Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC ...cancerres.aacrjournals.org/content/canres/76/11/3319.full.pdf · Therapeutics, Targets, and Chemical Biology Proteasomal

Therapeutics, Targets, and Chemical Biology

Proteasomal Inhibition by Ixazomib Induces CHK1and MYC-Dependent Cell Death in T-cell andHodgkin LymphomaDashnamoorthy Ravi1, Afshin Beheshti1, Nass�era Abermil1, Frank Passero1, Jaya Sharma1,Michael Coyle1, Athena Kritharis1, Irawati Kandela2, Lynn Hlatky3, Michail V. Sitkovsky4,Andrew Mazar2, Ronald B. Gartenhaus5, and Andrew M. Evens1

Abstract

Proteasome-regulated NF-kB has been shown to be importantfor cell survival in T-cell lymphoma and Hodgkin lymphomamodels. Several new small-molecule proteasome inhibitors areunder various stages of active preclinical and clinical develop-ment. We completed a comprehensive preclinical examination ofthe efficacy and associated biologic effects of a second-generationproteasome inhibitor, ixazomib, in T-cell lymphoma and Hodg-kin lymphoma cells and in vivo SCID mouse models. We dem-onstrated that ixazomib induced potent cell death in all celllines at clinically achievable concentrations. In addition, it sig-nificantly inhibited tumor growth and improved survival in T-celllymphoma and Hodgkin lymphoma human lymphoma xeno-graft models. Through global transcriptome analyses, proteaso-mal inhibition showed conserved overlap in downregulation ofcell cycle, chromatin modification, and DNA repair processesin ixazomib-sensitive lymphoma cells. The predicted activity for

tumor suppressors and oncogenes, the impact on "hallmarksof cancer," and the analysis of key significant genes fromglobal transcriptome analysis for ixazomib strongly favoredtumor inhibition via downregulation ofMYC andCHK1, its targetgenes. Furthermore, in ixazomib-treated lymphoma cells, weidentified that CHK1 was involved in the regulation of MYCexpression through chromatin modification involving histoneH3 acetylation via chromatin immunoprecipitation. Finally,using pharmacologic and RNA silencing of CHK1 or the associ-ated MYC-related mechanism, we demonstrated synergistic celldeath in combination with antiproteasome therapy. Altogether,ixazomib significantly downregulates MYC and induces potentcell death in T-cell lymphoma and Hodgkin lymphoma,and we identified that combinatorial therapy with anti-CHK1treatment represents a rational and novel therapeutic approach.Cancer Res; 76(11); 3319–31. �2016 AACR.

IntroductionUnderstanding the pertinent and functional biologic oncogen-

ic pathways of inhibition and mechanisms of resistance is criticalto optimally integrate novel therapeutics into cancer treatmentparadigms. Increased proteasomal activity is a frequentlyobserved phenomenon in malignant cells; hence, proteasomeinhibition may potentially block cell proliferation and result inthe inhibition of tumor progression (1). Bortezomib (Velcade)

was the first in class proteasomal targeted drug that is currentlyFDAapproved for the treatment of newly diagnosed and relapsed/refractory multiple myeloma and mantle cell lymphoma (2).Next-generation proteasome inhibitors, such as ixazomib, arecurrently under preclinical and clinical investigations. These novelagentswere developedwith anobjective of enhancing efficacy andimproving the tolerability of antiproteasomal therapeutics (3–5).Ixazomib is an investigational proteasome inhibitor currently inclinical trials in hematologic malignancies and early reportsindicate promising clinical activities in follicular and T-cell lym-phoma (6); however, its biologic activity in T-cell lymphoma andHodgkin lymphoma are largely unknown and the biologicmechanisms are not well defined.

T-cell lymphomas represent a heterogeneous group of aggres-sive non-Hodgkin lymphomas with overall poor prognosis (7).Further discovery of its tumor biology is needed and additionalnovel targeted therapeutic agents are desired.Hodgkin lymphomais generally a curablemalignancy by conventional chemotherapy;however, there continues to be a subset of patients with refractorydisease or relapse that succumbs to this disease. In addition, thereremains an unmet need to identify targeted, less toxic therapy forthe treatment of Hodgkin lymphoma to decrease the continuedacute and long-term toxicities associated with cytotoxic chemo-therapy and radiation (7–9). Moreover, multiple preclinical stud-ies strongly indicated that proteasome-dependentNF-kBmaybe a'master switch' of Hodgkin Reed Sternberg (HRS) cells and it has

1Division of Hematology Oncology andMolecular Oncology ResearchInstitute, Tufts Medical Center, Boston, Massachusetts. 2Chemistry ofLife Processes Institute, Northwestern University, Evanston, Illinois.3Center of Cancer Systems Biology, Tufts University School of Med-icine, Boston, Massachusetts. 4New England Inflammation and TissueProtection Institute, Northeastern University, Boston, Massachusetts.5University of Maryland, Baltimore, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

N. Abermil and F. Passero contributed equally to this article.

Corresponding Author: Andrew M. Evens, Division of Hematology/Oncology,Tufts Cancer Center, TuftsMedical Center, 800WashingtonAvenue, Boston, MA02111. Phone: 617-636-2694; Fax: 617-636-7060; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-2477

�2016 American Association for Cancer Research.

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been shown to be critical for cell survival in HRS and also T-celllymphoma models (10–15). Although proteasome is a rationaletherapeutic target for the treatment of T-cell lymphoma and Hodg-kin lymphoma, clinical studies in patients with thesemalignanciestreated with bortezomib were associated with modest clinicalbenefit (16, 17). With the development of ixazomib as second-generation proteasomal inhibitor with superior pharmacodynam-ic, pharmacokinetic, and tumor inhibitoryproperties in lymphomacompared with bortezomib (18, 19), interest in the treatment of T-cell lymphoma and Hodgkin lymphoma is renewed.

