C-Kit Overexpression in Gastrointestinal Stromal Tumors Lauren Sutton

C-Kit encodes an RTK whose ligand is Stem Cell Growth Factor (SCF) PROTO-ONCOGENE: C-kit, also known as CD117 What does it do? Receptor tyrosine kinase – binds to Stem Cell Growth Factor (SCF) Similar to the structure of PDGF Transmembrane RTK that is used as a cell marker to identify hematopoietic progenitor cells in the bone marrow (it’s expressed on the surface) Mutates and becomes constitutively active

C-kit plays vital roles in hematopoietic stem cells, pigmentation, and progenitor cells of the gut Identified in 1987 as the cellular homologue of a viral oncogene - HZ4 feline sarcoma virus. Highly conserved in evolution. C-kit plays a role in red blood cell formation, lymphocyte proliferation, mast cell development and function, melanin formation, and gamete formation. We’ll be focused on c-kit’s role in the interstitial cells of Cajal (ICCs), cells that are in the walls of the GI tract and are part of the autonomic nervous system. ICCs are like the pacemakers of the GI tract – they organize contractions. ICCs form from mesenchymal mesoderm precursor cells. C-kit is vital in ICC differentiation and maturation GISTs arise from ICCs (other colon tumors generally arise from epithelium). Kit-positive precursors that receive signaling become ICCs; Kit-positive precursors that do NOT receive signaling become smooth muscle cells C-kit is inactive in ICCs once completely differentiated Mice Experiments: C-kit is encoded by the W locus (also known as the dominant white spotting locus). Homozygous for W/W (mutation in c-Kit) is LETHAL. This interferes with stem cell proliferation = deficiencies in blood islands in embryonic yolk sac, deficiencies in the fetal liver, spleen and bone marrow = death from anemia in embryogenesis or shortly thereafter. Mice that have a homozygous loss-of-function mutation in SCF OR that have no SCF die of severe anemia in the last stages of gestation or very soon after birth. Heterozygotes have mild anemia, sterility, and abnormalities in mutation (white spotting)

C-Kit is part of RTK/Ras/MAPK Pathway Part of the RAS pathway When SCF binds, the RTK dimerizes  ligand-induced dimerization. Leads to transphosphorylation, phosphotyrosines that serve as SH2 docking sites. SCF  c-Kit dimerizes, transphosphorylates  Grb2 and SOS binds through SH2 domain  Ras is activated  Raf is activated  Mek is activated  Erk is activated  gene transcription. Ultimately leads to activating transcription factors like Jun and ETV1. Jun = required to go through G1 of cell cycle – leads to proliferation. ETV1 = regulates things like angiogenesis, cell growth, proliferation, apoptosis. Also participates in JAK/STAT pathway C-kit can be deregulated by monoubiquitylation by Cbl, an E3 ubiquitin ligase, as well as a serine/threonine kinase (PKC) that inhibits the kinase domain. We’ll see later that there’s also a juxtaposition membrane that serves as an inhibitory mechanism as well. It’s important to shut c-kit off! Of course, mutations cause abnormal expression genes and products, which can lead to cancer

C-Kit is an oncogene mutated in 85% of all GISTs 85% of GISTs result from gain-of-function mutations in c-kit. Most frequently mutated at Exon 11 (67% of cases) and Exon 9 (10% of cases) EXON 9: Mutation occurs in the extracellular binding motif  this is where the ligand binds. Leads to homodimerization and autophosphorylation, regardless of whether the ligand is bound or not. Not as sensitive to imatinib/Gleevec because they tend to develop resistance very quickly due to second site mutations. However, they have a better response when Gleevec is followed up with Sunitinib or Dasatinib, small-molecule RTK inhibitors. Sunitinib targets PDGF receptor, Src, and VEGF receptor; has a much broader spectrum of targets Have better relapse free survival and overall survival rates. Tumors tend to be larger and located in the small intestine. About 8% have a mutation in PDGFR

Mutations in the juxtamembrane domain on Exon 11 can cause cancer EXON 11: Mutation occurs in the cytoplasmic juxtamembrane domain  this is close to the transmembrane domain. Regulatory portion of the RTK. Plays an inhibitory role in regulating the receptor. When mutated, it can’t suppress receptor firing. Sits above the N-terminal lobe; when ligand binds, tyrosine residues in the JM domain get transphosphorylated, which leads to the JM domain moving out of the kinase domain. When mutated, the JM domain is partially activated, even when the ligand isn’t present. Can be subtle missense mutations or whole deletions of the exon (terrible survival rates). GISTs can also (very rarely) be a familial trait. An allele encoding a mutant c-kit (deletion in the juxtaposition domain) can be passed down in a dominant fashion. This results in a constitutively active c-kit. The phenotype doesn’t manifest until the patient is late in life, past childbearing years. Exon 11 mutations are far more resistant to Gleevec.

GIST was traditionally difficult to treat Chemotherapy and radiation are generally ineffective Surgical removal only works if there is no metastasis Average 5 year survival rate = 76% Men and women are equally affected by GISTs, and it typically presents in patients 50 to 80. GISTs typically arise in the stomach and small intestine, but can also occur in the rectum, colon, and esophagus. 25% of GISTs end up metastasizing. They usually only have a 10% response to chemotherapy. Because of this, chemotherapy is rarely used now as a treatment. Surgery is also only effective if the tumor hasn’t spread. The surgery is tricky, because they must be removed along with a small area of normal tissue around it. Otherwise, the capsule/lining around the tumor may break and cause spreading of the tumor.

Gleevec is an effective treatment We’ve talked about imatinib/gleevec in class in its treatment of CML/ALL. It is a receptor tyrosine kinase inhibitor that blocks the ATP binding site of the kinase domain. With Gleevec, 50% of patients have tumors shrink in size and a reduction in disease symptoms. 97% of patients with surgical removal and Gleevec also had no recurrence after 1 year! Overall, Gleevec extends the median survival from 15 months to 5 years. This image: Can see the dramatic response in A (was labeled using a glucose analog, the bladder shows the accumulation of dye, control); In B, can see initial response after one month (darker grey = increased water density, which shows apoptosis and degeneration), and after 9 months you can see three more light spots designated by yellow arrows within the same space = robustly growing subpopulations. These three regrowths carried a secondary point mutation.

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