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LEDGF/p75 is Dispensable for Hematopoiesis but

Essential for MLL-rearranged Leukemogenesis

Sara El Ashkar1, Juerg Schwaller2, Tim Pieters3,4, Steven Goossens3,4, Jonas

Demeulemeester1,5, Frauke Christ1, Siska Van Belle1, Sabine Juge2, Nancy Boeckx6,7,

Alan Engelman8, Pieter Van Vlierberghe3,4, Zeger Debyser1* and Jan De Rijck1*

1Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and

Pharmacological Sciences, KU Leuven, Leuven, Belgium. 2Department of Biomedicine, University Children’s Hospital (UKBB), University of Basel, Switzerland. 3Center for Medical Genetics, Ghent University, Ghent, Belgium. 4Cancer Research Institute Ghent (CRIG), Ghent, Belgium. 5Current address: The Francis Crick Institute, London, United Kingdom. 6Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium. 7Department of Oncology, KU Leuven, Leuven, Belgium 8Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School,

Boston, United States of America. *Shared senior authorship.

Correspondence should be addressed to Jan De Rijck:

Laboratory for Molecular Virology and Gene Therapy

Kapucijnenvoer 33, VCTB +5

3000 Leuven, Belgium

[email protected], [email protected]

Tel.: +32 16 374038, Fax.: +32 16 336336

Short Title:

Role of LEDGF in Hematopoiesis and MLL leukemia

Text word count: 3998

Abstract word count: 225

Number of Figures: 7

Reference count: 62

Blood First Edition Paper, prepublished online October 30, 2017; DOI 10.1182/blood-2017-05-786962

Copyright © 2017 American Society of Hematology

For personal use only.on December 4, 2017. by guest www.bloodjournal.orgFrom

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Key Points

• LEDGF/p75, an important cofactor required for MLL-rearranged leukemia, is not

essential for steady-state hematopoiesis.

• Loss of LEDGF/p75 blocks the development of MLL-rearranged leukemia supporting

the MLL-LEDGF/p75 interaction as a new therapeutic target.




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ABSTRACT

Mixed lineage leukemia (MLL) represents a genetically distinct and aggressive subset of

human acute leukemia carrying chromosomal translocations of the MLL gene. These

translocations result in oncogenic fusions that mediate aberrant recruitment of the

transcription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions

form a complex with Lens Epithelium-Derived Growth Factor (LEDGF/p75; encoded by

the PSIP1 gene) and MENIN. This complex contributes to the association of MLL and

MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that

both MENIN and LEDGF/p75 are required for efficient MLL-fusion–mediated

transformation and for the expression of downstream MLL-regulated genes like HOXA9

and MEIS1. In the light of developing a therapeutic strategy targeting this complex,

understanding the function of LEDGF/p75 in normal hematopoiesis is crucial. We

generated a conditional Psip1 knockout mouse model in the hematopoietic compartment

and examined the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the

initiation of MLL leukemogenesis. Psip1 knockout 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 decreased MLL 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 of MLL-

mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as novel

target for the treatment of MLL-rearranged leukemia.




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Introduction

The mixed lineage leukemia (MLL) gene is the mammalian homologue to Drosophila

Trithorax (trx) and is a master regulator of the clustered homeotic (HOX) genes1-5. MLL

is a frequent target of chromosomal rearrangements that are found in >70% of infant

acute leukemia6, ~10% of acute myeloid leukemia (AML) cases in adults7 and secondary

or therapy-related leukemias8. More importantly, MLL-rearranged (MLL-r) leukemia is

associated with poor clinical outcome9. The most common rearrangements are balanced

chromosomal translocations that fuse the N-terminus of the MLL gene in-frame with the

C-terminus of more than 130 different partners, producing oncogenic MLL-fusion

proteins (MLL-FPs)10,11.

While the exact molecular mechanism of most fusions remains unknown, MLL-FPs

mediate aberrant recruitment of transcription machinery to MLL target genes, driving

overexpression of HOX genes (e.g. HOXA9) and other co-regulators like MEIS1 and

PBX312-14. The extreme N-terminus of MLL forms a triple complex with MENIN and

LEDGF/p75 (Lens Epithelium-Derived Growth Factor/p75), a prerequisite for targeting

the MLL oncogenic complex to target genes (Figure 1A-B)15-17. The existence of this

complex is supported by structural studies where MENIN acts as a molecular adaptor

linking MLL with LEDGF/p7518-20.

LEDGF/p75 was first described as a transcriptional coactivator involved in stress

response21. It is an epigenetic reader of H3K36 di-and tri-methylation marks via its

PWWP domain and as such, preferentially associates with actively transcribed

chromatin22-25. The presence of the LEDGF/p75 PWWP domain was shown to be

important for the association of the MLL-fusion complex with chromatin16,26. In addition




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to its role in MLL-driven leukemia, LEDGF/p75 plays an essential role in the

pathogenesis of human immunodeficiency virus (HIV-1), where it functions as a

molecular tether targeting HIV-1 integration into the body of active genes27-31. This

research resulted in the development of a new class of HIV inhibitors, LEDGINs, that

block the LEDGF/p75-integrase interaction32. In analogy to HIV-1 integrase,

LEDGF/p75 binds the MLL-MENIN complex via its C-terminal integrase binding

domain ((IBD), Figure 1B)16,18. We and others demonstrated that Ledgf/p75 knockdown,

as well as overexpression of the IBD, dramatically reduces clonogenic growth and

decreases the expression of MLL target genes in vitro and in vivo16,33. Additionally, we

have shown that small peptides known to inhibit the LEDGF/p75–HIV-1 integrase

interaction, impair clonogenic growth of primary murine MLL-r cells20. These results

establish LEDGF/p75 as an important cofactor required for the leukemogenic function of

MLL-FPs and suggest the MLL-LEDGF/p75 interface as a potential therapeutic target.

