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Blood, 15 August 2006, Vol. 108, No. 4, pp. 1353-1362. Prepublished online as a Blood First Edition Paper on May 2, 2006; DOI 10.1182/blood-2006-01-011833. This Article Abstract Full Text (PDF) All Versions of this Article: blood-2006-01-011833v1 108/4/1353    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ferrari-Amorotti, G. Articles by Calabretta, B. Search for Related Content PubMed PubMed Citation Articles by Ferrari-Amorotti, G. Articles by Calabretta, B. Related Collections Neoplasia Oncogenes and Tumor Suppressors Gene Expression Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  NEOPLASIA Leukemogenesis induced by wild-type and STI571-resistant BCR/ABL is potently suppressed by C/EBP Giovanna Ferrari-Amorotti, Karen Keeshan, Michela Zattoni, Clara Guerzoni, Giorgio Iotti, Sara Cattelani, Nick J. Donato, and Bruno Calabretta From the Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA; the Department of Pathology, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia; and the Department of Bioimmunotherapy, University of Texas, M. D. Anderson Cancer Center, Houston.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Chronic phase–to–blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBP, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region–Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBP activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBP eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBP, which were more potent than those of K298E C/EBP, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBP in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBP has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBP activation.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Chronic myelogenous leukemia (CML) is a clonal disorder arising from neoplastic transformation of hematopoietic stem/progenitor cells.1 The typical course of CML involves progression from a protracted chronic phase marked by the accumulation of apparently normal neutrophils to a rapidly fatal blast crisis characterized by clonal expansion of differentiation-arrested myeloid or lymphoid precursor.2-4 CML is consistently associated with an acquired genetic abnormality, the Philadelphia chromosome, a shortened chromosome 22 resulting from a reciprocal translocation of the long arms of chromosomes 9 and 22.5,6 This translocation generates the breakpoint cluster region–Abelson murine leukemia (BCR/ABL) fusion gene, which is translated in the p210 or, less frequently, the p230 oncoprotein.7-9 Expression of p210 BCR/ABL is necessary and sufficient for the transformation of hematopoietic cells and for disease maintenance, as demonstrated by in vitro assays, leukemogenesis in mice, and the antileukemia effect of the BCR/ABL kinase inhibitor STI571 (imatinib mesylate [Gleevec]; Novartis, Basel, Switzerland).10-14 The mechanisms responsible for chronic phase–to–blast crisis transition remain poorly understood. A plausible model predicts that increased BCR/ABL expression during disease progression15-17 promotes secondary genetic and epigenetic changes essential for the expansion of clones with increasingly malignant characteristics.17,18 The BCR/ABL tyrosine kinase inhibitor imatinib is the first-line treatment for patients with CML.19 Most patients with newly diagnosed chronic-phase CML (CML-CP) treated with imatinib achieve durable responses,14,20 but treatment is less effective in the accelerated and blast-crisis phases of the disease.21 A small percentage of patients with CML-CP and most with advanced-phase disease have relapses on imatinib therapy.22 The most common mechanism of resistance involves specific point mutations in the kinase domain of BCR/ABL that interfere with STI571 binding.23-26 Amplification of the BCR/ABL gene and BCR/ABL-independent mechanisms of resistance have also been reported.26-28 Hematopoietic cell differentiation, which is defective in CML in blast crisis (CML-BC), is regulated by lineage-specific transcription factors, suggesting that the differentiation arrest of CML-BC cells depends, in part, on their altered expression/activity. The transcription factor CCAAT/enhancer-binding protein (C/EBP) induces differentiation and inhibits proliferation of many cell types, including myeloid cells.29 Within hematopoietic cells, C/EBP is expressed by granulocyte progenitors and precursors, but not by monocytes.30,31 Ectopic expression of C/EBP in bipotential myeloid progenitors induces granulopoiesis and blocks monocytic differentiation,32 and loss of C/EBP results in mice that retain monocytes but not mature granulocytes.33,34 A model of conditional knockout of C/EBP has further demonstrated the critical role of C/EBP in the transition of common myeloid progenitors into granulocyte-monocyte precursors.35 The induction of granulocyte differentiation by C/EBP is thought to depend on transcription activation,36-38 but the direct interaction of C/EBP with other proteins also has a profound influence on its function. For example, C/EBP interacts directly with the cyclin-dependent kinases CDK2 and CDK4 and prevents the assembly of functional CDK complexes that impede cell