CBL-C137, a Curaxin and Potent Suppressor Of NF-Kb Activity and Modulator Of p53 Function Is Active In Rituximab-Sensitive Or Resistant Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) Cells

Abstract The identification of critical signaling pathways necessary for the development, maintenance and progression of specific subtypes of DLBCL are necessary in order to develop novel therapeutics against them. Increased NFkB activity and p53 deregulation contribute to either the pathogenesis of some types of DLBCL (i.e. activated B-cell [ABC] subtype) or to rituximab and/or chemotherapy resistance in B-cell lymphoma. Optimal targeting of NFkB activity is an attractive therapeutic strategy that has been evaluated in pre-clinical and clinical models for over a decade with variable degrees of success. CBL-C137 is a novel and potent member of the curaxin family, capable of modulating p53 and NFkB activity and inducing cell death in several solid tumor cancer models. It has never been previously studied in lymphoid malignancies. In order to study and define the therapeutic potential of curaxins in B-cell lymphoma, we evaluated CBL-C137 in lymphoma pre-clinical models. A panel of rituximab sensitive or resistant lymphoma cell lines representing the two most common subtypes of DLBCL (i.e. ABC-DLBCL and germinal center B-cell [GCB] DLBCL) were exposed to CBL-C137 (0.5-16mM). Changes in cell viability; cell cycle distribution; apoptosis and p53/ NFkB p65 expression were evaluated by measuring ATP content, flow cytometry, and Western blotting, respectively. Subsequently, GCB- or ABC-DLBCL cells were exposed to CBL-C137 alone or in combination with various chemotherapy agents or other available (but less selective) NFkB inhibitors (i.e. lenalidomide or Ibrutinib) for 24 or 48 hrs. Changes in cell viability were determined using the cell titer glo assay. In addition, we conducted standardized 51Cr release assays on cells previously exposed to either CBL-C137 or DMSO to investigate the effects of NFkB inhibition on rituximab (or other anti-CD20) antibody-associated complement mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC). CBL-C137 induced dose- and time- dependent cell death in ABC-DLBCL greater than in GCB-DLBCL cell lines. The IC50 for CBL-C137 in ABC-DLBCL (rituximab/chemotherapy sensitive or resistant cells) ranged from 1.36 to 2.77mM. In contrast rituximab/chemotherapy GCB-DLBCL cells exhibited the highest IC50 (11.91mM, 95% C.I 7.1-19.8mM). In sensitive DLBCL cells, CBL-C137 induced both apoptosis and cell cycle arrest in G1 phase. Moreover, in vitro exposure to CBL-C137 decreased p53 and p65 in sensitive cells. CBL-C137 increased Lenalidomide, but not Ibrutinib, anti-lymphoma activity in the conditions tested. Finally, CBL-C137 did not affect rituximab or other anti-CD20 antibody-associated ADCC or CMC in DLBCL cells. Our data suggest that CBL-C137 is active in DLBCL pre-clinical models, primarily in ABC-DLBCL cell lines. In sensitive cells, CBL-C137 modulates p53 and NFkB activity and promotes death and/or cell cycle arrest. Ongoing studies are aimed to further define the anti-tumor effects of CBL-C137 in combination with other small molecules inhibitors targeting directly or indirectly NFkB activity in lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

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Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽

10.1182/blood.v122.21.377.377 ◽

2013 ◽

Vol 122 (21) ◽

pp. 377-377 ◽

Cited By ~ 3

Author(s):

Shruti Bhatt ◽

Daxing Zhu ◽

Xiaoyu Jiang ◽

Seung-uon Shin ◽

John M Timmerman ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cd20 Antibody ◽

Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

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Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽

10.1182/blood.v118.21.3721.3721 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3721-3721

Author(s):

Gerhard Niederfellner ◽

Olaf Mundigl ◽

Alexander Lifke ◽

Andreas Franke ◽

Ute Baer ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mechanisms Of Action ◽