Prior to developing small molecules targeting this interaction, understanding the role of

LEDGF/p75 in hematopoiesis is crucial. LEDGF/p75 and its splice variant LEDGF/p52,

which lacks the 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

hematopoiesis35,36. Surviving knockout mice displayed homeotic skeletal transformations,

suggesting a critical role in the control of Hox genes expression.

In this study, we established a conditional knockout mouse model to address the role of

Psip1 in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Mice with

Psip1 deletion are viable but showed defects in hematopoiesis, displayed reduced colony-




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forming unit (CFU) activity and decreased Hox genes expression in the hematopoietic

stem cells (HSC). While LEDGF/p75 is dispensable for the hematopoietic reconstitution,

it is essential for the initiation of MLL-r leukemia in vitro and in vivo.




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Material and Methods

Generation of Psip1 knockout mice

C57BL/6 Psip1-floxed mice (PsipFl/Fl) were engineered to harbor loxP sites flanking

exon 3 of the Psip1 gene (Figure S1A-B)31. To knockout Psip1 in the hematopoietic

system, PsipFl/Fl mice were crossed with Vav-iCre transgenic mice (a kind gift from Dr.

Jody Haigh, Flemish Interuniversity Institute for Biotechnology, Ghent, Belgium). PCR

genotyping confirmed Cre expression in PsipFl/Fl mice (Figure S1C). All primers are

listed in Table S1. All animal experiments were approved by the KU Leuven ethical

committee.

Animal experiments

To monitor peripheral blood counts at steady state, blood samples were collected from 8-

12 week-old mice in EDTA-containing Microtainer tubes (BD Biosciences) and analyzed

on Cell Dyn-3700 (Abbott Hematology). To extract lineage-depleted (lin-) progenitors,

bone marrow (BM) cells were isolated from femurs and tibias of 8-10 weeks old mice.

After red blood cell lysis, lineage-negative cells were enriched (EasySep™ Mouse

Hematopoietic Progenitor Cell Isolation Kit, STEMCELL technologies) and cultured in

RPMI-1640 (10% FCS, 50µg/ml gentamicin), supplemented with mIL-6 (10ng/ml), mIL-

3 (6ng/ml) and mSCF (100ng/ml, PeproTech).

Transduction of primary cells

Lentiviral and MSCV vector productions were previously described33. Lin- cells were

transduced by spinoculation (90 minutes at 2500rpm; 8μg/mL polybrene) on two




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consecutive days. Cells were washed with phosphate-buffered saline (PBS) 24�hours

after the second spinoculation and used as indicated for the different assays.

Flow cytometric analyses

To analyze the different hematopoietic compartments, single-cell suspensions were

prepared from BM cells. Antibodies used in FACS staining for hematopoietic

stem/progenitor cells and B-cell precursors are listed in Table S2 and Table S3,

respectively. For flow cytometric analysis of CFU colonies, cells were washed twice with

PBS and subsequently stained with Phycoerythrin (PE)-conjugated anti-Sca-1 and

Allophycocyanin (APC)-conjugated anti-cKit or PE-conjugated anit-Gr-1 and APC-

conjugated anti-Mac1.

Clonogenic growth in vitro

For myeloid colony formation assays, (transformed) lin- cells were plated in

methylcellulose (M3534, STEMCELL technologies). The number of colonies was scored

after 6-7 days. For replating experiments, colonies were harvested and washed with PBS.

Cells were counted in a Neubauer chamber using trypan blue for dead cell exclusion and

plated in fresh medium for the subsequent round. For pre-B lymphoid CFU assays, lin-

cells or spleen cells were plated in methylcellulose (M3231, STEMCELL technologies)

supplemented with mIL-7 (10ng/mL). May-Grünwald Giemsa staining was performed as

described earlier33.

Further information about the experimental procedures is provided in supplemental

methods.




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Results

Psip1 knockout mice have reduced peripheral blood cell counts

To study the role of LEDGF/p75 in normal hematopoiesis, we established a conditional

Psip1 knockout mouse model in the hematopoietic compartment by crossing Psip1-

floxed mice31 with Vav-iCre mice, which constitutively express Cre recombinase in HSC

at early embryonic stages37 (Figure S1A-C). Complete depletion of LEDGF/p75 was

confirmed by qRT-PCR in lineage-depleted (lin-) cells and by Western blot in whole

bone marrow (BM) cells using a LEDGF/p75-specific antibody (Figure 2A-B). Mice

harboring homozygous deletion of Psip1 in the hematopoietic system (referred to as

PsipFl/Fl Vav-Cre positive mice or PsipVav/Vav) were viable, fertile, phenotypically normal

and born at the expected Mendelian frequencies compared to 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 of PsipFl/Fl and PsipVav/Vav mice were compared.

Peripheral white blood cell (WBC) counts of PsipVav/Vav mice were ~65% lower than

PsipFl/Fl mice (Figure 2C). Significant reductions were also observed in the lymphocytes

and neutrophils. In contrast, PsipVav/Vav mice had normal red blood cell, hemoglobin and

platelet counts (Figure 2D).

Furthermore, we checked the cellularity of the spleens and thymuses of PsipFl/Fl and

PsipVav/Vav mice. Compared to the controls, the total numbers of splenocytes and

thymocytes were significantly reduced by ~55% and 60% in PsipVav/Vav, respectively

(Figure 2E). Collectively, these results reveal that Psip1 inactivation affects the

hematopoietic system at steady state.




