Translational Cancer Mechanisms and Therapy

Cotargeting of BCL2 with Venetoclax and MCL1with S63845 Is Synthetically Lethal In Vivo inRelapsed Mantle Cell LymphomaDana Prukova1, Ladislav Andera2,3, Zuzana Nahacka2,3, Jana Karolova1,4, Michael Svaton5,Magdalena Klanova1,4, Ondrej Havranek4,6, Jan Soukup7, Karla Svobodova8,Zuzana Zemanova8, Diana Tuskova1,4, Eva Pokorna1, Karel Helman9, Kristina Forsterova4,Mariana Pacheco-Blanco6, Petra Vockova1,4, Adela Berkova8, Eva Fronkova5,Marek Trneny4, and Pavel Klener1,4

Abstract

Purpose: Mantle cell lymphoma (MCL) is an aggressivesubtype of B-cell non-Hodgkin lymphomas characterized by(over)expression of BCL2. A BCL2-targeting drug, veneto-clax, has promising anticancer activity in MCL. We analyzedmolecular mechanisms of venetoclax resistance in MCL cellsand tested strategies to overcome it.

Experimental Design: We confirmed key roles of proa-poptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are,however, differentially expressed in cell lines comparedwith primary cells. First, NOXA protein is significantly over-expressed in most MCL cell lines. Second, deletions ofBIM gene harbored by three commonly used MCL cell lines(JEKO-1, MINO, and Z138) were not found by array com-

parative genomic hybridization using a validation set of 24primary MCL samples.

Results:WedemonstratedthatMCL1andNOXAplayimpor-tant roles in mediating resistance to venetoclax. Consequently,we tested an experimental treatment strategy based on cotarget-ing BCL2 with venetoclax and MCL1 with a highly specificsmall-molecule MCL1 inhibitor S63845. The combinationof venetoclax and S63845 demonstrated synthetic lethalityin vivo on a panel of five patient-derived xenografts establishedfrom patients with relapsed MCL with adverse cytogenetics.

Conclusions: Our data strongly support investigation ofvenetoclax in combination with S63845 as an innovativetreatment strategy for chemoresistant MCL patients withadverse cytogenetics in the clinical grounds.

IntroductionMantle cell lymphoma (MCL) is an incurable subtype of

B-NHL (1). Currently, the first-line treatment of MCL is basedon conventional chemotherapy and anti-CD20 mAb rituximab.

Relapsed or refractory patients usually receive either differentconventional chemotherapy agents (e.g., bendamustine, cis-platin etc.), or they are offered innovative nonchemotherapymolecules including B-cell receptor inhibitor ibrutinib orimmunomodulatory agent lenalidomide (2–4). After failureof a Bruton tyrosine kinase (BTK) inhibitor ibrutinib, however,patients' prognosis is usually dismal regardless of the subse-quent treatment. Complex karyotypes at diagnosis or at diseaserelapse are associated with especially dismal prognosis due tofrequent chemoresistance (5).

BCL2, one of the key antiapoptotic and prosurvival proteins, isoverexpressed virtually in all MCL tumors that are consideredBCL2-dependent. Venetoclax, a BCL2-specific BH3 mimetic, killsMCL cells indirectly, by displacing proapoptotic effectors includ-ing BIM (and other BH3-only proteins), from BCL2, and byblocking BCL2 from its inhibitory interaction with proapoptoticBAX/BAK1 proteins. Unbound BIM is then available to bind andactivate BAX/BAK1, which in turn disrupts mitochondrial outermembrane, thereby triggering programmed cell death indepen-dent of TP53-regulated genotoxic pathway (6). So far, venetoclaxhas been approved for the patients with chronic lymphocyticleukemia/small lymphocytic lymphoma and showed promisingantilymphoma activity in MCL and acute myelogenous leukemiapatients (7–11).

Despite promising data from early clinical trials, resistancesooner or later develops in a majority of patients with MCL onvenetoclax monotherapy (8). Mechanisms of constitutive or

1Institute of Pathological Physiology, First Faculty of Medicine, Charles Univer-sity, Prague, Czech Republic. 2Biocev, Institute of Biotechnology, Czech Acad-emyof Sciences, Vestec, Czech Republic. 3Institute of Molecular Genetics, CzechAcademy of Sciences, Prague, Czech Republic. 4First Medical Dept., CharlesUniversity and General University Hospital, Prague, Czech Republic. 5CLIP -Childhood Leukaemia Investigation Prague, Dept. of Paediatric Haematology/Oncology, Second Faculty of Medicine and Charles University Hospital in Motol,Prague, Czech Republic. 6Biocev, First Faculty of Medicine, Charles University,Vestec, Czech Republic. 7Department of Pathology and Molecular Medicine,Second Faculty of Medicine and Charles University Hospital in Motol, Prague,CzechRepublic. 8Center forOncocytogenetics, Institute ofMedical Biochemistryand Laboratory Diagnostics, Charles University and General University Hospital,Prague, Czech Republic. 9Faculty of Informatics and Statistics, University ofEconomics, Prague, Czech Republic.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:Pavel Klener, Charles University, UNemocnice 5, Prague12853, Czech Republic. Phone: 4202-2496-5933; Fax: 4202-2496-5916; E-mail:pavel.klener@gmail.com

