Letters to the Editor 1220 profiles of acute myeloid/T-lymphoid leukemia with silenced 17 Verhaak RG, Wouters BJ, Erpelinck CA, Abbas S, Beverloo HB, CEBPA and mutations in NOTCH1. Blood 2007; 110: 3706–3714. Lugthart S et al. Prediction of molecular subtypes in acute myeloid 15 Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, van Putten leukemia based on expression profiling. Haematologica WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single 2009; 94: 131–134. CEBPA mutations, define a subgroup of acute myeloid leukemia 18 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H with a distinctive profile that is uniquely associated et al. WHO Classification of Tumours of Haematopoietic and with a favorable outcome. Blood 2009; 113: 3088–3091. Lymphoid Tissues. 4th edn IARC: Lyon, France, 2008. 16 Bullinger L, Dohner K, Kranz R, Stirner C, Frohling S, Scholl C 19 Ioannidis JP, Allison DB, Ball CA, Coulibaly I, Cui X, Culhane AC et al. An FLT3 gene-expression signature predicts clinical outcome et al. Repeatability of published microarray gene expression in normal karyotype AML. Blood 2008; 111: 4490–4495. analyses. Nat Genet 2009; 41: 149–155.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

A transcriptome-wide approach reveals the key contribution of NFI-A in promoting erythroid differentiation of human CD34 þ progenitors and CML cells

Leukemia (2010) 24, 1220–1223; doi:10.1038/leu.2010.78; NFI-A or a control empty Vector as previously described.2 published online 29 April 2010 Forty-eight hours after transduction, GFP þ cells were sorted using FACSAria instrument (BD Biosciences, San Jose`, CA, USA), subjected to total RNA extraction and hybridized on Agilent whole oligo microarray (No. G4112F; -A (NFI-A) is a member of the NFI family Agilent Technologies, Palo Alto, CA, USA; detailed protocols of transcription factors, composed of four separate are summarized in Supplementary Information). (Nfia, Nfib, Nfic, Nfix) with distinct functions depending on To quantify the resulting amount of NFI-A in both the Vector the cell type and target promoter context.1,2 Recent studies and NFI-A samples, we performed a real-time PCR using primers showed a novel and considerable role for NFI-A in the specific for NFI-A (primers used are listed in Supplementary specification of hematopoietic lineages from CD34 þ human Table S2). As shown in Figure 1a, the relative amount of hematopoietic progenitor cells (HPCs). In particular, NFI-A was NFI-A is greatly increased in the NFI-A-transduced compared to shown to promote the erythroid lineage at the expense of the Vector-infected cells. Microarray results were then analyzed by granulocytic lineage, by direct repression of the G-CSF using the GeneSpring GX 10 software (Agilent Technologies). (G-CSFR) gene, and to activate b-globin transcription in normal Data transformation was applied to set all the negative raw erythroblasts and remarkably in K562 chronic myeloid leukemia values at 1.0, followed by a Quantile normalization. A filter on (CML) cells, which do not express endogenous b-globin.2 low gene expression was used to keep only the probes expressed Notably, NFI-A expression is shut off to allow terminal in at least one sample (flagged as Marginal or Present). Expressed granulocytic or monocytic differentiation by the activity of genes having a fold change of 41.5 between the Vector and microRNA-223 and microRNA-424, respectively,3,4 further NFI-A samples were selected. Interestingly, numerous genes indicating a common negative regulatory role for NFI-A in the were upregulated in the exogenous NFI-A-expressing myeloid lineage. sample, whereas relatively less genes were downregulated (for In line with these findings, we recently reported that the complete raw and normalized data see Supplemental exogenous NFI-A expression in K562 CML blast crisis cells Table S1), suggesting that NFI-A per se behaves both as a resulted in a dramatic restoration of the normal erythroid transcriptional activator and repressor in undifferentiated program and increased differentiation responsiveness to the CD34 þ cells. anti-metabolite cytosine arabinoside (Ara-C).2 Notably, during On careful analysis and classification of the data, we noted Ara-C-induced differentiation of K562 wild-type cells, no NFI-A among the modulated genes a consistent number of erythroid upregulation was observed compared to the physio- genes. Many of these genes reflected an intermediate-to-mature logical erythroid program of human HPCs, which is normally erythroid developmental stage, and were in general accordance accompanied by increased NFI-A accumulation.2 Altogether with recent genome-wide analyses performed on a variety of this evidence suggests a defective tumor suppressor-like func- in vitro erythroid differentiation systems.6–8 Out of this extensive tion of NFI-A in K562 CML cells. In fact, a recent study number of potential target genes, a list of 27 genes correspond- performed using array-comparative genomic hybridization and ing to well-characterized erythroid-affiliated genes was used for DNA sequencing reported structural alterations of the NFI-A cluster analysis of samples using the Euclidean distance as a gene associated with different chronic malignant myeloid measure of similarity (Figure 1b). As shown in Figure 1b, the diseases.5 NFI-A cells displayed considerable induction of erythroid cell In this study, to adequately dissect the molecular network membrane molecules, including CDH1, the Rh antigen family downstream of NFI-A, we performed gene expression profiling (RHAG; RHC/RHD), SLC4A1, AQP1, ADD3 and SPTB; mole- of normal human uncommitted CD34 þ HPCs efficiently cules involved in the hemoglobin biosynthetic pathway, expressing exogenous NFI-A at high level. CD34 þ HPCs were including ALAS2 and globin chains HBB, HBA and HBD; purified from fresh cord blood obtained from healthy donors, growth factors and growth factor receptors, including INHBA, pre-stimulated in serum-free medium supplemented with SCF EFBN2 and IGF2; signal transduction molecules, including 50 ng/ml, Flt3 ligand 50 ng/ml, IL-3 20 ng/ml and TPO 20 ng/ml JAK2, AKT2 and GAB1; transcription factors or DNA-binding overnight and transduced with a lentiviral vector encoding , including JUNB, HOXB8, KLF2, SSBP3 and TRIM10;

