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. 2013 Oct 15;19(20):5758-68.
doi: 10.1158/1078-0432.CCR-13-1323. Epub 2013 Aug 22.

Emergence of polyclonal FLT3 tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3-ITD-positive acute myeloid leukemia

Sharyn D Baker  1 , Eric I Zimmerman, Yong-Dong Wang, Shelley Orwick, Douglas S Zatechka, Jassada Buaboonnam, Geoffrey A Neale, Scott R Olsen, Eric J Enemark, Sheila Shurtleff, Jeffrey E Rubnitz, Charles G Mullighan, Hiroto Inaba
Affiliations

Emergence of polyclonal FLT3 tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3-ITD-positive acute myeloid leukemia

Sharyn D Baker et al. Clin Cancer Res. .

Abstract

Purpose: To evaluate the clinical activity of sequential therapy with sorafenib and sunitinib in FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-positive acute myelogenous leukemia (AML) and monitor the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations during treatment.

Experimental design: Six children with relapsed/refractory AML were treated with sorafenib in combination with clofarabine and cytarabine, followed by single-agent sorafenib if not a candidate for transplantation. Sunitinib was initiated after sorafenib relapse. Bone marrow samples were obtained for assessment of FLT3 TKD mutations by deep amplicon sequencing. The phase of secondary mutations with ITD alleles was assessed by cloning and sequencing of FLT3 exons 14 through 20. Identified mutations were modeled in Ba/F3 cells, and the effect of kinase inhibitors on FLT3 signaling and cell viability was assessed.

Results: Four patients achieved complete remission, but 3 receiving maintenance therapy with sorafenib relapsed after 14 to 37 weeks. Sunitinib reduced circulating blasts in two patients and marrow blasts in one. Two patients did not respond to sorafenib combination therapy or sunitinib. FLT3 mutations at residues D835 and F691 were observed in sorafenib resistance samples on both ITD-positive and -negative alleles. Deep sequencing revealed low-level mutations and their evolution during sorafenib treatment. Sunitinib suppressed leukemic clones with D835H and F691L mutations, but not D835Y. Cells expressing sorafenib-resistant FLT3 mutations were sensitive to sunitinib in vitro.

Conclusions: Sunitinib has activity in patients that are resistant to sorafenib and harbor secondary FLT3 TKD mutations. The use of sensitive methods to monitor FLT3 mutations during therapy may allow individualized treatment with the currently available kinase inhibitors.

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Conflict of interest statement

Conflicts of interest: All authors declare no potential conflict of interest.

Disclosure of Potential Conflict of Interest

The authors declared no conflict of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Figures

Figure 1
Figure 1
Clinical course of three patients treated sequentially with sorafenib and sunitinib. Day 0 indicates the initiation of treatment with sorafenib. Duration of treatment is indicated by the horizontal bars at the top of the graph (purple, sorafenib ± clofarabine and/or cytarabine; blue, sunitinib; orange, hydroxyurea). Vertical orange bars represent percentage of bone marrow blast cells assessed by flow cytometry. Abbreviations: Ara-C, cytarabine; Clo, clofarabine; RSV, respiratory syncytial virus; WBC, white blood cell.
Figure 2
Figure 2
Allele selection of the FLT3 D835H mutation in patient 1 during treatment with sequential sorafenib and sunitinib. The percentage of blasts samples that contained the reference allele (top) or alternative allele (bottom) for the FLT3 D835 mutation in exon 20 (rs121913488) and the rs17086226 variant in the intron downstream of exon 20 were monitored during treatment with sorafenib and sunitinib using deep amplicon sequencing.
Figure 3
Figure 3
Sorafenib-resistant FLT3 mutations are sensitive to sunitinib treatment. A) BaF3 cells were treated with sorafenib (left) or sunitinib (right) for 72 hr and cell viability was measured. Data represent the mean +/− SEM of 2–4 independent experiments (n = 12–24 replicates). B) BaF3 cells were treated for 1 hr with sorafenib or sunitinib. FLT3 was then immunoprecipitated from cell lysate, and Western blot analysis was performed for phosphorylated and total FLT3 (IB, immunoblot). C) Structure model of sorafenib and sunitinib interaction with wild-type FLT3 (adapted from PDB IDs 1RJB, 3G0E, and 1UWH using Pymol software) (blue, hinge region; green, activation loop; red, αC helix; yellow, juxtamembrane domain; orange, sorafenib; magenta, sunitinib). D) Surface and stick representation of sorafenib, sunitinib, and residues F691 and F830 (DFG motif).
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