FLT3 Inhibition: a Moving and Evolving Target in Acute Myeloid Leukaemia

REVIEW FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia

AYH Leung, C-H Man and Y-L Kwong

Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is a gain-of-function mutation common in acute myeloid leukaemia (AML). It is associated with inferior prognosis and response to chemotherapy. Single base mutations at the FLT3 tyrosine kinase domain (TKD) also leads to a gain of function, although its prognostic significance is less well defined because of its rarity. The clinical benefits of FLT3 inhibition are generally limited to AML with FLT3-ITD. However, responses are transient and leukaemia progression invariably occurs. There is compelling evidence that leukaemia clones carrying both ITD and TKD mutations appear when resistance to FLT3 inhibitors occurs. Interestingly, the emergence of double ITD and TKD mutants can be recapitulated in vitro when FLT3-ITD þ leukaemia cell lines are treated with mutagens and FLT3 inhibitors. Furthermore, murine xenotransplantation models also suggest that, in some cases, the FTL3-ITD and TKD double mutants actually exist in minute amounts before treatment with FLT3 inhibitors, expand under the selection pressure of FLT3 inhibition and become the predominant resistant clone(s) during the drug-refractory phase. On the basis of this model of clonal evolution, a multipronged strategy using more potent FLT3 inhibitors, and a combinatorial approach targeting both FLT3-dependent and FLT3-independent pathways, will be needed to improve outcome.

Leukemia (2013) 27, 260–268; doi:10.1038/leu.2012.195 Keywords: FLT3; internal tandem duplication; tyrosine kinase domain mutation; inhibition

INTRODUCTION Clinically, FLT3-ITD occurs most frequently in AML with normal 2,3 Acute myeloid leukaemia (AML) is a group of heterogeneous karyotype, trisomy 8, t(6;9) and t(15;17), and is generally 17 diseases sharing in common an abnormal increase in myeloblasts associated with leukocytosis on presentation. Although the in the circulation and bone marrow. Intensive chemotherapy and complete remission (CR) rate does not appear to be affected, 18 allogeneic haematopoietic stem cell (HSC) transplantation are the AMLs with FLT3-ITD have higher relapse rates and therefore 19 principal treatment modalities. However, these approaches have inferior disease-free and overall survivals, particularly in AML 20 21 22 reached an impasse, with a cure rate of only 30–40%.1 Internal with larger ITD size, higher allelic burden and multiple ITDs. tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) Therefore, FLT3 inhibition has become a legitimate therapeutic gene is a gain-of-function mutation, and represents one of the option, and clinical trials of FLT3 inhibitors in AML have been 23–25 most common genetic alterations in AML, occurring in nearly 30% ongoing for a decade. To date, more than 20 small molecule of cases.2,3 FLT3 is a class III receptor tyrosine kinase consisting of inhibitors against FLT3 have been reported, of which eight of 26 five extracellular immunoglobulin-like domains, a transmembrane them have been evaluated in clinical trials. Structurally, most of domain, a juxtamembrane domain and two tyrosine kinase these inhibitors are heterocyclic compounds, inhibiting FLT3 domains (TKD1 and TKD2).4 It is highly expressed in haemato- activity by competing with ATP for binding to the ATP-binding poietic stem and progenitor cells.5 Upon binding to FLT3 ligand, it pocket of the TKD. Functionally, they are generally multikinase dimerizes and autophosphorylates, resulting in activation of inhibitors. Their clinical activities appear to be mediated by FLT3 tyrosine kinase activity, which in turn activates the phosphoino- inhibition, so that their efficacies are limited to AML carrying FLT3- sitide-3-kinase/AKT and RAS/mitogen-activated protein kinase ITD, and correlated with inhibition of FLT3 phosphorylation and 27–31 pathways.6,7 FLT3-ITD involves an in-frame duplication of 3–400 hence its downstream signalling effectors. Although basepairs at the juxtamembrane or TKD1 domains. It results inhibition of FLT3 may be achievable, clinical efficacy is less than in alteration of cellular signalling, including the constitutive convincing, being limited by the invariable leukaemia progression 24 activation of FLT3 independent of ligand binding;8 activation of despite continuous treatment. Comprehensive reviews on 24,32–39 STAT5 (signal transducer and activator of transcription 5) via SRC the mechanisms of drug resistance have been published. kinase;9 phosphorylation of a transcription factor FOXO3A;10 The proposed mechanisms include persistent activation of FLT3 downregulation of the equilibrative nucleoside transporter 1 for signalling due to overexpression of FLT3 ligand and protein, cytarabine;11 and induction of reactive oxygen species production.12 activation of antiapoptotic signals and protection of leukaemia- These molecular consequences result in damage13 and defective initiating cells (LIC) by the bone marrow niche (Table 1). Although repair of DNA, and increased cellular proliferation and resistance to therapeutic strategies have been formulated on these proposi- apoptosis.14–16 Comprehensive description of normal and aberrant tions, they are based primarily on in vitro data and laboratory FLT3 signalling has been reviewed previously.4,14–16 observations.

