Page 144 - Haematologica May 2020

A. Sharma et al.
of various bones in the skull. In addition, MN1 KO mice apparently did not develop hematopoietic defects during the first few weeks of life until they died, suggesting that MN1 inhibition or deletion has a therapeutic window when targeted in leukemic cells.28 It has been previously reported that transient MN1 knockdown via siRNA in human leukemia cell lines reduces their proliferation;20 however, perpetual loss of MN1 expression has not been comprehensively studied in murine or human leukemias. Our study shows that CRISPR-Cas-mediated loss of MN1 substantially impairs leukemogenesis in MLL-r leukemias. MN1 loss prevents in vitro proliferation and colony-form- ing potential of MLL-AF9 and MLL-AF4-dependent cells. Correspondingly, we demonstrate that MN1 inactivation inhibits cell cycle, promotes apoptosis and induces differ- entiation in MLL-r leukemic cells. In addition, loss of MN1 significantly impairs MLL-AF9-mediated murine leukemo- genesis and reduces leukemic and tumor growth of human MLL-rearranged leukemias in vivo. Our results are supported by a previous study, which found that Mn1 co- operated with Mll-AF9 in leukemogenesis in an in vivo transplantation assay.6 The homing potential of MLL- AF9/Mn1null cells is not affected by Mn1 deletion. We also show that re-expression of MN1 in MLL- AF9/Mn1null cells restores the leukemic potential of MLL- AF9.
Both MN1 and MLL-AF9-induced leukemias rely on expression of the Hoxa9/Meis1 complex.2,25,33,35,37,38 On a genome-wide level chromatin binding of MN1 largely over-lapped with HOXA9 and MEIS1 binding, suggesting that the previously described function of MN1 as a co-fac- tor of HOXA9/MEIS1 is its major contribution to the leukemogenic activity of MLL-AF9, as illustrated in our study (Figure 7D). This is supported by our gene expres- sion data showing that HOXA9 target genes are primarily down-regulated upon MN1 deletion. Previous studies have found that both MLL-AF9 and MN1-induced leukemias are dependent on Dot1l.14,25,26 MLL fusions mis- direct DOT1L to the promoters of Hoxa cluster genes and Meis1 leading to H3K79 methylation and constitutive activation of these genes.2,25,33 It is important to note that MN1 transformed HSC have high expression of HOXA9 irrespective of DOT1L expression.14 In support of this observation, we show that loss of MN1 can overcome the effect of MLL-AF9 and DOT1L-directed dysregulation of HOXA cluster genes and MEIS1.
The leukemogenicity of MLL-positive leukemias is dependent on genes such as the Mll wild-type allele, AF9, Dot1l, JMJD1C, PU.1, CBX8, Hoxa7, Hoxa9 and S6K1, which mediate the leukemogenic activity of MLL-AF9
predominantly by Dot1l gene activation.26,33,37-41 Our data provide additional insights into MLL-rearranged leukemia through the addition of MN1 as a crucial co-factor regulat- ing the HOXA cluster, MEIS1 and their target genes. As U937 cells were moderately affected by MN1 deletion, we speculate that cells with high HOXA9/MEIS1 expression may also depend on MN1 like MLL-r cells. Therefore, we predict that MN1 is not only required for MLL-trans- formed leukemias, but for approximately 50% of AML cases, which show dysregulated expression of the clus- tered homeobox genes.
High MN1 expression has been reported as a poor prog- nostic marker in AML patients, and its overexpression induces ATRA resistance and AML in mouse cells.7-9,12, 13,23Also, it is most highly expressed in CMP, and scantily expressed in HSC and more differentiated cells.23 Interestingly, induced MN1 expression cannot transform stem cells, as previously described,23 and we hereby report that proliferation of CD34+ cells remain unaffected by depletion of MN1 expression. Hence, the expression char- acteristics of MN1 seem to be favorable for therapeutic targeting. A potential therapeutic index is indicated by lack of an inhibitory effect in normal stem/progenitor cells and a specific inhibitory effect in AML cells. Therefore, targeting MN1 by LNP/siRNA formulations holds a strong potential therapeutic avenue, as suggested by our previous proof-of-principle study in a PDX model of acute lym- phoblastic leukemia in vivo.32
In summary, we identify MN1 as an essential protein in MLL fusion gene leukemia in vivo and highlight its impor- tance as a co-factor of the HOXA9/MEIS1 complex. An LNP/siRNA formulation against MN1 effectively inhibits the colony-forming potential of MLL-r primary AML cells, suggesting that MN1 is a valid and druggable target in AML.
Acknowledgments
We thank Silke Glowotz, Annett Reinsch, Basem Othman, Kerstin Goerlich, Martin Wichmann, Nadine Kattre and Renate Schottmann for technical help. We also thank Dr. Matthias Ballmaier and the staff of the Cell Sorting Core Facility of Hannover Medical School for their excellent service (supported in part by the Braukmann-Wittenberg-Herz-Stiftung).
Funding
This study was supported by the Rudolf- Bartling Stiftung, an ERC grant under the European Union’s Horizon 2020 research and innovation program (n. 638035), Deutsche Krebshilfe (70112697) and DFG grants HE 5240/5-1, HE 5240/5-2, HE 5240/6-1 and HE 5240/6-2.
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