Gene Dosage Effect of CUX1 in a Murine Model Disrupts HSC Homeostasis and Controls the Severity and Mortality of MDS

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Regular Article

MYELOID NEOPLASIA Gene dosage eﬀect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS

Ningfei An,1 Saira Khan,1 Molly K. Imgruet,1 Sandeep K. Gurbuxani,1,2 Stephanie N. Konecki,1,3 Michael R. Burgess,4 and Megan E. McNerney1,2,5

1Department of Pathology, 2The University of Chicago Medicine Comprehensive Cancer Center, and 3Committee on Immunology, The University of Chicago, Chicago, IL; 4Department of Medicine, University of California San Francisco, San Francisco, CA; and 5Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL

KEY POINTS Monosomy 7 (27) and del(7q) are high-risk cytogenetic abnormalities common in myeloid malignancies. We previously reported that CUX1, a homeodomain-containing transcription l CUX1 deficiency leads to transient clonal factor encoded on 7q22, is frequently inactivated in myeloid neoplasms, and CUX1 myeloid expansion followed tumor suppressor activity is conserved from humans to Drosophila. CUX1-inactivating by HSC depletion, mutations are recurrent in clonal hematopoiesis of indeterminate potential as well as anemia, and trilineage myeloid malignancies, in which they independently carry a poor prognosis. To determine dysplasia. the role for CUX1 in hematopoiesis, we generated 2 short hairpin RNA-based mouse l CUX1 transcriptionally models with ∼54% (Cux1mid)or∼12% (Cux1low) residual CUX1 protein. Cux1mid mice de- regulates HSC velop myelodysplastic syndrome (MDS) with anemia and trilineage dysplasia, whereas quiescence, low proliferation, and CUX1 mice developed MDS/myeloproliferative neoplasms and anemia. In diseased mice, lineage specification. restoration of CUX1 expression was sufficient to reverse the disease. CUX1 knockdown bone marrow transplant recipients exhibited a transient hematopoietic expansion, followed by a reduction of hematopoietic stem cells (HSCs), and fatal bone marrow failure, in a dose-dependent manner. RNA-sequencing after CUX1 knockdown in human CD341 cells identified a 27/del(7q) MDS gene signature and altered differentiation, proliferative, and phosphatidylinositol 3-kinase (PI3K) signaling pathways. In functional assays, CUX1 maintained HSC quiescence and repressed proliferation. These homeostatic changes occurred in parallel with decreased expression of the PI3K inhibitor, Pik3ip1, and elevated PI3K/AKT signaling upon CUX1 knockdown. Our data support a model wherein CUX1 knockdown promotes PI3K signaling, drives HSC exit from quiescence and proliferation, and results in HSC exhaustion. Our results also demonstrate that reduction of a single 7q gene, Cux1,is sufficient to cause MDS in mice. (Blood. 2018;131(24):2682-2697)

Introduction and poor survival. Despite this clear clinical importance, the key tumor Monosomy 7 (27) and del(7q) are frequent, adverse-risk cyto- suppressor genes (TSGs) encoded on 7q have remained unclear. genetic abnormalities in diseases of hematopoietic stem and fi fi progenitor cells (HSPCs). 27/del(7q) occurs across the spectrum of We previously identi ed a haploinsuf cient, myeloid TSG on chro- 7 myeloid disorders, from bone marrow (BM) failure to overt leukemia, mosome band 7q22, CUX1. CUX1 is a nonclustered homeobox 8 and typically carries an adverse prognosis. Fourteen percent of adult transcription factor. The full-length p200 protein has 1 homeo- and 30% of pediatric myelodysplastic syndrome (MDS) patients domain and 3 cut repeat DNA-binding domains. CUX1 is highly have 27/del(7q).1,2 Among inherited and acquired BM failure conserved, ubiquitously expressed, and essential for survival in mice syndromes, 27/del(7q) develops in 40% to 80% of cases that and Drosophila.9-12 We demonstrated that haploinsufficiency of the evolve to MDS or acute myeloid leukemia (AML).3 27/del(7q) ortholog, cut, led to hemocyte overgrowth and tumor formation in occurs in 10% to 15% of de novo AML4 and 49% of therapy- Drosophila melanogaster. Similarly, CUX1 haploinsufficiency gave related myeloid neoplasms.5 In addition, 27/del(7q) is identified in human HSPC an engraftment advantage in mouse xenotransplants.7 33% of juvenile myelomonocytic leukemia (JMML) and 14% of chronic myelomonocytic leukemia (CMML), which are myelodysplastic/ The genetic evidence for CUX1 as a critical TSG is overwhelming. myeloproliferative neoplasms (MDS/MPN).2,6 Regardless of the CUX1-inactivating mutations have been reported in numerous disease subtype, 27/del(7q) typically portends chemoresistance analyses of myeloid neoplasms.7,13-17 CUX1 mutations that occur

A B 1.8 * * 1.6 * Cux1low Cux1mid 1.4 mm9 136.8 Mb 136.9 Mb 137.0 Mb 1.2 * Chr. 5 1.0 NM_001291233.1 0.8 * Cux1 NM_009986.4 0.6 NM_001291234.1 RNA level Cux1 RNA

relative to LT-HSC relative 0.4 NM_001291239.1 0.2 NM_001291240.1 0 Casp NM_198602.3 NM_001291238.1 MPPCMPMEPGMP T cellB cell LT-HSCST-HSC Monocyte Granulocyte C E RBC Platelets WBC p=0.06 15 3000 15 mid low

Ren Cux1 Cux1 ** 10 2000 10 Ren CUX1 p200 Cux1mid K/ µ L K/ µ L M/ µ L 5 1000 5 Actin

0 0 0 8 18 mos. 8 18 mos. 8 18 mos. D FG PB monocytes PB granulocytes 1.5 Ren Cux1mid 50 60 * 0.020 * Cux1low * 1.0 40 0.015 cells 40 + 30 0.5 0.010 Ren 20 mid 20 Cux1

relative to Ren Cux1 relative 10 0.005 % of CD45

0 Spleen/body weight LSK Myeloid 0 0 0.000 progenitors 8 18 mos. 8 18 mos. Ren Cux1mid HI J Peripheral blood Bone marrow Spleen ii iKL

M N Ren (low power)

ii ii ii O (low power) mid Cux1 iii iii iii P (high power) mid Cux1

Figure 1. Description of CUX1 knockdown mouse models and characterization of MDS in Cux1mid mice. (A) Cux1 has cell type–speciﬁc expression levels. Cells were sorted as follows from the BM of wild-type C57BL/6 mice: LT-HSC (LSK1/CD342/Flt32), ST-HSC (LSK1/CD341/Flt32), MPP (LSK1/CD341/Flt31), CMP (Lin2/Sca12/c-Kit1/CD341/CD16/ 32low), MEP (Lin2/c-Kit1/Sca12/CD342/CD16/322), and GMP (Lin2/c-Kit1/Sca12/CD341/CD16/32high). The following cells were sorted from the spleen: granulocytes (CD11b1/Gr11), monocytes (CD11b1/Gr12), B cells (B2201), and T cells (CD31). qRT-PCR for Cux1 mRNA was performed. The data represent the mean 6 SEM from 4 biological replicates. *Student

