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Down-Regulation of Homeobox Genes MEIS1 and HOXA in MLL-Rearranged Acute Leukemia Impairs Engraftment and Reduces Proliferation

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Down-Regulation of Homeobox Genes MEIS1 and HOXA in MLL-Rearranged Acute Leukemia Impairs Engraftment and Reduces Proliferation Down-regulation of homeobox genes MEIS1 and HOXA in MLL-rearranged acute leukemia impairs engraftment and reduces proliferation Kira Orlovskya, Alexander Kalinkovichb,1, Tanya Rozovskaiaa,1, Elias Shezenb, Tomer Itkinb, Hansjuerg Alderc, Hatice Gulcin Ozerd, Letizia Carramusaa, Abraham Avigdore, Stefano Voliniac, Arthur Buchbergf, Alex Mazog, Orit Kolletb, Corey Largmanh,2, Carlo M. Crocec,3, Tatsuya Nakamurac, Tsvee Lapidotb, and Eli Canaania,3 Departments of aMolecular Cell Biology and bImmunology, Weizmann Institute of Science, Rehovot, 76100 Israel; Departments of cMolecular Virology, Immunology and Medical Genetics and dBiomedical Informatics, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210; eHematology Division, Sheba Medical Center, Tel-Hashomer, 52621, Israel; Departments of fMicrobiology and Immunology and gBiochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107; and hVeteran Affairs Medical Center, San Francisco, CA 94121 Contributed by Carlo M. Croce, March 24, 2011 (sent for review January 31, 2011) Rearrangements of the MLL (ALL1) gene are very common in acute some translocation and MLL-AF4 (12). Subsequent gene ex- infant and therapy-associated leukemias. The rearrangements un- pression profiling of leukemic cells with MLL rearrangements derlie the generation of MLL fusion proteins acting as potent on- from patients showed up-regulation of MEIS1 and HOXA5– cogenes. Several most consistently up-regulated targets of MLL HOXA10, as well as other genes (13–15). Recently, application fusions, MEIS1, HOXA7, HOXA9, and HOXA10 are functionally re- of ChIP-seq determined that MEIS1, HOXA7, HOXA9, and lated and have been implicated in other types of leukemias. Each HOXA10 are among the 226 primary targets of MLL-AF4 in a of the four genes was knocked down separately in the human human ALL cell line (8). Up-regulation of similar Hoxa genes precursor B-cell leukemic line RS4;11 expressing MLL-AF4. The mu- and Meis1 were obtained in murine model systems (16–19). Many tant and control cells were compared for engraftment in NOD/ studies applying overexpression of HOXA9 and/or MEIS1 showed SCID mice. Engraftment of all mutants into the bone marrow the requirement for both proteins in induction of acute leukemia (BM) was impaired. Although homing was similar, colonization in mice (20, 21). HOXA9 and MEIS proteins function within the by the knockdown cells was slowed. Initially, both types of cells same protein complex (with PBX) and occupy simultaneously the were confined to the trabecular area; this was followed by a rapid same gene targets (22, 23). spread of the WT cells to the compact bone area, contrasted with The consistent up-regulation of several HOXA genes in MLL- a significantly slower process for the mutants. In vitro and in vivo associated leukemias raised the issue whether HOXA5-10 spec- BrdU incorporation experiments indicated reduced proliferation of ify a “HOX code” (18, 24), in which each of the members con- the mutant cells. In addition, the CXCR4/SDF-1 axis was hampered, tributes to pathogenicity and is required for it. Experiments to as evidenced by reduced migration toward an SDF-1 gradient and resolve the issue involved induction of leukemia in mice knocked loss of SDF-1–augmented proliferation in culture. The very similar out for HOXA9. The results varied with the particular MLL fu- phenotype shared by all mutant lines implies that all four genes sion, and with the way by which the MLL fusion was introduced are involved and required for expansion of MLL-AF4 associated into the mutant mice (18, 25, 26). Because murine leukemic cells, leukemic cells in mice, and down-regulation of any of them is not in particular those involved in AML, might not reproduce all of compensated by the others. the processes occurring in human ALL cells with MLL rear- rangements, we decided to examine the biological effects of leukemic cells’ migration | bone marrow colonization of leukemic cells knockdown of HOX A7,9,10 or MEIS1 in the human leukemic cell line RS4;11, expressing MLL-AF4. earrangements of the MLL/ALL1 gene occur in 20% of Racute lymphoblastic leukemias (ALL) and in 5–6% of acute Results myeloid leukemias (AML) (1, 2). A high percentage of ALLs Knockdown of HOXA Genes or MEIS1 Impairs Engraftment of RS4;11 with MLL rearrangement show biphenotypic traits. The epide- Cells. The precursor B-cell line RS4:11, a classical biphenotypic miology of MLL-associated leukemias is unique (3). They pre- cell line (27), was knocked down for the HOXA7, A9, A10 or dominate infant acute leukemia, and account for the majority of MEIS1 genes. This was done by lentiviral-mediated transduction therapy-related leukemias occurring in cancer patients treated of constructs encoding short hairpin (sh) RNAs, designed to si- with etoposide (VP-16) or doxorubicin (4). The very short la- lence the genes (Table S1). The shRNAs sequences were se- tency of the disease (3, 4) and its aggressive nature suggest the lected to minimize homology to nontarget mRNAs and avoid involvement of a powerful oncogene. Most MLL rearrangements perfect dsRNA stretches of >11 bp; also, no IFN response (28) are due to reciprocal chromosome translocations that link MLL was detected. We applied Western analysis to determine the to any of >100 partner genes (5). This results in production of oncogenic MLL proteins, comprising the N-terminal MLL poly- peptide fused in frame to the C-terminal fragment of a partner Author contributions: K.O., A.K., T.R., E.S., T.I., H.A., L.C., A.A., A.M., O.K., C.M.C., T.N., protein. The partner proteins most commonly found associated T.L., and E.C. designed research; K.O., A.K., T.R., E.S., T.I., H.A., L.C., A.A., and T.N. per- formed research; A.B., and C.L. contributed new reagents/analytic tools; K.O., A.K., T.R., with MLL (AF4, AF9, ENL, AF10, ELL) are transcriptional E.S., T.I., H.A., H.G.O., L.C., A.A., S.V., C.M.C., and T.N. analyzed data; and E.C. and T.L. elongation factors (6). Because these factors physically associate wrote the paper. (6, 7), the function of the most common partner polypeptides The authors declare no conflict of interest. within MLL fusion proteins appears to be the recruitment of the 1A.K. and T.R. contributed equally to this work. other elongation factors, and consequently the augmentation of 2Deceased October 8, 2009. – target genes transcription (8 11). 3To whom correspondence may be addressed. E-mail: [email protected] or eli. Initial gene expression analysis of leukemic cells from ALL [email protected]. patients indicated up-regulation of HOXA9 and HOXA10 and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. their essential cofactor MEIS1 in cells with the t(4;11) chromo- 1073/pnas.1103154108/-/DCSupplemental. 7956–7961 | PNAS | May 10, 2011 | vol. 108 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1103154108 Downloaded by guest on September 27, 2021 extent of silencing affected by the different shRNA constructs. HOXA genes (30), in MLL-rearranged cells knocked down for The identity of the HOXA/MEIS1 proteins was further con- HOXA9. To examine whether similar cross-modulation is asso- firmed by their comigration with the corresponding epitope- ciated with MEIS1, HOXA7, and HOXA10 knockdowns, we tagged (Flag or HA) proteins produced in 293T cells expressing analyzed the abundance of HOXA/MEIS1 RNAs by applying the former (Fig. 1A). In most cases we found more than one the technology of NanoString nCounter gene expression system, construct encoding unique shRNA, potent in down-regulation of which captures and counts individual mRNA transcripts by their >80% of the protein; cell lines showing the most efficient hybridization to a multicomplex probe library (31). Cultured lymphoid RS4;11 and SEM cells, both expressing MLL-AF4, knockdown were chosen for further work (Fig. 1A). As controls were treated with MEIS1, HOXA7, HOXA10, or scrambled we used RS4;11 cells infected with the lentiviral vector, and cells siRNAs and the extracted RNAs were reacted with a library expressing HOXA9 shRNA found to be nonpotent in elimina- containing probes for MEIS1 and HOXA and other mRNAs (SI tion of the protein. Two recent studies indicated down-regula- Materials and Methods). Although cells treated with a particular tion of MEIS1 RNA (29, 30), as well as of RNAs of multiple siRNA were down-regulated for its expression, the abundance of the other HOXA (except HOXA5) or MEIS1 RNAs was not reduced (Fig. 1B). We also considered the possibility that the shRNAs induced a general deleterious effect on the cells. However, we found this unlikely because shRNAs directed against different sequences of the same gene and downregulating its expression induced very similar biological and biochemical effects when contrasted with cells expressing shRNAs inefficient in silencing of the gene which behaved like vector-infected or noninfected cells (see Figs. 2 and 6A and Table S2). The transplanted NOD/SCID mice were killed at 1 d (16 h), 1–5 wk (mice injected with control cells), or 1–6 wk (mice injected with mutant cells). At 4 and 6 wk after injection of control and mutant cells, respectively, increasing numbers of animals appeared ill or succumbed to the malignancy. Initially, Fig. 1. Knockdown of MEIS1 and HOXA by lentiviral-mediated delivery of shRNAs or by treatment with siRNAs. (A) Western analysis of MEIS1 and HOXA7, A9, and A10 proteins in nuclear extracts of RS4;11 cells expressing shRNAs directed against one of the four genes (constructs MEIS1_3a, HOXA9_1, HOXA7_1, HOXA10_4a; 3,1,4 specify the gene’s sequence tar- geted, and a or b correspond to different plasmid clones of the same con- struct), or a vector construct, or a control construct.
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