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The MLL Fusion Gene, MLL-AF4, Regulates Cyclin-Dependent Kinase Inhibitor CDKN1B (P27kip1) Expression

The MLL Fusion Gene, MLL-AF4, Regulates Cyclin-Dependent Kinase Inhibitor CDKN1B (P27kip1) Expression

The MLL fusion , MLL-AF4, regulates -dependent kinase inhibitor CDKN1B (p27kip1) expression

Zhen-Biao Xia, Relja Popovic, Jing Chen, Catherine Theisler, Tara Stuart, Donna A. Santillan, Frank Erfurth, Manuel O. Diaz, and Nancy J. Zeleznik-Le*

Department of Medicine, Molecular Biology Program, and Oncology Institute, Cardinal Bernardin Center, Loyola University Medical Center, Maywood, IL 60153

Communicated by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, July 29, 2005 (received for review December 2, 2004) MLL, involved in many chromosomal translocations associated Recent studies suggest some potential mechanisms of MLL with acute myeloid and lymphoid leukemia, has >50 known fusion leukemogenesis. For example, fusion partner partner with which it is able to form in-frame fusions. dimerization domains and͞or activation domains fused to MLL Characterizing important downstream target genes of MLL and of can aberrantly activate downstream targets such as HOX genes MLL fusion may provide rational therapeutic strategies for and contribute to cell transformation (13, 14). This regulation is the treatment of MLL-associated leukemia. We explored down- mediated at the level of target gene . There is very stream target genes of the most prevalent MLL fusion protein, strict regulation of HOX during hematopoiesis, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell therefore misregulated expression of these genes is likely im- lines in different backgrounds. Overexpression of MLL-AF4 does portant in MLL leukemogenesis. not lead to increased proliferation in either cell line, but rather, cell During normal hematopoiesis, a tight balance is required be- growth was slowed compared with similar cell lines inducibly tween levels of mostly quiescent stem cells that can renew the expressing truncated MLL. We found that in the MLL-AF4-induced population and highly proliferating progenitor cells, before final cell lines, the expression of the cyclin-dependent kinase inhibitor differentiation along a particular lineage. This balance is regulated gene CDKN1B was dramatically changed at both the RNA and through regulators. and cyclin-dependent kinases protein (p27kip1) levels. In contrast, the expression levels of (CDKs) play important roles in this process (15). CDKs are CDKN1A () and CDKN2A () were unchanged. To explore opposed by CDK inhibitors (CDKIs) (16, 17). There are two related whether CDKN1B might be a direct target of MLL and of MLL-AF4, families of CDKIs: (i) the Cip͞Kip family (p21, p27, and p57), which we used chromatin immunoprecipitation (ChIP) assays and lucif- inhibits CDK2 and CDK1-containing complexes (cyclin A͞E– erase reporter gene assays. MLL-AF4 binds to the CDKN1B pro- CDK2, –CDK1) and (ii) the INK4 family (p15, p16, p18, moter in vivo and regulates CDKN1B promoter activity. Further, we and p19), which inhibits -containing complexes (cyclin confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as D–CDK4͞6). Expression of CDKIs generally causes growth arrest in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is and, when acting as tumor suppressors, may cause cell cycle arrest a downstream target of MLL and of MLL-AF4, and that, depending and . Numerous studies have also shown that CDKIs on the background cell type, MLL-AF4 inhibits or activates CDKN1B accumulate during cell differentiation (18, 19). However, the CD- expression. This finding may have implications in terms of leuke- KIs do not act similarly in all cell lineages. For example, in the mia resistance to chemotherapy in MLL-AF4 leukemias. myeloid lineage, expression of p27kip1 was required for differenti- ation of progenitors along this lineage, and p27-deficient marrow leukemia ͉ acute lymphoblastic leukemias ͉ cell cycle accumulated progenitor cells (20). In contrast, in the lymphoid lineage, expression of p27 inhibits differentiation, and p27 expres- LL is involved in chromosomal translocations associated sion must be decreased for normal T cell development (21). Mwith leukemia. Remarkably, MLL is involved in translo- The MLL fusion genes can cause leukemia in vivo (11, 12); cations with Ͼ50 different genes (1, 2). MLL is specifically however, the mechanism is unclear. It has been proposed that the cleaved shortly after translation into two peptides that nonco- fusions block hematopoietic cell differentiation and reduce cell valently associate with each other (3, 4). The amino-terminal death, thus contributing to leukemogenesis. Identification of addi- portion of MLL contains a region with AT-hooks that binds tional gene targets of MLL-AF4 regulation may allow the design of DNA, as well as a region with transcriptional repression activity rational therapeutic strategies. To identify potential direct targets of (5) that binds CpG-rich DNA (6) and recruits histone deacety- MLL-AF4, we generated cell lines with an inducible MLL-AF4 lases, the corepressor CtBP1, and polycomb group proteins (7). transgene. The carboxyl-terminal portion contains a transcriptional activa- In an epithelial cell background, we observed that MLL-AF4 tion domain (5), which interacts with CBP (2), and a SET down-regulated the CDKI p27 but not p21 or p16. Down-regulation domain, with histone methyltransferase activity (3, 8). Different of p27 occurred at both the RNA (CDKN1B) and protein levels. MLL fusion partners are associated with leukemias producing Furthermore, chromatin immunoprecipitation (ChIP) assays indi- blast cells of various lineages. MLL-AF9 results mainly in acute cated that MLL-AF4 binds to the CDKN1B promoter in vivo, and myeloid leukemia (AML), whereas MLL-AF4 causes almost reporter gene assays show that it represses transcription of the exclusively B-cell lineage acute lymphoblastic leukemias. These CDKN1B promoter in an epithelial cell line. Similarly, in a lymphoid findings suggest that MLL chimeras affect the phenotype of the cell background and in primary bone marrow progenitor cells, leukemia by influencing differentiation pathways of uncommit- ted cells or early progenitors. MLL-AF4, an MLL fusion protein Abbreviations: ChIP, chromatin immunoprecipitation; CDKs, cyclin-dependent kinases; that is associated with infant pro-B acute lymphoblastic leuke- CDKIs, CDK inhibitors; PI, propidium iodide; MEFs, murine embryonic fibroblasts. mias, is the most prevalent of the numerous MLL fusion proteins *To whom correspondence should be addressed at: Cardinal Bernardin Cancer Center, (9), and it is usually associated with a poor prognosis (10). Loyola University Medical Center, 2160 South First Avenue, 112-337, Maywood, IL 60153. Numerous data show that MLL fusion genes can transform E-mail: [email protected]. hematopoietic cells in vitro and cause leukemia in vivo (11, 12). © 2005 by The National Academy of Sciences of the USA

