KIT Kinase Domain Research Articles

KIT in oocytes: a key factor for oocyte survival and reproductive lifespan

The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).

Molecular docking study of Xanthyl Chalcone derivatives as potential inhibitor agents against KIT tyrosine kinase and KIT kinase domain mutant D816H

Molecular docking study of Xanthyl Chalcone derivatives as potential inhibitor agents against KIT tyrosine kinase and KIT kinase domain mutant D816H

Whole Exome Sequencing of Relapsed Double-Mutated CEBPA Acute Myeloid Leukemia Identified Mutated KIT and WNT10A As a Potential Co-Mutation with Negative Impact on Prognosis

Background: Acute myeloid leukemia (AML) with double-mutated CEBPA (dmCEBPA) occurred in about 10% of AML and it is usually associated with normal karyotype AML. It is classified as a favorable risk AML with an estimated 5 years overall survival in excess of 50%. Patients who achieved complete remission (CR) with induction chemotherapy will be consolidated with chemotherapy. Around 40% of the patients would suffer a relapse and required further salvage chemotherapy followed by allogeneic stem cell transplant. Co-mutations occurred frequently in dmCEBPA AML with TET2 being the most commonly co-mutated (34%) followed by GATA1 mutation (21%), WT1(13.7%), DNMT3a (9.6%), ASXL1 (9.5%), NRAS (8.4%) and other less common genes. Only TET2 mutation appeared to have negative impact on prognosis. In this study, we explore the relapse cases with Whole-Exome sequencing (WES) and compared with a control case (Chemo-responsive dmCEBPA AML who is in continuous complete remission (CCR)). We aimed to identify novel mutations not previously reported and with potential as new prognostic marker as well as therapeutic target for further exploration. Method: We identified 3 relapsed dmCEBPA AML cases from our leukemia cell bank and compared with a case of chemo-responsive dmCEBPA AML who is still in CCR at 8 years follow-up. Duration of prior CR for case #1, #2 and #3 were 10 months, 1 year 9 month and 1 year 10 months. WES was performed for all 4 cases. In each case, the variants were annotated using the IonReporter software and an additional customized script that annotates for variant population frequency, genotype-phenotype associations and computational missense prediction tools. Variants were filtered by removing all reference calls, intergenic variants and variants not present in the chemoresponsive case. Only exonic and non-synonymous variants were considered. Next, we look for variants with a phenotype-genotype association common with the AML phenotype by looking for variants with "Leukemia" terms listed in OMIM. In addition, in order to identify novel mutations not presently associated with AML, we identified variants that had a "Pathogenic" entry in ClinVar. Results: All 4 cases revealed the typical CEBPA mutants with VAF exceeding 40% except one with germline mutation. When compared with the chemo-responsive case, KIT was mutated in 2 out of 3 relapse cases. WNT10A was mutated in all 3 cases. The details of the mutated genes are presented in Table 1 (see below). WNT10A gene encodes for a secretory signaling protein in the WNT family of structurally related genes that are involved WNT-beta-catenin-TCF signaling pathway. WNT10A is strongly expressed in cell lines of promyelocytic leukemia. Beta-catenin, the downstream target of the WNT signaling pathway, is highly expressed in poor prognostic AML cells (Staal et al. Nature review Immun 2008). The Gly213 residue is highly evolutionary conserved in vertebrate with a PhyloP score of 9.8. Both missense variants Gly213Ser and Arg171Cys are computationally predicted be damaging (DANN score 0.99). However, these 2 variants are fairly common in the East Asian population (gnomAD_EAS MAF G213S 2.7% & R171C 1.5%). Both variants are ACMG guidelines classified as Variants of Unknown Significance (VUS). KIT gene encodes for the proto-oncogene c-kit. Mutations in c-kit are associated with mastocytosis and AML. Both variants occur within the kinase domain (exon 17) of KIT. Asp816Val variant has been linked to poorer prognosis and worse outcome on AML patients. Both missense variants are computationally predicted to be damaging (DANN score 0.99). Both Asp816Val and Asn822Lys are AMCG classified as Likely Pathogenic and VUS respectively. Conclusion: With stringent criteria of filtering in WES of relapsed dmCEBPA cases, when compared with the control, we found that KIT was mutated in 2 out of 3 cases, while WNT10A was mutated in all 3 cases. Though the sample number was small, these findings would warrant further evaluation in larger cohort of dmCEBPA AML and interrogation in pre-clinical model. Table 1. Disclosures No relevant conflicts of interest to declare.

