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The secondary FLT3-ITD F691L mutation induces resistance to AC220 in FLT3-ITD+ AML but retains in vitro sensitivity to PKC412 and Sunitinib

We report on a secondary FLT3 internal tandem duplication (FLT3-ITD) mutation in a 48-year-old female patient with FLT3-ITD+ acute myeloid leukemia (AML) mediating resistance to the FLT3 inhibitor AC220.

Initially, the patient presented with white blood cell (WBC) count of 27.03 g/l, platelets count of 101g/l, and 70% peripheral blood (PB) and 52% bone marrow (BM) blasts. Marrow cytogenetics showed a normal karyotype, and molecular analysis revealed mutation of NPM1 and an ITD mutation of 87 bp length within the juxtamembrane region of FLT3.

Induction therapy was initiated leading to a normalization of leukocyte counts and a reduction of blast cells in the PB and BM (Figure 1a). As no complete remission was achieved, salvage chemotherapy with high-dose cytarabine and mitoxatrone was started, and subsequently, an allogenic stem cell transplantation was performed using a human leukocyte antigen identical unrelated donor. A complete remission was achieved on day +28 post transplantation. On day +88, a relapse was diagnosed with >20% BM blasts. Subsequently, immunosupression was withdrawn and therapy with the FLT3 inhibitor Sorafenib (200 mg, twice daily) was initiated, leading to a second complete remission. Tyrosine kinase inhibitor (TKI) therapy, however, had to be stopped after 4 weeks, owing to severe side effects (skin rash and edema).

Figure 1
figure1

(a) Clinical course day 0 indicates time of first allogenic stem cell transplant. WBC, white blood cell count; BM, bone marrow; PB, peripheral blood. CTx+/−PKC412 indicates therapy according to the Ratify study protocol, a randomized, double-blind phase III study of induction (daunorubicin/cytarabine) and consolidation (high-dose cytarabine) chemotherapy combined with midostaurin or placebo in treatment-naive patients with FLT3-mutated AML. Time and duration of treatment are indicated below the time axis. (b) Identification and (c, d) characterization of FLT3-ITD F691L. FLT3 TK1 and TK2 were amplified from total RNA isolated from bone marrow mononuclear cells, using FLT3-specific primers. Sequence analysis before treatment with AC220 revealed FLT3-ITD wild-type sequence (b, upper panel) and a T-to-G mutation at codon 691, giving rise to a phenylalanine to leucine exchange at the time of relapse (b, lower panel). (c) Proliferation inhibition of Ba/F3 cells expressing FLT3-ITD or indicated mutants by AC220. Mutations were generated using site-directed mutagenesis. Proliferation was measured after 48 h using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-based assay. Data are presented as percentage of dimethylsulfoxide (DMSO)-treated cells and represent values of triplicates ±s.d. One representative of at least three independent experiments is shown. (d) Ba/F3 cells expressing FLT3-ITD or indicated mutants were treated with AC220 in serum-free and cytokine-free medium for 2.5 h. Immunoprecipitation of FLT3 followed by immunoblotting with phosphoFLT3 and FLT3 antibodies were performed. (e) Display of individual mutation patterns for different FLT3 inhibitors. After incubation for 48 h without and in the presence of indicated inhibitors, proliferation was measured using an MTS-based method. At least two independent experiments were performed for each FLT3-ITD construct. Representative results of one experiment per inhibitor and construct are expressed as individual cellular IC50 (half maximal inhibitory concentration) values. Individual dose response for AC220 is shown in Figure 1d. The extend of cellular resistance is indicated as strong (red), moderate (yellow) and none/weak (green), with a cellular IC50 range for weak/moderate/strong of <12.5/12.5–24.9/>25 nM, respectively, for PKC412 and AC220, and <250/250–499/>500 nM, respectively, for Sunitinib and Sorafenib. *, **Data for PKC412, Sunitinib and Sorafenib were obtained from previous experiments in our lab.2, 3

On day +252 post transplant, another relapse was diagnosed with 72.5% BM blasts. Cytogenetic and molecular analysis still showed a normal karyotype with the initially identified NPM1- and FLT3-ITD mutations, but no further alterations. The patient was enrolled onto a study with the FLT3 inhibitor AC220 (Quizartinib, 90–135 mg daily).1 Treatment was well tolerated and BM blasts were reduced to less than 0.5%. After 4 months of AC220 treatment, another relapse was noted with 60% blasts in the BM and 67% blasts in the PB. A conditioning treatment containing clofarabrine was initiated, followed by a second BM transplant from the identical donor.

