ITD Confer Resistance to Multiple Tyrosine Kinase Inhibitors

Home , Lestaurtinib, Midostaurin, Sunitinib

ORIGINAL ARTICLE Mutations of FLT3/ITD confer resistance to multiple tyrosine kinase inhibitors

AB Williams1, B Nguyen1,LLi1, P Brown1,3, M Levis1, D Leahy2 and D Small1,3

FMS-like tyrosine kinase 3 (FLT3) normally functions in the survival/proliferation of hematopoietic stem/progenitor cells, but its constitutive activation by internal tandem duplication (ITD) mutations correlates with a poor prognosis in AML. The development of FLT3 tyrosine kinase inhibitors (TKI) is a promising strategy, but resistance that arises during the course of treatment caused by secondary mutations within the mutated gene itself poses a significant challenge. In an effort to predict FLT3 resistance mutations that might develop in patients, we used saturation mutagenesis of FLT3/ITD followed by selection of transfected cells in FLT3 TKI. We identified F621L, A627P, F691L and Y842C mutations in FLT3/ITD that confer varying levels of resistance to FLT3 TKI. Western blotting confirmed that some FLT3 TKI were ineffective at inhibiting FLT3 autophosphorylation and signaling through MAP kinase, STAT5 and AKT in some mutants. Balb/c mice transplanted with the FLT3/ITD Y842C mutation confirmed resistance to sorafenib in vivo but not to lestaurtinib. These results indicate a growing number of FLT3 mutations that are likely to be encountered in patients. Such knowledge, combined with known remaining sensitivity to other FLT3 TKI, will be important to establish as secondary drug treatments that can be substituted when these mutants are encountered.

Leukemia (2013) 27, 48–55; doi:10.1038/leu.2012.191 Keywords: acute myeloid leukemia; mutant FLT3; drug resistance; tyrosine kinase inhibitors

INTRODUCTION stimulus and frequent mutation rate in AML, FLT3 has been FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine deemed as a highly desirable target for modulation. kinase that is expressed on early hematopoietic stem cells/ The impressive response of chronic myelogenous leukemia progenitors and is vital for development of normal levels of patients to BCR–ABL TKI generated enthusiasm for molecularly mature myeloid and lymphoid cells.1,2 FLT3 is normally activated targeted therapies in other malignancies dependent on constitu- by binding FLT3 ligand (FL), which prompts receptor dimerization tively activated kinase signaling. However, the development of and kinase activity with the subsequent activation of multiple resistance to imatinib due to the acquisition of point mutations in downstream signaling pathways, including Ras/MAP kinase, BCR–ABL also foreshadows a similar outcome now being reported AKT and STAT5.3–6 High coexpression of FLT3 with FL may lead in AML patients expressing a FLT3/ITD mutation being treated to dysregulation of activity via autocrine or paracrine mechanisms with FLT3 TKI.14–16 Resistance mutations often decrease the that promote leukemogenesis, as has been observed in MLL- affinity of a TKI for its target and necessitate the use of a rearranged infant ALL.7–9 Mutation of FLT3 leads to constitutive structurally unrelated inhibitor if one is available. This expectation kinase activation and transfection of these forms leads to transfor- has led to investigations attempting to identify a spectrum of mation of cytokine-dependent cell lines to factor independence. secondary mutations of FLT3/ITD in the laboratory, which confer FLT3 mutations represent one of the most common molecular resistance to FLT3 TKI prior to their emergence in the clinic. perturbations in acute myeloid leukemia (AML), accounting for Several groups have employed various techniques to identify FLT3 30–35% of de novo cases.10–12 FLT3 mutations generally occur in resistance mutations.17–21 In contrast to the wide array of BCR–ABL the juxtamembrane (JM) domain or in the kinase domain (KD). The resistance mutations, relatively few FLT3 resistance mutations JM mutations factor in approximately 23% of newly diagnosed have been identified, which may partially reflect the failure to cases of AML and occur as in-frame internal tandem duplications achieve sufficient levels of inhibition of FLT3 signaling in many (ITDs) of varying length, resulting in duplication of a sequence trials.22 In this study, we identified the F691L and Y842C mutations of typically 4–50 amino acids, often accompanied by a one or two previously identified as well as two novel mutations, F621L and amino-acid insert.10 The crystal structure of FLT3 shows that the A627P, that cause resistance to select TKI. These results suggest JM domain functions as an autoinhibitory mechanism to regulate that novel mutations arising in FLT3/ITD, perhaps by selection FLT3 kinase activity, and disruption by mutations destabilize its during the course of treatment with a TKI, may prove to be conformation.13 KD mutations constitute about 7–10% of AML refractory to FLT3 mutant AML management using most TKIs and cases and usually present as missense mutations of the activation emphasize the need for development of FLT3 inhibitors that can loop, most commonly at D835.11,12 Because of its proliferative overcome resistance due to mutations.

1Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD, USA and 3Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Correspondence: Dr D Small, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. E-mail: [email protected] Received 6 February 2012; revised 18 June 2012; accepted 26 June 2012; accepted article preview online 13 July 2012; advance online publication, 3 August 2012 Resistance to FLT3 tyrosine kinase inhibitors AB Williams et al 49 MATERIALS AND METHODS were conducted in accordance with the policy of the Johns Hopkins Reagents and antibodies University School of Medicine Animal Care and Use Committee. Lestaurtinib, midostaurin, sunitinib, sorafenib and AC220 were purchased from LC Labs (Westchester, PA, USA). KW2449 was from Kyowa Hakko Kirin Structural modeling of FLT3 TKI binding to resistance mutations Co., Ltd. (Tokyo, Japan). AGS324 was provided by Aviv Gazit. Recombinant human interleukin-3 was purchased from Pepro Tech, Inc. (Rocky Hill, NJ, Models of the FLT3 kinase complexed with lestaurtinib or midostaurin USA). FLT3 S-18 and STAT5 antibodies were from Santa Cruz Biotechnology were constructed by superimposing the crystal structure of Lck kinase with staurosporine bound (Protein Data Bank 1QJP)27and superimposed on (Santa Cruz, CA, USA), 4G10 anti-phosphotyrosine antibody and recombi- 13 nant protein A-agarose were from Upstate Biotechnology (Lake Placid, FLT3 (PDB code 1RJB). The resulting staurosporine placement relative to NY, USA) and CD135-phycoerythrin (PE)-conjugated and annexin V-PE FLT3 serves as a suitable substitute for lestaurtinib and midostaurin binding antibodies were from BD Pharmingen (San Jose, CA, USA). PhosphoMAP owing to their related pharmacophore and the high homology of Lck with FLT3. Further conﬁrmation was also provided by comparing lapatinib kinase, phosphoSTAT5, phosphoAKT, MAP kinase and AKT antibodies were 28 from Cell Signaling Technologies, Inc. (Beverly, MA, USA). Goat anti-mouse binding to EGFR (PDB code 1XKK). Likewise, FLT3 binding of sorafenib was and goat anti-rabbit horseradish peroxidase antibodies and the enhanced modeled by superposing the FLT3 kinase structure on the raf kinase with sorafenib bound (PDB code 3GCS)29 and superimposed on the FLT3 crystal chemiluminescence kit were from Amersham Biosciences (Arlington 13,29 Heights, IL, USA). structure. Images were generated and analyzed using PyMol.

