LEDGF/p75 is dispensable for hematopoiesis but … pdfs/95.full.pdfLEDGF/p75 in normal hematopoiesis...

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HEMATOPOIESIS AND STEM CELLS

LEDGF/p75 is dispensable for hematopoiesis but essentialfor MLL-rearranged leukemogenesisSara El Ashkar,1 Juerg Schwaller,2 Tim Pieters,3,4 Steven Goossens,3,4 Jonas Demeulemeester,1 Frauke Christ,1 Siska Van Belle,1 Sabine Juge,2

Nancy Boeckx,5,6 Alan Engelman,7 Pieter Van Vlierberghe,3,4 Zeger Debyser,1,* and Jan De Rijck1,*

1Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit (KU) Leuven, Leuven,Belgium; 2Department of Biomedicine, University Children’s Hospital (UKBB), University of Basel, Basel, Switzerland; 3Center for Medical Genetics, GhentUniversity, Ghent, Belgium; 4Cancer Research Institute Ghent (CRIG), Ghent, Belgium; 5Department of Laboratory Medicine, University Hospital Leuven, Leuven,Belgium; 6Department of Oncology, KU Leuven, Leuven, Belgium; and 7Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, HarvardMedical School, Boston, MA

KEY PO INT S

l LEDGF/p75, animportant cofactorrequired for MLL-rearranged leukemia,is not essential forsteady-statehematopoiesis.

l Loss of LEDGF/p75blocks thedevelopment of MLL-rearranged leukemiasupporting theMLL-LEDGF/p75interaction as a newtherapeutic target.

Mixed lineage leukemia (MLL) represents a genetically distinct and aggressive subsetof human acute leukemia carrying chromosomal translocations of the MLL gene. Thesetranslocations result in oncogenic fusions that mediate aberrant recruitment of the tran-scription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions form acomplex with lens epithelium-derived growth factor (LEDGF/p75; encoded by the PSIP1gene) and MENIN. This complex contributes to the association of MLL and MLL-fusionmultiprotein complexes with the chromatin. Several studies have shown that both MENINand LEDGF/p75 are required for efficient MLL-fusion–mediated transformation and for theexpression of downstream MLL-regulated genes such as HOXA9 and MEIS1. In light ofdeveloping a therapeutic strategy targeting this complex, understanding the function ofLEDGF/p75 in normal hematopoiesis is crucial. We generated a conditional Psip1 knockoutmouse model in the hematopoietic compartment and examined the effects of LEDGF/p75depletion in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Psip1knockout mice were viable but showed several defects in hematopoiesis, reduced colony-forming activity in vitro, decreased expression of Hox genes in the hematopoietic stem

cells, and decreasedMLL occupancy at MLL target genes. Finally, in vitro and in vivo experiments showed that LEDGF/p75 is dispensable for steady-state hematopoiesis but essential for the initiation ofMLL-mediated leukemia. These datacorroborate the MLL-LEDGF/p75 interaction as novel target for the treatment of MLL-rearranged leukemia. (Blood.2018;131(1):95-107)

IntroductionThe mixed lineage leukemia (MLL) gene is the mammalian ho-molog to Drosophila trithorax and is a master regulator of theclustered homeotic (HOX) genes.1-5 MLL is a frequent target ofchromosomal rearrangements that are found in .70% of infantacute leukemia,6 ;10% of acute myeloid leukemia (AML) cases inadults7 and secondary or therapy-related leukemias.8 More im-portantly, MLL-rearranged (MLL-r) leukemia is associated withpoor clinical outcome.9 The most common rearrangements arebalanced chromosomal translocations that fuse the N-terminus ofthe MLL gene in frame with the C terminus of .130 differentpartners, producing oncogenicMLL-fusion proteins (MLL-FPs).10,11

Although the exact molecular mechanism of most fusions re-mains unknown, MLL-FPs mediate aberrant recruitment oftranscription machinery to MLL target genes, driving over-expression of HOX genes (eg, HOXA9) and other coregulatorssuch as MEIS1 and PBX3.12-14 The extreme N-terminus of MLL

forms a triple complex with MENIN and lens epithelium-derivedgrowth factor/p75 (LEDGF/p75), a prerequisite for targeting theMLL oncogenic complex to target genes (Figure 1A-B).15-17 Theexistence of this complex is supported by structural studies inwhich MENIN acts as a molecular adaptor linking MLL withLEDGF/p75.18-20

LEDGF/p75 was first described as a transcriptional coactivatorinvolved in stress response.21 It is an epigenetic reader of H3K36di- and trimethylation marks via its Pro-Trp-Trp-Pro (PWWP)domain and as such preferentially associates with activelytranscribed chromatin.22-25 The presence of the LEDGF/p75PWWP domain was shown to be important for the associationof the MLL-fusion complex with chromatin.16,26 In addition to itsrole in MLL-driven leukemia, LEDGF/p75 plays an essential rolein the pathogenesis of HIV-1, where it functions as a moleculartether targeting HIV-1 integration into the body of activegenes.27-31 This research resulted in the development of a new

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class of HIV inhibitors, LEDGINs, that block the LEDGF/p75-integrase interaction.32 In analogy to HIV-1 integrase, LEDGF/p75 binds the MLL-MENIN complex via its C-terminal integrasebinding domain (IBD; Figure 1B).16,18 We and others demon-strated that Ledgf/p75 knockdown, as well as overexpression ofthe IBD, dramatically reduces clonogenic growth and decreasesthe expression of MLL target genes in vitro and in vivo.16,33

Additionally, we have shown that small peptides known to inhibitthe LEDGF/p75–HIV-1 integrase interaction impair clonogenicgrowth of primary murine MLL-r cells.20 These results establishLEDGF/p75 as an important cofactor required for the leuke-mogenic function of MLL-FPs and suggest the MLL-LEDGF/p75interface as a potential therapeutic target.