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in the context of normal chromosome 7 alleles independently carry 27/del(7q).7 We extended this finding by first analyzing The a poor prognosis.14,17 CUX1 is also recurrently mutated in clonal Cancer Genome Atlas AML data.32 CUX1 has 7 RefSeq isoforms; hematopoiesis of indeterminate potential (CHIP),18-21 indicating 3 encode the CUX1 DNA-binding transcription factor and the that CUX1 mutations provide HSPC with a competitive advantage others encode the Golgi-associated CASP protein.8,27 The and predisposition for transformation.22 Further, CUX1 is mutated in Cancer Genome Atlas AML RNA-sequencing data were pro- a variety of solid tumors; thus, it may regulate tumorigenesis more vided at the gene level, wherein CUX1 and CASP isoforms were generally.7,17 aggregated to a single gene. This analysis showed 60% residual CUX1/CASP in samples with a deletion spanning CUX1 (sup- We reported CUX1 global genomic targets by chromatin plemental Figure 1A). To look at this finding in more detail, we immunoprecipitation-sequencing in 3 human cell types, including parsed the CUX1 and CASP encoding isoforms from our published the K562 myeloid leukemia cell line.23 CUX1 preferentially bound RNA-sequencing data.7 The CUX1 isoform, NM_181552, is distal, active enhancers and controlled expression of cell-cycle expressed at 66% residual levels in 27/del(7q) patients (supple- regulators.23 CUX1 fit an analog model of dose-sensitive gene mental Figure 1B). We interrogated MDS and JMML microarray regulation,23 which may provide a nuanced means for CUX1 datasets.33,34 We divided patients into those with or without control of a disparate array of cellular programs (including pro- 27/del(7q) and removed CASP-specific probes. Across datasets, liferation, differentiation, and apoptosis) across a broad array of CUX1 probes showed a range of 20.3 to 20.9 log2 fold-change cell types, and even with a single cell type.24-26 reduction (supplemental Figure 1C-E). In other words, for the majority of patients with 27/del(7q), CUX1 levels do not fall below To identify the in vivo functions for CUX1 in mice, it was essential for 50% of the levels expressed in patients with diploid chromosome 7 us to take a nontraditional genetic approach because of the alleles. These results suggest that for patients with 27/del(7q), the complexity of the gene. Cux1 is large, ranked in the top 2.1% of remaining CUX1 allele is expressed and CUX1 is haploinsufficient. mammalian genes by size. Cux1 has 33 exons, more than ;97% of other murine genes. Cux1 shares exons with Casp, a gene encoding a Golgi-associated protein.8,27 In addition, Cux1 has Generation of CUX1 knockdown mouse models 27 3 alternative start sites and 7 RNA splice isoforms. Traditional We sought to determine the level of Cux1 in murine hemato- knockout mice had unanticipated alternative splicing that removed poietic lineages, and if the expression pattern is conserved the inserted STOP cassettes, causing hypomorphic protein ex- between humans and mice. By quantitative reverse transcription 12,28-30 pression and incomplete Cux1 knockouts. Previous models polymerase chain reaction (qRT-PCR), Cux1 is high in long-term 12,28-30 also had perinatal lethality and extrahematopoietic effects. HSC (LT-HSC) and lower in short-term HSC (ST-HSC) and my- To circumvent these issues, we engineered inducible Cux1 short eloid progenitors (MPs). Cux1 increases in differentiated gran- 31 hairpin RNA (shRNA) knock-in mice. We report that CUX1 ulocytes and monocytes, compared with granulocyte-monocyte knockdown led to MDS and MDS/MPN with features of human progenitors (GMPs) (Figure 1A). This pattern is similar to what we disease. Our results also demonstrate that reduction of a single observed in human counterparts.7 7q gene, Cux1,issufficient to cause myeloid disorders, and Cux1 is a critical regulator of hematopoietic stem cell (HSC) homoeostasis. To determine the in vivo role for CUX1 in hematopoiesis, we generated CUX1 knockdown mice using an shRNA approach.31 Methods We engineered 2 knock-in lines, expressing shRNAs of different specificity, to control for potential off-target effects. Cux1mid tar- Complete methods are provided in the supplemental Methods, gets exon 5, an exon shared by all Cux1 and Casp isoforms available on the Blood Web site. (Figure 1B). Cux1low targets the 39 untranslated region of exon 24, shared only by CUX1-encoding transcripts and not CASP- Results encoding isoforms (Figure 1B). The transgenes were knocked CUX1 is haploinsufficient in human in to the endogenous Col1a1 locus and under the control of a myeloid malignancies tetracycline response element (supplemental Figure 2). A second- We previously reported that CUX1 is expressed at ;50% levels generation reverse tet-transactivator, m2-rtTA, is expressed under in de novo AML and therapy-related myeloid neoplasms with the ubiquitous ROSA26 promoter. In the presence of doxycycline

Figure 1 (continued) t test, P , .05. (B) The Cux1 genomic locus is shown with all 7 RefSeq mRNA isoforms. shRNA (Cux1low and Cux1mid) targeting the indicated exons (red arrows) were used to generate transgenic mice. (C) A representative immunoblot for CUX1 protein in thymocytes isolated from Cux1low,Cux1mid,andRenmiceafter7daysofdox.Themean6 SD level of residual CUX1 protein quantiﬁed from 4 biological replicates for Cux1low was 12% 6 9% and 54% 6 17% for Cux1mid.(D)Cux1 mRNA level in sorted populations from Cux1mid and Cux1low BM compared with Ren littermate control. One representative replicate of 3 biological replicates is shown. LSK are Lin2/c-Kit1/Sca11), and myeloid progenitors are Lin2/Sca12/c-Kit1. (E) At 6 to 10 weeks of age, Cux1mid and Ren littermate control mice were started on continuous dox treatment and monitored for up to 18 months. Complete blood counts of mice at 8 and 18 months of dox are shown. (F) Flow cytometric analysis of peripheral blood (PB) samples for monocyte (CD11b1/Gr12) and granulocyte (CD11b1/Gr11) populations at 8 and 18 months of dox treatment. (G) Spleen weights after 18 months of dox. The mean 6 SD is shown. *P # .05, **P # .01, Wilcoxon rank test. (H-P) Representative morphology images are shown for mice after 18 months of dox. (H) Wright-Giemsa staining of the peripheral blood in (Hi) Ren and (Hii-iii) Cux1mid mice. (Hii) Cux1mid mice have an increase in circulating mature granulocytes (arrowheads). (Hiii) A granulocyte with pseudo Pelger-Huet anomaly is shown (yellow arrowhead). Red blood cells in Cux1mid mice have increased Howell-Jolly bodies (arrow) and increased reticulocytes with basophilic stippling (black arrowheads). (Ii-ii) H&E staining of the bone marrow of aged Cux1mid mice shows a marked increase in megakaryocytes. (Iiii) At high power, clusters of megakaryocytes (arrowheads) with dysplastic features can be appreciated, including micromegakaryocytes, nuclear hypolobation, and condensed, hyperchromatic nuclei. (Ji-ii) H&E staining shows an expansion of the red pulp at the expense of the white pulp. The white pulp is outlined by a yellow dashed line for clarity. (Jiii) High-power analysis of Cux1mid spleens illustrates increased megakaryocytes (arrowheads) with some micromegakaryocytes with condensed, hyperchromatic nuclei. (K-L) Additional examples of peripheral blood granulocytes with pseudo Pelger-Huet anomaly. (M) Neutrophil with hypersegmentation. (N) A representative giant platelet is shown (arrow). (O) Touch preparation of Cux1mid spleen reveals erythroid dysplasia as evidenced by binucleated erythroblasts (arrowheads) and abnormal nuclear contours (arrow). (P) H&E staining shows a myelomonocytic cell inﬁltrate (dashed yellow line) in the liver of Cux1mid mice. Images were taken with a Zeiss Axioskop microscope. Chr, chromosome; H&E, hematoxylin and eosin; mRNA, messenger RNA; PB, peripheral blood; SD, standard deviation; SEM, standard error of the mean.

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A B RBC Platelets WBC 100 X 15 * ** 3500 * 40 **** * Ren (n=19) 3000 80 X Cux1mid (n=11) 12 2500 30 60 Dox 2000 9 20 K/ µ L K/ µ L

M/ µ L 1500 40 low Cux1 (n=20) 1000 20 275 d dox 6 10 Percent survival Percent p < 0.001 500 0 3 0 0 0 100 200 300 400 500 1.53468 1.5 3 4 6 8 1.5 3 4 6 8 Days since birth Months post-dox Months post-dox Months post-dox C PB monocytes PB granulocytes PB B cells PB T cells 25 80 p=0.06 80 ***40 * * * *** 20 Ren cells 60 60 30 low + 15 Cux1 40 40 20 10 5 20 20 10 % of CD45 0 0 0 0 138 138 138 138 Months post-dox Months post-dox Months post-dox Months post-dox D Time of sacrifice E Spleen at sacrifice F Liver at sacrifice G BM Spleen ** Ren Cux1low *** 0.10 ** 600 0.06 0.08 )

6 400 0.04 0.06 ** 0.04 0.02 (x 10 200 0.02 Liver/body weight

Spleen/body weight 0.00 0.00 0 Total leukocyte count leukocyte Total Ren Cux1low Ren Cux1low Ren low Ren low Cux1 Cux1 H Bone marrow Spleen I Bone marrow ) 109 * * * ** 6 30 ** ** 8 10 20 107 10 106 Ren low Absolute cell count

Cux1 cells/hind legs Total 105 and hip bones (x 10 0 CD11b+ CD11b+ CD11b+ CD11b+ CD3+ B220+ Gr1- Gr1+ Gr1- Gr1+ J Bone marrow (8 mos. dox) ) 5 10 * 0.25 ** p=0.08 3 ** ** 0.2 2 * 0.15 1 0.1 1 0.05 0.1 0 0 Absolute cell count/hind legs and hip bones (x 10 LSK MP LT-HSC ST-HSC MPP CMP GMP MEP K 7 months dox followed by dox cessation L 7 months dox followed by dox cessation 20 * 20 * 80 * 25 ** * ** * n.s. * 20 Ren cells 15 15 cells 60 low + + Cux1 15 10 10 40 10 5 WBC K/ µ L 5 20 PB monocytes 5 PB granulocytes % of CD45 % of CD45 Hemoglobin (g/dL) 0 0 0 0 073 073 073 073 Weeks after dox stopped Weeks after dox stopped Weeks after dox stopped Weeks after dox stopped

Figure 2.

