Published OnlineFirst April 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1327
Cancer Genes and Networks Molecular Cancer Research The Pluripotency Regulator PRDM14 Requires Hematopoietic Regulator CBFA2T3 to Initiate Leukemia in Mice Lauren J. Tracey1,2, Travis Brooke-Bisschop2, Pascal W.T.C. Jansen3, Eric I. Campos1,2, Michiel Vermeulen3, and Monica J. Justice1,2
Abstract
PR domain–containing 14 (Prdm14) is a pluripotency leukemia. These results suggest a model whereby PRDM14 regulator central to embryonic stem cell identity and pri- recruits CBFA2T3 to DNA, leading to gene misregulation mordial germ cell specification. Genomic regions contain- causing progenitor cell expansion and lineage perturba- ing PRDM14 are often amplified leading to misexpression tions preceding T-ALL development. Strikingly, Prdm14- in human cancer. Prdm14 expression in mouse hematopoi- induced T-ALL does not occur in mice deficient for Cbfa2t3, etic stem cells (HSC) leads to progenitor cell expansion demonstrating that Cbfa2t3 is required for leukemogenesis. prior to the development of T-cell acute lymphoblastic Moreover, T-ALL develops in Cbfa2t3 heterozygotes with a leukemia (T-ALL), consistent with PRDM14's role in cancer significantly longer latency, suggesting that PRDM14-asso- initiation. Here, we demonstrate mechanistic insight into ciated T-ALL is sensitive to Cbfa2t3 levels. Our study high- PRDM14-driven leukemias in vivo.Massspectrometry lights how an oncogenic protein uses a native protein in revealed novel PRDM14–protein interactions including progenitor cells to initiate leukemia, providing insight into histone H1, RNA-binding proteins, and the master hemato- PRDM14-driven oncogenesis in other cell types. poietic regulator CBFA2T3. In mouse leukemic cells, CBFA2T3 and PRDM14 associate independently of the Implications: The pluripotency regulator PRDM14 requires related ETO family member CBFA2T2, PRDM14's primary the master hematopoietic regulator CBFA2T3 to initiate leu- protein partner in pluripotent cells. CBFA2T3 plays crucial kemia in progenitor cells, demonstrating an oncogenic role for roles in HSC self-renewal and lineage commitment, and CBFA2T3 and providing an avenue for targeting cancer- participates in oncogenic translocations in acute myeloid initiating cells.
Introduction Our lab previously identified PR domain–containing 14 (Prdm14) as a potent mammalian oncogene (3, 4). Prdm14 is a Leukemia is the most common cancer in children (1). Despite pluripotency regulator central to embryonic stem cell (ESC) advances in chemotherapies that improve survival, most relapsed identity (5–7) and primordial germ cell (PGC) specification (8, 9). patients succumb to the disease (1, 2). Cancer initiation is difficult Prdm14 is not expressed in adult tissues; however, its misexpres- to study in human patients, as by the time of diagnosis multiple sion, often as a result of genomic amplification, is found in genetic lesions are often present, making the initiating event multiple human cancers including leukemia (4, 10). A high level difficult to deduce (2). Mouse models of cancer offer powerful of expression of PRDM14 is associated with poor outcomes tools that allow for the study of cancer initiation to improve the in both non–small cell lung carcinoma (11) and breast understanding of disease progression. cancer (12, 13). Prdm14 was misexpressed in hematopoietic stem cells (HSC) by mating mice carrying a Rosa26-floxed-stop-Prdm14-IRES-GFP transgene to mice carrying Tg(Mx1-cre; R26PR;Mx1-cre; ref. 14). 1Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Upon inducing expression of Prdm14, R26PR;Mx1-cre mice rap- 2 Canada. Genetics and Genome Biology Program, The Hospital for Sick Children, idly succumb to a completely penetrant T-cell acute lymphoblas- 3 þ Toronto, Ontario, Canada. Faculty of Science, Department of Molecular Biology, tic leukemia (T-ALL) with a highly infiltrative CD8 immature Radboud Institute for Molecular Life Sciences, Oncode Institute, Radboud University Nijmegen, Nijmegen, the Netherlands. single positive T-cell immunophenotype (14). Prior to the onset of leukemia, abnormal HSC-like and lymphoid progenitor cells Note: Supplementary data for this article are available at Molecular Cancer accumulate in the bone marrow (BM) of the mice. Subsequent Research Online (http://mcr.aacrjournals.org/). work showed that the T-ALL is driven by activating mutations on Corresponding Author: Monica J. Justice, Hospital for Sick Children, The Peter Notch1 (15). In contrast, when Prdm14 is expressed in committed Gilgan Centre for Research and Learning, 686 Bay Street, Toronto, Ontario M5G T cells using a dLck-cre line (R26PR;dLck-cre), mice remain 0A4, Canada. Phone: 416-813-7654. Fax: 416-813-4931; E-mail: [email protected]. healthy and do not show signs of leukemia, suggesting that Prdm14 requires additional factor(s) present in progenitor cells Mol Cancer Res 2019;XX:XX–XX to act as an oncogene (14). However, the mechanism through doi: 10.1158/1541-7786.MCR-18-1327 which Prdm14 functions in progenitor cells to initiate cancer is 2019 American Association for Cancer Research. poorly understood.
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PRDM14 contains six zinc finger motifs that bind the same Protein extraction consensus DNA sequence in both mouse and human, and a PR Protein lysates from HEK293T cells and R26FLPR or R26FLPR; domain, which is related to the SET domain (6, 16). Certain PR Mx1-cre BM and thymus single-cell suspensions were extracted family proteins have histone methyltransferase activity; however, using NP-40 lysis buffer [50 mmol/L Tris-HCl pH 8.0, 150 mmol/L no methyltransferase activity has been reported for PRDM14 (15). NaCl, 1% NP-40, 2 mmol/L EDTA, protease inhibitors (Roche)]. Instead, it is likely that PRDM14 regulates gene expression After 30-minute incubation on ice the lysates were centrifuged through protein interaction partners. In support of this hypoth- for 15 minutes at 16,000 g. Protein concentrations were deter- esis, PRDM14 requires an interaction with the core-binding factor, mined using the Pierce Coomassie (Bradford) Protein Assay Kit runt domain, alpha subunit 2 translocated to 2 (CBFA2T2) (Thermo Fisher Scientific). protein to promote ESC self-renewal and to establish pluripotent PGCs (17, 18). In this context, the CBFA2T2–PRDM14 protein Mass spectrometry interaction stabilizes the complex on chromatin to regulate gene Label-free pull downs were performed in triplicate per sample expression. CBFA2T2 belongs to the eight-twenty-one (ETO) as described previously (23). Briefly, 1 mg of protein lysate per family of chromatin-associated proteins, which includes myeloid pull down was incubated with 10 mL of FLAG M2 beads translocation gene 8 (MTG8, ETO) and CBFA2T3 (MTG16, (Sigma-Aldrich) in a total volume of 600 mL NP-40 lysis buffer ETO-2). These proteins each contain 4 Nervy Homology Region containing 50 mg/mL ethidium bromide. After incubation for 1.5 (NHR) domains, and form homo- or hetero-oligomeric ETO hours on a rotation wheel at 4 C, the pull downs were sequen- complexes via the NHR2 domain (19–21). Each of the three ETO tially washed twice with 1 mL of lysis buffer (300 mmol/L NaCl family members participate in oncogenic translocations with and 0.5% NP40), twice with 1 mL PBS with 0.5% NP40 and