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In vivo ablation of Psip1 perturbs HSC compartments

Next, we analyzed the effects of Psip1 ablation on different hematopoietic compartments

by flow cytometric analysis of whole BM cells (Figure S2A-B). We first checked the

frequency of HSC (Lineage−Sca-1+ckit+ (LSK)) progenitors in total BM. FACS analysis

showed that the percentage of phenotypically defined HSC was significantly increased

after Psip1 deletion (Figure 3A). Sub-sectioning according to CD34 and CD135

expression showed that subpopulations containing primitive HSCs (Long term (LT)-

HSC, short term (ST)-HSC and multipotent progenitors (MPP)) were also increased in

PsipVav/Vav cells (Figure 3A). Further analysis of different subsets of the more

differentiated progenitors revealed that, while the percentage of the megakaryocyte-

erythroid progenitors (MEP) was comparable, the granulocyte-macrophage progenitors

(GMP) and common myeloid progenitors (CMP) were increased in Psip1-depleted cells

(Figure 3B). Similarly, the frequency of the common lymphoid progenitors (CLP) was

increased in Psip1-excised cells (Figure 3B).

Our data indicate that Psip1 knockout results in lower peripheral WBC 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, immature and mature B-cell

populations were reduced in PsipVav/Vav mice (Figure 3C). Together, these findings

suggest that loss of Psip1 affects the number of HSC and progenitor cells at steady state.




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Absence of Psip1 affects colony formation potential of HSC

Though loss of Psip1 resulted in higher number of HSC and progenitor cells, it was not

clear whether the functionality of the cells was affected. Accordingly, we sought to

compare the colony-forming capacity of Psip1 wild-type and knockout cells using

myeloid and pre-B lymphoid colony-forming unit (CFU) assays. Lin- cells were

harvested from PsipFl/Fl and PsipVav/Vav mice and serially plated in myeloid CFU culture.

After seven days (first plating), the number of colonies derived from PsipFl/Fl and

PsipVav/Vav mice was not significantly different (Figure 4A). However, the total cell

number harvested from the formed PsipVav/Vav colonies was reduced (8.9x106 PsipFl/Fl

cells vs. 4.6x106 PsipVav/Vav cells). In the second and third platings, the number of

colonies of PsipVav/Vav mice was significantly reduced compared to the wild-type control

(Figure 4A). Alternatively, we compared the colony-forming capacity of PsipFl/Fl and

PsipVav/Vav cells in pre-B lymphoid CFU assays. The number of pre-B colonies in Psip1-

depleted cells was reduced in lin- cells and spleen cells, compared to the controls (Figure

4B). To ensure that the decreased colony-forming potential in PsipVav/Vav cells was due to

Psip1 deletion and not Cre expression, we compared the CFU potential of lin- cells

harvested from C57BL/6 mice and Vav-iCre transgenic mice in the same background.

Cre-expression on its own did not affect the number of colonies formed upon replating,

nor did it alter the total cell number harvested from these colonies (data not shown).

Previous studies showed that germ-line deletion of Psip1 resulted in perinatal mortality

and homeotic skeletal transformations similar to Hox-cluster mutant mice35,36.

Additionally, MLL, MENIN and LEDGF/p75 have been shown to co-localize on HoxA7

and HoxA9 promoters and knockdown of LEDGF/p75 resulted in decreased HOXA9




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expression in MLL-FP transformed cells16,33. These results suggest that Hox genes might

be regulated by Psip1. Accordingly, we measured the expression of several HoxA and

HoxB cluster genes as well as other cofactors such as Meis1 in lin- cells harvested

directly from PsipFl/Fl and PsipVav/Vav mice and after plating in myeloid CFU assays.

HoxA9 and HoxA4 expression were significantly reduced only after first plating (Figure

4C). In contrast, expression of other HoxA genes (HoxA7, HoxA10 and HoxA11) and

HoxB4 was readily reduced at steady state. While the expression of HoxB8 was increased

after CFU plating in Psip1-depleted cells, Meis1 expression was comparable between

PsipFl/Fl and PsipVav/Vav cells before and after plating. May-Grünwald Giemsa staining on

the harvested colonies (after first plating) suggested a more differentiated morphology in

Psip1-depleted cells compared to the control (Figure 4D). In agreement, FACS analysis

of the harvested colonies showed reduced Sca-1 and cKit expression, suggesting

increased differentiation in PsipVav/Vav cells (Figure S3A-B). Furthermore, PsipVav/Vav cells

showed skewed differentiation towards the granulocytic lineage, as indicated by higher

Gr-1 expression when compared to 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

results suggest that Psip1 depletion affects the colony formation capacity of HSCs and

expression of Hox genes.

Specific loss of LEDGF/p75 and not LEDGF/p52 affects colony formation of HSCs

PSIP1 encodes LEDGF/p75 and the shorter LEDGF/p52 isoform. Since p52 lacks the

IBD, it is unable to interact with MLL (Figure 1B)34. To analyze the specificity of the

observed phenotype, we harvested lin- cells from C57BL/6J mice and transduced them




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with a lentiviral vector expressing a Ledgf/p75-specific miRNA or eGFP-miRNA as a

negative control. We observed ~40% knockdown for Ledgf/p75, while the p52 isoform

was not significantly affected as measured by qRT-PCR (Figure 5A). After seven days,

the effects of Ledgf/p75 knockdown were readily observed by the reduced number of

colonies (~70% reduction) compared to the control (Figure 5B). Moreover, knockdown

of Ledgf/p75 caused a ~2-fold reduction in HoxA9 expression (Figure 5C). In contrast,

depletion of Ledgf/p52 did not affect colony-formation potential of lin- cells or HoxA9

expression, even after three rounds in culture (Figure 5D-F). Altogether, our results

confirm that specific loss of LEDGF/p75 and not LEDGF/p52 affects the colony

formation capacity of HSCs.

