SCIENCE SIGNALING | RESEARCH ARTICLE

LEUKEMIA Copyright © 2017 The Authors, some MAFB enhances oncogenic Notch signaling in T cell rights reserved; exclusive licensee acute lymphoblastic leukemia American Association for the Advancement 1,2,3 2,3 1 2,3 1 of Science. No claim Kostandin V. Pajcini, * Lanwei Xu, Lijian Shao, Jelena Petrovic, Karol Palasiewicz, to original U.S. 2,3 2,3 2,3 3 4 Yumi Ohtani, Will Bailis, Curtis Lee, Gerald B. Wertheim, Rajeswaran Mani, Government Works Natarajan Muthusamy,4 Yunlei Li,5 Jules P. P. Meijerink,6 Stephen C. Blacklow,7 Robert B. Faryabi,2,3 Sara Cherry,8 Warren S. Pear2,3*

Activating mutations in the encoding the cell-cell contact signaling Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individu- ally enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease pene- trance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target , Downloaded from including and HES1, and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL. http://stke.sciencemag.org/

INTRODUCTION PEST mutations limit ICN degradation. Thus, these mutations either T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malig- increase nuclear Notch1 and/or inhibit NTC turnover, which ultimately nancy of immature T cell blasts that occurs in both children and adults leads to dysregulated (increased) Notch1-mediated transcription. The (1). Although current treatments are relatively successful, especially in enhanced Notch1 signaling resulting from either of these mutations children (2), ~20% of patients are not cured by current therapy (1). can be inhibited by g-secretase inhibitors (GSIs), which prevent release Activating mutations in NOTCH1 are frequent, occurring in >60% of the ICN from the plasma membrane (3). of T-ALLs (3). Expressing ICN1 in murine T-ALL models rapidly induces leukemia –

The intercellular Notch ligand interaction activates a con- in all mice; however, this form of Notch1 rarely occurs in human T-ALL on December 5, 2017 served signaling pathway that regulates multiple cellular functions (4). (3). In contrast, expressing mutated NOTCH1 alleles commonly asso- Physiologic Notch signaling occurs when a Notch receptor interacts ciated with human T-ALL results in T-ALL with a much longer latency with a ligand of the Jagged or Delta-like family in a neighboring cell. that is incompletely penetrant (8). These findings suggest that events This initiates a series of metalloproteinase- and g secretase–mediated that synergize with the weak Notch1 mutants and/or increase their cleavages that release the intracellular Notch (ICN) domain from the signaling strength likely contribute to T-ALL. In support of this idea, plasma membrane. The ICN then translocates to the nucleus where it coexpressing weak Notch1 alleles with mutations found in human pa- forms the Notch transcriptional complex (NTC) with the DNA binding tients, such as oncogenic Ras, decreases T-ALL latency and increases protein RBPJ and a member of the Mastermind-like (MAML) family. penetrance (8). This complex activates transcription of Notch target genes (5). The To identify potentially oncogenic hits that directly modify Notch1 NTC is short-lived and is tagged for degradation by proteins including signaling, we performed a gain-of-function complementary DNA the ubiquitin ligase Fbxw7, which recognizes PEST sequences residing (cDNA) screen to discover molecules that enhance the ability of weak in the ICN C terminus (6). oncogenicNOTCH1mutants.Amongthehitswerethetranscription Activating NOTCH1 mutations in T-ALL occur in the extracellular factors MAFB and ETS2. MAFB belongs to the Maf family of AP1 Notch1 regulatory region (NRR) and/or the C-terminal PEST domain transcription factors, all of which contain a basic (bZIP) (7). NRR mutations lead to ligand-independent signaling, whereas domain that binds two long palindromic sequences referred to as “MARE” sequences (9). MAFB also contains an N-terminal transcrip-

1 tional activation domain, which optimally induces Maf target gene Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 10 11 Chicago, IL 60612, USA. 2Abramson Family Cancer Research Institute, University of transcription by recruiting other transcriptional regulators ( , ). Pennsylvania, Philadelphia, PA 19104, USA. 3Department of Pathology and Laboratory MAFB serves many roles in embryonic development, including hema- Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 4The James, Ohio topoiesis, where it exerts important functions in macrophage differen- State University Comprehensive Cancer Center, Columbus, OH 43210, USA. 5Depart- MAFB ment of Pediatric Oncology/Hematology, Erasmus Medical Center, Rotterdam, Netherlands. tiation. In cancer, translocations of and its closely related 6Princess Maxima Center for Pediatric Oncology, Utrecht, Netherlands. 7Department of homolog, MAF, are frequent in multiple myeloma (12). ETS2 belongs Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, to a large and nearly ubiquitously expressed family of transcription 8 MA 02115, USA. Department of Microbiology, University of Pennsylvania, Philadelphia, factors that function in a wide variety of developmental roles (13). In PA 19104, USA. *Corresponding author. Email: [email protected] (K.V.P.); [email protected]. hematopoiesis, ETS2, like ETS1, with whom it shares close homology, edu (W.S.P.) is important for cortical thymocyte proliferation and survival (14–16).