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acquired venetoclax resistance remain poorly understood. It wasrepeatedly reported that similarly to approximately one third ofcurrently available MCL cell lines (JEKO-1, Z138, MINO, andREC-1), primary MCL cells also frequently harbor deletionsof BCL2-like11/BIM (12–14). It was even speculated that thehomozygous deletion of BIM plays an important role duringMCL lymphomagenesis (13). Because BIM represents a keymedi-ator of venetoclax proapoptotic activity, its homozygous deletionmight represent a valuable marker of venetoclax resistance. Upre-gulation of MCL1, another key antiapoptotic regulator besidesBCL2, was also reported as a plausible mechanistic rationalefor acquired resistance to venetoclax (15–17). Therefore, severalgroups including our own employed diverse strategies to indi-rectly inhibit MCL1 protein (in combination with venetoclax)including cyclin-dependent kinase inhibitor dinaciclib, plantalkaloid homoharringtonine or anthracycline daunorubi-cin (16, 18, 19). In 2017, Kotchny and colleagues reportedsingle-agent antitumor activity of a novel, highly specific MCL1inhibitor S63845 in many cancers including hematologicmalignancies (20). However, antilymphoma activity ofS63845 in MCL (considered a BCL2-dependent malignancy)was not studied. Antitumor activity of the combination ofvenetoclax and S63845 has been studied in acute leukemias,and there is one active trial testing the combination of BCL2-and MCL1-inhibitors (venetoclax and S64315) clinically inpatients with acute myeloid leukemias (GovTrial NumberNCT03672695; refs. 9–11, 21, 22).

Materials and MethodsCell lines, patient-derived xenografts, and primary lymphomasamples

MCL cell lines were purchased from DSMZ or ATCC with theexception of HBL2, which was a kind gift from Prof. MartinDreyling. The cell lines were authenticated in July 2016 byMultiplexion. UPF1G and UPF1H cell lines and all patient-derived lymphoma xenografts (PDX) were derived in ourlaboratory from patients with treatment-refractory MCL asdescribed previously (23). The cell lines were tested for Myco-plasma using a MycoAlert Mycoplasma Detection Kit (Lonza).All PDXs were confirmed by next-generation exome sequencingto keep majority of somatic mutations with the primary MCLcells from which they were derived (Supplementary Fig. S1;Supplementary Table S1; Supplementary Table S2). Primarylymphoma cells were obtained from patients with MCL accord-

ing to the Declaration of Helsinki. Informed written consentwas obtained from each subject. The experimental design wasapproved by the Ethics Committee of the General UniversityHospital Prague under number 63/16.

Next-generation exome sequencingSamples were sequenced by our facility on the NextSeq 500

(Illumina) instrument according to the manufacturer's protocolsand sequencing libraries were prepared using SureSelectXTHuman All Exon V6þ UTR Kit (Agilent Technologies). Sequencereads from PDX samples were first aligned against the mousereference genome mm10 combined with the human referencegenome hg19 and murine reads were filtered out from furtheranalysis by a custom script to reduce risk of contamination.Remaining reads were then aligned against the human referencegenome hg19. All alignments were performed by BWA (24).Genomic variants were called with samtools and VarScan2 (25, 26). Variant annotation was performed using SnpEff (27).Only nonsynonymous variants in the gene coding regions withcoverage of at least 10 reads with mapping quality and basequality higher than 20 in all related samples were comparedtogether based on their frequency. Variants present in patient'sgermline DNA at frequency higher than 0.05 were excluded fromanalysis in all cases. We compared variants with an allele fraction�0.2 in at least one of the compared samples that were present inat least 3 reads in both relapsed sample and derived PDX samples.All variant filtering was done in RStudio and frequencies andcounts of variants were plotted using the ggplot2 library (http://www.R-project.org; http://www.rstudio.com). These variantswere then manually reviewed in Integrative Genomics Viewer(http://www.broadinstitute.org/igv) and clear sequencing arte-facts or variants present but not called in the germline samplewere also excluded. List of 122 genes of special interest wascreated on the basis of recent publications of frequently mutat-ed genes in MCL samples, and variants present in these geneswere specifically selected and marked in resulting diagrams andtables (28–33). Copy number variants were predicted usingCNVkit with normalization to pooled normal samplessequenced on the same instrument using the same librarypreparation kits (34). Inferred segmental changes were calcu-lated using the fused lasso method and plotted in diagrams forrelapse and PDX samples (35).

FISHInterphase FISH analyses were performed on fixed cell suspen-

sions using commercially available probes fromAbbottMolecular(Vysis LSIMYCBA, LSI IGH/MYC/CEP8TCDF, LSICDKN2A/CEP9, LSI ATM/LSI TP53, LSI IGH/CCND1DF, LSI 13 RB1/LSI 13q34and LSI BCL2 BA). FISH assays were performed according to themanufacturers' protocols. At least 200 interphase nuclei wereanalyzed by two independent observers.

Real-time RT-PCRTotal RNA was isolated from cell lines in Ribozol (Amresco)

using phenol–chloroform extraction. Complementary DNAsynthesis was carried out from 1 mg of total RNA with High-Capacity cDNA Reverse Transcription Kit (random primers;Applied Biosystems). Real-time RT-PCR was performed usingTaqMan Gene Expression Assays (MCL1: Hs01050896_m1,NOXA: Hs00560402_m1, GAPDH: Hs02758991_g1) on ABI7900HT detection system (Applied Biosystems).

