Adam M. Burgoyne

CelAB, a Multifunctional Cellulase Encoded by Teredinibacter turnerae T7902T, a Culturable Symbiont Isolated from the Wood-Boring Marine Bivalve Lyrodus pedicellatus

ABSTRACT We characterized a multifunctional cellulase (CelAB) encoded by the endosymbiont Teredinibacter turnerae T7902T. CelAB contains two catalytic and two carbohydrate-binding domains, each separated by polyserine linker regions. CelAB binds cellulose and chitin, degrades multiple complex polysaccharides, and displays two catalytic activities, cellobiohydrolase (EC 3.2.1.91) and β-1,4(3) endoglucanase (EC 3.2.1.4).

Increased risk of additional cancers among patients with gastrointestinal stromal tumors: A population-based study

Most gastrointestinal stromal tumors (GISTs) are considered nonhereditary or sporadic. However, single‐institution studies suggest that GIST patients develop additional malignancies at increased frequencies. It was hypothesized that greater insight could be gained into possible associations between GISTs and other malignancies with a national cancer database inquiry.

FGFR1 and NTRK3 actionable alterations in "Wild-Type" gastrointestinal stromal tumors

AbstractBackgroundAbout 10–15% of adult, and most pediatric, gastrointestinal stromal tumors (GIST) lack mutations in KIT, PDGFRA, SDHx, or RAS pathway components (KRAS, BRAF, NF1). The identification of additional mutated genes in this rare subset of tumors can have important clinical benefit to identify altered biological pathways and select targeted therapies.MethodsWe performed comprehensive genomic profiling (CGP) for coding regions in more than 300 cancer-related genes of 186 GISTs to assess for their somatic alterations.ResultsWe identified 24 GIST lacking alterations in the canonical KIT/PDGFRA/RAS pathways, including 12 without SDHx alterations. These 24 patients were mostly adults (96%). The tumors had a 46% rate of nodal metastases. These 24 GIST were more commonly mutated at 7 genes: ARID1B, ATR, FGFR1, LTK, SUFU, PARK2 and ZNF217. Two tumors harbored FGFR1 gene fusions (FGFR1–HOOK3, FGFR1–TACC1) and one harbored an ETV6–NTRK3 fusion that responded to TRK inhibition. In an independent sample set, we identified 5 GIST cases lacking alterations in the KIT/PDGFRA/SDHx/RAS pathways, including two additional cases with FGFR1–TACC1 and ETV6–NTRK3 fusions.ConclusionsUsing patient demographics, tumor characteristics, and CGP, we show that GIST lacking alterations in canonical genes occur in younger patients, frequently metastasize to lymph nodes, and most contain deleterious genomic alterations, including gene fusions involving FGFR1 and NTRK3. If confirmed in larger series, routine testing for these translocations may be indicated for this subset of GIST. Moreover, these findings can be used to guide personalized treatments for patients with GIST. Trial registration NCT 02576431. Registered October 12, 2015

Gastrointestinal stromal tumors in the setting of multiple tumor syndromes

Purpose of review Knowledge related to gastrointestinal stromal tumor (GIST) in the setting of nonhereditary and hereditary multiple tumor syndromes continues to expand. This review describes associations between sporadic GIST and second malignancies, as well as new contributions to our knowledge about hereditary GIST multiple tumor syndromes. Recent findings Sporadic GIST patients have increased risk of developing synchronous/metachronous cancers, including nonhematologic and hematologic malignancies. Data suggest these associations are nonrandom, more prevalent in men and increase with age. New adrenal tumors have also been associated with nonhereditary Carneys triad. Meanwhile, understanding of the molecular basis of heritable GIST syndromes has improved. Several new familial GIST kindreds have been reported, including those with germline KIT and PDGFR&agr; mutations. Knowledge about succinate dehydrogenase (SDH) deficiency and mutations in hereditary GIST syndromes has expanded. It is now known that neurofibromatosis-1-associated GISTs are SDHB-positive, whereas Carney–Stratakis syndrome-associated GISTs are SDHB-deficient with underlying germline mutations in SDH subunits A–D. Summary Recognition and early diagnosis of GIST syndromes allows for improved comprehensive medical care. With additional understanding of the molecular pathogenesis of GIST multiple tumor syndromes, we can refine our screening programs and management of these patients and their families.

