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Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 45, pp. 30788 –30795, November 7, 2008 Printed in the U.S.A. Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish □ S S-Adenosylmethionine Synthesis Received for publication, May, 30, 2008, and in revised form, August 21, 2008 Published, JBC Papers in Press, August 27, 2008, DOI 10.1074/jbc.M804159200 ‡§ ‡§ ‡ ¶ § § Ramy R. Attia , Lidia A. Gardner , Engy Mahrous , Debra J. Taxman , Leighton LeGros , Sarah Rowe , ¶ ‡ ‡§1 Jenny P.-Y. Ting , Arthur Geller , and Malak Kotb ‡ § 2 From the University of Tennessee Health Science Center and the Research Service, Veterans Affairs Medical Center , Memphis, Tennessee 38104, the Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, and the Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524 We exploited the fact that leukemic cells utilize significantly goal has been to develop novel approaches that exploit physio- higher levels of S-adenosylmethionine (SAMe) than normal logical differences in metabolic needs between normal and leu- lymphocytes and developed tools that selectively diminished kemic cells to generate tools that would selectively diminish their survival under physiologic conditions. Using RNA inter- tumor cell growth in vivo, with minimal harm to normal host ference gene silencing technology, we modulated the kinetics of cells. Specifically, we sought to exploit significant differences in methionine adenosyltransferase-II (MAT-II), which catalyzes S-adenosylmethionine (SAMe) metabolism between normal SAMe synthesis from ATP and L-Met. Specifically, we silenced and leukemic T cells (4, 5). SAMe is an essential molecule in the the expression of the regulatory MAT-II subunit in Jurkat cells metabolism of every living species (6–10). As the main methyl and accordingly shifted the K of the enzyme 10–15-fold group donor, it methylates DNA, RNA, fatty acids, proteins, m L-Met above the physiologic levels of L-Met, thereby reducing enzyme and small molecules and regulates several transcription and activity and SAMe pools, inducing excessive apoptosis and translation processes, protein function, and membrane integ- diminishing leukemic cell growth in vitro and in vivo. These rity. SAMe is also involved in DNA mismatch repair, chromatin effects were reversed at unphysiologically high L-Met (>50 M), modeling, epigenetic modifications and imprinting, cell repli- indicating that diminished leukemic cell growth at physiologic cation, neurotransmission, and signaling (11). Additionally, L-Met levels was a direct result of the increase in MAT-II SAMe is an important precursor of the polyamines and a major K due to MAT-II ablation and the consequent reduction player in biological trans-sulfuration as well as folic acid and m L-Met null in SAMe synthesis. In our NOD/Scid IL-2R humanized one-carbon metabolism (8, 9, 11, 12). mouse model of leukemia, control shRNA-transduced Jurkat The importance of SAMe, together with the fact that its cells exhibited heightened engraftment, whereas cells lacking metabolism is constitutively elevated in malignant versus nor- MAT-II failed to engraft for up to 5 weeks post-transplant. mal cells, has for years made it an attractive target for cancer These stark differences in malignant cell survival, effected by chemotherapy (13–20). Unfortunately, chemical inhibitors of MAT-II ablation, suggest that it may be possible to use this SAMe synthesis have been difficult to generate in quantities approach to disadvantage leukemic cell survival in vivo with lit- needed for clinical use, and most were either unstable, reversi- tle to no harm to normal cells. ble, nonspecific, or highly toxic because no cell can survive total inhibition of SAMe synthesis. To this end, our approach has been to take advantage of the dependence on higher SAMe levels in leukemic cells to diminish rather than totally block Leukemia are among the deadliest and most common can- their ability to synthesize the needed amount of SAMe and cers. Despite advancements in novel individual and combina- tion drug treatment modalities, mortality rates remain high, thereby selectively halt their growth while sparing normal cells. The advent of novel biotools that can selectively silence pro- and some medications have serious adverse effects (2, 3). Our tein expression has made it possible to initiate studies to target the regulatory subunit of methionine adenosyltransferase * This work was supported, in whole or in part, by National Institutes of Health (MAT), which catalyzes the synthesis of SAMe from L-Met and Grant R01CA108792. This work was also supported by a Veterans Affairs ATP. All living organisms have at least one MAT enzyme (5, Merit Review Award and a Senior Research Career Scientist Award (to M. K.). The costs of publication of this article were defrayed in part by the 13). Mammals have liver-specific MAT-I/III and another payment of page charges. This article must therefore be hereby marked isozyme, MAT-II, that is expressed in all tissues (21, 22). MAT- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi- I/III are tetramer/dimer forms of a catalytic 1 subunit, and cate this fact. □ S they differ considerably in their kinetic and physical properties. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1. To whom correspondence should be addressed: Dept. of Molecular Genet- ics, Biochemistry and Microbiology, University of Cincinnati College of The abbreviations used are: MAT, methionine adenosyltransferase; SAMe, Medicine, 231 Albert Sabin Way, P.O. Box 670524, 2938 CVC Mail Loc-0524, S-adenosylmethionine; GFP, green fluorescence protein; shRNA, short Cincinnati, OH 45267-0524. Tel.: 513-558-5231; Fax: 513-558-1190; E-mail: hairpin RNA; HPLC, high pressure liquid chromatography; BM, bone mscbskotb@gmail.com. marrow. 30788 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 This is an Open Access article under the CC BY license. Targeting SAMe Metabolism in Leukemia It is believed that this differential oligomerization of MAT-1is data base to ensure their specificity for our target gene. The an important adaptation to cope with special metabolic pHSPG plasmid (Dr. Su, University of North Carolina) has a requirements in the liver, where SAMe levels need to be main- constitutively active polymerase III promoter in front of the tained at a certain range inasmuch as a deficiency or excess of multiple cloning site. We modified pHSPG by including a His- SAMe has been associated with serious pathology (23–25). In tone 1 promoter needed for shRNA formation (34), followed by livers of healthy subjects, MAT-III, which has a high K shRNA constructs of interest between the EcoRV and XbaI m L-Met (80–100 M), is the major isoform. By contrast, MAT-II is a sites, in the multiple cloning site. Downstream of this construct hetero-oligomer that has a catalytic2 subunit and a regulatory is a PGK-GFP cassette whose expression is driven by the mouse subunit with a K of 4–20 M (21, 26, 50). The 2 sub- mammary tumor virus promoter. The HSPG virus was chosen m L-Met unit, which has 84% sequence identity to 1, undergoes post- for its high transduction efficiency in hematopoetic cells (36, translational modifications resulting in expression of 2 (53 37). The design of different MAT-II-specific pHSPG-shRNA kDa) and 2 (51 kDa) forms (21). In fetal liver and certain adult drivenbytheHistone1promoterisshowninsupplementalFig.S1. liver diseases, including hepatocellular carcinoma, 1 subunit The effect of several control and MAT-II-specific pHSGP- expression is diminished and replaced by 2, along with the shRNA constructs on MAT-II expression were initially tested induction of MAT-II expression (27, 28). in transiently transfected Cos-1 cells. These studies showed MAT- subunits are highly conserved across many species that plasmid pHSGP-shRNA-1110 had the highest gene silenc- (22); by contrast MAT-II is only found in mammals, associ- ing activity and was not toxic to the Cos-1 cells, and thus we ated with MAT-II2. In several of our previous studies, we packaged it into HSPG-V1110 viral particles using HEK-293T showed that MAT-II plays a crucial physiological role by low- cells and the CaCl transfection method (35). We also gener- ering the K of MAT-II for L-Met from 55–65 M down to ated a control, empty virus without shRNA (V1302) and V1324 3.5–20 M (26, 29, 50). Inasmuch as the physiologic extrahe- that encodes an shRNA for an irrelevant, mouse plexin A1 gene patic concentration of L-Met are 5–10-fold lower than that in (37). the liver (30), we believe that the introduction of MAT-II to Transduction with HSPG-shRNA Retrovirus—We trans- lower the K of the extrahepatic enzyme may have been an duced Jurkat cells (10 cells) by adding 8 mg/ml polybrene plus essential evolutionary event that allowed MAT-II to function in 700 ml of viral supernatants and incubating for 20 min at room blood and other extrahepatic mammalian tissues, where L-Met temperature. The cells