Protein Kinase C Inhibitor Sotrastaurin Selectively Inhibits the Growth of CD79 Mutant Diffuse Large B-Cell Lymphomas

Published OnlineFirst February 15, 2011; DOI: 10.1158/0008-5472.CAN-10-2525

Cancer Therapeutics, Targets, and Chemical Biology Research

Tara L. Naylor1, Huaping Tang1, Boris A. Ratsch2, Andreas Enns2, Alice Loo1, Liqing Chen1, Peter Lenz3, Nigel J. Waters1, Walter Schuler4, Bernd Dorken€ 2, Yung-mae Yao1, Markus Warmuth1, Georg Lenz2, and Frank Stegmeier1

Abstract The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-kB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-kB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC

inhibition correlated with NF-kB pathway inhibition and were mediated by induction of G1-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities. Cancer Res; 71(7); 2643–53. 2011 AACR.

Introduction (doxorubicin), vincristine (Oncovin), prednisone] chemotherapy (R-CHOP). In contrast, ABC DLBCL represents the least Diffuse large B-cell lymphoma (DLBCL) represents the most curable subtype with 3-year overall survival rates of approxi- common subtype of malignant lymphoma and is diagnosed in mately 40% (5). A hallmark of the molecular pathogenesis of more than 20,000 patients each year in the United States. It is ABC DLBCL is the constitutive activation of the NF-kB path- heterogeneous with respect to morphology, biology, and clin- way, which occurs predominantly via the CBM (Carma1- ical presentation (1). By gene expression profiling, at least 3 Bcl10-Malt1) [CARD11 (caspase recruitment domain-contain- molecular subtypes of DLBCL can be distinguished: germinal ing protein 11, also known as CARMA1)/BCL10/MALT1] center B-cell–like (GCB) DLBCL, activated B-cell–like (ABC) signaling complex that promotes cell proliferation, differen- DLBCL, and primary mediastinal B-cell lymphoma (PMBL; tiation, and suppresses apoptosis (6, 7). refs. 2–4). The molecular DLBCL subtypes not only differ Physiologically, activation of the CBM complex in B cells with respect to their gene expression patterns but also have occurs in response to B-cell receptor (BCR) stimulation. significantly different overall survival rates. GCB DLBCL Antigen binding to the BCR induces receptor oligomerization and PMBL patients respond favorably to current standard- and thereby promotes Lyn-mediated phosphorylation of of-care combined therapy with the anti-CD20 antibody ritux- ITAM (immunoreceptor tyrosine-based activation motif) imab and CHOP [cyclophosphamide, hydroxydoxorubicin domains in the B-cell coreceptors CD79A and CD79B (8). Phosphorylated ITAM domains recruit and activate the protein tyrosine kinase SYK (spleen tyrosine kinase) at the plasma 1 Authors' Affiliations: Novartis Institutes for BioMedical Research, Cam- membrane that initiates downstream signaling through BTK bridge, Massachusetts; 2Department of Hematology, Oncology and Tumor Immunology, Charite, Berlin, Germany; 3Department of Physics, (Bruton's tyrosine kinase) and PLCg (phospholipase Cg) and Philipps-University Marburg, Marburg, Germany; and 4Novartis Institutes ultimately leads to the activation of PKC (protein kinase C). In for BioMedical Research, Basel, Switzerland B cells, PKCb is thought to be the predominant PKC isoform Note: Supplementary data for this article are available at Cancer Research mediating BCR-NF-kB activation, at least in part, through the Online (http://cancerres.aacrjournals.org/). phosphorylation of CARD11 (9, 10). Once activated at the Corresponding Author: Frank Stegmeier, Novartis Institutes for Bio- Medical Research, Oncology, 250 Massachusetts Avenue, 3C-282, plasma membrane, the CBM complex facilitates the activation Cambridge, MA 02139. Phone: 617-871-3736; Fax: 617-871-4000; E-mail: of the IKK (I kappa B kinase) complex, which phosphorylates [email protected] IkBa targeting it for proteosomal degradation, and thereby doi: 10.1158/0008-5472.CAN-10-2525 allows NF-kB transcription factors to enter the nucleus and 2011 American Association for Cancer Research. drive the expression of its target genes.