Leukemia Letters to the Editor 1221 CD34+ HPCs

CD34+ HPCs

50 Vector Vector NFI-A NFI-A 40 TRIM10 30 AQP1 20

NFI-A mRNA 10 KLF2 0

ALAS2 Vector NFI-A

GAPDH

Fold difference Gene Symbol Gene Description (NFI-A/Vector)

JAK2 Janus kinase 2 1.9 CDH1 Cadherin 1 2.0 JUNB JunB proto-oncogene 1.9 INHBA Inhibin, beta A 2.4 ADD3 Adducin 3 2.2 HBD Hemoglobin, delta 2.2 SPTB Spectrin beta, erythrocytic 2.2 EFNB2 Ephrin-B2 2.3 AKT2 v-akt homolog 2 1.5 RHAG Rh-associated glycoprotein 1.7 GAB1 GRB2-associated binding protein 1 2.1 HBB Hemoglobin, beta 3.8 HBA2 Hemoglobin, alpha2 5.3 ALAS2 Aminolevulinic acid synthase 2 2.9 RHCE Rh blood group, CcEe antigens 2.7 RHD Rh blood group, D antigen 5.4 HOXB8 B8 5.5 KLF2 Kruppel-like factor 2 4.9 HBA1 Hemoglobin, alpha1 5.2 SELENBP1 Selenium binding protein 1 6.6 IGF2 Insulin-like growth factor 2 8.1 SLC4A1 Erythrocyte membrane protein band 3 10.5 CSF2RB GM-CSF receptor, beta, low-affinity -2.0 ETS1 v-ets homolog 1 (avian) -1.9 AQP1 Aquaporin 1 29.5 SSBP3 Single stranded DNA binding protein 3 40.7 TRIM10 Tripartite motif-containing 10 16.3 Vector NFI-A

Figure 1 NFI-A regulates several erythroid-affiliated genes. (a) Real-time PCR of NFI-A expression in Vector- and NFI-A-transduced GFP þ sorted CD34 þ HPCs 48 h after transduction. Mean±s.e.m. (n ¼ 3). (b) Hierarchical clustering of selected erythroid genes in the Vector- and NFI-A expressing CD34 þ cells. Relative levels of gene expression are depicted with a color scale, red being the highest levels and green the lowest levels of expression. (c) Semiquantitative RT–PCR of transcripts from Vector- or NFI-A-transduced CD34 þ cells using GAPDH as a normalizer.

