Targeting the FMS-Like Tyrosine Kinase 3 in Acute Myeloid Leukemia

REVIEW Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia

R Swords1, C Freeman2 and F Giles3

Acute myeloid leukemia (AML) is a highly heterogenous disease with multiple signaling pathways contributing to its pathogenesis. A key driver of AML is the FMS-like tyrosine kinase receptor-3 (FLT3). Activating mutations in FLT3, primarily the FLT3-internal tandem duplication (FLT3-ITD), are associated with decreased progression-free and overall survival. Identification of the importance of FLT3-ITD and the FLT3 pathway in the prognosis of patients with AML has stimulated efforts to develop therapeutic inhibitors of FLT3. Although these inhibitors have shown promising antileukemic activity, they have had limited efficacy to date as single agents and may require use in combination with cytotoxic chemotherapies. Here, we review clinical and preclinical results for the clinically mature FLT3 inhibitors currently in development. We conclude that multitargeted FLT3 inhibitors may have more utility earlier in the course of disease, when in vitro evidence suggests that AML cells are less dependent on FLT3 signaling, perhaps because of upregulation of multiple other signaling pathways. More potent agents may have greater utility in relapsed and heavily pretreated patients, in whom high levels of circulating FLT3 ligand may necessitate use of an agent with a very favorable pharmacokinetic/ pharmacodynamic profile. Novel combination regimens are also discussed.

Leukemia (2012) 26, 2176–2185; doi:10.1038/leu.2012.114 Keywords: FLT3; tyrosine kinase inhibitors; acute myeloid leukemia; internal tandem duplication

INTRODUCTION internal tandem duplication (ITD) in the FMS-like tyrosine kinase 10 Adult acute myeloid leukemia (AML) accounts for B30% of receptor-3 gene (FLT3), partial tandem duplication in the mixed 11 12 leukemias and 440% of leukemia-related deaths.1 AML is a highly lineage leukemia gene, Wilms tumor gene mutations and 13 heterogenous disease characterized by several karyotypic abnor- overexpression of the BAALC gene. Other mutations that may be malities (Table 1).2,3 Karyotypic abnormalities associated with a associated with poor prognosis in patients with AML include relatively good prognosis are present in B15% of patients 10–11-translocation oncogene family member 2, isocitrate and include translocation of chromosomes 15 and 17, t(15;17) dehydrogenase-1, isocitrate dehydrogenase-2 and additional 14 (q22;q21), translocation of chromosomes 8 and 21, t(8;21) sex comb-like 1. Alternatively, CCAAT/enhancer-binding (q22;q22) and inversion of chromosome 16, inv(16)(p13q22)/ protein a (CEBPA) and nucleophosmin 1 (NPM1) mutations (in t(16;16)(p13;q22). Prognostically bad karyotypic abnormalities, FLT3-ITD-negative patients) have been associated with improved 14,15 also present in B15% of patients, include deletion of chromo- prognosis. some 7, deletion of chromosome 5q and more than three In this review, we evaluate the prognostic implications of gene chromosomal abnormalities.4,5 Other karyotypic abnormalities mutations and cytogenetic risk factors in patients with AML, that have an unclear effect on prognosis have been observed, focusing on the FLT3-ITD mutation. We discuss established and including trisomy 8 and trisomy 21d. These abnormalities are emerging approaches using FLT3 inhibitors for the treatment generally considered to be in the intermediate-risk prognosis of AML. category along with normal karyotype. The identiﬁcation of these markers led to the development of cytogenetic risk classiﬁcation systems by several cooperative groups internationally, including the Cancer and Leukemia Group B,5 Eastern Cooperative Oncology STRUCTURE/FUNCTION OF THE FLT3 RECEPTOR and Southwest Oncology Groups,6 the UK Medical Research FLT3 receptor Council2 and Fro¨hling et al.4 in Germany. Mutations in FLT3 are among the most commonly occurring Most patients with AML have poor rates of survival, particularly and clinically relevant mutations in patients with AML. FLT3 is a those with a poor-risk karyotype. Five-year survival rates are receptor tyrosine kinase expressed on the leukemic blasts of B55%, 24% and 5% in patients with favorable-, intermediate- and 70–100% of patients with AML16,17 that is increasingly recognized poor-risk cytogenetics, respectively.5 Even among patients with as having a role in the pathogenesis of this disease. Upon binding normal karyotype AML, however, clinical outcomes are of the FLT3 ligand, FLT3 dimerizes and undergoes a conformational heterogenous. Among other patient-related factors, mutations in change whereby its activation loop opens, revealing the ATP- key genes involved in AML pathogenesis lead to large interpatient binding pocket. The receptor becomes autophosphorylated, variability in prognostic risk (Table 2).7–9 There is a plethora of inducing cell growth and inhibiting apoptosis through the mutations associated with poor prognosis in AML. These include activation of a signaling cascade involving proteins such as the

