Supplementary Table, Materials and References from Activity of dasatinib against L576P KIT mutant melanoma: Molecular, cellular, and clinical correlates

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is a serine-threonine kinase that is overexpressed in gastric cancer (GC) and promotes tumor progression. Polyphyllin VII (PPVII), a pennogenin isolated from the rhizomes of Paris polyphylla, shows anticancer effects. Here, we explored the antitumor activity and mechanism of PPVII in GC. Ferroptosis was detected by transmission electron microscope, malondialdehyde, and iron determination assays. Autophagy and its upstream signaling pathway were detected by Western blot, and gene alterations. The binding of PPVII and TOPK was examined through microscale thermophoresis and drug affinity responsive target stability assays. An in vivo mouse model was performed to evaluate the therapeutic of PPVII. PPVII inhibits GC by inducing autophagy-mediated ferroptosis. PPVII promotes the degradation of ferritin heavy chain 1, which is responsible for autophagy-mediated ferroptosis. PPVII activates the Unc-51-like autophagy-activating kinase 1 (ULK1) upstream of autophagy. PPVII inhibits the activity of TOPK, thereby weakening the inhibition of downstream ULK1. PPVII stabilizes the dimer of the inactive form of TOPK by direct binding. PPVII inhibits tumor growth without causing obvious toxicity in vivo. Collectively, this study suggests that PPVII is a potential agent for the treatment of GC by targeting TOPK to activate autophagy-mediated ferroptosis.

Background: Capsular contracture is a serious complication that occurs after augmentation mammaplasty. The authors previously identified that carboxymethyl chitin had an inhibitory effect on capsule formation. This study was performed to elucidate the possible molecular mechanisms through which carboxymethyl chitin inhibits the formation of a capsule around silicone implants. Methods: In this study, the authors cultured human dermal fibroblasts and treated them with carboxymethyl chitin in vitro. The difference in proliferation between treated and untreated cells was analyzed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein levels of transforming growth factor beta-1 and alpha smooth muscle actin (α-SMA) were examined by Western blot analysis. Expression levels of type I and type III collagen were checked by enzyme-linked immunosorbent assay. In vivo, silicone implants were placed under the pectoralis muscle in 12 female rabbits. The thickness of the capsule was measured by histologic analysis, and the effect of carboxymethyl chitin on α-SMA, collagen type I and III expression levels was evaluated by real-time polymerase chain reaction analysis, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analysis. Results: In the in vitro study, we confirmed that carboxymethyl chitin inhibited the proliferation of fibroblasts. The protein expression levels of collagen type I, transforming growth factor beta-1, and α-SMA were inhibited by carboxymethyl chitin treatment. In vivo, carboxymethyl chitin treatment reduced capsular thickness and the expression of α-SMA and collagen types I and III in capsules around silicone implants. Conclusion: The authors’ results showed that carboxymethyl chitin could influence capsule formation around silicone implants by inhibiting the fibroblast activity, interrupting fibroblast-to-myofibroblast differentiation, and decreasing collagen synthesis. Clinical Relevance Statement: Carboxymethyl chitin influence capsule formation around silicone implants. Although more clinical studies are needed to verify the effect of carboxymethyl chitin on capsular contracture, the authors believe that it will play an effective role in the clinical application of reducing the occurrence of capsular contracture.

