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BackgroundApproximately 10-15 % of gastrointestinal stromal tumors (GISTs) lack gain of function mutations in the KIT and platelet-derived growth factor receptor alpha (PDGFRA) genes. An alternate mechanism of oncogenesis through loss of function of the succinate-dehydrogenase (SDH) enzyme complex has been identified for a subset of these "wild type" GISTs.MethodsPaired tumor and normal DNA from an SDH-intact wild-type GIST case was subjected to whole exome sequencing to identify the pathogenic mechanism(s) in this tumor. Selected findings were further investigated in panels of GIST tumors through Sanger DNA sequencing, quantitative real-time PCR, and immunohistochemical approaches.ResultsA hemizygous frameshift mutation (p.His2261Leufs*4), in the neurofibromin 1 (NF1) gene was identified in the patient's GIST; however, no germline NF1 mutation was found. A somatic frameshift mutation (p.Lys54Argfs*31) in the MYC associated factor X (MAX) gene was also identified. Immunohistochemical analysis for MAX on a large panel of GISTs identified loss of MAX expression in the MAX-mutated GIST and in a subset of mainly KIT-mutated tumors.ConclusionThis study suggests that inactivating NF1 mutations outside the context of neurofibromatosis may be the oncogenic mechanism for a subset of sporadic GIST. In addition, loss of function mutation of the MAX gene was identified for the first time in GIST, and a broader role for MAX in GIST progression was suggested.

Background: Most gastrointestinal stromal tumors (GISTs) harbor mutually exclusive gain of function mutations in the receptor tyrosine kinase (RTK) KIT (70-80%) or in the related receptor PDGFRA (~10%). These GISTs generally respond well to therapy with the RTK inhibitor imatinib mesylate (IM), although initial response is genotype-dependent. An alternate mechanism leading to GIST oncogenesis is deficiency in the succinate dehydrogenase (SDH) enzyme complex resulting from genetic or epigenetic inactivation of one of the four SDH subunit genes (SDHA, SDHB, SDHC, SDHD, collectively referred to as SDHX). SDH loss of function is generally seen only in GIST lacking RTK mutations, and SDH-deficient GIST respond poorly to imatinib therapy.Methods: Tumor and normal DNA from a GIST case carrying the IM-resistant PDGFRA D842V mutation was analyzed by whole exome sequencing (WES) to identify additional potential targets for therapy. The tumors analyzed were separate recurrences following progression on imatinib, sunitinib, and the experimental PDGFRA inhibitor crenolanib. Tumor sections from the GIST case and a panel of ~75 additional GISTs were subjected to immunohistochemistry (IHC) for the SDHB subunit.Results: Surprisingly, a somatic, loss of function mutation in exon 4 of the SDHB subunit gene (c.291_292delCT, p.I97Mfs*21) was identified in both tumors. Sanger sequencing confirmed the presence of this inactivating mutation, and IHC for the SDHB subunit demonstrated that these tumors were SDH-deficient. IHC for the SDHB subunit across a panel of ~75 GIST cases failed to detect SDH deficiency in other GISTs with RTK mutations.Conclusions: This is the first reported case of a PDGFRA mutant GIST exhibiting SDH-deficiency. A brief discussion of the relevant GIST literature is included. [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumors (GISTs) in adults are generally driven by somatic gain-of-function mutations in KIT or PDGFRA, and biological therapies targeted to these receptor tyrosine kinases comprise part of the treatment regimen for metastatic and inoperable GISTs. A minority (10-15%) of GISTs in adults, along with ~85% of pediatric GISTs, lacks oncogenic mutations in KIT and PDGFRA. Not surprisingly these wild type (WT) GISTs respond poorly to kinase inhibitor therapy. A subset of WT GISTs shares a set of distinguishing clinical and pathological features, and a flurry of recent reports has convincingly demonstrated shared molecular characteristics. These GISTs have a distinct transcriptional profile including over-expression of the insulin-like growth factor-1 receptor, and exhibit deficiency in the succinate dehydrogenase (SDH) enzyme complex. The latter is often but not always linked to bi-allelic inactivation of SDH subunit genes, particularly SDHA. This review will summarize the molecular, pathological, and clinical connections that link this group of SDH-deficient neoplasms, and offer a view toward understanding the underlying biology of the disease and the therapeutic challenges implicit to this biology. [ABSTRACT FROM AUTHOR]