three RUNX1 in acute myeloid leukemia (AML; ref. 22). times with 1 mL PBS. Bead-bound proteins were then subjected to Despite evidence that protein-binding partners are critical for trypsin digestion as described previously (24). Finally, tryptic PRDM14's function, PRDM14's functional interactors in a cancer peptides were acidified and desalted with C18 Stagetips prior to model have not been described. We hypothesize that when mass spectrometry (MS) analyses (25). Desalted tryptic peptides Prdm14 is expressed in progenitor cells, outside its normal milieu were separated on an Easy-nLC 1000 (Thermo Fisher Scientific) in a pluripotent cell, it requires a protein partner to facilitate connected online to a Thermo scientific Orbitrap Fusion Tribid oncogenesis. Here, we report the first PRDM14–protein interac- Mass spectrometer. MS and MS/MS spectra were recorded in a top tion in an in vivo cancer model and demonstrate how PRDM14 can speed modus with a run cycle of three seconds. Raw files were hijack a hematopoietic regulatory protein, CBFA2T3, in progen- analyzed using MAXQuant software version 1.5.1.0. with default itor cells to initiate leukemogenesis. The protein domains settings (24). Raw MS data were searched against the Uniprot involved in the interaction suggest a model whereby PRDM14 mouse proteome database, released 2015_12. Perseus version recruits CBFA2T3 to chromatin, which is stabilized in a large 1.3.0.4 (26) was used to further analyze the data. Briefly, the chromatin complex to initiate leukemia. We show that CBFA2T3 protein list was filtered for contaminants and reverse hits. Label- has a critical role in PRDM14-induced leukemia initiation, as mice free quantification (LFQ) values were transformed into log2 expressing Prdm14 in hematopoietic progenitor cells fail to devel- values and missing values were imputed by normal distribution. op T-ALL on a Cbfa2t3 null genetic background. Volcano plots were generated with an in-house R script.
Immunoprecipitation Materials and Methods Tissue protein lysates (500 mg) were incubated at 4 C over- Mouse strains/animal care night with Protein A Dynabeads (Invitrogen) and anti-FLAG (M2, All mouse experiments were carried out under the approval of Sigma), anti-ETO-2 (C20, Santa Cruz Biotechnology), or anti- the Animal Use Committee (AUC), and housed at The Centre for MTGR1 (KT42, Abcam) antibodies. HEK293T lysates were incu- Phenogenomics, which is accredited by the Association for Assess- bated at 4 C overnight with anti-FLAG (M2, Sigma) or anti-HA ment and Accreditation of Laboratory Animal Care International. (Thermo Fisher Scientific) magnetic beads. Beads were washed All mouse strains were maintained on a C57BL/6J congenic genetic three times with PBST (PBS, 0.1% Tween-20). Laemmli Sample background: B6.Gt(ROSA)26Sortm1(LSL-Prdm14-IRES-EGFP)Jus,abbrevi- Buffer 2X (Bio-Rad) was added to the beads and boiled for 5 ated "R26PR," B6.Gt(ROSA)26Sortm(LSL-3XFLAG-Prdm14-P2A-EGFP)Jus minutes at 95 C to obtain coimmunoprecipitated proteins. abbreviated "R26FLPR," (14, 15) B6.Tg(Mx1-cre)1Cgn/J abbrevi- ated "Mx1-cre" (from Dr. Margaret A. Goodell, Baylor College Density gradient of Medicine, Houston, TX), B6.Cbfa2t3tm1.1Swh abbreviated Protein lysate (100 mL) was loaded onto a linear 15%–40% "Cbfa2t3 / " (from Dr. Scott W. Hiebert, Vanderbilt University, glycerol gradient. The gradient was placed in a SW60 Ti swinging- Nashville, TN). Polyinosinic:polycytidylic acid (pIpC) administra- bucket rotor (Beckman Coulter) and spun in an Optima XPN-80 tion to activate Mx1-cre was performed at 8 weeks of age with one Ultracentrifuge (Beckman Coulter) at 44,000 rpm for 16 hours at intraperitoneal