1Sylvester Comprehensive Cancer Center, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA; 2Barts and The London NHS Trust, St Bartholomew’s Hospital, London, UK and 3HRB Clinical Research Facilities, National University of Ireland Galway and Trinity College Dublin, Dublin, Ireland. Correspondence: Dr FJ Giles, HRB Clinical Research Facility, National University of Ireland, University Road, Galway, Ireland. E-mail: [email protected] Received 28 December 2011; revised 22 March 2012; accepted 10 April 2012; accepted article preview online 27 April 2012; advance online publication, 22 May 2012 Role of FLT3 in AML R Swords et al 2177 Ras-GTPase-activating protein, phospholipase C, signal transducer Table 1. Karyotypic abnormalities in patients with AML, classiﬁed 18 5 and activator of transcription 5 and ERK1/2 (Figure 1). according to cytogenetic risk by the Cancer and Leukemia Group B, 19,20 Eastern Cooperative Oncology Group/Southwest Oncology Group6 Whereas, FLT3 ligand is ubiquitously expressed, the FLT3 and Medical Research Council3 receptor is localized primarily in hematopoietic and neural tissues.21 FLT3 is integral to hematopoiesis through its Cytogenetic risk group Abnormality Incidence, % regulation of proliferation, differentiation and apoptosis of hematopoietic progenitor cells. Knockout mice models have Favorable Inv(16) or t(16;16) 3.5–8.7 shown that lack of FLT3 ligand and FLT3 leads to reduced Del(9q) 1.6–2.8 hematopoietic precursors,22,23 demonstrating the importance of Del(16q) NR these proteins in hematopoietic expansion. t(8;21) 6.7–8.2 t(15;17) 4.4–12 FLT3 mutations Intermediate Normal karyotype 40–48 Several different mutations can occur in the FLT3 receptor in À 7 3.8–8.5 À Y 3.3–4.8 patients with AML. The most common of these is an ITD on exon Del(5q) 1.7–5.9 14 of the FLT3 gene, which varies from 3 to 4400 base pairs but is Del(7q) 1.6–8.5 typically in frame and occurs in the juxtamembrane region of the Del(9q) 1.6–2.8 receptor.24 The ITD disrupts the autoinhibitory function of the Del(12p) NR juxtamembrane domain, resulting in constitutive autophosphory- Del(20q) 1.3 lation of FLT3 and activation of its downstream effectors.25,26 þ 6NRThe FLT3-ITD mutation produces growth factor-independent þ 8 8.7–10.1 proliferation in leukemia cell lines and a fatal myeloproliferative 11 1.6 þ syndrome in murine models.27 Interestingly, however, when FLT3- þ 13 2.4 ITD was expressed in cells from knockout mice lacking FLT3 þ 21 2.3–2.8 28 t(6;9) 0.7–1.8 ligand, the receptor was only weakly activated. This suggests t(9;11) 2.2 that the ITD leads to gain of function mainly by inducing 11q23 3.7 hyperresponsivity of the FLT3 receptor to FLT3 ligand rather than through autoactivation of the receptor. a Unfavorable/adverse Complex 5.9–11.7 Activating mutations within the kinase domain of FLT3 are also Abnormal 3q 2.5 observed in patients with AML. The most common is mutation of Inv(3) or t(3;3) 1.0–2.0 the aspartate 835 (D835) mutation in the activation loop of the 5 1.6–5.9 À tyrosine kinase domain, which occurs in B7% of patients with À 7 3.8–8.5 29,30 Del(5q) 1.7–5.9 AML. Del(7q) 1.6–8.5 t(6;9) 0.7–1.8 t(9;22) 1.3 FLT3 IN THE CLINIC

a It has become clear that the presence of FLT3-ITD, which is Abbreviations: AML, acute myeloid leukemia; NR, not reported. Complex 31,32 karyotype deﬁned as X3 cytogenetic abnormalities. observed in B25% of patients with AML, is a key indicator of poor long-term prognosis. This has led to a concerted effort to

Table 2. Prognostically relevant mutations in patients with AML

Abnormality Prognostic outcome Study results P-value

Negative patient outcome FLT3-ITD (compared with non-ITD FLT3) Shorter median OS15 10 vs 15.5 months 0.015 Shorter median DFS15 8 vs 12.5 months 0.033 MLL partial tandem duplication Shorter duration of CR117 7.1 vs 23.2 months 0.01 (compared with MLL WT) Shorter median OS11 5 vs 12 months 0.006 Shorter median RFS11 4 months vs NR o0.001 Shorter 2-year EFS118 20% vs 66% 0.03 Overexpression of BAALC (compared Shorter median OS13 1.7 vs 5.8 years 0.02 with low BAALC expression) Shorter median EFS13 0.8 vs 4.9 years 0.03 Shorter median DFS13 1.4 vs 7.3 years 0.03

Positive patient outcome Mutated CEBPA (compared with Longer median duration of CR46 NR vs 26 months 0.01 nonmutated CEBPA) Longer median OS46 NR vs 19 months 0.05 NPM1 mutations (FLT3-ITD À pts Higher rates of CR44 86% vs 63% o0.001a compared with FLT3-ITD þ pts) Longer median OS43 1183 vs 321 days 0.014 Longer median EFS43 773 vs 234 days 0.001 Abbreviations: AML, acute myeloid leukemia; CEBPA, CCAAT/enhancer-binding protein a; CR, complete response; DFS, disease-free survival; EFS, event-free survival; FLT3, FMS-like tyrosine kinase receptor-3 gene; ITD, internal tandem duplication; MLL, mixed lineage leukemia; NPM1, nucleophosmin 1; NR, not reached; OS, overall survival; Pts, patients; RFS, relapse-free survival; WT, wild-type. aFor all comparisons: NPM1 þ /FLT3-ITD À vs NPM1 þ /FLT3-ITD þ vs NPM1-/FLT3-ITD þ vs NPM1-/FLT3-ITD À.