finally, the intracellular transporter SELENBP1 as reported by erythroid-like gene expression signature as observed for CD34 þ Komor et al.6 and Macaulay et al.7 and reported in Supplemen- HPCs (RT–PCR primers are listed in Supplementary Table S2), tary References 1À27. Collectively, we can postulate that the thus suggesting a defective pro-differentiating or tumor suppres- gene expression profile analysis uncovered an extensive net- sor-like role of NFI-A in these cells. In fact, the kinetics of the work of erythroid genes regulated after NFI-A expression, tested mRNA accurately reflected the transcript profiles that particularly erythroid membrane skeleton proteins and hemo- emerged from the microarray performed in primary cells. globin biosynthesis effectors. To validate the microarray data, The RT–PCR screening indicated several relevant NFI-A- we performed semiquantitative reverse transcription (RT)–PCR regulated genes. However, gene expression alone cannot analysis of a few selected genes in CD34 þ HPCs transduced establish whether a directly or indirectly with Vector or NFI-A. As shown in Figure 1c, enforced binds to its target genes. To address this question, we focused on expression of NFI-A parallels a vigorous accumulation of the two genes that have been characterized for their essential selected mRNAs, confirming the gene expression profiling function in erythroid cells: SLC4A1 and ALAS2. The SLC4A1 observations. gene encodes the major anion exchanger of the red cell To extend the significance of the microarray data in a CML (chloride-bicarbonate) also called band 3, or anion exchanger setting, we performed semiquantitative RT–PCR analysis of 10 1 (AE1), (an integral component of the cytoskeletal network modulated genes, selected for their biological relevance, in responsible for the unique functional properties of the erythro- K562 CML blast crisis cells transduced with the control Vector cyte membrane) whose alteration has been linked to the or NFI-A with or without Ara-C treatment (Figure 2a). NFI-A pathophysiology of dyserythropoietic conditions, (Supplemen- enforced expression in K562 cells potentiates the differentiating tary References 5,28). ALAS2 represents the erythroid specific effect of Ara-C2 by recapitulating or restoring the induction of an form of the ALAS enzyme, located in the mitochondria and

Leukemia Letters to the Editor 1222

Vector Vector ALAS2 HBA1 NFI-A NFI-A

Vector Vector TRIM10 HBA2 NFI-A NFI-A

Vector Vector AQP1 SPTB NFI-A NFI-A

Vector Vector KLF2 RHD NFI-A NFI-A

Vector Vector SLC4A1 RHCE NFI-A NFI-A

Vector Vector GAPDH GAPDH NFI-A NFI-A

SLC4A1 ALAS2 Vector 0.015 Vector 0.015 NFI-A NFI-A 0.010 0.010

0.005 0.005 (Relative Units) (Relative Units) NFI-A Occupancy NFI-A Occupancy 0.000 0.000 Ctr 48 96 Ctr 48 96 Hours Ara-C Hours AraC

Figure 2 Validation of NFI-A-induced upregulation of select genes from the microarray data in K562 CML cells. (a) Semiquantitative RT–PCR of transcripts from Vector- or NFI-A-transduced K562 cells treated or not with Ara-C for 96 h using GAPDH as a normalizer. (b) Real-time PCR of NFI-A binding to SLC4A1 and ALAS2 promoter regions of Vector- and NFI-A-transduced K562 cells treated with or without Ara-C. The relative occupancy of NFI-A present on the promoter (prom) specific regions is normalized to the Input (I) signal. The NFI-A-specific signal was then normalized to the signal obtained from the amplification of an intergenic region of the Tubulin gene (UR) following the algorithm: ((IPÀBO)/I)PROM/((IPÀBO)/I)UR. IP represents chromatin immunoprecipitated by the NFI-A antibody. BO (beads only) represents nonspecific signal in absence of antibody. Mean±s.e.m. (n ¼ 3).

