Oncogene (2010) 29, 5120–5134 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc REVIEW Drug resistance in mutant FLT3-positive AML

E Weisberg, M Sattler, A Ray and JD Griffin

Department of Medical Oncology/Hematologic Neoplasia, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

Mutant Fms-Like -3 (FLT3), which is of AML patients, however in o5% of patients with expressed in the leukemic cells of a subpopulation of acute myelodysplastic syndrome (Nakao et al., 1996; Horiike myeloid (AML) patients, represents an attrac- et al., 1997; Kiyoi et al., 1998; Kondo et al., 1999; tive target for the therapy of AML. There are several Rombouts et al., 2000). Gain-of-function FLT3 occurs FLT3 inhibitors presently in clinical trials with sufficient less frequently as point mutations in the activation efficacy and toxicity features to warrant further testing in loop (in B7% of AML cases), and is often found combination with standard therapies. However, the at position 835 (Yamamoto et al., 2001). Occurring transient and partial responses observed in AML patients less frequently are additional point mutations in the treated with FLT3 inhibitors, coupled with the discovery kinase domain, including N841I (Jiang et al., 2004) of drug-resistant leukemic blast cells in AML patients, and Y842C (Kindler et al., 2005). In addition, novel have made resistance to FLT3 inhibitors a growing activating point mutations have been identified in a concern. In this study, we provide an overview of the role 16 amino-acid stretch of the FLT3 juxtamembrane, of mutant FLT3 in AML, FLT3 inhibitors under clinical which generally have a weaker transforming potential and preclinical investigation, mechanisms of resistance to than FLT3-ITD and tyrosine kinase domain mutants FLT3 inhibitors, and possible therapeutic approaches to (Reindl et al., 2006). A diagram of FLT3 mutations is overcoming this resistance. shown in Figure 1. Oncogene (2010) 29, 5120–5134; doi:10.1038/onc.2010.273; Transplantation of murine bone marrow cells infected published online 12 July 2010 with a retrovirus expressing an FLT3-ITD mutant has been shown to result in the development of a rapidly Keywords: drug resistance; AML; FLT3; combination lethal myeloproliferative disease in mice (Kelly et al., therapy; stroma 2002a). This myeloproliferative syndrome occurs in the absence of any significant block in differentiation of granulocyte lineage cells, suggesting that the primary role of mutant FLT3 in AML might be to enhance the Introduction viability and growth of primitive myeloid cells. For the development of full and progressive AML, Acute myeloid leukemia (AML) is a hematological mutant FLT3 likely cooperates with other oncogenes malignancy marked by aberrant growth of myeloid associated with AML, such as PML/RARa (t(15;17) or precursor cells and a block in cellular differentiation at AML1/ETO (t(8;21), the major role of which is to block various stages. Most AML patients have underlying differentiation. Approximately 10% of AML patients genetic mutations that cause the production of precursor harbor translocations involving the mixed lineage leukemia cells characterized by either a block in differentiation, (MLL, HRX, ALL-1) gene, found on chromosome 11q23 aberrant and accelerated growth, or a combination (Dimartino and Cleary, 1999). Chromosomal translocations of both (Olsson et al., 1996; Tanaka et al., 1997; such as MLL-AF9 (t(9;11)), PML-RARalpha (t(15;17)) and Pabst et al., 2001). AML1-ETO (t(8;21)) result in the generation of chimeric Approximately 30% of AML patients, and a subset of fusion oncogenes, the acquisition of which represents the acute lymphoblastic leukemia (ALL) patients, express a first phase of leukemogenesis; additional mutations are mutated form of the class III required for complete leukemia development (Greaves (RTK), Fms-Like Tyrosine kinase-3 (FLT3) (STK-1, and Wiemels, 2003). For instance, activating FLT3 human Stem Cell Tyrosine Kinase-1; or FLK-2, Fetal mutations have been detected in some AMLs that Liver Kinase-2) (Stirewalt and Radich, 2003). Constitu- harbor MLL-AF9 translocations (Gilliland and Griffin, tively activated FLT3 occurs most frequently as internal 2002; Libura et al., 2003). tandem duplications (ITDs) within the juxtamembrane domain (Nakao et al., 1996), and is observed in B20–25% Mutant FLT3 and cell signaling Correspondence: Dr E Weisberg, Department of Medical Oncology, Dana Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, The survival advantage of leukemic cells driven by USA. E-mail: [email protected] mutant FLT3 is to a large extent because of its Received 17 April 2010; revised 2 June 2010; accepted 4 June 2010; activation of three major intracellular signaling path- published online 12 July 2010 ways, PI3K//PTEN/Akt/mTOR, RAS/Raf/MEK/ERK Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5121 and Jak/STAT (Kornblau et al., 2006). The significant et al., 2005), as well as STAT5 (Rocnik et al., 2006). cross-talk between the signaling mediators of these Mutant FLT3-positive AML cells are killed by rapa- pathways warrants consideration of the use of a mycin, because of its inhibition of mTOR, another multitargeted therapeutic approach for mutant FLT3- downstream effector of the PI3K/Akt signaling pathway positive AML. (Recher et al., 2005). Similarly, AML cells expressing FLT3-ITD mutations are associated with activation mutant FLT3 are killed by inhibition of the mutant of AKT, the downstream effector of PI3K (Brandts FLT3 chaperone, heat-shock protein 90 (Hsp90), which interferes with JAK/STAT, RAS/Raf/MEK/ERK and PI3K/Akt signaling (Al Shaer et al., 2008). A schematic FLT3 receptor: of FLT3 signaling is shown in Figure 2. Functional domains

Kinase inhibitors under clinical investigation for mutant Dimerization FLT3-positive AML activity & Ligand Binding Several inhibitors of FLT3 currently under clinical inves- tigation exhibit adequate efficacy and safety profiles to be considered for further development as therapeutics in combination with standard therapy for AML. Activating mutations For instance, a Phase III trial is presently recruiting Transmembrane patients for investigation of the N-indolocarbazole, Juxtamembrane (JM) PKC412 (; N-benzoyl-staurosporine, Novartis Internal Tandem Pharma AG, Basel, Switzerland), a broad-spectrum, orally Duplications (ITDs) Kinase bioavailable inhibitor that targets FLT3, platelet- (ATP binding) derived b (PDGFRb), c-KIT and c-FMS (Weisberg et al., 2002). The trial will investigate Kinase insert induction (daunorubicin/cytarabine) and consolidation (high-dose cytarabine) plus PKC412 or Kinase D835 placebo in newly diagnosed, mutant FLT3-positive (Phosphotransferase) AML patients under the age of 60 years. There is also an ongoing Phase I/II trial investigating the combina- Carboxy-terminal tion of PKC412 and 5-azacitadine in elderly AML tail region patients, and an ongoing Phase I trial investigating the Figure 1 Characteristics of documented FLT3 mutations. effects of RAD001 in combination with PKC412 in

