Human T-cell acute lymphoblastic leukemia (T-ALL) and B-cell chronic lymphoblastic leukemia (B-CLL) share an important feature: both malignancies can originate from aberrant immature precursor cells. Activating mutations in the NOTCH1 gene are observed in both human T-ALL (>50%) and B-CLL (10%). Several experimental models have established a link between NOTCH1 gain-of-function mutations and T-ALL development. Furthermore, acceleration of B-CLL disease is observed when Notch1 is mutated in a B-CLL-prone mouse model. However, the driver role of gain-of-function mutations that inactivate the PEST domain of Notch1, remains uncertain in both malignancies due to a lack of suitable experimental systems. Therefore, the present work explored the role of PEST mutations and the involvement of mutant hematopoietic stem and progenitor cells (HSPCs) in the process of leukemogenesis by performing CRISPR/Cas9/rAAV-mediated mutagenesis in the Notch1 gene of mouse HSPCs. To this end, insertion of a recurrent human PEST mutation in B-CLL (c.7544-7545, ∆CT) through homologous recombination was performed. Besides the intended PEST∆CT mutation, also unwanted genetic scars were present with high frequency in the PEST domain of HSPCs that were a result of non-homologous end joining (NHEJ) pathway. Approximately 6 months post HSPC transplantation into irradiated, immunodeficient recipient mice, monoclonal Notch1 PEST-mutated T-ALLs arose from HSPCs that carried genetic scars. B-CLL development from the targeted PEST∆CT-carrying HSPCs was not observed in this model. The T-ALLs acquired additional genomic aberrations (Chromosome 15 gain, mutations in Ikaros and Bcl11b and additional aberrations in Notch1). Hence, this work suggests that Notch1 PEST mutations can be the first oncogenic hit in mouse HSPCs that subsequently predispose developing thymocytes to a stepwise acquisition of additional mutations and thus, to malignant transformation. To avoid the introduction of unwanted genetic scars in the Notch1 locus, a Cas9D10A nickase mouse line was generated and the insertion of the PEST∆CT mutation was efficient in HSPCs while genetic scars were reduced to a minimum. This mouse line will serve as a useful tool to perform precise gene engineering to study a variety of malignancies.