expressed at high levels in red blood cells where it catalyzes the occupancy was observed on the proximal promoter regions of first step in the heme biosynthetic pathway. ALAS2 is mutated in both SLC4A1 and ALAS2 genes only in exogenous NFI-A- X-linked sideroblastic anemia and knockout of ALAS2 leads to expressing cells in the presence of Ara-C, indicating a direct severe anemia with an increase in nucleated definitive red cells binding and transcriptional activation during erythroid and accumulation of siderotic iron as reported in Supplementary differentiation (Figure 2b). References 29,30. To test whether NFI-A is actively recruited to In this study we established that NFI-A per se is able to induce the SLC4A1 and ALAS2 proximal promoters, we first scanned an erythroid transcriptional program in both CD34 þ HPCs and these regions in silico using UCSC genome browser (http:// leukemic K562 cells and acts directly at the proximal promoter genome.ucsc.edu/) combined with Chip-Mapper (http://bio. regions of two fundamental erythroid genes, SLC4A1 and chip.org/mapper/mapper-main), Alibaba 2.1 (http://darwin.nmsu. ALAS2. These results combined with our previous reports2–4 edu/~molb470/fall2003/Projects/solorz/aliBaba_2_1.htm) and show a critical role for NFI-A in the transcriptional activation of Mat-Inspector Professional (http://www.genomatix.de/products/ key erythroid molecules as well as its silencing as a prerequisite MatInspector/) software programs. Indeed, we found candidate for myelopoiesis. In conclusion, we along with others5 suggest NFI-A binding sites embedded in phylogenetically conserved that NFI-A alterations, such as aberrant genomic copy number regions approximately 750 and 75 bp upstream of the SLC4A1 or mutations, might entail a pathogenic relevance in hemato- (Supplementary Figure S1a) and ALAS2 (Supplementary logical disorders, including leukemia, hemoglobinopathies, Figure S1b) transcriptional start site, respectively. Chromatin myeloproliferative and dyserythropoietic disorders. immunoprecipitation was performed using an anti-NFI-A anti- body and analyzed by real-time PCR in Vector- and NFI-A transduced K562 CML cells in the absence or presence of Ara-C Conflict of interest as described2 (chromatin immunoprecipitation primers are listed in Supplementary Table S2). As a result, increasing NFI-A The authors declare no conflict of interest.

Leukemia Letters to the Editor 1223 Acknowledgements 2 Starnes LM, Sorrentino A, Pelosi E, Ballarino M, Morsilli O, Biffoni M et al. NFI-A directs the fate of hematopoietic pro- This work was supported by grants from the Italian Association genitors to the erythroid or granulocytic lineage and controls for Cancer Research (AIRC) to CN and CP; University of Roma ‘La beta-globin and G-CSF receptor expression. Blood 2009; 114: 1753–1763. Sapienza’ and Ministero dell’Istruzione, Universita` e Ricerca. 3 Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C et al. LM Starnes1,2,6, A Sorrentino3,6, M Ferracin4, A minicircuitry comprised of microRNA-223 and transcription M Negrini4, E Pelosi3, C Nervi1,2 and factors NFI-A and C/EBPalpha regulates human granulopoiesis. C Peschle5 Cell 2005; 123: 819–831. 1 4 Rosa A, Ballarino M, Sorrentino A, Sthandier O, De Angelis FG, Department of Histology and Medical Marchioni M et al. The interplay between the master trans- Embryology, University ‘La Sapienza’, Rome, Italy; 2 cription factor PU.1 and miR-424 regulates human monocyte/ Institute of cell biology and tissue engineering, macrophage differentiation. Proc Natl Acad Sci USA 2007; 104: San Raffaele Biomedical Park Foundation, 19849–19854. Rome, Italy; 3 5 Bernard F, Gelsi-Boyer V, Murati A, Giraudier S, Trouplin V, Department of Hematology, Oncology Ade´laı¨de J et al. Alterations of NFIA in chronic malignant myeloid and Molecular Medicine, Istituto Superiore di Sanita`, diseases. Leukemia 2009; 23: 583–585. Rome, Italy; 6 Komor M, Gu¨ller S, Baldus CD, de Vos S, Hoelzer D, Ottmann OG 4Department of Experimental and Diagnostic Medicine, et al. Transcriptional profiling of human hematopoiesis during University of Ferrara, Perrara, Italy and in vitro lineage-specific differentiation. Stem Cells 2005; 23: 5Division of Hematology-Oncology, IRCCS MultiMedica, 1154–1169. Milan, Italy 7 Macaulay IC, Tijssen MR, Thijssen-Timmer DC, Gusnanto A, E-mail: [email protected] Steward M, Burns P et al. Comparative gene expression profiling 6These authors contributed equally to this work. of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 2007; 109: 3260–3299. References 8 Cui K, Zang C, Roh TY, Schones DE, Childs RW, Peng W et al. Chromatin signatures in multipotent human hematopoietic stem 1 Gronostajski RM. Roles of the NFI/CTF gene family in transcription cells indicate the fate of bivalent genes during differentiation. and development. Gene 2000; 249: 31–45. Cell Stem Cell 2009; 4: 80–93.