Leukemic cell Plasma membrane

cytosolic Raf Ras targets FLT3 FLT3 receptor ligand SOS mTOR SHC GRB2 P MAPKK (MEK) P

AKT MAPK (ERK)

Bcl-X PP P L Nucleus STAT BAD PI3K BCl2 ITD or Survival/Proliferation point mutation JAK Mutagenic DNA repair

Apoptosis mitochondria BAD Bcl-XL FLT3 receptor

Figure 2 FLT3 signaling in a leukemic cell.

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5122 patients with relapsed, refractory or poor diagnosis et al., 2004) and the indolinone derivative SU11248 AML or myelodysplastic syndrome. (SU011248, , Sutent, Pfizer, New York, NY, USA) Phase I/II and Phase II clinical trials have been (Fiedler et al., 2005; Kancha et al., 2007; O’Farrell et al., completed for PKC412, investigating its effects in wild- 2003a, b), as well as the piperazinyl quinazoline MLN518 type and mutant FLT3-harboring AML or myelodys- (tandutinib; CT53518; Millennium, Cambridge, MA, USA) plastic syndrome patients. A Phase Ib clinical trial was (Kelly et al., 2002b; Cheng and Paz, 2008). There is carried out with newly diagnosed AML patients treated presently an ongoing Phase I/II trial investigating SU11248 with 50 mg PKC412 p.o. b.i.d. in sequential and combined with standard chemotherapy in mutant FLT3- simultaneous combinations with daunorubicin and positive AML patients over the age of 60 years. cytarabine induction and high-dose cytarabine consoli- Additional novel FLT3 inhibitors in early develop- dation (Stone et al., 2005b). Clinical responses were ment include the N-(4-(3-amino-1H-indazol-4yl)phenyl- complete (CR) in all the mutant FLT-positive patients N1(2-fluoro-5-methylphenyl) urea ABT-869 (Albert treated, and side effects were transient and/or reversible et al., 2006; Shankar et al., 2007; Zhou et al., 2008), (Stone et al., 2005b). In a Phase II clinical trial, which is a multitargeted inhibitor with activity toward PKC412 was generally well tolerated, with a FLT3, PDGFR, KIT and KDR; the benzimidalzole- decrease in peripheral blast counts observed in roughly quinoline CHIR-258 (TKI258; Chiron, Emeryville, CA, a third of PKC412-treated relapsed/refractory USA), which has among its targets FLT3, KIT, FMS, AML patients, and a median response duration of 13 VEGFR and FGFR (Lopes de Menezes et al., 2005) and weeks (Stone et al., 2004, 2005a). The hematological is a promising antimyeloma drug (Trudel et al., 2005) response rate in advanced AML patients treated presently in Phase I clinical trials for multiple myeloma, with PKC412 was comparable to that of chronic mixed solid tumors and AML; the hydroxystyryl- myeloid leukemia (CML) blast crisis patients receiving acrylonitrile LS104 (Kasper et al., 2008), which is in . a Phase I clinical trial for patients with refractory/ Also in clinical trials is the orally administered relapsed hematological malignancies; and AP24534 indolocarbazole alkaloid, CEP-701 (; Cepha- (Ariad, Cambridge, MA, USA), a multitargeted kinase lon) (Levis et al., 2002), which targets TrkA and inhibitor with activity against FLT3, KIT and FGFR vascular endothelial (VEGFR) (Rivera et al., 2008), and which is in Phase I clinical and inhibits the growth of FLT3-ITD-expressing cells trials for CML and other hematological malignancies. (Levis et al., 2002). Relapsed/refractory AML patients It is important to note that thus far, no selective FLT3 showed transient clinical responses to CEP-701 in early inhibitor has been developed that would allow the study clinical trials (Smith et al., 2004). Transient clinical of FLT3 exclusively as a target. responses were also observed in a Phase II trial for older (BMS-354825; Bristol Myers Squibb, AML patients not eligible for intensive chemotherapy New York, NY, USA) targets multiple proteins, and (Knapper et al., 2006). CEP-701 is presently being this property might very well increase its versatility investigated in the United Kingdom in combination in terms of disease targets. Dasatinib (BMS-354825; with chemotherapy in a Phase III clinical trial for AML Bristol Myers Squibb) is presently well known as being patients harboring wild-type FLT3 and mutant FLT3. a highly potent, orally active inhibitor of Abl that There is an ongoing Phase II trial investigating CEP-701 inhibits a wide panel of BCR-ABL point mutants that in AML patients, and an ongoing Phase I/II trial are resistant to imatinib (Shah et al., 2004; Burgess et al., investigating the combination of CEP-701 in combina- 2005; O’Hare et al., 2005). Dasatinib also inhibits SRC tion with cytarabine and idarubicin in younger patients and SRC-family kinases, including FGR, FYN, HCK, with relapsed or refractory AML. In addition to AML, LCK, LYN, SRC and YES (Das et al., 2006), as well as CEP-701 has also been investigated in a Phase II trial for a potent inhibitor of KIT, PDGFR and RTKs prostate cancer, and is under investigation in a Phase I (Melnick et al., 2006). Dasatinib received accelerated trial for high-risk neuroblastoma. approval by the US Food and Drug Administration Investigated in Phase I and II clinical trials for AML (FDA) for treatment of imatinib resistant/intolerant patients that are not candidates for approved therapy is adult CML, as well as drug-resistant Ph þ ALL. KW-2449 (Pratz and Levis, 2008; Pratz et al., 2009; A recent study showed activity of dasatinib, although Shiotsu et al., 2009), for the purpose of determining its at a relatively high concentration (B1 mM), toward maximum tolerated dose. In a Phase I trial, KW-2449 AML blasts and mutant FLT3 and mutant KIT treatment led to transient decreases in peripheral blast (Guerrouahen et al., 2010). Thus, inhibitors such as counts (Cortes et al., 2008; Pratz and Levis, 2008; Pratz dasatinib, which have a broad spectrum of protein et al., 2009). The dosing schedule of KW-2449 was targets, might be a consideration as a therapeutic reported to be suboptimal for Phase I testing, leading to alternative for treatment of mutant FLT3-positive termination of a Phase I trial. AML. Generally, at best, partial and transient clinical A Phase I trial, which has not yet started recruiting responses are observed in early phase clinical trials with patients, will investigate the combination of dasatinib inhibitors of FLT3. Among these are the broad- and all-trans retinoic acid in relapsed/refractory elderly spectrum inhibitors 3-substituted indolinone SU5416 AML patients. An ongoing Phase I/II trial is investigat- (Semaxanib, SuGen, New York, NY, USA) (Yee et al., ing dasatinib in patients with newly diagnosed core- 2002; Fiedler et al., 2003; Giles et al., 2003; O’Farrell binding factor AML.