cycle progression,39 but the CDK2/CDK4 interaction domain of C/EBP, which is located between amino acids 175 and 188, is not required for C/EBP regulation of granulocyte differentiation, which depends on its transcriptional activation function.40 C/EBP also interacts with and represses E2F, a key transcriptional regulator of genes involved in cell cycle progression, an effect possibly involved in C/EBP-dependent cell cycle arrest and induction of differentiation.41-43 Alteration of C/EBP function is a common feature of leukemia cells.44 C/EBP mutations have been reported in approximately 10% of patients with AML,45,46 and C/EBP expression is transcriptionally repressed in samples from patients with t(8;21) AML1-ETO–positive leukemia47 and is down-modulated in cell lines expressing the FLT3-ITD protein, the inv16 fusion protein, and the AML1-MDS1-EVI1 fusion gene.48-50 In BCR/ABL-expressing cell lines and in marrow cells from patients with CML-BC, but not with CML-CP, C/EBP expression is suppressed,51,52 suggesting that C/EBP down-regulation is important in the progression of CML.18,51 Given that ectopic expression of C/EBP in BCR/ABL-expressing cell lines can restore granulocyte differentiation,51,53 we sought to determine whether sustained C/EBP expression could permanently suppress leukemogenesis in mice injected with BCR/ABL-expressing cells and whether it would also exert its antileukemia effect in cells carrying STI571-resistant mutant BCR/ABL. We report here that ectopic expression/activity of C/EBP induced cell cycle arrest and morphologic, immunophenotypic, and molecular features of granulocyte differentiation in cells expressing wild-type or STI571-resistant mutant BCR/ABL. Similarly, conditional activation of C/EBP suppressed leukemogenesis in mice, whether it depended on wild-type or mutant BCR/ABL. C/EBP expression in CML-BC cells carrying the wild-type BCR/ABL or the T315I mutant rapidly induced neutrophilic differentiation, suggesting that therapeutic strategies relying on C/EBP activation may bypass STI571 resistance.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Plasmids uORF/spacer C/EBP-HA (p42 C/EBP), K298E C/EBP-HA, and 177-191 C/EBP-HA were previously described.40 Plasmids p42 C/EBP-ERTAM, K298E C/EBP-ERTAM, and 177-191 C/EBP-ERTAM were generated by polymerase chain reaction (PCR) as follows: the ligand-binding domain of the murine estrogen receptor (ER) was amplified by reverse transcription–PCR (RT-PCR) from 32Dc13 RNA and was point mutated (Gly525Arg) with the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The PCR product was then reamplified with an upstream oligomer containing a 5'-flapping BamHI site and a downstream oligomer containing a 3'-flapping EcoRI site. p42 C/EBP, K298E C/EBP, and 177-191 C/EBP were amplified by PCR from the respective plasmids with an upstream primer containing a 5'-flapping XhoI site and a downstream primer containing a 3'-flapping BamHI site after a mutated C/EBP stop codon. The p42 C/EBP or K298E C/EBP or 177-191 C/EBP and ERTAM PCR products were directionally cloned into the XhoI/EcoRI-digested MigR1 vector. Each plasmid was sequenced to verify the presence of the expected mutations. The plasmid pBabe Puro K-ERTAM was a kind gift of A. D. Friedman (Johns Hopkins University, Baltimore, MD). Cell cultures and retroviral infections 32D-BCR/ABL and derivative cell lines were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 10% WEHI-conditioned medium as a source of IL-3. For assays requiring the inhibition of BCR/ABL kinase activity, cells were cultured in IL-3–containing medium supplemented (2 µM) or not with the ABL-kinase inhibitor STI571 (Novartis). STI571-resistant 32D-BCR/ABL cell lines were established by exposing 32D-BCR/ABL cells to increasing concentrations of STI571 (0.1-2 µM). After selection, RNA from resistant cells was extracted, the cABL kinase domain region was amplified by RT-PCR, and the PCR product was sequenced to assess the presence of mutations. For retroviral infections, Phoenix cells (kind gift of G. P. Nolan, Stanford University School of Medicine, Stanford, CA) were transiently transfected with the indicated plasmids. The infectious supernatant was collected 48 hours later and was used to infect (a 48-hour procedure) 32D-BCR/ABL–expressing cells. Twenty-four hours later, infected cells were sorted (EPICS Profile Analyzer; Coulter, Hialeah, FL) for green florescent protein (GFP) expression and were kept in culture as described.40 Images were visualized using an Olympus CK2 microscope with a 40 x/0.65 numeric aperture objective, and were photographed using an Olympus SC35 type 12 camera (Olympus, Melville, NY). JPEG images were viewed using Adobe Photoshop (Adobe Systems, San Jose, CA), and contrast adjustments were made. Cell proliferation and differentiation assays For proliferation and differentiation assays, 32D-BCR/ABL–cells transduced with wild-type or mutant C/EBP-ERTAM were washed with phosphate-buffered saline (PBS) and treated with 4-hydroxytamoxifen (4-HT, 100 nM; Sigma, St Louis, MO). Viable cells were counted by trypan blue exclusion. Differentiation was monitored by May-Grünwald/Giemsa staining and by detection of the differentiation-related