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Leukemia (2011) 25, 1080–1094 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu

REVIEW Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy LS Steelman1,2, RA Franklin1, SL Abrams1, W Chappell1, CR Kempf2, J Ba¨secke3, F Stivala4, M Donia4, P Fagone4, F Nicoletti4, M Libra4, P Ruvolo5, V Ruvolo5, C Evangelisti6, AM Martelli6,7 and JA McCubrey1 1

Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA; Department of Physics, East Carolina University, Greenville, NC, USA; 3Department of Medicine, University of Go¨ttingen, Go¨ttingen, Germany; 4Department of Biomedical Sciences, University of Catania, Catania, Italy; 4Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX, USA; 6Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell’Apparato Locomotore, Universita` di Bologna, Bologna, Italy and 7IGM-CNR, Sezione di Bologna, C/o IOR, Bologna, Italy 2

The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/ MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health. Leukemia (2011) 25, 1080–1094; doi:10.1038/leu.2011.66; published online 15 April 2011 Keywords: resistance; therapeutic sensitivity; targeted therapy; Ras; Raf; ERK

Introduction The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway has key roles in the transmission of proliferative signals from membrane-bound receptors.1–12 Mutations can occur in the genes encoding pathway constituents (for example, Ras and Raf), upstream receptors (for example, Kit, Fms and Fms-like tyrosine kinase (Flt)-3) or chromosomal translocations (for example, BCR–ABL and TEL–platelet-derived growth factor receptor (PDGFR)), which activate this pathway. Chemotherapeutic drugs Correspondence: Dr JA McCubrey, Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, 600 Moye Boulevard, 5th Floor Brody Building 5N98C, Greenville, NC 27858, USA. E-mail: [email protected] Received 14 January 2011; revised 13 February 2011; accepted 10 March 2011; published online 15 April 2011

frequently used in leukemia therapy often activate this pathway.1,2 This pathway relays this information through interactions with various other proteins to the nucleus to control gene expression.1–12 This review will discuss how these pathways may be aberrantly regulated in leukemia and contribute to therapeutic sensitivity/resistance and in some cases poor prognosis.4–5,13 Inhibition of Ras (or Ras-related molecules), Raf, MEK and ERK may prove useful in leukemia treatment. These observations have propelled the pharmaceutical industry to develop inhibitors that target key components of this pathway and many have been evaluated or are currently being evaluated in clinical trials. The Ras/Raf/MEK/ERK signaling pathway consist of a kinase cascade that is regulated by phosphorylation and de-phosphorylation by specific kinases, phosphatases as well as GTP/GDP exchange proteins, adaptor proteins and scaffolding proteins. An overview of this pathway and common sites where mutated upstream receptors and products of chromosomal translocation may activate the pathway is presented in Figure 1. The sites of intervention of signal transduction inhibitors are also shown in this diagram. The membrane localization of these components is often critical for their activity, although some members of these pathways can function in other cellular regions (for example, mitochondrion (Raf-1), nucleus (ERK)). Indeed, one emerging observation in ERK1/2 signaling is the realization that this pathway generates specific biological responses dependent on where in the cell the signal originates. Ras subcellular localization can determine substrate specificity through distinct utilization of scaffold proteins.14 This substrate selectivity is governed by the participation of different scaffold proteins that distinctively couple ERK1/2, activated at defined subcellular domains, to specific substrates. Clearly, the subcellular localization of pathway components and the presence of various adaptor and scaffolding molecules are critical for the activity of these pathways. The localization of various signaling molecules also becomes important in determining the effects of certain inhibitors. The regulation and function of this pathway will be concisely reviewed as well as the effects of genetic mutations in these pathways that are important in human cancer and leukemia.

Overview of the Ras/Raf/MEK/ERK pathway After growth factor/cytokine/mitogen stimulation of the appropriate (cognate) receptor, a Src homology 2 domain containing protein (Shc) adaptor protein becomes associated with the

Roles of Ras/Raf pathway in therapeutic sensitivity LS Steelman et al

1081 Inhibitors in clinical trials or in therapy

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Figure 1 Overview of the Ras/Raf/MEK/ERK pathway and potential sites of therapeutic intervention with small molecule membranepermeable inhibitors. The Ras/Raf/MEK/ERK pathway is regulated by Ras, as well as various upstream growth factor receptors and nonreceptor kinases. The downstream transcription factors regulated by this pathway are indicated in diamond shaped outlines. This drawing depicts a relative common, yet frequently overlooked phenomenon in human cancer, autocrine transformation. Sites where various small molecule inhibitors suppress this pathway are indicated by black octagons. Drugs that have been evaluated to suppress this pathway (many in clinical trials, see below) are indicated in open boxes. Drugs that have been approved to treat cancer patients (not necessarily in leukemia patients) include: Sutent, Nexavar, Imatinib, Nilotinib, and Dasatinib. Drugs that have been in clinical trials to treat cancer patients (not necessarily in leukemia patients and not necessarily are they continuing to be evaluated in clinical trials) include: Midostaurin, Semaxanib, Lestaurtinib, Tandutinib, Zarnestra, Sarasar, PLX-4720, RAF265, Selumetinib, XL-518, RDEA119 and Bosutinib. GF, growth factor; GFR, growth factor receptor.

C-terminus of activated specific growth factor receptor (for example, FMS, Flt-3, PDGFR, insulin-like growth factor-1 receptor and many others).3–6 Shc recruits the growth factor receptor-bound protein 2 and the son of sevenless homolog protein, resulting in the loading of membrane-bound Ras with GTP.15 Ras can also be activated by growth factor receptor tyrosine kinases (RTK), such as insulin-like growth factor-1 receptor, via intermediates like insulin receptor substrate proteins that bind growth factor receptor-bound protein 2.15,16 Ras:GTP then recruits Raf to the membrane where it becomes activated, likely via a Src-family tyrosine (Y) kinase.2,17–19 At this point, we will be somewhat generic, although it should be pointed out that both Ras and Raf are members of multi-gene families and there are three Ras members (Ki-Ras, N-Ras and Ha-Ras)15 and three Raf members (B-Raf, Raf-1 (a.k.a C-Raf) and A-Raf).18,19 Raf is