14028–14033 ͉ PNAS ͉ September 27, 2005 ͉ vol. 102 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506464102 Downloaded by guest on September 27, 2021 MLL-AF4 also regulates CDKN1B expression, but in this case it is mM NaCl. Each buffer contained 10 ␮lof100ϫ protease inhibitors up-regulated. Our results suggest that MLL-AF4 regulates (catalogue no. P 8340; Sigma), 4 ␮lof1MDTT,2␮l of phosphatase CDKN1B expression directly, but that the outcome of this regula- inhibitor (catalogue no. P 5726; Sigma), and 2 ␮l of 1 M NaF per tion depends on the cell type. ml. Proteins were electrophoresed on SDS͞10% or 5% polyacryl- amide gels and transferred to polyvinylidene difluoride membrane. Materials and Methods Membranes were blocked with 5% milk and were incubated with Expression Plasmids. MLL(672) was generated by digestion of polyclonal rabbit anti-p21 (sc-397) or polyclonal rabbit anti-p27 pEGFP-MLL2Kb(22) with KpnI and ligating into pcDNA5͞ (sc-528) (1:1000) (Santa Cruz Biotechnology). Detection was per- FRT͞TO (Invitrogen). MLL(1250) was generated by digestion of formed with peroxidase-conjugated anti-rabbit Ig (1:3,000) (Am- MSCVneo-MLL-CBP(12) with PseI͞BamHI, followed by ligation ersham Pharmacia) and ECL detection as above. Membranes were of the MLL fragment into pcDNA5͞FRT͞TO-MLL(672). stripped and rehybridized as described in ref. 7. MLL-AF4 was generated by digestion of MSCVneo-MLL-AF4 (N.J.Z.-L., unpublished data) with BamHI, and ligation of AF4 into RNA Extraction, RT-PCR, and Real-Time RT-PCR. Total RNA was pcDNA5͞FRT͞TO-MLL(1250). The constructs were confirmed isolated with TRI Reagent (catalogue no. T 9424; Sigma) and by sequencing. converted to cDNA by using Invitrogen Superscript First Strand Synthesis system. CDKN2A, CDKN1A, and CDKN1B primers were Establishment of Cell Lines Expressing MLL and MLL-AF4 Proteins. By used as described in refs. 24–26. PCR was performed as follows: using the Flp-In T-REx 293 host cell line (Invitrogen), 9 ␮gof 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 30 cycles, then expression plasmid pOG44 was electroporated along with 1 ␮gof a final 72°C 10-min extension. The MLL-AF4 primers used were either pcDNA5͞FRT͞TO [MLL-AF4, MLL(672), MLL(1250), top, 5Ј-CACCTACTACAGGACCGCCAA-3Ј; and bottom, 5Ј- and MLL] or pCMV5͞RPT͞TO (vector alone). Hygromycin- GGGGTTTGTTCACTGTCACTGTCC-3Ј. PCR was performed resistant clones were obtained under 100 ␮g͞ml hygromycin selec- as follows: 94°C for 50 sec, 60°C for 50 sec, and 72°C for 1 min for tion. The cell lines used were ␤-galactosidase negative and zeocin 35 cycles. For quantitative RT-PCR, cDNA was prepared from sensitive and showed inducible expression by RT-PCR and Western MLL-AF4 induced and uninduced 293 and Jurkat cell lines and blotting. To generate inducible cell lines in the Jurkat background, from murine primary bone marrow progenitors infected with Flp-In Jurkat cells (Invitrogen) were used to generate a Flp-In MSCVneo-MLL-AF4 or MSCVneo retrovirus and cultured in T-REx Jurkat cell line, and inducible MLL-AF4 Jurkat lines were methylcellulose under G418 selection for 1 week as described in ref. developed in a similar manner. 12. All reactions were performed in triplicate, and CDKN1B expression level was measured by using SYBR green reagents and Cell Culture and Maintenance. Stably expressing 293 cell lines were the Applied Biosystems Prism 5700 sequencer. The relative maintained in Dulbecco’s modified Eagle’s medium (DMEM) plus CDKN1B expression level was determined by normalization to 10% FCS, 15 ␮g͞ml blasticidin, and 100 ␮g͞ml hygromycin. Stably murine (for bone marrow cells) or human (for 293 and Jurkat cells) expressing Jurkat cell lines were maintained in RPMI medium 1640 GAPDH. Primer sequences are available on request. with the same additives, except with 200 ␮g͞ml hygromycin. Cell lines were induced with 1 ␮g͞ml tetracycline. Leukemia cell lines Luciferase Assays. We seeded 1.0 ϫ 105 cells in six-well plates and were maintained in RPMI medium 1640 plus 10% FCS. Murine transfected empty vector, MLL, and MLL-AF4 cell lines with embryonic fibroblasts (MEFs) were cultured as described in ref. 23. CDKN1B promoter (Ϫ3568 to Ϫ12) (0.9 ␮g) (27), CDKN2A MllϪ/Ϫ MEFs were transfected with MLL or MLL-AF4-expressing promoter (Ϫ2000 to ϩ41 or Ϫ970 to Ϫ164) (0.9 ␮g) (M.O.D., plasmids and cultured 4 weeks with 200 ␮g͞ml hygromycin to create unpublished data) linked to the firefly luciferase gene or basic stable populations. vector (0.9 ␮g) plus pTKRL(Renilla) (100 ng), with Fugene6 (Roche) according to the manufacturer’s protocol. After 48 h, cells Cell Growth, Cell Cycle, and Apoptosis Assays. For growth rate were harvested. Dual-Luciferase Reporter assay system (Promega) analysis, cells were plated at 1 ϫ 105 cells with or without 1 ␮g͞ml was used according to manufacturer’s protocol. tetracycline. Viable and dead cells were assessed by counting with trypan blue exclusion at 24, 48, 72, 96, 120, and 144 h. For propidium ChIP Assay. ChIP was performed with Upstate Biotechnology ChIP iodide (PI) staining, Ϸ1 ϫ 106 cells were washed in PBS, fixed in assay (catalogue no. 17-295) following manufacturer’s protocol 4:1 (ice-cold methanol͞PBS), incubated in PI (5 ␮g͞ml PI in PBS) with some modifications. Either uninduced or induced 3 ϫ 106 cells with 100 ␮g͞ml RNase A at 37°C for 1 h, and analyzed on a Becton were crosslinked with 1% formaldehyde for 5 min at room tem- Dickinson FACSCalibur with CELLQUEST analysis software. An- perature, and the reaction was terminated with an excess of glycine. nexin V-FITC apoptotic detection (catalogue no. 556547; BD Chromatin was sonicated to an average size of 400 bp and was Pharmingen) was according to the manufacturer’s protocol. immunoprecipitated with MLL-specific polyclonal antibodies or preimmune serum (28), or with anti-FLAG antibody (catalogue no. Immunoprecipitation and Western Blot Analysis. FLAG-MLL pro- F 3165; Sigma). ChIP DNA was detected by using PCR and teins were expressed by tetracycline induction for 24 h. Cells were ethidium bromide staining after agarose gel electrophoresis. The lysed in IPH buffer [50 mM Tris⅐HCl,pH8.0͞150 mM NaCl͞5mM CDKN1B DNA fragments amplified promoter and coding regions, EDTA͞0.5% NP-40͞10 ␮l/ml protease inhibitor mix (Sigma)], spanning nucleotides 161–340 and 717–974, respectively (GenBank immunoprecipitated on anti-FLAG beads as described in ref. 7, and accession no. AF480891). washed four times with NETN buffer (20 mM Tris⅐HCl, pH 8.0͞100 mM NaCl͞1 mM EDTA͞0.5% Nonidet P-40). Proteins were Results resolved by SDS͞PAGE and detected with an enhanced chemilu- MLL and MLL-AF4 Expression in Conditional Cell Lines. To characterize MEDICAL SCIENCES minescence (ECL) kit (Amersham Pharmacia) according to the MLL fusion downstream target gene expression, we generated manufacturer’s protocols. Rabbit anti-MLL antibodies, anti-MLL- stable cell lines that conditionally expressed FLAG-tagged MLL- AT-hook, anti-MLL-RD, and anti-MLL-AD, are against MLL AF4, by using the Invitrogen Flp-In system (see Materials and amino acids 310–400, 1101–1400, and 2771–3114, respectively. Methods) in 293 human kidney epithelial and Jurkat lymphoblast Preimmune sera from the same rabbits were used as negative cell lines. We also generated cell lines based on the Flp-In T-REx controls. For Western blot analysis of p21 and p16, cells were lysed 293 cell line that conditionally express FLAG-MLL(672), FLAG- in hypotonic lysis buffer (HLB; 20 mM Hepes, pH 7.5͞10 mM MLL(1250), or full-length FLAG-MLL, or contain the empty KCl͞0.5 mM EDTA͞0.1% Triton X-100), followed by HLB͞500 expression vector (Fig. 1A). MLL expression was confirmed by