Division of Haematology and Bone Marrow Transplantation, Department of Medicine, Queen Mary Hospital, Hong Kong, China. Correspondence: Professor Y-L Kwong, Division of Haematology and Bone Marrow Transplantation, Department of Medicine, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. E-mail: [email protected] Received 11 May 2012; revised 26 June 2012; accepted 6 July 2012; accepted article preview online 16 July 2012; advance online publication, 3 August 2012 FLT3 inhibition AYH Leung et al 261 Table 1. Proposed mechanisms of resistance to FLT3 inhibitors

Proposed mechanisms Proposed means of targeting References

Overexpression of FLT3 ligands Targeting FLT3 ligand as a therapeutic strategy Sato et al.34 Overexpression of FLT3 protein Degrading FLT3 proteins by inhibition of chaperone heat-shock protein 90 Weisberg et al.37, Nishioka et al.81 and Al Shaer et al.82 Protection by bone marrow niche Reducing CXCL12 signalling in stromal cells by the HDM2 antagonist Nutlin-3a Kojima et al.32 via stabilization of p53 Disrupting interaction between LSCs and niche (for example, CXCR4 antagonists) Parmar et al.33 Over-riding microenvironment-mediated resistance to FLT3 inhibitors by combination Weisberg et al.83 treatment with dasatinib and JAK inhibitors Overexpression of antiapoptotic genes Targeting STAT pathway or survivin with inhibitors or shRNA Zhou et al.38 Targeting MCL-1 to restore sensitivity to FLT3 inhibitors Breitenbuecher et al.39 and Sto¨lzel et al.35 Emergence of resistant clones See text Abbreviations: FLT3, fms-like tyrosine kinase 3; JAK, janus kinase; LSC, leukaemia stem cells; shRNA, short hairpin RNA; STAT, signal transducer and activator of transcription.

Table 2. Clinical trials of FLT3 inhibitors in FLT3-mutated AML patients

Inhibitor Dosing schedulea Other drugs Number of patients Outcome References

ITD TKD mutation

Lestaurtinib 40–60 mg b.i.d. None 16 1 BR: 30%; HI: 16% Smith et al.30 Lestaurtinib 60 mg b.i.d., days 1–28 None 2 3 BR: 60%; HI: 60% Knapper et al.84 Lestaurtinib 80 mg b.i.d., days 7–112 Mitoxantrone, etoposide 103b 11b No improvement in survival Levis et al.49 cytarabine, days 1–5 Midostaurin 75 mg t.i.d. None 18 2 CR: 5%; BR: 35%; HI: 30% Stone et al.85 Midostaurin 50 mg b.i.d. None 12 6 BR: 67%; HI: 50% Fischer et al.28 Sunitinib 50–75 mg daily, days 1–28 None 2 2 CRi: 25%; PR: 75% Fiedler et al.51 80 mg b.i.d., days 29–58 Tandutinib 150–700 mg b.i.d. None 8 1 BR: 25%; HI: 25% DeAngelo et al.50 KW-2449 25–500 mg b.i.d., days 1–14 None 11 0 BR: 45% Pratz et al.52 Sorafenib 600 mg daily–400 mg b.i.d. None 7c 3b CRi: 11%; BR: 56% Zhang et al.53 Sorafenib 200–800 mg daily None 6 0 CR: 33%; BR: 66% Metzelder et al.86 Sorafenib 400–600 mg b.i.d., 14–28 days None 2 0 BR: 50% Pratz et al.87 Sorafenib 400 mg b.i.d., induction: 7 days; Idarubicin, cytarabine 13 2 CR þ CRp: 100% Ravandi et al.55 maintenance: 28 days AC220 200 mg daily None 62 0 CR þ CRp þ CRi: 45% Cortes et al.54 Sorafenib 400 mg b.i.d. None 13 0 CRi: 46%, nCRi: 46% Man et al.61 Sorafenib 400 mg b.i.d. None 65 0 CR þ CRi þ CMR: 38% Metzelder et al.88 HI þ BR þ PR: 61% Midostaurin 50 mg b.i.d. Daunorubicin Cytarabine 9 4 CR: 92% Stone et al. 56 Abbreviations: AML, acute myeloid leukaemia; b.i.d., twice daily; t.i.d., three times daily; BR, blast response; CMR, complete molecular response; CR, complete remission; CRp, CR without platelet recovery; CRi, CR with incomplete haematological recovery; FLT3, fms-like tyrosine kinase 3; HI, haematological improvement; ITD, internal tandem duplication; nCRi, near CRi but with focal blast prominence in trephine biopsy that could not be enumerated; PR, partial remission; TKD, tyrosine kinase domain. aUnless stated, continued until maximal response or unacceptable toxicity. All medications were orally administered. bTwo patients had both ITD and TKD mutation. cOne patient had both ITD and TKD mutation.