Type I ◽

Type Ii ◽

Cd20 Antibody ◽

Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

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Carfilzomib (CFZ): A Novel Proteasome Inhibitor That Induces Cell Cycle Arrest and Cell Death In Rituximab-Chemotherapy Resistant Lymphoma and Potentiates the Anti-Tumor Activity of Chemotherapeutic Agents

Blood ◽

10.1182/blood.v116.21.4908.4908 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4908-4908

Author(s):

Juan Gu ◽

Francisco J. Hernandez-Ilizaliturri ◽

Gregory P. Kaufman ◽

Cory Mavis ◽

Myron S. Czuczman

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Parp Cleavage ◽

Cycle Arrest ◽

Anti Tumor Activity

Abstract Abstract 4908 Rituximab-chemotherapy relapsed/refractory B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. Targeting the ubiquitin-proteasome system using bortezomib (BTZ) has resulted in significant anti-tumor activity and potentiates the effects of chemotherapy/biologic agents in multiple myeloma, and to a lesser degree, B-cell lymphoma. CFZ is as a novel proteasome inhibitor which is selective and structurally distinct from BTZ. In an attempt to characterize the biological activity of CFZ, we evaluated its anti-tumor activity in several lymphoma pre-clinical models. Rituximab-chemotherapy sensitive cell lines (RSCL), rituximab-chemotherapy resistant cell lines (RRCL), as well as primary tumor cells derived from patients with de novo or relapsed/refractory B-cell lymphoma, were exposed to escalating doses of CFZ or BTZ (1-7.5nM) alone or in combination with doxorubicin, paclitaxel, or gemcitabine for 24, 48 and 72hours. Cell viability was determined by cell titer glow luminescent assay and cell cycle was analyzed by FASCan DNA methodology. Patient-derived lymphoma cells were isolated from fresh biopsy tissue via negative selection using magnetic beads. Western blots were performed using cell lysates from CFZ, BTZ or control-treated cells to detect PARP-cleavage and/or changes in Bcl-2 family members. CFZ was more active than BTZ and exhibited dose-dependent and time-dependent cytotoxicity against RSCL, RRCL, and primary tumor cells. We found a 10-fold concentration difference between CFZ and BTZ activity. In vitro exposure of RRCL or RSCL to CFZ resulted in G2/M phase cell cycle arrest. In addition, CFZ exposure resulted in the up-regulation of Bak and Noxa levels and subsequent PARP cleavage in RRCL. Finally, CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumor activity of paclitaxel and gemcitabine in B-cell lymphoma cell lines. In summary, our data strongly suggest that CFZ is a novel and potent proteasome inhibitor which is able to: overcome resistance to some conventional chemotherapeutic agents, upregulate proapoptotic proteins to enhance cell death, and induce G2/M cell cycle arrest in lymphoma cells. Our preclinical data supports future clinical evaluation of CFZ in patients with refractory B-cell lymphoma. (Supported by USPHS grant R01 CA136907-01A1 from the National Cancer Institute). Disclosures: No relevant conflicts of interest to declare.

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Mechanisms of Sensitivity and Resistance to Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.1359.1359 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1359-1359

Author(s):

Ana A Tula-Sanchez ◽

Aaron Havas ◽

Peter Alonge ◽

Mary E Klein ◽

Taralyn Y Rogers ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Histone Deacetylase Inhibitors ◽