Before developing small molecules targeting this interaction,understanding the role of LEDGF/p75 in hematopoiesis is cru-cial. LEDGF/p75 and its splice variant LEDGF/p52, which lacksthe IBD domain, are encoded by PSIP1 gene (PC4 and SFRS1-interacting protein 1, Figure 1B).34 For reasons as yet unknown,ubiquitous deletion of Psip1 is characterized by high perinatal

lethality, limiting further analysis of adult hematopoiesis.35,36

Surviving knockout mice displayed homeotic skeletal transfor-mations, suggesting a critical role in the control of Hox genesexpression.

In this study, we established a conditional knockout mousemodel to address the role of Psip1 in postnatal hematopoiesisand the initiation of MLL leukemogenesis. Mice with Psip1deletion are viable but showed defects in hematopoiesis anddisplayed reduced colony-forming unit (CFU) activity and de-creased Hox genes expression in the hematopoietic stem cells(HSCs). Although LEDGF/p75 is dispensable for the hemato-poietic reconstitution, it is essential for the initiation of MLL-rleukemia in vitro and in vivo.

Materials and methodsGeneration of Psip1 knockout miceC57BL/6 Psip1-floxed mice (PsipFl/Fl) were engineered to harborloxP sites flanking exon 3 of the Psip1 gene (supplemental

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Figure 1. Interaction of the MLL-fusion complex with LEDGF/p75. (A) Schematic representation of the MLL-fusion complex. The most common MLL chromosomaltranslocations result in expression of theN-terminus ofMLL and the C-terminus of a fusion partner gene (MLL-fusion). The extremeN-terminus ofMLL forms a triple complexwithMENIN and LEDGF/p75. The latter recognizes H3K36 di- and trimethylated marks, whereby it tethers the MLL-fusion complex to its targets genes such as HOXA9,MEIS1, andPBX3. (B) Domain structure of wild-type MLL, MLL-fusion, LEDGF/p75, and LEDGF/p52. Wild-type MLL and MLL-fusions contain the MENIN binding domain (MBD) and theLEDGF/p75 binding domain (IBD-interacting motif [IBM]) at its extreme N-terminus. Further downstream, wild-type MLL contains 3 AT-hooks, 2 speckled nuclear localizationsignals (SNLs), a transcriptional repression domain (TRD), 4 PHD fingers, and a bromodomain. The transactivation domain (TAD) and SET domains are located at the MLLC-terminus. LEDGF/p75 harbors a PWWP (aa 1-93), a nuclear localization signal (NLS), 2 AT hook-like motifs (ATH), 3 charged regions (CRs), and the integrase binding domain(IBD) that binds to MLL. LEDGF/p52 shares similar structural domains but lacks the C-terminal IBD.

96 blood® 4 JANUARY 2018 | VOLUME 131, NUMBER 1 EL ASHKAR et al




Figure 1A-B, available on the Blood Web site).31 To knock outPsip1 in the hematopoietic system, PsipFl/Fl mice were crossedwith Vav-iCre transgenic mice (a kind gift from Jody Haigh,Flemish Interuniversity Institute for Biotechnology, Ghent,Belgium). Polymerase chain reaction (PCR) genotyping con-firmed Cre expression in PsipFl/Fl mice (supplemental Figure 1C).All primers are listed in supplemental Table 1. All animal ex-periments were approved by the Katholieke Universiteit Leuvenethical committee.

Animal experimentsTo monitor peripheral blood counts at steady state, bloodsamples were collected from 8- to 12-week-old mice in EDTA-containing Microtainer tubes (BD Biosciences) and analyzedon Cell Dyn-3700 (Abbott Hematology). To extract lineage-depleted (lin2) progenitors, bone marrow (BM) cells were iso-lated from femurs and tibias of 8- to 10-week-old mice. After redblood cell lysis, lineage-negative cells were enriched (EasySepMouse Hematopoietic Progenitor Cell Isolation Kit, STEMCELLTechnologies) and cultured in RPMI-1640 (10% fetal calf serum,50 mg/mL gentamicin), supplemented with murine interleukin-6(10 ng/mL), murine interleukin-3 (6 ng/mL), and murine stem cellfactor (100 ng/mL, PeproTech).

Transduction of primary cellsLentiviral and murine stem cell virus (MSCV) vector productionswere previously described.33 Lin2 cells were transduced byspinoculation (90 minutes at 2500 rpm; 8 mg/mL polybrene) on2 consecutive days. Cells were washed with phosphate-bufferedsaline (PBS) 24 hours after the second spinoculation and used asindicated for the different assays.

Flow cytometric analysesTo analyze the different hematopoietic compartments, single-cell suspensions were prepared from BM cells. Antibodies usedin fluorescence-activated cell sorter (FACS) staining for hema-topoietic stem/progenitor cells and B-cell precursors are listed insupplemental Tables 2 and 3, respectively. For flow cytometricanalysis of CFU colonies, cells were washed twice with PBS andsubsequently stained with phycoerythrin-conjugated anti-Sca-1and allophycocyanin-conjugated anti-cKit or phycoerythrin-conjugated anit-Gr-1 and allophycocyanin-conjugated anti-Mac1.

Clonogenic growth in vitroFor myeloid colony formation assays, (transformed) lin2 cellswere plated in methylcellulose (M3534, STEMCELL Technol-ogies). The number of colonies was scored after 6 to 7 days.For replating experiments, colonies were harvested andwashed with PBS. Cells were counted in a Neubauer chamberusing trypan blue for dead cell exclusion and plated in freshmedium for the subsequent round. For pre-B lymphoid CFUassays, lin2 cells or spleen cells were plated in methylcellulose(M3231, STEMCELL Technologies) supplemented with murineinterleukin-7 (10 ng/mL). May-Grunwald Giemsa staining wasperformed as described earlier.33

Further information about the experimental procedures is pro-vided in supplemental Methods.

