Enzastaurin Treatment Research Articles

PKCβ Facilitates Leukemogenesis in Chronic Lymphocytic Leukaemia by Promoting Constitutive BCR-Mediated Signalling

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCβII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCβII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCβII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCβII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCβ expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

Engine: Phase III Randomized Study of Enzastaurin/R-CHOP Versus Placebo/R-CHOP in Frontline High Risk Diffuse Large B Cell Lymphoma Patients with Novel Genomic Biomarker DGM1

Background: Progress in genome technology allows analysis of previously completed trials to identify subgroups potentially benefiting from therapy. Enzastaurin is a potent inhibitor of protein kinase C beta (PKC-β) and suppresses the phosphoinositide 3-kinase (PI3K)/AKT pathway. The safety and efficacy of Enzastaurin has been tested in more than 60 clinical trials including 2 major studies in DLBCL: (1) PRELUDE (A phase III maintenance trial of Enzastaurin vs Placebo, N=758) (Crump, 2016), and (2) S028 (A randomized phase II study of Enzastaurin/R-CHOP vs R-CHOP in frontline intermediate/high-risk DLBCL, N=101) (Hainsworth, 2016). DNA samples extracted from blood of patients from PRELUDE were retrospectively genotyped using whole genome SNP arrays. From the genome wide screening a novel genetic biomarker, DGM1, was identified showing high correlation with response to Enzastaurin treatment (Luo, ASH 2018). Importantly, these findings were replicated in the phase II S028 study. In the S028 study the hazard ratio (HR) for OS in high-risk (IPI ≥ 3) DGM1 positive (+) patients who received Enzastaurin/R-CHOP was 0.28 (0.1-0.81) when compared to subjects who received R-CHOP, a benefit favoring Enzastaurin (p=0.018). These data suggest that addition of Enzastaurin to R-CHOP may significantly improve outcome in frontline high-risk DGM1 (+) DLBCL. The ENGINE study was initiated to validate this finding in a prospective study. Study Design and Methods: Adult patients must have untreated CD20+ DLBCL, IPI ≥ 3. Patients are randomized 1:1 to Enzastaurin/R-CHOP or Placebo/R-CHOP for 6 cycles during combination phase. Each subject's treatment assignment will be unblinded after response assessment at the end of the combination phase. Subjects randomized to the investigational arm who have a complete or partial response will have the option to continue in the single agent phase to receive Enzastaurin for up to 2 additional years. The study intends to enroll approximately 235 patients with primary endpoint of OS in DGM1 (+) patients. The study is ongoing with 57 sites open in the US and China. As of 20 July 2019, 128 patients have been randomized. Clinical trial information: NCT03263026. Disclosures Nowakowski: Genentech, Inc.: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding. Brody:Acerta Pharma: Research Funding; Merck: Research Funding; Celldex Therapeutics: Research Funding; Genentech: Research Funding; Oncovir, Inc.: Research Funding; BMS: Research Funding; Kite Pharma: Research Funding. Lue:Kymera Therapeutics: Honoraria; Astex Pharmaceuticals: Honoraria. Luo:Denovo Biopharma LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:Denovo Biopharma LLC: Employment. Han:Denovo Biopharma LLC: Employment. Jivani:TRACON Pharmaceuticals, Inc.: Other: Stock; Denovo Biopharma LLC: Employment. Liu:Denovo Biopharma LLC: Employment. Li:Denovo Biopharma LLC: Employment. Sun:Novartis: Other: Stock; Denovo Biopharma LLC: Employment.

ENGINE: Phase III randomized study of enzastaurin/R-CHOP versus placebo/R-CHOP in frontline high-risk diffuse large B-cell lymphoma patients with novel genomic biomarker DGM1.

TPS7569 Background: Progress in genome technology allows analysis of previously completed trials to identify patients subgroups potentially benefiting from therapy. Enzastaurin is a potent inhibitor of protein kinase C beta (PKC- b) and suppresses the phosphoinositide 3-kinase (PI3K)/AKT pathway. The safety and efficacy of Enzastaurin has been tested in more than 60 clinical trials including 2 major studies in DLBCL: (1) PRELUDE (A phase III maintenance trial of Enzastaurin vs Placebo, N=758) ( Crump, 2016), and (2) S028 (A randomized phase II study of Enzastaurin/R-CHOP vs R-CHOP in frontline intermediate/high-risk DLBCL, N=101) ( Hainsworth, 2016). DNA samples extracted from blood of patients from PRELUDE were retrospectively genotyped using whole genome SNP arrays. From the genome wide screening a novel genetic biomarker, DGM1, was identified showing high correlation with response to Enzastaurin treatment ( Luo, ASH 2018). Importantly, these findings were replicated in the phase II S028 study. In the S028 study the hazard ratio (HR) for OS in high-risk (IPI ≥ 3) DGM1 positive (+) patients who received Enzastaurin/R-CHOP was 0.28 (0.1-0.81) when compared to subjects who received R-CHOP, a benefit favoring Enzastaurin (p=0.018). These data suggest that addition of Enzastaurin to R-CHOP may significantly improve outcome in frontline high-risk DGM1 (+) DLBCL. The ENGINE study was initiated to validate this finding in a prospective study. Methods: Adult patients must have untreated CD20+ DLBCL, IPI ≥ 3. Patients are randomized 1:1 to Enzastaurin/R-CHOP or Placebo/R-CHOP for 6 cycles during combination phase. Each subject’s treatment assignment will be unblinded after response assessment at the end of the combination phase. Subjects randomized to the investigational arm who have a complete or partial response will have the option to continue in the single agent phase to receive Enzastaurin for up to 2 additional years. The study intends to enroll approximately 235 patients with primary endpoint of OS in DGM1 (+) patients. The study is ongoing with 51 sites open in the US and China. As of 22 Jan 2019, 70 patients have been randomized. Clinical trial information: NCT03263026.