Xia et al. PNAS ͉ September 27, 2005 ͉ vol. 102 ͉ no. 39 ͉ 14029 Downloaded by guest on September 27, 2021 Fig. 1. Creation and characterization of conditional MLL and MLL-AF4 cell lines. (A) FLAG-tagged MLL and MLL-AF4 fusion proteins expressed in conditional cell lines: full-length MLL (Top), amino portions of MLL (Middle), and MLL-AF4 (Bottom). Numbers refer to amino acid number. MLL-AF4 retains the AT-hook and repression domain of MLL, plus amino acids 347-1221 of AF4. AT, AT hooks; RD1 and RD2, repression domains; PHD, plant homeodomain; AD, activation domain; CS, proteolytic cleavage site; SET, conserved domain with methyltransferase activity. (B) FLAG full-length MLL (a–c), FLAG-MLL-AF4 (c, d, and f), or two different amino-terminal FLAG-MLL proteins (e) were expressed in clonally selected stably transfected 293 cell lines or Jurkat cell lines (f) after induction with tetracycline (ϩ), but not without tetracycline (Ϫ) for 24 h. After immunoprecipitation with anti-FLAG beads, proteins were detected with anti-FLAG antibodies (a, d, e, and f), anti-MLL activation domain antibodies (b), or anti-AT plus anti-RD antibodies (c). (C) MLL-AF4 inhibits growth of inducible 293 and Jurkat cell lines. Cells were cultured in the absence (ϪTet) or presence (ϩTet) of 1 ␮g͞ml tetracycline, and cell numbers were counted every 24 h. Results are presented as mean (ϮSD) of three independent experiments. (D) MLL-AF4 expression does not affect apoptosis and cell cycle. Annexin and PI staining show that the cells were not significantly more apoptotic in 293 cell line with induced MLL-AF4 expression. (E) The relative percent cells in each stage of the cell cycle was not changed after induced MLL-AF4 expression in 293 and Jurkat (data not shown).