Cytogenetic and sequencing analysis before the treatment with the inhibitor AC220 revealed a normal karyotype with no change in the mutation status of NPM1 and FLT3-ITD. However, at the time of relapse following AC220 treatment, a novel FLT3-ITD F691L mutation was detected (Figure 1b). To determine whether resistance and subsequent relapse to AC220 were caused by this mutation, FLT3-ITD F691L was cloned and stably expressing cells with FLT3-ITD or FLT3-ITD F691L were established. Drug responses to AC220 were examined in these cells measuring proliferation and kinase activity (Figures 1c and d). The FLT3-ITD F691L mutation identified in our patient shifted AC220 responses more than100-fold compared with unmutated FLT3-ITD, both in a proliferation assay (Figure 1c) and in phosphoFLT3-ITD blots (Figure 1d).

Next, we analyzed the AC220-sensitivity profile of other secondary FLT3-ITD mutations previously identified in patients or cell-based resistance screens occurring with the FLT3 inhibitors PKC412, Sorafenib and Sunitinib.2, 3, 4, 5 Interestingly, we also found FLT3-ITD-F691I to be highly resistant to AC220, whereas other previously described resistance mutations (FLT3-ITD-Y842H, FLT3-ITD-A848P or FLT3-ITD-K676D) retained intermediate sensitivity to AC220 both in proliferation assays, as well as in immunoblots (Figures 1c and d).

Figure 1e summarizes the resistance profile of AC220 in comparison with other FLT3-ITD inhibitors (PKC412, Sorafenib and Sunitinib). All four compounds show unique and only partial overlapping sensitivity profiles. Importantly, the FLT3-ITD mutation identified in our patient retained intermediate sensitivity to both PKC412 and Sunitinib.2, 6

Shortly after the second BM transplantation, another relapse was diagnosed in our patient with 67.5% blasts in the BM. On the basis of our in vitro inhibitor studies described above, treatment with Sunitinib was started within an individual patient treatment program established at our institute. Initially, the patient seemed to respond well to Sunitinib treatment, as blasts in the PB were significantly reduced (Figure 1a). However, before the next scheduled BM biopsy, the patient deceased in a small community hospital owing to an intracranial bleeding.

Discussion

Here we report on a secondary FLT3-ITD mutation of the gate-keeper residue F691.

The identified mutation FLT3-ITD F691L was detected in an AML patient who relapsed upon treatment with AC220.

In vitro analyses confirmed that FLT3-ITD F691L mediates strong resistance towards the FLT3 inhibitor AC220. Thus, FLT3-ITD is clearly an important druggable target in FLT3-ITD-positive AML as recently shown by Smith et al.7

We analyzed the potency of AC220 toward several other known secondary FLT3-ITD-resistance mutations2, 7, 8 Interestingly, mutations in position 691 to leucine or isoleucine induce strong resistance to AC220, whereas the FLT3-ITD secondary mutations N676D, Y842H and A848P retained intermediate sensitivity to AC220. These data are in accordance with the analysis of Smith et al.7 Next, we were interested to determine whether alternative FLT3 inhibitors would be able to overcome resistance to the gatekeeper-mutation identified in our patient. Interestingly, both Sunitinib and PKC412 in contrast to AC220 display activity against FLT3-ITD F691L. We thus switched kinase inhibitor treatment from AC220 to Sunitinib in our patient. Indeed, a short-term response could be detected in that the load of blasts in the PB was significantly reduced.

Therefore, our case suggests that in patients with FLT3-ITD-positive AML the use of secondary TKI treatment may be beneficial given that resistance is mediated by specific secondary FLT3-ITD mutations. Resistance profiles of different FLT3 kinase inhibitors may then guide treatment decisions. Thus, in the future, analysis of secondary alterations in FLT3-ITD may have an important role in targeted therapy of FLT3-ITD-positive AML reminiscent of treatment strategies applied in BCR-ABL-positive CML.

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Correspondence to J Duyster.

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Albers, C., Leischner, H., Verbeek, M. et al. The secondary FLT3-ITD F691L mutation induces resistance to AC220 in FLT3-ITD+ AML but retains in vitro sensitivity to PKC412 and Sunitinib. Leukemia 27, 1416–1418 (2013). https://doi.org/10.1038/leu.2013.14

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