Articles and Brief Reports Hematopoiesis & Hematopoietic Stem Cells HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4

Céline Auvray, 1,2 Andrée Delahaye, 1-3 Françoise Pflumio, 4 Rima Haddad, 5 Sophie Amsellem, 6 Ayda Miri-Nezhad, 1,2 Loïc Broix, 1,2 Azzedine Yacia, 1,2 Frédérique Bulle, 1,2 Serge Fichelson, 1,2 * and Isabelle Vigon 1,2 *

1Inserm U1016, Institut Cochin, Paris; 2Université Paris Descartes, Sorbonne Paris Cité; 3Université Paris 13, INSERM U676, Service Histologie, Embryologie et Cytogénétique, Hôpital Jean Verdier, Bondy; 4Inserm U967, Université Paris 7, Commissariat à l’Energie Atomique, Fontenay-aux-Roses; 5INSERM U935, Faculté de Médecine Paris-Sud 11, le Kremlin-Bicêtre; 6Institut Gustave Roussy, Villejuif, France.

ABSTRACT *SF and IV contributed equally to this manuscript Background Expansion of hematopoietic stem cells represents an important objective for improving cell and Funding: this work was supported therapy protocols. Retroviral transduction of the HoxB4 homeogene in mouse and human by the French Ligue Nationale hematopoietic stem cells and hematopoietic progenitors is known to promote the cells’ expan - Contre le Cancer (LNCC) sion. A safer approach consists in transferring into hematopoietic stem cells [EL2009.LNCC/SF2], and by the taking advantage of the natural ability of homeoproteins to cross cell membranes. Thus, Association pour la Recherche HOXB4 transfer is operative for expanding human hematopoietic cells, but such expan - sur le Cancer (ARC) sion needs to be improved. [A08/1/1048]. Design and Methods Manuscript received on To that aim, we evaluated the effects of HOXC4, a protein encoded by a HOXB4 paralog gene, July 11, 2011. Revised by co-culturing HOXC4-producing stromal cells with human CD34 + hematopoietic cells. version arrived on September 13, 2011. Manuscript accepted Numbers of progenitors and stem cells were assessed by in vitro cloning assays and injection on October 18, 2011 into immuno-deficient mice, respectively. We also looked for activation or inhibition of target downstream . Correspondence: Serge Fichelson, Institut Cochin, Results Département Immunologie- We show that the HOXC4 homeoprotein expands human hematopoietic immature cells by 3 Hématologie, Maternité Port- to 6 times ex vivo and significantly improves the level of in vivo engraftment. Comparative tran - Royal 5ème étage, 123 Bd de scriptome analysis of CD34 + cells subjected or not to HOXB4 or HOXC4 demonstrated that Port-Royal, 75014 Paris, France. both homeoproteins regulate the same set of , some of which encode key hematopoietic Phone: international factors and signaling molecules. Certain molecules identified herein are factors reported to be +33.153104381. involved in stem cell fate or expansion in other models, such as MEF2C, EZH2, DBF4, DHX9, Fax: international YPEL5 and Pumilio. +33.143251167. E-mail: [email protected] Conclusions The present study may help to identify new HOX downstream key factors potentially involved The online version of this article in hematopoietic stem cell expansion or in leukemogenesis. has a Supplementary Appendix.

Key words: hematopoietic stem cell, hematopoiesis, stem cell transplantation, cell therapy.

Citation: Auvray C, Delahaye A, Pflumio F, Haddad R, Amsellem S, Miri-Nezhad A, Broix L, Yacia A, Bulle F, Fichelson S, and Vigon I. HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4. Haematologica 2012;97(2):168-178. doi:10.3324/haematol.2011.051235

©2012 Ferrata Storti Foundation. This is an open-access paper.

