scholarly journals Gene Expression and Epigenetic Analysis in Relapsed/Refractory Diffuse Large B Cell Lymphoma Provides Insights into Evolution of Treatment Resistance to R-CHOP

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Manishkumar S. Patel ◽  
Ellen K. Kendall ◽  
Sarah Ondrejka ◽  
Agrima Mian ◽  
Yazeed Sawalha ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Differentially Expressed Genes ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
P Value

Background Diffuse large B cell lymphoma (DLBCL) is curable in ~60-70% of patients using standard chemoimmunotherapy, but the prognosis is poor for relapsed/refractory (R/R) DLBCL. Therefore, understanding the underlying molecular mechanisms will facilitate early prediction and effective management of resistance to therapy. Recent studies of paired diagnostic-relapse biopsies from patients have relied on a single "omics" approach, examining either gene expression or epigenetic evolution. Here we present a combined analysis of gene expression and DNA methylation profiles of paired diagnostic-relapse DLBCL biopsies to identify changes responsible for relapse after R-CHOP. Methods Biopsies from 23 DLBCL patients were obtained at the time of diagnosis and relapse following frontline R-CHOP chemoimmunotherapy. The cohort had 18 (78.3%) male patients with median age of 62 (range, 35-86) years and median IPI of 2.5 (range, 1-5). The median time from diagnosis to relapse was 7 (range, 0-57) months. DNA and RNA were extracted simultaneously from formalin-fixed paraffin embedded (FFPE) biopsy samples. DNA methylation levels were measured through Illumina 850k Methylation Array for 22 pairs of diagnostic-relapse biopsies. RNA from diagnostic-relapse paired biopsies from 6 patients was sequenced using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB. Pearson's correlation with a Bonferroni correction to the p-value was used to calculate the correlation between regularized log transformed gene expression counts and methylation beta values. Results In a pairwise comparison of gene expression between diagnostic and R/R biopsy pairs, we found 14 differentially expressed genes (FDR<0.1 & Log2FC>|1|) consistent across all pairs. Compared to gene expression at diagnosis, five genes (CYP1B1, LGR4, ATXN1, CTSC, ZMAT3) were downregulated, and eight genes (ERBB3, CD19, CARD11, MT-RNR2, IGHG3, CCDC88C, ATP2A3, CENPE, and PCNT) were up-regulated in the R/R samples. Many of these genes have been previously implicated in oncogenesis, such as ERBB3, a member of the epidermal growth receptor family. Importantly, some of these genes have known roles in DLBCL biology, such as CD19, a member of the B-cell receptor complex, and CARD11, a gene in which several oncogenic mutations have been identified in DLBCL as a mediator of NF-KB activation. Gene set enrichment analysis revealed overexpression of immune signatures such as cytokine-cytokine receptor interaction, chemokine receptor-chemokine binding, and the IL-12-STAT4 pathway at diagnosis. At relapse, cell cycle, B-cell receptor, and NOTCH signaling pathways were overexpressed. Interestingly, in a pairwise comparison of methylation between diagnostic and R/R biopsy pairs, there were no differentially methylated probes (FDR<0.05), suggesting no coordinated epigenetic evolution between diagnostic and R/R pairs. For biopsy pairs that had both gene expression and methylation data (5 pairs), we correlated gene expression and methylation values. We found that none of the differentially expressed genes between the diagnostic and R/R biopsies were significantly correlated with methylation status (adjusted p-value<0.05). Conclusions By analyzing paired diagnostic and relapse DLBCL biopsies, we found that at the time of relapse, there are significant transcriptomic changes but no significant epigenetic changes when compared to diagnostic biopsies. Activation of B-cell receptor and NOTCH signaling, as well as the loss of immune signaling at relapse, cannot be attributed to coordinated epigenetic changes in methylation. As the epigenetic profile of the biopsies did not consistently evolve, these data emphasize the need for better understanding of the baseline methylation profiles at the time of diagnosis, as well as acquired somatic mutations that may contribute to the emergence of therapeutic resistance. Future studies are needed to focus on how activation of signaling pathways triggered by genomic alterations can be targeted in relapsed/refractory DLBCL. Disclosures Hsi: Seattle Genetics: Consultancy, Honoraria; Miltenyi: Consultancy, Honoraria; Abbvie: Research Funding; Eli Lilly: Research Funding; CytomX: Consultancy, Honoraria. Hill:Takeda: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding.

scholarly journals Ibrutinib-Resistant Mantle Cell Lymphoma Undergoes Metabolic Reprogramming Towards Oxphos

