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SummaryCancers arise through the acquisition of oncogenic mutations and grow through clonal expansion1, 2. Here we reveal that most mutagenic DNA lesions are not resolved as mutations within a single cell-cycle. Instead, DNA lesions segregate unrepaired into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterise this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can generate multiple alternative alleles in successive cell divisions, thereby increasing both multi-allelic and combinatorial genetic diversity. The phasing of lesions enables the accurate measurement of strand biased repair processes, the quantification of oncogenic selection, and the fine mapping of sister chromatid exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

Wnt/β-catenin and Notch signalling cooperate in regulating the transcription of various genes specifically in the small intestinal stem and progenitor cell compartments. We characterized Bmi1 functionally in this context and showed that it contributes to ISC self-renewal capacity. We postulated that it does so by regulating its classical locus Cdkn2a and probably also by supporting DNA damage repair. Yet another level of Notch and Wnt/β-catenin crosstalk was found in colorectal cancer where tumour-associated β-catenin induced Jagged 1 (Jag1) transcription, thus leading to Notch activation. We also investigated which is the contribution of intestinal epithelial Jag1-mediated Notch activation on tumour initiating activity. We found that intestinal-specific deletion of Jag1 greatly decreases tumour formation in the ApcMin/+ background, likely due to reduced stemness. Jag1 deletion in preformed spheroids abrogates stemness-related gene expression and proliferation leading to spheroid failure. Jag1 is dispensable for normal stem cells, which rely on Dll1/4 Notch ligands for their maintenance. Together, these results open a new path in personalised CRC therapy, presumably involving Notch inhibition from specific ligands. Las vías de señalización de Wnt/β-catenina y de Notch cooperan en la regulación transcripcional de varios genes específicamente en las células madre / progenitoras del epitelio intestinal. Hemos caracterizado la funcionalidad de Bmi1 en este contexto y demostrado que contribuye a la capacidad de auto-renovación de las células madre intestinales. Postulamos que lleva a cabo esta función mediante la regulación de su diana clásica, Cdkn2a, pero probablemente también llevando a cabo funciones alternativas ayudando a la reparación del daño en el ADN. Sin embargo, existe otro nivel de cooperación entre las vías de señalización de Wnt/β-catenina y de Notch en el contexto del cáncer colorectal. Aquí, la β-catenina asociada al tumor es capaz de inducir la transcripción de Jagged1 (Jag1), resultando en la activación de la vía de Notch. También hemos investigado cuál es la contribución a la iniciación tumoral de la activación de Notch mediada por Jag1 epitelial. Encontramos que delecionando Jag1 específicamente en el epitelio intestinal se reducía la formación tumoral en el modelo animal ApcMin/+, probablemente debido a una pérdida de las características de célula madre. La deleción de Jag1 en esferoides previamente formados abroga la expresión de genes de célula madre y la proliferación, llevando al colapso de los esferoides. Jag1 es dispensable para las células madre normales, que dependen de los ligandos de Notch Dll1/4 para su supervivencia. En conjunto, estos resultados abren un nuevo camino a las terapias personalizadas en el tratamiento del cáncer colorectal, presumiblemente mediante la inhibición de Notch a partir de ligandos específicos.

Additional file 3. Additional methods. Some of the filtering steps have been extended for clarification in this file.

Additional file 4. Review history.

Additional file 2. Additional figures. This file presents all supplementary figures referenced in the main text.

Additional file 2. Additional figures. This file presents all supplementary figures referenced in the main text.

Additional file 3. Additional methods. Some of the filtering steps have been extended for clarification in this file.

Additional file 4. Review history.

Cancers arise through the acquisition of oncogenic mutations and grow through clonal expansion. Here we reveal that most mutagenic DNA lesions are not resolved as mutations within a single cell-cycle. Instead, DNA lesions segregate unrepaired into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterise this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity. The phasing of lesions enables the accurate measurement of strand biased repair processes, quantification of oncogenic selection, and fine mapping of sister chromatid exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.