DNA constructs and cells RESULTS BaF3 or TF-1 cells were cultured in RPMI medium supplemented with Identification of resistance mutations 1 ng/ml recombinant human interleukin-3 or 1 ng/ml of granulocyte- macrophage colony-stimulating factor, respectively. FLT3/ITD cells were The scheme used to identify FLT3 resistance mutations is provided established from a patient sample as previously described.23 FLT3 point in Figure 1, and is based on a screen to identify resistance mutations were generated by site-directed mutagenesis in the pBabe Neo mutations in BCR–ABL.24 TF-1 cell colonies that grew in the vector containing FLT3/ITD complementary DNA using the QuickChange presence of FLT3 TKI were picked for analysis. TF-1 cells do not Site-Directed Mutagenesis (Stratagene, La Jolla, CA, USA) and used to normally express FLT3, so FLT3 was amplified by primers that transfect BaF3 cells using the Nucleofector II from Amaxa Biosystems covered the complementary DNA sequence and confirmed by (Walkersville, MD, USA). BaF3 cells were chosen for confirmation of sequencing. Cells were selected in methylcellulose at the resistance because we eventually wanted to transplant a resistant clone to syngeneic Balb/c mice and determine whether FLT3 TKI could overcome minimum FLT3 TKI concentrations determined to inhibit growth the mutation in vivo. of the parental FLT3/ITD cell colonies. These concentrations corresponded to 20 nM lestaurtinib, 20 nM midostaurin, 50 nM KW2449 and 100 nM AGS324 following a 2-week treatment. Selection for resistant clones Despite efforts to reduce the frequency of clones generating The cFUGW plasmid containing a FLT3/ITD complementary DNA was used resistance to FLT3 TKI caused by mechanisms unrelated to FLT3 to transform the XL1-Red mutator strain of E. coli as instructed by the mutations, only 9 out of 1500 clones analyzed harbored unique manufacturer (Stratagene).24 Transformed cells were allowed to propagate overnight at 37 1C on agar plates containing ampicillin at which point mutations of FLT3. Of the nine mutations, four were confirmed to plasmids were harvested and linearized by Not I digestion. The purified confer resistance when site-directed mutagenesis was used to products were used for packaging in lentivirus in NIH293T cells. The viral duplicate the isolated mutation in the FLT3/ITD backbone supernatant was used to transduce granulocyte-macrophage colony- followed by transfection into BaF3 cells. The confirmed FLT3 TKI stimulating factor-dependent TF-1 cells. Cells were then plated in cytokine- resistance mutations isolated were: F621L, A627P, F691L and deficient methylcellulose in the presence of a FLT3 TKI for 2 weeks at Y842C. Approximately 600 colonies that were selected in the which point FLT3 in clones of interest was sequenced. presence of lestaurtinib were sequenced and found not to contain resistance mutations. Of B400 colonies that were selected in Growth inhibition and apoptosis midostaurin, the A627P mutation was detected once as was the Cells were seeded in the presence or absence of inhibitor for 48 h with or F691L mutation. Of 300 colonies sequenced from KW2449 without 1 ng/ml of interleukin-3. Cell proliferation/viability was measured treatment, the F621L mutation was identified once and the as described using the MTT assay according to the manufacturer’s Y842C mutation 12 times. Of nearly 200 colonies grown in instructions (Roche Applied Science, Indianapolis, IN, USA) or induction AGS324, the F621L mutation was selected once and the Y842C 25 of apoptosis with annexin V/7-AAD (BD Biosciences, San Diego, CA, USA). mutation was seen 23 times. Each of the isolated mutations was then tested in BaF3 cells for Immunoprecipitation and western blotting the ability to engender resistance against six different FLT3 TKI as FLT3 was analyzed by performing immunoprecipitation, SDS-PAGE and western measured by MTT (growth/survival) assay. Lestaurtinib and blotting as previously described.26 Proteins were transferred to a polyvinyl midostaurin have undergone phase III clinical testing for treat- diflouride membrane (Millipore, Bedford, MA, USA) and probed for FLT3 using ment of FLT3 mutant AML. Lestaurtinib inhibited growth of BaF3 S-18 and 4G10 phosphotyrosine antibodies. FLT3 was then detected on a FLT3/ITD cells in the MTT assay with an IC50 of 2 nM (Figure 2a). The Li-Cor imager using Odyssey software (both from Li-Cor Biosciences, Lincoln, F621L and Y842C mutants displayed no resistance to this inhibitor, NE, USA). Other proteins were detected in whole cell lysates using but the F691L mutant exhibited a fourfold increase in the IC the indicated antibodies and a horseradish peroxidase-conjugated goat 50 (8 nM) to lestaurtinib. The A627P mutant resulted in such a large anti-rabbit secondary antibody followed by enhanced chemiluminescence. shift in the dose–response curve that the IC50 for lestaurtinib was not reached over the concentration range tested. Next, we Engraftment in Balb/c mice examined the effect of lestaurtinib on FLT3 activation in each BaF3 FLT3/ITD Y842C cells were transfected with the L3GFP plasmid (a gift mutant directly by western blotting. Lestaurtinib demonstrated from Dr Linzhao Cheng of Johns Hopkins University) containing genes for inhibitory activity on FLT3 autophosphorylation in all mutants, 5 luciferase and green fluorescent protein (GFP). For engraftment, 2 Â 10 though some residual phosphorylation could be observed at 10 nM cells were injected by tail vein into syngeneic female Balb/c mice (Jackson in the F621L and Y842C mutants (Figures 3a–e). The F691L mutant Laboratories, Bar Harbor, ME, USA) (day 0). Starting 3 days later, mice were treated with 20 mg/kg of lestaurtinib twice daily by subcutaneous displayed resistance to lestaurtinib at 10 nM but was partially injection, with 20 mg/kg sorafenib once daily or with a vehicle control. inhibited at 30 nM (Figure 3d). Thus, the western blotting results Mice were imaged by peritoneal injection of luciferin and visualized on an examining FLT3 inhibition by lestaurtinib for each mutant IVIS Spectrum imager (Caliper LifeSciences, Hopkinton, MA, USA) using correlated well with the MTT data except for the A627P mutant, Living Image software for analysis on days 3 and 8. All animal procedures which showed sensitivity to inhibition of FLT3 phosphorylation by

& 2013 Macmillan Publishers Limited Leukemia (2013) 48 – 55 Resistance to FLT3 tyrosine kinase inhibitors AB Williams et al 50

Miniprep and transfect Random Plate FLT3/ITD 293T Cells mutagenesis overnight Pool

cFUGW

XL1-Red Harvest virus and infect TF-1 Cells

Evaluate for resistance Sequence FLT3

Recapitulate resistance Grow clones in Select colonies in by site-directed liquid culture methylcellulose in the mutagenesis presence of inhibitor Figure 1. Scheme used to identify FLT3/ITD resistance mutations. XL1-Red cells were transformed with the cFUGW plasmid carrying a human FLT3/ITD and grown overnight in agarose. The harvested DNA was used to transduce hematopoietic cells, which were then selected for resistance based on their ability to grow colonies in methylcellulose in the presence of a FLT3 TKI for 2 weeks. The resulting colonies were expanded in liquid culture growth medium, analyzed and sequenced for FLT3 mutations.