responsible for serine/threonine (S/T) phosphorylation of MEK1.20 MEK1 phosphorylates ERK1 and 2 at specific T and Y residues.21 Activated ERK1 and ERK2 serine/threonine kinases phosphorylate and activate a variety of substrates, including p90 ribosomal six kinase-1 (p90Rsk-1).22 ERK1/2 has many downstream and even upstream substrates (see below). The number of ERK1/2 targets is high (460). Thus, suppression of MEK and ERK activities will have profound effects on cell growth. Downstream of ERK is p90Rsk-1. p90Rsk-1 can activate the cAMP response element-binding protein transcription factor, which can influence gene expression.22 Activated ERK can also translocate to the nucleus and phosphorylate additional transcription factors, such as Elk-1, cAMP response elementbinding protein, Fos and globin transcription factor 1 and others,23 which bind promoters of many genes, including growth factor and cytokine genes that are important in promoting growth and preventing apoptosis in hematopoietic cells. Thus, we have described in a simple fashion how the Ras/Raf/MEK/ERK cascade can transmit a signal from the cell membrane to the nucleus. Under certain circumstances, aberrant regulation of this pathway can contribute to abnormal cellular proliferation, which may lead to many abnormalities including: abrogation of cytokine dependence, secretion of autocrine cytokines, leukemic transformation and drug resistance.4,5,10–12,24–31 Activated ERK can also phosphorylate B-Raf, Raf-1 and MEK1, which alter their activity (Figure 1). Depending on the site phosphorylated on Raf-1, ERK phosphorylation can either enhance32 or inhibit33 Raf-1 activity. In contrast, when B-Raf34 or MEK1 (ref. 35) are phosphorylated by ERK, their activity decreases. ERK can also exert negative feedback by interfering with Ras activation by phosphorylating son of sevenless.36 These phosphorylation events serve to alter the stability and/or activities of the proteins. It is important that the reader realizes that certain phosphorylation events either inhibit or repress the activity of the affected protein. This often depends on the particular residue phosphorylated on the protein, which can confer a different configuration to the protein or target the protein to a different subcellular localization that may result in its proteasomal degradation. Furthermore, as previously mentioned, certain phosphorylation events will actually serve to shut off or slow down the pathway. Thus, protein phosphorylation by the Ras/Raf/MEK/ERK pathway is a very intricate process, which serves to finely tune the signal often originating either from a growth factor or mitogen. There are many phosphatases that remove key phosphates on molecules in this pathway.37 These include protein phosphatase 1 (PP1), PP2a and PP5. PP1 and/or PP2a remove the phosphate from S259 on Raf-1. PP5 removes the phosphate from S338 on Raf-1.38 The activity (velocity) of the phosphatases has been proposed to be important in carcinogenesis and interactions with other signaling pathways.39 There are also various scaffolding proteins that regulate the activity of this pathway. These include: Raf kinase inhibitory protein (RKIP), kinase suppressor of Ras, 14-3-3 and Mp-1.40–43 Many of these scaffolding proteins serve to modulate the activity of key components. Some scaffolding proteins (for example, Mp-1) serve to promote a complex between MEK and ERK to enhance signal transduction. In contrast, in some cases they may prevent activation (for example, RKIP), or keep the complex in an inactive state. The scaffolding proteins may be phosphorylated by other proteins (for example, ERK), which promote their removal from Raf-1 and result in enhanced signaling, which in some cases may promote tumor progression and metastasis.42,43 Leukemia


1082 Rationale for targeting the Ras/Raf/MEK/ERK pathway to improve leukemia therapy Effective targeting of signal transduction pathways activated by mutations, gene amplification or various leukemia therapies may be an appropriate approach to limit leukemia growth and drug resistance. The Ras/Raf/MEK/ERK pathway can be activated by mutations/amplifications of upstream growth factor receptors. The abnormal production of growth factors can frequently result in receptor activation, which in turns activates the Ras/Raf/MEK/ERK cascade (Figure 1). Furthermore, chemotherapeutic drugs used in leukemia therapy may induce the Ras/Raf/ MEK/ERK cascade, which may contribute to drug resistance (Figure 2).

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Doxorubicin (a.k.a. adriamycin) is a commonly prescribed antileukemia drug. Doxorubicin exerts its chemotherapeutic effects through multiple mechanisms. One mechanism is through its interactions with DNA and inhibition of topoisomerase II.4 The other mechanism of action is due to the generation of reactive oxygen species (ROS) that occurs via the interaction of doxorubicin with iron.1,2,4,44,45 Doxorubicin treatment results in the intracellular generation of superoxide anion, hydrogen peroxide and hydroxyl radicals.1,2,44,45 ROS appear to be important for some of the therapeutic effects of doxorubicin as scavenging oxygen radicals using anti-oxidants decreases the ability of doxorubicin to induce apoptosis.1,2,4,44,45 Although ROS are important for some of the activities of doxorubicin they are also the cause of some of the undesirable side effects of this drug1,2,4,44,45 (see Figure 2). ROS induce activation of Ras/Raf/MEK/ERK, c-Jun N-terminal kinase, p38, big mitogen-activated protein (MAP) kinase/ERK5 and phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR) signaling pathways. Oxidative stress-induced ERK1/2 activation is reported in a variety of cell types.1,10,45 In some cases, ROS can act directly on cytokine and growth factor receptors and induce the Ras/Raf/MEK/ERK signaling pathway.1,10,45 ROS can induce the ligand-independent activation of some cytokine and growth factor receptors and a subsequent increase in Ras and Raf/MEK/ERK activity.1,10 ROS are also known to inhibit PPs.1,4,45 Inhibition of phosphatase activity can result in activation of the Ras/Raf/MEK/ ERK signaling pathway.1,4 The Ras/Raf/MEK/ERK kinase signaling cascade can be activated at multiple points by ROS. Thus, this pathway is important in drug resistance. Targeting this pathway may be a novel therapeutic approach for drug resistant leukemia, which is often cross-resistant to multiple chemotherapeutic drugs. ROS can also result in increases in intracellular Ca2 þ , but ROS are also able to activate the calcium calmodulin-dependent kinases (CaM-Ks) in the absence of increases in intracellular Ca2 þ .45 CaM-KI and CaM-KII are expressed in many tissues, whereas the expression of CaM-KIV is more restricted. Multiple genes encode different isoforms of CaM-KII, which are designated CaM-KII-a, -b, -d and -g.45 Maximal activity of CaM-KI, CaM-KII and CaM-KIV requires phosphorylation. The mechanisms by which these enzymes are phosphorylated differs.45 CaM-KII undergoes autophosphorylation whereas CaM-KI and CaM-KIV are phosphorylated by CaM-KK. Once phosphorylated, these kinases retain catalytic activity even in the absence of increased intracellular Ca2 þ . PP1 and PP2A

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Figure 2 Induction of the CaM-K, Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/ mTOR and p53 pathways by chemotherapy. Chemotherapeutic drugs such as doxorubicin can generate ROS, which in turn can induce the CaM-K cascade and can result in activation of both the Ras/Raf/MEK/ ERK and Ras/PI3K/PTEN/Akt/mTOR pathways. Inappropriate or prolonged stimulation of these pathways can result in altering gene expression leading to chemotherapeutic drug resistance. In addition, ROS can mobilize the ATM/p53 pathway, which in turn can have effects on the DNA damage response, DNA repair and lead to gene mutations, which further contribute to chemotherapeutic drug resistance. Induction of p53 by chemotherapeutic drugs can also induce the expression of the discoidin domain receptor (DDR), which can result in Ras activation and subsequent stimulation of the Raf/ MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. Akt can phosphorylate and inactivate murine double minute 2 (MDM2), preventing its effects on p53 stability.

cleave the phosphate group from CaM-KII and CaM-KIV rendering them inactive.45 Inactivation of these phosphatases is one mechanism by which ROS can activate these kinases.45 ROS and Ca2 þ /calmodulin can activate the CaM-K cascade, which in turn can activate the Raf/MEK/ERK and PI3K/PTEN/Akt/ mTOR cascades, often via Ras (see Figure 2). An illustration of the effects of induction of Ras/Raf/MEK/ERK by leukemia therapies on cell cycle and apoptotic regulatory molecules and protein translation is presented in Figure 3. In the subsequent sections, we will discuss how the Ras/Raf/MEK/ ERK pathway may be activated by mutations as well as chemotherapeutic drugs used in leukemia therapy.