168 haematologica | 2012; 97(2) Human HSC expansion by HOXC4

Introduction fied herein (MEF2C, EZH2, DBF4, DHX9, YPEL5, Pumilio) are involved with stem cell fate or expansion in All blood cells derive from multipotent hematopoietic other models, thus corresponding to important targets for stem cells (HSC) that have the capacity of both long-term further studies. self-renewal and differentiation, according to body needs. Because of their scarcity, expansion of HSC repre - sents a major challenge for improving engraftment effi - Design and Methods ciency for cell or gene therapy applications. Ex vivo expansion of human hematopoietic cells currently relies Construction of the HOXC4 vector on the use of high concentrations of cytokines and The HOXC4 cDNA was a gift from Dr. Daga. 10 The mouse growth factors. However, the value of this practice is immunoglobulin k-chain leader sequence for protein secretion limited since it often leads to irreversible differentiation was inserted upstream of the HoxC4 sequence. That construct of HSC in the culture. An alternative approach consists in was then cloned into the TRIP vector plasmid, as described else - using transcription factors involved in HSC self-renewal where. 11 or maintenance. Among them, the HOXB4 homeopro - tein was identified as a major expansion factor of mouse Lentiviral vector production and transduction and human HSC after retroviral transduction of the HOXC4- , HOXB4- , or enhanced green fluorescent protein HoxB4 coding sequence. 1-3 Although that gene was first (EGFP )-containing lentiviral vectors were generated as described described as non-leukemogenic, a recent study revealed previously. 7 Vector particle concentrations were normalized that transduction of HoxB4 into the HSC of large animals according to the p24 human immunodeficiency virus (HIV-1) could lead to the late emergence of acute myeloid capsid protein content of the supernatants. Infectious particles leukemias. 4 Thus, retrovirus-mediated genetic alterations were also counted using serial dilutions of supernatants onto of HSC along with constitutive expression of human wild-type human embryonic kidney (HEK)-293T cells. Mouse HOXB4 can be hazardous for therapeutic applications. stromal MS-5 cells grown to 70% confluence were transduced To overcome this problem, we established an alternative for 24 h with 2.5 µg/mL of p24 from recombinant vectors. Cells expansion method taking advantage of the property of were then washed and cultured for at least five passages before homeoproteins to translocate spontaneously and use. reversibly through membranes and reach the cytoplasm and nucleus. 5,6 Long-term culture of human CD34 + imma - Cell lines ture cells in the presence of the homeoprotein induces The cell lines MS-5 wild-type, MS-5/GFP (MS-5 transduced expansion of HSC and hematopoietic progenitors of the with a lentiviral vector containing the EGFP cDNA, referred to myeloid and lymphoid lineages. Expanded cells have an as control), MS-5/HOXC4 and MS-5/HOXB4 (MS-5 transduced enhanced capacity to repopulate in vivo and to maintain with vectors containing the human HoxC4 or HoxB4 cDNA, their pluripotentiality. 7-9 respectively) were grown in alpha-minimum essential medium Nevertheless, whatever the technology used, the (α-MEM) containing 10% fetal calf serum (FCS) (Invitrogen, HOXB4-mediated expansion of HSC and progenitors is Cergy Pontoise, France). always somewhat lower in humans than in mice. We, therefore, decided to examine whether using HOXC4 Isolation, immuno-labeling and cultures of CD34 + cells would improve expansion efficacy. Actually, retroviral Human immature hematopoietic cell isolation, labeling, cul - transduction of HOXC4 , a paralog of the HOXB4 gene, ture and cloning assays were performed as already described, 7-9 had been shown to cause the expansion of human and fully presented in the Online Supplementary Design and hematopoietic progenitors. 10 In the present study, we Methods section. demonstrate that HOXC4 protein transfer into human CD34 + hematopoietic cells by way of co-culture with Cell expansion analysis MS-5 stromal cells engineered to actively secrete this The ‘relative fold expansion’ was calculated as the fold- homeoprotein, induces 3- to 6-fold expansion of HSC and increase in HSC expansion in the presence of MS-5/HOXC4 or hematopoietic progenitors. MS-5/HOXB4 cells, divided by that in the presence of MS-5/GFP The human genes regulated by homeoproteins during cells. The ‘absolute fold expansion’ was calculated as the total hematopoiesis are mostly unknown. We, therefore, chose number of HSC recovered per culture at day X, divided by that to search for potential effectors of HOXB4 and HOXC4 at day 0. using comparative transcriptome analysis of CD34 + human cells following exposure to these factors. We rea - Mouse assays soned that, since HOXB4 and HOXC4 display important Human cord blood CD34 + cells (5 ¥10 3 cells) were co-cultured molecular analogies and have similar temporal and spatial for 4 weeks with MS-5/GFP, MS-5/HOXB4, or MS-5/HOXC4. expression patterns during embryogenesis, both mole - Cells were collected and the whole CD34 + cells issued from the cules should influence the expression of the same set of initial 5 ¥10 3 cell input were sorted then injected into 3-Gy irra - genes. We show herein that the transcriptomes from diated NOD/LtSz-scid/scid (NOD-SCID) mice. More details on CD34 + cells exposed to HOXB4 or HOXC4 are virtually the numbers of injected cells are presented in the Online identical. Gene expression profiling revealed that various Supplementary Design and Methods section. Cord blood CD34 + sets of genes encoding key hematopoietic factors and sig - cells from day 0 (5 ¥10 3 cells, along with 10 5 CD34 neg irradiated naling pathway molecules (KLF10, HNRPDL, IKZF, and accessory cells) were used as controls. Seven to 8 weeks later, hypoxia, , IGF-1, 14-3-3 and angiopoietin-1 signaling) nucleated cells from the bone marrow of transplanted animals were either activated or repressed after cell exposure to were analyzed by flow cytometry for the presence of human these homeoproteins. Moreover, certain molecules identi - cells. Mice were considered positive when at least 1% of human

haematologica | 2012; 97(2) 169 C. auvray et al.

cells were detected among mouse bone marrow cells. Animal Microarray and hybridization experiments were approved by the Ethical Committee of the Human CD34 + cord blood (4 ¥10 6) cells were co-cultured for 24 French Agriculture Department. h with irradiated EGFP-, HOXB4- or HOXC4-transduced MS-5 cells. Cells were lysed and total RNA extracted using the Trizol Cytofluorometry RNA method (Invitrogen). Purified RNA samples were The presence of human cells in NOD-SCID mouse bone mar - processed for use on DNA arrays prepared by Dr. Gidrol’s team row was determined using phycoerythrin (PE)-conjugated anti - (CEA, Genopole Evry, France) from the Mediante database. The body to human CD45 (clone J33). These cells were immunophe - integrity of the RNA samples was verified using agarose gel elec - notyped with allophycocyanin (APC)-conjugated anti-CD34 trophoresis and the Bioanalyzer 2100 (Agilent, Massy, France). (clone 581), fluorescein isothiocyanate (FITC)-conjugated anti- RNA samples were reverse-transcribed using random primers, CD14 (clone RMO14), anti-CD15-FITC (clone 80H5), and phy - and aminoallyl-dUTP was incorporated for indirect labeling coerythrin-cyanine 5 (PC5)-conjugated anti-CD19 (clone J3-119) with Cy3 and Cy5. Three competitive hybridizations to the antibodies (Beckman Coulter, Villepinte, France). arrays were performed in duplicate using a dye-swap strategy. 5,6-carboxyfluorescein-diacetate-succinimidyl-ester GeneChip data analysis labeling Arrays were scanned and features were extracted using For 5,6-carboxyfluorescein-diacetate-succinimidyl-ester Genepix 6.0 software (MDC/Axon Instruments, Sunnyvale, CA, (CFSE) labeling, CD34 + cells were incubated with 2.5 mM CFSE USA). Data analysis, including intensity-dependent Lowess nor - (Molecular Probes, Eugene, OR, USA) for 10 min at 37°C then malization of the raw data, was performed using Bioconductor quenched with cold FCS. Labeled cells were then co-cultured software (R package version 1.9). Ingenuity Pathways Analysis with MS-5/HOXB4, MS-5/HOXC4, or MS-5/GFP and analyzed (IPA, Redwood City, CA, USA) was used to study the HOXB4- on days 3 and 6 using a FACSCalibur (Becton-Dickinson, Le Pont and HOXC4-mediated gene regulation profile in human CD34 + de Claix, France). cells. Microarray data are available in the Gene Expression Western blots Omnibus (GEO), accession number GSE24379. Methods for western blot analysis of cell products are present - ed in the Online Supplementary Design and Methods section. Gene expression analysis RNA samples were reverse-transcribed with SuperScript II (Invitrogen) reverse transcriptase by using random hexamers at 42°C. The cDNA obtained were used for semi-quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) of 28 genes, using the primers described in Online Supplementary Table S1 . Cycling conditions were: 95°C for 10 s, 60°C for 7 s, and 4 4 4 4