140 BaF3/ITD 120 120 120 F621L A627P 100 100 100 F691L Y842C 80 BaF3 ITD 80 BaF3/ITD 80 F621L 60 F621L 60 60 A627P A627P 40 F691L 40 F691L 40 Y842C Y842C 20 20 20 O.D. (% of Control) O.D. (% of Control) 0 0 O.D. (% of Control) 0 0 1020304050 0 1020304050 0 50 100 150 200 Lestaurtinib, nM Midostaurin, nM KW2449, nM

120 120 120 100 100 100 BaF3 ITD 80 80 80 BaF3/ITD BaF3/ITD F621L 60 F621L 60 F621L 60 A627P A627P A627P F691L 40 F691L 40 F691L 40 Y842C Y842C Y842C 20 20 20 OD (% of Control) O.D. (% of Control) 0 O.D. (% of Control) 0 0 0 1020304050 0 1020304050 0 100 200 300 400 500 Sorafenib, nM Sunitinib, nM AGS324, nM

120 100 BaF3/ITD 80 ITD/F621L 60 ITD/A627P ITD/F691L 40 ITD/Y842C 20

O.D. (% of Control) 0 0 1020304050 AC220, nM Figure 2. Growth of BaF3 FLT3/ITD resistance mutants in the presence of FLT3 tyrosine kinase inhibitors. Resistance was conﬁrmed by performing site-directed mutagenesis of FLT3/ITD and nucleoporation in BaF3 cells. The resulting clones were grown in the absence of interleukin-3 and analyzed by treatment in increasing concentrations of FLT3 TKI for 2 days, after which growth inhibition was assessed using the MTT assay. Means are representative of at least three independent experiments. Cells expressing FLT3 resistance mutations were treated with (a) Lestaurtinib, (b) Midostaurin, (c) KW2449, (d) Sorafenib, (e) Sunitinib, (f) AGS324 or (g) AC220.

lestaurtinib but resistance to growth inhibition. Recently, it was midostaurin revealed a similar pattern of resistance seen with shown that a mutation within BCR–ABL that inhibited its lestaurtinib, with the F621L and Y842C mutants being as sensitive phosphorylation also led to inhibition of STAT5 phosphorylation as the control BaF3/ITD cells (Figure 2b). Like the results obtained but no effect on MAP kinase, AKT or other pathways resulted.30 with lestaurtinib, the F691L mutant displayed partial resistance to This suggests a possible inherent property of the FLT3/ITD A627P midostaurin causing a fourfold increase in the IC50 (18 nM) for conformation to act as a scaffold that permits substrate binding growth inhibition compared with BaF3/ITD cells (IC50 of 4 nM). Like and activation that is compatible with reduced FLT3 kinase lestaurtinib, midostaurin was also ineffective at preventing phosphorylation. Treatment of each resistant mutant with proliferation in the A627P mutant. Midostaurin also exerted FLT3

Leukemia (2013) 48 – 55 & 2013 Macmillan Publishers Limited Resistance to FLT3 tyrosine kinase inhibitors AB Williams et al 51 inhibitory activity of each mutant, which mirrored the activity of by 48-fold for the A627P mutant (4200 nM), by 7-fold for the lestaurtinib, with only the F691L mutant exhibiting partial resistance F691L mutant (170 nM) and by 4-fold for the Y842C mutant (Figures 3a–e). Thus, the MTT data are in agreement with the (100 nM). Western blotting shows that KW2449 is ineffective western blotting results, which show inhibition of FLT3 autopho- at inhibiting FLT3 autophosphorylation of the F621L, F691L and sphorylation by midostaurin associated with inhibition of prolifera- Y842C resistant mutants at concentrations that inhibit FLT3/ITD tion of each mutant with the exception of the A627P mutant. activation in the BaF3 cells but it was able to inhibit FLT3 KW2449 recently underwent testing in phase II clinical trials phosphorylation in the A627P mutant (Figures 3a–e). targeting mutant FLT3. MTT results show growth inhibition of Sorafenib and sunitinib are TKI, both approved for use in renal BaF3/ITD cells with an IC50 of 25 nM, but each of the mutants cell carcinoma, that have activity against FLT3/ITD mutants demonstrated degrees of resistance to this inhibitor (Figure 2c). and are in clinical trials for FLT3/ITD AML. Sorafenib inhibited KW2449 IC50 values shifted by 2-fold for the F621L mutant (50 nM), proliferation of BaF3 FLT3/ITD-expressing cells with an IC50 of 8 nM, and was actually more potent at growth inhibition of the F621L cells (IC50 of 2 nM) (Figure 2d). The IC50 for growth inhibition by sorafenib was not reached at the concentrations tested for the F691L, A627P and Y842C mutants. In agreement with the MTT results, western blotting conﬁrmed that FLT3 autophosphorylation TKI, nM was inhibited by sorafenib in the BaF3/ITD and F621L mutant cells pFLT3 but was resistant to inhibition in each of the other mutant cell BaF3/ITD FLT3 lines (Figures 3a–e). Sunitinib inhibited growth of BaF3/ITD cells with an IC50 of 5 nM, pFLT3 but partial or complete resistance was observed for each of the F621L FLT3 mutant cell lines. The IC50 shifted to 30 nM for the F621L mutant and to 450 nM for each of the A627P, F691L and Y842C mutants. pFLT3 Western blotting conﬁrmed that sunitinib inhibited FLT3/ITD A627P FLT3 autophosphorylation in the BaF3/ITD cells, but had little to no effect on FLT3 activation in any of the resistant mutants (Figures 3a–e). pFLT3 F691L AGS324 inhibited proliferation of BaF3/ITD cells in the MTT FLT3 assay with an IC50 of 20 nM but the IC50 shifted to 100 nM in the F621L mutant and 4500 nM in the A627P, F691L and Y842C pFLT3 mutants. These results are in agreement with western blotting Y842C results, which show that AGS324 partially inhibited FLT3/ITD FLT3 autophosphorylation in BaF3/ITD cells at 20 nM and completely at 200 nM (Figures 3a–e). The F621L mutant was not affected at 20 nM Figure 3. FLT3 tyrosine kinase inhibitors fail to inhibit FLT3 phos- but at 200 nM was mostly inhibited. No FLT3 inhibitory activity by phorylation in resistant mutants. BaF3 FLT3/ITD resistance mutants AGS324 was observed at either concentration tested in the A627P, were treated with the indicated concentrations of FLT3 TKI for 1 h. Lysates (500 mg) were immunoprecipitated with FLT3 antibody (S-18) F691L or Y842E mutants. and probed for activation using 4G10 anti-phosphotyrosine antibody. While this paper was under review, a study was published that The membrane was then stripped and reprobed for FLT3 expression. reported several FLT3 mutations causing resistance to AC220 14 (a)BaF3FLT3/ITDcells,(b) FLT3/ITD F621L cells, (c)FLT3/ITDA627P in vitro and in patients. AC220 is one of the newest and most cells, (d) FLT3/ITD F691L cells and (e) FLT3/ITD Y842C cells. selective FLT3 TKI to achieve clinical trial status for AML. In light of