We utilized in vitro and in vivo tumor models to understand thebiologic mechanisms of action and antitumor activity of ixazo-mib in T-cell lymphoma and Hodgkin lymphoma, at clinicallyrelevant concentrations. Through global transcriptome and net-work analysis, we describe the impact of ixazomib on biologicpathways and tumor progression in T-cell lymphoma and Hodg-kin lymphoma cells. Furthermore, our investigation outlinesmechanisms of CHK1-dependent, MYC-regulated cell deathoccurring via ixazomib and the associated critical implicationsimpacting cell signaling and drug sensitivity. The impact ofixazomib on CHK1 andMYC is significant in the context of otherprevious studies indicating an existence of cooperative relation-ship between MYC and CHK1 as an important factor in drivinglymphomagenesis (20). Further sensitivity to CHK1 inhibitionhas also been reported in lymphoma cells with MYC overexpres-sion (21). Thus, with the ability to impactMYC and CHK1 as keydriving mechanisms of tumorigenesis in lymphoma, ixazomib isa potential appealing antilymphoma agent.

Materials and MethodsCell culture, reagents, and transfections

Hodgkin lymphoma cell lines L540 and L428 and, T-celllymphoma cell lines HH, Hut78, and Jurkat were grown inRPMI-1640 consisting of 10% heat-inactivated FBS and penicil-lin/streptomycin (Mediatech) under 5%CO2 and 37�C. Cell lineswere authenticated using short tandem repeat (STR) profilingservice provided by ATCC. Ixazomib was kindly provided byTakeda Pharmaceuticals, Inc. Belinostat, JQ1, and the CHK1inhibitor, AZD7762, were purchased from Selleck Chemicals.Nontargeting or MYC siRNAs were obtained fromGEHealthcare,and transfection was performed using Nucleofector device andreagent kit L (Lonza).

MTT and apoptosisMTT assay and Annexin V apoptosis detection were performed

as described previously (22). IC50 values and combination indicesfor drug treatment were derived using Calcusyn Version 2.1Software (Biosoft).

Lymphoma xenograftsFor examination of the in vivo effect of ixazomib, human

lymphoma xenografts derived from Jurkat (T-cell lymphoma) orL540 (Hodgkin lymphoma) grown in SCID mouse modelsdescribed previously (22)were treatedwith either saline (control)or ixazomib (0.36 or 0.72 mg/kg) by intraperitoneal (i.p.) injec-tions daily for 5 days for 3 weeks consisting 8 mice per group.Animals observation, tumor, and body weight measurements,and statistical analysis using GraphPad Prism 5.0, (GraphPadSoftware, Inc.), were performed as described previously (22). Toquantify tumor growth, we used nonlinear regression to fit the

tumor growth phase data with curves of the formV¼ bect, whereVis tumor volume and t is time (23). Here, c is interpreted as thetumor growth rate once the tumor starts to grow more or lessexponentially; b is interpreted as the extrapolated 'effective' vol-ume at the implant time, t ¼ 0.

Western blot analysisPreparation of protein lysates and Western blot analysis was

performed, as described previously (22). Primary antibodiesagainstMYC, total or phosphoCHK1 (Ser 345), total or acetylatedhistone H3, b-actin, HDAC3, p62, Cathepsin D total and cleavedcaspase-3, 8, 9, PARP, and b-actin were purchased from CellSignaling Technology.

Transcriptome analysesJurkat, L540, and L428 cells were treated in triplicates with 25

nmol/L ixazomib for 24 hours, RNA was isolated using RNeasyMinikit (Qiagen), and microarray experiment was performedusing Affymetrix Human Gene Chip 2.0 (Jurkat and L540), orHuman HT 12 Genechip Illumina (L428). The raw expressiondata from these experiments are available at NCBI Gene Expres-sion Omnibus (GEO) database, with following identifiersGSE66417 (Jurkat/L540) and GSE66415 (L428). Data for Jurkatand L540 cell lines were background adjusted and quantilenormalized using RMAExpress (24). Statistically relevant geneswere determined by applying LIMMA (25) with an false discoveryrate (FDR) < 0.05. Data for the L428 cell line (part of a largerdataset that was not used in this manuscript) was correctedthrough normalization of the housekeeping genes, quantile nor-malized, and then statistically relevant genes were determinedwith one-way ANOVA analysis with FDR < 0.05. Details on thepathway analysis, networks, and the unbiased method to deter-mine the key significant genes are previously published in refs.23, 26, 27.

Chromatin immunoprecipitation and PCR assaySample preparation was performed using Pierce Agarose ChIP

Kit, Thermo Scientific, qRT-PCR was performed using primer set(#PPH00100B) from SABiosciences, designed to amplify humanMYC promoter region proximal to transcription start site withSYBR Green Mastermix (Roche), and using Roche LightCycler I.For chromatin immunoprecipitation (ChIP), ChIP grade antibo-dies, Acetyl histone H3 K9 (#17-658) were purchased from EMDMillipore, anti-RNA polymerase II and normal rabbit IgG weresupplied in Pierce Agarose ChIP kit and used as positive andnegative control, respectively.