B C 72°C for 10 s, for a total of 40 cycles. B C x x P x x

A P o o F o o F H H G H H G / / / / / / 5 5 5 5 5 5 5 5 ------S S S S S S S S M M M M M M M M Results HoxC4 34 kDa HoxB4 43 kDa Recombinant MS-5/HOXC4 cells actively secrete 42 kDa 42 kDa b-actine b-actine the HOXC4 protein HoxB4 HoxC4 antibody antibody To transfer the human HOXC4 protein into HSC, we used a strategy consisting of co-culturing human CD34 + hematopoietic cells with MS-5 cells engineered to secrete 7 B MS5/HOXC4 hematopoietic cells hematopoietic cells HOXC4, as we reported previously with HOXB4. To cultured on cultured on this end, we first established the MS-5/HOXC4 stromal MS5/HOXC4 MS5/GFP cell line that could supply a continuous source of this homeoprotein. After lentiviral gene transduction, most MS-5 cells stably expressed the HOXC4 or the HOXB4 protein (as shown by western blotting, Figure 1A) or GFP protein control (fluorescence analysis, not shown ). MS- 5/HOXC4 cells were identical to non-transduced MS-5 cells and to MS-5/HOXB4 and MS-5/GFP cells in appear - ance, growth rate, and phenotypic markers. The pres - Human b tubulin ence of the HOXC4 homeoprotein in the cells co-cul - Human HOXC4 tured with secreting stromal cells was detected by west - ern blotting (Figure 1B). To avoid stromal cell contamina - tion, hematopoietic cells were co-cultured with Transwell permeable supports. Figure 1. Western blot analysis of ( A) HOXC4 protein by MS-5 engi - neered cells. HOXC4 was present at the expected size only in the MS- HOXC4 induces expansion of human 5 cells transduced by the HOXC4-coding vector; ( B) HOXC4 protein hematopoietic cells transduction into human hematopoietic target cells co-cultured with In order to establish the ability of HOXC4 to expand HOXC4-secreting MS-5 cells: whole hematopoietic cell lysate revealed the presence of HOXC4 at the expected size. human HSC and hematopoietic progenitors in vitro , we first analyzed the division profiles of CD34 + cells

170 haematologica | 2012; 97(2) Human HSC expansion by HOXC4

Day 3 Day 6 Figure 2. CD34 + cell division kinetic profiles during co-cul - 60 60 tures. Representative flow cytometry profiles of CFSE- +1 n 50 50 +3 +2 +1 labeled CD34 + cells co-cultured with MS-5/HOXB4 (red line), 40 40 MS-5/HOXC4 (yellow line), or s l

l MS-5/GFP (green line). The s s e t t +4 n c n position of the non-dividing

u 30 u 30 f o o

o cells corresponds to fluores -

C C r

e cence intensity of the cells at b 20 20 Day-0 (gray line). N = number

m +2 +5

u of cell divisions between day-0 N 10 10 and the first day-3 peak; the number above each peak rep - 0 0 resents additional numbers of 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 divisions, according to the fluo - CFSE CFSE rescence intensity. CFSE