Translational Relevance

BCL2-targeting agent venetoclax has promising anticanceractivity in mantle cell lymphoma (MCL), but remissions tendto be short, which calls for rational drug combinations. Wedemonstrated that MCL1 and NOXA play important roles inmediating resistance to venetoclax. Consequently, we pro-posed an experimental treatment strategy based on cotargetingBCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of vene-toclax and S63845demonstrated synthetic lethality in vivoon apanel of five patient-derived xenografts established frompatients with relapsed MCL with adverse cytogenetics.

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Western blottingWestern blotting was performed as described previously (19).

The antibodies were from Cell Signaling Technology: BIM(C34C5), BCL-XL (2764), Santa Cruz Biotechnology: BCL2(C21), MCL1 (S-19), Enzo: NOXA (114C307.1) and Abcam:b-Actin (AC15).

ImmunoprecipitationCellswere lysed inCHAPS lysis buffer (0.3%CHAPS, 1mmol/L

EDTA, 40 mmol/L HEPES pH 7.5 and 120 mmol/L NaCl) sup-plemented with protease and phosphatase inhibitor cocktail(Sigma) for 30 minutes. Protein concentrations of cell extractswere determined by Pierce BCA Protein Assay Kit (Thermo FisherScientific) and equal amounts of protein samples were incubatedwith BCL2 or MCL1 antibodies listed above or an isotype controlimmunoglobulin [rabbit IgG (Santa Cruz Biotechnology)] for 1hour at 4�C. Consequently, G-protein beads were added forovernight incubation. Immunoprecipitates were washed inCHAPS lysis buffer, resuspended in 2� Laemmli buffer (Bio-Rad)and analyzed by Western blotting.

Array comparative genomic hybridization/SNP microarrayanalysis

A microarray analysis [array-comparative genomic hybrid-ization/SNP (aCGH/SNP)] was performed with SurePrint G3Cancer CGHþSNP Microarray, 4 � 180K (Agilent Technolo-gies) to detect unbalanced chromosomal changes and copynumber neutral loss of heterozygosity. The final product wasscanned with the Agilent G2565CA Microarray Scanner System(Agilent Technologies) and analyzed with Agilent Cytoge-nomics v4.0.3.12 (Agilent Technologies).

Apoptosis measurementNumber of apoptotic and/or necrotic cells was determined

by flow cytometry (BD FACS Canto II) using Annexin VFITC (Apronex) and propidium iodide (Sigma). Percentageof apoptotic and/or necrotic cells was calculated using thefollowing formula: (measured apoptosis � basal apoptosis)/(100 � basal apoptosis) � 100 (%). Drug concentrations thatinduced apoptosis in 50% cells after 24 hours (IC50) weredetermined by nonlinear regression algorithms using GraphPad Prism software.

IHCSections from formalin-fixed paraffin-embedded (FFPE)

blocks from patients and from murine DLBCL xenografts werecut and stained by hematoxylin & eosin and Giemsa stains.IHC was performed using BCL2 (clone 124, Dako), BCL-XL(clone B4H6, Cell Signaling Technology), MCL1 (clone S-19,Santa Cruz Biotechnology), BIM (clone H-191, Santa CruzBiotechnology), and NOXA (clone 114C307, Santa Cruz Bio-technology) as described previously (19). Heat-induced pre-treatment of deparaffinized tissue sections in buffer pH 9.0(Dako, S2367) was applied. After blocking of endogenousperoxidase activity (3% solution of hydrogen peroxide), sam-ples were incubated overnight at 4�C with primary antibodiesanti-MCL1 (1:200) and anti–BCL-XL (1:200). Detection ofprimary antibody binding was performed for anti-MCL1 withpolymer system (secondary antibody and peroxidase; N-His-tofine Simple Stain MAX PO, Nichirei Biosciences) and foranti–BCL-XL with biotinylated secondary antibody and avi-

dine-peroxidase complex (LSABþ, Dako REAL Detection Sys-tems, HRP/DABþ, Rabbit/Mouse, Dako), followed by incuba-tion with solution of hydrogen peroxide and chromogen sub-strate DAB (3,30diaminobenzidine tetrahydrochloride, Dako).Nuclei were counterstained with Harris hematoxylin. Afterdehydration and clearing in xylene, slides were mounted inorganic solvent–based medium and evaluated under lightmicroscope. To differentiate BCL2, BCL-XL, MCL1, BIM, andNOXA-positive and -negative MCL cases, we applied the com-monly used cut-off value of 30% cells.

Establishment of MCL clones with knockdown or transgenicoverexpression of MCL1, BIM, and NOXAMCL1. MCL1 cDNA was cloned into Sleeping beauty plasmidspSBtet Pur (https://www.addgene.org/60507/) published inPMID: 25650551 and then electroporated together with transpo-sase coding plasmid (pCMV(CAT)T7-SB100) for insertion intogenome. Control plasmid (pSBtet Pur empty) had all inducibleelements, but was empty and did not produce any protein upondoxycycline addition. Three days after electroporation of cells ofinterest, puromycin was added in final concentration of 2 mg/mLand the cells selected for 6 days. Cells were then resuspended inmedia with doxycycline at final concentration 200 ng/mL andMCL1protein expressionwasmeasured after 48 hours byWesternblotting.