marker Gr-1 with a specific phycoerythrin (PE)–conjugated mouse monoclonal antibody (PharMingen, San Diego, CA). A PE-conjugated rat IgG2b isotype immunoglobulin (PharMingen) was also used as control for specific staining. Western and Northern blot analyses For Western blotting, cells were lysed (2 x 105 cells/20 µL) in Laemmli buffer, and proteins of interest were detected with anti-C/EBP (14AA) polyclonal antibody raised against the C-terminus of C/EBP (sc-61; Santa Cruz Biotechnology, Santa Cruz, CA), anti-C/EBP (15C8) monoclonal antibody raised against amino acids 1 to 14 of mouse C/EBP (ab 15047; Novus Biologicals, Littleton, CO), anti–G-CSFR (M20) polyclonal antibody (sc-694; Santa Cruz Biotechnology) with PY20H anti-PTyr/HRP (p11265; BD Transduction Laboratories, Lexington, KY) or with anti-GRB2 monoclonal antibody (610112, BD Transduction Laboratories). For Northern blot analysis, total RNA of untreated or 4-HT–treated cells was extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH), fractionated (10 µg/lane) onto denaturing 1% agarose/6.6% formaldehyde gels, transferred onto Hybond-nylon membrane (Amersham Pharmacia Biotechnologies, Piscataway, NJ) and hybridized to a P32-labeled 470 bp PstI fragment of murine myeloperoxidase (MPO) from plasmid puc19MPO.54 RNA levels 28S and 18S were monitored as control for equal loading. Mice Sublethally irradiated (4.5 Gy) 4- to 6-week-old C3H/HeJ (Jackson Laboratories, Bar Harbor, ME) male mice were injected intravenously through the lateral tail vein with 32D-BCR/ABL cells transduced with p42 C/EBP-ERTAM or K298E C/EBP-ERTAM or with 32D-BCR/ABLT315I STI571-resistant cells transduced with 177-191 C/EBP-ERTAM. Each mouse received 105 cells. Tamoxifen treatment 4-HT was dissolved in ethanol at 100 mg/mL, then diluted in autoclaved sunflower seed oil (Sigma) at 10 mg/mL. Each mouse was injected intraperitoneally with 1 mg 4-HT on consecutive days. Analysis of disease progression with acute and chronic activation of C/EBP For acute activation of C/EBP, sublethally irradiated C3H/HeJ male mice were injected intravenously (105 cells/mouse) with 32D-BCR/ABL cells expressing p42 C/EBP-ERTAM or K298E C/EBP-ERTAM and with STI571-resistant 32D BCR/ABLT315I cells expressing 177-191 C/EBP-ERTAM. Mice were monitored for leukemia progression with the use of peripheral blood taken retro-orbitally. After erythrocyte lysis, cells were plated in methylcellulose (1000 or 500 per plate) in the absence of cytokines, and colony formation was monitored after 5 days. When peripheral blood leukemia cell percentages reached 10% to 20%, mice were divided into 2 groups, 1 treated with 3 mg 4-HT injected intraperitoneally and the other treated with sunflower seed oil only. At the end of treatment, mice were killed, and GFP-positive bone marrow and spleen cells were used for colony-formation assays for the detection of Gr-1 marker expression and for DNA content analysis. For chronic activation of C/EBP, sublethally irradiated C3H/HeJ mice were injected intravenously with C/EBP-ERTAM–expressing 32D-BCR/ABL (wild-type or mutant) cells (105 cells/mouse). Forty-eight hours later, mice were divided into 2 groups, 1 treated with 1 mg 4-HT, the other with sunflower seed oil, and both injected intraperitoneally on consecutive days for 15 days. Mice were killed at the end of the injections, and bone marrow and spleen cells were used for morphologic examination (May-Grünwald/Giemsa staining) and for assessment of leukemic load by flow cytometry and methylcellulose colony formation assays of GFP-positive cells. Mice survival Sublethally irradiated C3H/HeJ male mice were injected intravenously with transduced 32D-BCR/ABL, p42 C/EBP-ERTAM cells (20 mice, 105 cells/mouse), K298E C/EBP-ERTAM cells (14 mice, 105 cells/mouse), or 177-191 C/EBP 32D-BCR/ABLT315I cells (14 mice, 105 cells/mouse). Forty-eight hours later, mice were divided in 2 groups, 1 treated with 1 mg 4-HT (intraperitoneally on consecutive days for 15 days) and the other with sunflower seed oil only. After the last injection, mice were monitored for survival. Detection of the T315I mutation in CML-BC cells Total RNA from peripheral blood blast cells of a patient with STI571- and BMS-354825–resistant CML-BC was used to generate a BCR/ABL-specific RT-PCR product including nucleotides corresponding to the Abl kinase domain with the use of a 5' BCR primer (5'-TGAAACTCCAGACTGTCCACA-3') and a downstream c-Abl 3' primer (5'-CTGGATTCCTGGAACATTGTTT-3'). Sequences of the PCR product revealed the presence of a T-to-C substitution generating the T315I mutation. Differentiation assay of CML-BC cells Bone marrow cells from a patient with CML-BC with 20% blasts carrying a double Ph chromosome were depleted of lineage-positive cells and enriched for CD34+ cells using the Stem Span protocol (Stem Cell Technology, Vancouver, BC, Canada). Cells were cultured for 24 hours in the presence of IL-6, Flt3 ligand, SCF, and IL-3, retrovirally transduced with wild-type or mutant C/EBP for 48 hours, selected for GFP positivity, and assessed for granulocyte differentiation (May-Grünwald/Giemsa staining) in the presence of 2 ng/mL human recombinant IL-3 or 25 ng/mL human recombinant G-CSF (R&D Systems, Minneapolis, MN). Peripheral blood blast cells (greater than 90%) from 3 other patients with CML-BC (2 of whom carried the T315I mutation) were expanded for 7 days in the presence of IL-6, Flt3 ligand, SCF, and IL-3 and were retrovirally transduced with the MigRI empty vector or with p42 C/EBP for 48 hours, selected for GFP positivity, and assessed for granulocyte differentiation in the presence of 25 ng/mL human recombinant G-CSF. CML patient samples, obtained from the Division of Medical Oncology, Thomas Jefferson Medical College, and the Department of Hematology, M. D. Anderson Hospital, were used with approval by the Institutional Review Board (IRB) of Thomas Jefferson Medical College.    