scholarly journals Characterization of Potent Paracaspase MALT1 Inhibitors for Hematological Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1187-1187
Author(s):  
Wu Yin ◽  
Zhe Nie ◽  
Karen Dingley ◽  
Michael Trzoss ◽  
Goran Krilov ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
B Cell Lymphomas ◽  
Anti Tumor Activity

Abstract Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a key mediator of the NF-κB signaling pathway, the main driver of a subset of B-cell lymphomas and functions by forming a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL). Previously, we described the discovery of novel and potent MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells. Here, we highlight the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models and the combination potential with agents including standard-of-care. Results: Novel small molecule MALT1 inhibitors were identified using Schrodinger's proprietary physics-based free energy perturbation (FEP+) modeling technology. These molecules demonstrate strong MALT1 protein binding affinity, potent inhibition of MALT1 enzymatic activity and anti-proliferative activity in the activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines such as OCI-LY3 and OCI-LY10. In combination with approved agents, these inhibitors demonstrate strong combination potential with Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib in ABC-DLBCL cell lines. In ABC-DLBCL CDX models, our representative MALT1 inhibitor induces tumor regression as a single agent and complete tumor regression in combination with ibrutinib. Our representative MALT1 inhibitor, when tested in LY2298 PDX models, demonstrates similar results. In addition, our representative MALT1 inhibitor was explored in a CDX model derived from a Mantle cell lymphoma REC-1 cell line, and demonstrates strong anti-tumor activity of ~78% tumor growth inhibition (TGI) as a single agent. Conclusions: Schrodinger's novel, potent MALT1 protease small molecule inhibitors are efficacious in in vitro B-cell lymphoma cell proliferation assays and in in vivo B-cell lymphoma xenograft models. These data suggest that targeting MALT1 may expand therapeutic options for patients with selected B-cell lymphomas, such as ABC-DLBCL, with the possibility of expanding into other B-cell lymphomas such as MCL. Furthermore, these small molecule MALT1 inhibitors demonstrate potential in combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphomas. Taken together, the data presented here strongly underscore the therapeutic potential of our MALT1 inhibitor and support further evaluation in clinical trials. Disclosures Weiss: Schrodinger: Current Employment; ARTham Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Evaluation of Idelalisib with B-Cell Receptor or Orthogonal Pathway Inhibitors in Diffuse Large B-Cell Lymphoma Cell Lines in Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1845-1845 ◽  
Author(s):  
Sarah Meadows ◽  
Anella Yahiaoui ◽  
Rick Sorensen ◽  
Zhi-Hua Cui ◽  
Robert Brockett ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bet Inhibitor ◽  
Large B Cell Lymphoma