BIM. Cell lines with stably integrated shRNA-gene/cDNAwere prepared as described previously (19). Briefly, packaginglentiviral vectors pMD2.G (Addgene, plasmid 12259), psPAX2(Addgene, plasmid 12260) together with pLKO.1 (SigmaAldrich)/pCDH-neo (SBI)/pLVX TetONE-puro vector containingthe gene of interest were transfected into HEK 293T/17. Condi-tioned medium was harvested 36 hours later, centrifuged, andprecipitated using PEG-it (System Biosciences) according to themanufacturer's instructions. Precipitated particles were resus-pended in PBS and stored in �80�C. Target cells were infectedwith equivalent multiplicity of infection for 24 hours and thetransductants were selected in the growthmediumcontaining 2 to3 mg/mL puromycin (LKO1-shRNAs, LVX TetONE-puro) or 2mg/mL G-418 (CDH-cDNAs).

NOXA. For siRNA-mediated silencing, we used NOXA duplex(GGUGCACGUUUCAUCAAUUTT) and anegative control siRNAas described previously (Eurogentec; refs. 36, 37). MCL cellswere electroporated with Amaxa nucleofector system usingSolution V (Lonza) using a program number 0-017. Twenty-four hours after nucleofection the knockdown efficiency wasvalidated by Western blotting, and the cells were exposed tovenetoclax or S63845.

Experimental therapy of lymphoma-bearing miceThe experimental design was approved by the Institutional

Animal Care and Use Committee (MSMT-11255/2015-4; 592/15). NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (referred to as NSGmice) were purchased from The Jackson Laboratory. All animalswere maintained in a pathogen-free environment in individuallyventilated cages and provided with sterilized food and water.Adult female NSG mice were used for all experiments. NSG micewere subcutaneously inoculated with 10 � 106 lymphoma cells.Therapy was initiated when all mice developed palpable tumors

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(¼day 1, D1). At D1 all mice were stratified so that all cohortscontained mice with comparable calculated tumor volumes.Venetoclax and S63845 were purchased fromMedchemExpress.Venetoclax (50 mg/kg, by oral gavage) was first diluted inethanol and then mixed with phosal-G/PEG-400 (1: 3: 6).Venetoclax was given on days 1, 2, 3, 6, and 7. S63845 (25mg/kg) was diluted in 50 mmol/L HCL and 20% hydroxyl-propyl-b-cyclodextrin, and administered intravenously on days1, 2, 3, 6, and 7. In case of damaged veins, S63845 wasadministered intraperitoneally. Tumor growth was recordeddaily using three perpendicular dimensions (in millimeters)with a digital caliper. Tumor volumes were calculated using thefollowing formula: p/6� length�width� height. Observationwas terminated (and experimental mice euthanized) whengrown subcutaneously tumors exceeded 2 cm in the largestdiameter. Tumors were excised and weighed, and the eutha-nized mice were dissected in search for any signs of advanced(disseminated) lymphoma (e.g., splenomegaly, abdominallymphoma spread, etc.).

Statistical analysisData from five in vivo experiments were analyzed, each

experiment covering different time periods with different num-bers of known data points (see Fig. 5). For the purpose ofassessing the statistical significance of treatment effectiveness,we made an assumption that the calculated differences betweenmean tumor sizes in the control group and groups treated withmonotherapies as well as between the latter groups and thosetreated with combined therapies were generated by a processwhich includes a deterministic linear trend in the form of yt ¼b0 þ b1t þ et, where yt denotes the data-generating stochasticprocess of the analyzed differences, t ¼ 1, 2, . . .T is a timevariable, T signifies the length of the experiment in days and etis the Gaussian IID white noise. Having concurrently performed34 statistical hypothesis tests about the zero value of b1, theBonferroni correction of 5% and 1% simultaneous significancelevels was utilized, resulting in individual significance levels of0.1471% and 0.0294%, respectively. The results are shownin Table 1.

ResultsSensitivity of established MCL lines and primary MCL cells tovenetoclax and S63845

Cytotoxic effects of venetoclax and S63845 was analyzedusing a panel of nine MCL cell lines (Fig. 1A–C) and ninefreshly isolated primary MCL cells (Fig. 1D and E). Patients'

baseline characteristics are displayed in Supplementary TableS3. Cells were arbitrarily considered as sensitive, if they dis-played >50% apoptosis 24 hours after exposure to 1 mmol/Lvenetoclax or 1 mmol/L S63845. Using these criteria five out ofnine cell lines were sensitive to venetoclax (Fig. 1A and C). OnlyMINO cell line was sensitive to S63845 (Fig. 1B and C). Incontrast to the cell lines, tested primary MCL samples were allsensitive to venetoclax, and five of nine were sensitive toS63845 (Fig. 1D–F).

Prevalence and impact of BIM deletion in established MCL celllines and primary MCL cells

Protein expression of BCL2, MCL1, BIM, and NOXA wasanalyzed by Western blotting using a panel of 9 MCL cell lines,9 primary MCL samples, and 5 PDXs (Fig. 2A–C). In addition,protein expression of BCL2, MCL1, BIM, and NOXA was evalu-ated semiquantitatively in FFPE tissue samples obtained fromMCL cell line–xenografted mice (Fig. 2D) and from patients withMCL at diagnosis or disease relapse (Fig. 2E).

