Mouse Models of Human Mantle Cell Lymphoma for the Study of Disease Biology and for Pre-Clinical Assessment of Experimental Treatment Approaches.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2759-2759
Author(s):  
Pavel Klener ◽  
Magdalena Klanova ◽  
Tomas Soukup ◽  
Jan Molinsky ◽  
Jan Zivny ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Single Agent ◽  
Cell Receptor ◽  
Ki 67 ◽  
Disease Biology ◽  
B Cell Receptor Signaling

Abstract Abstract 2759 Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. MCL animal models for the study of disease biology and for the testing of novel agents are scarce. We established and characterized various in vivo models of metastatic blastoid human MCL by tail vein injection of five MCL cell lines (Jeko-1, HBL-2, Mino, Rec-1, Granta-519) into the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ immunodeficient mice. Untreated animals were then observed to evaluate differences in the pattern of lymphoma growth and overall survival (OS) between different cell lines. We analyzed infiltration of selected murine organs (i.e. bone marrow [BM], spleen, liver, brain, kidneys, and enlarged lymph nodes [LN]) by immunohistochemistry (IHC) (CD20, Ki-67) at four different time-points related to OS. Extent of organ infiltration with human MCL cells was estimated using the Image-Pro Plus 5.1 software within 20 samples from different organ areas. Subsequently, we analyzed gene expression of Jeko-1 and Mino cells obtained from the xenografted animals (in vivo growing cells) compared to the cells cultured in vitro (controls).MCL cells isolated from various murine organs (the BM, liver, spleen, and LN) or in vitro cultured cells were magnetically sorted by CD45-microbeads. Gene expression analyses were carried out using Illumina BeadChips, and the data were functionally clustered with DAVID Bioinformatics tool. In addition, differences in surface expression of selected antigens were compared between in vivo vs. in vitro grown MCL cells by flow cytometry. Finally, we evaluated the anti-tumor activity of single-agent chemotherapy agents (cytarabine, fludarabine, bendamustine, and cisplatin), monoclonal antibodies (rituximab, ofatumumab, bevacizumab) or targeted agents (bortezomib, temsirolimus) in Jeko-1 and Mino bearing mice. Tumor engraftment was achieved in all the cell lines tested. The median overall survival (OS) of mice xenografted with 1–10×106 MCL cells ranged from 22 to 55 days depending on the cell line used. The principal site of engraftment and proliferation niche for all MCL cell lines was the bone marrow. MCL cells disseminated to other murine organs including the spleen, liver and brain. Development of enlarged lymph nodes (peripheral, intraabdominal) and/or extranodal MCL masses (subcutaneous tumors) were associated with Mino, while infiltration of the ovaries was inconstant finding in Jeko-1 xenografted mice. Mice xenografted with Jeko-1, HBL-2 and Granta-519 showed leukemization of peripheral blood before death. Gene expression studies of Jeko-1 and Mino in vivo growing cells revealed that the genes from the “B-cell receptor signaling” and the “oxidative-phosphorylation” pathways were the most upregulated or downregulated, respectively. In vivo growing Jeko-1 cells showed upregulation of CD31/PECAM, CD37, CD38, CD44, CD164, and downregulation of podoplanin and CXCR4. In vivo growing Mino cells had upregulation of CD23, but downregulation of CD37, CD40, CD44, CD54, CD138, CXCR4, CCR7 and podoplanin. Both Jeko-1 and Mino cells isolated from the BM (but not from the spleen, liver or LN) were significantly more sensitive to cytarabine (2–4 fold) and cisplatin (2 fold) than in vitro growing controls. Single-agent therapy of Jeko-1 and Mino bearing mice with either a chemotherapy agent, monoclonal antibody, or targeted agent resulted in significant prolongation of OS compared to untreated controls. Treatment of Jeko-1 and HBL2 bearing mice with single-agent cisplatin, single-agent cytarabine or combination of both agents revealed that the therapy with single-agent cisplatin was associated with the longest prolongation of OS. Moreover, IHC analyses of the BM, spleen and liver of the treated animals confirmed the most profound suppression of both MCL infiltration (CD20) and proliferation rate (Ki-67) in the single-agent cisplatin cohort compared to the other cohorts. In summary, the mouse models can be used for the study of MCL biology, as well as for preclinical assessment of experimental therapy of MCL including agents that cannot be properly tested in vitro (e.g. monoclonal antibodies, pro-drugs, anti-angiogenic agents, inhibitors of B-cell receptor signaling etc.). Financial Support: IGA-MZ NT13201-4/2012, GAUK 259211/110709, GAUK 446211, UNCE 204021, PRVOUK P24/LF1/3, PRVOUK 1–5101–280002 PVK, SVV-2012–254260507 Disclosures: No relevant conflicts of interest to declare.