Mutations affecting the Ras/Raf/MEK/ERK pathway in leukemia Mutations that affect activation of the Ras/Raf/MEK/ERK pathway in leukemia have been frequently detected. In acute myeloid


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Figure 3 Induction of the Ras/Raf/MEK/ERK pathway after leukemia therapy and subsequent effects on cell cycle progression, survival pathways and protein translation. After chemotherapy or radiotherapy there can be activation of signaling pathways, which can actually promote cell survival and may lead to therapy resistance. Chemotherapeutic drug treatment (shown in irregular black oval) frequently results in the induction of ROS (shown in black square). ROS can induce the calcium calmodulin kinase (CaM-K) cascade that can induce Ras, which can subsequently activate both the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades (most components of the two cascades, which promote signaling are show in white ovals, transcription factors are shown in white diamonds). Induction of the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways can result in the activation of many survival pathways, and regulate both cell cycle progression as well as protein translation. An emerging field in leukemia therapy is the role of these pathways in regulation of protein translation. These two pathways control the assembly of the eIF4 translation apparatus, downstream of mTORC1, which regulates the translation of many ‘weak’ mRNAs that are often involved in cell survival (for example, Mcl-1, c-Myc and vascular endothelial growth factor (VEGF)). Many of the phosphorylation events on apoptotic regulatory molecules such as Bad, Bim and caspase 9 may affect the activity or stability of the proteins, which can alter their subcellular distribution and target them to proteasomes or result in their degradation. These molecules are depicted in black as they are inactivated or degraded after phosphorylation by the Ras/Raf/MEK/ ERK and PI3K/PTEN/Akt/mTOR pathways. In contrast, some molecules such as Mcl-1 may be stabilized by phosphorylation by these cascades, although there are other pathways (for example, c-Jun N-terminal kinase (JNK)), which result in their inactivation. Also Raf-1 can be localized to the mitochondrial membrane and affect apoptotic regulatory molecules such as Bcl-2, Bcl-XL and Bad. Some of the phosphorylation events mediated by Akt actually serve to inhibit the activities of key proteins such as the Foxo transcription factors, the Tuberous sclerosis protein (TSC1) and (TSC2) tumor-suppressor proteins and the murine double minute (MDM2) ubiquitin ligase (depicted in black ovals). MDM2 serves to regulate p53 protein stability by ubiquitination, however, when it is phosphorylated by Akt it is inactivated. Moreover chemotherapeutic drugs and radiotherapy can induce the ataxia telangiectasia mutated (ATM) protein shown in a black oval, which can in turn phosphorylate and regulate p53. p53 can have complex positive and negative effects on cell growth (depicted in white diamond); it can regulate the expression of p21 cyclindependent kinase inhibitory protein-1 (p21Cip1), which controls cell cycle progression. p53 can also control the transcription of genes such as Puma, Noxa and Bax, which are involved in apoptosis (all of these molecules are shown in black ovals, as they tend to inhibit cell cycle progression or promote apoptosis). Alternatively, p53 can regulate the expression of the discoidin domain receptor (DDR) (shown in white oval), which in turn can regulate Ras and stimulate activation of the Ras/MEK/ERK pathway. Both Akt and ERK can phosphorylate p21Cip1 that alters its activity and ability to inhibit cell growth (shown as black phosphorylation sites) and subsequently influence cell growth and therapy resistance. p27Kip1 can also be phosphorylated by both Akt and ERK; however, the effects of these phosphorylation events are unclear. Akt phosphorylation of p27Kip1 may result in its cytoplasmic localization, while ERK phosphorylation of p27Kip1 may result in elevated levels of the protein. Hence, phosphorylation of proteins by ERK and Akt can have dramatic effects on cell proliferation and contribute to the therapy resistance of leukemias. This figure serves as an introduction as to how activation of these pathways by chemotherapy and radiotherapy may contribute to therapeutic resistance in leukemia, however, there are numerous other important proteins also regulated by these pathways, which contribute to therapy resistance.

Leukemia


1084 leukemia (AML) and myelodysplastic syndrome (MDS), these are often called class I mutations as they are believed to stimulate proliferation, whereas class II mutations consist of mutations (often chromosomal translocations) of transcription factors and the novel chimeric transcription factors prevent differentiation of the cells. In some cases, there are mutations/ gene rearrangements, which result in receptor gene activation (for example, FLT3, KIT, PDGFR and other receptors).46,47 The FLT3 gene is mutated in approximately 20–25% of AML patients48 and in o5% of MDS patients. It has been observed that one-third of MDS patients acquire mutations at either FLT3 or NRAS during the evolution into AML.49 Another study examined the mutations present in the NRAS, FLT3, KIT and MLL genes in 381 patients with MDS and 4130 patients with AML.50 Mutations at NRAS, FLT3, KIT and MLL were reported to be more frequent in secondary (s) AML compared with primary AML and MDS. The incidence of NRAS mutations increased from about 6.3% in MDS to about 12% in sAML. KIT816 mutations are rarer, in less than 2% of the patients examined. The presence of FLT3 length mutations increased in AML patients compared with MDS patients. Detection of the FLT-length mutation at initial diagnosis of MDS is associated with leukemic transformation and poorer prognosis.51 In addition, in this large study, the incidence of FLT3-length mutation was higher in relapsed AML than in all other classes of AML and MDS examined, documenting the significance of FLT3-length mutation in AML progression. NRAS mutations were among the most frequent mutation detected in MDS. These investigators demonstrated that NRAS mutations were associated with karyotype evolution, either the acquisition of monosomy 7 occurring during MDS transformation and associated with a poorer survival. In childhood acute lymphoblastic leukemia, there are mutations in FLT3, Ras (NRAS and KRAS), BRAF and genes encoding proteins, which modulate pathways activity (for example, mutations in the Shp2 phosphatase, a.k.a. PTPN11).52–55 It has been recently estimated that 35% of an unselected cohort of childhood acute lymphoblastic leukemia patients contained mutations in NRAS/KRAS2/PTPN11 or FLT3.56 These mutations were mutually exclusive, an event that the investigators state the importance of these mutations in leukemogenesis. Furthermore, the investigators observed an incidence of 21% mutations at this pathway in relapsed patients. In contrast, in these and other studies, the investigators did not observe mutations at BRAF.57 Overall, BRAF is not frequently mutated in childhood acute lymphoblastic leukemia, although there are some reports documenting activating BRAF mutations.54,58 Furthermore, many chromosomal translocations (for example, BCR–ABL, TEL–PDGFR) will activate the Ras/Raf/MEK/ERK pathway. Thus, there is a strong rational for developing inhibitors, which suppress Raf/MEK/ERK pathway activation.

Mutations in therapy-induced AML and MDS There are mutations in RAS, BRAF, CRAF and PTPN11 detected in t-AML and t-MDS. Previously, these patients were often treated with alkylating agents, topoisomerase II inhibitors or radiotherapy.59–61 An additional study has documented mutated alleles of CRAF in t-AML.62 These t-AMLs arose after chemotherapeutic drug treatment of breast cancer patients. The mutated CRAF genes were transmitted in the germ line, thus, they were not spontaneous mutations in the leukemia, but they may be Leukemia

associated with the susceptibility to induction of t-AML in the breast cancer patients studied. Also some cases of childhood acute lymphoblastic leukemia have arisen in cardio-faciocutaneous syndrome patients who have a germline BRAF mutation.63

Novel RAF mutations in human cancers Recently, it has been determined that there are genetic translocations of BRAF and CRAF in prostate and gastric cancer and melanoma.64,65 These translocations were detected using paired-end transcriptome sequencing to screen ETS rearrangement-negative prostate tumors for targetable gene fusions. They are currently believed to be associated with later stages of the cancer. Although these chromosomal translocations are rare, they offer a new mode of attacking their activity as they are sensitive to Raf and MEK inhibitors in in vitro studies. Whether similar translocations will be detected in leukemia remains to be determined.