Although imatinib mesylate (IM) has transformed the treatment of gastrointestinal stromal tumors (GIST), many patients experience primary/secondary drug resistance. In a previous study, we identified a gene signature, consisting mainly of Kruppel-associated box (KRAB) domain containing zinc finger (ZNF) transcriptional repressors that predict short-term response to IM. To determine if these genes have functional significance, a siRNA library targeting these genes was constructed and applied to GIST cells in vitro. These screens identified seventeen "IM sensitizing genes" in GIST cells (sensitization index (SI) <0.85 ratio of drug/vehicle) with a false discovery rate (FDR) <15%, including twelve ZNF genes, the majority of which are located within the HSA19p12-13.1 locus. These genes were shown to be highly specific to IM and another tyrosine kinase inhibitor (TKI), sunitinib, in GIST cells. In order to determine mechanistically how these ZNFs might be modulating response to IM, RNAi approaches were used to individually silence genes within the predictive signature in GIST cells and expression profiling was performed. Knockdown of the 14 IM-sensitizing genes (10 ZNFs) universally led to downregulation of six genes, including TGFb3, periostin, and NEDD9. These studies implicate a role of KRAB-ZNFs in modulating response to TKIs in GIST. [ABSTRACT FROM AUTHOR]

Simple Summary: Imatinib mesylate (IM, Gleevec) has remained first-line therapy for most gastrointestinal stromal tumor (GIST) patients since 2002. During this time, three additional inhibitors have been approved for treatment of IM-refractory tumors; however, disease stabilization for these agents is measured in months. Once all approved lines of therapy are ineffective, patients with advanced GIST are left without treatment options. Therefore, preventing or delaying development of acquired resistance to IM could have clinical benefits. Activation of the PI3K/AKT signaling pathway has been associated with resistance to IM in GIST. We evaluated MK-4440, a novel AKT inhibitor, in combination with IM in GIST cells and mouse models. Our studies demonstrate superior activity of MK-4440/IM combination in a panel of GIST cell lines caused by cell cycle arrest and elevated PDCD4 expression leading to increased cell death. Furthermore, dual inhibition of KIT and AKT provided impressive disease stabilization in IM-sensitive GIST growing in mice. The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting. [ABSTRACT FROM AUTHOR]

Purpose: Dedifferentiated liposarcoma (DDL) and leiomyosarcoma (LMS) are two common subtypes of soft-tissue sarcoma, a rare group of diseases for which new treatments are needed. Chemotherapy remains the standard option for advanced disease. Targeting cyclin-dependent kinase 4 and 6 (CDK4/6) in DDL and mTOR in LMS is of biologic interest. When combined, the CDK4 inhibitor ribociclib and the mTOR inhibitor everolimus have shown synergistic growth inhibition in multiple tumor models, suggesting that this combination could be beneficial in patients., Patients and Methods: This was a single arm, open label, multicenter phase II study of the combination of ribociclib and everolimus. Patients were enrolled into one of two cohorts: DDL or LMS with intact Rb. The primary endpoint was progression-free rate (PFR) at 16 weeks. Secondary endpoints included progression-free survival (PFS) and overall survival, safety and biomarker analyses., Results: In the DDL cohort, 33.3% [95% confidence interval (CI), 15.6%-55.3%] of patients were progression-free at 16 weeks. Median PFS in this cohort was 15.4 weeks (95% CI, 8-36 weeks) with 2 partial responses. In the LMS cohort the PFR at 16 weeks was 29.2% (95% CI, 12.6%-51.1%). Median PFS in this cohort was 15.7 weeks (95% CI, 7.7-NA). Most common toxicities included fatigue (66.7%), anorexia (43.8%), and hyperglycemia (43.8%). Concordance between Rb testing methodologies was poor., Conclusions: The combination of ribociclib and everolimus demonstrates activity in DDL with prolonged stable disease (≥16 weeks) meeting the primary endpoint. Notably partial responses were observed. The primary endpoint was not reached in the LMS cohort. The combination was well tolerated with expected side effects., (©2023 American Association for Cancer Research.)

Management of gastrointestinal stromal tumors (GISTs) has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of RTK inhibitors in advanced disease. Stratification of GISTs into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GISTs, resistance remains a significant clinical problem. Development of effective treatment strategies for refractory GISTs requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has the potential to identify critical signaling networks and reveal protein kinases essential in GISTs. Using multiplexed inhibitor beads and mass spectrometry, we explored the majority of the kinome in GIST specimens from the 3 most common molecular subtypes (KIT mutant, PDGFRA mutant, and succinate dehydrogenase deficient) to identify kinase targets. Kinome profiling with loss-of-function assays identified an important role for G2/M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT mutant and PDGFRA mutant GIST cell lines as well as notable efficacy of MK-1775 as a monotherapy in the engineered PDGFRA mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.