were spun at 2000 rpm for 3 h and then levels are 10–25 M (31–33). resuspended in fresh 1.5 ml of RPMI 1640 complete medium. We had reported that MAT-II expression and SAMe metab- We cultured the cells in 12-well tissue culture plates, repeated olism are considerably different in normal and malignant lym- the transduction after 24 and/or 48 h, and then assessed trans- phocytes (4, 5, 8). MAT-II expression in established and pri- duction efficiency by flow cytometry (FACSCalibur) to deter- mary human lymphocytic leukemia cells is significantly higher mine the percentage of GFP-positive cells. This procedure typ- than in quiescent or activated lymphocytes (4). MAT-II activity, ically yielded 90% GFP cells, which were then sorted using SAMe utilization rate, and SAMe pool size are, respectively, FACSAria (BD Biosciences) to obtain a 98% transduced cell 20-, 60-, and 60–100-fold higher in lymphocytic leukemia, than population. in normal lymphocytes (4). Based on these previous studies (4, MAT-II Subunits Expression Analysis—We lysed GFP 21, 26, 29), we predicted that if we specifically ablated MAT-II sorted cells by three cycles of freeze-thawing in extraction expression, we would shift MAT-II K by at least 10-fold buffer (50 mM Tris, pH 7.4, 50 mM NaCl, 5 mM MgCl ,4mM m L-Met 2 above physiologic L-Met levels, and that this would conse- dithiothreitol), containing a mixture of proteolytic inhibitors quently reduce SAMe pool size and selectively diminish the (Roche Applied Science) as described (5). Protein concentra- growth of leukemic cells in physiological fluids and extrahe- tion was determined in the cleared lysates by the bicinchoninic patic tissues. We report that MAT-II subunit specific shRNA acid method (38). Equal amounts of protein extracts were sep- successfully silenced the expression of the MAT-II regulatory arated on 10% SDS-PAGE and then transblotted onto nitrocel- subunit in the Jurkat leukemic T cell line and increased the lulose papers. Expression of 2 and  subunits was determined enzyme K by 10–15-fold, consequently depleting SAMe by Western blots and probed with antibodies to MAT-II and m L-Met pools, inducing excessive apoptosis, and diminishing the MAT-II proteins (4, 39). growth of these leukemic cells in physiologic L-Met concentra- We also used quantitative real time PCR to assess MAT- tions, both in vitro and in vivo in a humanized NOD/Scid II and MAT-II mRNA expression. We constructed cRNA null IL-2R mouse model of leukemia. standards for each subunit and generated standard curves for each run to quantify mRNA copy number/2 g of total EXPERIMENTAL PROCEDURES RNA. Briefly, we transformed Escherichia coli strain JM109 Leukemic Cells—Jurkat T cells (E6–1; ATCC, Manassas, VA) with pTargeT/MAT-II subunit or pTargeT/MAT-II were maintained in RPMI 1640 medium, supplemented with subunit (26) and purified those plasmids using the Wizard either 10% fetal bovine serum or 1% HL-1 supplement (L- PureFection DNA purification system (Promega). Correct Met), 2 mML-glutamine, 50 g/ml streptomycin, and 50 pTargeT/MAT-II and pTargeT/MAT-II plasmids were units/ml of penicillin. verified by sequencing, using a T7 promoter primer. T7 Generation of pHSPG-shRNA Retrovirus—We designed sev- Ribomax large scale RNA production system (Promega) was eral shRNA sequences to target MAT-II expression and used to generate MAT-II or MAT-II cRNA. The number BLAST-searched these sequences against the human genome of cRNA molecules were calculated as follows: N(molecules/ NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30789 Targeting SAMe Metabolism in Leukemia l)  [C*1825  10 ]/K, where C  cRNA g/l and K  In Vivo Animal Studies—All of the animals were treated bp (40). For MAT-II cRNA, K  1188 bp and C  0.658 according to Institutional Animal Care and Use Committee g/l; for MAT-II cRNA, K  1050 bp and C  0.632. regulations (protocol 1587). We irradiated highly immune defi- To test the efficiency of MAT-II silencing, we extracted cient NOD/ScidIL-2Rnull mice with 3.5GY, 24 h prior to 6 6 RNA from 10 untransduced or transduced cells using RNA- transplanting them via intraperitoneal injection of 15  10 STAT 60 (Tel-Test), removed residual contaminating genomic V1110 or V1302 Jurkat cells. We obtained sera from mice 12 DNA by DNase I treatment (Qiagen), purified the RNA using days post-injection and then once a week thereafter to quantify Qiagen RNeasy kit, and then converted 2 g of the purified levels of the surrogate tumor marker, soluble 2 microglobulin, RNA to cDNA using avian myeloblastosis virus reverse tran- using an enzyme-linked immunosorbent assay 2 microglobu- scriptase, 10 random hexamers, and 10 mM dNTP in the pres- lin kit (R & D). At 4 and 5 weeks post-transplant, we sacrificed ence of RNase inhibitor (Promega). Quantitative real time the transplanted mice (supplemental Table S1) and used fluo- PCRs were run using the fluorogenic SYBR Green quantitative rescence reflectance imaging to image GFP fluorescence in real time PCR system and the ABI prism 7900 Sequence Bio- spleen prior to extracting splenocytes and bone marrow cells to Detector (PE Biosystems). In each run, serial dilutions of MAT- assess tumor growth and engraftment by counting % human II or MAT-II cRNA (104–1010 cRNA molecules/l) were CD3/GFP-positive cells in each compartment by flow cytom- also converted to cDNA and used to generate the standard etry as detailed above. curve for each MAT-II subunit. The PCR mixture contained 25 Statistical Methods—We calculated statistical significance l of SYBR Green PCR Master mix (Applied Biosystems), 2.5 l with one-way analysis of variance and Newman-Keuls Multiple containing 1.5–12 pmol of each primer, and 5l of the template Comparison test for all the experiments except for the in vivo cDNA in a final volume of 50 l. The sequence of the MAT-II experiment where we used a Mann-Whitney test using the specific primers were (forward, 5-CACCTTACAGAGAGGA- GraphPad Prism program. AGA-3 and reverse, 5-CAGTCACAGCACTTTCTTC-3); and for MAT-II2 specific primers (forward, 5-AAAGTGGT- RESULTS TCGTGAAGCTGTTAAA-3 and reverse, 5-CCAAGGCTA- Successful Ablation of MAT-II Expression—We designed CCAGCACGTTAC-3). An 18 S RNA primer mix (Qiagen) several shRNA to ablate MAT-II expression, cloned them into was used for normalization. To calculate N for each test sample, the retroviral plasmid pHSPG, which also expresses GFP, and we first normalized the cycle threshold (CT) to the internal screened their gene silencing efficiency and nonspecific toxicity normalizer (18 S) and then determined N from the standard (supplemental Fig. S1). All MAT-II-specific shRNA con- curve using the equation Y  [MX  C]. structs were designed to target the two splice variants of MAT- Assay for MAT Activity—We assayed MAT activity in cell II (28). Initial screening was done in COS-1 cells, and con- extracts as described previously (21). For kinetic analyses, we structs with efficient gene silencing activity were then screened used different L-Met concentrations (1.25–80 M), using 14 in Jurkat cells (supplemental Fig. S1). Construct V1110 had the [ C]L-Met (57.9 mCi/mmol) and supplementing with cold highest MAT-II silencing activity (supplemental Fig. S1) and L-Met. Reaction velocity is expressed as units/mg protein, least toxicity and was thus packaged into infectious HSPG-GFP where 1 unit  1 nmol of adenosylmethionine/h (21). We cal- virus (36). culated K and V using the GraphPad Prism program. m max We transduced Jurkat cells with the various recombinant Cell Growth at Different L-Met Concentrations—We weaned HSPG-shRNA viruses and assessed MAT-II ablation at the the cells to grow in serum-free RPMI 1640 medium containing RNA and protein levels (supplemental Fig. S1). Controls 1% HL-1 supplement (Cambrex, NJ) plus 2 mML-glutamine, 50 included cells infected with an empty virus (V1302) or with a virus g/ml streptomycin, and 50 units/ml of penicillin (RPMI-HL1s encoding shRNA construct (V1324) directed to the mouse plexin medium). We seeded weaned cells (10 /ml) in RPMI-HL1s A1 gene (36). Although transduction efficiencies for all cells tested medium at L-Met 5–100 M, replaced the medium every 2 days, were 90%, significant reduction or complete ablation of MAT- recorded numbers of viable cells, and then replated the cells at II RNA and protein expression was only seen in V1110-trans- 10 /ml for further analyses. Cell necrosis and apoptosis were duced cells (Fig. 1). By contrast, expression of MAT-II2 was determined by a flow cytometric quantification of propidium essentially unaffected, thus indicating the MAT-II specificity of iodide and annexin-V (BD Pharmingen) stained cells, respec- V1110. In some cases, expression of MAT-II2 was insignificantly tively. In some studies, the cells were cultured at 10 ML-Met elevated, perhaps reflecting an attempt to increase enzyme synthe- for 3d, harvested, counted, and replated at 10 cells/ml in fresh sis to compensate for the lack of  expression and increase SAMe RPMI-HL1s