Psip1 is dispensable for hematopoietic reconstitution

Next, we evaluated the capacity of Psip1-depleted cells to reconstitute the hematopoietic

system after BM transplantation (BMT). Lin- cells harvested from PsipFl/Fl and PsipVav/Vav

mice were transplanted into lethally irradiated recipients. Subsequently, hematopoietic

regeneration was monitored by measuring peripheral blood counts 4, 8, 12 and 16 weeks

post-transplantation. Short-term reconstitution of PsipVav/Vav cells at 4 weeks did not differ

from PsipFl/Fl cells, suggesting that LEDGF/p75 is not essential for HSC homing,

engraftment or reconstitution. Nonetheless, at 12 weeks post-transplantation, a mild but

significant reduction in the number of peripheral WBC was observed (Figure 5G). This

reduction persisted at 16 weeks post-transplantation, in agreement with the effects

observed for LEDGF/p75 depletion on steady-state hematopoiesis (Figure 2C).




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Altogether, these data indicate that loss of LEDGF/p75 impairs, but does not abolish

hematopoietic reconstitution.

Psip1 gene signature overlaps with that of MLL-FP target genes

To better understand the observed defects in hematopoiesis, we performed gene-

expression profiling by RNA-Seq on lin- cells harvested from PsipFl/Fl and PsipVav/Vav

mice after one round in the myeloid CFU assay. We identified 44 downregulated

(comprising several known MLL target genes) and 170 upregulated genes using the

criteria of an absolute log2-fold change >1.5 and p-value <0.05 (Figure 6A and Table

S4). Downregulation of known MLL target genes such as HoxA9 and Eya1 in PsipVav/Vav

cells was validated by qRT-PCR (Figure S4A)38. It has been established that wild-type

MLL and MLL-FPs have partially overlapping target gene sets39, suggesting that they can

be recruited by different mechanisms. Gene set enrichment analysis (GSEA) revealed that

the principal signature in the PsipVav/Vav samples was not significantly up- or

downregulated when compared to a set of genes with wild-type MLL bound to the

promoter regions (p-value =0.21, Figure S4B) 38. However, those genes with evidence for

promoter-bound MLL-complexes (overlapping MLL, WDR5 and H3K4me2 ChIP-Seq

peaks) are significantly enriched among the downregulated genes (p-value =0.02, Figure

6B)38. A stronger enrichment was found when MLL-ENL target genes were tested

against the PsipVav/Vav ranked list (p-value <0.001, Figure 6C)38. Similar results were

obtained with a set of MLL-AF9–bound genes (p-value <0.001, Figure S4C)40. These

data suggest that LEDGF/p75 recruits wild-type MLL to genes that are also targeted by

MLL-FPs16,24. This is in line with conservation of the LEDGF/p75 interaction with MLL-




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FPs, while other tethering mechanisms are lost or disrupted in these fusions. To verify

this, we assessed MLL occupancy at Hox genes in lin- cells harvested from Psip1-

depleted cells through quantitative chromatin immunoprecipitation (ChIP-qPCR). In line

with earlier reports24,41, ChIP-qPCR analyses demonstrated that MLL occupancy at

HoxA9 and HoxA7 promoters was decreased ~2 fold in PsipVav/Vav cells compared to the

controls (Figure 6D).

HoxA9 overexpression rescues defective colony formation of PsipVav/Vav cells

One of the top hits revealed by GSEA was that of genes downregulated in MOLM-14

cells (AML) upon HOXA9 knockdown (q-value =0.001, Figure 6E)42. Constitutive

HOXA9 expression is one of the main drivers of MLL-r leukemia14. Moreover,

overexpression of HoxA9 was shown to be sufficient to elicit cellular transformation43.

As such, we aimed to determine whether the defects observed in Psip1-depleted cells

could be rescued by HoxA9 overexpression. Lin- cells harvested from PsipFl/Fl and

PsipVav/Vav mice were transduced with a retroviral vector expressing HoxA9 or a mock

control (Figure S4D) and plated for colony formation. While Psip1 knockout led to ~70%

reduction in the number of colonies, overexpression of HoxA9 restored CFU activity to

wild-type levels (Figure 6F). These data were corroborated in Ledgf/p75 knockdown cells

(Figure S4E). In contrast to PsipVav/Vav cells, acute knockdown of Ledgf/p75 readily

caused a significant reduction in HoxA9 expression associated with a 5-fold reduction in

colony formation (Figure S4F-G). This defect was rescued to wild-type levels upon

HoxA9 overexpression. Collectively, these results suggest that LEDGF/p75 is important

for proper HSC differentiation, likely through regulation of Hox genes expression.




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Psip1 is essential for inducing MLL-rearranged leukemia

Earlier studies reported that LEDGF/p75 is important to maintain MLL-mediated

transformation in human MLL-r cell lines and primary MLL-fusion transformed cells16,33.

The availability of Psip1 knockout cells allowed us to investigate whether LEDGF/p75 is

important for the initiation of MLL-r leukemia (Figure 7A). Lin- cells harvested

from PsipFl/Fl and PsipVav/Vav mice were transduced with an MLL-ENL fusion

(PsipFl/Fl.MLL-ENL and PsipVav/Vav.MLL-ENL, respectively) and their transformation

potential was compared in the CFU assay. While the number of colonies in

PsipFl/Fl.MLL-ENL cells increased after the third plating due to MLL-fusion–mediated

transformation, Psip1 knockout cells proved refractory to transformation; as evident by

the significant reduction in the number of colonies upon serial plating (Figure 7B).

Similar data were obtained in MLL-AF9–transformed cells (Figure S5). In contrast,

expression of an oncogenic E2A-HLF fusion (PsipFl/Fl.E2A-HLF and PsipVav/Vav.E2A-

HLF), which is independent of HoxA9 for transformation44, revealed comparable

transformation capacity after serial plating in PsipFl/Fl and PsipVav/Vav cells (Figure 7C).