Hedgehog pathway dysregulation contributes to the pathogenesis of human gastrointestinal stromal tumors via GLI-mediated activation of KIT expression

Gastrointestinal stromal tumors (GIST) arise within the interstitial cell of Cajal (ICC) lineage due to activating KIT/PDGFRA mutations. Both ICC and GIST possess primary cilia (PC), which coordinate PDGFRA and Hedgehog signaling, regulators of gastrointestinal mesenchymal development. Therefore, we hypothesized that Hedgehog signaling may be altered in human GIST and controls KIT expression. Quantitative RT-PCR, microarrays, and next generation sequencing were used to describe Hedgehog/PC-related genes in purified human ICC and GIST. Genetic and pharmacologic approaches were employed to investigate the effects of GLI manipulation on KIT expression and GIST cell viability. We report that Hedgehog pathway and PC components are expressed in ICC and GIST and subject to dysregulation during GIST oncogenesis, irrespective of KIT/PDGFRA mutation status. Using genomic profiling, 10.2% of 186 GIST studied had potentially deleterious genomic alterations in 5 Hedgehog-related genes analyzed, including in the PTCH1 tumor suppressor (1.6%). Expression of the predominantly repressive GLI isoform, GLI3, was inversely correlated with KIT mRNA levels in GIST cells and non-KIT/non-PDGFRA mutant GIST. Overexpression of the 83-kDa repressive form of GLI3 or small interfering RNA-mediated knockdown of the activating isoforms GLI1/2 reduced KIT mRNA. Treatment with GLI1/2 inhibitors, including arsenic trioxide, significantly increased GLI3 binding to the KIT promoter, decreased KIT expression, and reduced viability in imatinib-sensitive and imatinib-resistant GIST cells. These data offer new evidence that genes necessary for Hedgehog signaling and PC function in ICC are dysregulated in GIST. Hedgehog signaling activates KIT expression irrespective of mutation status, offering a novel approach to treat imatinib-resistant GIST.

Single Agent and Synergistic Activity of the “First-in-Class” Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma

Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo. Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual “first-in-class” PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. Mol Cancer Ther; 15(11); 2553–62. ©2016 AACR.

Duodenal-Jejunal Flexure GI Stromal Tumor Frequently Heralds Somatic NF1 and Notch Pathway Mutations

Purpose GI stromal tumors (GISTs) are commonly associated with somatic mutations in KIT and PDGFRA. However, a subset arises from mutations in NF1, most commonly associated with neurofibromatosis type 1. We define the anatomic distribution of NF1 alterations in GIST. Methods We describe the demographic/clinicopathologic features of 177 patients from two institutions whose GISTs underwent next-generation sequencing of ≥315 cancer-related genes. Results We initially identified six (9.7%) of 62 GISTs with NF1 genomic alterations from the first cohort. Of these six patients, five (83.3%) had unifocal tumors at the duodenal-jejunal flexure (DJF). Two additional patients with DJF GISTs had non-NF1 (KIT and BRAF) genomic alterations. After excluding one DJF GIST with an NF1 single nucleotide polymorphism, four (57.1%) of seven sequenced DJF tumors demonstrated deleterious NF1 alterations, whereas only one (1.8%) of 55 sequenced non-DJF GISTs had a deleterious NF1 somatic mutation (P < .001). One patient with DJF GIST had a germline NF1 variant that was associated with incomplete penetrance of clinical neurofibromatosis type 1 features along with a somatic NF1 mutation. Of the five DJF GISTs with any NF1 alteration, three (60%) had KIT mutations, and three (60%) had Notch pathway mutations (NOTCH2, MAML2, CDC73). We validated these findings in a second cohort of 115 GISTs, where two (40%) of five unifocal NF1-mutated GISTs arose at the DJF, and one of these also had a Notch pathway mutation (EP300). Conclusion Broad genomic profiling of adult GISTs has revealed that NF1 alterations are enriched in DJF GISTs. These tumors also may harbor concurrent activating KIT and/or inactivating Notch pathway mutations. In some cases, germline NF1 genetic testing may be appropriate for patients with DJF GISTs.

Tumor Symbiosis: Gastrointestinal Stromal Tumor as a Host for Primary Peritoneal Mesothelioma

