Letters to the Editor 1693 Ponatinib is active against imatinib-resistant mutants of FIP1L1-PDGFRA and KIT, and against FGFR1-derived fusion kinases

Leukemia (2012) 26, 1693--1695; doi:10.1038/leu.2012.8 and variable: no response was obtained in one case, whereas in the other patient, a short-lived response was followed by selection Imatinib, the small molecule inhibitor of BCR-ABL1, has revolutio- of the panresistant FIP1L1-PDGFRA-D842V mutation and blast nized treatment of chronic myeloid leukemia (CML), and of other 4,7 malignancies driven by deregulation of imatinib-sensitive tyrosine crisis. Myeloid/lymphoid neoplasms associated with eosinophilia kinases, for example PDGFRA, PDGFRB or KIT. Yet, selection of and a rearrangement of FGFR1, also known as the 8p11 myeloproliferative syndrome (EMS), are aggressive stem cell imatinib-resistant kinase domain mutations is curtailing CML 8 response rates. Ponatinib is a third-generation kinase inhibitor disorders. Although, the FGFR1 fusion kinase constitutes a with potent activity towards wild-type BCR-ABL1, as well as potential therapeutic target, the disease remains medically numerous imatinib-resistant BCR-ABL1 kinase domain mutants, untreatable today. Finally, various KIT mutations are pathogenic including the notorious T315I mutation.1 drivers in gastrointestinal stromal tumors (GIST), systemic masto- Patients with myeloid neoplasms with eosinophilia, and the cytosis (SM) and other malignancies. The KIT-D816V mutation FIP1L1-PDGFRA fusion gene, are exquisitely sensitive to imatinib occurs in SM and melanoma and is primarily resistant to imatinib. and many of them reach a durable molecular remission under In GIST, KIT-W557_K558del mutations are common and respond imatinib.2,3 Yet, rare cases of secondary resistance have also been well to imatinib treatment, but here also different secondary 9 reported, with the acquisition of a T674I mutation in seven mutations (T670I and D820A) are known that confer resistance. patients and a D842V mutation in one (for a recent overview, see Besides its activity against BCR-ABL1, the activity of ponatinib in 1 Metzgeroth et al.4).3--5 The FIP1L1-PDGFRA-T674I mutation has vitro also encompasses PDGFRA, KIT and FGFR1. Potent activity limited to absent sensitivity to nilotinib and dasatinib in vitro but towards oncogenic fusion or mutant kinases such as FIP1L1- responds well to sorafenib.6,7 However, the effect of sorafenib in PDGFRA, KIT-N822K and FGFR1OP2-FGFR1 has also been docu- 10 two patients with the FIP1L1-PDGFRA-T674I mutation was limited mented. Therefore, we investigated the effect of ponatinib on

Figure 1. Analysis of FIP1L1-PDGFRA mutant- and CUX1-FGFR1-expressing Ba/F3 cells. (a) The dose-response curve of Ba/F3 cells expressing FIP1L1-PDGFRA-T674I or FIP1L1-PDGFRA-D842V, when treated with ponatinib for 24 h. Inhibitor dilutions were made in dimethyl sulfoxide immediately before use. Shown is the relative proliferation compared with the untreated control. Wild-type Ba/F3 cells treated with ponatinib in the presence of IL-3 are also depicted. Dose-response curves were fitted using GraphPad Prism5 software (La Jolla, CA, USA). (b) Western blot analyses of 2 106 FIP1L1-PDGFRA mutant-transformed Ba/F3 cells after treatment with ponatinib for 90 min. The phosphorylation status of FIP1L1-PDGFRA and its downstream targets STAT5 and ERK1/2 decreases upon inhibitor treatment. (c) The dose-response curve of CUX1-FGFR1-expressing Ba/F3 cells, treated with ponatinib for 24 h. Shown is the relative proliferation compared with the untreated control. Wild-type Ba/F3 cells treated with ponatinib in the presence of IL-3 are also depicted. (d) Western blot analyses of 2 106 CUX1-FGFR1- transformed Ba/F3 cells after treatment with ponatinib for 90 min. The phosphorylation status of CUX1-FGFR1 and its downstream targets STAT5 and ERK1/2 decreases upon inhibitor treatment.