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ETS transcription factors are also dysregulated in multiple cancers, in- murine and human T-ALL cell lines and primagrafts (fig. S1, D and E). cluding Ewing’ssarcoma(17) and prostate cancer (18). Furthermore, analysis of two independent patient data sets (21, 22)iden- To determine the relevance of the findings of our screen, we inves- tified a range of MAFB expression in primary human T-ALL samples tigated the function of MAFB and ETS2 in multiple assays relevant to (fig. S1, F and G). T-ALL pathogenesis. We found that although both MAFB and ETS2 individually enhanced the signal strength of the Notch1 gain-of- MAFB and ETS2 combine to enhance Notch signaling function alleles, their coexpression resulted in synergistic activity that ETS2 was also identified as an enhancer in our screen (Fig. 1B), and its was comparable to the core NTC. Furthermore, MAFB alone increased closely related homolog, ETS1, is known to interact with MAFB (23). the penetrance and decreased the latency of T-ALL induced in mice We used Notch reporter assays to test the potential cross-talk between with weak Notch1 gain-of-function mutants, whereas inhibiting MAFB MAFB and ETS2. The two genes were transfected into U2OS cells along in both human and murine T-ALL cells inhibited their growth and de- with LPDP. Not only did MAFB and ETS2 synergistically increase re- creased the expression of a subset of Notch target genes. We found that porter activity but the signal was also comparable to that induced by the MafB interacts with ETS2 and, in doing so, cooperates with the NTC to MAML1 positive control (Fig. 1E). Although MAFB and ETS2 syner- augment Notch1-mediated transcription. We thus propose that MAFB gized in this reporter assay, MAFB appeared to be the limiting functions to amplify the signaling output of naturally occurring Notch1 component. A twofold decrease in the amount of MAFB decreased mutants, a finding consistent with its expression in a high percentage of the reporter signal by ~50%, whereas a fourfold drop in ETS2 DNA human T-ALLs. Not only do these data identify new proteins that en- concentration resulted in ~25% decrease in Notch1 reporter activity hance Notch1 function in T-ALL but they also identify MAFB as a (fig. S1C). These results support a robust, two-component enhance- potential new therapeutic target in T-ALL. ment of the LPDP mutant, with MAFB playing the major role. Downloaded from Consistent with MafB being the limiting component, both Ets1 and Ets2 are highly expressed in after b-selection thymocytes, whereas MafB RESULTS expression is much lower in these populations [www.immgen.org/ A cDNA screen detects enhanced Notch signaling databrowser/index.html (24)]. Our Notch1 gain-of-function screen relies on three genetic components transfected into U2OS cells: (i) a sensitive, synthetic Notch signaling re- MAFB increases penetrance and decreases latency of http://stke.sciencemag.org/ porter (TP1) containing 12 iterated RBPJ-binding sites driving lucifer- Notch-induced T-ALL ase expression in a pSP72 backbone; (ii) a cytomegalovirus (CMV) To assess the effect of MAFB in vivo, we assayed its ability to influence vector (pcDNA3) expressing a NOTCH1 mutant; and (iii) the cDNA the leukemogenic activity of LPDP in a retroviral transduction/bone library, containing 18,000 open reading frames in the Sport6 CMV marrow transplant (BMT) model (Fig. 2A) (25). Notch LPDPwas vector, individually preplated in 384-well plates (Fig. 1A). U2OS cells expressed from a murine stem cell virus retroviral vector (MigR1) co- were used because of their high transfectability and low basal Notch expressing green fluorescent protein (GFP) (26), whereas MAFB retro- signaling. We assayed several Notch mutants previously identified in viral particles coexpressed truncated nerve growth factor receptor 8 primary patient samples. We chose NOTCH1 L1601P-DeltaPest (NGFR) ( ), thus providing the ability to distinguish between them. on December 5, 2017 (LPDP) (8), which contains both heterodimerization domain (HD) Reconstituted mice were monitored at various times after BMT. At and PEST mutations, because it consistently yielded a luciferase signal 4 weeks after BMT, CD4+CD8+ T cells accumulated in the peripheral above background and produced a significant z score of 0.602 above blood of ICN and LPDP+MAFB mice (Fig. 2B). This population also background (fig. S1A). In contrast, the weaker NOTCH1 L1601P mu- expressed the GFP+ (ICN or LPDP) or GFP+NGFR+ (LPDP+ MAFB+) tant (8), which lacks a PEST mutation, only marginally increased surrogate markers, indicating that they originated from the trans- background Notch signaling in U2OS cells. Both mutants were consid- duced progenitors. Consistent with previous data, mice reconstituted erably weaker transcriptional activators than ICN1 (fig. S1A). with ICN1 succumbed to T-ALL with a 100% penetrance within 80 days From the screen, we identified the top 30 candidate genes, ranked by after BMT, with a median survival time of 58 days, whereas mice receiving relative fold induction of reporter activity (Fig. 1B and table S1). As LPDP alone developed T-ALL with a penetrance of 36% and latency greater expected, the positive control MAML1, a critical NTC component, than 100 days (Fig. 2C) (8, 25, 27). None of the mice receiving MAFB alone produced the greatest enhancement. Additional evidence that the succumbed to leukemia. In contrast, 16 of 16 mice reconstituted with screen was specific was the identification of ATP2A3, which encodes LPDP+MAFB succumbed to T-ALL with a median survival of 72 days. SERCA3, and ERO1L, both of which are known to function in the Consistent with the Kaplan-Meier plots, the white blood cell counts (19, 20). (WBCs) of the LPDP+MAFB mice were significantly higher than the LPDP-only transduced mice (Fig. 2D), as were the spleen weights and MAFB induces dose-dependent enhancement of percentage of infiltrating tumor cells in tissues compared to the LPDP- Notch signaling only mice (fig. S2). Together, these data show that MAFB strongly en- We independently validated the ability of several top candidate hits to hances the ability of weakly activating Notch1 mutants to induce T-ALL. activate the TP1 reporter (Fig. 1C). Verified hits comprised several transcription factors including MAFB and ETS2. Of the confirmed can- Suppressing MafB inhibits Notch signaling and T-ALL didates that we assayed, only MAFB showed a significant dose- cell growth dependent enhancement of Notch reporter activity in combination with To understand the function of MafB in T-ALL, we suppressed MafB LPDP (Fig. 1D). This was not a general effect of MAFB on transcriptional in T6E cells with fluorophore-conjugated small interfering RNAs reporters because MAFB had minimal effects on a nuclear factor kB (siRNAs). T6E cells were chosen because they express high levels (NF-kB) reporter (fig. S1B). These findings led us to focus on MAFB. of MafB (fig. S1D). At 48 hours after treatment, >90% of T6E cells Although MAFB was not expressed in U2OS cells, it was expressed in expressed the fluorophore (fig. S3A), and both siRNAs reduced

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 2of12 SCIENCE SIGNALING | RESEARCH ARTICLE

MafB expression by ~50% (Fig. 3A). There was a concomitant decrease (shScrmb)–treated cells, the loss of MafB significantly reduced p300 in- in the direct Notch1 targets Hes1, Myc, Notch3,andDeltex1 as well as teraction at both the Hes1 and the NDME (Fig. 3C). the Myc target CAD (Fig. 3B). In contrast, neither the expression of To gain further insights into the effect of MafB on Notch1-dependent Hey1 nor that of the negative control, GAPDH,changed(Fig.3Band transcription, we performed ChIP sequencing (ChIP-seq) for the histone fig. S3B). acetylation mark H3K27Ac on T6E cells treated with shScrmb or MafB To persistently inhibit MafB, we transduced T6E cells with retroviral shRNAs. We chose H3K27Ac because changes in Notch occupancy vectors expressing MafB short hairpin RNAs (shRNAs). Both shRNAs produce dynamic alterations in H3K27Ac levels at both enhancers and suppressed MafB as well as Hes1 and Myc expression (fig. S3, C to E). promoters of Notch1-dependent genes in T-ALL (31). To identify Because NTCs recruit p300 to activate transcription (28, 29), we per- Notch1-dependent genes in T6E cells, we performed a GSI-washout ex- formed chromatin immunoprecipitation (ChIP)–PCR for this protein periment (32) to compare expressed genes in the Notch-off to Notch-on on T6E cells treated with MafB shRNAs to determine p300 occupancy states (31, 32). Using stringent criteria (see Materials and Methods), we at the 5′ promoter region of Hes1 and at the 3′ Notch-dependent Myc identified 59 Notch1 positively regulated transcripts (table S2). We enhancer (NDME), both of which are known to recruit p300 in a Notch- then analyzed the effect of MafB knockdown on the putative enhancers, dependent manner (30). When compared to scrambled shRNA marked by H3K27Ac of these 59 Notch positively regulated transcripts.

A B Fold induction Fold induction Screen cDNA over empty over NOTCH1- gene symbol vector LPΔP Downloaded from • MAMLcontrol 27.69 6.71 • PKACA 15.32 3.72 Light KLF15 14.54 3.52 pcDNA3 TP1 HMGB2 12.62 3.06 TSPAN1 11.27 2.74 • SERCA3 11.20 2.72 METRNL 11.11 2.70 NOTCH1-LP P Notch luciferase reporter TYSND1 10.64 2.58 SUMO2 10.42 2.53 http://stke.sciencemag.org/ • ERO1L 10.37 2.51 BDH1 10.27 2.50 transfection MMP14 10.20 2.47 • ARF4 10.17 2.47 TSPAN4 10.09 2.46 U2OS • MAFB 9.61 2.33 cells TMPRSS2 9.56 2.32 RAD18 9.42 2.29 • ERO1-A 9.25 2.24 HMG4 9.23 2.24 MGAT1 9.21 2.24 Lysis CALLA 9.15 2.22 on December 5, 2017 Preplated cDNA reagent High-throughput readout MKP5 9.13 2.15 CAC 9.10 2.11 MAL2 9.09 2.11 MAFG 8.82 2.09 CED 8.78 2.01 OP1 8.69 1.99 HAI-2 8.47 1.97 • ETS2 8.45 1.97 ARH1 8.44 1.97 C D E Δ LP P LPΔP LPΔP 12.5 30 50 NS *** ** 40 10.0 ** 20 30 ** 7.5 **** ** ** 20 10 ** 5.0 * * ** 10 2.5 0 ** 0 0.0 P EV PΔ L MAML MAFB ETS2

MAFB + ETS2 Fig. 1. Screen to identify novel enhancers of Notch signaling. (A) Schematic of the methodology used in the cDNA gain-of-function screen. (B) List of candidate genes potentiating NOTCH1 LPDP activity. MAML1control is the MAML1 positive control. Black dots indicate candidates that were independently verified in reporter assays. (C) Luciferase induction using the Notch-responsive TP1 reporter and LPDP to validate candidates identified in the screen. Data were quantified relative to the empty vector (EV) control. (D) Luciferase assay using the TP1 reporter (40 ng per well) and LPDP (40 ng per well) and assaying dose dependency of Notch activation by ETS2 (40, 80, and 120 ng per well) or MAFB (40 and 80 ng per well). (E) Luciferase TP1 reporter assay with LPDP when MAFB and ETS2 were singly or cotransfected (at 40 ng each). To normalize the amount of plasmid per well, we added EV so that the total amount of DNA was 80 ng per well (not including reporters). Data are from three independent experiments. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001 by unpaired t tests. NS, not significant.