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(dox), GFP is turned on, along with the shRNA embedded within summary, the findings of anemia with trilineage dysplasia are the GFP 39 untranslated region. As a control, we used littermates consistent with a diagnosis of MDS, most similar to refractory expressing an shRNA targeting renilla luciferase (Ren.7131/2; cytopenia with multilineage dysplasia (RCMD).36-38 M2rtTA1/1 [Ren]).35 Cux1low (Cux1low1/2;M2rtTA1/1)hadmore 6 potent knockdown, with 12% 9% residual CUX1 protein in low 1 2 1 1 Spontaneous and fatal MDS/MPN in Cux1 mice thymocytes (Figure 1C). Cux1mid (Cux1mid / ;M2rtTA / )had mid low 54% 6 17% residual CUX1 protein. CUX1 protein was too low to Compared with Cux1 , the Cux1 line had more rapid and reliably detect in the BM and spleen by western blot. qRT-PCR of aggressive hematopoietic abnormalities, with a median survival low Lin2/c-Kit1/Sca11 (LSK) showed 58% 6 2% and 42% 6 2% re- of 275 days of dox (Figure 2A). Within 1 month, Cux1 mice sidual Cux1 transcripts in Cux1mid and Cux1low mice, respectively. had splenomegaly and an elevated red cell distribution width MPs (Lin2/c-Kit1/Sca12) from Cux1mid and Cux1low mice showed (supplemental Figure 4A-C). Over time, hemoglobin and red cell 31% 6 8% and 25% 6 18% residual Cux1 transcripts, respectively counts decreased, and the mice developed a severe, normocytic (Figure 1D). anemia (Figures 2B; supplemental Figure 4C). The platelet count was normal or elevated. The WBC count became elevated within mid Cux1 mice develop MDS with anemia and 3 months because of an expansion of monocytes (CD11b1/Gr12) trilineage dysplasia and granulocytes (CD11b1/Gr11; Figure 2B-C). B cells were We first characterized Cux1mid mice, which approximate CUX1 decreased and T cells were unchanged. haploinsufficiency. Because Cux1 is essential for development,12 we induced the transgene in 6- to 10-week-old mice. Cux1mid Unlike Cux1mid, Cux1low mice fail to gain weight and have fur loss mice had normal appearance and lifespan. Aged Cux1mid mice because of the role of CUX1 in hair follicle morphogenesis developed a normocytic anemia and thrombocytosis (Figure 1E; (supplemental Figure 5A-B).29,39 Almost all Cux1low mice were supplemental Figure 3A). Although the white blood cell count euthanized because of weakness and anemia after 9 months of (WBC) was unchanged, the monocyte and granulocyte fre- dox. At euthanization, Cux1low mice had hepatosplenomegaly quencies were increased (Figure 1F). By 18 months of dox, (Figure 2D-F). Cux1low mice had splenic and BM hypercellularity Cux1mid mice had mild splenomegaly (Figure 1G). Monocytes (Figure 2G), driven largely by an expansion of monocytes and were expanded in the BM, whereas B lymphocytes were reduced granulocytes (Figure 2H; supplemental Figure 5C). The T-cell (supplemental Figure 3D). CUX1 knockdown also led to an count was also increased in the BM and spleen, comprising an expansion of LSK and ST-HSC (supplemental Figure 3E). expansion of both CD41 and CD81 T cells (Figures 2I; supplemental Figure 5D). In comparison, the B-cell count was decreased Aged Cux1mid mice had red blood cell polychromasia, with in the BM (Figure 2I), consistent with the decreased B-cell fre- increased reticulocytes, basophilic stippling, and increased quency in the blood (Figure 2C). Thus, CUX1 knockdown leads to Howell-Jolly bodies (Figure 1H). Evidence of dysgranulopoiesis a spontaneous MPN, anemia, and premature mortality. included pseudo Pelger-Huet anomalies and hypersegmentation (Figure 1Hiii, K-M). Cux1mid BM had normal cellularity with We next quantified HSPCs. As with Cux1mid, Cux1low mice had an a marginal increase in erythroid precursors (P 5 .10; Figure 1I; expansion of the LSK compartment (Figure 2J; supplemental supplemental Figure 3D,F). Cux1mid BM had a marked increase in Figure 5F). Within this population, ST-HSC and multipotent megakaryocytes, many with dysplasia, including micromega- progenitors (MPPs) had increased numbers, whereas LT-HSC did karyocytes, nuclear hypolobation, and condensed hyperchromatic not. Among the MP, common myeloid progenitors (CMPs) and nuclei (Figure 1I). The myelomonocytic cells were maturing with GMPs exhibited increases numbers, whereas megakaryocyte- no morphologic increase in blasts. erythroid progenitors (MEPs) were unchanged (Figure 2J). The GMP expansion occurred as early as 1 month after CUX1 knock- Cux1mid splenic architecture was partially disrupted because of down (supplemental Figure 4D). The LSK expansion was not from redpulpexpansion(Figure1J).Although Ren mice had occasional decreased apoptosis (supplemental Figure 4E). These results are subcapsular megakaryocytes, Cux1mid mice had increased mega- consistent with a regulatory function for CUX1 in HSPC homeo- karyocytes throughout the spleen and some micromegakaryocytes stasis. Overall, this second mouse line confirms the role of with condensed, hyperchromatic nuclei (Figure 1J). Giant CUX1 in diverse hematopoietic lineages and that the level of platelets, which can accompany megakaryocyte dysplasia, were CUX1 deficiency affects the severity of hematopoietic and non- present (Figure 1N). The splenic touch preparation revealed hematopoietic phenotypes. dyserythropoiesis, including erythroblasts with binucleation or irregular nuclear contours (Figure 1O). There was myeloid cell To determine if restoration of CUX1 can reverse the disease infiltration of the livers of some Cux1mid mice (Figure 1P). In process, we reexpressed CUX1 in mice with established disease.

Figure 2. Cux1low mice develop a fatal MDS/MPN that is reversed by CUX1 reexpression. Adult Cux1low and littermate Ren mice were continuously treated with dox starting at 6 to 10 weeks of age. (A) Kaplan-Meier survival plot of Cux1low (n 5 20), Cux1mid (n 5 11), and Ren mice (n 5 19) shows that Cux1low mice have signiﬁcantly decreased survival (log-rank test). (B) Complete blood cell counts of peripheral blood samples collected over time demonstrate a progressive anemia and WBC expansion. (C) Flow cytometric analysis of PB granulocytes (CD11b1/Gr11), monocytes (CD11b1/Gr12), B cells (B2201), and T cells (CD31) over time. (D) Representative images of spleens collected at the time that Cux1low mice were euthanized because of disease. (E) Spleens and (F) livers were weighed and normalized to body weight. (G) Total leukocyte counts from BM (femurs, tibias, and hipbones) and spleens were enumerated at the time of euthanization. (H) Absolute monocyte and granulocyte cell counts are shown from the BM and spleen at the time of euthanization. (I) Absolute lymphoid cell counts from the BM are provided. (J) HSPC populations were quantiﬁed at the time of euthanization. (J, left) LSK1 (Lin2/Sca11/c-Kit1) cells and MP (Lin2/Sca12/c-Kit1) are shown. (Center) LSK were further gated for LT-HSC (LSK1/CD342/Flt32), ST-HSC (LSK1/CD341/Flt32), and MPP (LSK1/CD341/Flt31). (Right) Within the MP population, cells were separated into CMP (CD341/CD16/32low), GMP (Lin2/c-Kit1/Sca12/CD341/CD16/32high), and MEP (Lin2/c-Kit1/Sca12/CD342/CD16/322). (K) After 7 months of continuous dox exposure, dox treatment was ceased for a cohort of mice, and hematopoietic populations were assessed over time. PB indices show a normalization of RBC and WBC counts. (L) Flow cytometry shows a normalization of PB granulocyte frequency. Additional data are provided in supplemental Figure 5. The mean 6 SD is shown. Student t test *P # .05, **P # .01, ***P # .001.