Purpose: Most gastrointestinal stromal tumors (GIST) have activating mutations of KIT, PDGFRA , or uncommonly BRAF . Fifteen percent of adult and 85% of pediatric GISTs are wild type (WT), commonly having high expression of IGF-1R and loss of succinate dehydrogenase (SDH) complex function. We tested the efficacy of linsitinib, an oral TKI IGF-1R inhibitor, in patients with WT GIST., Patients and Methods: A multicenter phase II trial of linsitinib was conducted. The primary endpoint was objective response rate. Secondary endpoints were clinical benefit rate: complete response, partial response, and stable disease (SD) ≥ 9 months, and quantitative 2[18F]fluoro-2-deoxy-D-glucose (FDG) metabolic response (MR) at week 8. Serum levels for glucose, insulin, IGF-1R ligand IGF1, and binding proteins were obtained to explore correlations to patient outcomes and FDG-PET results., Results: Twenty patients were accrued in a 6-month period. Grade 3-4 toxicities possibly related to linsitinib were uncommon (8.5%). No objective responses were seen. Clinical benefit rate (CBR) at 9 months was 40%. Intense FDG uptake was observed at baseline, with partial MR of 12% and stable metabolic disease of 65% at week 8; these patients had RECIST 1.1 SD as their best response. Progression-free survival (PFS) and overall survival Kaplan-Meier estimates at 9 months were 52% and 80%, respectively. SDHA/B loss determined by IHC was seen in 35% and 88% of cases, respectively., Conclusions: Linsitinib is well tolerated in patients with WT GIST. Although the 9-month CBR was 40%, and PFS at 9 months was 52%, no objective responses were observed. Rapid accrual to this study demonstrates that clinical trials of experimental agents in selected subtypes of GIST are feasible., (©2019 American Association for Cancer Research.)

Purpose: Gastrointestinal stromal tumors (GIST) generally harbor activating mutations in the receptor tyrosine kinase KIT or in the related platelet-derived growth factor receptor alpha (PDGFRA). GIST treated with imatinib mesylate or second-line therapies that target mutant forms of these receptors generally escape disease control and progress over time. Inhibiting additional molecular targets may provide more substantial disease control. Recent studies have implicated the PI3K/AKT pathway in the survival of imatinib mesylate-resistant GIST cell lines and tumors., Experimental Design: Here, we performed in vitro and in vivo studies evaluating the novel combination of imatinib mesylate with the AKT inhibitor MK-2206 in GIST. Whole-transcriptome sequencing (WTS) of xenografts was performed to explore the molecular aspects of tumor response to this novel combination and to potentially identify additional therapeutic targets in GIST., Results: This drug combination demonstrated significant synergistic effects in a panel of imatinib mesylate-sensitive and -resistant GIST cell lines. Furthermore, combination therapy provided significantly greater efficacy, as measured by tumor response and animal survival, in imatinib mesylate-sensitive GIST xenografts as compared with treatment with imatinib mesylate or MK-2206 alone. WTS implicated two neural genes, brain expressed X-linked 1 and neuronal pentraxin I, whose expression was significantly upregulated in combination-treated tumors compared with tumors treated with the two monotherapies., Conclusions: These studies provide strong preclinical justification for combining imatinib mesylate with an AKT inhibitor as a front-line therapy in GIST. In addition, the WTS implicated the BCL-2/BAX/BAD apoptotic pathway as a potential mechanism for this enhanced combination effect. Clin Cancer Res; 23(1); 171-80. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: The insulin-like growth factor (IGF) pathway is implicated in the pathogenesis of hepatocellular carcinoma (HCC) and may be important in nonalcoholic fatty liver disease (NAFLD). The aim of this study is to determine expression levels of IGFs and receptors in NAFLD-associated HCC., Methods: Tissue microarrays were constructed from patients who underwent hepatectomy for HCC. Immunohistochemistry was performed using antibodies for IGF ligands and receptors. Immunostain results were scored by a pathologist blinded to clinical data., Results: Among 27 patients with HCC, the most common underlying liver diseases included NAFLD, hepatitis C, and alcoholic hepatitis. Expression levels of IGFs and receptors were not associated with patients' underlying liver disease. In all patients, IGF-2 expression was upregulated in tumor and adjacent non-neoplastic liver. Expression of IGF-1 was low in adjacent liver in 6 of 10 patients with cirrhosis, compared with 2 of 17 patients without cirrhosis (P = 0.025). Higher IGF-1 expression in liver adjacent to tumor was associated with poorer median survival of 22 months, compared with 72 months with equal or lower IGF-1 expression in adjacent liver relative to tumor (P = 0.006)., Conclusions: Our preliminary results demonstrate significant associations between IGF-1 expression and liver cirrhosis and survival after resection in patients with HCC, independent of their underlying liver disease.

