Mantle Cell Lymphoma Cell Death Research Articles

Combined Inhibition of Protein Kinase CSNK2 and BET Proteins As a Novel Therapeutic Strategy for Mantle Cell Lymphoma

Currently available therapeutic approaches for Mantle Cell Lymphoma (MCL), including the Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib, acalabrutinib and zanubrutinib, are not curative. Therefore, the discovery of novel molecules amenable of drug targeting is a research priority in the MCL field. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain (B) and extra terminal (ET) (BET) protein family that regulate chromatin modifications and assembly of transcriptional complexes. Inhibition of BET by available compounds have shown promising anticancer effect. In MCL, BET inhibitors, such as INCB054329 or JQ-1, have been shown to increase apoptosis through downregulation of the AKT-mTOR, ERK, and other B Cell Receptor (BCR)-triggered cascades. We and others have previously demonstrated that the S/T kinase CSNK2 supports chronic BCR activation in MCL by upregulating the AKT/PI3K and NF-κB pathways. Inactivation of CSNK2 results in efficacious induction of MCL cell death. Notably, BRD4 activity is regulated by CSNK2 and double inhibition of BRD4 and CSNK2 has shown significant activity in other tumor models. The most effective and tested CSNK2 chemical inhibitor is CX4945 (silmitasertib), but very recently a novel compound, SGC-CK2, has been developed. No data are available on the effects of SGC-CK2 on MCL cells. In this work, we have evaluated the effects of the combined inhibition of CSNK2 and BET proteins on MCL cell growth and survival signaling pathways. CSNK2 inhibitors employed were CX4945 and SGC-CK2. CSNK2 gene silencing was achieved with the generation of anti- CSNK2 short hairpin-RNA IPTG-inducible MCL cell clones. BET inhibitors used were JQ-1 and INCB054329. Cell survival, apoptosis and proliferation were investigated by AnnexinV/PI staining and FACS analysis, by Western blot (WB) detection of PARP cleavage and Mcl1 expression and by other cell proliferation assays. The synergic action of BET and CK2 inhibitors was assessed by the Chou-Talalay combination index method. CK2 expression and survival-related signaling components were analyzed by WB, qRT-PCR and protein expression assays. CX4945 and the novel inhibitor SGC-CK2 displayed a potent anti-tumor activity on MCL cells as judged by the effective induction of apoptosis and proliferation arrest. A synergistic effect of CSNK2 and BET inhibitors was observed in all the MCL cell lines tested, (Jeko-1, Rec-1, including Granta-519 that are known to be much less sensitive to ibrutinib) and with all the inhibitors tested. The same results were obtained in the CSNK2 gene silencing models. Preliminary data showed that the cooperative cytotoxic effect of CK2 inhibition and BET inhibition on MCL apoptosis was observed also in MCL patient derived B cells, but not in healthy B lymphocytes form buffy coat. CSNK2 inactivation was associated with strong down-regulation of c-Myc and Mcl-1 proteins and with hampered activation of NF-κB and PI3K/AKT-dependent signaling. Surprisingly, BET protein inhibition caused increased Mcl-1 protein expression and augmented Ser 529 p65-NF-kB phosphorylation in Granta-519 and Rec-1 cells. Remarkably, CK2 inactivation led to a robust reduction of the BET inhibitor-induced increase of Mcl1, and NF-kB Ser 529 phosphorylation, thus counteracting BET-inhibitors-evoked compensatory pathways that could favor apoptosis resistance. Therefore, combined CSNK2 and BET proteins inhibition could represent an innovative strategy for chemotherapy and BTKi-resistant MCL.