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Until recently, it was unclear whether the constitutive NF- BrdUrd (bromodeoxyuridine) incorporation (Roche). Caspase kB pathway activation in ABC DLBCL represents a primary 3/7 activity was measured using CaspaseGlo (Promega). pathogenetic event in lymphomagenesis or merely reflects the Detailed protocols are available in Supplementary Materials physiologic status of the tumor cell of origin. The identifica- and Methods. tion of oncogenic CARD11 mutations provided the first evidence for genetic deregulation of this pathway (11). Moreover, TaqMan mRNA expression, NF-kB nuclear recent studies have revealed somatically acquired genetic translocation, and IL-6/IL-10 secretion assay lesions in several NF-kB pathway regulators, including fre- In vivo tumor samples were harvested and snap frozen in quent loss-of-function mutations and deletions in the negative liquid nitrogen. Tissue samples were homogenized and lyzed regulator A20 (12, 13) and genetic abnormalities in CD79A and in RLT buffer with reagent DX, using the TissueLyser (Qiagen), CD79B (14). Thus, the vast majority of ABC DLBCLs seem to and mRNA expression was analyzed by TaqMan. NF-kB harbor genetic lesions that constitutively activate NF-kB translocation and interleukin (IL)-6/IL-10 secretion levels in pathway signaling. Previous studies showed that ABC DLBCL supernatants were assessed using Trans-AM (Active Motif) lines are sensitive to inhibition of CARD11, BCL10, MALT1, ELISA plates and Quantikine ELISA kits (R&D Systems), or IKKb, showing a clear dependence on NF-kB pathway respectively. Detailed protocols are available in Supplemen- signaling (6, 7, 15). These results contrast another study tary Materials and Methods. that proposed that ligand-independent "tonic" BCR signaling is a more general feature of B-cell lymphomas that renders Western blotting and gene expression analysis these cells dependent on downstream BCR signaling (16). To For Western blotting, 30 mg of protein from total cell lysate clarify the role of BCR signaling and assess the therapeutic was loaded onto 4% to 12% Bis-Tris gradient gels (Invitrogen). potential of PKC inhibitors in the treatment of DLBCL, we The following primary antibodies were used: a-CARD11, analyzed the response of DLBCL cell lines to treatment with a-cRel (Cell Signaling), a-p65 a-PARP, a-PKCb (Santa Cruz), the selective PKC inhibitor sotrastaurin (STN, also known as and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; AEB071), which is currently in phase II clinical trials for Sigma). Gene expression was measured using whole-genome psoriasis and solid organ transplantation (17–20). Agilent 4 44K gene expression arrays (Agilent Technologies) following the manufacturer's protocol. Materials and Methods Detailed protocols are available in Supplementary Materials and Methods. Cell culture and cell line generation TMD8, SU-DHL4, SU-DHL2, BJAB, U2932, K422, and HBL1 Tumor xenografts cells were grown in RPMI 1640 with 10% FBS, DB cells in RPMI Mice were maintained and handled in accordance with 1640 with 20% FBS, OCI-Ly3 in Iscove's modified Dulbecco's Novartis Biomedical Research Animal Care and Use Commit- medium (IMDM) with 20% FBS, and OCI-Ly10 in IMDM with tee protocols and regulations. Treatment was initiated when 20% human serum. Cell lines were authenticated by single tumor volume reached an average size of 160 mm2 (21 days nucleotide polymorphism profiling (fingerprinting). For the post–tumor implantation). STN solution was prepared weekly RNA interference experiments, cell lines were engineered to and dosed orally on a "tid" schedule. Tumor volume was express the murine ecotropic retroviral receptor for efficient determined by twice-weekly digital calipering and calculated retroviral transductions and the bacterial tetracycline repres- using the formula: length width2/2. Data were expressed as sor for doxycycline-inducible short hairpin RNA (shRNA) mean SEM, and differences were considered statistically expression as described (7). The targeting sequence of the significant at P < 0.05 by Student t test. PKCb shRNA was CGACCAACACTGTCTCCAAAT. HBL1 cells were engineered to stably express the activat- Results ing CARD11-L244P mutation by transduction with a lentiviral vector that constitutively expresses CARD11 L244P driven by CD79 mutant ABC DLBCL cell lines are sensitive to PKC either a CMV (cytomegalovirus) or UBC (ubiquitin C) promoter inhibition (pLenti6-CMV and pLenti6-UBC; Invitrogen). TMD8 cells were A recent study (16) proposed that the majority of DLBCL engineered to stably express the activating CARD11-L244P cell lines exhibit tonic BCR signaling that leads to dependence mutation by retroviral transduction. The CARD11-L244P cDNA on downstream kinases including SYK for their proliferation was inserted into a modified version of the inducible pRetro- and survival. This model suggested that cells with tonic BCR SUPER dual–promoter vector. Retroviral transductions were signaling may also be sensitive to PKCb inhibition, as this performed as previously described (7). kinase is a critical mediator of CBM complex activation downstream of SYK (9, 10). We therefore evaluated the effects Cell growth, flow cytometry, BrdUrd assay, and caspase of 2 ATP-competitive PKC inhibitors, the pan-PKC inhibitor 3/7 assay STN (18, 20–22) and the PKCa/b-selective compound BHA536 Cell growth assays were performed using CellTiter-Glo (Novartis; unpublished, see structure and selectivity data in Luminescent Cell Viability Assay Reagent (Promega). To Supplementary Fig. S1) on the proliferation of a panel of determine cell-cycle distribution, cells were analyzed by flow DLBCL cell lines. We included as control compounds the cytometry [fluorescence-activated cell sorting (FACS)] and IKKb-selective inhibitors AFN700 (23) and MLN120B (24).