Background Cell autophagy has been proposed to be involved in drug resistance therapy. However, how the long non-coding RNA (lncRNA) reduces risks of drug resistance in renal cancer (RC) cells needs a thorough inquiry. This study was assigned to probe the effect and mechanism of HOTAIR on sunitinib resistance of RC. Methods Clinical RC tissues and para-carcinoma tissues were obtained to detect the expressions of miR-17-5p, HOTAIR and Beclin1. Sunitinib-resistant cells (786-O-R and ACHN-R) were constructed using parental RC cells (786-O and ACHN). The resistance of 786-O-R and ACHN-R cells to sunitinib was examined. Western blot and qRT-PCR were assayed to obtain the expressions of miR-17-5p, HOTAIR and Beclin1. The effects of HOTAIR knockdown or miR-17-5p overexpression/knockdown on cell autophagy and sunitinib resistance were measured by MDC staining, immunofluorescence and Western blot. The sensitivity of RC cells to sunitinib and change in cell clone formation after sunitinib treatment were assessed by CCK-8 assay and colony formation assay, respectively. The relationships among HOTAIR, miR-17-5p and Beclin1 were verified by dual-luciferase reporter gene and RIP assay. The role of HOTAIR knockdown in sunitinib resistance was verified in nude mice. Results HOTAIR expression in sunitinib-resistant cells is higher than that in parental cells. Knockdown of HOTAIR in sunitinib-resistant cells lead to refrained sunitinib resistance and cell autophagy both in vivo and in vitro. Activation of autophagy could raise resistance to sunitinib in RC cells, while inhibition of autophagy could improve the sensitivity of sunitinib-resistant cells to sunitinib. HOTAIR could compete with miR-17-5p to regulate Beclin1 expression. Knockdown of miR-17-5p in parental cells increases cell resistant to sunitinib, and overexpression of miR-17-5p in sunitinib-resistant cells increases cell sensitive to sunitinib. Conclusion HOTAIR negatively targets miR-17-5p to activate Beclin1-mediated cell autophagy, thereby enhancing sunitinib resistance in RC cells.

Background: Exercise therapy plays an important role in the prevention and treatment of type 2 diabetes (T2DM). The mechanism of exercise therapy in the improvement of glycolipid metabolism of T2DM is very complex and not completely clear. Summary: Exercise training improves the whole body metabolic health in patients with T2DM, leading to an increase in glycolipid uptake and utilization, improved insulin sensitivity, optimized body mass index, and modulated DNA methylation, etc. Recent findings support that some cytokines such as irisin, osteocalcin, and adiponectin are closely related to exercise and metabolic diseases. This study briefly reviews the physiological mechanisms of exercise therapy in diabetes and the potential role of these cytokines in exercise. Key Messages: More high-quality, targeted, randomized controlled studies are needed urgently, from mechanism study to treatment direction, to provide a more theoretical basis for exercise therapy and to explore new therapeutic targets for diabetes.

Glioblastoma (GBM) is one of the most deadly brain tumors. The convenient access to The Cancer Genome Atlas (TCGA) database allows for large-scale global gene expression profiling and database mining for potential correlation between genes and overall survival of a variety of malignancies including GBM. Previous reports have shown that tumor microenvironment cells and the extent of infiltrating immune and stromal cells in tumors contribute significantly to prognosis. Immune scores and stromal scores calculated based on the ESTIMATE algorithm could facilitate the quantification of the immune and stromal components in a tumor. To better understand the effects of genes involved in immune and stromal cells on prognosis, we categorized GBM cases in the TCGA database according to their immune/stromal scores into high and low score groups, and identified differentially expressed genes whose expression was significantly associated with prognosis in GBM patients. Functional enrichment analysis and protein-protein interaction networks further showed that these genes mainly participated in immune response, extracellular matrix, and cell adhesion. Finally, we validated these genes in an independent GBM cohort from the Chinese Glioma Genome Atlas (CGGA). Thus, we obtained a list of tumor microenvironment-related genes that predict poor outcomes in GBM patients.

AIM:Chondrosarcoma is difficult to treat because of resistance to conventional chemotherapy and radiotherapy. This study evaluated the effects of ethanol in combination with doxorubicin in chondrosarcoma cells.MATERIALS & METHODS:JJ012, was treated with doxorubicin alone or in combination with ethanol. Effects on cellular proliferation, migration, invasion, apoptosis, and the cell cycle were evaluated.RESULTS:Treatment of JJ012 cells with 100 mM ethanol and doxorubicin resulted in reduced cell growth, invasion, and migration. In addition, doxorubicin uptake into the nucleus was enhanced and p53 mRNA expression was upregulated in JJ012 cells.CONCLUSION:Ethanol combined with doxorubicin increased doxorubicin uptake in the nucleus and enhanced the effects of doxorubicin in JJ012 cells.