There is now compelling clinical evidence that the resistance to full-blown AML when additional genetic alterations, such as FLT3 inhibitors may arise from the emergence of FLT3-ITD þ NUP98-HoxD13, are present.47 These data can be considered as clones carrying additional TKD mutations. A similar concept of evidence to suggest that FLT3-ITD may be a driver mutation, and evolution of resistant clones40 has been generated to explain that secondary mutations are required for frank leukaemogenesis. resistance to multikinase inhibitors in chronic myelogenous On the other hand, it has also been found that FLT3-ITD may occur leukaemia (CML)41 and solid organ malignancies.42 This review only at diagnosis and not relapse, or vice versa,44,45 suggesting focuses on the clinical and laboratory evidence of clonal evolution that ITD may be a secondary event developing in subclones in FLT3-ITD, and proposes testable models that may lead of leukaemia cells or acquired during leukaemia progression.48 to improved strategies to overcome drug resistance in FLT3- These observations underscore the intrinsic heterogeneity of AML, ITD þ AML. and that a particular genetic alteration may have distinctive pathogenetic roles depending on where it occurs in the hierarchy of leukaemic haematopoiesis.48 This heterogeneity may also FLT3-ITD: DRIVER MUTATION OR SECONDARY EVENT? account for the variable responses to FLT3 inhibitors observed A central theme in cancer genetics is the identiﬁcation of in clinical trials. the driver mutation that leads to transformation of the cancer-initiating cell. In over 80% of FLT3-ITD þ AML, the ITD is present at diagnosis and relapse, suggesting that it may be the CLINICAL EFFICACY OF FLT3 INHIBITORS driver associated with initiation of the leukaemia.43,44 Xenotrans- Several FLT3 inhibitors have been tested in clinical trials, including plantation experiments have also shown that FLT3-ITD þ cells lestaurtinib,49 midostaurin,28 tandutinib,50 sunitinib,51 KW-2449,52 dominate the engrafting leukaemic clones, implying that FLT3-ITD sorafenib53 and AC220(ref. 54) (Table 2). These trials have provided occurs at the level of LIC.45,46 Furthermore, knock-in of FLT3-ITD in important observations. As a single agent in patients with AML mice results in a myeloproliferative disease that transforms into shown to have FLT3-ITD and/or FLT3-TKD mutations, FLT3