Abstract Background: Idelalisib, a selective oral inhibitor of PI3Kd, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation Bruton's tyrosine kinase (BTK) inhibitor with improved selectivity compared with ibrutinib. We report here the results of the combination of a PI3Kd inhibitor and GS-4059 in a diffuse large B-cell lymphoma (DLBCL) xenograft model, demonstrating supportive data for our ongoing combination trial in B-cell malignancies (NCT02457598). Additionally, we investigated preclinical orthogonal combination approaches for DLBCL. Methods: Growth inhibition was assessed using CellTiter-Glo Assay after 96 h incubation with idelalisib and GS-4059. CB17-SCID mice were irradiated, implanted subcutaneously with TMD8, and treated BID PO with the PI3Kd inhibitor GS-649443, GS-4059, or coformulated combination when tumors reached 200 mm3. Lysates from tumors or cell cultures were analyzed by Simple Western (Protein Simple). Synergy for antiproliferative effects was assessed using Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Results: Idelalisib and GS-4059 potently inhibited the ABC subtype DLBCL cell line TMD8, which is a B-cell receptor (BCR)-dependent line that exhibits chronic activated B-cell signaling due to mutations in CD79A/CD79B and MYD88 (Kim Y. et al., Hum Pathol, 2014). When a clinically relevant single concentration of idelalisib or GS-4059 was added in combination to a dose responsive effect of the other, a shift in EC50 on cell viability was seen. GS-4059 (50 nM) shifted the EC50 of idelalisib from 141 nM to 5 nM, a 28-fold shift. Idelalisib (1 µM) shifted the EC50 of GS-4059 from 27 nM to 2 nM, a 14-fold shift. Evaluation of downstream signaling pathways implicated in malignant B-cell survival and proliferation showed enhanced inhibition of pAkt S437, pBTK Y223, pErk1/2 T202/Y204, and MYC with a combination of idelalisib and GS-4059, more than either single agent alone. When TMD8 xenografts were treated with a PI3Kd tool compound, GS-649443, GS-4059 or a combination of the 2 inhibitors, a statistically significant decrease in tumor volume was seen as well as tumor regression, when compared with single agent effects (Figure 1A). Evaluation of TMD8 tumor lysates showed strong suppression of pAkt S437, pBTK Y223, pS6RP S235/236, and MYC in tumors treated with both GS-649443 and GS-4059 (Figure 1B). pS6RP S235/236 and MYC, in formalin-fixed paraffin-embedded (FFPE) TMD8 tumors, were profoundly inhibited in tumors treated with combination therapy compared to the monotherapies (Figure 1C). Since the combination of a PI3Kd inhibitor and GS-4059 led to TMD8 tumor regression, an effect correlated to strong down-modulation of MYC, the combination of idelalisib with a bromodomain and extra-terminal (BET) family inhibitor was explored as a potential new orthogonal combination approach for DLBCL. A panel of DLBCL cell lines was evaluated for inhibition of cell viability by idelalisib in combination with GS-5829, a BET inhibitor currently being evaluated in a phase 1 clinical trial. At clinically relevant concentrations, the combination of idelalisib and GS-5829 showed synergistic effects on cell viability in 2 of 6 ABC subtype, 4 of 5 GCB subtype, and 2 of 2 double-hit DLBCL cell. As compared with combination with other agents that inhibit the BCR pathway (GS-4059) or the Bcl-2 pathway (ABT-199), the broadest activity across cell lines was seen with the combination of idelalisib and GS-5829. Conclusion: Idelalisib and GS-4059 demonstrated synergistic inhibition of the TMD8 xenograft with concomitant inhibition of MYC. Screening of other targeted agent combinations in a panel of DLBCL lines revealed broad preclinical activity for the BET inhibitor GS-5829 in combination with idelalisib. This represents a potential orthogonal approach for a new therapeutic strategy for the treatment of B-cell malignancies. Figure 1A Figure 1A. Figure 1B Figure 1B. Figure 1C Figure 1C. Disclosures Meadows: Gilead Sciences: Employment. Yahiaoui:Gilead Sciences: Employment. Sorensen:Gilead Sciences: Employment. Cui:Gilead Sciences: Employment. Brockett:Gilead Sciences: Employment. Keegan:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment.

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 ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3936-3936 ◽  
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.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2761-2761
Author(s):  
Natalie M Czuczman ◽  
Matthew J. Barth ◽  
Richa Dwar ◽  
Cory Mavis ◽  
Pavel Klener ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Clinical Models ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

