DNA damage induces p53-independent apoptosis through ribosome stalling.

In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response.

BACKGROUND: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies. METHODS: We optimized drug screen methods on 5-11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient's response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness. RESULTS: Drug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003). CONCLUSIONS: Our study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.

External Validation of the Colon Life Nomogram for Predicting 12-Week Mortality in Dutch Metastatic Colorectal Cancer Patients Treated with Trifluridine/Tipiracil in Daily Practice.

BACKGROUND: Predicting prognosis in refractory metastatic colorectal cancer (mCRC) patients is needed to guide decision making. The Colon Life nomogram was developed to predict 12-week mortality in refractory mCRC patients. The aim of this study is to validate the Colon Life nomogram in last line/refractory patients receiving trifluridine/tipiracil (FTD/TPI) in daily practice. METHODS: The validation cohort consists of 150 QUALITAS study patients, an observational substudy of the Prospective Dutch CRC cohort, who were treated with FTD/TPI between 2016 and 2019. Model performance was assessed on discrimination, calibration, and clinical usefulness. The additional prognostic value of baseline quality of life (QoL) and thymidine kinase (TK1) expression in tissue was explored. RESULTS: Of the 150 patients, 25 (16.7%) died within 12 weeks of starting FTD/TPI treatment. The C-statistic was 0.63 (95% C.I. 0.56-0.70). The observed/expected ratio was 0.52 (0.37-0.73). The calibration intercept and slope were -1.06 (-1.53 to -0.58) and 0.41 (0.01-0.81), respectively, which indicated overestimation of 12-week mortality by the nomogram. Decision curve analysis showed the nomogram did not yield a positive net benefit at clinically meaningful thresholds for predicted 12-week mortality. Addition of QoL to the nomogram improved the C-statistic to 0.85 (0.81-0.89). TK1 expression was associated with progression-free survival but not with overall survival. CONCLUSION: We demonstrated evident miscalibration of the Colon Life nomogram upon external validation, which hampers its use in clinical practice. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model including QoL.