& 2012 Macmillan Publishers Limited Leukemia (2012) 2176 – 2185 Role of FLT3 in AML R Swords et al 2178 FL FL

FL FL

RAS PI3K

GRB2

P P P P RAF1 GRB2 JAK SHC AKT

MEK1/2 FOXO mTOR

ERK1/2 STAT5a

Survival and/or proliferation Apoptosis Figure 1. Signaling pathways initiated by FLT3 ligand (FL) activation of the FLT3 receptor.18

identify therapeutic inhibitors of FLT3. Several of these agents are suggesting that FLT3 inhibitors may have a role in preventing currently in clinical trials and have shown promising antileukemic resistance to the cytotoxic chemotherapy regimens used in the activity. The presence of FLT3-ITD, however, is not the only treatment of AML. relevant factor in determining patient outcome. AML is a complex Although activating point mutations in the FLT3 tyrosine kinase disease characterized by multiple genetic abnormalities that likely domain have been grouped with FLT3-ITD in trial stratification interact with one another. Understanding how the myriad schemes and when discussing FLT3 in AML, these point mutations signaling pathways underlying the pathogenesis of AML interact have shown minimal prognostic implications in the absence with one another will be critical in determining how particular of FLT3-ITD.32,39 Mutations in the tyrosine kinase domain genetic abnormalities affect prognosis and response to therapy. of FLT3 frequently occur in patients with prognostically favorable NPM1 mutations, which further complicates analyses of their impact on patient outcomes.40,41 In fact, a study of 1107 FLT3 as a prognostic marker adult patients with AML showed improved survival in patients The presence of the FLT3-ITD mutation was determined to have a with FLT3 tyrosine kinase domain mutations vs patients with negative effect on long-term outcomes based on an analysis of wild-type FLT3.42 854 patients enrolled in Medical Research Council trials in the Frequently, patients with AML have mutations in more than United Kingdom.31 These findings were subsequently confirmed one gene, further complicating prediction of their prognoses. in numerous other trials.33–37 In general, patients with FLT3-ITD Mutations in NPM1 are the most commonly observed gene AML achieve complete remission rates comparable to those of abnormalities in patients with AML, occurring in B50% of patients with wild-type disease, but have significantly higher rates patients.41 Patients with an NPM1 mutation and wild-type FLT3 of relapse and shorter durations of disease-free and overall have the highest likelihood of long-term relapse-free, event-free survival (OS).31,33–35 In the Medical Research Council studies, and OS vs all other patients.43 However, NPM1 mutations predict patients with FLT3-ITD AML had a 74% relapse rate vs 48% for better outcomes only in the absence of FLT3-ITD.44 Patients who patients with FLT3-wild-type AML.31 The 5-year OS rate was 32% are NPM þ /FLT3-ITD þ , NPM1-/FLT3-ITD- or NPM1-/FLT3-ITD þ for patients with FLT3-ITD AML vs 44% for patients with FLT3-wild- have definitively worse prognoses. type AML;31 event-free survival was 20 vs 41 weeks, respectively C/EBP a is a transcription factor that is crucial for the (Po0.00001).32 differentiation of mature granulocytes. CEBPA gene mutations likely In vitro evidence suggests that FLT3-ITD may contribute to drug to induce differentiation arrest have been described in B10% of resistance. FLT3-ITD-expressing murine and human myeloid cell patients with AML.45 In a retrospective study of 135 patients with lines are resistant to cytosine arabinoside (ara-C), an essential AML, 22 CEBPA mutations were present only in patients (n ¼ 15) agent in AML chemotherapy regimens, and both cell lines have with intermediate-risk cytogenetics. OS was longer in patients with downregulated equilibrative nucleoside transporter 1 (ENT1), a CEBPA-mutated AML compared with those with CEBPA-nonmutated protein responsible for the cellular uptake of ara-C.38 Decreased AML (5-year OS, 53% vs 25%; P ¼ 0.04). In patients with a CEBPA levels of ENT1 may be related to the upregulation of hypoxia- mutation, the presence of FLT3-ITD mutation was associated with inducible factor 1 a subunit observed in these cells. Hypoxia- poorer 5-year OS compared with patients with wild-type FLT3 inducible factor 1 a subunit is known to downregulate the (20% vs 69%, P ¼ 0.03).45 Consistent with other studies,46 the promoter activity of the ENT1 gene. Pretreatment with the FLT3 presence of CEBPA mutations was identified as an independent inhibitor midostuarin (PKC412) rescued the ENT1 promoter activity predictor of good prognosis even after adjustment for cytogenetics and cellular uptake of ara-C in the human myeloid cell line, and FLT3 status in multivariate analysis.

