAK3-deficient severe et al. A family of cytokine-inducible inhibitors of signalling. combined immunodeficiency lymphocytes: JAK3-dependent Nature 1997; 387: 917–921. and independent pathways. Immunity 1996; 5: 605–615. 182 Kile BT, Alexander WS. The suppressors of cytokine signalling 162 David M, Petricoin III E, Benjamin C, Pine R, Weber MJ, Larner (SOCS). Cell Mol Life Sci 2001; 58: 1627–1635. AC. Requirement for MAP kinase (ERK2) activity in interferon 183 Lindeman GJ, Wittlin S, Lada H, Naylor MJ, Santamaria M, Zhang alpha- and interferon beta-stimulated gene expression through JG et al. SOCS1 deficiency results in accelerated mammary gland STAT proteins. Science 1995; 269: 1721–1723. development and rescues lactation in prolactin receptor-deficient 163 Winston LA, Hunter T. JAK2, Ras, and Raf are required for mice. Genes Dev 2001; 15: 1631–1636. activation of extracellular signal-regulated kinase/mitogen-acti- 184 Metcalf D, Alexander WS, Elefanty AG, Nicola NA, Hilton DJ, vated protein kinase by growth hormone. J Biol Chem 1995; 270: Starr R et al. Aberrant hematopoiesis in mice with inactivation of 30837–30840. the gene encoding SOCS-1. Leukemia 1999; 13: 926–934. 164 Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted 185 Starr R, Metcalf D, Elefanty AG, Brysha M, Willson TA, Nicola disruption of the mouse Stat1 gene results in compromised innate NA et al. Liver degeneration and lymphoid deficiencies in mice immunity to viral disease. Cell 1996; 84: 443–450. lacking suppressor of cytokine signaling-1. Proc Natl Acad Sci 165 Simpson SJ, Shah S, Comiskey M, de Jong YP, Wang B, USA 1998; 95: 14395–14399. Mizoguchi E et al. T cell-mediated pathology in two models of 186 Tanuma N, Nakamura K, Shima H, Kikuchi K. Protein-tyrosine experimental colitis depends predominantly on the interleukin phosphatase PTPepsilon C inhibits Jak–STAT signaling and 12/Signal transducer and activator of transcription (Stat)-4 path- differentiation induced by interleukin-6 and leukemia inhibitory way, but is not conditional on interferon gamma expression by T factor in M1 leukemia cells. J Biol Chem 2000; 275: cells. J Exp Med 1998; 187: 1225–1234. 28216–28221. 166 Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida 187 Irie-Sasaki J, Sasaki T, Matsumoto W, Opavsky A, Cheng M, N et al. Targeted disruption of the mouse Stat3 gene leads to Welstead G et al. CD45 is a JAK phosphatase and negatively early embryonic lethality. Proc Natl Acad Sci USA 1997; 94: regulates cytokine receptor signalling. Nature 2001; 409: 3801–3804. 349–354. 167 Danial NN, Pernis A, Rothman P. Jak–STAT signaling induced by 188 Rommel C, Clarke BA, Zimmermann S, Nun˜ez L, Rossman R, the v-abl oncogene. Science 1995; 269: 1875–1877. Reid K et al. Differentiation stage-specific inhibition of the 168 Danial NN, Rothman P. JAK–STAT signaling activated by Abl Raf–MEK–ERK pathway by Akt. Science 1999; 286: 1738–1741. oncogenes. Oncogene 2000; 19: 2523–2531. 189 Zimmermann S, Moelling K. Phosphorylation and regulation of 169 Migone TS, Lin JX, Cereseto A, Mulloy JC, O’Shea JJ, Franchini G Raf by Akt (protein kinase B). Science 1999; 286: 1741–1744. et al. Constitutively activated Jak–STAT pathway in T cells 190 Guan K, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD et al. transformed with HTLV-I. Science 1995; 269: 79–81. Negative regulation of the serine/threonine kinase B-Raf by Akt. 170 Weber-Nordt RM, Egen C, Wehinger J, Ludwig W, Gouilleux- J Biol Chem 2000; 275: 27354–27359. Gruart V, Mertelsmann R et al. Constitutive activation of STAT 191 Zhang BH, Tang E, Zhu T, Greenberg M, Vojtek A, Guan KL. proteins in primary lymphoid and myeloid leukemia cells and in Serum and glucocorticoid-inducible kinase SGK phosphorylates Epstein–Barr virus (EBV)-related lymphoma cell lines. Blood and negatively regulates B-Raf. J Biol Chem 2001; 276: 1996; 88: 809–816. 31620–31626. 171 Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell 192 Majewski M, Nieborowska-Skorska M, Salomoni P, Slupianek A, RG, Albanese C et al. Stat3 as an oncogene. Cell 1999; 98: Reiss K, Trotta R et al. Activation of mitochondrial Raf-1 is 295–303. involved in the anti-apoptotic effects of Akt. Cancer Res 1999; 172 Masuhara M, Sakamoto H, Matsumoto A, Suzuki R, Yasukawa H, 59: 2815–2819. Mitsui K et al. Cloning and characterization of novel CIS family 193 McCubrey JA, Steelman LS, Blalock WL, Lee JT, Moye PW, genes. Biochem Biophys Res Commun 1997; 239: 439–446. Chang F et al. Synergistic effects of PI3K/Akt on abrogation of 173 Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, cytokine-dependency induced by oncogenic Raf. Adv Enzyme Sprigg NS et al. Twenty proteins containing a C-terminal SOCS Regl 2001; 41: 289–323. box form five structural classes. Proc Natl Acad Sci USA 1998; 194 McCubrey JA, Lee JT, Steelman LS, Blalock WL, Moye PW, 95: 114–119. Chang F et al. Interactions between the PI3K and Raf signaling 174 Minamoto S, Ikegame K, Ueno K, Narazaki M, Naka T, pathways can result in the transformation of hematopoietic cells. Yamamoto H et al. Cloning and functional analysis of new Cancer Detect Prevent 2001; 25: 375–393.