Accepted article preview online 17 January 2012; advance online publication, 3 February 2012

& 2012 Macmillan Publishers Limited Leukemia (2012) 1681 --1729 Letters to the Editor 1694 imatinib-resistant mutations of FIP1L1-PDGFRA, of KIT and on an imatinib-insensitive FGFR1 fusion. Ba/F3 cells expressing the imatinib-resistant FIP1L1-PDGFRA- T674I or panresistant FIP1L1-PDGFRA-D842V mutant were cultured for 24 h in the presence of increasing ponatinib concentrations. Ponatinib strongly inhibited growth of the FIP1L1-PDGFRA-T674I mutant-expressing cells with an IC50 of 9nM. It was also active against the FIP1L1-PDGFRA-D842V mutant but with a higher IC50 (154 nM) (Figure 1a). The IC50 of ponatinib for BCR-ABL1-T315I- and FIP1L1-PDGFRA-expressing Ba/F3 was 16 nM and 0.6 nM, respectively, consistent with previous reports (data not shown).1,10 IL-3-driven growth of wild-type Ba/F3 cells was highly resistant to ponatinib (IC50 of 2 mM) (Figure 1a). With western blotting, we demonstrate a strong inhibition of the constitutive autophosphorylation of either FIP1L1-PDGFRA-T674I or FIP1L1-PDGFRA-D842V by ponatinib starting from 10 nM and 500 nM, respectively. Also, the FIP1L1-PDGFRA downstream targets STAT5 and ERK1/2 were inactivated at similar concentrations (Figure 1b). Next, we explored the activity of ponatinib against CUX1-FGFR1, a recently described oncogenic FGFR1 fusion kinase, not responding to imatinib.11 The growth of CUX1-FGFR1- expressing Ba/F3 cells was inhibited by ponatinib with an IC50 of 56 nM (Figure 1c). Again, this correlated nicely with decreasing tyrosine phosphorylation of the fusion protein and its downstream targets STAT5 and ERK1/2 (Figure 1d). Furthermore, we investigated cell-based models of imatinib- resistant KIT mutant-driven malignancies. Ba/F3 cells were used expressing KIT-D816V, KIT-Y823D, KIT-W557_K558del þ T670I and KIT-W557_K558del þ D820A. Treatment for 24 h with increasing ponatinib concentrations strongly inhibited cell growth of Ba/F3 cells expressing the KIT double mutants KIT-W557_K558del þ T670I and KIT-W557_K558del þ D820A with an IC50 of 15 nM and 2nM, respectively. For the primary imatinib-resistant KIT mutants, an IC50 of 62 nM (KIT-Y823D) and 405 nM (KIT-D816 V) was recorded (Figure 2a). With western blotting, a complete inhibition Figure 2. Effect of ponatinib on KIT mutant-expressing Ba/F3 cells. of KIT phosphorylation was demonstrated for the KIT double (a) The dose-response curve of Ba/F3 cells expressing different KIT mutants upon ponatinib treatment at 100 nM, with a correspond- mutants, upon ponatinib treatment for 24 h. Shown is the relative ing decreasing phosphorylation of the downstream targets ERK1/2 proliferation compared with the untreated control. Wild-type Ba/F3 and AKT (Figure 2b). Although KIT-Y823D phosphorylation was cells treated with ponatinib in the presence of IL-3 are also depicted. sensitive to ponatinib, this was less the case for its downstream- (b) Western blot analyses of 4 106 KIT mutant-transformed Ba/F3 cells signaling intermediates ERK1/2 and AKT (Figure 2b). In line with after treatment with ponatinib for 2 h. The phosphorylation status of the growth experiment, ponatinib had no effect on KIT-D816V KIT-W557_K558del þ T670I and its downstream targets ERK1/2 and phosphorylation (data not shown). AKT decreases with increasing ponatinib concentrations. A similar result was obtained for KIT-W557_K558del þ D820A (data not shown). Thus, ponatinib is highly active in vitro towards the major For KIT-Y823D, KIT and ERK1/2 activity decreases upon ponatinib imatinib-resistant FIP1L1-PDGFRA-T674I mutation and, at the treatment, which had only a limited effect on AKT phosphorylation. higher end of the clinically achievable concentration range, against FIP1L1-PDGFRA-D842V.12 Although the number of eligible patients is low, their prognosis is uniformly dismal, which urges conformation of the kinase.15 The KIT-D816V mutation alters the clinical testing of ponatinib in this setting.4 We also demonstrate inactive--active equilibrium by stabilizing the activated kinase strong inhibition of the CUX1-FGFR1 fusion by ponatinib at conformation. Thus, binding of type-II inhibitors, such as clinically achievable levels. This provides additional credence to its ponatinib, is hindered leading to a reduced inhibitory activity.12,15 activity against FGFR1-derived oncogenic fusions in cell lines.10 In summary, our data indicate that ponatinib, which is currently Of interest, we previously showed sensitivity of the CUX1-FGFR1 under investigation in phase II clinical trials for imatinib-resistant fusion to dovitinib, but with ponatinib the non-toxic range is CML, is active in vitro against CUX1-FGFR1, FIP1L1-PDGFRA-T674I, broader.11 Taken together, this suggests that ponatinib could have FIP1L1-PDGFRA-D842V and against specific KIT mutants. Its a wider therapeutic index than dovitinib in the treatment of EMS. potential in the therapeutic management of EMS, primary or Lastly, the inhibitory potential of ponatinib was evaluated for secondary imatinib-resistant GIST, or imatinib-resistant FIP1L1- different imatinib-resistant KIT mutants. The imatinib-resistant PDGFRA-positive disease, needs further evaluation. double mutant KIT-W557_K558del þ T670I was shown to be sensitive to sunitinib and sorafenib, whereas KIT-W557_K558del þ CONFLICT OF INTEREST 13,14 D820A was not previously investigated. Here, we demonstrate The authors declare no conflict of interest. a good response of both mutants to low nanomolar doses of ponatinib, and in this context ponatinib adds to the diversity of ACKNOWLEDGEMENTS treatment options. Also the primary imatinib-resistant KIT-Y823D This work was supported by grants from KU Leuven (GOA/11/010 to JC, PV) and mutant, known to be sensitive to sorafenib, was inhibited by FWO-Vlaanderen (G.0509.10 to PV) and the Interuniversity Attraction Poles (IAP) ponatinib. In contrast, ponatinib lacks therapeutic efficacy towards granted by the Federal Office for Scientific, Technical and Cultural Affairs, Belgium the imatinib-resistant KIT-D816V mutation, typically occurring in (JC and PV). EL is a postdoctoral fellow from FWO-Vlaanderen. PV is a senior clinical SM. Ponatinib is a type-II inhibitor targeting the inactive DFG-out investigator from FWO-Vlaanderen.

