Rational for the Use of a Targeted-Therapy Using ABT-737 In Mantle-Cell Lymphoma

Abstract Abstract 770 Mantle-cell lymphoma (MCL) is still considered as incurable, thus new therapeutic approaches are needed. The anti-apoptotic protein Bcl-2 is known to be over-expressed in MCL and implicated in drug resistance. Therefore, there is a strong rational for the use of a Bcl-2-targeted therapy in MCL. The BH3-only mimetic ABT-737 (Abbott) is a potent specific inhibitor of Bcl-2, Bcl-xL and Bcl-w but not of Mcl-1. ABT-737 have been investigated in several hematologic malignancies cell lines, such as chronic lymphoid leukemia (CLL) and diffuse large B cell lymphoma, and showed promising results. In this study, we investigated the anti-tumoral effect of ABT-737 in the setting of MCL using MCL cell lines and MCL patients' samples. Our aims were also to identify prognostic biomarkers that may predict MCL tumor cells sensitivity to ABT737 and to study the anti-MCL activity of ABT737 alone or in combination. Five well characterized MCL cell lines were used: JEKO-1, MINO, REC-1, GRANTA-519 and UPN-1. Cytotoxicity of ABT-737 was assessed by flow cytometry using APO 2.7 staining. MINO and GRANTA 519 cell lines were highly sensitive (EC50= 20 nM after 24 hours of treatment) and apoptosis occurred rapidly within 2 hours following treatment as demonstrated by caspase 3 cleavage. In contrast, the three other cell lines (JEKO-1, REC-1 and UPN-1) were resistant (EC50> 4μM) to ABT-737. Western Blot analysis revealed that a major difference between sensitive cell lines (MINO and GRANTA-519) compared to other cell lines was their Bcl-2high/Mcl-1low profile. We also investigated ABT-737-induced apoptosis in primary MCL tumor cells (n=12). Seven patients' samples were sensitive to ABT-737 (median EC50 of 20 nM), whereas the 5 others samples were resistant (EC50 not achieved). As observed in MCL cell lines, western blot analysis revealed that primary tumor cells showing a Bcl-2high/Mcl-1low profile were associated with sensitivity to ABT-737. In contrast a Mcl-1high profile was associated with resistance to ABT-737. Taken together our investigation in both MCL cell lines and primary tumor cells from patients suggested that expression level Mcl-1 could influence ABT-737-induced apoptosis. Flavopiridol is a cyclin dependant kinase inhibitor and known to induce a transcriptional down-regulation of Mcl-1 in multiple myeloma and CLL. We therefore hypothesized that addition of Flavopiridol to ABT-737 could enhance induced-apoptosis. We first confirmed that two hours of treatment with flavopiridol induced a strong down-regulation of Mcl-1 at both mRNA and protein levels. Using suboptimal dose of ABT-737 (25 nM) and Flavopiridol (100 nM) for 24 hours, we observed a strong synergistic anti-MCL as evidence by a combination indice <1 according to the Chou-Talalay method. In conclusion, ABT-737 at low nanomolar concentration induces a strong apoptosis in the subgroup of Bcl-2high/Mcl-1low MCL cells profile which represents around 50% of the MCL patients. Thus, this subgroup of patients appears to be good candidate for clinical trials evaluating ABT-737 alone. In the subgroup of Mcl-1high patients, a specific down-regulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes with ABT-737. Indeed, our results strongly support the use of ABT-737 in MCL based on the concept of a targeted therapy according to the Bcl-2/Mcl-1 tumor cell profile. ABT-263, an orally bioavailable BH3 mimetic compound of the same class than ABT-737, is currently under investigation in various hematological malignancies, thus our investigation provides a biological rational for future clinical trials evaluating ABT-263 in combination or not with Flavopiridol in MCL patients. Disclosures: No relevant conflicts of interest to declare.

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Bortezomib Is Synergistic with Rituximab and Cyclophosphamide in Inducing Apoptosis of Mantle Cell Lymphoma Cells In Vitro and In Vivo.

