Introduction: MLL rearranged leukemias are associated with a poor prognosis and response to conventional therapies is limited. Therefore, identification of new targets resulting in the development of novel compounds are urgently needed. However, the lack of authentic model systems due to retroviral MLL fusion protein overexpression and usage of pure mouse models that do not mimic the nature of the original human disease compromise efficient pharmacological studies. We generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system a human MLL-rearranged (MLLr) leukemia model derived from umbilical cord blood cells, that is based on patient specific complete translocations of the MLL, AF4 or AF9 genes and shares phenotypical, morphological and molecular features of patient leukemic cells. Thus, it allows us to reproduce downstream effects caused by MLL fusion proteins on patient-like pathologic levels. Gene set enrichment analysis (GSEA) and expression analysis identified Myc as a critical target in our indefinite in vitro growing human MLLr model. Here, we report the impact of the indirect Myc inhibitor JQ1 on cell proliferation and apoptosis in our authentic human MLLr leukemia model.

Methods: We used CRISPR/Cas9 to induce MLL-AF4 and -AF9 translocations in primary hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood. MLLr cells were characterized via RNA sequencing, compared to primary patient cells and subjected to GSEA and differential gene expression analysis. MLLr cells were used to analyze the consequences of the Myc inhibitor JQ1 on proliferation and apoptosis determined by flow cytometry.

Results: Patient-specific MLL-AF4 and -AF9 cells were generated with a translocation efficiency of 100% after a cell culture period of 30 days. In liquid culture MLLr cells displayed a distinct survival advantage with indefinite growth potential whereas control cells (HSPCs nucleofected with Cas9 alone) terminally differentiated and eventually died out after 60 days of culture. Our human MLLr model based on complete translocations of the MLL and AF4 or AF9 genes, respectively, shared molecular oncogenic features of patient leukemic cells determined by RNA sequencing: Analysis of differentially expressed genes revealed a typical MLLr leukemia signature, with upregulation of HOXA cluster and downregulation of HOXB cluster. Interestingly, further analysis of the impact of MLL fusion protein on transcriptional programs revealed Myc as a highly important target. Strikingly, treatment with the indirect Myc inhibitor JQ1 resulted in a dose-dependent reduced proliferation (50 nM: 61-65%) and induction of apoptosis (100 nM: 23-46%) of MLLr cells whereas control cells were not affected.

Conclusions: Our genome editing approach to generate indefinite growing MLL-AF4 and -AF9 translocated cells faithfully models MLLr leukemia and provides an experimental platform to identify molecular targets and testing of new therapies. Myc inhibitor JQ1 shows robust antiproliferative capacity and promotes cell death serving as a potential new therapeutic target for the treatment of MLLr leukemia.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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