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

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

Blood ◽

10.1182/blood-2018-99-118480 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5379-5379

Author(s):

Sumera Khan ◽

Kyle Runckel ◽

Cory Mavis ◽

Matthew J. Barth ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Parp Cleavage ◽

Synergistic Activity ◽

In Vitro Exposure ◽

Induction Of Apoptosis

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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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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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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Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Blood ◽

10.1182/blood.v122.21.5150.5150 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5150-5150

Author(s):

Indira D Joshi ◽

Mitchell R Smith

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Single Agent ◽

Annexin V ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Low Level ◽

Induced Apoptosis

Abstract Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL. Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2, and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures: No relevant conflicts of interest to declare.

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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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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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Phospho-SFKs (Y416) As a Potential Predictive Marker for Dasatinib Therapy in B-Cell Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3727.3727 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3727-3727

Author(s):

Xiaoxian Zhao ◽

Eric D. Hsi

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Predictive Marker ◽

Lymphocytic Leukemia ◽

Lymphoma Cell ◽

Raji Cells ◽

B Lymphoma ◽

Induced Apoptosis

Abstract Abstract 3727 The Src family of protein tyrosine kinases (SFKs) plays an important role in regulating multiple signaling networks including B-cell receptors (BCR) mediated pathways and abnormal SFK kinase activation promotes B lymphoma cell survival. Dasatinib is an oral BCR/ABL1 and SKF inhibitor useful in the treatment of imatinib-resistant CML and Ph+ALL. Given its broad inhibitory activity, dasatinib may be useful in the treatment of other hematologic malignancies and having a biologic predictor of response would be helpful in rational selection of this targeted therapeutic. We hypothesized this agent could have therapeutic potential against lymphoma patients with p-SFK (Y416) expression. Constitutive p-SFK (Y416) expression (indicating active SFK signaling) was detected in both B-lymphoma cell lines and a subset of primary lymphoma tissues including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma and small lymphocytic leukemia (SLL). Dasatinib induced apoptosis of B-lymphoma Raji cells correlated with high level expression of constitutive p-SFK (Y416) and dasatinib rapidly reduced the global level of tyrosine phosphorylations including p-SFK (Y416) in Raji cells. 19 of 28 lymphoma cases (67.9%) were positive for p-SFK (Y416) by Western blot analysis. Dasatinib displayed in vitro dose-dependent (10–200 nM) killing activity against 17 of those 19 p-SFK (Y416) cases (89.5%). In contrast, only 2 of 9 p-SFK (Y416) negative cases (22.2%) had response to dasatinib exposure. Thus presence of p-SFK (Y416) was associated with in vitro response to dasatinib (p <0.0001). Similar to tested Raji cells, dasatinib induced apoptosis of primary B-cell lymphoma cells was accompanied with de-phosphorylation of p-SFK (Y416) and cleavage of caspase-3. 6 of 9 tested CLL cases were p-SFK (Y416) positive. Dasatinib displayed in vitro killing activities against 5 of 6 positive cases with a range of killing from 12% to 53% (mean 26.5%) of malignant B-cells. Meanwhile, one of three negative cases showed response to dasatinib (17% killing). We conclude that p-SFK (Y416) may be a useful predictive marker of response to dasatinib. Potential uses include pharmacodynamic monitoring or integral biomarker for selecting appropriate patients with B-cell malignancies for clinical trials. Disclosures: No relevant conflicts of interest to declare.

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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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