Blood ◽

10.1182/blood.v110.11.4514.4514 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4514-4514

Author(s):

Liang Zhang ◽

Yuankai Shi ◽

Xiaohong Han ◽

Jing Yang ◽

Jianfei Qian ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Tumor Cells ◽

Cell Lines ◽

Primary Tumor ◽

Cell Lymphoma ◽

Fixed Dose ◽

Mantle Cell ◽

Induced Apoptosis

Abstract Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor clinical outcome. Although frontline therapy induces a high rate of complete remission, relapse is inevitable and new regimens are needed for relapsed MCL. The proteasome inhibitor bortezomib (BTZ) induces apoptosis and sensitizes MCL cells to chemotherapy in relapsed MCL, but as a single agent, response rate is low, duration of response is short and side effects are severe. Here we evaluated whether BTZ is additive or synergistic with cyclophosphamide (CTX) and rituximab (RTX). Material and Methods: Four human MCL cell lines SP53, MINO, Grant 519, and Jeko-1 and freshly isolated primary tumor cells from three MCL patients were treated with BTZ, CTX, RTX individually or in combination of RTX and CTX (RC), or BTZ plus RTX and CTX (BRC regimen). Cell proliferation and apoptosis were evaluated to determine if there was additive or synergistic effect of the BRC regimen. Western blot analysis was used to elucidate the molecular mechanism by which BTZ, RTX, CTX, RC and BRC induces apoptosis in MCL cells. In addition, in vivo experiments using severe combined immunodeficiency mice with human mantle cell lymphoma xenografts were performed to examine the in vivo efficacy of the regimen to control the growth of and eradicate MCL cells. Results: BTZ and CTX as single agents inhibited the growth of MCL cell lines in a dose-dependent manner (P < 0.01). The IC50 (inhibitory concentration at 50%) for BTZ and for CTX were between 10 and 20 nM and between 5 and 20 mM, respectively. Increasing doses of BTZ with a fixed dose of RTX (10 μg/mL) and CTX (10 mM) resulted in markedly synergistic growth inhibition of MCL cells (P < 0.01). The BRC regimen induced apoptosis in about 69.7% of MCL cell lines and 92.6% of primary tumor cells (P < 0.05 and P < 0.01, compared with those induced by BTZ, RTX, CTX or RC). Furthermore, western blotting analysis showed that BRC induced apoptosis earlier via activation and cleavage of caspases-8, -9, and -3, and PARP as compared with BTZ, RTX, CTX or RC. The pan-caspase inhibitor z-VAD-FMK completely blocked apoptosis induced by BRC. In vivo studies demonstrated that BRC regimen eradicated subcutaneous tumors in MCL-bearing SCID mice and significantly prolonged the long-term event-free survival up to 10 weeks in 70% of the mice, whereas all tumor-bearing mice receiving BTZ, RTX, CTX or RC or PBS (control) died of aggressive MCL within 6 weeks. Conclusion: Cytoreductive chemotherapy with both BTZ and anti-CD20 antibody effectively inhibited the growth and induced apoptosis of MCL cells in vitro and in vivo. Bortezomib-rituximab-cyclophosphamide (BRC) regimen may offer a better therapeutic modality for MCL patients. Thus, our data lay the basis for a clinical trial in relapsed MCL using the BRC combination treatment.

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Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

Blood Advances ◽

10.1182/bloodadvances.2018016048 ◽

2018 ◽

Vol 2 (16) ◽

pp. 2039-2051 ◽

Cited By ~ 16

Author(s):

Jimmy Lee ◽

Liang Leo Zhang ◽

Wenjun Wu ◽

Hui Guo ◽

Yan Li ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Resistant Cell ◽

Intrinsic Resistance ◽

Mantle Cell ◽

Myc Gene ◽

Bona Fide ◽

Ibrutinib Resistance ◽

Induced Apoptosis

Abstract The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

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The Tak-1 Inhibitor AZ-Tak1 Inhibits XIAP, Activates Caspase-9, and Induces Apoptosis in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v114.22.1692.1692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1692-1692 ◽

Cited By ~ 1

Author(s):

