Letters to the Editor 1804 2 Grand FH, Hidalgo-Curtis CE, Ernst T, Zoi K, Zoi C, McGuire C et al. RARS-T, a mechanism analogous to UPD9p and JAK2 V617F Frequent CBL mutations associated with 11q acquired mutation. Leukemia 2009; 23: 610–614. uniparental disomy in myeloproliferative neoplasms. Blood 2009; 6 Jasek M, Gondek LP, Bejanyan N, Tiu R, Huh J, Theil KS et al. TP53 113: 6182–6192. mutations in myeloid malignancies are either homozygous 3 Makishima H, Cazzolli H, Szpurka H, Dunbar A, Tiu R, Huh J et al. or hemizygous due to copy number-neutral loss of heterozygosity Mutations of e3 ligase cbl family members constitute or deletion of 17p. Leukemia 2010; 24: 216–219. a novel common pathogenic lesion in myeloid malignancies. J Clin 7 O’Keefe C, McDevitt MA, Maciejewski JP. Copy neutral loss of Oncol 2009; 27: 6109–6116. heterozygosity: a novel chromosomal lesion in myeloid malignan- 4 Delhommeau F, Dupont S, Della Valle V, James C, Trannoy S, cies. Blood 2010; 115: 2731–2739. Masse A et al. Mutation in TET2 in myeloid cancers. N Engl J Med 8 Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R 2009; 360: 2289–2301. et al. Somatic mutations altering EZH2 (Tyr641) in follicular and 5 Szpurka H, Gondek LP, Mohan SR, Hsi ED, Theil KS, Maciejewski diffuse large B-cell lymphomas of germinal-center origin. Nat Genet JP. UPD1p indicates the presence of MPL W515L mutation in 2010; 42: 181–185.

Targeting 72 enhances inhibitor-induced apoptosis in myeloma

Leukemia (2010) 24, 1804–1807; doi:10.1038/leu.2010.168; factor-kB), migration (phosphoinositide 3-kinase) and antiapop- Published online 12 August 2010 totic (nuclear factor-kB) pathways. Secondly, it has a central role in the UPR, which is responsible for ensuring the correct folding of nascent immunoglobulin, as well as providing survival signals Recent years have seen the development of a number of new for clonal expansion. Although targeting these pathways using small molecule therapies for the treatment of myeloma and as a inhibitors of Hsp90 has proved successful in myeloma preclinical result of their introduction there have been significant improve- models, preliminary results in patients suggest that combinations ments in patient survival. However, patients continue to relapse, of agents will be required for clinical use. and the current challenge is to develop therapies able to To provide a rationale for the development of effective effectively treat these refractory cases. An emerging feature of mechanism-based clinical combinations, we have tested the the use of single, highly specific molecules is that they can be hypothesis that the induction of the family member, clinically ineffective because of the redundancy in the pathway Hsp72, after treatment with an Hsp90 inhibitor, protects targeted and compensatory changes induced by drug exposure. myeloma cells from apoptosis by enabling cells to increase Consequently therapeutic efficacy is reliant on the development their ability to fold damaged proteins. Hence inhibition of the of effective combinations targeting synergistic pathways. function of Hsp72 may enhance the apoptosis induced by A simplified view of the function and biology of plasma cells is Hsp90 inhibition. We have therefore investigated the role of that they exist to produce immunoglobulin and this can provide a Hsp72 in myeloma using two chemical compounds, which have differential marker, which can be targeted. The requirement for been shown to inhibit the protective effects of Hsp70, triptolide immunoglobulin production means that the myeloma plasma cell and KNK437. To confirm the impact of Hsp72 inhibition, we is reliant upon the unfolded protein response (UPR) and have verified these findings using small hairpin RNA (shRNA) molecular chaperones for survival.1 Heat shock protein (Hsp) specifically directed against Hsp72. 90 is a molecular that has an important role in the Our results show that after treatment with the Hsp90 inhibitor pathophysiology of myeloma and represents a relevant therapeu- 17-AAG for 24 h, myeloma cells increase the protein level of tic target.2,3 It fulfils two essential roles in plasma cells: firstly it is Hsp72. Combination treatment with KNK437 or triptolide a major pro-survival chaperone, which associates with and blocks this increase (Figure 1a) and results in a decrease in stabilizes an extensive range of signal transducers important to proliferation compared with treatment with 17-AAG alone. plasma cell survival and proliferation (insulin-like - Using a series of 16 concentrations of these agents applied in 1, vascular endothelial growth factor, RAF and p42/44 mitogen- combination for a period of 4 days, we demonstrate that the activated protein kinase), regulation (AKT and nuclear concomitant application of 17-AAG with either of these