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Synergistic activity of the Src/Abl inhibitor bosutinib in combination with imatinib

Leukemia (2010) 24, 1223–1227; doi:10.1038/leu.2010.79; against platelet-derived growth factor receptor and c-Kit. In published online 6 May 2010 addition, structural and modeling data suggest that bosutinib binds an intermediate/active conformation of Bcr-Abl, whereas imatinib binds only the inactive conformation of Bcr-Abl.2 Chronic myeloid leukemia (CML) is a hematopoietic disorder Moreover, in a recently published in vitro screening study, caused by a translocation occurring between 22 bosutinib was tested against a panel of the most frequent imatinib- and chromosome 9, leading to the formation of the Philadelphia resistant Bcr-Abl mutants and was found to have activity against chromosome (Ph) and the BCR-ABL fusion gene. The oncogenic the large majority of them.3 Bosutinib also possesses additional protein tyrosine kinase expressed by this gene fusion, Bcr-Abl, is inhibitory activities absent in the imatinib inhibition profile.4 the underlying cause of CML. Considering the differences in the binding features and in the The Abl kinase inhibitor imatinib was registered for clinical selectivity profiles between bosutinib and imatinib, a combina- use in 2001 and is now the current first-line treatment for CML. tion of these two drugs might be more effective than single agent Beside the activity against Abl, imatinib can also inhibit platelet- treatment in reducing the risk of selecting resistant clones. derived growth factor receptor-b and c-Kit, which is believed to In this paper, we report the synergistic cooperation between contribute to a number of side effects such as edema, muscle bosutinib and imatinib in several Bcr-Abl-positive cell lines as cramps, skin rash, pigmentation and low-grade inhibition of well as in samples derived from patients with CML. normal hemopoiesis. In addition, imatinib is unable to eradicate Bosutinib was provided by Wyeth, whereas imatinib was the leukemic cells and resistance to it can develop, especially in synthesized by Dr Enrico Rosso (University of Venice, Italy). The advanced patients, thus leading to short-term remissions.1 antiproliferative activity for the single agents as well as for the Resistant clones carrying point mutations on Bcr-Abl protein combined treatment was assessed by thymidine incorporation assay. sequence can be found in approximately 50% of patients with Bosutinib was tested in combination with imatinib on a panel advanced disease. of Bcr-Abl-positive cell lines across a wide range of concentra- Recently, several new inhibitors have been developed with the tions and at different bosutinib/imatinib ratios (1:3, 1:10 and aim of increasing both potency and selectivity against Abl. Among 1:33). The concentrations tested were equally distributed 5 them, bosutinib (SKI-606; Wyeth, Pearl River, NY, USA) is a dual around the IC50 values as suggested by Chou and co-workers. Src/Abl inhibitor that possesses an in vitro activity in the low The combination indices (CI) calculated are reported in Table 1 nanomolar range on several Bcr-Abl-positive cell lines and it is and proliferation studies are presented in Figure 1a. For all the presently in phase III clinical trials.2 Bosutinib is devoid of activity cell lines tested, the CI indicates an extremely positive

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