exposed to HOXC4, by comparison with HOXB4 or pared to cells co-cultured with MS-5/GFP (6/8 versus 1/8 none. Cell division kinetics during cultures was assessed positive animals, respectively) ( P<0.05). By contrast, by measuring fluorescence after CFSE cell staining equivalent engraftment ability was observed when com - (Figure 2). At day 3, CD34 + cells co-cultured with MS- pared to cells cultured in the presence of MS-5/HOXB4 5/HOXC4 cells yielded at least one more cell generation (5/8 positive animals), Moreover, the repopulating capac - than CD34 + cells co-cultured with MS-5/GFP cells, and at ity in terms of proportion of human cells per mouse was day 6 a mean of two or three supplementary divisions clearly greater in cultures with MS-5/HOXC4 than in cul - was observed, alike with MS-5/HOXB4. Thus, HOXC4 tures with MS-5/GFP (7.8% versus 1.8%, respectively) induced the expansion of CD34 + cells, indicating that this (Figure 4A). These findings demonstrate that HOXC4 homeoprotein rapidly activates the transcription of genes induces clear-cut ex vivo expansion of SRC in culture, involved in the regulation of cell division. while maintaining their capacity of long-term reconstitu - To evaluate cell proliferation in vitro , hematopoietic tion in animals. Phenotypic analysis revealed that the cells in long-term cultures were assessed every week induction of SRC expansion in vitro by HOXC4 did not over 4-5 weeks for the number of: (i) total cells, (ii) total change their differentiation program in vivo : the propor - CD34 + cells, (iii) total clonogenic progenitors (colony- tions of human myeloid cells (CD14 +CD15 +), B lymphoid forming cells), (iv) total immature progenitors (long-term cells (CD19 +), and CD34 + cells were not altered when culture-initiating cells), and (v) HSC in vivo defined as compared to control cells (Figure 4B). Finally, we looked immature SCID-repopulating cells (SRC). These values for a potential synergistic effect of HOXB4 and HOXC4 were used to calculate the expansion rates of these dif - on HSC and progenitor cells. We found that hematopoi - ferent populations. For total cells, CD34 + cells, colony- etic cells exposed to both HOXB4 and HOXC4 (by co- forming cells and long-term culture-initiating cells, - culture with MS-5/HOXB4/C4, a cell line transduced tive (Figure 3A-D) and absolute (Figure 3E-H) expansion with both HOXB4 and HOXC4 cDNA) just displayed levels observed in the presence of MS-5/HOXC4 cells moderate improvement in the rate of cell expansion, were comparable to those observed with MS-5/HOXB4. compared with HSC exposed to HOXB4 or to HOXC4 For total cells, CD34 + cells, and colony-forming cells, the alone ( Online Supplementary Figure S1 ). This suggested an best relative expansion rates were observed at weeks 2 additive effect of HOXB4 and HOXC4 activities rather and 3 of co-culture. Absolute expansion of total cells was than true synergy. maximal at week 4 of co-culture, probably due to the progressive accumulation of maturing cells in the cul - Determination of early molecular targets of HOXB4 tures. Of note, colonies from colony-forming cells gener - and HOXC4 in immature hematopoietic cells ated in the presence of HOXC4 were similar in size and Transcriptome experiments in cell composition to those from control cultures. At the molecular level, the mechanisms of action of Hox Finally, the expansion rates of the progenitors were genes during hematopoiesis remain elusive. Although equivalent to those obtained in the presence of MS- reports have described the involvement of Hox genes in 5/HOXB4 using a similar co-culture method ( Online some molecular pathways in mice, 12,13 the HOX protein tar - Supplementary Figure S1 ). gets in human hematopoietic cells are mostly unidentified. To determine the effect of HOXC4 on SRC expansion, Since HOXB4 and HOXC4 are encoded by paralogous CD34 + cells (5000 cells plated at day 0) were expanded genes that display similar effects on HSC and progenitors, for 4 weeks in the presence of HOXC4 and the progeny we presumed that both factors regulate the same target was injected to NOD-SCID mice. Bone marrow from genes during human HSC expansion. To identify early fac - injected mice was analyzed for human stem cell engraft - tors and signaling pathways involved in cell expansion con - ment 7-8 weeks after injection. SCR co-cultured with secutive to the action of HOXB4 or HOXC4, we per - MS-5/HOXC4 were significantly expanded when com - formed a differential transcriptome analysis of RNA sam -

haematologica | 2012; 97(2) 171 C. auvray et al.

ples from cord blood-derived CD34 + cells cultured for 24 h observed in the arrays were confirmed by RT-qPCR per - in the presence of MS-5/HOXB4 or MS-5/HOXC4 cells. formed on a series of 28 selected genes encompassing all MS-5/GFP cells were used as a control in separate co-cul - the scale of changes ( Online Supplementary Figure S3 ). tures to exclude non-specific changes in gene expression Overall, the transcriptomes from hematopoietic cells which may be associated with the presence of lentivirus- exposed to HOXB4 or HOXC4 were virtually similar. transduced MS-5 cells. RNA were hybridized with micro- Indeed, direct comparison of transcriptomes of CD34 + array slides containing probes that represented 21,353 dif - cells exposed either to HOXB4 or HOXC4 did not show ferent human genes. Duplicate, both-sense experiments any difference in gene expression profiles. This was con - were performed to compare transcriptomes either directly firmed by indirect transcriptome comparison which just (cells exposed to HOXB4 versus HOXC4) or indirectly (cells revealed some insignificant differences. exposed to HOXB4 versus GFP versus HOXC4), as illustrat - When applying a fold-change of 0.5, we found that 422 ed in Online Supplementary Figure S2 . genes were up-regulated and 167 genes down-regulated after exposure to HOXB4/C4 with regard to GFP. A list of Differential analysis of hematopoietic cell transcriptomes these genes is available in Online Supplementary Table S2 Global variations of gene expression obtained by (and Table S2 xlsx). hybridization of probe-containing slides with the cDNA Further analysis of the transcripts whose expression was from the various CD34 + cultured cells are deposited in altered after contact with HOXB4/C4 proteins ( versus GFP GEO database (accession number: GSE24379). HOXB4 or control) comprised: first, identifying cardinal functions HOXC4 was considered to have an influence on gene and canonical molecular pathways that involve the identi - expression when the P-value between HOX and control fied genes and second, comparing these gene expression micro-array results was less than 0.05 and the log 2-fold patterns with those reported in the literature in other cell change was higher than 0.5. Changes in gene expression models.

