Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation

Leukemia (2013) 27, 278–285 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation Y Bai1,4, G Bandara1,4, E Ching Chan1, I Maric2, O Simakova2, SN Bandara1, W-P Lu3, SC Wise3, DL Flynn3, DD Metcalfe1, AM Gilfillan1 and TM Wilson1 Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34 þ -derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FceRImediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits. Leukemia (2013) 27, 278–285; doi:10.1038/leu.2012.218 Keywords: mastocytosis; switch pocket; KIT; KIT D816V; tyrosine kinase inhibitor; mast cell INTRODUCTION KIT is a member of the tyrosine kinase-containing family of growth factor receptors, which is expressed on restricted hematopoietic cell lineages including mast cells.1 Activation of KIT, following ligation by stem cell factor (SCF), is critical for mast cell growth, survival, differentiation and homeostasis, and may contribute to mast cell homing to resident tissues.2 The extent of antigenmediated mast cell activation within these tissues also has the capacity to be upregulated by SCF-dependent KIT activation.3 Hence, inappropriate KIT activation profoundly influences mast cell-driven pathology. Systemic mastocytosis is characterized by the pathological accumulation of neoplastic mast cells in various tissues accompanied by activating mutations in KIT, most notably the aspartic acid to valine substitution at residue 816 (KIT D816V).4 Clinically, this clonal accumulation often results in persistent mast cell mediator-related symptoms and, in rare cases, aggressive neoplastic overgrowth with end-organ failure. In these latter presentations, lifespan is significantly reduced and cytoreductive therapy is required.5 To date, tyrosine kinase inhibitors targeting the KIT D816V mutation have demonstrated only modest efficacy in advanced disease states and are often accompanied by significant side effects, likely the result of off-target kinase inhibition.6–9 Most tyrosine kinase inhibitors target the ATP binding pocket, which is often conserved among kinases and subject to competition from high intracellular ATP concentrations. For reasons not clearly understood, certain ATP-competitive inhibitors do not potently inhibit oncogenic forms of kinases, which adopt aggressive, constitutively active conformations independent of normal regulatory mechanisms. KIT kinase can be aggressively activated in this manner and KIT mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.10 KIT has an interior pocket located between the N and C lobes of the kinase, which functions as a switch pocket (SP). KIT also has two pendent switch ligands, which compete for occupancy of this SP.11 If the inhibitory switch (exon 11 juxtamembrane domain) of KIT occupies this SP, the kinase adopts an inactive conformation. If the activating switch (exon 17 activation loop) occupies this SP, the kinase adopts a catalytically active conformation. Thus, changes in phosphorylation of both the inhibitory and activating switches govern the overall activity of KIT by regulation of the occupancy of its SP by its pendent inhibitory and activating switches. Primary mutations and/or deletions in the exon 11 inhibitory switch render KIT constitutively active and such mutations drive the progression of gastrointestinal stromal tumors.12 In mast cell leukemia/mastocytosis, the primary KIT mutation resides in the activating switch, and in particular, the D816V exon 17 mutation is most aggressive. Classical ATP-competitive KIT inhibitors, including imatinib, sunitinib, sorafenib and regorafenib, do not block this aggressive mutant form of KIT. The SP inhibitors, DP-2976 and DP-4851, were developed by rational design to block 1 Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; 2Department of Laboratory Medicine, Clinical Center, Bethesda, MD, USA and 3Deciphera Pharmaceuticals, Lawrence, KS, USA. Correspondence: Dr TM Wilson, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 12S235A, 10 Center Drive, Bethesda, MD, 20892-1881, USA. E-mail: 4 These authors share first authorship. Received 29 June 2012; revised 17 July 2012; accepted 19 July 2012; accepted article preview online 31 July 2012; advance online publication, 21 August 2012 Novel mechanism to suppress mast cell activation and neoplastic proliferation Y Bai et al 279 access to the KIT SP and prevent KIT D816V from adopting the catalytically active conformation. In addition, because SP inhibitors are not ATP competitive, they are resilient even to high 4–5 mM concentrations of cellular ATP. Here we describe the ability of two KIT SP inhibitors, DP-2976 and DP-4851, to potently and selectively inhibit both wild-type (WT) and KIT D816V activation, and consequently proliferation of both neoplastic and non-neoplastic mast cell populations. Furthermore, these inhibitors effectively blocked the ability of SCF to enhance antigen-mediated mast cell activation. These studies demonstrate that by targeting the SP, this class of KIT inhibitors possesses a novel mechanism of inhibition whose dual suppression of KIT D816V neoplastic proliferation and SCFenhanced mast cell activation advocate future clinical development. MATERIALS AND METHODS Reagents and antibodies DP-2976 and DP-4851 were synthesized within Deciphera Pharmaceuticals LLC (Lawrence, KS, USA). PKC 412 and imatinib were purchased from LC laboratories (Woburn, MA, USA). All compounds were prepared and stored as a 10 mM stock solution in dimethylsu (...truncated)