Targeting XIAP Via Degradation of MDM-2 By MX69 in Aggressive Lymphomas

Abstract Background: The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistance cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. MX69, a dual inhibitor of Mdm2 and XIAP that indirectly downregulates XIAP, is undergoing pre-clinical testing. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decrease XIAP translation and activation of caspase 3, 7 and 9 as well as PARP cleavage leading to apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69. Materials and Methods: A panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Changes in Mdm2, p53, XIAP and PARP expressions were determined following MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was evaluated by Annexin V/propidium iodine staining. Subsequently, cell lines were exposed to MX69 (0-80 uM), in combination with Doxorubicin (0-1uM), Cytarabine(0-50uM), Vincristine (0-10nM), Etoposide(0-50uM), Carboplatin (0-20uM), Ixazomib (0-1.5uM), Ibrutinib (0-20uM) and Venetoclax (0-10uM) for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, MX69 single agent exposure induced cell death in a dose and time-dependent manner in all cell lines tested. Western blotting studies confirmed downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro exposure to MX69. Induction of apoptosis was observed by flow cytometry in all cell lines tested. The combination of MX69 with Doxorubicin, Cytarabine, Vincristine, Ixazomib, Carboplatin, Etoposide, Ibrutinib, and Venetoclax resulted in significant synergistic activity. The strongest CI of synergy was observed when cell lines were exposed to MX69 and Venetoclax, Ixazomib, Etoposide or Ibrutinib. Conclusion: Our data suggests that in vitro exposure of a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) to MX69 resulted in anti-tumor activity. Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior SiRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as Venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are been planned. Targeting Mdm2 and XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

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Metformin Induces p53-Dependent Mitochondrial Stress in Therapy-Sensitive and -Resistant Lymphoma Pre-Clinical Model and Primary Patients Sample with B-Cell Non-Hodgkin Lymphoma (NHL)

Blood ◽

10.1182/blood.v126.23.4008.4008 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4008-4008

Author(s):

Juan J Gu ◽

Qunling Zhang ◽

Cory Mavis ◽

Myron S. Czuczman ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Board ◽

In Vitro Exposure ◽

Induction Of Apoptosis

Abstract Introduction: Metformin, a guanidine originally derived for Galega officinalis (French lilac), has been widely prescribed to type II diabetics since 1950 and well known for its safety toxicity profile. Recently, metformin use was associated with a decrease in risk cancer development and lower cancer-related mortality among breast, colorectal, prostrate, lung, hepatic and ovarian cancer patients. Our group previously reported that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). The mechanism(s) of action underlying the antitumor effect of metformin remains not to be fully elucidated. Previous research done in our laboratory revealed that metformin inhibited cell proliferation through repressing PCNA and p21 proteins. Here, we report that metformin activates tumor suppressor p53 by suppressing MDMX in pre-clinical lymphoma models. Methods: A panel of rituximab-sensitive (RSCL), rituximab-resistant (RRCL) cell lines and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of metformin (0-64mM). Changes in cell viability were determined by Presto Blue (Sigma) assay in cell lines and Titer Glo in patient samples. Changes in MDMX, MDM2 and p53 expression levels were determined by western blotting after exposure cells to metformin. MDMX-MDM2-p53 interactions and p53 ubiquitination following in vitro exposure to metformin were determined by immunoprecipitation of p53 and probing for MDMX, MDM2, ubiquitin and p53 in RSCL and RRCLs. Loss of Dψm or induction of apoptosis following metformin exposure were assessed by DiOC6 or Annexin V/PI staining and flow cytometry. Oxidative stress induced by metformin was measured by flow cytometry using dihydrorhodamine-123 (DHR-123) dye. Result: Metformin induced a dose-and time- dependent cell death in cell lines and primary patient tumor cells. In vitro exposure of lymphoma cell to metformin resulted in a decrease in MDMX levels. Immunoprecipitation studies demonstrated that following exposure to metformin, MDMX bound less to p53 leading to less p53 ubiquitination. In vitro exposure of RSCL or RRCL to metformin resulted in the expression of p53 regulated BH3 single domain proteins (Noxa and Puma). Moreover, metformin repressed mitochondrial potential, induced reactive oxidative species (ROS) generation and triggered apoptosis. Conclusion: Our data suggests that metformin had anti-tumor activity against RSCL, RRCL and primary tumor cells isolated from lymphoma patients. The down-regulation of MDMX and re-activation p53 function following metformin exposure may contribute to the disruption in the mitochondria potential, generation of ROS and induction of apoptosis observed in our models RSCL and RRCL. Our finding highlights a potential role for metformin in the treatment of B-cell malignancies. (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 Funds) Disclosures Czuczman: MorphoSys: Consultancy; Boehringer-Ingelheim: Other: Advisory Board; Celgene: Employment; Immunogen: Other: Advisory board.