A 57-year-old man with a history of polycythemia vera presented for routine primary care follow-up and was found to have anemia with hemoglobin level of 12 mg/dL from 16 mg/ dL. He was referred to gastroenterology for upper and lower endoscopies, which revealed an ulcerated submucosal mass in the gastric antrum. Endoscopic ultrasound (EUS) with core needle biopsy revealed a spindle cell neoplasm on hematoxylin and eosin (H&E) staining. Immunohistochemical analyses were positive for CD117 (KIT) and DOG-1, consistent with a gastrointestinal stromal tumor (GIST). Staging computed tomography (CT) of the abdomen/pelvis demonstrated a 15 × 12 × 13 cm gastric mass with effacement of the left liver. He was started on neoadjuvant imatinib (Gleevec, Novartis). Follow-up CT after 3 months of therapy demonstrated a 19% increase in tumor volume and new omental nodules (Fig. 1a). Tumor sequencing revealed a PDGFRA D842V mutation, which is known to be imatinib resistant. Thus, he was switched to dasatinib, but this was poorly tolerated. Therefore, he underwent diagnostic laparoscopy and peritoneal biopsies, which revealed primary peritoneal mesothelioma in addition to the known gastric GIST. He was then referred to our center for possible cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC) to treat his mesothelioma. To determine his candidacy for this approach, repeat diagnostic laparoscopy was performed. This revealed the large gastric GISTwith invasion, rather than abutment, of hepatic segments 2 and 3. Moreover, there were numerous peritoneal implants coating the surface of the GIST (Fig. 1b). The peritoneal cancer index (PCI) regions of the upper abdomen (e.g., regions 1– 3) and along the small bowel (e.g., regions 9–12) were most affected. As compared to the upper abdomen, the pelvis and lower quadrants were essentially spared of mesothelioma. The patient had a PCI score of 17. We proceeded with an exploratory laparotomy, subtotal gastrectomy with an en bloc left lateral sectionectomy and omentectomy for the GIST followed by complete peritoneal cytoreduction (CC0) and HIPEC for the mesothelioma. Final histopathology (Fig. 2a) revealed numerous peritoneal calretinin immunostain-positive tumor deposits that were histopathologically consistent with epithelioid peritoneal mesothelioma (Fig. 2b) with an underlying highly vascularized KIT (C-KIT, CD117) immunostainpositive spindle cell tumor consistent with GIST (Fig. 2c).

Abstract LB-298: The novel triple PI3K-CDK4/6-BRD4 inhibitor SRX3177 harnesses synthetic lethality relationships to orthogonally disrupt cancer cell signaling

Dysregulation of the cell cycle is a hallmark of nearly all cancers, and efforts to target signaling pathways regulating cell growth and proliferation have driven much of cancer drug discovery. Despite advances in novel therapeutics, however, most patients with advanced neoplasms do not achieve long-term survival with single agent targeted therapy. Here, we describe a novel “triple inhibitor” (i.e., SRX3177) that simultaneously targets three oncogenes promoting cancer cell growth: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic regulator BRD4. This rationally-designed, thieno-pyranone scaffold-based small molecule inhibitor uses known synthetic lethality relationships to orthogonally disrupt three targets within the cancer cell with one agent. Single agent CDK4/6 inhibitors such as palbociclib, which is FDA-approved in combination with hormone therapy in estrogen-receptor positive breast cancer, suffer from being cytostatic in nature, requiring combinations to be more effective and avoid development of resistance. Concurrent PI3K inhibition can prevent resistance to CDK4/6 inhibition, and combined CDK4/6 and PI3K inhibition leads to synthetic lethality reported in a number of cancer types, including breast cancer and mantle cell lymphoma. Moreover, blocking the chromatin reader protein BRD4 downregulates MYC and cyclin D1 transcription, further promoting cell cycle arrest in G1. Thus, we designed SRX3177 as a triple inhibitor of PI3K, CDK4/6, and BRD4 to maximally block cell cycle progression and cancer cell growth. SRX3177 is a potent ATP competitive CDK4/6 inhibitor (IC50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC50: BD1 = 32.9 nM, BD2 = 88.8 nM). In a panel of mantle cell lymphoma, neuroblastoma, and hepatocellular carcinoma cell lines, SRX3177 has maximal IC50 values of 578 nM, 385 nM, and 495 nM respectively. This represents a 19 to 82-fold increase in potency compared to palbociclib. SRX3177 is 5-fold more potent in cancer cells than the combination of similar potency single PI3K, CD4/6, and BRD4 inhibitors (i.e., BKM120 + palbociclib + JQ1). SRX3177 is also 40-fold less toxic to normal epithelial cells than the co-treatment with single inhibitors. Furthermore, SRX3177 induces cell cycle arrest and apoptosis in propidium iodide and annexin V assays, respectively. Finally, SRX3177 inhibits Akt and Rb phosphorylation (downstream of PI3K and CDK4/6 signaling, respectively) and blocks BRD4 binding to chromatin. Thus, our triple inhibitor SRX3177 is efficacious, is more potent and less toxic to normal cells than administration of three individual inhibitors, and has robust pharmacodynamic effects on its targets. Taken together, our data support the development of SRX3177 as a novel therapeutic agent for multiple cancers. Citation Format: Adam M. Burgoyne, Francisco M. Vega, Alok Singh, Shweta Joshi, Joseph R. Garlich, Guillermo A. Morales, Tatiana G. Kutateladze, Donald L. Durden. The novel triple PI3K-CDK4/6-BRD4 inhibitor SRX3177 harnesses synthetic lethality relationships to orthogonally disrupt cancer cell signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2017-LB-298