Improved Survival with Enzastaurin Treatment in Diffuse Large B-Cell Lymphoma Patients with the Novel Genetic Biomarker, DGM1

BackgroundDrugs that have benefited a subset of patients but discontinued for development may be rescued through identification of a biomarker predictive of response. Enzastaurin, a potent and selective inhibitor of protein kinase C-β, improved PFS in high-risk DLBCL patients in a randomized phase 2 trial when combined with RCHOP, but not when administered as maintenance therapy in DLBCL patients achieving CR. Using data from both trials, we identified a biomarker potentially predictive of enzastaurin benefit.MethodsBiomarker discovery was conducted on Eli Lilly's (Lilly) PRELUDE study, a phase 3 maintenance trial that enrolled approximately 750 DLBCL patients who achieved a complete response to R-CHOP front-line therapy. Patients were randomized to enzastaurin or placebo maintenance for up to three years. A genome-wide screen was performed on DNA extracted from blood samples from patients participating in this study and results were evaluated for correlation to efficacy endpoints through bioinformatic analysis. Confirmation of the biomarker identified in the phase 3 study was performed by independent analysis of the biomarker in a separate completed Lilly enzastaurin study in patients with DLBCL. This study was a phase 2 trial in 101 newly diagnosed DLBCL patients randomized to treatment with R-CHOP or R-CHOP plus enzastaurin. Patients receiving R-CHOP plus enzastaurin and achieving a CR or PR after induction were eligible to continue with single agent enzastaurin for up to 3 years.ResultsDenovo Genomic Marker 1 (DGM1), a germline polymorphism on chromosome 8, was identified using Lilly's phase 3 samples as highly correlated and potentially predictive of response to enzastaurin. Although there was no difference in overall survival (OS) in the ITT population, biomarker analysis found that DGM1+ patients receiving enzastaurin had significantly improved OS compared to DGM1- patients receiving enzastaurin (HR 0.27, p=0.0002) in the PRELUDE trial (Figure 1).These findings were replicated in the phase 2 study biomarker analysis: DGM1+ patients receiving R-CHOP plus enzastaurin had significantly improved OS (HR 0.1, p-0.005) compared to DGM1- patients (Figure 2). The original analysis of the phase 2 study found a trend towards improved, but not statistically significant, OS in patients with high-risk DLBCL receiving R-CHOP plus enzastaurin. Biomarker analysis of this population demonstrated significant improvement in OS (HR 0.28, p=0.018) for high-risk DLBCL DGM1+ patients receiving R-CHOP plus enzastaurin compared to high-risk DLBCL DGM1+ patients receiving R-CHOP alone (Figure 3). DGM1+ status was not predictive of efficacy in the control (non-enzastaurin) arm (Figure 4).ConclusionThese data are supportive of DGM1 as a potentially predictive biomarker for enzastaurin efficacy. The mechanism of DGM1 impact in DLBCL is under study. Based on this data, a biomarker driven phase 3 trial (ENGINE Trial) of R-CHOP plus enzastaurin versus R-CHOP in DGM1+ and DGM1- patients with newly diagnosed high-risk DLBCL is underway. [Display omitted] DisclosuresLuo:Denovo Biopharma LLC: Employment, Equity Ownership. Sun:Denovo Biopharma LLC: Employment, Equity Ownership. Smith:Portola: Research Funding; Merck Sharpe Dohme and Corp: Consultancy, Research Funding; Acerta Pharma BV: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding. Han:Denovo Biopharma LLC: Employment, Equity Ownership. Shazer:Denovo Biopharma LLC: Employment, Equity Ownership.