Western blotting, and MLL-AF4 expression was confirmed by both the mRNA level (Fig. 2A, lane 6, and B and C) and protein RT-PCR (data not shown) and Western blotting (Fig. 1B). MLL- (p27) level (Fig. 2D, lane 1) after induced expression of MLL-AF4 AF4 RNA is expressed from 2 to 48 h after tetracycline induction compared with cells inducibly expressing MLL(672), MLL(1250), as determined by RT-PCR (data not shown). MLL-AF4 protein is or full-length MLL. CDKN1A (Fig. 2A, lane 4) and CDKN2A (Fig. observed by 12 h after induction and remains detectable 48 h after 2A, lane 2) levels were not affected by induction of MLL-AF4 in this induction (data not shown). All protein expression was detectable cell line. Because p27 targets CDK1͞cyclin B and CDK2͞cyclin A in the nuclear fraction or by immunoprecipitation from whole cell or complexes to regulate the cell cycle, over-expression of extract. The FLAG full-length MLL and MLL-AF4 proteins are p27 usually causes cell cycle arrest at the G0͞G1 phase or causes cell detectable from whole cell lysates only after immunoprecipitation growth to slow (29). Because we showed that MLL-AF4 down- in these cell lines because even the induced levels of expression are regulates CDKN1B, we wanted to test whether MLL-AF4 expres- quite low. We also confirmed that MLL-AF4 localized in the sion could alter the percentage of cells in any phase of the cell cycle. nucleus by immunostaining (data not shown). We found that cell We performed cell-cycle analysis by PI staining and determined growth slowed after induction of MLL-AF4, but not after induction that the percentage of cells in each stage of the cell cycle does not of MLL(1250) (Fig. 1C Upper) or MLL(672) (data not shown), seem to be significantly affected after induction of MLL-AF4 at relative to the empty vector control in the epithelial cell background time points from 2 to 16 h (Fig. 1 D and E) and also through 6 days and in the Jurkat lymphoid cells (Fig. 1C Lower). However, this (data not shown). These data suggested that p27 down-regulation slower growth was not due to increased apoptosis in either cell line by MLL-AF4 does not result in cell-cycle arrest at a specific phase (Fig. 1 D and E and data not shown). in this cell line.