injection of 250 mg. Mice were monitored for signs 4 C. The gradient was immediately fractionated into 21 fractions of cancer including enlarged lymph nodes and abdomen, lethargy, of 200 mL. and labored breathing. Tissues from moribund mice were dissected and processed for protein, RNA, flow cytometry, and/or blood Western blot analysis analyses. Samples for pathology analysis were fixed in 10% neutral Protein lysates were resolved on a 4%–12% Polyacrylamide buffered formalin (NBF) overnight. Tissues were paraffinembed- Gradient Gel (Bio-Rad) and transferred to a polyvinylidene ded and sectioned, and slides stained with hematoxylin and eosin difluoride (PVDF) membrane (Bio-Rad) for blotting. Membranes (H&E). Images were acquired with an Eclipse E100 (Nikon) were blocked in 5% skim milk for 1 hour and subsequently microscope and DS-Qi1Mc (Nikon) camera. incubated with primary antibodies diluted in 5% skim milk for
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2 hours. After washing with PBST, membranes were incubated 2% FBS (Wisent), 10 mmol/L HEPES, pH 7.2]. One million cells with horseradish peroxidase (HRP)-conjugated secondary anti- of each sample were stained with fluorochrome-conjugated anti- bodies diluted in 5% skim milk for 1 hour. Membranes were bodies for 30 minutes on ice in the dark, and washed with SM. washed again with PBST, and chemiluminescence Clarity Western Samples were analyzed on a LSR II flow cytometer (BD ECL Substrate (Bio-Rad) was added for signal detection with Biosciences) in the Flow and Mass Cytometry Facility at the Amersham Hyperfilm ECL (GE Healthcare). The following pri- Hospital for Sick Children. Data collection and analysis were mary antibodies were used: FLAG (M2, Sigma), ETO-2 (C20, performed with FACSDiva (BD Biosciences) and FlowJo v10 (BD) Santa Cruz Biotechnology), MTGR1 (Abcam), HA (Sigma). The software. The following antibodies were used: CD4-Alexa 700 following secondary antibodies were used: Goat Anti-Rat IgG (eBioscience), CD8a-eFluor 450 (eBioscience), CD24-PE H&L HRP (Abcam), Rabbit Anti-Goat IgG H&L HRP (Abcam), (eBioscience), TCRBeta-APC (eBioscience), 7-AAD (BioLegend). Peroxidase AffiniPure Goat Anti-Mouse IgG Light Chain Specific (Jackson ImmunoResearch). Statistical analysis A t test was performed on LFQ values and the FDR-corrected P Protein domain interactions value was calculated to determine significant proteins after Full-length coding sequences of mouse PRDM14 and CBFA2T3 AP/MS. Kaplan–Meier survival curves were compared using a were amplified using CloneAmp HiFi PCR Premix (ClonTech) log-rank (Mantel–Cox) test for statistical significance in Prism with the following primer sequences (50 to 30): PRDM14, GAC- (GraphPad). An unpaired t test was performed to identify changes GATGACGACAAGTTCCGCATGGCCTTACCGCCCTCTGGT and in quantified flow cytometry data in Prism (GraphPad). AACGGGCCCTCTAGACTCGAGCTAGCAGGTTTTATGAAGCCT; CBFA2T3, CCAGACTACGCGGGAATAAGGATGTCCCAGGCAT- CCACCACC and GGGTTTAAACGGGCCCTCTAGATCAGCGG- Results GGCACAGCAGCGTC. In-Fusion HD Cloning Kit (Takara Bio) PRDM14 interacts with CBFA2T3 in leukemia cells was used to clone fragments into the pcDNA3.1þ (Invitrogen) To determine the molecular mechanism for PRDM14-induced mammalian expression vector. The resulting plasmids were used cancer initiation, we sought to identify PRDM14 protein– to amplify and subclone the deletion constructs into pcDNA3.1þ interacting partners. Prdm14 expression was induced in 8-week- vectors for expression with the following primer sequences (50 to old R26FLPR;Mx1-cre animals by injection of polyinosinic:poly- 30): PRDM14-DZF, GACGATGACGACAAGTTCCGCATGGCCT- cytidylic acid (pIpC). Immunoprecipitation (IP) of FLAG- TACCGCCCTCTGGT and AACGGGCCCTCTAGACTCGAGCTA- PRDM14 was conducted on BM from moribund R26FLPR; CTCATAGCCATTTCCGTACCA; PRDM14-DN-PR, GACGATGAC- Mx1-cre with anti-FLAG magnetic beads. Bead-bound proteins GACAAGTTCCGCAAGTTCCTGGGCGTTCCCATG and AACG- were digested with trypsin and subjected to quantitative LC/MS- GGCCCTCTAGACTCGAGCTAGCAGGTTTTATGAAGCCT; PRD- MS for identification. AP/MS analysis was performed using meth- M14-PR, GACGATGACGACAAGTTCCGCGGCTTCAACTTCACA- ods that are optimized for low protein inputs. Control BM from GAGGAG and AACGGGCCCTCTAGACTCGAGCTACTCATAG- R26FLPR animals not exposed to cre and therefore not expressing CCATTTCCGTACCA; CBFA2T3- DNterm, CCAGACTACGCGG- Prdm14 was also subjected to LC/MS-MS to eliminate proteins GAATAAGGCAGCTGCTGCTGGACGCCAGC and GGGTTTAAA- pulled down through nonspecific antibody binding. LFQ analysis CGGGCCCTCTAGATCAGCGGGGCACAGCAGCGTC; CBFA2- revealed several strong interactions with proteins involved in T3-DCterm, CCAGACTACGCGGGAATAAGGATGTCCCAGGCAT- NOTCH1 activation (SGK223/PEAK1), splicing (THRAP3, CCACCACC and GGGTTTAAACGGGCCCTCTAGACTATTCGT- RBM10), and chromatin binding (CBFA2T3, linker histone var- GCTGGGCCAGGTACTG; CBFA2T3-NHR1, CCAGACTACGCG- iants HIST1h1d/H1.3 and HIST1h1b/H1.5, CBFA2T2; Fig. 1A; GGAATAAGGTGTCTCTTGATGAACGGCAGC and GGGTTTAAA- Supplementary Table S1). The protein with the strongest fold CGGGCCCTCTAGACTATTCGTGCTGGGCCAGGTACTG. Accu- change over control was ETO family member CBFA2T3. PRDM14 rate plasmid sequence was confirmed by Sanger sequencing. To interaction with both ETO family members was confirmed using coexpress constructs, HEK293T cells were cultured in DMEM reciprocal co-IP on thymus tissue with FLAG-PRDM14 and (Wisent) with penicillin/streptomycin and 10% FBS. Cells were endogenous CBFA2T3 (Fig. 1B) or CBFA2T2 (Fig. 1C). The transfected with Lipofectamine 2000 (Invitrogen) as per the man- leukemias that develop in PRDM14-expressing mice are highly ufacturer's instructions and lysed 24 hours posttransfection. infiltrative, saturating all hematopoietic tissues, with monoclonal immature pre-T cells (14, 15). Therefore, thymus tissue was used RT-PCR to confirm the interaction in a second tissue type, producing high RNA extraction was performed with RNeasy Mini Kit (Qiagen). protein yields to reduce the number of animals required, and RNA was reverse transcribed using SuperScript VILO cDNA Syn- providing a more homogeneous wild-type control tissue than BM. thesis Kit (Invitrogen). cDNA was amplified with AccuStart II PCR Co-IP with IgG alone confirmed proteins were not being recov- SuperMix (Quantabio). Primers used (50 to 30) were as follows: ered through nonspecific binding to the antibody (Fig. 1B and C). Cbfa2t3, TGGAAGCACCTCAACAGTCTTC and GTGGTTGAGTT- The strength of the interaction with CBFA2T3 and its importance CCTCACGGT; Tbp, CCTTGTACCCTTCACCAATGAC and ACA- in hematopoietic cell regulation led us to further investigate its GCCAAGATTCACGGTAGA. relationship with PRDM14. To further characterize the interaction with PRDM14, we ran Flow cytometry R26FLPR;Mx1-cre thymus protein lysates through a density gra- Single-cell suspensions were prepared from thymus, spleen, dient to separate potential protein complexes. Western blot and BM tissues. Red blood cells were lysed with Red Blood Cell analysis showed a partial cofractionation of PRDM14 (64 kDa) Lysis Solution (Miltenyi Biotec) and washed with staining media and CBFA2T3 (68 kDa) interacting in a fraction corresponding to [SM: Hanks' Balanced Salt Solution (Thermo Fisher Scientific), approximately 400–500 kDa in size, suggesting that additional