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A Ren (low power) Cux1low (low power) Cux1low (high power) Cux1low (high power) Ai Aii Aiii Aiv Peripheral blood

Cux1low C Ren (low power) Cux1low (high power) Cux1low (high power) Av

3 7 5

2 8 Bone marrow

4 D 1 6 Spleen

B Cux1low Bi E Lymph node Lymph

Bii Biv F

Biii Liver

G Kidney

H Lung

Figure 3.

DOSE-DEPENDENT CUX1 REGULATION OF ANEMIA AND MDS blood® 14 JUNE 2018 | VOLUME 131, NUMBER 24 2687 From www.bloodjournal.org by guest on July 30, 2018. For personal use only.

By 7 months of dox, Cux1low mice have anemia and leukocytosis, One cohort was euthanized after 3 months for analysis and to becoming moribund by 8 to 9 months; thus, we withdrew dox harvest BM for secondary transplantation. Compared with Ren after 7 months for a cohort of mice. Within 3 weeks, the pe- BMTs, Cux1mid BMTs had shorter survival (median, 370 days of ripheral blood indices began to normalize (Figure 2K). By 7 weeks dox; Figure 4B). Cux1low primary BMT recipients had more rapid after dox withdrawal, hemoglobin levels rise to control levels, disease onset (median, 262 days of dox), which was not sig- the WBC decreases to normal levels, and the mice gain weight nificantly different from nontransplanted Cux1low mice (median, (Figures 2K-L; supplemental Figure 6). All hematopoietic pop- 275 days of dox). Secondary Cux1low BMT recipients had the ulations assessed, including HSPC, also normalize (supplemental most rapid onset of disease (217 days), although this did not Figure 6). Thus, the disease process in Cux1low mice is dependent achieve statistical significance compared with primary Cux1low on continued CUX1-deficiency and is reversible upon CUX1 BMTs. Secondary Cux1mid BMTs survived at least 350 days, at restoration. which time they were euthanized for analysis.

Trilineage dysplasia and myelomonocytic organ Both Cux1mid and Cux1low BMTs had transient WBC expansions, inﬁltration in Cux1low mice whereas Cux1low BMTs also had transient increased platelet Hematopathologic assessment of Cux1low mice demonstrated in- counts (Figure 4C). In contrast, RBC counts were decreased in creased circulating myelomonocytic cells, occasional myeloblasts Cux1mid and Cux1low BMTs during the time course (Figure 4C). (1% to 2% of WBC), and granulocytic dysplasia (Figure 3Ai-iv). Mice were euthanized because of weakness and/or anemia, at Red cells exhibited polychromasia and anisopoikilocytosis, which time Cux1low BM recipients had pancytopenia with a and giant platelets were observed (Figure 3Av). Erythroblast macrocytic anemia (Figure 4C-D). Of the 4 Cux1mid BM recipients dysplasia was evident in splenic touch preparations, similar to that became diseased, 2 were anemic, all were thrombocyto- Cux1mid (Figure 3B). Cux1low BM demonstrated decreased eryth- penic, and none had an elevated WBC count (Figure 4C). ropoiesis (supplemental Figure 5E) and maturing myelomonocytic cells with no morphologic increase in blasts (Figure 3C). Mega- At 3 months posttransplant, Cux1low and Cux1mid BM and spleen karyocytes were increased, many with dysplasia (Figure 3C). The cellularity was normal or increased, similar to nontransplanted splenic architecture was completely effaced in Cux1low mice mice (Figure 4E). In contrast, the BM and spleens tended to (Figure 3D). The expanded red pulp comprised maturing ery- be hypocellular in Cux1low and Cux1mid BMTs at the time of throid and myeloid cells and a remarkable expansion of mega- euthanization (Figure 4E). Both Cux1low and Cux1mid BMT re- karyocytes to a greater extent than Cux1mid. Myelomonocytic cells cipients had remarkable dysplasia in megakaryocyte, erythroid, were observed inﬁltrating lymph nodes and nonhematopoietic and granulocyte lineages (supplemental Figure 7). In sum, the organs, including liver periportal and sinusoidal areas, lung, and cytopenias and trilineage dysplasia in Cux1mid and Cux1low BMT kidney (Figure 3E-H). recipients are consistent with MDS, akin to RCMD.

In summary, low levels of CUX1 led to a striking expansion of At 3 months of dox, Cux1low BMT recipients had increased LSK, mature granulocytes and monocytes in hematopoietic and non- ST-HSC, MP, and MPP (Figure 4F). In stark contrast, most stem hematopoietic tissues, accompanied by anemia and trilineage and myeloid progenitor populations were significantly depleted dysplasia. In human disease, CMML often has hepatosplenomegaly, in Cux1low BMT at euthanization (Figure 4G). Cux1mid BMT had normocytic anemia, giant platelets, micromegakaryocytes with decreased myeloid progenitors at euthanization. Decreased abnormal lobation, and myeloid infiltrate of lymph nodes.38 JMML HSPC numbers may partly explain the BM failure in these mice. patients also often have hepatosplenomegaly, normocytic anemia, Indeed, secondary Cux1mid and Cux1low BMT recipients had a and myelomonocytic infiltrates of the liver, lung, and lymph nodes38; significant reduction in LT-HSC, consistent with impaired HSC self- thus,thediseaseinCux1low mice is classified as a MDS/MPN, akin to renewal (Figure 4H). Although Cux1low secondary BMT exhibited CMML-1 and JMML.36,38 pancytopenia, Cux1mid secondary BMT did not within 350 days of dox (supplemental Figure 8E-F). The finding that Cux1mid sec- BM transplants develop transient hematopoietic ondary BMT had decreased LT-HSC suggests that these mice may expansion followed by CUX1 dose-dependent ultimately develop anemia over longer times. These data indicate BM failure that CUX1 knockdown leads to a transient HSPC expansion fol- To determine the hematopoietic-intrinsic role for CUX1, we lowed by depletion, via a hematopoietic-intrinsic mechanism. performed BM transplants (BMTs) of Cux1mid, Cux1low, or Ren BM into lethally irradiated, wild-type recipients. We waited 4 weeks These findings led us to test the hypothesis that CUX1- for the transplants to engraft before starting dox (Figure 4A). knockdown HSPCs outcompete control cells in short-term

Figure 3. Myelomonocytic expansion and myelodysplasia in Cux1low mice. Representative morphology images are shown from mice at the time of euthanization because of disease. (A) Wright-Giemsa staining of the peripheral blood in (Ai) Ren and (Aii-Av) Cux1low mice. (Aii) The increased white blood cell count is primarily composed of mature granulocytes (arrowheads), monocytes (arrow), and (Aiii) occasional circulating myeloblasts (arrows). (Aiv) Dysplasia in the granulocyte lineage as evidenced by pseudo Pelger- Huet anomaly (arrows). (Av) Red blood cells in Cux1low mice have anisopoikilocytosis: (1) macrocyte; (2) microcyte; (3) target cell; (4) spherocyte; (5) tear drop cell; (6) and red cell fragment. (7) Polychromatophilic reticulocyte. (8) Giant platelet. (B) Touch preparation of Cux1low spleen shows erythroid dysplasia, including (Bi) binucleated erythroblast (arrow) and nuclear budding (arrowheads), (Bii) nuclear budding, and (Biii) abnormal nuclear contours. (Biv) Pseudo Chediak-Higashi granule in a maturing granulocyte. (C) H&E of Cux1low BM demonstrates numerous megakaryocytes. Nine megakaryocytes (yellow arrowheads) are shown in a single high-power ﬁeld, several of which are atypically small, with hypolobation and condensed, hyperchromatic nuclei. (D) Compared with Ren, Cux1low spleen H&E staining demonstrates complete effacement of normal splenic architecture with expansion of red pulp and increased megakaryocytes. White pulp is outlined with a yellow dashed line for clarity. There is a small portion of residual white pulp in the bottom left corner of the Cux1low spleen (D, middle panel). Micromegakaryocytes are present, as well as megakaryocytes with hypolobation, abnormally widely spaced nuclear lobes, and hyperchromatic nuclei. H&E staining shows a myelomonocytic cell inﬁltrate in the following organs of Cux1low mice: (E) lymph nodes; (F) liver periportal and sinusoidal areas; (G) kidney; and (H) lung. Images were taken with a Zeiss Axioskop microscope.