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Targeted inhibition of PI3Kα/δ is synergistic with BCL-2 blockade in genetically defined subtypes of DLBCL

Blood ◽

10.1182/blood-2018-08-872465 ◽

2019 ◽

Vol 133 (1) ◽

pp. 70-80 ◽

Cited By ~ 30

Author(s):

Kamil Bojarczuk ◽

Kirsty Wienand ◽

Jeremy A. Ryan ◽

Linfeng Chen ◽

Mariana Villalobos-Ortiz ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Single Agent ◽

Xenograft Model ◽

Myeloid Cell ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Synergistic Activity ◽

Genetic Bases

Abstract Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1–dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2–mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.

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Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines,

Blood ◽

10.1182/blood.v118.21.3734.3734 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3734-3734

Author(s):

Cory Mavis ◽

Sarah Frys ◽

Juan Gu ◽

John Gibbs ◽

Myron S. Czuczman ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

In Vitro Exposure ◽

Anti Tumor Activity

Abstract Abstract 3734 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Entinostat (MS-275) is a novel and potent DAC class I inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant (r/r) B-cell lymphoma, we studied the anti-tumor activity of entinostat as a single agent or in combination with the proteasome inhibitor bortezomib (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or r/r B-cell lymphoma. In addition, we characterized the mechanisms responsible for entinostat's anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of entinostat (0.1 to 20uM) +/− BTZ (1–10nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from entinostat +/− BTZ exposed cells and changes in members of Bcl-2 and cell cycle family proteins were evaluated by Western blotting. Finally, to characterize entinostat's mechanisms-of-action, lymphoma cells were exposed to entinostat with or without pan-caspase (Q-VD-OPh, 5mM) and changes in cell viability were detected. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=32). In addition, in vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Moreover synergistic activity was observed by combining entinostat with BTZ in vitro. The pharmacological interactions between entinostat and proteasome inhibitor could be explained in part by each agent's effects on the expression levels of cell cycle proteins. In vitro exposure of lymphoma cells to entinostat resulted in p21 upregulation and p53 down-regulation, whereas BTZ exposure lead to up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL. Together this data suggests that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways. Our data suggests that entinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of BTZ. A better understanding in the molecular events (caspase-dependent and -independent) triggered by entinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these novel agents in future clinical trials. Disclosures: Czuczman: Millennium: Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy.

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Panobinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) In Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines

Blood ◽

10.1182/blood.v116.21.3936.3936 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3936-3936 ◽

Cited By ~ 3

Author(s):

Francisco J. Hernandez-Ilizaliturri ◽

Cory Mavis ◽

Ilir Maraj ◽

Mohammad Muhsin Chisti ◽

John Gibbs ◽

...

Keyword(s):

Western Blot ◽

B Cell ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Anti Tumor Activity

Abstract Abstract 3936 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Panobinostat (LBH589) is a novel and potent DAC class I and II inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant B-cell lymphoma., We studied the anti-tumor activity of panobinostat as a single agent or in combination with the proteasome inhibitor (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed B-cell lymphoma. In addition, we characterized the mechanisms responsible for panobinostat anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of panobinostat (0.5-5nM) +/− BTZ (1-5nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Subsequently, protein lysates were isolated from panobinostat +/− BTZ exposed cells and changes in members of Bcl-2 family proteins were evaluated by Western blot. Finally, to characterize panobinostat's mechanisms-of-action, lymphoma cells were exposed to panobinostat with or without pan-caspase (Q-VD-OPh, 5mM) or autophagy (3-methyladenine [3MA] 5mM) inhibitors and changes in cell viability were detected as above. Optimal experimental conditions were confirmed by Western blot. Panobinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=25). In addition, synergistic activity was observed by combining panobinostat with BTZ in vitro. The pharmacological interactions between panobinostat and proteasome inhibitor could be explained in part by the effects each agent has on the expression levels of Bcl-2 family members. In vitro exposure of lymphoma cells to panobinostat resulted in Bcl-XL down-regulation, whereas BTZ exposure causes up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished panobinostat anti-tumor activity in RSCL but not in RRCL. On the other hand, exposure of RRCL to 3MA, significantly inhibited the anti-tumor activity of panobinostat in RRCL. Together this data suggest that, panobinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and -independent pathways. Our data suggests that panobinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of a proteasome inhibitor (BTZ). A better understanding in the molecular events (caspase-dependent and -independent) triggered by panobinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these exciting agents in future clinical trials. (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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Effects of Proteasome Inhibition in the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Blood ◽

10.1182/blood-2018-99-116510 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2942-2942

Author(s):