TKI, nM TKI, nM

ITD pSTAT5 F621L pSTAT5 STAT5 STAT5 pAKT pAKT AKT AKT

pMAPK pMAPK MAPK MAPK

A627P pSTAT5 STAT5 F691L pSTAT5 STAT5 pAKT AKT pAKT AKT pMAPK pMAPK MAPK MAPK Y842C pSTAT5 STAT5

pAKT AKT

pMAPK MAPK Figure 4. FLT3 signaling remains intact in some resistant mutants when treated with a FLT3 tyrosine kinase inhibitor. BaF3 FLT3/ITD cells expressing the unmutated ITD, the F621L/ITD, the A627P/ITD, the F691L/ITD or the Y842C/ITD mutation were treated with the indicated concentrations of FLT3 TKI for 1 h. Cells were lysed in NP-40 buffer for 30 mins on ice. Lysates were separated on 8% SDS-PAGE gels, transferred to polyvinyl diﬂouride membranes using antibodies for pSTAT5, pAKT, pMAPK and their total protein counterparts and analyzed by enhanced chemiluminescence.

& 2013 Macmillan Publishers Limited Leukemia (2013) 48 – 55 Resistance to FLT3 tyrosine kinase inhibitors AB Williams et al 52 Day 3 Day 8

16000000 Control Control 14000000 Sorafenib 12000000 Lestaurtinib 10000000 8000000 6000000 Sorafenib, 20 mg/kg 4000000 once daily Average Flux (p/s) 2000000 p = 0.0007 0 -2000000 Day 3 Day 8 Lestaurtinib, 20 mg/kg twice daily

1.25

1.00

0.75

0.50

0.25 Spleen Weight (g) p<0.0001

0.00 Control Sorafenib Lestaurtinib Figure 5. FLT3/ITD Y842C confers resistance in vivo.(a) BaF3 FLT3/ITD Y842C luc þ cells were transplanted via tail vein in syngeneic Balb/c mice on day 0. Bioluminescence was monitored by peritoneal injection of luciferin on day 3 to monitor engraftment. Treatment with sorafenib (20 mg/kg, once daily), lestaurtinib (20 mg/kg, twice daily) or lestaurtinib vehicle control began on day 3 and continued until day 8 to assess FLT3 TKI activity. (b) The treatments described in (a) were continued until day 15 when mice in the control group became sick at which point spleen weights were measured in all groups. Values expressed are the means±s.e.m.