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A B 100 Ren 1° (n=5) Ren, low p=0.004 mid Cux1 1° (n=12), 262 d Cux1 or 75 p=0.044 Cux1mid 1° (n=6), 370 d Cux1low 2° 50 Ren 2° (n=5) transplant p=0.013 1° transplantDox Collect tissue Cux1low 2° (n=6), 217 d Long-term 25 mid

Percent survival Percent Cux1 2° (n=8) WT follow 0 C57BL/6 0 100 200 300 400 Day 0 1 mo. 4 mos. 8 mos. Days of dox

C WBC Platelets RBC 15 1500 15 ** * 10 ** 1000 10 * Ren * Cux1mid K/  L K/  L

M/  L * * low 5 * * 500 * 5 ** Cux1 * ** ** 0 0 0 Wks post dox: 0 162030 42 sac 0 1 6 20 30 42 sac 0 1 6 20 30 42 sac

D 1° BMT PB at E 1° BMT BM 1° BMT spleen time of euthanization 3 mos. euthanization 3 mos. euthanization ) ) 6 50 70 100 p=0.09 45 p=0.09 6 70 50 p=0.15 ** * 40 * * 45 60 80 40 60 40 30 35 50 60 50 35 20 30 30 40 40 40 10 25 25 0 30 20 20 30 20 Leukocyte count (x 10 Leukocyte Leukocyte count (x 10 Leukocyte

RDW % MCV fL low low low low Ren mid Ren mid Ren Ren mid

Cux1Cux1 Cux1 Cux1 Cux1 Cux1 Cux1

F 1° BMT bone marrow after 3 mos. dox * 1000 * 10 ** 100 100 )

5 1 10 Ren 1° 10 * Cux1mid 1° low (x 10 0.1 1 Cux1 1° 1

Absolute cell count 0.1 0.01 0.1 LSK MP CMP GMP MEP MPP LT-HSC ST-HSC

G 1° BMT bone marrow at euthanization p p p 1000 * 10 =0.07 * =0.07 100 **=0.1 * ** 100 ) 1 10 Ren 1° 5 mid * Cux1 1° 10 Cux1low 1° (x 10 0.1 1 1

Absolute cell count 0.1 0.01 0.1 LSK MP CMP GMP MEP MPP LT-HSC ST-HSC

Figure 4. Transient hematopoietic expansion is followed by HSC exhaustion and BM failure in CUX1-knockdown BM transplant recipients. (A) Schematic of the BM transplant (BMT) time course. BM from Cux1mid, Cux1low, or Ren mice was transplanted into lethally irradiated C57BL/6 wild-type recipients. Four weeks after transplant, recipient mice were treated continuously with dox. Most mice were monitored long-term, up to 350 to 370 days of dox or time of euthanization because of disease. A cohort of mice was euthanization after 3 months of dox for analysis and to harvest BM for secondary transplantation. Secondary transplant recipients were given dox starting on the day of transplantation. (B) Kaplan-Meier plot shows decreased survival of Cux1mid and Cux1low BM transplant recipients (log-rank test). (C) Complete blood count analysis shows a transient WBC and platelet count expansion in Cux1 knockdown BM transplant recipients, whereas WBC, platelets, and RBCs were decreased by the time of euthanization. The

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H I 1:10 Competitive transplant 2° BMT BM WBC B cells T cells Monocytes Granulocytes * 1 * 1.5 8 *** *** 6 )

5 1.0 ** 0.1 ** 4 *** (x 10 0.5 ** ** 2 * *** * CD45.2/CD45.1 * Absolute cell count 0.01 0 0 LT-HSC 25815 25815 25815 25815 25815 Weeks post transplant Ren 2° Ren Cux1mid 2° Cux1mid Cux1low 2° Cux1low

Figure 4. (Continued). competitive BMT. Because CUX1 is associated with CHIP, we We next determined if ineffective erythropoiesis was due to transplanted Cux1low,Cux1mid, or Ren BM (CD45.2) at a 1:10 erythroblast apoptosis. After 1 month of dox, the frequency of ratio with competitor BM (CD45.1) to model low-frequency Annexin V1 cells was similar in Cux1low and Ren erythroblast clonal hematopoiesis.18-21 In these experiments, dox was given populations, suggesting the anemia is not due to decreased on the day of transplant to assess HSPC engraftment. Within erythroblast survival (supplemental Figure 5G). In summary, 2 months, both Cux1mid and Cux1low outcompeted control cells, these data indicate that CUX1 is novel transcriptional regulator speciﬁcally in the myeloid lineages (Figure 4I); thus, CUX1 of red cell development regulating at least 2 stages of eryth- knockdown provides a myeloid clonal advantage for HSPCs. ropoiesis: (1) maintenance of HSCs and (2) progression into the orthochromatophilic erythroblast stage.

CUX1 knockdown leads to CUX1 regulates proliferative and ineffective erythropoiesis phosphatidylinositol 3-kinase (PI3K) pathways and To better understand the cause of the anemia, we explored JMML and 27/del(7q) MDS gene signatures erythropoiesis in the mouse models. During erythroid matura- These ﬁndings led to us to determine the genes regulated tion, CD711/Ter119med (RI) pro-erythroblasts gradually lose by CUX1 in primary human HSPC, the normal counterparts CD71 expression as they progress through basophilic (RII), thought to give rise to myeloid malignancies.41 We transduced polychromatophilic (RIII), and ultimately orthochromatophilic (RIV) human CD341 cells with shRNA targeting CUX1 or a control stages followed by enucleation.40 We gated on all erythroid cells shRNA,7 resulting in approximately 50% and 80% residual CUX1 and assessed the proportion of erythroblasts within each of these transcripts (Figure 6A). RNA-sequencing analysis identiﬁed stages. Aged Cux1mid mice had a mild decrease in the BM RII 339 differentially expressed genes (5% false discovery rate), population and normal splenic RI-RIV frequencies (Figure 5A-B). 81% of which decreased, indicating that CUX1 is primarily at In contrast, Cux1low splenic erythroblasts had an increased fre- transcriptional activator in this cell type (Figure 6B-C). quency in RII and a decrease in RIV, with relatively normal BM populations (Figure 5C-D). The splenic RII expansion and RIV Gene set enrichment analysis42 determined that genes prefer- reduction was recapitulated in Cux1mid and Cux1low BMTs 3 months entially expressed in quiescent HSPC43 were downregulated after transplant (Figure 5E-F). Thus, in nontransplanted Cux1low, after CUX1 knockdown (Figure 6D). Conversely, genes prefer- as well as in Cux1mid or Cux1low BM recipients, there is a partial entially expressed in proliferative HSPC43 were upregulated. This block in progression to the RIV orthochromatophilic erythro- suggests a transcriptional role for CUX1 in regulating HSPC blast stage. quiescent vs proliferative states. Genes upregulated by activation of the PI3K/AKT/mTOR pathway (ie, the “Hallmark PI3K Interestingly, at the time of disease in BMT recipients, the RII and AKT MTOR Signaling” gene set), were enriched in upregulated RIV frequencies normalized (Figure 5F). At this time, the total genes, which is notable because increased PI3K signaling is erythroblast cell count was decreased in the BM of transplant associated with HSC exit from quiescence and proliferation.44 recipients (Figure 5G). Because HSC exhibited evidence of The Gene Ontology pathway, “Negative regulation of mye- exhaustion by this time (Figure 4), decreased erythropoiesis at loid cell differentiation” was downregulated, indicating CUX1 euthanization may be largely due to upstream HSC defects, represses myeloid differentiation. This is in accordance with the masking downstream erythroblast maturation defects. myeloid expansion we observed after CUX1 knockdown in vivo.