ResultsIn vitro and in vivo efficacy with therapy

Cell viability following exposure to ixazomib (12.5–1,000nmol/L) for 72 hours in T-cell lymphoma cell lines (Jurkat,Hut78, HH) and Hodgkin lymphoma cell lines (L428, L540)resulted in a dose-dependent decrease in cell viability in allcell lines (Fig. 1A and B). L540 cells were observed to bemost sensitive with associated 50% inhibitory effective dose(ED) concentration ED50 of 25 nmol/L compared with HH(41 nmol/L), Hut78 (52 nmol/L), Jurkat (38 nmol/L), andL428 (117 nmol/L). Treatment with ixazomib resulted inaccumulation of ubiquitinylated protein as would be expectedwith proteosomal inhibition (Fig. 1C). In addition, there was

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induction of apoptosis in all lymphoma cell lines as detected byWestern blot analysis of caspase-3, -7, -8, and -9, and PARPcleavage, except in L428 (Fig. 1C); this was confirmed withixazomib-treated lymphoma cells using Annexin/PI stainingand flow cytometry (Fig. 1D).

The in vivo efficacy of ixazomib was investigated using tumorxenografts derived using either Jurkat or L540 cell lines. Resultsfrom Jurkat-derived xenograft experiments showed that after 4weeks of ixazomib administration, there was a significant reduc-tion in the average approximate size of the tumor (1,094 � 76mm3) with 0.36 mg/kg or (582 � 45 mm3) with 0.72 mg/kgcompared with the average size of tumor (1,465 � 224 mm3) inthe vehicle-treated control group (P < 0.0001; Fig. 1E). Kaplan–Meier survival analysis showed a significant improved survival inSCID mice consisting Jurkat xenograft treated with ixazomibcompared with control group (P < 0.0001; Fig. 1F). Treatmentwith ixazomib in L540 xenograft tumor-bearing SCID mice alsoresulted in a significant reduction in the average size of the tumor(821 � 54 mm3) with 0.36 mg/kg or (290 � 5 mm3) with 0.72mg/kg compared with vehicle-administered control group (1,128� 162mm3), at the end of 4weeks (P¼ <0.001; Fig. 1G). Kaplan–Meier survival analysis showed a significant increase in survivalwith ixazomib treatment compared with control groups (P <0.0001; Fig. 1H).

We also analyzed detailed tumor growth kinetics by comparingtumor volumes measured before, during, and at the completionof treatment. Ixazomib resulted in decreased average tumorgrowth rate, 0.045 � 0.0060 with 0.36 mg/kg and significantly(P¼ 0.002; 0.017� 0.0060with 0.72mg/kg), compared with thegrowth rate 0.049 � 0.0045 in the vehicle-administered controlgroup (Fig. 1I) consistent with inhibition of tumor progression inL540. This indicates there will be steady decrease in tumor growthwith increasing doses of ixazomib. The kinetics of tumor growthin the Jurkat xenograft, however, showed no significant differ-ences in growth rate (0.076� 0.012with 0.36mg/kg and 0.056�0.015with 0.72mg.kg) comparedwith the growth rate of 0.052�0.006 vehicle-administered control group, suggesting that ixazo-mib is causing a lag timedelay in the tumor growth rather than anysignificant change in the growth rate. It should be noted that thisprediction is obscured by large variances in the growth rates oftumor observed in the ixazomib-treated cohorts (Fig. 1I).Although ixazomib treatment appeared to affect the kinetics ofthe tumor growth by causing a lag time delay in the Jurkat-derived xenografts, comparison of average tumor size resultedin overall reduction in the tumor burden and improved survivalbenefit (Fig. 1E and H), indicating antitumor activity of ixazo-mib in this model. The results from in vitro and in vivo experi-ments demonstrate that proteasomal inhibition via ixazomib isassociated with strong activity against T-cell lymphoma andHodgkin lymphoma.

Transcriptome analyses of the impact of proteasomalinhibition on biologic pathways and "hallmarks of cancer"

Global transcriptome analyses following ixazomib exposureshowed significant gene changes with a 1.2 fold change for Jurkat(508 genes), L540 (4765 genes), and L428 (423 genes; Fig. 2A)with anFDR<0.05. L540with the highest significantly differentialexpressed genes was the most ixazomib-sensitive Hodgkin lym-phoma line (Fig. 1A) with significant inhibition of tumor growth(Fig. 1I). Gene expression with ixazomib compared with untreat-ed control showed a total of 40 overlapping genes with a 1.2 fold

change difference in Jurkat, L540, and L428 cells, with 326overlapping genes in Jurkat and L540, and 212 overlappinggene sets in L428 and L540 Hodgkin lymphoma cells (Fig. 2B).The overlap based on common significantly regulated genes inJurkat, L540, and L428 cells treated with ixazomib showedconserved upregulation of ubiquitin proteasome componentsPSMB3, PSMB6, PSMC4, PSMD6, UBE2H, UBFD1, and lyso-some-associated factors ATG4A, CD68, PSAP, and SQSTM1(also known as p62; Fig. 2C; ref. 9), and these gene expressionchanges were confirmed by qRT-PCR–based assay (Supplemen-tary Fig. S1).