Results Top Abstract Introduction Materials and methods Results Discussion References   STI571-dependent granulocyte differentiation of 32D-BCR/ABL cells is blocked by an inducible C/EBP transcription repressor Myeloid precursor 32Dc13 cells are dependent on IL-3 for proliferation and undergo differentiation on treatment with G-CSF.55 Upon expression of BCR/ABL, these cells become growth factor independent and fail to differentiate, a phenotype associated with the down-modulation of C/EBP.51,52 Ectopic expression of C/EBP in 32D-BCR/ABL cells can overcome the block in differentiation by allowing the cells to respond to G-CSF.51 Treatment with the BCR/ABL kinase inhibitor STI571 leads to increased C/EBP expression and reverses, in part, the block in granulocyte differentiation induced by BCR/ABL,52 suggesting that STI571-dependent activation of C/EBP expression and the induction of differentiation in STI571-responsive cells are functionally linked. To assess the requirement of C/EBP activity in STI571-induced differentiation of 32D-BCR/ABL cells, a fusion protein consisting of the 89-amino acid Kruppel-associated box (KRAB) transrepression domain (K), the C/EBP DNA–binding domain(), and the 4-HT–responsive murine ER ligand–binding domain, K-ERTAM was expressed in 32D-BCR/ABL cells. Upon 4-HT treatment of these cells, STI571-dependent G-CSF–induced granulocyte differentiation was suppressed, as assessed by monitoring of the levels of the granulocyte surface marker Gr-1 (Figure 1A). Western blot analysis confirmed that C/EBP expression was induced by G-CSF and STI571 treatment and showed that activation of the transrepressor K-ERTAM had no effect on C/EBP expression but inhibited its activity,as indicated by the suppression of G-CSFR induction (Figure 1B). View larger version (40K): [in this window] [in a new window]   Figure 1.. Induction of differentiation in STI571-treated 32D-BCR/ABL cells is dependent on C/EBP activity. Gr-1 expression at 48 hours (A) and C/EBP and G-CSFR levels at the indicated times (B) in STI571-treated 32D-BCR/ABL cells upon activation of K-ERTAM, a C/EBP transcription repressor. Levels of K-ERTAM were detected with anti-C/EBP (14AA) raised against the C-terminus; endogenous C/EBP was detected with anti-C/EBP15C8 raised against amino acids 1 to 14 of mouse C/EBP. GRB2 levels were measured as control of equal loading.   The induction of C/EBP in STI571-treated 32D-BCR/ABL cells and the demonstration that the STI571 induction of differentiation markers is suppressed by functional inhibition of C/EBP-regulated transcription suggest that ectopic expression/activity of C/EBP might suppress the leukemogenic potential of STI571-sensitive and -resistant BCR/ABL-expressing cells. Effect of wild-type and mutant C/EBP on proliferation and differentiation of STI571-sensitive and -resistant 32D-BCR/ABL cells To assess the effects of ectopic C/EBP expression in cells transformed by BCR/ABL and to identify the domains necessary for its activity, 32D-BCR/ABL cells were transduced with retroviruses expressing only GFP (the MigRI empty vector); p42 C/EBP-ERTAM, a 4-HT–inducible form of wild-type C/EBP; K298E C/EBP-ERTAM, a 4-HT–inducible form of a basic region/DNA-binding domain mutant that maintains the alpha helical structure but is deficient in DNA binding40,56; and 177-191 C/EBP-ERTAM, a 4-HT–inducible internally deleted mutant that fails to interact with CDK2/CDK439 (Figure 2A). Immunoblots of 32D-BCR/ABL cell lysates confirmed that the C/EBP fusion proteins were equally expressed (Figure 2B, lanes 6-8) and that their levels were more abundant in endogenous protein in untreated and G-CSF–treated (3 days) parental and BCR/ABL-expressing 32Dcl3 cells (Figure 2B, lanes 1-4). 4-HT–dependent activation of p42 or mutant C/EBP-ERTAM in 32D-BCR/ABL caused a marked decrease in cell number (Figure 2C). Cell morphology was assessed daily by May-Grünwald/Giemsa staining of cytospins (Figure 3A). Activation of p42 C/EBP-ERTAM and 177-191 C/EBP-ERTAM led to the rapid appearance of many cells with nuclear segmentation and of terminally differentiated neutrophils; by contrast, activation of K298E C/EBP-ERTAM did not induce differentiation (Figure 3A). Moreover, activation of p42 C/EBP-ERTAM and 177-191 C/EBP-ERTAM, but not of K298E C/EBP-ERTAM, led to increased expression of Gr-1, a marker of granulocyte differentiation (Figure 3B). Total RNA and whole cell lysates of 4-HT–treated 32D-BCR/ABL cells expressing the various chimeric proteins were prepared daily for 3 days. Total RNA was subjected to Northern blotting for MPO expression. 