MLL-AF4 Represses Endogenous CDKN1B (p27) Expression, but Not MLL-AF4 Represses CDKN1B Promoter Activity in an Epithelial Cell CDKN2A (p16) or CDKN1A (p21), in Epithelial Cells. CDKIs are im- Background. Because induction of MLL-AF4 results in decreased portant in regulation of cell cycle transit. Because induced expres- levels of CDKN1B mRNA, we tested whether this repression occurs sion of MLL-AF4 caused the cells to grow more slowly, we through the CDKN1B promoter by using luciferase reporter gene determined whether MLL-AF4 alters expression of CDKIs in these assays. 293 stable cell lines, inducible for full-length MLL or cell lines. We used inducible MLL(672), MLL(1250), full-length MLL-AF4, or containing empty vector, were transfected with a MLL, and MLL-AF4 epithelial cell lines to compare their relative firefly luciferase reporter plasmid containing the CDKN1B pro- effect on expression of CDKIs. CDKN1B shows down-regulation at moter, the CDKN2A promoter, or basic vector, plus Renilla expres-

14030 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506464102 Xia et al. Downloaded by guest on September 27, 2021 Fig. 3. MLL-AF4 represses CDKN1B, but not CDKN2A, promoter activity in 293 epithelial cells. Reporter constructs basic pGL3, pGL3-CDKN1B (Ϫ3568 to Ϫ12) (A), pGL3–2kbCDKN2A, or pGL3–0.8kbCDKN2A (B), were cotransfected with pTKRL (Renilla) as internal control, in MLL-AF4-, MLL-, or vector-inducible 293 cell lines. Luciferase activity was detected in the absence (dark bars) or presence (light bars) of tetracycline. The data were normalized to the internal control and the basic vector. The data were from at least three independent experiments (mean Ϯ SD).