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5123 Another multi-targeted inhibitor, the biaryl urea can either be used effectively alone or combined with compound, (BAY 43-9006, Nexavar; Bayer other agents to suppress disease progression and pro- AG, Leverkusen, Germany), which was initially developed long lifespan. as a RAF inhibitor and has been FDA approved for There are multiple factors that have been identified as advanced renal cancer and advanced liver cancer, has possible underlying causes of resistance to FLT3 inhibitors. activity against VEGFR-2, VEGFR-3, PDGFRb,andKIT, A schematic of resistance mechanisms is shown in Figure 3. FLT3-ITD and D835G (Auclair et al., 2007; Lierman et al., 2007; Zhang et al., 2008). Sorafenib potently inhibits FLT3-ITD with an IC50 in the low nanomolar range FLT3 mutations that interfere with drug binding (Auclair et al., 2007; Kancha et al., 2007). In a Phase I An important mechanism of resistance to FLT3 clinical trial for patients with refractory or relapsed AML, inhibitors is acquired point mutations in the molecular sorafenib reduced the percentage of leukemia blasts in the targets (Shah et al., 2002; Cools et al., 2003). For bone marrow and peripheral blood of FLT3-ITD-positive example, resistance to PKC412 in patients has been AML patients (Zhang et al., 2008). Compassionate use of attributed to preexisting or acquired mutations in the sorafenib as monotherapy before or after allogeneic stem kinase domain of FLT3 (Heidel et al., 2006). This is cell transplantation in relapsed or refractory FLT3-ITD- reminiscent of the most frequent mechanism of resis- positive AML induced clinically significant and rapid tance to the ABL inhibitor, imatinib: point mutations in responses in patients (Metzelder et al., 2009). the BCR-ABL gene. Such point mutations result in A phase II trial investigating the efficacy of sorafenib amino-acid changes in the catalytic domain of the BCR- plus standard primary therapy for newly diagnosed ABL protein that reduce the ability of imatinib to bind elderly AML patients has been completed. There are and inhibit the tyrosine kinase activity of the enzyme several ongoing Phase I or Phase I/II trials testing (Gorre et al., 2001). sorafenib in combination with other agents, such as A screening assay used to study resistance profiles of idarubicin, cytarabine, clofarabine or vorinostat in three FLT3 inhibitors, PKC412, SU5614 and sorafenib, poor-risk or refractory or relapsed AML. One Phase I showed nonoverlapping mechanisms of resistance for trial, which has not yet started recruiting patients, the three inhibitors (von Bubnoff et al., 2009), which is will investigate granulocyte colony stimulating factor in contrast to the overlapping resistance profiles (G-CSF) and Plerixafor plus sorafenib for mutant displayed for ABL inhibitors, namely imatinib, FLT3-positive AML. and dasatinib, which, for instance, all show resistance to A novel and potent second-generation FLT3 inhibi- the T315I gatekeeper mutation (Deininger et al., 2005; tor, AC220 (Chao et al., 2009; Zarrinkar et al., 2009), Von Bubnoff et al., 2006; Ray et al., 2007). Specifically, exhibits higher potency and selectivity when compared FLT3-ITD-expressing cells resistant to PKC412 only with first-generation FLT3 inhibitors CEP-70, MLN- showed mutations within tyrosine kinase domain 1 at 518, PKC412, sorafenib and sunitinib. In animal residue N676 (von Bubnoff et al., 2009), which has models, AC220 showed efficacy at 1 mg/kg administered previously been identified in a PKC412-resistant AML orally, once daily. AC220 is presently under investiga- patient as the sole cause of drug resistance in the patient tion in an ongoing Phase II trial investigating its efficacy (Heidel et al., 2006). FLT3-ITD-expressing cells resis- as monotherapy in FLT3-ITD-positive AML, as well as tant to SU5614 were characterized mainly by mutations an ongoing Phase I trial studying its effects in relapsed/ in tyrosine kinase domain 2, predominantly in the D835 refractory AML patients harboring wild-type as well as residue (von Bubnoff et al., 2009), and FLT3-ITD- mutant FLT3. expressing cells resistant to sorafenib were unique to A listing of these inhibitors, as well as additional sorafenib in their resistance mechanisms, and included novel FLT3 inhibitors that are in preclinical develop- the F691L mutation in tyrosine kinase domain 1 and muta- ment, is provided in Table 1. tions in the Y842 residue in tyrosine kinase domain 2 (von Bubnoff et al., 2009). PKC412, sunitinib and sorafenib, respectively, were generally able to override SU5614-resistant mutations in tyrosine kinase domain 2; Causes of resistance to FLT3 inhibitors however, the Y842D mutation was highly resistant to sorafenib (von Bubnoff et al., 2009). In an independent The development of resistance in leukemia patients to study, the FLT3 inhibitor, SU5614, exhibited 7- to 26- treatment with targeted tyrosine kinase inhibitors is a fold more activity toward FLT3-ITD-expressing cells growing area of concern. Generally, FLT3 inhibitors than FLT3-ITD-positive cells additionally harboring tested thus far clinically induce only partial and specific protein tyrosine kinase domain mutations transient responses in patients when used as single (D835N and Y842H) acquired during selection in agents. As an example, Phase I testing of KW-2449 culture with increasing concentrations of SU5614 showed only transient inhibition of FLT3 in patients to (Bagrintseva et al., 2004). o20% of baseline (Pratz et al., 2009). It is anticipated Different inhibitors show varying potencies toward that the limited clinical efficacy of FLT3 inhibitors may ITD mutations and activation loop mutants. For be due to their inability to elicit sustained, complete instance, sorafenib is more effective against FLT3-ITD responses in patients (Chu and Small, 2009). This than FLT3-D835Y, whereas sunitinib is equipotent toward suggests a need for development of novel agents that both mutants (Kancha et al., 2007). MLN518 shows widely