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 3of12 SCIENCE SIGNALING | RESEARCH ARTICLE

Suppression of MafB decreased H3K27Ac by at least 1.4-fold in 37 of 59 er than endogenous murine MafB protein (Fig. 4A). In both cell lines, (62%) Notch1-responsive genes (Fig. 3D and table S2). These included the expression of the Notch1 target genes Hes1 and Myc was significant- the well-known direct Notch targets Dtx1, Myc, Notch3, NRarp, Notch1, ly increased (Fig. 4, B and C), and this was accompanied by increased and IL2RA (Fig. 3E, fig. S3F, and table S2). Although Hes1 expression cell proliferation (fig. S4A). significantly decreased in the GSI-washout condition, the decrease in We tested whether increased MAFB expression could compensate H3K27Ac abundance did not meet our 1.4-fold threshold, because it for decreased Notch1 signaling in T-ALL cells treated with GSIs. To de- decreased by only 1.2-fold; nevertheless, there was a decrease in termine the appropriate dose of GSI treatment for T6E cells, we titrated H3K27Acloadingatthe5′ site of the Hes1 transcription start site (fig. the concentration of GSI and determined that the range 0.1 to 0.01 mM S3F). Furthermore, shMafB is not a global inhibitor of transcription, reduced Hes1 expression by ~50% (fig. S4B). We next performed a GSI- given that many genes, such as Trib1, which is not a Notch1 target, washoutexperiment(33). As expected, GSI treatment down-regulated showed no change in H3K27Ac (fig. S3F). Together, these data suggest the Notch targets Hes1 and Myc, which were up-regulated after GSI that inhibiting MafB decreases Notch-mediated transcription at a sig- washout (Fig. 4, D and E, black bars). However, the effects of both nificant number of direct Notch target genes (binomial P value = 0.06). the GSI treatment and the washout were blunted in T6E cells expressing To test the fitness of T-ALL cells treated with MafB-shRNAs, we per- HA-MAFB (Figs. 4, D and E, gray bars). We next assayed cell growth formed an in vitro competition assay comparing the shMafB-transduced under GSI treatment. As anticipated, MigR1 cells treated with DMSO T6E cells to cells receiving the shScrmb control (Fig. 3F). Unlike cells continued to proliferate, whereas those treated with GSI experienced transduced with scrambled control vectors, the shMafB-transduced cells growth arrest as early as 48 hours aftertreatment.Ontheotherhand, were outcompeted by the shScrmb cells (Fig. 3G). These findings suggest the MAFB-overexpressing cells continued to expand (Fig. 4F). These that inhibiting MafB represses T-ALL cell growth by inhibiting the ex- findings support a role for MAFB to enhance Notch1 function and sug- Downloaded from pression of crucial Notch1 targets, such as Myc, Dtx1,andHes1. gest that T-ALL tumors expressing high amounts of MAFB may be more resistant to GSI-based therapies. Increased MAFB expression promotes growth and subverts the effects of GSI in T-ALL cells MAFB enhances Notch signaling by interacting with ETS To better understand the effect of increased MAFB expression in T-ALL MAFB and ETS2 were initially detected by our luciferase Notch reporter cells, we retrovirally expressed human-MAFB in T6E cells. Because of screen; thus, we tested whether MAFB directly binds to the Rbpj/Notch1 http://stke.sciencemag.org/ the poor quality of commercial anti-MafB antibodies, we appended 5′ sites in the TP1 reporter. For this, we used oligo-IP (OIP) assays, where HA or FLAG molecular tags to the MAFB coding sequence. We a biotinylated oligonucleotide containing the consensus Rbpj/Notch1 selected cell lines with different amounts of MAFB overexpression; (CSL) binding sequence (CGTGGGAA) found in the TP1 reporter HA-MAFB was slightly higher, whereas FLAG-MAFB was much high- was added to the lysate of U2OS cells transfected with vector control,

A C 100 on December 5, 2017

50 ICN MAFB-only T-ALL free (%) LPΔP-only LPΔP+MAFB *** 0 0 100 200 300 B ICN MafB LPΔP LPΔP+MAFB D Days posttransplant 200

160

120 GFPNGFR *** 80

40 59.0

CD4CD8 0 ICN MafB LPΔPLPΔP+MAFB Fig. 2. MAFB enhances T-ALL onset of a weak Notch gain-of-function allele. (A) Schematic representation of experimental design for retroviral transduction of 5-fluorouracil (5-FU)–treated BM progenitors. When only a single cDNA was expressed (MAFB-only, ICN-only, or LPDP-only), an equivalent dose of the retroviral vector (either MigR1 or MigR1- NGFR) was used to normalize the total retroviral titers. (B) Peripheral blood analysis of the four cohorts of transplanted mice (ICN, n =8;MAFBn =9;LPDP, n = 14; and LPDP+MAFB, n = 15) 4 weeks after BMT. Representative populations shown were gated for live, singlet, and Lin− cells. NGFR is the surrogate marker for MAFB transduction, and GFP is the surrogate marker for Notch-mutant transduction. (C) Kaplan-Meier plot indicating percent T-ALL–free mice (that is, insert space between free and mice) after BMT. Additional animals that succumbed to non–T-ALL conditions, such as irradiation poisoning (MAFB-only, n =1;LPDP-only, n = 1) and BM failure or anemia (LPDP-only, n = 1), were excluded from the analysis. (D) WBC counts in the peripheral blood of the mice in each of the experimental cohorts. A diagnostic threshold for tumor onset of 40 million/ml of WBCs and circulating double-positive (DP) T cells in the peripheral blood was used as a benchmark for T-ALL, as previously described (8, 25). ***P ≤ 0.0005 by Mantel-Cox test (C) or unpaired t test (D).

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 4of12 SCIENCE SIGNALING | RESEARCH ARTICLE

MAFB, ETS2,orMAFB and ETS2 (Fig. 5A). Of particular relevance, we Although Ets1 deficiency had minimal effects on T-ALL onset in cells identified the core DNA sequence for ETS factor binding (C/A)GGAA transduced with ICN1, the loss of a single Ets factor significantly decreased (G/A) (34) interspaced between the Rbpj binding sequences of the TP1 re- T-ALL penetrance in recipient mice, with median survival increasing from porter (underlined in Fig. 5A). The OIP assay revealed ETS2 binding to the 72 days in wild-type (WT) BM donors transduced with LPDP+MAFB to − − TP1 oligo; however, MAFB bound only in the presence of exogenous ETS2, 160 days in ETS1 / BM donors transduced with LPDP+MAFB (fig. S5B). which is only marginally expressed in U2OS cells (Fig. 5B). This same de- These results suggest that the amount of Ets may influence disease latency, pendency of MAFB on ETS factors was confirmed for ETS1 (fig. S5A). even in the presence of MafB. These data also suggest that lack of Ets factors will limit MafB- dependent enhancement of Notch signaling and delay T-ALL onset MAFB interacts with ETS2 through its bZIP domain and in vivo. We tested this using the BMT model described in Fig. 2; however, functions to recruit transcriptional coactivators − − for these studies, we transplanted the BM from ETS1 / donor hemato- To determine whether MAFB directly binds ETS factors in T-ALL cells, poietic progenitors (35) that were transduced with ICN1 or LPDP+MAFB. we transduced T6E cells with epitope-tagged MAFB and performed IPs

siScrmb shScrmb A B C 1.0 1.5 * ** MafB-siRNA#1 MafB-shRNA#3 0.8 *** 1.0 *** 0.6

0.4 0.5 Downloaded from MafB expression % of P300 input 0.2 Relative expression

0.0 0.0 A

Hes1 iRNA#2 NDME untreated s GAPDH Scrmb-siRN Myc MafB-siRNA#1MafB- D E 2.2 chr5: 120,675,000 20 kb mm10 shScrmb Notch-dependent transcripts

2.2 http://stke.sciencemag.org/ MafB-dependent H3K27Ac MafB-independent H3K27Ac shMafB#3

37 (62.7%) Dtx1

2.2 chr15: 62,100,000 500 kb mm10 22 shScrmb 2.2 shMafB#3

F Myc Pvt1 Myc NDME Harvest RNA T6E for expression

FACS on December 5, 2017 Transduce with Recover for 24 hours analysis shRNA in culture Seed in equal numbers for competition

G shScrmb-GFP shScrmb-GFP shScrmb-GFP shScrmb-NGFR shMafB#1-NGFR shMafB#3-NGFR 80 80 80