medium with 10ML-Met. On day 6, the cells were synthesis. harvested, washed, and replated in fresh medium containing 20 Effect of MAT-II Silencing on MAT-II Activity, Kinetics, M or 50 ML-Met. The cell growth/death were determined SAMe Pool Size, and Cellular Growth—We next tested whether every 3 days thereafter, replating 10 viable cells/ml each time. ablation of MAT-II expression will alter MAT-II K and Measurement of SAMe Levels—We quantified SAMe levels in m L-Met reduce SAMe pools in Jurkat cells. As predicted, the K a neutralized 2 N perchloric acid-soluble extract by HPLC as pre- m L-Met viously detailed (4). We calculated the SAMe concentration increased from 3.5–6 M in untransduced and control virus- (pmol/10 cells) from a standard curve using different concentra- transduced Jurkat cells, up to 56–62 M in V1110 cells (Fig. tions of SAMe standard (USB Corp.) and then converted the val- 2A). Further, at the high end of physiological L-Met levels (20 ues toM based on cell volume for Jurkat cells 0.76 ml/10 cells (4). M), MAT-II specific activity was 4–5-fold lower in V1110 cells 30790 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 Targeting SAMe Metabolism in Leukemia than in untransduced or control virus-transduced Jurkat cells expressing MAT-II (Fig. 2B). Importantly, in the absence of MAT-II, SAMe was unde- tectable in V1110 cells cultured at 10 ML-Met (not shown), and even at 20 ML-Met SAMe pool size was85% less than in untransduced or control-transduced V1302 cells (9.5  0.5 M versus 62  4 M; p  0.05) (Fig. 2C). Significant 45–50%, reductions in SAMe pool size (p  0.01) were also found even when V1110 cells were maintained at 50 ML-Met, which is 2.5-fold higher than the upper range of physiological L-Met levels. The sharp reduction in SAMe pool size, consequent to MAT- II ablation, diminished the growth and viability of V1110 cells. At 20 ML-Met, V1110 cells ceased to grow, and at 50 M their growth was reduced by 33–36% compared with control- FIGURE 1. Successful ablation of MAT-II subunit using viral encoded transduced V1302 cells (p  0.01) (Fig. 3). By contrast, at 100 shRNA. MAT-II 2 and  protein expression (60 g of protein/well) and mRNA levels were analyzed in untransduced or 98% transduced cells as detailed ML-Met, V1110 cells grew at essentially the same rates and for under “Experimental Procedures.” Quantitative real time PCR was conducted the same duration in culture as the untransduced or control- using RNA extracted from cells maintained in 100 ML-Met RPMI medium, and RNA copy number was calculated as detailed under “Experimental Procedures.” transduced Jurkat cells (p  0.05) (Fig. 3). Thus, at unphysi- The results (means S.E.) of two combined experiments, each done in triplicate, ologically high L-Met levels approaching MAT-II2 K with- are shown. Statistical differences were determined as described under “Experi- out  (i.e. K  50–60 M), V1110 leukemic cells appear to mental Procedures.” MAT-II expression was significantly ablated in V1110 cells. ***, p  0.001. MAT-II2 was slightly elevated or unaffected (p 0.05). have satisfied their SAMe needs and grew normally. These results indicate that diminished V1110 cell growth at physiologic L-Met levels was a direct conse- quence of specific MAT-II silenc- ing, which caused an upper shift in K of MAT-II and a significant m L-Met reduction of SAMe synthesis and pools. Cell Death and Apoptosis of Leu- kemic Cells Lacking MAT-II—Sig- nificant reduction in growth rates of V1110 cells at physiological L-Met levels was associated with a signifi- cant increase in both cell necrosis and apoptosis (Fig. 4). At physio- logic L-Met levels (5, 10, and 20 M), the extent of necrosis in the V1110 cells was significantly higher (20– 55%) than in untransduced or con- trol V1302 cells (p  0.001). Mini- mal cell death was seen at 50 M L-Met; however, V1110 cells exhib- ited 70–80% higher levels of apo- ptosis than the control leukemic cells even at this unphysiological L-Met concentration (p 0.01) (Fig. 4). This is likely attributed to the 50% reduction in SAMe pool size even at 50 ML-Met (Fig. 2). Taken together, these findings indicate FIGURE 2. MAT-II ablation modulates MAT-II kinetics, reduces its specific activity, and diminishes intra- that the growth and viability of cellular SAMe levels. A, MAT-II Lineweaver-Burk kinetic plots of 1/V, where V is units of enzyme activity/mg of V1110 cells lacking MAT-II protein (units/mg; 1 unit 1 nmol of adenosylmethionine/h). MAT assays were performed using extracts from expression remained at a selective untransduced, V110- or V1302-transduced cells in the presence of 2.5– 80 ML-Met, as detailed under “Exper- imental Procedures.” The data for each cell extract represents the means S.D. of three separate experiments, disadvantage, even at more than each assayed in triplicate. B, MAT specific activity (units/mg of protein) at 20ML-Met. C, SAMe levels (pmol/10 twice the higher end of physiologi- cells) as determined by HPLC in neutralized perchloric acid extracts from cells that were maintained at 50 or 20 ML-Met. *, p  0.05; **, p  0.01; ***, p  0.001. cal L-Met levels. NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30791 Targeting SAMe Metabolism in Leukemia Attempts to Rescue Cells by Incrementally Elevating L-Met Levels—Following up on selective pressure imposed on the via- bility of MAT-II-deficient Jurkat cells in physiologic L-Met conditions, we next tested whether V1110 cells can be rescued by incremental elevation of L-Met in culture. All of the cells grew at lower rates at the low end (5–10 M) of physiologic L-Met levels compared with 20 M. We therefore maintained V1110 and control Jurkat cells at 10 ML-Met for 6 days in culture and then increased L-Met levels to either 20 or 50 M to determine whether these cells can be rescued (Fig. 5). The growth of control untransduced and control V1302 Jurkat cells increased by almost 80 and 90% when L-Met levels were raised to 20 or 50 M, respectively. By contrast, under the same con- ditions, V1110 cells could not be rescued with 20ML-Met (p 0.01) and exhibited diminished growth even at 50 ML-Met (p  0.05) (Fig. 5). These data suggest that the initial insult to the V1110 cells, caused by low SAMe levels due to the absence of MAT-II, is irreversible at the high end of physiologic L-Met levels (20 M) and minimally reversible at 50 ML-Met. Failure to rescue V1110 cells is consistent with the above observed increased apoptosis of these cells, even at 50 ML-Met (Fig. 4). MAT-II Ablation Diminishes Leukemic Cell Growth in an null NOD/Scid IL-2R Mouse Leukemia Model—To rule out possible in vitro culture artifacts, we investigated whether Jur- FIGURE 3. MAT-II ablation reduces leukemic cell growth in physio- logic L-Met levels. We seeded 5 10 of indicated cells at 5–100 ML-Met kat cells that do not express MAT-II would survive and mul- and monitored their growth every 2 days using the trypan blue exclusion tiply in vivo in our severely immunodeficient NOD/Scid method, adding fresh medium each time. *, p  0.05; **, p  0.01; ***, p null 0.001. IL-2R mouse model (41). These mice allow heightened engraftment with xenogeneic cells because, besides lacking T, B, and NK cells, they are also defi- cient in complement and macro- phage function (41). After mildly irradiating these mice, we trans- planted them intraperitoneally with 15  10 V1110 or control V1302 cells (n  15 mice/group). Irradi- ated, noninjected mice served as additional controls. We monitored tumor engraftment and growth weekly for 5 weeks post-transplant by determining the percentage of human CD3 and GFP expression in bone marrow (BM)- and spleen-de- rived cells and by measuring serum levels of the surrogate tumor marker, 2-microglobulin (2) (42). We also imaged GFP expres- sion in whole spleens of sacrificed mice for a semi-quantitative meas- ure of tumor burden (Fig. 6). During the first 3 weeks, none of the transplanted mice showed sig- nificant engraftment, but after 4 weeks, 57% of mice transplanted with control V1302 cells began to show variable levels of engraftment FIGURE 4. MAT-II ablation induces high levels of apoptosis and necrosis of leukemic cells. A, propidium iodide-stained necrotic cells (%) at 20 ML-Met. B, propidium iodide-stained necrotic cells (%) at 50 ML-Met. in spleen (3–44% human CD3 / C, necrotic cells (%) at 6 days in 5–50 ML-Met. D, annexin-V-stained apoptotic cells (%) at 20 ML-Met. GFP cells; mean 16  7.6%) and in D, annexin-V stained apoptotic cells (%) at 50 ML-Met. F, apoptotic cells (%) at 6 days in 5–50 ML-Met. *, p 0.05; **, p  0.01; ***, p  0.001. BM (3–12% human CD3 /GFP 30792 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 Targeting SAMe Metabolism in Leukemia DISCUSSION In this study, we showed that by silencing the expression of the regulatory  subunit of MAT-II, we exerted significant growth disadvantage upon human Jurkat leukemic T cells under physiologic L-Met levels in vitro and in vivo. In the liver, L-Met levels are 80–100 