Leukemia (2012) 2176 – 2185 & 2012 Macmillan Publishers Limited Role of FLT3 in AML R Swords et al 2179 Table 3. FLT3-targeted therapies currently in clinical development

Agent Company Molecular Efﬁcacy in FLT3 þ AML Most frequent any grade AEs Current targets clinical phase in AML

Midostaurin Novartis FLT3-WT, FLT3 In combination with standard induction GI toxicity, fatigue, pyrexia, Phase 3 (PKC412) mutant, c-KIT, chemotherapy in pts with FLT3-mutant and -WT dyspnea, hypokalemia, febrile VEGFR-2, AML: CR (50 mg twice daily dose; N ¼ 40): 92% neutropenia and pneumonia PDGFR (FLT3-ITD), 74% (FLT3-WT); similar rates of 1-year (85% vs 81%) and 2-year (62% vs 59%) OS in FLT3-ITD vs -WT AML54 Lestaurtinib Cephalon FLT3, JAK2 In combination with standard induction Elevated alkaline phosphatase, Phase 2 (CEP701) chemotherapy in pts with FLT3-mutant AML in GI toxicity, transaminitis, ﬁrst relapse (N ¼ 224): no difference in CR/CRp or hyperbilirubinemia and fatigue OS observed with lestaurtinib vs placebo60 Sorafenib Bayer FLT3, c-KIT, In combination with standard induction Febrile neutropenia, Phase 2 NRAS, RAF chemotherapy in pts with FLT3-mutant and -WT pneumonia in neutropenia, AML (N ¼ 197): no differences in EFS, OS or rates sepsis, diarrhea, rash and of CR with sorafenib vs placebo69 mucositis, hypertensiona Quizartinib Ambit FLT3, FLT3-ITD, Monotherapy in pts with relapsed/refractory GI toxicity, peripheral edema Phase 2 (AC220) Biosciences non-ITD- FLT3-ITD AML (N ¼ 53 evaluable): composite CR and dysguesia mutated FLT3, rate was 43% (all but one were CRi)74 c-KIT Tandutinib Millennium FLT3, PDGFR-b, Monotherapy in pts with AML or MDS (N ¼ 40): Muscular weakness, GI toxicity, Phase 1 (MLN518) Pharmaceuticals c-KIT antileukemic response occurred in two of eight QT prolongation, periorbital pts with FLT3-ITD mutations; only SD was and peripheral edema observed in pts with FLT3-WT76 Abbreviations: AE, adverse event; AML, acute myeloid leukemia; CR, complete remission; CRi, CR with incomplete blood count recovery; CRp, CR with incomplete platelet recovery; EFS, event-free survival; FLT3, FMS-like tyrosine kinase receptor-3; GI, gastrointestinal; ITD, internal tandem duplication; JAK2, Janus-activated kinase 2; MDS, myelodysplastic syndromes; OS, overall survival; PDGFR, platelet-derived growth factor receptor; Pts, patients; SD, stable disease; VEGFR-2, vascular endothelial growth factor receptor-2; WT, wild-type. aGrade 3/4 AEs reported.

A recent study of 481 patients with AML treated at the MD pathogenesis of AML, including c-KIT, vascular endothelial growth Anderson Cancer Center suggested that FLT3-ITD mutations led to factor receptor-2 and platelet-derived growth factor receptor.50 In worse survival in patients with normal karyotype AML but did not a phase 1 study of various advanced solid tumors, doses up to have a prognostic impact in patients with AML who had favorable- 75 mg thrice daily were tolerable and associated with FLT3 or poor-risk karyotypes.32 In addition, both the length47 and inhibition in vivo.51 Subsequent studies using in vitro kinase assays location10 of ITD insertions can negatively affect the prognostic and cell lines expressing FLT3-ITD demonstrated that midostaurin outcome of patients, as can the absence of the wild-type allele in inhibited activated FLT3 in the nanomolar range and that FLT3 FLT3-ITD-positive patients,48 whereas stem cell transplantation can was the major target of midostaurin in these leukemia cells.50 improve patient survival.49 These data emphasize the complex The 75-mg thrice daily dose was evaluated in a phase 2 study of interactions between the numerous karyotypic abnormalities, 20 patients with FLT3-mutant AML. Midostaurin at this dose was patient-related factors and mutations found in patients with AML. generally well tolerated by these patients, with X50% reduction in peripheral blasts achieved in 14 patients (70%). In all, 7 of these FLT3 inhibitors 14 patients achieved a X2-log reduction in blasts lasting X4 weeks, and 1 patient achieved a near-complete remission (bone Recognition of the importance of FLT3 in the pathogenesis and marrow cellularity of 10%).52 These promising results prompted prognosis of patients with AML led to investigations aimed at the initiation of a phase 2b trial of midostaurin at 50 or 100 mg identifying inhibitors of this tyrosine kinase. Several small- twice daily in patients with myelodysplastic syndromes or FLT3- molecule inhibitors of FLT3 currently in development demonstrate mutant or -wild-type AML. In this trial, midostuarin was associated various degrees of FLT3 inhibition and clinical efficacy (Table 3). with bone marrow/peripheral blood blast reduction in 71% of These agents are typically classified by their affinity for FLT3, with patients with FLT3-mutant AML and in 42% of patients with FLT3- midostaurin, lestaurtinib (CEP701) and sorafenib grouped as wild-type AML.53 Although blast reductions were achieved in a multitargeted tyrosine kinases, and other less clinically advanced majority of patients, only one patient achieved a partial agents such as quizartinib (AC220) and tandutinib (MLN518) response.53 The authors concluded that the clinical activity of considered more specific for FLT3. It has become clear, however, midostaurin in this population supported further evaluation of that predicting responses to FLT3 inhibitors is a complicated midostaurin in combination with chemotherapy. endeavor. Drug-related factors including potency for FLT3 Midostaurin in combination with daunorubicin and cytarabine inhibition, the ability to target other enzymes and pathways, was associated with high rates of complete remission in newly pharmacokinetic properties, the FLT3 ligand’s ability to influence diagnosed patients with FLT3-wild-type (20 of 27; 74%) or -mutant the activity of kinase inhibitors and the ability to target the (12 of 13; 92%) AML in a phase 1b trial.54 OS rates for patients with leukemic stem cell must be considered, as must patient-related FLT3 mutations were comparable to that of patients with the more factors such as disease state, concomitant medications and favorable FLT3-wild-type allele. The first randomized phase 3 trial comorbidities. testing the impact of an FLT3 inhibitor in frontline therapy (Cancer and Leukemia Group B 10603; RATIFY) has just completed accrual. Midostaurin This international, double-blind, placebo-controlled trial for newly Midostaurin is a broad-spectrum tyrosine kinase inhibitor of wild- diagnosed adults (o60 years) with FLT3-mutated AML evaluates type and mutated FLT3 as well as other kinases involved in the standard induction and postremission chemotherapy with