60 60 60 cells 40 40 40

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0 0 0 Day 0 Day 3 Day 6 Day 9 Day 12 Day 0 Day 3 Day 6 Day 9 Day 12 Day 0Day 3Day 6Day 9Day 12 Fig. 3. MafB-deficient T-ALL cells down-regulate Notch targets and perform poorly in competitive culture conditions. (A) MafB expression measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in T6E cells 48 hours after treatment with indicated siRNAs relative to that in cells cultured with transfection reagent (untreated). *P ≤ 0.05, **P ≤ 0.005 by unpaired t test. (B) Relative expression analysis by qRT-PCR of Notch signaling and Myc signaling (CAD) targets and expression controls (GAPDH) in MafB-siRNA#1–treated cells relative to each in Scrmb-siRNA–treated cells. (C) ChIP assay measuring the occupancy of P300 at the Hes1 promoter and at the NDME in MafB-shRNA#3–treated T6E cells. Genomic DNA sequence of GAPDH is used as the nonspecific control. ***P <0.0005byunpairedt test. (D) Pie chart representation of Notch- dependent stratified on the basis of differential H3K27Ac abundance measured by ChIP-seq analysis of T6E cells transfected with shScrmb or shMafB#3. Of the 59 Notch-dependent transcripts, as determined by a GSI-washout experiment, 37 have nearby MafB-dependent H3K27Ac regions, which display a fold change of ≤−1.5 (dark gray portion of the pie chart). (E) Genome tracts of individual examples of MafB-dependent H3K27Ac for Notch target genes (Dtx1, top; Myc, bottom) in T6E cells transfected with shScrmb or shMafB#3. kb, kilobase. (F) Schematic representation of the experimental design for MafB shRNA–treated T-ALL cells. The cells were sorted for their respective markers and subsequently cultured in equal numbers directly afterward. Every 3 days, the percentage of the cellular population bearing each marker was determined in an aliquot of the competition culture. FACS, fluorescence-activated cell sorting. (G) Left: Competition assay of T6E cells treated with GFP+ or NGFR+ shScrmb plated in equal numbers (2.5 × 105) at day 0 (no significant difference; n = 3). Middle and right: Competition assay of T6E cells treated with GFP+ shScrmb or one of two NGFR+ MafB shRNAs plated in equal numbers (2.5 × 105)atday0(n =3).P = 0.02 and 0.03, respectively.

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 5of12 SCIENCE SIGNALING | RESEARCH ARTICLE to identify binding partners (Fig. 5C). MAFB strongly bound both ETS1 also markedly decreased p300 binding; however, the bZIP domain was and ETS2, which are highly homologous (16). Previous studies of the not required for the PCAF interaction (Fig. 5E). Previous ChIP-seq MAFB homolog, MAFA, showed that it forms complexes with impor- analysis showed that ETS binding sites are enriched within 250 base tant transcriptional coactivators with histone acetyltransferase activity, pairs (bp) of Notch1/RBPJ binding sites, thus placing ETS factors including the P300/CBP-associated factor (PCAF) complex (36)and in the immediate vicinity of the NTC (40). We tested whether MAFB p300 (37), both of which are recruited to NTCs where they activate can interact with components of the NTC, specifically the cleaved transcription (38, 39). To test whether MAFB interacts with PCAF ICN1, as detected by the Val1744 antibody and RBPj. We found that and P300, we performed IPs in T6E cells expressing FLAG-tagged MAFB coimmunoprecipitated in T6E cells with cleaved Notch1 (Fig. MafB. Our results indicate that MafB interacts with both PCAF and 5F) and RBPj (Fig. 5G) and that this interaction with the NTC was lost P300 in T-ALL cells (Fig. 5D), suggesting a mechanism through which when the MAFB bZIP domain was deleted (Fig. 5G). These findings it enhances Notch signaling. further highlight the ability of MAFB-ETS complexes to cooperate with We next set out to determine how MAFB interacts with ETS2. We the NTC to activate transcription. used site-directed mutagenesis to remove the bZIP domain from MAFB To determine the effect of the MAFB DbZIP mutant on Notch tran- (Fig. 5C) (9, 11, 23). Using T6E cell lysates, we first verified the deletion scriptional activity, we tested its effect in T6E cells using the TP1 reporter. and the size of the mutant (Fig. 5C, right). We then performed IP The MAFB DbZIP mutant decreased reporter activity by more than 60% experiments (Fig. 5E), which showed that deleting the MAFB bZIP do- (Fig. 5H). When ETS2 was cotransfected in the assay with the MAFB main markedly diminished the MAFB-ETS2 interaction. MAFB DbZIP DbZIP mutant, the overall Notch activity was increased, but the loss of the bZIP domain did not significantly en- Downloaded from ABHes1 C Myc hance Notch signaling more than when

a 100 **** 50 * ETS2 or MAFB were added alone, thus sug-

EF1 *** AFB 40 * 80 gesting that MAFB-mediated enhancement to of Notch transcription occurs through its MigR1 FLAG-MHA-MAFB 30 60

MafB relative bZIP domain. We next assayed the MAFB 20 40 GAPDH deletion mutant in vivo using the BMT http://stke.sciencemag.org/ 10 20 model. As before, MAFB greatly decreased s. quantity 0 b 0 the latency and increased the penetrance of A Abs. quantity relative to EF1a to relative quantity Abs. T-ALL development. In contrast, the MAFB MigR1 -MAFB MigR1 HA-MAFB D HA-MAFB LAG-MAFB bZIP mutant markedly limited the ability FLAG F D E of MAFB to enhance LPDP leukemo- 30.0 Hes1 125 Myc a genicity (Fig. 5I). Together, these data * NS F1 F1a

E show that the bZIP domain of MAFB is

100

o *** * 20.0 NS required for MAFB to enhance LPDPac- *** 75 ** ** tivity both in vitro and in vivo. on December 5, 2017 relative to E relative to

ty 50 ti tity relative t tity

n 10.0

a MAFB enhances Notch signaling in

qu 25 human T-ALL cells Abs. quan Abs. 0 0 To determine whether MafB served a simi- h I I h O SI s S s a G a W lar role in human T-ALL, we determined -DMSO - MigR1-GSI MigR1-GS the levels of MAFB in three T-ALL cell lines MigR1-Wash MigR1-Wash MigR1-DMSO -MAFB-DMSHA-MAFB-G MigR1-DMSO -MAFB HA-MAFB- -MAFB A A HA HA-MAFB-W H H (Fig. 6A). Of these, KOPT-K1 had the high- F 4 est levels of MAFB expression. KOPT-K1 cells were treated with shRNAs designed )

6 MAFB 3 MigR1-DMSO against human , which decreased HA-MAFB-DMSO MAFB expression by more than 50% (Fig. MigR1-GSI MAFB 2 HA-MAFB-GSI *** 6B). The effect of suppressing in human KOPT-K1 cells was similar to mu- 1 rine T6E cells as the expression of the di- Cell number ( × 10 rect Notch target genes HES1, DTX1,and MYC 0 was suppressed (Fig. 6C). Suppression 0 4 y 3 of MAFB did not affect the expression of Day Day 1 Day 2 Da Day ETS factors or ZMIZ1 nor did it alter the Fig. 4. Ectopic MAFB expression in T-ALL cells enhances Notch target gene expression and limits the effect of GSI occupancy of ETS2 at the 5′ HES1 promoter treatment. (A) Western blotting for MafB (with anti-MafB antibody; Abcam) and loading control (GAPDH) in whole-cell in T-ALL cells (fig. S6, A to D). Likewise, lysates (10 mg) from T6E cells expressing Flag-MAFB or HA-MAFB. MigR1, empty vector. (B and C) Hes1 (B) and Myc (C) ex- KOPT-K1 cells treated with anti–MAFB- pression measured by qRT-PCR in T6E cells overexpressing MAFB. EF1a, elongation factor 1a.(D and E) Hes1 (D) and Myc (E) expression measured by qRT-PCR in T6E cells treated with dimethyl sulfoxide (DMSO) or GSI (0.1 mM) for 24 hours HU-shRNAs were at a disadvantage in and subsequently washed out for 6 hours. Black bars, control cells; gray bars, MAFB-overexpressing cells. (A to E) n =3to cell growth assays when compared to cells 6experiments.(F) Proliferation of MigR1-transfected control or MAFB-overexpressing T6E cells (2.5 × 105)platedina treated with shScrmb (Fig. 6D). medium containing DMSO or GSI (10 nM). n = 2 experiments with three technical replicates per experiment. *P ≤ 0.05, We next tested whether ETS factors **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00001 by unpaired t test. also played a similar role in human T-ALL