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Figure 1. PRDM14 interacts with CBFA2T3 in leukemia cells. A, Volcano plot of LFQ mass spectrometry data from R26FLPR;Mx1-cre leukemic BM performed technically in triplicate. Datapoints represent individual proteins with significant hits in the top right corner having –log FDR
>1.301 and log2 fold change (FC) >8 over control R26FLPR animals. B, Co-IP in leukemic thymus confirms protein interaction between PRDM14 and CBFA2T3, which is not due to unspecific IgG binding. Representative image from four experiments. C, Co-IP in leukemic thymus confirms protein interaction between PRDM14 and CBFA2T2, which is not due to unspecificIgG binding. Representative image from four experiments. D and E, Western blot probing for FLAG (PRDM14), CBFA2T3, and CBFA2T2 after separation of protein complexes via density gradient. Top arrows indicate approximate size of complexes running at fraction 5 or between fractions 13 and 15 based on purified protein standards, and side arrows indicate expected size of the indicated protein. D, FLAG (PRDM14) and CBFA2T3 proteins, but not CBFA2T2, peak in fraction 13. Fractions 12–14 were pooled and FLAG-PRDM14 was pulled down with aCBFA2T3 antibody confirming an association in those fractions. Representative images from four experiments. E, In control R26FLPR tissue not expressing PRDM14, CBFA2T3 peaks in fraction 5. Representative images from three experiments.
complex members or CBFA2T3 oligomerization may contribute fying enzymes that bind NHR3-4 (27). PRDM14 interacts to the formation of multimeric protein associations (Fig. 1D). with other proteins primarily via its PR domain (17). To CBFA2T2, PRDM14's primary interaction partner in pluripotent identify the protein domain at which the PRDM14–CBFA2T3 cells (17, 18), peaked in the first fractions (5 and 7), but did not interaction occurs, a series of deletion constructs lacking cofractionate with PRDM14 or CBFA2T3 in the later fractions, individual functional domains was created for each protein indicating that it does not participate abundantly in the same (Fig. 2A). To test the interacting domains, tagged expression CBFA2T3-containing complex in murine leukemia cells. Co-IP plasmids were cotransfected into HEK293T cells followed by performed with an aCBFA2T3 antibody from lysates from frac- protein extraction and co-IP with either anti-FLAG tions 12–14 confirmed an association between FLAG-PRDM14 (PRDM14) or anti-HA (CBFA2T3) magnetic beads. PRDM14 and CBFA2T3 in those peak fractions. In contrast, a density bound to full length CBFA2T3 without its C-terminal zinc gradient carried out in R26FLPR control thymus lacking cre fingers, but the N-PR region was required for interaction. revealed that CBFA2T3 shows a peak in fraction 5 with no PRDM14's PR domain alone was sufficient to pull down detectable signal in later fractions when PRDM14 is absent, CBFA2T3 (Fig. 2B). CBFA2T3 bound full length PRDM14 demonstrating that CBFA2T3 is predominantly found as a mono- without the C-terminal half of the protein, but the N-ter- mer in wild-type thymus (Fig. 1E). minal half was required for interaction. Furthermore, the NHR1 domain of CBFA2T3 alone was sufficient to pull down PRDM14 and CBFA2T3 interact through the PR and NHR1 PRDM14 (Fig. 2C; Supplementary Fig. S1), even though this domains small region of the protein appeared to be less stable than CBFA2T3 contains four NHR domains that bind diverse the larger components. Together, these data demonstrate that types of proteins, enabling it to act as a bridge between PRDM14 and CBFA2T3 interact through the PR and NHR1 transcription factors that bind NHR1 and chromatin-modi- domains, respectively.
