Inhibition of Intracellular Dipeptidyl Peptidases 8 and 9 Enhances Parthenolide’S Anti-Leukemic Activity

ORIGINAL ARTICLE Inhibition of intracellular dipeptidyl peptidases 8 and 9 enhances parthenolide’s anti-leukemic activity

PA Spagnuolo1, R Hurren2, M Gronda2, N MacLean2, A Datti3,4, A Basheer1, F-H Lin2, X Wang2, J Wrana3 and AD Schimmer2,5

Parthenolide is selectively toxic to leukemia cells; however, it also activates cell protective responses that may limit its clinical application. Therefore, we sought to identify agents that synergistically enhance parthenolide’s cytotoxicity. Using a high- throughput combination drug screen, we identiﬁed the anti-hyperglycemic, vildagliptin, which synergized with parthenolide to induce death of the leukemia stem cell line, TEX (combination index (CI) ¼ 0.36 and 0.16, at effective concentration (EC) 50 and 80, respectively; where CI o1 denotes statistical synergy). The combination of parthenolide and vildagliptin reduced the viability and clonogenic growth of cells from acute myeloid leukemia patients and had limited effects on the viability of normal human peripheral blood stem cells. The basis for synergy was independent of vildagliptin’s primary action as an inhibitor of dipeptidyl peptidase (DPP) IV. Rather, using chemical and genetic approaches we demonstrated that the synergy was due to inhibition of the related enzymes DPP8 and DPP9. In summary, these results highlight DPP8 and DPP9 inhibition as a novel chemosensitizing strategy in leukemia cells. Moreover, these results suggest that the combination of vildagliptin and parthenolide could be useful for the treatment of leukemia.

Leukemia (2013) 27, 1236–1244; doi:10.1038/leu.2013.9 Keywords: Parthenolide; vildagliptin; dipeptidyl peptidase; acute myeloid leukemia

INTRODUCTION MATERIALS AND METHODS Acute myelogenous leukemia (or acute myeloid leukemia; AML) is Cell culture a heterogeneous group of aggressive malignant diseases charac- Leukemia (OCI-AML2, TEX) and lymphoma cell lines (OCI-Ly17) were terized by the clonal expansion of undifferentiated myeloid cultured in Iscove’s Modified Dulbecco’s Medium (IMDM). TEX cells (a gift precursors (that is, blasts).1 AML induction chemotherapy, from Dr J. Dick, Toronto, Ontario, Canada) were maintained in IMDM, 15% consisting of 3 days of daunorubucin (45–90 mg/m2) and 7 days fetal bovine serum (FBS), 1% penicillin-streptomycin, 2 mM L-glutamine, of continuous intravenous infusions of cytarabine (100 mg/m2), 20 ng/ml stem cell factor, 2 ng/ml interleukin-3. Unless otherwise noted, all 2 media were supplemented with 10% FBS, 100 units/ml of streptomycin results in complete response rates up to 80%; however, the and 100 mg/ml of penicillin (all from Hyclone, Logan, UT, USA). Cells were majority of patients ultimately relapse.3 Thus, there is a need for incubated in a humidified air atmosphere containing 5% CO2 at 37 1C. improved chemotherapeutics. Primary human AML samples were isolated from the peripheral blood of Parthenolide, the bioactive component of the medicinal consenting AML patients, who had at least 80% malignant cells in their plant Feverfew (Tanacetum parthenium), is a sesquiterpene peripheral blood and cultured at 37 1C in IMDM, 20% fetal calf serum, lactone with specific toxicity to AML cells compared with 100 units/ml of streptomycin and 100 mg/ml of penicillin. Normal normal hematopoietic cells.4 The mechanism of parthenolide- granulocyte colony stimulating factor (GCSF)-mobilized peripheral blood induced death appears multi-factorial. Parthenolide has mononuclear cells were obtained from volunteers donating peripheral been shown to inhibit NFkB activation,5 increase reactive blood stem cells for allotransplant. The collection and use of human tissue 5,6 7 for this study was approved by the local ethics review board (University oxygen species, inhibit STAT3 activation and increase Health Network, Toronto, Ontario, Canada). p53 phosphorylation.8 However, parthenolide also induces anti-oxidant, cellular protective responses that likely reduce its overall cytotoxicity, such as increases in the transcription High-throughput screen A high-throughput screen was performed as previously described.10,11 Briefly, factor NF-E2-related factor 2 and its transcriptional target 4 heme oxygenase 1.9 Here, we sought to identify agents that TEX cells (1.0 10 /well) were seeded in 96-well polystyrene tissue culture synergistically enhance parthenolide’s cytotoxicity. Specifically, plates. After seeding, cells were treated with aliquots (10 mM final concentration) of the chemical library with a final dimethyl sulfoxide we screened an in-house library of on-patent and off-patent (DMSO) concentration of 0.05%. After 72 h incubation, cell proliferation and drugs for agents that increased the cytotoxicity of parthenolide. viability were measured by the MTS assay. Liquid handling was performed by From this screen, we identified vildagliptin, an oral anti- a Biomek FX Laboratory Automated Workstation (Beckman Colter). hyperglycemic agent.