FLT3 mutations and leukemia therapy Inhibitors (for example, midostaurin, sutent, semaxanib, lestaurtinib, tandutinib and others), which target Flt-3 have been developed and examined in the treatment of leukemia patients with FLT3 mutations.46 Some of these inhibitors may have additional targets besides Flt-3 (for example, protein kinase C, vascular endothelial growth factor receptor, Kit, PDGFR and others). Some of these inhibitors may induce Bim that promotes apoptosis.46 Sorafenib is a multi-kinase inhibitor, which also inhibits Flt-3. Sorafenib has many targets including Flt-3, Raf, Kit and PDGFR.66–67 Previously, it was shown that sorafenibinduced apoptosis in AML cells through Bim.68

RKIP as a therapeutic target in cancer including leukemia RKIP was originally identified as a scaffolding protein, which inhibits the function of Raf-1.43,69,70 Overexpression of RKIP inhibits MEK, ERK and Ap-1 transcription factor activity. Similarly, inhibition of RKIP stimulates MEK, ERK and Ap-1 activity. Phosphorylation of RKIP on S153 by protein kinase C71 or ERK72 results in dissociation of RKIP from Raf-1 and relief of RKIP’s suppressive effects on Raf-1. RKIP can bind to the N-terminal region of Raf-1 preventing binding of MEK to Raf-1 and downstream activation.73,74 Although RKIP can regulate ERK activation, ERK can also regulate RKIP expression, potentially via phosphorylation on S99 and indirectly by regulating the transcription factor Snail by phosphorylation, which can suppress RKIP transcription.75,76 RKIP has been subsequently shown to interact with other proteins (for example, nuclear factor-binding immunoglublin-k chain enhancer in B cells and inhibitory kB kinase) and be regulated by other proteins (for example, Snail), which are involved in regulation of the epithelial–mesenchymal transition.77,78 RKIP can also antagonize nuclear factor-binding immunoglublin-k chain enhancer in B cell activation in response to tumor necrosis factor-1 and interleukin-1b stimulation.78–80 RKIP expression is characterized as a metastasissuppressor protein. The transcription factor Snail is a repressor of RKIP transcription in metastatic prostate cancer.78 Furthermore, ERK can phosphorylate and regulate Snail expression, thus documenting the complicated feedback loops between Snail and Raf/MEK/ERK expression. Low levels of RKIP are frequently


1085 observed in metastatic prostate and breast cancer samples whereas higher levels are observed in non-metastatic cells. Increased expression of RKIP can sensitize therapy-resistant cells to various therapeutic approaches (for example, tumor necrosis factor-related apoptosis-inducing ligand and chemotherapeutic drugs).81 In contrast, suppression of RKIP renders therapysensitive cells, therapy-resistant.82 Proteasome inhibitors can suppress the therapy resistance by downregulation of nuclear factor-binding immunoglublin-k chain enhancer in B cell and Snail and increased expression of RKIP.83 Although there have not been many studies published regarding RKIP and leukemia, it was recently observed that RKIP had a role in some cases of t-AML containing mutant CRAF. Blast cells from patients with the CRAF (Raf-1) germline mutations also had loss of RKIP.84 The importance of RKIP was determined by transfection experiments with either small interfering RNA directed against RKIP or expression vectors overexpressing RKIP.84 The levels of RKIP were determined to influence the levels of CRAFmediated transformation as high levels of RKIP suppressed CRAF-mediated transformation, while low levels enhanced CRAF-mediated transformation.84

Control of apoptotic regulatory molecules by the Ras/Raf/ MEK/ERK pathway The Ras/Raf/MEK/ERK pathway regulates the activity of many proteins involved in apoptosis (Figure 3). Deregulated expression of apoptotic regulatory molecules can lead to drug resistance.10–12,30,31 ERK phosphorylates transcription factors that influence the transcription of the Bcl-2 family of genes as well as other important genes involved in the regulation of apoptosis.27,85 Furthermore, increased expression of Bcl-2 and Bcl-XL and decreased expression of Bax is observed in some drug-resistant leukemias.86–89 Many of the effects of the Ras/Raf/ MEK/ERK pathway on apoptosis are mediated by ERK phosphorylation of key apoptotic effector molecules (for example, Bcl-2, Mcl-1, Bad, Bim, cAMP response element-binding protein, caspase-9 and many others).90–93 Certain Bcl-2 inhibitors (for example, Abt-737) may sensitize chronic lymphocytic leukemia and chronic myeloid leukemia cells to chemotherapy, implicating the Bcl-2 family members, which are sensitive to Abl-737 (for example, Bcl-2 and Bcl-XL but not Mcl-1) in their drug resistance.94–95 Bcl-2 inhibitors sensitize B lymphoma cells to rituximab, a chimeric monoclonal antibody, which targets CD20.96 Bcl-2 inhibitors also render various lymphoid malignancies susceptible to proteasome inhibitors.97 Resistance to the chemotherapeutic drug fludarabine, in some situations, may also be mediated by other Bcl-2 family members such as increased expression of Mcl-1, which is associated with a poor prognosis.98 This may be regulated by microRNAs. Some of the effects of the microRNAs are on the suppression of genes, such as PTEN and Bim.99 Development of specific Mcl-1 inhibitors is in progress based on the crystal structure of Mcl-1.100,101 Mcl-1 confers less protection against chemotherapy than Bcl-2 in certain experimental situations. This has been examined with Em-Bcl-2 and Em-Mcl-1 mice.102 This may be due to the effects of ERK as well as mTOR and their phosphorylation of the apoptotic molecules as well as regulation of protein translation (Figure 3). A key molecule downstream of the Ras/Raf/MEK/ERK cascade is the BH3-domain containing anti-apoptotic protein Bim. Bim’s activity is regulated by phosphorylation at different residues by ERK, Akt and c-Jun N-terminal kinase.92,103–105 When Bim is

phosphorylated by ERK and Akt, it is targeted for proteosomal degradation and also inhibits Bim’s interaction with Bax, a death executioner protein. In contrast, when Bim is phosphorylated by c-Jun N-terminal kinase, it has enhanced pro-apoptotic activity.106 Bim is also transcriptionally regulated by Foxo-3a, (a transcriptional factor), which is also a target of Akt. Normally, when Akt is active, it phosphorylates and inhibits the activity of Foxo-3a (Figure 3). On cytokine withdrawal of hematopoietic cells, Foxo-3a is not phosphorylated and enters the nucleus and stimulates the transcription of genes, such as Bim and other, which results in apoptosis.107 Bim levels can also be regulated by epigenetic silencing. This may be important in the sensitivity of ALL cells to glucocortoids.108 The glucocorticoid resistance observed in the xenografts and patient biopsies in this study correlated with decreased histone H3 acetylation. The investigators demonstrated that the histone deacetylase inhibitor vorinostat relieved Bim repression and also exerted anti-leukemic effects when combined with dexamethasone in vitro and in vivo. These studies suggest a new approach to overcome glucocorticoid resistance and improve therapy for high-risk pediatric ALL patients. Bim protein levels are also regulated by stromal cell interactions. When leukemia cells were attached to stroma and b1 integrin was activated, suppression of Bim expression and apoptosis occurred and drug resistance increased.109 Bim was targeted to the proteasome and degraded.109 Proteasomal inhibitors suppressed Bim degradation and rendered the cells sensitive to therapy. The investigators of this study suggested that b1 integrin-mediated stromal interactions with leukemia cells and subsequent Bim protein degradation may contribute to minimal residual disease. The tumor microenvironment can regulate drug resistance, perhaps due to the presence of cytokines, which stimulate anti-apoptotic Bcl-2 and other factors preventing prevent apoptosis.110–112