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 703 195 Gelfanov VM, Burgess GS, Litz-Jackson S, King AJ, Marshall MS, inducing or microtubule-disrupting agents. Apoptosis 2006; 11: Nakshatri H et al. Transformation of interleukin-3-dependent 1275–1288. cells without participation of Stat5/Bcl-xL: cooperation of akt 215 Inoshita S, Takeda K, Hatai T, Terada Y, Sano M, Hata J et al. with raf/erk leads to p65 nuclear factor kB-mediated antiapop- Phosphorylation and inactivation of myeloid cell leukemia 1 by tosis involving c-IAP2. Blood 2001; 15: 2508–2517. JNK in response to oxidative stress. J Biol Chem 2002; 277: 196 von Gise A, Lorenz P, Wellbrock C, Hemmings B, Berberich- 43730–43734. Siebelt F, Rapp UR et al. Apoptosis suppression by Raf-1 and 216 Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. MEK1 requires MEK and phosphatidylinositol 3-kinase dependent Glycogen synthase kinase-3 regulates mitochondrial outer signals. Mol Cell Biol 2001; 21: 2324–2336. membrane permeabilization and apoptosis by destabilization of 197 Shelton JG, Steelman LS, Lee JT, Knapp SL, Blalock WL, Moye MCL-1. Mol Cell 2006; 21: 749–760. PW et al. Effects of the RAF/MEK/ERK and PI3K/AKT signal 217 Yu J, Zhang L. The transcriptional targets of p53 in apoptosis transduction pathways on the abrogation of cytokine-dependence control. Biochem Biophys Res Commun 2005; 331: 851–858. and prevention of apoptosis in hematopoietic cells. Oncogene 218 Flotho C, Valcamonica S, Mach-Pascual S, Schmahl L, Corral J, 2003; 22: 2478–2492. Ritterbach H et al. RAS mutations and clonality analysis in 198 Shelton JG, Blalock WL, White ER, Steelman LS, McCubrey JA. children with juvenile myelomonocytic leukemia (JMML). Ability of the activated PI3K/Akt oncoproteins to synergize with Leukemia 1999; 13: 32–37. MEK1 and induce cell cycle progression and abrogate the 219 Stirewalt DL, Kopecky KJ, Meshinchi S, Applebaum FR, Slovak cytokine-dependence of hematopoietic cells. Cell Cycle 2004; ML, Willman CL et al. FLT3, RAS, and TP53 mutations in 3: 503–512. elderly patients with acute myeloid leukemia. Blood 2001; 97: 199 Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. 3589–3595. Bad, a hetro-dimeric partner for Bcl-xL and Bcl-2 displaces Bax 220 Garnett MJ, Marais R. Guilty as charged: B-Raf is a human and promotes cell death. Cell 1995; 80: 285–291. oncogene. Cancer Cell 2004; 6: 313–319. 200 Pugazhenthi S, Nesterova A, Sable C, Heidenreich KA, Boxer LM, 221 Zebisch A, Staber PB, Delavar A, Bodner C, Hiden K, Fischereder Heasley LE et al. Akt/protein kinase B up-regulates Bcl-2 K et al. Two transforming C-RAF germ-line mutations identified in expression through cAMP-response element-binding protein. patients with therapy-related acute myeloid leukemia. Cancer J Biol Chem 2000; 275: 10761–10766. Res 2006; 166: 3401–3408. 201 Pugazhenthi S, Miller E, Sable C, Young P, Heidenreich KA, 222 Christiansen DH, Andersen MK, Desta F, Pedersen-Bjergaard J. Boxer LM et al. Insulin-like growth factor-I induces Bcl-2 Mutations of genes in the receptor tyrosine kinase (RTK)/ promoter through the transcription factor c-AMP-response RAS–BRAF signal transduction pathway in therapy-related element-binding protein. J Biol Chem 1999; 274: 27529–27535. myelodysplasia and acute myeloid leukemia. Leukemia 2005; 202 Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg 19: 2232–2240. ME. Cell survival promoted by the Ras–MAPK signaling pathway 223 Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. by transcription-dependent and independent mechanisms. Mutations of the BRAF gene in human cancer. Nature 2002; 417: Science 1999; 286: 1358–1362. 949–954. 203 Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y et al. 224 Fischer A, Hekman M, Kuhlmann J, Rubio I, Wiese S, Rapp UR. Akt phosphorylation of BAD couples survival signals to the cell- B- and C-RAF display essential differences in their binding to Ras. intrinsic death machinery. Cell 1997; 91: 231–241. J Biol Chem 2007; 282: 26503–26516. 204 Harada H, Becknell B, Wilm M, Mann M, Huang LJS, Taylor SS 225 Wan PT, Garnett MJ, Ros SM, Lee S, Niculescu-Duvaz D, Good et al. Phosphorylation and inactivation of BAD by mitochondria- VM et al. Mechanism of activation of the Raf–MEK signaling anchored . Mol Cell 1999; 3: 413–422. pathway by oncogenic mutations of B-Raf. Cell 2004; 116: 205 Sunayama J, Tsuruta F, Masuyama N, Gotoh Y. JNK antagonizes 856–867. Akt-mediated survival signals by phosphorylating 14-3-3. J Cell 226 Busca R, Abbe P, Mantous F, Aberdam E, Peyssonnaux C, Biol 2005; 170: 295–304. Eychene A et al. Ras mediates the cAMP-dependent activation of 206 She QB, Solit DB, Ye Q, O’Reillly KE, Lobo J, Rosen N. The BAD extracellular signal-regulated in melanocytes. EMBO J 2000; 19: protein integrates survival signaling by EGFR/MAPK and PI3K/Akt 2900–2910. kinase pathways in PTEN-deficient tumor cells. Cancer Cell 227 Rushworth LK, Hindley AD, O’Neil E, Kolch W. Regulation and 2005; 8: 287–297. role of Raf-1.B-Raf heterodimerization. Mol Cell Biol 2006; 26: 207 Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG et al. 2262–2272. Differential targeting of prosurvival Bcl-2 proteins by their BH3- 228 Garnett MJ, Rana S, Paterson H, Barford D, Marais R. Wild-type only ligands allows complementary apoptotic function. Mol Cell and mutant B-RAF activate C-RAF through distinct mechanisms 2005; 17: 393–403. involving heterodimerization. Mol Cell 2005; 20: 963–969. 