& 2012 Macmillan Publishers Limited Leukemia (2012) 2176 – 2185 Role of FLT3 in AML R Swords et al 2180 Table 4. Novel combination therapies with FLT3 inhibitors currently in clinical trials

Trial Phase Patient population Therapy Rationale Primary endpoint

NCT01130662 1 Elderly (X60 years) pts Midostaurin þ Decitabine is a low-intensity treatment for elderly pts with MTD with newly diagnosed or decitabine AML, and combined treatment with midostaurin may relapsed/refractory AML provide improved antitumor beneﬁts without additional toxicity NCT00819546 1 Pts with relapsed, Midostaurin þ Dual inhibition of mTOR and FLT3 pathways may reduce MTD refractory or poor- RAD001 growth and survival of tumor cells and prevent resistance prognosis AML or MDS NCT01093573 1/2 Elderly (X60 years) pts Midostaurin þ Combined treatment with midostaurin and the low-intensity MTD CR, with AML 5-azacitidine therapy 5-azacitidine may be associated with improved PR and response rates without additional toxicity HI NCT00943943 1 Pts with relapsed or Sorafenib þ Combination therapy with peripheral blood stem cell MTD refractory AML with FLT3 G-CSF þ mobilizers may make AML stem cells more accessible to mutations and elderly pts plerixafor sorafenib with FLT3-mutated AML who are ineligible for standard frontline chemotherapy NCT01371981 3 Pts with newly diagnosed Sorafenib þ Treatment with the proteasomal inhibitor bortezomib in EFS AML, with or without FLT3- bortezomib combination with sorafenib may be associated with higher ITD mutations rates of tumor cell death and improvements in pt survival compared with the active comparator regimen (ADE) and may prevent resistance Abbreviations: ADE, cytarabine/daunorubicin/etoposide; AML, acute myeloid leukemia; CR, complete response; EFS, event-free survival; FLT3, FMS-like tyrosine kinase receptor-3; G-CSF, granulocyte colony-stimulating factor; HI, hematologic improvement; ITD, internal tandem duplication; MDS, myelodysplastic syndromes; MTD, maximum tolerated dose; PR, partial response; pts, patients.

placebo or midostaurin at 50 mg twice daily on days 8 through Whereas complete remission rates correlated with FLT3 21 of each chemotherapy cycle and 1 year of midostaurin inhibition in the lestaurtinib combination trial, this response maintenance therapy.55 outcome was not well correlated with drug levels.60 This may have been related to elevations in plasma FLT3 ligand and a-1 acid glycoprotein levels in response to chemotherapy.61 Samples from Lestaurtinib patients in four separate lestaurtinib trials demonstrated that FLT3 ligand levels rise substantially after induction chemotherapy. This Lestaurtinib (CEP701) is a staurosporine analog that has activity was particularly apparent in relapsed patients, who had mean against FLT3,56 Janus kinase 2(ref. 57) and several other tyrosine FLT3 ligand levels of 1148 pg/ml on day 15 of induction therapy vs kinases. Lestaurtinib is currently in phase 2 trials alone or in 488 pg/ml in newly diagnosed patients. FLT3 ligand levels combination with idarubicin and cytarabine in AML, including rose even higher with subsequent courses of chemotherapy, to relapsed/refractory AML and AML with FLT3-activating mutations. a mean of 3251 pg/ml after the fourth course. In vitro experiments Clinical data have demonstrated that lestaurtinib is well tolerated demonstrated that FLT3 ligand at similar concentrations and active in both patients with relapsed/refractory AML58 and to those observed in patients diminished FLT3 inhibition and newly diagnosed elderly patients with AML.56 Lestaurtinib has cytotoxicity for all FLT3 inhibitors tested (lestaurtinib, midostaurin, demonstrated clinical activity against both wild-type and mutated sorafenib, KW-2449 and quizartinib). FLT3 (FLT3-ITD and FLT3-D835 mutations).56 Like midostaurin, the Although statistical comparisons were not done, there was a activity of lestaurtinib as a single agent was limited primarily to trend toward improved outcomes among younger patients bone marrow and peripheral blood blast reduction. The modest treated with lestaurtinib in the phase 2 trial.60 Taken together, but clinical activity observed with the single agent and the in vitro these findings suggest that FLT3 inhibitors may have better synergy of this agent with chemotherapy in killing FLT3-mutant efficacy in younger patients and those with newly diagnosed AML cells59 supported further evaluations of lestaurtinib in disease and minimal prior chemotherapy, in whom FLT3 ligand combination with cytotoxic regimens. upregulation may not be as prevalent. The Medical Research Surprisingly, in a multicenter trial of patients with relapsed AML Council is currently conducting two similar studies with randomized to re-induction chemotherapy alone or chemother- lestaurtinib (AML 15 and 17). These studies include younger apy followed by lestaurtinib, the addition of lestaurtinib did not patients, both relapsed and newly diagnosed, who may have less result in higher response rates or longer OS times in these patients FLT3 ligand upregulation. Early results suggested a high rate of with advanced disease.60 A major strength of this phase 2 study is target inhibition and complete remission in evaluable samples.62 that it incorporated correlative studies in which all samples were assayed for FLT3 plasma inhibitory activity, FLT3 ligand levels and a-1 acid glycoprotein levels. The plasma inhibitory activity assay has become a standard in the field as a surrogate for determining Sorafenib FLT3 inhibition in patients receiving FLT3 inhibitors. Plasma A multitargeted tyrosine kinase inhibitor of FLT3, c-KIT, NRAS, RAF inhibitory activity analysis revealed that only 58% of patients and others, sorafenib is approved for the treatment of advanced receiving lestaurtinib achieved target FLT3 inhibition by day 15 of renal cell and heptatocellular carcinomas.63 Small phase 1 studies therapy. Complete remission rates were significantly higher in and case reports showed reduction in bone marrow blasts and these patients than in patients without FLT3 inhibition at aplasia acceptable tolerability with single-agent sorafenib in patients assessment (39% vs 9%; P ¼ 0.004). with AML.64–67

