An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

The injury response to DNA damage in live tumor cells promotes antitumor immunity.

Although immune checkpoint blockade (ICB) has strong clinical benefit for treating some tumor types, it fails in others, indicating a need for additional modalities to enhance the ICB effect. Here, we identified one such modality by using DNA damage to create a live, injured tumor cell adjuvant. Using an optimized ex vivo coculture system, we found that treating tumor cells with specific concentrations of etoposide, mitoxantrone, or doxorubicin markedly enhanced dendritic cell­mediated T cell activation. These immune-enhancing effects of DNA damage did not correlate with immunogenic cell death markers or with the extent of apoptosis or necroptosis; instead, these effects were mediated by live injured cells with activation of the DNA-PK, ATR, NF-κB, p38 MAPK, and RIPK1 signaling pathways. In mice, intratumoral injection of ex vivo etoposide­treated tumor cells in combination with systemic ICB (by anti-PD-1 and anti-CTLA4 antibodies) increased the number of intratumoral CD103+ dendritic cells and circulating tumor-antigen­specific CD8+ T cells, decreased tumor growth, and improved survival. These effects were absent in Batf3−/− mice and in mice in which the DNA-damaging drug was injected directly into the tumor, due to DNA damage in the immune cells. The combination treatment induced complete tumor regression in a subset of mice that were then able to reject tumor rechallenge, indicating that the injured cell adjuvant treatment induced durable antitumor immunological memory. These results provide a strategy for enhancing the efficacy of immune checkpoint inhibition in tumor types that do not respond to this treatment modality by itself.

Transite: A Computational Motif-Based Analysis Platform That Identifies RNA-Binding Proteins Modulating Changes in Gene Expression.

RNA-binding proteins (RBPs) play critical roles in regulating gene expression by modulating splicing, RNA stability, and protein translation. Stimulus-induced alterations in RBP function contribute to global changes in gene expression, but identifying which RBPs are responsible for the observed changes remains an unmet need. Here, we present Transite, a computational approach that systematically infers RBPs influencing gene expression through changes in RNA stability and degradation. As a proof of principle, we apply Transite to RNA expression data from human patients with non-small-cell lung cancer whose tumors were sampled at diagnosis or after recurrence following treatment with platinum-based chemotherapy. Transite implicates known RBP regulators of the DNA damage response and identifies hnRNPC as a new modulator of chemotherapeutic resistance, which we subsequently validated experimentally. Transite serves as a framework for the identification of RBPs that drive cell-state transitions and adds additional value to the vast collection of publicly available gene expression datasets.

The role of multi-purpose solutions in prevention and removal of lipid depositions on contact lenses.

The sorption and desorption of radiolabeled dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CH) were measured on 5 types of commercial contact lenses. The lenses were soaked in vitro in an artificial tear fluid for 16h. The effects of borate buffered saline and two commercial multi-purpose lens-care solutions (MPSs) on reducing the lipid (DPPC and CH) sorption and increasing the lipid removal were examined. The results showed that silicone hydrogel (SiHy) lenses accumulated the most lipids, sorbing over an order of magnitude more than polymacon, a conventional hydrogel lens. Pre-soaking the SiHy lenses for 16h in MPSs reduced the DPPC sorption by up to 13% and the CH sorption by up to 11%, compared to controls that were not pre-soaked. However neither these reductions nor those on polymacon were statistically significant (p>0.05). In sorption experiments without presoaking, subsequent exposure to the MPSs removed some DPPC from the lenses (0-3.1% for SiHy lenses and 14-55% for polymacon), but CH removal was 0.0-0.8% for SiHy lenses and 0.6-28% for polymacon lenses. Some of these removals were statistically significant (p<0.05).

IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

Quantitation of cholesterol and phospholipid sorption on silicone hydrogel contact lenses.

The introduction of silicone hydrogel (SiHy) contact lenses to the consumer marketplace necessitates study of the susceptibility of these lenses to spontaneous deposition by hydrophobic lipid components of ocular tears. The use of radioisotopes to measure lipid sorption on SiHy contact lenses gives precise and accurate results but requires institutional infrastructure and compels efficient lipid removal from the lens. This study compares three methods of quantitating phospholipid and cholesterol sorption on SiHy lenses using radiolabeled cholesterol and phosphatidylcholine that were sorbed on lenses from an artificial tear fluid. A triple extraction technique using n-propanol gives the most reliable results. Comparison of sorption on SiHy lenses shows that balafilcon A and senofilcon A lenses sorb similar amounts, while lotrafilcon B lenses sorb comparatively less.