B Cell Lymphoma ◽

Resistant Cell ◽

G1 Arrest ◽

Cycle Arrest

Abstract Abstract 1359 Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin Lymphoma (NHL) throughout the world. DLBCL is an aggressive, heterogeneous disease with two major recognized cell-of-origin subtypes: “germinal center” (GCB) and “activated B-cell like” (ABC), the latter having the worse prognosis. Overall, DLBCL remains fatal for about 30% patients due to relapse or lack of response to initial therapy. Resistant/relapsed DLBCL patients could benefit from the addition of new promising antiproliferative drugs, such as histone deacetylase inhibitors (HDACIs), to current chemotherapy regimens. So far, Vorinostat and Romidepsin, two structurally different HDACIs, have been approved for the treatment of hematological cancers. Despite their proven antiproliferative, pro-apoptotic effects, response to these drugs against DLBCL in clinical trials have been variable, ranging from complete/partial responses to stable disease to no response. The mechanisms of action of these drugs are still poorly understood, mainly because the function of their target deacetylases are cell context-specific. Therefore, characterization of the specific anticancer mechanisms of action of HDACIs in DLBCL could potentially lead to development of novel combinatorial drug regimens effective against resistant/relapsed DLBCL patients. To define HDACI action in DLBCL, we treated DLBCL-derived cell lines with PXD101, (Belinostat); a hydroxamate HDACI, like Vorinostat. We demonstrated that PXD101 is able to produce 24h growth inhibition (IC50) at submicromolar concentrations regardless of the DLBCL subtype. The 24h IC50values were used in all the subsequent experiments. Cell cycle and apoptosis analysis by flow cytometry indicated that PXD101 produces cytotoxic effects on two of the GCB cell lines; DB and OCILY19 underwent G2/M cell cycle arrest at 24 hours followed by apoptosis at 48 and 72 hours of treatment. Immunoblotting of PARP and caspase-3 cleavage further confirmed apoptosis. More importantly, when cells were treated for only 8 hours with PXD101 and then the drug was removed for 24 hours, cells showed apoptosis rates similar to those observed with 48h of continuous treatment; suggesting that once that these cell lines are exposed to the drug they rapidly commit to cell death. Thus, we have classified the DB and OCILY19 cell lines as models for sensitivity to the apoptotic effects of HDACI. In contrast, PXD101 induced cytostatic effects on the GCB cell line SUDHL4 and ABC cell lines U2932 and SUDHL8. All three cell lines showed G1 phase cell cycle arrest with little apoptosis. The G1 arrest is reversible after 48 hours of drug removal. Because of the lack of cell death and the reversibility of cell cycle arrest, we have classified these cell lines as models of HDACI resistance. Previous studies have shown that induction of p21 is responsible for G1 arrest in cells treated with HDACIs. Western blot analysis showed that none of the cell lines, except U2932, express p21, but upon PXD101, p21 protein levels were induced at 24, 48 and 72 hours of PXD101 treatment in SUDHL4 and U2932. In contrast, p21 was induced to a lesser extent in OCILY19 and DB, but its expression was not sustained beyond 24 hours of treatment. Since we also observed a corresponding loss in Rb phosphorylation, we tested the effect of PXD101 on cyclin dependent kinase 2 (CDK2) activity. This enzyme complex is responsible for entry into S phase and is inhibited by association with p21. In all three resistant cell lines CDK2 activity was reduced after only 24 hours of treatment with PXD101. The loss in activity was correlated with increased association with p21, as determined by immunoprecipitation. These results indicate that sustained upregulation of p21 by HDACIs such as PXD101 plays a role in bringing about G1 arrest that may protect DLBCL cells from apoptosis. Combined treatment with therapeutics that prevent p21 upregulation and G1 arrest may work synergistically with HDACIs to trigger apoptosis in HDACI-resistant cell lines. To that end, we have begun analysis of the cyclin-dependent kinase inhibitor, flavopiridol, and have shown that it prevents both p21 upregulation and G1 arrest in the HDACi-resistant DLBCL cell lines. Studies to measure synergism with PXD101 in bringing about cell death are currently underway. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

Blood ◽

10.1182/blood-2020-134778 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-34

Author(s):

Maciej Szydlowski ◽

Filip Garbicz ◽

Ewa Jabłońska ◽

Patryk Górniak ◽

Beata Pyrzynska ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Raji Cells ◽

Pim Kinases ◽

Large B Cell Lymphoma ◽

Cd20 Antibody ◽

Anti Cd20 ◽

Large B Cell

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

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The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.2732.2732 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2732-2732

Author(s):

Enrico Derenzini ◽

Ilaria Iacobucci ◽

Elisa Brighenti ◽

Federica Cattina ◽

Richard Eric Davis ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Cycle Arrest ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 2732 The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Disclosures: No relevant conflicts of interest to declare.