208 Putcha GV, Le S, Frank S, Besirli CG, Clark K, Chu B et al. JNK- 229 Rajagopalan H, Bordelli A, Lengauer C, Kinzler KN, Vogelstein B, mediated BIM phosphorylation potentiates BAX-dependent Velculescu VE. Tumorigenesis: Raf/Ras oncogenes and mis- apoptosis. Neuron 2003; 38: 899–914. match-repair status. Nature 2002; 418: 934. 209 Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI et al. 230 Libra L, Malaponte G, Navolanic PM, Gangemi P, Bevelacqua V, Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl- Proietti L et al. Analysis of BRAF mutation in primary and 2, until displaced by BH3-only proteins. Genes Dev 2005; 19: metastatic melanoma. Cell Cycle 2006; 4: 968–970. 1294–1305. 231 Kornblau SM, Womble M, Qiu YH, Jackson CE, Chen W, 210 Nosaka T, Kawashima T, Misawa K, Ikuta K, Mui AL, Kitamura T. Konopleva M et al. Simultaneous activation of multiple signal STAT5 as a molecular regulator of proliferation, differentiation transduction pathways confers poor prognosis in acute myelo- and apoptosis in hematopoietic cells. EMBO J 1999; 18: genous leukemia. Blood 2006; 108: 2358–2365. 4754–4765. 232 Shelton JG, Steelman LS, Abrams SL, Bertrand FE, Franklin RA, 211 Wang K, Gross A, Waksman G, Korsmeyer SJ. Mutagenesis of the McMahon M et al. The epidermal growth factor receptor as a BH3 domain of BAX identifies residues critical for dimerization target for therapeutic intervention in numerous cancersFwhat’s and killing. Mol Cell Biol 1998; 18: 6083–6089. genetics got to do with it? Expert Opin Ther Targets 2005; 9: 212 Ernst P, Fisher JK, Avery W, Wade S, Foy D, Korsmeyer SJ. 1009–1030. Definitive hematopoiesis requires the mixed-lineage gene. Dev 233 Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Cell 2004; 6: 437–443. Bannigan BW et al. Activating mutations in the epidermal growth 213 Zhao S, Konopleva M, Cabreira-Hansen M, Xie Z, Hu W, Milella factor receptor underlying responsiveness of non-small cancer to M et al. Inhibition of phosphatidylinositol 3-kinase depho- gefitinib. N Engl J Med 2004; 350: 2129–2139. sphorylates BAD and promotes apoptosis in myeloid leukemias. 234 Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing Leukemia 2004; 18: 267–275. EGFR mutations in lung cancer activate anti-apoptotic pathways. 214 Vrana JA, Cleaveland ES, Eastman A, Craig RW. Inducer-and Science 2004; 305: 1163–1167. cell type-specific regulation of antiapoptotic MCL-1 in myeloid 235 Sequist LV, Haber DA, Lynch TJ. Epidermal growth factor leukemia and multiple myeloma cells exposed to differentiation- receptor mutations in non-small cell lung cancer; predicting

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 704 clinical response to kinase inhibitors. Clin Cancer Res 2005; 11: 254 Nakahara Y, Nagai H, Kinoshita T, Uchida T, Hatano S, Murate T 5668–5670. et al. Mutational analysis of the PTEN/MMAC1 gene in 236 Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I et al. non-Hodgkin’s lymphoma. Leukemia 1998; 12: 1277–1280. EGF receptor mutations are common in lung cancers from 255 Herranz M, Urioste M, Santos J, Martinez-Delgado JB, Rivas C, ‘never smokers’ are associated with sensitivity of tumors to Benitez J et al. Allelic losses and genetic instabilities of PTEN gefitinib and erlotinib. Proc Natl Acad Sci USA 2004; 101: and p73 in non-Hodgkin lymphomas. Leukemia 2000; 14: 13306–13311. 1325–1327. 237 Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M et al. 256 Cheong JW, Eom JI, Maeng HY, Lee ST, Hahn JS, Ko YW et al. KRAS mutations and primary resistance of lung adenocarcinomas Phosphatase and tensin homologue phosphorylation in the to gefitinib or erlotinib. PLoS Med 2005; 2: 57–61. C-terminal regulatory domain is frequently observed in acute 238 Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A myeloid leukaemia and associated with poor clinical outcome. Br et al. BRaf mutation predicts sensitivity to MEK inhibition. Nature J Haematol 2003; 122: 454–456. 2006; 439: 358–362. 257 Vazquez F, Grossman SR, Takahashi Y, Rokas MV, Nakamura N, 239 Shelton JG, Steelman LS, Abrams SL, White ER, Akula SM, Sellers WR. Phosphorylation of the PTEN tail acts as an inhibitory Franklin RA et al. Conditional EGFR promotes cell cycle switch by preventing its recruitment into a protein complex. J Biol progression and prevention of apoptosis in the absence of Chem 2001; 276: 48627–48630. autocrine cytokines. Cell Cycle 2005; 4: 822–830. 258 Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute 240 McCubrey JA, Shelton JG, Steelman LS, Franklin RA, Sreevalsan myeloid leukemia cells requires PI3 kinase activation. Blood T, McMahon M. Conditionally active v-ErbB:ER transforms NIH- 2003; 102: 972–980. 3T3 cells and converts human and mouse cells to cytokine- 259 Grandage VL, Gale RE, Linch DC, Khwaja A. PI3-kinase/Akt is independence. Oncogene 2004; 23: 7810–7820. constitutively active in primary acute myeloid leukaemia cells 241 Konopleva M, Shi Y, Steelman LS, Shelton JG, Munsell M, Marini and regulates survival and chemoresistance via NF-kB, Mapki- F et al. Development of a conditional in vivo model to evaluate nase and p53 pathways. Leukemia 2005; 19: 586–594. the efficacy of small molecule inhibitors for the treatment of 260 Liu TC, Lin PM, Chang JG, Lee JP, Chen TP, Lin SF. Mutation Raf-transformed hematopoietic cells. Cancer Res 2005; 65: analysis of PTEN/MMAC1 in acute myeloid leukemia. Am 9962–9970. J Hematol 2000; 63: 170–175. 