Only Hematopoietic Stem and Progenitor Cells from Cord Blood Are Susceptible to Malignant Transformation by MLL-AF4 Translocations

Abstract

Mixed lineage leukemia (MLL) (KMT2A) rearrangements (KMT2Ar) play a crucial role in leukemogenesis. Dependent on age, major differences exist regarding disease frequency, main fusion partners and prognosis. In infants, up to 80% of acute lymphoid leukemia (ALL) bear a MLL translocation and half of them are t(4;11), resulting in a poor prognosis. In contrast, in adults only 10% of acute myeloid leukemia (AML) bear t(9;11) with an intermediate prognosis. The reasons for these differences are poorly understood. Recently, we established an efficient CRISPR/Cas9-based KMT2Ar model in hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood (huCB) and faithfully mimicked the underlying biology of the disease. Here, we applied this model to HSPCs from adult bone marrow (huBM) to investigate the impact of the cell of origin and fusion partner on disease development. Both genome-edited infant and adult KMT2Ar cells showed monoclonal outgrowth with an immature morphology, myelomonocytic phenotype and elevated KMT2Ar target gene expression comparable to patient cells. Strikingly, all KMT2Ar cells presented with indefinite growth potential except for MLL-AF4 huBM cells ceasing proliferation after 80 days. We uncovered FFAR2, an epigenetic tumor suppressor, as potentially responsible for the inability of MLL-AF4 to immortalize adult cells under myeloid conditions.

Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia

“…Plotted are the mean ± SEM (n = 3 for negative controls, n = 2 for MEF2D sgRNAs). (E) Volcano plots of RNA-seq data in MA4- (left) or MA9- (right) transformed human CD34 + HSPCs versus normal HSPCs isolated from BM, where KMT2A translocation was induced via CRISPR-Cas9 ( Secker et al., 2020 ). (F) Competition-based proliferation assays in MA9-FLT3 ITD (left) or MA9-NRAS G12D (right) cells, which were generated by retroviral transduction of KMT2A-AF9 followed by FLT3 FLT3 ITD and NRAS G12D into human CD34 + HSPC cells, respectively ( Wei et al., 2008 ; Wunderlich et al., 2013 ) (n = 3, mean ± SEM).…”

Creation of Philadelphia chromosome by CRISPR/Cas9-mediated double cleavages on BCR and ABL1 genes as a model for initial event in leukemogenesis

“…45 Subsequently, EWSR::FLI1 and NPM1::ALK were generated by simultaneous cleavages at the target sites with the CRISPR/Cas9 system. [46][47][48][49] Similarly, leukemogenic MLL fusion genes including MLL::AF4, [50][51][52] MLL::AF9, 50,[53][54][55] and MLL::ENL 56 were generated by double cleavages using CRISPR/Cas9 in human hematopoietic cells and in the murine 32D myeloid progenitor cell line. For the development of incorrect ligation to take place between two DSBs, each DSB is supposed to be proximally located in the nucleus.…”

The high-performance technology CRISPR/Cas9 improves knowledge and management of acute myeloid leukemia

“…The genome-edited KMT2Ar cells obtained from infant and adult showed indefinite growth potential except for MLL-AF4 huBM cells whose proliferation stopped after 80 days. FFAR2 (an epigenetic tumor suppressor) could be responsible for the failure of MLL-AF4 to immortalize adult cells under myeloid conditions 11 .…”