Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Somatic mutations in GRM1 in cancer alter metabotropic glutamate receptor 1 intracellular localization and signaling.

The activity of metabotropic glutamate receptors (mGluRs) is known to be altered as the consequence of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease. However, little attention has been paid to this receptor family's potential link with cancer. Recent reports indicate altered mGluR signaling in various tumor types, and several somatic mutations in mGluR1a in lung cancer were recently described. Group 1 mGluRs (mGluR1a and mGluR5) are coupled primarily to Gαq, leading to the activation of phospholipase C and to the formation of diacylglycerol and inositol 1,4,5-trisphosphate, leading to the release of Ca(2+) from intracellular stores and protein kinase C (PKC) activation. In the present study, we investigated the intracellular localization and G protein-dependent and -independent signaling of eight GRM1 (mGluR1a) somatic mutations. Two mutants found in close proximity to the glutamate binding domain and cysteine-rich region (R375G and G396V) show both decreased cell surface expression and basal inositol phosphate (IP) formation. However, R375G shows increased ERK1/2 activation in response to quisqualate stimulation. A mutant located directly in the glutamate binding site (A168V) shows increased quisqualate-induced IP formation and, similar to R375G, increased ERK1/2 activation. Additionally, a mutation in the G protein-coupled receptor kinase 2/PKC regulatory region (R696W) shows decreased ERK1/2 activation, whereas a mutation within the Homer binding region in the carboxyl-terminal tail (P1148L) does not alter the intracellular localization of the receptor, but it induces changes in cellular morphology and exhibits reduced ERK1/2 activation. Taken together, these results suggest that mGluR1a signaling in cancer is disrupted by somatic mutations with multiple downstream consequences.

Binding, Neutralization and Internalization of the Interleukin-13 Antibody, Lebrikizumab.

INTRODUCTION: IL-13 is the primary upregulated cytokine in atopic dermatitis (AD) skin and is the pathogenic mediator driving AD pathophysiology. Lebrikizumab, tralokinumab and cendakimab are therapeutic monoclonal antibodies (mAb) that target IL-13. METHODS: We undertook studies to compare in vitro binding affinities and cell-based functional activities of lebrikizumab, tralokinumab and cendakimab. RESULTS: Lebrikizumab bound IL-13 with higher affinity (as determined using surface plasma resonance) and slower off-rate. It was more potent in neutralizing IL-13-induced effects in STAT6 reporter and primary dermal fibroblast periostin secretion assays than either tralokinumab or cendakimab. Live imaging confocal microscopy was employed to determine the mAb effects on IL-13 internalization into cells via the decoy receptor IL-13Rα2, using A375 and HaCaT cells. The results showed that only the IL-13/lebrikizumab complex was internalized and co-localized with lysosomes, whereas IL-13/tralokinumab or IL-13/cendakimab complexes did not internalize. CONCLUSION: Lebrikizumab is a potent, neutralizing high-affinity antibody with a slow disassociation rate from IL-13. Additionally, lebrikizumab does not interfere with IL-13 clearance. Lebrikizumab has a different mode of action to both tralokinumab and cendakimab, possibly contributing to the clinical efficacy observed by lebrikizumab in Ph2b/3 AD studies.

Evaluation of molecular subtypes and clonal selection during establishment of patient-derived tumor xenografts from gastric adenocarcinoma.

Patient-derived xenografts (PDX) have emerged as an important translational research tool for understanding tumor biology and enabling drug efficacy testing. They are established by transfer of patient tumor into immune compromised mice with the intent of using them as Avatars; operating under the assumption that they closely resemble patient tumors. In this study, we established 27 PDX from 100 resected gastric cancers and studied their fidelity in histological and molecular subtypes. We show that the established PDX preserved histology and molecular subtypes of parental tumors. However, in depth investigation of the entire cohort revealed that not all histological and molecular subtypes are established. Also, for the established PDX models, genetic changes are selected at early passages and rare subclones can emerge in PDX. This study highlights the importance of considering the molecular and evolutionary characteristics of PDX for a proper use of such models, particularly for Avatar trials.