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5124 Table 1 FLT3 inhibitors in clinical and preclinical development for treatment of AML FLT3 inhibitor Chemical name IC50 (mutant IC50 (wt FLT3* References FLT3, cell or mutant FLT3 proliferation, nM) phosphorylation, nM)

PKC412 N-Indolocarbazole 10–12 13–30 Weisberg et al., 2002; Zarrinkar et al., 2009 CEP-701 Indolocarbazole alkaloid 2.1–5 1.5–3 Levis et al., 2002; Zarrinkar et al., 2009 SU5416 3-Substitued indolinone 250 100 Yee et al., 2002 SU11248 Indolinone derivative 10–60 34–50 O’Farrell et al., 2003a; Zarrinkar et al., 2009 MLN518 Piperazinyl quinazoline 10–60 B33–100 Kelly et al., 2002b; Zarrinkar et al., 2009 KW-2449 — 11–24 144 Shiotsu et al., 2009; Pratz et al., 2009 AC220 N-(5-Tert-butyl-isoxazol-3-yl)-N0-{4-[7- 0.56 1.1 Chao et al., 2009; (2-morpholin-4-yl-ethoxy)imidazo[2,1-b] Zarrinkar et al., 2009 [1,3]benzothaizol-2-yl]phenyl}urea dihydrocholirde ABT-869 N-(4-(3-Amino-1H-indoazol-4-yl)phenyl)- 4–6 o1 Albert et al., 2006; N1-(2-fluoro-5-methylphenyl)urea Shankar et al., 2007 CHIR-258 Benzimidalzole-quinoline 13 1 Lopes de Menezes et al., 2005 Sorafenib Biaryl urea 0.87–0.88 2–2.28 Auclair et al., 2007; Zarrinkar et al., 2009 LS104 Hydroxystyryl-acrylonitrile 3–4000 B100 Kasper et al., 2008 AP24534 — 13 1 Rivera et al., 2008 AG1296 Quinoxaline 500–800 1000 Tse et al., 2001, 2002 D-65476 (5-Butanotate-1 H-2-indolyl)(1H-2- 200–300 B300 Teller et al., 2002 indolyl)-methanone Compounds 11–14 Tricyclic quinoxaline 1000–3000 B300–3000* Gazit et al., 2003 GTP-14564 1-Phenyl-3-H-8-oxa-2,3-diaza-cyclopen- p1000 300 Murata et al., 2003 ta[a]inden Ki23819 Quuinoline urea o1–3 1–3 Komeno et al., 2005 KRN383 Quinoline-urea derivative o2.9 o5.9 Nishiyama et al., 2006 FI-700 2,4,5-Trisubstituedpyrimidine 14 20 Kiyoi et al., 2007 Ki11502 Quinoline 500–600 37.54 Nishioka et al., 2008 C-4 modified analogs 5-(1,3,4-Oxadiazol-2-yl)pyrimidene 3.2–588 4.8–515 Ishida et al., 2008 derivatives C-5 modified analogs 5-(1,3,4-Oxadiazol-2-yl)pyrimidine 19–78 ND Ishida et al., 2008 derivatives NVP-AST487 N,N0-Diphenyl urea 120 o5 Weisberg et al., 2008b Compounds 1g-n Pyrimido-diazepines ND 2–16* Gracias et al., 2008 Compounds 6–16 4-Amino-6-piperazin-1-yl-pyrimidine- 52 to >1000 1–1370 Gaul et al., 2007 5-carbaldehydoximes Compounds 20–29 4-Amino-6-piperazin-1-yl-pyrimidine- 18–260 58–554 Gaul et al., 2007 5-carbaldehydoximes Compounds 25 2-Acylaminothiopene-3-carboxamides 340 42 Patch et al., 2006

Abbreviations: AML, acute myeloid leukemia; FLT3, Fms-Like Tyrosine kinase-3; ND, not determined. Asterisk refers to wt FLT3 phosphorylation.

variable activity in terms of inhibition of FLT3, STAT5 molecular pathways that lead to disease development. and ERK phosphorylation when tested against a panel Importantly, FLT3 mutations have been found in only a of FLT3 activation loop mutants, including D835Y, fraction of the AML cells, suggesting that they may be D835A, D835E, D835H, D835N, D835V, D835del and disease-promoting rather than initiating. Dysregulated I836del, with IC50s ranging from 500 nM to more than pathways different from FLT3 signaling may therefore 10 mM (Clark et al., 2004). In contrast, PKC412 shows contribute to the growth of AML cells even during relatively uniform and potent activity toward FLT3 FLT3-targeted approaches. activation loop mutants (Barry et al., 2007). FOXO3A. AML patients harboring FLT3 mutations Aberrant activation of growth and viability pathways have been found to have higher levels of phosphoprotein Previous models have suggested that AML is caused by expression of the forkhead transcription factor, FOX- two types of mutations (two hits): one that could O3A (Kornblau et al., 2010). This correlates with a promote growth and viability (type I), and another that poorer prognosis: higher rates of primary drug resis- could block terminal differentiation (type II). However, tance and decreased duration of remission (Kornblau results from full genome sequencing indicate that AML et al., 2010). Indeed, FLT3-ITD has been implicated in is initiated by up to 20 different mutations and this the negative regulation of the FOXO3A transcription simplified model may not sufficiently explain the factor and promotion of cell viability by blocking

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5125 Drug-resistant leukemic cell Plasma membrane

P-glyco- FLT3 FLT3 protein STAT receptor ligand mutation + activity Drug N-Ras + resistance IAPs + transformation BCl2 + P27kip1, Bim P family proteins BAD P c-Cbl Nucleus ITD or Foxo3A + point mutation + Pim-1 mutation apoptosome leukemic cell- stromal cell interactions FLT3 receptor cytokines

FLT3 stromal inhibitor cells Figure 3 Drug-resistant leukemia cell.