Regulation of Bim and Foxo3A by the Raf/MEK/ERK pathway BCR–ABL-transformed hematopoietic precursor cells have been shown to have low levels of Bim and to be refractory to the induction of apoptosis after cytokine withdrawal.113–114 BCR– ABL induces ERK activation and hence suppression of ERK by either imatinib or dasatinib results in prevention of Bim phosphorylation and Bim is not targeted to the proteosome and is active. Thus, these drugs are proposed to exert some of their inhibitory effects by induction or enhancement of Bim activity. Knockdown of Bim by small interfering RNA abrogates imatinib-induced cell death in chronic myeloid leukemia cells.115 Imatinib will induce Foxo-3a in BCR–ABL-transformed cells, which subsequently induces Bim transcription and apoptosis.116 Imatinib induces the transcription of Bim and Bmf and also induces posttranslational modifications of Bim and Bad favoring apoptosis. Bcl-2 inhibitors can overcome resistance to imatinib because of decreases in Bim and Bad levels.115 Mutant receptors such as Flt-3 may induce Foxo-3a inactivation, which can lead to resistance to Flt-3 inhibitors and result in a poor prognosis.51,117,118 Recently, it was demonstrated that Foxo-3a is inactive in AML cells and is localized in the cytoplasm.119 Treatment of AMLs with MEK and PI3K/Akt inhibitors did not result in the nuclear translocation of Foxo-3a where it could potentially induce cell cycle inhibitory and apoptotic genes. In contrast, it was shown that inhibitory kB kinase controlled Foxo-3A expression. Treatment of cells with the inhibitory kB kinase-specific Leukemia


1086 inhibitor NEMO resulted in the nuclear translocation of Foxo-3a and induced the expression of p21Cip-1 and Fas by inhibitory kB kinase.119

Regulation of the eIF4F translation complex by the Ras/Raf/ MEK/ERK pathway Certain mRNAs encoding genes implicated in survival (for example, Mcl-1, c-Myc, Bcl-XL, survivin, cyclin D, vascular endothelial growth factor, ornithine decarboxylase, matrix metalloproteinase 3 and -9) have 50 untranslated regions (UTRs) possessing tertiary structures that are difficult to translate and are hence considered weak mRNAs.120,121 These 50 UTR are G þ C rich and highly structured and can affect cellular transformation and metastatic progression. Efficient translation of these survival mRNAs requires the assembly of a translation complex, which attaches to the 50 UTR.122 eIF4E is a component of the eIF4F complex, which stimulates ribosome recruitment. This translation complex is comprised of eIF4E, the RNA helicase eIF4A and its activator eIF4B that serves to unwind the 50 UTR. Other proteins are also involved, eIF4G serves as a scaffold necessary for assembly of the complex and eIF2a is a negative regulator, which can block translation if it is phosphorylated by protein kinase R. eIF4E is the critical rate-limiting factor in the formation of the complex. Ectopic overexpression of eIF4E can transform certain cells in culture and cooperate with Em-Myc in the induction of lymphomatogenesis and induce chemotherapeutic drug resistance.123 However, overexpression of eIF4E, which is downstream of mTOR and also regulated by ERK does not confer sensitivity to the mTOR inhibitor rapamycin, in fact, the tumors are rapamycin-resistant. Furthermore, overexpression of eIF4E in cancers, which were previously rapamycin-sensitive made them rapamycin-resistant.123 Another kinase downstream in the Ras/Raf/MEK/ERK pathway that regulates the translation complex is p90Rsk-1, which phosphorylates ribosomal protein S6 (rpS6) at S235/236, the same site as p70S6K.124 Phosphorylation of rpS6 at these sites promotes its association with the 7-methyguanosine cap complex. eIF4B exerts a dominant role in recruiting the 40S ribosomal subunit to the mRNA. On mitogen stimulation, eIF4B is phosphorylated on S422 by p70S6K in a rapamycin-sensitive manner. It turns out that p90Rsk1 also phosphorylates eIF4B on the same residue.125 However, the phosphorylation by the two different kinases occurs by very different kinetics.125 Both p70S6K and p90Rsk-1 are members of the cyclic adenosine monophosphate kinase, cyclic guanosine monophosphate kinase, protein kinase C family (AGC) of protein kinases family, and require phosphotidylinositide-dependent kinase 1 phosphorylation for activation.125 These studies further demonstrate the importance of the Ras/Raf/MEK/ERK cascade in the translational machinery and also the redundancy in regulation of rpS6 and eIF4B phosphorylation, which is critical for the translation of many critical RNAs involved in growth control. The ability of eIF4e to enter the eIF4F complex is regulated by the eIF4E-binding proteins, which are regulated by mTOR phosphorylation. On phosphorylation of 4-BP by mTOR, eIF4E disassociates from the eIF4E-binding proteins and eIF4E can enter the eIF4F complex and stimulate transcription of the survival mRNAs with the unique 50 UTRs. Recently, it has been shown that cyclopenta[b]benzofuran flavagines such as silvestrol can modulate eIF4A activity and inhibit translation initiation.125 Silvestrol and related compounds may be able to enhance chemosensitivity in cancers, which grow in response to deregulated PTEN and downstream eIF4E.125 Leukemia

One of the key targets of the eIF4F complex is Mcl-1.126 High levels of Mcl-1 and eIF4E are detected in myelomas, which may contribute to their drug resistance.127 ERK and p38MAPK can increase phosphorylation of eIF4E via MAP kinase-interacting serine/threonine-protein kinase1/2 (MNK1/2). ERK can phosphorylate and activate MNK1/2.128 Activated MNK1 can phosphorylate and activate eIF4E on S209.129 Constitutively activated MNK1 can induce lymphatogenesis in a fashion similar to eIF4E.130–131 MNK1/2 is reported to be dispensable for normal mammalian development.132 Recent studies have indicated that combined MNK1 and MNK2 deficiency (in mouse crosses lacking both genes) delays tumor progression in both PTEN-deficient T cells and in glioblastoma.133 Thus, MNK1/2 is potentially an important target for cancer therapy and drug resistance. The ras homology protein enriched in brain (Rheb) is a GTPase and an activator of mTOR. Rheb is highly expressed in some human cancers.134–138 Increased Rheb expression is associated with a poor prognosis in breast and head and neck cancers.138 Rheb activity is dependent on farnesylation. The effects of enhanced expression of Rheb on the induction of lymphomas in the Em-Myc mouse system were examined.139,140 Introduction of Rheb into the Em-Myc mouse model enhanced the development of lymphomas. These lymphomas were more resistant to chemotherapeutic drugs such as doxorubicin. Rheb expression induced cellular senescence and treatment with rapamycin prevented the induction of senescence.139 In contrast, c-Myc expression blocked the induction of senescence. However, the combination of c-Myc expression and Rheb, prevented the induction of senescence induced by Rheb and the apoptosis induced by c-Myc, which may explain the increased incidence of lymphomas in the Rheb/EmMyc mice than in Em-Myc mice.140 This group has shown that the abilities of Ras, Akt and Rheb to activate cellular senescence are dependent at least in part by their ability to activate mTOR. This effect may be due to eIF4B activation as eIF4B also induces cellular senescence. MEK inhibitors also inhibit cellular senescence in some systems.139 Thus, in some scenarios mTOR and MEK inhibitors may promote cell growth by preventing cellular senescence. Similar to previous experiments performed with introduction of eIF4E into Em-Myc mice, the Rheb-Em-Myc mice also expressed high levels of the Mcl-1 protein. Lymphomas that overexpress Rheb are sensitive to both farnesyl transferase inhibitors and rapamycin.140 The effects of farnesyl transferase inhibitors on PTEN-deficient tumor cells are dependent on functional Rheb activity. In summary, in some cases, Rheb can be an oncogenic regulator of mTORC1 and eIF4E and Rheb is a direct target of farnesyl transferase inhibitors in cancer. By performing high-throughput screening assays, 4EGI-1 was identified as an eIF4E-binding protein-1 mimetic and a potent inhibitor of the interactions between eIF4E and eIF4G. 4EGI-1 has been shown in some systems to inhibit the growth of BCR–ABL-transformed Ba/F3 cells but not the parental interleukin-3-dependent cells line (Ba/F3).141 The specificity of 4EGI-1 was also examined on AML blasts and normal CD34 þ hematopoietic precursor cells and it was demonstrated that 4EGI1-induced apoptosis in the AML blasts but had much less effects on the differentiation and survival of normal CD34 þ cells.142 The antiviral drug ribavirin can block the binding of eIF4E to mRNA.143–144 The effects of this compound were examined in some patients with relapsed M4/M5 AML who were no longer eligible for chemotherapy. Although the number of patients in this preliminary trial were low (n ¼ 11), one complete and two partial responders were observed.145