1School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada; 2Princess Margaret Cancer Center, Ontario Cancer Institute, Toronto, Ontario, Canada; 3SMART laboratory for High-Throughput Screening Programs, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada and 4Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy. Correspondence: Dr PA Spagnuolo, School of Pharmacy, University of Waterloo, Health Science Campus, 10A Victoria Street South; room 3006, Kitchener, Ontario N2G 1C5, Canada. E-mail: [email protected] 5ADS is a Leukemia and Lymphoma Society Scholar in Clinical Research. Received 18 May 2012; revised 27 November 2012; accepted 7 January 2013; accepted article preview online 15 January 2013; advance online publication, 8 February 2013 Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1237 Cell growth and viability media containing protamine sulfate (5 mg/ml) and 1 ml of virus cocktail Cell growth and viability was measured using the 3-(4,5–dimethylthiazol– (which contains the short hairpin RNA (shRNA) sequence and a puromycin 2–yl)-5-(3-carboxymethoxyphenyl)-2-(4–sulfophenyl)-2H-tetrazolium inner antibiotic resistance gene) and incubated overnight at 37 1C. The following salt (MTS) reduction assay (Promega, Madison, WI, USA), according to day, the virus was removed by centrifugation and the cells were the manufacturer’s protocol and as previously described.11 Cells were washed and resuspended in fresh media containing puromycin (1 mg/ seeded in 96-well plates and treated with drug for 72 h. Optical density was ml). Two days later, puromycin-selected live cells were plated for viability measured at 490 nm. Cell viability was also assessed by the trypan blue and growth assays. The shRNA-coding sequences were: DPP8 (Accession exclusion assay and by Annexin V and propidium iodide (PI) staining no. NM_017743) shRNA clone 4170 50-GCACCACATGATTTCATGTTT-30, (Biovision, Mountainview, CA, USA), as previously described.11 DPP8 shRNA clone 6740 50-CGGAATTGCTTCTTACGATTA-30; DPP9 0 Clonogenic growth assays with primary AML cells were performed, as (Accession no. NM_139159.2) shRNA clone 2315: 5 -GCTGGTGAATAAC previously described.11 Briefly, primary AML mononuclear cells were isolated TCCTTCAA-30, DPP9 shRNA clone 2871: 50-CCCAACGAGAGACACAGTATT-30. by Ficoll separation (Histopaque-1077, Sigma-Aldrich, St Louis, MO, USA) from fresh peripheral blood samples of AML patients with 480% blasts in their Engraftment assay peripheral blood. Fresh AML mononuclear cells (4 105 cells/ml) were Engraftment assays were performed similar to previously described.10 incubated with vehicle control, parthenolide (5 mM), vildagliptin (10 mM), or þ parthenolide and vildagliptin for 48 h, washed and then plated in duplicate by Briefly, a CD34 AML patient sample was treated ex vivo with 10 mM volume at 105 cells/ml per 35 mm dishes (Nunclon, Rochester, NY, USA) in vildagliptin, 2.5 mM parthenolide, a combination of these two agents or 1 MethoCult GF H4434 medium (StemCell Technologies, Vancouver, British DMSO control for 24 h at 37 C and 5% CO2. Cells were then injected into Columbia, Canada) containing 1% methylcellulose in IMDM, 30% FBS, 1% the right femur of sub-lethally irradiated NOD/SCID mice. Following a bovine serum albumin, 3 U/ml recombinant human erythropoietin, 10 4 M2- 4-week period, mice were killed and the left bone marrow flushed. Marrow engraftment of AML cells was assessed by measuring the percentage of mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant human stem cell þ þ factor, 10 ng/ml recombinant human granulocyte macrophage-colony human CD45 /CD19 /CD33 cells by flow cytometry. stimulating factor and 10 ng/ml recombinant human interleukin-3. After 7 days of incubation at 37 1Cwith5%CO2 and 95% humidity, the numbers of Statistical analysis colonies were counted on an inverted microscope. A cluster of 10 or more cells Unless otherwise stated, the results are presented as mean±s.d. Data were were counted as one colony. analyzed using GraphPad Prism 4.0 (GraphPad software, La Jolla, CA, USA). Pp0.05 was accepted as being statistically significant. Drug combination Drug combination studies data were analyzed using Calcusyn software (Biosoft, Cambridge, UK). The combination index (CI) was used to evaluate the interaction between parthenolide and vildagliptin. TEX cells were treated with increasing concentrations of parthenolide or vildagliptin and 72 h after incubation cell RESULTS viability was measured by the MTS assay. The Calcusyn median effect A screen of on-patent and off-patent drugs for compounds that model was used to calculate the CI values and evaluate whether the enhance parthenolide’s anti-leukemic activity identifies combination of parthenolide and vildagliptin was synergistic, antagonistic vildagliptin or additive. CI values of o1 indicate synergism, CI ¼ 1 indicate additivity To identify agents that synergistically enhance the cytotoxicity of and CI 41 indicate antagonism.12,13 parthenolide, we screened an in-house chemical library of 313 on- patent and off-patent drugs focused on anti-microbials and Quantitative real-time PCR metabolic regulators with a wide therapeutic index and well- First-strand complementary DNA was synthesized from 1 mg of DNase- characterized pharmacokinetics. TEX leukemia cells were treated treated total cellular RNA using random primers and SuperScript II reverse with aliquots of the chemical library (final concentration 10 mM) transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufac- with or without parthenolide at its EC 30 concentration (2.5 mM; turer’s protocols. Real-time PCR assays were performed in triplicate with Supplementary Figure 1). After 72-h incubation, cell growth and 5 ng of RNA equivalent complementary DNA, SYBR Green PCR Master mix viability was assessed using the MTS assay. The top six library (Applied Biosystems, Foster City, CA, USA) and 400 nM of gene-specific primers. Reactions were processed and analyzed on an ABI 7900 Sequence compounds with the greatest reduction in viability when in Detection System (Applied Biosystems). Forward/reverse PCR primer pairs combination with parthenolide were then assessed in secondary for human complementary DNAs were as follows: dipeptidyl peptidase confirmatory assays (Supplementary Figure 2). As we were (DPP) IV: forward, 50-gaattatccggtcgggtttt-30; reverse, 50-gtgacatcactgcccac interested in drug synergy, library compounds that imparted atc-30; DPP8: forward, 50-agagctggatggactcctga-30; reverse, 50-tcgtg 450% toxicity in the absence of parthenolide were excluded as actttggggaaaaac-30; DPP9: forward, 50-gttcccgaaggtggagtaca-30, reverse, possible hits. Therefore, the compound with the greatest 50-atccagacgttggtgacctc-30; and 18S, forward, 50-aggaattgacggaagggcac-30; reduction in viability when in combination with parthenolide reverse, 50-ggacatctaagggcatcaca-30. Relative mRNA expression was 14 was vildagliptin (Figure 1a). determined using the DDCT method as previously described. Synergy between vildagliptin and parthenolide was validated using the CI analysis (Figure 1b). The combination of parthenolide Surface DPP activity and the presence of DPIV/CD26 and vildagliptin synergistically reduced growth and viability with Surface activity of DPP in leukemia and lymphoma cell lines was CI values of 0.64, 0.36 and 0.16, at EC 30, 50 and 80, respectively. In determined using the Gly-Pro-p-nitroanalide substrate (Sigma-Aldrich), as addition, we confirmed reductions in growth and viability using previously described.15 Briefly, cells were washed in PBS and whole-cell the MTS assay and demonstrated the drug combination increased suspensions (5 105 cells/200 ml in PBS) were prepared and placed in 96-well cell death by Annexin V/PI staining (Figure 1c). We also evaluated plates. Gly-Pro-p-nitroanalide substrate, which is converted to p-nitroanalide the combination of parthenolide and vildagliptin in OCI-AML2 inthepresenceofDPPenzymes,wasthenaddedtoafinalconcentrationof leukemia cells. Similar to the effects in TEX cells, the combination 0.24 mM. The suspension was measured spectrophotometrically at 405 nm at 37 1C at 0 and 60 min. DPP enzyme activity was calculated from the increase of vildagliptin and parthenolide inhibited cell growth greater than of absorption between 0 and 60 min. either agent alone (Figure 1d). A two-way analysis of variance Surface CD26 expression was measured by flow cytometry. Cells (ANOVA) confirmed a significant interaction between the main (5.0 105) were suspended in 1 ml PBS þ 0.05% FBS and incubated with effects of vildagliptin and parthenolide (Po0.001). an anti-CD26/DPPIV monoclonal antibody at 37 1C for 45 min and read on a Vildagliptin’s ability to enhance the cytotoxicity of common FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA). clinical therapeutics was also assessed. Here, vildagliptin (10 mM) was incubated with increasing concentrations of cytarabine (Ara RNAi knockdown of DPP8 and DPP9 C), doxorubicin or vinblastine for 72 h in TEX cells. Cell growth and Lentiviral infections were performed as described by Xu et al16. Briefly, viability was determined using the MTS assay. Vildagliptin did not OCI-AML 2 cells (5 106) were centrifuged and resuspended in 6.5 ml enhance the cytotoxicity of these chemotherapeutics (Figure 1e).