Leukemia (2012) 2176 – 2185 & 2012 Macmillan Publishers Limited Role of FLT3 in AML R Swords et al 2181 In a phase 1/2 trial of sorafenib in combination with cytarabine burden, inhibition of FLT3 activity frequently did not result in cell and idarubicin-based induction in 51 newly diagnosed patients death, suggesting that AML in patients with earlier stage disease with AML, complete remissions were achieved by 38 patients and lower FLT3-ITD allele burden may not be addicted to FLT3 (75%). Complete remission rates were particularly high among signaling for survival and proliferation. This hypothesis will be patients with FLT3-ITD AML (14 of 15; 93%).68 evaluated further in the prospective, phase 3 trial of midostaurin However, these positive results were not confirmed in a in combination with cytarabine/daunorubicin, in which younger European placebo-controlled, randomized trial of sorafenib patients with FLT3-mutant AML were stratified by FLT3-ITD allele combined with standard induction, consolidation and mainte- ratio at baseline.55 nance in 197 elderly patients with AML. The combination was well In newly diagnosed patients with AML, targeting additional tolerated, but no benefit for sorafenib was observed in complete tyrosine kinases that contribute to the pathogenesis of AML (other remission, event-free survival or OS, even in patients with mutant than FLT3) may be more important than the potency of the agent FLT3-ITD AML (although this subgroup was small).69 Trials with toward FLT3-ITD receptors. Patients with FLT3-wild-type AML, for sorafenib are ongoing in patients with AML, including two trials example, had substantial responses to midostaurin in the phase focused exclusively on patients with FLT3-ITD disease, a phase 2 1b study,54 suggesting that they derived clinical benefit from the trial in elderly patients and a phase 3 study in pediatric patients. multitargeted profile of this agent. Lestaurtinib has also shown in vitro cytotoxicity toward FLT3-wild-type AML, whereas sunitinib and quizartinib display minimal cytotoxicity toward FLT3-wild-type Quizartinib (AC220) samples.78 Likewise, the in vitro cytotoxicity dose–response curves Quizartinib is a second-generation FLT3 inhibitor that is highly of lestaurtinib were similar in diagnostic vs relapsed samples, selective for FLT3-ITD70 as well as non-ITD FLT3 mutations and the 71 unlike those of sorafenib and, even more so, quizartinib, for which c-KIT kinase. Quizartinib was developed expressly to be an FLT3 relapsed samples, in which potent FLT3 inhibition may be more inhibitor and is more potent and specific in vitro than the broadly 70 important, were considerably more responsive than samples targeted agents such as midostaurin, lestaurtinib and sorafenib. obtained at diagnosis. Other in vitro studies have confirmed the Pharmacokinetic data from a dose-escalation study showed that association between the nonselectivity of the FLT3 inhibitors quizartinib is well tolerated at doses up to 450 mg and has good midostaurin and lestaurtinib and increased leukemic cell death.81 oral bioavailability and a long effective half-life (1.5 days) and that Resistance to FLT3 inhibitors can occur, primarily through quizartinib exposure is maintained between doses in patients with 82 83 72 upregulation of the FLT3 receptor, point mutations or AML. increased FLT3 ligand levels.60,61 In vitro screens have identified Quizartinib has shown activity in a phase 1 trial in patients with 73 four mutations in the ATP-binding pocket of FLT3 that confer relapsed/refractory AML (N ¼ 76). Responses according to the varying degrees of resistance to FLT3 inhibitors.83 FLT3 clones with International Working Group criteria were observed in 23 patients mutations at alanine 627, asparagine 676 or phenylalanine remain (30%), and complete responses were observed in 10 (13%). The sensitive to higher concentrations of most inhibitors, but a most commonly reported possibly drug-related adverse events glycine-to-arginine mutation at position 697 conferred resistance were gastrointestinal events, peripheral edema and dysguesia to all nine tyrosine kinase inhibitors tested and, when it arises, (mostly grade 1 or 2). The maximum tolerated dose was may necessitate alternative therapeutic approaches. This crucial established as 200 mg continuous dosing in this study as grade mutation at codon 697 has not been detected to date in patients 3 QTc prolongation was observed at the 300-mg continuous with AML. dosing dose. A phase 2 study in relapsed/refractory patients with Upregulation of FLT3 in the presence of FLT3 inhibitors, on the FLT3-ITD AML is currently recruiting patients, with interim data other hand, has been observed in vitro82 and in vivo.56 In addition, supporting doses of quizartinib 135 and 90 mg in men and the lestaurtinib combination trial illustrated that FLT3 ligand women, respectively, due to QTc prolongation at a starting dose of 74 upregulation is another important clinical feature of resistance. It 200 mg. is clear that FLT3 ligand can interfere with tyrosine kinase inhibition of FLT3.61 Myeloablative radiation and chemotherapy Tandutinib (MLN518) induce marked increases in FLT3 ligand.84–86 This is especially Tandutinib is more selective for FLT3 than midostaurin or problematic in heavily treated patients with relapsed disease. lestaurtinib, although it does have some activity against platelet- Further understanding of the mechanisms through which patients derived growth factor receptor-b and c-KIT.75 Although tandutinib develop primary or secondary resistance to FLT3 inhibitors will demonstrated some antileukemic activity as a single agent and help to guide how these agents should be incorporated into the in combination with chemotherapy in phase 1 studies,76,77 dose- treatment algorithm for AML. limiting muscle weakness (reversible) and high rates of nausea In single-agent studies with FLT3 inhibitors, responses were and vomiting were observed. This may be related to the slow limited to peripheral blast reduction. None of the inhibitors clearance and resultant high plasma levels of the drug. Low currently in development led to sustained cytogenic responses on potency and unfavorable pharmacokinetics have thus limited the their own, making combination therapy the preferred strategy for clinical utility of the drug for the treatment of AML. using these agents in patients with AML. Combination therapies may result in more durable responses through induction of faster and perhaps deeper responses and by thwarting FLT3-resistance Lessons learned from FLT3 laboratory/clinical studies mechanisms. In the trial of midostaurin plus induction (daunor- The prognostic implications of FLT3-ITD and response to FLT3 ubin/cytarabine) and consolidation (high-dose cytarabine) inhibitors are dependent on numerous patient- and drug-related chemotherapy, for example, disease-free survival and OS in factors, including allele burden,32,78 the presence of other FLT3-ITD patients was comparable to that of the more favorable mutations,32 stage of disease and drug pharmacokinetics. FLT3-wild-type prognostic group.54 Among these, allele burden of FLT3-ITD is a key predictor of response to FLT3 inhibitor therapy.79,80 In vitro studies of FLT3-ITD- positive cell lines demonstrated that relapsed samples and ALTERNATIVE STRATEGIES FOR FLT3 INHIBITION samples with a high mutant allele burden were more likely to After it was discovered that activated FLT3 is a marker of poor be responsive to cytotoxicity from FLT3 inhibition compared with prognosis in AML, it was anticipated that inhibiting this target the samples obtained at diagnosis or those with a low mutant would significantly change the natural history of this disease. allele burden.78 In these samples with lower FLT3-ITD allele Unfortunately, none of the currently available FLT3 inhibitors have