M, but in other tissues they range from 10 to 25 M. Thus, the high K hepatic isozyme, MAT-III, can be fully functional in that organ without MAT-II. By con- trast, MAT-II, the only SAMe-synthesizing enzyme in extrahe- patic tissue can only function when associated with its MAT- II regulatory subunit, which plays an important role in lowering MAT-II K to values close to physiological m L-Met L-Met levels outside the liver (26, 29, 43, 50). Without the subunit, MAT-II would function at minimal capacity and would not be able to provide normal extrahepatic cells with adequate SAMe levels needed for their growth and survival. In resting human T cells, MAT-II is expressed at low levels, and mitogenic activation of these cells does not induce further expression of this subunit; by contrast, MAT-II is constitu- tively expressed at a much higher level in established and pri- mary leukemic T cells (4, 5). Induction of MAT-II expression has been reported in several types of cancer and has been shown to confer a proliferative advantage to human hepatomas (44). The role of MAT-II in leukemic cells is even more crucial because leukemic T cells, both freshly explanted ALL-2 cells and the Jurkat cell line, utilize SAMe at significantly higher rates than normal resting or activated T cells (4, 5). The consti- tutive high expression of MAT-II in leukemic cells allows high levels of SAMe synthesis needed to meet the growth require- ments of malignant cells. In our quest to diminish SAMe in leukemic cells, it was unreasonable to target the MAT-II2 cat- alytic subunit, because that would be quite detrimental and toxic to normal cells that do not express the hepatic MAT-I/III isozyme, i.e. the majority of extrahepatic cells. However, target- ing MAT-II expression seemed much more appropriate and FIGURE 5. Differential rescue of Jurkat cells  MAT-II expression by practical, because this would not block SAMe synthesis com- incrementally increasing L-Met levels in culture. Untransduced and pletely but would reduce it to where the rapid growth and pro- V1302- and V1110-transduced Jurkat cells (5  10 ) were cultured at 10 M liferation of malignant lymphocytes and possibly other cancer- L-Met for 6 days, and then L-Met was raised to either 20 M (A)or50 M (B). Viable cells were counted over 15 days, with the addition of fresh medium ous cells can no longer be sustained. every 3 days. V1110 cells ceased to grow when L-Met was raised to 20 M and We succeeded in silencing the MAT-II regulatory subunit exhibited diminished growth even at 50 M.*, p  0.05; **, p  0.01; ***, p 0.001. without significantly affecting the expression of the MAT-II2 subunit. Under these conditions, our in vitro and in vivo studies provided strong evidence that MAT-II ablation is detrimental cells; mean 4  1.6%). Engraftment was much less evident in to the growth of malignant T cells and that this is directly due to spleens of mice transplanted 4 weeks earlier with V1110 cells drastic reductions in SAMe levels needed to support their rapid (0.96  0.32% CD3 /GFP cells; p  0.004). This trend con- growth. Inasmuch as it is quite feasible to modulate L-Met levels tinued through 5 weeks post-transplant, with significantly in vivo through dietary control (45), we believe these results are higher human CD3 /GFP cells in spleen and BM of mice of particular interest, especially in the context of our quest to injected with V1302 versus V1110 cells (supplemental Table 1S eventually translate these research findings into clinical appli- and Fig. 6). At 5 weeks, 25% of V1110 cells transplanted mice cations. Indeed, there is a large body of evidence that the growth started to show low levels of CD3 /GFP engraftment in of several types of tumors is dependent on high L-Met and that spleen (1.1 0.4%) and BM (2.4 1%). In stark contrast, 75% of L-Met deprivation causes cell cycle arrest in the G phase (46, mice transplanted with V1302 cells showed heightened 47). Several clinical studies demonstrated that diminishing engraftment in spleen (30  10%; p  0.02) and BM (11  4%; p  0.04) (Fig. 6). Additionally, levels of the surrogate tumor L-Met in cancer patients by maintaining them on a Met- or marker 2 were 2-fold higher in mice transplanted with con- choline-free diet and/or administering methioninase exerts trol V1302 leukemic cells than those transplanted with V1110 selective growth disadvantage pressure on cancer cells in vitro cells (supplemental Fig. S2). and in vivo, inducing mitotic and cell cycle arrest, apoptotic NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30793 Targeting SAMe Metabolism in Leukemia approach in the treatment of certain blood malignancies, and we are cur- rently exploring these possibilities. The growth of Jurkat cells with and without MAT-II expression was reduced at 10 M compared with 20 ML-Met; however, when we attempted to rescue these cells by incrementally increasing L-Met levels in culture, we found that the growth of cells expressing MAT-II was fully restored at 20 M, whereas cells lacking MAT-II could not be rescued at this concentration. Even at unphysiologically high 50 M L-Met we still observed significant apoptosis in cells lacking MAT-II. We therefore predict that it may be possible to adjust L-Met levels in vivo in a way that would rescue nor- mal cells while still inducing death and apoptosis of leukemic cells that do not express this important regu- latory subunit. We ruled out the possibility that our in vitro observations are due to culture artifacts, because when we compared the ability of Jurkat cells that do and do not express MAT-II to engraft and survive in an in vivo environment of physiologic L-Met levels in our hyperimmune defi- null cient NOD/ScidIL-2R mouse model, we found significant differ- ences in the in vivo tumor growth and engraftment, even in the absence of host immune rejection inasmuch as these mice were not reconstituted with a human FIGURE 6. MAT-II ablation diminishes leukemic cell growth in vivo. Hyperimmune-deficient NOD/Scid null 6 IL-2R mice were irradiated with 3.5GY 24 h prior to intraperitoneal transplant with 15 10 of the indicated immune system (41). Control trans- cells. Controls included irradiated but not injected mice and V1302 transplanted mice. Tumor engraftment duced Jurkat cells grew well in this and growth were monitored by measuring % human CD3 /GFP cells in mice spleens and bone marrow at 4 mouse model, whereas cells lacking and 5 weeks post-transplant with either control V1302 or V1110 cells. Flow cytometry histograms of CD3 / GFP in spleen (A) or bone marrow (B) 5 weeks post-transplant. C, GFP expression in whole spleens of same MAT-II expression showed signif- mice prior to processing their splenocytes. D, number of mice engrafted with V1302 or V1110 leukemic cells at icant diminution in engraftment for 4 and 5 weeks post-transplant. E and F show % CD3 /GFP cells in spleens (E) or bone marrow (F) of mice transplanted with either V1302 or V1110 leukemic cells, 5 weeks post-transplant. The statistical differences up to 5 weeks post-transplant. were calculated using a Mann-Whitney test. *, p  0.05; **, p  0.01; ***, p  0.001. Although these results are very promising, we are currently design- death, and widespread necrosis in tumors (48). Induction of ing studies to treat leukemic cells in vivo, after they have Jurkat cells apoptosis and death consequent to silencing MAT- engrafted. II is also in agreement with previous studies showing In summary, we had predicted that an approach that targets increased apoptosis and necrosis of tumor cells cultured in the SAMe metabolism would provide a good adjunctive therapeu- absence of L-Met (1, 49). We observed significant apoptosis in tic tool for the treatment of leukemia because of the their exces- Jurkat cells lacking MAT-II at physiologic L-Met levels, and sive requirement for this central metabolic compound. Our in this indicates that direct depletion of SAMe may be more effec- vivo data suggest that this approach, by itself, may have a more tive than L-Met depletion in inducing tumor cell apoptosis in drastic effect on diminishing leukemic cell growth than we had vivo. We believe that targeting MAT-II expression in combi- originally anticipated. The stark difference in the in vivo sur- nation with restricting dietary L-Met alone or in conjunction vival and engraftment between the V1110- and V1302-trans- with chemotherapeutic agents may prove to be a powerful duced cells in mice lacking proper immune defenses lead us to 30794 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 Targeting SAMe Metabolism in Leukemia 29720–29725 be even more optimistic that it will be possible to generate tar- 27. Garcia-Trevijano, E. R., Latasa, M. U., Carretero, M. V., Berasain, C., geted therapeutics and dietary protocols to selectively diminish Mato, J. M., and Avila, M. A. 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Chem. 274, 6,696,279 NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30795 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 45, pp. 30788 –30795, November 7, 2008 Printed in the U.S.A. Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish □ S S-Adenosylmethionine Synthesis Received for publication, May, 30, 2008, and in revised form, August 21, 2008 Published, JBC Papers in Press, August 27, 2008, DOI 10.1074/jbc.M804159200 ‡§ ‡§ ‡ ¶ § § Ramy R. Attia , Lidia A. Gardner , Engy Mahrous , Debra J. Taxman , Leighton LeGros , Sarah Rowe , ¶ ‡ ‡§1 Jenny P.-Y. Ting , Arthur Geller , and Malak Kotb ‡ § 2 From the University of Tennessee Health Science Center and the Research Service, Veterans Affairs Medical Center , Memphis, Tennessee 38104, the Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, and the Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524 We exploited the fact that leukemic cells utilize significantly goal has been to develop novel approaches that exploit physio- higher levels of S-adenosylmethionine (SAMe) than normal logical differences in metabolic needs between normal and leu- lymphocytes and developed tools that selectively diminished kemic cells to generate tools that would selectively diminish their survival under physiologic conditions. Using RNA inter- tumor cell growth in vivo, with minimal harm to normal host ference gene silencing technology, we modulated the kinetics of cells. Specifically, we sought to exploit significant differences in methionine adenosyltransferase-II (MAT-II), which catalyzes S-adenosylmethionine (SAMe) metabolism between normal SAMe synthesis from ATP and L-Met. Specifically, we silenced and leukemic T cells (4, 5). SAMe is an essential molecule in the the expression of the regulatory MAT-II subunit in Jurkat cells metabolism of every living species (6–10). As the main methyl and accordingly shifted the K of the enzyme 10–15-fold group donor, it methylates DNA, RNA, fatty acids, proteins, m L-Met above the physiologic levels of L-Met, thereby reducing enzyme and small molecules and regulates several transcription and activity and SAMe pools, inducing excessive apoptosis and translation processes, protein function, and membrane integ- diminishing leukemic cell growth in vitro and in vivo. These rity. SAMe is also involved in DNA mismatch repair, chromatin effects were reversed at unphysiologically high L-Met (>50 M), modeling, epigenetic modifications and imprinting, cell repli- indicating that diminished leukemic cell growth at physiologic cation, neurotransmission, and signaling (11). Additionally, L-Met levels was a direct result of the increase in MAT-II SAMe is an important precursor of the polyamines and a major K due to MAT-II ablation and the consequent reduction player in biological trans-sulfuration as well as folic acid and m L-Met null in SAMe synthesis. In our NOD/Scid IL-2R humanized one-carbon metabolism (8, 9, 11, 12). mouse model of leukemia, control shRNA-transduced Jurkat The importance of SAMe, together with the fact that its cells exhibited heightened engraftment, whereas cells lacking metabolism is constitutively elevated in malignant versus nor- MAT-II failed to engraft for up to 5 weeks post-transplant. mal cells, has for years made it an attractive target for cancer These stark differences in malignant cell survival, effected by chemotherapy (13–20). Unfortunately, chemical inhibitors of MAT-II ablation, suggest that it may be possible to use this SAMe synthesis have been difficult to generate in quantities approach to disadvantage leukemic cell survival in vivo with lit- needed for clinical use, and most were either unstable, reversi- tle to no harm to normal cells. ble, nonspecific, or highly toxic because no cell can survive total inhibition of SAMe synthesis. To this end, our approach has been to take advantage of the dependence on higher SAMe levels in leukemic cells to diminish rather than totally block Leukemia are among the deadliest and most common can- their ability to synthesize the needed amount of SAMe and cers. Despite advancements in novel individual and combina- tion drug treatment modalities, mortality rates remain high, thereby selectively halt their growth while sparing normal cells. The advent of novel biotools that can selectively silence pro- and some medications have serious adverse effects (2, 3). Our tein expression has made it possible to initiate studies to target the regulatory subunit of methionine adenosyltransferase * This work was supported, in whole or in part, by National Institutes of Health (MAT), which catalyzes the synthesis of SAMe from L-Met and Grant R01CA108792. This work was also supported by a Veterans Affairs ATP. All living organisms have at least one MAT enzyme (5, Merit Review Award and a Senior Research Career Scientist Award (to M. K.). The costs of publication of this article were defrayed in part by the 13). Mammals have liver-specific MAT-I/III and another payment of page charges. This article must therefore be hereby marked isozyme, MAT-II, that is expressed in all tissues (21, 22). MAT- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi- I/III are tetramer/dimer forms of a catalytic 1 subunit, and cate this fact. □ S they differ considerably in their kinetic and physical properties. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1. To whom correspondence should be addressed: Dept. of Molecular Genet- ics, Biochemistry and Microbiology, University of Cincinnati College of The abbreviations used are: MAT, methionine adenosyltransferase; SAMe, Medicine, 231 Albert Sabin Way, P.O. Box 670524, 2938 CVC Mail Loc-0524, S-adenosylmethionine; GFP, green fluorescence protein; shRNA, short Cincinnati, OH 45267-0524. Tel.: 513-558-5231; Fax: 513-558-1190; E-mail: hairpin RNA; HPLC, high pressure liquid chromatography; BM, bone mscbskotb@gmail.com. marrow. 30788 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 This is an Open Access article under the CC BY license. Targeting SAMe Metabolism in Leukemia It is believed that this differential oligomerization of MAT-1is data base to ensure their specificity for our target gene. The an important adaptation to cope with special metabolic pHSPG plasmid (Dr. Su, University of North Carolina) has a requirements in the liver, where SAMe levels need to be main- constitutively active polymerase III promoter in front of the tained at a certain range inasmuch as a deficiency or excess of multiple cloning site. We modified pHSPG by including a His- SAMe has been associated with serious pathology (23–25). In tone 1 promoter needed for shRNA formation (34), followed by livers of healthy subjects, MAT-III, which has a high K shRNA constructs of interest between the EcoRV and XbaI m L-Met (80–100 M), is the major isoform. By contrast, MAT-II is a sites, in the multiple cloning site. Downstream of this construct hetero-oligomer that has a catalytic2 subunit and a regulatory is a PGK-GFP cassette whose expression is driven by the mouse subunit with a K of 4–20 M (21, 26, 50). The 2 sub- mammary tumor virus promoter. The HSPG virus was chosen m L-Met unit, which has 84% sequence identity to 1, undergoes post- for its high transduction efficiency in hematopoetic cells (36, translational modifications resulting in expression of 2 (53 37). The design of different MAT-II-specific pHSPG-shRNA kDa) and 2 (51 kDa) forms (21). In fetal liver and certain adult drivenbytheHistone1promoterisshowninsupplementalFig.S1. liver diseases, including hepatocellular carcinoma, 1 subunit The effect of several control and MAT-II-specific pHSGP- expression is diminished and replaced by 2, along with the shRNA constructs on MAT-II expression were initially tested induction of MAT-II expression (27, 28). in transiently transfected Cos-1 cells. These studies showed MAT- subunits are highly conserved across many species that plasmid pHSGP-shRNA-1110 had the highest gene silenc- (22); by contrast MAT-II is only found in mammals, associ- ing activity and was not toxic to the Cos-1 cells, and thus we ated with MAT-II2. In several of our previous studies, we packaged it into HSPG-V1110 viral particles using HEK-293T showed that MAT-II plays a crucial physiological role by low- cells and the CaCl transfection method (35). We also gener- ering the K of MAT-II for L-Met from 55–65 M down to ated a control, empty virus without shRNA (V1302) and V1324 3.5–20 M (26, 29, 50). Inasmuch as the physiologic extrahe- that encodes an shRNA for an irrelevant, mouse plexin A1 gene patic concentration of L-Met are 5–10-fold lower than that in (37). the liver (30), we believe that the introduction of MAT-II to Transduction with HSPG-shRNA Retrovirus—We trans- lower the K of the extrahepatic enzyme may have been an duced Jurkat cells (10 cells) by adding 8 mg/ml polybrene plus essential evolutionary event that allowed MAT-II to function in 700 ml of viral supernatants and incubating for 20 min at room blood and other extrahepatic mammalian tissues, where L-Met temperature. The cells were spun at 2000 rpm for 3 h and then levels are 10–25 M (31–33). resuspended