242 Blalock WL, Pearce M, Steelman LS, Franklin RA, McCarthy SA, 261 Dahia PL, Aguiar RC, Alberta J, Kum JB, Caron S, Sill H et al. Cherwinski H et al. A conditionally-active form of MEK1 results PTEN is inversely correlated with the cell survival factor Akt/PKB in autocrine transformation of human and mouse hematopoietic and is inactivated via multiple mechanisms in haematological cells. Oncogene 2000; 19: 526–536. malignancies. Hum Mol Genet 1999; 8: 185–193. 243 Hoyle PE, Moye PW, Steelman LS, Blalock WL, Franklin RA, 262 Aggerholm A, Gronbaek K, Guldberg P, Hokland P. Mutational Pearce M et al. Differential abilities of the Raf family of protein analysis of the tumour suppressor gene MMAC1/PTEN in kinases to abrogate cytokine-dependency and prevent apoptosis malignant myeloid disorders. Eur J Haematol 2000; 65: 109–113. in murine hematopoietic cells by a MEK1-dependent mechanism. 263 Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H Leukemia 2000; 14: 642–656. et al. Pten dependence distinguishes haematopoietic stem cells 244 McCubrey JA, Steelman LS, Hoyle PE, Blalock WL, Weinstein- from leukaemia-initiating cells. Nature 2006; 441: 475–482. Oppenheimer C, Franklin RA et al. Differential abilities of 264 Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT et al. activated Raf oncoproteins to abrogate cytokine-dependency, PTEN maintains haematopoietic stem cells and acts in prevent apoptosis and induce autocrine growth factor lineage choice and leukaemia prevention. Nature 2006; 441: synthesis in human hematopoietic cells. Leukemia 1998; 12: 518–522. 1903–1929. 265 Palomero T, Sulis ML, Cortina M, Real PJ, Barnes K, Ciofani M 245 Kubota Y, Ohnishi H, Kitanaka A, Ishida T, Tanaka T. Constitutive et al. Mutational loss of PTEN induces resistance to NOTCH1 activation of PI3K is involved in the spontaneous proliferation of inhibition in T-cell leukemia. Nat Med 2007; 13: 1203–1210. primary acute myeloid leukemia cells: direct evidence of PI3K 266 Steelman LS, Navolanic PM, Sokolosky ML, Taylor JR, Lehmann activation. Leukemia 2004; 18: 1438–1440. BD, Chappell WH et al. Suppression of PTEN function increases 246 Cuni S, Perez-Aciego P, Perez-Chacon G, Vargas JA, Sa´nchez A, breast cancer chemotherapeutic drug resistance while conferring Martı´n-Saavedra FM et al. A sustained activation of PI3K/NF- sensitivity to mTOR inhibitors. Oncogene 2008, (in press). kappaB pathway is critical for the survival of chronic lymphocytic 267 Rhei E, Kang L, Bogomolniy F, Federici MG, Borgen PI, Boyd J. leukemia B cells. Leukemia 2004; 18: 1391–1400. Mutation analysis of the putative tumor suppressor gene PTEN/ 247 Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroeck B, MMAC1 in primary breast carcinomas. Cancer Res 1997; 57: Gout I, Fry MJ et al. Phosphatidylinositol-3-OH kinase as a direct 3657–3659. target of Ras. Nature 1994; 370: 527–532. 268 Singh B, Ittmann MM, Krolewski JJ. Sporadic breast cancers 248 Hu L, Shi Y, Hsu JH, Gera J, Van Ness B, Lichtenstein A. exhibit loss of heterozygosity on chromosome segment 10q23 Downstream effectors of oncogenic ras in multiple myeloma close to the Cowden disease locus. Genes Cancer cells. Blood 2003; 101: 3126–3135. 1998; 21: 166–171. 249 Gire V, Marshall C, Wynford-Thomas D. PI-3-kinase is an 269 Feilotter HE, Coulon V, McVeigh JL, Boag AH, Dorion-Bonnet F, essential anti-apoptotic effector in the proliferative response of Duboue B et al. Analysis of the 10q23 chromosomal region and primary human epithelial cells to mutant RAS. Oncogene 2000; the PTEN gene in human sporadic breast carcinoma. Br J Cancer 19: 2269–2276. 1999; 79: 718–723. 250 Sun H, King AJ, Diaz HB, Marshall MS. Regulation of the protein 270 Tsutsui S, Inoue H, Yasuda K, Suzuki K, Higashi H, Era S et al. kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3- Reduced expression of PTEN protein and its prognostic implica- kinase, Cdc42/Rac and Pak. Curr Biol 2000; 10: 281–284. tions in invasive ductal carcinoma of the breast. Oncology 2005; 251 Ninomiya Y, Kato K, Takahashi A, Ueoka Y, Kamikihara T, Arima 68: 398–404. T et al. K-Ras and H-Ras activation promote distinct conse- 271 Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, quences on endometrial cell survival. Cancer Res 2004; 64: Yang H et al. Ubiquitination regulates PTEN nuclear import and 2759–2765. tumor suppression. Cell 2007; 128: 141–156. 252 Jucker M, Sudel K, Horn S, Sickel M, Wegner W, Fiedler W et al. 272 Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP et al. Expression of a mutated form of the p85alpha regulatory subunit Essential role for nuclear PTEN in maintaining chromosomal of phosphatidylinositol 3-kinase in a Hodgkin’s lymphoma- integrity. Cell 2007; 128: 157–170. derived cell line (CO). Leukemia 2002; 16: 894–901. 273 Luo JM, Yoshida H, Komura S, Ohishi N, Pan L, Shigeno K et al. 253 Mu¨ller CI, Miller CW, Hofman W-K, Gross ME, Walsh CS, Possible dominant-negative mutation of the SHIP gene in acute Kawamata N et al. Rare mutations of the PIK3CA gene in myeloid leukemia. Leukemia 2003; 17: 1–8. malignancies of the hematopoietic system as well as endome- 274 Luo JM, Liu ZL, Hao HL, Wang FX, Dong ZR, Ohno R. Mutation trium, ovary, prostate and osteosarcomas, and discovery of a analysis of SHIP gene in acute leukemia. Zhongguo Shi Yan Xue PIK3CA pseudogene. Leuk Res 2007; 31: 27–32. Ye Xue Za Zhi 2004; 12: 420–426.