Peter Besmer

Recruitment of Stem and Progenitor Cells from the Bone Marrow Niche Requires MMP-9 Mediated Release of Kit-Ligand

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit+ stem/progenitors. In MMP-9-/- mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus

Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility1. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy1,2. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W19H has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis, c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus3 and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor4,5. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.

Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family--oncogenic activation of v-kit involves deletion of extracellular domain and C terminus.

The protein kinase domains of v‐kit, the oncogene of the acute transforming feline retrovirus HZ4‐FeSV (HZ4‐feline sarcoma virus), CSF‐1R (macrophage colony stimulating factor receptor) and PDGFR (platelet derived growth factor receptor) display extensive homology. Because of the close structural relationship of v‐kit, CSF‐1R and PDGFR we predicted that c‐kit would encode a protein kinase transmembrane receptor (Besmer et al., 1986a; Yarden et al., 1986). We have now determined the primary structure of murine c‐kit from a DNA clone isolated from a brain cDNA library. The nucleotide sequence of the c‐kit cDNA predicts a 975 amino acid protein product with a calculated mol. wt of 109.001 kd. It contains an N‐terminal signal peptide, a transmembrane domain (residues 519‐543) and in the C‐terminal half the v‐kit homologous sequences (residues 558‐925). c‐kit therefore contains the features which are characteristic of a transmembrane receptor kinase. Comparison of c‐kit, CSF‐1R and PDGFR revealed a unique structural relationship of these receptor kinases suggesting a common evolutionary origin. The outer cellular domain of c‐kit was shown to be related to the immunoglobulin superfamily. The sites of expression of c‐kit in normal tissue predict a function in the brain and in hematopoietic cells. N‐terminal sequences which include the extracellular domain and the transmembrane domain as well as 50 amino acids from the C‐terminus of c‐kit are deleted in v‐kit. These structural alterations are likely determinants of the oncogenic activation of v‐kit.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation

Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.

Tumor mitotic rate, size, and location independently predict recurrence after resection of primary gastrointestinal stromal tumor (GIST)

Gastrointestinal stromal tumor (GIST) is the most frequent sarcoma of the intestinal tract and often shows constitutive activation of either the KIT or PDGFRA receptor tyrosine kinases because of gain‐of‐function mutation. Although the efficacy of tyrosine kinase inhibitors in metastatic GIST depends on tumor mutation status, there have been conflicting reports on the prognostic importance of KIT mutation in primary GIST.

Novel V600E BRAF mutations in imatinib‐naive and imatinib‐resistant gastrointestinal stromal tumors

BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild‐type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib‐naive wild‐type GISTs from 61 patients, including 15 children and 28 imatinib‐resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49‐ to 55‐years‐old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF‐mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.

Gastrointestinal stromal tumors in children and young adults: a clinicopathologic, molecular, and genomic study of 15 cases and review of the literature.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the intestinal tract that typically occur in adults over the age of 40 years. GISTs in younger patients are rare and not well characterized. The objective was to define the characteristics of GISTs in children and young adults (<30 years old). Clinicopathologic and molecular features, including KIT/PDGFRA genotype, in GISTs from 5 children and 10 young adults were analyzed. Gene expression analysis was performed on 5 gastric tumor samples from 2 children, 2 gastric tumors from young adults, and 10 gastric GISTs from older adults using an U133A Affymetrix platform (22,000 genes). All five pediatric GISTs occurred in girls, involved the stomach as multiple nodules, showed predominantly an epithelioid morphology, often involved lymph nodes, and lacked KIT or PDGFRA mutations. Although all five patients developed recurrence (four in the liver, three in the peritoneum, and two in both sites), four are still alive with disease. Of the 10 GISTs in young adults, half occurred in the small bowel and had spindle cell morphology, and one case had lymph node metastasis. KIT mutations were identified in seven cases, four in exon 11 and three in exon 9. Seven patients developed recurrence, and at last follow-up two patients had died of disease. Gene expression analysis showed high expression of PHKA1, FZD2, NLGN4, IGF1R, and ANK3 in the pediatric and young adult versus older adult cases. GISTs that occur in children are a separate clinicopathologic and molecular subset with predilection for girls, multifocal gastric tumors, and wild-type KIT/PDGFRA genotype. In contrast, GISTs in young adults are a more heterogeneous group, including cases that resemble either the pediatric or the older adult-type tumors. The distinct gene expression profile suggests avenues for investigation of pathogenesis and potential therapeutic strategies.

Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells.

The pleiotropic effects of the Kit receptor system are mediated by Kit‐Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3′‐kinase (PI3‐kinase), p21ras and mitogen‐activated protein kinase (MAPK). To define the role of PI3‐kinase, p21ras and MAPK in Kit‐mediated cell proliferation, survival and adhesion in bone marrow‐derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c‐kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL‐induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3‐kinase, p21ras and MAPK, and induced expression of c‐fos, junB, c‐myc and c‐myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3‐kinase activation, diminished c‐fos and junB induction, and impaired KL‐induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3‐kinase, p21ras and MAPK activation, and induction of c‐myc, c‐myb, c‐fos and c‐jun mRNA, while KL‐induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3‐kinase in Kit‐mediated cell adhesion, wortmannin blocked Kit‐mediated cell adhesion at concentrations known to specifically inhibit PI3‐kinase. We conclude, that association of Kit with p85PI3‐K, and thus with PI3‐kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit‐mediated mitogenesis and survival, but not cell adhesion.

Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Δ/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition, mast cell numbers in the dorsal skin were increased. Therefore KitV558Δ/+ mice reproduce human familial GISTs, and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly, these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal.

L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition

Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non‐chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation‐carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KITL576P mutant cells showed sensitivity for dasatinib (previously known as BMS‐354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib‐sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KITL576P mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.