Figure 4 (continued) mean 6 SEM is shown. (D) Blood cell indices at the time of euthanization show a macrocytic anemia. The mean 6 SD is shown. (E) BM from hind legs and hips, and spleen leukocyte cellularity at the indicated time points. Cell counts for Cux1mid spleens were not determined for the 3-month time point. (F) Cux1low BM transplant recipients have greater numbers of HSPC after 3 months of dox, as determined by ﬂow cytometry. The mean 6 SD is shown. (G) At the time that diseased mice were euthanized because of anemia and weakness, HSPC were decreased in Cux1 knockdown BM transplant recipients compared with Ren BM transplant recipients. The mean 6 SD is shown. (H) Absolute LT-HSC from the BM of secondary BMT recipients is shown at the time of euthanization for disease in Cux1low recipients and after 350 days of dox for Cux1mid recipients. (I) Competitive BM transplantation was performed with Cux1low,Cux1mid, or Ren BM (CD45.2) at a 1:10 ratio with competitor BM (CD45.1). Dox was given on the day of transplant. One replicate of 3 biological replicates is shown, n 5 6 mice per experimental group. Flow cytometry of the peripheral blood shows that Cux1mid and Cux1low HSPCs outcompete controls in the total WBC population and myeloid lineages. The mean 6 SD is shown. All data were analyzed with Wilcoxon rank test, *P # .05, **P # .01, ***P # .001.

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A Cux1mid bone marrow 18 mos. dox E BMT at 3 mos. of dox Ren Cux1mid BM 5 100 * 100 ** ** 10 RII RII RI RI 19.3 0.91 24.5 1.74 80 80 104 60 Ren 60 RIII RIII 3 3.54 mid 10 1.80 40 Cux1 40 0 CD71 RIV RIV 20 20 -103 6.07 6.56 % of cells erythroid % of cells erythroid 0 0 -103 0 103 104 105 -103 0103 104 105 RI RII RIII RIV RI RII RIII RIV Ter119 Spleen ** mid *** B Cux1 spleen 18 mos. dox 100 * mid 80 Ren Cux1 100 60 Ren 5 RI 10 RI RII RII mid 13.1 1.57 21.5 80 Ren 40 Cux1 1.40 mid low 104 60 Cux1 20 Cux1 RIII RIII 103 2.15 3.46 40 0 % of cells erythroid 0 RIV RIV 20 CD71 11.8 5.16 RI RII RIII RIV 3 % of cells erythroid -10 0 -103 0103 104 105 -103 0103 104 105 RI RII RIII RIV F BMT at euthanization Ter119 BM * 100 * C Cux1low bone marrow at euthanization Ren Cux1low 50 100 * 105 RI RII RI RII 0.64 20.5 2.59 4.90 80 0 104 60 RIII RIII Ren 1.11 1.06 % of cells erythroid 40 low 103 Cux1 0 RIV 20 RI RII RIII RIV 12.4 RIV 4.23 CD71 3 0

-10 % of cells erythroid Spleen 3 3 4 5 3 3 4 5 -10 010 10 10 -10 010 10 10 100 * RI RII RIII RIV Ter119 80 Ren Cux1mid 60 Cux1low D Cux1low spleen at euthanization 40 low 20 Ren Cux1 ** ** 100 0

5 RI RII RI RII % of cells erythroid 10 0.14 9.22 1.38 31.2 80 4 60 RI RII RIII RIV 10 RIII RIII Ren 2.00 3.81 40 low 103 Cux1 G BMT at euthanization 20 0 RIV RIV BM Spleen 26.3 9.09 0 )

3 6

-10 % of cells erythroid

CD71 50 p=0.06 100 -103 0103 104 105 -103 0103 104 105 RI RII RIII RIV Ter119 40 80 30 60 20 40 10 20 0 0 Erythroblast count (X 10 count (X Erythroblast Ren mid low Ren mid low Cux1Cux1 Cux1Cux1

Figure 5. CUX1 knockdown leads to a partial block in erythropoiesis. Flow cytometric analysis of BM and spleen erythroblast populations was performed for: (A-B) Cux1mid mice treated with dox for 18 months; (C-D) Cux1low mice at the time of euthanization for disease; (E) primary BMT recipients 3 months after transplantation; and (F) primary BMT recipients at the time of euthanization. (G) Absolute count of all RI through RIV erythroblasts in the BM and spleens of BMT recipients at the time of euthanization is provided. Age-matched, littermate control Ren mice were used for all experiments. The erythroid cell populations from least to most differentiated are indicated as RI through RIV. RI, pro- erythroblasts; RII, basophilic erythroblasts; RIII, polychromatophilic erythroblasts; RIV, orthochromatophilic erythroblasts. (A-D) Representative ﬂow analysis. The mean 6 SD is shown. All data were analyzed with Wilcoxon rank test, *P # .05, **P # .01, ***P # .001.

To test whether CUX1-regulated genes are associated with patients.33 Strikingly, genes that are signiﬁcantly decreased human disease, we compared the transcriptional proﬁle in 27/del(7q) MDS compared with normal karyotype MDS of CUX1-knockdown HSPC to gene signatures from MDS were also decreased after CUX1 knockdown in human HSPCs

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A B C Human CD34+ HSC CXCL13 339 genes 150 FDR=0.05 30 81% go down 125 LILRA3 C15orf48 100 20 controlshCUX1-B shRNAshCUX1-B rep1 rep2shCUX1-A rep1shCUX1-A rep1 rep2 75 MAFB control shRNA rep2 50 AGR2 60 million reads

–log10(p value) 10 40 CUX1 counts per 25 PRG3 0 KLRK1 20 Count 0 0 shRen –1 0 1 shCUX1-B –10 –5 0 5 10 shCUX1-A Row Z-Score log fold change D Venezia: Quiescent Venezia: Proliferative 0.0 0.35 -0.2 0.25 0.15 -0.4 0.05 -0.6

Enrichment score E Up in Down in Up in Down in BM with 20 min. serum CUX1 KD CUX1 KD CUX1 KD CUX1 KD KLC4 HDAC5 CLK3 P2RX4 UBXN6 GADD45B SOCS3 HLA-DQA1 TNFRSF1B CDC20 ECT2 MKI67 TFDP1 mid low

Ren Cux1 Cux1 p-AKT

GO: Negative regulation of myeloid Hallmark PI3K AKT Actin cell differentiation MTOR Signaling 0.40 0.0 0.30 F –0.2 0.20 1.5 –0.4 1.5 *** 0.10 *** p=0.09 –0.6 0 1.0 1.0 Enrichment score

Up in Down in Up in Down in 0.5 0.5 CUX1 KD CUX1 KD CUX1 KD CUX1 KD TIAM1 PLCG1 PIK3R3 VAV3 HOXA5 FSTL3 LYN LILRB3 NFKB1A C1QC LILRB4 DLL1 MAFB LILRB1 CCL3 Pik3ip1 relative to Ren 0 0 LSK Myeloid progenitors Pellagatti: Down in Ren mid -7/del(7q) vs NK MDS Up in CUX1 KD Cux1 Cux1low 0.0 0.40 0.30 -0.2 0.20 -0.4 0.10 -0.6 0 Enrichment score

Up in Down in Up in Helsmoortel: Down in CUX1 KD CUX1 KD JMML JMML JMML TCN1 CLC CXCR1 ARSB PRG3 AGR2 ENPP3 HRH4 LPGAT1 MS4A2 RFX8 DUSP27 HPGD CDK15 GATM NRP1 RGL1 GPNMB IFIT1 MS4A7 HNMT HMOX1 PLA2G7 C1QB IGSF6 C1QC SMPDL3A FABP3 C1QA PID1 MAFB FEZ1 SELL IL18RAP AP1S3 KCNH1 DHRS9 THSD7A HGF KIAA1462 SPAG17 TRPM6 ALS2 SGPP1 SLC7A8 SLC18A2

Figure 6.

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(Figure 6D). Because of the myelomonocytic phenotype of the We consistently observed that Cux1low led to an excess of CFU- MPD in Cux1low mice, we next assessed if CUX1-regulated granulocyte/monocyte (CFU-GM) and a paucity of burst-forming genes are enriched for a JMML gene signature. Using micro- units (BFU-Es, erythroid; Figure 7C). Cux1mid also had decreased array data from JMML patients and healthy controls, we found BFU-Es. Although Cux1mid BM did not demonstrate increased CFU- that genes upregulated in JMML are enriched for genes GM in this assay, we note that the majority of increased CFU in upregulated after CUX1 knockdown (Figure 6D). Thus, the passages 2 and 3 of serial replating of Cux1mid BM were CFU-GM CUX1 gene signature is associated with JMML and 27/del(7q) (Figure 7A). These data concur with the in vivo myelomonocytic ex- MDS disease states, consistent with the JMML and MDS pansion and anemia of Cux1low and Cux1mid mice and indicate that phenotypes of the CUX1 knockdown mice. CUX1 levels control cell fate of early stem and/or progenitor cells.