as nuclear targeting sequences, are all present in the amino- terminal portion of the protein. These domains are present in wild-type MLL, as well as in all MLL fusions, including MLL-AF4. Because MLL-AF4 could regulate p27 expression acting through the promoter, we hypothesized that wild-type MLL might also regulate p27 expression. MEFs, either wild type (ϩ͞ϩ) or mutant (Ϫ͞Ϫ) for Mll (23) were assessed for p27 protein expression levels (Fig. 2E). We found an Mll-dependent effect on p27 expression, whereas p21 protein levels were unchanged. Surprisingly, however, the absence of wild-type Mll decreased p27 protein levels, whereas in the 293 epithelial cells, induced MLL-AF4 expression decreased p27 expression. This observation was also substantiated by microar- ray analysis reported by others using these same cells, where RNA ϩ ϩ Ϫ Ϫ Fig. 2. MLL-AF4 decreases p27 expression in 293 epithelial and MEF cells. (A) levels for CDKN1B were significantly higher in Mll / versus Mll / MLL-AF4 expression decreases CDKN1B RNA in 293 cells. Shown is RT-PCR for MEFs (30). Furthermore, CDKN1B͞p27 expression was rescued in CDKN2A (lanes 1 and 2), CDKN1A (lanes 3 and 4), and CDKN1B (lanes 5 and 6), MllϪ/Ϫ MEFs stably transfected with MLL, whereas MLL-AF4 in conditionally expressed MLL-AF4 293 cell line with (ϩ) or without (Ϫ) further reduced CDKN1B expression in these cells as measured by tetracycline. (B) Semiquantitative RT-PCR was used to determine changes of quantitative RT-PCR (Fig. 2F). relative expression in CDKN2A, CDKN1A, and CDKN1B, normalized to GAPDH. The relative CDKN1B expression (CDKN1B͞GAPDH) was significantly reduced after induction of MLL-AF4 compared with noninduced. Reduced expression MLL-AF4 Induces p27 Expression in Lymphoid Cells. The MLL-AF4 was not seen with CDKN2A or with CDKN1A. The data were derived from at translocation almost always results in acute leukemia with lymphoid least three independent experiments (mean Ϯ SD). (C) CDKN1B was decreased phenotype. Therefore, it is relevant to determine whether MLL- 8-fold by MLL-AF4-expressing 293 cells as compared with uninduced 293 cells AF4 regulates p27 expression in lymphoid cells. After induction of by quantitative RT-PCR. CDKN1B expression levels were normalized to GAPDH MLL-AF4 expression in Jurkat cells, p27 RNA and protein expres- expression and performed in triplicate. (D) p27kip1 protein levels are reduced sion were increased (Fig. 4 A and B). This finding was corroborated after MLL-AF4 expression in 293 cells. p27 protein expression was reduced 24 h by Affymetrix microarray hybridization of MLL-AF4- vs. control- after induction of MLL-AF4 (lane 1) in 293 cells compared with the noninduced induced Jurkat RNA, where p27 was identified as one of the most cells (lane 2). p27 protein levels were not changed with induced expression of MLL(672), MLL(1250) clones 1 and 2, or full-length MLL (lanes 3–10). The upper significantly increased genes (data not shown). Furthermore, in two arrow indicates loading control. (E) p27 protein level was reduced in MllϪ/Ϫ cell lines derived from patients with the MLL-AF4 translocation, MEFs compared with wild-type Mllϩ/ϩ MEFs (upper arrow). p21 protein levels MV4–11 and RS4;11, p27 was expressed at a high level, but p27 were not changed (lower arrow). (F) MLL rescues CDKN1B expression, and expression was at a lower level in Mono Mac 6 and THP-1, cell lines MLL-AF4 further reduces CDKN1B expression in murine MllϪ/Ϫ MEFs. RNA was with the MLL-AF9 translocation (Fig. 4A). To determine whether Ϫ Ϫ Ϫ Ϫ isolated from Mll / MEFs (lane 1) or from Mll / MEFs transfected with the MLL-AF4 fusion could also regulate p27 expression in primary MLL-AF4-expressing (lane 2) or MLL-expressing (lane 3) plasmids and grown hematopoietic cells, we infected murine bone marrow progenitor under hygromycin selection. Data are presented as fold-change in CDKN1B Ϫ Ϫ cells with MSCVneo-MLL-AF4 retrovirus and analyzed cells after expression levels relative to untransfected Mll / MEFs as determined by quantitative RT-PCR normalized to GAPDH and performed in triplicate. 1 week under G418 selection. CDKN1B expression was increased 2-fold compared with control by quantitative RT-PCR (Fig. 4C).

sion construct (for normalization). MLL or MLL-AF4 expression MLL-AF4 Binds the Endogenous CDKN1B Promoter in 293 Cells and in was induced with tetracycline 24 h after transfection. MLL-AF4 MLL-Fusion Leukemia Cell Lines. MLL-AF4 is able to regulate MEDICAL SCIENCES expression repressed activity of the CDKN1B promoter (Fig. 3A) CDKN1B promoter activity (Fig. 3) and to affect the levels of but not the CDKN2A promoter (Fig. 3B). In contrast, empty vector endogenous CDKN1B RNA and protein (Figs. 2 and 4). These control and full-length MLL had no significant effect on CDKN1B findings suggest that CDKN1B might be a direct transcriptional promoter activity in these cells (Fig. 3A). target of MLL-AF4 and of MLL. Therefore, we performed ChIP assays by using inducible MLL-AF4 cells and two cell lines devel- p27 Expression Is Regulated by Wild-Type MLL. The MLL protein oped from patients with MLL-AF4 translocation leukemias (31) to domains responsible for target DNA binding, including the AT determine whether MLL-AF4 was bound to the CDKN1B pro- hooks and CXXC motifs present in the repression domain, as well moter in vivo. After crosslinking, chromatin was sheared to an