the importance of AC220, we tested it against the mutations similar to published results in which MAP kinase remained fully identiﬁed in our screen. Figure 2g shows that AC220 inhibited activated despite inhibition of FLT3/ITD A627E autophosphoryla- growth of the F621L mutant as potently as it did against BaF3 tion by midostaurin.31 The FLT3/ITD F691L mutant displayed a low FLT3/ITD cells (IC50o1nM). However, A627P and F691L mutant level of resistance to both lestaurtinib and midostaurin as was cells were completely unresponsive to treatment with AC220. evident in the ability of these two FLT3 TKI to inhibit downstream The Y842C mutation caused the IC50 to shift to B20 nM. Likewise, signaling pathways only at the higher 30 nM concentrations but the response of each mutant cell line to growth inhibition by not at 10 nM (Figure 4d). Otherwise, STAT5, AKT and MAP kinase AC220 was paralleled by the responsiveness of each mutation to phosphorylation was largely unaffected when this mutant was inhibition of FLT3 autophosphorylation and downstream signaling treated even with high concentrations of KW2449, sorafenib, pathways (Figures 3 and 4). sunitinib or AGS324. Downstream signaling of the FLT3/ITD Y842C mutant showed a high level of resistance to treatment with KW2449, sorafenib, sunitinib and AGS324, but remained sensitive FLT3 signaling in resistant mutants remains intact in the presence to lestaurtinib and midostaurin (Figure 4e). Figure 4e shows that of FLT3 TKI the ability of this mutation to signal through STAT5, AKT and MAP In light of the evidence that many AML patients expressing kinase in the presence of TKI mirrored the results seen when FLT3 mutant FLT3 acquire TKI resistance that cannot be accounted for activation was probed. Lestaurtinib and midostaurin blocked all by mutations within FLT3 itself, we examined FLT3 signaling three signaling pathways in this mutant, which was consistent pathways to determine whether resistance to FLT3 inhibition with the effects seen on FLT3 autophosphorylation. correlated with retention of FLT3 signaling pathways in the mutants identiﬁed in the present study. Figure 4a shows that in BaF3/ITD cells, each FLT3 TKI that was tested inhibited FLT3/ITD FLT3/ITD Y842C causes resistance in vivo autophosphorylation and this resulted in termination of signaling The FLT3/ITD Y842C mutation produced high-level resistance to through STAT5, AKT and MAP kinase pathways. Against the F621L KW2449, sorafenib, sunitinib and AGS324 in vitro but remained mutant (Figure 4b), all three signaling pathways were completely sensitive to lestaurtinib and midostaurin. We evaluated this inhibited by lestaurtinib, midostaurin and sorafenib, whereas mutation further in vivo by transplanting luciferase-positive cells KW2449 and AGS324 could only effect partial inhibition of in syngeneic Balb/c mice and treating with a FLT3 TKI that was downstream signaling events even at the higher concentrations. ineffective in vitro (sorafenib) and one that was effective Sunitinib exhibited almost no inhibitory activity against any of the (lestaurtinib). Bioluminescence emanating from transplanted signaling pathways in this FLT3 mutant. The A627P mutant luc þ cells upon intraperitoneal injection of luciferin was used to displayed strong activation of MAP kinase and AKT but a rather quantify the level of FLT3/ITD engraftment in mice. Figure 5 shows weak phosphorylated STAT5 signal (Figure 4c). Despite inhibition that injection of BaF3 FLT3/ITD Y842C luc þ cells in mice led to of FLT3/ITD autophosphorylation caused by treatment with engraftment in the bone marrow that was visible by biolumines- several of the TKI, all pathways remained activated in the cence by day 3 (Figure 5a). Drug or lestaurtinib vehicle treatment presence of each FLT3 TKI. This supports the idea that other of each group began at day 3 and continued until day 8. mechanisms related to mutation at 627 contribute to resistance, Bioluminescence increased dramatically in the control group from