Figure 1 (a) Myeloma cells were treated with either: 17-AAG (5 mM), triptolide (T, 100 nM) and KNK437 (K, 100 nM) alone or in combination for 24 h, and Hsp72 expression analyzed by western blot. (b) Myeloma cells were treated with a 16-point concentration range of each agent. Agents were applied for 4 days, in three regimens: concomitantly on day 1, agent 1 applied 24 h before agent 2 and the inverse, as indicated. The combination index (CI) methods of Chou and Talalay were used to assess for synergistic effects. Graphs show the CI plotted against the survival fraction. Values o1 indicate synergism, values between 1 and 1.2 indicate additive effects, values 41.2 indicate antagonism. (c) Myeloma cells were left untreated (C), treated with triptolide alone (T) or in combination with 17-AAG (17) for 24 h and the % of cell death analyzed: Upper panel: trypan blue, middle and lower panels: annexin V/PI results for H929 and U266 cell lines. (d) Left panel: reverse transcriptase-PCR (RT-PCR) analysis of CHOP expression after treatment with 17-AAG or triptolide or a combination of both agents. Right panel: cleavage of XBP1 was analyzed by RT-PCR using LUX primers. The ratio of spliced to unspliced is shown. (e) Upper panel: western blot analysis of cell death pathways after treatment with drugs alone or in combination for 24 h. Lower panel: analysis of the dependence of drug-induced cell death on caspase- dependent cell death pathways. Cells were pretreated with Z-VAD-FMK (Z) (50 mM) followed by the drugs alone or in combination 24 h. Samples were analyzed by annexin V/PI staining and the % of apoptotic cells displayed. Calculated P-values showed that there was a significant difference n between T and T þ Z(P ¼ 0.019) and between 17 þ T and 17 þ T þ Z(P ¼ 0.034). indicates statistically significant P-values o0.05. (f) Upper panel: myeloma cells transfected with P, C1 or C4 shRNA were treated with 17-AAG and cell proliferation measured by WST-1 assay over a 4-day period. Lower panel: western blot analysis of Hsp72 expression after transduction with Hsp72 shRNAs: P ¼ control, scrambled shRNA plasmid transfected cells. C1 and C4 ¼ transfected cells expressing two different Hsp72 shRNAs. All experiments were performed in triplicate and, unless otherwise stated representative data using H929 cells is shown throughout.

Leukemia Letters to the Editor 1805 adHsp72 H929 CHOP XBP1 Splicing 12 2 10 Actin 8 1 17-AAG 6 0

C 17 K 17 +K 4 Ratio 2 -1

Fold Change 0 -2 Hsp72 024624 C24624

U266 12 2 10 Actin Triptolide 8 1 alone 6 0 4 -1 C 17 T 17 +T 2

Fold Change -2 0 024624 C2 4 624 b 2.5 12 2 2 10 Antagonistic 1 1.5 17-AAG + 8 6 0 Triptolide Ratio Ratio H929 1 Additive 4 -1 2 0 0.2 0.4 0.6 0.8 1 -2 Fold Change 0.5 0 0 24624 C 2 46 24 Synergistic 0 Time (h) Time (h)

Combination Inddex (CI) -0.5 e 17-AAG Triptolide 17-AAG + Survival Fraction Triptolide 2.5 024624 0246 24 0 2 4 6 24 Time (h) 2 Antagonistic Caspase 9 1.5 U266 1 Additive 0 0.2 0.4 0.6 0.8 1 Caspase 8 0.5 Synergistic 0

Combination Index (CI) -0.5 Survival Fraction Caspase 3

17-AAG and Triptolide concomitantly 17-AAG 24 h then Triptolide Actin Triptolide 24 h then 17-AAG

100 c 06 24 Time (h) 90 80 * * 100 90 70 80 60 70 60 H929 50 50 40 40 30 U266 30

20 % Cell Death

% Cell Death 10 20 0 10 0

06 24 Time (h)