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GCB cell lines were generally insensitive to both IKKb and with STN and BHA536 (Fig. 1A). Notably, OCI-Ly3 and PKC inhibitors, with half-maximal growth inhibitory concen- SU-DHL2 have reported mutations in CARD11 and A20, trations (IC50) greater than 10 mmol/L in SU-DHL4 and DB respectively, and the fact that these oncogenic lesions are cells (Fig. 1A and B and Table 1), which is consistent with thought to function downstream of PKCb in CBM-NF-kB the notion that this subtype does not exhibit NF-kB pathway signaling (Supplementary Fig. S2) provides a potential mole- activation (6, 7). In contrast, most ABC DLBCL cell lines were cular rationale for their insensitivity. U2932 cells, which dis- sensitive to IKKb inhibitors (Fig. 1B), but their response played intermediate sensitivity to PKC inhibitors (Fig. 1A), to pharmacologic PKC inhibitors varied strongly (Fig. 1A). were recently reported to harbor a TAK1 mutation, although OCI-Ly3 and SU-DHL2 cells were insensitive to PKC inhibition, the oncogenic nature of this mutation has not yet been with IC50 values greater than 10 mmol/L in the growth assays confirmed experimentally (12). In contrast, HBL1, TMD8,

Figure 1. PKC inhibitors and PKCb knockdown selectively impair the proliferation of CD79 mutant ABC DLBCL cell lines. CD79A/B mutant ABC DLBCL cell lines (blue; OCI-LY10, HBL1, TMD8), ABC DLBCL cell lines with downstream NF-kB pathway mutations (green; OCI-LY3, U2932, SU-DHL2), and GCB DLBCL cell lines (red; SU-DHL4, DB) were treated for 5 days with serial dilutions of (A) the pan-PKC inhibitor STN, the PKCa/b-selective inhibitor BHA536, (B) the IKKb inhibitor AFN700, or (C) the BTK inhibitor PCI-32765. Relative cell growth (average of at least 3 independent experiments) was measured by Cell Titer Glo and normalized to dimethyl sulfoxide (DMSO)-treated cells. D, shRNA-mediated knockdown of PKCb is toxic to CD79A/B mutant ABC DLBCL but not to ABC DLBCL with mutant CARD11 or GCB DLBCL cell lines. PKCb was knocked down by retroviral vectors expressing an shRNA together with green fluorescent protein (GFP). Live GFPþ cells were enumerated by FACS on the indicated days post–retroviral infection and normalized to the value at day 2 following retroviral infection. Western blot analysis shows shRNA-mediated knockdown of PKCb. Dox, doxycycline.

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Table 1. PKC, BTK, andSYK inhibitorsselectively inhibit the proliferation of CD79 mutant ABC DLBCL cell lines

ABC GCB Ly10 HBL1 TMD8 U2932 DHL2 Ly3 DB K422 BJAB DHL4 Cell line Target Compound CD79 CD79 CD79 TAK1 A20 CARD11 NR NR NR NR NF-κB mut Syk R406 ND 3 4 4 >20 >20 >20 >20 >20 5 BTK PCI-32765 ND 0.0060 0.0006 20 >.1 >20 >20 ND 15 15 Sotrastaurin 1.3 0.5 0.2 10 >20 >20 >20 >20 >20 >20 BHA536 0.5 0.3 0.2 10 15 >20 15 11 20 7.5 PKC LY333531 2.1 3 ND 5 >20 >20 11 >10 ND >10 Enzastaurin ND 5 15 10 >20 15 >20 ND 20 10 AFN700 0.3 2.5 0.3 15 15 0.4 15 10 15 15 IKKβ MLN120B 10 10 10 >40 >40 10 >40 >40 >40 >20 Proteasome velcade 0.01 0.01 ND 0.01 ND 0.010 0.004 0.020 ND ND

NOTE: The table displays the half maximal growth inhibitory concentrations (IC50) for the indicated compounds across several DLBCL

cell lines. IC50 values are expressed in micromolar concentrations and were derived from dose–response curves similar to the ones described in Figure 1. Abbreviations: ND, not determined; NR, not reported.