Dihydroorotate dehydrogenase inhibition acts synergistically with tyrosine kinase inhibitors to induce apoptosis of mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is a non‐Hodgkin lymphoma that remains incurable with the treatment options available today. In the present study, we have identified the dihydroorotate dehydrogenase (DHODH), an essential enzyme for the de novo biosynthesis of pyrimidine‐based nucleotides, to be overexpressed in MCL in comparison to healthy peripheral blood mononuclear cells (PBMC). In vitro inhibition of the DHODH activity using a newly developed DHODH inhibitor, namely (R)‐HZ05, can induce MCL cell death in the nanomolar range independently than the P53 status of the investigated cell lines. Moreover, the combination of (R)‐HZ05 with tyrosine kinase inhibitor shows the synergistic activity on cell death. Pre‐clinical investigation on the efficacy of (R)‐HZ05 shows that it can be prolonged animal lifespan similar to ibrutinib. (R)‐HZ05 use in combination with tyrosine kinase inhibitor demonstrated a superior efficacy on tumor burden reduction and survival than either drug alone. We have demonstrated that the depletion of the pyrimidine nucleotide pool, using DHODH inhibitor, represents a new therapeutic strategy that may benefit MCL patients.

Preclinical Anti-Lymphoma Activity of the BCL-2 BH4 Domain Antagonist Bda-366 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell non-Hodgkin lymphoma. MCL is genetically characterized by t(11;14) translocation, which leads to cyclin D1 overexpression. Despite the introduction of high-dose chemotherapy followed by autologous stem cell transplantation and novel agents including the Bruton's tyrosine kinase inhibitor ibrutinib, the proteasome inhibitor bortezomib and the immunomodulator lenalidomide, MCL still remains an incurable disease. BCL-2 is often amplified and/or overexpressed in MCL; high-level copy number gains of the BCL2 locus at 18q21.3 are most frequent. The novel BCL-2BH4 domain antagonist BDA-366 converts anti-apoptotic BCL-2 into the pro-apoptotic molecule (Han et al., Cancer Cell 2015). Considering the promising clinical activity of the BH3 mimetics venetoclax (Davids et al., JCO 2017), we investigated anti-lymphoma effects of BDA-366 in MCL. A total of eight MCL cell lines (Z-138, JVM-2, Granta-519, MINO, JeKo-1, REC-1, MAVER-1 and NCEB-1) were used. Z-138, Granta-519, MINO and NCEB-1 have high-level DNA amplification of BCL-2 . BDA-366 induced dose- and time-dependent phosphatidylserine externalization in MCL cells, with 72-h ED50 (effective concentration inducing 50% cell killing as measured by annexin V positivity) values ranging from 9.3 to 240 nM. 72-h IC50 values (concentration at which cell growth is inhibited by 50%) were also determined, which ranged from 5 to 30 nM. BDA-366 showed smaller differences in sensitivity than venetoclax, raising the possibility that BDA-366 may exhibit broader anti-lymphoma activity against heterogeneous MCL backgrounds. Basal levels of BCL-2 (determined by flow cytometry) did not significantly affect MCL cell susceptibility (ED50) to BDA-366 (r = -0.08, P = 0.85). The presence of high-level DNA amplification of BCL-2 was not associated with BDA-366 sensitivity either. BDA-366 did not increase total BCL-2 levels but it actively promoted the exposure of pro-apoptotic BH3 domain of BCL-2, supporting its proposed mode of action. BDA-366 did not significantly affect cell cycle distribution; S and G2/M phase cells appeared to be susceptible to BDA-366-induced apoptosis. BDA-366 induced loss of mitochondrial membrane potential, caspase-3 cleavage and BAX activation, indicating mitochondrial apoptosis. p53 knockdown (>85% knockdown efficiencies, as determined by Western blotting) desensitized p53 wild-type cell lines Z-138 and JVM-2 to BDA-366 (ED50 values of 29 nM and 10 nM in Z-138; 303 nM and 108 nM in JVM-2), implying the interference of p53 with BCL-2 signaling. BDA-366 synergized with venetoclax to induce apoptosis in venetoclax-sensitive Z-138 (averaged combination index (aCI) = 0.25) and MINO cells (aCI = 0.14), indicating that the combination may abrogate BCL-2-mediated survival signaling in MCL. The combination effects of BDA-366 with doxorubicin or AraC were additive (aCI ~ 1.0). BDA-366 induced apoptosis in primary leukemia cells from patients with B-cell chronic lymphoproliferative disorders. BDA-366 appeared to take longer time to fully induce apoptosis than ABT-199, perhaps reflecting the distinct mode of action of BDA-366 that changes BCL-2 conformation. From a clinical viewpoint, the slower pharmacodynamics in inhibiting BCL-2 and inducing MCL cell death may reduce the risk of tumor lysis syndromes in patients receiving BDA-366. Collectively, BCL-2 BH4 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresNo relevant conflicts of interest to declare.