Daniela Buglio ◽

Sangeetha Palakurthi ◽

Katharine F. Byth ◽

Anas Younes

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Small Molecule ◽

Cell Lymphoma ◽

Small Molecule Inhibitor ◽

Caspase 9 ◽

Lymphoma Cells ◽

Molecule Inhibitor ◽

Mantle Cell ◽

Induced Apoptosis

Abstract Abstract 1692 Poster Board I-718 Transforming growth factor-b-activated kinase 1 (TAK1) is a key regulator of NF-kB activation. TAK1 can be activated by a variety of pro-inflammatory cytokines and T and B cell receptors. Recent experiments demonstrated that deletion of TAK1 results in inactivation of both JNK and NF-kB signaling resulting in massive apoptotic death of hematopoietic cells in mice. In this study, we examined the expression pattern of TAK1 and its role as a potential therapeutic target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). These lines expressed relatively low levels of the tumor suppressor protein A20. Mino and SP53 expressed high level of p-p38. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. It also inhibits Jak2 but at a much higher concentration (IC50=0.18 mM). AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1-0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when primary mantle cell lymphoma cells were examined. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, AZ-Tak1 decreased the level of SMAC/DIABOLO and cytochrome –C in the mitochondria, which was associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and induction of apoptosis. Furthermore, and consistant with its ability to inhibit Jak2 activity, AZ-Tak1 reduced STAT2 and STAT6 levels. AZ-Tak1 demonstrated no significant effect on bcl-2 family members. Finally, co-treatment with HDAC inhibitors demonstrated synergistic effect with low concentrations of AZ-Tak1. Collectively, our data demonstrate that targeting TAK1 by the small molecule inhibitor AZ-Tak1 induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma. Disclosures Palakurthi: Astra Zeneca: Employment. Byth:Astra Zeneca : Employment.

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The Bioactive Polyphenol Curcumin (diferuloylmethane) In Human Apolipoprotein A-1 Nanodisks Enhances Apoptosis and G1 Cell Cycle Arrest In Mantle Cell Lymphoma Compared with Free Curcumin

Blood ◽

10.1182/blood.v116.21.3934.3934 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3934-3934

Author(s):

Amareshwar T.K. Singh ◽

Mistuni Ghosh ◽

C. Shad Thaxton ◽

Trudy M. Forte ◽

Robert O. Ryan ◽

...

Keyword(s):