& 2013 Macmillan Publishers Limited Leukemia (2013) 1236 – 1244 Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1238 a 120 b 2.0 100 1.5 80

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% Viable 40 0.5 20 Combination Index 0 0.0 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0.8 Compound Number Fraction Effect (FA)

c TEX TEX 100 * 50

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+ * 40 VILD 75 /PI + 30 50 20

25 10 % Growth Inhibtion % Dead (ANN 0 0 CTL PTL CTL PTL

d OCI-AML2 OCI-AML2 50 CTL 100 ) * + * 40 VILD /PI

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e Ara C Doxorubicin Vinblastine 20 120 120 NO VILD 100 100 00 VILD 80 80 80 60 60 60 40 40 % Viable 40 20 20 20 0 0 0 1 1 1 0.1 10 0.1 10 0.1 10 0.01 0.01 0.01 0.001 0.001 0.001 0.0001 0.0001 0.0001 0.00001 0.00001 0.00001 Ara C [m] Doxorubicin [m] Vinblastine [m] Figure 1. Vildagliptin synergizes with parthenolide in AML cell lines. (a) Vildagliptin (arrow) is identiﬁed, using a high-throughput screen, as a novel anti-cancer agent that enhances parthenolide’s anti-cancer activity. TEX cells were incubated with 10 mM of library compounds (n ¼ 313) with and without parthenolide (2.5 mM) for 72 h. After incubation, cell growth and viability were measured by the MTS assay. (b) CI versus fractional effects (Fa) plot to assess synergy of parthenolide (PTL) and vildagliptin (VILD) in TEX cells. CI values o1 indicate synergism. One of two representative isobologram experiments performed in triplicate is shown. (c) TEX or (d) OCI-AML2 cells were treated with vildagliptin (0, 10 mM) and parthenolide (0, 2.5 mM) for 72 h or vildagliptin (0, 10 mM) and parthenolide (0, 5 mM) for 24 h. After incubation, cell growth was measured by the MTS assay (at 72 h) and cell viability was measured by Annexin/PI staining (24 h). (e) TEX cells were treated with 10 mM vildagliptin along with increasing concentrations of cytarabine (Ara C), doxorubicin or vinblastine for 72 h. After treatment, cell growth and viability were measured by the MTS assay. Unless otherwise stated, data are the mean±s.d. from three independent experiments performed in duplicate (*Po0.001).

Therefore, vildagliptin appears to selectively enhance the cyto- incubation, cell viability was measured by Annexin V and PI toxicity of parthenolide. staining. Vildagliptin enhanced parthenolide’s cytotoxicity in five primary AML patient samples (Figures 2a and b). A two-way ANOVA confirmed significant interaction effects between vilda- Vildagliptin enhances parthenolide’s cytotoxicity in primary AML gliptin and parthenolide (Po0.05). In addition, we demonstrated patient samples enhanced cytotoxicity with the drug combination in the The ability of vildagliptin to enhance parthenolide’s cytotoxicity CD34 þ /CD38 fraction of AML stem cells (Figure 2b). In contrast was also evaluated in primary AML patient samples and normal to the effects on the malignant cells, the combination had less hematopoietic cells. Primary cells were treated for 24 h with effect on the viability of normal hematopoietic cells. Two of three vildagliptin, parthenolide or a combination of the drugs. After tested samples showed no increase in toxicity with the drug

Leukemia (2013) 1236 – 1244 & 2013 Macmillan Publishers Limited Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1239