Pecularities of Raf kinase inhibitors Most of the described experiments below have been performed in non-hematopoietic cells. However, they are presented in this review as Raf and MEK have also been considered as targets in leukemia and this information should be kept in mind when considering targeting this pathway. It has been known for quite some time now, that treatment with certain inhibitors (for example, Raf inhibitors), can result in hyper-activation of wildtype (WT) Raf.8,146–148 It has recently become clear that it will be essential to determine the genetic status at both BRAF and RAS before treatment with B-Raf selective inhibitors proceeds.149 Class I B-Raf inhibitors (active conformation inhibitors) such as PLX4720 and 885-A (a close analog of SB590885) will inhibit BRAF mutants, however, these ATP-competitive B-Raf inhibitors will not inhibit WT BRAF or mutant RAS. In fact these B-Raf inhibitors can activate Raf-1 in these cells in the presence of active Ras. 885-A could induce B-Raf binding to Raf-1 and to a lesser extent PLX-4720 also could when the ERKmediated negative feedback loop on B-Raf was inhibited with a MEK inhibitor. These binding events were determined to be dependent on the presence of oncogenic or growth factor activated Ras, which may be required for translocation from the cytoplasm to the membrane and assembly into the signaling complex. Thus, this has therapeutic implications as in cells with mutant RAS, if the patient is treated with certain B-Raf inhibitors, B-Raf can bind and activate Raf-1 and promote the oncogenic pathway. In fact, even kinase-dead BRAF mutations, which are found in human cancer, can dimerize with Raf-1, when stimulated by the mutant Ras protein and activate the Raf/ MEK/ERK cascade. Also kinase-dead BRAF mutants form a constitutive complex with Raf-1 in mutant RAS D04 cells, which activated MEK constitutively. This activation was independent of inhibitor treatment. Thus, it is inactive B-Raf that forms a complex with Raf-1 and results in MEK activation. Additional experiments were also performed with various conditional mouse mutants that demonstrated that only inactive B-Raf-bound Raf-1 in a mutant Ras background. These studies provide a model of how resistance to these inhibitors could develop in patients treated with these B-Raf selective inhibitors. Such inhibitor treatment could result in additional cancers if the drug treatment select for cells in the patients, which have either a RAS mutation or a mutation in an upstream receptor or signaling molecule that activates Ras. Hence, B-Raf selective inhibitors should not be administered to patients with RAS mutations or mutations in upstream signaling components. Also these results indicate a model where cells could become resistant to pan Raf inhibitors. If there was a gatekeeper mutation in Raf-1, which prevented inhibitor binding, the mutant B-Raf could stimulate the mutant Raf-1 to allow inhibitor-resistant growth. Pan-Raf inhibitors such as ZM336372, and RAF265 also induced B-Raf binding to Raf-1 but did not activate ERK. This is likely because they also inhibit Raf-1. Sorafinib induced B-Raf binding to Raf-1 and Raf-1 activation. However, sorafinib inhibited ERK activity. Other studies on B-Raf inhibitors documented that other B-Raf inhibitors could induce Raf activity and that Raf activity can be stimulated in B-Raf-null tumor lines though Raf-1 dimerization.150 However, activation of MEK/ERK by the Raf inhibitors was inhibited in cells containing the CRAF gatekeeper mutation, which cannot bind the Raf inhibitors. Finally, a third study suggested that the binding of the Raf inhibitors to one Raf protein results in the transactivation of the other Raf protein in

the dimer.151 These observations are dependent on Ras being active.151 Overall these results document the complexity with Raf inhibitor therapy, clearly for B-Raf selective inhibitors to be therapeutic useful previous screening of patients for Ras mutations will be required, as well as perhaps additional screening during treatment. Otherwise resistance may develop and in some cases further stimulation of the Raf/MEK/ERK cascade.

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Genes involved in sensitivity and resistance to MEK inhibitors MEK inhibitors such as PD0329501 inhibit both MEK1 and MEK2 by locking the enzymes in a closed and catalytically inactive conformation.152 In MEK inhibitor-sensitive cells, MEK inhibitor treatment results in a dose-dependent decrease in activated ERK1,2 levels as well as cyclin D expression. In contrast, there is an increase in p27Kip1 levels, RB hyposphosphorylation and accumulation of cells in G1 phase of the cell cycle.153 By analyzing transcriptional pathway signatures, a panel of 18 genes has been determined to be important in terms of MEK addiction and response to the MEK inhibitor selumetinib (AZD6244, ARRY-142886).154 Importantly, this transcriptional signature was determined to be reproducible across different cell panels of diverse tumor origins, even when the profiling was performed in different laboratories using different technology platforms. The genes they determined to be transcriptional common events to MEK function are termed MEK-functionalactivation signature and include: dual-specificity phosphatases (DUSP4/6), sprouty homologue 2 (SPRY2), pleckstrin homologylike domain family A member 1 (PHLD1), all the above three genes were previously identified to be transcriptional targets of MEK/ERK and involved in the downregulation of the pathway. Other transcriptional targets in the signature included the ETS variant transcription factors ETV4, ETV5 and ELF1, and other MEK family members such as MAP3K3. Finally, some genes in the MEK inhibitor responsive signature were linked to regulation of MAPK singling, cell cycle and tumor progression were identified which included tribbles 2 (TRIB2) galectin 3 (LGALS3) and the transcription factors KANK1 (ANKRD15) and TS1. The investigators also identified a 13-gene signature of genes, which are involved in resistance to the MEK inhibitors when MEK is active other than phosphatidyl inositol 3 kinase catalytic subunit (PIK3CA). These genes were determined to be reproducibly predictive of resistance and entitled compensatory-resistance genes. Some of these genes have a common linkage to transforming growth factor-b signaling. Others are known to regulate signaling pathways and may offer alternative routes to cell proliferation (for example, Frizzled homolog 2 (FZD2)). Some genes may enhance cell survival and chemoresistance by controlling hypoxia and angiogenesis (serine protease SERPINE1), lysyl oxidase LOX, collagens (COL5A1 and COL1A2), cell cycle (GOG1 switch 2 (GOS2) proliferation/ apoptosis (cysteine-rich transmembrane BMP regulator 1 (CRIM1)) and immune evasion (CD274). Although most of these studies have been performed in non-hematopoietic cells, certain leukemias are known to be sensitive to MEK inhibitors although the genetic mechanisms for the sensitivities have not been as well elaborated. We present this information on MEK inhibitor sensitivity in non-hematopoietic cancers as an example of what may in the future be determined to result in MEK inhibitor sensitivity in leukemia. In a different genomic screening analysis, genes associated with either BRAF mutations and sensitivity to MEK inhibitors or Leukemia