Cell Cycle ◽

Drug Delivery ◽

Cyclin D1 ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Cell Lymphoma ◽

Parp Cleavage ◽

Cycle Arrest ◽

Mantle Cell ◽

Induced Apoptosis

Abstract Abstract 3934 Background: Mantle cell lymphoma (MCL) is a pre–germinal center neoplasm characterized by cyclin D1 overexpression resulting from translocation of the cyclin D1 gene on 11q13 to the promoter of the immunoglobulin heavy chain locus on 14q32. Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. The bioactive polyphenol curcumin (Curc), derived from the rhizome of Curcuma longa Linn, has been shown to have pleiotropic activities related to its complex chemistry and its influence on multiple signaling pathways including NF-kB, Akt, Nrf2 and pathways involved in metastasis and angiogenesis. Curc has been shown to cause growth arrest and apoptosis of BKS-2 immature B-cell lymphoma by downregulating growth and survival promoting genes (Clin Immunol 1999; 93:152). However, because of poor aqueous solubility Curc has had limited clinical utility, so investigators have explored nanoparticle drug delivery approaches (J Nanobiotech 2007, 5:3, MCT 2010; 9:2255). We reasoned that effective and targeted drug delivery by nanoparticles required appropriate receptors to facilitate binding. We therefore screened lymphoma cell lines for receptors that recognize apolipoprotein (apo) A-1. We hypothesized that a novel discoidal nanoparticle (ND) consisting of apoA-1, phospholipid and Curc (Curc ND) would bind to such receptors to facilitate drug delivery. Methods: We compared biologic activity of free Curc vs. Curc-ND in MCL cell lines expressing receptors for apoA-1. Cell lines were grown and maintained in culture, treated, and apoptosis and cell cycle progression was measured by flow cytometry. Relevant signaling intermediates and presence of apoA-1 receptors were measured by immunoblotting using specific antibodies. Results: Granta and Jeko cells (both MCL cell lines) expressed apoA-1 receptors including class B scavenger receptor (SR-B1) and the ATP-binding cassette transporter of the sub-family G1 (ABCG1). To compare the pro-apoptotic effect of free Curc and Curc-ND, Granta cells were incubated with free Curc, Curc-ND, empty ND, and medium alone (untreated). Compared to medium alone, empty ND had no effect while free Curc (20 μM) induced apoptosis. Curc-ND produced a dose-dependent increase in apoptosis, with ∼70% apoptosis at 20 μM. To investigate the mechanism of Curc-ND induced apoptosis, apoptosis-related proteins were studied in cultured Granta cells. A time-dependent decrease in caspase-9 levels was observed following incubation with Curc-ND or free Curc. The decrease in caspase-9 seen with Curc-ND, however, occurs much earlier (between 2–4 h of incubation) than for free-Curc. Caspase-3 was undetectable after 16 h with either treatment. Loss of this band implies activation of caspase-3, which was confirmed by PARP cleavage, wherein a decrease in the 116 kD band was accompanied by an increase in the 85 kD cleavage product. Unlike free Curc, Curc-ND induced PARP cleavage even at 16 h of incubation, suggesting sustained drug release. Curc-ND downregulated cyclin D1, decreased Akt phosphorylation and enhanced cleavage of caspases-9 and -3, and PARP. In addition, Curc-ND induced G1 cell cycle arrest to a greater extent than free Curc in Granta and Jeko cells (Granta: Control 34% G1, Curc 37% G1, Curc-ND 46% G1; Jeko: Control 39% G1, Curc 49% G1, Curc-ND 54% G1). Conclusion: We have shown that the MCL cell lines Granta and Jeko express apoA-1 receptors, making them likely targets for discoidal nanoscale delivery vehicles stabilized with Apo-A1. These nanodisks, when carrying the polyphenol Curc, can result in increased caspase -dependent apoptosis, cell cycle arrest, downregulation of cyclin-D1 and decreased p-Akt. These data suggest that the pleiotropic polyphenol Curc has cell killing/arrest activity in MCL and that Curc-ND may be a potential therapeutic with drug targeting ability. Disclosures: Forte: Lypro Biosciences: Employment.

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Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies

Blood ◽

10.1182/blood-2011-04-346601 ◽

2012 ◽

Vol 119 (2) ◽

pp. 476-487 ◽

Cited By ~ 72

Author(s):

Mamta Gupta ◽

Andrea E. Wahner Hendrickson ◽

Seong Seok Yun ◽

Jing Jing Han ◽

Paula A. Schneider ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Transcriptional Activation ◽

Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Clinical Samples ◽

Lymphoid Malignancies ◽

Mantle Cell ◽

Induced Apoptosis

Abstract The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

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Inhibition of Cyclin Dependent Kinase 9 Synergistically Enhanced Venetoclax Activity in Mantle Cell Lymphoma Cells

Blood ◽

10.1182/blood.v128.22.1593.1593 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1593-1593

Author(s):