Leukemia (2013) 48 – 55 & 2013 Macmillan Publishers Limited Resistance to FLT3 tyrosine kinase inhibitors AB Williams et al 53 A627 While nearly 100 mutations have been clinically described in chronic myelogenous leukemia patients that are at least partially resistant to imatinib,41 FLT3 TKI monotherapy has not selected for nearly as many mutations. There are a number of reasons for this F621 F691 with the major ones likely being: (1) a lack of success of first- 90 generation FLT3 TKI in achieving thorough inhibition of FLT3 kinase activity. As more recent FLT3 TKI are more successful in vivo, the frequency of selecting for resistance mutations is increasing;14–16 (2) in FLT3/ITD AML, it appears there are 8–10 other mutations that contribute to leukemogenesis and so Y842 resistance is easily selected for via pathways that have nothing to do with resistance to inhibiting FLT3. Nevertheless, resistance mutations are likely to have an increasingly important role as FLT3 TKI that achieve targeted levels of inhibition are developed. Identification of FLT3 resistance mutations is still at an early A627 stage. One of the first in vitro screens identified resistance to midostaurin at four residues within the ATP-binding cleft of FLT3/ ITD.17 These included substitutions at A627, N676, F691 and G697 that conferred varying levels of resistance. A later study also F691 F621 identified mutations at N676 or F691 as mediators of resistance to 90 midostaurin or sorafenib, respectively, and selection in SU5614 led to mutations mapping exclusively to TKD2.18 A recent saturation mutagenesis screen found mutations at D835, Y842 and F691 that caused resistance to AC220, and the authors also detected D835 and F691 in FLT3 mutant AML patients treated with AC220, in Y842 effect further validating the in vitro screening protocols.14 One of the most frequent FLT3/ITD resistance mutations discovered to date is the selection within the ITD allele of point mutations in the second half of the KD (TKD2). Activating point mutations within the KD of FLT3 occur in the absence of an ITD Figure 6. Structural modeling of FLT3 TKI with resistant mutants. mutation at a rate of 7–10% in AML patients, and some of these (a) Lestaurtinib/midostaurin (magenta) binding to FLT3 was based 42 on staurosporine bound to Lck kinase and superimposed on the are insensitive to some FLT3 TKI. Thus, the acquisition of such a FLT3 crystal structure. FLT3 TKI resistance mutations (red) were mutation in a FLT3/ITD allele confers TKI resistance. The most identified in the ATP-binding cleft (F621L, A627P or F691L) or the common activating point mutations occur on the activation loop activation loop (Y842C). The N-terminus is shown in pale cyan, the and they respond with differential sensitivity to various FLT3 TKI, C-terminus in cyan, the juxtamembrane in yellow (partially removed though they all respond to both lestaurtinib and midostaurin. for greater clarity) and the activation loop in green. (b) Sorafenib Emerging data are now implicating the acquisition of point (magenta) binding to FLT3 was based on its atomic coordinates in mutations in TKD2 within a FLT3/ITD allele as a major source of complex with p38 Map Kinase. Images were generated using PyMol resistance to sorafenib or AC220.14–16 and are shown as orthogonal views and views rotated 901. Different FLT3 TKI appear to affect both the frequency of selection for resistance mutations and the residues affected. Compared with other FLT3 TKI, lestaurtinib and midostaurin are a mean flux of 22 800 photons/s counts on day 3 to 11 670 000 by effective against more known FLT3 mutations, especially those day 8. As expected, based on the in vitro data, twice daily occurring on the activation loop; in fact, no resistance mutations administration with lestaurtinib led to a significant reduction occurring in the second half of the KD have been reported in in the progression of the FLT3/ITD Y842C cells (P ¼ 0.0007). patients as being resistant to these two agents. In contrast, many In contrast, engraftment continued unabated in mice treated with of the mutations located on or near the activation loop show sorafenib to levels similar to the control group (day 8 mean level widely varying sensitivity to different FLT3 TKI. In this regard, of 10 000 000 photons/s). Figure 5b shows that lestaurtinib also lestaurtinib and midostaurin bear similarity to nilotinib and significantly reduced mean spleen weights compared with control dasatanib, while the remaining FLT3 TKI more closely resemble and sorafenib-treated mice (0.09 g vs 1.02 g and vs 0.93 g, imatinib. For example, treatment with imatinib generates numer- Po0.0001 vs both). ous mutations throughout BCR–ABL that confer imatinib resistance, but only a handful of resistance mutations emerge while patients are on nilotinib or dasatanib, and these tend to be DISCUSSION dominated by the T315I substitution.41 This has caused some to A number of FLT3 TKI have progressed to clinical trials for FLT3 postulate that lestaurtinib and midostaurin may act as class I AML.32 Management of AML, however, is complicated by the fact kinase inhibitors, which target the activated FLT3 conformation, that it is a multi-mutational disease with typically 8–10 somatic while other FLT3 TKI act as class II inhibitors that bind to inactive mutations per AML patient. This results in FLT3 inhibition alone FLT3, in analogy to structural studies of BCR–ABL in complex with often being insufficient to induce cell death of most FLT3- imatinib.43–45 With both FLT3/ITD and BCR–ABL, far more point mutated AML blasts.33,34 In contrast, early stage chronic myeloid mutations have been identified that are capable of conferring leukemia is driven primarily by expression of the oncogenic resistance to class II TKI compared with class I inhibitors. Thus, one BCR–ABL fusion protein, and targeting BCR–ABL kinase activity might propose that FLT3/ITD AML patients who develop a FLT3 KD has been remarkably successful as a strategy to improve survival mutation that causes resistance to a class II inhibitor might still in chronic myelogenous leukemia patients.35–38 However, respond to a class I inhibitor. In fact, the A848P mutation within selection for point mutations in BCR–ABL conferring resistance the FLT3/ITD allele that was selected for in one AML patient while via reduced binding is a mechanism that reduces efficacy, leading being treated with sunitinib and sorafenib was shown to still be to relapse.39,40 responsive to midostaurin in vitro.46 Another characteristic of FLT3