Unlabelled: We investigated the correlation between metabolic response by (18)F-FDG PET and objective response, glucose transporter type 4 (GLUT4) expression, and KIT/PDGFRA mutation status in patients with gastrointestinal stromal tumor undergoing neoadjuvant imatinib mesylate therapy., Methods: (18)F-FDG PET was performed at baseline, 1-7 d, and 4 or 8 wk after imatinib mesylate initiation. Best objective response was defined by version 1.0 of the Response Evaluation Criteria in Solid Tumors (RECIST). Mutational analysis and tumor GLUT4 expression by immunohistochemistry were done on tissue obtained at baseline or surgery., Results: (18)F-FDG PET showed high baseline tumor glycolytic activity (mean SUV(max), 14.2; range, 1.3-53.2), decreasing after 1 wk of imatinib mesylate (mean, 5.5; range, -0.5-47.7, P < 0.001, n = 44), and again before surgery (mean, 3.0; range, -0.5-36.1, P < 0.001, n = 40). At week 1, there were 3 patients with complete metabolic response (CMR), 33 with partial metabolic response (PMR), 6 with stable metabolic disease (SMD), and 2 with progressive metabolic disease (PMD). Before surgery, there were 3 with CMR, 33 with PMR, 4 with SMD, and none with PMD. The best response according to RECIST was 2 with partial response, 36 with stable disease, and 1 with progressive disease (n = 39). Of the patients with a posttreatment decrease in GLUT4 expression, 1 had CMR, 15 had PMR, 2 had SMD, and 1 had PMD at week 1, whereas before surgery 1 patient had CMR, 16 had PMR, 2 had SMD, and none had PMD. Among 27 patients with KIT exon 11 mutations, 1 had CMR, 22 had PMR, 3 had SMD, and 1 had PMD at week 1, whereas 1 had CMR, 22 had PMR, 2 had SMD, and 2 were unknown before surgery; among 4 patients with a wild-type genotype, 2 had PMR and 2 SMD at week 1, whereas 1 had CMR, 2 had PMR, and 1 had SMD before surgery., Conclusion: After imatinib mesylate initiation, metabolic response by (18)F-FDG PET was documented earlier (1-7 d) and was of much greater magnitude (36/44) than that documented by RECIST (2/39). Immunohistochemistry data suggest that GLUT4 may play a role in (18)F-FDG uptake in gastrointestinal stromal tumor, GLUT4 levels decrease after imatinib mesylate therapy in most patients with PMR, and the biologic action of imatinib mesylate interacts with glycolysis and GLUT4 expression. A greater than 85% metabolic response was observed as early as days 1-7 in patients with exon 11 mutations.

Cell signaling plays a central role in the etiology of cancer. Numerous therapeutics in use or under development target signaling proteins; however, off-target effects often limit assignment of positive clinical response to the intended target. As direct measurements of signaling protein activity are not generally feasible during treatment, there is a need for more powerful methods to determine if therapeutics inhibit their targets and when off-target effects occur. We have used the Bayesian Decomposition algorithm and data on transcriptional regulation to create a novel methodology, Differential Expression for Signaling Determination (DESIDE), for inferring signaling activity from microarray measurements. We applied DESIDE to deduce signaling activity in gastrointestinal stromal tumor cell lines treated with the targeted therapeutic imatinib mesylate (Gleevec). We detected the expected reduced activity in the KIT pathway, as well as unexpected changes in the p53 pathway. Pursuing these findings, we have determined that imatinib-induced DNA damage is responsible for the increased activity of p53, identifying a novel off-target activity for this drug. We then used DESIDE on data from resected, post-imatinib treatment tumor samples and identified a pattern in these tumors similar to that at late time points in the cell lines, and this pattern correlated with initial clinical response. The pattern showed increased activity of ETS domain-containing protein Elk-1 and signal transducers and activators of transcription 3 transcription factors, which are associated with the growth of side population cells. DESIDE infers the global reprogramming of signaling networks during treatment, permitting treatment modification that leverages ongoing drug development efforts, which is crucial for personalized medicine.