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

The SMAC Mimetic Lcl-161 Demonstrates Anti-Tumor Activity As a Single Agent and Decreases the Apoptotic Threshold of Chemotherapy Agents in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 61-61
Author(s):  
Kyle Runckel ◽  
Cory Mavis ◽  
Joseph Skitzki ◽  
Myron S. Czuczman ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
De Novo ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Abstract 61 The loss of response to apoptotic stimulus in lymphoma is a major obstacle in the treatment of primary and refractory B-cell malignancies. While the role of the anti-apoptotic Bcl-2 family proteins in the pathogenesis, maintenance, and progression of many sub-types of B-cell lymphoma is well characterized, the impact of the expression level(s) of other key regulatory proteins of cell death pathways (i.e. inhibitor of apoptosis [IAP] proteins) is less defined. The role of IAP proteins in the acquirement of resistance to rituximab or chemotherapy in B-cell lymphoma is unclear. Overexpression of IAP proteins and loss of expression of its antagonist, the second mitochondria-derived activator of caspases (SMAC) correlates with inferior clinical outcomes in a range of malignancies. Perhaps related to the acquisition of resistance, we found that rituximab-resistant cell lines (RRCL) have a deregulation of pro-apoptotic (Bak/Bax) and anti-apoptotic (Mcl-1, Bcl-XL) Bcl-2 family protein expression along with increased expression of the IAP protein survivin. Small molecule SMAC mimetics like LCL-161 are promising agents for lowering the threshold of tumor cell apoptosis, and represent a potential new avenue of therapy for de novo and refractory drug-resistant lymphoma. To this end, we evaluated the anti-tumor activity of LCL-161 in a range of rituximab-sensitive (RSCL), RRCL, and primary lymphoma cells. Cells were exposed to escalating doses of LCL-161 alone or in combination with various chemotherapy agents (i.e. etoposide, doxorubicin, vincristine, gemcitabine, carboplatin, oxaliplatin, bortezomib and cytarabine) for 48 and 72 hrs. Changes in cell viability and ATP content were determined by the CellTiter-Glo viability assay. Protein lysates were obtained from RSCL and RRCL to determine baseline levels of IAP protein family members. LCL-161 displayed significant anti-tumor activity against Burkitt's lymphoma (BL), diffuse large B-cell (DLBCL) and mantle cell lymphoma (MCL) cell lines. Activity was observed in both RSCL and RRCL cell lines. IC50 values for LCL-161 alone were between 35uM and 45uM for the DLBCL lines. Responses were slightly lower in BL and MCL compared to DLBCL cell lines. Synergistic activity between LCL-161 and several chemotherapy agents (e.g. gemcitabine, cytarabine, carboplatin, vincristine, etoposide and bortezomib) commonly used in the management of aggressive lymphoma was seen at physiologically-relevant doses. In vitro exposure of lymphoma cells to LCL-161 decreased the cytotoxic threshold of chemotherapy by 50%, while ex vivo studies with primary patient lymphoma samples showed a decrease of nearly 60%. In vivo studies using a xenograft SCID murine model are planned. In summary, LCL-161 has shown activity both as a single agent, and when combined with several chemotherapy agents in BL, MCL, and DLBCL cell lines as well as primary patient samples. Additionally, LCL-161 exhibits significant cytotoxic activity against RRCLs suggesting an ability to antagonize IAP proteins. This data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of de novo and refractory aggressive B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Triterpenoids Display Single Agent Anti-tumor Activity in a Transgenic Mouse Model of Chronic Lymphocytic Leukemia and Small B Cell Lymphoma

PLoS ONE ◽  
2007 ◽  
Vol 2 (6) ◽  
pp. e559 ◽  
Author(s):  
Christina L. Kress ◽  
Marina Konopleva ◽  
Vanesa Martínez-García ◽  
Maryla Krajewska ◽  
Sophie Lefebvre ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
B Cell ◽  
Transgenic Mouse ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Transgenic Mouse Model ◽  
Lymphocytic Leukemia ◽  
Anti Tumor Activity

scholarly journals Shared idiotype expression by chronic lymphocytic leukemia and B-cell lymphoma

Blood ◽  
1990 ◽  
Vol 76 (9) ◽  
pp. 1825-1829 ◽  
Author(s):  
M Chatterjee ◽  
M Barcos ◽  
T Han ◽  
XL Liu ◽  
Z Bernstein ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
B Cell Lymphomas ◽  
Surface Immunoglobulin ◽  
Leukemias And Lymphomas ◽  
Repetitive Pattern