4-HT treatment had no effect on MPO expression in cells transduced with the MigRI vector or with K298E C/EBP-ERTAM, but MPO mRNA levels markedly increased by day 2 in cells expressing p42 C/EBP-ERTAM and 177-191 C/EBP-ERTAM (Figure 3C). View larger version (34K): [in this window] [in a new window]   Figure 2.. Effects of C/EBP on 32D-BCR/ABL cells numbers. (A) Schematic diagram of wild-type and mutant C/EBP-ERTAM chimeric proteins. (B) Levels of endogenous C/EBP and ectopic C/EBP-ERTAM in 32Dcl3 (lanes 1-2), in 32D-BCR/ABL cells (lanes 3-4), or in 32D-BCR/ABL cells transduced with the MigRI empty vector (lane 5) or wild-type or mutant C/EBP-ERTAM (lanes 6-8). The anti-C/EBP blot was exposed for 2 minutes (lanes 1-4) or 10 seconds (lanes 5-8). (C) Cell counts of 4-HT–treated MigRI- or C/EBP-ERTAM–transduced 32D-BCR/ABL cells. Values represent mean ± SD of 3 different experiments.   G-CSFR protein levels were also markedly increased in cells expressing p42 C/EBP-ERTAM and 177-191 C/EBP -ERTAM, but not in cells transduced with the MigRI vector or with K298E C/EBP-ERTAM (Figure 3D). Thus, C/EBP activation in 32D-BCR/ABL–expressing cells promotes granulocyte differentiation and mimics, in part, the effects of STI571. C/EBP-dependent differentiation, but not cell cycle arrest, requires DNA binding and transactivation ability (as demonstrated by the effects of the K298E C/EBP mutant), whereas the CDK2/CDK4-binding domain is not required for C/EBP-dependent differentiation or cell cycle arrest. To assess whether activation of C/EBP can overcome the block in granulocyte differentiation of STI571-resistant cells, we exposed parental and 177-191 C/EBP-ERTAM–expressing 32D-BCR/ABL cells to increasing concentrations (0.05-2 µM) of STI571. When the selection was complete, total RNA was extracted from resistant cells, and the segment encoding the c-Abl kinase domain was amplified by RT-PCR and sequenced to assess the presence of mutations. The Y253H mutation was identified in 32D-BCR/ABL cells, whereas the T315I mutation was found in 177-191 C/EBP-ERTAM–expressing 32D-BCR/ABL cells. To confirm that the mechanism of resistance was BCR/ABL dependent, the 2 cell lines and control STI571-sensitive cells were treated with 2 µM STI571, and cell lysates were assessed for levels of tyrosine-phosphorylated BCR/ABL by antiphosphotyrosine Western blotting. As expected, levels of tyrosine-phosphorylated proteins (including BCR/ABL) were markedly down-modulated in STI571-sensitive cells but not in the STI571-resistant cell lines (not shown). STI571-resistant 32D-BCR/ABL cells were then retrovirally transduced with the MigRI empty vector, p42 C/EBP-ERTAM,or K298E C/EBP-ERTAM to determine whether C/EBP activation bypasses STI571 resistance. Compared with MigRI-transduced cells, activation of p42 or mutant C/EBP caused a marked decrease in the number of 32D-BCR/ABL cells carrying the Y253H or the T315I mutation (Figure 4A). Cell morphology was assessed daily by May-Grünwald/Giemsa staining of cytospins (Figure 4B). After 3 days of 4-HT treatment, most p42 C/EBP-ERTAM and 177-191 C/EBP-ERTAM–expressing cells were morphologically differentiated, as indicated by the appearance of segmented nuclei typical of mature neutrophils; by contrast, features of morphologic differentiation were not detected in cultures of MigRI-transduced cells or of cells expressing the K298E mutant (Figure 4B). Activation of p42 and 177-191 C/EBP-ERTAM, but not K298E C/EBP-ERTAM, led to increased levels of Gr-1 (Figure 4C) and G-CSFR (Figure 4D). View larger version (67K): [in this window] [in a new window]   Figure 3.. Effects of C/EBP on differentiation of 32D-BCR/ABL cells. Morphology (A), Gr-1 levels (B), MPO (C), and G-CSFR expression (D) in 4-HT–treated 32D-BCR/ABL cells retrovirally transduced with MigRI or C/EBP-ERTAM.   CEBP activation suppresses in vivo leukemogenesis of STI571-sensitive and -resistant 32D-BCR/ABL cells To assess whether C/EBP can suppress BCR/ABL-dependent leukemogenesis in mice, 32D-BCR/ABL cells, 32D-BCR/ABLY253H cells expressing p42 C/EBP-ERTAM, and 32D-BCR/ABLT315I cells expressing 177-191 C/EBP-ERTAM were injected intravenously (105 cells/mouse) into sublethally irradiated syngeneic mice; 2 days later, mice were treated with vehicle or 4-HT (1 mg/d for 15 consecutive days). At the end of the treatment, mice were killed and cells were harvested from bone marrow and spleen. May-Grünwald/Giemsa staining of bone marrow cell suspensions of 4-HT–treated mice revealed the presence of normal myeloid and nonmyeloid cells, suggesting that the bone marrow was not infiltrated by BCR/ABL-transformed cells. By contrast, samples of vehicle-treated mice showed various proportions of blastlike cells characterized by high nuclear-cytoplasmic ratios and round nuclei (Figure 5A-C, left panel). Bone marrow samples of vehicle-treated mice displayed a high proportion of GFP-positive cells (32D-BCR/ABL–injected mice, 70% ± 1.16%, n = 2; 32D-BCR/ABLY253H–injected mice, 16.5% ± 2.0%, n = 2; 32D-BCR/ABLT315I–injected mice, 21.4% ± 4.5%, n = 2), whereas in 4-HT–treated mice, GFP positivity of marrow cells was negligible (32D-BCR/ABL–injected mice, 1.04% ± 1.6%, n = 3; 32D-BCR/ABLY253H–injected mice, 0.14% ± 0.3%, n = 2; 32D-BCR/ABLT315I–injected mice, 0.94% ± 0.2%, n = 3) (Figure 5A-C, middle panels). The clonogenic potential of bone marrow cells was evaluated by methylcellulose assays performed in the absence of cytokines. Five days after plating, cells of vehicle-treated mice formed numerous colonies consistent with the presence of growth factor–independent BCR/ABL-expressing cells; by contrast, few colonies formed from bone marrow cells of 4-HT–treated mice injected with 32D-BCR/ABL p42 C/EBP-ERTAM cells (Figure 5A, right panel), and no colonies developed from bone marrow of 4-HT–treated mice injected with STI571-resistant 32D-BCR/ABL cells (Figure 5B-C, right panels). View larger version (46K): [in this window] [in a new window]   Figure 4.. Effects of C/EBP on STI571-resistant 32D-BCR/ABL cells. Cell counts (A), morphology (B), Gr-1 levels (C), and G-CSFR expression (D) in STI571-resistant 32D-BCR/ABL cells upon 4-HT activation of C/EBP-ERTAM. Cell count values represent mean ± SD of 3 different experiments.   The same analysis was performed on splenocytes harvested from vehicle or 4-HT–treated mice. Spleens of control mice were markedly larger than those of 4-HT–treated mice, suggesting they were infiltrated by BCR/ABL-transformed cells. Microscopic examination of spleen cell suspensions from 4-HT–treated mice showed that most cells were lymphocytes, whereas the splenocyte suspension from vehicle-treated mice was enriched in cells with a blastlike morphology (Figure 5A-C, left panel). Fluorescence-activated cell sorter (FACS) analysis displayed a cohort of GFP-positive cells in control mice (32D-BACR/ABL–injected mice, 23.4% ± 2.8%, n = 2; 32D-BCR/ABLY253H–injected mice: 5% ± 1.0%, n = 2; 32D-BCR/ABLT315I–injected mice, 4.7% ± 1.1%, n = 3), whereas no GFP-positive cells were detected in the spleen suspensions of 4-HT–treated mice (Figure 5A-C, middle panels). Colony-formation assays further demonstrated the presence of BCR/ABL-expressing cells in spleen cell suspensions of vehicle-treated, but not of 4-HT–treated, mice (Figure 5A-C, right panels). Because the leukemia load in mice injected with 32D-BCR/ABLY253H and 32D-BCR/ABLT315I cells was lower than that in mice injected with 32D-BCR/ABL cells, clonogenic assays were also performed by seeding splenocytes and bone marrow cells from mice injected with STI571-resistant 32D-BCR/ABL cells at 19 000 cells/plate, a number chosen to approximate the GFP positivity of cell suspensions from mice injected with STI571-sensitive 32D-BCR/ABL cells. No colonies formed from samples of 4-HT–treated mice (not shown), indicating that C/EBP is equally effective in STI571-sensitive and -resistant cells. View larger version (55K): [in this window] [in a new window]   Figure 5.. In vivo effects of C/EBP activation in STI571-sensitive and -resistant 32D-BCR/ABL cells. Mice were injected with C/EBP-ERTAM-expressing 32D-BCR/ABL cells (A), C/EBP-ERTAM–expressing 32D-BCR/ABLY253H cells (B), or 177-191 C/EBP-ERTAM 32D-BCR/ABLT315I cells (C) and were treated with vehicle alone or with 4-HT for 15 consecutive days. (left panels) Morphology, (middle panels) GFP positivity, and (right panels) colony formation of bone marrow and spleen cells isolated at the end of treatment. Colony number values represent mean ± SD of 3 different experiments.   Because the T315I BCR/ABL mutant is completely insensitive to STI571 and other ATP-binding competitive inhibitors of the BCR/ABL kinase, including BMS-354825,57-59 we further tested the antileukemia effect of C/EBP in 32D-BCR/ABLT315I cells. Thus, mice (7 per group) injected with 105 177-191 C/EBP-ERTAM 32D-BCR/ABLT315I cells were treated 2 days later with vehicle only or with 1 mg/d 4-HT for 15 consecutive days and then were assessed for survival. Untreated mice were all dead 25 days after the injection of leukemia cells; by contrast, only 1 mouse in the 4-HT–treated group died of leukemia (51 days after injection), whereas the remaining 6 were alive and without signs of disease 225 days after injection (Figure 6A). The effect of C/EBP was also assessed in mice in which bone marrow and spleen were heavily infiltrated by leukemia cells. Thus, 32D-BCR/ABLT315I cells expressing 177-191 C/EBP-ERTAM were injected intravenously in sublethally irradiated mice and were allowed to develop leukemia. View larger version (34K): [in this window] [in a new window]   Figure 6.. Effect of C/EBP activation on mice injected with 32D-BCR/ABLT315I cells. (A) Survival of mice injected with 177-191 C/EBP-ERTAM 32D-BCR/ABLT315I cells (105 cells/mouse) and treated 2 days later with vehicle only or with 4-HT (1 mg/d for 15 consecutive days). (B) Methylcellulose colony formation assays, (C) Gr-1 immunostaining, and (D) DNA content of GFP-positive cells isolated from bone marrow and spleen of mice injected with 177-191 C/EBP-ERTAM 32D-BCR/ABL cells (105 cells/mouse) and treated 14 days later with vehicle only or with 4-HT (1 mg/12 hours, for 3 injections). Colony number values represent mean ± SD of 3 different experiments.   