Custom scoring based on ecological topology of gut microbiota associated with cancer immunotherapy outcome.

The gut microbiota influences the clinical responses of cancer patients to immunecheckpoint inhibitors (ICIs). However, there is no consensus definition of detrimental dysbiosis. Based on metagenomics (MG) sequencing of 245 non-small cell lung cancer (NSCLC) patient feces, we constructed species-level co-abundance networks that were clustered into species-interacting groups (SIGs) correlating with overall survival. Thirty-seven and forty-five MG species (MGSs) were associated with resistance (SIG1) and response (SIG2) to ICIs, respectively. When combined with the quantification of Akkermansia species, this procedure allowed a person-based calculation of a topological score (TOPOSCORE) that was validated in an additional 254 NSCLC patients and in 216 genitourinary cancer patients. Finally, this TOPOSCORE was translated into a 21-bacterial probe set-based qPCR scoring that was validated in a prospective cohort of NSCLC patients as well as in colorectal and melanoma patients. This approach could represent a dynamic diagnosis tool for intestinal dysbiosis to guide personalized microbiota-centered interventions.

Stochastic transcription in the p53-mediated response to DNA damage is modulated by burst frequency.

Discontinuous transcription has been described for different mammalian cell lines and numerous promoters. However, our knowledge of how the activity of individual promoters is adjusted by dynamic signaling inputs from transcription factors is limited. To address this question, we characterized the activity of selected target genes that are regulated by pulsatile accumulation of the tumor suppressor p53 in response to ionizing radiation. We performed time-resolved measurements of gene expression at the single-cell level by smFISH and used the resulting data to inform a mathematical model of promoter activity. We found that p53 target promoters are regulated by frequency modulation of stochastic bursting and can be grouped along three archetypes of gene expression. The occurrence of these archetypes cannot solely be explained by nuclear p53 abundance or promoter binding of total p53. Instead, we provide evidence that the time-varying acetylation state of p53's C-terminal lysine residues is critical for gene-specific regulation of stochastic bursting.

The clinical impact of using complex molecular profiling strategies in routine oncology practice.

Molecular profiling and functional assessment of signalling pathways of advanced solid tumours are becoming increasingly available. However, their clinical utility in guiding patients' treatment remains unknown. Here, we assessed whether molecular profiling helps physicians in therapeutic decision making by analysing the molecular profiles of 1057 advanced cancer patient samples after failing at least one standard of care treatment using a combination of next-generation sequencing (NGS), immunohistochemistry (IHC) and other specific tests. The resulting information was interpreted and personalized treatments for each patient were suggested. Our data showed that NGS alone provided the oncologist with useful information in 10-50% of cases (depending on cancer type), whereas the addition of IHC/other tests increased extensively the usefulness of the information provided. Using internet surveys, we investigated how therapy recommendations influenced treatment choice of the oncologist. For patients who were still alive after the provision of the molecular information (76.8%), 60.4% of their oncologists followed report recommendations. Most treatment decisions (93.4%) were made based on the combination of NGS and IHC/other tests, and an approved drug- rather than clinical trial enrolment- was the main treatment choice. Most common reasons given by physicians to explain the non-adherence to recommendations were drug availability and cost, which remain barriers to personalised precision medicine. Finally, we observed that 27% of patients treated with the suggested therapies had an overall survival > 12 months. Our study demonstrates that the combination of NGS and IHC/other tests provides the most useful information in aiding treatment decisions by oncologists in routine clinical practice.

Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells.

Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.

Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage.

A functional DNA damage response is essential for maintaining genome integrity in the presence of DNA double-strand breaks. It is mainly coordinated by the kinases ATM, ATR, and DNA-PKcs, which control the repair of broken DNA strands and relay the damage signal to the tumor suppressor p53 to induce cell cycle arrest, apoptosis, or senescence. Although many functions of the individual kinases have been identified, it remains unclear how they act in concert to ensure faithful processing of the damage signal. Using specific inhibitors and quantitative analysis at the single-cell level, we systematically characterize the contribution of each kinase for regulating p53 activity. Our results reveal a new regulatory interplay in which loss of DNA-PKcs function leads to hyperactivation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence. This interplay determines the outcome of treatment regimens combining irradiation with DNA-PKcs inhibitors in a p53-dependent manner.

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells.

Levels of residual structure in disordered interaction domains determine in vitro binding affinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage. These results establish that residual structure is an important determinant of signaling fidelity in cells.

Differential protein occupancy profiling of the mRNA transcriptome.

BACKGROUND: RNA-binding proteins (RBPs) mediate mRNA biogenesis, translation and decay. We recently developed an approach to profile transcriptome-wide RBP contacts on polyadenylated transcripts by next-generation sequencing. A comparison of such profiles from different biological conditions has the power to unravel dynamic changes in protein-contacted cis-regulatory mRNA regions without a priori knowledge of the regulatory protein component. RESULTS: We compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells. Briefly, we developed a bioinformatics workflow to identify differential crosslinking sites in cDNA reads of 4-thiouridine crosslinked polyadenylated RNA samples. We identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells at an estimated false discovery rate of 10%. 73% of all reported differential protein-RNA contact sites cannot be explained by local changes in exon usage as indicated by complementary RNA-seq data. The majority of differentially crosslinked positions are located in 3' UTRs, show distinct secondary-structure characteristics and overlap with binding sites of known RBPs, such as ELAVL1. Importantly, mRNA transcripts with the most significant occupancy changes show elongated mRNA half-lives in MCF7 cells. CONCLUSIONS: We present a global comparison of protein occupancy profiles from different cell types, and provide evidence for altered mRNA metabolism as a result of differential protein-RNA contacts. Additionally, we introduce POPPI, a bioinformatics workflow for the analysis of protein occupancy profiling experiments. Our work demonstrates the value of protein occupancy profiling for assessing cis-regulatory RNA sequence space and its dynamics in growth, development and disease.