& 2013 Macmillan Publishers Limited Leukemia (2013) 260 – 268 FLT3 inhibition AYH Leung et al 262 inhibitors induced a response rate ranging between 46 and 100%. inhibitor and chemotherapy in FLT3-mutated AML is ongoing,56 In addition to disease heterogeneity, the varying responses might and it remains to be seen if a beneficial effect on relapse may be have been due to different definition of responses and the demonstrated clinically. number of patients studied. However, the majority of responses was restricted to reduction of blast counts and haematological improvement. CR, or complete remission with incomplete FLT3-TKD MUTATIONS: IMPACTS ON AML haematological recovery (CRi), only occurred in 5–35% of cases. FLT3-TKD mutations occur mostly in exon 20, with substitution Furthermore, these remissions were not durable. Hence, it has of aspartic acid by tyrosine at codon 835 the most common been suggested that FLT3 inhibitors may best be used as a (Table 4). FLT3-TKD mutations stabilize the activation loop of the bridging therapy to bring patients into remission for a subsequent open ATP-binding configuration, so that it is similar to ITD in haematopoietic stem cell transplantation. leading to constitutive FLT3 activation. However, FLT3-TKD Interestingly, FLT3 inhibitors might induce responses in mutations occur much less frequently, and do not appear to have leukaemias apparently without FLT3 mutations (Table 3). However, the same adverse prognostic implications as FLT3-ITD. When responses were mainly reduction in blast counts, and CR/CRi has transduced into murine haematopoietic stem cells, FLT3-TKD not been reported. These responses were most likely related to mutations result in lymphoproliferative diseases instead of the multikinase activities of these drugs instead of FLT3 inhibition. myeloid neoplasms, indicating that the signalling pathways To overcome the transient responses induced by FLT3 deranged may be different from those of ITD.9,57 Furthermore, inhibitors, the addition of concomitant or sequential chemother- coexistence of FLT3-ITD and TKD mutations (referred to as the apy appears to be an attractive option. In one study of relapsed ‘double mutant’ hereafter) was infrequent (0.2–2%), suggesting AML patients with FLT3 mutation (ITD, N ¼ 104; TKD, N ¼ 11) that TKD mutations occur rarely, and that the two mutations treated with chemotherapy followed by lestaurtinib,49 a CR/CRi might not necessarily have cooperative advantages in the absence rate of 26% was observed, comparable with that of 21% in of selection pressure. patients without FLT3 mutation. There was no difference in survivals. In another study of 15 cases of relapsed AML with FLT3 mutation (ITD, N ¼ 13; TKD, N ¼ 2) treated with concomitant LOSS OF RESPONSE TO FLT3 INHIBITORS: CLINICAL EVIDENCE chemotherapy and sorafenib,55 a CR/CRi rate of 100% was found, OF EMERGENCE OF RESISTANT CLONES which was significantly better than that of 66% in 36 cases Despite variable initial responses, AML treated with FLT3 inhibitor without FLT3 mutation. There was, however, no apparent monotherapy almost always progresses. This observation is difference in survivals. In a more recent study in which 13 cases reminiscent of CML treated by the multikinase inhibitor imatinib, of newly diagnosed AML with FLT3 mutation (ITD, N ¼ 9; TKD, where emergence of imatinib-resistant clones may occur in up to N ¼ 4) was treated with chemotherapy and either concomitant or 30% of patients.58 Whereas in CML resistance can often be shown sequential midostaurin,56 the CR/CRi rate was 92%, which was to be due to mutations in the ABL1 part of the BCR-ABL1 fusion higher than that of 74% in 27 cases without FLT3 mutation. Similar gene, the genetic basis of non-responsiveness to FLT3 inhibitors is to previous studies, there was again no difference in survivals. not fully understood. Leukaemic cells from a patient with FLT3-ITD What these studies showed was that FLT3 inhibitors could safely treated with midostaurin showed, at relapse but not on be combined with chemotherapy, leading to comparable or better presentation, the presence of a single amino-acid substitution CR/CRi rates. However, the clinical problem of AML with FLT3 N676K within the TKD,59 which appeared to confer resistance to aberration is not a low CR rate, but increased relapses. None of the midostaurin in vitro. The appearance of an A848 TKD mutation was previous studies showed survival improvement, suggesting that also demonstrated in a patient with chronic myelomonocytic the addition of chemotherapy failed to prevent relapses in such leukaemia treated with alternating courses of sunitinib and cases. Furthermore, owing to protocol design, the mechanistic sorafenib.60 In a heavily pretreated cohort of 13 patients with bases of relapses following an initial response had not been FLT3-ITD, sorafenib monotherapy resulted in significant clearance investigated. Therefore, data from these studies were unhelpful in of marrow blasts in 12 of them.61 Comparison of FLT3 genotype the rational design of strategies to overcome the increased relapse before and after sorafenib treatment in six patients demonstrated, risks conferred by FLT3 aberrations. Prospective trial involving in addition to ITD, the presence of TKD D835H mutation in one larger number of patients examining the effect of combined FLT3 and D835Y mutation in three patients at leukaemia progression.

Table 3. Clinical trials of FLT3 inhibitors in FLT3-wildtype AML patients

Inhibitor Dosing schedule Other drugs Number Outcome References of cases

Semaxanib 145 mg/m2, twice weekly Â 8 doses, i.v. None 64 PR: 6%; HI: 2% Giles et al.89 Semaxanib 145 mg/m2, twice weekly Â 8 doses, i.v. None 43 PR: 16% Fiedler et al.27 Sunitinib 50–75 mg daily, days 1–28, oral None 10 PR: 20% Fiedler et al.51 Lestaurtinib 60 mg b.i.d., days 1–28, oral None 24 BR: 16% Knapper et al.84 80 mg b.i.d., days 29–58, oral Lestaurtinib 80 mg b.i.d., days 7–112, oral Mitoxantrone 111 Not different from patients Levis et al.49 Etoposide with mutated FLT3 Cytarabine Days 1–5 Midostaurin 50 mg b.i.d., oral None 30 BR: 50%; HI: 43% Fischer et al.28 100 mg b.i.d., oral None 27 BR: 33%; HI: 26% Sorafenib 600 mg daily–400 mg b.i.d., oral None 7 BR: 43% Zhang et al.53 Sorafenib 400–600 mg b.i.d., 14–28 days, oral None 13 No response Pratz et al.87 Sorafenib 400 mg b.i.d., 7 days of induction, 28 days of Idarubicin 36 CR þ CRp: 75% Ravandi et al.55 maintenance, oral Cytarabine Midostaurin 50 mg b.i.d., oral Daunorubicin 27 CR: 74% Stone et al.56 Cytarabine Abbreviations: AML, acute myeloid leukaemia; b.i.d., twice daily; BR, blast response; CR, complete remission; CRp, CR without platelet recovery; FLT3, fms-like tyrosine kinase 3; HI, haematological improvement; i.v., intravenous; PR, partial remission.