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 6of12 SCIENCE SIGNALING | RESEARCH ARTICLE

cells. First, we confirmed that three human cell lines expressed ETS1 and ETS2 (Fig. 6E). Next, we tested whether MAFB binds A EBS and CSL sites in TP1 reporter B 5' Biotin - AATGGGCGGAAGGGCACCGTGGGAAAATAGTAGATCCCGA ETS2 in human T-ALL cells. We transduced 33’’ TCGGGATCTACTATTTTCCCACGGTCGGGATCTACTATTTTCCCACGG TGCCCTTCCGCCCATT-TGCCCCTTCCGCCCATT-BiBiotinotin 2 Transfected TS2+FLAG-MAFB KOPT-K1 cells with FLAG-tagged MAFB WB: EV ETS FLAG-MAFBE component and performed IPs to detect interactions ETS2

OIP and found that MAFB strongly bound both

ETS2 and RBPJ in KOPT-K1 cells (Fig. 6F).

FLAG

U2OS cell lysate Biotin-tagged oligo Streptavidin beads

This suggests that a similar MAFB-ETS2

C s 01 pathway enhances Notch signaling in human

ETS2 %

l

o T-ALL. FLAG or HA-tagged MAFB HA-IP of T6E Lysate NCTAD FLAG da

Stp bZIP

Tag i

HA-tagged MAFB-ΔbZIP HA-MAFB gn NCTAD HA-ΔbZIP GAPDH Stp Tag DISCUSSION bZIP The finding that human and murine T-ALLs D E F G ΔbZIP ΔbZIP B B AFB with activating NOTCH1 mutations fre- 1 -MAF -MAFB LAG-M AG quently require persistent Notch signaling MigR1 HA-MAFB MigR1 FLAG-MAFB MigR1 HA HA-MAF MigR MigR1 F L WB: HA-MAFB F ETS2 PCAF ETS2 for growth and survival led to the idea that Notch1 RBPJ Notch is a druggable target in this disease. Al- Downloaded from

IP 10% loading ETS1 P300 PCAF IP IP IP HA though multiple studies support this idea, FLAG 10% loading

HA FLAG 10% loading P300 one paradox is that the common NOTCH1 Notch1 RBPJ PCAF mutations in T-ALL patients are weak onco- ETS2 ETS2 10% loading genic drivers in murine T-ALL models. This ETS1 P300 PCAF GAPDH FLAG raises the question of whether there are addi- http://stke.sciencemag.org/ P300 GAPDH GAPDH GAPDH tional molecules that potentiate Notch

MAFB signaling, which could identify additional HA bZIP therapeutic targets. GAPDH Here, we set out to identify proteins that enhance the activity of the common H I LP P-only LP P+MAFB Notch1 mutations in T-ALL patients. *** LP P+MAFB- bZIP Our screen identified multiple genes,

12 100 some of which were previously verified 19 20

to be important in Notch signaling ( , ). on December 5, 2017 10 We chose to focus on MAFB and ETS2. 8 ** These transcription factors have well- 6 50 NS established roles in myeloid and lymphoid ** 4 ** development (15, 41), respectively; how-

2 T-ALL free (%) ever, their involvement in T-ALL is poorly understood. In reporter assays, MAFB 0 0 V 2 2 E FB 0 50 100 150 200 and ETS2 demonstrated strong synergy bZIP ETS MAML Δ MA Days posttransplant and enhanced Notch activity to a level bZIP+ETS2AFB- MAFB+ETSΔ M on par with MAML1, a core component

AFB- M of the NTC. In our functional studies, Fig. 5. MAFB interacts with ETS proteins and recruits cofactors to Notch/RBPj binding sites. (A) Schematic repre- MAFB seems particularly important be- sentation of the experimental design for the OIP assay in U2OS cells. A biotin-tagged oligo from the TP1 Notch reporter was cause expressing MAFB along with the used as the bait to immunoprecipitate proteins interacting at the CSL or EBS regions. (B) Representative results of OIP assay weak oncogenic NOTCH1 mutant LPDP in U2OS cells transfected with MigR1, ETS2, Flag-MAFB, or ETS2 + Flag-MAFB and probed with ETS2 and Flag antibodies in BM progenitors accelerated T-ALL afterOIP.Tenpercentloadingofnuclearlysatesindicates protein abundance; GAPDH is the loading control. (C)Schematic onset and increased T-ALL penetrance. representation of the tagged MafB and MafB DbZIP mutant. Right: Western blotting (WB) on mixed lysates of HA-MAFB and Inhibiting MAFB, by either siRNA or HA–MAFB DbZIP immunoprecipitated from doubly transduced T6E cells to show relative protein size and HA-pulldown shRNA, down-regulated the expression efficiency. (D) Representative results of HA- or FLAG-IP assessing MAFB interactions with ETS1, ETS2, P300, and PCAF in T6E of multiple direct Notch targets, including – T-ALL cells. (E) Representative HA-IP in T6E cells transduced with MigR1, HA-MAFB, or HA MAFB DbZIP and probed for HA, MYC, DTX1,andHES1, in both murine ETS2, PCAF, and P300. The background bands in the HA 10% loading control likely result from the blot being probed and and human T-ALL cells and also decreased restriped multiple times. (F) Representative HA-IP in T6E cells transduced with HA-MafB and probed for cleaved-Notch1 (Val1744). (G) Representative FLAG-IP in T6E cells transduced with MigR1, FLAG–MAFB DbZIP, or FLAG-MAFB and probed for the proliferation of murine and human RBPJ and FLAG. (H) Notch TP1 reporter assay in T6E cells transfected with LPDP and the indicated plasmids. EV is the vector T-ALL cells. In contrast, overexpressing control. **P ≤ 0.005 by unpaired t test; n =3.(I) Kaplan-Meier plot comparing percent of T-ALL–free survival after BMT MAFB enhanced the expression of these with progenitors transduced with LPDP-only, LPDP+MAFB, or LPDP+MAFBDbZIP domain deletion mutant. LPDP, n =6; Notch targets and promoted T-ALL LPDP+MAFB, n =12;LPDP+MAFB DbZIP, n = 10. Animals that succumbed to non–T-ALL conditions, such as BM failure/ growth. Thus, we propose that the com- anemia (LPDP-only, n = 1), were excluded from the analysis. ***P ≤ 0.0001 by Mantel-Cox test. bination of MAFB and ETS2 has the

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 7of12 SCIENCE SIGNALING | RESEARCH ARTICLE potential to amplify the activity of weak activating Notch1 muta- MAFB is a multidomain that can bind DNA di- tions. Although multiple Notch1 targets are inhibited by blocking rectly and form protein-protein interactions (9, 44). In our T-ALL MAFB, additional analyses are needed to determine whether MAFB in- studies, MAFB did not appear to bind DNA independently; instead, fluences all direct Notch1 target genes. It is unlikely that withdrawal of it appears to either bind cooperatively with ETS transcription factors MafB broadly represses transcription because the expression of many or be tethered to DNA through the ETS transcription factors. ETS genes, such as Trib1, was not affected by MafB knockdown. binding sites are common and in close proximity with RBPJ binding How MAFB boosts the signaling of mutant Notch receptors was not sites (40). Because MAFB is known to directly interact with ETS1 (23), immediately obvious. A previous work characterized MafB as a differ- we believe that MAFB is recruited near sites of NTC formation through entiation factor, with roles in monocyte-to-macrophage differentiation a similar interaction with ETS2 or ETS1. We found that MafB interacts (42). In T cells, MAFB expression is low (43); however, data from pub- with both P300 and PCAF, which are important for Notch-induced lished databases and our own analysis of cell lines, patient primagrafts, transcriptional activation (38), and that the loss of MafB decreases the and primary tumors show that MAFB is frequently expressed in T-ALL occupancy of p300 at the Hes1 and Myc Notch-dependent enhancers. cells and primary patient samples. There was no correlation with Notch1 The bZIP domain of MafB is required for this activity, thus bringing mutation subtypes, and there was no evidence of MAFB or ETS1/2 ge- PCAF and P300 and by connection, the Notch/RBPj/MAML complex netic amplification in primary T-ALLs (21, 22). Thus, we hypothesize into a larger protein complex capable of high transcriptional output. that in these tumors, MAFB expression, driven by unknown mechan- This MAFB-ETS2 synergy and its ability to enhance Notch signaling isms, potentiates both Notch signaling and its oncogenic capacity. Our are also conserved in human T-ALL cells. A recent work showed that findings also suggest that in other tumors with weakly activating Notch1 the T cell transcription factor Zmiz1 interacts with Notch1 and regu- mutations, such as chronic lymphocytic leukemia, additional cofactors lates the expression of oncogenic Notch targets such as Myc (45), indi- Downloaded from analogous to MAFB may be required to potentiate oncogenic Notch1 cating that enhancing Notch signaling by cofactors is an important step signals. in T-ALL onset. In our studies, suppressing MafB did not affect Zmiz1 expression. Circulating double-positive A B T cells, which express high levels of ETS 2.0 2.5 ** ** factors, are observed in the BMT model – 8 http://stke.sciencemag.org/ 2.0 of NOTCH1 LPDP induced T-ALL ( ); 1.5