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 705 275 Hollestelle A, Elstrodt F, Nagel JHA, Kallemeijn WW. Phospha- pathway and its therapeutical implications for human acute tidylinositol-3-OH kinase or Ras pathway mutations in human myeloid leukemia. Leukemia 2006; 20: 911–928. breast cancer cell lines. Mol Cancer Res 2007; 5: 195–201. 295 Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, 276 Staal SP. Molecular cloning of the Akt oncogene and its human Mauchauffe M et al. A TEL–JAK2 fusion protein with constitutive homologues AKT1 and AKT2: amplification of AKT1 in a primary kinase activity in human leukemia. Science 1997; 278: human gastric adenocarcinoma. Proc Natl Acad Sci USA 1987; 1309–1312.. 84: 5034–5037. 296 Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF 277 Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, receptor beta to a novel ets-like gene, tel in chronic myelomo- Hamilton TC et al. AKT2, a putative oncogene encoding a nocytic leukemia with t(5;12) chromosomal translocation. Cell member of a subfamily of protein-serine/threonine kinases, is 1994; 77: 307–316. amplified in human ovarian carcinomas. Proc Natl Acad Sci USA 297 Golub TR, Barker GF, Stegmaier K, Gilliland DG. The TEL gene 1992; 89: 9267–9271. contributes to the pathogenesis of myeloid and lymphoid 278 Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomar DA, Watson leukemias by diverse molecular genetic mechanisms. Curr Top DK et al. Amplification of AKT2 in human pancreatic cells and Microbiol Immunol 1997; 220: 67–79. inhibition of AKT2 expression and tumorigenicity by anti-sense 298 Quentmeier H, Reinhardt J, Zabgorski M, Drexler HG. FLT3 RNA. Proc Natl Acad Sci USA 1996; 93: 3636–3641. mutations in acute myeloid leukemia cell lines. Leukemia 2003; 279 Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S et al. 17: 120. High frequency of mutations of the PIK3CA gene in human 299 Frohling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis cancers. Science 2004; 304: 554. K et al. Prognostic significance of activating FLT3 mutations in 280 Carpten JD, Faber AL, Horn C, Donoho GP, Briggs SL, Robbins younger adults (16–60 years) with acute myeloid leukemia and CM et al. A transforming mutation in the pleckstrin homology normal cytogenetics: a study of the AML Study Group Ulm. Blood domain of AKT1 in cancer. Nature 2007; 448: 439–444. 2002; 100: 4372–4380. 281 Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, 300 Shurtleff SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC, Bignell G et al. Patterns of somatic mutation in human cancer Hancock ML et al. TEL/AML1 fusion resulting from a cryptic genomes. Nature 2007; 446: 153–158. t(12;21) is the most common genetic lesion in pediatric ALL and 282 Tibes R, Kornblau SM, Qiu Y, Mousses SM, Robbins C, Moses T defines a subgroup of patients with an excellent prognosis. et al. PI3K/Akt pathway activation in acute myeloid leukaemias is Leukemia 1995; 9: 1985–1989. not associated with AKT1 pleckstrin homology domain mutation. 301 Weisser M, Haferlach C, Hiddemann W, Schnittger S. Br J Haematol 2008; 140: 344–347. The quality of molecular response to chemotherapy is 283 Lin J, Adam RM, Santiestevan E, Freeman MR. The phosphatidy- predictive for the outcome of AML1-ETO-positive AML and is linositol 30-kinase pathway is a dominant growth factor-activated independent of pretreatment risk factors. Leukemia 2007; 21: cell survival pathway in LNCaP human prostate carcinoma cells. 1177–1182. Cancer Res 1999; 59: 2891–2897. 302 van der Reijden BA, Dauwerse HG, Giles RH, Jagmohan- 284 Fry MJ. Phosphoinositide 3-kinase signalling in breast Changur S, Wijmenga C, Liu PP et al. Genomic acute myeloid cancer: how big a role might it play? Breast Cancer Res 2001; leukemia-associated inv(16)(p13q22) breakpoints are tightly 3: 304–312. clustered. Oncogene 1999; 18: 543–550. 285 Lin X, Bohle AS, Dohrmann P, Leuschner I, Schulz A, Kremer B 303 Roti G, Rosati R, Bonasso R, Gorello P, Diverio D, Martelli MF et al. Overexpression of phosphatidylinositol 3-kinase in human et al. Denaturing high-performance liquid chromatography: a lung cancer. Langenbecks Arch Surg 2001; 386: 293–301. valid approach for identifying NPM1 mutations in acute myeloid 286 Krasilnikov M, Adler V, Fuchs SY, Dong Z, Haimovitz-Friedman leukemia. J Mol Diagn 2006; 8: 254–259. A, Herlyn M et al. Contribution of phosphatidylinositol 3-kinase 304 Look AT. Oncogenic transcription factors in the human acute to radiation resistance in human melanoma cells. Mol Carcinog leukemias. Science 1997; 278: 1059–1064. 1999; 24: 64–69. 305 Shih LY, Liang DC, Fu JF, Wu JH, Wang PN, Lin TL et al. 287 Martinez-Lorenzo MJ, Anel A, Monleon I, Sierra JJ, Pin˜eiro A, Characterization of fusion partner genes in 114 patients with de Naval J et al. Tyrosine phosphorylation of the p85 subunit of novo acute myeloid leukemia and MLL rearrangement. Leukemia phosphatidylinositol 3-kinase correlates with high proliferation 2006; 20: 218–223. rates in sublines derived from the Jurkat leukemia. Int J Biochem 306 Warrell Jr RP, de The´ H, Wang ZY, Degos L. Acute promyelocytic Cell Biol 2000; 32: 435–445. leukemia. N Engl J Med 1993; 329: 177–189. 