ResultsPsip1 knockout mice have reduced peripheralblood cell countsTo study the role of LEDGF/p75 in normal hematopoiesis, weestablished a conditional Psip1 knockout mouse model in thehematopoietic compartment by crossing Psip1-floxed mice31

with Vav-iCre mice, which constitutively express Cre recombi-nase in HSC at early embryonic stages37 (supplementalFigure 1A-C). Complete depletion of LEDGF/p75 was confirmedby quantitative reverse transcription (qRT)-PCR in lin2 cells andby western blot in whole BM cells using a LEDGF/p75-specificantibody (Figure 2A-B). Mice harboring homozygous deletionof Psip1 in the hematopoietic system (referred to as PsipFl/Fl

Vav-Cre-positive mice or PsipVav/Vav) were viable, fertile, pheno-typically normal, and born at the expectedMendelian frequenciescomparedwith their PsipFl/Fl Vav-Cre-negative littermates (PsipFl/Fl)(data not shown).

Next, we determined whether knockout of Psip1 affects steady-state hematopoiesis. Peripheral blood cell counts from a pool ofPsipFl/Fl and PsipVav/Vav mice were compared. Peripheral whiteblood cell (WBC) counts of PsipVav/Vav mice were ;65% lowerthan PsipFl/Fl mice (Figure 2C). Significant reductions were alsoobserved in the lymphocytes and neutrophils. In contrast,PsipVav/Vav mice had normal red blood cell, hemoglobin, andplatelet counts (Figure 2D).

Furthermore, we checked the cellularity of the spleens andthymuses of PsipFl/Fl and PsipVav/Vav mice. Compared with thecontrols, the total numbers of splenocytes and thymocytes weresignificantly reduced by ;55% and 60% in PsipVav/Vav, re-spectively (Figure 2E). Collectively, these results reveal thatPsip1 inactivation affects the hematopoietic system at steadystate.

In vivo ablation of Psip1 perturbsHSC compartmentsNext, we analyzed the effects of Psip1 ablation on differenthematopoietic compartments by flow cytometric analysis ofwhole BM cells (supplemental Figure 2A-B). We first checked thefrequency of HSC (Lineage2Sca-11ckit1 [LSK]) progenitors intotal BM. FACS analysis showed that the percentage of phe-notypically defined HSC was significantly increased after Psip1deletion (Figure 3A). Subsectioning according to CD34 andCD135 expression showed that subpopulations containingprimitive HSCs (long-term HSC, short-term HSC and multipotentprogenitors) were also increased in PsipVav/Vav cells (Figure 3A).Further analysis of different subsets of the more differentiatedprogenitors revealed that, although the percentage of themegakaryocyte-erythroid progenitors was comparable, thegranulocyte-macrophage progenitors, and common myeloidprogenitors were increased in Psip1-depleted cells (Figure 3B).Similarly, the frequency of the common lymphoid progenitorswas increased in Psip1-excised cells (Figure 3B).

Our data indicate that Psip1 knockout results in lower peripheralWBC counts. We evaluated earlier steps in B-cell development.FACS analysis showed a significant decrease in the pre-pro-B-cell and pre-B-cell precursors in the BM of excised mice(Figure 3C). Consistent with the peripheral WBC counts, im-mature andmature B-cell populations were reduced in PsipVav/Vav

ROLE OF LEDGF IN HEMATOPOIESIS AND MLL LEUKEMIA blood® 4 JANUARY 2018 | VOLUME 131, NUMBER 1 97




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Figure 2. Knockout of Psip1 reduces peripheral blood cell counts. (A-B) qRT-PCR of lineage-depleted BM cells (A) and western blot of whole BM cells (B) validating completeLEDGF/p75 depletion in Psip1 knockout (PsipVav/Vav) mice comparedwith the floxed control mice (PsipFl/Fl). Messenger RNA (mRNA) expression in (A) and protein expression in (B)were normalized toGapdh and b-tubulin, respectively. (C-D) Peripheral blood cell counts measured in PsipFl/Fl and PsipVav/Vavmice (8-12 weeks of age). Mean values and standarderror of the mean (SEM) are indicated. HGB, hemoglobin; RBC, red blood cells; ns, nonsignificant. (E) Total cell number extracted from spleens and thymuses of PsipFl/Fl andPsipVav/Vav mice (8-10 weeks of age). Mean values and SEM are indicated. P values (*) show significance (**P , .01, ***P , .001, Student t test).





mice (Figure 3C). Together, these findings suggest that loss ofPsip1 affects the number of HSC and progenitor cells at steadystate.

Absence of Psip1 affects colony formationpotential of HSCAlthough loss of Psip1 resulted in higher number of HSC andprogenitor cells, it was not clear whether the functionality ofthe cells was affected. Accordingly, we sought to compare thecolony-forming capacity of Psip1 wild-type and knockout cellsusing myeloid and pre-B lymphoid CFU assays. Lin2 cells wereharvested from PsipFl/Fl and PsipVav/Vav mice and serially plated inmyeloid CFU culture. After 7 days (first plating), the number ofcolonies derived from PsipFl/Fl and PsipVav/Vav mice was not sig-nificantly different (Figure 4A). However, the total cell numberharvested from the formed PsipVav/Vav colonies was reduced(8.93 106 PsipFl/Fl cells vs 4.63 106 PsipVav/Vav cells). In the secondand third platings, the number of colonies of PsipVav/Vav micewas significantly reduced compared with the wild-type control(Figure 4A). Alternatively, we compared the colony-forming

capacity of PsipFl/Fl and PsipVav/Vav cells in pre-B lymphoid CFUassays. The number of pre-B colonies in Psip1-depleted cells wasreduced in lin2 cells and spleen cells, comparedwith the controls(Figure 4B). To ensure that the decreased colony-forming po-tential in PsipVav/Vav cells was due to Psip1 deletion and not Creexpression, we compared the CFU potential of lin2 cells har-vested from C57BL/6 mice and Vav-iCre transgenic mice in thesame background. Cre expression on its own did not affectthe number of colonies formed upon replating, nor did it alterthe total cell number harvested from these colonies (data notshown).