Leukemia (2013) 260 – 268 & 2013 Macmillan Publishers Limited FLT3 inhibition AYH Leung et al 263 Table 4. Common FLT3-TKD mutations at diagnosis

TKD mutations Cases/total Impact on CR Impact on OS Impact on DFS ITD þ TKD/total References

Exon 20 7/97 NA Nil NA NA Abu-Duhier et al.90 D835 30/429 Nil Nil Nil 1/429 Yamamoto et al.91 D835/836 75/979 Nil Inferior (age p60 years) Nil 17/979 Thiede et al.92 D835 16/208 Nil NA Inferior 4/208 Moreno et al.93 D835 8/109 Nil Nil NA 3/109 Andersson et al.94,a D835/836 8/256 NA Nil NA 7/256 Auewarakul et al.95 D835 9/143 Nil NA NA 2/149 Wang et al.96 Exon 20b 127/1107 Nil Superior Superior 27/1107 Mead et al.97,c D835, DI836 147/3082 NA Nil Nild 17/1596d Bacher et al.98,d D835/I836 16/217 Nil Nil Inferior 3/217 Whitman et al.99,e Abbreviations: AML, acute myeloid leukaemia; CR, complete remission; DFS, disease-free survival; EFS, event-free survival; FLT3, fms-like tyrosine kinase 3; ITD, internal tandem duplication; NA, not applicable; OS, overall survival; TKD, tyrosine kinase domain. aPatients age 460 years. bIncluding codons 835 and 836, and signiﬁcantly associated with inv(16). cInclude FLT3-ITD þ cases. dIn all, 9/13 TKD mutant clones at diagnosis disappeared at relapse; inferior EFS among t(15;17) patients and associated with the microgranular variant; FLT3-ITD examined in 1596 patients. eAge o60 years, cytogenetically normal AML, excluded FLT3-ITD þ in survival analyses.