Figure 2. Vildagliptin enhances parthenolide’s cytotoxicity in primary AML. (a) Primary AML cells, (b) the CD34 þ /38 fraction of AML stem cells and (c) normal hematopoietic cells were treated with parthenolide (5 mM), vildagliptin (10 mM), the combination of the two agents, or DMSO control for 24 h. After incubation, viability was measured by the Annexin/PI assay. Data are the mean±s.d. from one independent # experiment performed in duplicate. (*Po0.05; Po0.001; **Po0.0001). (d) CD34 þ AML patient sample was treated with parthenolide (5 mM), vildagliptin (10 mM) the combination or a DMSO control for 24 h and then injected into the right femur of preconditioned NOD/SCID mice. After 4 weeks, bone marrow from the left femurs was extracted and assessed for human CD45 þ /CD33 þ /CD19 cells (*Po0.005). combination (P40.05 by two-way ANOVA), whereas a third combination decreased leukemia stem cell engraftment com- sample showed only a small enhancement (Figure 2c). Thus, the pared with either drug alone and completely eliminated the þ þ combination of vildagliptin and parthenolide appeared to CD45 /CD19 /CD33 AML cells in the left femur (F2,24 ¼ 8.01; selectively target AML cells over normal hematopoietic cells. Po0.005; Figure 2d). Collectively, these results demonstrate that However, trials in patients with AML will ultimately be required to the combination of vildagliptin and parthenolide can target AML assess the therapeutic window of this combination. stem cells. We further assessed the activity of this drug combination on AML stem cells using clonogenic growth assays. Primary AML cells were treated with parthenolide and vildagliptin alone or in Vildagliptin’s enhancement of parthenolide’s cytotoxicity is combination, and the cells were then plated into colony formation independent of its known target DPPIV assays. Vildagliptin enhanced parthenolide’s ability to reduce the Vildagliptin’s known target as an oral anti-hyperglycemic is clonogenic growth of the tested primary AML samples by 29–39% DPPIV,17 but the mechanism by which it synergized with parthe- (Supplementary Table 1). In addition, we tested the effects of the nolide was unclear. Therefore, we asked whether vildagliptin’s parthenolide/vildagliptin combination by engrafting primary AML synergy with parthenolide was mediated through the known cells into immune-deficient mice. Ex vivo AML patient cells were target DPPIV. To evaluate the potential role of DPPIV in mediating treated with the combination, individual drugs, or a DMSO control the synergistic effects, we measured the expression of cell surface and injected into the right femur of preconditioned NOD-SCID DPPIV in TEX cells by flow cytometry. DPPIV is absent from the mice. Following a 4-week engraftment period, mice were killed surface of TEX and OCI-AML2 cells. In contrast, we determined that and the percentage of AML cells in the non-injected femur was OCI-Ly17 cells, a human lymphoma cell line, express DPPIV measured by flow cytometry. The parthenolide/vildagliptin (Figure 3a); however, these cells do not show synergy with this

& 2013 Macmillan Publishers Limited Leukemia (2013) 1236 – 1244 Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1240

a 100 100 100 Tex AML2 OCI-Ly17 80 80 80

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0 0 0 100 101 102 103 100 101 102 103 100 101 102 103 DPPIV DPPIV DPPIV

b OCI-Ly17 c 0.10 DPP activity µ 140 Vildagliptin ( M) 0.08 120 0 10 100 0.06 80 60 0.04 Absorbance % Viable 40 0.02 20 0 0.00 0.0 2.5 OCI-LY17 TEX Parthenolide (µM) Cell line

d TEX OCI-AML2 20 30 Sitagliptin (µM) 0 15 10 20 10 10 5 % Growth inhibition % Growth 0 0 0.0 2.5 0.0 2.5 Parthenolide (µM) Parthenolide (µM) Figure 3. Vildagliptin’s synergy with parthenolide is independent of DPPIV. (a) The expression of cell surface DPPIV was measured in TEX, OCI- AML2 and OCI-Ly17 by ﬂow cytomtery. (b) OCI-Ly17 cells were incubated with 0 or 2.5 mM parthenolide and 0 or 10 mM vildagliptin for 72 h. Cell growth and viability were measured by the MTS assay. (c) DPP activity was measured in TEX and OCI-Ly17. TEX and OCI-Ly17 cells were incubated with the Gly-Pro-p-nitroanalide substrate for 60 min at 37 1C. The DPPIV-catalyzed conversion of Gly-Pro-p-nitroanalide to p-nitroanalide was measured spectrophotometrically at 405 nm. (d) TEX and OCI-AML2 cells were incubated with 0 or 2.5 mM parthenolide and 0or10mM sitagliptin for 72 h. Cell growth and viability were measured by the MTS assay. Data are the mean±s.d. from three independent experiments performed in duplicate.

drug combination (Figure 3b). Confirming the findings by flow and 9 share sequence homology, and vildagliptin inhibits the cytometry, a DPPIV enzymatic assay also demonstrated no DPPIV enzymatic activity of DPP8 (KI ¼ 810 nM), DPP9 (KI ¼ 95 nM) and 20 activity on TEX cells (Figure 3c). DPPIV (KI ¼ 3nM). We therefore assessed whether the inhibition Finally, to confirm that inhibition of DPPIV is not related to of DPP8 or DPP9 was responsible for vildagliptin’s enhancement vildagliptin’s enhancement of parthenolide’s activity, we assessed of parthenolide’s cytotoxicity. the combination of parthenolide and sitagliptin, a more selective As an initial step, we evaluated the expression of DPPs IV, inhibitor of DPPIV.18 Here, TEX or OCI-AML2 cells were treated with 8 and 9 in TEX, OCI-AML 2 cells and normal bone marrow. parthenolide and/ or sitagliptin and following 72 h of incubation, Both AML cell lines expressed DPP8 and DPP9, but had cell growth and viability were measured by the MTS assay. In little expression of DPPIV compared with normal marrow contrast to the synergistic combination with vildagliptin, the (Figure 4a). We also compared four publicly available micro- selective DPPIV inhibitor sitagliptin did not increase the anti- array data sets21–24 to determine whether DPP8 and DPP9 were leukemic activity of parthenolide (Figure 3d, two- way ANOVA, elevated in CD34 þ /CD38 leukemia cells compared with normal interaction effect: F2,6 ¼ 0.36; P ¼ 0.786). Thus, taken together, hematopoietic cells. Most but not all data sets demon- these results demonstrate that vildagliptin’s synergy with parthe- strated increased expression of DPP8 or DPP9 in AML stem cells nolide occurs through mechanisms unrelated to DPPIV inhibition. compared with normal hematopoietic cells (Supplementary Table 2). Next, we assessed the effects of parthenolide treatment on Vildagliptin’s synergy with parthenolide is mediated through expression of DPP8 and DPP9 in OCI-AML2 and TEX cells. inhibition of intracellular DPP8 and DPP9 Treatment with parthenolide at concentrations that synergize The DPPIV gene family consists of several members that include with vildagliptin did not alter the mRNA expression of either DPP8 DPPs IV, 8 and 9 and fibroblast activation protein.19 DPPs IV, 8 or DPP9 in OCI-AML2 or TEX cells (Figure 4b).