BAFF receptor antibody for mantle cell lymphoma therapy

ABSTRACT Mantle cell lymphoma (MCL) is an aggressive form of B cell non-Hodgkin’s lymphoma and remains incurable under current treatment modalities. One of the main reasons for treatment failure is the development of drug resistance. Accumulating evidence suggests that B cell activating factor (BAFF) and BAFF receptor (BAFF-R) play an important role in the proliferation and survival of malignant B cells. High serum BAFF levels are often correlated with poor drug response and relapse in MCL patients. Our study shows that BAFF-R is expressed on both MCL patient cells and cell lines. BAFF-R knockdown leads to MCL cell death showing the importance of BAFF-R signaling in MCL survival. Moderate knockdown of BAFF-R in MCL cells did not affect its viability, but sensitized them to cytarabine treatment in vitro and in vivo, with prolonged mice survival. Anti-BAFF-R antibody treatment promoted drug-induced MCL cell death. Conversely, the addition of recombinant BAFF (rhBAFF) to MCL cells protected them from cytarabine-induced apoptosis. We tested the efficacy of a humanized defucosylated ADCC optimized anti-BAFF-R antibody in killing MCL. Our data show both in vitro and in vivo efficacy of this antibody for MCL therapy. To conclude, our data indicate that BAFF/BAFF-R signaling is crucial for survival and involved in drug resistance of MCL. Targeting BAFF-R using BAFF-R antibody might be a promising therapeutical strategy to treat MCL patients resistant to chemotherapy.

PRMT5 Inhibition Drives Therapeutic Vulnerability to BCL-2 Inhibition with Venetoclax and Provides Rationale for Combination Therapy in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50<10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50>1uM). All lines reached an IC50 <1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores > 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Ibrutinib inhibits antibody dependent cellular cytotoxicity induced by rituximab or obinutuzumab in MCL cell lines, not overcome by addition of lenalidomide

BackgroundThe Bruton’s Tyrosine Kinase (BTK)-inhibitor ibrutinib is highly active in mantle cell lymphoma (MCL) but may inhibit response to anti-CD20 antibody as previously shown in CLL models. We investigated how antibody-dependent cellular cytotoxicity (ADCC) induced by type I/II anti-CD20 antibodies was affected by treatment with ibrutinib in MCL. Furthermore, we investigated if lenalidomide, a potential sensitizer to anti-CD20 treatment, could prevent an inhibitory effect of ibrutinib.MethodsAnti-CD20 (rituximab/obinutuzumab) opsonized MCL cell lines were co-cultured with ibrutinib (± lenalidomide)—exposed effector cells, and analyzed for evaluation of cell death.ResultsCell death induced by rituximab was reduced with 75% at 0.5 µM ibrutinib and with 52% at 0.1 µM ibrutinib when induced by obinutuzumab, even by addition of lenalidomide. Moreover, obinutuzumab was associated with higher rate of cell death compared to rituximab.ConclusionIbrutinib negatively affects anti-CD20 induced cell death in MCL, not reversed by lenalidomide. Explorations of sequential administration and selective BTK-inhibitors may reveal the optimal combination of novel agents in MCL.

