Understanding the cellular and molecular mechanisms of EVI1-expressing KMT2A-rearranged acute myeloid leukaemia

The translocation t(9;11)(p22;q23), leading to the leukaemogenic hybrid gene and fusion protein KMT2A-MLLT3, is one of the most common KMT2A-rearrangements (KMT2A-r) and is associated with acute myeloid leukaemia (AML). In some KMT2A-r AML the tumour cells express the stem cell gene EVI1 which has been proposed to be a marker of poor outcome. By characterisation of an inducible iKMT2A-MLLT3 transgenic mouse line, our group previously modelled aggressive AML with aberrant Evi1 expression1, however the underlying cellular and molecular mechanisms remained unclear. To address this point, we crossed the iKMT2A-MLLT3 line with a Evi1-IRES-GFP reporter line to trace Evi1 expression. The consequence of aberrant Evi1 expression in KMT2A-MLLT3 AML was investigated in colony formation- and flow cytometric assays, transplantations as well as high-resolution imaging experiments. To obtain molecular-mechanistic insights, we performed single-cell and bulk RNA-sequencing experiments of enriched haematopoietic stem and progenitor cells (HSPC). Ongoing efforts complement these with chromatin conformation- and immunoprecipitation experiments.
We found that about 30% of the quiescent long-term haematopoietic stem cells (LT-HSC) as well as the multipotent progenitors (MPP1) express abundant Evi1 (“Evi1high”) levels. Evi1highKMT2A-MLLT3+ bone marrow (BM) cells produced invasive colonies in vitro, and high Evi1 expression accelerated MPP1- but not LT-HSC-derived AML progression in vivo. Interestingly, a single injection of the haematopoietic growth factor thrombopoietin (mTPO) doubled the Evi1+ cell fraction and particularly increased cycling of Evi1+LT-HSC. Notably, mTPO-treatment resulted in significant acceleration of KMT2A-MLLT3-driven AML development upon transplantation of HSPC into irradiated recipients. Gene expression signatures of emerging AML cells were characterized by up-regulation of “TPO-signalling” and pathways downstream of the mTPO-receptor called Mpl. To understand the early effects of mTPO on HSPC, we performed multiplexed scRNA-sequencing on purified HSC, which revealed differentially expressed genes linked to invasive disease. To explore the relevance of these observations for the human disease, we interrogated several public databases indicating that EVI1+KMT2A-r AML patients express putative EVI1 targets but also MPL suggesting a similar origin. Knockdown and pharmacological intervention studies will show whether this origin-dependent epigenetic footprint of Evi1+KMT2A-MLLT3 AML can be exploited to develop more selective therapies for this rare aggressive cancer.
Additionally, we generated embryonic stem (ES) cells for two new lines. The iSII-KMT2A-MLLT3-P2A-dtTomato line will allow tracing of the fusion expression and molecular analysis by pull-down of the SII-tagged fusion protein from chromatin. The iRBM15-MKL1 line will allow the dissection of this rare AMKL disease’s mechanism.