As previously reported, three commonly used MCL cell lines(JEKO-1, MINO, and Z138) lack BIM protein expressionas a consequence of biallelic BIM gene deletion (Fig. 2A). Weconfirmed that lack of BIM protein observed in these cell lines isassociated with resistance (JEKO-1, Z138) or decreased sensitivity(MINO) to venetoclax (Fig. 1A and C). First, we functionallyanalyzed the role of BIM in venetoclax-triggered apoptosis bytransgenic BIM overexpression and shRNA-mediated BIM knock-down in selectedMCL cell lines.We confirmed that ectopic (over)expression of BIM in JEKO-1 and Z138 partially restored vene-toclax-sensitivity of these cell lines (Fig. 3A and D). In analogy,knockdown of BIM expression in the highly venetoclax-sensitivecell lines HBL2 and MAVER-1 partially inhibited venetoclax-induced apoptosis (Fig. 3B and E).

Some groups reported that deletions of BIM gene arefrequently seen in primary MCL samples (12–14). We hypoth-esized that a deletion of BIM might predict venetoclax resis-tance in MCL patients in the clinical grounds. Unexpectedly,analysis of BIM protein expression either by Western blottingusing a panel of nine primary MCL samples and five PDXcells, or by IHC using a panel of 37 FFPE tissue samplesobtained from patients with MCL did not reveal a singlesample with undetectable protein expression (Fig. 2B, C, andE; Supplementary Table S4). To confirm our findings atthe genomic level, we implemented aCGH on a validationset of 24 primary MCL samples obtained from peripheralblood of leukemized patients and on two MCL cell lines withpreviously reported deletion of BIM (JEKO-1 and MINO).

Table 1. Statistical analysis of in vivo experiments

VFN-M1 VFN-M2 VFN-M3 VFN-M5R1 VFN-M8

CTRL vs. VTX 0.0643 0.0004� 0.0000��� 0.0001��� 0.0000���

CTRL vs. S63845 0.0000��� 0.0120 0.0000��� 0.0000��� 0.0000���

VTX vs. VTX þ S63845 0.0000��� 0.0000��� 0.0001��� 0.0005� 0.0000���

S63845 vs. VTX þ S63845 0.0001��� 0.0000��� 0.0000��� 0.0000��� 0.0000���

NOTE: P values of partial t tests about zero slope ofmean tumor size differences (statistical significance: � 5% and ��� 1% simultaneous significance level;N indicates anegative slope). Except for the linear trend ofmean tumor size differences between treatments with CFZ and VTXþCFZ combination in the VFN_M2 experiment, allslopes were positive, suggesting that each monotherapy was effective in reducing the tumor size, each combination being more efficient than the correspondingmonotherapies. Twenty-six of 34 P values in Table 1 show statistical significance at 1% simultaneous significance level, and four P values indicating statisticalsignificance at 5% simultaneous significance level. These results suggest a statistically significant difference between the development of tumor size in control groupsand those treated with monotherapies as well as between the groups treated with combined therapies and the one treated with corresponding monotherapies. Inother words, there is not only practically significant but also statistically significant difference in the effectiveness of different treatments.

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Although deletion of BIM gene was confirmed in both testedMCL cell lines, only one of 24 analyzed primary MCL cellsamples had detectable monoallelic loss of BIM (in this case,the whole long arms of chromosome 2 were deleted within acomplex karyotype; Supplementary Fig. S3; SupplementaryTable S3).

Expression of NOXA in MCL cells and its functional impact onMCL1-mediated venetoclax resistance

Discrepancies in the protein expression of NOXA betweenprimary MCL cells and established cell lines have been reportedby other groups (38). Here, we confirmed that not only primaryMCL cells, but also PDXs express significantly lower levels ofNOXA protein compared to majority of the established MCL celllines (Fig. 2; Supplementary Table S4). NOXA is a BH3 onlyprotein that specifically binds and blocksMCL1.We assumed thatrelative overexpression of NOXA observed in themajority ofMCLcell lines compared with primary MCL cells and PDXs mightrender MCL cell lines (that do not harbor deletions of BIM)hypersensitive to venetoclax. Indeed, siRNA-mediated NOXAgene and protein knockdown decreased venetoclax-triggered apo-ptosis (Fig. 3C and F).

Mechanisms of resistance of MCL cells to venetoclaxBesides biallelic deletions of BIM (which plausibly represent