Cotargeting of BCL2 with Venetoclax and MCL1 with S63845 Is Synthetically Lethal In Vivo in Relapsed Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

The Pathophysiological Significance and Therapeutic Targeting of PPM1D-Mediated Signaling in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin lymphoma genetically characterized by balanced t(11:14) translocation and cyclin D1 overexpression. A great majority of patients with MCL remain incurable. The selection of chemoresistant subclones occurs during therapies, leading to relapse of MCL. Many chemotherapeutic agents induce DNA damage and activate the tumor suppressor p53. Recently, non-genotoxic p53 activators like MDM2 (master regulator of p53) inhibitors and XPO1 (nuclear exporter for p53) inhibitors have entered clinical trials. p53 stabilization, however, has not always led to p53-mediated transcriptional activation in MCL. PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. PPM1D has been thought to be an oncoprotein that inhibits p53. PPM1D overexpression or amplification has been reported in various cancers, including lung, breast, kidney and ovarian cancers. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 (GSK) (Nat Chem Biol 2014) in MCL.RESULTS Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart B-lymphocytes. PPM1D mRNA levels positively correlated with CCND1 (Cyclin D1) mRNA levels (r = 0.33, P = 0.0014; n = 92) and with proliferation signature averages (r = 0.54, P < 0.0001; n = 92) in a series of MCL samples. Increased PPM1D expression at diagnosis was itself associated with a poorer prognosis in MCL patients (median overall survival of 3.9 years and 1.4 years for cases in the lowest and highest PPM1D expression tertiles, respectively; P = 0.0047). PPM1D levels in MCL were as high as those in aggressive lymphomas including Burkitt’s lymphoma and diffuse large B-cell lymphoma, and were significantly higher than those in indolent lymphomas including chronic lymphocytic leukemia/small lymphocytic lymphoma (P = 0.0076) and follicular lymphoma (P = 0.011). Eight MCL (three p53 wild-type (WT) and five mutant (MUT)) cell lines were exposed to GSK. GSK inhibited the cell growth, being more prominent in p53 WT cells (48.3 ± 9.8% versus 14.8 ± 4.7% growth inhibition, P = 0.036). In sensitive cells, GSK caused a significant loss of mitochondrial membrane potential in addition to increased annexin V positivity, indicating apoptosis induction. Stable p53-specific shRNA-expressing cells were generated in p53 WT Z-138 and JVM-2 cells, with > 85% knockdown efficiency. p53 knockdown cells were less susceptible to GSK than control cells, indicating that GSK utilizes p53-mediated signaling to eradicate MCL cells. This idea is supported by the Western blot data that GSK treatment increased total and phosphorylated p53 levels and those of p53 targets p21 and PUMA. Basal and GSK-induced levels of PPM1D and its target proteins NFkB-p65, p38 MAPK, and histone H2A.X did not predict MCL cell sensitivity to GSK. GSK and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 WT MCL cells. Importantly, GSK sensitized MCL cells to bortezomib (BTZ) and doxorubicin (DOX) irrespective of p53 mutational status. BTZ and DOX are clinically active agents against MCL. In p53 WT cells, GSK/BTZ and GSK/DOX combinations potently activated p53-mediated apoptosis signaling. In p53 MUT cells, combination treatment did not activate p53 signaling. Instead, p38 signaling appeared to be actively involved in the GSK/BTZ lethality, as the selective p38 inhibitor SB203580 significantly attenuated bortezomib- and GSK/bortezomib-induced lethality. Activation of p38 MAPK has been found to cause MCL cell death. SB203580 did not protect MCL cell from doxorubicin- or GSK/doxorubicin-induced lethality.CONCLUSION PPM1D inhibition increases levels of phosphorylated p53 at Ser15 in a non-genotoxic manner, which enhances p53-mediated transcription in MCL cells. In addition, PPM1D inhibition shows anti-lymphoma effects in p53 MUT cells, partially through activation of p38 signaling. Although a single-agent activity of GSK was modest against MCL cells, especially against those with mutant p53, its p53-independent potentiation effect on BTZ and DOX may support the use of PPM1D inhibitors as part of a combination therapeutic strategy for MCL. Collectively, PPM1D inhibition may offer a novel therapeutic strategy for MCL. DisclosuresNo relevant conflicts of interest to declare.