ATP-Competitive Inhibitors Midostaurin and Avapritinib Have Distinct Resistance Profiles in Exon 17-Mutant KIT.

KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17-mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations. SIGNIFICANCE: This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.

Computational and pharmacological investigation of novel 1,5-diaryl-1,4-pentadien-3-one derivatives for analgesic, anti-inflammatory and anticancer potential.

Objective(s):The novel 1,5-diaryl-1,4-pentadien-3-one derivatives were studied for analgesic, anti-inflammatory and anticancer potential to establish their role in pain, inflammatory disorders and cancer.Materials and Methods:Two 1,5- diaryl-1,4-pentadien-3-one derivatives: (1E,4E)- 5-(4-fluoro phenyl)-1-(4-methoxyphenyl)- 2-methylpenta-1,4-dien-3-one (A2K2A17) and (1E,4E)-5-(4-nitrophenyl)-1-(4-nitrophenyl)-2-ethylhexa-1,4-dien-3-one (A11K3A11) were synthesized and characterized via 1H NMR and 13C NMR techniques. Molecular docking, anti-inflammatory, analgesic and anticancer activities were performed using Auto Doc Vina, carrageenan mediated paw edema and formalin induced chronic inflammation, acetic acid induced writhings and hotplate assay and brine-shrimp lethality assay. Results:A2K2A17 and A11K3A11 showed high computational affinities (binding energy > -9.0 Kcal/mol) against COX-1, kappa receptor and braf kinase domain. A2K2A17 and A11K3A11 exhibited moderate docking affinities (binding energy > -8.0 Kcal/mol) against COX-2, human capsaicin receptor, tumor necrosis factor, lipoxygenase, colony stimulating factor, delta receptor, cyclin dependent protein kinase-2, mitogen activated kinase, mu receptor and kit kinase domain. A2K2A17 and A11K3A11 possess low docking affinities (binding energy > -7.0 Kcal/mol) against purinoceptor, platelets-derived growth Factor-1 and vascular-endothelial growth factor. In analgesic activity, A2K2A17 (1-30 mg/kg) and A11K3A11 (1-10 mg/kg) decreased acetic acid induced writhes and prolonged the latency time (P<0.01, P<0.001 vs saline group) respectively. A2K2A17 (10-30 mg/kg) and A11K3A11 (1-10 mg/kg) reduced carrageenan as well as formalin mediated edema (P<0.01, P<0.001). A2K2A17 found effective for cytotoxicity assay with LC50 value 1.5 µg/ml. Conclusion:The in silico, in vitro and in vivo studies on A2K2A17 and A11K3A11 reports their computational binding affinities against targets as well as the analgesic, anti-inflammatory and the anticancer effects.

Oncogenic Kit signalling on the Golgi is suppressed by blocking secretory trafficking with M-COPA in gastrointestinal stromal tumours

Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as “AMF-26”), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs.

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR.

Identification of a secondary mutation in the KIT kinase domain correlated with imatinib-resistance in a canine mast cell tumor

Imatinib-resistance is a major therapeutic problem in human chronic myeloid leukemia, human gastrointestinal stromal tumors, and canine mast cell tumors. In the present study, we identified the secondary mutation c.2006C>T in c-KIT exon 14 in a mast cell tumor obtained from a dog carrying c.1663-1671del in exon 11 and showing resistance to imatinib. The mutation in exon 14 resulted in substitution of threonine with isoleucine at position 669, which was located at the center of the ATP binding site as a gatekeeper and played an important role in binding to imatinib. Transfectants were constructed to survey the functions of these mutations in exons 11 and 14. The transfectant with mutant KIT encoded by c-KIT carrying c.1663-1671del showed constitutive ligand-independent phosphorylation that was suppressed by imatinib, indicating a gain-of-function mutation. Furthermore, the transfectant with mutant KIT encoded by c-KIT carrying both c.1663-1671del and c.2006C>T caused ligand-independent phosphorylation, which was not suppressed by imatinib. From these results, we concluded that the mutation c.2006C>T in c-KIT exon 14 was an imatinib-resistance mutation in a canine mast cell tumor. These findings revealed, for the first time, a mechanism of imatinib resistance in a clinical case of canine mast cell tumor.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