and OCI-Ly10 cells were very sensitive to both PKC inhibitors, is in clinical development for several cancers including DLBCL with IC50 values ranging from 0.2 to 1.0 mmol/L (Fig. 1A), (25), had only a modest effect on the growth of CD79 mutant suggesting that these cell lines are dependent on BCR signal ABC DLBCL cell lines (Supplementary Fig. S3B). While enzas- transduction upstream of CARD11. To further test this taurin caused a limited inhibitory effect at 5 to 10 mmol/L, the hypothesis, we examined the sensitivity of the DLBCL cell higher concentrations also affected the proliferation of several line panel to small molecule inhibitors targeting other kinases GCB cell lines without significant discrimination of CD79 that mediate signaling from the BCR to CBM complex. Strik- mutant cell lines (Supplementary Fig. S3B). These findings ingly, HBL1 and TMD8 cells were also the most sensitive lines suggest that STN and BHA536 have superior activity in CD79- in response to treatment with BTK and SYK inhibitors (Fig. 1C, mutated cell lines compared with enzastaurin. Supplementary Fig. S3A, and Table 1). These findings show PKCb is thought to be the major PKC isoform that mediates that HBL1, TMD8, and OCI-Ly10 cells are "addicted" to BCR/ BCR signaling. We therefore reasoned that the growth inhi- CBM/NF-kB pathway signaling and raised the possibility that bitory effect of STN and BHA536 in CD79-mutated cells is these cells may harbor genetic lesions that activate the NF-kB mediated through inhibition of PKCb rather than other PKC pathway upstream of PKCb. Indeed, during the course of our isoforms. To test this hypothesis, we examined the effect of a study, Davis and colleagues identified mutations in the BCR PKCb-selective shRNA across a panel of ABC and GCB lym- coreceptor CD79A/B that lead to chronic activation of BCR phoma lines. Strikingly, knockdown of PKCb was toxic to signaling (14). We confirmed that each of the 3 ABC DLBCL CD79 mutant ABC DLBCL cells but did not affect the pro- cell lines that were sensitive to PKC inhibition harbored liferation of CARD11 mutant or that of GCB DLBCL cell lines mutations in the ITAM motif of CD79A/B (Supplementary (Fig. 1D), closely recapitulating the effects of the pharmaco- Fig. S4; data not shown). Consistent with the short-term logic PKC inhibitors. Collectively, our findings show that growth assays, CD79 mutant cell lines (HBL1 and TMD8) also CD79-mutated ABC DLBCL cell lines are specifically sensitive showed significant growth inhibition in a clonogenic assay to BCR signaling inhibitors and identify PKCb as a novel whereas the CARD11 mutant cell line (OCI-LY3) was not therapeutic target for this genetically defined subpopulation affected by STN (Supplementary Fig. S5). of DLBCL. To exclude the possibility that CD79 mutant cell lines are just generally hypersensitive to pharmacologic agents, we PKC inhibitors reduce NF-kB pathway signaling in tested a variety of "cytotoxic" agents, such as cisplatin, Taxol CD79 mutant cells (paclitaxel), and Velcade (bortezomib), across our lymphoma Constitutive activation of the NF-kB pathway is a mole- cell line panel. Importantly, none of these drugs showed cularhallmarkofABCDLBCLcellsandisrequiredfortheir preferential or enhanced killing of HBL1, TMD8, and OCI- proliferation and survival (2, 6, 7). We therefore evaluated Ly10 cells compared with the other B-cell lymphoma lines whether the growth inhibitory effect in response to PKC (Supplementary Fig. S3C, Table 1; data not shown), further inhibition is mediated through modulation of NF-kBpath- supporting the notion that the differential growth inhibition in way signaling. STN treatment of HBL1 and TMD8 cells response to PKC inhibitors is a specific effect. inhibited the nuclear translocation of both p65 and c-Rel During our evaluation of additional PKC inhibitors, we (Fig.2AandB),whichareNF-kBtranscriptionfactorsub- surprisingly found that the PKC inhibitor enzastaurin, which units that have been implicated in mediating NF-kBpath-

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Figure 2. The PKC inhibitor STN attenuates NF-kB signaling in CD79 mutant ABC DLBCL cell lines. The CD79 mutant ABC DLBCL cell lines HBL1 and TMD8 were treated with STN (5 mmol/L) or DMSO for 24 hours. A, cytoplasmic (Cyto) and nuclear (Nuc) fractions from cell lysates were separated on SDS-PAGE gels and immunoblotted with antibodies to cRel and p65. B, the activity of the NF-kB transciption factors p65 and c-Rel in response to STN treatment was determined from total protein lysates by a oligonucleotide capture ELISA assay. DNA binding is expressed relative to lysates from DMSO-treated cells. C, gene expression profiling of the ABC DLBCL cell line HBL1 after treatment with the IKKb inhibitor MLN120B and the PKC inhibitor STN at the indicated time points (6, 12, 24, and 48 hours). Gene expression changes were assessed using DNA microarrays and depicted according to the color scale shown. A gene was selected as an NF-kB target gene in HBL1 cells if MLN120B decreased the expression of the gene by at least 50% at 3 of the 4 time points. This gene signature was subsequently applied to the gene expression data following treatment with the PKC inhibitor STN. Note that a 2-color array was used to normalize the compound-treated samples to control-treated (DMSO) samples. D, decrease in the NF-kB target gene signature following incubation with the IKKb inhibitor MLN120B and the PKC inhibitor STN. Gene expression measurements for the component genes in the NF-kB signature (C) were averaged for each of the indicated conditions. way activation in ABC DLBCL (26). To monitor the tran- NF-kB pathway activation leads to the induction of various scriptional response to PKC inhibition,wetreatedtheCD79 cytokines, such as IL-6 and IL-10, which promote proliferation mutant cell line HBL1 with STN and assessed relative and survival of B cells (27–29). IL-6 and IL-10 also responded changes in gene expression at several time points by to STN treatment in our gene expression profiling studies, whole-genome expression arrays (Fig. 2C and D). Applica- with the most pronounced downregulation at the 6 and tion of 2 independent NF-kB pathway signatures to the gene 12 hour time points (Supplementary Fig. S6C). We therefore expression profiling data revealed that STN treatment used IL-6 and IL-10 as markers to study NF-kB pathway caused a significant downregulation of NF-kB target genes, modulation by PKC inhibitors in more detail. STN treatment strongly supporting the notion that PKC inhibition results in resulted in a dose-dependent decrease in IL-6 mRNA expres- suppression of the NF-kB pathway (Fig. 2C and D, and sion in the CD79 mutant cell lines OCI-Ly10, HBL1, and TMD8 Supplementary Fig. S6A and B). (Fig. 3A). In contrast, IL-6 mRNA expression was unaffected in

