scholarly journals Inhibition of PIM Kinases Targets Synthetic Vulnerabilities and Enhances Antigen Presentation in B-Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2858-2858
Author(s):  
Patrizia Mondello ◽  
Chiara Tarantelli ◽  
Luciano Cascione ◽  
Alberto Arribas ◽  
Andrea Rinaldi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Antigen Presentation ◽  
Cell Lines ◽  
Mhc Class Ii ◽  
High Throughput Screening ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Pim Kinases

The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Em-PIM and Em-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. (Figure 1A)The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin (Figure 1B), suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. (Figure 1C) Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. (Figure 1D) Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. (Figure 1E) In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Disclosures Younes: Xynomics: Consultancy; Biopath: Consultancy; Genentech: Research Funding; AstraZeneca: Research Funding; Syndax: Research Funding; BMS: Research Funding; HCM: Consultancy; Celgene: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Takeda: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Pharmacyclics: Research Funding. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants.

scholarly journals BET Bromodomain Inhibitor OTX015 Affects the Expression of Micrornas Involved in the Pathogenesis of Diffuse Large B-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4495-4495 ◽  
Author(s):  
Luciano Cascione ◽  
Eugenio Gaudio ◽  
Elena Bernasconi ◽  
Chiara Tarantelli ◽  
Andrea Rinaldi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Models ◽  
Expression Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, accounting for 30%-40% of all cases. Despite a major improvement in the cure rate, a large number of DLBCL patients lack therapeutic options. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA, including microRNA (miRNA), contribute to DLBCL pathogenesis and represent potential therapeutic targets. OTX015 targets bromodomain and extra-terminal (BET) proteins, which are epigenetic readers contributing to gene transcription. It has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) and promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). To better understand the mechanism of action of OTX015, we studied molecular changes induced by this compound in DLBCL cell lines. Methods. Total RNA was extracted from 2 DLBCL cell lines, the germinal center B-cell (GCB) type DOHH2 and activated B-cell-like (ABC)-type SU-DHL-2, following treatment with 500 nM OTX015 or DMSO for 4h or 8h. RNA samples were labeled with cyanine-3 dye using the Agilent microRNA Complete Labeling System & Hyb Kit and hybridized to the Agilent Human microRNA microarray v.3. Raw expression values were obtained with Agilent Feature Extraction Software, log-transformed and normalized by the quantile method. Data were filtered to exclude relatively invariant features and those below the detection threshold. Limma (Linear Models for Microarray data analysis) was employed using R/Bioconductor and the filtered dataset. Baseline miRNA profiling was obtained from 22 DLBCL cell lines with the Nanostring nCounter Human v2 miRNA Expression Assay kit. Baseline gene expression profiling (GEP) was obtained in these cell lines with the Illumina HumanHT-12 v4 Expression BeadChip. Selected miRNA changes were validated by real-time PCR. Validated miRNA targets were retrieved using the miRWalk database (Dweep et al, 2011). Gene Set Enrichment Analysis (GSEA) software was used to assess enrichment of miRNA targets in the GEP datasets. Results. miRNA profiling of the GCB and ABC DLBCL cell lines exposed to OTX015 identified four downregulated miRNAs and eight which were upregulated. Among them, the oncomirs miR-92a-1-5p (log2 FC, -2.01; P=0.004) and miR-21-3p (log2 FC, -0.37; P=0.0045) were downregulated, while the tumor suppressor miR-96-5p (log2 FC, 0.39; P=0.041) was upregulated. Interestingly, changes of these miRNAs matched GEP variations of validated target genes (e.g., miR-92a-1-5p: CDKN1A, log2 FC, 0.81, CDKN2A, log2 FC, 0.81; miR-96-5p: MYC, log2 FC, -0.57, MYD88, log2 FC, -0.35). We then evaluated if these three miRNAs play a role in OTX015-sensitivity by obtaining baseline miRNA and GEP profiling data in 22 DLBCL cell lines. Compared to 8 cell lines with lower sensitivity to OTX015 (IC50 >500 nM), the 14 sensitive cell lines (IC50 <500 nM) presented lower miR-96-5p expression levels (log ratio, 2.12; P=0.026) and their GEPs were significantly enriched for validated miR-96-5p targets (normalized enrichment score, 1.4; P=0.026), suggesting miR-96-5p levels may predict response to OTX015. Conclusions. Changes in the expression levels of biologically relevant miRNAs may contribute to response to OTX015. miR-92a-1-5p, the oncomir which was most strongly downregulated by OTX015, is a member of the MYC target MIR17HG (mir-17-92 cluster), involved in the pathogenesis and chemo-resistance of lymphomas, mainly contributing to PI3K/AKT/mTOR pathway activation. Since the cell cycle transcriptional regulator E2F1 is targeted by mir-17-92, OTX015 may contribute to cell cycle arrest and to downregulation of the E2F1 target gene reported with BRD inhibitors in DLBCL cell lines. miR-21-3p, also downregulated by OTX015, is a well-known oncomir, and forced miR-21-3p expression in transgenic mice results in the development of leukemias and lymphomas. miR-96-5p, upregulated by OTX015, targets oncogenes such as RAS or MYC, and low expression has been reported in mantle cell lymphoma. Interestingly, low miR-96-5p baseline levels were associated with higher sensitivity to OTX015, an observation meriting validation in other tumor models and evaluation in clinical studies. Disclosures Stathis: Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.

