Page 135 - Haematologica May 2020

MLL-rearranged AML requires MN1
to be co-operating partner of several oncogenic fusion genes (NUP98–HOXD13,18 CALM–AF10,19 MLL–AF96 and MLL–ENL)20 and mutated RUNX1,21 and as a common tar- get of insertional mutagenesis in a hematopoietic stem cell (HSC) gene therapy trial,22 thereby promoting leukemogen- esis.
Interestingly, MN1-induced AML is also dependent on Hoxa cluster genes and Meis1.23 Multipotent progenitor cells (MPP) and common myeloid progenitors (CMP) have been identified as the cell of origin in MN1-induced AML, while granulocyte-macrophage progenitors (GMP) cannot be transformed.23 We found that the differential expression of Hoxa9, Hoxa10 and Meis1 in MPP/CMP compared to GMP cells was responsible for the ability of MN1 to trans- form the more immature, but not the more mature, progen- itor cells.23 One important difference between MN1 and MLL-r leukemia is that MN1 cannot activate Hox gene expression by itself, while MLL-AF9 can.23,24 Therefore, MN1 is unable to transform GMP cells, while MLL-AF9 can transform myeloid progenitor cells down to the differentia- tion state of a GMP. Both MLL-AF9- and MN1-induced leukemias depend on the H3K79 methyltransferase DOT1L.14,25, 26 In addition, deletion of Mll and Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes, and impaired the leukemogenic activity of MN1 in vivo.14 However, it is not known whether MLL-AF9 transformed cells also depend on MN1.
High MN1 expression confers resistance to all-trans retinoic acid (ATRA)-induced differentiation and chemotherapy-induced cytotoxicity.7,27 Recent studies have shown that pyrimethamine [a dihydrofolate reductase (DHFR) inhibitor] and DOT1L inhibitors possess anti- leukemic effects in MN1hi AML cells.14,27 However, the mechanism of MN1-induced AML and drug resistance is still not completely understood due to its little structural/functional similarity to any other protein.14 Mn1 null mice have severe defects in bones of the cranial skele- ton, yet the effects of its deletion in hematopoiesis/leukemia are not known.28 Here, we show that CRISPR–Cas9-mediated deletion of MN1 in MLL-r leukemias, and consequently treatment of MLL-r leukemias with an anti-MN1 siRNA, led to strong anti-leukemic effects, including increased terminal myeloid differentiation and suppression of leukemic growth in vitro and in vivo.
Methods
Viral vectors, vector production, and CRISPR
Briefly, helper-free recombinant retrovirus harvested from super- natants of the transfected ecotropic Phoenix packaging cell line was used to transduce 5-fluoro-uracil treated mouse bone marrow cells to generate an immortalized MLL-AF9 cell line, as described before.15,29 CRISPR lentiviral vector L40C-CRISPR.EFS.dTomato (Addgene # 89392) was kindly provided by Dr. Dirk Heckl, Hannover Medical School, Hannover, Germany.30 CRISPR-Cas9 target sites in the Mn1/MN1 gene were selected using the CCTop selection tool.31 Single guide RNA (sgRNA) were cloned in the CRISPR-Cas9 vector. The list of sgRNA is summarized in Online Supplementary Table S1.
Cell lines and primary cells
Bone marrow from healthy donors was obtained from the transplantation unit of Hannover Medical School. Mononuclear
cells from peripheral blood or bone marrow were isolated by den- sity gradient centrifugation. CD34+ cells were isolated using the CD34 microbead kit (Miltenyi Biotech, Bergisch Gladbach, Germany), as per manufacturer’s protocol. AML patient samples were taken from the AML-myelodysplastic syndromes (AML- MDS) repository of Hannover Medical School. Written informed consent was obtained for the use of patient samples in accordance with the Declaration of Helsinki, and the study was approved by the institutional review board of Hannover Medical School (ethi- cal vote 1187-2011).
Mouse transplantation and homing assay
NOD-SCID and NOD/SCID/IL2rgnull (NSG) mice were bred and maintained in pathogen-free conditions in the animal labora- tory of Hannover Medical School, Hannover, Germany. All animal experiments were approved by the Lower Saxony state office for consumer protection, Oldenburg, Germany.
siRNA and lipid nanoparticles
All siRNAs (MN1 and control/AHA1) were purchased from Axolabs GmbH (Kulmbach, Germany). siRNAs were packaged in lipid nanoparticles (LNP), as described previously.32
Gene expression and chromatin immunoprecipitation sequencing analysis
We performed gene expression profiling of in vitro cultured MLL-AF9/Mn1wt versus MLL-AF9/Mn1null cells in triplicate. RNA was extracted using the standard trizol method and was fur- ther used for gene expression profiling. Gene expression profiling using extracted RNA from MLL-AF9/MNn1wt and MLL- AF9/MNn1null cells was performed on Affymetrix GeneChip Mouse 430 2.0 arrays (43,000 probes). The whole dataset can be found at GEO (GSE130631) for public access.
Chromatin immunoprecipitation sequencing (Chip-Seq) DNA binding data were taken for H3K79me2 from GSE55038,33 MLL- AF9 from GSE29130,25 Hoxa9 from GSE33518,34 and MN1 and MEIS1 from our previous publication.23
Statistical analysis
Pairwise comparisons were performed by Student t-test for con- tinuous variables. Two-sided significance was set at P<0.05. Comparison of survival curves were performed using the log rank test. Statistical analyses were performed with Microsoft Excel (Microsoft, Munich, Germany) and GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
More detailed information on the materials and methods used can be found in the Online Supplementary Appendix.
Results
MLL-AF9 cell proliferation depends on Meningioma 1
To study the effects of MN1 gene inactivation in leukemia cells, we generated CRISPR-Cas9 engineered MN1null murine and human leukemia cells and character- ized these cells in vitro and in vivo (Figure 1A). MN1 was deleted in murine cells transformed by MLL-AF9, HOXA9, HOXA9MEIS1, E2A-HLF and 10 human leukemia cell lines: THP-1, MV-4-11, NB4, OCI-AML2, OCI-AML3, U937, K562, Kasumi-1, HL-60 and HEL. Ninety-six to 288 transduced cells per cell line were single cell sorted and the outgrowing clones were evaluated for MN1 deletion by qualitative RT-PCR (qRT-PCR), western blot and sequencing (Online Supplementary Tables S2 and S3 and Online Supplementary Figure S1A-F). While up to
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