Abstract Antiidiotype (Id) antibodies identify unique determinants within the surface immunoglobulin (Ig) that are present on B-cell tumors. Anti-Ids have been used for diagnosis and therapy of B-cell lymphoma and leukemia. A panel of 29 anti-Id monoclonal antibodies (MoAbs) that recognize shared idiotypes (SIds) on B-cell lymphomas was tested for reactivity with both B-cell leukemias and lymphomas. Ten of 40 (25%) cases of chronic lymphocytic leukemia (CLL) reacted with at least one of the 29 anti-SId MoAbs. Three cases reacted with more than one anti- SId MoAb, but there was no repetitive pattern of a single anti-SId MoAb reacting with a large proportion of CLL cases. In contrast, for B-cell lymphoma, in which 11 of 31 (36%) cases reacted, one anti-SId (B4–1) reacted with five of the positive cases; all were diffuse histology. Restricted anti-SId reactivity may lead to important insights into the etiology of certain B-cell lymphomas. In addition, these anti-SIds may obviate the need to develop “tailor-made” antibodies for individual patients.

Genomic Rearrangements Involving Programmed Death Ligands Are Recurrent In Primary Mediastinal Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 635-635 ◽  
Author(s):  
David D. W. Twa ◽  
Fong Chun Chan ◽  
Susana Ben-Neriah ◽  
Bruce W. Woolcock ◽  
King L. Tan ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Transcript Levels ◽  
Barr Virus ◽  
Large B Cell Lymphoma ◽  
Programmed Death ◽  
Large B Cell

Abstract Introduction Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive malignancy commonly diagnosed in young adult females. In recent years, mutational and gene expression profiling has established genotypic and phenotypic similarity of PMBCL with both classical Hodgkin and diffuse large B-cell lymphoma (DLBCL). In-depth analyses of genomes and transcriptomes have highlighted several inactivating mutations (SOCS1, TP53), chromosomal amplifications (2p, 9p, Xp, Xq) and translocations (CIITA) thought to be integral in establishing and/or maintaining the PMBCL phenotype. Programmed death ligands (PDL) 1 (CD274) and 2 (PDCD1LG2), which are located on chromosome 9p24.1, are two emerging genes of interest that have been shown to be altered in PMBCL and can induce T-cell anergy by binding to the receptor, programmed death 1. Here, we describe the recurrence of chromosomal rearrangements of the PDL locus in various B-cell lymphomas and explore the association of these rearrangements with transcript levels. Methods To establish the frequency of CD274 and PDCD1LG2 aberration, we conducted fluorescence in situ hybridization (FISH) on 551 clinical samples and 20 established cell lines using in-house break-apart probes. Epstein-Barr virus encoded RNA in situ hybridization was also carried out on the clinical cohort. The clinical cases, sourced from the British Columbia Cancer Agency’s Centre for Lymphoid Cancer tissue repository, consisted of 125 PMBCLs, 216 DLBCLs, 130 primary DLBCL of the central nervous system (PCNSL), 12 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL) and 68 follicular lymphomas (FL) with diagnoses based on the WHO classification. The DLBCL cohort could be further subdivided into 134 nodal DLBCLs and 82 testicular DLBCLs (T-DLBCL). Quantitative real-time PCR (qRT-PCR) was subsequently conducted on 17 cell lines and a clinical sub-cohort of 76 samples, for which fresh-frozen material was available, to determine the effect of mutations on transcript expression. We then characterized the PDL aberrations of two clinical PMBCL cases and three cell lines (DEV, L-428, L-1236), at base pair resolution, by applying the bioinformatic tools, nFuse, deFuse and destruct to both newly produced and previously published whole genome (WGS) and whole transcriptome (RNA-seq) libraries. Results FISH revealed a PDL locus (9p24.1) break-apart frequency of 20% (25/125) in PMBCL. There were no differences in any known clinical parameters or frequency of Epstein-Barr virus positivity between positive and negative PDL break-apart cases. Break-apart frequencies in other malignancies were calculated to be 3% in DLBCL, 7% in T-DLBCL and 1% in PCNSL; no positive cases were identified in either NLPHL or FL. The proportion of break-apart positive cases was significantly higher in PMBCL as compared to the other lymphomas surveyed (P < 0.05). Further, in agreement with the published literature, we observed an amplification frequency of the PDL locus in 36% (45/125) of PMBCLs. qRT-PCR established that PDCD1LG2 transcript levels were significantly higher in cases with 9p24.1 locus rearrangements compared to copy number neutral (P = 0.0003), gain (P = 0.001) and amplified cases (P = 0.005). Likewise, CD274 transcript levels were significantly higher in rearranged cases compared to copy number neutral cases (P = 0.03). Following the analysis of WGS and RNA-seq libraries, we were able to characterize four novel fusion transcripts involving the 9p24.1 locus: PDCD1LG2-NRG1 (PMBCL clinical case), PDCD1LG2-IGHV7-81 (L-1236), CIITA-PDCD1LG2 (DEV) and KIAA1432-CLDN14 (L-428). Aberrations involving both NRG1 and CIITA have previously been implicated in breast cancer and B-cell lymphomas, respectively. We also identified a translocation in another PMBCL clinical case with breakpoints in the intergenic spaces near LRMP and CD274, though this rearrangement did not produce a fusion transcript. Conclusion Taken together, our findings show that rearrangement of the PDL locus is recurrent in PMBCL, characteristic of PMBCL and leads to overexpression of PDL transcripts. Given the well-referenced function of PDLs in repressing the anti-tumor response, these data suggest that targeting the PDL axis in a subgroup of B-cell lymphomas holds clinical promise. Disclosures: No relevant conflicts of interest to declare.