A B C Relative expansion of CFC Relative expansion of CD34 + cells Relative expansion of total cells 600 600 450 HoxB4 400 500 500 HoxB4 HoxB4 HoxC4 350 HoxC4 HoxC4 400 300 400 250 % % % 300 300 200 150 200 200 Control 100 Control Control 100 50 100 0 0 0 Week 2 Week 3 Week 4 Week 2 Week 3 Week 4 Week 2 Week 3 Week 4 D E F Relative expansion of LTC-IC Absolute expansion of total cells Absolute expansion of CD34 + cells 1200 60 160 GFP 1000 GFP HoxB4 140 HoxB4 50 HoxB4 e e

120 HoxC4 s HoxC4 s

800 a

a 40 HoxC4 e e r

r 100 % c c n n 600 i i 80 30

d d l l o

o 60 F

400 F 20 40 200 Control 20 10 0 0 0 Total number of LTC-IC Week 2 Week 3 Week 4 Week 2 Week 3 Week 4 G H Absolute expansion of CFC Absolute expansion of LTC-IC 140 35 Figure 3. Relative and absolute expan - + 120 GFP 30 GFP sion of total cells, CD34 cells, colony- HoxB4 HoxB4 forming cells (CFC), and long-term cul - 100 +

e 25 e ture-initiating cells (LTC-IC). CD34 cells s s HoxC4 HoxC4 a a e e 80 were co-cultured with MS-5/HOXB4,

r 20 r c c MS-5/HOXC4, or MS-5/GFP. Histograms n n i i

60 15 d

d (A-D) represent the fold expansion rela - l l o o F F 40 10 tive to MS-5/GFP (defined as 100%). Error bars represent ± standard devia - 20 5 tion. The Y axis of ( E) to ( H) indicates 0 0 fold expansion over the initial numbers Week 2 Week 3 Week 4 Total number of LTC-IC of cells studied.

172 haematologica | 2012; 97(2) Human HSC expansion by HOXC4

A 18 17 Figure 4. Ex vivo expansion of SRC co-cultured with MS- 16 5/HOXB4, MS-5/HOXC4, or MS- 15 5/GFP control cells. (A) 14 Proportion of human cells in the 13 bone marrow of NOD-SCID mice 12 transplanted with the total proge- 11 ny from 5000 initial human CD34+ cells, co-cultured for 4 10 weeks with MS-5/HOXB4 (n = 8), 9 MS-5/HOXC4 (n = 8), or MS- 8 5/GFP (n = 8). Each symbol rep- 7 resents results from a single 6 transplanted mouse. The three horizontal lines indicate the aver-

bone marrow (5000 cells) 5 age percentages of chimerism 4 among injected mice. (B) % % of human CD45+ cells in mouse 3 Representative flow cytometry 2 profiles showing multilineage 1 repopulation of engrafted human + 0 CD34 cells after 4 weeks of co- cultures with MS-5/GFP (i-iii), MS- GFP HoxB4 HoxC4 5/HOXB4 (iv-vi), or MS-5/HOXC4 (vii-ix) cells. Dot plots i, iv, and vii define gates for lymphoid and myeloid cells. The X axis indicates forward angle scatter (FSC), Y axis B MS-5/eGFP indicates side angle scatter 103 103 (SSC). Dot plots ii, v, and viii are

1023 gated on CD19 lymphoid cells. 102 102 Dot plots iii, vi, and ix are gated on CD14/CD15 myeloid cells. 101 101 Percentages indicate positive cells for the defined antigens. 100 100

0 0 1023 100 102 102 103 100 102 102 103 MS-5/SP-HOXB4 103 103 1023 102 102

101 101

0 0 SSC-Height

CD19 CD19 (PC5) 10 10 CD14-15 CD14-15 (FITC) 0 0 1023 100 102 102 103 100 102 102 103 MS-5/SP-HOXC4

103 103 1023 102 102

101 101

100 100 0 100 102 102 103 100 102 102 103 FSC-Height CD34 (APC)

Identification of cardinal functions, pathways and stem cell reg- eration”, “RNA post-transcriptional modifications”, ulatory factors whose expression was deregulated by “Protein synthesis” and “DNA replication, recombination HOXB4/C4 and repair” (Online Supplementary Figure S4A). Moreover, To understand the biological effects of HOXB4/C4 on numerous genes involved in cell cycle progression (such human HSC, all the 422 up-regulated and 167 down-regu- as Cyclins A2, D2, E2; CDC CDK2; Myc; Max; HDAC2) lated genes were submitted to ingenuity pathway analysis. were positively regulated following cell exposure to IPA analysis showed that the two major cell functions HOXB4/C4 whereas some genes involved in cell cycle called “Cell cycle” and “Cancer” were mainly involved in reduction (p57/Kip2; Cyclin D binding protein 1) were the HOXB4/C4-treated cells as well as, to a lesser extent, negatively regulated (Online Supplementary Table S3). important functions such as “Cellular growth and prolif- We also used ingenuity pathway analysis to identify

haematologica | 2012; 97(2) 173 C. auvray et al.

the molecular pathways implicated in HOXB4/C4-medi - teins family or related genes (listed in Online ated stem cell expansion. Thus, we identified key mole - Supplementary Table S5 ), among which Hspa1a , was up- cules belonging to specific pathways such as “Role of regulated by HOXB4/C4. This observation underlines BRCA1 in DNA damage response”, “Hypoxia signaling”, the potential importance of Hsp molecules in the regula - “Cell cycle G1/S and G2/M checkpoints”, or “Myc medi - tion of hematopoiesis by HOX proteins. ated apoptosis signaling” ( Online Supplementary Figure Finally, the expression of molecules previously known S4B and Table S4 ). to be involved in stem cell fate or expansion in other The percentages of genes whose expression was mod - models (MEF2C, EZH2, DBF4, DHX9, YPEL5, Pumilio) ified in each of these pathways are presented in Online was also regulated by HOXB4/C4 in our model. Supplementary Figure S4C : the expression of between 18% (for BRCA1 ) and 8% (for angiopoietin ) of the total Comparison of gene expression changes with those reported in genes from each pathway was up- or down-regulated. other models Elsewhere, it must be emphasized that the expression The study of gene regulation by HOXB4 or HOXC4 in of 14 genes belonging to the heat-shock-encoding pro - a variety of cellular models may reveal important com -