The Brd-Inhibitor OTX015 Is Active in Pre-Clinical Models of Mature B-Cell Lymphoid Tumors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1657-1657 ◽  
Author(s):  
Paola Bonetti ◽  
Michela Boi ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stahis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Rt Pcr ◽  
Down Regulation ◽  
Myc Gene ◽  
Dose Dependent

Abstract Abstract 1657 Background: Bromodomain-containing proteins play an important role in gene expression regulation, via chromatin structure remodelling. Antitumor activity has been reported in acute and chronic hematological malignancies using inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a large panel of cell lines derived from mature B-cell lymphoid tumors. Material and Methods: Established human cell lines derived from 13 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphoma (MCL), three splenic marginal zone lymphoma (SMZL) and from three multiple myeloma (MM) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72 hours exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: OTX015 demonstrated anti-proliferative activity in DLBCL cell lines (median IC50 0.192μM; range 0.069–12.68μM). Similar results were obtained on SMZL (median IC50 0.165μM, range 0.105–0.24μM), and on MM cell lines (median IC50 0.449μM; range 0.06–0.7μM). Conversely, MCL cell lines appeared less sensitive to OTX015 (median IC50 2.01μM; range 1.22- >15μM). Among DLBCL cell lines, there was no significant difference based upon the cell of origin of the cell lines. OTX105 caused a cell cycle arrest in G1 in a dose-dependent manner in 5/5 DLBCL and 3/3 MM cell lines, without an increase in cell death. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in 1/1 sensitive DLBCL cell line. In order to understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after 24h treatment with increasing doses. We observed a dose-dependent suppression of MYC mRNA by OTX015 in 4/5 DLBCL and in 2/2 MM cell lines. In DLBCL, down-regulation of MYC mRNA was observed within 1h after treatment with OTX015, suggesting a direct effect of the compound on the MYC gene. To determine whether the suppression of MYC gene by OTX015 was reversible, DLBCL cell lines were treated for 2h with OTX015 and then the inhibitor was removed from the media. MYC mRNA suppression appeared reversible, as shown in DLBCL cell lines, which, after 2h exposure to OTX015, showed a time-dependent restoration of MYC mRNA expression to untreated levels after 2–3h. In one of the most sensitive DLBCL cell lines no MYC mRNA down-regulation was observed after treatment, suggesting that alternative pathways can be affected by BRD-inhibition. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several cell lines representative of mature B-cell tumors. An apparently reversible down-regulation of MYC mRNA was commonly observed, appearing as a possible mechanism of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in mature B-cell origin malignancies. A phase I trial is scheduled to start in 2012. Disclosures: Bonetti: OncoEthix SA: Research Funding. Inghirami:OncoEthix SA: Research Funding. Noel:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Bertoni:OncoEthix SA: Research Funding.