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3017-3017
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Petra Hillmann ◽  
Filippo Spriano ◽  
Ivo Kwee ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Apoptosis Induction ◽  
In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13569-e13569
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle Bongero ◽  
Christine McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

scholarly journals Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Research Report ◽  
Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

scholarly journals Venetoclax Synergizes with Radiation Therapy for Treatment of B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 467-467
Author(s):  
Shyril O'Steen ◽  
Amelia Waltman ◽  
Garrett Booth ◽  
Aimee L Kenoyer ◽  
Margaret Nartea ◽  
...  
Keyword(s):  
B Cell ◽  
Survival Time ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Combination Group ◽  
Mean Survival Time

Abstract Introduction: An estimated 19,970 Americans died of non-Hodgkin lymphoma (NHL) in 2015, with diffuse large B-cell lymphoma (DLBCL) accounting for roughly 30% of newly diagnosed NHL. Our study focuses on three NHL subtypes: germinal center (GCB)-DLBCL, the most common DLBCL subtype; activated (ABC)-DLBCL, a particularly aggressive and high-risk subtype; and mantle cell lymphoma (MCL), considered incurable. Constitutive B-cell receptor signaling is implicated in the pathogenesis of ABC-DLBCL and MCL and may couple with aberrant apoptotic BCL-2 pathway proteins. The BCL-2 inhibitor venetoclax is a promising targeted agent that promotes apoptosis in a variety of NHL subtypes, but is almost never curative as a single agent. Radiotherapy promotes apoptosis by creating DNA strand breaks, and we hypothesized that the combination of radiotherapy and venetoclax would act synergistically in NHL to increase the probability of cures. Methods: We tested in vitro killing efficacy of sublethal 137Cesium irradiation combined with venetoclax in 15 cell lines, representing a diversity of NHL subtypes. Cells were treated with 137Cesium and venetoclax in 8 x 8 dose combination matrices, incubated 72-120 hrs, then assayed for viability with Celltiter-Glo (Promega). The degree of treatment antagonism, additivity, or synergism was determined using isobolographic analyses. For in vivo studies, we tested combinations of venetoclax with either 137Cesium total body irradiation (TBI), or CD20 pre-targeted radioimmunotherapy (PRIT), in threetumor models chosen for divergent single agent sensitivities. Tumor xenografts of Rec-1 (MCL), U2932 (ABC-DLBCL), and SU-DHL-6 (GCB-DLBCL) were produced by subcutaneous flank injection of 10 x 106 cells in male and female NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice. When tumor volumes were 50 mm3, mice (n = 8-12/group) were treated with either venetoclax (100-200mg/kg daily for 10-30 days), diluent control, TBI (single dose, 6-10 Gy 137Cesium), or a combination of venetoclax and TBI. In PRIT studies, mice were coinjected with 300µg unlabeled streptavidin-conjugated anti-CD20 antibody (murine IgG2a) and 400µg HB8181 (IgG2a isotype control to block non-specific binding) in place of TBI. Twenty-one hours later, 5.8 nmol biotin-galactose "clearing agent" was administered, followed in 3 hours by 1.2 nmol DOTA-biotin labeled with 400, 800, or 1200 µCi of 90Y (14.8, 29.6, or 44.4 MBq, respectively). Results: In vitro, 10 of 15 lymphoma cell lines responded synergistically to combined radiotherapy and venetoclax, including GCB-DLBCL, ABC-DLBCL and MCL lines (p < .04 in 10 cell lines). In vivo, each of 3 lymphoma models responded synergistically to combination therapy. In mice bearing Rec-1 xenografts, venetoclax alone did not affect mean survival time (p = .32), 8 Gy TBI lengthened survival by 44% compared to controls (p < .0001), but TBI combined with venetoclax tripled survival time compared to controls (p < .0001, combination group > TBI alone). The SU-DHL-6 model produced similar results. In the U2932 model, tumors disappeared during venetoclax monotherapy, but recurred in all mice, such that mean survival time doubled compared to controls (p = .0001). Six Gy TBI had no effect (p = .73), but combining TBI with venetoclax tripled survival time compared to controls (p = .0003, combination group > venetoclax alone). Using PRIT in place of TBI produced yet greater efficacy. In Rec-1 bearing mice, venetoclax had no effect alone (p = .12), 800µCi PRIT lengthened survival time 111% beyond controls (p = .0001), while the combination extended survival 483% beyond controls and cured 40% (p = .001, combination group > PRIT alone). In the U2932 xenograft model, venetoclax alone doubled survival time compared to controls (p < .0001) and 800µCi PRIT alone doubled survival and cured 30% (Fig. 1, p < .0001). Combination treatments cured 100% (Fig. 1). Conclusion: In vitro and in vivo results support our hypothesis that radiotherapy combines effectively with venetoclax to treat NHL. Despite differences in single agent sensitivity, xenograft models of GCB-DLBCL, ABC-DLBCL and MCL all responded synergistically to combinations of either TBI or PRIT with venetoclax. PRIT combinations with venetoclax produced cures (Fig. 1) without detectable toxicity, and merit clinical preference. Ongoing studies examine predictive biomarkers and optimal treatment protocols for therapeutic efficacy. Disclosures Gopal: Paid Consultancy- Gilead, Janssen, Seattle Genetics, Spectrum, Research funding- Gilead, Janssen, Pfizer, BMS, Merck, Teva, Takeda, Spectrum, Seattle Genetics: Consultancy, Honoraria, Research Funding.

