Activity of oncolytic vaccinia virus vectors in ovarian cancer.

PhDOncolytic vaccinia virus has great potential in the treatment of cancer and two engineered strains have entered clinical trials. As the advent for oncolytic vaccinia virus as an approved therapy beckons, it is critical to consider some of the barriers that may hinder this progress. These include suboptimal delivery of the virus to tumour sites, incomplete destruction of the tumour mass, and a lack of full understanding of the way in which oncolytic vaccinia kills its target cells. This thesis attempts to address these issues, with a particular focus on ovarian cancer. As ovarian cancer is generally restricted to the peritoneal cavity, intraperitoneal delivery may be preferable over intravenous delivery. Here, it is shown that Lister-dTK, an engineered vaccinia strain, is able to selectively replicate in ovarian tumours, including metastases to the liver following intraperitoneal delivery. To determine whether Lister-dTK could potentially be used in combination with current therapies for ovarian cancer, the effect of cisplatin and Lister-dTK together was assessed in vitro but showed no improvement in overall cell death. In an attempt to further improve the anti-tumour efficacy of Lister-dTK, the extracellular matrix protein (ECM) decorin was expressed from the virus. Decorin interacts with various signalling pathways and is proposed to enhance virus spread. However, abrogation of EGFR and TGFβ signalling could not be demonstrated in vitro, nor could improved virus spread. In an intraperitoneal model of ovarian cancer, Lister-mDCN did not demonstrate enhanced efficacy over a control virus. To determine the mechanisms of ovarian cancer cell death induced by Lister-dTK, the roles of apoptosis, autophagy and necrosis were investigated. Whilst some features of both apoptosis and autophagy were observed, inhibition of these pathways did not attenuate Lister-dTK. It is proposed that necrosis is the primary cause of cell death but that this process may occur in a regulated manner

Generation of human parallel chimeric antigen receptor (pCAR) T cells to achieve synergistic T cell co-stimulation

Dual co-stimulation may be harnessed using parallel chimeric antigen receptors (pCARs) in which two distinct co-stimulatory units are adjacently localized on the plasma membrane. This protocol summarizes construct design, human T cell isolation, retroviral transduction, tissue culture expansion, and preclinical testing of pCAR T cells, exemplified by receptors that co-target avb6 integrin and ErbB dimers. For complete details on the use and execution of this protocol, please refer to Muliaditan et al. (2021)

Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c.

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing

Size-dependent increase in RNA Polymerase II initiation rates mediates gene expression scaling with cell size

Cell size varies during the cell cycle and in response to external stimuli. This requires the tight coordination, or “scaling”, of mRNA and protein quantities with the cell volume in order to maintain biomolecules concentrations and cell density. Evidence in cell populations and single cells indicates that scaling relies on the coordination of mRNA transcription rates with cell size. Here we use a combination of single-molecule fluorescence in situ hybridisation (smFISH), time-lapse microscopy and mathematical modelling in single fission yeast cells to uncover the precise molecular mechanisms that control transcription rates scaling with cell size. Linear scaling of mRNA quantities is apparent in single fission yeast cells during a normal cell cycle. Transcription rates of both constitutive and regulated genes scale with cell size without evidence for transcriptional bursting. Modelling and experimental data indicate that scaling relies on the coordination of RNAPII transcription initiation rates with cell size and that RNAPII is a limiting factor. We show using real-time quantitative imaging that size increase is accompanied by a rapid concentration independent recruitment of RNAPII onto chromatin. Finally, we find that in multinucleated cells, scaling is set at the level of single nuclei and not the entire cell, making the nucleus the transcriptional scaling unit. Integrating our observations in a mechanistic model of RNAPII mediated transcription, we propose that scaling of gene expression with cell size is the consequence of competition between genes for limiting RNAPII

Chemotherapy-induced apoptosis, autophagy and cell cycle arrest are key drivers of synergy in chemo-immunotherapy of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological malignancies in the UK. Recent evidence has shown that there is potential for immunotherapies to be successful in treating this cancer. We have previously shown the effective application of combinations of traditional chemotherapy and CAR (chimeric antigen receptor) T cell immunotherapy in in vitro and in vivo models of EOC. Platinum-based chemotherapy synergizes with ErbB-targeted CAR T cells (named T4), significantly reducing tumor burden in mice. Here, we show that paclitaxel synergizes with T4 as well, and look into the mechanisms behind the effectiveness of chemo-immunotherapy in our system. Impairment of caspase activity using pan-caspase inhibitor Z-VAD reveals this chemotherapy-induced apoptotic pathway as an essential factor in driving synergy. Mannose-6-phosphate receptor-mediated autophagy and the arrest of cell cycle in G2/M are also shown to be induced by chemotherapy and significantly contributing to the synergy. Increased expression of PD-1 on T4 CAR T cells occurred when these were in culture with ovarian tumor cells; on the other hand, EOC cell lines showed increased PD-L1 expression following chemotherapy treatment. These findings provided a rationale to look into testing PD-1 blockade in combination with paclitaxel and T4 immunotherapy. Combination of these three agents in mice resulted in significant reduction of tumor burden, compared to each treatment alone. In conclusion, the mechanism driving synergy in chemo-immunotherapy of EOC is multifactorial. A deeper understanding of such process is needed to better design combination therapies and carefully stratify patients