scholarly journals Karonudib Has Potent Anti-Tumor Effects in Preclinical Models of B-Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Morten P Oksvold ◽  
Ulrika Warpman Berglund ◽  
Helge Gad ◽  
Baoyan Bai ◽  
Trond Stokke ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Medical Research ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Knock Out ◽  
Non Profit

Although chemo-immunotherapy has improved survival in B-cell lymphoma patients, refractory and relapsed disease still represents a major challenge, urging for development of new therapeutics. A new approach is to target nucleotide metabolism. Karonudib (TH1579), was developed to inhibit MutT-homologue-1/Nudix hydrolase 1 (MTH1/NUDT1), an enzyme that prevents oxidized nucleotides to be incorporated into DNA. Under normal conditions with low reactive oxygen species (ROS) burden, MTH1 is not essential for cell survival. This contrasts cancer cells which frequently upregulate MTH1 to compensate for increased ROS with corresponding higher oxidized nucleotide levels, and therefore become more susceptible for MTH1 inhibition. Here, our aim was to perform preclinical testing of karonudib in B-cell lymphoma. Using two different gene expression datasets, we demonstrate that NUDT1, the gene encoding MTH1, was highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma as compared to follicular lymphoma and peripheral blood B cells from healthy donors, hence demonstrating a rationale for targeting MTH1 in aggressive B-cell lymphoma. We tested the efficacy of karonudib (0.06-1 µM) in vitro in a range of B-cell lymphoma cell lines using CellTiterGlo and by flow cytometry detection of active caspase-3 and TUNEL to identify apoptotic cells. Karonudib strongly reduced viability in all B-cell lymphoma cell lines tested (n = 12) and induced apoptosis at concentrations well tolerated by peripheral blood B cells from healthy donors. Cell cycle analysis and microscopy revealed that most cells arrested in prometaphase in the presence of karonudib. Failed spindle assembly led to mitotic arrest and subsequent apoptosis. Prometaphase arrest was seen in TP53 mutant as well as in TP53 wild type cell lines, confirming that karonudib induced apoptosis independent of TP53 mutational status. To test the efficacy of karonudib in vivo, we utilized two different lymphoma xenograft models, including an ABC DLBCL patient-derived model. Mice were treated with karonudib (90 mg/kg) or vehicle b.i.d, three times a week and tumor growth was monitored by in vivo imaging system or MR. In both models, karonudib as single agent completely controlled tumor growth, and significantly prolonged survival (p&lt;0.0001, as compared to control mice). The specificity of MTH1 inhibitors has been debated and TH588, the first generation MTH1 inhibitor, was recently shown to bind b-tubulin and induce mitotic arrest in MTH1 knock out cell lines (Patterson et al, Cell Syst 2019). To elucidate the mechanism of karonudib in B-cell lymphoma, we generated MTH1 knock out cells using CRISPR/Cas9, and compared the functional effects of karonudib in these clones with the original lymphoma cells. We demonstrated on-target effect of the inhibitor as the MTH1 knock out clones were less sensitive towards karonudib. However, MTH1 knock out clones exhibited a similar cell cycle arrest as the wild type cells after karonudib treatment. This clearly indicates that karonudib can induce cell cycle arrest independent of MTH1, and hence has a dual mechanism of action. Our preclinical data suggest that karonudib is a promising drug with potential therapeutic use in B-cell lymphoma, and may be particular effective in aggressive lymphoma types. Disclosures Warpman Berglund: Oxcia AB: Other: shareholders; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Gad:Oxcia: Other: shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Pham:Oxcia AB: Other: Shareholder. Sanjiv:Oxcia AB: Other: Shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Helleday:Oxcia AB: Other: Shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Targeting NF-κB with First in Class N-Quinoline Benzenesulfonamides (NQBS) Reveals Potent Activity in Models of Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4435-4435
Author(s):  
Matko Kalac ◽  
Michael Mangone ◽  
Alison Rinderspacher ◽  
Shi-Xian Deng ◽  
Luigi Scotto ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
B Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Research Funding ◽  