A paradigm shift has occurred in the last decade from chemotherapy to targeted therapy for the management of many patients with advanced sarcoma. This work identifies a combination of targeted agents and doxorubicin that are effective against small cell sarcoma cell lines. Three small cell sarcoma cell lines were studied: RD18 (rhabdomyosarcoma), A204 (undifferentiated sarcoma) and TC 71 (Ewing's sarcoma). Each cell line was exposed to increasing concentrations of vorinostat (HDAC inhibitor), 17-DMAG (HSP90 inhibitor), abacavir (anti-telomerase) or sorafenib (tyrosine kinase inhibitor) alone, combined with one another, or combined with doxorubicin. Cell viability, cell cycle analysis and apoptosis were assessed by MTS assay, propidium iodide-Annexin V staining, and caspase 3/7 activity, respectively. The Chou and Talalay combination index (CI) was used to determine whether the effects were additive (CI = 1), synergistic (CI < 1) or antagonistic (CI > 1). In monotherapy, targeted agents achieved 30-90% reductions in viability, with the exception of abacavir. Dual-targeted combination therapies with vorinostat, sorafenib and 17-DMAG demonstrated synergy. Abacavir was antagonistic with every other drug and was not further studied. Both vorinostat and 17-DMAG synergized with doxorubicin, achieving 60% cell killing compared to 12% with doxorubicin alone. No synergy was observed for sorafenib with doxorubicin. The triple therapy vorinostat, 17-DMAG and doxorubicin did not show synergy, but increased the subG1 population at 24H, from 30% to 70% compared to monotherapies with an increase in apoptosis. This work provides evidence of synergy of combinations of vorinostat, 17-DMAG and sorafenib in small cell sarcoma. In addition to doxorubicin, these combinations enhance doxorubicin cytotoxicity at therapeutically relevant concentrations.

e13519 Background: While a change of paradigm occurred in the last decade from chemotherapy to targeted therapy for cancer treatment, this work investigates the optimal combination of targeted agents with doxorubicin in sarcoma. Methods: Three sarcoma cell lines were studied RD18 (rhabdomyosarcoma), A204 (undifferentiated sarcoma) and TC 71 (Ewing's sarcoma) and exposed to increasing concentrations of vorinostat (HDAC inhibitor), 17-DMAG (Hsp90 inhibitor), abacavir (anti-telomerase) and sorafenib (tyrosine kinase inhibitor) alone, combined 2 by 2, then with doxorubicin. Viability was assessed by MTS assay. The Chou and Talalay combination index (CI) was used to determine additive (CI=1), synergistic (CI<1) or antagonistic effect (CI>1). Cell cycle analysis, measure of apoptosis by Annexin V and caspase 3/7 activity were studied using flow cytometry analysis and luminescent assay. Results: In monotherapy, the agents showed 30% to 90% decrease in viability but abacavir, which remained less active. Combination therapies with vorinostat, sorafenib and 17-DMAG showed strong synergism. Abacavir was found antagonistic with each drug. Either vorinostat or 17DMAG synergized with doxorubicin, achieving 60% cell killing compared to doxorubicin alone 12% (p=0.007). However, no synergy was observed for sorafenib with doxorubicin. The triple therapy vorinostat, 17DMAG and Doxorubicin did not show synergism but transiently increased the subG1 population at 24H, 70% compared to 30% in monotherapy with an increase in early caspase-independent apoptosis (11% to 36%, p= 0.0008). Conclusions: This work provides evidence of synergism of dual combinations of vorinostat, 17DMAG and sorafenib. In adjunction to doxorubicin, these combinations enhance doxorubicin cytotoxicity at therapeutically relevant concentrations. [Table: see text]

Rhabdoid features in gastrointestinal stromal tumors (GISTs) are rare. To the authors’ knowledge, only 51 cases have been reported. Most of these reports consist of case series in which the rhabdoid GISTs comprise a small proportion of the tumors studied. Information regarding site of origin and clinical behavior is sparse. Although the stomach is the only site of origin documented, most reports do not include this data. Malignancy has not been reported, though follow-up is inadequate in most cases to comment on tumor behavior. Exon 11 mutations comprise all previously described KIT mutations, the majority of which are deletions. The authors present the case of a malignant small-intestinal rhabdoid GIST that recurred twice following resection and treatment with tyrosine kinase inhibitors. The tumor harbored a KIT exon 11, 579-580 LY insertion that, to the authors’ knowledge, has not been previously reported. This case is the first rhabdoid GIST described in the small intestine and is the first to show documented evidence of malignancy.

Background: Although imatinib mesylate has revolutionized the management of patients with gastrointestinal stromal tumor (GIST), resistance and progression almost inevitably develop with long‐term monotherapy. To enhance imatinib‐induced cytotoxicity and overcome imatinib‐resistance in GIST cells, we examined the antitumor effects of the pro‐apoptotic Bcl‐2/Bcl‐xL inhibitor ABT‐737, alone and in combination with imatinib.Methods: We treated imatinib‐sensitive, GIST‐T1 and GIST882, and imatinib‐resistant cells with ABT‐737 alone and with imatinib. We determined the anti‐proliferative and apoptotic effects by cell viability assay, flow cytometric apoptosis and cell cycle analysis, immunoblotting, and nuclear morphology. Synergism was determined by isobologram analysis.Results: The IC50 of single‐agent ABT‐737 at 72 h was 10 μM in imatinib‐sensitive GIST‐T1 and GIST882 cells, and 1 μM in imatinib‐resistant GIST48IM cells. ABT‐737 and imatinib combined synergistically in a time‐ and dose‐dependent manner to inhibit the proliferation and induce apoptosis of all GIST cells, as evidenced by cell viability and apoptosis assays, caspase activation, PARP cleavage, and morphologic changes. Isobologram analyses revealed strongly synergistic drug interactions, with combination indices <0.5 for most ABT‐737/imatinib combinations. Thus, clinically relevant in vitro concentrations of ABT‐737 have single‐agent antitumor activity and are synergistic in combination with imatinib.Conclusion: We provide the first preclinical evidence that Bcl‐2/Bcl‐xL inhibition with ABT‐737 synergistically enhances imatinib‐induced cytotoxicity via apoptosis, and that direct engagement of apoptotic cell death may be an effective approach to circumvent imatinib‐resistance in GIST.

Gastrointestinal stromal tumors (GISTs) rarely metastasize to the skin. We describe 5 patients with GIST with subcutaneous and cutaneous metastases. The mean age at metastasis was 54 years (range 30-68 years) with a male predominance (4:1). Primary tumors occurred in the stomach (n = 3), small bowel (n = 1), and abdomen, not otherwise specified (n = 1). The average time from primary tumor resection to the resection of skin metastases was 59 months (range 11-155 months). The metastases occurred in the scalp (n = 2), cheek (n = 1), and abdomen (n = 2) with 3 patients presenting with solitary nodules and 2 patients with multiple nodules. The average size was 2 cm (range 0.6-4 cm). Histologically, 2 cases were spindled and 3 cases demonstrated mixed epithelioid and spindle cell morphology. All were confirmed to have CD117 reactivity. KIT genotyping was performed in 4 of 5 cases. Two cases harbored a mutation in exon 11, and the remaining 2 cases were wild type in exons 9, 11, 13, and 17. All 5 patients had multiple concurrent or subsequent abdominal and/or hepatic metastases. In 4 patients with an average follow-up of 32 months (range 6-75 months), after the resection of the metastases, 2 were alive with disease and 2 died of disease. Cutaneous metastases seem to be a late complication of GIST, but their presence does not necessarily herald a rapid demise of the patient.