Previous studies showed that germ line deletion of Psip1resulted in perinatal mortality and homeotic skeletal transfor-mations similar to Hox-cluster mutant mice.35,36 Additionally,MLL, MENIN, and LEDGF/p75 have been shown to colocalize onHoxA7 and HoxA9 promoters, and knockdown of LEDGF/p75resulted in decreasedHOXA9 expression inMLL-FP transformedcells.16,33 These results suggest that Hox genes might be reg-ulated by Psip1. Accordingly, we measured the expression of

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Figure 3. Psip1 excision perturbs theHSC compartments. (A) The proportion of BM corresponding to theHSC-containing LSK progenitorsmeasured in PsipFl/Fl and PsipVav/Vav

mice (4-7 animals per group, 8-10 weeks of age). Subsectioning according to CD34 and CD135 expression yielded phenotypic assessments of LT-HSC, ST-HSC, and MPPfractions. LT-HSC, long-term hematopoietic stem cells; MPP, multipotent progenitors; ST-HSC, short-term hematopoietic stem cells. (B) BM frequencies of the more dif-ferentiated progenitors gated in the LSK population, subsectioned based on CD16/32 and CD34 expression to compare common myeloid progenitors (CMPs), granulocyte-macrophageprogenitors (GMPs), andmegakaryocyte-erythroid progenitors (MEPs). TheCLP fraction is gated based onCD127 expression. CLP, common lymphoid progenitors.(C) Percentage of BM cells at different steps in B-cell development. The frequencies of pre-pro-B, pro-B, pre-B, immature, and mature B cells in PsipFl/Fl and PsipVav/Vav mice areshown (4-7 animals per group). Mean values and SEM are shown. P values (*) show significance (*P , .05, **P , .01, ***P , .001, Student t test).





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Figure 4. Psip1 knockout affects colony formation potential of HSC in vitro. (A) Number of colonies in 3 consecutive rounds of a myeloid CFU assay per 104 lin2 BM cellsharvested from PsipFl/Fl and PsipVav/Vav mice (10 weeks of age). (B) Pre-B CFU assays for PsipFl/Fl and PsipVav/Vav cells. Lin2 cells and spleen cells were harvested and plated inmethylcellulose for 7 to 10 days. The number of colonies formed was normalized to the control (PsipFl/Fl) cells. (C) qRT-PCRmeasuring expression levels (normalized toGapdh) ofdifferent genes in lin2 cells harvest from PsipFl/Fl or PsipVav/Vav cells (before first plating) and after 1 round in myeloid CFU assay (after first plating). (D) Comparison of cellmorphology of PsipFl/Fl and PsipVav/Vav cells after 1 round in the CFU assay via May-Grunwald Giemsa staining. A representative picture is shown. (E) FACS analysis for Gr-1expression in PsipFl/Fl and PsipVav/Vav cells (n5 8) harvested after 1 round in CFU assay. Mean and SEM values are indicated. Error bars (panels A, B, C, and E) represent standarddeviation of triplicate measurements. Statistical differences were determined using Student t test; *P , .05, **P , .01, ***P , .001.





several HoxA and HoxB cluster genes as well as other cofactorssuch as Meis1 in lin2 cells harvested directly from PsipFl/Fl andPsipVav/Vav mice and after plating in myeloid CFU assays. HoxA9and HoxA4 expression were significantly reduced only after firstplating (Figure 4C). In contrast, expression of other HoxA genes(HoxA7, HoxA10, and HoxA11) and HoxB4 was readily reduced

at steady state. Although the expression ofHoxB8was increasedafter CFU plating in Psip1-depleted cells, Meis1 expression wascomparable between PsipFl/Fl and PsipVav/Vav cells before andafter plating. May-Grunwald Giemsa staining on the harvestedcolonies (after first plating) suggested a more differentiatedmorphology in Psip1-depleted cells compared with the control

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Figure 5. Specific knockdown of Ledgf/p75 affects CFU activity of HSC. (A) qRT-PCR measuring Ledgf/p75 and Ledgf/p52 mRNA expression levels in lin2 cells harvestedfrom 8-week-old C57BL/6J mice after transduction with lentiviral vector expressing a Ledgf/p75-specific miRNA (Ledgf/p75 KD) or eGFP-miRNA as a control. Expression levelswere normalized to Gapdh. (B) CFU assay per 104 cells after Ledgf/p75 knockdown. (C) HoxA9 expression levels (normalized to Gapdh) as measured by qRT-PCR in Ledgf/p75knockdown cells. (D) qRT-PCR measuring Ledgf/p75 and Ledgf/p52 expression levels and serial plating of a myeloid CFU assay (E) after Ledgf/p52 knockdown (KD). Expressionlevels in panel D were normalized toGapdh. (F) qRT-PCR measuring HoxA9 expression levels (normalized toGapdh) in Ledgf/p52 knockdown cells after first plating in the CFUassay (E). (G) Peripheral WBCs measured after BM transplantation in lethally irradiated recipients transplanted with a total of 13 106 lin2 cells harvested from 8- to 10-week-oldPsipFl/Fl and PsipVav/Vav mice. WBC was monitored 4, 8, 12, and 16 weeks posttransplantation. Mean values and SEM are indicated. Error bars (panels A-F) represent standarddeviation of triplicate measurements. Differences in panels A-G were determined using Student t test; *P , .05, **P , .01, ***P , .001.