Nuclear Translocation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract The first two authors contributed equally to this work Identifying pharmacologic strategies to inhibit the activation of NF-κB and its target genes has been a major research pursuit. To date, no direct inhibitors of the NF-κB subunits have been explored in the clinic. Based on the constitutive activation of NF-κB in diffuse large B-cell lymphoma (DLBCL), we used this disease model to develop drugs targeting NF-κB. Using a fluorescence-based high throughput screening (HTC) approach, a unique N-quinoline-benzenesulfonamide (NQBS) scaffold was identified as potential small molecule inhibitor of the NF-κB pathway. A confocal microscopy based HTC assay performed in human umbilical vein endothelial cells (HUVEC) identified hit compounds that contained a unique NQBS core structure. The assay screened for compounds that inhibited nuclear translocation of NF-κB subunits in TNFα-induced HUVEC cells. To date over 100 NQBS analogs have been synthesized with varying potency and cytotoxicity in inhibiting growth of DLBCL lines (OCI-Ly10, RIVA, HBL-1 and OCI-Ly3). Cytotoxicity assays demonstrated that the most potent compounds exhibit IC50s in the 0.5 to 1.5 µM range. These most potent NQBS analogs identified as CU-O42 CU-O47 and CU-O75 were also able to induce apoptosis and caspase activation. Apoptosis was preceded by exclusion of the NF-κB proteins from the nucleus. To analyze the localization of NF-κB proteins within the cell compartments before and after the treatment with CU-O42, CU-O47 and CU-O75, we used confocal microscopy, electromobility shift (EMSA) and ELISA assays. Control cells tested positive for p50/p65 both within the cytoplasm and the nucleus. Following treatment with CU-O42 NF-κB was sequestered within the cytoplasm of the cell which occurred as early as 3h after exposure. In addition, all three analogs reduced the nuclear levels of NF-κB in a concentration-dependent manner when measured by EMSA and ELISA. Furthermore, CU-O47 and CU-O75 were able to inhibit TNFα induced luciferase expression in a HEK293T cell model where luciferase is controlled by an NF-κB promoter. A KINOMEscan platform (examining the activity of over 450 different kinases) showed that no NQBS analog screened (CU-O42 and CU-O75) inhibited any of the kinases in the assay. In addition, a proteasome inhibition assay tested negative for trypsin-like and chromotrypsin-like protease activity (CU-O42, CU-O47 and CU-O75). Stabilization of the inactive trimer of p50, p65 and IκBα was hypothesized as a potential mechanism of action of CU-O42 and CU-O75 through Internal Coordinate Mechanics (ICM) software. This binding hypothesis was further corroborated by cellular thermal shift assays (CETSA) with an increase of the IκBα melting temperatures (2.5-3°C) in whole cell lysates following rapid (30min) exposure to CU-O42 and CU-O75. Using a genome-wide regulatory network perturbation analysis (DeMAND) based on the RNA-Seq data collected from OCI-Ly10 cells treated with CU-O75, we identified IκBα as one of the potential targets of the compounds. Gene set enrichment analysis demonstrated NF-κB target gene downregulation using IC20 of CU-O75 at 24h (p=0.045). In vivo experiments were conducted in two models: (1) xenografts with human DLBCL cell lines of both ABC and GC subtype; and (2) myc cherry luciferase mouse model where mice spontaneously develop aggressive lymphomas. In both models, CU-O42 was able to inhibit tumor growth. Interestingly, in the xenograft model, malignant cell growth was inhibited in both ABC (HBL-1) and GC (OCI-Ly1) cells when compared to controls (p=0.01 and p=0.02). However, overall survival of mice with ABC xenografts treated with CU-042 significantly exceeded the survival of mice with GC xenografts (p<0.01) suggesting a more sustainable response in this subtype of disease, consistent with its dependency on NF-κB. Identification of a unique NQBS scaffold has led to the chemical synthesis of over 100 structural analogs with a potent inhibition on NF-κB nuclear translocation. They display potent activity across a panel of lymphoma cell lines, producing a survival benefit in mice implanted with an ABC-subtype of lymphoma. ICM, CETSA and DeMAND suggest that this is a direct effect mediated on the proteins within the p65/p50/IκBα complex. These findings point to a novel mechanism of action and warrant further research into potential clinical translation of this class of small molecules. Disclosures Califano: Thermo Fischer Scientific: Consultancy; Ipsen pharmaceuticals: Consultancy; Cancer Genetics Inc: Consultancy; Therasis Inc: Employment. O'Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria, Research Funding; Acetylon Pharmaceuticals, INC: Consultancy.