The role of immunohistochemistry in the assessment of KIT status in melanomas, especially acral lentiginous/mucosal, is not well established. Although the reported prevalence of KIT mutations in acral lentiginous/mucosal melanomas is relatively low, detection of mutations in KIT can have profound therapeutic implications. We evaluated the efficacy of immunohistochemistry to predict mutations in KIT. One hundred seventy-three tumors, comprising primary and metastatic melanomas (141 acral lentiginous/mucosal, 5 nodular, 4 lentigo maligna, 3 superficial spreading, 2 uveal, 1 melanoma of soft parts, 8 metastases from unclassified primaries, and 9 metastases from unknown primaries) were studied. Immunohistochemical expression of KIT using an anti-CD117 antibody and KIT mutational analysis by gene sequencing of exons 11, 13, and 17 were performed. Eighty-one percent of acral lentiginous/mucosal melanomas, primary and metastatic, showed KIT expression by at least 5% of the tumor cells. The overall frequency of activating KIT gene mutations in acral lentiginous/mucosal melanomas was 15% (14 out of 91 cases), being the L576P mutation in exon 11 the most frequently detected (4 of 14 cases). Cases showing less than 10% positive tumor cells were negative for KIT mutations. Eighty-two percent (12 of 14) of cases positive for KIT mutation showed KIT expression in more than 50% of the cells. An association between immunohistochemical expression of KIT and mutation status was found (P=0.007). Immunohistochemical expression of KIT in less than 10% of the cells of the invasive component of acral lentiginous/mucosal melanomas appears to be a strong negative predictor of KIT mutation and therefore can potentially be used to triage cases for additional KIT genotyping.

PURPOSE:To assess whether a combination of in vivo anatomic and functional imaging can help quantify expression of somatostatin receptor type 2 (SSTR2)-based reporters after in vivo gene transfer.MATERIALS AND METHODS:All animal experiments were approved by an institutional animal care and use committee. Six nude mice bearing two subcutaneous L3.6pl (human pancreatic cancer) tumors were injected intratumorally with an adenovirus containing a human somatostatin receptor type 2 gene chimera (Ad-HA-SSTR2) or a control adenovirus containing green fluorescent protein (Ad-GFP). Two days later, magnetic resonance (MR) imaging was performed to derive tumor weight and analyze morphology. Intravenous injection of Food and Drug Administration-approved indium 111 octreotide was followed by gamma camera imaging (planar imaging and single photon emission computed tomography [SPECT]) the next day. Region-of-interest analysis followed. The procedure was also performed in six nude mice with slow-growing MDA-MB-435 (human breast carcinoma) tumors, which allowed serial imaging 3 days and 2 weeks after adenovirus injection. After imaging, excised tumor weight and biodistribution were assessed. Statistical analyses included a Student t test and linear regression.RESULTS:With both tumor types, ex vivo and image-based in vivo biodistribution demonstrated greater uptake (percentage of injected dose per gram) in tumors infected with Ad-HA-SSTR2 than in those infected with Ad-GFP (P < .05). Furthermore, in vivo and ex vivo biodistribution analysis correlated (ex vivo vs planar and MR imaging: r = 0.87, P < .05, n = 24; ex vivo vs SPECT and MR imaging: r = 0.84, P < .05, n = 24). Moreover, in vivo biodistribution distinguished greater expression at the earlier time point in MDA-MB-435 tumors infected with Ad-HA-SSTR2 from waning expression at the later time point (P < .05).CONCLUSION:A combination of in vivo functional and anatomic imaging methods can help quantify gene expression after in vivo gene transfer.

AbstractPoint mutations in the KIT receptor tyrosine kinase gene have recently been identified in mucosal, acral lentiginous, and chronically sun-damaged melanomas. We have identified the first human melanoma cell line with an endogenous L576P mutation, the most common KIT mutation in melanoma (∼30-40%). In vitro testing showed that the cell viability of the L576P mutant cell line was not reduced by imatinib, nilotinib, or sorafenib small molecule KIT inhibitors effective in nonmelanoma cells with other KIT mutations. However, the viability of the mutant cells was reduced by dasatinib at concentrations as low as 10 nM (P = 0.004). Molecular modeling studies found that the L576P mutation induces structural changes in KIT that reduce the affinity for imatinib (ΔΔGbind = −2.52 kcal/mol) but not for dasatinib (ΔΔGbind = +0.32 kcal/mol). Two metastatic melanoma patients with the L576P KIT mutation were treated with dasatinib, including one patient previously treated with imatinib. Both patients had marked reduction (>50%) and elimination of tumor F18-fluorodeoxyglucose (FDG)-avidity by positron emission tomography (PET) imaging after dasatinib treatment. These data support the selective inhibitory effect of dasatinib against cells harboring the most common KIT mutation in melanoma, and thus has therapeutic implications for acrallentiginous, chronic sun-damaged, and mucosal melanomas. [Mol Cancer Ther 2009;8(8):2079–85]