Hedgehog inhibitors selectively target cell migration and adhesion of mantle cell lymphoma in bone marrow microenvironment.

The clinical benefits of a Hedgehog (Hh) inhibitor, LDE225 (NPV-LDE-225, Erismodegib), have been unclear in hematological cancers. Here, we report that LDE225 selectively inhibited migration and adhesion of mantle cell lymphoma (MCL) to bone marrows via very late antigen-4 (VLA-4) mediated inactivation of focal adhesion kinase (FAK) signaling. LDE225 treatment not only affected MCL cells, but also modulated stromal cells within the bone marrow microenvironment by decreasing their production of SDF-1, IL-6 and VCAM-1, the ligand for VLA-4. Surprisingly, LDE225 treatment alone did not suppress cell proliferation due to increased CXCR4 expression mediated by reactive oxygen species (ROS). The increased ROS/CXCR4 further stimulated autophagy formation. The combination of LDE225 with the autophagy inhibitors further enhanced MCL cell death. Our data, for the first time, revealed LDE225 selectively targets MCL cells migration and adhesion to bone marrows. The ineffectiveness of LDE225 in MCL is due to autophagy formation, which in turn increases cell viability. Inhibiting autophagy will be an effective adjuvant therapy for LDE225 in MCL, especially for advanced MCL patients with bone marrow involvement.

Identification of USP14 and UCHL5 As Druggable Oncotargets in Ibrutinib-Resistant Mantle Cell Lymphoma

Background: The clinical impact of ibrutinib is irrefutable and has been hailed as a revolutionary treatment for several cancers, including mantle cell lymphoma (MCL). Despite high response rates noted with ibrutinib therapy in MCL (ORR, 68%), complete responses are low and durability of remission is suboptimal in MCL vs. CLL or Waldenstrom macroglobulinemia (WM) patients (Cheah et al Ann Oncol 2015). This relative lack of efficacy may be due to MCL cells having a higher proliferation rate and being less indolent in nature than CLL or WM cells. Tumor cells with a high proliferation index rely on optimally functioning protein metabolism/homeostasis systems. In MCL, this observation has been fortuitously exploited with the use of bortezomib, a 20S proteasome inhibitor. Indeed the ubiquitin proteasome system is a validated target and consists of several individual components whose function in MCL has yet to be mapped out. We have recently reported that inhibition of the 19S proteasomal deubiquitinating enzymes (DUBs), USP14 and UCHL5; with the small molecule inhibitor b-AP15 is lethal to drug-resistant WM tumor cells. This intriguing observation, prompted us to define the role of USP14/UCHL5 and their inhibition in a diverse panel of B-cell lymphoma cell lines, including MCL models as well as ibrutinib-resistant MCL cells.Materials: b-AP15 (and its clinical grade analog, VLX1570) was obtained from Vivolux AB and bortezomib from Sellekchem. Human NHL (DLBCL, MCL, Burkitt), Hodgkin lymphoma (HL), multiple myeloma (MM) cell lines as well as established drug-resistant subclones were used in drug-sensitivity screening experiments (total n=25).Results: USP14 and UCHL5 mRNA expression was queried in the CCLE database (Broad-Novartis) and showed highest expression in plasma cell and B-lymphoid tumor cell types. This was also observed at the protein level, wherein analysis of The Human Protein Atlas database revealed moderate to high intensity staining for USP14 and UCHL5 localizing to the cytoplasmic/membranous and nuclear compartments in high-grade NHL tissues. Next, we examined B-lymphoid tumor cell sensitivity to pharmacologic inhibition of USP14/UCHL5 with either b-AP15 or VLX1570 (72hr fluorescein diacetate hydrolysis assay or CellTiter Glo). We observed that MCL cells, including those resistant to ibrutinib (Jeko/IR cell line) were highly sensitive to VLX1570 (median IC50 12 - 25nM). In contrast, MM cells were not as sensitive (IC50's ranging between 58 - 90nM). Nine DLBCL cell lines exposed to equivalent time/concentrations of b-AP15 showed a median IC50 of 311nM (range, 70 - 910nM) and 3 HL models exhibited an IC50 of 531nM (range, 420 - 783nM). No difference in sensitivity to VLX1570 was noted between ABC-type vs. GCB-type DLBCL models. These functional results aligned with prior genomic inquiry where USP14 and UCHL5 gene expression was noted to be higher in MCL cell lines compared to DLBCL and non-hematologic cancer models analyzed (FDR=0.0019, p=0.001), suggesting their more central role in maintaining MCL tumor viability. Indeed MCL tumor survival (particularly ibrutinib-resistant cells) appears linked to USP14/UCHL5 function as treatment of Jeko/IR cells with VLX1570 (250nM, 12hrs) caused marked apoptosis (71.8% annexin-V+ staining) and cleavage of PARP-1. Moreover, the combination of ibrutinib and VLX1570 synergistically induced MCL cell death (median CI: 0.3). Mechanistic studies demonstrated that VLX1570 modulates several proteins critical for MCL survival including cyclin-D1, Myc and CXCR4/CD184. Reduction in cyclin-D1 was also noted to coincide with cell cycle arrest of VLX1570-treated MCL cells. Altogether, these findings suggest that VLX1570 modulates critical cellular signaling pathways utilized by MCL tumor cells, particularly those that are involved in resistance to ibrutinib.Conclusions: MCL remains a challenging malignancy to treat with no effective treatments for ibrutinib-relapsed disease. Our data supports the role of the proteasome-associated DUBs, USP14 and UCHL5, as critical factors involved in ibrutinib-resistant MCL tumor survival. VLX1570 is a potentially effective agent for the treatment of ibrutinib-resistant MCL. DisclosuresGullbo:Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