1088 with RTK mutations that conferred resistance to MEK inhibitors were determined.155 The importance of this study is the formulation of the concept that different oncogenic mutations in genes in the same pathway can result in divergent sets of gene expression. Briefly, in cells with BRAF mutations, the cells express a panel of genes, which are MEK/ERK dependent and sensitive to MEK inhibitors. Whereas in cells with RTK mutations, the cells express a different panel of genes, which are MEK inhibitor insensitive. The investigators determined that Raf/MEK/ERK signaling is activated in BRAF-mutant cells and downregulated in RTK-mutant cells. In the BRAF-mutant cells, the Raf/MEK/ERK pathway was resistant to feedback downregulation by downstream phosphatases, whose transcription is sometimes dependent on activation of this pathway. This results in two different subtypes of cellular transformation originating from mutations at genes in the same pathway. In the BRAFmutant cells, they express elevated activated ERK and these cells are insensitive (or less sensitive) to negative feedback regulation by phosphatases as the mutant BRAF protein is resistant to the negative feedback initiated by activated ERK. In contrast, in the RTK-mutant cells, they express a different panel of genes and the Ras/Raf/MEK/ERK pathway is downregulated and sensitive to feedback regulation. This study identified a panel of 52 genes whose expression was dependent on MEK/ERK activity in BRAF-mutant (V600E) cells. Some of the genes identified included: cyclin D1 (CCN1), transcription factors (MYC, FOS, FOSL1, EGF1, IER3, ETV1, ETV5, EV4 and MAFF, which were previously identified as ERK-target genes. Interestingly, this study documented the importance of feedback regulation of the Ras/Raf/MEK/ERK pathway as some of the genes identified were phosphatases including: DUSP6, DUSP4, SPRY2, SPRY4 and SPRED2. The investigators also documented the presence of 36 genes in their profile, which had not previously been associated with Raf/MEK/ ERK signaling. Thus, in RTK-mutant cells, there is modest activation of the Ras/Raf/MEK/ERK pathway, which is regulated by phosphatases, whereas in the BRAF-mutant cells, there is a high level of activation of the Raf/MEK/ERK including the induction of phosphatase gene expression, however, the mutant BRAF is not subject to downregulation by ERK. Recently, it has been determined that certain mutations in MEK1 will cause resistance to both MEK and B-Raf selective inhibitors.156 By performing a MEK1 random mutagenesis screen in vitro, mutations in MEK1 were identified which conferred resistance to the MEK inhibitor AZD6244 (selumetinib). The complementary DNA library containing the MEK1 mutations were introduced into the MEK inhibitor-sensitive line A375 melanoma cells, which harbor the BRAFV600E mutation and then cultured the cells in the presence of MEK inhibitors (either selumetinib or CI-1040) for 4 weeks. Over 1000 MEK inhibitorresistant clones emerged, pooled and the MEK1 gene was characterized en masse by massively parallel sequencing. MEK inhibitor-resistant alleles segregated into two different classes, those with mutations either within or affecting the allosteric MEK inhibitor-binding pocket or those outside of the MEK inhibitor-binding pocket. Binding of the MEK inhibitors to the binding pocket prevents the structural reorganization of the MEK1 protein and generates a catalytically inactive MEK1 conformation. Arylamine MEK inhibitors lock MEK1 in a closed inactive conformation whereby the activation loop induces helix C to be rotated and displaced.157 However, the MEK1 mutants, which prevent MEK inhibitor-binding cause resistance by preventing this altered conformation of MEK. It has been known that treatment of cells with this class of MEK inhibitors results in the detection of activated MEK1/2 but inactivated Leukemia

downstream ERK1/2. The other class of MEK inhibitor-resistant mutants contained mutations present in two different regions of MEK1. Some were present in the N-terminal negative regulatory domain known as helix A (for example, Q56P) while others were present in a proline proximal to the C helix that abuts helix A (for example, P124S), which may enhance MEK1 activity. The mutations at Q56P and P124S in MEK1 are similar in biological consequences to the mutations detected in cardio-facio-cutaneous syndrome. The investigators concluded that resistance to MEK inhibitors can arise via reduction of drug binding (first group) or enhanced MEK1 activation (second group). The effects of these mutations on resistance to MEK inhibitors was confirmed by introducing the specific mutations into a MEK1 complementary DNA by side directed mutagenesis, introduction into A375 melanoma cells (normally sensitive to MEK inhibitors) and then determining the biochemical responses to MEK inhibitor treatment. The presence of a MEK1 mutation was determined in a melanoma patient sample from a patient treated with selumetinib, which had undergone relapse. This mutation occurred in P124L, which is functionally similar to the P124S mutation indentified by the cell line screening approach. The P124L mutation was only present in the patient’s post-relapse sample and not present in the pretreatment tumor sample. Melanoma cultures (M307) were derived from the relapse patient’s selumetinib-resistant melanoma from their left axillary lymph node. These cells contained the MEK1 P124L mutation and were indeed more resistant to selumetinib (GL50 exceed 2 mM) than treatment-naı¨ve melanoma cultures containing the BRAF V600E mutation (GL50 10–50 nM). The MEK1 mutation present in M307 cells derived from the melanoma patient treated with selumetinib who had undergone relapse was determined to confer resistance to the selective B-Raf inhibitor (PLX4720) as these cells were highly resistant to the B-Raf inhibitor. Thus, MEK1 inhibitor-resistant mutations may also confer cross-resistance to B-Raf inhibitors. Finally, these investigators determined that administration of B-Raf and MEK1 inhibitors simultaneously prevented the emergence of resistant clones suggesting that this might be a therapeutic option. Furthermore, the concept of dual targeting of MEK and PI3K pathways is arising because of the cross-regulation of many targets of this pathway (for example, transcription factors, apoptotic regulatory molecules and proteins involved in the regulation of protein translation).158–160

Mutations within the RTK/Ras/Raf/MEK/ERK pathway that alter sensitivity to MEK inhibitors Mutations at the BRAF, KRAS, EGFR genes or the chromosomal fusion between ALK and ROS are detected in approximately 50% of non-small cell lung cancer (NSCLC). NSCLC cells with mutations at BRAF where shown to be more sensitive to MEK inhibitors than NSCLC with mutations in EGFR, KRAS, or the chimeric fusion between ALK and ROS.158,161 This was determined by screening a large panel of cell lines (n ¼ 87) and tumors (n ¼ 916). These results support the hypothesis that mutations at BRAF are driver mutations, which are critical for the initiation of the tumor. In this study, cells with mutations at EGFR were resistant to MEK inhibitors. This may have resulted from the ability of EGFR to activate the PI3K/PTEN/Akt/mTOR pathway, which has some crucial overlapping targets as the Raf/ MEK/ERK pathway. As expected BRAF-mutant cells were not sensitive to EGFR inhibitors. Thus, NSCLC patients with BRAF mutations should not be treated with EGFR inhibitors and


1089 NSCLC patients with EGFR mutations should not be treated with MEK (or BRAF) inhibitors as the respective therapies would be ineffective. The investigators developed a mass spectrometry based genotyping method for the detection of hotspot mutations in BRAF, KRAS and EGFR as NSCLC patients, which harbor BRAF mutations should be treated differently than NSCLC patients with EGFR tumors. Interestingly, the investigators demonstrated that EGFR mutations are not necessarily associated with cigarette smoking in NSCLC, whereas BRAF mutations are associated with either current or former cigarette smoking. Mutations in neurofibromin-1 (NF1) or MEK1 may result in cells, which are dependent on MEK signaling and sensitive to MEK inhibitors. MEK1 mutations are observed at a low frequency in NSCLC.162