288 Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H 307 Ritter M, Kattmann D, Teichler S, Hartmann O, Samuelsson MK, et al. Mechanisms of tamoxifen resistance: increased estrogen Burchert A et al. Inhibition of retinoic acid receptor signaling by receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. Ski in acute myeloid leukemia. Leukemia 2006; 20: 437–443. J Natl Cancer Inst 2004; 96: 926–935. 308 Mikesch JH, Steffen B, Berdel WE, Serve H, Muller-Tidow C. 289 Frogne T, Jepsen JS, Larsen SS, Fog CK, Brockdorff BL, Lykkesfeldt Themerging role of Wnt signaling in the pathogenesis of acute AE. Antiestrogen-resistant human breast cancer cells require myeloid leukemia. Leukemia 2007; 21: 1638–1647. activated protein kinase B/Akt for growth. Endocr Relat Cancer 309 Liu Y, Chen L, Ko TC, Fields AP, Thompson EA. Evi1 is a survival 2005; 12: 599–614. factor which conveys resistance to both TGFbeta- and 290 Kirkegaard T, Witton CJ, McGlynn LM, Tovey SM, Dunne B, Lyon taxol-mediated cell death via PI3K/AKT. Oncogene 2006; 25: A et al. AKT activation predicts outcome in breast cancer patients 3565–3575. treated with tamoxifen. J Pathol 2005; 207: 139–146. 310 Trubia M, Albano F, Cavazzini F, Cambrin GR, Quarta G, 291 Nyakern M, Tazzari PL, Finelli C, Bosi C, Follo MY, Grafone T Fabbiano F et al. Characterization of a recurrent translocation et al. Frequent elevation of Akt kinase phosphorylation in blood t(2;3) (p15–22;q26) occurring in acute myeloid leukaemia. marrow and peripheral blood mononuclear cells from high-risk Leukemia 2006; 20: 48–54. myelodysplastic syndrome patients. Leukemia 2006; 20: 311 Raimondi SC, Dube ID, Valentine MB, Mirro Jr J, Watt HJ, Larson 230–238. RA et al. Clinicopathologic manifestations and breakpoints of the 292 Mantovani I, Cappellini A, Tazzari PL, Papa V, Cocco L, Martelli t(3;5) in patients with acute nonlymphocytic leukemia. Leukemia AM. Caspase-dependent cleavage of 170-kDa P-glycoprotein 1989; 3: 42–47. during apoptosis of human T-lymphoblastoid CEM cells. J Cell 312 Fallini B, Bigerna B, Pucciarini A, Tiacci E, Mecucci C, Morris Physiol 2006; 207: 836–844. SW et al. Aberrant subcellular expression of nucleophosmin and 293 Nyakern M, Cappellini A, Mantovani J, Martelli AM. Synergistic NPM–MLF1 fusion protein in acute myeloid leukaemia carrying induction of apoptosis in human leukemia T cells by the Akt t(3;5): a comparison with NPMc+AML. Leukemia 2006; 20: inhibitor perifosine and etoposide through activation of intrinsic 368–371. and Fas-mediated extrinsic cell death pathways. Mol Cancer Ther 313 Fornerod M, Boer J, van Baal S, Morreau H, Grosveld G. 2006; 5: 1559–1570. Interaction of cellular proteins with the leukemia specific fusion 294 Martelli AM, Nyakern M, Tabellini G, Bortul R, Tazzari PL, proteins DEK-CAN and SET-CAN and their normal counterpart, Evangelisti C et al. Phosphoinositide 3-kinase/Akt signaling the nucleoporin CAN. Oncogene 1996; 13: 1801–1808.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 706 314 Garcon L, Libura M, Delabesse E, Valensi F, Asnafi V, Berger C CD34+ cells: implications for leukemogenesis in gene therapy. et al. DEK-CAN molecular monitoring of myeloid malignancies Leukemia 2007; 21: 754–763. could aid therapeutic stratification. Leukemia 2005; 19: 333 Hecht JL, Aster JC. Molecular biology of Burkitt’s lymphoma. 1338–1344. J Clin Oncol 2000; 18: 3707–3721. 315 Shikami M, Miwa H, Nishii K, Takahashi T, Shiku H, Tsutani H 334 Liu JN, Deng R, Guo JF, Zhou JM, Feng GK, Huang ZS et al. et al. Myeloid differentiation antigen and cytokine receptor Inhibition of myc promoter and telomerase activity and induction expression on acute myelocytic leukaemia cells with of delayed apoptosis by SYUIQ-5, a novel G-quadruplex inter- t(16;21)(p11;q22): frequent expression of CD56 and interleukin- active agent in leukemia cells. Leukemia 2007; 21: 1300–1302. 2 receptor alpha chain. Br J Haematol 1999; 105: 711–719. 335 Turner SD, Alexander DR. Fusion tyrosine kinase mediated 316 Choi HW, Shin MG, Sawyer JR, Cho D, Kee SJ, Baek HJ et al. signaling pathways in the transformation of haematopoietic cells. Unusual type of TLS/FUS-ERG chimeric transcript in a pediatric Leukemia 2006; 20: 573–582. acute myelocytic leukemia with 47,XX,_10,t(16;21)(p11;q22). 336 Alvarez-Larran A, Cervantes F, Bellosillo B, Giralt M, Julia A, Cancer Genet Cytogenet 2006; 167: 172–176. Hernandez-Boluda JC et al. Essential thrombocythemia in young 317 Nakamura T. NUP98 fusion in human leukemia: dysregulation of individuals: frequency and risk factors for vascular events and the nuclear pore and homeodomain proteins. Int J Hematol 2005; evolution to myelofibrosis in 126 patients. Leukemia 2007; 21: 82: 21–27. 1218–1223. 318 Romana SP, Radford-Weiss I, Ben Abdelali R, Schluth C, Petit A, 337 Metzgeroth G, Walz C, Score J, Siebert R, Schnittger S, Haferlach Dastugue N et al. NUP98 rearrangements in hematopoietic C et al. Recurrent finding of the FIP1L1–PDGFRA fusion gene in malignancies: a study of the Groupe Francophone de Cytogene- eosinophilia-associated acute myeloid leukemia and lympho- tique Hematologigue. Leukemia 2006; 20: 696–706. blastic T-cell lymphoma. Leukemia 2007; 21: 1183–1188. 