& 2013 Macmillan Publishers Limited Leukemia (2013) 48 – 55 Resistance to FLT3 tyrosine kinase inhibitors AB Williams et al 54 TKI that might be important in limiting the number of resistance related and unrelated to FLT3 mutations, as has been mutations that emerge during treatment relates to the degree reported.20,49–51 The relative rate of occurrence of different of selectivity of the inhibitor. Broad-spectrum inhibitors such as mechanisms of resistance in patients treated on FLT3 TKI is not lestaurtinib would be expected to produce more toxicity at high known, but mutations are being detected at a greater rate as concentrations in AML patients, but such inhibitors might reduce successful inhibition of FLT3 kinase activity is achieved. Until the number of resistance mutations that emerge during treatment recently, only three resistance mutations had been reported, while compared with a more selective inhibitor. AML patients were being treated with a TKI and only the FLT3/ITD Modeling of FLT3 with inhibitors bound shows that F691 is the N676K mutation actually imparted resistance to inhibition of FLT3 only residue in this study likely to make direct contact with FLT3 phosphorylation by midostaurin.52 While this manuscript was in TKI (Figure 6). This position is analogous to the ‘gatekeeper’ T315 preparation, additional resistance mutations were identified residue in BCR–ABL, which when mutated to isoleucine, confers clinically in FLT3 mutant AML patients treated with the FLT3 TKI pan-resistance to BCR–ABL TKI, including imatinib, nilotinib and sorafenib or AC220. The recently detected mutations include dasatinib, by inhibiting drug binding. Our results suggest that substitutions at D835 and one of the mutations identified in our mutation to leucine (F691L) probably would not result in steric screen, F691L, all of which occurred as secondary mutations within hindrance because of the smaller size of the substituted side the FLT3/ITD allele. The increasing number of AML patients who chain. However, mutation to leucine may result in weaker are found to express FLT3 resistance mutations following TKI interactions with inhibitors, thus decreasing their affinity for FLT3. treatment provides compelling evidence that mutations such as Although F621 and A627 appear unlikely to make direct contact FLT3/ITD F621L, A627P, F691L and Y842C found in the present with FLT3 TKI in these models, both of these residues lie within the screen and others may lead to significant therapeutic challenges. nucleotide-binding loop and their mutation may affect transitions As the list continues to grow, a knowledge of which FLT3 TKI are from active to inactive forms. In fact, mutation to proline at still active against these mutations will be required to better position A627, located at the roof of the ATP-binding cleft, is manage the emergence of resistance cases. The results presented predicted to restrict a backbone phi conformation in the b1-b2 here indicate that some FLT3 TKI will be less effective against region that is normally allowed by alanine. The F621L mutation several FLT3 resistance mutants and that even lestaurtinib displayed the lowest level of TKI resistance, which may not be and midostaurin, which target the greatest range of mutations, surprising considering that F621 is further outside of the cleft and will exhibit reduced potency against some clones. Thus, there is a that leucine is less bulky than phenylalanine. Figure 6 shows that need to profile FLT3/ITD mutations for their ability to confer neither staurosporine nor sorafenib is likely to contact F621, which resistance to FLT3 TKI and a need to expand the repertoire of is consistent with the biological data showing inhibition of kinase inhibitors available for clinical use against emerging TKI-resistant activity in this mutation. clones. Y842 is located on the activation loop of the carboxy terminal kinase lobe, so assessing the effects of a residue so far removed from the ATP-binding site is complicated by the lack of crystal CONFLICT OF INTEREST structures of different activated FLT3 conformations. However, the The authors declare no conflict of interest. activation loops in related kinases adopt a wide range of conformations. In the autoinhibited state, the unphosphorylated activation loop is folded between the amino- (N) and carboxy- (C) terminal kinase lobes and prevents ATP binding by contacting the ACKNOWLEDGEMENTS JM-binding motif that is embedded between the N and C lobes. In We are grateful to Dr Linzhao Cheng (Johns Hopkins University) for providing us with activated kinases, the tyrosine-phosphorylated activation loop the L3GFP vector used to visualize FLT3/ITD engraftment and to members of the lab for numerous thoughtful discussions. This work was supported by grants from the swings outward, thus permitting access of ATP and substrate to NCI (CA90770 and CA90668), Leukemia and Lymphoma Society and Giant Food the active site. The Y842C mutation likely alters the ensemble of Pediatric Cancer Research Fund. DS is also supported by the Kyle Haydock activation loop conformations such that ATP can still enter the Professorship. nucleotide-binding site but sorafenib is less able to align itself in its preferred orientation. Structural modeling shows that sorafenib does in fact extend further into the ATP cleft than midostaurin and AUTHOR CONTRIBUTIONS occupies an adjacent site within FLT3. Sorafenib also clashes with ABW designed experiments, performed research, analyzed data and wrote the the JM domain in a way that midostaurin does not. manuscript; LL and BN performed research; ML analyzed data; PB and DL analyzed The FLT3/ITD Y842C mutant might pose significant challenges data and wrote the manuscript; DS designed experiments, supervised the project, to treatment with some TKI because it was highly resistant to both analyzed data and wrote the manuscript. sunitinib and sorafenib. It is important to note that five AML patients have been identified with activating mutations of FLT3 47,48 Y842C or N841. This repeated occurrence strongly suggests REFERENCES that these mutations are likely to be more frequently encountered as a mechanism of resistance to select FLT3 TKI monotherapy 1 Small D, Levenstein M, Kim E, Carrow C, Amin S, Rockwell P et al. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34 þ human bone (such as sorafenib and sunitinib). 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