Clinical Case Signs and Symptoms Patient Profile: Has vomit blood Problem swallowing Low red blood cell count (anemia) Abdominal pain/discomfort Feels full after eating a small amount of food Weight loss Patient Profile: Male 60 yrs old Lets look at a clinical case. Louis Beck has decided he needs to pay a visit to his doctor because he has seen blood in his vomit, has had problem swallowing, low red blood cell, abdominal pain and discomfort, feels full after eating a small amount of food, and has lost a lot of weight. Mr. Beck is 60 yrs and after preforming a blood test his doctors find he has anemia and they decide to preform an endoscopy a tumor is seen in his stomach. An incisional biopsy is taken of Mr. Beck’s tumor and his histological slide looks like this. If you take a close look the cells are spindle shaped: elongated and skinny. Mr. Beck has a non-epithelial tumor in his stomach, but the doctors have no way at that time to differentiate between the different kinds of non-epithelial tumors and therefore can not choose the right treatment. So what type exact non-epithelial tumor does he have?

c-KIT discovery in Gastrointestinal Stromal Tumors (GIST) led to new diagnostic tools KIT staining in Normal cells KIT staining in GIST Just in 1987, Axel Ullrich and his research team had discovered the human proto-oncogene c-KIT in chromosome 4. But it was not until 11 years later that researchers independently in Japan and Sweden discover that a non-epithelial tumor called gastrointestinal stromal tumor expresses KIT. Therefore, a immunostain that tests positive for c-KIT can differentiate between GIST and other non-epithelial tumors. More than 80% of GIST cases carry mutations in KIT So that is exactly what Mr. Beck’s doctors did. They took his histological slide and immunostained them with antibody specific for the protein KIT. Above you can compare how normal cells from the GI tract look like and the excessive amount of c-KIT expression in neoplastic cells. And the pictures below show what a positive and negative test for KIT looks like. If it is negative then it is not GIST but other intestinal sarcomas like Leiomyosarcoma. Mr. Beck’s results were positive which means he has a gastrointestinal stromal tumors. KIT (+) Test GIST KIT (-) Test Leiomyosarcoma

c-KIT null mutant mice are sterile, have extensive white-spotting, and die of anemia So what is KIT and what does it do? Well when you knock out c-KIT in mice and produce homozygous null mutants for KIT there are sterile, have extensive white-spotting and severe anemia. How did we discover this? Well, in an experiment, the researchers produced a mouse model that carried the mutation K641E that is found in both sporadic and familial GIST. Figure A shows the “white-spotting” mouse with an uniformly white coat and black eyes which differentiates it form albino mice. In Figure B flow cytometry of the bone marrow revealed that in homozygote mutants Kit expression was almost undetectable compared to the the wild-type mice and even the heterozygote mice. Hence why null mutants develop severe anemia (absence of hematopoietic erythroid cells). The heterozygote, as suspected, had an intermediate expression of KIT. Figure C shows the testis of 1 month old mouse and how there is only 1 layer of germ cells and a complete lack of spermatozoa. Figure D is the testis of 3 month old mouse showing Leydig cell hyperplasia (i.e. enlargement of the Leydig cells in the testicles)  Infertility Figure E: Ovaries composed of spindle-shaped cells (short arrow). And lastly Figure F is a diagram showing the decrease of dermal mast cells in null mutants A

ICC (Interstitial cells of Cajal) do not require c-KIT for embryogenesis So that was the broad picture of normal biological role of KIT. Now lets zoom in and look at its role in Interstitial cells of cajal from were GIST develops. So in 1996 a group of French scientists wanted to identify which cell express KIT during embryogenesis, so marked c-kit expressing cells with the reporter gene lacZ (which was introduced by gene targeting at the Kit locus in mouse embryonic stem cells) and followed their fate during embryogenesis. What they discovered is that ICC express Kit during embryogenesis but are dependent on its expression for migration, proliferation, and survival during embryogenesis. Figure A is a visualization of KIT via immunofluorescence and as you can see they are found in both the external longitudinal muscle (lm) and the inner circular muscle (cm) of the small intestine (where they are suppose to be). Fig. B&C: Shows the presence of ICC (blue stains) in the stomach’s muscles and the pyloric part of a heterozygote for KIT . Fig D: Shows the ICC lacZ expression colon of homozygous null mice which is identical to the pattern observed in the heterozygote, so KIT is not necessary for embryonic ICC.