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Figure 2. PRDM14 and CBFA2T3 interact through the PR and NHR1 domains, respectively. A, Deletion constructs lacking key functional domains were constructed for PRDM14 (FLAG-tagged) and CBFA2T3 (HA-tagged). Numbers indicate beginning and ending amino acid positions for each corresponding full-length protein. Western blot results after co-IP with anti-FLAG (B) or anti-HA (C) antibodies. The constructs important for facilitating interaction are detected in the bottom panels. The NHR1 panel of the HA blot in (C) shows a longer exposure due to decreased expression. Representative images from three experiments. PRDM14-FL (67 kDa), PRDM14-DZF (42 kDa), PRDM14-DN-PR (25 kDa), PRDM14-PR (21 kDa), CBFA2T3-FL (70 kDa), CBFA2T3-DNterm (45 kDa), CBFA2T3-DCterm (25 kDa), CBFA2T3-NHR1 (13 kDa). UT, untransfected; FL, full length.
Altering levels of Cbfa2t3 can prevent or slow T-ALL that Cbfa2t3 is essential for Prdm14-induced T-ALL (Fig. 3C). þ þ development Unexpectedly, R26PR;Mx1-cre;Cbfa2t3 / mice showed a signif- Cbfa2t3 is important for HSC self-renewal and is most highly icant delay in leukemia onset with 6/12 mice succumbing to expressed in hematopoietic stem and progenitor cells (27–30). disease in 32–136 days post-pIpC injection, and 6/12 mice Therefore, we hypothesized that a misappropriation of its role in remaining healthy by 150 days post-pIpC injection, in contrast þ þ BM when PRDM14 is ectopically present is critical for T-ALL to R26PR;Mx1-cre;Cbfa2t3 / mice. These data suggest that the pathogenesis. To test this idea, we placed the R26PR and Mx1- leukemia that develops due to expression of Prdm14 is sensitive to cre constructs together on a Cbfa2t3 / genetic background levels of Cbfa2t3. (R26PR;Mx1-cre;Cbfa2t3) prior to pIpC induction (Fig. 3A and þ/ B) and assessed leukemia phenotypes over time. Strikingly, T-ALL has a similar disease phenotype in both Cbfa2t3 and þ þ þ þ although R26PR;Mx1-cre;Cbfa2t3 / animals (11/12) develop Cbfa2t3 / backgrounds a very penetrant T-ALL as early as 16 days post-pIpC injection, The significant difference in survival between Prdm14-expres- þ þ þ R26PR;Mx1-cre;Cbfa2t3 / animals (11/11) did not develop any sing mice on a Cbfa2t3 / or Cbfa2t3 / background led us to signs of leukemia over 150 days post-pIpC injection, suggesting consider that the cell type may also differ in leukemias that arose
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Figure 3. Deletion of Cbfa2t3 prevents T-ALL development in R26PR;Mx1-cre mice. A, Mouse cross of R26PR;Mx1- cre mice with Cbfa2t3 / mice and experimental timeline. B, RT-PCR detecting Cbfa2t3 and Tbp expression (internal control) in Cbfa2t3þ/þ and Cbfa2t3þ/ mice, but not Cbfa2t3 / . C, Kaplan– Meier survival curve of R26PR;Mx1- cre;Cbfa2t3 mice presented as percent survival by days post-pIpC injection (Prdm14 expression) for each genotype. Significance was determined by comparing the three curves using a log-rank (Mantel– Cox) test for statistical significance. None of the R26PR;Mx1-cre; Cbfa2t3 / mice died by 150 days (n ¼ 11); R26PR;Mx1-cre;Cbfa2t3þ/ mice died between 32 and 136 days post-pIpC (n ¼ 12, mean ¼ 100 days); R26PR;Mx1-cre;Cbfa2t3þ/þ mice died between 16 and 74 days post-pIpC (n ¼ 12, mean ¼ 38 days). Tbp, TATA-box binding protein.