Xiaoxian Zhao ◽

Juraj Bodo ◽

Ruoying Chen ◽

Lisa Durkin ◽

Andrew J. Souers ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

Cyclin Dependent Kinase ◽

Bcl2 Family ◽

Mantle Cell ◽

Induced Apoptosis ◽

Cdk9 Inhibitor

Abstract Better therapeutic strategies are needed for patients with mantle cell lymphoma (MCL), an aggressive and largely incurable subtype of Non-Hodgkin Lymphoma. Concurrent expression of anti-apoptotic BCL2 family proteins in lymphoma cells contribute to their evasion of apoptosis. Therefore, targeting only one anti-apoptotic protein may lead to or uncover resistance associated with activity of other anti-apoptotic BCL2 family members. A variety of cyclin-dependent kinase (CDK) inhibitors are undergoing clinical trials either as a single agent or in combination with other approved drugs. CDK9, a portion of the elongation factor P-TEFb, phosphorylates Ser-2 in the C-terminal domain of RNA Polymerase II, which is required for transcript elongation. The effect of CDK9 inhibition is observed most immediately on those proteins with rapid turnover rates such as the BCL2 family protein MCL1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. Here we report the responses of 4 MCL cell lines (Mino, Jeko-1, CCMCL1 and JVM2) and 5 primary MCL samples (representing de novo and relapsed cases, including two relapsed cases after ibrutinib failure and a relapsed case harbor Myc rearrangement) to venetoclax and a novelCDK9 inhibitor A-1467729. Exposure of Mino and Jeko-1 cells to venetoclax rapidly induced apoptosis (IC50 at 5 hours were 235 and 955 nM, respectively). In contrast, CCMCL1 and JVM2 cells were not sensitive to venetoclax with IC50s > 3000 nM. However, CCMCL1 cells were more responsive to A-1467729 alone than the other 3 lines, while JVM2 cells were much less sensitive to A-1467729. All primary samples were sensitive to venetoclax, ex vivo, at the doses between 1 - 100 nM, although their IC50s were variable (range: 2-90 nM). A-1467729 at doses of 1-20 nM had modest single agent effects on the primary samples; however, its combination with venetoclax synergistically induced apoptosis and decreased the IC50 of venetoclax by 2-10 times in all cell lines and primary samples. The strongest synergy was observed in Jeko-1 cells with all combined indexes < 0.1. Studies on mechanisms through immunoblotting and immunohistochemical staining demonstrated that A-1467729 quickly down-regulated phospho-RNA Polymerase II (Ser2) and MCL1 protein levels. CCMCL1 cells lack BCL2 expression, while JVM2 displayed higher expression of MCL1 than other cells. The expression levels of BCL2 and MCL1 in primary samples were case-dependent as well. The expression pattern and level of anti-apoptotic BCL2 family proteins in cell lines and primary cases may be responsible for their variable reactions to these two agents. To further confirm that CDK9 inhibition was affecting cell viability at least partially through its function on MCL1, A-1210477, a MCL1 inhibitor, was applied to the same study. Strong synergistic apoptotic induction was also observed when A-1210477 was combined with venetoclax, especially in MCL1-"dependent" CCMCL1 cells as evidenced by flow cytometry based apoptotic assay and PARP cleavage. Further mechanism studies aiming the effects of CDK9 inhibitor/venetoclax on MCL1/BIM association is being under investigation. MCL mouse xenograft study for such a combined effect has been planned. In summary, the combination of a CDK9 inhibitor and venetoclax showed synergistic induction of apoptosis in both MCL cell lines and primary patient samples. These findings support further evaluation of the efficacy of such a combination in MCL, including ibrutinib-resistant MCL. Disclosures Souers: AbbVie: Employment. Phillips:AbbVie Inc.: Employment. Hsi:HTG Molecular Diagnostics: Consultancy; Abbvie: Honoraria, Research Funding; Seattle Genetics: Honoraria; Cellerant: Honoraria, Research Funding; Eli Lilly: Research Funding; Onyx Pharmaceuticals: Honoraria.

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Arsenic Trioxide in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v106.11.4814.4814 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4814-4814 ◽

Cited By ~ 1

Author(s):

Yok Lam Kwong ◽

Chit Chow ◽

Cyrus R. Kumana ◽

Gopesh Srivastava ◽

Wing Yan Au

Keyword(s):