Eosinophil Differentiation and Viability in Hypereosinophilia Is Impaired By Genetic and Pharmacologic Targeting of LCP1 and PKC Beta

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Massive overproduction of hyperactive eosinophils leads to enhanced tissue infiltration and, in some cases, severe organ damage. Clonal malignancies with HE involve the FIP1L1-PDGFRA (F/P)-positive neoplasms and other neoplasms harboring PDGFRA and PDGFRB rearrangements, all of which are sensitive to imatinib. However, most subtypes do not carry a defined genetic aberration and, therefore, lack specific treatment.In eosinophils, modulation of the actin cytoskeleton is mandatory for priming and migratory function. In this study, we aimed to target the actin-binding protein LCP1, also termed L-plastin, to reduce eosinophilic burden in HE. LCP1 is a direct target of the serine/threonine kinase PKCß, which phosphorylates LCP1 at serine 5, essential for its full activity. We confirmed high LCP1 expression and Ser5 phosphorylation dependent on F/P activity in the HES cell line Eol-1 and 32D-F/P cells. The PKCβ selective inhibitor enzastaurin led to dephosphorylation of LCP1 in Eol-1 cells, and this was associated with significantly reduced proliferation (p < 0.0001), metabolic activity (p < 0.0001), and colony formation (11.8 fold reduced) as well as enhanced apoptosis (p < 0.0001) and impaired migration to SDF-1 (p < 0.001). Apoptosis was associated with ß-Catenin and c-Jun protein stabilization and increased expression of p73. Reduced surface expression of CD11b was detected (1.5 fold reduction; p=0.0043), which may explain the impaired migration capacity to SDF-1. While enzastaurin did not alter F/P-induced STAT3, STAT5, and ERK1/2 phosphorylation, it specifically inhibited STAT1Tyr701 and AKTSer473 but not AKTThr308 phosphorylation, and shRNA knock-down experiments confirmed that this process was mediated by LCP1.The mTORC2 complex triggers AKTSer473 phosphorylation, and PDK1 is the main Ser/Thr kinase responsible for AKTThr308 phosphorylation. Knockdown of LCP1 protein or reduction of its phosphorylation by enzastaurin treatment resulted in mTORC2 activity loss, while PDK1 activity remained unchanged, suggesting differential phosphorylation of AKTSer473 and AKTThr308. Importantly, AKT substrate activity was significantly reduced upon loss ofAKTSer473 phosphorylation, and SIN1T86 phosphorylation as an indicator of mTORC2 activity was dependent on LCP1 protein, suggesting that targeting of LCP1 reduces mTORC2 activity.Biologically, both enzastaurin treatment and LCP1 knockdown led to impaired IL5-induced eosinophil differentiation of HoxB8-immortalized murine bone marrow progenitor cells (10.1 fold decrease; p=0.021). Moreover, enzastaurin reduced eosinophil differentiation (1.38 fold decrease; p=0.005) and survival (1.6 fold decrease; p=0.0078) of primary peripheral blood-derived samples from HE patients in vitro.In conclusion, our data demonstrate that HE involves active LCP1, which triggers mTORC2 activity and eosinophil migration, and that the PKCβ inhibitor enzastaurin may provide a novel treatment approach for hypereosinophilic disorders. DisclosuresReiter:Incyte: Consultancy, Honoraria. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy.

The PKC-β selective inhibitor, Enzastaurin, impairs memory in middle-aged rats.

Enzastaurin is a Protein Kinase C-β selective inhibitor that was developed to treat cancers. Protein Kinase C-β is an important enzyme for a variety of neuronal functions; in particular, previous rodent studies have reported deficits in spatial and fear-conditioned learning and memory with lower levels of Protein Kinase C-β. Due to Enzastaurin’s mechanism of action, the present study investigated the consequences of Enzastaurin exposure on learning and memory in 12-month-old Fischer-344 male rats. Rats were treated daily with subcutaneous injections of either vehicle or Enzastaurin, and behaviorally tested using the spatial reference memory Morris Water Maze. Rats treated with Enzastaurin exhibited decreased overnight retention and poorer performance on the latter testing day, indicating a mild, but significant, memory impairment. There were no differences during the probe trial indicating that all animals were able to spatially localize the platform to the proper quadrant by the end of testing. RNA isolated from the hippocampus was analyzed using Next Generation Sequencing (Illumina). No statistically significant transcriptional differences were noted. Our findings suggest that acute Enzastaurin treatment can impair hippocampal-based learning and memory performance, with no effects on transcription in the hippocampus. We propose that care should be taken in future clinical trials that utilize Protein Kinase C-ß inhibitors, to monitor for possible cognitive effects, future research should examine if these effects are fully reversible.