These data were corroborated in vivo by BMT. PsipFl/Fl.MLL-ENL and PsipVav/Vav MLL-

ENL cells were transplanted into lethally irradiated mice. Whereas recipients transplanted

with PsipFl/Fl.MLL-ENL cells succumbed to leukemia within 60-85 days after

transplantation, animals transplanted with PsipVav/Vav.MLL-ENL or control cells (PsipFl/Fl

cells transduced with pMSCV.Neo) did not show any sign of the disease, even at 285 days

post-transplantation (Figure 7D). PCR analysis for MLL-ENL fusion in whole BM cells

harvested from PsipFl/Fl.MLL-ENL moribund mice or PsipVav/Vav.MLL-ENL and control




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animals 180 days after BMT confirmed expression of the MLL-ENL fusion in PsipVav/Vav

cells despite absence of disease progression (Figure 7E).

In accordance with previous studies16, our data show that LEDGF/p75 can recruit both

wild-type MLL and MLL-FPs to genes driving MLL-r leukemia. In contrast, it has been

recently suggested that LEDGF/p75 is required to tether the wild-type complex, but not

MLL-FP at target gene loci24. To further investigate this, we introduced two mutations

(F148A and F151A) in the consensus IBD-interacting motif (IBM)45 of the MLL-ENL

fusion (mut.MLL-ENL), that specifically abrogate the interaction with LEDGF/p75 and

evaluated its oncogenic activity in vitro. In contrast to the wild-type MLL-ENL fusion,

the mut.MLL-ENL was unable to transform myeloid progenitors (Figure 7F).

Collectively, these data support a role for LEDGF/p75 in targeting both wild-type MLL

as well as its oncogenic counterpart onto the chromatin and that the function of

LEDGF/p75 in the MLL-FP complex is important to establish cellular transformation.




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Discussion

Oncogenic fusion proteins resulting from chromosomal translocations of the MLL gene

induce aggressive acute leukemias in children and adults, emphasizing the need for

new therapeutic strategies. It is clear that epigenetic states play an important role in the

proliferative capacity of transformed cells. Targeting epigenetic readers and writers

has become an attractive anti-cancer strategy as evidenced by the identification and

development of BET protein inhibitors46. However, careful understanding of the

proteins involved is necessary to avoid toxicity and to assess therapeutic windows.

MLL forms a ternary complex with MENIN and the epigenetic reader LEDGF/p75.

Several studies show that the latter tethers wild-type MLL and/or MLL-FP complexes

to chromatin (Figure 1A)16,24,41. Several approaches have been taken to interfere with

the function of MLL-fusion complexes, including DOT1L or BET protein inhibitors

that target the fusion moiety of MLL-FPs47-50. On the other hand, others directly target

the MLL fragment of the fusion. In this regard, the development of MLL-MENIN

interaction inhibitors proved the feasibility of targeting the MLL-MENIN-LEDGF/p75

complex51,52. Furthermore, detailed structural characterization of the MLL-MENIN-

LEDGF/p75 complex and strategies exploiting inhibitory peptides also support the

MLL-LEDGF/p75 interaction as a potential new therapeutic target19,20. However, since

the role of LEDGF/p75 (more specifically, the MLL-LEDGF/p75 interaction) in

hematopoiesis has not been studied, the specificity of potential MLL-LEDGF/p75

interaction inhibitors has not been addressed. This knowledge is required to fully

appreciate the druggability of this interaction.




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Since mice with systemic Psip1 deletion were shown to suffer from perinatal

lethality35,36, we investigated adult hematopoiesis by knocking out Psip1 in the

hematopoietic system. These mice did not exhibit any evident phenotypic abnormalities,

suggesting that Psip1 is not essential for HSC maintenance or survival in adult mice.

Nonetheless, Psip1 knockout mice displayed specific hematopoietic defects including ~2-

fold decrease in peripheral WBC counts, perturbed numbers of HSCs and progenitor cells

and impaired colony-forming capacity upon serial plating.

Earlier gene targeting studies of Mll underscored its importance for HSC development

during embryogenesis and adult hematopoiesis53-55. Mll deletion resulted in

hematopoietic defects and reconstitution failure, suggesting that LEDGF/p75 is not

exclusively required for all physiological functions of MLL. Other mechanisms might

exist that tether the MLL complex to its target genes, as has been suggested before56,57.

We observed that expression changes in PsipVav/Vav samples are more pronounced at

MLL-FP-bound genes than at MLL wild-type–bound genes. This result confirms that

those genes targeted by the MLL-MENIN-LEDGF/p75 complex are also crucial for the

development of MLL-r leukemia. In contrast to the p52 isoform, LEDGF/p75 contains the

IBD domain, which specifically interacts with MLL and several other cellular proteins45.

However, among these interaction partners, only MLL is known to affect HOX

expression. Although, we did not observe decreased expression of HoxA9 in the lin-

fraction in contrast to other members of the HoxA cluster, our RNA-Seq data revealed

downregulation of HoxA9 targets in Psip1-depleted cells after one week in culture and

the fact that the observed phenotypes could be rescued upon HoxA9 overexpression

suggests that the HoxA9-axis is an important component of the observed differentiation




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defects and decreased CFU formation. Targeted disruption of HoxA9 was shown to

display similar hematopoietic defects to Psip1 knockout mice such as lower peripheral

lymphocytes, reduced spleen and thymus cellularity, defects in B-cell production and

impaired CFU capacity58,59.