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 41-41
Author(s):  
Krystle Nomie ◽  
Liang Zhang ◽  
Yixin Yao ◽  
Yang Liu ◽  
Shaojun Zhang ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Enrichment Analysis ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Gene Set Enrichment Analysis ◽  
Rna Seq ◽  
Advisory Committees ◽  
Oncogenic Pathways

Abstract Introduction Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma subtype and constitutive activation of the B-cell receptor pathway is a hallmark of B-cell lymphomas. Bruton's tyrosine kinase (BTK) is a critical component of the B-cell receptor pathway, and ibrutinib, a first-in-class, once-daily, and oral covalent inhibitor of BTK, was developed to reduce/silence B-cell receptor pathway activity, leading to clinically remarkable anti-tumor activity. In our prior multiple-center Phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68% (Wang et al., NEJM, 2013), surpassing the effectiveness of other therapies. Although ibrutinib is extremely efficacious in patients with relapsed/refractory MCL, the one-year overall survival rate of ibrutinib-exposed patients who relapse is only 22%. Methods Patient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with reverse phase protein array (RPPA) analysis of ibrutinib-sensitive and -resistant MCL cell lines. Metabolic assays including the measurement of mitochondria respiration rates with the Seahorse analyzer and reactive oxygen species (ROS) levels, targeted metabolomics, and ATP analysis. Functional studies targeting this molecular pathway were conducted, including in vitro cell viability and apoptosis assays, as well as in vivo efficacy studies in an ibrutinib-resistant MCL patient-derived xenograft mouse model. Results WES data analysis identified frequent inactivating somatic alterations in ATM, KMT2D, and TP53 in both the ibrutinib-sensitive and -resistant tumors. CDKN2A (5/7, 71%) was frequently deleted, and the deletion was only observed in the ibrutinib-resistant tumors (p = 0.010). The RNA-seq analysis identified a total of 63 protein-coding genes as the most differentially expressed genes (DEGs) between the ibrutinib-resistant and -sensitive groups, with a fold change of ≥ 2 or ≤ -2 and the false discovery rate (FDR q-value) ≤ 0.01. Among the DEGs, 26 genes were upregulated in ibrutinib-resistant tumors. In addition, gene set enrichment analysis (GSEA) revealed the marked upregulation of oncogenic pathways including c-MYC, mTOR (mTORC1), Wnt, and NF-ĸb signaling, followed by cell cycle, apoptosis, BCR signaling and DNA repair in the ibrutinib-resistant tumors. Notably, in addition to these oncogenic pathways, the metabolic pathways, including oxidative phosphorylation (OXPHOS), were significantly enriched in the ibrutinib-resistant tumors (normalized enrichment score > 3 and FDR q-value < 1e-5). In further support of this finding, metabolomics analysis and the measurement of ATP production and mitochondrial respiration indicated that the OXPHOS pathway is the predominant metabolic pathway employed by ibrutinib-resistant MCL cells. To determine the effects of targeting these pathways, OXPHOS was inhibited with a novel electron transport complex I inhibitor (IACS-010759, developed by MD Anderson Cancer Center) in both MCL cell lines and ibrutinib-resistant MCL patient-derived xenograft (PDX) models. Single agent IACS-010759 treatment at 10 mg/kg oral gavage for 5 consecutive days/week completely prevented tumor growth compared with the vehicle control as shown by measuring tumor volume (n = 5, p < 0.0001) and human β2M levels (n = 5; p < 0.0001) throughout treatment. No apparent toxicities were observed in the IACS-010759-treated MCL PDX mice. Conclusion This current study warrants the exploitation of active cancer metabolic pathways, especially OXPHOS, to improve the clinical outcomes of MCL and additional lymphoma, which is actively being investigated in a Phase I lymphoma clinical trial (NCT03291938). Disclosures Wang: AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria.

scholarly journals Evidence of a Synergistic Cross-Talk between the B Cell Receptor (BCR) and Nicotinamide Phosphoribosyl Transferase (NAMPT) in Richter's Syndrome Patient-Derived Xenograft Models: Therapeutic Implications

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 250-250
Author(s):  
Vincenzo Gianluca Messana ◽  
Nicoletta Vitale ◽  
Matteo Rovere ◽  
Lucia Renzullo ◽  
Francesca Arruga ◽  
...  
Keyword(s):  
B Cell ◽  
Disease Progression ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Metabolic Reprogramming ◽  
Rate Limiting ◽  
Pdx Models ◽  
Richter’S Syndrome