Table 1A. Genes whose expression was modulated by exposure of CD34 + cells to HOXB4 or HOXC4, and whose expression was also modified in the study by Deneault et al. 14 Gene symbol Gene (full name) Score # Accession N. ‡ Type of Protein Type of regulation* location molecule EGR1 early growth response 10 NM_001964 down nucleus transcription regulator MEF2C myocyte enhancer factor 2C 10 NM_002397 down nucleus transcription regulator NCL nucleolin 10 NM_005381 up nucleus other TRIM28 tripartite motif-containing 28 10 NM_005762 up nucleus transcription regulator BPTF bromodomain PHD finger 9 AL83375 down nucleus transcription regulator TES testis derived transcript (3 LIM domains) 9 NM_015641 up plasma membrane other KLF10 Kruppel-like factor 10 8 NM_005655 up nucleus transcription regulator GATA3 GATA binding protein 3 7 NM_002051 down nucleus transcription regulator HNRPDL heterogeneous nuclear ribonucleoprotein K 7 NM_005463 up nucleus other HNRPK heterogeneous nuclear ribonucleoprotein D-like 7 NM_002140 up nucleus other HOXA9 homeobox A9 7 NM_002142 up nucleus transcription regulator RNPS1 RNA binding protein S1, serine-rich domain 7 NM_006711 up nucleus other CEBPB CCAAT/enhancer binding protein (C/EBP), beta 6 NM_005194 up nucleus transcription regulator TSC22D1 TSC22 domain family, member 1 6 NM_006022 down nucleus transcription regulator ZMYND11 MYND domain containing 11 6 NM_006624 up nucleus other HBP1 HMG-box transcription factor 1 5 NM_012257 down nucleus transcription regulator IKZF1 ikaros family zinc finger 1 5 NM_006060 up nucleus transcription regulator LYAR Ly1 antibody reactive homolog (mouse) 5 NM_017816 up plasma membrane other RNF10 ring finger protein 10 5 NM_014868 up unknown other SUPT16H suppressor of Ty 16 homolog (S. cerevisiae) 5 BC021561 up nucleus transcription regulator SYF2 SYF2 homolog, RNA splicing factor (S. cerevisiae) 5 NM_015484 down nucleus other

#Scores are those established in the study by Deneault et al. ‡Accession numbers are from the PubMed database. The main functions of these genes are described in Online Supplementary Table S6. *Pattern of gene regulation observed in our study.

Table 1B. Representative examples of genes whose expression was modulated by HOXB4 in our study and which are homologous to those report - ed in the study by Deneault et al. Genes from the present report Regulation by HOXB4 Genes from Deneault et al. Score Common Functions NFkB inhibitors Negative NFkB1 9 HSC regulation RBBP8 Positive RBBP7 6 Rb-binding proteins CITED2 (score 4) Positive CITED4 5 Cbp/p300-interacting transactivators EZH2 Positive EZH1 5 Role in hematopoietic development EXOSC2 Positive EXOSC8 5 Ribosome RNA processing

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Table 2. Genes whose expression was altered in our study as a result of CD34 + cell exposure to HOXB4/C4, compared with data from the liter - ature acquired using different cell models. Reference Cell Populations Studied Gene Symbols* 12 Primary murine HSC: genes regulated after HoxB4 transduction CEBPB, UHRF1, HSPA1A, JMJD1A, MGEA5, HBP1 26 Murine primitive hematopoietic progenitor cell line, EML overexpressing HOXB4 (microarrays and (ChIP)-chip) EGR1,LGALS3, TES,TPM4, TSC22D1 27 Zebrafish, murine, and human HSC: study of common regulators AHNAK, DACH1, EGR1 28 Murine HSC: study of development regulators CDKN1C, WDR77, GATA3, LGALS1 29 Murine long-term versus short-term HSC and long-term CD53, CDKN1C, DACH1, TEK HSC versus multipotent progenitors 30 Genes up-regulated by NUP/HOXA10 and NUP/HOXD13 fusion proteins ARRB1, HOXA9, MEF2C, TSC22D1 31 Genes up-regulated in murine HSC ATM , CHD4 , CUL4A , G3BP1 , PSAP

32 Human G 0 HSC versus dividing HSC TNFSF4 , SOD2, MCM6 , CCNA2 , CDC2 33 Murine long-term versus short-term HSC FLT3 , MPL , HSPA1A 34 Human HSC Rho lo versus Rho hi ME2, PRKCH, SFXN1, TK1, ASF1B, EZH2, GSR, SLC40A1 , HBG1, HIST1H2BD, HIST1H2BK, HLA-DPB1, HNRPD, MCM5, MCM6 35 Murine hematopoietic cell line LGALS3, TES, TPM4, TSC22D1 21 Murine HSC and progenitors developing from ES overexpressing HOXB4 AASDHPPT, ANGPT1, CCND2, CD53, CDC6, CITED2, CMBL, (microarrays and (ChIP)-chip) DDX56, ELOVL6, GATA3, HBP1, IFRD2, RRK1, MARS, MYC, NAT10, NCBP, NFKBIZ, NOLC1, NPC2, PAICS, PFDN5, PRKDC, PRMT1, PRPF4, SOD2, TMEM97, TPM4, UCHL5, UHRF1, UHRF1, WDR77, XPOT, YPEL5 *Underlined bold-type genes are common to those listed in Table 1A. The main functions of these genes are presented in Online Supplementary Table S6. Bold type genes have to do with at least two studies listed here and ours; main functions of these genes are presented in Online Supplementary Tables S6 and S7.