Despite initial efficacy of imatinib mesylate in most gastrointestinal stromal tumor (GIST) patients, many experience primary/secondary drug resistance. Therefore, clinical management of GIST may benefit from further molecular characterization of tumors before and after imatinib mesylate treatment. As part of a recent phase II trial of neoadjuvant/adjuvant imatinib mesylate treatment for advanced primary and recurrent operable GISTs (Radiation Therapy Oncology Group S0132), gene expression profiling using oligonucleotide microarrays was done on tumor samples obtained before and after imatinib mesylate therapy. Patients were classified according to changes in tumor size after treatment based on computed tomography scan measurements. Gene profiling data were evaluated with Statistical Analysis of Microarrays to identify differentially expressed genes (in pretreatment GIST samples). Based on Statistical Analysis of Microarrays [False Discovery Rate (FDR), 10%], 38 genes were expressed at significantly lower levels in the pretreatment biopsy samples from tumors that significantly responded to 8 to 12 weeks of imatinib mesylate, that is, >25% tumor reduction. Eighteen of these genes encoded Krüppel-associated box (KRAB) domain containing zinc finger (ZNF) transcriptional repressors. Importantly, 10 KRAB-ZNF genes mapped to a single locus on chromosome 19p, and a subset predicted likely response to imatinib mesylate-based therapy in a naïve panel of GIST. Furthermore, we found that modifying expression of genes within this predictive signature can enhance the sensitivity of GIST cells to imatinib mesylate. Using clinical pretreatment biopsy samples from a prospective neoadjuvant phase II trial, we have identified a gene signature that includes KRAB-ZNF 91 subfamily members that may be both predictive of and functionally associated with likely response to short-term imatinib mesylate treatment.

Nbs1, a member of the Mre11-RAD50-Nbs1 complex, is phosphorylated by ATM, the product of the ataxia-telangiectasia mutated gene and a member of the phosphatidylinositol 3-kinase-related family of serine-threonine kinases, in response to DNA double-strand breaks (DSBs) to regulate DNA damage checkpoints. Here we show that BCR/ABL stimulated Nbs1 expression by induction of c-Myc-dependent transactivation and protection from caspase-dependent degradation. BCR/ABL-related fusion tyrosine kinases (FTKs) such as TEL/JAK2, TEL/PDGFbetaR, TEL/ABL, TEL/TRKC, BCR/FGFR1, and NPM/ALK as well as interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) also stimulated Nbs1 expression. Enhanced ATM kinase-dependent phosphorylation of Nbs1 on serine 343 (S343) in response to genotoxic treatment was detected in leukemia cells expressing BCR/ABL and other FTKs in comparison to normal counterparts stimulated with IL-3, GM-CSF, and SCF. Expression of Nbs1-S343A mutant disrupted the intra-S-phase checkpoint, decreased homologous recombinational repair (HRR) activity, down-regulated XIAP expression, and sensitized BCR/ABL-positive cells to cytotoxic drugs. Interestingly, inhibition of Nbs1 phosphorylation by S343A mutant enhanced the antileukemia effect of the combination of imatinib and genotoxic agent.

Breakpoint cluster region/Abelson (BCR/ABL) tyrosine kinase enhances the ability of leukemia cells to infiltrate various organs. We show here that expression of the helix-loop-helix transcription factor Id1 is enhanced by BCR/ABL in a signal transducer and activator of transcription 5 (STAT5)-dependent manner. Enhanced expression of Id1 plays a key role in BCR/ABL-mediated cell invasion. Down-regulation of Id1 in BCR/ABL leukemia cells by the antisense cDNA significantly reduced their invasive capability through the Matrigel membrane and their ability to infiltrate hematopoietic and nonhematopoietic organs resulting in delayed leukemogenesis in mice. The Id1-promoted cell invasiveness was seemingly mediated by matrix metalloproteinase 9 (MMP9). Transactivation of MMP9 promoter in BCR/ABL cells was dependent on Id1 and abrogation of the MMP9 catalytic activity by a metalloproteinase inhibitor or blocking antibody decreased invasive capacity of leukemia cells. These data suggest that BCR/ABL-STAT5-Id1-MMP9 pathway may play a critical role in BCR/ABL-mediated leukemogenesis by enhancing invasiveness of leukemia cells.