(Figure 4D). In agreement, FACS analysis of the harvested col-onies showed reduced Sca-1 and cKit expression, suggestingincreased differentiation in PsipVav/Vav cells (supplementalFigure 3A-B). Furthermore, PsipVav/Vav cells showed skeweddifferentiation toward the granulocytic lineage, as indicated byhigher Gr-1 expression when compared with PsipFl/Fl cells(Figure 4E). Of note, Mac-1 expression was comparable in PsipFl/Fl

and PsipVav/Vav cells (data not shown). Taken together, these resultssuggest that Psip1 depletion affects the colony formation capacityof HSCs and expression of Hox genes.

Specific loss of LEDGF/p75 and not LEDGF/p52affects colony formation of HSCsPSIP1 encodes LEDGF/p75 and the shorter LEDGF/p52 isoform.Because p52 lacks the IBD, it is unable to interact with MLL(Figure 1B).34 To analyze the specificity of the observed phe-notype, we harvested lin2 cells from C57BL/6J mice andtransduced them with a lentiviral vector expressing a Ledgf/p75-specific microRNA (miRNA) or eGFP-miRNA as a negativecontrol. We observed;40% knockdown for Ledgf/p75, whereasthe p52 isoform was not significantly affected as measured byqRT-PCR (Figure 5A). After 7 days, the effects of Ledgf/p75knockdown were readily observed by the reduced numberof colonies (;70% reduction) comparedwith the control (Figure 5B).Moreover, knockdown of Ledgf/p75 caused approximately atwofold reduction in HoxA9 expression (Figure 5C). In contrast,depletion of Ledgf/p52 did not affect colony-formation potentialof lin2 cells or HoxA9 expression, even after 3 rounds in culture(Figure 5D-F). Altogether, our results confirm that specific loss ofLEDGF/p75 and not LEDGF/p52 affects the colony formationcapacity of HSCs.

LEDGF/p75 is dispensable forhematopoietic reconstitutionNext, we evaluated the capacity of Psip1-depleted cells to re-constitute the hematopoietic system after BM transplantation(BMT). Lin2 cells harvested from PsipFl/Fl and PsipVav/Vavmice weretransplanted into lethally irradiated recipients. Subsequently,hematopoietic regeneration was monitored by measuring pe-ripheral blood counts 4, 8, 12, and 16 weeks posttransplantation.Short-term reconstitution of PsipVav/Vav cells at 4 weeks did notdiffer from PsipFl/Fl cells, suggesting that LEDGF/p75 is not es-sential for HSC homing, engraftment, or reconstitution. None-theless, at 12 weeks posttransplantation, a mild but significantreduction in the number of peripheral WBC was observed(Figure 5G). This reduction persisted at 16 weeks post-transplantation, in agreement with the effects observed forLEDGF/p75 depletion on steady-state hematopoiesis (Figure 2C).Altogether, these data indicate that loss of LEDGF/p75 impairs,but does not abolish hematopoietic reconstitution.

Psip1 gene signature overlaps with that of MLL-FPtarget genesTo better understand the observed defects in hematopoiesis, weperformed gene expression profiling by RNA-sequencing (RNA-seq) on lin2 cells harvested from PsipFl/Fl and PsipVav/Vavmice after1 round in the myeloid CFU assay. We identified 44 down-regulated (comprising several knownMLL target genes) and 170upregulated genes using the criteria of an absolute log2-foldchange .1.5 and P value , .05 (Figure 6A; supplementalTable 4). Downregulation of known MLL target genes such as

HoxA9 and Eya1 in PsipVav/Vav cells was validated by qRT-PCR(supplemental Figure 4A).38 It has been established that wild-type MLL and MLL-FP have partially overlapping target genesets,39 suggesting that they can be recruited by differentmechanisms. Gene set enrichment analysis (GSEA) revealed thatthe principal signature in the PsipVav/Vav samples was not sig-nificantly up- or downregulated when compared with a set ofgenes with wild-type MLL bound to the promoter regions(P5 .21, supplemental Figure 4B).38 However, those genes withevidence for promoter-bound MLL complexes (overlappingMLL, WDR5, and H3K4me2 chromatin immunoprecipitation[ChIP]-seq peaks) are significantly enriched among the down-regulated genes (P 5 .02, Figure 6B).38 A stronger enrichmentwas found when MLL-ENL target genes were tested against thePsipVav/Vav ranked list (P, .001, Figure 6C).38 Similar results wereobtained with a set of MLL-AF9–bound genes (P , .001, sup-plemental Figure 4C).40 These data suggest that LEDGF/p75recruits wild-type MLL to genes that are also targeted by MLL-FP.16,24 This is in line with conservation of the LEDGF/p75interaction with MLL-FP, whereas other tethering mechanismsare lost or disrupted in these fusions. To verify this, we assessedMLL occupancy at Hox genes in lin2 cells harvested fromPsip1-depleted cells through ChIP-qPCR. In line with earlierreports,24,41 ChIP-qPCR analyses demonstrated that MLL occu-pancy at HoxA9 and HoxA7 promoters was decreased abouttwofold in PsipVav/Vav cells compared with the controls (Figure 6D).

HoxA9 overexpression rescues defective colonyformation of PsipVav/Vav cellsOne of the top hits revealed by GSEA was that of genesdownregulated in MOLM-14 cells (AML) upon HOXA9 knock-down (q value 5 0.001, Figure 6E).42 Constitutive HOXA9expression is one of the main drivers of MLL-r leukemia.14

Moreover, overexpression of HoxA9 was shown to be sufficientto elicit cellular transformation.43 As such, we aimed to de-termine whether the defects observed in Psip1-depleted cellscould be rescued byHoxA9 overexpression. Lin2 cells harvestedfrom PsipFl/Fl and PsipVav/Vav mice were transduced with a ret-roviral vector expressingHoxA9 or a mock control (supplementalFigure 4D) and plated for colony formation. Although Psip1knockout led to ;70% reduction in the number of colonies,overexpression of HoxA9 restored CFU activity to wild-typelevels (Figure 6F). These data were corroborated in Ledgf/p75knockdown cells (supplemental Figure 4E). In contrast to PsipVav/Vav

cells, acute knockdown of Ledgf/p75 readily caused a significantreduction in HoxA9 expression associated with a fivefold re-duction in colony formation (supplemental Figure 4F-G). Thisdefect was rescued to wild-type levels upon HoxA9 over-expression. Collectively, these results suggest that LEDGF/p75 isimportant for proper HSC differentiation, likely through regulationof Hox genes expression.