mon pathways and factors that are regulated by these Discussion homeoproteins. To find key genes involved in stem cell renewal, Deneault et al. 14 performed a wide review of the There is a large body of evidence demonstrating the literature and established a database of nuclear factors in important role of HOX factors in hematopoiesis. The four which the selected genes were scored on the basis of their Hox paralog group 4 members, Hoxa4 , b4 , c4 , and d4 , level of expression, particularly in immature cell fractions. encode proteins with highly conserved homeodomains. According to this criterion, genes encoding these factors Hoxb4 gene disruption only induces very subtle hematopoi - were ranked from 1 to 10 relative to their potential impor - etic defects, 15 possibly because of a compensatory activity tance for stem cell maintenance and growth. We com - of the other three Hox genes. Recently, Iacovino et al. 16 pared the HOXB4/C4 up- and down-regulated genes list - showed that each Hox paralog group 4 member similarly ed in Online Supplementary Table S2 with the genes select - promotes proliferation and inhibits differentiation of ed by Deneault et al. whose score was 5 or more. In this hematopoietic progenitors derived from the c-kit +/CD41 + way we identified 21 genes in common with those from cell fraction from murine embryoid bodies, leading to Deneault’s study (Table 1A). The main functions of these expansion of undifferentiated hematopoietic cells in vitro . genes are described in Online Supplementary Table S6 . This indicates a potentially identical self-renewal activity Among these, KLF10 and HNRPDL are of particular inter - for all members of the Hox paralog group 4 genes in est since they belong to a series of 18 genes known to hematopoiesis derived from murine embryonic stem cells. confer a repopulation advantage to HSC. In our study, In addition to a possible redundancy within the Hox paralog HOXB4/C4 also induced changes in the expression of group 4, there are several examples of inter-paralog redun - some genes closely related to genes reported by Deneault dancies in hematopoiesis. This is demonstrated by the fact et al. (Table 1B). Among them, six (namely two genes that mice that underwent simultaneous disruption of sever - encoding NF kB inhibitors, RBBP , Cited , EZH2 , and al homeogenes such as Hoxb3 + Hoxb4 17 and Hoxa9 + Hoxb3 EXOSC ) belong to families that encode key factors for + Hoxb4 18 have important deficits in HSC proliferative stem cell regulation. capacity. However, these findings did not ascertain that the We also compared our data with published data from four Hox paralog group 4 members regulate the prolifera - divergent hematopoietic and embryonic stem cell mod - tion of human HSC via the same mechanisms. els, particularly studies including gene expression analy - sis of cells that over-expressed HOXB4. To that aim, we HOXB4 and HOXC4 proteins have similar effects examined 12 studies reporting the modulation of gene on the behavior of human hematopoietic progenitors expression in HSC. (Table 2). These genes are of partic - and stem cells ular relevance since they were described to be part of We have previously shown that the continuous pres - the HSC signature design in several models. ence of the HOXB4 protein in long-term co-cultures