Upstream regulator analysis performed using Ingenuity Path-way Analysis (IPA) and in conjunction with published datareporting impact on tumor progression, (Fig. 3; SupplementaryTable S1) summarized the effects of ixazomib on tumor dynam-ics. Ixazomib treatment in Jurkat and L540 appeared to haveshifted the balance of upstream regulators associated with tumorpromotion and tumor suppression in favor of tumor inhibition(Fig. 3A), supporting the tumor-inhibitory effects of ixazomibobserved with tumor xenografts (Fig. 1I). Conversely, theresponses in L428 with ixazomib appeared to favor tumor pro-gression (Fig. 3). Next, the status of upstream regulators wasmapped on their functional contextual relationship to classical"hallmarks of cancer" (28, 29). Results from this evaluation showthat ixazomib overwhelmingly impacts 9 of 12 "hallmarks ofcancer," (Fig. 4A), providing clues to tumor-inhibitory functionsof ixazomib in L540 (Fig. 3A). In contrast, ixazomib treatment inL428 appears to promote the "self-sufficiency in growth signal"favoring tumor progression (Fig. 4B). Although additionalexperiments for a functional validation of these predictionsare required, it must be noted that there was an apparent lack ofapoptotic activity in L428 cells despite proteasomal inhibitionwith ixazomib (as detected as accumulation of ubiquitinylatedprotein; Fig. 1C). In fact, these cells demonstrated the lowestsensitivity to ixazomib compared with other cell lines tested(Fig. 1A and B). Interestingly, MYC inhibition representedamong several "hallmarks of cancer" present in L540 is notablyabsent in L428 with ixazomib treatment (Fig. 4) and furthervalidated by qRT-PCR–based assay (Supplementary Fig. S1).MYC is functionally associated with several oncogenic mechan-isms associated with tumor progression (30), and inhibition ofMYC with ixazomib treatment could be an important event inthe antitumor activity of ixazomib.

Gene set enrichment analysis (GSEA) revealed conserved upre-gulation of biologic pathways representing protein localization,proteasome, vesicle transport, apoptosis, oxidative stress, NF-kB,and catabolic processes with ixazomib in both L540 and L428cells (Fig. 5A and Supplementary Fig. S2; Supplementary TablesS2 and S3). This included conservation of biologic pathways withgene expression analysis using different and higher doses ofixazomib in L428 and L540 (data not shown). Although biologicpathways associated with DNA repair, mitotic cell cycle, andmicrotubule organizationoppositely regulatedbetweenL540 andL428 Hodgkin lymphoma cells, such patterns of biologicresponses to ixazomib were consistently observed with GSEAperformed using multiple reference databases (KEGG, Reactomeand Biocarta; Supplementary Fig. S2; Supplementary Tables S2and S3). In addition, GSEA predicted overall downregulation ofMYC pathway with ixazomib in L540 or Jurkat (SupplementaryFig. S1C) as validated by qRT-PCR–based assay (SupplementaryFig. S1).

Antiproteasomal Therapy in Lymphoma

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Figure 1.Proteasomal inhibition induces potent in vitro cell death and inhibits in vivo tumor growth in human lymphoma xenografts. A and B, dose-dependent increase incytotoxicity with ixazomib in T-cell lymphoma and Hodgkin lymphoma lymphoma cell lines by MTT assay at 72 hours. C, Western blot analysis of ixazomib-treatedT-cell lymphoma or Hodgkin lymphoma cell lines show accumulation of ubiquitinylated protein and activation of apoptotic markers at 24 hours. D, bar graphshows dose-dependent significant increase in Annexin-V–positive apoptotic cells in ixazomib-treated lymphoma cells. � , P < 0.05; �� , P < 0.0001. Tumor-bearing SCID mice treated with ixazomib (0.72 mg/kg) showed significant reduction in the tumor volume (represented as line graph, P < 0.001, two-way repeatedANOVA), andKaplan–Meier analysis showed significant increase in survival comparedwith controls (P<0.001) in the experiments performedwith Jurkat (E and F) orL540 (G and H)-derived tumor xenografts. I, tumor growth rate plot showing the effect of ixazomib treatment on tumor progression over the entireduration of assessment, with significant decrease in growth rate (P < 0.001) in L540, while in Jurkat, lag in tumor growth was noted.

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Sixteen common key significant genes were determined forJurkat and L540 cells treated with ixazomib, and these werenonoverlapping with L428 (Supplementary Table S4). These keygenes are considered as "core responsive genes" and were deter-mined by comparing common genes involved in the predictedupstream regulator analysis and biofunction analysis through IPAwith the genes involved in the GSEA analysis (Fig. 5B). Surpris-

ingly, the majority of these genes also have existing knowninteractions (Fig. 5B). Analysis of these 16 key genes (shownin Fig. 5B) using DAVID functional annotation and classificationtool (31) identified 22 functional clusters that included mitoticcell cycle, apoptosis, proteolysis, metabolism, and lysosomalactivity as key biologic responses affected by ixazomib in Jurkatand L540 (Supplementary Table S5).

Figure 2.Gene expression profiling identifies critical genes, signaling pathways, and regulators affected by antiproteasomal therapy. A, average signal log2 fold-change comparing Ixazomib 24 hours versus control for 25 nmol/L ixazomib treatment in Jurkat, L540, and L428 cells. Whiskers show the range of the outliers, withmax andmin values as O and the 1 and 99th percentile outliers as X. Individual data points are shown on the left of box plots as filled circles. Dotted red lines show the1.2 fold-change cutoff. B, Venn diagrams of the genes with 1.2 up- and downregulated fold changes for comparisons between ixazomib 24 hours versuscontrol for Jurkat, L540, and L428 cells. C, scatter plot of average signal log2 fold-change comparing common significantly regulated genes between ixazomib24 hours versus control for 25 nmol/L ixazomib treatment in Jurkat, L540, and L428 cells. Dotted red lines show the 1.2 fold-change cutoff.