Abstract 4020: Inhibition of KIT in vivo modifies immune cell populations to improve the efficacy of checkpoint inhibitors in syngeneic mouse tumor models

Abstract The KIT receptor tyrosine kinase plays critical roles in GIST and a range of other tumor types, and when activated by somatic mutation serves as a potent oncogenic driver in these neoplasms. Whereas small molecule inhibitors of the tyrosine kinase domain of KIT have demonstrated considerable success in treatment of GIST patients with some KIT mutations, there remains a need to develop additional KIT-directed therapies for treatment of GIST patients that fail to respond to these agents, or that develop resistance during therapy. Furthermore, KIT is prominently expressed on mast cells, which promote an immune evasive environment within the tumor microenvironment by influencing the function of other immune cells, such as myeloid derived suppressor cells (MDSC’s). However, the potential impact of KIT inhibition on anti-tumor immunity has not been studied extensively to date. KTN0158 is a humanized anti-KIT IgG1 monoclonal antibody that specifically binds to the extracellular domain of KIT, and is being developed as a potential therapy for cancer and other mast cell-related diseases such as neurofibromatosis type 1 (NF1). KTN0158 inhibits KIT signaling and function in vitro and in vivo and has demonstrated anti-tumor activity in dogs with mast cell tumors expressing either wild-type or mutant KIT. In order to explore the potentially broad anti-tumor benefit of KIT inhibition by an antibody, a series of syngeneic tumor models were evaluated for sensitivity and immune cell profile changes in response to a surrogate antibody that recognizes mouse KIT (ACK2), dosed as either a single agent or in combination with T cell checkpoint inhibitors. Within this panel of tumor models, ACK2 strongly enhanced the anti-tumor activity provided by an anti-CTLA4 antibody in the colon26 model, while exhibiting no anti-tumor activity when dosed as a single agent. The colon26 cell line does not express detectable levels of KIT, and does not exhibit sensitivity to ACK2 or SCF treatment in proliferation assays performed in vitro, suggesting that this effect of ACK2 is not due to a direct effect on the tumor cells. Strikingly, ACK2 treatment was associated with pronounced effects on immune cell populations, including a profound decrease in both intra-tumoral and splenic monocytic MDSC counts. These observations suggest that KIT inhibition in vivo has the potential to modulate immune cell populations including MDSC's and mast cells, thereby targeting immune suppressive innate immune cells in the tumor and sensitizing the tumor to immune checkpoint inhibitors. Furthermore, the data provide a rationale to investigate KTN0158 in combination with immune checkpoint inhibitors across a range of human tumor types. Citation Format: Andrew J. Garton, Lori Lopresti-Morrow, Scott Seibel, Theresa LaVallee, Richard Gedrich. Inhibition of KIT in vivo modifies immune cell populations to improve the efficacy of checkpoint inhibitors in syngeneic mouse tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4020.

Familial systemic mastocytosis with germline KIT K509I mutation is sensitive to treatment with imatinib, dasatinib and PKC412

Mastocytosis are myeloproliferative neoplasms commonly related to gain-of-function mutations involving the tyrosine kinase domain of KIT. We herein report a case of familial systemic mastocytosis with the rare KIT K509I germ line mutation affecting two family members: mother and daughter. In vitro treatment with imatinib, dasatinib and PKC412 reduced cell viability of primary mast cells harboring KIT K509I mutation. However, imatinib was more effective in inducing apoptosis of neoplastic mast cells. Both patients with familial systemic mastocytosis had remarkable hematological and skin improvement after three months of imatinib treatment, suggesting that it may be an effective front line therapy for patients harboring KIT K509I mutation.

Clinical relevance of molecular aberrations in paediatric acute myeloid leukaemia at first relapse.