10569 Background: The chemokine CXCL12, and its receptor CXCR4, are expressed in over 23 different types of cancers, and have been associated with the metastatic phenotype and inferior clinical outcomes. Given the poor prognosis after failure of front-line therapy for patients with metastatic Ewing's sarcoma (EWS) and rhabdomyosarcoma (RMS), we examined the functional role of CXCR4 in these small cell sarcoma in vitro. Methods: Human EWS and RMS cell lines and tissue were used to study CXCR4 expression and activation. Immunoblotting techniques were used to evaluate CXCR4 activation and inhibition. Cell viability, cell cycle, apoptosis, chemosensitivity, migration, and invasion assays were utilized to assess the effects of the small peptide CXCR4 antagonist, CTCE-9908 (Chemokine Therapeutics) on cultured cells. Results: CXCR4 was highly expressed on 46 % of human RMS tumor samples. CXCR4 underwent phosphorylation after stimulation with CXCL12 in EWS and RMS cell lines with downstream activation of Akt, p42/44 MAPK, JAK2 and PLCγ1 in select cell lines. CTCE-9908 was able to specifically inhibit downstream signaling of Akt, p42/44 MAPK and JAK2. Decrease in cell proliferation (20–30%, p < .05), increase in cell apoptosis (20–40 %, p < .05) and cell cycle arrest was also observed with CXCR4 blockade. CTCE-9908 significantly inhibited migration and invasion (68–93 %, p < .05) in our cell lines. Conclusions: CXCR4-CXCL12 axis may be important in EWS and RMS metastasis. These results provide evidence that CTCE-9908 may be a novel therapy for these sarcomas. No significant financial relationships to disclose.