Author Correction: Evaluation of molecular subtypes and clonal selection during establishment of patient-derived tumor xenografts from gastric adenocarcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic characterization of intrinsic and acquired resistance to cetuximab in colorectal cancer patients.

Anti-EGFR antibodies are effective in therapies for late-stage colorectal cancer (CRC); however, many tumours are unresponsive or develop resistance. We performed genomic analysis of intrinsic and acquired resistance to anti-EGFR therapy in prospectively collected tumour samples from 25 CRC patients receiving cetuximab (an EGFR inhibitor). Of 25 CRC patients, 13 displayed intrinsic resistance to cetuximab; 12 were intrinsically sensitive. We obtained six re-biopsy samples at acquired resistance from the intrinsically sensitive patients. NCOA4-RET and LMNA-NTRK1 fusions and NRG1 and GNAS amplifications were found in intrinsic-resistant patients. In cetuximab-sensitive patients, we found KRAS K117N and A146T mutations in addition to BRAF V600E, AKT1 E17K, PIK3CA E542K, and FGFR1 or ERBB2 amplifications. The comparison between baseline and acquired-resistant tumours revealed an extreme shift in variant allele frequency of somatic variants, suggesting that cetuximab exposure dramatically selected for rare resistant subclones that were initially undetectable. There was also an increase in epithelial-to-mesenchymal transition at acquired resistance, with a reduction in the immune infiltrate. Furthermore, characterization of an acquired-resistant, patient-derived cell line showed that PI3K/mTOR inhibition could rescue cetuximab resistance. Thus, we uncovered novel genomic alterations that elucidate the mechanisms of sensitivity and resistance to anti-EGFR therapy in metastatic CRC patients.

Adipose-derived stem cells.

Human adipose tissue has been shown to contain a population of cells that possesses extensive proliferative capacity and the ability to differentiate into multiple cell lineages. These cells are referred to as adipose tissue-derived stem cells (ADSCs) and are generally similar, though not identical, to mesenchymal stem cells (also referred to as marrow stromal cells). ADSCs for research are most conveniently extracted from tissue removed during an elective cosmetic liposuction procedure but may also be obtained from resected adipose tissue. This chapter describes surgical procedures associated with improved ADSC recovery and the processes by which aspirated adipose tissue is washed and digested with collagenase to yield a heterogeneous population from which ADSCs can be expanded. The large volume of tissue obtained from a liposuction procedure (average approximately 2 L), combined with the relatively high frequency of ADSC within the digestate, yields substantially more stem cells than can be realized from marrow without extensive expansion in culture.

Merestinib (LY2801653) inhibits neurotrophic receptor kinase (NTRK) and suppresses growth of NTRK fusion bearing tumors.

Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.

Genomic Aberrations that Activate D-type Cyclins Are Associated with Enhanced Sensitivity to the CDK4 and CDK6 Inhibitor Abemaciclib.

Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3'UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.

Fat tissue: an underappreciated source of stem cells for biotechnology.

Adipose tissue can be harvested in large amounts with minimal morbidity. It contains numerous cells types, including adipocytes, preadipocytes, vascular endothelial cells and vascular smooth muscle cells; it also contains cells that have the ability to differentiate into several lineages, such as fat, bone, cartilage, skeletal, smooth, and cardiac muscle, endothelium, hematopoietic cells, hepatocytes and neuronal cells. Cloning studies have shown that some adipose-derived stem cells (ADSCs) have multilineage differentiation potential. ADSCs are also capable of expressing multiple growth factors, including vascular endothelial growth factor and hepatocyte growth factor. Early, uncontrolled, non-randomized clinical research, applying fresh adipose-derived cells into a cranial defect or undifferentiated ADSCs into fistulas in Crohn's disease, has shown healing and an absence of side effects. The combination of these properties, and the large quantity of cells that can be obtained from fat, suggests that this tissue will be a useful tool in biotechnology.

Plasticity of human adipose stem cells toward endothelial cells and cardiomyocytes.

Recent preclinical and clinical studies have suggested that adult stem cells have the ability to promote the retention or restoration of cardiac function in acute and chronic ischemia. Published clinical studies have used autologous donor cells, including skeletal muscle myoblasts, cultured peripheral blood cells, or bone marrow cells. However, our research and that of others indicates that human adipose tissue is an alternative source of cells with potential for cardiac cell therapy. These findings include the presence of cells within adipose tissue that can differentiate into cells expressing a cardiomyocytic or endothelial phenotype, as well as angiogenic and antiapoptotic growth factors. This potential is supported by preclinical studies in large animals.