Psip1 is essential for inducing MLL-rearrangedleukemiaEarlier studies reported that LEDGF/p75 is important to maintainMLL-mediated transformation in human MLL-r cell lines andprimary MLL-fusion transformed cells.16,33 The availability ofPsip1 knockout cells allowed us to investigate whether LEDGF/p75 is important for the initiation of MLL-r leukemia (Figure 7A).Lin2 cells harvested from PsipFl/Fl and PsipVav/Vav mice weretransducedwith anMLL-ENL fusion (PsipFl/Fl.MLL-ENL andPsipVav/Vav.MLL-ENL, respectively) and their transformation potential was





compared in the CFU assay. Although the number of coloniesin PsipFl/Fl.MLL-ENL cells increased after the third plating be-cause of MLL-fusion–mediated transformation, Psip1 knockoutcells proved refractory to transformation, as evident by thesignificant reduction in the number of colonies upon serialplating (Figure 7B). Similar data were obtained in MLL-AF9–transformed cells (supplemental Figure 5). In contrast, ex-pression of an oncogenic E2A-HLF fusion (PsipFl/Fl.E2A-HLF

and PsipVav/Vav.E2A-HLF), which is independent of HoxA9 fortransformation,44 revealed comparable transformation capacityafter serial plating in PsipFl/Fl and PsipVav/Vav cells (Figure 7C).

These data were corroborated in vivo by BMT. PsipFl/Fl.MLL-ENLand PsipVav/Vav MLL-ENL cells were transplanted into lethally ir-radiated mice. Whereas recipients transplanted with PsipFl/Fl.MLL-ENL cells succumbed to leukemia within 60 to 85 days after

Ifi202bHoxa9CtseGm9025Eya1Psma8Mcpt1Rps24−ps3Psip1Gm8822Mcpt2Mcpt4C1qaPtk7St8sia1F13a1HnmtSix1Tspan13Dock9Nos2Ifi47Mef2cPtprv2810417H13RikSh2d1b1CtskMyrfTchhPeg12Arnt2Pls3Kif21aSfmbt2Ccnd1MarcoFgfr1Naip5Meis3Prickle2H2afy2Xkr6Adra2cAstn2Adamts1Elavl2Uchl1Fzd8Abcc8Ndn

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actin

Figure 6. Psip1 knockout gene expression signature overlaps with that of MLL-FP target genes. (A) Heat map of RNA-seq data showing the top differentially expressedgenes in PsipFl/Fl and PsipVav/Vav cells harvested after 1 round in the CFU assay. GSEA showing that Psip1-regulated genes are enriched for genes with promoter regions bound by(B) MLL complex and (C) MLL-ENL fusion. (D) Quantitative ChIP assay for PsipFl/Fl and PsipVav/Vav cells using Mll antibody. The promoter regions amplified by qPCR are indicatedbelow the respective panels. (E) GSEA showing the correlation between the principal signature in the PsipVav/Vav RNA-seq samples and genes downregulated in MOLM-14 cells(AML) upon knockdown ofHoxa9. (F) CFU assay per 104 cells harvested from PsipFl/Fl and PsipVav/Vavmice. Cells were transduced with pMSCV-HoxA9-pgk-neo or mock vector andplated for colony formation. Error bars indicate standard deviations of triplicate measurements. Differences were determined using Student t test; *P , .05.





PsipFl/Fl

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Figure 7. LEDGF/p75 is essential for MLL-rearranged transformation. (A) Schematic representation of the experimental setup. BM cells were harvested from 10-week-oldPsipFl/Fl and PsipVav/Vavmice. After depletion of lineage-committed progenitors, cells were transducedwithMLL-ENL or E2A-HLF fusions and assessed for leukemogenic activitiesin vitro in CFU assays and in vivo in BMT. CFU per 104 PsipFl/Fl or PsipVav/Vav cells transduced with MSCV encodingMLL-ENL (B) and E2A-HLF (C) fusions. Representative images oftetrazolium-stained colonies after the third plating are shown. Error bars represent standard deviation of triplicate measurements. Differences were determined using Studentt test; **P, .01; ***P, .001. (D) Kaplan-Meier survival curve for lethally irradiated recipients transplanted with PsipFl/Fl or PsipVav/Vav cells transduced withMLL-ENL (PsipFl/Fl.MLL-ENL and PsipVav/Vav.MLL-ENL, respectively) or control cells (PsipFl/Fl cells transduced withmock vector). Number of transplanted animals (n) per group is indicated. (E) PCR analysisof whole BM cells of moribund mice transplanted with PsipFl/Fl.MLL-ENL cells and whole BM cells of PsipVav/Vav.MLL-ENL or control animals (described in panel D) harvested at180 days posttransplantation. Primers were designed to specifically detect the MLL-ENL fusion gene. A vertical line has been inserted to indicate a repositioned gel lane.(F) Replating CFU transformation assay for lin2 cells transduced with MLL-ENL fusion or MLL-ENL mutated in the LEDGF/p75-binding domain (mut.MLL-ENL). Error barsrepresent standard deviation of triplicate measurements. Differences were determined using Student t test; **P , .01.





transplantation, animals transplanted with PsipVav/Vav.MLL-ENLor control cells (PsipFl/Fl cells transduced with pMSCV.Neo) didnot show any sign of the disease, even at 285 days post-transplantation (Figure 7D). PCR analysis for MLL-ENL fusion inwhole BM cells harvested from PsipFl/Fl.MLL-ENL moribund miceor PsipVav/Vav.MLL-ENL and control animals 180 days after BMTconfirmed expression of the MLL-ENL fusion in PsipVav/Vav cellsdespite absence of disease progression (Figure 7E).

