Biology subjects

Draper, B.

Publications and source records attributed to Draper, B..

4 recordsLinked to original sources

bioRxiv2024

Knockdown proteomics reveals USP7 as a regulator of cell-cell adhesion in colorectal cancer via AJUBA

Ubiquitin-specific protease 7 (USP7) is implicated in many cancers including colorectal cancer in which it regulates cellular pathways such as Wnt signalling and the P53-MDM2 pathway. With the discovery of small-molecule inhibitors, USP7 has also become a promising target for cancer therapy, and therefore systematically identifying USP7 deubiquitinase interaction partners and substrates has become an important goal. In this study, we selected a colorectal cancer cell model that is highly dependent on USP7 and in which USP7 knockdown significantly inhibited colorectal cancer cell viability, colony formation, and cell-cell adhesion. We then used inducible knockdown of USP7 followed by LC-MS/MS to quantify USP7 dependent proteins. We identified the Ajuba LIM domain protein as an interacting partner of USP7 through co-IP, its substantially reduced protein levels in response to USP7 knockdown, and its sensitivity to the specific USP7 inhibitor FT671. The Ajuba protein has been shown to have oncogenic functions in colorectal and other tumours, including regulation of cell-cell adhesion. We show that both knockdown of USP7 or Ajuba results in a substantial reduction of cell-cell adhesion, with concomitant effects on other proteins associated with adherens junctions. Our findings underlie the role of USP7 in colorectal cancer through its protein interaction networks and show that the Ajuba protein is a component of USP7 protein networks present in colorectal cancer.

systems biology↗

bioRxiv2024

Functional avidity of anti-B7H3 CAR-T constructs predicts antigen density thresholds for triggering effector function

Chimeric Antigen receptor T cell (CAR-T) treatments for solid cancers have been compromised by limited expansion and survival in the tumor microenvironment following interaction with antigen-expressing target cells. Using B7H3 as a model antigen with broad clinical applicability, we evaluated the relationship between the antibody/antigen affinity of three clinical candidate binders and the three following functional characteristics: functional avidity, prolonged cytotoxicity in tumoroid re-stimulation assays, and in vivo anti-tumoral responses. BEHAV3D video-microscopy assessed distinct CAR-T cell behaviors at single cell resolution. T cell exhaustion did not dictate effector function. Rather, we demonstrated a threshold avidity of CAR-T / tumor cell interaction, characterized by longer cumulative CD8+ CAR-T / tumor target interaction times, and required for adequate CAR-T cell expansion to result in sustained tumor control upon re-challenge. These results provide new insights into design of CAR-T cells for antigen-dim cell targeting, and avoidance of antigen-dim tumor relapse.

bioengineering↗

bioRxiv2022

OPS-γδ: allogeneic opsonin-secreting γδT cell immunotherapy for solid tumours mediates direct and bystander immunity

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous {beta}T cells. These approaches are limited by tonic signalling of synthetic constructs and costs associated with manufacture of bespoke patient products. {gamma}{delta}T cells are an emerging alternative chassis for cellular therapy, possessing innate anti-tumour activity, potent antibody-dependent cytotoxicity (ADCC) and minimal alloreactivity. We present an immunotherapeutic platform technology built around the V{gamma}9V{delta}2 {gamma}{delta}T cell chassis, harnessing specific characteristics of this cell type and offering an allo-compatible means of delivering cellular therapy that recruits bystander immunity. We engineered {gamma}{delta}T cells to secrete synthetic opsonins and stabilized IL15 (stIL15). Using GD2 as a model antigen we show how opsonin-secreting V{gamma}9V{delta}2 (OPS-{gamma}{delta}) have enhanced cytotoxicity and also confer this benefit on lymphoid and myeloid bystander cells. Reflecting the secreted nature of the engineered efficacy modules, the entire product rather than just the gene-modified fraction exhibited enhanced activation and cytotoxic profiles, superior persistence and proliferative capacity even upon repeated tumour challenge. Secretion of stIL15 abrogated the need for exogenous cytokine supplementation during expansion and further mediated functional licensing of bystander NK cells. Compared to unmodified {gamma}{delta}T cells, stIL15-OPS-{gamma}{delta} cells exhibited superior in-vivo control of subcutaneous tumour and persistence in the blood. stIL15-OPS-{gamma}{delta} cells were further efficacious in 3D patient-derived osteosarcoma models, where efficacy could be boosted with the addition of immunomodulatory aminobisphosphonate drug, zoledronic acid. Together the data identify stIL15-OPS-{gamma}{delta} cells as a novel allogeneic platform combining direct cytolysis with bystander activation to effect solid tumour control. One Sentence SummaryArmoured, opsonin-secreting OPS-{gamma}{delta} cell immunotherapy is built on the innate strengths of the V{gamma}9V{delta}2 cell chassis for allogeneic solid tumour targeting.

immunology↗

bioRxiv2019

The roles of Amh in zebrafish gonad development and sex determination

Fetal mammalian testes secrete Amh (Anti-Mullerian hormone), which inhibits female reproductive tract (Mullerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes PCOS (polycystic ovarian syndrome). Zebrafish (Danio rerio) lacks Mullerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh. To discover the roles of Amh in the absence of Mullerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated non-vitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-seq data partitioned 21-day post-fertilization (dpf) juveniles into two groups that each contained mutant and wild type fish. Group21-1 up-regulated ovary genes compared to Group21-2, which were likely developing as males. By 35dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly under-expressed granulosa and theca genes and testes under-expressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes.\n\nArticle SummaryAnti-Mullerian hormone (Amh) inhibits female reproductive duct development, signals oocyte reserve, and marks polycystic ovarian syndrome. Zebrafish lacks Mullerian ducts and the typical Amh receptor, questioning evolving roles of Amh. We made knockout mutations in zebrafish amh. Most mutants were female and the few males often had oocytes in their testes, showing that Amh promotes male development. Mutant reproductive ducts functioned, but testes were enormous and ovaries accumulated immature oocytes, showing that Amh regulates germ cell proliferation and maturation. Transcriptomics revealed that Amh controls development of steroid-producing gonad cells. Amh in zebrafish preserved ancestral roles despite losing Mullerian ducts and the Amh receptor.

genetics↗