very rare events in MCL) upregulation of MCL1 was associated

with venetoclax resistance in diffuse large B-cell lymphoma andacute myeloid leukemia cells (16, 39). Mechanistically, MCL1may serve as a buffer for BIM released upon binding of venetoclaxto BCL2 (16). Indeed, immunoprecipitation of REC-1 cells beforeand after venetoclax treatment showed not only expected greatreduction of BIM bound to BCL2, but also substantial increase ofBIM bound to MCL1, both as a consequence of venetoclaxtreatment (Fig. 4A–D). This molecular mechanism (i.e., bufferingof BIM by MCL1) also provides a plausible explanation for theobserved venetoclax resistance of REC-1 cells. Next, we estab-lished MAVER-1 and HBL2 venetoclax-resistant clones by long-term cultivation with gradually increasing venetoclax concentra-tions up to 1 mmol/L. HBL2 venetoclax-resistant (VTX-R) cells hadsignificant upregulation of both MCL1 and NOXA proteins,but only MCL1 mRNA was significantly upregulated, specifically22.6-times more MCL1 mRNA in VTX-R compared with CTRL.NOXA mRNA remained unchanged (i.e., <2-fold total mRNAchange) indicating posttranscriptional mechanism of NOXA pro-tein deregulation in HBL2 VTX-R cells. MAVER-1 VTX-R hadmarkeddownregulationofNOXAwithunchangedMCL1protein.Messenger RNA levels of NOXA and MCL1 were not significantly(>2-fold) changed between VTX-R and CTRLMAVER-1 cells (datanot shown), again suggesting posttranslational mechanism ofNOXA dysregulation.

Interestingly, transgenic overexpression of MCL1 was accom-panied by a similar pattern of NOXA protein level deregulation as

VTX 1 µmol/L S63845 1 µmol/L IC50 (nmol/L)VTX S63845

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Sensitivity of MCL cell lines and primary cells to venetoclax and S63845.A, B,D, and E, Cytotoxic activity of venetoclax and S63845 toward established MCL celllines (A and B) and primary MCL cells (D and E). Y-axes show numbers of Annexin-Vþ/PIþ cells 24 hours after exposure to 1 mmol/L venetoclax or S63845. Barsrepresent means� SDs of two independent experiments. C and F, Calculated IC50 for the cell lines (C) and primary lymphoma samples (F). Basal apoptosis ofunexposed patients' cells for PT1-PT9 was 40%, 24%, 37%, 26%, 50%, 15%, 40%, 36%, and 44%. PT, patient.

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in the respective HBL2 and MAVER-1 VTX-R clones (compareFig. 4E andG). MAVER-1MCL1-UP clone displayed upregulationof MCL1 accompanied by decreased levels of NOXA protein,which was associated with significant inhibition of both veneto-clax and S63845-triggered apoptosis (Fig. 4E and F). In contrast,HBL2 MCL1-UP clone displayed upregulation of NOXA and itssensitivity to venetoclax and S63845 compared with empty vectortransfected HBL2 cells was onlymildly attenuated (Fig. 4E and F).

Cotargeting of BCL2 and MCL1 on a panel of five PDX murinemodels of aggressive MCL

MCL1 protein appears to be a critical molecule that attenuatesvenetoclax-induced apoptosis withMCL1 overexpression leadingto venetoclax resistance (Fig. 4). Concurrent blockage of BCL2with venetoclax and MCL1 with a highly specific small-moleculeinhibitor S63845 might thus represent an effective treatmentstrategy in MCL.

In vivo experiments were implemented on a panel of five PDXmodels derived from patients with treatment-refractory MCL(Supplementary Fig. S1; Supplementary Table S1; SupplementaryTable S2). First, single-agent S63845 (25 mg/kg) showed prom-ising in vivo efficacy in all five PDXmodels. In the case of VFN-M3,efficacy of S63845 was even significantly higher than that ofvenetoclax (Fig. 5; Supplementary Table S5). Second, the com-bination of venetoclax and S63845 was in all five PDX modelsassociatedwith excellent anti-MCL synergy comparedwith single-agent approaches.

DiscussionIn the current study, we evaluated molecular mechanisms of

sensitivity of MCL cells to BCL2-targeting agent venetoclax. Wedemonstrated critical role of the functional status of anti-apoptotic MCL1 protein in conferring both inherited and

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Expression of BIM and NOXA in MCL cell lines and primary cells. A–C,Western blot analysis of BCL2, MCL1, BIM, and NOXA on a panel of established MCL cell lines(A), primary lymphoma samples (B), and PDXs (C). MAVER-1 was used as interassay control. D and E, Semiquantitative intensity (Y-axes) of staining of BCL2,MCL1, BIM, and NOXA on a panel of formalin-fixed paraffin-embedded sections of MCL cell line–based subcutaneous xenografts (D) and patient (PT) primarysamples (E). For details, see Supplementary Methods.

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acquired resistance to venetoclax. As MCL1 and its interactionswith NOXA appear as principal molecules that attenuatesvenetoclax-induced apoptosis, we proposed a treatment strat-egy aimed at concurrent inhibition of BCL2 by venetoclax andMCL1 by S63845, which proved to be highly synergistic in MCLin vivo.

From our data as well as from other published communica-tions, there is an apparent bias in the expression of BCL2 andMCL1-targeting BH3-only proteins BIM and NOXA betweenestablished cell lines and primary MCL cells. At the genomiclevel, biallelic loss of BIM gene was repeatedly reported in MCLcell lines, but never in primary MCL samples (12–14). In theoriginal article by Tagawa and colleagues, only heterozygous(not homozygous) deletions of BIM were found in as few as 5of 27 (18.5%) MCL patients (12). In another study, Mestre-

Escorihuela and colleagues focused mainly on analysis of celllines and the only information provided about BIM status inprimary MCL samples was based on IHC analysis by tissuemicroarray with loss of BIM protein expression detected in 7 of22 (33%) patient samples (14). Katz and colleagues focused ongenetic proof of concept of biallelic BIM gene deletion duringMCL lymphomagenesis, however, did not analyze primaryMCL cells (13).