The synergistic effect of BCR signaling inhibitors combined with an HDAC inhibitor on cell death in a mantle cell lymphoma cell line.

Mantle cell lymphoma (MCL) is a B cell malignancy characterized by aberrant expression of cyclin D1 due to a t(11;14) translocation. MCL is refractory to conventional chemotherapy, and treatment remains challenging. We investigated the efficacy of the histone deacetylase (HDAC) inhibitor vorinostat combined with one of several B-cell receptor (BCR) signaling inhibitors on MCL cell death and the underlying mechanisms, using MCL cell lines. The Bruton's tyrosine kinase inhibitor PCI-32765 and the spleen tyrosine kinase inhibitor R406 showed synergistic effects with vorinostat on growth inhibition. Treatment with PCI-32765 or R406 alone induced 27.3 ± 2.1 or 25.1 ± 3.2% apoptosis. When combined with vorinostat, these apoptotic fractions significantly increased to 50.8 ± 4.9 and 63.1 ± 5.0%, respectively. Activation of caspase-3 and poly-(ADP-ribose) polymerase cleavage were markedly increased. We performed gene expression profiling following treatment with the combination of vorinostat and individual BCR signaling inhibitors using a microarray, and differentially expressed genes were identified. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the nuclear factor (NF)-κB signaling pathway was significantly enriched following treatment with the combination of vorinostat and R406. Protein expression analysis confirmed the down-regulation of NF-κB1/p105 and cyclin D1, suggesting inhibition of the NF-κB pathway. Taken together, the combination of an HDAC inhibitor and a BCR signaling inhibitor may be a novel therapeutic strategy for MCL.

Combination of lenalidomide with vitamin D3 induces apoptosis in mantle cell lymphoma via demethylation of BIK.