In accordance with previous studies,16 our data show thatLEDGF/p75 can recruit both wild-type MLL and MLL-FPs togenes driving MLL-r leukemia. In contrast, it has been recentlysuggested that LEDGF/p75 is required to tether the wild-typecomplex, but not MLL-FP at target gene loci.24 To further in-vestigate this, we introduced 2 mutations (F148A and F151A) inthe consensus IBD-interacting motif45 of the MLL-ENL fusion(mut.MLL-ENL) that specifically abrogate the interaction withLEDGF/p75 and evaluated its oncogenic activity in vitro. Incontrast to the wild-type MLL-ENL fusion, the mut.MLL-ENL wasunable to transform myeloid progenitors (Figure 7F). Collec-tively, these data support a role for LEDGF/p75 in targeting bothwild-typeMLL and its oncogenic counterpart onto the chromatinand that the function of LEDGF/p75 in the MLL-FP complex isimportant to establish cellular transformation.

DiscussionOncogenic fusion proteins resulting from chromosomal trans-locations of the MLL gene induce aggressive acute leukemias inchildren and adults, emphasizing the need for new therapeuticstrategies. It is clear that epigenetic states play an important rolein the proliferative capacity of transformed cells. Targetingepigenetic readers and writers has become an attractive anti-cancer strategy as evidenced by the identification and develop-ment of BET protein inhibitors.46 However, careful understandingof the proteins involved is necessary to avoid toxicity and to assesstherapeutic windows. MLL forms a ternary complex with MENINand the epigenetic reader LEDGF/p75. Several studies show thatthe latter tethers wild-type MLL and/or MLL-FP complexes tochromatin (Figure 1A).16,24,41 Several approaches have been takento interfere with the function of MLL-fusion complexes, includingDOT1L or BET protein inhibitors that target the fusion moiety ofMLL-FPs.47-50 On the other hand, others directly target the MLLfragment of the fusion. In this regard, the development of MLL-MENIN interaction inhibitors proved the feasibility of targetingthe MLL-MENIN-LEDGF/p75 complex.51,52 Furthermore, de-tailed structural characterization of the MLL-MENIN-LEDGF/p75complex and strategies exploiting inhibitory peptides alsosupport the MLL-LEDGF/p75 interaction as a potential newtherapeutic target.19,20 However, because the role of LEDGF/p75(more specifically, the MLL-LEDGF/p75 interaction) in hema-topoiesis has not been studied, the specificity of potentialMLL-LEDGF/p75 interaction inhibitors has not been addressed.This knowledge is required to fully appreciate the “druggability” ofthis interaction.

Because mice with systemic Psip1 deletion were shown to sufferfrom perinatal lethality,35,36 we investigated adult hematopoiesisby knocking out Psip1 in the hematopoietic system. These micedid not exhibit any evident phenotypic abnormalities, sug-gesting that Psip1 is not essential for HSC maintenance or sur-vival in adult mice. Nonetheless, Psip1 knockout mice displayed

specific hematopoietic defects including an approximate twofolddecrease in peripheral WBC counts, perturbed numbers of HSCsand progenitor cells, and impaired colony-forming capacity uponserial plating.

Earlier gene targeting studies of Mll underscored its impor-tance for HSC development during embryogenesis and adulthematopoiesis.53-55 Mll deletion resulted in hematopoietic de-fects and reconstitution failure, suggesting that LEDGF/p75 isnot exclusively required for all physiological functions of MLL.Other mechanisms might exist that tether theMLL complex to itstarget genes, as has been suggested previously.56,57 We ob-served that expression changes in PsipVav/Vav samples are morepronounced at MLL-FP–bound genes than at MLL wild-type–bound genes. This result confirms that those genes targeted bythe MLL-MENIN-LEDGF/p75 complex are also crucial for thedevelopment of MLL-r leukemia. In contrast to the p52 isoform,LEDGF/p75 contains the IBD domain, which specifically interactswith MLL and several other cellular proteins.45 However, amongthese interaction partners, only MLL is known to affect HOXexpression. Although we did not observe decreased expressionof HoxA9 in the lin2 fraction in contrast to other members of theHoxA cluster, our RNA-seq data revealed downregulation ofHoxA9 targets in Psip1-depleted cells after 1 week in culture andthat the observed phenotypes could be rescued upon HoxA9overexpression suggests that the HoxA9 axis is an importantcomponent of the observed differentiation defects and de-creased CFU formation. Targeted disruption of HoxA9 wasshown to display similar hematopoietic defects to Psip1knockout mice such as lower peripheral lymphocytes, reducedspleen and thymus cellularity, defects in B-cell production, andimpaired CFU capacity.58,59

Despite its dispensability for normal hematopoiesis, our datashow that LEDGF/p75 is essential for the initiation of MLL-rleukemia. AlthoughMLL-r leukemia is mainly induced by gain offunction of the MLL-FPs, the remaining wild-type allele is typi-cally expressed. Some studies suggested a functional role ofthe latter in cellular transformation.60,61 On the other hand, MLL-AF6 human leukemic cells (ML2 cells) do not express the wild-type MLL protein.38 Although all MLL-FPs retain the wild-typeN-terminus, genome-wide analysis has shown that wild-typeMLL and MLL-FPs have distinct chromatin-binding profiles.39

