Published OnlineFirst February 15, 2011; DOI: 10.1158/0008-5472.CAN-10-2525 Cancer Therapeutics, Targets, and Chemical Biology Research Protein Kinase C Inhibitor Sotrastaurin Selectively Inhibits the Growth of CD79 Mutant Diffuse Large B-Cell Lymphomas 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. Ó2011 AACR. Introduction (doxorubicin), vincristine (Oncovin), prednisone] chemother- apy (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 pro- tein 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 Ó2011 American Association for Cancer Research. drive the expression of its target genes. www.aacrjournals.org 2643 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst February 15, 2011; DOI: 10.1158/0008-5472.CAN-10-2525 Naylor et al. 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 evi- dence 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 Æ 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