Cyclin D1 ◽

Mantle Cell Lymphoma ◽

Arsenic Trioxide ◽

Cell Lymphoma ◽

Down Regulation ◽

Synergistic Interactions ◽

Mantle Cell ◽

Induced Apoptosis ◽

Mtt Assays

Abstract Background. Mantle cell lymphoma (MCL) is incurable for many patients. Arsenic trioxide (As2O3) has activity in vitro against lymphoid malignancies. The effects of As2O3 on MCL in vitro and in patients with refractory disease were investigated. Materials and methods. Mantle cell lymphoma (MCL) lines (Jeko-1, Granta-519) were treated with As2O3, in combination with mitoxantrone (MTZ) and ascorbic acid (AA). Consenting patients with refractory MCL were treated with oral-As2O3 (10 mg/day), AA (1 g/day) and chlorambucil (4 mg/day) as outpatients until maximum response or the disease judged refractory. Responses were defined by standard NCI criteria. In patients showing an initial response, vincristine (2 mg intravenously) and prednisolone (30 mg/day) might also be added. After achievement of maximum response, patients were maintained with As2O3 (10 mg/day) and AA (1 g/day) for two weeks every month, for a planned two years. Results. As2O3 and MTZ but not AA induced a dose dependent apoptosis in the MCL lines, as shown by flow cytometry and MTT assays. As2O3, MTZ and AA were tested in various combinations in MTT assays. Synergistic interactions were observed only in the combinations As2O3 (1 uM) + MTZ (0.2 mg/L), and As2O3 (1 uM) + MTZ (0.2 mg/L) + AA (100 uM). Western blotting showed that As2O3-induced apoptosis was associated with a dose and time dependent down-regulation of cyclin D1. However, quantitative polymerase chain reaction showed no change in cyclin D1 gene transcription during As2O3-induced apoptosis. Eleven patients (10 men, 1 women) at a median age of 69 (51–70) years with refractory MCL were studied, at a median of 33 (8–85) months from diagnosis. At the time of As2O3 treatment, they had already had a median of 2 (1 – 4) relapses managed with a median of 2 (1 – 6) previous chemotherapeutic regimens. Eight of eleven patients were evaluable (for the other three, one died after 4 days of treatment, and the two were still receiving therapy). At a median follow up of 9 (4 – 20) months, 4 patients had reached complete remission (CR) or probable CR (CRu), two were in good partial remission, and two had died with progressive disease. Conclusion. As2O3 induced apoptosis of MCL cells by post-transcription down-regulation of cyclin D1. Synergistic interactions were observed with AA and cytotoxics. Oral-As2O3, AA and chlorambucil were an active regimen for relapsed and therapy-refractory MCL, and treatment results compared favorably with other salvage regimens. This entirely oral regimen has several attractions, including outpatient treatment, low toxicity and cost.

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Anti-CD20 Antibody GA101 Shows Higher Cytotoxicity but Is Competitively Displaced by Rituximab in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v114.22.2704.2704 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2704-2704

Author(s):

Daniel A. Heinrich ◽

Christian Klein ◽

Kristina Decheva ◽

Marc Weinkauf ◽

Grit Hutter ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Deutschland Gmbh ◽

Mantle Cell ◽

Cd20 Antibody ◽

Anti Cd20

Abstract Abstract 2704 Poster Board II-680 Background: Mantle cell lymphoma (MCL) is characterized by a poor long-term prognosis with a median survival of 3–5 years. Type I anti-CD20 antibody rituximab has demonstrated a clear anti-proliferative effect in MCL and achieves increased response rates in combination with chemotherapy. GA101, a third-generation IgG1 anti-CD20 antibody displays improved ADCC and superior direct cell death induction by virtue of glycoengineering compared to rituximab and its targeting a type II epitope on CD20, respectively. Methods: Using a panel of MCL cell lines (Rec-1, HBL-2, Jeko-1, Granta-519, JVM-2 and Z-138) we determined the effect of GA101 alone as well as in combination with rituximab on cell viability and proliferation. Karpas-422 (Diffuse Large B-Cell Lymphoma) was used as a control cell line. MCL and Karpas-422 cells were treated with GA101 or rituximab at concentrations of 1 – 20μg/ml and rituximab. Cell viability was analyzed by trypan-blue exclusion tests at 0h, 24h, 48h and 72h. The panel of MCL cell lines and Karpas-422 were then treated with GA101 and rituximab each at 1 and 10 μg/ml to determine potential synergism of antibody combinations. Accordingly, a fractional product calculation was performed: synergism > 0,1; antagonism < −0,1. In addition, Western-blot and RNA-array-analyses were performed to elucidate potential intra-cellular downstream pathway mechanisms. Results: After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (65–75% cell reduction in Granta-519 and 35–40% in Rec-1). Intermediate results were gained for Z-138, HBL-2, Jeko-1 and JVM-2 and Karpas-422 (15–20%). rituximab mono-exposure at 12,5 μg/ml showed a 25% reduction of cell count in Granta-519, 20% in HBL-2 and < 5% in Rec-1, Jeko-1 and Z-138. Combination experiments suggested the competitive binding of the two antibodies. Thus, GA101 plus rituximab combination experiments resulted in a lower cytotoxicity than GA101 alone, according to fractional product calculations. Conclusions: Although GA101 is competitively displaced by rituximab, GA101 demonstrates higher efficacy in MCL cell lines than rituximab, even at a more than 10-fold lower concentration. Currently RNA-array- and Western blot analysis are being performed to identify the critical pathways responsible for the superior cytotoxicity of GA101. Disclosures: Klein: Discovery Oncology, Roche Diagnostics GmbH: Employment. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