Xia et al. PNAS ͉ September 27, 2005 ͉ vol. 102 ͉ no. 39 ͉ 14031 Downloaded by guest on September 27, 2021 MLL-AF4; however, MLL-AF4 did not bind to the CDKN1B coding region in the MLL-AF4-induced cell line (Fig. 5A). Anti- FLAG antibody did not show binding, but this may be because the FLAG epitope was not accessible in vivo. In similar experiments with the leukemia cell lines, MV4–11 and RS4;11, which express endogenous MLL-AF4, we observed binding to the CDKN1B promoter, whereas no binding was observed with preimmune sera or in the absence of antibody (Fig. 5B). MLL binding to the CDKN1B promoter was also observed in cell lines expressing the MLL-AF9 fusion, Mono Mac 6 and THP-1. The MLL fusion proteins and͞or MLL bind to the CDKN1B promoter in these cell lines because both endogenous proteins are expressed and recog- nized by the anti-MLL antibodies used. Discussion Our data demonstrate that an oncogenic protein, MLL-AF4, can regulate expression of p27kip1. Interestingly, MLL-AF4 causes Fig. 4. p27 expression is increased in MLL-AF4-expressing hematopoietic either a decrease or an increase in p27 expression, depending on cells. (A) p27 protein expression is increased in MLL-AF4-expressing hemato- the cell type. Furthermore, p27 expression can also be modu- poietic cell lines. (Upper) Western blot analysis shows that induced expression lated by wild-type MLL. MLL has previously been shown to bind of MLL-AF4 results in increased p27 expression in Jurkat cells (compare lanes to the promoters of Hoxc8 and HOXA9, targets of MLL regu- 1 and 2) and that p27 is increased in human patient-derived leukemia cell lines lation (3, 8), but no other direct targets have previously been expressing the MLL-AF4 translocation (MV4–11 and RS4;11) (lanes 5 and 6) but identified. We therefore explored whether MLL͞MLL-AF4 was not in lines expressing the MLL-AF9 translocation (Mono Mac 6 and THP-1) bound to the CDKN1B promoter. Our data indicate that MLL- (lanes 3 and 4). (Lower) Blots were stripped and rehybridized with antibody AF4 binds to the CDKN1B promoter in both the inducible recognizing actin. (B) CDKN1B is up-regulated by MLL-AF4 expression in Jurkat cells. Semiquantitative RT-PCR for CDKN1B in MLL-AF4 Jurkat cell line com- MLL-AF4 epithelial cell line and in patient-derived MLL fusion- pared with parental Jurkat cell line (control) induced with tetracycline. Two- expressing leukemia cell lines. While this manuscript was under fold dilutions of cDNA were used as template for RT-PCR. The relative CDKN1B revision, it was reported by another group (32) that MLL and expression (CDKN1B͞GAPDH) was significantly increased after induction of menin cooperatively regulate expression of the CDKIs p27kip1 MLL-AF4. (C) CDKN1B is up-regulated by MLL-AF4 expression in primary and p18Ink4C in murine embryonic fibroblasts. Similar to our murine bone marrow progenitor cells. Bone marrow progenitors were in- findings, it was observed that MLL positively regulates p27 fected with control (MSCVneo) or MLL-AF4-expressing (MSCVneo-MLL-AF4) expression by directly binding to the p27 . retrovirus. After G418 selection for 1 week, RNA was isolated and analyzed by p27 is one of the CDKIs involved in cell-cycle progression and can quantitative RT-PCR for CDKN1B expression. Expression levels were normal- also act as a tumor-suppressor gene. Functionally, loss of CDKN1B ized to GAPDH expression and performed in triplicate. The relative CDKN1B expression was increased 2-fold after MLL-AF4 expression. enhances the growth of mice (33) and results in hyperplasia of most organs, including spleen and thymus, causes a selective expansion of progenitor cells populations (CFU-GM and CFU-E or BFU-E) average size of 400 bp and immunoprecipitated with antibodies (34), and increases the risk of lymphoma development (35). Fur- thermore, in the absence of CDKN1B, altered cell kinetics was against the amino-terminal portion of MLL (AT and RD) (Fig. observed among progenitors (36). In addition, a number of recent 1Bc) (28) or with anti-FLAG antibodies. MLL-AF4 binds to the studies have demonstrated the prognostic significance of p27 in CDKN1B promoter (Fig. 5A) in 293 cells induced to express human cancer. Decreased levels of total p27 protein are associated with high tumor grade and stage in human breast, colorectal, and gastric , among others (37–40). The expression of p27 during hematopoiesis varies at different stages of hematopoietic cell differentiation. For example, stem cells express high levels of p27, and they are quiescent or cycling at a very low level. Bone marrow progenitor cells express lower levels of p27 and cycle rapidly (29). Terminally differentiated cells often accu- mulate p27 and are growth arrested; however, the growth arrest itself is not sufficient to cause differentiation (29). Recently, a number of studies show that p27 may be involved in cell differen- tiation (41), and although the cells differentiate, they accumulate p27, which cooperates with other proteins to modulate cell differ- entiation (20, 42). There are several features of MLL-AF4 leukemias that suggest that regulation of p27 by the fusion protein may be important in this disease. Infants with MLL-AF4 leukemia, which is the predominant type of infant leukemia, have a poor prognosis as compared with Fig. 5. MLL-AF4 binds to the endogenous CDKN1B promoter. ChIP assays other types of acute lymphoblastic leukemias (43). If MLL-AF4 were performed by using antibodies to MLL (AT ϩ RD) or FLAG, or preimmune potentiates expression of p27 in bone marrow progenitor cells with sera. Immunoprecipitated chromatin was analyzed by PCR with primers spe- this translocation, this could have significant ramifications in terms cific for either the CDKN1B promoter (A Left; B)ortheCDKN1B coding region of the cycling of these cells. Increased p27 expression would likely (A Right). (A) MLL-AF4 binds to the CDKN1B promoter (Left) but not to the CDKN1B coding region (Right) in induced MLL-AF4 293 cells. (B) Endogenous result in lower cycling of these cells, causing them to be resistant to MLL͞MLL-AF4 binds to the CDKN1B promoter in MV4–11 and RS4;11 cells. most current types of chemotherapy regimens. Recently, condi- Endogenous MLL͞MLL-AF4 binds to the endogenous CDKN1B promoter in tional MLL-AF4-expressing U937 myelomonocytic leukemia cell MV4–11 and RS4;11 cells (Upper). Endogenous MLL͞MLL-AF9 binds to the lines were created by another group (44). Similar to our results, it endogenous CDKN1B promoter in Mono Mac 6 and THP-1 cells (Lower). was found that MLL-AF4 expression resulted in increased doubling