Leukemia (2012) 1681 --1729 & 2012 Macmillan Publishers Limited Letters to the Editor 1695 AUTHOR CONTRIBUTIONS analysis of patient-specific genomic DNA fusion junctions. Leukemia 2009; 23: EL designed the study, performed research, analyzed the data and wrote the paper; 332 --339. SS performed research and analyzed the data; BDW and MDB provided the KIT 6 Stover EH, Chen J, Lee BH, Cools J, McDowell E, Adelsperger J et al. The small constructs and analyzed the KIT data; JC provided the FIPL1-PDGFRA mutants and molecule tyrosine kinase inhibitor AMN107 inhibits TEL-PDGFRbeta and FIP1L1- revised the article for intellectual content; PV designed the study, analyzed the data PDGFRalpha in vitro and in vivo. Blood 2005; 106: 3206 --3213. and wrote the paper. 7 Lierman E, Michaux L, Beullens E, Pierre P, Marynen P, Cools J et al. FIP1L1- PDGFRalpha D842 V, a novel panresistant mutant, emerging after treatment of E Lierman1,3, S Smits1, J Cools1,2, B Dewaele3, FIP1L1-PDGFRalpha T674I eosinophilic leukemia with single agent sorafenib. Leukemia 2009; 23: 845 --851. M Debiec-Rychter1,3 and P Vandenberghe1,3 1 8 Cross NC, Reiter A. Fibroblast growth factor receptor and platelet-derived growth Center for Human Genetics, KU Leuven, factor receptor abnormalities in eosinophilic myeloproliferative disorders. Acta Leuven, Belgium; Haematol 2008; 119: 199 --206. 2 Department of Molecular and Developmental Genetics, 9 Debiec-Rychter M, Sciot R, Le Cesne A, Schlemmer M, Hohenberger P, van CME - VIB11, Leuven, Belgium and Oosterom AT et al. KIT mutations and dose selection for imatinib in patients 3Center for Human Genetics, University Hospital Leuven, with advanced gastrointestinal stromal tumours. Eur J Cancer 2006; 42: Leuven, Belgium 1093 --1103. E-mail: [email protected] 10 Gozgit JM, Wong MJ, Wardwell S, Tyner JW, Loriaux MM, Mohemmad QK et al. Potent activity of ponatinib (AP24534) in models of FLT3-driven acute myeloid leukemia and other hematologic malignancies. Mol Cancer Ther 2011; 10: 1028 --1035. REFERENCES 11 Wasag B, Lierman E, Meeus P, Cools J, Vandenberghe P. The kinase inhibitor 1 O’Hare T, Shakespeare WC, Zhu X, Eide CA, Rivera VM, Wang F et al. AP24534, TKI258 is active against the novel CUX1-FGFR1 fusion detected in a patient with a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T-lymphoblastic leukemia/lymphoma and t(7;8)(q22;p11). Haematologica 2011; T315I mutant and overcomes mutation-based resistance. Cancer Cell 2009; 16: 96: 922 --926. 401 --412. 12 von Bubnoff N, Gorantla SP, Engh RA, Oliveira TM, Thone S, Aberg E et al. 2 Cools J, DeAngelo DJ, Gotlib J, Stover EH, Legare RD, Cortes J et al. A tyrosine The low frequency of clinical resistance to PDGFR inhibitors in myeloid neoplasms kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target with abnormalities of PDGFRA might be related to the limited repertoire of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med 2003; 348: of possible PDGFRA kinase domain mutations in vitro. Oncogene 2011; 30: 1201 --1214. 933 --943. 3 Gotlib J, Cools J. Five years since the discovery of FIP1L1-PDGFRA: what we have 13 Prenen H, Cools J, Mentens N, Folens C, Sciot R, Schoffski P et al. Efficacy of the learned about the fusion and other molecularly defined eosinophilias. Leukemia kinase inhibitor SU11248 against gastrointestinal stromal tumor mutants 2008; 22: 1999 --2010. refractory to imatinib mesylate. Clin Cancer Res 2006; 12: 2622 --2627. 4 Metzgeroth G, Erben P, Martin H, Mousset S, Teichmann M, Walz C et al. Limited 14 Guo T, Agaram NP, Wong GC, Hom G, D’Adamo D, Maki RG et al. Sorafenib inhibits clinical activity of nilotinib and sorafenib in FIP1L1-PDGFRA positive chronic the imatinib-resistant KITT670I gatekeeper mutation in gastrointestinal stromal eosinophilic leukemia with imatinib-resistant T674I mutation. Leukemia 2012; 26: tumor. Clin Cancer Res 2007; 13: 4874 --4881. 162 --164. 15 Zhou T, Commodore L, Huang WS, Wang Y, Thomas M, Keats J et al. Structural 5 Score J, Walz C, Jovanovic JV, Jones AV, Waghorn K, Hidalgo-Curtis C et al. mechanism of the Pan-BCR-ABL inhibitor ponatinib (AP24534): lessons for Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by overcoming kinase inhibitor resistance. Chem Biol Drug Des 2011; 77: 1 --11.