319 Romana SP, Poirel H, Leconiat M, Flexor MA, Mauchauffe M, 338 Jost E, Do ON, Dahl E, Maintz CE, Jousten P, Habets L et al. Jonveaux P et al. High frequency of t(12;21) in childhood Epigenetic alterations complement mutation of Jak2 tyrosine B-lineage acute lymphoblastic leukemia. Blood 1995; 86: kinase in patients with BCR/ABL-negative myeloproliferative 4263–4269. disorders. Leukemia 2007; 21: 505–510. 320 Inukai T, Yokota S, Okamoto T, Nemoto A, Akahane K, 339 Mesa RA, Tefferi A, Lasho TS, Loegering D, McClure RF, Powell Takahashi K et al. Clonotypic analysis of acute lymphoblastic HL et al. Janus kinase (V617F) mutation status, signal transducer leukemia with a double TEL–AML1 fusion at onset and relapse. and activator of transcription-3 phosphorylation and impaired Leukemia 2006; 20: 363–365. neutrophil apoptosis ion myelofibrosis with myeloid metaplasia. 321 DiMartino JF, Cleary ML. MLL rearrangements in hematological Leukemia 2006; 20: 1800–1808. malignancies: lessons from clinical and biological studies. Br J 340 Steensma DP, Caudill JS, Pardanani A, McClure RF, Lasho TL, Haemotol 1999; 106: 614–626. Tefferi A. MPL W515 and Jak2 V617 mutation analysis in patients 322 Jansen MW, van der Velden VH, van Dongen JJ. Efficient and with refractory anemia with ringed sideroblasts and an elevated easy detection of MLL–AF4, MLL–AF9 and MLL–ENL fusion gene platelet count. Leukemia 2006; 20: 971–978. transcripts by multiplex real-time quantitative RT-PCR in TaqMan 341 Vannucchi AM, Pancrazzi A, Bogani C, Antonioli E, Guglielmelli and LightCycler. Leukemia 2005; 19: 2016–2018. P. A quantitative assay for Jak2 (V617F) mutation in myelopro- 323 DeBraekeleer M, Morel F, Le Bris MJ, Herry A, Douet-Guilbert N. liferative disorders by ARMS-PCR and capillary electrophoresis. The MLL gene and translocations involving chromosomal band Leukemia 2006; 20: 1055–1060. 11q23 in acute leukemia. Anticancer Res 2005; 25: 1931–1944. 342 DeKeersmaecker K, Cools J. Chronic myeloproliferative disorders: 324 Foa R, Vitale A, Mancini M, Cuneo A, Mecucci C, Elia L et al. a tyrosine kinase tale. Review. Leukemia 2006; 20: 200–205. E2A-PBX1 fusion in adult acute lymphoblastic leukaemia: 343 Bellosillo B, Martinez-Aviles L, Gimeno E, Florensa L, Longaron biological and clinical features. Br J Haematol 2003; 120: R, Navarro G et al. A higher Jak2 V617F-mutated clone is 484–487. observed in platelets than in granulocytes from essential 325 Prima V, Gore L, Caires A, Boomer T, Yoshinari M, Imaizumi M thrombocythemia patients, but not in patients with polycythemia et al. Cloning and functional characterization of MEF2D/DAZAP1 vera and primary myelofibrosis. Leukemia 2007; 21: 1331–1332. and DAZAP1/MEF2D fusion proteins created by a variant 344 Nishii K, Nanbu R, Lorenzo VF, Monma F, Kato K, Ryuu H et al. t(1;19)(q23;p13.3) in acute lymphoblastic leukemia. Leukemia Expression of the Jak2 V617F mutation is not found in de novo 2005; 19: 806–813. AML and MDS but is detected in MDS-derived leukemia of 326 Gotoh A, Broxmeyer HE. function of BCR/ABL and related proto- megakaryoblastic nature. Leukemia 2007; 21: 1337–1338. oncogenes. Curr Opin Hematol 1997; 4: 3–11. 345 Wong CLP, Ma ESK, Wang CLN, Lam HY, Ma SY. Jak2 V617F 327 Pane F, Cimino G, Izzo B, Camera A, Vitale A, Quintarelli C et al. due to a novel TG-CT mutation at nucleotides 1848–1849: Significant reduction of the hybrid BCR/ABL transcripts after diagnostic implication. Leukemia 2007; 21: 1344–1346. induction and consolidation therapy is a powerful predictor of 346 Ohyashiki K, Aota Y, Akahane D, Gotoh A, Ohyashiki JH. Jak2 treatment response in adult Philadelphia-positive acute lympho- (V617F) mutational status as determined by semiquantitative blastic leukemia. Leukemia 2005; 19: 628–635. sequence-specific primer-single molecule fluorescence detection 328 Fornerod M, Boer J, van Baal S, Morreau H, Grosveld G. assay is linked to clinical features in chronic myeloproliferative Interaction of cellular proteins with the leukemia specific fusion disorders. Leukemia 2007; 21: 1097–1099. proteins DEK-CAN and SET-CAN and their normal counterpart, 347 Hermouet S, Dobo I, Lippert E, Boursier M-C, Ergand L, Perrault- the nucleoporin CAN. Oncogene 1998; 7: 3199–3292. Hu F et al. Comparison of whole blood vs purified blood 329 Chen X, Pan Q, Stow P, Behm FG, Goorha R, Pui CH et al. granulocytes for the detection and quantitation of Jak2(V617F). Quantification of minimal residual disease in T-lineage acute Leukemia 2007; 21: 112801130. lymphoblastic leukemia with TAL-1 deletion using a standardized 348 Inami M, Inokuchi K, Okabe M, Kosaka F, Mitamura Y, real-time PCR assay. Leukemia 2002; 15: 166–170. Yamaguchi H et al. Polycythemia associated with the Jak2V617F 330 Speleman F, Cauwelier B, Dastugue N, Cools J, Verhasselt B, mutation emerged during treatment of chronic myelogenous Poppe B et al. A new recurrent inversion, inv(7)(p15q34), leades leukemia. Leukemia 2007; 21: 1103–1104. to transcriptional activation of HOXA10 and HOXAll in a subset 349 Schnittger S, Bacher U, Kern W, Haferlach C, Haferlach T. Jak2 of T-cell acute lymphoblastic leukemia. Leukemia 2005; 19: seems to be a typical cooperating mutation in therapy-related 358–366. t(8;21)/AML1-ETO-positive AML. Leukemia 2007; 21: 183–184. 