scholarly journals PRAME Expression Is Correlated with Treatment Outcome and Specific Features of the Tumor Microenvironment in Classical Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1509-1509
Author(s):  
Katsuyoshi Takata ◽  
Lauren C. Chong ◽  
Avinash Thakur ◽  
Tomohiro Aoki ◽  
Anja Mottok ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Sequencing Data ◽  
And Function ◽  
Inhibitor Treatment

Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Carfilzomib (CFZ): A Novel Proteasome Inhibitor That Induces Cell Cycle Arrest and Cell Death In Rituximab-Chemotherapy Resistant Lymphoma and Potentiates the Anti-Tumor Activity of Chemotherapeutic Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4908-4908
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Gregory P. Kaufman ◽  
Cory Mavis ◽  
Myron S. Czuczman
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Cycle Arrest ◽  
Anti Tumor Activity

Abstract Abstract 4908 Rituximab-chemotherapy relapsed/refractory B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. Targeting the ubiquitin-proteasome system using bortezomib (BTZ) has resulted in significant anti-tumor activity and potentiates the effects of chemotherapy/biologic agents in multiple myeloma, and to a lesser degree, B-cell lymphoma. CFZ is as a novel proteasome inhibitor which is selective and structurally distinct from BTZ. In an attempt to characterize the biological activity of CFZ, we evaluated its anti-tumor activity in several lymphoma pre-clinical models. Rituximab-chemotherapy sensitive cell lines (RSCL), rituximab-chemotherapy resistant cell lines (RRCL), as well as primary tumor cells derived from patients with de novo or relapsed/refractory B-cell lymphoma, were exposed to escalating doses of CFZ or BTZ (1-7.5nM) alone or in combination with doxorubicin, paclitaxel, or gemcitabine for 24, 48 and 72hours. Cell viability was determined by cell titer glow luminescent assay and cell cycle was analyzed by FASCan DNA methodology. Patient-derived lymphoma cells were isolated from fresh biopsy tissue via negative selection using magnetic beads. Western blots were performed using cell lysates from CFZ, BTZ or control-treated cells to detect PARP-cleavage and/or changes in Bcl-2 family members. CFZ was more active than BTZ and exhibited dose-dependent and time-dependent cytotoxicity against RSCL, RRCL, and primary tumor cells. We found a 10-fold concentration difference between CFZ and BTZ activity. In vitro exposure of RRCL or RSCL to CFZ resulted in G2/M phase cell cycle arrest. In addition, CFZ exposure resulted in the up-regulation of Bak and Noxa levels and subsequent PARP cleavage in RRCL. Finally, CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumor activity of paclitaxel and gemcitabine in B-cell lymphoma cell lines. In summary, our data strongly suggest that CFZ is a novel and potent proteasome inhibitor which is able to: overcome resistance to some conventional chemotherapeutic agents, upregulate proapoptotic proteins to enhance cell death, and induce G2/M cell cycle arrest in lymphoma cells. Our preclinical data supports future clinical evaluation of CFZ in patients with refractory B-cell lymphoma. (Supported by USPHS grant R01 CA136907-01A1 from the National Cancer Institute). Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3734-3734
Author(s):  
Cory Mavis ◽  
Sarah Frys ◽  
Juan Gu ◽  
John Gibbs ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
In Vitro Exposure ◽  
Anti Tumor Activity

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

Apoptotic and Non-Apoptotic Cellular Pathways Executed by Bortezomib (BTZ) in Rituximab-Resistant Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4967-4967
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
Karen E Thudium ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mitotic Catastrophe ◽  
M Cell ◽  
Lymphoma Cells