scholarly journals Chemical Genomics Reveals JAK STAT Activation As a Mechanism of Resistance to HDAC Inhibitors in B Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 271-271
Author(s):  
Matthew S. McKinney ◽  
Anne W Beaven ◽  
Andrea Moffitt ◽  
Jason Landon Smith ◽  
Eric Lock ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Resistant Cell ◽  
Hdac Inhibition ◽  
Stat Pathway

Abstract Background: HDAC inhibitors (HDACi) are being investigated as treatment for relapsed/refractory non Hodgkin lymphoma (NHL) and other cancers. However, the mechanisms underlying sensitivity and resistance to HDAC inhibition in lymphomas have not been fully characterized. We probed the cellular and molecular response to HDACi in vitro and in vivo in order to determine factors that dictate the response to HDACi and to enable design of approaches to incorporate HDACi into novel combination therapeutics. Methods: High-throughput cytotoxicity screening was performed using two different HDAC inhibitors, LBH589 (panobinostat) and SAHA (vorinostat) in 52 lymphoid cell lines characterized through RNA-seq and microarray gene expression profiling. This screen revealed a greater than 50-fold range in concentration needed to induce cytotoxicity for the 2 different HDAC inhibitors and there was moderate correlation between the 2 compounds in this panel (Pearson correlation r = 0.76, p < 0.01). By pairing this chemosensitivity data with gene expression profiles of the screened cell lines, we developed a gene expression classifier for LBH589 that identified resistant and sensitive cell line groups. This predictor was applied to B-cell NHL cell lines tested with LBH589 in the Cancer Cell Line Encyclopedia (CCLE) and we found that the sensitive and resistant cell line groups distinguished by this method differed more than 5-fold in IC50 (0.021 vs. 1.24 nM, P < 0.01 by Wilcoxon rank sum), thus validating the ability of this approach to distinguish HDACi resistant cell lines. We further initiated a clinical trial of LBH589 in relapsed/refractory diffuse large B cell lymphoma patients combined with RNAseq profiling of their tumors prior to embarking on treatment. We treated nine patients with LBH589, and application of our response predictor to scaled RNAseq gene expression data revealed 4 predicted responders and 5 predicted non-responders. Two of the predicted responders had a clinical response to LBH589, whereas none of the predicted non-responders had a clinical response, thus our classifier was able to identify all of the LBH589-responsive patients from this cohort (P = 0.08 by Fisher's exact test). Analysis of differentially expressed molecular pathways in HDACi sensitive and resistant samples by gene set enrichment revealed the JAK-STAT pathway as the most differentially expressed pathway associated with HDACi resistance (at P < 0.001 and FDR < 0.20). We further identified a number of distinct mutations including STAT3, SOCS1 and JAK1 that were associated with activation of the JAK-STAT pathway by gene expression signatures and the LBH589 response signature in DLBCL cell lines and patient samples by analysis of RNA-seq data. Phosphoprotein analysis by Western blot and Sis-inducible-element (SIE) luciferase reporter assays were used to confirm JAK-STAT activation in these samples and we found that overexpression of STAT3 Src-homology domain mutations activated JAK-STAT3 signaling in isogenic cell lines and fostered resistance to LBH589 in vitro. Conversely, using in vivo DLBCL xenograft models, we found that combining JAK-STAT and HDAC inhibition by treatment with LBH589 and ruxolitinib resulted in synergistic reduction of tumor cell viability and tumor growth with tolerable toxicity in mice. Conclusions: Sustained JAK-STAT activation appears to mediate resistance to HDAC inhibition in DLBCL and other NHLs and several recurrent genetic lesions drive JAK-STAT activation in these diseases. This process can be overcome by JAK 1/2 inhibition with ruxolitinib and these findings demonstrate a role for combination therapy with HDAC inhibitors and small molecules targeting the JAK-STAT pathway in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Gene Expression Studies Reveal a Potential Mechanism of Glivec Resistance in Patients with Ph+ ALL Treated.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4374-4374
Author(s):  
To Ha Loi ◽  
David D.F. Ma
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
B Cell ◽  
Cell Lines ◽  
Unexpected Finding ◽  
B Cell Differentiation ◽  
Down Regulation ◽  
Blast Cells