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Figure 3. Growth inhibitory effect of the PKC inhibitor STN correlates with NF-kB pathway inhibition. A, STN treatment (16 hours) leads to a dose-dependent reduction of IL-6 mRNA levels in CD79 mutant cell lines (HBL1, TMD8, and OCI-Ly10) but not in the CARD11 mutant cell line OCI- Ly3. IL-6 mRNA expression was determined using TaqMan qPCR

assays by the DDCt method relative to DMSO-treated cells. TBP was used as the endogenous control gene. B, IL-6 mRNA levels are reduced in a dose-dependent manner by treatment with the PKC inhibitors BHA536 and LY333531. C and D, the secretion of IL-6 is inhibited by the IKKb inhibitor MLN120B (C) and the PKC inhibitor STN (D) in a dose- dependent manner. IL-6 secretion was determined 48 hours posttreatment with the Quantikine ELISA assay, using supernatant from treated cells (normalized to DMSO-treated cells).

the CARD11 mutant cell line OCI-Ly3 (Fig. 3A), which is CARD11 (L244P), are insensitive to PKC inhibition. This consistent with the fact that CARD11 functions downstream finding suggested that the L244P mutation in the coiled- of PKC (Supplementary Fig. S2). Treatment of TMD8 cells with coil domain of CARD11 might obviate the requirement for 2 additional PKC inhibitors [BHA536 and LY333531, a pan- PKCb-mediated activation of the CBM complex (Supple- PKC inhibitor also known as ruboxistaurin (30)] also reduced mentary Fig. S2). Indeed, we confirmed in an engineered IL-6 mRNA expression in a dose-dependent manner (Fig. 3B), cellular system that in contrast to PMA (phorbol-12-myr- providing further evidence that IL-6 modulation is an on- istate-13-acetate)/ionomycin-induced pathway activation, target effect of PKC inhibition. The fact that IL-6 is secreted NF-kB pathway activation in response to CARD11-L244P from cells allowed us to monitor IL-6 levels in cell culture is not sensitive to PKC inhibition (Supplementary Fig. S8). supernatants. As expected, treatment with IKKb inhibitors We therefore wanted to test whether expression of CARD11- strongly reduced IL-6 secretion in all ABC DLBCL cell lines L244P in a CD79 mutant cell line confers resistance to PKC tested (Fig. 3C). The PKC inhibitor STN also strongly inhibited inhibitors. To this end, HBL1 cells were stably transduced IL-6 and IL-10 secretion in TMD8 and HBL1 cells (IC50 < 0.2 with lentiviral vectors expressing CARD11-L244P under the mmol/L) but again did not affect their levels in the CARD11 control of the CMV or UBC promoter. Expression of the mutant cell line OCI-Ly3 (Fig. 3D and Supplementary Fig. S7). mutant CARD11 allele was confirmed by Western blotting Importantly, the concentrations required to inhibit NF-kB and sequencing of the reverse-transcribed mRNA transcripts signaling (as measured by IL-6 secretion) in the sensitive cell (Fig. 4A and B). The expression levels of exogenous CARD11- lines closely correlated with the growth inhibitory IC50 values. L244P in the CMV construct was comparable with that of Moreover, the lack of NF-kB pathway modulation by PKC endogenous CARD11 (Fig. 4A and B), and only 2- to 3-fold inhibitors in OCI-Ly3 cells is in agreement with the insensi- above endogenous levels when expressed from the UBC tivity of this cell line in growth assays. Collectively, these promoter (Fig. 4A). Strikingly, expression of CARD11- findings show that PKC inhibitors can suppress NF-kB path- L244P rendered HBL1 cells to be resistant to the growth way signaling in cells with CD79 mutations and support the inhibitory effects of STN (Fig. 4C). Similar rescue of STN notion that downregulation of NF-kB pathway signaling is a sensitivity was observed in TMD8 cells (Supplementary major contributor to the growth inhibitory effect observed in Fig. S9C). A CARD11 mutant construct lacking the CARD these cells. domain, which is crucial to facilitate CBM complex assembly (31), could not confer resistance to PKC inhibitors (Supple- mentary Fig. S9A). The CARD11-L244P expression lines, Oncogenic CARD11 mutations confer resistance to PKC however, were still responsive to IKKb inhibitors (Fig. 4C inhibitors and Supplementary Fig. S9B), thereby excluding the possi- Our cell line profiling experiments with STN indicated bility that the resistance to PKC inhibitors was due to that OCI-Ly3 cells, which have an activating mutation in emergence of a nonspecific resistance mechanism.