Mutations at PIK3CA confer resistance to MEK inhibitors in KRAS-mutant tumors Mutations at KRAS often confer sensitivity to MEK inhibitors. MEK inhibitor-sensitive tumors are usually WT at PIK3CA. Many tumors exist that have altered expression of both the Ras/Raf/ MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. These tumors will usually be resistant to MEK inhibitors. This was examined experimentally by using pairs of isogenic cell lines (HCT116 and DLD-1) with mutant KRAS and either WT or mutant PIK3CA.163 Cells containing mutant PIK3CA were insensitive to MEK inhibitors whereas the KRAS mutant cells, which had WT PIK3CA in these tumors were sensitive to MEK inhibitors. Clinically, there are tumors (for example, melanomas) that have mutations at KRAS or BRAF and also have mutations at PIK3CA or deletions at PTEN. For successful inhibition of cell growth in these tumors, inhibition of both pathways is necessary. However, not all MEK inhibitor-resistant cells have PIK3CA or PTEN mutations. In the study by Halilovic et al., elevated activated Akt levels were detected in the MEK inhibitor-resistant lines in the absence of PIK3CA and PTEN mutations, suggesting that Akt was activated by a PI3K/PTEN-independent mechanism. Clearly, there are additional genes that confer inhibitorresistance. These results have suggested that these two pathways have a common set of downstream components. One downstream component is eIF4E-binding protein-1.160 Another important downstream target that both of these pathways converge on is cyclin D1. Increased cyclin D1 expression will confer B-Raf inhibitor resistance.163

Interactions between KRAS and PIK3CA mutations can result in conferring resistance to rapamycin Cancers containing PIK3CA mutations are often sensitive to the mTOR inhibitor rapamycin and the modified rapamycins (Rapalogs). However, PIK3CA-mutant cells that also have mutations at KRAS are resistant to rapalogs.164 This may be due to complicated feedback loops between the Ras/Raf/MEK/ ERK and PI3K/PTEN/Akt/mTOR pathways wherein either mTORC1 inhibition leads to ERK1/2 activation by a p70S6K/ PI3K/Ras-dependent pathway or by the KRAS mutants activating p90Rsk-1, which serves to activate eIF4B and rpS6 thereby bypassing mTOR-dependent activation. Although many of the examples in the previous sections were from studies with solid cancers, they serve to illustrate the complexity of sensitivity and resistance to targeted therapy with Raf and MEK inhibitors.

Resistance to MEK inhibitors in leukemia Although mutations in certain upstream cytokine receptors or components of the Ras/Raf/MEK/ERK pathway often alter sensitivity to MEK inhibitors in leukemic cells, recently it has been shown that certain mutations will confer resistance to MEK inhibitors.165 NF1 is a GTPase activating protein, which normally terminates Ras signaling via stimulation of Ras-GTP hydrolysis. NF1 is inactivated in children with NF1 and these children have a 200- to 500-fold increase in juvenile myelomonocytic leukemia.166,167 Dr Shannon et al. have developed mouse models to examine the genes involved in transitions from MPD to AML. MPDs initiated by inactivating NF1 in mouse bone marrow are resistant to MEK inhibitors as are myeloid progenitor cells from WT mice; however, subsequent AMLs induced in the NF1-deficient mice by retroviral insertional mutagenesis indicated that the NF1-deficient AMLs were sensitive to MEK inhibitors. These results indicated that the NF1-mutant AMLs probably acquired additional mutations, which made them sensitive to Raf/MEK/ERK pathway signaling. However, resistance to the MEK inhibitors could develop in some NF1deficient AML cells. The resistant cells were determined to be present in the original NF1-deficient-sensitive clones as determined by the sites of retroviral integration. That is, there were subsequent mutations in the original retroviral-induced NF1deficient AML, which resulted in resistant AMLs and the resistant cells were selected on culture in media containing the MEK inhibitor. The sites of retroviral insertion were subsequently cloned in the resistant cells. The candidate genes frequently implicated in resistance to the MEK inhibitors were identified. One gene identified was Rasgrp1. Some MEK inhibitor-resistant clones displayed approximately 1000-fold increase in copy number of Rasgrp1, which resulted in an average of 10-fold increase in mRNA specific for this gene. Ras activation was constitutive in the MEK inhibitor-resistant cells, which formed cytokine-independent blast colonies in methylcellulose. Infection of MEK inhibitor-resistant cells with a lentivirus containing a short hairpin RNA specific for Rasgrp1 reduced the RasGRP1 protein and restored sensitivity to the MEK inhibitor. Another gene identified whose altered expression resulted in resistance to the MEK inhibitor is Mapk14, which encodes p38a. In this case, the retrovirus integrated in the opposite reading frame to Mapk14, which resulted in inactivating one Mapk14 allele. Drugs such as SB202190 (a specific p38a inhibitor) antagonized the inhibitor effects of the MEK inhibitors on the MEK inhibitor-sensitive NF1-deficient AMLs, but had only minimal effects on the MEK inhibitor-resistant NF1-deficient AMLs. The presence of resistant mutants present in the original leukemias has also been observed with chronic myeloid leukemias and ALLs.168,169 The genes often detected in the resistant clones included genes involved in developmental programs, cell cycle control and DNA damage response. In contrast, mutations/overexpression of genes involved in drug transport or metabolism are not commonly observed, at least in the drug-resistant ALLs.

Switching Raf isoforms can confer resistance to Raf inhibitors In BRAF-mutant melanoma cells that had been maintained in medium containing the B-Raf inhibitor AZ628 shifted their dependence from B-Raf to Raf-1.170 Some of additional genetic mutations may be preexisting in the tumor cell population and on culture of the cells or tumor in the presence of the Raf inhibitor; the ‘mutant-resistant’ cells may take over the population. Leukemia


1090 Conclusions The Ras/Raf/MEK/ERK cascade has important roles in promoting hematopoietic cell growth, regulating apoptosis, as well as the induction of chemotherapeutic drug resistance. Commonly used anti-leukemia chemotherapeutic drugs can induce this pathway and may contribute to subsequent drug resistance. Recent studies have also indicated that ERK and p90Rsk-1 can regulate certain aspects of protein translation in conjunction with the PI3K/PTEN/Akt/mTOR pathway. Mutations that activate this cascade are among the most frequently detected in human cancer. Inhibitors of Ras, Raf and MEK have been developed and analyzed clinically. Although many of these inhibitors show promise, there are also complications. This review has discussed some of the genes that confer resistance/sensitivity to Ras, Raf and MEK inhibitors. Sometimes mutations at other signaling pathways such as PI3K/PTEN/Akt/mTOR (for example, PIK3CA) will inhibit the sensitivity to the inhibitors, which target the Ras/ Raf/MEK/ERK pathway. Similarly, mutations at KRAS will eliminate the sensitivity of PIK3CA-mutant tumors to rapamycin. Other mutations have also been identified which either confer resistance or sensitivity to MEK inhibitors. Certain inhibitors (for example, Raf inhibitors) may activate the Raf/MEK/ERK pathway that may be deleterious in cells, which have KRAS mutations. Thus, although we now have more precise bullets in our arsenal to treat leukemia than classical chemotherapy, we also have discovered mechanisms by which the leukemic cells can protect penetration and subsequent death by these bullets.

Conflict of interest

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11

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The authors declare no conflict of interest.

Acknowledgements This work was supported in part by grants from Fondazione del Monte di Bologna e Ravenna, MinSan 2008, ‘Molecular therapy in pediatric sarcomas and leukemias against IGF-1 receptor system’ (PRIN 2008 and FIRB 2010 (RBAP10447J)) to AMM.

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