Leukemia (2013) 1236 – 1244 & 2013 Macmillan Publishers Limited Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1241

a Expression of DPP enzymes b TEX OCI-AML2 1.2 2.0 DP4 1.2 DPP8 DP8 1.0 1.0 DPP9 1.5 DP9 0.8 0.8 1.0 0.6 0.6 0.4 0.4 0.5 0.2 0.2 Relative expression Relative Relative expression Relative Relative expression Relative (Arbitrary Units- AU) (Arbitrary Units- AU) (Arbitrary Units- AU) 0.0 0.0 0.0 BMTEX AML2 CTL PTL CTL PTL Cell type Treatment I II III

c DPP8 mRNA DPP8 - Parthenolide alone DPP8 - Parthenolide + Vildagliptin 75 100 1.00 * PTL ** PTL + VILD 75 * 0.75 50 ** 50 0.50 * 25 0.25 25 % Growth inhibition % Growth % Growth inhibition % Growth Relative expression Relative (Arbitrary Units- AU) 0.00 0 0 GFP 8-417.0 8-674.0 GFP 8-417 8-647 GFP 8-417 8-647 Clone ID Clone ID Clone ID

d DPP9 mRNA DPP9 - Parthenolide alone DPP9 - Parthenolide + Vildaglitpin *** 1.00 100 100 ** PTL PTL + VILD 0.75 75 75 ** * 0.50 50 * 50

0.25 25 25 % Growth inhibition % Growth % Growth inhibition % Growth Relative expression Relative (Arbitrary Units- AU) 0.00 0 0 GFP 9-2871.0 9-2315 GFP 9-2871 9-2315 GFP 9-2871 9-2315 Clone ID Clone ID Clone ID Figure 4. Knockdown of DPP8 or DPP9 enhances parthenolide-mediated cell death, but does not fully recapitulate the effects of vildagliptin. (a) DPPIV, DPP8 and DPP9 mRNA expression were measured in TEX and OCI-AML2 cells using qRT-PCR and normalized to the expression in human bone marrow. (representative figure of two independent experiments) (b) TEX and OCI-AML2 cells were incubated with 5 mM parthenolide (PTL) for 24 h and DPP8 and DPP9 mRNA expression was measured using qRT-PCR (c) OCI-AML2 cells were infected with shRNA- targeting DPP8 or control sequences. Target knockdown was confirmed by qRT-PCR (Panel I: representative figure of two independent experiments). Cells were then treated with 2.5 mM parthenolide for 72 h. Cell growth and viability were measured by the MTS assay and death was confirmed by trypan blue staining (Panel II). Cells were also treated with 2.5 mM parthenolide and 0 or 10 mM vildagliptin for 72 h. Cell growth and viability were measured by the MTS assay and death was confirmed by trypan blue staining (Panel III)). (d) OCI-AML2 cells were infected with shRNA-targeting DPP9 or control sequences in lentiviral vectors. Target knockdown was confirmed by qRT-PCR (Panel I: representative figure of two independent experiments). Cells were then treated with 2.5 mM parthenolide for 72 h and cell growth and viability was measured by the MTS assay and death was confirmed by trypan blue staining. (Panel II). Cells were also treated with 2.5 mM parthenolide and 0 or 10 mM vildagliptin for 72 h. Cell growth and viability were measured by the MTS assay and death was confirmed by trypan blue staining. (Panel III). Unless otherwise stated, data are the mean±s.d. from three independent experiments performed in duplicate. (*Po0.05; **Po0.01; ***Po0.005).

Finally, we knocked down DPP8 and DPP9 to determine the treated with parthenolide and vildagliptin, a significant increase in potential role of these targets in mediating vildagliptin’s synergy cell death was still observed when compared with parthenolide with parthenolide. We knocked down DPP8 and DPP9 in OCI- treatment alone (Panel III: DPP8: clone 8–417, t4 ¼ 6.5, Po0.05; AML2 cells using shRNA in lentiviral vectors. We were unsuccessful clone 8–647, t4 ¼ 6.8, Po0.01; DPP9: clone 9–2315, t4 ¼ 4.7, in knocking down these proteins in TEX cells. Knockdown of DPP8 Po0.01; clone 9–2871, t4 ¼ 20.4; Po0.005; Figures 4c and d). or DPP9 with two independent shRNA clones per target was Thus, these results suggest that knockdown of DPP8 or DPP9 can confirmed using quantitative reverse transcriptase PCR (qRT-PCR) enhance the cytotoxicity of parthenolide, but the single knock- (Panel I: Figures 4c and d). Next, we treated DPP8 or DPP9 down of either is insufficient to fully recapitulate the effects of knockdown cells with parthenolide. Knockdown of DPP8 and vildagliptin. DPP9 increased sensitivity to parthenolide compared with cells We therefore hypothesized that vildagliptin enhances parthe- infected with vector controls (Panel II: one-way ANOVA: DPP8: nolide’s cytotoxicity by inhibiting both DPP8 and DPP9. To test F2,8 ¼ 11.73, Po0.01; Figure 4c; DPP9: F2,8 ¼ 18.61, Po0.01; this hypothesis, DPP8 and DPP9 were simultaneously knocked Figure 4d). However, the enhancement of cytotoxicity did not down in OCI-AML2 cells using shRNA in lentiviral vectors. Target fully mimic the effect of vildagliptin. Moreover, when these single knockdown was confirmed using qRT-PCR (Figure 5a). Significant knockdown cells (that is, devoid in either DPP8 or DPP9) were enhancement of cytotoxicity was observed when DPP8 and DPP9