When leukemia cell percentages reached 15% to 20%, mice were treated with vehicle alone or with 1 mg 4-HT every 12 hours for a total of 3 injections. At 36 hours, mice were killed, leukemia cells in bone marrow and spleen were sorted by GFP positivity, and their proliferation potential and differentiation were tested. Compared with GFP-positive cells of vehicle-treated mice, activation of 177-191 C/EBP-ERTAM by 4-HT induced a marked decrease in colony formation (841 ± 101 vs 14 ± 7 from bone marrow GFP-positive cells; 860 ± 79 vs 49 ± 17 from spleen GFP-positive cells) (Figure 6B) but led to an increased number of differentiating cells expressing the Gr-1 antigen (4.5% ± 3.6% vs 30.4% ± 7.9% in bone marrow and 1.5% ± 0.4% vs 25.3% ± 0.4% in spleen) (Figure 6C). Compared with vehicle treatment only, activation of 177-191 C/EBP-ERTAM in 32D-BCR/ABLT315I cells injected in mice led to a dramatic G0/G1 arrest of GFP-positive cells in bone marrow (vehicle-treated mice: G0/G1 phase, 45.4% ± 2.25%; S phase, 35% ± 2.0%; G2/M phase, 19% ± 1.1%; 4-HT–treated mice: G0/G1 phase, 75.3% ± 11.4%; S phase: 12.5% ± 6.4%; G2/M phase: 9.2% ± 4.0%) and spleen (vehicle-treated mice: G0/G1 phase, 58.5% ± 0.28%; S phase, 25.3% ± 1.0%; G2/M phase, 13% ± 0.4%; 4-HT–treated mice: G0/G1 phase, 76.5% ± 11.4%; S phase, 12.5% ± 6.0%; G2/M phase, 9.2% ± 4.0%) (Figure 6D). Thus, the activation of functional C/EBP induced differentiation, suppressed the proliferation of 32D-BCR/ABLT315I cells injected in mice, and enhanced the survival of leukemic mice. Differentiation induction and proliferation inhibition are both required for the antileukemia effect of C/EBP We further assessed whether C/EBP suppression of in vivo leukemogenesis was dependent on both proliferation inhibition and differentiation induction. Thus, 105 32D-BCR/ABL cells expressing p42 C/EBP-ERTAM or K298E C/EBP-ERTAM were injected into sublethally irradiated syngeneic mice and, 2 days later, were treated with vehicle only or 1 mg/d 4-HT for 15 consecutive days. Activation of K298E C/EBP-ERTAM prolonged the survival of leukemic mice (treated: mean, 60.5 days; median, 62 days; n = 7; untreated: mean, 25 days; median, 25 days; n = 7; difference between means, 35.5 days; P = .001; difference between medians, 37 days; P = .001, Wilcoxon 2-sample exact test). However, the activation of p42 C/EBP-ERTAM had a more potent effect (treated: mean, 230 days; median, 83 days; n = 10; untreated: mean, 20.2 days; median, 21 days; n = 10; difference between means, 209.8 days; P < .001; difference between medians, 62 days; P < .001, Wilcoxon 2-sample exact test). Notably, 4 of 10 treated mice were still alive more than 15 months after the last injection, indicating that they were cured of their disease (Figure 7A-B). On comparing the effect of the 2 C/EBP proteins, the differentiation-inducing function of p42 C/EBP, but not of K298E C/EBP, may explain the more potent antileukemia effect of the former. Thus, we tested cell cycle inhibition and differentiation induction by p42 C/EBP and K298E C/EBP in 32D-BCR/ABL injected in mice. Leukemia was allowed to develop in mice injected with 32D-BCR/ABL cells expressing p42 or K298E C/EBP-ERTAM. When percentages of leukemia cells in the peripheral blood were 15% to 20%, mice were treated with vehicle only or with 1 mg/d 4-HT for 3 days. On day 4, mice were killed, bone marrow and splenocytes were harvested, GFP-positive cells were sorted, and Gr-1 positivity and DNA content were evaluated. 4-HT activation of p42 C/EBP-ERTAM led to markedly increased Gr-1 expression in bone marrow (31.3% ± 11.7% [n = 2] vs 4.5% ± 3.6% [n = 2] in vehicle-treated mice) and spleen (36.2% ± 4.6 [n = 2] vs 1.5% ± 0.4 [n = 2] in vehicle-treated mice) GFP-positive cells (Figure 7C). By contrast, 4-HT activation of K298E C/EBP-ERTAM did not induce an increase of Gr-1 levels in bone marrow (3.8% ± 1.1%) or spleen (1.2% ± 0.5%) GFP-positive cells (Figure 7C). DNA content analysis of bone marrow and spleen GFP-positive cells from mice injected with p42 or K298E C/EBP-ERTAM–expressing 32D-BCR/ABL cells revealed that 4-HT induced in both increased numbers of G0/G1 cells (bone marrow: G0/G1, 57.2% ± 2.0% and 59.2% ± 2.2%, n = 2, respectively; spleen: G0/G1, 64.25% ± 1.0% and 63.5% ± 2.1%, n = 2, respectively) compared with treatment with vehicle (bone marrow: G0/G1, 45.4% ± 2.3%, n = 2; spleen: G0/G1, 57.5% ± 0.5%, n = 2). Thus, the more potent effect of p42 C/EBP-ERTAM in suppressing leukemogenesis by 32D-BCR/ABL cells likely depends on its ability to induce the differentiation of leukemia cells in mice. View larger version (26K): [in this window] [in a new window]   Figure 7.. Effect of C/EBP activation on survival of mice injected with 32D-BCR/ABL cells and on in vivo differentiation of 32D-BCR/ABL cells. (A) Survival of mice (n = 10) injected with p42 C/EBP-ERTAM 32D-BCR/ABL cells (105/mouse) and left untreated or treated with 4-HT (1 mg/d for 15 consecutive days). (B) Survival of mice (n = 7) injected with K298E C/EBP-ERTAM 32D-BCR/ABL cells (105/mouse) and left untreated or treated with 4-HT (1 mg/d for 15 consecutive days). (C) Gr-1 levels in GFP-positive 32D-BCR/ABL cells expressing p42 C/EBP-ERTAM or K298E C/EBP-ERTAM and treated with 4-HT.   