Abstract Detail

Abstract

A recent study by Boon et al. was published in Science and proposed a novel form of p53-independent apoptosis (1). Teir findings indicate that, in response to chemotherapy-induced DNA damage, SLFN11 depletes tRNAUUA, resulting in ribosome stalling at UUA codons. This process inhibits overall protein translation while ZAKα transmits a stress signal that activates caspase-3, initiating apoptosis. Generally, when cells undergo extensive DNA damage, p53 will be activated to induce apoptosis (2). This mechanism is exploited in radiotherapy and chemotherapy, which induce severe DNA damage in cancer cells through irradiation and drugs respectively to trigger p53-dependent apoptosis and eliminate cancer cells. Clinically, however, it has been observed that p53 mutations are present in approximately 50% of cancers (3), yet the same treatment strategies still induce cancer cell apoptosis, suggesting that cell death was caused by an alternative pathway that is p53-independent. First, Boon et al. employed CRISPR-Cas9 to knockout p53 in HAP1 and A549 cell lines, revealing that chemotherapeutic agents still induced apoptosis via caspase activation, even in the absence of functional p53. Their research confirmed a global decrease in protein synthesis alongside caspase-3 activation, which persisted despite caspase inhibition, indicating an independent mechanism of translation inhibition. Second, the authors observed that after treating cell lines with chemotherapeutic agents, there was a global reduction in protein synthesis, accompanied by activation of caspase-3. To exclude the possibility that this phenomenon was caused by caspase-3 itself, the authors co-treated the cells with a pan-caspase inhibitor and the chemotherapeutic agents. The results still showed a decrease in protein translation levels, indicating that the inhibition of protein translation occurs simultaneously with, but independently of, caspase-3 activation. Furthermore, by analyzing the screening results of ribosome profiling and verifying with qPCR, the authors confirmed that etoposide treatment led to a reduction in the abundance of tRNAUUA, causing a delay in translation due to the inability of tRNAUUA to bind promptly to the ribosome. Third, the authors conducted a genetic screening, focusing on genes with relatively high expression levels that influence tRNA transcription or degradation. The results identified tRNAse SLFN11 and the kinase GCN2 as key factors. A significant reduction in the abundance of tRNAUUA and a concomitant decrease in overall translation levels were linked to the overexpression of SLFN11 and GCN2 in cells. Subsequent experiments demonstrated that, in the absence of SLFN11, there was no significant decrease in tRNAUUA abundance, nor was there an inhibition of overall translation. On the other hand, in cells deficient in both GCN2 and SLFN11, DNA damage-induced phosphorylation of eIF2α was absent. Additionally, GCN2 deficiency did not affect etoposide-induced UUA stalling, despite the overall inhibition of translation. This finding is consistent with previous knowledge that GCN2 responds to amino acid starvation or ribosomal perturbations but does not directly respond to DNA damage. Consequently, it can be inferred that SLFN11 likely acts upstream of GCN2. In summary, significant DNA damage activates SLFN11 in cells, leading to a reduction in tRNAUUA abundance. This reduction induces ribosomal UUA stalling, which in turn triggers GCN2-dependent global translation inhibition. Finally, the authors investigated how the aforementioned events lead to caspase-3 activation and apoptosis. In their screen for genes responsive to ribosomal abnormalities during etoposide-induced caspase activation, they identified an enzyme sensing ribosomal stress toxicity, ZAKα. In their experiments, ZAKα's response to etoposide-induced apoptosis was highly dependent on SLFN11. However, the reduction in global translation and ribosomal stalling induced by SLFN11 did not depend on ZAKα, indicating that ZAKα acts downstream of SLFN11. Further review revealed that, in addition to tRNAse SLFN11 and kinase GCN2, some MAPK pathway members were also abnormally active. The authors then explored the relationship between ZAKα and these MAPK pathway components. It is known that ZAKα itself is an upstream component of the MAPK pathway, a finding that was confirmed through additional experiments. The authors found that ZAKα is essential for its substrate MAP2K4 and downstream signaling, leading to the activation of JNK and caspase-3, thereby inducing apoptosis. Thus, the core mechanism of p53-independent apoptosis was elucidated: SLFN11 responds to DNA damage by depleting tRNAUUA, directly causing ribosomal stalling at UUA codons. This results in global translation inhibition on one hand, while on the other hand, ZAKα senses the ribosomal stress and transmits this signal downstream to activate caspase-3, initiating apoptosis.

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