& 2013 Macmillan Publishers Limited Leukemia (2013) 1236 – 1244 Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1242 single oral dose.20 Renal elimination and analysis showed that 22–29% of excreted vildagliptin is unchanged (that is, not metabolized) and 4.5% of unchanged drug was excreted in the feces.25 In healthy patients, a 200-mg single oral dose achieved 95% DPPIV inhibition and no adverse reactions or toxicities were observed; AUC of 5.7±0.6 mg.h/ml (equivalent of 18 mM), Cmax 1.3±0.26 mg/ml and peak plasma concentrations at 1.5 h were reported.26 Further, in a two-year trial with patients with type 2 diabetes mellitus, 50 mg vildagliptin tablets twice daily were associated with minimal adverse effects (for example, dizziness, nausea in less than 12% of the study participants, n ¼ 396).27 Thus, given the favorable pharmacokinetic data for the treatment of hyperglycemia, vildagliptin concentrations (10 mM) necessary to synergize with parthenolide to induce leukemia cell cytotoxicity appear pharmacologically achievable and generally well-tolerated. Vildagliptin’s inhibition of DPPIV is well-documented in its role as an oral anti-hyperglyemic. However, as an anti-cancer agent we determined that vildagliptin’s activity, in combination with parthenolide, is independent of DPPIV. Instead, we demonstrate that vildagliptin’s synergy with parthenolide occurs through inhibition of intracellular DPP8 and DPP9. A limitation to this study is that we could not evaluate expression of DPPs 8 and 9 by immunoblotting. All commercially available antibodies cross- reacted with many non-specific proteins and the identity of the DPP8 and DPP9 proteins could not be determined with certainty. However, DPPIV shares 21 and 20% sequence homology with DPP8 and DPP9, respectively,28 and vildagliptin inhibits the enzymatic activity of all enzymes of the DPPIV gene family.20 Therefore, given the sequence similarity and cross-reactivity of vildagliptin with all members of the DPPIV gene family of enzymes, vildagliptin’s synergy with parthenolide through vildagliptin’s inhibition of DPP8 and DPP9 is a highly plausible mechanism. The DPPIV family of enzymes are ubiquitously expressed and are found in immune and epithelial cells of the brain, blood, lung, Figure 5. Knockdown of DPP8 and DPP9 recapitulates the effects of spleen and testes.29–31 DPPIV cleaves a number of substrates vildagliptin. Cells containing DPP8 knocked down were infected including chemokines, glucagon-like peptides, stromal-derived with DPP9 or control shRNA in lenti-virual vectors. (a) DPP8 and factor 1 (CXCL12) and RANTES.17 In addition to its role in DPP9 mRNA were quantified in these double knockout cells using qRT-PCR. (representative figure of two independent experiments) regulating insulin levels by inactivating the incretin hormones, (b) OCI-AML2 double knockout cells were treated with 0 or 2.5 mM DPPIV, as a component of CD26, exerts a level of cellular protec- parthenolide and 0 or 10 mM vildagliptin for 72 h. Cell growth and tion. For example, DPPIV protects against ischemia-induced tissue viability were measured by the MTS assay and confirmed by trypan damage by promoting adenosine metabolism through its binding blue staining. Data are the mean±s.d. from three independent of adenosine deaminase.32,33 However, the intracellular func- experiments performed in duplicate. (*Po0.05) (GFP: t4 ¼ 57.1, tions of DPP8 and DPP9 are less clear. A role in modifying cell– Po0.05; DPP9–2315/DPP8–417: t4 ¼ 2.4, P40.05, DPP9–2871/ extracellular matrix interactions has been suggested, but there is DPP8–674: t4 ¼ 1.9, P40.05). limited in vivo or human data and few DPP8 and DPP9 substrates have been identified.34 A recent study demonstrated a role in cellular proliferation, as DPP8 and DPP9 affect EGF receptor double knockdown cells were treated with parthenolide, and the signaling through inhibition of AKT phosphorylation.19 Although magnitude of effect matched that of vildagliptin (Figure 5b, the function of DPP8 and DPP9 remain to be elucidated, these Supplementary Figure 3). Moreover, the addition of vildagliptin enzymes are elevated in numerous hematological malignancies. did not produce further cell death in DPP8 and DPP9 knockdown Patients with B-cell chronic lymphocytic leukemia had elevated cells treated with parthenolide (Figure 5b). Thus, these results mRNA and protein levels of DPP8 and DPP9;35 plasma DPP activity indicate that vildagliptin’s synergy with parthenolide is mediated was elevated in 80 and 62% in patients with lymphoid leukemia through the inhibition of both DPP8 and DPP9. and AML, respectively.36 These elevated DPP levels suggest these enzymes may have a role in leukemia pathogenesis, and are thus valuable therapeutic targets. DISCUSSION The mechanism by which vildagliptin’s inhibition of DPP8 and Combination screening determined vildagliptin synergistically DPP9 enhances parthenolide’s cytotoxicity remains to be eluci- enhanced parthenolide’s anti-leukemic activity. This combination dated. However, vildagliptin’s inhibition of DPP8 and DPP9 was was effective in primary AML samples including the stem cell specific to parthenolide, as the cytotoxicity of other conventional fraction. Mechanistically, we determined that synergy was through therapeutics (that is, cytarbine, vinblastine and doxorubicin) was vildagliptin’s inhibition of intracellular DPP8 and DPP9. not enhanced by the addition of vildagliptin. Potentially, Vildagliptin is used clinically as an oral anti-hyperglycemic for vildagliptin influences one or more of the cell signaling path- the treatment of diabetes. As such, the pharmacokinetics of ways perturbed by parthenolide. Indeed, parthenolide has been vildagliptin has been well-studied. It is rapidly absorbed (tmax ¼ demonstrated to inhibit NFkB by binding to IkB-kinase, modify 1.1 h) and rapidly excreted (elimination t1/2 ¼ 1.6–2.5 h) with a p50 and p65 NFkB subunits, activate p53, block STAT 3 phos- reported absolute bioavailability of B85%, following a 100-mg phorylation and increase the production of reactive oxygen