Downregulation of MYC via proteosomal inhibitionAnalysis of upstream regulators and GSEA with ixazomib

transcriptome predicted MYC inhibition in L540 and Jurkatcells, but notably absent in L428 (Figs. 3 and 4) and validatedby qRT-PCR–based assay (Supplementary Fig. S1). MYC is animportant regulator of cell cycle, DNA repair, and replication,chromatin modification, inflammatory responses, lysosomalautophagy, metabolism, and oxidative stress (32, 33), andthese pathways are also enriched with ixazomib treatment(Supplementary Fig. S2). Therefore, we next investigated thebiologic responses of these enriched pathways using ixazo-mib-treated panel of lymphoma cells. Results from our experi-ments showed that MYC protein levels decreased with increas-ing concentrations of ixazomib in all lymphoma cells, exceptin the relatively less sensitive L428 cell line (Fig. 6A) consis-tent with transcriptional downregulation of MYC expression,as determined by qRT-PCR–based assay (Supplementary Fig.S1). A dose-dependent increase in lysosomal activity-associ-ated Cathepsin D cleavage was observed in most of the celllines, with L428 showing increase in lysosomal autophagy-related p62. Lysosomal activation could be relevant to MYCdownregulation with ixazomib as previous data showed thatMYC was a substrate in Cathepsin D–dependent proteolysis(34); presumably the activation of lysosomal proteolysis by

ixazomib could be relevant to downregulation of MYC proteinlevels.

Proteasomal inhibition has been previously shown to inhibitDNA damage response to IR via inhibition of CHK1 phosphor-ylation (35) and our results from GSEA show downregulation ofgenes associated with DNA damage, DNA repair and cell-cycleprogression with ixazomib (Fig. 5 and Supplementary Fig. S2;Supplementary Tables S2 and S3). We examined inhibition ofCHK1 phosphorylation as a marker for these responses (36) andobserved inhibition of CHK1 phosphorylation in all cell lineswith ixazomib treatment except in L428 (Fig. 6A). Histone H3acetylation, a marker of chromatin modification, which is regu-lated by CHK1 (37) and affects MYC expression (38), was alsodecreased with increasing concentrations of ixazomib in Jurkat,HH, and Hut78 and L540 cells, without significant effect in L428cells (Fig. 6A). Collectively, these results suggest that biologicresponses of MYC, lysosome, cell cycle, and chromatin modifi-cation correlate with each other and are conserved in ixazomib T-cell lymphoma and Hodgkin lymphoma cell lines, while suchconservation was lacking in L428 cells, exactly as predicted fromGSEA (Fig. 5) and DAVID analysis (Supplementary Table S5)

Lysosomal activity represents as alternate means for proteindegradation and therefore, excess protein accumulation resultingfrom proteasomal inhibition is expected to trigger lysosomal

Figure 3.Common upstream regulatorsdetermined by IPA software fromthe significant genes withantiproteasomal treatment. A, aschematic of the activation states ofthe upstream regulators illustratingthe balance between the tumorpromoters (text in yellow) and tumorsuppressors (text in white andunderlined) with a predictedactivation (circled in orange) orpredicted inhibition (circled in blue). B,scatter plot of the common significantupstream regulators (with activationz-scores > 2 or < �2) betweenixazomib 24 hours treatment versuscontrols for Jurkat, L540, and L428cells. Nonsignificant genes for L428are also shown as gray dots. Dottedred lines show the cutoff forsignificantly regulated upstreamregulators.

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function (39). Because MYC is also a substrate for lysosomalproteolysis (34), we subsequently investigated the role of lyso-somal activity and its relation to MYC. Analysis via electronmicroscopy revealed characteristic features representing lysosom-

al maturation in ixazomib-treated cells (Fig. 6B); however, sub-sequent Western blot analysis did not show evidence of autop-hagy as determined on the basis of mTOR and Beclin phosphor-ylation, and LC3A cleavage (data not shown). Further inhibition

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Prediction Legends

Figure 4.Effects of antiproteasomal treatment on the "hallmarks of cancer." A and B, a schematic of the "hallmarks of cancer" overlaid with activation states of the upstreamregulators illustrating the balance between the tumor promoters (text in red or blue) and tumor suppressors (text in red or blue, and underlined) with apredicted activation (text in red) or predicted inhibition (text in blue), showing the effect on "hallmarks of cancer" inhibition (shaded in dark/light green) or activation(shaded in red/orange) in the ixazomib treatment versus controls for L540 and L428 cells.






of lysosomal proteasewith E64d/Pepstatin CHK1didnot result inMYCaccumulation but rather led to accelerated reduction inMYCprotein and histone H3 acetylation, and enhanced apoptosis,detected as cleaved caspase-3 andPARP in ixazomib-treated Jurkatand L428 cells (Fig. 6C). There was no corresponding HDACaccumulation with decreased histone H3 acetylation with lyso-somal inhibition (Fig. 6C), suggestingHDAC-independent reduc-tion of histone H3 acetylation with ixazomib. Taken together,inhibition of proteasomal and lysosomal proteolysis, instead ofresulting in MYC accumulation resulted in further loss of MYCprotein, indicating a possible role for transcriptional regulation ofMYC expression with ixazomib treatment.

Next, we focused our investigation on the cell-cycle regulatoryCHK1 in the context of MYC function, because MYC and CHK1interaction is a known oncogenic mechanism in lymphomagen-esis (20), the role of CHK1 in transcriptional regulation viahistoneH3 acetylation is known (37), and histoneH3 acetylationis required for MYC transcription (33). We examined genesregulated by MYC and CHK1 in ixazomib-treated cells andobserved strong overlap in the target gene sets regulated by MYCand CHK1. There was overall downregulation ofMYC-associatedgenes in both L540 (53%) and Jurkat (5%) and upregulation ofMYC-associated genes in L428 (6%; Fig. 6D and E). Similarly,

there was overall downregulation of CHK1-associated genes inboth L540 (47%) and Jurkat (11%) and upregulation of CHK1-associated genes in L428 (7%; Fig. 6D and E). By comparingMYCand CHK1-regulated genes, we noted the presence of overlappinggene sets in L540 (20%), Jurkat (7%), and L428 (33%). Consid-ering the strong overlap and the pattern of differentially expressedgene sets representing both MYC and CHK1, we narrowed ourinvestigation to determine the biologic relationship betweenthese oncogenes.