Ectopic expression of p42 C/EBP induces differentiation of CML-BC cells The effects of C/EBP were also investigated in human CML-BC cells. Thus, wild-type or mutant C/EBP was retrovirally transduced in CD34+ cells of a patient with CML-BC with 20% blasts carrying a double Ph chromosome and no mutations in the BCR/ABL kinase domain and GFP-positive cells assessed for granulocyte differentiation in the presence or in the absence of G-CSF. In the presence of G-CSF, differentiation was essentially complete by day 4 (Figure 8A, upper panel, second and fourth rows), whereas in the absence of G-CSF the process was slower and most p42 C/EBP and 177-191 C/EBP–expressing cells underwent granulocyte differentiation by day 7 (not shown). By contrast, few MigRI-transduced cells or cells expressing the K298E C/EBP mutant showed morphologic features of differentiation (Figure 8A, first and third rows). Of note, C/EBP expression was low in bone marrow mononuclear cells but increased 3- to 4-fold after STI571 treatment (Figure 8A, lower panel). Peripheral blood blast cells of another CML-BC patient without mutations in the BCR/ABL kinase domain were also induced to differentiate by ectopic expression of p42 C/EBP (not shown). The ability of C/EBP to induce granulocyte differentiation of CML-BC cells was also tested with the use of peripheral blood blasts from a patient with CML-BC resistant to STI571 and to BMS-354825 and, predictably, carrying the T315I mutation. Blast cells from this patient with CML-BCT315I were retrovirally transduced with MigRI or wild-type p42 C/EBP for 48 hours, selected for GFP positivity, and assessed for granulocyte differentiation. In the presence of G-CSF, most p42 C/EBP-expressing cells underwent granulocyte differentiation by day 5; by contrast, few MigRI-transduced cells showed features of differentiation in the presence of G-CSF (Figure 8B, upper panel). C/EBP expression was low in untreated cells and, as expected, did not increase upon STI571 treatment (Figure 8B, lower panel). Peripheral blood blast cells of another patient with CML-BC carrying the T315I mutation were also induced to differentiate by ectopic expression of p42 C/EBP (not shown). View larger version (59K): [in this window] [in a new window]   Figure 8.. Effect of C/EBP in CML-BC cells. (Top panel) Induction of granulocyte differentiation by constitutive expression of C/EBP in CML-BC cells. Bone marrow CD34+ cells (A) or peripheral blood blasts (B) from 2 CML-BC patients were retrovirally transduced with the MigRI empty vector or with wild-type or mutant C/EBP. After sorting, GFP-positive cells were cultured in the presence of IL-3 or G-CSF and were assessed (May-Grünwald/Giemsa staining of cytospins) for morphologic differentiation. (Bottom panel) Western blot analysis shows C/EBP expression in untreated and STI571-treated cells of the 2 patients with CML-BC. GRB-2 levels were measured as control of equal loading.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   CML therapy had been revolutionized by the development of STI571, the first ATP-binding competitive inhibitor of the BCR/ABL kinase.24 STI571 is highly effective in patients with CML-CP; BCR/ABL transcripts remain detectable in a cohort of STI571-treated CML-CP patients,60 but the long-term consequences of this finding are unclear. Resistance to STI571 develops in most patients with advanced-stage CML and in a few with CML-CP, often as a consequence of mutations in the BCR/ABL kinase domain.26,61 Given that the development of STI571 resistance reduces the therapeutic options in CML patients, it is important to identify molecular targets of STI571 that may mimic its effects. When activated, some of these targets might bypass STI571 resistance, raising the possibility of novel therapeutic strategies that use effector molecules functioning downstream of the drug target. One such target of STI571 is the transcription factor C/EBP because its expression is repressed by BCR/ABL and is restored by BCR/ABL kinase inhibition. Enhanced C/EBP expression after STI571 treatment of BCR/ABL-expressing cells is likely to be important for the drug's effects because markers of granulocyte differentiation are not induced by STI571 in cells conditionally expressing a C/EBP transcription repressor (Figure 1). Although this repressor is also expected to inhibit C/EBP- and C/EBP-dependent transcription, these effects are likely to be less important than those on C/EBP because C/EBP is induced late during myeloid differentiation52 and C/EBP is less potent than C/EBP in inducing the differentiation of BCR/ABL-expressing cells.62 Thus, we assessed the antileukemia effects of C/EBP using STI571-sensitive and -resistant BCR/ABL-expressing cells in vitro and in mice in which C/EBP was conditionally activated. For these studies, we tested the effect of wild-type p42 C/EBP and of 2 mutants, K298E and 177-191 C/EBP, that are deficient in DNA-binding/transcription activation and CDK2/CDK4 interaction, respectively. In vitro, p42 C/EBP and both mutants all induced a marked decrease in cell number, indicating that neither transcription activation nor CDK2/CDK4 interaction is required for the cell cycle effects. By contrast, the induction of granulocyte differentiation by C/EBP depended on its transcripti