Multipotential differentiation of adipose tissue-derived stem cells.

Tissue engineering offers considerable promise in the repair or replacement of diseased and/or damaged tissues. The cellular component of this regenerative approach will play a key role in bringing these tissue engineered constructs from the laboratory bench to the clinical bedside. However, the ideal source of cells still remains unclear and may differ depending upon the application. Current research for many applications is focused on the use of adult stem cells. The properties of adult stem cells that make them well-suited for regenerative medicine are (1) ease of harvest for autologous transplantation, (2) high proliferation rates for ex vivo expansion and (3) multilineage differentiation capacity. This review will highlight the use of adipose tissue as a reservoir of adult stem cells and draw conclusions based upon comparisons with bone marrow stromal cells.

Mucosal regeneration of a duodenal defect using small intestine submucosa.

Intestinal tissue engineering has the potential of developing new treatment strategies for patients with a deficit in intestinal surface area. The purpose of this study was to investigate the capacity of small intestine submucosa (SIS) to regenerate intestinal epithelia in a rodent model for a duodenal defect. A duodenotomy was created in 20 Sprague-Dawley rats and was repaired with a circular patch of SIS. The repaired sites were retrieved 1, 2, 4, and 12 weeks after implantation. The size of the residual mucosal defect was measured. The retrieved tissues were fixed in formalin and processed by standard histologic techniques. The animals tolerated the procedure well. The mean size of the mucosal defects significantly decreased with time. Complete epithelialization of the defects was noted within 4 weeks in three of five samples. Histologically, the defects were lined with crypts and villi, but the muscularis layer did not regenerate. In the rodent model, SIS can be used as a patch to repair a duodenotomy. Mucosal regeneration was observed in the area of the defect. Further studies will determine whether SIS may be used to preserve or increase mucosal surface area in patients whose bowel length is compromised.

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.

BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold.

Osteogenic differentiation of bone marrow stromal cells (BMSC) in a three-dimensional (3-D) scaffold has not been well studied. In this work, we studied expression of bone-related genes during differentiation of rabbit BMSCs in response to bone morphogenetic protein (BMP)-2 in both 2-D and 3-D culture systems. When BMSCs were cultured on films (2-D) of biodegradable poly(lactide-co-glycolide) (PLGA), increases in mRNA expression of type I collagen (Col I) and vascular endothelial growth factor (VEGF) became evident after 1 week. However, expression of both genes was only mildly stimulated by BMP-2. Expression of the osteopontin gene was highly stimulated by BMP-2 treatment. Expression of chordin, a BMP antagonist, increased significantly after 7 days. The increase was abrogated by BMP-2 treatment. BMP-2 was also able to stimulate mineralization of cultured BMSCs. After cells were switched to 3-D PLGA scaffolds for 24 h, expression of osteopontin and VEGF were markedly increased while expression of type I collagen and chordin remained unchanged. Expression of Col I did not increase with time in a 3-D culture as it did when cells were cultured on a 2-D film. We further explored the possibility of engineering bone tissue in vitro by seeding BMSCs into PLGA scaffolds. Cellular differentiation and bone formation in the scaffolds were analyzed histologically at 2 weeks and 2 months. Secretion of ECM by cells was evident at both 2-week and 2-month scaffolds, and was enhanced by rhBMP-2. Striking differences in 2-month scaffolds were observed between BMP-treated and untreated cells. A woven bone-like structure appeared in the scaffolds treated with BMP-2. The structure was verified to be bone-related by: (1) the presence of organized collagen fibrils; (2) the presence of mineral; and (3) morphological features of trabecular bone. Although collagen was abundant in the untreated 2-month scaffolds, it was disorganized. The untreated scaffolds also lacked mineral deposits, which were present in 2-D cultured cells even in the absence of BMP-2. Our results indicate that the requirement of osteo-inductive agents, such as BMP-2, is essential for bone tissue engineering.