Expression and activity of eIF6 trigger malignant pleural mesothelioma growth in vivo.

eIF6 is an antiassociation factor that regulates the availability of active 80S. Its activation is driven by the RACK1/PKCβ axis, in a mTORc1 independent manner. We previously described that eIF6 haploinsufficiency causes a striking survival in the Eμ-Myc mouse lymphoma model, with lifespans extended up to 18 months. Here we screen for eIF6 expression in human cancers. We show that Malignant Pleural Mesothelioma tumors (MPM) and a MPM cell line (REN cells) contain high levels of hyperphosphorylated eIF6. Enzastaurin is a PKC beta inhibitor used in clinical trials. We prove that Enzastaurin treatment decreases eIF6 phosphorylation rate, but not eIF6 protein stability. The growth of REN, in vivo, and metastasis are reduced by either Enzastaurin treatment or eIF6 shRNA. Molecular analysis reveals that eIF6 manipulation affects the metabolic status of malignant mesothelioma cells. Less glycolysis and less ATP content are evident in REN cells depleted for eIF6 or treated with Enzastaurin (Anti-Warburg effect). We propose that eIF6 is necessary for malignant mesothelioma growth, in vivo, and can be targeted by kinase inhibitors.

The serine‐threonine kinase p90RSK is a new target of enzastaurin in follicular lymphoma cells

Follicular lymphoma is the second most common non-Hodgkin's lymphoma and, despite the introduction of rituximab for its treatment, this disease is still considered incurable. Besides genetic alterations involving Bcl-2, Bcl-6 or c-Myc, follicular lymphoma cells often display altered B-cell receptor signalling pathways including overactive PKC and PI3K/Akt systems. The effect of enzastaurin, an inhibitor of PKC, was evaluated both in vitro on follicular lymphoma cell lines and in vivo on a xenograft murine model. Using pharmacological inhibitors and siRNA transfection, we determined the different signalling pathways after enzastaurin treatment. Enzastaurin inhibited the serine-threonine kinase p90RSK which has downstream effects on GSK3β. Bad and p70S6K. These signalling proteins control follicular lymphoma cell survival and apoptosis; which accounted for the inhibition by enzastaurin of cell survival and its induction of apoptosis of follicular lymphoma cell lines in vitro. Importantly, these results were replicated in vivo where enzastaurin inhibited the growth of follicular lymphoma xenografts in mice. The targeting of p90RSK by enzastaurin represents a new therapeutic option for the treatment of follicular lymphoma.

Protein Kinase Cβ Is Required for Lupus Development in Sle Mice

To evaluate the requirement for protein kinase Cβ (PKCβ) in the development of lupus in mice, and to explore the potential of targeting PKCβ as a therapeutic strategy in lupus. Congenic mice bearing the disease loci Sle1 or Sle1 and Sle3, which represent different stages of severity in the development of lupus, were crossed with PKCβ-deficient mice. The effect of PKCβ deficiency in lupus development was analyzed. In addition, the effects of the PKCβ-specific inhibitor enzastaurin on the survival of B cells from mice with lupus and human 9G4-positive B cells as well as the in vivo effect of enzastaurin treatment on the development of lupus in Sle mice were investigated. In Sle mice, PKCβ deficiency abrogated lupus-associated phenotypes, including high autoantibody levels, proteinuria, and histologic features of lupus nephritis. Significant decreases in spleen size and in the peritoneal B-1 cell population, reduced numbers of activated CD4 T cells, and normalized CD4:CD8 ratios were observed. PKCβ deficiency induced an anergic B cell phenotype and preferentially inhibited autoreactive plasma cells and autoantibodies in mice with lupus. Inhibition of PKCβ enhanced apoptosis of both B cells from Sle mice and human autoreactive B cells (9G4 positive). Treatment of Sle mice with the PKCβ-specific inhibitor enzastaurin prevented the development of lupus. This study identifies PKCβ as a central mediator of lupus pathogenesis, suggesting that PKCβ represents a promising therapeutic target for the treatment of systemic lupus erythematosus. Moreover, the results indicate the feasibility of using a PKCβ inhibitor for the treatment of lupus.

Enzastaurin has anti-tumour effects in lung cancers with overexpressed JAK pathway molecules

Background:Enzastaurin, an oral serine–threonine kinase inhibitor, was initially developed as an ATP-competitive selective inhibitor against protein kinase Cβ. However, the mechanism by which enzastaurin contributes to tumourigenesis remains unclear.Methods:We analysed the anti-tumour effects of enzastaurin in 22 lung cancer cell lines to ascertain the potential for enzastaurin-based treatment of lung cancer. To identify molecules or signalling pathways associated with this sensitivity, we conducted a gene, receptor tyrosine kinases phosphorylation and microRNA expression profiling study on the same set of cell lines.Results:We identified eight genes by pathway analysis of molecules having gene-drug sensitivity correlation, and used them to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. Pathway analysis revealed that the JAK/STAT signalling pathway was one of the main ones involved in sensitivity to enzastaurin. Overexpression of JAK1 was observed in the sensitive cells by western blotting. Simultaneous administration of enzastaurin and JAK inhibitor inhibited enzastaurin-induced cell growth-inhibitory effect. Furthermore, lentiviral-mediated JAK1-overexpressing cells were more sensitive to enzastaurin than control cells.Conclusion:Our results suggested that the JAK1 pathway may be used as a single predictive biomarker for enzastaurin treatment. The anti-tumour effect of enzastaurin should be evaluated in lung cancer with overexpressed JAK pathway molecules.

Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells.

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and β generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC β inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC β as another PKC β inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC β, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.

Role of protein kinase C β and vascular endothelial growth factor receptor in malignant pleural mesothelioma: Therapeutic implications and the usefulness of Caenorhabditis elegans model organism

Purpose:To examine the role of both protein kinase C (PKC)-β and vascular endothelial growth factor receptor (VEGFR)-2 in malignant pleural mesothelioma (MPM) using respective inhibitors, enzastaurin and KRN633.Materials and Methods:MPM cell lines, control cells, and a variety of archived MPM tumor samples were used to determine the protein expression levels of PKC-β, VEGFR-2, VEGF, and p-AKT. Effects of enzastaurin and KRN633 on phosphorylation status of key signaling molecules and viability of the mesothelioma cells were determined. The common soil nematode, Caenorhabditis elegans, was treated with enzastaurin to determine its suitability to screen for highly potent kinase inhibitors.Results:PKC-β1, PKC-β2 and VEGFR-2/KDR were overexpressed in MPM cell lines and MPM tumor tissues. Enzastaurin treatment resulted in significant loss in viability of VEGF induced cell proliferation; however, the effect of KRN633 was much less. Enzastaurin also dramatically decreased the phosphorylation of PKC-β, its downstream target p-AKT, and surprisingly, the upstream VEGFR-2. The combination of the two drugs at best was additive and similar results were obtained with respect to cell viability. Treatment of C. elegans with enzastaurin resulted in clear phenotypic changes and the worms were hypermotile with abnormal pattern and shape of eggs, suggesting altered fecundity.Conclusions:PKC-β1 and VEGFR-2 are both excellent therapeutic targets in MPM. Enzastaurin was better at killing MPM cells than KRN633 and the combination lacked synergy. In addition, we show here that C. elegans can be used to screen for the next generation inhibitors as treatment with enzastaurin resulted in clear phenotypic changes that could be assayed.

Modulation of 4E-BP1 Function as a Critical Determinant of Enzastaurin-Induced Apoptosis

Enzastaurin (LY317615.HCl) is currently in a phase III registration trial for diffuse large B-Cell lymphoma and numerous phase II clinical trials. Enzastaurin suppresses angiogenesis and induces apoptosis in multiple human tumor cell lines by inhibiting protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K)/AKT pathway signaling. PI3K/AKT pathway signaling liberates eukaryotic translation initiation factor 4E (eIF4E) through the hierarchical phosphorylation of eIF4E binding proteins (4E-BP). When hypophosphorylated, 4E-BPs associate with eIF4E, preventing eIF4E from binding eIF4G, blocking the formation of the eIF4F translation initiation complex. Herein, we show that enzastaurin treatment impacts signaling throughout the AKT/mTOR pathway leading to hypophosphorylation of 4E-BP1 in cancer cells of diverse lineages (glioblastoma, colon carcinoma, and B-cell lymphoma). Accordingly, enzastaurin treatment increases the amount of eIF4E bound to 4E-BP1 and decreases association of eIF4E with eIF4G, thereby reducing eIF4F translation initiation complex levels. We therefore chose to evaluate whether this effect on 4E-BP1 was involved in enzastaurin-induced apoptosis. Remarkably, enzastaurin-induced apoptosis was blocked in cancer cells depleted of 4E-BP1 by siRNAs, or in 4EBP1/2 knockout murine embryonic fibroblasts cells. Furthermore, eIF4E expression was increased and 4E-BP1 expression was decreased in cancer cells selected for reduced sensitivity to enzastaurin-induced apoptosis. These data highlight the importance of modulating 4E-BP1 function, and eIF4F complex levels, in the direct antitumor effect of enzastaurin and suggest that 4E-BP1 function may serve as a promising determinant of enzastaurin activity.

Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

AbstractAbstract 2893 Background:The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis. Methods:Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment. Results:Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling’, (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis’ (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling’ (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer’ (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation’ (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death’ (BCL6, EEF1D, PAK6, RAD50), 'cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), 'cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression’ (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways. Conclusion:In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL. Disclosures:Dreyling:Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.

RNA-Expression and Proteome Analysis Identify Complementary but Not Identical Molecular Targets in Enzastaurin-Treated Mantle Cell Lymphoma.