in fresh 1.5 ml of RPMI 1640 complete medium. We had reported that MAT-II expression and SAMe metab- We cultured the cells in 12-well tissue culture plates, repeated olism are considerably different in normal and malignant lym- the transduction after 24 and/or 48 h, and then assessed trans- phocytes (4, 5, 8). MAT-II expression in established and pri- duction efficiency by flow cytometry (FACSCalibur) to deter- mary human lymphocytic leukemia cells is significantly higher mine the percentage of GFP-positive cells. This procedure typ- than in quiescent or activated lymphocytes (4). MAT-II activity, ically yielded 90% GFP cells, which were then sorted using SAMe utilization rate, and SAMe pool size are, respectively, FACSAria (BD Biosciences) to obtain a 98% transduced cell 20-, 60-, and 60–100-fold higher in lymphocytic leukemia, than population. in normal lymphocytes (4). Based on these previous studies (4, MAT-II Subunits Expression Analysis—We lysed GFP 21, 26, 29), we predicted that if we specifically ablated MAT-II sorted cells by three cycles of freeze-thawing in extraction expression, we would shift MAT-II K by at least 10-fold buffer (50 mM Tris, pH 7.4, 50 mM NaCl, 5 mM MgCl ,4mM m L-Met 2 above physiologic L-Met levels, and that this would conse- dithiothreitol), containing a mixture of proteolytic inhibitors quently reduce SAMe pool size and selectively diminish the (Roche Applied Science) as described (5). Protein concentra- growth of leukemic cells in physiological fluids and extrahe- tion was determined in the cleared lysates by the bicinchoninic patic tissues. We report that MAT-II subunit specific shRNA acid method (38). Equal amounts of protein extracts were sep- successfully silenced the expression of the MAT-II regulatory arated on 10% SDS-PAGE and then transblotted onto nitrocel- subunit in the Jurkat leukemic T cell line and increased the lulose papers. Expression of 2 and  subunits was determined enzyme K by 10–15-fold, consequently depleting SAMe by Western blots and probed with antibodies to MAT-II and m L-Met pools, inducing excessive apoptosis, and diminishing the MAT-II proteins (4, 39). growth of these leukemic cells in physiologic L-Met concentra- We also used quantitative real time PCR to assess MAT- tions, both in vitro and in vivo in a humanized NOD/Scid II and MAT-II mRNA expression. We constructed cRNA null IL-2R mouse model of leukemia. standards for each subunit and generated standard curves for each run to quantify mRNA copy number/2 g of total EXPERIMENTAL PROCEDURES RNA. Briefly, we transformed Escherichia coli strain JM109 Leukemic Cells—Jurkat T cells (E6–1; ATCC, Manassas, VA) with pTargeT/MAT-II subunit or pTargeT/MAT-II were maintained in RPMI 1640 medium, supplemented with subunit (26) and purified those plasmids using the Wizard either 10% fetal bovine serum or 1% HL-1 supplement (L- PureFection DNA purification system (Promega). Correct Met), 2 mML-glutamine, 50 g/ml streptomycin, and 50 pTargeT/MAT-II and pTargeT/MAT-II plasmids were units/ml of penicillin. verified by sequencing, using a T7 promoter primer. T7 Generation of pHSPG-shRNA Retrovirus—We designed sev- Ribomax large scale RNA production system (Promega) was eral shRNA sequences to target MAT-II expression and used to generate MAT-II or MAT-II cRNA. The number BLAST-searched these sequences against the human genome of cRNA molecules were calculated as follows: N(molecules/ NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30789 Targeting SAMe Metabolism in Leukemia l)  [C*1825  10 ]/K, where C  cRNA g/l and K  In Vivo Animal Studies—All of the animals were treated bp (40). For MAT-II cRNA, K  1188 bp and C  0.658 according to Institutional Animal Care and Use Committee g/l; for MAT-II cRNA, K  1050 bp and C  0.632. regulations (protocol 1587). We irradiated highly immune defi- To test the efficiency of MAT-II silencing, we extracted cient NOD/ScidIL-2Rnull mice with 3.5GY, 24 h prior to 6 6 RNA from 10 untransduced or transduced cells using RNA- transplanting them via intraperitoneal injection of 15  10 STAT 60 (Tel-Test), removed residual contaminating genomic V1110 or V1302 Jurkat cells. We obtained sera from mice 12 DNA by DNase I treatment (Qiagen), purified the RNA using days post-injection and then once a week thereafter to quantify Qiagen RNeasy kit, and then converted 2 g of the purified levels of the surrogate tumor marker, soluble 2 microglobulin, RNA to cDNA using avian myeloblastosis virus reverse tran- using an enzyme-linked immunosorbent assay 2 microglobu- scriptase, 10 random hexamers, and 10 mM dNTP in the pres- lin kit (R & D). At 4 and 5 weeks post-transplant, we sacrificed ence of RNase inhibitor (Promega). Quantitative real time the transplanted mice (supplemental Table S1) and used fluo- PCRs were run using the fluorogenic SYBR Green quantitative rescence reflectance imaging to image GFP fluorescence in real time PCR system and the ABI prism 7900 Sequence Bio- spleen prior to extracting splenocytes and bone marrow cells to Detector (PE Biosystems). In each run, serial dilutions of MAT- assess tumor growth and engraftment by counting % human II or MAT-II cRNA (104–1010 cRNA molecules/l) were CD3/GFP-positive cells in each compartment by flow cytom- also converted to cDNA and used to generate the standard etry as detailed above. curve for each MAT-II subunit. The PCR mixture contained 25 Statistical Methods—We calculated statistical significance l of SYBR Green PCR Master mix (Applied Biosystems), 2.5 l with one-way analysis of variance and Newman-Keuls Multiple containing 1.5–12 pmol of each primer, and 5l of the template Comparison test for all the experiments except for the in vivo cDNA in a final volume of 50 l. The sequence of the MAT-II experiment where we used a Mann-Whitney test using the specific primers were (forward, 5-CACCTTACAGAGAGGA- GraphPad Prism program. AGA-3 and reverse, 5-CAGTCACAGCACTTTCTTC-3); and for MAT-II2 specific primers (forward, 5-AAAGTGGT- RESULTS TCGTGAAGCTGTTAAA-3 and reverse, 5-CCAAGGCTA- Successful Ablation of MAT-II Expression—We designed CCAGCACGTTAC-3). An 18 S RNA primer mix (Qiagen) several shRNA to ablate MAT-II expression, cloned them into was used for normalization. To calculate N for each test sample, the retroviral plasmid pHSPG, which also expresses GFP, and we first normalized the cycle threshold (CT) to the internal screened their gene silencing efficiency and nonspecific toxicity normalizer (18 S) and then determined N from the standard (supplemental Fig. S1). All MAT-II-specific shRNA con- curve using the equation Y  [MX  C]. structs were designed to target the two splice variants of MAT- Assay for MAT Activity—We assayed MAT activity in cell II (28). Initial screening was done in COS-1 cells, and con- extracts as described previously (21). For kinetic analyses, we structs with efficient gene silencing activity were then screened used different L-Met concentrations (1.25–80 M), using 14 in Jurkat cells (supplemental Fig. S1). Construct V1110 had the [ C]L-Met (57.9 mCi/mmol) and supplementing with cold highest MAT-II silencing activity (supplemental Fig. S1) and L-Met. Reaction velocity is expressed as units/mg protein, least toxicity and was thus packaged into infectious HSPG-GFP where 1 unit  1 nmol of adenosylmethionine/h (21). We cal- virus (36). culated K and V using the GraphPad Prism program. m max We transduced Jurkat cells with the various recombinant Cell Growth at Different L-Met Concentrations—We weaned HSPG-shRNA viruses and assessed MAT-II ablation at the the cells to grow in serum-free RPMI 1640 medium containing RNA and protein levels (supplemental Fig. S1). Controls 1% HL-1 supplement (Cambrex, NJ) plus 2 mML-glutamine, 50 included cells infected with an empty virus (V1302) or with a virus g/ml streptomycin, and 50 units/ml of penicillin (RPMI-HL1s encoding shRNA construct (V1324) directed to the mouse plexin medium). We seeded weaned cells (10 /ml) in RPMI-HL1s A1 gene (36). Although transduction efficiencies for all cells tested medium at L-Met 5–100 M, replaced the medium every 2 days, were 90%, significant reduction or complete ablation of MAT- recorded numbers of viable cells, and then replated the cells at II RNA and protein expression was only seen in V1110-trans- 10 /ml for further analyses. Cell necrosis and apoptosis were duced cells (Fig. 1). By contrast, expression of MAT-II2 was determined by a flow cytometric quantification of propidium essentially unaffected, thus indicating the MAT-II specificity of iodide and annexin-V (BD Pharmingen) stained cells, respec- V1110. In some cases, expression of MAT-II2 was insignificantly tively. In some studies, the cells were cultured at 10 ML-Met elevated, perhaps reflecting an attempt to increase enzyme synthe- for 3d, harvested, counted, and replated at 10 cells/ml in fresh sis to compensate for the lack of  expression and increase SAMe RPMI-HL1s medium with 10ML-Met. On day 6, the cells were synthesis. harvested, washed, and