100% Treatment Early apoptosis 80% Late apoptosis 60% f 120

H929 Viable 40% H929 P 100 H929 C1

% Total cells 20% 80 H929 C4 0% 60 40 20 % Cell Proliferation 0246 Time (h) 0 1 3 5 0 10 15 0.1 100% 0.3 0.7 0.75 0.05 0.25 0.45 0.65 Early apoptosis 0.05 0.08 80% Late apoptosis 17-AAG (uM) 60% Viable PC1C4 40% U266 20% Hsp72 % Total cells 0% Actin

Treatment 48 72 48 7296 48 72 96 Time (h)

Leukemia Letters to the Editor 1806 compounds induces either additive or synergistic, antiprolifera- Hsp90 inhibitors in myeloma, we tested the effect of 17-AAG on tive effects (Figure 1b). These effects are not seen in normal, the proliferation of cells transfected with Hsp72 shRNAs human mononuclear cells (data not shown). We go on to show compared with those transfected with a scrambled shRNA. that both KNK437 and triptolide act synergistically with 17-AAG We demonstrate that Hsp72 knockdown induced a considerable to increase the induction of apoptosis, as assessed by trypan increase, of up to 40%, in the susceptibility of myeloma cells to blue exclusion and annexin V/PI staining (Figure 1c). Cell cycle 17-AAG-induced apoptosis (Figure 1f). analysis demonstrated that single-agent treatment with 17-AAG In this work, we demonstrate that the combination of Hsp90 induced a G2 cell cycle arrest. However, the combination of inhibitors with inhibitors of Hsp72 induction, act synergistically triptolide with 17-AAG overcomes this arrest creating an to kill myeloma cells. This observation is compatible with the increase in the sub-G1 cell population, indicative of increased current understanding of the function of Hsp72, which interacts cell death (data not shown). with members of the intrinsic apoptotic pathway, such as Bax, to To determine the mechanism underlying the increase in prevent release of pro-apoptotic molecules from the mitochon- apoptosis and decrease in cell proliferation induced by these dria and with Apaf-1, blocking the recruitment of procaspase 9 inhibitors, we examined the cellular stress/UPR pathway and the to the apoptosome. In addition, it also associates with and caspase-dependent apoptotic pathways. Previously we have inhibits precursor forms of caspases 3 and 7.5 Thus inhibition of shown that Hsp90 inhibition in myeloma cells results in the Hsp72 results in an increase in caspase-dependent cell death. In induction of endoplasmic reticulum stress and UPR activation, addition, we show that combining inhibitors of Hsp90 with leading to cell death via activation of the ER resident inhibitors of Hsp72 induction enhances cell death through transmembrane protein kinase (PERK) branch of the UPR and modulation of the endoplasmic reticulum stress pathway and the delivery of a proapoptotic signal via CCAAT/enhancer the UPR. We demonstrate increased expression of CHOP binding protein homologues protein (CHOP). In this study, we mRNA, suggesting that the combination efficiently induces show that treatment with 17-AAG or triptolide alone induced cell death via the PERK branch of the UPR, which is initiated upregulation of CHOP mRNA levels over a 24-h period through the effects of Bax on mitochondria and the subsequent (Figure 1d). Concomitant treatment with both agents resulted initiation of the mitochondrial-dependent cell death signaling in a sustained increase in CHOP mRNA expression, above those cascades. When this pathway is activated by 17-AAG alone, seen with either agent alone, suggesting that this is one it is normally interrupted downstream of CHOP by the mechanism by which the combination enhances apoptosis. Hsp70-DnaJ chaperone complex which inhibits Bax transloca- Activation of the Inositol requiring enzyme 1 (IRE1) branch of tion. However, this inhibitory effect is overcome by the the UPR results in the splicing of X-box binding protein 1 (XBP1) combined use of Hsp inhibitors and contributes to their to its active form, spliced XBP1 (XBP1s), which delivers a synergistic effects. Further evidence of cell death signaling prosurvival signal.4 We show that 17-AAG induced rapid, via the UPR comes from the demonstration that the drug transient cleavage of XBP1,whereas triptolide induced XBP1 combination induces earlier and stronger XBP1 cleavage than cleavage that was sustained for up to 24 h (Figure 1d). Cleavage either drug alone. However, this early pro-survival effect is not of XBP1 induced by the drug combination was readily detected, maintained and subsequent downregulation of XBP1 promotes sustained and stronger than that induced by single agent increased cell death. therapy. However, by 24 h XBP1s was undetectable and a We confirm that the effects on cell death seen with the decrease in the total level of XBP1 was noted. These results chemical inhibitors of Hsp72 induction are a direct effect of suggest that by 24 h, the combination of agents has tipped the Hsp72 silencing using shRNA. shRNA gene silencing of Hsp72 survival/apoptosis balance of UPR signaling towards cell death increased the susceptibility of myeloma cells to 17-AAG- with an increase in the levels of pro-apoptotic CHOP and a induced cell death in a manner similar to that seen with the decrease in the level of prosurvival XBP1s. chemical inhibitors. This result is in line with work in other cell 17-AAG activates both the intrinsic (caspases 9 and 3) and types including colon and ovarian carcinoma cells, confirming extrinsic caspase (caspases 8 and 3) pathways within 4 h of the potential of Hsp72 as a molecular target.6 treatment (Figure 1e). Triptolide alone induces cleavage of A number of high throughput screens have identified more caspases 9, 8 and 3 but only after 24 h of treatment. As specific Hsp72 small molecule inhibitors which are suitable for expected, the combination of agents induced cleavage of both clinical use.7–9 Using these drugs either in combination with sets of caspases at 4 h which is sustained. Importantly, the Hsp90 inhibitors or other drugs which induce cellular stress and combination of 17-AAG and triptolide induced a more upregulate Hsp72, offers a way to increase clinical efficacy. Our significant cleavage of caspases 9 and 3 than of caspase 8, work specifically demonstrates that combinations of specific suggesting that the combination potentiates cell death via small molecule inhibitors of Hsp’s 90 and 72 may be sustained and enhanced activation of predominantly the particularly effective against immunoglobulin producing, intrinsic caspase-dependent cell death pathway. The validity myeloma cells, as these cells intrinsically rely heavily upon of this observation was confirmed using the caspase inhibitor heat shock proteins to deal with their protein load and redirect Z-VAD-FMK, which showed that only a minority of cell death and degrade misfolded proteins. occurs via a caspase-dependent mechanism in response to 17-AAG treatment alone, whereas the majority of cell death in response to triptolide alone or the combination occurs via a Conflict of interest caspase-dependent mechanism (Figure 1e). As triptolide is primarily an inhibitor of Heats Shock Factor 1 The authors are employees of The Institute of Cancer Research, (HSF1)-mediated heat shock response and not a specific which has a commercial interest in Hsp90 inhibitors. The inhibitor of Hsp72, we determined the specific effects of authors have been involved in funded research collaborations Hsp72 knockdown using shRNA. Using a transient expression on Hsp90 inhibitors with Vernalis Ltd. and on chaperone and system a number of shRNAs were found to successfully stress pathways with AstraZeneca. Intellectual property arising knockdown expression of Hsp72 in H929 cells (Figure 1f). To from the Hsp90 programme has been licensed to Vernalis and verify whether knockdown of Hsp72 increased the efficacy of Novartis. PW has been a consultant to Novartis.