In our study, aCGH analysis of 24 primary MCL samplesobtained from patients with high-risk disease according to MCLinternational prognostic index identified only a single patientwith a monoallelic BIM deletion (in the context of loss of entirelong arms of chromosome 2 within a complex karyotype; Sup-plementary Table S3; Supplementary Fig. S3). Similarly to thegenomic analysis, IHC analysis of 37 FFPE tissue samples

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BIM and NOXA regulate sensitivity to venetoclax. A, Transgenic overexpression of BIM in JEKO-1 and Z138 cell lines. "CTRL" stands for the original cell lines withBIM gene deletion. B, shRNA-mediated BIM gene knockdown in HBL2 and MAVER-1 cell lines. "Empty" stands for empty vector–transfected cells. C, siRNA-mediated NOXA gene knockdown in HBL2 and MAVER-1 cell lines. "siCTRL" stands for cells transfected with noncoding siRNA. D, Cytotoxic activity of venetoclax(1 mmol/L, 24 hours) in JEKO-1 and Z138 cell clones with transgenic overexpression of BIM (JEKO-1:BIM, Z138:BIM). DOX, doxycycline. E, Cytotoxic activity ofvenetoclax (25 nmol/L, 24 hours) in HBL2 and MAVER-1 cell clones with shRNA-mediated knockdown of BIM expression. F, Cytotoxic activity of venetoclax(25 nmol/L, 24 hours) in HBL2 and MAVER-1 cell clones with siRNA-mediated knockdown of NOXA expression. Y-axes show numbers of Annexin-Vþ/PIþ cells.Bars represent means� SDs of two independent experiments.

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obtained from MCL patients did not identify a single case withundetectable BIMprotein (Supplementary Table S4).On the basisof our results, and other published data, it appears that biallelicdeletions of BIM or loss of BIM protein expression are extremely

rare events in primary MCL cells. It might be further speculatedthat MCL cell lines with loss of BIM might be derived from suchrare cases reflecting extremely aggressive, highly rearranged dis-eases. On the other hand, all five PDX murine models of MCL

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Molecular mechanisms of resistance tovenetoclax. A, Immunoblot of the totalREC-1 protein lysate 24 hours afterexposure to venetoclax (1 mmol/L)compared with untreated REC-1 cells(CTRL) shows unchanged levelstotal BIM and NOXA proteins.B, Immunoprecipitation of REC-1 cellswith BCL2 antibody 24 hours afterexposure to venetoclax (1 mmol/L)compared with untreated REC-1 cells(CTRL) shows decrease of BCL2-boundBIM after exposure to venetoclax.C, Immunoprecipitation of REC-1 cellswith MCL1 antibody 24 hours afterexposure to venetoclax (1 mmol/L)compared with untreated REC-1 cells(CTRL) shows increase of MCL1-boundBIM (released from BCL2) after exposureto venetoclax. D,Numbers of Annexin-Vþ/PIþ cells 24 hours after exposure ofREC-1 cells to venetoclax (1 mmol/L),S63845 (5 mmol/L) and the combinationof both agents. Y-axes show numbers ofAnnexin-Vþ/PIþ cells 24 hours afterexposure to 1 mmol/L venetoclax,or the combination of both agents.E, Immunoblots of HBL2 and MAVER-1clones with transgenic overexpression ofMCL1 (MCL1-UP) compared with cellstransfected with an empty vector(empty). F,Numbers of Annexin-Vþ/PIþ

cells 24 hours after exposure of the MCL1-UP clones to venetoclax (100 nmol/L) orS63845 (5 mmol/L). G, Immunoblots ofHBL2 and MAVER-1 clones with in vitroacquired resistance to venetoclax(VTX-R) compared with the originalcell lines (CTRL). H,Numbers ofAnnexin-Vþ/PIþ cells 24 hours afterexposure of the VTX-R clones tovenetoclax (100 nmol/L) or S63845(5 mmol/L; Y-axes). Downregulation ofMCL1 and BCL-XL in HBL2, as well asdownregulation of MCL1 and BIM inMAVER-1, are the most plausibleconsequence of the apoptotic process.

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derived from patients with relapsed/refractory disease in ourlaboratory retained BIM protein expression by Western blotting.Therefore, one should consider that loss of BIM in MCL cell linesmight be a consequence of in vitro–induced changes associatedwith a cell line derivation.

Another molecule with markedly different protein expressionbetween primary MCL samples and MCL cell lines is NOXA (38).As few as 4 of 72 primary MCL samples were reported to express

NOXA compared with ubiquitous overexpression of NOXAdetectable in most (if not all) MCL cell lines (14). In our study,NOXA protein expression was markedly lower in primary MCLsamples (and in PDX cells) in comparison with MCL cell lines, asanalyzed by both approaches, IHC and Western blotting (Fig. 2;Supplementary Table S3).

In summary, the two critical proapoptotic proteins, BIM andNOXA, are differentially expressed betweenMCLprimary samples

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Experimental therapy of MCL-bearing mice.A–E, Growth curves of the individualtreatment cohorts constructed from thecalculated tumor volumes (means�standard deviations). For details seeMethods. X are days, where day 1 (D1) marksinitiation of the therapy, Y shows calculatedtumor volumes from three perpendiculartumor dimensions. CTRL, untreated controls;VTX, venetoclax. � Themice wereeuthanized despite small subcutaneoustumors because they developed generalizedinability to thrive. Postmortem examinationdemonstrated spleen enlargement in amajority of mice; see SupplementaryTable S4.