FOXO3A-mediated apoptosis (Scheijen et al., 2004). of drug-resistant cells characterized by continued Specifically, FLT3-ITD expression in the murine Ba/F3 activation of parallel downstream PI3K/Akt and/or cell line leads to phosphorylation of FOXO3A, which Ras/MEK/MAPK signaling pathways, which likely stimulates translocation of FOXO3A from the nucleus compensates for the loss of FLT3 activity in terms into the cytoplasm and correlates with inhibition of of survival and growth (Piloto et al., 2007). Inhibition FOXO target genes cylcin-dependent kinase inhibitor of these signaling pathways in part reverses resistance to p27kip1 and proapoptotic Bim (Scheijen et al., 2004). FLT3 inhibition (Piloto et al., 2007). N-Ras mutations FLT3-ITD expression also correlates with phosphoryla- have also been found to evolve as part of the mechanism tion and activation of ERK1/2, STAT5 and Akt of resistance in FLT3 inhibitor-resistant cells (Piloto (Scheijen et al., 2004; Brandts et al., 2005). et al., 2007).

STAT5/Pim pathway. Activation of the STAT5 tran- Apoptosome. Apoptosome inhibition represents a me- scription factor is thought to be important for the chanism whereby mutant FLT3-positive leukemic cells transformation of myeloid leukemia cells. In particular, can exhibit chemoresistance. Constitutively activated the STAT5 target gene, PIM, may contribute to kinases, such as mutant FLT3, BCR-ABL and TEL- transformation. In vitro PIM-1 or PIM-2 cooperate with PDGFRb, are able to inhibit the activity of the Apaf-1/ the MYC proto-oncogene and induce cell growth and caspase-9 apoptosome, which mediates mitochondria- viability (Wernig et al., 2008). Thus, Pim may be another related programmed cell death by altering the interac- putative target for mutant FLT3-positive AML, as it has tion between the apoptosome and the apoptosome been found to be significantly downregulated on FLT3 inhibitor, Hsp90b (Kurokawa et al., 2008). inhibition (Kim et al., 2005). Pim-1 expression also correlated with resistance to FLT3 inhibition (Kim et al., Mediators of viability signaling pathways. Aberrant 2005) and has been identified as having a key role in the expression of antiapoptotic proteins is another mechan- antiapoptotic effects of FLT3-ITD signaling through ism of resistance to FLT3 inhibitors. For example, phosphorylation of BAD (Kim et al., 2006). These data increased protein levels of the inhibitor of apoptosis suggest that FLT3-mediated upregulation of Pim-1 (IAP), survivin, as well as activation of STAT1, STAT3 contributes to the antiapoptotic effects of FLT3 signal- and STAT5, were observed in ABT-869-resistant MV4- ing. Evidence exists as well in support of the importance 11 cells (Zhou et al., 2009), and antiapoptotic proteins of Pim-2 expression for the survival of mutant FLT3- were upregulated in PKC412-resistant MV4-11 cells expressing cells (Adam et al., 2006). (Stolzel et al., 2010). Similarly, overexpression of antiapoptotic proteins of the BCL2 family has been PI3K/Akt and Ras/MEK/MAPK pathways. Prolonged found to mediate resistance to FLT3 inhibition in FLT3 inhibitor treatment leads to the development hematopoietic cells with activating FLT3 mutations

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5126 (Kohl et al., 2007) and the antiapoptotic protein, MCL- will be important to understand how mutant CBL leads 1, was observed to be induced by a non-juxtamembrane to activation of FLT3 and related pathways to develop ITD that has integrated into the b-2 sheet of the first new therapeutic approaches. kinase domain (FLT3_ITD627E) (Brietenbuecher et al., 2009). In association with expression of FLT3-ITD FLT3 ligand and tyrosine kinase domain mutation dual mutants, FLT3 ligand expression was observed to be upregulated which occurs in 1–2% of mutant FLT3-positive in MV4-11 cells made resistant to the FLT3 inhibitor, patients, there is induction of tyrosine kinase inhibitor ABT-869, for a prolonged period of time (Zhou et al., and daunorubicin resistance through hyperactivation 2009). This elevated expression led to increased protein of STAT5 and consequent upregulation of Bcl-x(L) levels of the IAP, survivin, as well as activation of (Bagrintseva et al., 2005). Similarly, strong phospho- STAT1, STAT3 and STAT5. In addition, blocking rylation of Bcl-2 antagonist of cell death (BAD) has FLT3 ligand with an FLT3 ligand-neutralizing antibody been observed in mutant FLT3– and constitutively was observed to enhance the proapoptotic activity of activated wild-type FLT3-expressing AML cells; siRNA ABT-869 against ABT-869-resistant cells (Zhou et al., knockdown of BAD expression in FLT3-ITD-expres- 2009). Another study of resistance in MV4-11 cells made sing cells confers resistance to CEP-701 (Kim et al., resistant to PKC412 revealed acquisition of clonal 2006). alternations at chromosome 13q, additional FLT3 signaling, and changes in gene expression before and Gain-of-function mutations in CBL after treatment with PKC412, including that of FLT3 Normally, FLT3 signaling is negatively regulated in part ligand (Stolzel et al., 2010). by the E3 ubiquitin ligase CBL. Phosphorylated FLT3 The concentration of FLT3 ligand has been observed or related RTKs are recognized by the tyrosine kinase- to be elevated in the plasma of cancer patients that binding domain (TKB) of CBL, leading to mono- or have been treated with chemotherapy or radiotherapy polyubiquitination of the target protein. This modifica- (Lyman et al., 1995; Wodnar-Filipowicz et al., 1996; tion results in altered function or reduced stability Zwierzina et al., 1999; Bojko et al., 2002). In the case (Thien and Langdon, 2005). Recently, CBL has been of AML, this is predicted to impede the efficacy of FLT3 identified as a frequent target of gain-of-function inhibitors given after chemotherapy. This phenomenon mutations in several myeloid neoplasias, including has been replicated in animal models: Significantly juvenile and chronic myelomonocytic (JMML elevated FLT3 ligand levels were observed in the serum and CMML), AML, myelodysplastic syndromes and of mice treated with the chemotherapeutic agent myeloproliferative neoplasms (Caligiuri et al., 2007; busulphan (Molyneux et al., 2008). Sargin et al., 2007; Dunbar et al., 2008; Grand et al., 2009; Loh et al., 2009; Makishima et al., 2009; Reindl et al., 2009; Sanada et al., 2009). Mutations between the Protection by the stromal cell microenvironment TKB and ring-finger domains, associated with acquired Bone marrow stroma and stromal cell-derived factors 11q uniparental disomy (Dunbar et al., 2008; Grand have been implicated in the long-term survival and et al., 2009; Makishima et al., 2009; Sanada et al., 2009), growth of various hematological malignancies, includ- lead to a loss of the E3 ligase activity (Sargin et al., ing precursor B-ALL ((Ashley et al., 1994; Bradstock 2007). Even though these mutations require the expres- et al., 1996). Bone marrow stroma has also been shown sion of a functional RTK for factor-independent growth to prevent programmed cell death of AML and chronic (Sargin et al., 2007; Grand et al., 2009), it is not known lymphocytic leukemia cells (Lagneaux et al., 1998, 1999; whether a loss of E3 activity is sufficient for this Konopleva et al., 2002). In addition, splenic stroma and phenotype. In this context, it should be noted that mice splenocytes support the viability and proliferation of with CBL gene disruption are not known to be associated both normal and malignant hematopoiesis (Shaked with ligand-independent RTK activation (Murphy et al., et al., 2005; Despars and O’Neill, 2006). Furthermore, 1998; Naramura et al., 1998). Thus, even though mutated leukemic lymphoblasts that are coupled to bone marrow CBL gains a new function by supporting transformation stroma through gap junction communication are believed of hematopoietic cells, the loss of its E3 ligase activity to be held in a quiescent, nondividing state, which is may be essential but not sufficient for this phenotype. In thought to contribute to resistance to antimitotic agents normal signaling, CBL is not known to be involved in (Paraguassu´-Braga et al., 2003). ligand-independent receptor activation and it is likely that A flow cytometry-based assay system demonstrated this new putative function alters its interaction with enhancement of CD34( þ )CD38(–)CD123( þ ) leukemic signaling partners. stem and progenitor cell survival in response to Transforming forms of CBL have the potential to treatment with Ara-c and the FLT3 inhibitor, interact with different RTKs and other signaling AG1296, respectively, when cells were drug treated molecules. For example, Sanada et al. (2009) recently under defined ‘niche-like’ (or microenvironmental sup- suggested that cells in the absence of CBL are hyperpro- port) conditions (Mony et al., 2008). The defined ‘niche- liferative in response to , , like’ microenvironment in this study was comprised of 3 (IL-3) and FLT3 ligand. Targeting FLT3 serum-free medium supplemented with IL-3, IL-6, stem alone may therefore still lead to activation of CBL- cell factor and Ang-1, and also included immobilized dependent pathways that increase growth or viability. It fibronectin. Contrary to the drug resistance observed