MYC promoter binding and importance of CHK1We investigated the effect of CHK1 inhibition on histone H3

acetylation and MYC in L428 cells treated with ixazomib. Weobserved that CHK1 inhibitor (AZD7762) alone, or in combina-tion with ixazomib, strongly downregulated histone H3 acetyla-tion, phosphorylationofCHK1-dependent Threonine 11 (37), onhistone H3, and MYC protein (Fig. 7A). Furthermore, inhibitionof histone acetyl transferase (HAT) by C646 also resulted indownregulation ofMYC (Supplementary Fig. S3), indicating thatCHK1 and histone H3 acetylation are upstream in the regulationof MYC expression. Therefore, we performed ChIP using anti-acetylated histone H3 and PCR assay for its binding on the MYCpromoter region. We observed that ixazomib treatment resulted

Figure 5.GSEA of transcriptomic network and the significant key genes in responses to antiproteasomal treatment. A, network representation of GSEA for GO C5gene sets for ixazomib 24 hours versus control for Jurkat, L540, and L428 cells, with L540 overlaid with either Jurkat or L428. Leading edge analysis with aFDR < 0.05 determined significant gene sets enriched for each group. The size of each node reflects the amount of molecules involved for each gene set.The edge thickness (green lines for L540 and blue lines for either Jurkat or L428) represents the number of genes associated with the overlap of two genesets (or nodes) that the edge connects. Clusters in each groupingwere named according to common function. Upregulated gene sets, red; downregulated gene sets,blue. B, common significant key genes with the lymphoma cells treated with ixazomib. The interactions of the common key significant genes with symbolsrepresenting the biologic function and colors denoting up or downregulation in gene expression are described in the inserted legend. C, schematic of the activationstates of the common significant key genes illustrating the balance between the tumor promoters (text in blue) and tumor suppressors (text in black andunderlined) with the status of up (circled in red) or downregulation (circled in green) in the gene expression experiment with ixazomib.

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Figure 6.MYC downregulation by ixazomib. A, Western blot analysis show the dose-dependent effects of ixazomib on MYC protein and the events associatedwith lysosomalactivity, cell cycle, and chromatin modification responses. B, electron microscopic findings with ixazomib treatment show presence of vacuolar structures withlight todense amorphous granulations, representing lysosomal bodies (arrows), localizedpredominantly in the cytoplasmandoccasionally present in theperinuclearor nuclear region. C, cytoplasm; N, nucleus. C, inhibition of lysosomal proteases with E64/Pepstatin potentiates downregulation of MYC and acetylation ofhistone H3, and increases caspase-3 and PARP cleavage, detected by Western blot analysis. D, box plot based on average signal log2 fold-change comparingixazomib 24 hours versus control for 25 nmol/L ixazomib treatment in Jurkat, L540, and L428 cells. Dotted red lines show the 1.2 fold-change cutoff for thedifferentially expressed MYC- and CHK1-related genes. E, bar graph shows representation in percentages of MYC- or CHK1-regulated differentially expressedgenes within the entire set of MYC- or CHK1-regulated genes among the entire transcriptome and overlap between these differentially expressed gene sets inixazomib-treated Jurkat, L540, and L428 cells.






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Figure 7.CHK1 and histone H3 acetylation-dependent MYC response to ixazomib. A, inhibition of CHK1 potentiates downregulation acetylation and phosphorylationof histone H3, and MYC protein with ixazomib. B, ChIP with anti-acetylated histone H3 and PCR for MYC promoter normalized with input DNA, show decreasedpromoter occupancy in Jurkat, and increased promoter occupancy in L428 cells with ixazomib treatment. C, ChIP with anti-acetylated histone H3 and PCR for MYCpromoter normalized with input DNA, show that AZD7762 (i.e., CHK1 inhibitor) decreased acetylated histone H3 promoter occupancy in L428 cells, withixazomib treatment. D–F, bar graphs show significant increase in Annexin V positivity (P < 0.001) in ixazomib-treated L428 cells in the presence of AZD7762compared with all doses of ixazomib alone. Western blot analysis show treatment with JQ1 (i.e., bromodomain inhibitor) downregulates MYC and increasescaspsase-3 and PARP cleavage with ixazomib in Jurkat and L428 cells as well as via with MYC or CHK1 RNAi silencing results in increased PARP cleavage.G–J, treatment of ixazomib in combination with AZD7762 or JQ1 for 72 hours, in L428 and the analysis of results from MTT assay show synergistic effect (CI < 1),represented as median dose effect curve and combination indices graph, determined using Calcusyn software.


