Dissecting the early steps of MLL induced leukaemogenic transformation using a mouse model of AML

Silvia Basilico,Berthold Göttgens,Konstantinos Tzelepis,Kim Wong,George Giotopoulos,David J Adams,Alison Kennedy,Xiaonan Wang,Larissa S Carnevalli,Sarah J Kinston,Brian J P Huntly,Fernando J Calero-Nieto,George S Vassiliou,Pedro M Quiros

doi:10.1038/s41467-020-15220-0

Abstract

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.

Highlights

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells
Cells were transduced with either MSCV-MLLENL-GFP or control MSCV-GFP and were serially re-plated in methylcellulose (CFU) in the absence of Flt3L and β-estradiol but in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF) and erythropoietin (EPO)
Our results show that MLL-ENL can induce a leukaemic transcriptional program in Hoxb8-FL cells, but only if the cells are taken out of their Flt3L and β-estradiol self-renewal culture condition, reminiscent of previous studies showing that MLLENL did not induce acute myeloid leukaemia (AML) in mice when transduced into highly purified HSCs9 and MLL-AF9 did not cause AML in either HSCs or CMPs when myeloid differentiation was compromised by C/ EBPα deletion[29]

Summary

Results

Development of a clonal mouse model of MLL-ENL-driven AML. Highly purified HSPC populations are recognised to be heterogeneous[10,17]. Our results show that MLL-ENL can induce a leukaemic transcriptional program in Hoxb8-FL cells, but only if the cells are taken out of their Flt3L and β-estradiol self-renewal culture condition, reminiscent of previous studies showing that MLLENL did not induce AML in mice when transduced into highly purified HSCs9 and MLL-AF9 did not cause AML in either HSCs or CMPs when myeloid differentiation was compromised by C/ EBPα deletion[29]. Having identified that early molecular changes foreshadow the MLL-ENL preleukaemic transcriptional program, we explored whether any of these early events represent genetic vulnerabilities associated with MLL-ENL expression by performing a genome-wide CRISPR-Cas[9] drop-out screen in both the Parental (Flt3L and β-estradiol dependent) as well as the METransformed cells cultured in IL-3 (Fig. 5a). The genome-wide screen, performed using at least two biological replicates per cell line, was analysed using MaGECK44 and this revealed 465, 1624 and 1798 depleted genes (FDR < 0.25)

D7 D4 D7 GM-CSF Flt3L

Mitotic cell cycle

Discussion

Methods

Code availability