High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3022-3022
Author(s):  
Jonathan Scott Rink ◽  
Sol Misener ◽  
Osman Cen ◽  
Shuo Yang ◽  
Leo I. Gordon ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Density Lipoprotein ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Cholesterol Homeostasis ◽  
B Cell Lymphomas ◽  
Bcr Signaling

Abstract Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas. Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk. Results: The Burkitt's lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death. Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. Disclosures Thaxton: Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

scholarly journals TCL1: A Shared Tumor-Associated Antigen for Immunotherapy Against B-Cell Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 105-105
Author(s):  
Jinsheng Weng ◽  
Seema Rawal ◽  
Hyun Jun Park ◽  
Rakesh Sharma ◽  
Sattva S. Neelapu
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
B Cell Lymphomas ◽  
Therapeutic Vaccines ◽  
Normal Tissues ◽  
Tumor Associated Antigen

Abstract Abstract 105 Rituximab-based chemotherapy regimens have improved complete response rates, progression-free survival, and overall survival of B-cell non-Hodgkin lymphoma patients. However, most patients relapse and die of their lymphoma. To further improve clinical outcome, novel strategies that eradicate minimal residual disease (MRD) after induction therapy are needed. Therapeutic vaccines may induce antitumor antibody and T-cell responses and may eradicate MRD by complementary mechanisms. A recent randomized phase III clinical trial showed that patient-specific idiotype vaccination improves disease-free survival when administered in the setting of MRD in follicular lymphoma (FL), providing proof of principle that therapeutic vaccines can improve clinical outcome (Schuster et al, J Clin Oncol, 2011). However, generation of a custom-made vaccine formulation for each patient can be expensive and time consuming. To overcome these difficulties, identification of novel shared lymphoma-associated antigens is necessary. The T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene is a co-activator of Akt and promotes cell proliferation and survival. Ectopic expression of TCL1 in B cells in transgenic mice results in the development of B-cell malignancies and TCL1 was reported to be aberrantly expressed in multiple human B cell malignancies. Here, we determined whether TCL1 can serve as a novel shared tumor-associated antigen in B cell lymphomas. We analyzed the expression pattern of TCL1 in human B cell lymphomas and normal tissues by real-time PCR, flow cytometry, immunohistochemistry, and Western blotting. TCL1 mRNA transcripts were hyperexpressed in multiple types of primary B cell lymphomas [chronic lymphocytic leukemia (CLL), n=7; mantle cell lymphoma (MCL), n=6; FL, n=12, diffuse large B-cell lymphoma (DLBCL), n=5] as compared with B cells derived from normal donors. TCL1 mRNA was not detected in other normal tissues that included T cells, adipose tissue, esophagus, ovary, spleen, bladder, heart, placenta, brain, kidney, prostate, liver, muscle, thyroid, colon, lung, intestine, trachea, thymus, and cervix. Very low TCL1 mRNA was detected in testis. By flow cytometry, immunohistochemistry, and/or Western blotting we observed that the TCL1 protein is hyperexpressed in CLL, MCL, DLBCL, Burkitts lymphoma, and FL, but not splenic marginal zone lymphoma. To determine whether TCL1 is immunogenic, we synthesized