Outcome for relapsed paediatric acute myeloid leukaemia (AML) remains poor. Strong prognostic factors at first relapse are lacking, which hampers optimization of therapy. We assessed the frequency of molecular aberrations (FLT3, NRAS, KRAS, KIT, WT1 and NPM1 genes) at first relapse in a large set (n=198) of relapsed non-French-American-British M3, non-Down syndrome AML patients that received similar relapse treatment. We correlated molecular aberrations with clinical and biological factors and studied their prognostic relevance. Hotspot mutations in the analysed genes were detected in 92 out of 198 patients (46·5%). In 72 of these 92 patients (78%), molecular aberrations were mutually exclusive for the currently analysed genes. FLT3-internal tandem repeat (ITD) (18% of total group) mutations were most frequent, followed by NRAS (10·2%), KRAS (8%), WT1 (8%), KIT (8%), NPM1 (5%) and FLT3-tyrosine kinase domain (3%) mutations. Presence of a WT1 aberration was an independent risk factor for second relapse (Hazard Ratio [HR]=2·74, P=0·013). In patients who achieved second complete remission (70·2%), WT1 and FLT3-ITD aberrations were independent risk factors for poor overall survival (HR=2·32, P=0·038 and HR=1·89, P=0·045 respectively). These data show that molecular aberrations at first relapse are of prognostic relevance and potentially useful for risk group stratification of paediatric relapsed AML and for identification of patients eligible for personalized treatment.

Flumatinib, a selective inhibitor of BCR‐ABL/PDGFR/KIT, effectively overcomes drug resistance of certain KIT mutants

Activating mutations in KIT have been associated with gastrointestinal stromal tumors (GISTs). The tyrosine kinase inhibitor imatinib mesylate has revolutionized the treatment of GISTs. Unfortunately, primary or acquired resistance to imatinib does occur in GISTs and forms a major problem. Although sunitinib malate, a multi-kinase inhibitor, has shown effectiveness against imatinib-resistant GISTs, recent studies have indicated that some imatinib-resistant GISTs harboring secondary mutations in the KIT activation loop were also resistant to sunitinib. Therefore, new drugs capable of overcoming the dual drug resistance of GISTs probably have potential clinical utility. In this study, we investigated the efficacy of flumatinib, an inhibitor of BCR-ABL/PDGFR/KIT, against 32D cells transformed by various KIT mutants and evaluated its potency to overcome the drug resistance of certain mutants. Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P). Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D. Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug-resistance conferred by activation loop mutations. These findings suggest that flumatinib could be a promising therapeutic agent against GISTs resistant to both imatinib and sunitinib because of secondary mutations in the activation loop.

Familial Mastocytosis: Identification Of KIT K509I Mutation and Its In Vitro Sensitivity To Imatinib, Dasatinib and PKC412

BackgroundMast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug. AimsTo characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors. Patients and methodsFour individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter]), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture. ResultsCase 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen. ConclusionsWe herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013. Disclosures:No relevant conflicts of interest to declare.

Genome-wide functional screening identifies CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

Abstract 3394: Ponatinib, a pan-BCR-ABL inhibitor, potently inhibits key activating and drug-resistant KIT mutants found in GIST.