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MLN2238, a Novel and Potent Proteasome Inhibitor, Induces Caspase-Dependent Cell Death, Cell-Cycle Arrest, and Potentiates the Anti-Tumor Activity of Chemotherapy Agents In Rituximab-Chemotherapy Sensitive or Resistant B-Cell Lymphoma Cell Lines

Blood ◽

10.1182/blood.v116.21.3939.3939 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3939-3939

Author(s):

Juan Gu ◽

Patil Ritesh ◽

Cory Mavis ◽

George Deeb ◽

John Gibbs ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Anti Tumor Activity

Abstract Abstract 3939 The use of proteasome inhibitors such as bortezomib (BTZ) has generated much excitement as a potential therapeutic approach capable of effectively treating resistant/refractory lymphoid neoplasm. Clinical outcomes in multiple myeloma and relapsed mantle cell lymphoma demonstrate that these novel agents can overcome resistance demonstrated by a lack of antitumor activity to traditional salvage chemotherapeutic agents. Our group of investigators have demonstrated that proteasome inhibition using BTZ can increase pro-apoptotic Bcl-2 family member expression and restore chemotherapy sensitivity in rituximab-chemotherapy resistant cell lines (RRCL). To further develop therapeutic strategies targeting the proteasome system, we studied the anti-tumor activity and mechanisms-of-action of MLN2238, a novel irreversible proteasome inhibitor, in pre-clinical lymphoma models. Experiments were conducted in rituximab-chemotherapy sensitive cell lines (RSCL), RRCL, and in tumor cells derived from patients with de novo or relapsed/refractory B-cell lymphoma. Cells were exposed in vitro and/or ex vivo to escalating doses of MLN2238 or BTZ (0.1-10nM) +/− caspase inhibitors (zVAD-fmk or Q-VD-OPh) for 24, 48 and 72h. Differences in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay; changes in ATP content (apoptosis) were determined using the Cell Titer Glow assay. Effects on cell cycle were analyzed by the FASCan DNA method. In addition, lymphoma cells were exposed to MLN2238 or BTZ +/− doxorubicin, gemcitabine or paclitaxel and cell viability was evaluated as described above. In vitro, MLN2238 exhibited more potent concentration- and time-dependent cytotoxicity and inhibition of cell proliferation in RSCL, RRCL, as well as primary lymphoma cells than BTZ. In vitro exposure of RSCL and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL in a dose-dependent manner. Co-incubation of RSCL with bortezomib, or MLN2232 and either pan-caspase inhibitor led to a significant decrease in BTZ- or MLN2232-induced cell death. In contrast, neither zVAD-fmk nor Q-VD-OPh was capable of blocking BTZ- or MLN2232-induced cell death of RRCL. Our data suggest that BTZ and MLN2238 are also capable of inducing caspase-independent cell death in RRCL. To this regard, we found differences that RRCL are more likely to be in S phase in resting conditions when compared to RSCL. In vitro exposure of RRCL cells to MLN2232 (and to a much lesser degree BTZ) reduced RRCL S-phase and induced arrest at G2/M phase. Collectively, these data suggest that MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy sensitive or resistant cell models and potentiates the anti-tumor activity of chemotherapy agents. MLN2232 appears to posses several mechanisms-of-action (induction of apoptosis and/or cell cycle arrest) and has the potential of becoming a novel and potent target-specific therapeutic agent in the future treatment of therapy-resistant B-cell lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute). Disclosures: No relevant conflicts of interest to declare.

