Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. T he rapid growth in large cancer-sequencing efforts using exome and genome sequencing, as well as elucidation of copy number variations of many cancers, has provided important information about the genetic landscapes and somatic mutations that occur in most cancers. The various catalogs of somatic mutations, chromosomal translocations, and aberrant gene expressions have provided valuable insights about distinct patient populations exhibiting gain-of-function mutations that function as causal "driver" genes for subtypes of different cancers (1-3). These studies have revealed numerous oncogenic mutations in receptor tyrosine kinases (RTKs), which result in constitutive ligand-independent tyrosine kinase activation, cell transformation, and oncogenesis. Consequently, more than 20 kinase inhibitors and half a dozen therapeutic antibodies that block the action of RTKs or the action of critical components of their intracellular signaling pathways have been developed during the past decade and successfully applied in the clinic for treatment of many cancers.A variety of gain-of-function somatic mutations in the receptor tyrosine kinase KIT, including point mutations, in-frame deletions, and in-frame duplications, have been identified in human cancers, including gastro-intestinal-stromal tumors (GISTs), acute myeloid leukemia (AML), mast cell leukemia (MCL), and melanomas (4). Activation of the receptor tyrosine kinase KIT by its ligand stem cell factor (SCF) plays a central role in development of germ cells, hematopoietic cells, interstitial pacemaker cells, and other cells (5). Like other members of the type ΙΙΙ family of RTKs, the extracellular ligand binding domain of KIT contains five Ig-like modules (designated as D1, D2, D3, D4, and D5) connected to a single transmembrane helix, followed by a cytoplasmic region containing a regulatory juxtamembrane (JM) domain, a tyrosine kinase domain (TKD) with a kinase insert region and a C-terminal tail. Tyrosine autophosphorylation sites in the kinase insert region and the...

Somatic oncogenic mutations in the receptor tyrosine kinase KIT function as major drivers of gastrointestinal stromal tumors and a subset of acute myeloid leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression of disease eventually occurs in virtually all patients. In this report, we describe inhibitory KIT antibodies that bind to the membrane-proximal Ig-like D4 of KIT with significant overlap with an epitope in D4 that mediates homotypic interactions essential for KIT activation. Crystal structures of the anti-KIT antibody in complex with KIT D4 and D5 allowed design of affinity-matured libraries that were used to isolate variants with increased affinity and efficacy. Isolated antibodies showed KIT inhibition together with suppression of cell proliferation driven by ligand-stimulated WT or constitutively activated oncogenic KIT mutant. These antibodies represent a unique therapeutic approach and a step toward the development of "naked" or toxin-conjugated KIT antibodies for the treatment of KIT-driven cancers.phosphorylation | therapeutic antibodies | cancer therapy | cell signaling | protein kinase