Abstract Ponatinib (AP24534) is a multi-targeted tyrosine kinase inhibitor with potent pan-BCR-ABL activity being investigated in patients with CML. Ponatinib also inhibits the kinase activity of KIT, a clinically proven oncogenic driver. Approximately 80% of gastrointestinal stromal tumors (GIST) contain primary activating KIT mutations, the majority of which cluster in exon 11 (V560D, Δ557-8) or more rarely in exon 9 (AY502-3ins). Imatinib is approved for the 1st line treatment of GIST; however, patients frequently relapse due to the acquisition of secondary resistance mutations located in either the KIT ATP-binding pocket (T670I, V654A) or the activation (A) loop (D816H, D820A, N822K, A829P). Sunitinib is registered for 2nd line treatment of GIST but does not effectively inhibit A-loop mutants. Here we explored the activity of ponatinib against major primary and secondary KIT mutants found in GIST. The drug sensitivity of KIT mutants was determined using engineered Ba/F3 cells harboring mutant forms of KIT. Imatinib potently inhibited the viability of cells expressing exon 11 mutant KIT (IC50&amp;lt; 30 nM). The introduction of secondary mutations into a mutant exon 11 background (Δ557-8) dramatically reduced imatinib's activity (IC50 180 nM - 10 μM). Sunitinib was highly active (IC50&amp;lt; 15 nM) versus KIT primary mutants and secondary ATP-binding pocket mutants. However, its activity was substantially reduced by A-loop mutations (IC50&amp;gt; 200 nM). In contrast, ponatinib was highly potent (IC50&amp;lt; 15 nM) across a range of primary and secondary KIT mutants, including T670I and all 4 A-loop mutants tested. The potency of ponatinib was slightly reduced versus the KIT exon 9 primary mutant (IC50= 56 nM) or when V654A was present as a secondary mutation (IC50= 59 nM). Importantly, in patients dosed once daily with 45 mg ponatinib, peak (145 nM) and trough (64 nM) plasma concentrations achieved are predicted to lead to substantial inhibition of all KIT mutants tested here. To rationalize ponatinib's activity we solved the structure of the native KIT kinase domain co-crystallized with ponatinib. Ponatinib bound the inactive (DFG-out) conformation of KIT, filling the ATP-binding pocket as well as two adjacent hydrophobic pockets, analogous to its binding to ABL. Ponatinib can accommodate the gatekeeper T670I mutation by virtue of its triple bond circumventing the steric bulk of the larger isoleucine residue. Ponatinib's activity against A-loop mutants can be rationalized by its tight binding to the inactive conformation of KIT, even in the presence of A-loop mutations. In conclusion, ponatinib possesses potent activity versus the major clinically relevant KIT mutants, inhibiting their activity with IC50s that fall within clinically achievable plasma concentrations. These data therefore support the evaluation of ponatinib in patients with GIST, particularly since KIT A-loop mutants represent an unmet medical need. Citation Format: Andrew P. Garner, Rana Anjum, Sadanand Vodala, Alexa Schrock, Tianjun Zhou, Tim Clackson, Joseph M. Gozgit, Victor M. Rivera. Ponatinib, a pan-BCR-ABL inhibitor, potently inhibits key activating and drug-resistant KIT mutants found in GIST. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3394. doi:10.1158/1538-7445.AM2013-3394

Mutations in C-KIT exon 11 in canine cutaneous mast cell tumors

Background The c-KIT proto-oncogene encodes the receptor tyrosine kinase KIT, which has been shown to play important roles in the cellular maturation, survival, proliferation, and migration of several cell types including mast cells. Mast cell tumors (MCTs) are the most common cutaneous tumor in the dog. MCTs exhibit wide variation in biological behavior. KIT mutations and aberrant KIT expression have been identified in canine MCTs. Unlike human mastocytosis patients, in which point mutations primarily occur in the kinase domain of KIT, internal tandem duplications (ITD) have been identified in the juxtamembrane domain of KIT in canine MCTs. Among several KIT mutations identified, an ITD in exon 11 has been analyzed most consistently and is significantly associated with malignant behaviour of affected tumors. The goal of this pilot study was to describe ITD KIT mutation in six high grade MTCs and four low grade MCTs.

Gastrointestinal Stromal Tumors Short Review on KIT/PDGFRA Gene Mutations and Molecular Therapy

Gastrointestinal stromal tumors (GISTs) are most frequent mesenchimal neoplasms of the gastrointestinal tract. Current knowledge demonstrates that the KIT and PDGFRA gene mutations play a central part in the pathogenesis. Mutations can be subdivided into primary and secondary mutations. Secondary mutations usually occur in KIT kinase domains after tyrosine kinase inhibitors treatment resulting in resistance to drugs. Besides surgery, therapy with tyrosine kinase inhibitors has led to develop novel treatments for patients now. However, secondary resistance has become a significant concern, illustrating the need to increase collaboration between surgical management and molecular therapy.

The KIT D816V Allele Burden in Systemic Mastocytosis Is Strongly Associated with Disease Subtype.