Whereas MLL-FPs exert their oncogenic activity through acti-vation of a particular gene expression program, integrativeanalyses of MLL binding and expression profiling showedthat MLL-FPs share only a small subset of target genes with thewild-type protein,38 suggesting different chromatin-tetheringmechanisms. Earlier work claimed that LEDGF/p75 specificallyassociates with wild-type MLL and MLL-FP and colocalizes ontarget genes.16 In contrast, recent studies from the same groupand others showed that knockdown of LEDGF/p75 led to in-creased MLL-FP recruitment.24,41 The authors suggested an in-dependent role for LEDGF/p75 in the wild-type complex, whichin turn competes with MLL-FP for chromatin-binding sites.Similar to Zhu et al, we observed a significant reduction of wild-type MLL binding to HoxA9 and HoxA7 promoter regions uponLEDGF/p75 depletion. Although our Psip1 knockout mice revealthat LEDGF/p75 is essential for MLL-r leukemia and that theabsence of LEDGF/p75 affects Hox expression and hemato-poiesis likely due to diminished MLL recruitment, it was unclearwhether the leukemogenic defect is due to diminished tethering





of wild-type MLL and/or MLL-FP to target genes. Based onrecent structural data, we mutated the IBD-interacting motif inMLL-ENL, which is responsible for the interaction with LEDGF/p75.45 CFU assays revealed impaired transformation capacityin lin2 cells. These data corroborate the function of LEDGF/p75in the tethering the MLL-FP, as has been suggested earlier.16

In summary, we demonstrated that LEDGF/p75 is not essential foradult hematopoiesis, whereas MLL-r leukemia cannot be initiatedin its absence. These data open new perspectives to develop smallmolecule inhibitors of theMLL-LEDGF/p75 interaction, validating anovel target with a sufficient therapeutic window to treat MLLleukemia and possibly other MLL-dependent diseases.62

AcknowledgmentsThe authors thank Martin Michiels and Sara T’Sas for the technical as-sistance and Thomas Milne for the technical advice.

This work was supported by grants from the Katholieke UniversiteitLeuven Interdisciplinair onderzoeksprogramma (IDO) program (IDO/12/008-3E130241), the Flemish Research Foundation - Flanders (G.0595.13and G065614N) (Z.D.), the Odysseus program (P.V.V.), the Flemishagency for Innovation by Science and Technology (SBO 140038[ChromaTarget]), and the US National Institutes of Health, National In-stitute of Allergy and Infectious Diseases (AI039394) (A.E.). S.E.A. is apostdoctoral fellow supported by the IDO program. J.S. is supported bythe Swiss National Science Foundation (31003A_149714), the SwissCancer League (KFS-3487-08-2014), and the Gertrude Von MeissnerFoundation (Basel, Switzerland). T.P. and S.G. are postdoctoral fellowsfunded by the Belgian “Stand Up To Cancer” Foundation.

AuthorshipContribution: S.E.A. and J.D.R. designed the experiments and wrotethe manuscript. S.E.A., J.D.R., T.P., S.G., S.J., and S.V.B. performed

experiments and analyzed data. A.E. generated PsipFl/Fl mice. J.D. per-formed bioinformatics analyses. N.B. performed and analyzed May-Grunwald Giemsa stainings. J.S., F.C., P.V.V., Z.D., and J.D.R. supervisedthe project. All authors read and approved the final manuscript.

Conflict-of-interest disclosure: The authors declare no competinginterests.

The current affiliation for J.D. is The Francis Crick Institute, London,United Kingdom.

ORCID profiles: S.E.A., 0000-0001-9410-5687; J.S., 0000-0001-8616-0096; T.P., 0000-0002-2985-0104; S.G., 0000-0002-5693-8570; J.D.,0000-00002-2660-2478; F.C., 0000-0001-9580-3874; P.V.V., 0000-0001-9063-7205; Z.D., 0000-0002-3982-1565.

Correspondence: Jan De Rijck, Laboratory for Molecular Virology andGene Therapy, Kapucijnenvoer 33, 3000 Leuven, Belgium; e-mail: [email protected]; and Zeger Debyser, Laboratory for Molecular Vi-rology and Gene Therapy, Kapucijnenvoer 33, 3000 Leuven, Belgium;e-mail: [email protected].

FootnotesSubmitted 26 May 2017; accepted 20 October 2017. Prepublishedonline as Blood First Edition paper, 30 October 2017; DOI 10.1182/blood-2017-05-786962.

*Z.D. and J.D.R. shared senior authorship.

The online version of this article contains a data supplement.

There is a Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked “advertisement” in accordance with 18 USC section 1734.

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http://orcid.org/0000-0001-9410-5687

http://orcid.org/0000-0001-8616-0096

http://orcid.org/0000-0001-8616-0096

http://orcid.org/0000-0002-2985-0104

http://orcid.org/0000-0002-5693-8570

http://orcid.org/0000-00002-2660-2478

http://orcid.org/0000-00002-2660-2478

http://orcid.org/0000-0001-9580-3874

http://orcid.org/0000-0001-9063-7205

http://orcid.org/0000-0001-9063-7205

http://orcid.org/0000-0002-3982-1565

mailto:[email protected]



https://doi.org/10.1182/blood-2017-05-786962

https://doi.org/10.1182/blood-2017-05-786962

http://www.bloodjournal.org/content/131/1/4



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online October 30, 2017 originally publisheddoi:10.1182/blood-2017-05-786962

2018 131: 95-107

Debyser and Jan De RijckChrist, Siska Van Belle, Sabine Juge, Nancy Boeckx, Alan Engelman, Pieter Van Vlierberghe, Zeger Sara El Ashkar, Juerg Schwaller, Tim Pieters, Steven Goossens, Jonas Demeulemeester, Frauke MLL-rearranged leukemogenesisLEDGF/p75 is dispensable for hematopoiesis but essential for

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