CUX1 knockdown induced genes upregulated by PI3K signal- The elevated PI3K activity upon CUX1 knockdown led us to test ing (Figure 6D), implying increased PI3K activity. Indeed, the hypothesis that CUX1 knockdown promotes HSC exit from Cux1low BM cells have increased phosphorylation of the PIK3 quiescence.44 Cell-cycle analyses showed a significant reduction substrate, AKT (Figure 6E). These data align with reports that in the fraction of Cux1mid and Cux1low LT-HSC in the quiescent, 17,45 CUX1/Cut suppresses PI3K activity, by transcriptionally ac- G0, state and a concordant increase in the fraction of cells in S/G2 tivating expression of the PI3K inhibitor, PIK3IP1.17 In agree- (Figure 7D-E). ST-HSC, MPP, and GMP also had an increase in ment, we determined CUX1 knockdown leads to decreased frequency of cycling cells in S/G2 (Figure 7D-E). These findings Pik3ip1 in Cux1low LSK and MP (Figure 6F). Although Cux1mid show that CUX1 knockdown promotes LT-HSC exit from qui- LSKs had a slight decrease in Pik3ip1, we did not reliably detect escence and cell cycling and are in agreement with the ex- increased phospho-AKT in Cux1mid whole BM. Perhaps BM pansion of downstream ST-HSC in vivo and the quiescent and heterogeneity masks putatively increased phospho-AKT in proliferative gene signatures in human HSPC. rare HSPC in Cux1mid mice. Overall, these findings fit a model wherein CUX1 knockdown leads to decreased PI3KIP1, increased PI3K activity, and resulting in increased stem/progenitor Discussion cell-cycle entry. In our studies, all transplant and nontransplant CUX1 knockdown mouse models had anemia and multilineage dysplasia in CUX1 regulates HSPC homeostasis common, demonstrating a hematopoietic-intrinsic role for CUX1 To further understand the role of CUX1 in HSPC differentiation reduction in MDS (Table 1). The nontransplanted Cux1low mice and self-renewal, we performed colony-forming unit (CFU) and additionally exhibit myelomonocytic expansion, comparable serial replating assays. Because CUX1 knockdown alters HSPC to MDS/MPN. In many ways, the Cux1low phenotypes were populations, we used BM from dox-na¨ıve mice and provided dox stronger than those of Cux1mid; for example, BMT survival, AKT only during the assay. Cux1low BM had a significant increase in phosphorylation, and fur loss, indicating a dosage effect. A prior CFU on passage 1 (Figure 7A; supplemental Figure 9). CUX1 study of mice homozygous for deletion of the CUX1 homeo- knockdown colonies were larger than Ren, particularly Cux1low at domain (Cux1HD/HD) reported only 5.4% were viable and few passage 1 and Cux1mid at passage 2 (supplemental Figure 9). survived beyond 4 week of age, precluding long-term analysis.12 Indeed, the number of cells enumerated at the end of most However, as with Cux1low mice, Cux1HD/HD mice had hair loss, passages was greater for Cux1low and Cux1mid, consistent with cachexia, and myeloid hyperplasia; elevated tumor necrosis increased progenitor proliferation (Figure 7B). Both Cux1low and factor was thought to explain some of the extrahematopoietic Cux1mid BM had increased CFU in passages 2 and 3 and effects.12 We speculate that the myelomonocytic expansion extinguished by passage 4 (Figure 7A). It is unclear why Cux1mid in Cux1low mice is also due to hematopoietic-extrinsic in- CFU increased only after the first passage. Because of the de- flammatory cytokines stemming from ubiquitous, low CUX1 creased potency of Cux1mid shRNA, perhaps Cux1mid lagged levels. Cux1low in achieving sufficient CUX1 knockdown, resulting in a delayed phenotype. Overall, the CFU results are in agreement Hematopoietic abnormalities have not been reported for with the in vivo data demonstrating temporarily increased HSPC Cux1HD/wt heterozygous mice.12 However, based on our work, number and proliferation but decreased long-term self-renewal the phenotype would not be apparent until 18 months; it is (Figure 4). In addition, these in vitro assays further confirm the unclear how long heterozygous mice were aged in the prior cell-intrinsic role for CUX1 regulation of HSPCs. study. In a second Cux1 knock-out mouse model, with deletion of only the first cut repeat domain, no hematopoietic To test if CUX1 is acting at an early stem/progenitor stage to abnormalities were found in heterozygous or homozygous regulate lineage specification, we identified colony morphologies. mice at 2-3 weeks of age.28 This may be due to the young age

Figure 6. CUX1 regulates genes associated with MDS and JMML gene signatures and PI3K/AKT signaling. Primary human CD341 HSPC were transduced with shCUX1-A, shCUX1-B, or nonspeciﬁc control shRNA. After 7 days, GFP1 CD341 cells were sorted and RNA collected for RNA-sequencing analysis, with 2 biological replicates. (A) CUX1 transcript levels after CUX1 knockdown as determined by RNA-sequencing. (B) The volcano plot shows gene expression changes after CUX1 knockdown. Red indicates differentially expressed genes with a 5% false discovery rate (FDR). The horizontal dashed line represents a nominal P value 5 .05. Vertical dashed lines indicate 6 one-fold change. (C) Heat map of the 339 differentially expressed genes identiﬁed after CUX1 knockdown, with a 5% FDR. (D) Gene set enrichment analysis of RNA-sequencing data. Gene sets used were: “Quiescent” and “Proliferative”43; GO gene set from the MSig Database,42 27/del(7q) MDS vs normal karyotype MDS,33 and JMML.34 Genes listed are those that had nominal P values of ,.05 (CUX1 knockdown vs control) and were within the GSEA “Core enrichment” results. (E) Whole BM was collected from Cux1mid, Cux1low,or Ren control mice after 5 days of dox, cultured in serum-free media for 2 hours, and stimulated with serum for 20 minutes. Whole cell lysates were probed for phospho-AKT by western blot. One representative blot of 3 biological replicates is shown. (F) qRT-PCR results from LSK and myeloid progenitors sorted from the indicated mice. One of 3 biological replicates is shown.

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A B C )

5 p=0.06 200 ** *** ** 15 ** ** 200 *** ** *** ** ** Ren 150 150 Cux1mid 10 Cux1low 100 100 *

5 CFU count * 50 50 CFU (GM) count

0 Total cell number (X10 0 0 P1 P2 P3 P4 P1 P2 P3 P4 CFU- BFU- CFU- GM E GEMM

D LT-HSC ST-HSC MPP GMP 105 G1 S G2 G1 S G2 G1 S G2 105 G1 S G2 1.34 69.8 22.0 1.27 62.0 64.5 24.3 3.78 4 87.9 0 23.5 7.04 10 104 103 3 Ren 10 102 G0 G0 G0 0 G0 1 10 3.91 3.54 2.88 -103 4.17

105 G1 S G2 G1 S G2 G1 S G2 105 G1 S G2 89.4 3.99 0 61.8 29.9 3.60 50.8 33.5 10.0 65.6 24.1 4.24 4 10 104 mid 103 103 2 Cux1 10 G0 G0 G0 0 G0 1 3.62 10 3.32 2.35 1.69 -103 105 G1 S G2 G1 S G2 G1 S G2 105 G1 S G2 88.3 1.15 0 62.5 26.4 3.96 36.1 34.8 19.9 52.8 29.7 8.31 4 10 104 low 103 103 2

Cux1 10 G0 G0 G0 0 G0 1 10 2.68 2.05 0.87 -103 4.66 Ki-67 0 50K 150K 0 50K 150K 0 50K 150K 0 50K 150K DAPI

E LT-HSC ST-HSC MPP GMP 15 6 4 * * 2.0 * ** * * * Ren * * 3 1.5 Cux1mid 10 4 * Cux1low * 2 1.0 5 * 2 1 0.5 Relative to Ren 0 0 0 0 G0 G1 S/G2 G0 G1 S/G2 G0 G1 S/G2 G0 G1 S/G2 Cell cycle stage