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Figure 4. Expression of activated CARD11 renders HBL1 cells insensitive to STN. HBL1 cells were stably transduced with constructs that express activated CARD11 (L244P) under the control of a CMV or UBC promoter. A, expression of exogenous CARD11-L244P was confirmed by sequencing the reverse- transcribed mRNA of engineered HBL1 cells (TCT ! TCC encodes the L244P codon change). OCI- Ly3 cells, which harbor homozygous mutation of CARD11-L244P, were used as a control. B, Western blot to compare CARD11 expression in parental and engineered HBL1 cells. Note that exogenous CARD11-L244P is untagged and will migrate similarly to endogenous CARD11. C, parental HBL1 and engineered HBL1- CARD11 mutant cells were treated for 5 days with the PKC inhibitor STN (left) or the IKKb inhibitor MLN120B (right). Relative cell growth was determined using the Cell Titer Glo assay and is expressed as a percentage of DMSO-treated cells. Error bars, SD. D, parental HBL1 and engineered HBL1-CARD11 mutant cells were washed and then immediately treated for 48 hours with the PKC inhibitor STN (left) or the IKKb inhibitor MLN120B (right). IL-6 levels in cell supernatants were determined by the Quantikine ELISA assay and are expressed as a percentage of IL-6 secretion from DMSO-treated cells. Error bars, SD.

Next, we analyzed whether the growth rescue by PKC inhibition induces G1 arrest and/or cell death in CARD11-L244P expression was due to the restoration of CD79 mutant cells downstream NF-kB signaling. While IL-6 secretion was To gain additional insights into the nature of the growth strongly inhibited by STN in the parental HBL1 cells, IL- inhibitory effects in response to PKC inhibition, we next 6 secretion was no longer modulated in cells engineered to examined whether STN treatment induces cell-cycle arrest express CARD11-L244P (Fig. 4D). Cells expressing the or apoptosis in sensitive lines. Treatment with STN induced CARD11 constructs, however, still downregulated IL-6 in astrongG1 arrest in both HBL1 and TMD8 cells, as evi- response to IKKb inhibitor treatment (Fig. 4D). Together, denced by an increase in the G1 population and concomitant these "rescue" experiments further show that the growth decreasesinthefractionofcellsinSandG2/M phase (Fig. 5A inhibitory effect of STN in CD79-mutated cells is due to on- and B, Supplementary Fig. S10). The G1 arrest was further target inhibition and strongly suggests that NF-kB pathway confirmed by a BrdUrd assay, where STN caused a dose- downregulation is a critical mediator of the growth inhi- dependent inhibition of S-phase entry in HBL1 and TMD8 bitory effect. cells (Supplementary Fig. S11). Importantly, the cell-cycle

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Figure 5. The PKC inhibitor STN

induces a G1 arrest and/or cell death in CD79 mutant ABC DLBCL cell lines. HBL1 (A) and TMD8 (B) cells were treated with 5 mmol/L STN for 48 hours and cell- cycle distribution was assessed by measuring DNA content of propidium iodide–stained cells (flow cytometry). 2N and 4N denote the DNA content of diploid

cells in G1 and G2 phase, respectively. A and B, STN

treatment induces G1 arrest in a dose-dependent manner. The graph displays the percentage of cells in each cell-cycle phase

(G1,S,G2/M, S, and sub-G1) relative to DMSO-treated cells. C and D, parental TMD8, engineered TMD8-GFP-CARD11 mutant (CARD*), and TMD8-GFP cells (control) were treated for 5 days with 1.25 mmol/L STN. Constitutive CARD11* (L244P) expression (C) restores cell-cycle

progression through G2/M and S phase and (D) rescues cell death induced by STN as determined by flow cytometry.