Gastrointestinal stromal tumor (GIST) represents the majority of soft tissue sarcomas arising from the GI tract. Most GISTs encode activating mutations in the kit gene, an important genetic event in tumorigenesis. Imatinib mesylate (imatinib, Gleevec™; Novartis, Basel, Switzerland) has revolutionized the management of patients with advanced disease by targeting the aberrant kinase activity of Kit. Mutation status of kit in GIST has emerged as a prognostic and predictive factor for imatinib response. At diagnosis, most kit mutations are within exon 11 or less commonly within exon 9, 13, or 17 (Chen et al., 2004). Regarding exon 13, early reports suggested that tumors harboring these mutations responded suboptimally to imatinib therapy. However, the efficacy of imatinib in this subpopulation has been difficult to analyze due to its rarity with an estimated incidence of approximately 1% (Desai et al., 2007). Chen and colleagues showed that primary mutation of the Kit kinase domain 1 (exon 13, V654A) correlated to imatinib resistance and rapid progression (Heinrich et al., 2006). Desai and colleagues demonstrated that 80% of patients with imatinib-resistant nodules after initial response to imatinib harbored secondary mutations in kit exons 13 and 17 (Lasota et al., 2008). Thus, kit exon 13 can be altered by primary or secondary mutation that can confer resistance to imatinib. However, the differential effect on imatinib binding and clinical response for each particular mutation within kit exon 13 is not fully understood. We encountered a patient with GIST harboring a kit exon 13 mutation (K642E) who experienced a robust and prolonged response to imatinib. This 66-year-old Caucasian man presented to The University of Texas — M. D. Anderson Cancer Center in August 2001 with recurrent GIST measuring approximately 3.5cm at the gastroesophageal junction and several small intra- and extra-peritoneal implants (Fig. 1A). Pathologic evaluation revealed a highly cellular, spindle cell tumor with brisk mitotic activity. The tumor was diffusely reactive for Kit, CD34, and focally reactive for smooth muscle actin while being negative for desmin, S-100, and pan-cytokeratin. He was treated with 600mg daily imatinib, and experienced a decrease in tumor burden by 1 year (Fig. 1B). However, after 45 months of imatinib therapy, he had multi-focal progression within the abdominal cavity and a pleural implant (Fig. 1C). He underwent surgical resection to debulk his tumor burden. Characteristic histopathologic features of treatment response to imatinib were not present in these recurrent tumor areas. Mutational analysis of his tumor at the time of imatinib initiation revealed a missense mutation of kit exon 13, K642E (Fig. 1D). This mutation is the most common codon affected in kit exon 13 (Desai et al., 2007). Upon resistance and progression of disease, mutational analysis revealed a secondary kit exon 13 mutation (V654A) along with the primary K642E mutation (Fig. 1E). This finding corroborates those seen by Chen and others regarding the V654A mutation and imatinib resistance (Chen et al., 2004; Singer et al., 2002). Importantly, molecular modeling of the K642E mutant predicts that the negatively charged glutamic acid (in contrast to the positively charged lysine) results in altered interactions of the control c-helix with the Kit juxtamembrane domain which, in turn, leads to an uncontrolled phosphorylation of the kinase, without effecting binding of imatinib (Fig. 1F). However, the 654 position of Kit lies within the ATP binding pocket and directly participates in imatinib binding. Thus, substitution of a valine for a smaller alanine in the 654 position leads to a “void” in the binding site and, hence, a less efficient drug binding. The calculated free energy of binding (ΔGbind) between wild-type Kit and imatinib is −10.22kcal/mol (Tamborini et al., 2006). The ΔGbind for the K642E mutant is marginally less at −10.03kcal/mol. The ΔGbind for the Kit V654A mutant is −8.7kcal/mol or a one-fold decrease in drug affinity. The ΔGbind for the Kit K642E/V654A double mutant is −8.04kcal/mol indicating a 1–2 fold decrease in affinity for imatinib. Thus, the two mutations work together to enhance the resistance to imatinib as compared to each mutation alone. In conclusion, this GIST patient harboring kit exon 13 mutation K642E benefited from imatinib. Treatment selected for the double mutation that conferred resistance and eventuated death. Despite the minimal 11 codons separating these loci, the resultant phenotype is imatinib-sensitive for K642E and imatinib-resistant for K642E/V654A. As GIST kit mutation reporting becomes more prevalent, documentation of the specific mutation is important to distinguish those patients with probable imatinib resistance from those that may benefit from imatinib for many years.

Endogenous and exogenous somatostatin receptors are commonly targeted for imaging using radiopharmaceutical analogues of somatostatin. Ligand binding activates receptor-mediated signaling. We assessed whether somatostatin receptor type 2A (SSTR2A) imaging can be uncoupled from signal transduction. In both human fibrosarcoma (HT1080) and human embryonic kidney (HEK293) cells, reverse transcriptase–polymerase chain reaction and enzyme-linked immunosorbent assay found similar levels of expression of hemagglutinin A tagged SSTR2A (HA-SSTR2A) or the same fusion protein with a deletion of the C-terminus beyond amino acid 314 (HA-SSTR2Δ314). Scatchard analysis demonstrated similar degrees of ligand binding by the wild-type or mutant receptor to 111 In-octreotide in both cell pairs. Cyclic guanosine monophosphate (cGMP) production and inhibition of forskolin-induced cylic adenosine monophosphate (cAMP) production were evaluated at the signaling level, and growth inhibition was evaluated at the cellular level before and after stimulation. Unlike wild-type receptor, HA-SSTR2Δ314 was deficient in inhibiting forskolin-induced cAMP production ( p < .05) and in inciting cGMP ( p < .05) production; furthermore, at the cellular level, HA-SSTR2Δ314 was deficient in inhibiting cell proliferation ( p < .05). Yet tumors expressing HA-SSTR2Δ314 could be imaged in vivo. Thus, in vivo imaging of SSTR2 can be uncoupled from cAMP and cGMP signaling as well as growth inhibition.