ICC require c-KIT for normal postnatal development and survival However, ICC require c-KIT for a controlled and normal postnatal development and survival. Fig A: The Kaplan-Meier curve shows the significant decrease survival for homozygote mutants because they succumb to gastric or intestinal obstruction by week 30. Fig B & C: Comparison of abdominal content (more specially the small bowel) between a WT KIT and null mutant Fig D: 6-month null mutant stomach and cecum enlarged due to ICC hyperplasia Fig E:Is a molecular close-up of what is going on with KIT. We can see both the expression and phosphorylation of KIT in GIST from two different null mutant mice. M is the control. The top half are protein lysates that were immunoprecipitated with anti-KIT antibody demonstrating the over-expression of KIT and the bottom have was immunoprecipitated with an anti-phosphotyrisine antibody demonstration the over-phosphorylation of KIT.

c-KIT (a RTK) is involved in many signal transduction pathways that lead to the hallmarks of cancer As a matter of fact, C-KIT is a receptor tyrosine kinase that is present in the surface of several cells types like we saw. It ligand Stem Cell Factor (SCF) (aka Steele) is a cytokine that is important in hematopoiesis, spermatogenesis, and melanogenesis. C-KIT belongs to the Type III RTK family which includes PDGF-alpha and beta. When SCF binds to KIT, it activates and triggers KIT’s dimerization, relief of auto-inhibitory interactions and trans-phosphorylates within the dimer. From there it recruits, phosphorylates and activates downstream signaling proteins. So how is this connected to cancer? Well several of the pathways downstream of KIT affect cell survival, migration, proliferation, and cell cycle progression- all which lead to the hallmarks of cancer. One of the downstream pathways of KIT is the MAPK pathway that we learned in class. KIT’s phosphorylated tyrosine attaches to GRB2’s SH2 domain and GRB2’s SH3 domain attaches to SOS (son of sevenless) which binds and activates Ras which activates Raf and the kinases cascade continues with MEK and MAPK. So imagine the implications of a mutated KIT and the signaling pathways it is involved….

Different mutations on different parts of the c-KIT gene cause it be constitutively active That is what happens in GIST: mutations in C-KIT gene happen that cause it to be constitutively active The mutations associate with GIST are gain-of-function and cause KIT to become ligand independent and constitutively active; therefore Kit is an oncogene . We call these multiple mutation sites in KIT “HOT SPOTS”. Some correspond to the extracellular juxtamembrane domain (exon 8 and exon), and the intracellular juxtamembrane (exon 11), and the activation loop of the kinase domain (exon 17). The Extracellular juxtamembrane domain (exon 8 & 9) consist of the 4th and 5th immunoglobin (Ig)-like loops that are involved in correctly orienting receptor monomers, stabilizing the dimers, and mediating dimer interaction. Exon 8 is an important mutation in AML and exon 9 is mutated in almost 10% of all GIST. The most common site of mutation in GIST with a 65% is exon 11 which codes for the intracellular juxtamembrane domain (JMD) which is a key autoregulatory domain that stabilizes the inactive conformation of the kinase domain. When there is no ligand and KIT should be inactive, JMD folds back into the active site of the kinase. Most GIST develop form sporadic mutations and only 5% of GIST are familial. Since both sporadic and familial mutations in GIST are inherited in an autosomal dominant way each child from an affected parent, who has a mutation in their germ line, has a 50% percent chance of developing and usually develops it at an earlier age than the previous person. A mutation in exon 11 is commonly found in familial GIST. Most mutations that occur in exon 11 are point mutations, tandem duplication (alanine and tyrosine) deletions, and insertions. (65%)

c-KIT mutations in GIST disrupt the auto-inhibitory mechanisms Inactive; Auto-inhibited Active Here we can compare the differences between an inactive c-KIT and an activate c-KIT. The purple region is the activation loop (encoded by exon 17 which found in AML (acute myeloid leukemia)). In an inactive state, the activation loop obstructs access of substrates to the active site. The yellow, less loopy region in the JMD (juxtamembrane domain) folds back into the active site of the kinase domain when KIT is inactive, and when JMD is relief from its autoinhibiton it folds out and it results in kinase activation by favoring conformations in which the activation loop is shifted away from the active site allowing space for ATP to align. Once ATP is bound catalysis can begin.

Gleevec: first line treatment for metastatic GIST So now that we know the mutations that make KIT not function properly and the structure of KIT, we can help treat Mr. Beck, our patient that we saw in the beginning of our presentation. Mr. Beck has a mutation in exon 11 that is causing KIT to be constitutively active by preventing the JMD (juxtamembrane domain) to interact with the active site cleft. His tumor is to big to me surgically removed, so what are his options? -Imatinib, or Gleevec, is the first line treatment for GIST. It was first used to treat CML (chronic myeloid leukemia) by inhibiting the tyrosine kinase activity of the BCR/ABL oncoprotein like we learned in class. And Imatinib has revolutionized the treatment and prognosis for patients with GIST, who before Imatinib was available would succumb to metastatic GIST in just 18 months. Imatinib has become the poster child for targeted therapy in sarcomas. Partial response or disease stabilization is achieved in ~80 % of patients with a median survival of 2 years. But how can be Imatinib highly selective when the human genome encodes up to 800 serine/threonine/tyrosine kinases all which bind ATP? Targeting directly the binding site would be challenging and not at all selective. However, regions proximal to the ATP binding pocket offer an appealing option. What Imatinib does it that it binds to the inactive conformation of the kinase in which the activation loop is folded into the ATP-binding site hence blocking it. Imatinib selective targets c-KIT via c-KIT’s hydrophobic pocket in which Imatinib forms H-bonds with specific amino acids like Thr670 (Tyrosine), Glu640 (Glutamic acid), Cys673 (Cysteine), and Asp 810 (Aspartic acid). ATP