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5127 with Ara-c and AG1296 in the presence of a ‘niche-like’ tyrosine kinase domain-1 and the residual tyrosine kinase microenvironment, AML cells were more sensitive to domain-1), length of the ITD and influence on patient inhibitors of signaling pathways, such as the MAPK prognosis (Kayser et al., 2009). The integration of the inhibitor, U0126, the JAK-STAT inhibitor, AG490, and ITD in the b-1 sheet was found to be a negative the PI3K inhibitor, Wortmannin, under the same ‘niche- prognostic indicator in terms of survival and remission, like’ conditions. and thus is believed to have a role in resistance to In nilotinib-treated mice, high tumor burden/residual chemotherapy (Kayser et al.,2009). disease has been identified in tissues characterized as significant sources of hematopoiesis-promoting stroma; FLT3-ITD and genomic instability. Little is known the pattern of leukemia distribution in mice was about the molecular mechanisms that regulate genomic consistent with what is observed in imatinib/nilotinib- instability in AML, but active forms of FLT3 are treated CML patients (Weisberg et al., 2008c). Stromal thought to promote the occurrence of additional protection of leukemic cells from the inhibitory effects mutations. These mutagenic events are caused by a of nilotinib and PKC412 has been observed, and two-phase process, whereby initially DNA is damaged, stromal-secreted viability factors have been identified, and then in a second step the damage is failed to be including IL-6 and GM-CSF, as possibly having a role appropriately repaired. In addition to exogenous fac- in stromal-mediated cytoprotection of tyrosine kinase tors, oxidative stress has been recognized as a significant inhibitor-treated leukemia cells (Weisberg et al., 2008c, contributor to DNA damage. Intracellular reactive 2009). These results may help to explain why FLT3 oxygen species (ROS) have the potential to damage inhibitors such as CEP-701 and PKC412 are clinically DNA, leading to direct oxidation or DNA strand breaks able to deplete blasts in the peripheral blood, but are less (Rodrigues et al., 2008). Consistent with this model, effective against blasts in the marrow (Smith et al., 2004; FLT3-ITD has been shown to induce ROS production, Stone et al., 2005a, b). as well as DNA damage and decreased fidelity of DNA repair (Sallmyr et al., 2008). Cells transformed by FLT3- Pgp ITD and other oncogenic tyrosine kinases are also Antiapoptotic p-glycoprotein (pgp), a membrane efflux associated with increased mutagenic single-strand an- pump, is another putative target for drug-resistant, nealing (SSA), a repair process that involves sequence mutant FLT3-positive AML. Pgp expression in FLT3- repeats (Cramer et al., 2008; Fernandes et al., 2009). ITD-positive cells reduces the activity of inhibitors such Mutagenic SSA depends on the transforming kinase as herbimycin A and AG1296 (Hunter et al., 2004). activity but may be increased by stromal-derived factors, Contrary to this, however, PKC412 showed activity even in the presence of kinase inhibitors (Fernandes against pgp- and FLT3-ITD-positive cells (Hunter et al., et al., 2009). An increased rate of mutagenic events is 2004), which suggests that pgp expression only contri- likely to be the cause of drug resistance associated with butes to drug resistance involving certain select inhibitors. disease progression in AML. The particular defect that is involved in the mechanism causing drug resistance may be difficult to pinpoint and it is possible that a Mutant FLT3 and resistance to chemotherapy.Thereis combination of events are causal factors. evidence in support of mutant FLT3 itself contributing to resistance of AML to chemotherapeutic agents. For instance, in a study in which FLT3-ITD was introduced into myeloid cell lines, FLT3-ITD expression was Approaches to overriding drug resistance observed to correlate with Ara-c resistance due to decreased cellular uptake of Ara-c (Jin et al., 2009). Drug Combined treatment using FLT3 inhibitors and resistance was accompanied by downregulation of a chemotherapeutic agents transporter responsible for Ara-C cellular uptake, equili- It has been shown that, in at least some primary AML brative nucleoside transporter 1 (ENT1), possibly cells, there is continued phosphorylation of ERK, mediated by upregulation of hypoxia-inducible factor 1 STAT5 or AKT following inhibition of FLT3-ITD, alpha subunit (HIF1A) (Jin et al., 2009). Pretreatment which may contribute to the limited efficacy of FLT3 with PKC412 reversed the reduced cellular uptake of Ara-c inhibitors used as single agents for the treatment of in FLT3-ITD-expressing myeloid cells (Jin et al., 2009). mutant FLT3-positive AML (Siendones et al., 2007). In Also supporting the notion of resistance of FLT3-ITD addition, it has recently been shown that FLT3-ITD to chemotherapy is an investigation assessing mutant inhibition of autophosphorylation does not always FLT3 expression in a group of Korean patients, in which result in cell death, which raises the question of the FLT3-ITD was detected in a subset of patients that failed importance of FLT3 signaling for some mutant FLT3- to achieve remission (Bang et al., 2008). In contrast, 93 positive AML (Pratz et al., 2010). patients that were in remission tested negative for FLT3- The effectiveness of combined treatment using an ITD expression (Bang et al., 2008). FLT3 inhibitor and chemotherapeutic agents may, at Finally, an extensive characterization of ITDs in a least in part, rest in the ability of FLT3 inhibitors, group of AML patients was carried out in terms of their such as PKC412, to inhibit repair of chemotherapy- site of insertion (that is, juxtamembrane domain, induced DNA damage, in effect reversing the drug- juxtamembrane domain hinge region, b-1 sheet of the resistant phenotype of mutant FLT3-expressing cells

