Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co‐culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue‐like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two‐dimensional (2D) substrates and review the popular approaches currently available that enable the development of three‐dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold‐free or scaffold‐based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Our study provides the first in vivo and in patient evidence supporting the role of CHD1 in DSB repair and in response to DNA damaging therapy. We uncover mechanistic insights that CHD1 modulates the choice between HR and NHEJ DSB repair and suggest that CHD1 loss may contribute to the genomic instability seen in this subset of PCas.

Welti et al; Targeting BET family proteins in CRPC 2 Statement of Translational RelevanceAdvanced prostate cancer invariably progresses to lethal castration resistant prostate cancer (CRPC). Resistance to current androgen receptor (AR) targeting therapies is associated with the development of AR aberrations including the constitutively active AR splice variant 7 (AR-V7). Currently, no clinically available therapies effectively inhibit aberrant AR signaling. BRD4, a bromodomain and extraterminal (BET) family protein, is a critical AR coregulator. We show that BRD4 expression associates with patient outcome and AR driven transcription in lethal prostate cancer. Moreover, BET inhibitors (BETi) reduce AR splicing and AR-V7 expression by regulating alternative splicing, abrogating AR signaling and inhibiting growth of CRPC patient derived models. Clinical studies with BETi in CRPC should pursue pharmacodynamics studies evaluating abrogation of AR splicing and persistent AR signaling to optimize the development of these drugs for the treatment of CRPC.Research. Experimental Design: We determined associations between BET expression, AR driven transcription and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer (PC) models.Results: Nuclear BRD4 protein expression increases significantly (p=<0.01) with castration resistance in same patient treatment naïve (median H-score; interquartile range: 100; 100-170) and CRPC (150; 110-200) biopsies, with higher expression at diagnosis associating with worse outcome (HR 3.25, 95% CI 1.50-7.01; p=<0.001). BRD2, BRD3 and BRD4 RNA expression in CRPC biopsies correlates with AR driven transcription (all p=<0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of PC cells and patient derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (p=<0.001) of a patient derived xenograft model of CRPC with AR amplification and AR-V7 expression. Conclusion:BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof of mechanism pharmacodynamic studies.