Despite its dispensability for normal hematopoiesis, our data show that LEDGF/p75 is

essential for the initiation of MLL-r leukemia. While MLL-r leukemia is mainly induced

by gain-of-function of the MLL-FPs, the remaining wild-type allele is typically

expressed. Some studies suggested a functional role of the latter in cellular

transformation60,61. On the other hand, MLL-AF6 human leukemic cells (ML2 cells) do

not express the wild-type MLL protein38. Although all MLL-FPs retain the wild-type N-

terminus, genome-wide analysis has shown that wild-type MLL and MLL-FPs have

distinct chromatin binding profiles39. Whereas MLL-FPs exert their oncogenic activity

through activation of a particular gene expression program, integrative analyses of MLL

binding and expression profiling showed that MLL-FPs share only a small subset of

target genes with the wild-type protein38, suggesting different chromatin-tethering

mechanisms. Earlier work claimed that LEDGF/p75 specifically associates with wild-

type MLL and MLL-FP and co-localizes on target genes16. In contrast, recent studies

from the same group and others showed that knockdown of LEDGF/p75 led to increased

MLL-FP recruitment24,41. The authors suggested an independent role for LEDGF/p75 in

the wild-type complex, which in 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 upon LEDGF/p75 depletion. While our Psip1

knockout mice reveal that LEDGF/p75 is essential for MLL-r leukemia and that the




21

absence of LEDGF/p75 affects Hox expression and hematopoiesis likely due to

diminished MLL recruitment, it was unclear whether the leukemogenic defect is due to

diminished tethering of wild-type MLL and/or MLL-FP to target genes. Based on recent

structural data, we mutated the IBM in MLL-ENL which is responsible for the interaction

with LEDGF/p7545. CFU assays revealed impaired transformation capacity in lin- cells.

These data corroborate the function of LEDGF/p75 in the tethering the MLL-FP, as has

been suggested earlier16.

In summary, we demonstrated that LEDGF/p75 is not essential for adult hematopoiesis,

while MLL-r leukemia cannot be initiated in its absence. These data open new

perspectives to develop small molecule inhibitors of the MLL-LEDGF/p75 interaction,

validating a novel target with a sufficient therapeutic window to treat MLL leukemia and

possibly other MLL-dependent diseases62.




22

Acknowledgements

We thank Martin Michiels and Sara T’Sas for the technical assistance and Prof. Thomas

Milne for the technical advice. This work was supported by grants from the KU Leuven

IDO program (Interdisciplinair onderzoeksprogramma) (IDO/12/008-3E130241) and the

Flemish FWO (G.0595.13 to Z.D, G065614N and Odysseus program to P.V.V), the

Flemish agency for Innovation by Science and Technology (IWT) SBO 140038

(ChromaTarget) and the US National Institutes of Health (AI039394 to A.N.E.). S.EA is

a postdoctoral fellow supported by the IDO program. J.S. is supported by the Swiss

National Science Foundation (31003A_149714), the Swiss Cancer League (KFS-3487-

08-2014) and the Gertrude Von Meissner Foundation (Basel, Switzerland). T.P. and S.G

are postdoctoral fellows funded by the Belgian “Stand Up To Cancer” Foundation.

Authorship

Contributions:

S.EA and J.DR designed the experiments and wrote the manuscript. S.EA, J.DR, T.P,

S.G, S.J and S.VB performed experiments and analyzed data. A.E. generated PsipFl/Fl

mice. J.D performed bioinformatics analyses. N.B performed and analyzed May-

Grünwald Giesma stainings. J.S, F.C, P.VV, Z.D and J.DR supervised the project. All

authors read and approved the final manuscript.

Conflict-of-interest disclosure:

The authors have no competing interests to disclose.

Correspondence:




23

Jan De Rijck, Laboratory for Molecular Virology and Gene Therapy, Kapucijnenvoer 33,

3000 Leuven, Belgium; email: [email protected] and Zeger Debyser, Laboratory

for Molecular Virology and Gene Therapy, Kapucijnenvoer 33, 3000 Leuven, Belgium;

email: [email protected]




24

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28

Figure Legends

Figure 1. Interaction of the MLL-fusion complex with LEDGF/p75.

(A) Schematic representation of the MLL-fusion complex. The most common MLL

chromosomal translocations result in expression of the N-terminus of MLL and the C-

terminus of a fusion partner gene (MLL-Fusion). The extreme N-terminus of MLL forms

a triple complex with MENIN and LEDGF/p75. The latter recognizes H3K36di-and tri-

methylated marks, whereby it tethers the MLL-fusion complex to its targets genes such as

HOXA9, MEIS1 and PBX3.

(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 the

LEDGF/p75 binding domain (IBD-interacting motif (IBM)) at its extreme N-terminus.

Further downstream, wild-type MLL contains three AT-Hooks, two speckled nuclear

localization signals (SNLs), a transcriptional repression domain (TRD), four PHD fingers

and a bromodomain. The transactivation domain (TAD) and SET domains are located at

the MLL C-terminus. LEDGF/p75 harbors a Pro-Trp-Trp-Pro domain (PWWP; aa 1–91),

a nuclear localization signal (NLS), two AT hook-like motifs (ATH), three charged

regions (CR), and the integrase binding domain (IBD) which binds to MLL. LEDGF/p52

shares similar structural domains but lacks the C-terminal IBD.

Figure 2. Knockout of Psip1 reduces peripheral blood cell counts.

(A-B) qRT-PCR of lineage-depleted bone marrow (BM) cells (A) and Western blot of

whole BM cells (B) validating complete LEDGF/p75 depletion in Psip1 knockout




29

(PsipVav/Vav) mice compared to the floxed control mice (PsipFl/Fl). mRNA expression in

(A) and protein expression in (B) were normalized to Gapdh and β-Tubulin, respectively.

(C-D) Peripheral blood cell counts measured in PsipFl/Fl and PsipVav/Vav mice (8-12 weeks

of age). Mean values and SEM are indicated. WBC; white blood cells, RBC; red blood

cells, HGB; hemoglobin, ns; non-significant.

(E) Total cell number extracted from spleens and thymuses of PsipFl/Fl and PsipVav/Vav

mice (8-10 weeks of age). Mean values and SEM are indicated. P values (*) show

significance (**p< 0.01, ***p< 0.001, Student’s t test).