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5128 and diminishing the risk of relapse (Seedhouse et al., structure of second mitochondria-derived activator of 2006). Indeed, preclinical studies have shown that the caspase (Smac), which mediates programmed cell death FLT3 inhibitor, SU11657, increases the median survival through the intrinsic apoptotic pathway (Du et al, 2000) of leukemic mice when used in combination with and binds to and inhibits the IAP family of proteins doxorubicin (Lee et al., 2007). In related manner, (Liu et al., 2000; Wu et al., 2000). FLT3 inhibitors such as PKC412 and CEP-701 are Recent study has indicated that IAP inhibitors can being investigated in late-stage clinical trials for AML in target multiple IAPs (that is, XIAP, c-IAP) to enhance conjunction with the use of standard chemotherapy. the activity of different proapoptotic signaling pathways (Galban et al., 2009). However, the role of XIAP in intrinsic versus extrinsic death pathways is unclear; Utilization of signaling pathway inhibitors recent studies suggest that it has a more significant role The combination of selective inhibitors of mediators of in regulating death receptor-mediated apoptosis than major signaling pathways with FLT3 inhibition offers intrinsic pathway-mediated cell death (Sensintaffar considerable potential clinical benefit. For example, the et al., 2010). dual PI3K/PDK-1 inhibitor, BAG956 (Weisberg et al., Accordingly, effective IAP inhibitors have been devel- 2008a), the farnesyl-transferase inhibitor, lonafarnib oped, such as LBW242 (Weisberg et al.,2007),which (Mollgard et al., 2008) and rapamycin (Mohi et al., bind to and inhibit multiple IAPs (that is, XIAP, c-IAP) 2004) have the ability to effectively combine with to enhance the activity of different proapoptotic signaling PKC412 to kill drug-sensitive and insensitive mutant pathways. The development of the proapoptotic IAP FLT3-expressing cells. The rapamycin derivative, inhibitor, LBW242 (Novartis), a 3-mer and Smac RAD001, augments the antileukemic activity of the mimetic, was based on the ability of the N-terminal FLT3 kinase inhibitor, sunitinib (Ikezoe et al., 2006), as seven amino acids of Smac to neutralize the BIR3 does the MEK1/2 kinase inhibitor, AZD6244 (ARRY- domain of XIAP (Liu et al.,2000;Wuet al.,2000). 142886) (Nishioka et al., 2008). The multiple kinase LBW242 was the result of a rational drug design effort inhibitor, KP372-1, inhibits the kinase activity of AKT, based on Smac 4-mer, and has been found to display nM PDK1 and FLT3, and kills AML cells in a manner potency against XIAP and CIAP1 in a competitive mediated by Pim-1 downregulation (due to FLT3 binding assay with Smac 7-mer. LBW242 also kills cells inhibition), mitochondrial dysfunction, and inhibition in a manner strictly dependent on caspases, and death is of phosphorylation of downstream PI3K/AKT signaling accompanied by PARP cleavage, annexin positivity and components, such as p70S6 kinase (Zeng et al., accumulation of cells in subG1. The ability of LBW242 2006b). to synergize with PKC412 has been demonstrated in vivo against progressive mutant FLT3-positive leukemia and Targeting bone marrow microenvironment to override stromal-mediated chemoresistance in vitro In addition to identifying and developing potent kinase (Weisberg et al.,2007). inhibitors representative of novel and unique structural Another strategy for overriding stromal-mediated classes with the ability to override drug resistance due to chemoresistance is the use of inhibitors of the chemo- changes in the target protein, there is a push toward kine receptor, CXCR4, which mediates the migration of gaining a better understanding of the mechanisms hematopoietic cells to the bone marrow and has a key underlying drug resistance in CML and AML as they role in leukemic cell–stromal cell interactions. Mutant relate to the leukemic cell microenvironment. Clinical FLT3 activates CXCR4 signaling, and CXCR4 inhibi- trial data with tyrosine kinase inhibitors show that tion partially overcomes stromal-mediated cytoprotec- although the peripheral blood of patients responds well, tion of FLT3 inhibitor-treated AML cells (Zeng et al., bone marrow responds less well (Smith et al., 2004; 2009). In vivo, the CXCR4 inhibitor, AMD3465, Stone et al., 2005a, b). It appears possible that small lowered leukemia burden and enhanced survival numbers of leukemic CD34 þ cells can persist in the through mobilization of AML cells and progenitor cells marrow microenvironment of leukemia patients after into circulation, and potentiation of the antileukemic years of therapy with kinase inhibitors. Stromal cells effects of the FLT3 inhibitor, sorafenib, and chemother- have been implicated, as they provide viability signals to apy (Zeng et al., 2009). The polypeptide, RCP168, which leukemic cells that protect them from inhibitor effects. antagonizes SDF-1a- or stromal cell-induced chemo- Indeed, the quantity of leukemic stem cells that rely on taxis of leukemic cells, was also shown to significantly stroma to survive is predictive of disease outcome augment chemotherapy-induced apoptosis in stroma- (Kumagai et al., 1996). Thus, deregulated signaling cocultured mutant FLT3-expressing cells (Zeng et al., molecules associated with viability/apoptotic signaling 2006a). Another promising strategy would be to target represent attractive targets for therapeutic intervention, pathways that regulate receptor recycling. A recent and several strategies have emerged that may be paper (Grundler et al., 2009) suggests that PIM kinase effective in preventing drug resistance due to this. mediates surface expression of CXCR4, which suggests One approach involves combining targeted inhibitors that targeting PIM/FLT3 would be a more efficient with small molecule inhibitors of key components of therapy for targeting the bone marrow microenviron- major signaling pathways affecting the viability/expan- ment. Thus, the use of CXCR4 inhibitors may be a sion of leukemic cells, such as agents that block the potentially useful approach to overriding stromal- activity of IAPs. These inhibitors are based on the mediated chemoresistance.