Abstract Abstract 4967 Rituximab-chemotherapy relapsed/refractory (r/r) B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. A better understanding of the mechanism(s)-of-action of BTZ and other proteasome inhibitors (PI) is likely to aid in the identification of biomarkers that can be used to determine clinical responsiveness and/or help in the rational development of novel PI-based therapeutic combinations (e.g. incorporating biologics, small molecules and/or chemotherapy) in r/r B-cell lymphoma. Previously we demonstrated that rituximab resistance was associated with increased proteasome activity leading to a de-regulation in the apoptotic threshold of lymphoma cells to multiple chemotherapy agents. Pharmacological and genetic (e.g. siRNA silencing of BAK/BAX) inhibition of apoptosis partially affected BTZ activity in rituximab-resistant (RSCL) but not in rituximab-sensitive cell lines (RSCL) suggesting the existence of alternative pathways of cell death associated with PI exposure. To this end we evaluated the contribution of cellular senescence, cell cycle inhibition, or mitotic catastrophe to the anti-tumor activity of BTZ as a single agent or in combination with chemotherapeutic agents in RSCL, RRCL and in primary tumor cells. Lymphoma cells were exposed to BTZ (10-25nM) for 24–48 hrs. Cell senescence was determined by SA-β-gal staining using a senescence assay kit and inverted phase-contrast microscopy was performed. Changes in cell cycle were analyzed by the FACScan DNA method and changes in cell cycle regulatory proteins (i.e. cdc2, cyclinA/B, p21, CDK2/4/6) were analyzed by Western blotting. Mitotic index was determined by Wright-Giemsa stain and positive cells were counted under a Nikon microscope. Mitotic catastrophe was determined by confocal microscopy by staining with α-tubulin antibody. Finally, changes in ATP content was determined by the Cell Titer Glo assay. Baseline differences were observed between RSCL and RRCL in terms of cell morphology, proliferation rate and senescence. RRCL (Raji2R and Raji4RH) were considerably larger in size, had a slower proliferation rate and an exhibited a 3-fold increase the number of cells in senescence than RSCL. In vitro exposure of RSCL and RRCL to BTZ attenuated the number of cells in senescence by 50–75%. Cell cycle analysis demonstrated that RRCL had more cells in S phase when compared to RSCL. In vitro exposure to BTZ-induced G2/M arrest in RRCL, but not in RSCL. Overexpression of G2/M cell cycle regulatory proteins cyclin B and cdc2 were observed in RRCL and in tumor cells isolated from r/r B-cell lymphoma patients. Mitotic catastrophe with multi-nucleated cells were only detected in RRCLs exposed to BTZ. In vitro and ex vivo exposure of RSCL and RRCL to BTZ potentiated the cytotoxic effects of paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL and primary tumor cells isolated from r/r lymphoma patients in a dose-dependent manner. Our results suggested that BTZ activates several death pathways in B-cell lymphoma pre-clinical models. In addition to apoptosis, BTZ is capable in triggering mitotic catastrophe in rituximab-chemotherapy lymphoma cells with decreased levels of pro-apoptotic proteins. Moreover, sensitization of RRCL to drug therapy involves interplay between cellular senescence attenuation, G2/M cell cycle regulation, and mitotic catastrophe. Hence, proteasome inhibition may provide a novel therapeutic approach for treating apoptosis-resistant B-cell lymphoma. Research, supported in part as a subproject of NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute. Disclosures: Hernandez-Ilizaliturri: Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy. Czuczman:Millennium: Honoraria, Research Funding.