Leukemia (2013) 1236 – 1244 & 2013 Macmillan Publishers Limited Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1243 4–8 species. However, targeting of one these pathways alone is 12 Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the com- insufficient to induce cell death. For example, treatment of AML bined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: blasts with NFkB decoy oligonucleotides did not elicit an 27–55. apoptotic response; apoptosis did occur when cytarabine was 13 Chou TC. Preclinical versus clinical drug combination studies. Leuk Lymphoma added in combination with the decoy oligonucleotides;37 and 2008; 49: 2059–2080. cellular anti-oxidant status determines sensitivity to parthenolide, 14 Eberhard Y, Gronda M, Hurren R, Datti A, MacLean N, Ketela T et al. Inhibition of SREBP1 sensitizes cells to death ligands. Oncotarget 2011; 2: 186–196. as only myeloma and hepatoma cells with low levels of catalase38 37 15 Sato K, Aytac U, Yamochi T, Yamochi T, Ohnuma K, McKee KS et al. CD26/ or glutathione, respectively, were sensitive to parthenolide- dipeptidyl peptidase IV enhances expression of topoisomerase II alpha and sen- induced cell death. Finally, a number of studies demonstrate that sitivity to apoptosis induced by topoisomerase II inhibitors. Br J Cancer 2003; 89: numerous drugs (for example, mTOR inhibitors, TRAIL, paclitaxel, 1366–1374. sulindac) with various targets combine with parthenolide to 16 Xu GW, Ali M, Wood TE, Wong D, Maclean N, Wang X et al. The ubiquitin- induce cancer cell death,9,39,40 which further supports the notion activating enzyme E1 as a therapeutic target for the treatment of leukemia and that multiple cellular processes are involved in parthenolide- multiple myeloma. Blood 2010; 115: 2251–2259. induced cell death. Therefore, it is likely that the inhibition of DPP8 17 Kirby M, Yu DM, O’Connor S, Gorrell MD. Inhibitor selectivity in the clinical and DPP9 by vildagliptin adds further stress to parthenolide- application of dipeptidyl peptidase-4 inhibition. Clin Sci 2010; 118: 31–41. 18 Sauve M, Ban K, Momen MA, Zhou YQ, Henkelman RM, Husain M et al. Genetic treated leukemia cells, thus enhancing its overall cytotoxicity. deletion or pharmacological inhibition of dipeptidyl peptidase-4 improves In summary, a high-throughput screen identified vildagliptin, an cardiovascular outcomes after myocardial infarction in mice. Diabetes 2010; 59: oral anti-hyperglycemic drug used in the treatment of diabetes, as 1063–1073. a drug capable of synergistically enhancing parthenolide’s anti- 19 Yao TW, Kim WS, Yu DM, Sharbeen G, McCaughan GW, Choi KY et al. A novel role leukemia activity. Mechanistically, vildagliptin’s basis for synergy of dipeptidyl peptidase 9 in epidermal growth factor signaling. Mol Cancer Res with parthenolide was through vildagliptin’s inhibition of 2011; 9: 948–959. the intracellular enzymes DPP8 and DPP9. Future studies are 20 Burkey BF, Hoffmann PK, Hassiepen U, Trappe J, Juedes M, Foley JE. Adverse needed to evaluate the safety of the parthenolide/vildagliptin effects of dipeptidyl peptidases 8 and 9 inhibition in rodents revisited. Diabetes combination. Obes Metab 2008; 10: 1057–1061. 21 Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011; 17: 1086–1093. CONFLICT OF INTEREST 22 Gentles AJ, Plevritis SK, Majeti R, Alizadeh AA. Association of a leukemic stem cell The authors declare no conflict of interest. gene expression signature with clinical outcomes in acute myeloid leukemia. JAMA 2010; 304: 2706–2715. 23 Kikushige Y, Shima T, Takayanagi S, Urata S, Miyamoto T, Iwasaki H et al. TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells. Cell Stem ACKNOWLEDGEMENTS cell 2010; 7: 708–717. We thank Dr John Dick (University Health Network, Toronto, ON) for the use of TEX 24 Majeti R, Becker MW, Tian Q, Lee TL, Yan X, Liu R et al. Dysregulated gene cells. This study was supported by research grants to ADS by the Leukemia/ expression networks in human acute myelogenous leukemia stem cells. Proc Natl Lymphoma Society and PAS by The University of Waterloo. PAS was supported by the Acad Sci USA 2009; 106: 3396–3401. Canadian Institutes for Health Research postdoctoral fellowship and the Lady Tata 25 He H, Tran P, Yin H, Smith H, Batard Y, Wang L et al. Absorption, metabolism, and Memorial Trust Award. excretion of [14C]vildagliptin, a novel dipeptidyl peptidase 4 inhibitor, in humans. Drug Metab Dispos 2009; 37: 536–544. 26 Hu P, Yin Q, Deckert F, Jiang J, Liu D, Kjems L et al. Pharmacokinetics and pharmacodynamics of vildagliptin in healthy Chinese volunteers. J Clin Pharmacol REFERENCES 2009; 49:39–49. 1 Lowenberg B, Downing JR, Burnett A. Acute myeloid leukemia. N Engl J Med 1999; 27 Rosenstock J, Niggli M, Maldonado-Lutomirsky M. Long-term 2-year safety and 341: 1051–1062. efficacy of vildagliptin compared with rosiglitazone in drug-naive patients with 2 Burnett A, Wetzler M, Lowenberg B. Therapeutic advances in acute myeloid type 2 diabetes mellitus. Diabetes Obes Metab 2009; 11: 571–578. leukemia. J Clin Oncol 2011; 29: 487–494. 