331 Wadman IA, Osada H, Gru¨tz GG, Agulnick AD, Westphal H, 350 Verstovsek S, Silver RT, Cross NC, Tefferi A. Jak2V617F Forster A et al. The LIM-only protein Lmo2 is a bridging molecule mutational frequency in polycythemia vera: 100%,490%. assembling an erythroid, DNA-binding complex which includes Leukemia 2006; 20: 2067. the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J 1997; 351 Mesa RA, Tefferi A, Li CY, Steensma DP. Hematologic and 16: 3145–3157. cytogenetic response to lenalidomide monotherapy in acute 332 Pike-Overzet K, de Ridder D, Weerkamp F, Baert MR, Verstegen myeloid leukemia arising from Jak2(V617F) positive, MM, Brugman MH et al. Ectopic retroviral expression of LMO2, del(5)(q13q33) myelodysplastic syndrome. Leukemia 2006; 20: but not IL2Rgamma, blocks human T-cell development from 2063–2064.

Leukemia Signaling and apoptotic pathways and leukemia LS Steelman et al 707 352 Renneville A, Quesnel B, Charpentier A, Terriou L, Crinquette A, 363 Bellosillo B, Besses C, Florensa L, Sole´ F, Serrano S. Jak2 V617F Lai JL. High occurrence of Jak2 V617 mutation in refractory mutation, PRV-1 overexpression and endogenous erythroid anemia with ringed sideroblasts associated with marked throm- colony formation show different coexpression patterns among bocytosis. Leukemia 2006; 20: 2067–2070. Ph-negative chronic myeloproliferative disorders. Leukemia 353 Ceesay MM, Lea NC, Ingram W, Westwood NB, Ga¨ken J, 2006; 20: 736–737. Mohamedali A et al. The Jak2 V617F mutation is rare in RARS but 364 Desta F, Christiansen DH, Andersen MK, Pedersen-Bjergaard J. common in RARS-T. Leukemia 2006; 20: 2060–2061. Activating mutations of Jak2V617F are uncommon in t-MDS and 354 Di Ianni M, Moretti L, Del Papa B, Gaozza E, Bell AS, Falzetti F t-AML and are only observed in atypic cases. Leukemia 2006; 20: et al. A microelectronic DNA chip detects the V617F Jak-2 547–548. mutation in myeloproliferative disorders. Leukemia 2006; 20: 365 Vizmanos JL, Ormaza´bal C, Larra´yoz MJ, Cross NC, Calasanz MJ. 1895–1897. Jak2 V617F mutation in classic chronic myeloproliferative 355 Florensa L, Bellosillo B, Besses C, Puigdecanet E, Espinet B, diseases: a report on a series of 349 patients. Leukemia 2006; Pe´rez-Vila E et al. Jak2 V617F mutation analysis in different 20: 534–535. myeloid lineages (granulocytes, platelets, CFU-MK, BFU-E and 366 Kratz CP, Bo¨ll S, Kontny U, Schrappe M, Niemeyer CM, Stanulla CFU-GM) in essential thrombocythemia patients. Leukemia M. Mutational screen reveals a novel Jak2 mutation, L611S, in a 2006; 20: 1903–1905. child with acute lymphoblastic leukemia. Leukemia 2006; 20: 356 Fiorini A, Farina G, Reddiconto G, Palladino M, Rossi E, Za T 381–383. et al. Screening of Jak2 V617F mutation in multiple myeloma. 367 James C, Delhommeau F, Marzac C, Teyssandier I, Coue´dic JP, Leukemia 2006; 20: 1912–1913. Giraudier S et al. Detection of Jak2 V617F as a first intention 357 Park MJ, Shimada A, Asada H, Koike K, Tsuchida M, Hayashi Y. diagnostic test for erythrocytosis. Leukemia 2006; 20: 350–353. Jak2 mutation in a boy with polycythemia vera, but not in other 368 Ohyashiki K, Aota Y, Akahane D, Gotoh A, Miyazawa K, Kimura pediatric hematologic disorders. Leukemia 2006; 20: 1453–1454. Y et al. The Jak2 V617F tyrosine kinase mutation in myelodys- 358 Chen CY, Lin LI, Tang JL, Tsay W, Chang HH, Yeh YC et al. plastic syndromes (MDS) developing myelofibrosis indicates the Acquisition of Jak2, PTPN11, and RAS mutations during disease myeloproliferative nature in a subset of MDS patients. Leukemia progression in primary myelodysplastic syndrome. Leukemia 2005; 19: 2359–2360. 2006; 20: 1155–1158. 369 Antonioli E, Guglielmelli P, Pancrazzi A, Bogani C, Verrucci M, 359 Murati A, Ade´laı¨de J, Gelsi-Boyer V, Etienne A, Re´my V, Fezoui Ponziani V et al. Clinical implications of the Jak2 V617F mutation H et al. t(5;12)(q23–31;p13) with ETV6–ACSL6 gene fusion in in essential thrombocythemia. Leukemia 2005; 19: 1847–1849. polycythemia vera. Leukemia 2006; 20: 1175–1178. 370 Chen CY, Lin LI, Tang JL, Tsay W, Chang HH, Yeh YC et al. 360 Yip SF, So CC, Chan AY, Liu HY, Wan TsK, Chan LC. The lack of Acquisition of Jak2, PTPN11, and RAS mutations during disease association between Jak2 V617F mutation and myelodysplastic progression in primary myelodysplastic syndrome. Leukemia syndrome with or without myelofibrosis. Leukemia 2006; 20: 2006; 20: 1155–1158. 1165. 371 Zhang B, Groffen J, Heisterkamp N. Increased resistance to a 361 McClure R, Mai M, Lasho T. Validation of two clinically useful farnesyltransferase inhibitor by N-cadherin expression in Bcr/Abl- assays for evaluation of Jak2 V617F mutation in chronic P190 lymphoblastic leukemia cells. Leukemia 2007; 21: myeloproliferative disorders. Leukemia 2006; 20: 168–171. 1189–1197. 362 Melzner I, Weniger MA, Menz CK, Mo¨ller P. Absence of the Jak2 372 Tagliafico E, Tenedini E, Manfredini R, Grande A, Ferrari F, V617F activating mutation in classical Hodgkin lymphoma Roncaglia E et al. Identification of a molecular signature and primary mediastinal B-cell lymphoma. Leukemia 2006; 20: predictive of sensitivity to differentiation induction in acute 157–158. myeloid leukemia. Leukemia 2006; 20: 1751–1758.

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