Abstract Background: A rare complication of Chronic Lymphocytic Leukemia (CLL), Richter's Syndrome (RS) represents the transformation of a pre-existing CLL into a Diffuse Large B-cell Lymphoma (DLBCL), generally associated with poor prognosis. Current therapeutic approaches are limited and do not significantly reduce disease progression. For these reasons there is intense investigation to identify potentially druggable molecular circuits, opening the way to innovative combination therapies. Among the several oncogenic signalling pathways that may contribute to disease progression, the B Cell Receptor (BCR) is a main driver and an actionable target. We previously showed that BCR ligation in CLL cells increases expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD pathway starting from nicotinamide, a finding in line with the notion of oncogenic-driven metabolic reprogramming. In this context, increased NAMPT expression leading to heightened NAD+ levels could sustain proliferation through the modulation of the activity of several intracellular enzymes, including sirtuins (NAD-dependent deacetylases). Aim: This work explores the connections between BCR signalling and the NAMPT/NAD/sirtuin axis in RS cells, asking the question of whether BCR and NAMPT can be simultaneously targeted. Methods: RS-PDX cells were freshly purified from tumor masses grown in mice and immediately used for short ex-vivo experiments. For in vivo experiments, NSG mice were injected subcutaneously with RS-PDX cells, which were left to engraft until a palpable mass was evident. Mice were then randomized to receive duvelisib, OT82 or a combination of the two for two consecutive weeks. Control mice were similarly treated with vehicle only. Mice were under a niacin-free diet. Results: By using our 4 RS patient-derived xenografts (PDX) models, we invariably observed high levels of NAMPT expression. High levels of NAMPT expression were also observed in 15 primary RS lymph node biopsies analyzed by RNA sequencing. BCR engagement through αIgM polyclonal antibodies significantly up-regulated NAMPT expression, as determined by qRT-PCR and protein analysis, with a concomitant increase in intracellular NAD+ levels. We then asked whether RS cells are sensitive to NAMPT inhibition, alone or in combination with drugs that target the BCR pathway. As most RS patients would likely have been treated during the preceding CLL phase with a BTK inhibitor, possibly developing resistance, we turned to PI3K inhibitors, which are less commonly used for CLL therapy. As NAMPT inhibitors (NAMPTi) we used both FK866 and OT-82, which are validated small molecules. Results indicated that the combination of the dual PI3K-δ/γ inhibitor duvelisib, with either FK866 or OT-82 induces dramatic apoptosis in all 4 models tested, as confirmed by annexinV/PI staining, by caspase 3 activation and by a significant drop in ATP and NAD+ levels. Importantly, two RS-PDX models (RS9737 and RS1316) were fully resistant to NAMPTi used alone, likely due to high levels of nicotinate phosphoribosyltransferase (NAPRT), which is the rate-limiting enzyme in the NAD salvage pathway that starts from nicotinic acid. However, addition of duvelisib, which was mildly effective when used alone, was followed by marked apoptosis even in these two models. Molecular dissection of the pathway showed that the combination of duvelisib and NAMPTi was followed by complete inhibition of the PI3K pathway, which was only partially blocked by duvelisib alone, even at high doses. The connection between NAMPT and PI3K is represented by cytoplasmic sirtuins, particularly SIRT2, which activate AKT through de-acetylation. Immunoprecipitation and two-dimensional gel electrophoresis showed that in the presence of NAMPTi, the amount of acetylated, i.e., inactive, AKT increased considerably. Consistently, treatment of RS-PDX mice with a combination of duvelisib and OT-82 was followed by significantly higher responses and longer animal survival. Conclusions: These results highlight a crosstalk between BCR signalling and NAMPT/sirtuin axis in RS models, showing the increased efficacy of the dual targeting (i.e., PI3K-δ/γ and NAMPT), and supporting this novel and promising therapeutic strategy for the treatment of RS patients. Disclosures Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

scholarly journals Integrative DNA Methylation and Gene Expression Analysis Reveals Candidate Biomarkers Associated with Dichotomized Response to Chemoimmunotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-22
Author(s):  
Ellen K. Kendall ◽  
Manishkumar S. Patel ◽  
Sarah Ondrejka ◽  
Agrima Mian ◽  
Yazeed Sawalha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Research Funding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Enrichment Analysis ◽  
B Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
Gene Sets

