HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development

Home , Common gamma chain, HOXA9, IL22RA2, Janus kinase, Janus kinase 3, Klf4

Published OnlineFirst March 1, 2018; DOI: 10.1158/2159-8290.CD-17-0583

RESEARCH ARTICLE

Charles E. de Bock1 ,2 , Soﬁ e Demeyer 1 ,2 , Sandrine Degryse 1 ,2 , Delphine Verbeke 1 ,2 , Bram Sweron 1 ,2 , Olga Gielen 1 ,2 , Roel Vandepoel 1 ,2 , Carmen Vicente 1 ,2 , Marlies Vanden Bempt 1 ,2 , Antonis Dagklis 1 ,2 , Ellen Geerdens 1 ,2 , Simon Bornschein 1 ,2 , Rik Gijsbers 3 , Jean Soulier 4 , Jules P. Meijerink 5 , Merja Heinäniemi 6 , Susanna Teppo 7 , Maria Bouvy-Liivrand 6 , Olli Lohi 7 , Enrico Radaelli 1 ,2 , and Jan Cools 1 ,2

ABSTRACT Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/ STAT5 signaling and HOXA9 overexpression, two events identifi ed as signifi cantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a signifi cant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC- seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specifi c signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.

SIGNIFICANCE: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616–31. ©2018 AACR.

1 KU Leuven, Center for Human Genetics, Leuven, Belgium . 2 VIB, Center Note: Supplementary data for this article are available at Cancer Discovery for Cancer Biology, Leuven, Belgium. 3 Laboratory for Viral Vector Technol- Online (http://cancerdiscovery.aacrjournals.org/). ogy and Gene Therapy, Department of Pharmaceutical and Pharmacological Corresponding Author: Jan Cools, Laboratory for Molecular Biology of 4 Sciences, KU Leuven, Leuven, Belgium. U944 INSERM and Hematology Leukemia, VIB-KU Leuven Center for Cancer Biology, KU Leuven Cam- Laboratory, St-Louis Hospital, APHP, Hematology University Institute, Uni- pus Gasthuisberg O&N 4, 3000 Leuven, Belgium. Phone: 32-16-33-00-82; 5 versity Paris-Diderot, Paris, France. Princess Máxima Center for Pediatric E-mail: [email protected] Oncology, Utrecht, the Netherlands. 6 Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland. 7 Tampere Centre doi: 10.1158/2159-8290.CD-17-0583 for Child Health Research, University of Tampere and Tampere University ©2018 American Association for Cancer Research. Hospital, Tampere, Finland.

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INTRODUCTION is bound by its ligand IL7, there is phosphorylation of both JAK1 and JAK3. This leads to the recruitment and phospho- T-cell acute lymphoblastic leukemia (T-ALL) is an aggres- rylation of STAT5, which then translocates to the cell nucleus sive hematologic disease, which arises from the malignant to regulate gene expression. The binding of IL7 to its receptor transformation of developing T-cell progenitors. The best- is essential for the survival and proliferation of double-neg- characterized genetic defects in T-ALL include inactivation ative thymocytes and is also important in the homeostasis, of cell-cycle regulators (p16/p15), overexpression of tran- differentiation, and activity of mature T cells (11, 12). The scription factors (TLX1, TLX3, TAL1, HOXA), mutations JAK3 mutations found in patients with T-ALL are able to cir- that activate the NOTCH1 and PI3K signaling cascades, and cumvent this tightly controlled signaling process and activate hyperactivation of signaling pathways by mutant kinases STAT5 in the absence of cytokine, transform cells in vitro, and such as JAK3 mutants or the NUP214–ABL1 fusion (1–3). In drive the development of T-ALL in vivo using a murine bone T-ALL, there are an average of 10 to 20 damaging genomic marrow transplant leukemia model (13). lesions per case (4–6). These mutations target critical cel- Although the validation of JAK3 mutations as bona fide lular pathways including lymphoid development, cell-cycle “driver” mutations using a one-oncogene murine model is regulation, tumor suppression, lymphoid signaling, and drug significant, from a clinical perspective, patients have addi- responsiveness (6, 7). Recent studies using next-generation tional mutations that may cooperate for malignant cell trans- sequencing have identified mutations in the IL7R/JAK3/ formation. For example, it has been shown that BCR–ABL STAT5 pathway in 28% of T-ALL cases with JAK3 tyrosine and NUP98–HOXA9 can cooperate synergistically in acute kinase mutations being the most frequent mutations found myeloid leukemia, and modeling this cooperation identified in up to 16% of T-ALL cases (4, 5, 8, 9). compounds that selectively target leukemic stem cells (14, In normal T-cell biology, the JAK3 and JAK1 kinases are 15). More recently, the existence of multiple “mini-driver” essential components of the heterodimeric IL7 receptor com- mutations has also been suggested that might substitute for plex. JAK3 binds the common gamma chain (IL2RG), and a major driver change (16), and therefore given the high num- JAK1 binds the IL7Rα chain (10). When the receptor complex ber of genetic changes within T-ALL, it is likely that leukemia

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RESEARCH ARTICLE de Bock et al. development and progression require synergistic modulation (Fig. 1D), suggesting that HOXA9 is the most important of downstream signaling pathways by cooperating oncogenic upregulated gene of the HOXA cluster in T-ALL. lesions in a polygenic manner. Some cooperating lesions in T-ALL have already been iden- JAK3 Mutations and HOXA9 Coexpression tified and includeArf inactivation in combination with Lmo2 Transform Hematopoietic Stem and Progenitor overexpression significantly accelerating the development of Cells and Lead to Rapid Leukemia Development the T-cell malignancies and acquisition of Notch1 mutations In Vivo Using a Bone Marrow Transplant Model (17). Similar work in our laboratory found that loss of the The most common JAK3 mutation found within T-ALL phosphatase PTPN2 enhanced the kinase activity of the cases is the M511I mutation just upstream of the pseu- NUP214–ABL1 fusion or JAK1 mutants, thereby sensitiz- dokinase domain (5, 9, 13). Therefore, we next sought ing cells to leukemogenic transformation (18, 19). However, to determine whether mutant JAK3 signaling (using the the vast majority of co-occurring lesions and their ability to JAK3M511I mutant) can cooperate with HOXA9 in the trans- cooperate in driving tumorigenesis still require functional formation of hematopoietic stem progenitor cells (HSPC) validation. and leukemia development. Two separate retroviral vectors In the current work, we set about identifying cooperating containing JAK3M511I (also expressing GFP) or HOXA9 oncogenes in the context of a JAK3 mutant–driven leukemia (also expressing mCHERRY) were used for the cotransduc- and functionally validating these in vivo using a two-oncogene tion of murine HSPCs ex vivo and yielded a mixture of model. We found that T-ALL cases with JAK3 mutations are nontransduced, single-transduced, or double-transduced often HOXA+, specifically expressing high levels of HOXA9, cells as assessed by GFP and mCHERRY fluorescence (Fig. and together can drive an aggressive leukemia using mouse 2A). HSPCs transduced with both JAK3M511I and HOXA9 models. rapidly transformed to cytokine-independent growth and expanded over 20 days in the absence of all cytokines. RESULTS All nontransduced and single-transduced cells either were outcompeted and/or did not grow (Fig. 2B). The vari- JAK3 Mutations Are Significantly Associated ous JAK3 mutations activate downstream STAT5 signaling + with HOXA T-ALL Cases with the majority also requiring binding to a functional Based on results of large-scale sequencing studies in T-ALL cytokine receptor complex (13, 23). Constitutively active (4, 5, 8, 9, 20), it is becoming clear that some mutations STAT5BN642H mutations have also been identified and func- frequently co-occur, whereas others are mutually exclusive. tionally characterized in T-ALL (24, 25). Therefore, the We analyzed genetic data for 155 T-ALL cases (Vicente data- same experimental setup was repeated using STAT5BN642H set; ref. 5) in which IL7R/JAK/STAT5 mutations occur in and HOXA9 retroviral expression constructs. Again, only 28% of the cases and are found more frequently in HOXA+, the double-transduced STAT5BN642H and HOXA9 cells immature, and TLX1/3-positive cases. Focusing specifically expanded in the absence of all cytokines, phenocopying on JAK3 mutations alone, these account for the most fre- the JAK3M511I and HOXA9 result (Fig. 2C). Taken together, quent type of mutation found in 16% of the cases (5). these results show that oncogenic JAK3/STAT5 activation Upon analyzing different T-ALL subgroups, these JAK3 muta- is necessary and sufficient for cooperation with HOXA9 in tions were found to significantly associate with those T-ALL ex vivo HSPC cell transformation. cases designated as HOXA+ (P value = 0.018) and negatively Given this evident cooperation in HSPC transforma- associate with those designated as TAL1/LMO2–positive (P tion ex vivo, we next investigated the cooperation between value = 0.002; Fig. 1B). The designation of HOXA+ included JAK3M511I and HOXA9 during in vivo leukemia development. cases with chromosomal rearrangements known to either Mouse HSPCs were isolated and transduced with retroviral directly or indirectly upregulate genes within the HOXA vectors containing JAK3M511I or HOXA9 or a mixture of cluster (i.e., CALM-AF10, SET-NUP214, NUP98-RAP1GDS, and both and then injected into sublethally irradiated synge- TCRB-HOXA; refs. 5, 21, 22). Analysis of a second independ- neic recipient mice (BALB/c or C57BL/6). JAK3M511I+HOXA9 ent cohort of T-ALL cases (Liu dataset; ref. 9) showed 22% of recipient BALB/c mice rapidly developed a leukemia char- the patients had IL7R/JAK/STAT5 mutations (data included acterized by a rapid increase in peripheral white blood cell mutations within JAK1, JAK3, IL7R, PTPN2, FLT3, STAT5B, (WBC) counts in excess of 10,000 cells/mm3 within 30 days and SH2B3; Fig. 1A). These mutations were enriched within compared with JAK3M511I only (Fig. 2D). For each recipi- the HOXA+, TLX1/3+, and LMO2/LYL1+ cases. Furthermore, ent, the double transduced JAK3M511I and HOXA9 leukemic focusing specifically on JAK3-mutant cases again revealed clone outcompeted all single transduced clones (Fig. 2E). a significant positive association with HOXA+ and negative This rapid leukemia development resulted in significantly association with TAL1/TAL2/LMO2 cases (Fig. 1B). Moreo- decreased disease-free survival (DFS; median DFS = 25 days, ver, associated gene expression data showed that HOXA+ P value < 0.0001) when compared with JAK3M511I alone cases have overall higher JAK3 expression levels compared (median DFS = 126.5 days; Fig. 2F). In contrast, clones with the majority of other T-ALL cases (Fig. 1C). Activation coexpressing TAL1 and JAK3M511I were counter-selected of the HOXA cluster in T-ALL typically results in upregula- in vivo with mice succumbing to a JAK3M511I-only disease tion of both HOXA9 and HOXA10. Analysis of primary T-ALL (Fig. 2F; Supplementary Fig. S1) and reconciled with JAK3 samples (Supplementary Table S1) revealed strong HOXA9 mutations negatively associating with TAL1 in T-ALL expression in 3 of 5 JAK3-mutant cases, that was up to (Fig. 1A). Notably, when the same experimental approach was 10-fold higher when compared with HOXA10 and HOXA11 used for HOXA10 together with JAK3M511, there was only a

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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE

ABHOXA+ P = 0.018 TAL1/LMO2+ P = 0.002 IL7R/JAK/STAT5 pathway mutation (Vicente dataset) JAK3 wild-type JAK3 mutant Immature (n = 130) (n = 25) (n = 15) + HOXA + + Other HOXA HOXA 12% Other (n = 10) 15% 24% TLX1/TLX3+ 36% (n = 41) 32% 32% TAL1+ TLX1/3+ 41% (n = 54) 8% + 0528 0 100 TLX1/3 TAL1/LMO2+ TAL1/LMO2+ Percentage

IL7R–JAK–STAT5 pathway mutation (Liu dataset) HOXA+ P = 0.006 + LMO2_LYL1 TAL1/TAL2/LMO2 P = 0.0047 (n = 18) JAK3 wild-type JAK3 mutant LMO1/2 (n = 244) (n = 20) (n = 10) HOXA+ HOXA+ 11% TLX1/3 HOXA+ (n = 33) TLX1/3 25% TLX1/TLX3+ Other 27% 35% (n = 72)a 13%

TAL1/TAL2 10% 7% LMO2 (n = 95) LMO2 LYL1 42% LYL1 20% NKX2_1 10% (n = 14) Other TAL1/TAL2/ TAL1/TAL2/ LMO2 02250 100 LMO2 Percentage

CD 150 HOXA9 HOXA9 JAK3 HOXA10 60 80 100 HOXA11 60 50 40 40 10

20 CNRQ 20 8 RNA-seq FPKM RNA-seq FPKM 6 0 0 4 2 TLX1 TLX3 TLX1 TLX3 HOXA HOXA TAL1/2 TAL1/2 LMO1/2 NKX2_1 LMO1/2 0 NKX2_1 Unknown Unknown LMO2_LYL1 LMO2_LYL1 X16 X11 X12 X15 X17 X13 X10 X09 863I 382L 389E 839U 9H60 1Q94 O623 7Q05 XB37 XB47 XB41 XB34 XA32 XC57

Mut JAK3

Figure 1. JAK3 mutations and ectopic expression of HOXA9 are significantly associated in clinical T-ALL cases. A, Frequency of IL7R/JAK/STAT mutations in 155 T-ALL cases (Vicente dataset) and 264 T-ALL cases (Liu dataset) within different defined transcription factor subgroups. Dotted line denotes average percentage of IL7R/JAK/STAT mutations across entire dataset. B, Association between JAK3 mutations only and defined transcription factor subgroups within the Vicente and Liu datasets. C, RNA-seq FPKM levels for JAK3 and HOXA9 in the Liu dataset. D, Real-time qPCR of primary T-ALL samples for HOXA9, HOXA10, and HOXA11.

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Cellular A transformation assay

5′LTR JAK3 (M511I) IRES GFP 3′LTR

Bone marrow 5′LTR HOXA9 IRES mCHERRY 3′LTR transplant Retroviral spinfection of Lin− cells HOXA9 JAK3 (M511I)

Tail vein injection sublethally irradiated mice (BALB/c; C57BL/6)

JAK3 (M511I) + HOXA9 B HOXA9 1.5 × 108 JAK3 (M511I) Day 1 Day 10 Day 20 Nontransduced 19 25 14 53 3 89

1.0 × 108 umber

5.0 × 107 Cell n 45 11 10 22 0.14 8 0 0510 15 20 25 C 8 1.5 × 10 STAT5B (N642H) + HOXA9 Day 1Day 10 Day 20 HOXA9 17 37 21 61 3 92 STAT5B (N642H ) 1.0 × 108 Nontransduced mber

5.0 × 107 Cell nu 33 13 5 13 0.35 4 0 0510 15 20 25 Days post-transduction DEF 600,000 JAK3 (M511I)/HOXA9 (n = 9) 100 JAK3 (M511I)/HOXA9 100 500,000 JAK3 (M511I) (n = 10) HOXA9 JAK3 400,000 80 )

3 300,000 200,000 JAK3 (M511I)

mm 60 100,000 JAK3 (M511I)/HOXA9 100,000 50 40 JAK3 (M511I)/TAL1* 80,000 BALB/c ***

60,000 Fluorophore (%)

WBC (cells/ WBC P < 0.0001 20 40,000 Disease-free survival (%) 20,000 0 0 0 0100 200300 050100 150200 250 End Start Days post-transplant Days post-transplant

GHJAK3 (M511I)/HOXA9 (n = 9) I 400,000 JAK3 (M511I) (n = 5) JAK3 (M511I) 100 JAK3 (M511I)/HOXA9 100 HOXA9 (n = 5) HOXA9 300,000 HOXA9 JAK3 (M511I)/HOXA9 200,000 JAK3 ) 80 STAT5B (N642H)/ 3 200,000

m HOXA9

150,000 60 50

C (cells/m 40 C57BL/6 100,000 *** WB

Fluorophore (%) P < 0.0001

50,000 20 Disease-free survival (%)

0 0 0 0100 200300 0 100 200 300 End Days post-transplant Start Days post-transplant

Figure 2. Coexpression of JAK3M511I and HOXA9 transforms hematopoietic progenitor cells and leads to rapid leukemia development in vivo. A, Schematic of the two retroviral expression vectors used in the transduction of murine HSPCs for the ex vivo transformation assay and bone marrow transplant model. Dual transduction results in single, double, and nontransduced cells for direct clonal competition analyses ex vivo and in vivo. B, Cell proliferation of HSPCs transduced with both JAK3M511I and HOXA9 over 20 days in the absence of all cytokines with matching FACS profiles. C, Cell proliferation of HSPCs transduced with both STAT5N642H and HOXA9 over 20 days in the absence all cytokines with matching FACS profiles. D, WBC counts in recipient BALB/C mice that received lin– cells transduced with JAK3M511I and HOXA9 retrovirus or JAK3M511I-only retrovirus. E, Clonal analysis by FACS for GFP and mCHERRY reporters representing JAK3M511I and HOXA9, respectively, at injection start and end stages. F, Kaplan–Meier DFS for JAK3M511I/ HOXA9 mice (median DFS = 25 days, P value < 0.0001), JAK3M511I alone (median DFS = 126.5 days), and JAK3M511I/TAL1 (median DFS = 144.5 days). DFS determined by WBC <30,000 cells/mm3. G and H, Reciprocal experiments in C57BL/6 mice coexpressing JAK3M511I and HOXA9 for WBC and clonal analysis by FACS for GFP and mCHERRY reporters representing JAK3M511I and HOXA9 respectively at injection start and end stages. I, Kaplan–Meier DFS for STAT5BN642H/HOXA9 (median DFS = 12 days), JAK3M511I/HOXA9 (median DFS = 40 days), JAK3M511I (median DFS = 150 days), and HOXA9 only (median DFS = 182 days) recipient C57BL/6 mice.

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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE moderate and less profound cooperative effect compared with development. Second, there is a degree of transcription factor HOXA9 (median DFS = 100 days; Supplementary Fig. S1). specificity for cooperation withJAK3 mutations, because JAK3 To negate any mouse strain–specific effects, the same experi- mutations do not cooperate with the oncogenic transcription mental setup was repeated on a C57BL/6 background. Similar factor TAL1. Third, the resulting leukemia remains dependent to BALB/c, recipient C57BL/6 mice receiving cotransduced on activated JAK3 signaling. JAK3M511I/HOXA9 had a rapid increase in WBC compared with JAK3M511I or HOXA9 alone (Fig. 2G), and in all cases, the A Novel Retroviral Vector to Direct Transgene double-transduced clone was the major clone at end stage (Fig. Expression within Developing Lymphoid Cells M511I 2H). This resulted in a significant decrease in DFS (median Demonstrates Cooperation of JAK3 /HOXA9 in DFS = 40 days) compared with JAK3M511I (median DFS = 150 T-ALL Development days) or HOXA9 (median DFS = 182 days) only recipient mice The previous data illustrate the potential of combined (Fig. 2I). Moreover, when JAK3M511I was substituted for the con- expression of JAK3M511I and HOXA9 to generate mixed mye- stitutive active STAT5BN642H and cotransduced with HOXA9, loid/lymphoid leukemia originating from multipotent hemat- this further reduced the DFS (median DFS = 12 days). opoietic precursor cells. However, HOXA9 expression is often At end-stage disease, the leukemia development in vivo activated in T-ALL by T cell–specific enhancers that become was characterized by both myeloid and lymphoid expan- active only in lymphoid-committed progenitors (e.g., TCRB- sion. Phenotypic FACS staining and pathology analysis of HOXA9; refs. 21, 22). To convincingly demonstrate coop- the JAK3M511I/HOXA9-driven leukemia revealed that leuke- eration between JAK3M511I and HOXA9 during T-cell leukemia mic mice had polyclonal expansion of cells displaying either development, and to specifically model lymphoid-specific acti- myeloid (CD16/32+, CD11b+, Gr1+/−) or lymphoid (CD8+) line- vation of HOXA9 expression as observed in human T-ALL, we age markers and occurred at different frequencies (Fig 3A). At developed a strategy to limit HOXA9 expression to developing end-stage disease, histopathologic and immunohistochemical lymphoid cells. We tested a previously published strategy with analysis revealed the development of a complex hematolym- 3′ UTR mir-223 sites (27), but in the context of JAK3M511I/ phoid neoplasm characterized by the coexistence of different HOXA9, this strategy was unable to prevent overt myeloid populations of atypical cells displaying both lymphoid and expansion and eventual acute myeloid leukemia (AML) expan- myeloid differentiation. There was significant leukemic infil- sion that we speculate to indicate endogenous miR-223 satu- tration into spleen, lymph nodes, and bone marrow organs by ration in the presence of high levels of transcript driven by the atypical lymphocytes (CD3+, TdT-) and myeloid/granulocytic strong MSCV promoter (Supplementary Fig. S5). (myeloperoxidase-positive) cells (Fig. 3B; Supplementary Fig. We therefore developed a retroviral vector in which one or S2). Western blot analysis confirmed expression of JAK3 and two oncogenes are initially cloned in an antisense orientation, HOXA9 in vivo with robust phosphorylation of STAT5 (Fig. and thus remain completely inactive. The expression of the 3C). These cells proliferated for up to 30 days ex vivo in the oncogenes is activated only upon Cre-mediated inversion of absence of all cytokines with clonal selection of those clones the LOX66/71 flanked construct (Fig. 4A). By use of hemat- expressing high levels of HOXA9 and JAK3M511I as assessed opoietic cells isolated from cell type–specific Cre mice, we could by GFP and mCHERRY expression (Fig 3D and E). Moreo- then direct expression of the oncogenes to specific hematopoi- ver, sorted JAK3M511I/HOXA9 CD8+ T-ALL clones were able etic cell types. The novel vector with JAK3M511I+HOXA9 was to engraft secondary recipient mice with higher frequency first validated in Ba/F3 cells and found to transform the cells compared with age-matched JAK3M511I CD8+ leukemic clones into cytokine-independent growth only when Cre-recombinase (Supplementary Fig. S3). was present and did not display ectopic “leaky” expression To determine whether the resulting leukemia remained or cell transformation in Ba/F3 wild-type cells over an 8-day dependent on activated JAK/STAT5 signaling directly, period (Fig. 4B). Western blot analysis confirmed the expres- JAK3M511I/HOXA9 leukemic mice were treated for 20 days with sion of JAK3 and HOXA9 in the Cre-positive Ba/F3 cells and the JAK3 inhibitor tofacitinib (40 mg/kg/day; Fig. 3F). Within highlighted that although there was less JAK3M511I expression 3 days of treatment with tofacitinib, there was a significant compared with constitutive expression of JAK3, both had decrease in the levels of pSTAT5 compared with vehicle-treated equivalent levels of phospho-STAT5 (Fig. 4C). The novel retro- mice (Fig 3G). After 20 days of tofacitinib treatment, there viral vector strategy was also validated in vivo using a well-estab- was a significant attenuation in leukemia WBC expansion lished NOTCH1-ICN bone marrow transplant model, but reduced splenomegaly (Fig 3H–J). Staining of the spleen at now with ICN cloned in the antisense direction between the end stage also showed a significant reduction in the number LOX66/71 sites. When the viral vector was transduced on cells of proliferative cells as assessed by Ki67 staining (Fig. 3K). derived from CD4-Cre mice, the recipient mice succumbed to Tofacitinib is approved for rheumatoid arthritis and known the anticipated CD4+/CD8+ T-ALL leukemia (median overall to cause decreased numbers of neutrophils, lymphocytes, and survival = 43 days; Supplementary Fig. S6). natural killer (NK) cells but rarely to an extent that leads to Using this new strategy, we next tested whether combined serious adverse effects in patients (26). Indeed, in our own expression of JAK3M511I and HOXA9 via CD2-Cre–mediated studies, wild-type mice treated at 40 mg/kg/day showed a activation in developing lymphoid cells (ref. 28; Fig. 4D) could minor decrease in NK-cell frequency but no significant altera- generate T-cell leukemia. Mice that received transplantation tions in CD4+ or CD8+ T-cell frequencies (Supplementary Fig. with inducible JAK3M511I-P2A-HOXA9 HSPCs developed S4). Taken together, these results first provide strong valida- T-ALL–like leukemia with decreased latency when compared tion that ectopic expressions of HOXA9 together with JAK3/ with JAK3M511I-P2A-BFP only (median DFS = 135 days vs. 244 STAT5 activation are bona fide cooperating events inleuk emia days). Notably, both JAK3M511I and JAK3M511I-P2A-HOXA9

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A B MPO CD3 2 0 100 0 3 48 Bone M127- Day 63 marrow

93 4 0 0 8 41 14% 82 0 12 0 0.5 0 Spleen 38% HOXA9 JAK3 (M511I) 8 10 88 0 99 0.5 Lymph Gr1 CD8 node CD16/32 CD4 CD135 CD11b

C DEDay 1 Day 35 Spleen 1.5 × 107 Spleen BM Spleen Bone marrow pJAK3 1.0 × 107 pJAK1

JAK3 Bone marrow

Cell number 6 pSTAT5 (Y694) 5.0 × 10

STAT5 0 HOXA9 0246810 HOXA9 (mCherry) Day JAK3 (GFP) F G MFI = 557 Day 14 Day 35 Primary JAK3(M511I)/HOXA9 MFI = 696 Vehicle

Vehicle MFI = 296

Tofa MFI = 424

Tofacitinib (40 mg/kg/day) pSTAT5-APC

HI JK P = 0.006 P < 0.001 Ki67 6 500 1 × 10 ** 1,000 *** Vehicle

) 400 3 Vehicle 300 800 ) 200 3 100 600 50 100,000 Tofacitinib

40 400 Tofacitinib Weight (mg)

30 WBC (cells/mm

WBC ( × 1,000) (cells/mm 20 200 10 0 Day 14 Day 35 10,000 0 P = 0.08

Vehicle Vehicle Tofacitinib ofacitinib T

Figure 3. Constitutive expression of JAK3M511I and HOXA9 leads to development of both AML and T-ALL in vivo. A, Representative peripheral blood FACS of expanding leukemic clones expressing JAK3M511I and HOXA9. B, Histopathology for MPO and CD3 staining of bone marrow, spleen, and lymph nodes. Scale bars: bone marrow, 40 μm; spleen and lymph nodes, 80 μm. C, Western blot analysis of spleen and bone marrow leukemic cells. D and E, Ex vivo growth assay of leukemia clones and matching FACS with JAK3M511I and HOXA9 levels measured by GFP and mCherry expression, respectively. F, Schematic of in vivo treatment of primary bone marrow transplant recipients. Mice were treated daily for 20 days from day 14 after transplantation with either 40 mg/kg/day tofacitinib (oral gavage) or vehicle control. G, Phospho-flow cytometry of pSTAT5 levels in JAK3M511I/HOXA9 leukemic cells in peripheral blood after 3-day tofacitinib (tofa) treatment compared with vehicle control. H, Change in WBC count before and after the treatment. I, Total WBC counts at day 35. J, Spleen weights of tofacitinib-treated vs. vehicle-treated mice at day 35. K, Ki67 staining of spleens.

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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE

ABBa/F3 Ba/F3_Cre 10 JAK3(M511I) 10 JAK3(M511I) [JAK3(M5111I)-P2A-BFP]Lox66/71 [JAK3(M5111I)-P2A-BFP]Lox66/71 Lox66 Lox71 [JAK3(M511I)-P2A-HOXA9]Lox66/71 [JAK3(M511I)-P2A-HOXA9]Lox66/71 5’LTR5’LTR IRES GFP 3’3’LTLTR 8 8

6 6

JAJAK3(K3(M511I)-P2A-BFP(M511I)-P) 2A-BFP 4 4

JAJAK3(K3(M511I)-P2A-HOXA9(M511I)-P) 2A-HOXA9 2 2 Cell proliferation (fold change) Cell proliferation (fold change) 0 0 02468 02468 Days (-IL3) Days (-IL3)

Lox66/71 Lox66/71 Lox66/71 Lox66/71

CD CD2-Cre donor MSCV-JAK3(M511I)[JAK3(M511I)-P2A-BFP][JAK3(M511I)-P2A-HOXA9]MSCV-JAK3(M511I)[JAK3(M511I)-P2A-BFP][JAK3(M511I)-P2A-HOXA9] phospho-JAK1 phospho-JAK3

JAK3

phospho-STAT5 Retroviral spinfection of Lin− cells

STAT5

HOXA9

GFP Injection into sublethally irradiated wild-type recipients

β-actin Leukemia latency/disease progression assessment IL3: − ++−−− Cre: −− − +++ EF DMLox LoxP 100 JAK3(M511I)-P2A-BFP (n = 12) 5’LTR JAJAK3K3 (M511I)(M51( 1I)) P2AA HOXA9HOXA9 IRES GFP 3’LTR3’LTR JAK3(M511I)-P2A-HOXA9 (n = 13) F1 R1 F2 R2

50 Disease-free survival K3-P2A-BFP K3-P2A-BFP K3-P2A-BFP K3-P2A-HOXA9 K3-P2A-HOXA9 K3-P2A-HOXA9

**P = 0.0014 0 M12: JA M12: JA M13: JA M14: JA M18: M20: JA M20: M57: JA M57: 0 100 200 300 Days post-transplant F1+R1

100% CD8+ 23% Mixed CD8+/DN F2+R2

77% CD8+

JAK3(M511I)-P2A-BFP JAK3(M511I)-P2A-HOXA9

Figure 4. Novel retroviral strategy to restrict JAK3M511I/HOXA9 expression to developing lymphoid cells results in significantly decreased leukemia latency. A, Schematic of new retroviral vector incorporating antiparallel LOX66/71 sites for unidirectional inversion in the presence of Cre-recombinase. B and C, Cell proliferation of Ba/F3 or Ba/F3_Cre-recombinase cells transduced with the retroviral constructs encoding JAK3M511I or JAK3M511I_P2A_ HOXA9 in between the LOX66/71 sites. C, Western blot analysis of the transduced Ba/F3. D, Model of improved bone marrow transplant utilizing Cre-donor mice in combination with novel retroviral vector. E, Kaplan–Meier DFS cure for mice with hCD2-iCre–driven inversion of JAK3M511I and HOXA9 and the cell surface phenotype frequency for the resulting leukemia (DFS = WBC <30,000 cells/mm3). F, Genomic PCR detection of the inversion of the retroviral construct.

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RESEARCH ARTICLE de Bock et al. mice now developed a CD8+ or CD4−CD8− T-ALL, without with the constitutively active STAT5BN642H mutant was observed, myeloid expansion (Fig. 4E). For all mice, CD2-mediated but not with wild-type STAT5 (Supplementary Fig. S10). recombination was confirmed by PCR (Fig. 4F). These data Global analysis of the regions with cobinding of HOXA9 and not only illustrate that JAK3 mutants cooperate with HOXA9 STAT5 found an association with diminished levels of repres- to induce leukemia when expressed in common lymphoid sive H3K27me3 marks at upregulated genes and concomitantly progenitors, but also demonstrate the utility and efficacy of associated with increased levels of active H3K27Ac marks (Fig. this novel Cre-inducible retroviral vector for cell type–specific 5E). Integrating the ChIP-seq analysis with the RNA-seq differ- expression in the hematopoietic system. entially expressed genes also revealed that STAT5 and HOXA9 loading was higher in upregulated genes compared with down- STAT5 and HOXA9 Co-occupy Similar Genetic regulated or unchanged genes (Fig. 5F), illustrating that the Loci Resulting in Increased JAK/STAT Signaling presence of HOXA9 leads to increased gene activation. Indeed, in Leukemia Cells gene set enrichment analysis (GSEA) pathway analysis com- To determine the underlying molecular mechanism driving paring CD8+ JAK3M511I T-ALL versus CD8+ T cells and CD8+ the enhanced oncogenic potential of JAK3M511I in the presence JAK3M511I/HOXA9 versus CD8+ JAK3M511I found a significant of HOXA9, RNA-sequencing (RNA-seq) analysis was carried and increasingly positive enrichment for JAK/STAT signal- out on age-matched sorted leukemic CD8+ JAK3M511I, CD8+ ing (Fig. 5G; Supplementary Table S3). These data show that JAK3M511I/HOXA9 as well as sorted normal thymic CD8+ T ectopic expression of HOXA9 enhances STAT5 transcriptional cells (Supplementary Table S2). The addition of HOXA9 led to activity in CD8+ leukemic cells rather than inducing a de novo skewed upregulation of genes when compared with JAK3M511I- gene expression program complimentary to STAT5. only T-ALL (Fig. 5A). Notably, principal component analysis PIM1 kinase is overexpressed in a number of different can- revealed distinct clustering of myeloid and T-cell leukemias cers and is an emerging cancer drug target (34, 35). Consistent (Supplementary Fig. S7). The normal CD8+ T cells formed a with our RNA-seq and ChIP-seq data, we confirmed increased tight cluster away from the JAK3M511I/HOXA9 CD8+ T ALL cells Pim1 expression in JAK3M511I/HOXA9 leukemic mouse sam- and in between the JAK3M511I-only cells. Furthermore, within ples by qPCR (Fig. 6A). Analysis of primary human T-ALL the myeloid cell cluster, the JAK3M511I/HOXA9-driven AML cells patient samples showed the highest levels of PIM1 expression clustered separately from MEIS1/HOXA9 and MLL-AF9 AML in HOXA+ cases and those cases with IL7R/JAK3/JAK1 muta- (29), suggesting different mechanisms driving these leukemias, tions (Fig. 6B). Furthermore, PIM1 RNA and protein levels and indeed there was no increased MEIS1 expression in our were rapidly downregulated in a HOXA9+ patient-derived leukemia, suggesting that the cooperation is independent of xenograft (PDX) sample with active JAK/STAT signaling after MEIS1. FLT3 ITD-driven leukemia (30) clustered closer to the treatment with the JAK1 inhibitor ruxolitinib (Fig. 6C). This JAK3M511I/HOXA9 and could in part reflect the downstream provided further evidence for a functional signaling net- activation of STAT5 in both leukemias (Supplementary Fig. S7). work of PIM1 regulation downstream of JAK/STAT signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) Exploiting this observation, we next sought to determine analysis of CD8+ JAK3M511I/HOXA9 leukemia showed sig- whether dual inhibition of PIM1 and JAK1 would be benefi- nificant co-occupancy for both STAT5 and HOXA9 across the cial in JAK/STAT-mutant T-ALL samples. Here, we observed genome. Regions cobound by STAT5 and HOXA9 were asso- a synergistic response in JAK3-mutant T-ALL samples when ciated with H3K27Ac and H3K4me3 marks indicating colo- treated with a JAK kinase inhibitor (ruxolitinib) in combina- calization at proximal promoters rather than distal enhancers tion with a PIM1 inhibitor (AZD1208) within ex vivo cell cul- (Fig. 5B and C). Comparison of the STAT5 signal in JAK3M511I ture and a significant attenuation in leukemia burdenin vivo versus JAK3M511I/HOXA9 showed strong overlap, suggest- using a PDX sample (Fig. 6D and E). Taken together, these ing that HOXA9 does not significantly reposition STAT5 data suggest that the ectopic expression of HOXA9 cobinds to de novo sites (Supplementary Fig. S8). Specific analysis with STAT5 to enhance the JAK/STAT, and targeting of both of canonical STAT5 target genes illustrated the strong co- PIM1 and JAK1 provides a strong therapeutic benefit. occupancy of both STAT5 and HOXA9. For example, PIM1 kinase, a known target gene of both STAT5 (31) and HOXA9 Chromatin Accessibility by ATAC-seq Analysis (32), showed co-occupancy of the same DNA region for Reveals a Role for AP1 Activation both STAT5 and HOXA9. Similarly, Bcl2, Osm, Cish, and Il7r To further investigate and identify additional factors and also showed co-occupancy by STAT5 and HOXA9 (Fig. 5D; mechanisms underlying the JAK3/HOXA9 cooperation, chro- Supplementary Fig. S8). Analysis of relevant human T-ALL matin accessibility was assessed at a global level using Assay samples also showed strong co-occupancy of HOXA9 with for Transposase-Accessible Chromatin using sequencing STAT5 peaks at known STAT5-regulated genes including (ATAC-seq) comparing JAK3M511I with JAK3M511I/HOXA9 leu- BCL6, MYC, SOCS, and PIM1 (Supplementary Fig. S9). kemia. This revealed significant changes of chromatin archi- This strong STAT5/HOXA9 colocalization prompted the tecture with appearing peaks associated with upregulated question of whether a direct protein–protein interaction occurs gene expression and loading of STAT5 and HOXA9 (Fig. 7A between STAT5 and HOXA9. Indeed, a direct interaction between and B). Surprisingly, many upregulated sites were bound by HOXA9 and STAT5 is suggested based on available mass spec- neither STAT5 or HOXA9. We interpreted this finding as acti- trometry data (33). However, we were unable to confirm a robust vation by secondary events downstream of activated STAT5/ protein–protein interaction between HOXA9 and STAT5 using HOXA9 signaling. Motif scanning using i-cisTarget revealed coimmunoprecipitation, nor in an independent assay using a these peaks were highly enriched for FOS and JUN motifs recombinant strepII-tagged HOXA9. Only a weak interaction (Fig. 7C). RNA-seq data further confirmed increased levels of

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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE

A B STAT5 HOXA9 H3K27Ac H3K27me3 H3K4me3 H3K4me1 C STAT5/HOXA9 50 overlapping 45 HOXA9 40 peaks 35 30 Other 20 Enhancers

) 18%

adj 15 ( P

10 80% 10 Log Promoters

5 centered at the summits

0 S TA T5 ChIP-seq peaks Score-ranked −8 −6 −4 −20 2468 Summit Summit Summit Summit Summit Summit Log2 fold change − 5 kb + 5 kb − 5 kb + 5 kb − 5 kb + 5 kb − 5 kb + 5 kb − 5 kb + 5 kb − 5 kb + 5 kb D 5 kb STAT5 signal HOXA9 signal F 12 3 Up Up [0-100] 10 2.5 H3K27Ac ) Down ) Down

H3K27me3 [0-50] 8 Neutral 2 Neutral H3K4me3 [0-1000] 6 1.5 H3K4me1 [0-200] 4 1 JAK3(M511I) [0-100] Signal ( × 10 Signal ( × 10 STAT5 2 0.5

[0-100] H3K27Ac −4kb −2kb +2kb +4kb −4kb −2kb +2kb +4kb [0-50] TSS H3K27me3 TSS H3K4me3 [0-1000] [0-200]

HOXA9 H3K4me1 + [0-100] G JAK3(M511I) JAK3(M511I) STAT5 + HOXA9 HOXA9 [0-40]

Pim1 + 50 kb CD8 JAK3(M511I)JAK3(M511I) Dataset: CD8 vs. JAK3(M511I) Gene set: JAK/STAT SIGNALING PATHWAY [0-100] IL2RB H3K27Ac IL7R KEGG HSA04630 [0-50] PIK3R5 H3K27me3 IFNGR1 H3K4me3 [0-1000] JAK3 H3K4me1 [0-200] IL4R JAK3(M511I) PIM1 STAT5 [0-100] STAT3 NES: 1.525 IL21R PIK3R1 P: 0.007 SOCS3 FDR q-value: 0.135 STAT4 H3K27Ac [0-100] TYK2 [0-50] CBLB H3K27me3 CREBBP H3K4me3 [0-1000] IL10RB [0-200] IL6R H3K4me1 STAT2 0 17,500 + HOXA9 STAT5 [0-100] PIK3CG JAK3(M511I) JAK3(M511I) BCL2L1 Rank in ordered dataset HOXA9 [0-40] SOS2 SOCS1 IL10RA Bcl2 Kdsr Vps4b CISH IL12RB1 Dataset: JAK3(M511I) vs. E CRLF2 PIAS3 JAK3(M511I) + HOXA9 OSM Gene set: JAK/STAT SIGNALING PATHWAY Dataset: JAK3(M511I)/HOXA9 vs. Dataset: JAK3(M511I)/HOXA9 vs. IFNG CSF2RA KEGG HSA04630 JAK3(M511I) JAK3(M511I) IL15RA IL3RA Gene set: Appearing Gene set: Disappearing MYC H3K27Ac peaks H3K27me3 peaks IL2RA SOCS2 IL20RB NES: 1.659 CSF3R CSF2RB P: 0.018 SPRY2 SPRY4 FDR q-value: 0.098 LIF NES: 1.32 NES: 1.43 LIFR IL13RA1 P: 0.029 P: 0.018 CSF2 FDR q-value: 0.032 FDR q-value: 0.038 IL5RA IL12A IL15 0 17,500 EPOR IL10 Rank in ordered dataset IL22RA2 IL6 0 25,000 0 25,000 −2.020.0 .0 Rank in ordered dataset Rank in ordered dataset

Figure 5. HOXA9 and STAT5 co-occupy similar genomic regions and increase JAK/STAT signaling. A, Volcano plot of differential gene expression analysis of age-matched CD8+ T-ALL JAK3M511I/HOXA9 vs. JAK3M511I-only leukemia cells. B, JAK3M511I/HOXA9 leukemia ChIP-seq density heat maps centered on STAT5 signal alongside HOXA9 and associated H3K27Ac, H3K27me3, H3K4me3, and H3K4me1 histone marks. C, Frequency of STAT5/HOXA9 co-occupied DNA regions with promoters (H3K4me3-positive) and enhancers (H3K27Ac-positive/H3K4me3-negative). D, Individual ChIP-seq tracks for canonical STAT5 target genes Pim1 and Bcl2. E, GSEA analysis of RNA-seq differentially expressed genes for JAK3M511I/HOXA9 vs. JAK3M511I and loss of the repressive H3K27me3 chromatin marks or gain in H3K27ac activation mark. F, Transcription factor loading of STAT5 and HOXA9 in upregulated genes, downregulated genes, or genes that do not change. G, Heat map and GSEA pathway of JAK/STAT signaling pathway genes in CD8+ control T-cell vs. JAK3M511I vs. JAK3M511I/HOXA9 leukemia.

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RESEARCH ARTICLE de Bock et al.

A B C PDX(XE89) : HOXA9-positive )

2 CORE ENRICHMENT 11 JAK3/JAK1/IL7R mutation 2h 4h 6h 4 * * 10 1.5 3

9 DMSO RUXO DMSO RUXO DMSO RUXO 1.0 2 8 pSTAT5

1 7 0.5 PIM1

6 PIM mRNA (CNRQ) expression (CNRQ) PIM1 expression 0 0 209193_at PIM1 (RMA, Log β-actin TAL1 TLX1 TLX3 + T cell HOXA

CD8 IMMATURE 2h DMSO2h RUXO4h DMSO4h DMSO6h DMSO6h RUXO + JAK3(M511I)

CD8 + JAK3(M511I)/HOXA9

CD8 D DND41 JAK3 mutant (PDX) JAK3 wild-type (PDX) (CI = 0.16) 150 XC65 X11 XC57 X10 150 150 150 150 (CI = 0.53) (CI = 0.29) (CI = n/a) (CI = n/a)

100 100 100 100 100 Viability Viability Viability 50 Viability 50 50 50 50 Proliferation

0 0 0 0 0 −+−+ AZD1208 −+−+ AZD1208 −+−+ −+−+ AZD1208 −+−+ −−++ Ruxolitinib −−++ Ruxolitinib −−++ −−++ Ruxolitinib −−++

E XC65-Day 7 Ruxolitinib AZD1208 BLI - absolute difference (Day 7 vs. Day 1) Vehicle (50 mg/kg/day) (30 mg/kg/day) Combination ** 4

7 * 6.0 10 p/s) × 3 ; 9 2

4.0 × 107 1 ROI ( × 10 ROI 0 i 2.0 × 107 Ruxo AZD Vehicle Comb

Figure 6. PIM1 expression in increased in JAK3M511I/HOXA9 leukemia. A, qPCR analysis of PIM1 levels in normal CD8+ T cells, CD8+ JAK3M511I, and CD8+ JAK3M511I/HOXA9 cells B, Analysis of clinical T-ALL samples for PIM1 expression levels and those cases with JAK3/IL7R/STAT mutations (red dots). C, Analysis of PIM1 RNA and protein expression in HOXA9-positive PDX samples ex vivo after 1 μmol/L ruxolitinib over 6 hours. D, Combinatorial treatment with small molecular inhibitors against JAK kinases (ruxolitinib) and PIM1 kinase (AZD1208) in DND41(IL7R mutant) and JAK3-mutant PDX samples. E, In vivo combination treatment with ruxolitinib and AZD1208 (7 days) of the JAK3-mutant XC65 PDX sample.

members of the AP1 complex including FOS and JUN (Fig. tions in T-ALL, this has often been in the context of a single 7D) and increased expression of AP1 target genes (Fig. 7E). oncogene, either within transgenic (e.g., TLX1; ref. 37) or bone Although some of these target genes such as Fasl were bound marrow transplant mouse models (e.g., JAK3M511I; ref. 13). How- by STAT5 and HOXA9, many others such as App, Klf4, and Csf1 ever, patients present with multiple mutations at diagnosis, and had very weak or no binding (Fig. 7F) and represent examples we therefore hypothesized that mutations that significantly of genes activated by AP1 independent of STAT5. associate with one another are potentially cooperating events in driving T-ALL. In a recent analysis, significant associations were found between mutations in the IL7R/JAK signaling path- DISCUSSION way and epigenetic regulators (5). Here, we extend this analysis The development of T-ALL is a stepwise process via the accu- and find that those cases withJAK3 mutations are significantly mulation of somatic mutations (36). Although we and others associated with cases designated HOXA+. In particular, we have identified and confirmed various important driver muta- show that HOXA9 expression levels are significantly elevated

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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE

A B JAK3(M511I) JAK3(M511I)/ JAK3(M511I) JAK3(M511I)/ Dataset: JAK3(M511I)/HOXA9 vs. JAK3(M511I) HOXA9 HOXA9 Gene set: Appearing ATAC-seq peaks ATAC-seq ATAC-seq STAT5 ChIP STAT5 ChIP HOXA9 ChIP

NES: 1.83 P <0.001 FDR q-value <0.001 Disappearing ATAC-seq peaks

025,000 Rank in ordered dataset Appearing ATAC-seq Dataset: JAK3(M511I)/HOXA9 vs. JAK3(M511I) peaks Gene set: Disappearing ATAC-seq peaks

NES: −1.69 C P <0.001 FOSL2 (NES = 6.34) JUN (NES = 6.29) FDR q-value <0.001 2.0 2.0

1.0 1.0 Bits Bits

025,000 Rank in ordered dataset 0.0 0.0 1 5′ 2 3 4 5 6 7 8 9 3′ 5′ 1 2 3 4 5 6 7 8 9 3′ 10 11 12 D

18 Fosl2 18 Jun E AP1 target genes 16 16 15 14 14 12 CD8+ 12 10 JAK3(M511I) Normalized count s Normalized counts 8 10 10 + JAK3(M511I)/ + HOXA9 CD8 CD8

JAK3(M511I) JAK3(M511I) 5 Normalized counts JAK3(M511I)/HOXA9 JAK3(M511I)/HOXA9 18 Fos 18 Jund 16 16 0 14 14 12 APP CSF1 FGL2 FASL GZMB KLF4 10 12 CEBPB CEBPA

Normalized count s 10

Normalized count s 8 + +

CD8 CD8

JAK3(M511I) JAK3(M511I)

JAK3(M511I)/HOXA9 JAK3(M511I)/HOXA9 F 5 kb 50 kb JAK3(M511I) STAT5 JAK3(M511I) STAT5 HOXA9 HOXA9 JAK3(M511I)/HOXA9 JAK3(M511I)/HOXA9 STAT5 STAT5 JAK3(M511I) ATAC JAK3(M511I) ATAC JAK3(M511I)/HOXA9 ATAC JAK3(M511I)/HOXA9 ATAC Fasl App 5 kb 5 kb JAK3(M511I) STAT5 JAK3(M511I) STAT5 HOXA9 HOXA9 JAK3(M511I)/HOXA9 JAK3(M511I)/HOXA9 STAT5 STAT5 JAK3(M511I) ATAC JAK3(M511I) ATAC JAK3(M511I)/HOXA9 ATAC JAK3(M511I)/HOXA9 ATAC

Klf4 Csf1

Figure 7. ATAC-seq reveals role for AP1 complex in JAK3M511I/HOXA9-driven leukemia. A, GSEA results showing appearing and disappearing ATAC-seq peaks correlated with upregulated and downregulated genes. B, HOXA9 and STAT5 bound genomic regions relative to appearing and disappearing ATAC-seq peaks. C, i-cisTarget results for DNA-binding motifs within the appearing ATAC-seq peaks. D, Relative expression of members of the AP1 complex including Fos, Jun, Jund, and Fosl2. E, Relative expression of AP1 target genes in normal CD8+ cells and JAK3M511I or JAK3M511I/HOXA9 leukemias. F, ChIP-seq analysis tracks for AP1 targets such as Fasl (bound by STAT5 and HOXA9) and Csf1, App, and Klf4 not bound by STAT5 or HOXA9 (all track heights = 0–100).

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RESEARCH ARTICLE de Bock et al. compared with either HOXA10 or HOXA11 in HOXA+ cases, bone marrow transplant studies presented here showed high and coexpression of HOXA9 and JAK3M511I leads to the rapid leukemia penetrance for MSCV-based HOXA9 expression and development of leukemia in vivo. reconcile with a recent study using a similar retroviral strategy In our initial mouse model, the developing leukemia (52) highlighting the role of promoter strength and viral titers in vivo was characterized by overt myeloid and lymphoid in dictating leukemia latency. expansion due to the constitutive expression of both JAK3M511I Nevertheless, what is now well understood is that the ability and HOXA9 within the HSPCs. Traditionally, the modeling for HOXA9 to drive rapid leukemia development and cellular of T-cell leukemia in mice and the role on oncogenes have transformation is dependent on the expression of different often used transgenic mice using tissue-specific promoters to cofactors. Most of these cofactors belong to the “three loop limit the expression within developing lymphoid cells (e.g., amino-acid-loop extension” homeobox family (48). Early work CD2-LMO2, ref. 38; LCK-TLX1, ref. 37). However, generat- over a decade ago showed an essential role for MEIS1 as a ing transgenic knock-in mice remains technically laborious cofactor of HOXA9-driven AML in mice (49, 53), and since and time consuming. To this end, we developed a retroviral this time, many additional HOXA9-interacting factors have expression strategy for lineage-specific expression using the been identified including STAT5 in AML (33, 48). Indeed, bone marrow transplant system that is compatible with the STAT5B activation is important in AML, and loss of STAT5B numerous well-characterized Cre mice now available, and decreases the leukemia-initiating cell frequency in HOXA9/ we demonstrated the efficacy of this retroviral vector to rap- MN1-induced leukemias (54). In our current work, we find idly generate mouse T-ALL models. Moreover, this system a striking growth advantage and cellular transformation of allows a more physiologic comparison of cellular competition HSPCs transduced with JAK3M511I and HOXA9 that is pheno- between wild-type and transgene-expressing cells during dif- copied by constitutive active STAT5BN642H coexpressed with ferentiation and potential leukemia development. HOXA9. Moreover, in our bone marrow transplant model, we From a mechanistic perspective, the integrated ChIP-seq see the rapid development of both AML and T-ALL clones in and RNA-seq of JAK3M511I/HOXA9-driven leukemias revealed vivo showing that sustained STAT5 activation and HOXA9 that HOXA9 expression leads to a significant amplification expression are bona fide cooperative events in driving leuke- of STAT5 transcriptional output and to addiction of leukemia development in both lineages. Significantly, the levels mia cells to STAT5 activation. This reconfirms STAT5 as a of MEIS1 expression are very low in our STAT5/HOXA9- major central player in T-ALL pathogenesis and identifies driven leukemias and, together with the principal component STAT5 target genes (such as PIM1) as potential targets for analysis segregating HOXA9/MEIS1 AML away from both therapy. PIM1 is a constitutive serine threonine kinase over- JAK3M511I/HOXA9 AML and T-ALL, suggest that MEIS1 is not expressed in a number of cancers and regulates a number important in our JAK3M511I/HOXA9 leukemia models. Fur- of different oncogenic processes including cell survival and thermore, we showed that the resulting JAK3M511I/HOXA9- apoptosis, making it an attractive target for inhibition (39). driven leukemia was sensitive to tofacitinib, suggesting these Indeed, PIM1 inhibition has been shown to be effective in a leukemias remain dependent on the STAT5 signaling and are triple-negative breast cancer model in vivo (40), and more not purely HOXA9-driven leukemias. Taken together, our recently also in T-ALL cell lines and human T-cell leukemia virus data identify STAT5 as a novel cofactor of HOXA9 in T-ALL. type 1–associated adult T-cell leukemia and T-cell lymphoma Interestingly, transformed progenitor cells expressing (41, 42). Secondly, HOXA9 through cobinding with STAT5 also HOXA9 and MEIS1a but not HOXA9 induced the expression leads to the upregulation of FOS and JUN and AP1 target genes. of FLT3 and IL7R (50), both of which activate STAT5 down- Members of the AP1 complex are overexpressed in numerous stream, suggesting that our leukemia model in part bypasses lymphoid malignancies, including T-cell leukemia, and have the requirement of MEIS1 via direct activation of STAT5. The been recently reviewed (43) and therefore could further contrib- link between HOX cluster activation and activated STAT sign- ute to the enhanced leukemogenesis. The addition of HOXA9 aling is also observed in acute megakaryoblastic leukemia with with JAK3 mutations thereby upregulates the AP1 complex at HoxR cells also carrying an activating mutation of the MPL the transcriptional levels directly rather than the canonical AP1 receptor driving the phosphorylation of STAT5 (55). Similarly, activation by the upstream MAPK signaling pathway. MLL intragenic abnormalities in AML upregulate HOXA9 and HOXA9 has a well-established role in both hematopoiesis also correlated with FLT3 mutations that activate STAT5 (56). and leukemia. HOXA9 loss-of-function studies in mice show In conclusion, we show that HOXA9 and JAK3/STAT5 are deficits in committed myeloid, erythroid, and B-cell progeni- bona fide cooperating factors in driving leukemia develop- tors and perturbations in T-cell development (44, 45). Con- ment and that the cooperation is situated at the transcrip- versely, overexpression of HOXA9 in transgenic mice using tional level where STAT5 and HOXA9 co-occupy similar lymphoid-specific regulatory elements results in mild CD8 genomic regions. Furthermore, our results have significance expansion (46). Early evidence for HOXA9 in the development not only for T-ALL but also for other leukemias where acti- of leukemia was the identification of recurrent translocation vated STAT5 would potentially cooperate with HOXA9. t(7;11) producing the NUP98–HOXA9 fusion (47) with subsequent studies showing MLL fusions and NPM1 also resulting in HOXA9 overexpression (48). Early studies of ectopic METHODS HOXA9 expression in mice showed weak leukemogenic poten- Expression Plasmids and Retrovirus Production tial with either AML developing after a long latency using The MSCV-JAK3M511I-IRES-GFP retroviral vector is as previ- retroviral transduction/transplantation (49, 50) or a T-cell ously described (13). The human HOXA9 cDNA was synthesized malignancy (preT LBL) using a transgenic approach (51). Our by Genscript and cloned into the MSCV-IRES-mCHERRY vector.

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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE

The LOX66/71-IRES GFP vector was generated by cloning in the Ex Vivo Cell Culture Transformation Assay LOX66/71 sites directly into the multiple cloning site of MSCV-GFP The Lin– cells were spin infected with two retroviral constructs retroviral vector. Retrovirus production using 293T cells and retrovi- leading to lineage-negative cells with all four combinations— ral transduction of Ba/F3 cells and hematopoietic lineage–negative nontransduced, single transduced JAK3M511I only, HOXA9 only, and cells were performed as previously described (13, 57). double transduced JAK3M511I/HOXA9. The day after transduction, the cells were split into media containing only 20% FBS/RPMI, removing RNA Extractions and qPCR the SCF, IL6, and IL3 that were present. The cells were then left to RNA was extracted from tissue and cells using either the illustra grow in this cytokine-free media and split when appropriate. There RNAspin Mini Kit (GE Healthcare Life Sciences) or the Maxwell 16 was no sorting of cells prior to cytokine withdrawal. LEV Simply RNA Purification Kit (Promega) as per the manufac- turer’s instructions. cDNA synthesis was carried out using GoScript In Vivo and Ex Vivo Treatment of PDX Samples (Promega) and real-time quantitative performed using the GoTaq PDX samples were transplanted in 8-week-old NSG mice through qPCR master mix (Promega) with the ViiA7 Real Time PCR system tail-vein injection. Human leukemic cell expansion was monitored (Applied Biosystem). Quality control, primer efficiency, and data through human CD45 staining on blood samples. Single cells were analysis were carried out using qbase+ software (Biogazelle). All gene isolated from the spleen, which at time of sacrifice contained> 85% expression was normalized using two housekeeping reference genes. human CD45+ cells. Spleen cells were seeded in 96-well plate (5 × Primers used for qPCR are listed in Supplementary Table S4. 105 cells/well) and incubated with vehicle (DMSO) or inhibitor. Cell viability was assessed at 48 hours using ATP-Lite. CompuSyn was RNA-seq used to calculate the combination index. For in vivo treatment stud- The single-end RNA-seq data were first cleaned (i.e., removal of ies, the XC65 was transduced overnight with lentivirus pCH-SFFV- adapters and low-quality parts) with the fastq-mcf software after eGFP-P2A-fLuc. The GFP-positive cells were then sorted using the S3 which a quality control was performed with FastQC. The reads were Sorter (Bio-Rad) and retransplanted back into recipient NSG mice. then mapped to the Mus Musculus (mm10) genome with Tophat2. Upon confirmation that XC65 was greater than> 95% GFP positive, To identify the gene expression, HTSeq-count was used to count leukemic cells were isolated from the spleen and reinjected into a the number of reads per gene. These read count numbers were then larger cohort of NSG mice for acute 7-day in vivo treatment. Ruxoli- normalized to the sample size. Differential gene expression analysis tinib (HY-50858, MedChem Express) was dissolved in 0.5% methylcel- was performed with the R-package DESeq2 (Supplementary Table lulose, AZD1208 was dissolved in 50% PEG400/0.5% methylcellulose, S2). Pathway enrichment and other gene set enrichment analy- and both were administered by oral gavage. ses were done with GSEA. GSEA datasets were downloaded from http://download.baderlab.org/EM_Genesets/. The data are accessible Flow Cytometry Analyses through GEO Series accession number GSE109653 (https://www.ncbi. Single-cell suspensions were prepared from peripheral blood, bone nlm.nih.gov/geo/query/acc.cgi?acc=GSE109653). marrow, spleen, thymus, and lymph nodes. Cells were analyzed on either a FACS Canto flow cytometer (BD Biosciences) or MACSQuant Western Blotting Vyb (Miltenyi) with the following antibodies: CD3e, CD8a, CD45, Cells were lysed in cold lysis buffer containing 5 mmol/L CD4, CD45R, CD44, CD25, CD11b, Gr1/Ly6G, CD127, CD117, and

NA3VO4 and protease inhibitors (Complete EDTA-free, Roche). TCRbeta. These antibodies were conjugated to phycoerythrin (PE), The proteins were separated on NuPAGE NOVEX Bis-Tris 4%–12% allophycocyanin (APC), or peridinin chlorophyll protein Cyanine5.5 gels (Life Technologies) and transferred to PVDF membranes. (PerCP-Cy5), VioBlue, PE-Vio770, or APC-Vio770. For intracellular Subsequent Western blot analysis was performed using antibod- phospho-STAT5 staining, cells were first fixed in a 1:1 ration with ies directed against phospho-JAK1/JAK3 (SC-16773), STAT5 (SC- IC fix buffer (Invitrogen; #00-8222-49) and then permeabilized in 1081), JAK1 (Millipore; 05-1154), phospho-STAT5 (Cell Signaling ice-cold 100% methanol. Staining was then carried out in PBS/2% Technology; #9359), HOXA9 (Millipore; 07-178), and beta-actin FCS with 1:20 dilution pSTAT5-APC (eBioscience; #4334964) or (Sigma; A1978). Anti–phospho-JAK1 antibody was used to detect IgG1 kappa isotype (eBioscience; #17-4714-41) control. Data were both phosphorylated JAK1 and JAK3. Western blot detection was analyzed with the FlowJo software (Tree Star). performed with secondary antibodies conjugated with horseradish peroxidase (GE Healthcare). Bands were visualized using a cooled ChIPmentation, ChIP-Seq, and CUT&RUN ChIP-Seq charge-coupled device camera system (ImageQuant LAS-4000; GE ChIPmentation ChIP-seq on mouse leukemic samples was carried Health Care). out as described with modifications (58). ALL-SIL cells were origi- nally sourced from DSMZ in 2004 and authenticated using short Murine Bone Marrow Transplantation tandem repeat analysis and PCR for the NUP214–ABL1 fusion in Male C57BL/6 or BALB/c mice were purchased from Charles River 2017. Briefly, 20 to 40 million cells were washed in PBS and cross- Laboratories. Male mice were sacrificed, and bone marrow cells were linked with 1% formaldehyde for 10 minutes at room temperature harvested from femur and tibia. Lineage-negative cells were enriched and then quenched by addition of glycine (125 mmol/L final con- (STEMCELL Technologies) and cultured overnight in RPMI with centration). For nuclei isolation, cells were resuspended in 1× RSB

20% FCS with IL3 (10 ng/mL; Peprotech), IL6 (10 ng/mL; Peprotech), buffer (10 mmol/L Tris, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2) SCF (50 ng/mL; Peprotech), and penicillin–streptomycin. The follow- and left on ice for 10 minutes to swell. Cells were collected by cen- ing day, 1 × 106 cells were transduced by spinoculation (90 minutes at trifugation and resuspended in RSBG40 buffer (10 mmol/L Tris, pH

2,500 rpm) with viral supernatant and 8 μg/mL polybrene. The fol- 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, 10% glycerol, 0.5% NP40) lowing day, the cells were washed in PBS and injected (1 × 106 cells/ with 1/10 v/v of 10% detergent (3.3% w/v sodium deoxycholate, 0.3 mL) into the lateral tail vein of sublethally irradiated (5 Gy) 6.6% v/v Tween-40). Nuclei were collected by centrifugation and syngeneic female recipient mice. Mice were housed in individually resuspended in L3B+ buffer (10 mmol/L Tris-Cl, pH 8.0, 100 mmol/L ventilated cages and monitored daily. End-point analysis for DFS was NaCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA, 0.1% Na-Deoxycholate, defined at WBC> 30,000 cells/mm3. In vivo treatment of mice with 0.5% N-Lauroylsarcosine, 0.2% SDS). Chromatin was fragmented tofacitinib was carried out as previously described (13). to 200 to 400 bp using 20 cycles (30 seconds on, 30 seconds off,

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RESEARCH ARTICLE de Bock et al.

High Setting) using the Bioruptor (Diagenode). Chromatin immunoprecipitation was carried out overnight in the presence of anti- REFERENCES bodies (H3K27me3 07-449 Millipore or H3K27me3 Cat# 39155 . 1 Van Vlierberghe P, Ferrando A. The molecular basis of T cell acute Active Motif Lot#06517018; H3K4me3 Cat# 39159 Active Motif lymphoblastic leukemia. J Clin Invest 2012;122:3398–406. Lot#15617005; H3K4me1 Cat#39297 Active Motif Lot#01714002; 2. Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward Active Motif H3K27Ac Cat#4729 Lot#GR251958-1, or H3K27Ac T-cell acute lymphoblastic leukemia. Blood 2017;129:1124–33. Cat#39134 Active Motif Lot#20017009; STAT5 Cell Signaling 3. Girardi T, Vicente C, Cools J, De Keersmaecker K. The genetics and Cat#8363 Lot9; HOXA9 Sigma Atlas Antibodies Cat#HPA061982 molecular biology of T-ALL. Blood 2017;129:1113–23. Lot#86503) coupled to magnetic protein A/G beads (Millipore). 4. 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HOXA9 and Mutant JAK3 Cooperation in Leukemia RESEARCH ARTICLE

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HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development

Charles E. de Bock, Sofie Demeyer, Sandrine Degryse, et al.

Cancer Discov 2018;8:616-631. Published OnlineFirst March 1, 2018.

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