Abstract 1915Poster Board I-938 Introduction:Protein kinase C beta (PKCbeta), a pivotal enzyme in B-cell signaling and survival, is over-expressed in most cases of mantle cell lymphoma (MCL) and results in activation of PI3K/AKT pathway. Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through PKCbeta/PI3K/AKT pathways, induces apoptosis, reduces proliferation, and suppresses tumor-induced angiogenesis. Aim: To optimize the treatment options with this promising inhibitor the goal of this study was to elucidate the molecular pathways altered by Enzastaurin treatment in MCL. Methods:Four documented MCL cell lines (Granta 519, HBL-2, Jeko-1, Rec-1) were harvested after 2-8h Enzastaurin exposure at a previously defined dose of 10μM and analyzed by RNA-array and proteome analysis as previously described (2D-polyacrylamide-gel-electrophoresis (2D-PAGE); Weinkauf 2009). Regulated molecules were mapped in a functional interaction network and candidates representing different pathways were verified by Western blotting. Results:Enzastaurin exposure led to significant reduction of cell viability in all cell lines (15-20%). This was also reflected in distinct alterations of the observed protein patterns in 2D-PAGE after enzastaurin exposure. Of a total of 977 concurrent protein spots 115 (12%) spots exhibited significantly (>3fold) altered protein levels after 4h of enzastaurin exposure. Mass spectrometry of 62 protein spots (39 increase; 23 decreased) identified 108 different candidate proteins, which were used to create a protein interaction network identifying the affected functional pathways. The results of the 2D-PAGE analysis were verified by Western blot in selected candidate proteins of apoptosis (VIM, PLEC1), DNA-repair (RAD50, PCNA, RFC1) and gene expression (EEF1D, SMC1A) pathways.In parallel, RNA-expression array analysis identified 180 different genes regulated early after enzastaurin treatment. Again these genes were mapped to an interaction network, highlighting enzastaurin involvement in e.g. NFkB-, apoptosis and B-cell- and death receptor signaling pathways. Interestingly, the involved genes complemented the regulated proteins in the functional pathways. Network analysis of both screenings (2D-PAGE-based proteomics and RNA-expression array) classified the candidate molecules in functional groups, including DNA repair and replication (e.g. RAD50, PCNA), apoptosis (e.g.PSMC4, VIM), signal transduction (e.g. GRB2, EF1D) and gene expression/mRNA processing (e.g.EEF1D, SFRS7). Thus, combined analysis of both screening methods resulted in a more comprehensive network than each respective analysis. Conclusion:Enzastaurin-treatment affects three main cellular control mechanisms as highlighted by two independent screening approaches (proteomics and expression array analysis). Interestingly, these two layers of molecular response (protein and RNA respectively) resulted in minimal overlap of identified molecules at this early (4h) time, but indicated common pathways nonetheless. Ongoing experiments now incorporate this knowledge to select optimal combination partners of enzastaurin. Disclosures: Dreyling:Lilly Deutschland GmbH: Research Funding.

Protein Kinase C Modulates Bcl-2 Family Protein Levels in Cutaneous T Cell Lymphoma.

Abstract 2755Poster Board II-731Cutaneous T cell lymphomas (CTCL) represent a spectrum of several distinct extranodal non-Hodgkin's lymphomas and are characterized by an invasion of the skin by malignant, clonal CD4+ lymphocytes. Although current treatments may achieve remission, early chemotherapeutic treatment of CTCL has not demonstrated survival benefits and no treatments have proven to be curative. Our lab has previously demonstrated that treatment with the Protein Kinase C (PKC) inhibitor Enzastaurin increases apoptosis in CTCL which directly led to the clinical trial of Enzastaurin for CTCL. PKCs phosphorylate a number of substrates important in modulating cell survival, including Bcl-2 family members. To investigate the effects of PKC signaling on Bcl-2 family member levels in CTCL we treated cells with Enzastaurin and measured downstream effects. Inhibition of PKC signaling by Enzastaurin led to a decrease of Bcl-xL protein levels but not Bcl-2 levels. Apoptosis was assessed by flow cytometry and cells cultured in Enzastaurin showed an increase in cell death which correlated with the decrease in anti-apoptotic Bcl-2 family member levels. Enzastaurin treatment also resulted in a decrease of both Bad and Bcl-2 phosphorylation as well as a decrease in Bcl-xL but not Mcl-1 or Bcl-2 mRNA levels. To determine if PKC modulates Mcl-1 levels through phosphorylation and inactivation of GSK3β in CTCL, cells were exposed to Enzastaurin for 24 hours and immunoblots were performed for Mcl-1, total GSK3β and phosphor-Ser9 GSK3β. Enzastaurin blocked basal phosphorylation as well as TPA-induced phosphorylation of GSK3β. However, Mcl-1 levels were not affected by Enzastaurin. This suggests that PKC modulates GSK3β activity but that this is independent of Mcl-1. To determine if PKC negative regulation of GSK3β inhibits apoptosis we treated cells with Enzastaurin and the GSK3β inhibitor ARA014418. Surprisingly, the addition of the GSK3β inhibitor did not reverse the effects of Enzastaurin but instead increased apoptosis. This suggests that PKC does not deliver survival signals in CTCL by negatively regulating GSK3β. Treatment with Enzastaurin alone as well as treatment with both Enzastaurin and GSK3β inhibitors resulted in an upregulation of β catenin. However, a higher percentage of β catenin was unphosphorylated when both Enzastaurin and the GSK3β inhibitor were added. Interestingly, the combination of both inhibitors resulted in a dramatic increase in β catenin / TCF transcription as measured by TOPGLOW luciferase assay.The downregulation of Bcl-2 family member levels by PKC inhibition strongly suggests that PKC modulation of Bcl-2 family members is important for CTCL survival. PKC may be affecting Bcl-2 family members by modulating phosphorylation or by indirectly activating or repressing transcription. Additionally, β catenin upregulation may play a role in apoptosis mediated by inhibition of PKC. β catenin TCF/LEF complexes can activate transcription of several genes linked to apoptosis, including c-Jun, fra-1, p53 and Myc. β catenin may also be functioning to repress transcription of Bcl-2 family members such as Bcl-xL. Elucidating the underlying mechanism will help to identify new drug targets for CTCL which may be applicable to a broad spectrum of malignancies. Disclosures:Rosen:Eli Lilly: Consultancy.