Molecular characterization of deletions of the long arm of chromosome 5 (del(5q)) in 94 MDS/AML patients

Leukemia (2012) 26, 1695--1697; doi:10.1038/leu.2012.9 the WHO criteria to be included in this subset of MDS, which appears to be rare.3,4 For over 15 years, many studies have focused on defining the Deletion of the long arm of chromosome 5 (del(5q)) is a common common deleted region of chromosome 5.5--10 Initially, the finding in myelodysplastic syndrome (MDS) and in acute myeloid Knudson two-hit model was thought to occur in these large leukemia (AML). First described in 1974 by Van den Berghe et al.,1 deletions. Now, the haploinsufficiency of one or more genes the 5q- syndrome, more frequently found in old-aged females, is seems the model to explain 5q deletion consequences.11 characterized by erythroid hypoplasia, macrocytic anemia, normal In this work, our objective was to characterize breakpoints to elevated platelets count, preponderance of monolobulated of del(5q) among 70 MDS and 24 AML patients using megakaryocytes, isolated 5q deletion and low rate of progression fluorescence in situ hybridization (FISH). We identified not only to AML. Now, the 5q- syndrome is recognized as a clinical and the common deleted region but also the common retained biological entity of MDS according to the revised World Health regions in these 94 MDS/AML patients. We differentiated patients Organization (WHO) classification in 2008.2 The WHO classification with isolated 5q deletion and those with additional chromosomal recognized that the 5q- syndrome is narrowly defined as de novo abnormalities. MDS with an isolated cytogenetic abnormality involving deletions Patients were distributed in two groups (Supplementary Table). between bands q21 and q32 of chromosome 5. The criteria for Group 1 consisted of patients with an apparently isolated del(5q) inclusion have evolved from the description by Van den Berghe (46 patients: 42 MDS and 4 AML). In this group, we integrated five et al.1 to normal to increased megakaryocytes with hypolobated patients with MDS associated with isolated del(5q), according to nuclei, o5% blasts, no Auer rods in bone marrow and normal or the criteria of the revised WHO classification (bone marrow: increased platelets and o5% blasts in blood. Furthermore, normal to increased megakaryocytes with hypolobated nuclei, additional cytogenetic abnormalities or 5% or more blasts in the o5% blasts, no Auer rods; blood: platelets normal or increased, blood or marrow is exclusionary for the diagnosis.2 Therefore, o5% blasts) (I25-I29). Group 2 included patients with del(5q) and most of the patients (95%) with an isolated 5q deletion do not fit additional cytogenetic abnormalities (48 patients: 28 MDS and 20

Accepted article preview online 17 January 2012; advance online publication, 31 January 2012

& 2012 Macmillan Publishers Limited Leukemia (2012) 1681 --1729