Figure 7. CUX1 regulates HSPC numbers, proliferation, and lineage specification. Total BM cells isolated from dox-na¨ıve Cux1mid, Cux1low, or littermate Ren mice were mixed with M3434 methylcellulose medium (25 000 cells per well) and dox for CFU assays. At each passage (P1-P4), cells were collected and 25 000 cells were replated, with 7 to 11 days between each passage. (A) Total colony numbers and (B) cell counts were quantified at the end of each passage (n 5 3). (C) Specific colonies (CFU-GM, BFU-E, and CFU granulocyte, erythrocyte, monocyte, and megakaryocyte [CFU-GEMM]) were identified by morphology days 10 through 12 in a separate CFU assay. (D) BM cells were collected from Cux1mid, Cux1low, and Ren littermate controls after 3 days of dox. Lin2 cells were cultured in StemSpan SFEM medium (Stemcell Technologies) supplemented with 100 ng/mL of murine stem cell factor, throbopoietin, and Flt3 and dox for 3 days before flow analysis. Representative flow cytometric analysis of HSPC cell-cycle analysis is shown. (E) The bar graph represents the mean of 3 biological replicates 6 SEM. All data were analyzed with Student t test, *P # .05, **P # .01, ***P # .001. of the mice and/or residual hypomorphic CUX1 protein in that regulating lineage specification and differentiation. We propose model. a model wherein at the top of the hematopoietic hierarchy, CUX1 promotes LT-HSC quiescence through transcriptional activation of Our studies show that CUX1 is essential for maintaining HSC Pik3ip1, thereby limiting growth factor–induced signaling path- quiescence, suppressing HSPC proliferation and self-renewal, and ways. CUX1 knockdown leads to increased PI3K activity, a central

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Table 1. Phenotype comparison of CUX1 knockdown mouse models

Phenotype Cux1mid Cux1low Cux1mid BMT Cux1low BMT

CUX1 protein (% of normal) ;54 ;12 ;54 ;12

Disease MDS (RCMD) MDS/MPN (CMML/JMML) MDS (RCMD) MDS (RCMD)

Survival (days of dox) Normal 275 370 262

Splenomegaly Mild Yes No No

BM cellularity Normal Increased Decreased Decreased

RBCs Decreased Decreased Slightly decreased Decreased

Platelets Increased Normal or increased Decreased Decreased

WBCs Normal Increased Normal Slightly decreased

Granulocytes Increased PB % Increased Increased PB % Increased PB %

Monocytes Increased Increased Increased PB % Normal

B cells Decreased Decreased Slightly decreased Decreased

T cells Normal Increased Normal Normal

Dysplasia Trilineage Trilineage Trilineage Trilineage

LT-HSCs Normal Normal Decreased in second Decreased in ﬁrst and second transplant transplants

activator of the HSC cell cycle,44 promoting LT-HSC exit from Oncogenic Ras signaling may provide the enhanced self- quiescence and proliferation. These LT-HSCs give rise to ST-HSC, renewal capacity necessary for sustained CUX1-deficient HSC expanding the ST-HSC pool and downstream progenitors. clonal expansion.54 Of note, effectors of Ras include the PI3K/ Eventually, the HSC exhaust and are depleted, leading to BM AKT/mTOR and Raf/MEK/ERK axes. Our finding that CUX1 failure. These findings are consistent with prior reports that in- knockdown enhances PI3K signaling raises the prospect that creased PI3K signaling leads to transient HSC proliferative ex- CUX1 haploinsufficiency cooperates with Ras by potentiating pansion followed by exhaustion.46-48 Ras signaling via increased PI3K activity.

In MPP, loss of CUX1 causes increased proliferation and a myeloid There is evidence for other pathogenic genes on 7q,55-61 pro- differentiation bias, increasing downstream myeloid progenitor voking the notion of a contiguous gene syndrome, wherein numbers, and ultimately myelomonocytic expansion. CUX1 is also haploinsufficiency of multiple TSGs contributes to disease.62 necessary for normal erythroid and megakaryocyte development Nonetheless, our data provide strong evidence that CUX1 because CUX1 deficiency led to dysplasia and cytopenia in both knockdown alone is sufficient for MDS and MDS/MPN, especially lineages. Acting at multiple stages of hematopoiesis, CUX1 re- because the disease was also reversible upon CUX1 restoration. It duction generally leads to the mobilization of myeloid progenitors will be critical to identify how combined haploinsufficiency of with differentiation defects. Increased proliferation and aberrant 7q genes cooperate in neoplasia, however. differentiation are the cardinal features of myeloid malignancies, and CUX1 deficiency drives both. Cut, in Drosophila, is exquisitely dose sensitive and changes in cut levels can result in dramatically different cellular programs, in- 49,50 Our results shed light on how del(7q) and somatic CUX1 cluding proliferation, differentiation, and apoptosis, even within 18,19 inactivating mutations give a clonal advantage in CHIP. CUX1 the same cell type.24-26 Transcription factor dosage can be critical fi haploinsuf ciency may provide human HSC with a proliferative in myeloid differentiation and transformation, as evidenced, for advantage that enables mutant HSC to outcompete wild-type example, by Pu.1.63,64 In future work, it will be important to de- counterparts, resulting in clonal expansion, as we observed in com- termine the threshold of CUX1 necessary for maintaining myeloid petitive transplantation assays. That CUX1 knockdown eventually TSG activity and if changes in CUX1 levels also dictate hemato- causes HSC exhaustion provides an intriguing biological expla- poietic cell fate, as described for cut in other models.25,26 nation for the transient 27 or del(7q) that has been observed in some children and adults.51,52 A second genetic alteration may be required to increase the long-term survival and/or self-renewal of 27/del(7q) clones, thereby enabling transformation. Acknowledgments The authors thank Angela Stoddart, Jeffrey Kurkewich, and Kevin Shannon for critical reading of the manuscript. The authors also thank 2 “ We reported that in one-half of 7/del(7q) cases, the second Christine Labno and The University of Chicago’s Integrated Light Mi- hit” is a mutation that results in Ras pathway activation.53 croscopy Core Facility, Pieter Faber and the High Throughput Genomics

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Core Facility, and David Leclerc in the Flow Cytometry Facility for special reverse transcription polymerase chain reaction. S.N.K. performed ap- assistance and services. Linda Degenstein and the Transgenic Mouse optosis assays. S.K.G. performed hematopathologic analyses and edited and Embryonic Stem Cell Facility helped generate the Cux1mid mice. the manuscript. M.R.B. generated the shCux1.581 LMNcherry construct. Lentivirus was packaged and concentrated at the Northwestern University RNA/DNA Delivery Core with special assistance from Conﬂict-of-interest disclosure: The authors declare no competing ﬁ- Alex Yemelyanov. nancial interests.

This work was supported by grants from the National Institutes of Health ORCID proﬁle: M.E.M., 0000-0002-8260-3598. (NIH) National Cancer Institute (K08CA181254) (M.E.M.), The University of Chicago Medicine Comprehensive Cancer Center Support Grant Correspondence: Megan E. McNerney, Department of Pathology, The (P30CA014599), NIH National Institute of General Medicine Sciences University of Chicago, Knapp Center for Biological Discovery Room (T32GM007281) (M.K.I.), NIH National Institute of Allergy and Infectious 5128, 900 E. 57th St, Chicago, IL, 60637; e-mail: megan.mcnerney@ Diseases (T32AI007090) (S.N.K.), and NIH National Center for Advancing uchospitals.edu. Translational Sciences (UL1 TR000430); V Foundation for Cancer Re- search V Foundation Scholar Award; American Cancer Society In- stitutional Research Grant (IRG-58-004); Cancer Research Foundation Young Investigator Award; and the University of Chicago Cancer Footnotes Research Foundation Auxiliary Board. Submitted 6 October 2017; accepted 21 March 2018. Prepublished online as Blood First Edition paper, 28 March 2018; DOI 10.1182/blood- 2017-10-810028. Authorship Contribution: M.E.M. conceived the experiments, analyzed the RNA-seq The online version of this article contains a data supplement. data, and wrote the manuscript. N.A. performed the mouse experiments and in vitro assays, analyzed data, and edited the manuscript. S.K. The publication costs of this article were defrayed in part by page charge performed human hematopoietic stem and progenitor cell experiments, payment. Therefore, and solely to indicate this fact, this article is hereby western blots, and mouse genotyping. M.K.I. performed quantitative marked “advertisement” in accordance with 18 USC section 1734.

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2018 131: 2682-2697 doi:10.1182/blood-2017-10-810028 originally published online March 28, 2018

Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS

Ningfei An, Saira Khan, Molly K. Imgruet, Sandeep K. Gurbuxani, Stephanie N. Konecki, Michael R. Burgess and Megan E. McNerney

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