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results in the expansion of the most immature HSC iden - angiopoietin-1 signaling). The involvement of these sig - tifiable in human, i.e. long-term culture-initiating cells and naling pathways is of particular importance, as discussed SRC, and of more mature progenitors. 7 In order to below. First, hypoxia has been implicated in the control of improve in vitro HSC expansion, and to circumvent poten - the metabolism and growth of numerous stem cells, and tial drawbacks such as those observed by Zhang et al. ,4 HSC in particular. 19 Second, it has been suggested that we transduced the HOXC4 protein into human HSC HOXB4 induces stem cell expansion through c-myc using the same method as for HOXB4. 7 We provide evi - expression, resulting in enhancement of HSC self-renew - dence that, like HOXB4, the human HOXC4 protein al. 20 In agreement, the study by Oshima et al. 21 and ours behaves as a growth factor able to enhance the number of showed that c-myc (and its partner in our study) was hematopoietic progenitors and stem cells. We had shown up-regulated in HSC exposed to HOXB4 (or to HOXC4). that in presence of the HOXB4 protein, the number of Third, IGF-1 signaling is required for the maintenance or SRC was amplified by approximately 2.1-fold after 4 growth of various stem cells, particularly mesenchymal weeks of cultures when compared to the number of SRC stem cells, 22 spermatogonia, 23 and hematopoietic stem from CD34 + cells cultured in the absence of HOXB4. and progenitors cells, and IGF-1 is involved in normal ery - Here, we demonstrate that SRC submitted to HOXC4 or thropoiesis, granulopoiesis, and lymphopoiesis. Fourth, to HOXB4 are expanded and maintained similarly. angiopoietin-1 was identified as an indispensable factor HOXC4-expanded primitive HSC could not be distin - for the quiescent and anti-apoptotic states of HSC, medi - guished from non-expanded HSC as they retained full ated by binding to its Tie2 on stem cells. 24 multilineage repopulating ability, and the mature cells Collectively, these findings are coherent with the derived from HOXC4-expanded HSC had normal pheno - hypothesis that HOXB4 and HOXC4 stimulate the initia - types. Hence, HOXC4 homeoprotein, like HOXB4, may tion of stem cell expansion by activating a number of be useful in clinical applications involving cell transfer. early genes involved in the cell cycle, which, in turn, Regarding our experimental model, one may wonder allow the progression of the cell cycle. HOXB4 and whether HOXB4- or HOXC4-dependent expansion is HOXC4 also modulate the expression of other genes or due to the homeoprotein activity in HSC or to other fac - pathways that have important functions in the tors attributed to HOX-expressing MS-5 cells. We previ - hematopoietic process: in particular, they up-regulate the ously showed that MS-5 cells engineered to secrete genes coding for the growth hormone, Ikaros, and several HOXB4 had no phenotypic changes. 7,9 In the present members of the ATP-binding cassette family, and down- study, we extended this observation to HOXC4 -trans - regulate mpl and GATA3 genes. Moreover, HOXB4 and duced MS-5 cells as well. This strongly argues for a direct HOXC4 modulate the expression of N-FATC2IP, which role of the secreted homeoproteins on human HSC. regulates N-FAT expression, and KU-70, DOCK2, and Additional studies using purified recombinant HOX pro - Ezrin, whose products interact with the hematopoietic- teins would be needed to confirm the proteins’ direct specific factor VAV-1. effect on HSC. Unfortunately, these proteins are difficult to purify, and improvements in the production and stabi - Some genes regulated by HOXB4 and HOXC4 lization of homeoproteins are required to allow the use of proteins in our study are also found in other purified proteins instead of co-cultures. models of stem cell regulation A comparison of our results with those of Deneault et HOXB4 and HOXC4 regulate the same early genes al. 14 revealed 21 genes (Table 1A) with similar expression in an identical manner patterns (score ≥5). Further examination of these genes We chose to study gene expression after short-term revealed that some are well known for their role in stem exposure of CD34 + cells to the homeoproteins because cell metabolism. Notably, KLF10 and HNRPDL belong to we believed that the main events regulating HSC expan - the gene set defined by these authors as able to induce sion would be rapidly initiated by the action of HOX substantial enhancement of HSC activity. KLF10 is partic - transcription factors on target genes. It must be empha - ularly relevant since it encodes a zinc-finger transcription sized that only a small fraction of the CD34 + cells are factor that induces early expression of transforming actually true repopulating cells. The gene expression growth factor- b. Thus, it may take part in microenviron - changes evaluated here could, therefore, just in part ment activity on stem and leukemia cells in bone marrow. reflect the changes present in these repopulating cells. Importantly, KLF10 is a target of VHL, 25 a factor involved The similarity of the gene expression regulation patterns in the ubiquitination and degradation of the hypoxia- observed in HOXB4- and HOXC4-treated cells suggests inducible factor (HIF), which plays a central role in oxy - that they operate through the same mechanisms of action gen-mediated regulation of gene expression, particularly on the same target genes. during hematopoiesis. Milsom et al. 15 suggested that HOXB4 could exert its HOXB4 and HOXC4 regulate genes important effects by inhibiting tumor necrosis factor (TNF)- α signal - in various cell growth pathways ing in HSC. Actually, the use of a gene disruption mouse Analysis of important pathways revealed that HOXB4 model for Fanconi anemia allowed that team to conclude and HOXC4 induce notable variations in the expression that over-expression of HOXB4 reversed the hypersensi - of key molecules involved in cell growth, differentiation, tivity of Fanconi anemia HSC to TNF- α and rescued the and transformation. Importantly, variations were in vivo engraftment capacity of these cells, probably medi - observed in the processes and pathways regulating the ated by the down-regulation of TNF- α receptor expres - cell cycle (checkpoint regulations, BTG-containing pro - sion in HoxB4 -transduced cells. In contrast to that study, tein regulation, RAN signaling), translation initiation our model did not allow us to conclude that TNF- α path - (EIF1 signaling), tumor suppression (BRCA2 and sig - way genes are regulated by HOXB4 or HOXC4, possibly naling), and HSC metabolism (hypoxia, Myc, IGF-1 and because our human HSC expansion model is notably dif -

176 haematologica | 2012; 97(2) Human HSC expansion by HOXC4

ferent from the Fanconi anemia mouse stem cell rescue In conclusion, we have demonstrated that the HOXC4 model by Milsom et al. , and therefore involves different homeoprotein displays clear-cut positive effects on pathways. Importantly, we noted that the modified growth and maintenance of human HSC and hematopoi - expression of 33 genes in our study is shared by genes in etic progenitors, similar to those previously reported with the study by Oshima et al. described as potential direct HOXB4 in other models. This is corroborated by almost targets of HOXB4. 21 identical transcriptomes of HOXB4- and HOXC4-treated Gene expression variations reported in various publica - cells. Target genes of homeoproteins may represent good tions often lead to divergent or contradictory results candidates for the development of new stem cell therapy because the models used in the studies are different. Our strategies using the products of HOX target genes instead model is the only one that takes into consideration the of homeoproteins themselves. These not yet clearly iden - effects of HOXB4 and HOXC4 proteins on gene expres - tified factors could be safer and more potent for the sion in human HSC. The results presented here show that expansion, self-renewal, and maintenance of human gene expression variations in our transcriptomes con - HSC. Some of them may also be involved in leukemic cerned important genes and pathways shared with other stem cell expansion or maintenance. studies, but also genes and pathways apparently specific to our model. Importantly, we found that the expression of genes involved in stem cell fate was modulated by the Authorship and Disclosures exposure of HSC to HOXB4 or HOXC4. Among these genes, some ( Mef2C , Ezh2 , Dbf4 , Dhx9 , Ypel5 , Pumilio ) are The information provided by the authors about contributions from also involved in the fate of stem cells from various tissues persons listed as authors and in acknowledgments is available with in several species of mammals, fishes, and insects. We are the full text of this paper at www.haematologica.org. currently performing functional studies of some of these Financial and other disclosures provided by the authors using the genes to determine their potential involvement in expan - ICMJE (www.icmje.org) Uniform Format for Disclosure of sion of HSC or regulation of hematopoiesis. Competing Interests are also available at www.haematologica.org.

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