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Novel CRM-1 Inhibitors for Therapy In Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.2734.2734 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2734-2734

Author(s):

Kejie Zhang ◽

Lan V Pham ◽

Liang Zhang ◽

Archito T. Tamayo ◽

Zhishuo Ou ◽

...

Keyword(s):

B Cells ◽

Western Blot ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Parp Cleavage ◽

Mantle Cell ◽

Dose Dependent

Abstract Abstract 2734 Chromosomal Region Maintenance 1 (CRM1) overexpression has been associated with cancer progression and mortality in several human cancers, suggesting that activation of nuclear export may play a role in human neoplasia and may serve as a novel target for the treatment of cancers. This overexpression of CRM1 may be related to the export of most tumor suppressor and growth regulatory proteins out of the nucleus, thereby functionally inactivating them. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that is not yet curable. The objective of our study was to investigate the status of CRM1 in MCL, both in MCL cell lines and primary MCL cells, in comparison to normal B cells, and to evaluate the therapeutic efficiency of CRM1 inhibition in MCL in vitro and in vivo, and to elucidate the mechanism of CRM1 inhibitor-mediated MCL cell apoptosis. We used 8 established MCL cell lines and primary cells from 4 patients with relapsed/refractory MCL. KPT185 and KPT276 are novel, highly selective, drug-like small molecular CRM1 inhibitors. Western Blot analysis showed that CRM1 was expressed in both the cytoplasm and nuclei of 8 MCL cell lines. CRM1 was mainly detected in nuclei of normal resting B cells; In contrast, CRM1 was primarily detected in the cytoplasm of freshly isolated primary MCL cells from patients with relapsed/refractory MCL. In 3H-thymidine incorporation assays, inhibition of CRM1 by KPT185 resulted in a significant dose-dependent growth inhibition of 8 MCL cells, with IC50 values range between 10 nM to 120 nM. The blastoid-variant MCL cell lines (Z-138 and Rec-1) were significantly more sensitive to KPT185 than the non-blastoid variant MCL cell lines. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that KPT185 induced MCL cells apoptosis in both time- and dose-dependent manners, but had no effect on cell cycle arrest. MCL cells treated with KPT185 for 12 hours showed caspase 3 activation and PARP cleavage. As shown in Western blot and confocal microscopy, blocking CRM1 activity by KPT185 in MCL cells up-regulated the protein expression of p53, a known CRM1-mediated export protein, and also induced CRM1 translocation to the nucleus and decreased CRM1 expression. In severe combined immunodeficient (SCID) mice bearing palpable Z-138 tumors, treatment with KPT-276 (similar structure to KPT-185 but improved animal pharmacokinetics), 50mg/kg or 150 mg/kg PO QDx5 each week, or cyclophosphamide 100 mg/kg on days 1–3, was initiated. Tumor growth was significantly inhibited (>75%) in all of treatment groups compared with vehicle control. Neutropenia and other cytotoxic-agent specific effects have not been observed in treated animals. In conclusion, CRM1 inhibitors inhibited growth of MCL cells in vitro and in vivo, and induced apoptosis of MCL cells via inhibition of CRM1 expression and blockage of its translocation with functional nuclear proteins. Our data suggest that novel CRM1 inhibitors provide a potential therapy for patients with relapsed/refractory MCL. Disclosures: No relevant conflicts of interest to declare.

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