AbstractAbstract 2859Point mutations in the kinase domain of the receptor tyrosine kinase KIT at position 816 (D816V >95%; D816H, D816Y, others <5%) play a central role in the pathogenesis, diagnosis and targeted treatment of systemic mastocytosis (SM). However, accurate numbers on the overall frequency of KIT D816V in the diverse SM subtypes remain largely unknown because of i) the rarity of disease, ii) highly variable mast cell burden in bone marrow (BM) and peripheral blood (PB) and iii) inadequate sensitivity of disparate assays. We therefore developed an allele-specific quantitative RT-PCR (RQ-PCR) with an enhanced sensitivity of 0.01–0.1% which was superior to D-HPLC (mutation detection rate: 0.5%-1%) or conventional sequencing (mutation detection rate: 10–20%). Classification of the patients into disease subtypes was performed according to the WHO 2008 classification. Irrespective of subtype, KIT D816 mutations were identified in 143/144 (99.3%) of patients (D816V, n=141, 98.6%; D816H, n=1; D816Y, n=1) with SM (indolent SM [ISM], n=62, 43.1%; smoldering SM [SSM], n=7, 4.9%; SM with associated hematological non-mast cell disease [SM-AHNMD], n=15, 10.4%, and aggressive SM/mast cell leukemia [ASM/MCL], n=59, 41%). As control groups, seven patients with cutaneous mastocytosis, 20 healthy individuals and 80 patients with chronic myeloid leukemia in molecular remission were negative for the KIT D816V mutation in BM and PB. All patients with SSM (n=7), SM-AHNMD (n=14) and ASM/MCL (n=56) were positive in BM and also in PB. In contrast, 25/51 (49%) ISM patients were positive in BM but negative in PB. The KIT D816V RQ-PCR was subsequently used for quantitative assessment of the KIT D816V allele burden, expressed as the ratio KIT D816V/total KIT, in a total of 207 samples from PB (n=126) and BM (n=81) of 141 KIT D816V positive patients. In BM, the median allele burden was 8.9% (range 1.1–48.3%) in ISM, 29.7% (range 13.6–39.4%, p=0.007) in SSM, 23.3% (range 1.8–45.8%, p=0.09) in SM-AHNMD and 30.7% (range 1–99%, p<0.0001) in ASM/MCL. In PB, the allele burden clearly allowed differentiation of ISM (median 0, range 0–15.2%) vs. SSM (median 8.8%, range 0.3–42.5%, p=0.001), ISM vs. SM-AHNMD (median 11.5%, range 0.2–64.9%, p<0.001) and ISM vs. ASM/MCL (median 33%, range 0.53–74.0%; p<0.001). The vast majority of ISM patients (47/51; 92%) had an allele burden in PB <10%. ISM patients with an allele burden ≥10% collectively showed overlapping symptoms of diarrhea with (n=2) or without (n=1) histologically confirmed bowel infiltration, mild cytopenia (n=1), splenomegaly (n=1) and/or abdominal lymphadenopathy (n=1) potentially indicating more advanced disease. The wide heterogeneity of the allele burden in SSM and SM-AHNMD patients reflects the clinical heterogeneity in these subtypes. Of interest, the correlation of clinical findings and allele burden also revealed that patients with ASM-CMML had a significantly higher allele burden than ASM patients without monocytosis (45.5% vs. 30.9%, p=0.0029). In order to evaluate the use of RQ-PCR for monitoring of residual disease, serial measurements of the allele burden in PB were performed in patients after chemotherapy with cladribine (n=1) or allogeneic stem cell transplantation (SCT, n=2). The patient on cladribine revealed a decrease from 42% to 5% within 9 months which was associated with a decrease of serum tryptase levels from 610 μg/l to 129 μg/l and a decrease of mast cells in BM from 70–80% to 10%. When the patient relapsed 18 months after completion of chemotherapy with progressive organomegaly and thrombocytopenia, the allele burden had also risen to 35%. In the two allografted patients, allele burden was 35% and 45%, respectively, prior to SCT. Both patients are currently in complete remission without detectable KIT D816V mutation 17 and 12 months after allogeneic SCT. We therefore conclude that the measurement of the KIT D816V allele burden in SM patients from cDNA is a useful complementary tool for diagnosis and subclassification of SM. In the new treatment era of successful use of tyrosine kinase inhibitors and allogeneic SCT, it may also become very useful for monitoring of response to treatment in addition to established parameters like C-findings, serum tryptase and BM histology. Disclosures:No relevant conflicts of interest to declare.