replated in fresh medium containing 20 Effect of MAT-II Silencing on MAT-II Activity, Kinetics, M or 50 ML-Met. The cell growth/death were determined SAMe Pool Size, and Cellular Growth—We next tested whether every 3 days thereafter, replating 10 viable cells/ml each time. ablation of MAT-II expression will alter MAT-II K and Measurement of SAMe Levels—We quantified SAMe levels in m L-Met reduce SAMe pools in Jurkat cells. As predicted, the K a neutralized 2 N perchloric acid-soluble extract by HPLC as pre- m L-Met viously detailed (4). We calculated the SAMe concentration increased from 3.5–6 M in untransduced and control virus- (pmol/10 cells) from a standard curve using different concentra- transduced Jurkat cells, up to 56–62 M in V1110 cells (Fig. tions of SAMe standard (USB Corp.) and then converted the val- 2A). Further, at the high end of physiological L-Met levels (20 ues toM based on cell volume for Jurkat cells 0.76 ml/10 cells (4). M), MAT-II specific activity was 4–5-fold lower in V1110 cells 30790 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 Targeting SAMe Metabolism in Leukemia than in untransduced or control virus-transduced Jurkat cells expressing MAT-II (Fig. 2B). Importantly, in the absence of MAT-II, SAMe was unde- tectable in V1110 cells cultured at 10 ML-Met (not shown), and even at 20 ML-Met SAMe pool size was85% less than in untransduced or control-transduced V1302 cells (9.5  0.5 M versus 62  4 M; p  0.05) (Fig. 2C). Significant 45–50%, reductions in SAMe pool size (p  0.01) were also found even when V1110 cells were maintained at 50 ML-Met, which is 2.5-fold higher than the upper range of physiological L-Met levels. The sharp reduction in SAMe pool size, consequent to MAT- II ablation, diminished the growth and viability of V1110 cells. At 20 ML-Met, V1110 cells ceased to grow, and at 50 M their growth was reduced by 33–36% compared with control- FIGURE 1. Successful ablation of MAT-II subunit using viral encoded transduced V1302 cells (p  0.01) (Fig. 3). By contrast, at 100 shRNA. MAT-II 2 and  protein expression (60 g of protein/well) and mRNA levels were analyzed in untransduced or 98% transduced cells as detailed ML-Met, V1110 cells grew at essentially the same rates and for under “Experimental Procedures.” Quantitative real time PCR was conducted the same duration in culture as the untransduced or control- using RNA extracted from cells maintained in 100 ML-Met RPMI medium, and RNA copy number was calculated as detailed under “Experimental Procedures.” transduced Jurkat cells (p  0.05) (Fig. 3). Thus, at unphysi- The results (means S.E.) of two combined experiments, each done in triplicate, ologically high L-Met levels approaching MAT-II2 K with- are shown. Statistical differences were determined as described under “Experi- out  (i.e. K  50–60 M), V1110 leukemic cells appear to mental Procedures.” MAT-II expression was significantly ablated in V1110 cells. ***, p  0.001. MAT-II2 was slightly elevated or unaffected (p 0.05). have satisfied their SAMe needs and grew normally. These results indicate that diminished V1110 cell growth at physiologic L-Met levels was a direct conse- quence of specific MAT-II silenc- ing, which caused an upper shift in K of MAT-II and a significant m L-Met reduction of SAMe synthesis and pools. Cell Death and Apoptosis of Leu- kemic Cells Lacking MAT-II—Sig- nificant reduction in growth rates of V1110 cells at physiological L-Met levels was associated with a signifi- cant increase in both cell necrosis and apoptosis (Fig. 4). At physio- logic L-Met levels (5, 10, and 20 M), the extent of necrosis in the V1110 cells was significantly higher (20– 55%) than in untransduced or con- trol V1302 cells (p  0.001). Mini- mal cell death was seen at 50 M L-Met; however, V1110 cells exhib- ited 70–80% higher levels of apo- ptosis than the control leukemic cells even at this unphysiological L-Met concentration (p 0.01) (Fig. 4). This is likely attributed to the 50% reduction in SAMe pool size even at 50 ML-Met (Fig. 2). Taken together, these findings indicate FIGURE 2. MAT-II ablation modulates MAT-II kinetics, reduces its specific activity, and diminishes intra- that the growth and viability of cellular SAMe levels. A, MAT-II Lineweaver-Burk kinetic plots of 1/V, where V is units of enzyme activity/mg of V1110 cells lacking MAT-II protein (units/mg; 1 unit 1 nmol of adenosylmethionine/h). MAT assays were performed using extracts from expression remained at a selective untransduced, V110- or V1302-transduced cells in the presence of 2.5– 80 ML-Met, as detailed under “Exper- imental Procedures.” The data for each cell extract represents the means S.D. of three separate experiments, disadvantage, even at more than each assayed in triplicate. B, MAT specific activity (units/mg of protein) at 20ML-Met. C, SAMe levels (pmol/10 twice the higher end of physiologi- cells) as determined by HPLC in neutralized perchloric acid extracts from cells that were maintained at 50 or 20 ML-Met. *, p  0.05; **, p  0.01; ***, p  0.001. cal L-Met levels. NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30791 Targeting SAMe Metabolism in Leukemia Attempts to Rescue Cells by Incrementally Elevating L-Met Levels—Following up on selective pressure imposed on the via- bility of MAT-II-deficient Jurkat cells in physiologic L-Met conditions, we next tested whether V1110 cells can be rescued by incremental elevation of L-Met in culture. All of the cells grew at lower rates at the low end (5–10 M) of physiologic L-Met levels compared with 20 M. We therefore maintained V1110 and control Jurkat cells at 10 ML-Met for 6 days in culture and then increased L-Met levels to either 20 or 50 M to determine whether these cells can be rescued (Fig. 5). The growth of control untransduced and control V1302 Jurkat cells increased by almost 80 and 90% when L-Met levels were raised to 20 or 50 M, respectively. By contrast, under the same con- ditions, V1110 cells could not be rescued with 20ML-Met (p 0.01) and exhibited diminished growth even at 50 ML-Met (p  0.05) (Fig. 5). These data suggest that the initial insult to the V1110 cells, caused by low SAMe levels due to the absence of MAT-II, is irreversible at the high end of physiologic L-Met levels (20 M) and minimally reversible at 50 ML-Met. Failure to rescue V1110 cells is consistent with the above observed increased apoptosis of these cells, even at 50 ML-Met (Fig. 4). MAT-II Ablation Diminishes Leukemic Cell Growth in an null NOD/Scid IL-2R Mouse Leukemia Model—To rule out possible in vitro culture artifacts, we investigated whether Jur- FIGURE 3. MAT-II ablation reduces leukemic cell growth in physio- logic L-Met levels. We seeded 5 10 of indicated cells at 5–100 ML-Met kat cells that do not express MAT-II would survive and mul- and monitored their growth every 2 days using the trypan blue exclusion tiply in vivo in our severely immunodeficient NOD/Scid method, adding fresh medium each time. *, p  0.05; **, p  0.01; ***, p null 0.001. IL-2R mouse model (41). These mice allow heightened engraftment with xenogeneic cells because, besides lacking T, B, and NK cells, they are also defi- cient in complement and macro- phage function (41). After mildly irradiating these mice, we trans- planted them intraperitoneally with 15  10 V1110 or control V1302 cells (n  15 mice/group). Irradi- ated, noninjected mice served as additional controls. We monitored tumor engraftment and growth weekly for 5 weeks post-transplant by determining the percentage of human CD3 and GFP expression in bone marrow (BM)- and spleen-de- rived cells and by measuring serum levels of the surrogate tumor marker, 2-microglobulin (2) (42). We also imaged GFP expres- sion in whole spleens of sacrificed mice for a semi-quantitative meas- ure of tumor burden (Fig. 6). During the first 3 weeks, none of the transplanted mice showed sig- nificant engraftment, but after 4 weeks, 57% of mice transplanted with control V1302 cells began to show variable levels of engraftment FIGURE 4. MAT-II ablation induces high levels of apoptosis and necrosis of leukemic cells. A, propidium iodide-stained necrotic cells (%) at 20 ML-Met. B, propidium iodide-stained necrotic cells (%) at 50 ML-Met. in spleen (3–44% human CD3 / C, necrotic cells (%) at 6 days in 5–50 ML-Met. D, annexin-V-stained apoptotic cells (%) at 20 ML-Met. GFP cells; mean 16  7.6%) and in D, annexin-V stained apoptotic cells (%) at 50 ML-Met. F, apoptotic cells (%) at 6 days in 5–50 ML-Met. *, p 0.05; **, p  0.01; ***, p  0.001. BM (3–12% human CD3 /GFP 30792 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 Targeting SAMe Metabolism in Leukemia DISCUSSION In this study, we showed that by silencing the expression of the regulatory  subunit of MAT-II, we exerted significant growth disadvantage upon human Jurkat leukemic T cells under physiologic L-Met levels in vitro and in vivo. In the liver, L-Met levels are 80–100 M, but in other tissues they range from 10 to 25 M. Thus, the high K hepatic