Leukemia Letters to the Editor 1807 Acknowledgements activation of the unfolded protein response pathway in myeloma plasma cells. Blood 2007; 110: 2641–2649. We acknowledge NHS funding to the NIHR Biomedical Research 2 Powers MV, Workman P. Inhibitors of the heat shock response: Centre. This work was supported by the Kay Kendall Leukaemia Fund, biology and pharmacology. FEBS Lett 2007; 581: 3758–3769. 3 Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Kung AL, Myeloma UK, Luck-Hille Foundation, Cancer Research UK Program Davies FE et al. Antimyeloma activity of heat shock protein-90 Grant C309/A8274 and PW is a Cancer Research UK Life Fellow. inhibition. Blood 2006; 107: 1092–1100. 4 Davenport EL, Morgan GJ, Davies FE. Untangling the unfolded protein response. Cell Cycle 2008; 7: 865–869. EL Davenport1, A Zeisig1, LI Aronson1, HE Moore1, 5 Komarova EY, Afanasyeva EA, Bulatova MM, Cheetham ME, S Hockley1, D Gonzalez1, EM Smith1, MV Powers2, SY Sharp2, Margulis BA, Guzhova IV. Downstream caspases are novel targets P Workman2, GJ Morgan1 and FE Davies1 for the antiapoptotic activity of the molecular chaperone hsp70. 1Section of Haemato-Oncology, The Institute of Cancer Cell Stress Chaperones 2004; 9: 265–275. Research, Sutton, Surrey, UK and 6 Powers MV, Clarke PA, Workman P. Dual targeting of HSC70 and 2Cancer Research UK Centre for Cancer Therapeutics, The HSP72 inhibits HSP90 function and induces tumor-specific apop- Institute of Cancer Research, Sutton, Surrey, UK tosis. Cancer Cell 2008; 14: 250–262. E-mail: [email protected] 7 Evans CG, Chang L, Gestwicki JE. Heat shock protein 70 (Hsp70) as an emerging drug target. J Med Chem 2010; 53: 4585–4602. 8 Leu JI, Pimkina J, Frank A, Murphy ME, George DL. A small molecule References inhibitor of inducible heat shock protein 70. Mol Cell 2009; 36: 15–27. 9 Powers MV, Jones K, Barillari C, Westwood I, van Montfort RL, Workman 1 Davenport EL, Moore HE, Dunlop AS, Sharp SY, Workman P, P. Targeting HSP70: the second potentially druggable heat shock protein Morgan GJ et al. Heat shock protein inhibition is associated with and molecular chaperone? Cell Cycle 2010; 9: 1542–1550.