Synergistic Antilymphoma Effect of Protein Kinase C Beta (PKCβ) and mTOR Inhibition in Mantle Cell Lymphoma.

Abstract 4780 IntroductionThe protein kinase C (PKC) family of enzymes are serine/threonine kinases essential to the cell signal cascades effecting cellular growth, proliferation and apoptosis. Accordingly PKCβ overexpression correlates with poor clinical prognosis in diffuse large cell lymphoma. The pivotal role of PKCb in neoplastic transformation renders it a potential therapeutic target in the therapy of hematologic malignancies. AimTo determine drugs which are efficiently inhibiting cell proliferation in combination with enzastaurin in MCL. MethodsFive MCL cell lines (HBL-2, GRANTA 519, Jeko-1, Z138, Rec-1) and patient samples were cultured in the presence of LY317615 (PKCb inhibitor), rapamycin (mTOR inhibitor) and LY294002 (PI3K inhibitor). Cell proliferation and viability was assessed by cell count and WST-1 proliferation assay. Analysis of cell cycle profile and apoptosis was performed by flow cytometry (PI and Annexin V FITC staining). mRNA expression was measured before and after treatment (8h) by microarray and real time PCR in cell lines. Protein expression was analysed by Western blot. ResultsIn a panel of mantle cell lymphoma cell lines, with IC50 values ranging from 2 to 5 microM for enzastaurin treatment, a refractory to enzastaurin cell line (Rec-1) was characterized. Treatment of the cell lines with enzastaurin induced apoptosis and lead to accumulation of cells in the G2, M phase in susceptible cell lines (Hbl-2, Jeko-1), whereas cell cycle profile remained unaltered in the refractory cell line (Rec-1).While enzastaurin induced increased phosphorylation of mTOR and MEK and decrease of p90RSK phosphorylation in all MCL cell lines, mTOR phosphorylation was twice as high in the refractory cell line (Rec-1). In line with this observation the combination of enzastaurin with rapamycin lead to a synergistic effect on the inhibition of cell proliferation in the Rec-1 cell line as well as in an additional MCLpatient sample. Protein expression levels (low CCND1, phAkt, php90RSK, phPDK) achieved in Rec-1 after treatment with enzastaurin were also characteristic for the cell lines more sensitive to rapamycin. In contrast in some cell lines combination of enzastaurin and the PI3K inhibitor (LY294002) displayed antagonism. Further mRNAand proteinexpression analysis of patient samples are ongoing to determine molecular predictors of drug sensitivity. ConclusionIn our study a combination of rapamycin and enzastaurin acted synergistically in MCL cell lines and a patient samples whereas the combination with a PI3K inhibitor displayed partial antagonism. Based on this results we have identified the underlying signal pathways to develop new synergistic molecular combinations in MCL. Disclosures:Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Rieken:Lilly Deutschland GmbH: Research Funding. Weinkauf:Lilly Deutschland GmbH: Research Funding. Dreyling:Lilly Deutschland GmbH: Research Funding.

Enzastaurin in Previously Treated Waldenstrom's Macroglobulinemia: An Open-Label, Multicenter, Phase II Study.