Mantle cell lymphoma (MCL) is a currently incurable B-cell malignancy. Lenalidomide (Len) has been demonstrated to be one of the most efficient new treatment options. Because Len and 1α,25-dihydroxyvitamin (VD3) synergize to kill breast cancer cells, we investigated whether VD3 could increase the ability of Len to induce MCL cell death. While MCL cells were weakly sensitive to Len (1 μM), the addition of VD3 at physiological dose (100 nM) strongly increased cell death, accompanied by slowdown in cell cycle progression in MCL cell lines (n=4 out of 6) and primary samples (n=5 out of 7). The Len/VD3 treatment markedly increased the expression of the BH3-only BCL2-interacting killer (Bik) without affecting the expression of other Bcl-2 molecules. Immunoprecipitation assays demonstrated that Bik was free from anti-apoptotic partners, Bcl-2 and Bcl-xL, in treated cells. Moreover, silencing of BIK prevented apoptosis induced by Len/VD3, confirming the direct involvement of Bik in cell death. Bik accumulation induced by Len/VD3 was related to an increase in BIK mRNA levels, which resulted from a demethylation of BIK CpG islands. The sensitivity of MCL cells to Len/VD3 was similar to the response to 5-azacytidine, which also induced demethylation of BIK CpG islands. These preclinical data provide the rationale to investigate the role of VD3 in vivo in the response to Len.

Novel targeted therapies for mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy characterized by short median survival despite intensive therapies. The clinical behavior of MCL may be due to the complex pathophysiology of the disease which includes its genetic hallmark, the chromosomal translocation t(11;14) resulting in aberrant expression of cyclin D1, alteration in the DNA damage response, and constitutive activation of key anti-apoptotic pathways such as phosphatidyl-inositol 3-kinase (PI3K)/Akt and nuclear factor-kB (NF-kB). Collectively, these changes result in cell cycle dysregulation and give rise to profound genetic instability. Given this complex pathophysiology, the limited number of options for patients with relapsed/refractory MCL, and the difficulty in achieving long-lasting remissions with conventional approaches, it is essential to explore new treatment options targeting the numerous dysregulated pathways that are operable in MCL. We have recently reported that milatuzumab, a fully humanized anti-CD74 monoclonal antibody (mAb), in combination with anti-CD20 mAbs has significant preclinical and clinical activity in MCL. Here we discuss these results, provide additional insights into milatuzumab-mediated MCL cell death, and report preliminary data on the activity of other targeted biologic agents including PCI-32765 and CAL-101 currently undergoing evaluation at our institution and others.

Potentiation of Cannabinoid-Induced Cytotoxicity in Mantle Cell Lymphoma through Modulation of Ceramide Metabolism

Ceramide levels are elevated in mantle cell lymphoma (MCL) cells following treatment with cannabinoids. Here, we investigated the pathways of ceramide accumulation in the MCL cell line Rec-1 using the stable endocannabinoid analogue R(+)-methanandamide (R-MA). We further interfered with the conversion of ceramide into sphingolipids that promote cell growth. Treatment with R-MA led to increased levels of ceramide species C16, C18, C24, and C(24:1) and transcriptional induction of ceramide synthases (CerS) 3 and 6. The effects were attenuated using SR141716A, which has high affinity to cannabinoid receptor 1 (CB1). The CB1-mediated induction of CerS3 and CerS6 mRNA was confirmed using Win-55,212-2. Simultaneous silencing of CerS3 and CerS6 using small interfering RNA abrogated the R-MA-induced accumulation of C16 and C24. Inhibition of either of the enzymes serine palmitoyl transferase, CerS, and dihydroceramide desaturase within the de novo ceramide pathway reversed ceramide accumulation and cell death induced by R-MA treatment. To enhance the cytotoxic effect R-MA, sphingosine kinase-1 and glucosylceramide synthase, enzymes that convert ceramide to the pro-proliferative sphingolipids sphingosine-1-phospate and glucosylceramide, respectively, were inhibited. Suppression of either enzyme using inhibitors or small interfering RNA potentiated the decreased viability, induction of cell death, and ceramide accumulation induced by R-MA treatment. Our findings suggest that R-MA induces cell death in MCL via CB1-mediated up-regulation of the de novo ceramide synthesis pathway. Furthermore, this is the first study were the cytotoxic effect of a cannabinoid is enhanced by modulation of ceramide metabolism.