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Evaluation of the Anti-Tumor Activity of MLN4924, A Novel NEDD8 Activating Enzyme Inhibitor, in Pre-Clinical Models of Rituximab Chemotherapy-Sensitive or -Resistant B-Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2761.2761 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2761-2761

Author(s):

Natalie M Czuczman ◽

Matthew J. Barth ◽

Richa Dwar ◽

Cory Mavis ◽

Pavel Klener ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Clinical Models ◽

Chemotherapy Agents ◽

Anti Tumor Activity

Abstract Abstract 2761 Clinical outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) remains poor with currently available therapies. Recently, the ubiquitin-proteasome system (UPS) appears to play an important role in the development of resistance in MCL and some subtypes of DLBCL. Targeting UPS represents a rational approach in an attempt to eradicate drug-resistant lymphoma clones. MLN4924 is a novel, potent and selective inhibitor of the NEDD8-activating enzyme (NAE) that is necessary for the modification of cullin-RING ubiquitin ligases. We evaluated the anti-tumor activity of MLN4924 against a panel of rituximab-sensitive (RSCL) or rituximab/chemotherapy–resistant (RCRCL) DLBCL and Burkitt lymphoma cell lines, cytarabine-sensitive or -resistant (AraCR) MCL cell lines, and primary tumor cells freshly isolated from lymphoma patients (n=13). Lymphoma cells were exposed to escalating doses of MLN4924 alone or in combination with selected chemotherapy agents for up to 72 hrs. Changes in the cell viability or ATP content were determined by alamar Blue reduction or CellTiterGlo assays, respectively. Induction of apoptosis and changes in the levels of NFkB and UPS regulatory proteins were analyzed by Western blotting. Cell cycle alterations were determined by propidium iodide staining and NFkB activity was quantified by flow cytometry using the Imagestream technology. MLN4924 demonstrated time- and dose-dependent anti-lymphoma activity in all cell lines tested. The IC50 in RSCLs were Raji=400nM, RL=1uM and U2932=>3uM. All RCRCLs were less responsive to MLN4924 as a single agent with IC50 concentrations 4–10× those of their respective sensitive parental cell lines. The MCL cell lines Mino, MinoAraCR, Z-138, HBL-2 and HBL-2AraCR were most sensitive to MLN4924 anti-tumor effects (IC50=250nM) with no significant difference between cytarabine-sensitive and -resistant cell lines; while the MCL cell lines Rec-1, Rec-1AraCR, Jeko-1 and Jeko-1AraCR were less sensitive (IC50=500–1000nM). A variable degree of anti-tumor activity was also observed in primary lymphoma cells. In addition to single-agent activity, MLN4924 plus selected anti-lymphoma chemotherapy agents (bortezomib, bendamustine and cytarabine) demonstrated synergy in cytarabine-sensitive and (to a lesser degree) cytarabine-resistant MCL cell lines. Combinations with additional chemotherapeutic agents (doxorubicin and vincristine) resulted in additive effects. Exposure of MCL cells to MLN4924 resulted in G1 cell cycle arrest. In vitro exposure of the more sensitive MCL cell lines Mino and MinoAraCR to MLN4924 resulted in an increase in p-IkBα and down-regulation of both total and nuclear NFkB. The less sensitive cell lines Rec-1 and Rec-1AraCR demonstrated little to no change in NFkB activation following exposure to MLN4924. Additional studies are ongoing to further define the molecular mechanisms of the anti-tumor activity observed following NAE inhibition by MLN4924 in these pre-clinical models and to further evaluate the activity of MLN4924 in in vivo SCID mouse models of B-cell lymphoma. Our data suggests that MLN4924, a novel NAE inhibitor, is active against B-cell lymphomas, particularly MCL, and is a promising agent warranting further investigation in relapsed/refractory aggressive B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

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