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in decreased MYC amplification relative to untreated Jurkat con-trol cells (Fig. 7B), indicating reduced MYC promoter occupancyby acetylated histone H3 as a possible mechanism for ixazomib-dependent downregulation ofMYC expression in the Jurkat cells.In contrast, we noted increased PCRamplification ofMYC in L428cells, indicating higher promoter occupancy by acetyl histone H3,suggesting an active transcription processwith ixazomib (Fig. 7B).Our conclusions based on the results from the ChIP experimentshowed decreased and increased promoter occupancy in Jurkatand L428 (Fig. 7B), respectively, which are consistent with thepatterns of MYC-dependent gene expression observed in thesecells (Fig. 6A andD), indicate thatMYC function is regulated at thelevel of transcription with ixazomib treatment. Because weobserved that ixazomib treatment resulted in downregulation ofMYC andCHK1phosphorylation in all lymphoma cells, except inL428 cells, we chose to examine the consequences of blockingCHK1 on MYC expression in L428 cells. Previous analyses dem-onstrated a direct role for CHK1 kinase activity in histone H3phosphorylation (Threonine 11) and stabilization of histone H3acetylation to facilitate transcription via retention of relaxedchromatin confirmation (37). Therefore, we performedChIP PCRassay for MYC, in the presence of AZD7762 (CHK1 inhibitor) todetermine whether CHK1-dependent histone modificationaffects MYC expression. The results from our experiments showdecreased MYC promoter occupancy by acetylated-Histone com-pared with untreated control, or in the presence of ixazomib inL428 cells (Fig. 7C). Taken together, these results demonstratedthat CHK1 inhibition leading to decreased histoneH3 acetylationand MYC expression (Fig. 7A) is associated with decreased pro-moter occupancy onMYC gene (Fig. 7C), suggesting that CHK1 isactively involved in the transcriptional control of MYC in theixazomib-treated lymphoma cells.

CHK1 or MYC inhibition synergizes with antiproteasomaltherapy

Considering that CHK1 phosphorylation andMYC expressionwere not affected by ixazomib treatment in L428 (Fig. 6A and D),and L428 showed least sensitivity to ixazomib (by MTT andApoptosis; Fig. 1B and C) compared with other lymphoma cells,we predicted that a combination CHK1 inhibitor and ixazomib islikely to downregulate MYC and restore ixazomib sensitivity inthe L428 Hodgkin lymphoma cells. Therefore, we treated L428cells with increasing doses of ixazomib alone and in presence ofCHK1 inhibitor, AZD7762, and observed a significant increase inAnnexin—positive apoptosis (P < 0.001) in the combination ofixazomib with AZD7762 compared with controls (Fig. 7D). Nextconsidering thatMYCdownregulation is predicted as a prominentmechanism in ixazomib sensitivity, and we used JQ1 (bromo-domain inhibitor, blocks MYC transcription) to inhibit MYCexpression and observed potentiation of apoptosis, detected ascleaved PARP, both in Jurkat (T-cell lymphoma) and L428 Hodg-kin lymphoma cells in the presence of ixazomib (Fig. 7E). Fur-thermore, these results were independently confirmed usingRNAi-mediated silencing of MYC or CHK1 (Fig. 7F). Consideringthat MYC downregulation and cotargeting of MYC could be clin-ically relevant for ixazomib-based treatments,wefinally investigatedto determine whether such targeted drug combinations would besynergistic. Our results based on MTT assays show that targetingCHK1 with AZD7762 (ED50 1.1 mmol/L) in the presence of ixazo-mib was synergistic (CI < 1) in L428Hodgkin lymphoma cells (Fig.7G and H) and similarly, combination of JQ1 (ED50 5.5 mmol/L)

with ixazomib also resulted in synergistic effect (CI < 1) in L428(Fig. 7I and J), suggesting that AZD7762 or JQ1-dependent MYCdownregulation will lead to synergistic cell death in ixazomib-refractory L428 cells, with dominant MYC function.

DiscussionThe prominent substrates regulated by proteasome-dependent

turnover includes key proteins involved in the regulation ofreplication, transcription, translation, cell cycle, tumor suppres-sion, signal transduction, apoptosis, metabolism among others(40). NF-kB has been shown to be constitutively active in Hodg-kin lymphoma cell lines and also critical for HRS cell survival inxenograft studies (10, 11). Furthermore,NF-kBhasbeen shown tobe highly overexpressed and responsible for tumorigenesis inboth HRS and T-cell lymphoma models (14, 15). Despite thesepromising preclinical data, proteosomal inhibition with borte-zomib had only modest clinical activity in Hodgkin lymphoma(16, 17), while more encouraging clinical activity has beenreported in T-cell lymphoma (41, 42). To exploit the strong andfunctional presence of NF-kB in Hodgkin lymphoma and T-celllymphoma, which is regulated by the proteasome, we examinedthe effects of the novel second-generation orally bioactive protea-some inhibitor, ixazomib, which has superior pharmacokinetics/pharmacodynamics compared with bortezomib (18). Consider-ing also that early reports from ixazomib lymphoma clinicalstudies have been encouraging (6), the results from our analysesprovide the biologic basis and the mechanisms of activity ofixazomib activity.

Results herein demonstrated that ixazomib induced potent celldeath and inhibited tumor growth in T-cell lymphoma andHodgkin lymphoma cells at clinically achievable nanomolarconcentrations. Transcriptome analysis showed conserved andconsistent biologic responses with ixazomib in both T-cell lym-phoma and Hodgkin lymphoma cells. Furthermore, the activa-tion status of tumor suppressors and inhibition of oncogenesindicated strong potential for the inhibition of tumor progressionwith ixazomib in T-cell lymphoma and Hodgkin lymphoma andsubstantial impact were predicted on overall global "hallmarks ofcancer." Although we observed conserved biologic regulationswith ixazomib treatment in Jurkat and L540 cells, we also notedthat a lack of conservation with biologic responses, associatedwith minimal impact on the 'hallmarks of cancer' was related topoor sensitivity to ixazomib in L428 cells.

