Genetic dependencies associated with transcription factor activities in human cancer cell lines.

Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology.

Confounding Factors in Targeted Degradation of Short-Lived Proteins.

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

Exploration of cell-free DNA (cfDNA) recovery for touch deposits.

Although touch deposit DNA is widely used in forensic casework, its cellular and acellular contents and their biological origins are poorly understood. There is evidence that the cell-free component of DNA deposited by handling may contribute substantial genetic information; however, most research into touch DNA recovery does not separate cellular and cell-free fractions or seek to characterize their contents. This work is an important early step in developing methods to isolate the cfDNA from biological material deposited by handling. Size-filtration as a separation technique was determined to be prone to DNA loss, even on optimized control samples of pure ladder DNA. Centrifugal separation was optimized to determine minimum speed and time required to reliably remove all cellular debris from the material collected by rinsing donor hands. To determine if the centrifugal force risked rupturing shed corneocyte cells and releasing cellular DNA into the supernatant, DNA levels were measured, and cells were visualized microscopically before and after centrifugation of hand rinses. Heated buccal cells were used as a positive control to demonstrate cell rupture would be detected with these methods. Following the determination of a suitable separation technique, an investigation into purification methods for cfDNA was conducted. DNA recovery using three kits for plasma cfDNA, one for PCR clean-up and one for genomic DNA were assessed on both ladder DNA to simulate cfDNA fragments and on collected hand deposit supernatants from both unwashed and washed hands. Purification methods designed for recovery of short DNA fragments from plasma yielded the highest recovery percentage across sample types, with BioChain cfPure performing the best. Donors' hands were shown to shed high levels of cfDNA, which were better recovered with a method for short fragments than with a traditional genomic technique often used on touch DNA samples.