Abstract In Ph+ ALL, patients respond to Glivec but nearly always relapse due to acquired resistance. The biological processes associated with Glivec resistance in Ph+ ALL are yet to be fully elucidated. The aim of this study is to use microarray and Q-PCR technology to dissect changes in signaling pathways of blast cells in Ph+ ALL patients treated with Glivec. Peripheral blood (PB) samples were taken before treatment and on consecutive days after administration of Glivec from two Ph+, c-ALLA+ and CD19+ ALL patients. Blast cells were isolated and their gene expression assayed using 19K cDNA microarrays. Over 400 differentially expressed genes were identified with at least a 1.5-fold up- or down-regulation in treated cells compared to cells collected pre-treatment. Based on these gene expression results, three main gene ontology groups were further evaluated: Apoptosis, Proliferation and B cell differentiation. The up-regulation of Bim and Bcl-6, and down regulation of Cyclin D2, confirms the induction of apoptosis via the FOXO3a pathway in cells treated with Glivec in vivo. Interestingly, both the proliferation genes, Tcl1-A and PKCe, and B cell differentiation associated genes, including CD79a, ETS1 and a cohort of IGH and Igl and k genes, were up-regulated during therapy. These gene expression changes observed in vivo were confirmed by Q-PCR in the Ph+ cell lines K562 (derived from CML blast crisis) and SUP-B15 (derived from ALL) treated with Glivec. The unexpected finding of increased expression of pro-proliferative genes and B cell differentiation genes by microarray revealed potential links with early B cell development and B cell receptor (BCR) signaling. Evidence for apoptosis and proliferation of Ph+ cell lines treated with Glivec were then examined by FACs. After 5 days of treatment with Glivec, 90% of K562 and 50% SUP-B15 cells underwent apoptosis. Furthermore, cell cycle analysis revealed the existence of a population of cells in G2 phase even after 6 days of Glivec treatment in SUP-B15 but not K562 cells, thus providing evidence of a population of cells undergoing proliferation during Glivec treatment in vitro. In summary, our in vivo observations supported by in vitro experiments suggest that Glivec induces the majority of Ph+ ALL blasts to undergo apoptosis. However, as treatment is prolonged, a population of ALL cells escapes death and undergoes proliferation and differentiation. We hypothesise that Glivec induced differentiation and proliferation of Ph+ cells may result in the clonal enrichment of cells resistant to Glivec.