1.5 however, these mice develop T-ALL at a

1.0 1.0 low frequency. From the results of our

0.5 0.5 study, we predict that when MAFB is 0.0 expressed at sufficient levels, it creates an 0.0 MafB expression relative to EF1a MafB expression relative to EF1a h1 h2 environment where transcriptional cofac- s U2OS DND41 CUTLL1 KOPT-K1 -HU-s HU- shScrmb B- tors are recruited at a higher-density near MAFB MAF sites of Notch transcriptional targets, thus C shScrmb D

shScrmb raising Notch1 signaling output to levels on December 5, 2017 MAFB-HU-sh2 1.5 1.0 MAFB-HU-sh1 *** that exceed the threshold for oncogenic

) MAFB-HU-sh2 6 transformation. 1.0 × 10 In summary, our findings show that 0.5 the MAFB-EST2 interaction enhances 0.5 Notch1 signaling in a leukemic setting Cell number( by supporting the higher expression of

Relative expression to Sh-Scrmb 0.0 1 0.0 Notch targets. These findings suggest that s y1 Myc He He Day 0Day 1Day 2Day 3 Deltex the MAFB-ETS2 axis may serve as a ther- apeutic target in T-ALL. Our findings also E F -MAFB G gR1 A Mi FL raise issues regarding the efficacy of GSI LL T KOPT-K1 CU DND41 FLAG-MAFB therapy in tumors with increased MAFB ETS1 abundance, because our data show that IP ETS2 cells expressing high amounts of MAFB ETS2 RBPJ were less sensitive to GSI treatment. Thus, GAPDH strategies that decrease MAFB expression ETS2 or formation of the MAFB-ETS2 complex RBPJ may enhance the sensitivity of Notch- 10% loading GAPDH dependent T-ALL cells to GSI therapy.

Fig. 6. Loss of MAFB suppresses Notch target gene expression and cell proliferation in human T-ALL. (A) MAFB mRNA expression in human T-ALL cell lines (U2OS cells used as negative control). (B) MAFB mRNA expression after MAFB MATERIALS AND METHODS knockdown in KOPT-K1 cells by each of the two shRNAs. (C) Notch target gene expression in sorted KOPT-K1 cells 72 hours after transduction with shRNA against MAFB relative to KOPT-K1 cells treated with Scrmb-shRNA. (D) Proliferation of sorted Cell culture KOPT-K1 cells after transduction with shRNAs against MAFB. Growth is compared to cells treated with Scrmb-shRNA (n =3). T-ALL cell lines were cultured in RPMI (E) Representative Western blotting for ETS1 and ETS2 in human T-ALL cell lines. GAPDH is the loading control. (F)Repre- 1640 (Invitrogen) supplemented with sentative FLAG-IP in KOPT-K1 cells transduced with MigR1 or FLAG-MAFB and probed for FLAG, ETS2, and RBPJ. Ten per- 10% fetal bovine serum (FBS) (HyClone), cent loading of lysates indicates RBPJ and ETS2 abundance. GAPDH is the loading control. **P ≤ 0.005, ***P ≤ 0.001. 2mML-glutamine, 1% nonessential amino

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 8of12 SCIENCE SIGNALING | RESEARCH ARTICLE acids (Gibco), 1% sodium pyruvate (Gibco), and 2-mercaptoethanol Human T-ALL data analysis and statistical analysis [0.0005% (v/v); Sigma), with antibiotics. U2OS cells were maintained Normalized microarray expression analysis for the Haferlach data set in Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% FBS was obtained from the Oncomine database, and Zuurbier data set (Gibco) and antibiotics. Cells were grown at 37°C in 5% CO2.Retroviral was provided by J. P. P. Meijerink. Each set was separately analyzed transduction, sorting, and expression analysis of T-ALL cells were per- for the expression of MafB, Notch1 receptor, and Notch1 target genes: formed as described previously (3). The GSI, compound E (Calbiochem, Hes1, Deltex,andMyc. Each sample that displayed higher than twofold 565790), was titrated on T6E cells for 12 hours at concentrations ranging Notch receptor and Notch target expression was parsed from the total from 1 mM to 1 nM. GSI-washout experiments were performed as de- data set. MafB expression was cross-referenced for any individual sam- scribed previously (32) after 24 hours of culture in 0.1 mMGSI. ple that showed a Notch enhancement signature. The comparative Notch1 Mafb values of , Notch targets, and were plotted as a log2 median Constructs and retroviruses value for all the samples that show Notch enhancement in the Haferlach MigR1 (46), MigR1-NGFR, MigR1-Notch1-L1601P, and MigR1-Notch1 and Zuurbier data sets. Statistical analysis was performed using Prism 6 LPDP are described previously (8). Sport6-MafB (780 bp; MafB coding (GraphPad). Survival curves were computed using Kaplan-Meier analy- sequence) and Sport6-ETS2 (822 bp; ETS2 coding sequence) were obtained sis, and comparison of survival curves was performed using Mantel-Cox from the library screen. The 5′ HA or FLAG tags added to MafB were gen- test provided through Prism. Predicted experimental mouse numbers for erated by PCR. MafB deletions were generated using QuikChange II the Notch1-LPDP and MafB experiment were powered (0.8). z score for (Agilent Technologies), and primer design was based on the QuikChange Notch1 gain-of-function screen was determined by the following Primer Design program (www.genomics.agilent.com). formula: z score = 1 − [(3SD− +3SD+)/(Av+ − Av−)]. An unpaired t test P value of less than 0.05 was considered to be significant in all Downloaded from Luciferase screen and reporter assays experiments, unless noted otherwise; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ The cDNA screening strategy involved the use of three key components: 0.0005, ****P ≤ 0.00001. (i) a pcDNA3 plasmid encoding a modestly strong NOTCH1 gain-of- function mutant, LPDP, driven from a CMV promoter (40 ng of cDNA Quantitative PCR per well), (ii) a Notch firefly luciferase reporter (TP1) containing 12 CSL RNA was extracted using Qiagen RNeasy Mini and Micro Kits. binding sites (50 ng of cDNA per well), and (iii) a preplated cDNA cDNA was synthesized from RNA with the SuperScript III kit (In- http://stke.sciencemag.org/ library cloned into the Sport6 plasmid (40 ng of cDNA per well). A vitrogen). Transcripts were amplified with SYBR Green PCR reagent MAML1 cDNA was the positive control for each screen plate, whereas (Applied Biosystems), and quantitative PCR (qPCR) was performed empty vector and a DTX1 cDNA were background and negative on the ABI Prism 7900HT system (Applied Biosystems). mRNA controls (40 ng of cDNA per well), respectively. DNA spotting was per- quantities, either in absolute or relative quantification, were nor- formed using a Matrix PlateMate (Thermo Fisher Scientific) for the malized to elongation factor 1a and GAPDH, respectively. Primer3 control wells. The Matrix WellMate (Thermo Fisher Scientific) was software was used for primer design, and sequences are provided used to dispense the transfection mix containing the reporter and in table S3.