isozyme, MAT-III, can be fully functional in that organ without MAT-II. By con- trast, MAT-II, the only SAMe-synthesizing enzyme in extrahe- patic tissue can only function when associated with its MAT- II regulatory subunit, which plays an important role in lowering MAT-II K to values close to physiological m L-Met L-Met levels outside the liver (26, 29, 43, 50). Without the subunit, MAT-II would function at minimal capacity and would not be able to provide normal extrahepatic cells with adequate SAMe levels needed for their growth and survival. In resting human T cells, MAT-II is expressed at low levels, and mitogenic activation of these cells does not induce further expression of this subunit; by contrast, MAT-II is constitu- tively expressed at a much higher level in established and pri- mary leukemic T cells (4, 5). Induction of MAT-II expression has been reported in several types of cancer and has been shown to confer a proliferative advantage to human hepatomas (44). The role of MAT-II in leukemic cells is even more crucial because leukemic T cells, both freshly explanted ALL-2 cells and the Jurkat cell line, utilize SAMe at significantly higher rates than normal resting or activated T cells (4, 5). The consti- tutive high expression of MAT-II in leukemic cells allows high levels of SAMe synthesis needed to meet the growth require- ments of malignant cells. In our quest to diminish SAMe in leukemic cells, it was unreasonable to target the MAT-II2 cat- alytic subunit, because that would be quite detrimental and toxic to normal cells that do not express the hepatic MAT-I/III isozyme, i.e. the majority of extrahepatic cells. However, target- ing MAT-II expression seemed much more appropriate and FIGURE 5. Differential rescue of Jurkat cells  MAT-II expression by practical, because this would not block SAMe synthesis com- incrementally increasing L-Met levels in culture. Untransduced and pletely but would reduce it to where the rapid growth and pro- V1302- and V1110-transduced Jurkat cells (5  10 ) were cultured at 10 M liferation of malignant lymphocytes and possibly other cancer- L-Met for 6 days, and then L-Met was raised to either 20 M (A)or50 M (B). Viable cells were counted over 15 days, with the addition of fresh medium ous cells can no longer be sustained. every 3 days. V1110 cells ceased to grow when L-Met was raised to 20 M and We succeeded in silencing the MAT-II regulatory subunit exhibited diminished growth even at 50 M.*, p  0.05; **, p  0.01; ***, p 0.001. without significantly affecting the expression of the MAT-II2 subunit. Under these conditions, our in vitro and in vivo studies provided strong evidence that MAT-II ablation is detrimental cells; mean 4  1.6%). Engraftment was much less evident in to the growth of malignant T cells and that this is directly due to spleens of mice transplanted 4 weeks earlier with V1110 cells drastic reductions in SAMe levels needed to support their rapid (0.96  0.32% CD3 /GFP cells; p  0.004). This trend con- growth. Inasmuch as it is quite feasible to modulate L-Met levels tinued through 5 weeks post-transplant, with significantly in vivo through dietary control (45), we believe these results are higher human CD3 /GFP cells in spleen and BM of mice of particular interest, especially in the context of our quest to injected with V1302 versus V1110 cells (supplemental Table 1S eventually translate these research findings into clinical appli- and Fig. 6). At 5 weeks, 25% of V1110 cells transplanted mice cations. Indeed, there is a large body of evidence that the growth started to show low levels of CD3 /GFP engraftment in of several types of tumors is dependent on high L-Met and that spleen (1.1 0.4%) and BM (2.4 1%). In stark contrast, 75% of L-Met deprivation causes cell cycle arrest in the G phase (46, mice transplanted with V1302 cells showed heightened 47). Several clinical studies demonstrated that diminishing engraftment in spleen (30  10%; p  0.02) and BM (11  4%; p  0.04) (Fig. 6). Additionally, levels of the surrogate tumor L-Met in cancer patients by maintaining them on a Met- or marker 2 were 2-fold higher in mice transplanted with con- choline-free diet and/or administering methioninase exerts trol V1302 leukemic cells than those transplanted with V1110 selective growth disadvantage pressure on cancer cells in vitro cells (supplemental Fig. S2). and in vivo, inducing mitotic and cell cycle arrest, apoptotic NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30793 Targeting SAMe Metabolism in Leukemia approach in the treatment of certain blood malignancies, and we are cur- rently exploring these possibilities. The growth of Jurkat cells with and without MAT-II expression was reduced at 10 M compared with 20 ML-Met; however, when we attempted to rescue these cells by incrementally increasing L-Met levels in culture, we found that the growth of cells expressing MAT-II was fully restored at 20 M, whereas cells lacking MAT-II could not be rescued at this concentration. Even at unphysiologically high 50 M L-Met we still observed significant apoptosis in cells lacking MAT-II. We therefore predict that it may be possible to adjust L-Met levels in vivo in a way that would rescue nor- mal cells while still inducing death and apoptosis of leukemic cells that do not express this important regu- latory subunit. We ruled out the possibility that our in vitro observations are due to culture artifacts, because when we compared the ability of Jurkat cells that do and do not express MAT-II to engraft and survive in an in vivo environment of physiologic L-Met levels in our hyperimmune defi- null cient NOD/ScidIL-2R mouse model, we found significant differ- ences in the in vivo tumor growth and engraftment, even in the absence of host immune rejection inasmuch as these mice were not reconstituted with a human FIGURE 6. MAT-II ablation diminishes leukemic cell growth in vivo. Hyperimmune-deficient NOD/Scid null 6 IL-2R mice were irradiated with 3.5GY 24 h prior to intraperitoneal transplant with 15 10 of the indicated immune system (41). Control trans- cells. Controls included irradiated but not injected mice and V1302 transplanted mice. Tumor engraftment duced Jurkat cells grew well in this and growth were monitored by measuring % human CD3 /GFP cells in mice spleens and bone marrow at 4 mouse model, whereas cells lacking and 5 weeks post-transplant with either control V1302 or V1110 cells. Flow cytometry histograms of CD3 / GFP in spleen (A) or bone marrow (B) 5 weeks post-transplant. C, GFP expression in whole spleens of same MAT-II expression showed signif- mice prior to processing their splenocytes. D, number of mice engrafted with V1302 or V1110 leukemic cells at icant diminution in engraftment for 4 and 5 weeks post-transplant. E and F show % CD3 /GFP cells in spleens (E) or bone marrow (F) of mice transplanted with either V1302 or V1110 leukemic cells, 5 weeks post-transplant. The statistical differences up to 5 weeks post-transplant. were calculated using a Mann-Whitney test. *, p  0.05; **, p  0.01; ***, p  0.001. Although these results are very promising, we are currently design- death, and widespread necrosis in tumors (48). Induction of ing studies to treat leukemic cells in vivo, after they have Jurkat cells apoptosis and death consequent to silencing MAT- engrafted. II is also in agreement with previous studies showing In summary, we had predicted that an approach that targets increased apoptosis and necrosis of tumor cells cultured in the SAMe metabolism would provide a good adjunctive therapeu- absence of L-Met (1, 49). We observed significant apoptosis in tic tool for the treatment of leukemia because of the their exces- Jurkat cells lacking MAT-II at physiologic L-Met levels, and sive requirement for this central metabolic compound. Our in this indicates that direct depletion of SAMe may be more effec- vivo data suggest that this approach, by itself, may have a more tive than L-Met depletion in inducing tumor cell apoptosis in drastic effect on diminishing leukemic cell growth than we had vivo. We believe that targeting MAT-II expression in combi- originally anticipated. The stark difference in the in vivo sur- nation with restricting dietary L-Met alone or in conjunction vival and engraftment between the V1110- and V1302-trans- with chemotherapeutic agents may prove to be a powerful duced cells in mice lacking proper immune defenses lead us to 30794 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 45 •NOVEMBER 7, 2008 Targeting SAMe Metabolism in Leukemia 29720–29725 be even more optimistic that it will be possible to generate tar- 27. Garcia-Trevijano, E. R., Latasa, M. U., Carretero, M. V., Berasain, C., geted therapeutics and dietary protocols to selectively diminish Mato, J. M., and Avila, M. A. 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Chem. 274, 6,696,279 NOVEMBER 7, 2008• VOLUME 283 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 30795

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