We postulated that examining the biologic differences inresponse to ixazomib in the differentially sensitive lymphomacells could provide clues to determine the associated biologicmechanisms of actions to ixazomib treatment. Constitutiveexpression of NF-kB has been commonly observed in Hodgkinlymphoma cells including L428 (43). Furthermore, we observedinduction of NF-kB genes with ixazomib treatment in bothixazomib-sensitive L540 and -resistant L428Hodgkin lymphomacells (Fig. 5A), andwe subsequently verified that NF-kB activationoccurs in ixazomib-treated multiple T-cell lymphoma and Hodg-kin lymphoma cells as shown by Western blot assays (data notshown) thus ruling out a significant role for NF-kB in resistance toixazomib. Among the significant upstream regulators affectedwith ixazomib, inactivation of MYC, which was predicted inJurkat and L540 cells, were absent in L428. MYC is central tomajor regulatory pathways including cell cycle, DNA repair,replication, transcription and translation, and metabolism (44)






with global impact on major "hallmarks of cancer" progression(30). MYC is transcriptionally regulated by cell-cycle regulatoryproteins (p21, CHK1, CDC2) through chromatin modificationvia histone acetylation (37) and BRCA1 pathways (45).

We demonstrated that ixazomib affected many of the afore-mentioned MYC-related biologic pathways and expression ofMYC protein (Fig. 6A). Overexpression of MYC occurs in 30%of all human cancers and frequently predicts for aggressive bio-logic behavior, advanced stage of disease, increased likelihood ofrelapse, and poor clinical outcome (46, 47). Studies in transgenicmouse models showed that even brief MYC inactivation is suf-ficient to induce tumor regressions (48) and that targeting MYCmay destabilize apoptosis regulatory network and induce celldeath in lymphoma (49). In multiple myeloma, a correlationbetween MYC overexpression (50) and poor response to treat-ment with bortezomib has been reported, and in mantle celllymphoma, cotargeting MYC with (CPI203) was shown to over-come bortezomib resistance (51). Although these studies show acorrelation between MYC and resistance to proteasome inhibi-tion, results from our experiments demonstrated the biologicmechanism for MYC-dependent resistance to proteasome inhi-bition, and moreover, how to circumvent these mechanisms toimprove therapeutic response.

In L428 cells, poor sensitivity to ixazomib correlated with lackof MYC regulation and histone H3 acetylation. Both histoneacetylation andMYC affect similar downstream biologic process-es including, chromatin modification, gene expression, DNAreplication and repair, cell cycle, cytoskeletal function, and pro-tein trafficking (52). Chromatin modification involving histoneacetylation is mediated by a family of HATs and HDACs withopposing enzymatic activities, and histone acetylation is animportant mechanism in the regulation of MYC gene expression(52). Furthermore, the cell-cycle regulatory CHK1 is known toregulate histone acetylation via histoneH3phosphorylation (37),which leads toHDAC3 disassociation favoringMYC transcription(38). Thus, in our experiments, CHK1 inhibition alone or in thepresence of ixazomib, decreased MYC promoter occupancy (byacetylated histoneH3) and reducedMYC protein in L428 (Fig. 7Aand D). Furthermore, by using pharmacologic inhibitors andRNAi for CHK1 and MYC, we demonstrated that MYC down-regulation was required for inducing cell death with ixazomib. In

addition, CHK1 inhibition has been reported to enhance cyto-toxicity with lenalidomide selectively in MYC-dependent lym-phoma (53), supporting the role for CHK1 dependency in MYC-driven tumors. Altogether, results from our analyses showed thatixazomib alone or in combination with CHK1 inhibition has thepotential to significantly downregulate MYC and induce potentcell death in T-cell lymphoma and Hodgkin lymphoma models,and it represents a novel combinatorial therapeutic platform forthe treatment of these cancers.

Disclosure of Potential Conflicts of InterestA.M. Evens has received speakers bureau honoraria from and is a consultant/

advisory board member for Millennium. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: D. Ravi, M.V. Sitkovsky, A.P. Mazar, A.M. EvensDevelopment of methodology: D. Ravi, A. Beheshti, I. Kandela, L. Hlatky,A.M. EvensAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):D. Ravi, A. Beheshti, N. Abermil, F. Passero, J. Sharma,M. Coyle, I. KandelaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D. Ravi, A. Beheshti, F. Passero, J. Sharma, I. Kandela,R.B. Gartenhaus, A.M. EvensWriting, review, and/or revision of the manuscript: D. Ravi, A. Beheshti,N. Abermil, M. Coyle, A. Kritharis, R.B. Gartenhaus, A.M. EvensAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Ravi, A.P. Mazar, A.M. EvensStudy supervision: D. Ravi, A. Beheshti, L. Hlatky, A.M. EvensOther (discussions of the need to focus on the manipulation of the mechan-isms of cell death to improve the immunogenicity of lymphomas):M.V. Sitkovsky

Grant SupportResearch funding and ixazomib drug were provided by Takeda Pharmaceu-

tical Inc. (A.M. Evens) and NIH grant R01 CA164311 (A.M. Evens andR.G. Gartenhaus).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 4, 2015; revised January 27, 2016; accepted February 29,2016; published OnlineFirst March 17, 2016.

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2016;76:3319-3331. Published OnlineFirst March 17, 2016.Cancer Res Dashnamoorthy Ravi, Afshin Beheshti, Nasséra Abermil, et al. MYC-Dependent Cell Death in T-cell and Hodgkin LymphomaProteasomal Inhibition by Ixazomib Induces CHK1 and

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