RUNX1 regulates a transcription program that affects the dynamics of cell cycle entry of naive resting B cells

RUNX1 is a transcription factor that plays key roles in hematopoietic development and in hematopoiesis and lymphopoiesis. In this article, we report that RUNX1 regulates a gene expression program in naive mouse B cells that affects the dynamics of cell cycle entry in response to stimulation of the BCR. Conditional knockout of Runx1 in mouse resting B cells resulted in accelerated entry into S-phase after BCR engagement. Our results indicate that Runx1 regulates the cyclin D2 (Ccnd2) gene, the immediate early genes Fosl2, Atf3, and Egr2, and the Notch pathway gene Rbpj in mouse B cells, reducing the rate at which transcription of these genes increases after BCR stimulation. RUNX1 interacts with the chromatin remodeler SNF-2-related CREB-binding protein activator protein (SRCAP), recruiting it to promoter and enhancer regions of the Ccnd2 gene. BCR-mediated activation triggers switching between binding of RUNX1 and its paralog RUNX3 and between SRCAP and the switch/SNF remodeling complex member BRG1. Binding of BRG1 is increased at the Ccnd2 and Rbpj promoters in the Runx1 knockout cells after BCR stimulation. We also find that RUNX1 exerts positive or negative effects on a number of genes that affect the activation response of mouse resting B cells. These include Cd22 and Bank1, which act as negative regulators of the BCR, and the IFN receptor subunit gene Ifnar1 The hyperresponsiveness of the Runx1 knockout B cells to BCR stimulation and its role in regulating genes that are associated with immune regulation suggest that RUNX1 could be involved in regulating B cell tolerance

AMPK activation protects against prostate cancer by inducing a catabolic cellular state

Emerging evidence indicates that metabolic dysregulation drives prostate cancer (PCa) progression and metastasis. AMP-activated protein kinase (AMPK) is a master regulator of metabolism, although its role in PCa remains unclear. Here, we show that genetic and pharmacological activation of AMPK provides a protective effect on PCa progression in vivo. We show that AMPK activation induces PGC1α expression, leading to catabolic metabolic reprogramming of PCa cells. This catabolic state is characterized by increased mitochondrial gene expression, increased fatty acid oxidation, decreased lipogenic potential, decreased cell proliferation, and decreased cell invasiveness. Together, these changes inhibit PCa disease progression. Additionally, we identify a gene network involved in cell cycle regulation that is inhibited by AMPK activation. Strikingly, we show a correlation between this gene network and PGC1α gene expression in human PCa. Taken together, our findings support the use of AMPK activators for clinical treatment of PCa to improve patient outcome

CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells

AMPK activation protects against diet induced obesity through Ucp1-independent thermogenesis in subcutaneous white adipose tissue

Obesity results from a chronic imbalance between energy intake and energy output but remains difficult to prevent or treat in humans. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important regulator of energy homeostasis1,2,3 and is a molecular target of drugs used for the treatment of metabolic diseases, including obesity4,5. Here we show that mice expressing a gain-of-function AMPK mutant6 display a change in morphology of subcutaneous white adipocytes that is reminiscent of browning. However, despite a dramatic increase in mitochondrial content, Ucp1 expression is undetectable in these adipocytes. In response to a high-fat diet (HFD), expression of skeletal muscle–associated genes is induced in subcutaneous white adipocytes from the gain-of-function AMPK mutant mice. Chronic genetic AMPK activation results in protection against diet-induced obesity due to an increase in whole-body energy expenditure, most probably because of a substantial increase in the oxygen consumption rate of white adipose tissue. These results suggest that AMPK activation enriches, or leads to the emergence of, a population of subcutaneous white adipocytes that produce heat via Ucp1-independent uncoupling of adenosine triphosphate (ATP) production on a HFD. Our findings indicate that AMPK activation specifically in adipose tissue may have therapeutic potential for the treatment of obesity

CAR-T cell. the long and winding road to solid tumors

Adoptive cell therapy of solid tumors with reprogrammed T cells can be considered the "next generation" of cancer hallmarks. CAR-T cells fail to be as effective as in liquid tumors for the inability to reach and survive in the microenvironment surrounding the neoplastic foci. The intricate net of cross-interactions occurring between tumor components, stromal and immune cells leads to an ineffective anergic status favoring the evasion from the host's defenses. Our goal is hereby to trace the road imposed by solid tumors to CAR-T cells, highlighting pitfalls and strategies to be developed and refined to possibly overcome these hurdles