A Phase II Study of MLN8237 (Alisertib) Alone and in Combination with Rituximab in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphomas (NHL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3082-3082 ◽  
Author(s):  
Kami J. Maddocks ◽  
Jonathon B. Cohen ◽  
Beth Christian ◽  
Joseph M Flynn ◽  
Samantha M. Jaglowski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Partial Response ◽  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Grade 3

Abstract Introduction: Aurora A kinase is a serine-threonine kinase important in the cell cycle. Overexpression of Aurora A kinase has been associated with the development of NHL with Aurora A kinase activity amplified in aggressive NHL. Alisertib is an orally bioavailable Aurora A kinase inhibitor that targets a key molecule in the cell cycle of dividing cells that leads to mitotic spindle defects and apoptosis. Due to demonstrated pre-clinical and early clinical efficacy of Alisertib in the treatment of B-cell NHL, we conducted a phase 2 trial with Alisertib alone and in combination with rituximab in patients with relapsed aggressive and indolent NHL. Methods: Patients ≥ 18 years with relapsed or refractory B-cell NHL including follicular lymphoma (FL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia (LPL/WM), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma (BL) who had received one prior therapy and transformed NHL (tNHL) at any time after diagnosis of transformation were eligible. Additional eligibility included ECOG Performance Status 0-2, adequate renal and liver function, absolute neutrophil count (ANC) ≥ 1000/µL and platelets ≥ 75,000/µL. Patients with indolent NHL (FL, MZL or LPL/WM) were accrued to Cohort A and all other patients (MCL, DLBCL, BL and tNHL) were accrued to Cohort B. Patients treated on Cohort A received Alisertib at a dose of 50 mg twice daily (BID) for days 1-7 of 21 day cycles with rituximab (375 mg/m2) added on day 1 of cycles 5-12 in patients who failed to achieve a complete response (CR) after cycle 4. Patients treated on Cohort B received Alisertib at a dose of 50 mg BID for days 1-7 of 21 day cycles with rituximab added on day 1 of cycles 3-10 in patients who failed to achieve a partial response (PR) or CR after cycle 2 and on day 1 of cycles 5-12 in any patient remaining on monotherapy who had not achieved a CR after cycle 4. Response was assessed according to the International Harmonization Project criteria (Cheson, JCO 2007) and patients were evaluated by imaging after cycle 2, 4, 6 and after every 3 cycles thereafter. Toxicity was assessed per NCI CTCAE v4.0. Results: Eleven patients were enrolled in this trial, 1 patient in Arm A with FL and 10 patients in Arm B with DLBCL (n=6), primary mediastinal DLBCL (n=1), and tNHL (n=3). The median age was 58 years (range 35-77); 10 patients had Stage III-IV disease; the median number of prior therapies was 4 (range 2-6); and 5 patients had prior autologous stem cell transplant. Patients received a median of 2 treatment cycles (range 1-5). Two patients in Cohort B received rituximab added on day 1 of cycle 3 for SD. Grade 3/4 adverse events included grade 3 neutropenia (36%), grade 3 fatigue and dehydration (18%), grade 3 mucositits (9%), grade 3 cystitis (9%), grade 3 pancreatitis (9%), grade 3 pneumonitis (9%) and grade 3 thromboembolic disease (9%). One patient required dose reduction for mucositis, 1 patient went off study for pneumonitis, 1 patient went off study due to patient preference and 8 patients came off study for PD. The overall response rate was 9%, with 1 patient achieving a partial response (PR). This patient had Stage IV CD5+ DLBCL with 2 prior therapies. The patient had SD after 2 cycles and rituximab was added, with response improving to a PR after 5 cycles. The patient went off study after 5 cycles to proceed with allogeneic stem cell transplantation and remains in remission 262 days post-transplant. Conclusions: In this heavily pre-treated, high-risk population of patients with relapsed/refractory NHL, the oral agent Alisertib was overall well tolerated with one responding patient with refractory CD5+ DLBCL able to proceed to allogeneic transplant who remains in continuous remission. Enrollment continues to both cohorts A and B in this trial to the defined interim analysis point of 16 patients in each arm, with a requirement of 5 responses in each arm to continue the study. Disclosures Maddocks: Pharmacyclics, Seattle Genetics, MorphoSys: Advisory Board Other, Research Funding. Cohen:Pharmacyclics: Consultancy; Seattle Genetics: Consultancy; BMS: Research Funding; Janssen: Research Funding. Christian:Immunomedics: Research Funding.

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