Figure 3. Psip1 excision perturbs the HSC compartments.

(A) The proportion of BM corresponding to the HSC-containing LSK (Lineage−Sca1+c-

kit+) progenitors measured in PsipFl/Fl and PsipVav/Vav mice (4-7 animals per group, 8-10

weeks of age). Sub-sectioning according to CD34 and CD135 expression yielded

phenotypic assessments of LT-HSC, ST-HSC and MPP fractions. LT-HSC; long-term

hematopoietic stem cells, ST-HSC; short-term hematopoietic stem cells, MPP; multi-

potent progenitors.

(B) BM frequencies of the more differentiated progenitors gated in the LSK population,

sub-sectioned based on CD16/32 and CD34 expression to compare CMP, GMP and

MEP. The CLP fraction is gated based on CD127 expression. CLP; common lymphoid

progenitors, CMP; common myeloid progenitors; GMP; granulocyte-macrophage

progenitors, MEP: megakaryocyte-erythroid 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 are




30

shown (4-7 animals per group). Mean values and SEM are shown. P values (*) show

significance (ns; non-significant, *p< 0.05, **p< 0.01, ***p< 0.001, Student’s t test).

Figure 4. Psip1 knockout affects colony formation potential of HSC in vitro.

(A) Number of colonies in three consecutive rounds of a myeloid CFU assay per 104

lineage-depleted (lin-) BM cells harvested from PsipFl/Fl and PsipVav/Vav mice (10 weeks

of age).

(B) Pre-B CFU assays for PsipFl/Fl and PsipVav/Vav cells. Lin- cells and spleen cells were

harvested and plated in methylcellulose for 7-10 days. The number of colonies formed

was normalized to the control (PsipFl/Fl) cells.

(C) qRT-PCR measuring expression levels (normalized to Gapdh) of different genes in

lin- cells harvest from PsipFl/Fl or PsipVav/Vav cells (before 1st plating) and after one round

in myeloid CFU assay (after 1st plating).

(D) Comparison of cell morphology of PsipFl/Fl and PsipVav/Vav cells after one round in the

CFU assay via May-Grünwald Giemsa staining. A representative picture is shown.

(E) FACS analysis for Gr-1 expression in PsipFl/Fl and PsipVav/Vav cells (n=8) harvested

after one round in CFU assay. Mean and SEM values are indicated (**p < 0.01).

Error bars in panels A, B, C and E represent standard deviation of triplicate

measurements. Statistical differences were determined using Student's t-test; *p< 0.05,

**p < 0.01, ***p < 0.001.




31

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 lin- cells

harvested from 8-weeks C57BL/6J mice after transduction with lentiviral vector

expressing a Ledgf/p75-specific miRNA (Ledgf/p75 KD) or eGFP-miRNA as a control.

Expression levels were 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/p75 knockdown cells.

(D-E) qRT-PCR measuring Ledgf/p75 and Ledgf/p52 expression levels (D) and serial

plating of a myeloid CFU assay (E) after Ledgf/p52 knockdown (KD). Expression levels

in (D) were normalized to Gapdh.

(F) qRT-PCR measuring HoxA9 expression levels (normalized to Gapdh) in Ledgf/p52

knockdown cells after first plating in the CFU assay (described in E). Error bars represent

standard deviation of triplicate measurements.

(G) Peripheral white blood cell counts (WBC) measured after bone marrow

transplantation in lethally irradiated recipients transplanted with a total of 1x106 lin- cells

harvested from 8-10 weeks PsipFl/Fl and PsipVav/Vav mice. WBC was monitored 4, 8, 12

and 16 weeks post-transplantation. Mean values and SEM are indicated.

Error bars in panels A-F represent standard deviation of triplicate measurements.

Differences in panels A to G were determined using Student's t-test; ns; non-significant,

*p< 0.05, **p < 0.01, ***p < 0.001.




32

Figure 6. Psip1 knockout gene expression signature overlaps with that of MLL-FP

target genes.

(A) Heat map of RNA-sequencing data showing the top differentially expressed genes in

PsipFl/Fl and PsipVav/Vav cells, harvested after one round in the CFU assay.

(B-C) Gene set enrichment analysis (GSEA) showing that Psip1 regulated genes are

enriched for genes with promoter regions bound by MLL complex (B) and MLL-ENL

fusion (C).

(D) Quantitative chromatin immunoprecipitation (ChIP) assay for PsipFl/Fl and PsipVav/Vav

cells using Mll antibody. The promoter regions amplified by qPCR are indicated below

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

of Hoxa9.

(F) CFU assay per 104 cells harvested from PsipFl/Fl and PsipVav/Vav mice. Cells were

transduced with pMSCV-HoxA9-pgk-neo or mock vector and plated for colony

formation.

Error bars indicate standard deviations of triplicate measurements. Differences were

determined using Student's t-test; *p< 0.05.

Figure 7. LEDGF/p75 is essential for MLL-rearranged transformation.

(A) Schematic representation of the experimental setup. Bone marrow cells were

harvested from 10-weeks old PsipFl/Fl and PsipVav/Vav mice. After depletion of lineage-

committed progenitors, cells were transduced with MLL-ENL or E2A-HLF fusions and




33

assessed for leukemogenic activities in vitro in colony formation unit (CFU) assays and

in vivo in bone marrow transplantations (BMT).

(B-C) Colony-forming units (CFU) per 104 PsipFl/Fl or PsipVav/Vav cells transduced with

MSCV encoding MLL-ENL (B) and E2A-HLF (C) fusions. Representative images of

Tetrazolium-stained colonies after the third plating are shown. Error bars represent

standard deviation of triplicate measurements. Differences were determined using

Student's t-test; **p < 0.01; ***p < 0.001.