Resistance due to secondary mutations force researchers to seek alternative treatments Can this cancer ever be eradicated with kinase inhibitors alone? The answer is probably no. Despite the success of Imatinib, about ½ of patients with metastatic GIST develop resistance almost always due to a second mutation in the same allele of KIT as the primary mutation. Common mutations act on the activation loop or the kinase domain (exons 13&14). These mutations prevent Imatinib to bind to the inactive conformation of KIT or influence the conformation of its binding site (H-bond lost with tyrosine). Thus second line inhibitors like Sunitib, which binds to the inactive conformation instead of the inactive, are used. Nonetheless, patients rapidly relapse on drug withdrawal especially if discontinued within 3 years. Targeting downstream signaling pathways that are required for KIT-dependent growth like PI3K and mTOR which are part of a pathway that appears to be critical in survival of GIST. The MAPK pathway is activated in primary GIST, so using drug that inhibit BRAF like Sorafenib (induces cell cycle arrest and apoptosis in clear-cell renal carcinoma) and Vermurafenib and Dabranenib (both used in melanoma targeted therapy) are being explored as a possibility. Also studies in GIST show that many cells in GIST become quiescent and do not undergo apoptosis when treated with Imatinib and therefore can resume proliferation. Thus, targeting mediators of cell survival is being explored as an adjuvant and alternative therapy. Lastly, Mutational Analysis which is now technically straightforward and relative inexpensive compared to chemo is a great need for GIST patients. For example, patients’ sensitivity to Imatinib varies depending on the exon mutation. Patients with a mutation in exon 11 and exon 9 have a stronger and more durable response to gleevec, and those with exon 9 mutation benefit more from a higher dose.

Sources Bernex, F., P. De, C. Kress, C. Elbaz, C. Delouis, and J. J. Panthier. "Spatial and Temporal Patterns of C-kit-expressing Cells in WlacZ/+ and WlacZ/WlacZ Mouse Embryos." Development (Cambridge, England). U.S. National Library of Medicine, Oct. 1996. Web. 07 Apr. 2017. <https://www.ncbi.nlm.nih.gov/pubmed/8898216>. Rubin, B. P., Cristina R. Antonescu, and James P. Scott-Browne. "A Knock-In Mouse Model of Gastrointestinal Stromal Tumor Harboring Kit K641E." Cancer Research65.15 (2005): 6631-639. American Association for Cancer Research. American Association for Cancer Research, 1 Aug. 2005. Web. 9 Apr. 2017. Manley, P.W., S.W. Cowan-Jaob, and D. Fabbro. "Imatinib: A Selective Tyrosine Kinase Inhibitor." ScienceDirect. European Journal of Cancer, 2002. Web. 12 Apr. 2017. Tarn, Chi, Erin Merkel, Adrian A. Canutescu, Wei Shen, Yuliya Skorobogatko, Martin J. Heslin, Burton Eisenberg, Ruth Birbe, Arthur Patchefsky, Roland Dunbrack, J. Pablo Arnoletti, Margaret Von Mehren, and Andrew K. Godwin. "Analysis of KIT Mutations in Sporadic and Familial Gastrointestinal Stromal Tumors: Therapeutic Implications through Protein Modeling." Clinical Cancer Research. American Association for Cancer Research, 15 May 2005. Web. 12 Apr. 2017. <http://clincancerres.aacrjournals.org/content/11/10/3668.long>. Ashman, Leonie K., and Renate Griffith. "Expert Opinion on Investigational Drugs." Taylor and Francis Online. Expert Opinion on Investigational Drugs, 6 Nov. 2012. Web. 13 Apr. 2017. <http://www.tandfonline.com/doi/full/10.1517/13543784.2013.740010>. Klinac, Dragana, Elin Solomonovna Gray, Mel Ziman, and Michael Millward. "Advances in Personalized Targeted Treatment of Metastatic Melanoma and Non-Invasive Tumor Monitoring." Frontiers in Oncology. Frontiers in Oncology, 28 Feb. 2013. Web. 13 Apr. 2017. <http://journal.frontiersin.org/article/10.3389/fonc.2013.00054/full>. Lynch, Henry T., Jane F. Lynch, and Trudy G. Shaw. "Hereditary Gastrointestinal Cancer Syndromes." Gastrointestinal Cancer Research : GCR. International Society of Gastrointestinal Oncology, July-Aug. 2011. Web. 13 Apr. 2017. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3283002/>. "Signs and Symptoms of Gastrointestinal Stromal Tumors." American Cancer Society. American Cancer Society, n.d. Web. 13 Apr. 2017. <https://www.cancer.org/cancer/gastrointestinal-stromal-tumor/detection-diagnosis-staging/signs-symptoms.html>. "GIST Overview." GIST Support International. GIST Support International, n.d. Web. 13 Apr. 2017. <http://www.gistsupport.org/>.