& 2012 Macmillan Publishers Limited Leukemia (2012) 2176 – 2185 Role of FLT3 in AML R Swords et al 2182 met this expectation. This may be explained by the numerous MEK inhibitors. The MEK/MAPK pathway is downstream of FLT3 FLT3-resistance mechanisms previously discussed in addition to and is critical for the control of hematopoietic cell differentiation the extremely heterogeneous nature of AML.52,56,58,83,87,88 Another and proliferation.27,106 Consistent with this, MEK inhibition impairs important explanation is the failure of available FLT3 inhibitors to the growth of FLT3-ITD cells107 and leads to apoptosis in primary adequately inhibit their target. To overcome these problems, AML blasts.108 Recent reports have demonstrated that MEK/MAPK better drug delivery systems (for example, monocloncal FLT3 inhibition can release cells from FLT3-ITD-mediated differentiation anitbodies) need to be developed and rational combination blockade.109 These findings suggest that MEK is a rational target therapies need to be fully evaluated before we consider for combination studies with available FLT3 inhibitors. abandoning FLT3 as a target for the treatment of AML. Arsenic trioxide. Emerging preclinical reports of activity with ATO combined with a MEK inhibitor in primary AML110 led to FLT3 monoclonal anitbodies the discovery that ATO in combination with an FLT3 inhibitor (AG1296) could potently impair the growth of FLT3-ITD cell Monoclonal antibodies (mAbs) have shown significant efficacy in 111 112 various cancers.89 In AML, mAbs may be able to circumvent lines. Given that ATO is well tolerated in the clinic, this some of the common resistance mechanisms observed with FLT3- exciting combination is certainly worth exploring in clinical trials.