MDM2 Antagonist Nutlin-3 Displays Antiproliferative and Proapoptotic Activity in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkin's lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL. MCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed. Nutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 micromol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 micromol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO. These findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

The Functional Impact of PKCβ/PI3K/AKT Signalling on Translational Initiation in MCL

Introduction: Mantle cell lymphoma (MCL) is an aggressive form of B-cell non-Hodgkin's lymphoma (NHL). It is characterized by the t(11;14)(q13;q32) translocation, which results in the overexpression of cyclinD1, a cyclin regulated by the PI3K/AKT pathway. Activation of the PI3K/AKT pathway has been shown to be involved in the pathogenesis of MCL. In addition overexpression of the protein kinase C beta (PKCβ) has been described for most cases of MCL, inhibited by enzastaurin which in turn induces apoptosis and reduces proliferation through the PKCβ/PI3K/AKT pathways. 4EBP1 is described as one of the downstream targets of PI3K/mTOR pathway linking translation initiation with PI3K/mTOR signalling as a EIF4E binding protein and playing therefore critical role in the control of protein synthesis, survival and cell growth. Targeting 4EBP1 and/or EIF4E via the PI3K/AKt/mTOR signalling or directly will affect tumor tissue.Aim of the study: The aim of the study was to determine the functional impact of PKCβ/PI3K/AKt/mTOR signaling on the translation initiation factor EIF4E, its binding protein and regulated proteins in MCL cell lines.Methods: MCL cell lines were treated with inhibitors of the PKCβ/PI3K/AKt/mTOR pathways (enzastaurin, LY294002, rapamycin) for up to 48h.The impact of the drugs on the proliferation rate of the cells was accessed after 48h by WST-assay and/or cell count. mRNA expression levels were determined using Taqmanassays. Protein phosphorylation status and protein expression were identified by westernblot. For downregulation of EIF4E in the cells sodium arsenite was used. Specific silencing of EIF4E was achieved by transfection of cells with siRNA against EIF4E.Results: The MCL celllines (5) responded to the treatment with the inhibitors of the PI3K/AKt/mTOR pathway at a IC50 for rapamycin between 5nM-50nM and for the PI3Kinhibitor between 0,31μM-5μM. Treatment of the cells with the PI3K/AKt/mTOR inhibitors induced dephosphorylation of 4EBP1 in a time-and dosedependent manner while a potential effect of the PI3K and mTOR inhibitors on the EIF4E expression and its target genes (cyclinD1, BCL2) could not be shown consistently. 4 out of 5 MCL cell lines were susceptible to enzastaurin with an IC50 between 2μM-5μM. In the not responding to enzastaurin and most resistant to rapamycin cell line (Rec-1) no 4EBP1 proteinexpression was detectable. Dephosphorylation of 4EBP1 achieved by treatment of the cells with sodium arsenit was accompanied by downregulation of EIF4E, cyclinD1 and BCL2 proteins but also stop of proliferation. The potential involvement of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death in MCL cell lines was shown by silencing the expression of the eIF4E gene by transfection with siRNA specifically targeting the eIF4E gene expression leading to downregulation of cyclinD1, 4EBP1 proteins and cell proliferation.Conclusion: Eventhough treatment of the cells with the PI3K/AKt/mTOR inhibitors induced dephosphorylation of 4EBP1 a potential effect of the PI3K and mTOR inhibitors on the EIF4E expression and its target genes (cyclinD1, BCL2) could not be shown consistently. Instead dephosphorylation of 4EBP1 accomponied by downregulation of EIF4E or targeted downregulation of eIF4E gene expression lead to downregulation of cyclinD1 and BCL2 proteins as well as cell death in MCL. Therefore targeting the downstream targets of the PI3K/AKt/mTOR signalling 4EBP1 and/or EIF4E directly seems to be a promising anticancer strategy.