arrest occurs at concentrations that correlate well with the STN inhibits tumor growth of CD79-mutated DLBCL growth inhibitory IC50 values. While G1 arrest seemed to in vivo be the predominant response of CD79-mutated cells to STN, To address whether CD79-mutated ABC DLBCLs are we noted the emergence of a sub-G1 peak in TMD8 cells, also sensitive to PKC inhibition in an in vivo setting, especially at later time points (48 hours; Fig. 5B and Sup- we established a subcutaneous TMD8 xenograft model. plementary Fig. S10), suggesting that TMD8 cells may be We optimized the dosing regimen for STN to achieve sus- undergoing cell death upon prolonged STN treatment. We tained NF-kB pathway inhibition as judged by downregula- also detected an increase in PARP cleavage, a cellular marker tion of IL10 mRNA over the entire dose period (Fig. 6B, data of cell death/apoptosis, in TMD8 cells but not in HBL1 cells notshown).DailyoraldosingofSTN(80mg/kg,tid)resulted (Supplementary Fig. S12C). Expression of CARD11-L244P in in statistically significant inhibition of tumor growth com- TMD8 cells could rescue both the G1 arrest (Fig. 5C) and cell paredwithvehicle-treatedanimals, corresponding to 17% death (Fig. 5D), strongly indicating that these effects of STN tumor volume change, treated over control group (Fig. 6A). are on-target and mediated by inhibition of CBM-NFkB This dosing regimen was well tolerated with less than 5% signaling. reduction in mouse body weight (data not shown). Together, To further examine whether STN induces an apoptotic these results show an in vivo dependence of CD79-mutated response in TMD8 cells, we monitored the activity of the tumors on PKC signaling and support the potential thera- key effector caspases (3 and 7) in response to compound peutic value of STN in CD79-mutated ABC DLBCL. treatment. STN treatment induced only a modest increase in caspase 3/7 activity, especially when compared with Discussion strong apoptosis inducers such as the DNA-damaging agent camptothecin (Supplementary Fig. S12A and B). Despite recent improvements in the therapy of DLBCL, These findings suggest that the cell death observed in especially the introduction of the anti-CD20 antibody ritux- TMD8 cells at later time points is likely a secondary effect imab (32), a significant number of patients will still succumb of prolonged G1 arrest rather than acute apoptosis induc- to their disease and new therapeutic strategies for DLBCL tion. Collectively, our data indicate that the major response patients are critically warranted. Our increasing molecular of CD79-mutated DLBCL cell lines to STN seems to be G1 understanding of the signaling pathways that contribute to cell-cycle arrest. lymphoma pathogenesis provides several novel therapeutic

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Sotrastaurin Inhibits CD79 Mutant DLBCL

Figure 6. Treatment with the PKC inhibitor STN results in significant inhibition of in vivo tumor growth. A, in a subcutaneous TMD8 xenograft model in SCID (severe combined immunodeficient) mice, STN treatment (80 mg/kg, tid) resulted in inhibition of tumor growth (percent tumor volume change, treated over control group ¼ 17%) relative to animals treated with vehicle. Data are expressed as mean SEM. B, mRNA expression of IL-10 was measured by TaqMan qPCR at 1 and 8 hours after the first STN dose.

options. By screening several selective PKC inhibitors includ- The results from a clinical trial of the pan-PKC inhibitor ing STN across a panel of B-cell lymphoma lines, we discov- enzastaurin in DLBCL patients were recently reported (25). In ered that these compounds selectively inhibit a subset of ABC the phase II study by Robertson and colleagues, only 7% of DLBCL cell lines, whereas most lymphoma cell lines screened patients achieved a long-term remission. We surprisingly were insensitive to PKC inhibition. Intriguingly, we identified found that enzastaurin only modestly inhibits the growth of that all cell lines with CD79A/B mutations exhibit strong CD79 mutant ABC DLBCL cell lines. Moreover, the antipro- dependence on PKC signaling. During the course of our liferative effects observed at 5 to 10 mmol/L enzastaurin do not studies and consistent with our results, Davis and colleagues seem to be specific for CD79 mutant cell lines, as enzastaurin found that CD79 mutations are associated with chronic active inhibits the proliferation of many cancer cell lines, including BCR signaling (14) and confer dependence on BTK and SYK. GCB DLBCL cell lines, at these higher concentrations. Given We provide several lines of evidence to show that the selective the lack of selective antiproliferative effect, it is possible that killing of CD79 mutant ABC DLBCL cells by STN is due to on- the growth inhibitory effects at higher enzastaurin concentra- target PKC inhibition. First, selective inhibition of growth is tions may be attributed to previously described off-target observed with several different PKC inhibitors and the cellular activities of this compound (33) rather than specific PKC IC50 of PKC/NF-kB pathway inhibition strongly correlates inhibition. Together, these findings suggest that STN has with the antiproliferative effect. Second, cell lines that harbor superior activity than enzastaurin and may provide a larger mutations in downstream signaling components, such as therapeutic window for this genetically defined subpopulation CARD11, are insensitive to selective PKC inhibitors. Moreover, of DLBCL. It is also important to note that patients in this expression of mutant CARD11 in the CD79 mutant HBL1 cell phase II study with enzastaurin were neither stratified for line renders these cells completely resistant to the growth molecular DLBCL subtype (ABC DLBCL or GCB DLBCL) nor inhibitory effects of STN. PKCb is thought to be the major PKC screened for mutations in the NF-kB pathway. Given the fact isoform that relays the signal from BCR activation to the CBM that CD79 mutations occur in about 20% of ABC DLBCL and complex. Consistent with this notion, we found that BHA536, a 10% of all DLBCL (including non-ABC subtypes), it is possible PKCa/b-selective inhibitor that does not significantly inhibit that the few observed complete responses may have occurred the novel or atypical PKC isoforms (Supplementary Fig. S1), in patients harboring CD79 mutations or other genetic lesions also strongly inhibits the proliferation of CD79 mutant cells. that lead to chronic active BCR signaling. In addition, we genetically confirmed that PKCb is the critical Our study clearly shows the therapeutic potential of PKC isoform, as shRNA-mediated knockdown of PKCb was selec- inhibitors in patients with CD79 mutant DLBCL. However, tively toxic to CD79-mutated cell lines. A recent study showed future clinical trials in DLBCL should incorporate methods that a substantial fraction of DLBCL cell lines exhibits tonic such as gene expression profiling and mutational screens to BCR signaling and proposed that tonic pathway activation discern patients according to their genetic aberrations. Our confers dependency on downstream BCR signaling (16). How- data provide first insights into potential mechanisms of ever, our data and a recent study by Davis and colleagues (14) resistance that may emerge in patients treated with specific clearly show that a genetically defined subpopulation of pathway inhibitors. We detected that the CARD11 mutant cell DLBCL, such as those carrying CD79A/B mutations, is sensi- line OCI-Ly3 is insensitive to STN and other PKC inhibitors. tive to pharmacologic PKC and BTK inhibitors. Together, In addition, expression of the constitutively active CARD11 these findings strongly suggest that mutational activation mutant L244P rendered a CD79 mutant cell line resistant to (chronic signaling) but not tonic BCR pathway activation PKC inhibitors. These findings indicate that patients with leads to PKC and BTK dependency. CARD11 mutations are unlikely to respond to PKC inhibitors