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5129 Antibody and siRNA therapy duplication of amino-acid residues Y591-Y597, which Target-specific antibodies represent one approach to encodes the switch and zipper regions of the juxtamem- overriding drug resistance by potentially enhancing the brane domain of FLT3 (Meshinchi et al., 2008). The efficacy of FLT3 inhibitors. For instance, the novel longer length of ITDs in these patients was associated FLT3-directed antibody, IMC-EB, has been shown to with a worse relapse-free survival (Meshinchi et al., inhibit growth of leukemia cells expressing wild-type or 2008). constitutively activated FLT3 (Youssouflan et al., 2010). Importantly, pediatric AML primary samples with Another therapeutic approach would be the use RNA FLT3-ITD mutations were found to be preferentially interference to knockdown FLT3 expression. For killed by FLT3 inhibition (Brown et al., 2004). FLT3 example, FLT3-targeted siRNA has been shown to inhibitors, such as PKC412, have been tested mainly in induce apoptosis in FLT3-ITD-expressing cells and to adults and have shown clinical activity, although as increases the activity of the FLT3 inhibitor, MLN518 described above, this is generally in the form of transient (Walters et al., 2005). However, the difficulties to reductions in blast counts in peripheral blood or bone efficiently deliver these molecules in vivo have not yet marrow. There are presently ongoing Phase I/II clinical been overcome. trials studying the safety and preliminary efficacy of oral PKC412 in relapsed or refractory pediatric leukemia (children under the age of 18 years), including patients Drug resistance in mutant FLT3-positive pediatric AML with mutant FLT3-positive AML. Drug development studies in pediatric AML, however, are generally As pediatric AML has a poor prognosis, novel therapies complicated by the small number of patients available are needed. A potentially clinically significant target in for such studies, as well as the lack of interest on the part pediatric AML is the FLT3 tyrosine kinase, as FLT3- of pharmaceutical companies to carry out pediatric ITD or FLT3 point mutants are found in 17–24% of studies due to the existence of a small market. This pediatric AML cases and FLT3-ITD mutations confer a reality makes it crucial to optimize new therapeutic particularly poor prognosis in pediatric AML patients strategies for AML that are being explored in adults, (Stirewalt and Radich, 2003). Constitutively activated and that may soon be of interest and importance to FLT3 occurs most frequently as ITD mutations, and pediatric patients. is observed in B15% of pediatric AML patients and 20–25% of adult AML patients (Nakao et al., 1996). As with adults, the prevalence of point mutation of the Conclusion activation loop domain in pediatric AML patients is 7% (Meshinchi et al, 2003). There is a pressing need for the identification and In a large pediatric study involving 630 children with development of new therapeutic approaches that could de novo AML, FLT3-ITD mutations were found in 12% lead to improved clinical responsiveness in AML of the patients, and FLT3 activation loop domain patients. A better understanding of mechanisms of B mutations were observed in 7% of patients, with resistance to FLT3 inhibitors, such as those associated poorer progression-free survival associated with FLT3- with stromal-mediated cyto-protection and upregulation ITD (Meshinchi et al., 2006). In a study involving 234 of antiapoptotic signaling factors, will aid in the pediatric AML patients, FLT3-ITD mutations were discovery and design of more effective treatment found in 11.5% of patients who were classified as strategies. Novel approaches to overriding drug resis- significantly older with lower remission induction rates tance include antibody therapy, and the combined use of and lower 5-year probability rates of event-free survival FLT3 inhibitors with inhibitors of viability signaling or (Zwaan et al., 2003). High ratios between mutant and components of PI3K/Akt, MAPK and JAK/STAT wild-type FLT3 appeared to worsen prognosis in these pathway signaling. Each novel strategy carries with it patients (Zwaan et al., 2003). However, although FLT3- the potential to bring clinicians closer to overriding ITD proved to be a strong adverse prognostic factor existing challenges in the treatment of mutant FLT3- in this study, it did not appear to be associated positive AML. with increased cellular drug resistance in vitro (Zwaan et al., 2003). Clinical outcome has been found to be worse for pediatric AML patients harboring CD34( þ )CD33(À) precursors that are FLT3-ITD Conflict of interest positive, suggesting that FLT3-ITD expression in this primitive cell population may be coupled to resistance The authors declare no conflict of interest. (Pollard et al., 2006). The physical characteristics of the ITD in mutant FLT3-positive pediatric patients may have a role in Acknowledgements response to therapy. In a study of 77 FLT3-ITD- positive pediatric patients, 77% had a single ITD, 21% This work is supported in part by the National Institutes had 2 ITDs and 3% had 3 ITDs; the presence of >1 of Health Grants (CA134660-02, MS; and CA36167 and ITD was not found to be clinically significant DK50654, JDG), and a Leukemia and Society (Meshinchi et al., 2008). In all cases, there was SCOR grant (JDG).

Oncogene Mechanisms of resistance to FLT3 inhibition E Weisberg et al 5130 References

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