Notch1 mutant plasmids in combination with transfection reagent on December 5, 2017 (FuGENE6, Promega), which were added to the wells (4000 U20S siRNA and shRNA cells per well) after a 30-min incubation. Luminescence was measured RNA interference was carried out with siRNA duplexes designed 48 hours after plating using Britelite plus (PerkinElmer) luciferase re- and then screened for specific and effective knockdown of target agent with LJL BioSystems Analyst HT96-384. Methods for screen val- genes. Fluorescence-conjugated duplexes (n =3)targetingMafB idation are provided in the Supplementary Materials. were ordered directly as ON-TARGETplus siRNA from Thermo Fisher Scientific/Dharmacon. siRNA methods are provided in the Luciferase screen validation Supplementary Materials. For shRNA suppression, two different For the validation of screen candidates and independent luciferase as- MafB-targeting shRNAs and a shScrmb were purchased from Open says, 4000 U2OS cells per well were seeded on Corning opaque 384-well Biosystems (sequences are provided in table S4). For siRNA control plates, and FuGENE6 transfection mix was prepared in Opti-MEM transfections, nontargeting siRNA and siGLO Green were pur- (Gibco) serum-free medium with three plasmid components: (i) 50 ng chased from Thermo Fisher Scientific/Dharmacon. For transfec- per well of Notch TP1 firefly reporter plasmid, (ii) 40 ng per well of tions of cells, siRNA duplexes were resuspended in siRNA buffer pcDNA3-LPDP plasmid, and (iii) 40 ng per well of screen-component (Dharmacon) and added to the cell growth medium for 12 hours cDNAsclonedintopCMVSport6(forexample,MafBorETS2). with siIMPORTER transfection reagent (Millipore) as per the manu- pcDNA3-MAML1 and Flag-CMV-DTX1 plasmids (40 ng per well) facturer’s instructions. Forty-eight to 72 hours after transfection, RNA served as positive and negative controls, respectively. Five nanograms was harvested from cells with RNeasy Mini kit (Qiagen), and 500 ng of pRL-TK Renilla luciferase was used as internal control plasmid. After of total RNA was used in qPCR analysis. For shRNA suppression, 20 min at room temperature, 20 ml of the transfection reaction mix was two different MafB-targeting shRNAs and a shScrmb were purchased added to the cells by a multichannel pipette. Twenty-four wells were ana- from Open Biosystems (sequences are provided in table S3 and the lyzed for each individual transfection sample set. After a 48-hour incuba- Supplementary Materials) and were subcloned into the pLMP-GFP tion with the transfection mix, 35 ml per well of Britelite plus luciferase or pLPM-NGFR vector. For viral transduction, cells were centrifuged reagent was added by a multichannel pipette; luminescence was with viral supernatant and hexadimethrine bromide (8 mg/ml) (Sigma) measured by LJL BioSystems Analyst HT96-384 (LJL BioSystems Inc.). at 2500 rpm for 90 min at 25°C. FACS-purified shRNA-treated cells Stop & Glo buffer and Renilla luciferase reagent (Promega) were used to were analyzed for growth or gene expression at the indicated times assess transfection efficiency. At least three individual repeats were per- aftertransduction.Forgrowthcurvesorcompetitionassays,5×105 formed for each experiment. sorted cells were seeded.

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 9of12 SCIENCE SIGNALING | RESEARCH ARTICLE

ChIP-seq analysis Western blotting and IP analyses ChIP-seq reads were aligned to mm10 using Burrows-Wheeler Aligner Whole-cell lysates were prepared with radioimmunoprecipitation assay (47) and filtered to remove PCR duplicates and multimapped reads. All (RIPA) buffer or FLAG-IP lysis buffer (50 mM tris, 150 mM NaCl, reads were postfiltered by known ENCODE blacklist regions. Peak call- 1 mM EDTA, 0.5% NP-40, and 10% glycerol), with protease inhibitor ing was performed using MAC2 (version 2.0.9) (48) with the following tablets (cOmplete, Roche). Protein concentration was determined with – – 1 parameters: no model, shift size = ( /2 estimated fragment length), the Bio-Rad protein assay dye reagent (Bio-Rad). Proteins were separated − –keep-dup = 1, and a false discovery rate (FDR) threshold of 1 × 10 6. using SDS–polyacrylamide gel electrophoresis and wet-transferred to ChIP-seq display files were generated using SAMtools, BEDTools, polyvinylidene difluoride membranes. Blots were visualized with Super- and UCSC utilities. Scaling for all ChIP-seq tracks in figures is equal Signal West Pico Chemiluminescent or SuperSignal West Femto Chemi- to local fragment coverage × (1,000,000/total count). luminescent substrate (Thermo Fisher Scientific). Antibodies used for The peaks were associated to their most proximal gene as defined in Western blotting were cleaved Notch1 (Val1744) antibody (Cell Signaling Ensembl GRCm38 transcript model using BEDTools and HOMER- Technology, no. 2421), RBPJSUH (Cell Signaling Technology, D10), annotatePeaks (version 4.8) (49). H3K27Ac signal was compared be- MafB (Santa Cruz Biotechnology, P20), rabbit monoclonal antibody tween the GSI and GSI-washout conditions on the 1500 (bp) (Abcam, ab66506), GAPDH (Santa Cruz Biotechnology, FL-335), ETS1 regions flanking the peak summits. Regions (1500 kb) flanking the (Santa Cruz Biotechnology, C-20), ETS2 (Sigma, E3783), PCAF (Santa summits of peaks were merged across GSI and GSI-washout con- Cruz Biotechnology, E-8), P300 (Thermo Fisher Scientific, RW109), ditions using BEDTools-merge (version 2.25.0). In each library, peak- HA (Covance, MMS-101P), FLAG (Sigma, M2), and secondary anti- filtered H3K27Ac signal was quantified and normalized to fragment per mouse horseradish peroxidase (HRP) (Pierce) or anti-rabbit HRP (Pierce). kilobase per million reads (FPKM). The logarithmic fold change of IP experiments were performed using HA-probe agarose beads Downloaded from H3K27Ac load on with nonzero read counts in at least one condition (Santa Cruz Biotechnology) or anti-FLAG agarose beads (Sigma, wascalculatedaslog2FPKM of the GSI versus GSI-washout condition M1). Samples were loaded, and Western blot analysis was performed with a pseudocount of 1, and the regions with absolute fold change as described above. OIP experiments were performed as described pre- 51 greaterthanorequaltolog2(0.5) were indicated in table S2 and viously ( ). Oligonucleotide sequences for the OIP experiments are MafB-dependent. provided in table S3. For each pulldown, 0.5 to 1 mg of fresh protein lysate was used and combined with 25 to 30 ml of beads for incubation http://stke.sciencemag.org/ Mice and BM transduction overnight at 4°C with rotation. Beads were washed four times in lysis BM transductions and transplantation into lethally irradiated recipients buffer with increasing concentration of NaCl [200 nM (2×) and 300 nM were performed as described previously (26, 50). Recipient mice used in (2×)]. The IP experiments depicted in Figs. 5 and 6 were conducted in these experiments were 6- to 8-week-oldC57BL/6femalemiceobtained similar fashion; however, the cells were lysed with FLAG-IP lysis buffer from Charles River Laboratories. In cases where two retroviruses (for and washed only three times, once withlysisbufferandtwicewithlysis example, MigR1-LPDP and MigR1-NGFR-MafB) were used to trans- buffer with higher NaCl concentration (150 nM). Protein was released duce the BM progenitors, the total viral titer and multiplicity of infec- from washed beads by addition of 2× Laemmli buffer and boiled for

tion were adjusted to reflect previously used T-ALL Notch-mutant virus 10 min at 95°C. on December 5, 2017 titers (8). Mice were maintained on antibiotics in the drinking water for 2 weeks after BMT, and peripheral blood was drawn every 2 weeks to Microarray analysis monitor blood counts and evaluate the presence of circulating im- Mouse Gene 2.0 ST Affymetrix array CEL files were imported, normal- mature T cells by flow cytometry. Mice with WBC counts of >4.0 × ized, and summarized using robust multichip average and median- 106/ml and a body condition score of ≤2 were euthanized, and tissues polish algorithms, respectively, using the Bioconductor “oligo” were harvested for flow cytometry and histology (hematoxylin and eo- package (52). Limma (53) was used for differential gene expression sin) analysis. All mice were housed in specific pathogen–free facilities at on triplicate samples of GSI and GSI-washout conditions. Package the University of Pennsylvania. Experiments were performed according mogene20sttranscriptcluster.db in Bioconductor was used for annota- to the guidelines from the National Institutes of Health with approved tion. Genes with greater than twofold change and a Benjamini-Hochberg protocols from the University of Pennsylvania Animal Care and Use FDR of <0.1 were called as differentially expressed. Committee.