These mutations were not identified by a sensitive allele-specific B-lymphoid cell line Ba/F3 transduced with FLT3-ITD, prolonged polymerase chain reaction before sorafenib treatment. Similarly, treatment with the FLT3 inhibitors midostaurin, semaxinib and emergence of FLT3-ITD clone carrying the TKD D835/I836 sorafenib resulted in TKD mutations of significantly higher mutation was recently reported in 14 of 58 patients with FLT3- frequencies if the line had a prior exposure to the mutagen ITD þ AML treated with FLT3 inhibitors.62 Genetic analysis of six N-ethyl-N-nitrosourea (ENU), as compared with the control FLT3-ITD þ AML patients treated with sorafenib and standard line. This observation implied that genotoxic chemotherapy chemotherapy also demonstrated TKD D651G(H), G619C, I687F might predispose to the development of TKD mutation when and E858K mutations at relapse in three patients.63 With AC220 subsequent selection pressure by FLT3 inhibitors is imposed. This monotherapy, emergence of TKD D835 and F691 mutations was suggestion is supported by data from next-generation demonstrated in all eight patients who subsequently progressed sequencing, which showed that additional gene mutations could with continuous therapy.64 These observations provided be induced by chemotherapy during AML treatment.66 unequivocal clinical evidence that the loss of response to FLT3 Importantly, each FLT3 inhibitor induced its distinctive TKD inhibitors was associated with the emergence of TKD mutations. mutant pattern, suggesting non-overlapping resistance profile to FLT3 inhibitors in this cellular model. Intriguingly, in another study in which MOLM-13 cells were treated with increasing RESISTANCE TO FLT3 INHIBITORS: OBSERVATION IN MURINE concentrations of midostaurin or HG-7-85-01 for up to 13 weeks, MODELS no TKD mutations could be demonstrated.37 Therefore, the In clinical studies where FLT3-TKD mutations have appeared in occurrence of TKD mutation may depend not only on FLT3-ITD, addition to ITD during disease progression, it has remained but also on prior treatment regimens and the FLT3 inhibitors used. unclear whether the double mutants are present before or are acquired after treatment with FLT3 inhibitors. One possibility is that the double mutant clone may have been too small to be THE DRUG-RESISTANT MODEL identified from the pre-treatment examples. To test this hypoth- On the basis of these observations, we propose a model that may esis, patients with FLT3-ITD þ AML treated with sorafenib, who provide a theoretical framework on which future therapeutic responded initially but progressed with the additional appearance intervention can be rationally designed (Figure 1). At leukaemia of ITD/TKD double mutants, were studied. Xenotransplantation initiation, FLT3-ITD occurs as a driver event in some cases of AML. of leukaemia samples obtained on presentation and apparently The resulting genomic instability leads to additional mutations at lacking TKD mutations in immunodeficient mice had resulted in the TKD, although these are rare events. Intensive chemotherapy engraftment of double ITD/TKD mutants, often overwhelming used for induction and consolidation induces and/or enhances the ITD þ clones.61 This observation demonstrated that double mutant occurrence of gene mutations and provides the basis for clonal clones albeit rare were present before treatment with FLT3 inhibitor, selection. At subsequent relapses and salvage treatment with FLT3 but were selected upon monotherapy with FLT3 inhibitor, which inhibitors, the FLT3-ITD þ myeloblasts either undergo apopto- might have accounted for the subsequent leukaemia progression. sis38,67,68 and/or differentiation,61,69 resulting in prompt reduction or clearance of blasts. However, the double mutant clones are mostly insensitive to the inhibitors. In particular, the FLT3-TKD TKD MUTATIONS RESISTANT TO FLT3 INHIBITORS: mutations at the activation loop or gatekeeper residue confer OBSERVATIONS IN THE LABORATORY allosteric hindrance to the binding of the inhibitors and stabilize Data supporting TKD mutations as a mechanism for resistance to the activation loop of the open ATP-binding configuration.64,70,71 FLT3 inhibitors come predominantly from laboratory observations. The TKD mutations to be selected may depend on the specific On the basis of a cell line derived from a patient with relapsed FLT3 inhibitors used and the sequence specificity of prevailing FLT3-ITD þ AML (MOLM-13), FLT3-TKD D835Y mutation could be FLT3-ITD. Once expanded, the double mutant clones progress induced by 6–7 weeks of in vitro treatment with increasing rapidly and survive on alternative signalling pathways that are concentrations of tandutinib. The resultant line became resistant either FLT3 dependent or independent, leading to clinical to subsequent treatment with AC220 and sorafenib.65 It was resistance. Studies of Ba/F3 cells transduced with FLT3-ITD-TKD possible that the TKD mutation might have pre-existed in a small double mutants demonstrated resistance to conventional leukaemia subclone in MOLM-13 cell line, which was acquired chemotherapy in addition to tyrosine kinase inhibitors, which during chemotherapy and selected upon tandutinib treatment. was associated with the activation of STAT5 and its downstream This proposition was supported by the finding that, in a mouse targets involved in DNA repair and antiapoptosis.71

& 2013 Macmillan Publishers Limited Leukemia (2013) 260 – 268 FLT3 inhibition AYH Leung et al 264

Figure 1. Proposed model of clonal evolution that accounts for resistance to FLT3 inhibitor. Leukaemic clones with FLT3-ITD may acquire TKD mutation during genotoxic chemotherapy. Clones carrying both FLT3-ITD and TKD (double mutant), less sensitive to FLT3 inhibitors, are selected during treatment and lead to leukaemia progression. nCRi, near CRi.

Table 5. New FLT3 inhibitors

Inhibitor IC50 Study outcome References

109 AUZ454 Not mentioned IC50 in FLT3-ITD or FLT3-D835Y-Ba/F3 around 1 nmol/l; 100-fold decrease in IC50 Weisberg et al. ATH686 compared with PKC412 in PKC412-resistant cells Ponatinib 2 nmol/l Inhibited MV4-11 and primary FLT3-ITD þ AML; inhibited TKD as well Gozgit et al.110 Linifanib 0.55 nmol/l Inhibited Ba/F3-FLT3-ITD cells in vitro and in xenograft; also inhibited AKT and GSK3b Hernandez-Davies et al.111 LY2457546 10 nmol/l Inhibited the proliferation of MV4-11 cells with B90-fold increased activity compared Burkholder et al.112 with sunitinib Canertinib Not mentioned Inhibited proliferation and induced apoptosis of FLT3-ITD þ cell lines and primary AML Nordiga˚rden et al.113 (CI-1033) SKLB1028 55 nmol/l Resulted in rapid and complete tumor regression of MV4-11 in mice Cao et al.114 20c 10 nmol/l Resulted in complete tumor regression of MV4-11 xenograft mouse model Li et al.115 BPR1J-097 1–10 nmol/l Induced apoptosis in MOLM-13 and MV4-11 in vitro and in xenograft Lin et al.116 VX-322 o1 nmol/l Inhibited cell lines expressing FLT3-ITD or c-KIT mutation and prolonged survival Davies et al.117 and Heidary et al.118 of FLT3-ITD knock-in mouse model

Abbreviations: AML, acute myeloid leukaemia; FLT3, fms-like tyrosine kinase 3; IC50, half maximal inhibitory concentration; ITD, internal tandem duplication; TKD, tyrosine kinase domain.