14032 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506464102 Xia et al. Downloaded by guest on September 27, 2021 times of these cells, although expression levels of cell-cycle regu- during a critical stage in the hematopoietic differentiation program. lators including p27 were not reported. An increase in p27 in the leukemia stem cell population would likely Much attention is currently focused on trying to identify and result in a less effective response to standard chemotherapy regi- understand leukemia stem cells (45). Some progress has been made mens. Alternative strategies that target p27 in leukemia stem cells in chronic myelogenous leukemia (CML), where it was found that may prove effective in treatment of this disease. Wild-type MLL the leukemia stem cells are mostly quiescent (46). Murine models also regulates p27 expression. MLL may be involved in regulating of this disease are useful in trying to determine which strategies the balance between quiescence and proliferation in normal he- might be most effective in destroying the leukemia stem cell to cure matopoietic stem cells. disease. Unfortunately, the leukemia stem cell for MLL-AF4 leukemia has not yet been identified, nor have any reliable animal We thank Dr. T. Sakai (Kyoto Prefectural University of Medicine) and models been created that recapitulate the human disease. This E. A. Williamson (University of California Los Angeles School of limitation is in contrast to other MLL fusion leukemias, where Medicine) for providing pLG3-CDKN1B and Dr. S. Korsmeyer (Dana– animal models have been successfully created (11, 12). We have Farber Cancer Institute, Boston) for providing MEFs. We thank Drs. Michael Thirman and Stephen Nimer for helpful advice regarding the demonstrated that CDKN1B is a direct target of MLL-AF4. Our paper. This work was funded by National Institutes of Health͞National working hypothesis is that MLL-AF4 aberrantly regulates expres- Cancer Institute Grants CA78438 and CA40046 (to N.J.Z.-L.) and sion of p27 as compared with wild-type MLL, which might result in CA81269 and CA104300 (to M.O.D.), and the Dr. Ralph and Marian an inappropriate either increase or decrease of p27 expression Falk Medical Research Trust (to N.J.Z.-L.).

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