overlapping 15-mer peptides spanning the entire length of the TCL1 protein and stimulated PBMC from HLA-A*0201+ (HLA-A2+) normal donors to generate peptide-specific T cells. We found that TCL165–79 peptide (TQIGPSLLPIMWQLY) consistently induced T cells that secreted significant amount of IFN-γ from 3 normal donors. By intracellular cytokine assay, we determined that the TCL165–79 peptide-specific IFN-g was produced by CD8+ T cells but not CD4+ T cells. Using a panel of peptide-pulsed EBV-transformed B-cell lines that were mismatched at the MHC Class I locus and MHC Class I blocking antibodies, we determined that HLA-A2 is the restriction element of the TCL165–79 peptide-specific T cells. Consistent with this, TCL165–79 peptide-specific CD8+ T cells lysed TCL1 expressing HLA-A2+ MCL cell lines but did not lyse, TCL1 expressing HLA-A2− myeloma cell line. Using a panel of peptides truncated progressively by one amino acid at the N- and C-termini of the TCL165–79peptide, we determined that TCL171–78(LLPIMWQL) is the minimal epitope that bound to HLA-A2. Furthermore, cytotoxic T lymphocytes (CTL) specific to this peptide efficiently killed lymphoma cell lines and primary lymphoma cells from CLL, MCL, DLBCL, and FL in an HLA-A2-restricted manner. Using TCL171–78 peptide-specific HLA-A2 tetramers, we found that TCL1 epitope-specific CTLs were present in the peripheral blood and/or tumor-infiltrating lymphocytes of B-cell lymphoma patients (n=8). More importantly, these TCL1 epitope-specific CTLs could be expanded in vitro and lysed autologous and allogeneic tumor cells in an HLA-A2-restricted manner. In conclusion, our results suggest that TCL1 is naturally processed and presented on the surface of primary lymphoma cells for recognition by CTL and can serve as a novel, shared tumor-associated antigen for therapeutic vaccine development against common B-cell lymphomas including CLL, MCL, DLBCL, and FL. Disclosures: Neelapu: Biovest International, Inc.: Research Funding.

scholarly journals Epstein Barr virus-positive B-cell lymphoma is highly vulnerable to MDM2 inhibitors in vivo

2021 ◽  
Author(s):  
Xiaoshan Zhang ◽  
Ran Zhang ◽  
Chenghui Ren ◽  
Yi Xu ◽  
Shuhong Wu ◽  
...  
Keyword(s):  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
T Cell Therapy ◽  
Barr Virus ◽  
Epstein Barr

Epstein-Barr virus (EBV)-positive B-cell lymphomas are common in immunocompromised patients and remain an unmet medical need. Here we report that MDM2 inhibitors (MDM2i) navtemadlin and idasanutlin have potent in vivo activity in EBV+ B-cell lymphoma established in immunocompromised mice. Tumor regression was observed in all 5 EBV+ xenograft-associated B-cell lymphomas treated with navtemadlin or idasanutlin. Molecular characterization showed that treatment with MDM2i resulted in activation of p53 pathways and downregulation of cell cycle effectors in human lymphoma cell lines that either were EBV+ or had undetectable expression of BCL6, a transcriptional inhibitor of the TP53 gene. Moreover, treatment with navtemadlin resulted in tumor regression and prevented systemic dissemination of EBV+ lymphoma derived from 2 juvenile patients with posttransplant lymphoproliferative diseases, including one whose tumor was resistant to virus-specific T-cell therapy. These results provide proof-of-concept for targeted therapy of EBV+ lymphoma with MDM2i and the feasibility of using EBV infection or loss of BCL6 expression to identify responders to MDM2i.

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