(D) Kaplan-Meier survival curve for lethally irradiated recipients transplanted with

PsipFl/Fl or PsipVav/Vav cells transduced with MLL-ENL (PsipFl/Fl.MLL-ENL and

PsipVav/Vav.MLL-ENL, respectively) or control cells (PsipFl/Fl cells transduced with mock

vector). Number of transplanted animals (n) per group is indicated.

(E) PCR analysis of 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

(D)) harvested at 180 days post transplantation. 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 lin- cells transduced with MLL-ENL fusion

or MLL-ENL mutated in the LEDGF/p75–binding domain (mut.MLL-ENL). Error bars

represent standard deviation of triplicate measurements. Differences were determined

using Student's t-test; **p < 0.01.




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GMP

0.0

0.5

1.0

1.5

0

5

10

15

0

5

10

15

20

0

2

4

6

8

10

0.00

0.01

0.02

0.03

0.04

0.05

BM

Fre

quen

cy (%

)

Pre-Pro B-cells

Pro B-cells

Pre B-cells

Immature B-cells

Mature B-cells

*** **

*

******

**

*** ***

*

A

B

C

PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav

PsipFl/Fl PsipVav/VavPsipFl/Fl PsipVav/VavPsipFl/Fl PsipVav/VavPsipFl/Fl PsipVav/Vav

PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav PsipFl/Fl PsipVav/Vav

ns

ns

Figure 3




1st Plating 2nd Plating

0

100

200

300

400

CFU

/104 c

ells

3rd Plating

A

DPsipFl/Fl

PsipVav/Vav

*****

PsipFl/Fl

PsipVav/Vav

E

0

20

40

60

80

Gr-

1 po

sist

ive

cells

(%

) **

PsipFl/Fl

PsipVav/Vav

-103 0 103 1040

20

40

60

80

100

PsipFl/Fl

PsipVav/Vav

Negative control

Gr-1 expression

Figure 4R

elat

ive

expr

essi

on

0.0

0.5

1.0

1.5

Meis1

0.0

0.5

1.0

1.5HoxA4

**

HoxA7

***

*

HoxA10

*

***

HoxA11

*

*

*

*

**

HoxB4 HoxB8

Rel

ativ

e ex

pres

sion

Meis1HoxA4 HoxA7 HoxA10 HoxA11 HoxB4 HoxB8

Before 1st plating

After 1st plating

C

PsipFl/Fl

PsipVav/Vav

HoxA9

HoxA9

**

0.0

0.5

1.0

1.5

Rel

ativ

e C

FU n

umbe

r

Lin- Spleen

***

PsipFl/Fl

PsipVav/Vav

B




Ledgf/p52 KD

Rel

ativ

e ex

pres

sion

Control

0

20

40

60

150

200

250

300

350

CFU

/104

cells

D E

1st Plating 2nd Plating 3rd Plating

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

(Hox

A9/

Gap

dh)

ControlLedgf/p75 KD

0

100

200

300

400

500

CFU

/104 c

ells

A CB

***

*

0

10

20

30

4 weeks 8 weeks

WB

C (1

03 /µl)

12 weeks 16 weeks

** *nsns

G

Figure 5

PsipFl/Fl

PsipVav/Vav

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

Ledgf/p75 Ledgf/p52

**

0.0

0.5

1.0

1.5

Ledgf/p75 Ledgf/p52

0.0

0.5

1.0

1.5

**

Rel

ativ

e ex

pres

sion

(Hox

A9/

Gap

dh)

F




0

100

200

300

400

500

600

700

800

CFU

/104 c

ells

Mock HoxA9

C

*

**

Ifi202bHoxa9CtseGm9025Eya1Psma8Mcpt1Rps24−ps3Psip1Gm8822Mcpt2Mcpt4C1qaPtk7St8sia1F13a1HnmtSix1Tspan13Dock9Nos2Ifi47Mef2cPtprv2810417H13RikSh2d1b1CtskMyrfTchhPeg12Arnt2Pls3Kif21aSfmbt2Ccnd1MarcoFgfr1Naip5Meis3Prickle2H2afy2Xkr6Adra2cAstn2Adamts1Elavl2Uchl1Fzd8Abcc8Ndn

−6 −3 0 3

Fold change (log2)

PsipFl/Fl PsipVav/VavA

PsipFl/Fl

PsipVav/Vav

0.00

0.05

0.10

0.15

0.2

0.3

0.4

0.5

0.6

% In

put

HoxA9 HoxA7 βactin

**

PsipFl/Fl

PsipVav/Vav

Figure 6

ED

B

F,

,




PsipFl/Fl.MLL-ENLPsipVav/Vav.MLL-ENL

0

100

200

300

4001000

1200

1400

1600

1800

2000

CFU

/104 c

ells

1st Plating 3rd Plating

0 20 40 60 80 260 280 300

0

50

100

Control (n=5)

Days post-transplantation

Perc

ent s

urvi

val

PsipFl/Fl.MLL-ENL (n=8)

PsipVav/Vav.MLL-ENL (n=8)

400 bp500 bp

PsipFl/F

l .MLL-E

NL

PsipVav

/Vav .MLL-E

NL

Control

PsipFl/Fl

or PsipVav/Vav

mice

A

B C

D

E

0

200

400

6001000

1200

1400

1600

1st Plating 2nd Plating 3rd PlatingC

FU/1

04 cel

ls

PsipFl/Fl.E2A-HLFPsipVav/Vav.E2A-HLF

2nd Plating

***** ***

BMT

CFU

in vivo

in vitroLineage-depletedBM cells

Transduction withMLL-ENL

or E2A-HLF

Figure 7

0

500

1000

1500

2000MLL.ENLmut.MLL.ENL

1st Plating 2nd Plating

CFU

/104 c

ells

3rd Plating

****

F




doi:10.1182/blood-2017-05-786962Prepublished online October 30, 2017;

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

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