Flow cytometry Flow cytometry was performed on BD LSR II, cell sorting was performed SUPPLEMENTARY MATERIALS on BD Aria II, and FlowJo software was used for analysis. The peripheral www.sciencesignaling.org/cgi/content/full/10/505/eaam6846/DC1 blood (bimonthly), spleen, thymus, and BM (terminal analysis) were har- Fig. S1. Validation of Notch1 gain-of-function screen and MAFB expression in murine and vested, and cell suspensions were generated from mice transduced as de- human T-ALL. scribed above. Antibodies used in staining of the tissues include: CD45.2 Fig. S2. Analysis of spleen and peripheral blood after BMT. (104, BD Biosciences), CD25 (PC61, BioLegend), CD44 (IM7, Fig. S3. Validation of MafB siRNA and shRNA reagents. Fig. S4. Ectopic MAFB expression enhances T6E cell growth. eBioscience), CD3 (17A2, eBioscience), CD4 (RM4-5, eBioscience), Fig. S5. MAFB interacts with DNA through ETS factors, and the loss of ETS1 delays onset of CD8 (53-6.7, eBioscience), CD19 (1D3, eBioscience), CD11b (M1/70, T-ALL. eBioscience), F480 (BM8, eBioscience), and Gr-1 (RB6-8C5, eBioscience). Fig. S6. Zmiz1 expression in T6E cells is not affected by the loss of MafB. 4′,6-Diamidino-2-phenylindole (DAPI) was used for Live/Dead determi- Table S1. Notch signaling enhancement of Notch-mutant LPDP by candidate genes from the cDNA library. nation. Transduced T6E cell lines were sorted on the basis of internal GFP TableS2.MafBsuppressionaffectsasubsetofNotchsignalinggenetargetsinT6E fluorescence or surface staining with anti-NGFR–biotinylated antibody cells. generated in-house from the 8737 hybridoma line. Table S3. List of oligo sequences and primer sets for qPCR and OIP.

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 10 of 12 SCIENCE SIGNALING | RESEARCH ARTICLE

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48. Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson, B. E. Bernstein, C. Nusbaum, R. M. Myers, (to W.S.P. and S.C.B.), R01HL134971 (to K.V.P.), and R01AI047833 (to W.S.P.)]. This work was M. Brown, W. Li, X. S. Liu, Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008). also supported by a grant from the KiKa foundation Stichting Kinderen Kankervrij [KIKA- 49. S. Heinz, C. Benner, N. Spann, E. Bertolino, Y. C. Lin, P. Laslo, J. X. Cheng, C. Murre, H. Singh, 2010-082 (to Y.L.)]. Author contributions: K.V.P. designed and performed most of the C. K. Glass, Simple combinations of lineage-determining transcription factors prime experiments, interpreted the results, wrote the manuscript, and assembled the figures. L.X. cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 performed in vivo mouse experiments and BMTs. L.S. and K.P. performed the experiments and (2010). generated the data for Fig. 6. J.P. performed the H3K27Ac experiments for Fig. 3. Y.O. and 50. J. C. Pui, D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, T. Kadesch, W.B. performed the array shown in table S2. C.L. performed the experiments shown in fig. S4 R. R. Hardy, J. C. Aster, W. S. Pear, Notch1 expression in early lymphopoiesis influences (B and C). G.B.W. analyzed the histology and tumor infiltration in leukemic transplants. B versus T lineage determination. Immunity 11, 299–308 (1999). R.M. and N.M. provided the ETS1 knockout BM and helped design the experiments shown in 51. D. M. Gerhardt, K. V. Pajcini, T. D’altri, L. Tu, R. Jain, L. Xu, M. J. Chen, S. Rentschler, fig. S5. Y.L. and J.P.P.M. analyzed the human T-ALL data sets shown in fig. S2. S.C.B. helped O. Shestova, G. B. Wertheim, J. W. Tobias, M. Kluk, A. W. Wood, J. C. Aster, P. A. Gimotty, design the experiments and edited the manuscript. R.B.F. helped design the ChIP-seq and J. A. Epstein, N. Speck, A. Bigas, W. S. Pear, The Notch1 transcriptional activation domain analyzed the results for the experiment in Fig. 3. S.C. helped design the Notch gain-of-function is required for development and reveals a novel role for Notch1 signaling in fetal screen, provided the cDNA library, and assembled and analyzed the data shown in Fig. 1. hematopoietic stem cells. Genes Dev. 28, 576–593 (2014). W.S.P. designed the experiments in this study, interpreted all the data, and helped write 52. B. S. Carvalho, R. A. Irizarry, A framework for oligonucleotide microarray preprocessing. the manuscript. Competing interests: The authors declare that they have no competing Bioinformatics 26, 2363–2367 (2010). interests. Data and materials availability: The H3K27Ac ChIP-seq and Affymetrix data have 53. G. K. Smyth, Linear models and empirical bayes methods for assessing differential been deposited to Gene Expression Omnibus (accession no. GSE104993). expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004). Submitted 6 January 2017 Acknowledgments: We thank K. Toscano and S. Yu for technical assistance; P. Gimmoty for Resubmitted 9 February 2017 statistical consultation; J. Aster, M. Chiang, A. Ferrando, and D. Gerhardt for insightful Accepted 13 October 2017 comments; J. Aster for reagents; J. Soulier and C. Mullighan for the advice on patient T-ALL Published 14 November 2017 Downloaded from data sets; and the following cores at the University of Pennsylvania that contributed to this 10.1126/scisignal.aam6846 study: Mouse husbandry (University Laboratory Animal Resources), the Abramson Cancer Center Flow Cytometry Core (P30-CA016520), and the Abramson Family Cancer Research Citation: K. V. Pajcini, L. Xu, L. Shao, J. Petrovic, K. Palasiewicz, Y. Ohtani, W. Bailis, C. Lee, Institute Cores. This work benefited from data assembled by the Immunological Genome G. B. Wertheim, R. Mani, N. Muthusamy, Y. Li, J. P. P. Meijerink, S. C. Blacklow, R. B. Faryabi, (Immgen) Project Consortium. Funding: This work was supported by a Leukemia and S. Cherry, W. S. Pear, MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic Lymphoma Society Fellow Award and T32HL007843 (K.V.P.) and the NIH [grants P01CA119070 leukemia. Sci. Signal. 10, eaam6846 (2017). http://stke.sciencemag.org/ on December 5, 2017

Pajcini et al., Sci. Signal. 10, eaam6846 (2017) 14 November 2017 12 of 12 MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia Kostandin V. Pajcini, Lanwei Xu, Lijian Shao, Jelena Petrovic, Karol Palasiewicz, Yumi Ohtani, Will Bailis, Curtis Lee, Gerald B. Wertheim, Rajeswaran Mani, Natarajan Muthusamy, Yunlei Li, Jules P. P. Meijerink, Stephen C. Blacklow, Robert B. Faryabi, Sara Cherry and Warren S. Pear

Sci. Signal. 10 (505), eaam6846. DOI: 10.1126/scisignal.aam6846

New targets, better mouse model for leukemia T cell acute lymphoblastic leukemias (T-ALLs) are often caused by mutations in the gene encoding Notch1, which

mediates cell-cell contact signaling in embryonic development and adult tissue maintenance. However, mice expressing Downloaded from these mutants frequently fail to develop T-ALL. Pajcini et al. found that the transcription factors MAFB and ETS2 increased the expression of Notch1 target genes in mouse and human T-ALL cells by recruiting histone acetyltransferases. Expressing MAFB enhanced the development of Notch1-mutant T-ALL in mice. Because Notch1 is critical for the maintenance of various healthy adult tissues, developing a way to inhibit MAFB or its interacting partners may be a more targeted therapy for leukemia patients. http://stke.sciencemag.org/

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