Blood First Edition Paper, prepublished online March 4, 2015; DOI 10.1182/blood-2014-09-599258
MEIS1 Regulates an HLF-Oxidative Stress Axis in MLL-fusion Gene Leukemia
Short title: Meis1 regulates oxidative stress in leukemia via HLF
Jayeeta Roychoudhury1, Jason P. Clark1, Gabriel Gracia-Maldonado1, Zeenath Unnisa1†, Mark
Wunderlich2, Kevin A. Link2, Nupur Dasgupta3, Bruce Aronow4, Gang Huang2, James C. Mulloy2, Ashish R. Kumar1
1Division of BM Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA
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2Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital文章结构
Medical Center, Cincinnati, OH, 45229, USA
3Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH,
45229, USA
童话故事的特点4Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati,
OH, 45229, USA
†Current address: University of Rochester Medical Center, Rochester, NY, 14642, USA乒乓球技巧
*Correspondence:
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Ashish Kumar
Cincinnati Children’s Hospital Medical Center,
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3333 Burnet Ave,老实人是什么意思
Cincinnati, OH 45229
ashish.kumar@cchmc
Abstract
Leukemias with MLL-translocations are often found in infants and are associated with poor outcome
s. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia however remain elusive. Here, we ud inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL-leukemia. We demonstrate that Meis1 is esntial for maintenance of established leukemia. Additionally, in both the murine model and human leukemia cells, we found that Meis1-loss led to incread oxidative stress, oxygen flux and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reverd the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. The results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia.
Key points
•Meis1 is required for the maintenance of MLL-fusion gene leukemia. HLF is a key downstream mediator of Meis1.
•Meis1 and HLF regulate restrict oxidative stress. Induction of oxidative phosphorylation may be therapeutic in leukemia.
Introduction
Reciprocal translocations of the 11q23 locus lead to acute leukemias of both myeloid and lymphoid lineages. The leukemias are usually resistant to conventional chemotherapies. The translocation generates an oncogenic fusion protein comprid of an amino terminus derived from MLL (now called KMT2A), the gene at 11q23, fud to a carboxy terminus derived from one of veral different genes. Recent studies have revealed that the MLL-fusion proteins then join higher order protein complexes containing various transcriptional activators and/or elongating factors such as DOT1L, PAFc and p-TEFB 1,2. Recruitment of the complexes to genomic loci was shown to be associated with chromatin modifications and subquent changes in gene expression 3. Regardless of the fusion partner involved in the translocation and of the conquential protein-complex asmbled, the downstream genes activated by the various MLL-fusion proteins are largely identical. Data from patient-derived leukemia cells and from experimental models reveal that MLL-fusion proteins upregulate the expression of posterior HOX-A genes and of the HOX cofactor MEIS14-6. Further, retroviral overexpression of a Hox gene (with few exceptions) along with Meis1 in murine hematopoietic cells induces an aggressive leukemia in transplanted mice 7,8. Thus, MLL-fusion protein induced upregulation of HOX and MEIS1 genes is considered central to the pathology of the leukemias.
Studies to test the requirement of HOX-A and MEIS1 genes in MLL-fusion leukemias have yielded mixed results. While shRNA knockdown of HOXA9 was shown to inhibit leukemia, mice lacking Hoxa9 protein developed leukemia induced by MLL-fusion proteins at the same rate and frequency as tho mice bearing normal Hoxa9 9,10. The discrepancies could be due to technical differences in the experimental models. Recent studies using larger patient cohorts
however show that some MLL-fusion gene leukemias lack expression of all the HOX genes 11,12. On the other hand, MEIS1 is universally expresd in all MLL-fusion leukemias, including tho lacking HOX expression. Mice lacking Meis1 are embryonically lethal and Meis1-/- fetal liver cells were shown to be resistant to transformation by MLL-fusion proteins 13. This suggests that Meis1 is esntial for the initiation of leukemia. In order to be considered a therapeutic target however, an esntial role for MEIS1 in maintenance of established leukemia needs to be rigorously demonstrated. The mechanisms by which MEIS1 facilitates MLL-fusion induced leukemia are also unknown. Several previous studies aimed at determining the oncogenic mechanisms of Meis1 have been carried out, using retroviral expression of Hoxa9 and Meis1 or NUP98-HOXD13 and Meis1 in murine hematopoietic cells 7,13-16 . The results suggest that the Meis1/Hox complex induced leukemic transformation via intermediates such as Flt3, Trib2 and Ccl3. However, the cellular context
in which MEIS1 is found naturally overexpresd eg. MLL-fusion induced leukemia is quite different from that of the retroviral models. We recently reported on the role of Meis1 in normal hematopoietic stem cell function using inducible Meis1-knockout mice 17. In the current study, we ud the mice to investigate the role of Meis1 in leukemia. We found that Meis1 was critical for the maintenance of established leukemia and identified HLF as a downstream mediator of Meis1. More importantly, our studies expod a vulnerability in leukemia to oxidative stress that has potential for novel therapy.
Materials & Methods
Additional methods are included in the supplement.
Cell Lines
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Leukemia cell lines were cultured in Iscove’s modified Dulbecco’s medium (IMDM, GE Healthcare) supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin. For mou leukemic cells 10 ng/ml of SCF, IL-3, GM-CSF, IL-6 were added to the medium (PeproTech). 293T cells were cultured in DMEM supplemented with 10% FCS. OP9 stromal cells were maintained in α-Minimum Esntial medium supplemented with 20% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/
ml streptomycin (Sigma). For co-culture studies, OP9 cells were eded overnight and puromycin lected cells were added the next day. Patient samples
Deidentified patient samples were obtained under an IRB-approved protocol at Cincinnati Children’s Hospital Medical Center.
Animal studies
Transgenic Meis1f/f/CreER mice were generated by crossing Meis1f/f mice with Rosa26CreER mice 17. Meis1f/f/CreER mice were crosd with MLL-AF9+/- mice. All mice ud in this study were 6-8 weeks of age. All animal experiments ud were approved by the institutional animal care and u committee. For transplantation experiments, BoyJ mice were lethally irradiated and injected intravenously with up to 106 CD45.2+ leukemic cells mixed with 2.5 X 104 CD45.1+ mononuclear cells. Transplanted mice were treated with tamoxifen (Sigma) (4mg