targeted agents. For example, improved specificity for FLT3 might 113 offer less clinical toxicity in addition to the therapeutic benefits of hsp90 inhibitors. hsp90 is a molecular chaperone for FLT3, and hsp90 inhibition leads to FLT3 polyubiquitination and proteasomal ligand blockade, the potential for immunoconjugation and 114 antibody-dependent cell-mediated cytoxicity.87 Several putative degradation. As hsp90 targets misfolded proteins generated by FLT3 mAbs have been evaluated in the laboratory and are starting mutations, it is likely that FLT3-ITD proteins are unstable and to be used in the clinic. require chaperoning by hsp90 in leukemic cells. On the basis of A number of fully human immunoglobulin anti-FLT3 mAbs, this rationale, the hsp90 inhibitor 17-AAG combined with an FLT3 inhibitor was an effective strategy in preclinical AML studies and is derived from fragments originally isolated from naı¨ve human 115,116 monovalent antibody fragment phage display libraries, have been worth exploring in phase 1 trials. developed.90–93 Agents such as IMC-EB10, D4-3 and IMC-NC7 bind to FLT3 with high affinity.90 IMC-EB10 inhibits FLT3 ligand- CONCLUSIONS/FUTURE DIRECTIONS initiated phosphorylation and activation of FLT3 in ITD-mutant FLT3 cell lines.92 Target inhibition was demonstrated through Midostaurin and lestaurtinib are multitargeted kinase inhibitors in vitro interruption of MAPK, PI3K/AKT and signal transducer and that include FLT3 as one of their targets, whereas quizartinib is a activator of transcription 5 signaling, and prolonged survival highly selective FLT3 inhibitor, but is less cytotoxic to FLT3-ITD was seen in IMC-EB10-treated mice with both wild-type and primary samples in vitro. These drug properties will likely dictate ITD-mutated FLT3. IMC-EB10 was also effective in leukemic blasts where these agents belong in the treatment algorithm. Quizartinib resistant to lestaurtinib.87,94 A phase 1 clinical trial is currently may be useful in relapsed patients, in whom high FLT3 ligand underway with this agent (ClinicalTrials.gov Identifier: levels may necessitate a highly potent agent able to prevent NCT00887926). ligand binding to FLT3. In newly diagnosed patients, broader To address the limitation of intracellular penetration of pure spectrum agents such as midostaurin or lestaurtinib may be more FLT3 mAbs, mAb–cytotoxin conjugates have been developed. appropriate. Novel agents that may have a role in the treatment of Novel auristatin conjugates (using EB10 and a dolastatin analog, AML might include FLT3-ITD-specific small molecules and mAbs monomethyl auristatin F, linked with a protease-sensitive dipep- targeting FLT3. Another interesting approach might be to develop tide90,95) specifically select for FLT3 þ cells, maintaining stability in and incorporate mAbs targeting FLT3 ligand into the treatment the plasma but releasing the cytotoxic moiety once internalized algorithm for AML. Such antibodies may counteract the effects of and released by the cell lysosome. These agents and others96,97 FLT3 ligand upregulation during induction and consolidation are expected to be in clinical development soon. chemotherapy. AML is a heterogenous disease and, especially in early disease, multiple signaling pathways can contribute to pathogenesis. Novel combination strategies Combination approaches with novel agents and optimized algorithms may further improve outcomes in patients with AML. Currently available FLT3 inhibitors have been combined effectively Upregulation of alternate kinase pathways can be a factor in with established cytotoxic agents, both in the laboratory and in resistance to FLT3 inhibitors, and agents targeting the Janus the clinic.59,98,99 Ongoing clinical trials are exploring combination kinase /signal transducer and activator of transcription, phosphoi- therapy with FLT3 inhibitors and agents with various mechanisms nositide 3 kinase and mTOR families, for example, as well as other of action, including nucleoside analogs, mTOR inhibitors, agents designed to interfere with the proteasome and other cell proteasome inhibitors and C-X-C chemokine receptor type 4 processes may have a role in combination with FLT3 inhibitors. (CXCR4) inhibitors (Table 4). Other promising approaches Several of these combinations are currently being evaluated in might include MEK inhibitors, arsenic trioxide (ATO) or heat shock clinical trials. protein 90 (hsp90) inhibitors in combination with an FLT3 To fully eradicate AML in a patient, it may be necessary to inhibitor. eradicate AML stem cells. Novel approaches to AML treatment include the incorporation of stem cell mobilizers such as CXCR4 CXCR4 inhibitors. CXCR4 and its ligand (chemokine stromal-derived inhibitors and granulocyte-macrophage colony-stimulating factor factor 1a) are critical components of the stromal–leukemia cell into the treatment regimen. These agents may make AML stem interface. CXCR4 has an important role in cell adhesion-mediated cells more accessible for eradication by FLT3 inhibitors and drug resistance100,101 and, interestingly, is found more abundantly chemotherapeutic agents. Proof of concept for this approach has in FLT3-ITD AML samples than in FLT3-wild-type samples.102 been established in a mouse model of AML,105 and combination CXCR4 inhibition results in disruption of AML-niche interactions therapies with FLT3 inhibitors and stem cell mobilizers are and sensitizes leukemic blasts to cytotoxic chemotherapy.103–105 currently being evaluated in clinical trials. Thus, CXCR4/FLT3 combination therapy might preferentially target The optimal timing and duration of FLT3 inhibitor therapy in FLT3-ITD cells. This combination is currently being evaluated in a the treatment algorithm for AML remain an open question. phase 1 clinical trial. One potential strategy may be to use the FLT3 inhibitor early in

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