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and established cell lines (Fig. 2). Using clones with transgenic(over)expression or shRNA/siRNA-mediated knockdown of BIMand NOXA, we confirmed that they are indeed key mediators ofvenetoclax proapoptotic activity (Fig. 3). As a consequence, MCLcell lines unfortunately represent unreliable models for asses-sing proapoptotic activity of BH3 mimetics including veneto-clax. Loss of BIM makes some cell lines (JEKO-1, Z138, MINO)"falsely" venetoclax resistant, and conversely, overexpression ofNOXA makes other cell lines (HBL2, MAVER1, GRANTA-519)"falsely" hypersensitive to venetoclax. From this perspective,PDX models, which both retain BIM expression, and do notoverexpress NOXA, represent better models than cell lines,more closely reflecting primary MCL samples in this context.In addition, it was repeatedly demonstrated that microenvi-ronmental factors may induce resistance to venetoclax, namelyby PI3K–AKT–mTOR pathway–mediated upregulation of BCL-XL and MCL1, further suggesting that PDX models likelyrepresent the most relevant preclinical models for evaluationof anticancer activity of BH3 mimetics (39–41).

Molecular mechanisms of acquired resistance to venetoclaxin MCL remain poorly understood. Upregulations of MCL1 andBCL-XL were reported as plausible mechanisms in diffuse largeB-cell lymphoma (39). Our data pinpointed MCL1 as a prin-cipal mediator of VTX-R. First, MCL1 upregulation wasobserved in both MCL clones (HBL2-VTX-R, and MAVER1-VTX-R) derived from two most venetoclax-sensitive cell linesHBL2 and MAVER1 (Fig. 4G and H). Second, we confirmedthat MCL1 can act as a buffer for BIM (and potentially otherBH3-only proteins) released from BCL2 by venetoclax (Fig. 4A–C) (16). And third, stable overexpression of MCL1 in HBL2and MAVER1 cell lines was associated with significantlydecreased sensitivity to venetoclax (Fig. 4E and F). The datathus provided sound mechanistic explanation for potentialsynthetic lethality between venetoclax and S63845 in MCL.We have confirmed that both venetoclax- and S63845-inducedcytotoxicity is a caspase-dependent apoptotic process (Supple-mentary Fig. S4A and S4B). Interestingly, venetoclax-resistantHBL2 clones were "cross"-resistant not only to S63845, but alsoto two different BCL-XL inhibitors WEHI-539 and A-1155463(Fig. 4H; Supplementary Fig. S4E and S4F). The data thussuggest a more complex disruption of mitochondrial apoptosis(in addition to MCL1-NOXA deregulation) as a result of in vitroacquired venetoclax resistance.

Even though MCL is generally considered a BCL2-dependentmalignancy, we showed very promising antilymphoma efficacy ofS63845 monotherapy (Fig. 5; Supplementary Table S4). Concur-

rent inhibition of MCL1 and BCL2 with S63845 and venetoclax,respectively, was associated with significantly increased antilym-phoma efficacy compared with the single-agent approaches. Thecombination of venetoclax and S63845 was well tolerated, andinduced long-term lymphoma-free survival of MCL xenografts infive different PDXmodels derived from patients with chemother-apy-refractory diseases.

In conclusion, the data strongly support investigation ofS63845 in combination with venetoclax for targeted eradicationof chemotherapy-resistant MCL cells including patients withadverse cytogenetics in the clinical grounds.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D. Prukova, O. Havranek, P. KlenerDevelopment of methodology: Z. Nahacka, J. Karolova, O. Havranek,M. Pacheco-Blanco, E. Fronkova, P. KlenerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Prukova, M. Klanova, J. Soukup, Z. Zemanova,D. Tuskova, E. Pokorna, K. Forsterova, P. Vockova, M. Trneny, P. KlenerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Prukova, L. Andera, J. Karolova, M. Svaton,J. Soukup, K. Svobodova, Z. Zemanova, E. Pokorna, K. Helman, A. Berkova,E. Fronkova, M. Trneny, P. KlenerWriting, review, and/or revision of the manuscript: D. Prukova, L. Andera,M. Svaton, M. Klanova, O. Havranek, M. Pacheco-Blanco, E. Fronkova,M. Trneny, P. KlenerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Pacheco-Blanco, P. KlenerStudy supervision: P. Klener

AcknowledgmentsThis work was supported by Ministry of Health of the Czech Republic

grant AZV 17-28980A (all rights reserved), Charles University Center ofExcellence grant UNCE/MED/016 and PRIMUS/17/MED/9, Ministry ofEducation, Youth and Sports grants PROGRES Q26/LF1, PROGRES Q28/LF1, SVV 260374. Institutional support to L. Andera and Z. Nahacka. CzechAcademy of Sciences RVO:86652036 and RVO:68378050. Institutionalsupport to Z. Zemanova, A. Berkova, and K. Svobodova was RVO-VFN64165.Institutional support to E. Fronkova and M. Svaton was LO1604 and00064203.

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 October 8, 2018; revised January 30, 2019; accepted April 16, 2019;published first April 19, 2019.

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