Shalin K. Kothari ◽

Cory Mavis ◽

Juan Gu ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Scid Mice ◽

Parp Cleavage ◽

Synergistic Activity ◽

Clinical Models ◽

Anti Tumor Activity

Abstract Background: At the molecular level, mantle cell lymphoma (MCL) is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) and cell cycle (cyclin D1) regulatory proteins. Perhaps related to this, the clinical outcome of MCL continues to be poor specially for those patients with disease progression after high dose chemotherapy and autologous stem cell rescue and/or BTK inhibitors, stressing the need to develop novel therapeutic strategies or optimize current available options. Venetoclax (V), a highly selective Bcl-2 inhibitor, has shown modest activity against relapsed/refractory MCL. Over-expression of Mcl-1 has been postulated to be a mechanism of resistance to V limiting its anti-tumor activity in subtypes of lymphoma including MCL. The lethality by proteasome inhibitors (PIs) has been associated with changes in the Bcl-2 family members (Bax, Noxa, Mcl-1 and Bcl-XL) in lymphoma pre-clinical models, making them ideal agents to combine with V. To this end, we studied the anti-tumor activity of combining PIs with V in MCL pre-clinical models. Materials and Methods: A panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell lines (Jeko araC, HBL-2 araC, and Mino araC) were exposed to V, Bortezomib (BTZ), carfilzomib (CFZ), or ixazomib (IXZ) for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of V (0.001uM-5uM) and CFZ (1.5625nM-50nM), BTZ (3.125nM-100nM) or IXZ (3.125nM-100nM). In addition, primary tumor cells isolated from B-cell lymphoma patients (N=21) including MCL patients were exposed to V +/- BTZ or CFZ for 48 hrs. Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn software program. Induction of apoptosis was detected by Annexin V/Propidium iodine staining and PARP cleavage. Changes in Bcl-2 and cell cycle regulatory proteins were evaluated by Western blotting in HBL-2 cells. For in vivo experiments, 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 HBL-2 cells via tail vein injection (IV). Subsequently, SCID mice were treated with V (100mg/kg/dose via gastric lavage on days 3-7, 10-14 and 17-21) or IXZ (6mg/kg/dose IV days 3, 6, 10, 13, 17 and 20) or combination of both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro exposure of MCL cell lines to either V, BTZ, CFZ, and IXZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both V with CFZ or IXZ at known sub-therapeutic and therapeutic doses of individual agents measured by ATP content and apoptosis potential. Anti-tumor activity was observed in cytarabine sensitive and resistant cell lines. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. In vitro exposure of MCL cell lines with the lowest IC50 (HBL-2) to V and PIs (BTZ, CFZ, or IXZ) resulted in the upregulation of Noxa, BIM, Mcl-1 cleavage form (pro-apoptotic) and downregulation of Bcl-XL leading to PARP cleavage and apoptosis. In vivo treatment of MCL bearing SCID mice with V resulted in significant anti-tumor activity when compared to single agent IXZ treated or control animals. Of interest, MCL bearing SCID animals treated with V and IXZ exhibited a better disease control and the survival was longer than SCID animals treated with V or IXZ single agent (P<0.05). Conclusion: Our data suggests that V exhibits strong synergistic activity with PIs, especially with CFZ (in vitro) or IXZ (in vitro and in vivo). Together, our data supports the evaluation of V in combination with readily available novel PIs (IXZ or CFZ) in relapsed/refractory MCL. (Supported by LRF grant 555463, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) Disclosures No relevant conflicts of interest to declare.

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Karonudib has potent anti-tumor effects in preclinical models of B-cell lymphoma

Scientific Reports ◽

10.1038/s41598-021-85613-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Morten P. Oksvold ◽

Ulrika Warpman Berglund ◽

Helge Gad ◽

Baoyan Bai ◽

Trond Stokke ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Dlbcl Patient ◽

Cycle Arrest ◽

Induced Apoptosis ◽

Tumor Biopsies

AbstractChemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.

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Characterization of two new high-grade B-cell lymphoma cell lines with MYC and BCL2 rearrangements that are suitable for in vitro drug sensitivity studies

Leukemia & Lymphoma ◽

10.1080/10428194.2018.1508663 ◽

2018 ◽

Vol 60 (4) ◽

pp. 1043-1052

Author(s):

Marie-Sophie Dheur ◽

Hélène A. Poirel ◽

Geneviève Ameye ◽

Gaëlle Tilman ◽

Pascale Saussoy ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Drug Sensitivity ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

High Grade ◽

Sensitivity Studies

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Repurposing dasatinib for diffuse large B cell lymphoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905239116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 16981-16986 ◽

Cited By ~ 5

Author(s):

Claudio Scuoppo ◽

Jiguang Wang ◽

Mirjana Persaud ◽

Sandeep K. Mittan ◽

Katia Basso ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Kinase Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Multikinase Inhibitor ◽

Large B Cell Lymphoma ◽

Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

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Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

Journal of Virology ◽

10.1128/jvi.77.3.2134-2146.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2134-2146 ◽

Cited By ~ 195

Author(s):

Vicky M.-H. Sung ◽

Shigetaka Shimodaira ◽

Alison L. Doughty ◽

Gaston R. Picchio ◽

Huong Can ◽

...

Keyword(s):

Hepatitis C ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Culture System ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

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Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2417.2417 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2417-2417

Author(s):

Olga Ritz ◽

Jochen K Lennerz ◽

Karolin Rommel ◽

Karola Dorsch ◽

Elena Kelsch ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Proximal Promoter ◽

Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

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