28 Rummey C, Metz G. Homology models of dipeptidyl peptidases 8 and 9 3 Shipley JL, Butera JN. Acute myelogenous leukemia. Exp Hematol 2009; 37: with a focus on loop predictions near the active site. Proteins 2007; 66: 649–658. 160–171. 4 Guzman ML, Rossi RM, Karnischky L, Li X, Peterson DR, Howard DS et al. The 29 Yu DM, Ajami K, Gall MG, Park J, Lee CS, Evans KA et al. The in vivo expression of sesquiterpene lactone parthenolide induces apoptosis of human acute myelo- dipeptidyl peptidases 8 and 9. J Histochem Cytochem 2009; 57: 1025–1040. genous leukemia stem and progenitor cells. Blood 2005; 105: 4163–4169. 30 Maes MB, Dubois V, Brandt I, Lambeir AM, Van der Veken P, Augustyns K et al. 5 Guzman ML, Rossi RM, Neelakantan S, Li X, Corbett CA, Hassane DC et al. An orally Dipeptidyl peptidase 8/9-like activity in human leukocytes. J Leukoc Biol 2007; 81: bioavailable parthenolide analog selectively eradicates acute myelogenous 1252–1257. leukemia stem and progenitor cells. Blood 2007; 110: 4427–4435. 31 Abbott CA, Yu DM, Woollatt E, Sutherland GR, McCaughan GW, Gorrell MD. 6 Zunino SJ, Ducore JM, Storms DH. Parthenolide induces significant apoptosis and Cloning, expression and chromosomal localization of a novel human dipeptidyl production of reactive oxygen species in high-risk pre-B leukemia cells. Cancer peptidase (DPP) IV homolog, DPP8. Eur J Biochem 2000; 267: 6140–6150. Lett 2007; 254: 119–127. 32 Moro PJ, Quilici J, Giorgi R, Cuisset T, By Y, Boussuges A et al. Mononuclear cell 7 Carlisi D, D’Anneo A, Angileri L, Lauricella M, Emanuele S, Santulli A et al. adenosine deaminase and CD26/dipeptidylpeptidase-IV activities are sensitive Parthenolide sensitizes hepatocellular carcinoma cells to TRAIL by inducing the markers of reperfusion during percutaneous transluminal angioplasty. Int J Cardiol expression of death receptors through inhibition of STAT3 activation. J Cell Physiol 2011; e-pub ahead of print 5 November 2011; PMID: 22062893. 2011; 226: 1632–1641. 33 Tan EY, Richard CL, Zhang H, Hoskin DW, Blay J. Adenosine downregulates DPPIV 8 Gopal YN, Chanchorn E, Van Dyke MW. Parthenolide promotes the ubiquitination on HT-29 colon cancer cells by stimulating protein tyrosine phosphatase(s) and of MDM2 and activates p53 cellular functions. Mol Cancer Ther 2009; 8: 552–562. reducing ERK1/2 activity via a novel pathway. Am J Physiol Cell Physiol 2006; 291: 9 Hassane DC, Sen S, Minhajuddin M, Rossi RM, Corbett CA, Balys M et al. Chemical C433–C444. genomic screening reveals synergism between parthenolide and inhibitors of the 34 Yu DM, Wang XM, McCaughan GW, Gorrell MD. Extraenzymatic functions of the PI-3 kinase and mTOR pathways. Blood 2010; 116: 5983–5990. dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration 10 Skrtic M, Sriskanthadevan S, Jhas B, Gebbia M, Wang X, Wang Z et al. Inhibition of and apoptosis. FEBS J 2006; 273: 2447–2460. mitochondrial translation as a therapeutic strategy for human acute myeloid 35 Sulda ML, Abbott CA, Macardle PJ, Hall RK, Kuss BJ. Expression and prognostic leukemia. Cancer cell 2011; 20: 674–688. assessment of dipeptidyl peptidase IV and related enzymes in B-cell chronic 11 Spagnuolo PA, Hu J, Hurren R, Wang X, Gronda M, Sukhai MA et al. The anti- lymphocytic leukemia. Cancer Biol Ther 2010; 10: 180–189. helmintic flubendazole inhibits microtubule function through a mechanism 36 de Andrade CF, Bigni R, Pombo-de-Oliveira MS, Alves G, Pereira DA. CD26/DPPIV distinct from Vinca alkaloids and displays preclinical activity in leukemia and cell membrane expression and DPPIV activity in plasma of patients with acute myeloma. Blood 2010; 115: 4824–4833. leukemia. J Enzyme Inhib Med Chem 2009; 24: 708–714.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1236 – 1244 Vildagliptin synergizes with parthenolide PA Spagnuolo et al 1244 37 Romano MF, Lamberti A, Bisogni R, Tassone P, Pagnini D, Storti G et al. 39 Yip-Schneider MT, Nakshatri H, Sweeney CJ, Marshall MS, Wiebke EA, Schmidt CM. Enhancement of cytosine arabinoside-induced apoptosis in human myeloblastic Parthenolide and sulindac cooperate to mediate growth suppression and inhibit leukemia cells by NF-kappa B/Rel- speciﬁc decoy oligodeoxynucleotides. the nuclear factor-kappa B pathway in pancreatic carcinoma cells. Mol Cancer Ther Gene Ther 2000; 7: 1234–1237. 2005; 4: 587–594. 38 Wang W, Adachi M, Kawamura R, Sakamoto H, Hayashi T, Ishida T et al. Parthe- 40 Zhang S, Lin ZN, Yang CF, Shi X, Ong CN, Shen HM. Suppressed NF-kappaB and nolide-induced apoptosis in multiple myeloma cells involves reactive oxygen sustained JNK activation contribute to the sensitization effect of parthenolide to species generation and cell sensitivity depends on catalase activity. Apoptosis TNF-alpha-induced apoptosis in human cancer cells. Carcinogenesis 2004; 25: 2006; 11: 2225–2235. 2191–2199.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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