on the two genetic backgrounds. However, the difference was Lymphoblasts could also be detected in the meninges and blood restricted to penetrance and the timing of onset, because few vessel linings within the brain (Fig. 4F). differences in leukemia subtype or pathology were noted between We have previously shown that cells misexpressing þ þ moribund R26PR;Mx1-cre;Cbfa2t3 / and R26PR;Mx1-cre; PRDM14 have a block in T-cell development leading to an þ þ Cbfa2t3 / mice. All moribund animals presented with lethargy, accumulation of CD8 immature single-positive (ISP) T cells þ þ distended abdomens, and labored breathing and upon dissection (CD4 CD8 CD24 TCRb lo/ ) at the expense of double-positive þ þ had grossly enlarged thymi, spleens, and livers. However, (DP) T cells (CD4 CD8 ; ref. 14). We confirmed this result using þ only 67% of R26PR;Mx1-cre;Cbfa2t3 / mice (4/6) developed flow cytometry immunophenotyping of R26PR;Mx1-cre; þ þ þ enlarged lymph nodes compared with 100% of R26PR;Mx1-cre; Cbfa2t3 / mice and showed that R26PR;Mx1-cre;Cbfa2t3 / þ þ þ Cbfa2t3 / mice (11/11). Complete blood counts in R26PR;Mx1- mice develop a corresponding accumulation of CD8 ISP T cells þ cre;Cbfa2t3 / mice showed a significant increase in white blood in the thymus (Fig. 5A and B). Taken together, these results cell counts (48–192 109/L, mean 90.74 26.37) over control indicate that the T-ALL that develops in R26PR;Mx1-cre; þ animals (2.1–8.2 109/L, mean 5.63 1.38; Fig. 4A). Although Cbfa2t3 / mice is similar in cell type to that in R26PR;Mx1- þ þ þ þ R26PR;Mx1-cre;Cbfa2t3 / mice showed a significant decrease in cre;Cbfa2t3 / mice but with a significantly delayed onset. Strik- red blood cell numbers from control animals (5.71–8.3 1012/L, ingly, the absence of Cbfa2t3 prevents T-ALL when Prdm14 is mean 7.62 0.49 in moribund animals versus 8.8–9.9 1012/L, misexpressed in HSCs. mean 9.49 0.24 in control animals), those in R26PR;Mx1-cre; þ Cbfa2t3 / mice were more variable, and did not reach signifi- cance (4.6–11.4 1012/L, mean 8.25 1.10 in moribund Discussion animals versus 8.4–9.3 1012/L, mean 8.95 0.20 in control Our work suggests that the presence of CBFA2T3 in hemato- animals; Fig. 4B). Hemoglobin, hematocrit, and platelet values poietic progenitors is required for PRDM14 to initiate T-ALL. The were statistically unchanged between moribund and control mice protein–protein interaction between PRDM14 and CBFA2T3 that (Fig. 4C–E). Histopathologic analyses of both R26PR;Mx1-cre; occurs in conserved family motifs suggests a model where this þ þ þ Cbfa2t3 / and R26PR;Mx1-cre;Cbfa2t3 / mice revealed a loss of complex, which likely contains multiple CBFA2T3 oligomers, normal architecture in the BM, spleen, thymus, liver, and kidney provides a platform that recruits chromatin-modifying enzymes associated with an extensive infiltration of lymphoblast cells. to initiate leukemia (Fig. 6). The master hematopoietic regulator
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Cbfa2t3 Is Essential for Prdm14-Induced T-ALL
Figure 4. R26PR;Mx1-cre;Cbfa2t3þ/ moribund mice develop T-ALL with a similar disease phenotype compared with R26PR;Mx1-cre;Cbfa2t3þ/þ moribund mice. White blood cell (A; WBC), red blood cell (B; RBC), hemoglobin (C), platelet (D), and hematocrit counts (E) in peripheral blood of control (n ¼ 4) and moribund (n ¼ 5) mice. Significance was determined using an unpaired t test. F, Representative images of hematoxylin and eosin–stained sections of fixed BM, thymus, spleen, kidney, liver, and brain tissues. All images were taken with a 40 objective. Scale bar, 100 mm( , P < 0.0006; , P < 0.05; ns, not significant).