Preclinical evaluation of KZR-261, a novel small molecule inhibitor of Sec61.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3582-3582
Author(s):  
Eric Lowe ◽  
R. Andrea Fan ◽  
Jing Jiang ◽  
Henry W. B. Johnson ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Expression Profiling ◽  
Single Agent ◽  
Tumor Types ◽  
Anti Tumor Activity

3582 Background: Secreted and transmembrane proteins play key roles in malignant transformation and growth, including in autocrine growth factor expression, receptor oncogene signaling, and immune system evasion. Biogenesis of these proteins involves translocation of the nascent polypeptides into the endoplasmic reticulum (ER) through the Sec61 channel, providing an untapped therapeutic target for a broad spectrum of malignancies. Here we describe preclinical activity of KZR-261 and related inhibitors of Sec61-dependent protein secretion. Methods: Sec61 inhibition with KZR-261 and related analog KZR-834 were evaluated using cell lines overexpressing proteins of interest tagged with luciferase. In vitro anti-tumor activity was assessed against a panel of 346 cell lines across 25 tumor types. Quantitative proteomic profiling by mass spec and gene expression profiling by RNAseq were conducted following treatment in multiple solid and heme tumor cell lines. Anti-tumor efficacy was evaluated in athymic nude mice implanted with the cancer cell lines H82 (SCLC), HT29 (CRC), BxPC3 (Pancreatic), 22RV1 (Prostate), H929 (Myeloma) and RL (NHL). Activity was also evaluated in a MC38 syngeneic colon tumor model. Results: KZR-261 and KZR-834 exhibited nanomolar potency against many therapeutic targets, including immune checkpoints, VEGF-A, VEGFR and EGFR. Broad in vitro anti-cancer activity was observed with KZR-834, which potently decreased cell viability across both solid and heme tumor types including CRC, Pancreatic, HNSCC, HCC, Lymphoma and Myeloma. Global proteomic analysis observed more than 1.5 fold downregulation of < 10% of detected Sec61 client proteins following treatment, while gene expression profiling revealed upregulation of ER stress response genes in sensitive versus resistant cell lines. Analysis of the TCGA database also found these genes upregulated in a number of different tumor types. In vivo, weekly IV administration was well tolerated and induced a dose dependent anti-tumor response at doses below the MTD in solid and heme xenograft models. In the syngeneic MC38 model, administration of KZR-834 in combination with anti-PD1 antibody resulted in greater anti-tumor activity than either single agent. Conclusions: Novel Sec61 inhibitors potently block expression of secreted and membrane proteins, translating into anti-tumor activity against many tumor types in vitro and in vivo, suggesting broad therapeutic potential. Clinical trials are being planned with KZR-261 to understand safety and early efficacy of this novel compound and therapeutic target.

scholarly journals RNA Interference Screen Implicates TNFAIP3 and FOXO1 in MALT1 Inhibition Resistance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 837-837
Author(s):  
Lorena Fontan ◽  
Rebecca Goldstein ◽  
Gabriella Casalena ◽  
Himaly Shinglot ◽  
Ilkay Us ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Library Screening ◽  
Sensitive Cell ◽  
Shrna Library

