Approaches to the Development of In Vivo Propagation Systems for Hepatitis C Virus (HCV)

Hepatitis C virus (HCV) is a recently discovered major human pathogen infecting 170 million individuals worldwide. It was the first hepatotropic virus to be isolated by molecular biology techniques. Since its discovery in 1989 considerable progress has been made in the identification of the viral functional regions, the interactions between viral products, and viral pathogenesis. However, little is known about its replication cycle and the only experimental in vivo HCV infection model is limited to the chimpanzee. Thus, the development of a cheap and accessible in vitro or in vivo culture system has become a priority. The work in this thesis explores a variety of different approaches to an in vivo culture system using the severely combined immunodeficient (SCID) mouse. These animals lack functional T and B cells and will accept xenografts from a range of species including humans. Hepatocyte derived cell lines and primary human hepatocytes (PHHC) were transplanted intrasplenically and under the renal capsules after performing a partial hepatectomy. The rationale rests on the hypothesis that these cells would attain a biologically functional state of differentiation in vivo, increasing their sensitivity to HCV infection and acting as targets for HCV replication in an in vivo culture system. HCV positive serum was used to infect the cells prior to transplantation or 3 days post transplantation. PHHCs were also mixed with anti- met antibody and interleukin 6 (IL6) prior to transplantation to increase their survivability and proliferation in vivo; silica was administered intraperitoneally to deplete host macrophage cells. All the animals transplanted with hepatocyte derived cell lines developed extremely large metastatic tumours which were hemorrhagic, additionally these cells were not susceptible to HCV infection, irrespective of the route of infection and source of the HCV positive serum. PHHCs failed to engraft irrespective of treatment and were HCV reverse transcription polymerase chain reaction (RTPCR) negative

Temperament and self-based correlates of cooperative, competitive and individualistic learning preferences

People vary in the extent to which they prefer cooperative, competitive or individualistic achievement tasks. In the present research, we conducted two studies designed to investigate correlates and possible roots of these social interdependence orientations, namely approach and avoidance temperament, general self-efficacy, implicit theories of intelligence, and contingencies of self-worth based in others’ approval, competition, and academic competence. The results indicated that approach temperament, general self-efficacy, and incremental theory were positively, and entity theory was negatively related to cooperative preferences (|r| range from .11 to .41); approach temperament, general self-efficacy, competition contingencies, and academic competence contingencies were positively related to competitive preferences (|r| range from .16 to .46); and avoidance temperament, entity theory, competitive contingencies, and academic competence contingencies were positively related, and incremental theory was negatively related to individualistic preferences (|r| range from .09 to .15). The findings are discussed with regard to the meaning of each of the three social interdependence orientations, cultural differences among the observed relations, and implications for practicioners

JAK/STAT3 represents a therapeutic target for colorectal cancer patients with stromal-rich tumors

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression

Sequential ATR and PARP inhibition overcomes acquired DNA damaging agent resistance in pancreatic ductal adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDAC) remains the most lethal cancer. While DNA damaging agents such as platinum and PARP inhibitors have derived clinical benefits, acquired resistance invariably develops. Hence there is an urgent need for novel therapeutic strategies to overcome acquired resistance. Methods Clinically relevant resistance in PDAC patient-derived cell lines was achieved by extended exposure to chemotherapy agents. Synergy scoring, clonogenicity, flow cytometry, immunofluorescence, and transcriptomic analysis were used to investigate the efficacy of ATR (ceralasertib) and PARP (olaparib) inhibitors in overcoming acquired resistance. Results Acquired resistance was associated with transcriptomic shifts in cell cycle checkpoint regulation, metabolic control, DNA damage response (DDR), programmed cell death, and the replication stress response. Combination treatment with ceralasertib, and olaparib was synergistic in all models of acquired resistance. Sequential use of ceralasertib prior to olaparib was highly effective at low dose for DDR proficient models, whereas DDR deficient models responded better with olaparib treatment first. Conclusions We provide in vitro evidence of a novel therapeutic strategy to overcome acquired resistance to PARP inhibitor and platinum in PDAC, using sequential exposure to ceralasertib and olaparib. A sequential regimen should be investigated clinically to circumvent dose limiting toxicity seen in concurrent combinations

Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF–PDE8A protein–protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF–cAMP/PKA signaling axes and treating KRAS–c-RAF dependent PDAC. Through disrupting the c-RAF–PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS–c-RAF dependent PDAC

Genomic and molecular analyses identify molecular subtypes of pancreatic cancer recurrence

Pancreatic cancer (PC) remains a highly lethal malignancy, and most patients with localized disease that undergo surgical resection still succumb to recurrent disease. Pattern of recurrence after pancreatectomy is heterogenous, with some studies illustrating that site of recurrence can be associated with prognosis.1 Another study suggested that tumors that develop local and distant recurrence can be regarded as a homogenous disease with similar outcomes.2 Here we investigate novel molecular determinants of recurrence pattern after pancreatectomy for PC

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarkerdriven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient–derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.Stephan B. Dreyer ... Karin S. Kassahn ... et al

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P &lt; 0.001) and PARP inhibitor therapy (P &lt; 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P &lt; 0.018) and WEE1 inhibitor (P &lt; 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P &lt; 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy