TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia by Jianhua Yu, Maxim Ershler, Li Yu, Min Wei, Björn Hackanson, Akihiko Yokohama, Takeki Mitsui, Chunhui Liu, Hsiaoyin Mao, Shujun Liu, Zhongfa Liu, Rossana Trotta, Chang-gong Liu, Xiuping Liu, Kun Huang, Jan Visser, Guido Marcucci, Christoph Plass, Alexander V. Belyavsky, and Michael A. Caligiuri Blood Volume 113(22):5558-5567 May 28, 2009 ©2009 by American Society of Hematology

Methylation of the TSC-22 promoter in mouse T or NK LGL leukemia. Methylation of the TSC-22 promoter in mouse T or NK LGL leukemia. (A) Examination of sequences within the murine TSC-22 promoter shows a CpG island located from −300 to +100 (G + C, > 50%; length, > 200; observed/expected ratio, > 0.6). The vertical lines are indicative of CG dinucleotides; TSS indicates transcription start site. (B) Leukemic cells were enriched by flow cytometry (≥ 95%) from spleens of the representative mice with T or NK LGL leukemia. Data of the L1210 T-cell lymphoma cell line and of nonleukemic cells (enriched from WT mice or IL-15tg mice with polyclonal T- and NK-cell expansion) were included for comparison. Combined bisulfite restriction analysis (COBRA) of DNA from these malignant and nonmalignant populations provided evidence of TSC-22 promoter methylation (arrow) in L1210 cell line and in the T or NK LGL leukemia cells (Leu) but not in control WT cells (WT) or in cells from IL-15tg mice with polyclonal T- and NK-cell expansion (Tg). Numbers below the agarose gel represent the intensity of the methylated band, normalized by the upper unmethylated band. (C) Bisulfite sequencing of the TSC-22 promoter region from WT, IL-15tg polyclonal, and T or NK LGL leukemia from mouse spleens. Each row of ovals represents the sequence of an individual clone. Unshaded ovals indicate unmethylated CpG site; shaded ovals, methylated CpG site; TSS, transcription start site, which is designed as +1. (D) COBRA analysis of DNA from the TSC-22 promoter region in 5 murine cell lines derived from hematologic malignancies. The 2 digested fragments (arrows) correspond to methylated DNA separated on a PAGE gel. Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology

Reduced or absent TSC-22 expression in hematologic malignant tissue is reversed by the demethylation agent 5-Aza in vitro. Reduced or absent TSC-22 expression in hematologic malignant tissue is reversed by the demethylation agent 5-Aza in vitro. (A) Expression of TSC-22 detected by regular RT-PCR in WT, polyclonal IL-15tg (Tg), and primary T or NK LGL leukemia (Leu) mouse splenocytes. (B) RT-PCR of the murine lymphoid tumor cell lines YAC-1 and EL-4 indicated that TSC-22 mRNA was undetectable in untreated cells. Treatment with the demethylation agent 5-Aza restored TSC-22 expression in a dose-dependent fashion. Treatment with trichostatin A (TSA, 300 nM), a histone deacetylase inhibitor, did not have a significant effect on TSC-22 transcription. Amplification of HPRT was included as an internal control. Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology

TSC-22 promoter hypermethylation attenuates gene expression. TSC-22 promoter hypermethylation attenuates gene expression. (A) Luciferase reporter assays in 293T cells transfected with variable lengths of the mouse (m) TSC-22 promoter indicate that strong promoter activity occurs within the −320-bp proximal region. (B) In vitro methylation of the −320-bp promoter of TSC-22 with Sss1 DNA methyltransferase (shaded bar) indicates that methylation of the −320-bp promoter region significantly inhibits TSC-22 promoter activity, which was detected by luciferase assays, compared with an unmethylated control construct (unshaded bar; P < .05). Error bars indicate standard deviations for triplicates in 1 of 3 representative experiments (A,B). Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology

In vivo 5-Aza therapy reveres silenced TSC-22 gene expression and prolongs survival in mice bearing T or NK LGL leukemia. In vivo 5-Aza therapy reveres silenced TSC-22 gene expression and prolongs survival in mice bearing T or NK LGL leukemia. (A) In vivo treatment of mice moribund with T or NK LGL leukemia using PBS (“untreated,” ▵) or 5-Aza (▴) for 4 consecutive days shows a significant reversal, with up-regulation of TSC-22 expression as assessed by real-time RT-PCR (P < .05). Horizontal bars indicate mean values. The TSC-22 expression levels of the untreated samples were normalized to 1. (B) One million primary T or NK LGL leukemia cells from IL-15tg mice were adoptively transferred into sublethally irradiated WT FvB mouse recipients. When engrafted mice showed progressive disease, they received daily subcutaneous injections with either 1 or 2 cycles of the indicated dose of 5-Aza or PBS for 4 consecutive days. Mice receiving 2 cycles of therapy had 4 days of rest between cycles at doses of 12 μg or 8 days of rest between cycles at doses of 32 μg. As shown by the Kaplan-Meier curve, mice given increasing doses and/or cycles of 5-Aza displayed prolonged survival compared with control mice treated with PBS (P < .001 among all groups). Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology

Ectopic expression of TSC-22 in malignant cells inhibits cell proliferation and delays tumor formation in vivo. Ectopic expression of TSC-22 in malignant cells inhibits cell proliferation and delays tumor formation in vivo. (A) Growth of YAC-1 cells transfected with a pMSCV vector containing TSC-22 exhibited significant inhibition of proliferation in vitro compared with YAC-1 cells transfected with the empty vector alone (P < .05). This experiment was repeated 3 times with similar results. (B) In vivo tumorigenesis resulting from subcutaneous injection of YAC-1 cells ectopically expressing TSC-22 or the pMSCV vector alone into NOD-SCID mice. Tumor size was measured daily. Overexpression of TSC-22 in YAC-1 cells delays the onset of tumor formation in vivo by an average of 2 days (P < .05). (C) The weights of tumors collected from mice killed at the end of the in vivo experiment (day 17). The average weight of tumors formed from YAC-1 cells ectopically expressing TSC-22 was less than that of tumors formed from the YAC-1 cells transfected with the vector alone (P < .05). (D) Photograph of the representative tumor pairs excised from killed mice that had been injected with YAC-1 cells ectopically expressing the pMSCV vector alone (top) or TSC-22 (bottom). (E) Western blot detection of the TSC-22 protein levels in tumors derived from the injection of YAC-1 cells ectopically expressing the pMSCV vector alone or TSC-22 into NOD-SCID mice. Presence of MYC-tag indicates ectopic TSC-22 expression. The tumorigenicity assay in panels B-E was performed twice with a total of 16 mice. Injected transfected YAC-1 tumor cells were first purified to 99% or more purity by cell sorting for GFP. Error bars in panels A-C indicate SDs (n ≥ 3) in one representative experiment. Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology

Generation of TSC-22–deficient mice. Generation of TSC-22–deficient mice. (A) Schematic representation of the targeting construct and homologous recombination in the TSC-22 region. Restriction enzyme sites of KpnI, NheI, and XbaI at the TSC-22 locus are indicated as K, N, and X, respectively. The probes used in Southern screening of embryonic stem cell and the expected sizes of endogenous and mutated fragments obtained by KpnI single digestion or XbaI and NheI double digestion are shown. (B) Southern blot analysis of KpnI-digested DNA extracted from the littermates derived from a cross between heterozygous TSC-22 mice by using probe 1 (A). The 7.5-kb band indicates the existence of the TSC-22 allele, whereas the 4.0-kb band shows the allele loss. The double digestion of XbaI and NheI followed by hybridization with probe 2 (A) gives rise to a 9.5-kb fragment for the existence of the TSC-22 allele and a 7.5-kb fragment for the allele loss (not shown). (C) Northern blotting (left panel) and Western blotting (right panel) to confirm the lack of TSC-22 expression in the TSC-22−/− mice. Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology

Lin−CD117+ BM cells from TSC-22−/− mice show higher proliferation than those from TSC-22+/+ mice in vivo. Lin−CD117+ BM cells from TSC-22−/− mice show higher proliferation than those from TSC-22+/+ mice in vivo. (A) Representative example of BM cells obtained from TSC-22+/+ and TSC-22−/− mice after ingesting BrdU-containing drinking water for 7 consecutive days. BM cells were stained for Lin markers, CD117, and BrdU and then were analyzed by flow cytometry. The region shaded in gray represents Lin−CD117+ BM cells from TSC-22−/− mice and shows higher BrdU incorporation than identical BM cells obtained from TSC-22+/+ mice (open area, black line). (B) Summary of data represented in panel A but performed on 3 pairs of littermate mice. There is significantly higher BrdU incorporation in the TSC-22−/− Lin−CD117+ BM cells compared with those of TSC-22+/+ mice (P < .05, n = 3). Error bars indicate standard errors for 3 independent experiments. Jianhua Yu et al. Blood 2009;113:5558-5567 ©2009 by American Society of Hematology