HH-GV-678, a novel selective inhibitor of Bcr-Abl, outperforms imatinib and effectively overrides imatinib resistance

Leukemia (2010) 24, 1807–1809; doi:10.1038/leu.2010.169; a Published online 12 August 2010

Imatinib mesylate (STI-571, Gleevec, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA) is now used worldwide for the treatment of chronic myelogenous leukemia (CML) with b 0 13 10 30 100 300 1000 (nM) great success; however, CML often progresses or relapses HH-GV-678 p-Bcr-Abl because of the development of imatinib resistance after long-term treatment. The main cause for imatinib resistance p-Stat5 is the acquisition of point mutations in the genetic sequences p-Erk1/2 encoding the Abl kinase domain.1 Therefore, the discovery eIF4E and development of a new generation of Bcr-Abl inhibitors capable of overriding imatinib resistance is of utmost Imatinib p-Bcr-Abl importance. On the basis of the crystallographic structure of the imatinib–Abl Bcr-Abl complex, we identified a novel inhibitor of Bcr-Abl called HH-GV-678 (Figure 1a). The kinase activities of c-Abl, PDGFRb c p-Bcr-Abl (platelet derived growth factor -b) and c-Kit were HH-GV-678 Bcr-Abl inhibited by HH-GV-678 with IC50 values of 1.2±0.7, 307.6±259.0 and 665.5±306.8 nM; however, this drug had no p-Bcr-Abl effect on the kinase activities of vascular endothelial growth Imatinib factor receptor 2/kinase insert domain receptor (VEGFR2/KDR), Bcr-Abl Flt3, Ret, VEGFR3, c-Src, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). d p-Bcr-Abl Imatinib had IC50 values of 100.9±91.8, 201.8±23.6 and HH-GV-678 361.8±40.0 nM for c-Abl, PDGFRb and c-Kit, respectively. Bcr-Abl Consistent with the in vitro results of a kinase assay, p-Bcr-Abl HH-GV-678 completely blocked cellular Bcr-Abl autophosphory- Imatinib lation and Stat5 and Erk1/2 phosphorylation in K562 leukemia Bcr-Abl cells, with much more potent activity than did imatinib (30 vs 300 nM) (Figure 1b). However, HH-GV-678 inhibited the Figure 1 Effects of HH-GV-678 and imatinib on cellular phosphorylations of c-Kit and PDGFRb with less potency phosphorylation of Bcr-Abl in cells expressing nonmutated or mutated (Supplementary Figure 1) and had no effect on the phosphoryla- Bcr-Abl. (a) Chemical structure of HH-GV-678. (b) K562 cells expressing nonmutated Bcr-Abl, (c) 32D cells expressing H396P tion of EGFR, VEGFR, c-Src or HER2 (Supplementary Bcr-Abl mutant, and (d) 32D cells expressing T315I Bcr-Abl mutant Figure 2). These data indicate that HH-GV-678 is a selective were incubated with HH-GV-678 or imatinib for 3 h. Total cell lysates inhibitor of the c-Abl kinase and has much stronger activity than were analyzed by western blotting, and the levels of phospho- and imatinib. un-phosphoproteins were determined using specific antibodies.

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