Abstract 3867Poster Board III-803 BackgroundEnzastaurin is an oral serine/threonine kinase inhibitor that targets the PKC and PI3K/AKT pathways. Enzastaurin has demonstrated activity in preclinical models of multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM), and clinical studies suggest encouraging activity and a well-tolerated safety profile in a variety of hematologic cancers. We are conducting a multicenter, two (parallel) cohort, two-stage, phase II trial to determine whether further study of single-agent enzastaurin is warranted in patients with previously treated WM or MM. The primary objective is to assess the response rate (RR); secondary objectives include assessment of time to progression (TTP), safety, biomarkers, and the impact of adding dexamethasone to enzastaurin in patients with progressive disease (PD). Preliminary results for the WM cohort are reported here. MethodsEligible patients with WM and 1-5 prior therapies were enrolled and treated with 250 mg oral enzastaurin twice daily (1125-mg loading dose on day 1) in 28-day cycles. Patients continued for 8 cycles or until PD or unacceptable toxicity occurred. At the investigator's discretion, dexamethasone (20-40 mg po QD, days 1-4, 9-12, and 17-20 for 4 cycles; days 1-4 of each cycle thereafter) was added to enzastaurin in patients with PD. According to the Simon two-stage design, if 2 of the first 10 patients (in stage 1) experienced a minor response (MR) or better, then the study would be expanded to 29 patients (stage 2). Best response was determined according to the response assessment recommendations of the Third International Workshop on WM (IWWM). Adverse events were graded according to CTCAE version 3.0. ResultsTwenty-nine patients (7 females, 22 males) with WM were enrolled. The median age was 65.6 years (range: 51.7-82.3 years) and 93% of patients had an Eastern Cooperative Oncology Group performance status of 0. Patients had a median of 2 prior systemic therapies and 26 patients (89.7%) had prior rituximab. Patients completed a median of 4 cycles. Six patients received ≥6 cycles of enzastaurin treatment. Twenty patients remain on study. There were no drug-related discontinuations. None of the patients had a complete response (CR). One patient had a partial response (PR) and 7 patients had a minor response (MR), for a RR (CR+PR+MR) of 27.6%. Immunoglobulin M decreased by ≥25% in 11 patients. Three (10.3%) patients had a PD. One patient had a drug-related grade 3 wound complication; there were no other drug-related grade ≥3 toxicities. One patient died on study due to infection unrelated to enzastaurin. ConclusionsAlthough the results are preliminary, enzastaurin appears to have activity and is well tolerated in patients with previously treated WM. The WM cohort was expanded to allow up to 50 patients to be treated on study. Disclosures:Ghobrial:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Research Funding, Speakers Bureau. Lin:Eli Lilly and Company: Employment. Yuan:Eli Lilly and Company: Employment. Benhadji:Eli Lilly and Company: Employment. Wooldridge:Eli Lilly and Company: Employment. Leblond:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

Enzastaurin induces H2AX phosphorylation to regulate apoptosis via MAPK signalling in malignant glioma cells

Enzastaurin is an acyclic bisindolylmaleimide derived from staurosporine that acts as an ATP competitor, and interferes with the activity of protein kinase C (PKC) isoforms. Our previous studies have shown that clinically achievable concentrations of this agent induce apoptosis in many glioma cell lines. Our goal in this study was to expand on the previous results and to determine the signalling mechanisms responsible for enzastaurin-induced inhibition of cell growth and induction of apoptosis. To address these issues, cell cycle progression following enzastaurin treatment was analysed by fluorescence-activated cell sorting (FACS) in parallel with analyses of growth and apoptosis signalling pathways. Enzastaurin treatment activated H2AX and Chk2 phosphorylation, and enhanced phosphorylation of mitogen-activated protein kinase (MAPK) family kinases. Inhibition of MAP kinases by chemical inhibitors reduced H2AX and Chk2 phosphorylation and decreased apoptosis induced by enzastaurin. These data call attention to a novel signalling pathway (MAPK/H2AX) to regulate apoptosis in malignant glioma cells.

Combination of Enzastaurin and Pemetrexed Inhibits Cell Growth and Induces Apoptosis of Chemoresistant Ovarian Cancer Cells Regulating Extracellular Signal-Regulated Kinase 1/2 Phosphorylation

New strategies in the therapy for malignant diseases depend on a targeted influence on signal transduction pathways that regulate proliferation, cell growth, differentiation, and apoptosis by the activation of serine/threonine kinases. Enzastaurin (LY317615.HCl), a selective inhibitor of protein kinase Cbeta (PKCbeta), is one of these new drugs and causes inhibition of proliferation and induction of apoptosis. Pemetrexed, a multitarget inhibitor of folate pathways, is broadly active in a wide variety of solid tumors. Therefore, the effect of enzastaurin and the combination treatment with pemetrexed was analyzed when applied to the drug-sensitive ovarian cancer cell line HEY and various subclones with drug resistance against cisplatin, etoposide, docetaxel, and paclitaxel, as well as pemetrexed, and gemcitabine. In these novel chemoresistant subclones, the expression of the enzastaurin targets PKCbetaII and glycogen synthase kinase 3beta (GSK3beta) was analyzed. Exposition to enzastaurin showed various inhibitory effects on phosphorylated forms of GSK3beta and the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. Cell proliferation experiments identified the cell line-specific half-maximal inhibitory concentration values of enzastaurin and a synergistic inhibitory effect by cotreatment with the antifolate pemetrexed. Induction of apoptosis by enzastaurin treatment was investigated by Cell Death Detection ELISA and immunoblot analyses. Simultaneous treatment with pemetrexed resulted in an enhanced inhibition of proliferation and induction of apoptosis even in partial enzastaurin-resistant cells. Therefore, the combinational effect of enzastaurin and pemetrexed can have promise in clinical application to overcome the fast-growing development of resistance to chemotherapy in ovarian cancer.