Table 6. Multiple targeting of FLT3-ITD þ AML

Combination Speciﬁc agents Study outcome References

mTOR and FLT3 Rapamycin and Rapamycin and PKC412 synergistically inhibited the proliferation of PKC412-resistant Mohi et al.100 PKC412 Ba/F3-FLT3-ITD/T691I mTOR and FLT3 RAD001 and sunitinib Rapamycin analog RAD001 enhanced the inhibitory effect of sunitinib in AML cell Ikezoe et al.101 lines (MV4-11, Kasumi-1) and primary leukaemia cells NF-kB and FLT3 AS602868 Inhibited both FLT3 and IKK2, and suppressed the growth and induced cell death of Griessinger et al.78 Ba/F3-FLT3-ITD/TKD and FLT3-ITD þ MV4-11 lines MEK/ERK and FLT3 AZD6244 and sunitinib AZD6244 enhanced the inhibitory and apoptotic effect of sunitinib in MV4-11, Nishioka et al.102 MOLM13 and primary AML cells PI3K/PDK-1 and mTOR BAG956 and BAG956 and rapamycin resulted in signiﬁcant killing of PKC412-resistant Ba/F cells Weisberg et al.103 rapamycin (Ba/F3-FLT3-N676D or -G697R) NF-kB and FLT3 CDDO-Me and PKC412 Synergistic inhibitory and apoptotic effects on Ba/F3-FLT3-ITD, MV4-11 and MOLM-14 Ahmad et al.104 PIM1 and FLT3 AR00459339 Inhibited FLT3-ITD þ cell lines and primary AML and sorafenib-resistant FLT3-ITD/ Fathi et al.105 D835H mutants. Weak synergism with FLT3 inhibitor PRL-3 and FLT3 SAHA and ABT-869 Synergistic inhibitory and apoptotic effects on FLT3-ITD þ cell lines Zhou et al.106 CDKs, JAK2 and FLT3a TG02 Inhibited MV4-11 growth in vitro and in xenograft Goh et al.107 JAK2 and FLT3 Pacritinib Inhibited the proliferation of primary AML samples and linifanib/ABT-869-resistant Lou et al.108 MV4-11 cells Aurora kinase and FLT3 CCT137690 Suppressed the growth of AC220/sorafenib-resistant FLT3-ITD/TKD mutants Moore et al.65 Abbreviations: AML, acute myeloid leukaemia; CDK, cell death kinase; ERK, extracellular signal-regulated kinase; IKK2, I-kB kinase-2; MEK, mitogen-activated protein kinase/ERK kinase; mTOR, mammalian target of rapamycin; NF-kB, nuclear factor-kB; PI3K, phosphoinositide kinase-3. aIncluding FLT3-TKD D835Y mutation.

As predicted by this model, we hypothesize that a number of inhibitors that can target both ITD and TKD mutations may be strategies may be evaluated clinically, for which candidates have more efﬁcacious. Although clinical data are lacking, laboratory emerged from laboratory research: (1) increasing the potency of studies have shown promising results (Table 5). The multikinase FLT3 inhibition; (2) development of new multikinase inhibitors inhibitor ponatinib, which also inhibits BCR-ABL1 in CML and can targeting simultaneously FLT3 and alternative pathways; and overcome the hitherto highly resistant T315I mutation,72 has been (3) combination therapy with inhibitors against FLT3 and shown to induce apoptosis, and inhibit cellular proliferation and alternative pathways. FLT3 signalling in murine myeloid cells transduced with FLT3-ITD and FLT-TKD N676D, F691I, G697R mutants.73 In this respect, high- throughput screening for tyrosine kinase inhibitors against both MORE POTENT FLT3 INHIBITORS FLT3-ITD and TKD may provide more candidates for laboratory and The emergence of TKD mutant clones in FLT3-ITD þ AML patients clinical trials. A caveat of this approach is that, in some cases, the treated with FLT3 inhibitors suggests that more potent FLT3 resistant ITD/TKD double mutant clones have actually

