A new pathway to cell death

When Thijn Brummelkamp, a cancer biologist at the Netherlands Cancer Institute, learned that cells use an alternative way to make triglycerides when they cannot do it through the usual pathway, he perked up. “We got fascinated by that and thought: There may be more than one pathway for other important things that cells do,” he said. Following that logic, his group investigated the classic cell death signal: p53. They knew that cells can still undergo apoptosis in its absence, but how they did it remained a mystery, until now.

In a study published in Science, Brummelkamp’s group reported that in cells with mutated p53, DNA damage stopped protein translation by stalling ribosomes, which caused immense stress in cells, and eventually, death (1). Their findings highlight a previously unknown mechanism of DNA damage-associated cell death, which could have implications for cancer treatment.

“How you get p53-independent apoptosis is a long-standing mystery,” said Katrin Karbstein, a biochemist at The Herbert Wertheim University of Florida Scripps Institute for Biomedical Innovation and Technology who was not involved with the study. “They've actually explained the mechanism using single cell translation analyses and ribosome profiling.”

Normally, ribosomes get stalled by things like bacterial toxins. In this case, the cell lets the ribosome stall on purpose in order to activate a signaling pathway [that ultimately leads to apoptosis].
– Thijn Brummelkamp, the Netherlands Cancer Institute

To examine how cells might die without p53, Brummelkamp’s group damaged the DNA of p53-mutated HAP1 cells using etoposide, cisplatin, and hydroxyurea. Before undergoing apoptosis, HAP1 cells had a global decrease in protein translation. Ribosome profiling revealed that ribosomes accumulated at the translation start site, specifically at rare leucine UUA codons, which stopped protein production.

To understand why this stalling happened, the researchers measured the levels of the tRNA that matched the UUA codon. They found that the amount of this tRNA decreased after DNA damage, causing ribosomes to stall. “Normally, ribosomes get stalled by things like bacterial toxins,” Brummelkamp said. “In this case, the cell lets the ribosome stall on purpose in order to activate a signaling pathway [that ultimately leads to apoptosis].”

A genetic screen of HAP1 cells treated with etoposide showed strong expression of Schlafen family member 11 (SLFN11), a tRNA-cutting enzyme that plays a critical role in stopping protein production when DNA is damaged. Etoposide-treated HAP1 cells deficient for SLFN11 had trouble turning translation off, had no ribosome stalling at UUA codons, and showed normal levels of the tRNA for UUA.

SLFN11 expression is linked to a positive chemotherapy response in cancer patients (2). In future work, Brummelkamp’s group plans to study when and where ribosome stalling happens in patients receiving cancer treatment. “We are also interested in knowing when SLFN11 gets inactivated in cancer, at what moment during tumorigenesis this happens, and how it contributes to the treatment response,” he said.