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Naylor et al.

or other upstream inhibitors. The implications of mutations of efficacy of STN in CD79 mutant DLBCL in a clinical trial. the negative regulator of NF-kB signaling A20 are currently Moreover, our findings illustrate the necessity to incorporate unclear (34). A20 is frequently inactivated in different subtypes gene expression profiling and genetic sequencing to stratify of lymphoma (12, 13). It is thought to predominantly function patients on the basis of their molecular NF-kB pathway lesions in a negative feedback loop to dampen NF-kB pathway to maximize response rates for PKC inhibitors in DLBCL. signaling, but it is unclear whether loss of A20 by itself is sufficient to drive oncogenic NF-kB signaling or still requires Disclosure of Potential Conflicts of Interest upstream signaling. Interestingly, the only cell line (SU-DHL2) No potential conflicts of interest were disclosed. in our lymphoma panel that harbors biallelic loss of A20 was insensitive to SYK, BTK, and PKC inhibitors. Several putative Acknowledgments A20 substrates, such as NEMO and MALT1, are thought to function downstream of PKCb (34, 35). Thus, it is possible that We thank Maurice van Eis, Bruno Cenni, Guido Koch, and Francisco Adrian complete loss of A20 function can override the need for for sharing reagents and technical advice. We are grateful to Jennifer Rocnik and upstream pathway activation. Marion Dorsch for their critical reading of the manuscript. BCR signaling is complex and leads to the activation of several downstream effector pathways, including the PI3K/ Author Contribution mTOR, Ras/Raf/Mek/ERK, and CBM/NF-kB pathway. The BCR T.L. Naylor, H. Tang, B.A. Ratsch, A. Loo, N.J. Waters, L. Chen, and A. Enns signaling network therefore presents several opportunities for performed research and analyzed data. P. Lenz performed statistical analyses. rationale combination therapies, such as "horizontal combina- W. Schuler contributed vital new reagents. B. Dorken, Y-m. Yao, and M. Warmuth provided intellectual input and helped write the article. F. Stegmeier, tions" of PKC and PI3K/mTOR pathway inhibitors (36). Con- T.L. Naylor, and G. Lenz designed the research, analyzed data, and wrote the sistent with this notion, preliminary studies suggest that STN article. shows significant synergy when combined with the mTOR- selective inhibitor RAD001 (data not shown; refs. 37, 38). In Grant Support addition to maximizing the antitumor activity, combination therapies may also provide a valuable strategy to delay or G. Lenz was supported by the German Research Foundation (DFG), the Deutsche Krebshilfe, and the Else-Kroner-Fresenius€ Stiftung. prevent the emergence of resistance (36). Several targeted The costs of publication of this article were defrayed in part by the therapies for the PI3K/mTOR pathway are currently in clinical payment of page charges. This article must therefore be hereby marked development (39), and future studies should further evaluate advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the benefit of combining these agents with selective PKC inhibitors in preclinical and clinical studies. In conclusion, Received July 13, 2010; revised December 10, 2010; accepted January 2, 2011; our study provides a strong rationale for investigating the published OnlineFirst February 15, 2011.

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www.aacrjournals.org Cancer Res; 71(7) April 1, 2011 2653

Protein Kinase C Inhibitor Sotrastaurin Selectively Inhibits the Growth of CD79 Mutant Diffuse Large B-Cell Lymphomas

Tara L. Naylor, Huaping Tang, Boris A. Ratsch, et al.

Cancer Res 2011;71:2643-2653. Published OnlineFirst February 15, 2011.

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