Leukemia (2013) 260 – 268 & 2013 Macmillan Publishers Limited FLT3 inhibition AYH Leung et al 265 STAT5-mediated PIM (proviral integretion site for Moloney murine leukemia virus 1) expression, which is an antiapoptotic effector. Recent reports have demonstrated that PIM inhibition suppresses the growth of FLT3-ITD þ cell lines and may function synergistically with FLT3 inhibitors in vitro.79 On the basis of the inhibitory activity of arsenic trioxide on ERK activity, a downstream effector of FLT3 activation, combination of arsenic trioxide and the FLT3 inhibitor AG1296 has been evaluated.80 The combination synergistically suppresses the growth and induces apoptosis in FLT3-ITD þ cell lines. Interestingly, the synergism is specific to FLT3-ITD and is not identified in leukaemia cell lines bearing wild-type FLT3. Although these therapeutic concepts have yet to be tested in clinical trials, they provide proof-of-principle observations that combinatorial targeting of multiple pathways may be exploited for treatment of drug-resistant double mutants. More candidate targets should be identified either by rational design of regimen or high-throughput chemical screening. Figure 2. Potential targets for therapeutic intervention that may overcome resistance to FLT3 inhibitors. Multiple targeting of FLT3 (green cross) and FLT3-dependent or -independent signalling CONCLUSIONS pathways (red crosses) may be necessary for optimal therapeutic effects. The spectacular success of multikinase inhibitors in CML represents a paradigm for the treatment of other neoplastic demonstrated persistently suppressed FLT3 signalling, clearly diseases. The constitutive activation of FLT3 by ITD and the indicating that these cells escape FLT3 inhibition by surviving on effective targeting by FLT3 inhibitors also represents a logical alternative mechanisms during leukaemia progression.61 therapeutic option. However, the clinical application has been limited by the invariably transient response followed by leukaemia progression. Compelling clinical and laboratory evidence has demonstrated that the resistance is due to the emergence of NOVEL MULTIKINASE INHIBITORS AGAINST FLT3 AND resistant clones carrying both FLT3-ITD and TKD mutations. ALTERNATIVE PATHWAYS Importantly, the response to FLT3 inhibition and the specific Because the resistant ITD/TKD double mutant clones may be able mutations arising during leukaemia progression are likely to be to survive on FLT3-independent pathways, a number of studies heterogeneous, depending on the specific AML subtypes, the have reported the development of novel multikinase inhibitors sequence of the prevailing ITD as well as the specific FLT3 capable of inhibiting FLT3 and alternative pathways (Table 6). In inhibitors used for treatment. Despite the heterogeneity, a particular, aurora kinases are highly conserved serine–threonine number of recurrent issues have emerged that need to be protein kinases that play important roles in mitosis. Selective addressed to improve the treatment outcome of patients with in vitro inhibition of aurora kinases has been shown to disrupt cell FLT3-ITD þ AML. These include a detailed dissection of the relative cycle and cytokinesis, leading to cell death. Aurora kinase importance of ITD and TKD mutations (or mutations in other parts inhibitors have emerged as potential therapeutic agents in AML of FLT3) in the leukaemogenic process: the alternative survival in clinical trials, but the overall response rate was only around pathways adopted by the double mutant clones, the development 74 20%. Recently, a novel kinase inhibitor CCT137690 targeting of potent FLT3 inhibitors against the double-resistant clones and both FLT3 and aurora kinase has been shown to suppress the the establishment of a robust platform whereby specific agent(s) growth of MOLM-13 cells bearing double FLT3-ITD/TKD D835 capable of suppressing the mutant clones can be identified. The 65 mutations resistant to AC220 and sorafenib. In addition, the latter can be accomplished by either rational design of therapeutic nuclear factor-kB pathway has been shown to be activated in strategies or high-throughput chemical screening. The clinical 75 LIC, and pharmacological inhibition of I-kB kinase-2, the enzyme benefits conferred by these efforts will have to be further that phosphorylates and hence inactivates the inhibitory subunit evaluated. of nuclear factor-kB, has been shown to enhance the cytotoxicity of chemotherapy in AML blasts.76,77 FLT3 activation has also been shown to stimulate the nuclear factor-kB pathway.78 On the basis of these premises, a dual FLT3-I-kB kinase-2 inhibitor AS602868 CONFLICT OF INTEREST has recently been shown to inhibit cellular proliferation and The authors declare no conflict of interest. induce apoptosis in Ba/F3 cells bearing FLT3-ITD and TKD, as well as in the FLT3-ITD þ human MV4-11 cell line.78 Despite the apparent efficacies of these novel agents in the laboratory, it REFERENCES remains to be determined whether inhibition of aurora kinases and nuclear factor-kB may be useful clinically in suppressing the 1 Rowe JM, Tallman MS. How I treat acute myeloid leukemia. 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