Abstract Recent studies have identified small molecule inhibitors of the paracaspase activity of MALT1, a protease and scaffolding protein involved in the B-cell receptor (BCR) signaling pathway, that are effective killing lymphomas in vitro and in vivo in xenograft models of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL). ABC-DLBCL is characterized by constitutive NF-κB activity. This activation has been attributed to mutations in various protein components of the B-cell receptor (BCR) as well as Toll-like receptor (TLR) pathways. However, not all ABC-DLBCL cell lines and primary patient samples were equally sensitive to MALT1 inhibitors in vitro. In order to discover genetic modifiers of response to MALT1 inhibition we used an shRNA library screening approach. MALT1 inhibition sensitive cell line HBL-1 was infected with DECIPHER barcoded shRNA library Module 1 and cells were treated with vehicle or 300 nM of MALT1 inhibitor MI-2 for 22 days. At this time cells were harvested and genomic DNA extracted. PCR was used to amplify barcodes and gel purified bands were extracted and sequenced. Cellecta's Deconvoluter software was used to quantify the number of reads per shRNA, reads were normalized to total number of reads and fold change between vehicle and MI-2 treated cells was calculated. Among the top positively and negatively enriched hairpins, we found a significant number of genes involved in the BCR pathway including: positively regulated shRNAs against TNFAIP3 and FOXO1 and negatively regulated hairpins against BTK, CD79B and PI3K genes PIK3C2A and PIK3C2D. Interestingly, TNFAIP3 and FOXO1 are negative regulators of the BCR pathway while BTK, CD79B and PI3K genes are positive regulators of this pathway. In order to validate these results and given the abundance of inhibitors of different proteins in the BCR pathway, we run a focused combination screen using MALT1 inhibitor MI-2 and inhibitors against other proteins in the pathway in 4 MALT1 sensitive cell lines. Combinations with PI3K inhibitors were most synergistic (combination index (CI) ranging 0.12-0.67), while BTK and PKC inhibitors showed an additive effect (CI ranging 0.7-0.9). These results were confirmed using a second MALT1 inhibitor, mepazine. In order to characterize the molecular mechanism by which MALT1 inhibition cooperates with PI3K, we focused on the FDA approved drug Idelalisib. In vitro treatment of cells with MI-2 and Idelalisib showed that effect on cell growth was a combination of decreased proliferation and increased apoptosis. Moreover, we found a decrease in AKT phosphorylation followed by a decrease in FOXO1 T24 phosphorylation and an accumulation of FOXO1 protein. This result correlates with our finding that FOXO1 knockdown favors MALT1 inhibition resistance. In vivo treatment of TMD8 xenografts with a combination of MI-2 and Idelalisib showed a stronger effect than either drug used as a single agent or vehicle, confirming the increased efficacy of the combination over either drug alone. In summary, we have used an shRNA library screening in order to determine which proteins and pathways cooperate with MALT1 inhibition to kill ABC-DLBCL and to evaluate combinatorial treatments in an unbiased manner. This same approach has pointed out TNFAIP3 and FOXO1 as possible biomarkers of response. This is especially interesting since these two proteins are mutated in a proportion of ABC-DLBCL patients and could affect response to treatment not only against MALT1 inhibitors but potentially any BCR targeted therapy. Disclosures Melnick: Janssen: Research Funding.

Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 616-623 ◽  
Author(s):  
Andrew G. Polson ◽  
Shang-Fan Yu ◽  
Kristi Elkins ◽  
Bing Zheng ◽  
Suzanna Clark ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Non Hodgkin Lymphoma ◽  
Antibody Drug

Abstract Targeting cytotoxic drugs to cancer cells using antibody–drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor, and are attractive targets for the use of ADCs because they are B-cell–specific, expressed in non-Hodgkin lymphomas (NHL), and are trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stable-linker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of nonHodgkin lymphoma cell lines in vitro. Further, these 2 ADCs are equally effective as low doses in xenograft models of follicular, mantle cell, and Burkitt lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, anti-CD79b antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like major histocompatibility complex class II–positive compartment MIIC. These results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.

Phosphatidylinositol-3 Kinase Delta (PI3Kδ) Inhibitor AMG 319 Is a Potent, Selective and Orally Bioavailable Small Molecule Inhibitor That Suppresses PI3K-Mediated Signaling and Viability in Neoplastic B Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4964-4964 ◽  
Author(s):  
Angus Sinclair ◽  
Daniela Metz ◽  
Tim Cushing ◽  
Liqin Liu ◽  
Rachael Brake ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Single Agent ◽  
Chemotherapeutic Agents ◽  
B Cell Malignancies ◽  
Molecule Inhibitor ◽  
Human B Cell

Abstract Abstract 4964 Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models. Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 <10 nM). AMG 319 also potently suppressed the phosphorylation of AKT (pAKTS473) in primary murine splenocytes (IC50<5 nM) after BCR cross linking and demonstrated a less than 10 fold shift in human whole blood B cells using a similar BCR pAKT assay in vitro. In a cell based selectivity screen, AMG 319 was selective for PI3Kδ against other PI3K class I isoforms (200 to >5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%. Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies. In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.

Sign in / Sign up

Export Citation Format

Share Document