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. While 60% of DLBCL patients achieve complete remission with frontline therapy, relapsed/refractory (R/R) DLBCL patients have a poor prognosis with median overall survival below one year, necessitating investigation into the biological principles that distinguish cured from R/R DLBCL. Recent analyses have identified unfavorable molecular signatures when accounting for gene expression, copy number alterations and mutational profiles in R/R DLBCL. However, an integrative analysis of the relationship between epigenetic and transcriptomic changes has yet to be described. In this study, we compared baseline methylation and gene expression profiles of DLBCL patients with dichotomized clinical outcomes. Methods: Diagnostic DLBCL biopsies were obtained from two patient cohorts: patients who relapsed or were refractory following chemoimmunotherapy ("R/R"), and patients who entered durable clinical remission following therapy ("cured"). The median age for R/R and cured cohorts were 62 (range 35-86) years vs. 64 (range 28-83) years (P= 0.27). High-intermediate or high IPI scores were present in 14 vs. 6 patients (P= 0.08) in the R/R and cured cohorts, respectively. All patients were treated with frontline R-CHOP or R-EPOCH. DNA and RNA were extracted simultaneously from formalin-fixed, paraffin embedded biopsy samples. An Illumina 850k Methylation Array was used to identify DNA methylation levels in 29 R/R patients and 20 cured patients. RNA sequencing was performed on 9 R/R patients and 7 cured patients at diagnosis using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB. Results: At the time of diagnosis, we found significant epigenetic and transcriptomic differences between cured and R/R patients. Comparing cured to R/R samples, there were 8,159 differentially methylated probes (FDR&lt;0.05). Differentially methylated regions between R/R and cured cohorts overlap with genes previously identified as mutation hotspots in DLBCL. Upon comparing transcriptomic profiles between R/R and cured, 267 genes were found to be differentially expressed (Log2FC&gt;|1| and FDR&lt;0.05). Gene set enrichment analysis revealed gene sets related to cell cycle, membrane trafficking, Rho and Rab family GTPase function, and transcriptional regulation were upregulated in the R/R samples. Gene sets related to innate immune signaling, Type I and II interferon signaling, fatty acid and carbohydrate metabolism were upregulated in the cured samples. To identify genes likely to be regulated by specific changes in methylation, we selected genes that were both differentially expressed and differentially methylated between the R/R and cured cohorts. In the R/R samples, 13 genes (ARMC5, ARRDC1, C12orf57, CCSER1, D2HGDH, DUOX2, FAM189B, FKBP2, KLF5, MFSD10, NEK8, NT5C, and WDR18) were significantly hypermethylated and underexpressed when compared to cured specimens, suggesting that epigenetic silencing of these genes is associated with lack of response to chemoimmunotherapy. In contrast, 12 genes (ATP2B1, C15orf41, FAM102B, FAM3C, FHOD3, FYTTD1, GPR180, KIAA1841, LRMP, MEF2A, RRAS2, and TPD52) were significantly hypermethylated and underexpressed in cured patients, suggesting that epigenetic silencing of these genes is favorable for treatment response. Many of these epigenetically modified genes have been previously implicated in cancer biology, including roles in NOTCH signaling, chromosomal instability, and biomarkers of prognosis. Conclusions: This is the first integrative epigenetic and transcriptomic analysis of diagnostic biopsies from cured and R/R DLBCL patients following chemoimmunotherapy. At the time of diagnosis, both the methylation and gene expression profiles significantly differ between patients that enter durable remission as opposed to those who are R/R to therapy. Soon, the hypomethylating agent CC-486 (i.e. oral azacitidine) will be explored in combination with mini-R-CHOP for older DLBCL patients in whom DNA methylation is likely increased. These data support the use of hypomethylating agents to potentially restore sensitivity of DLBCL to chemoimmunotherapy. Disclosures Hsi: Eli Lilly: Research Funding; Abbvie: Research Funding; Miltenyi: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; CytomX: Consultancy, Honoraria. Hill:Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Takeda: Research Funding; Beigene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding.

scholarly journals Discovery of DNA G-Quadruplexes As a New Target for B-Cell Receptor Signaling Inhibition in Diffuse Large B-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3502-3502
Author(s):  
Ying-Zhi Xu ◽  
Thomas Raney ◽  
Samantha L. Kendrick
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Cd Spectroscopy ◽  
B Cell Receptor ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Abstract Extensive gene expression profiling and RNA interference studies revealed the frequently chemo-resistant activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on constitutive B-cell receptor (BCR) signaling. As such, the clinical importance of BCR signaling inhibition is well appreciated and thus far led to the development of kinase and protease inhibitors. However, this therapeutic approach fails to achieve complete, sustained responses in DLBCL patients because of inherent resistance due to additional genetic lesions in other components of the BCR pathway or acquired kinase mutations. The emerging field of DNA secondary structures support that guanine (G)-rich stretches of DNA capable of adopting G-quadruplex (G4) motifs act as transcription regulatory units, or switches, that can turn gene expression on or off. Targeting G4s is likely to overcome activating kinase mutations by limiting the amount of gene available for translation into protein. Here, we explore a drug discovery effort based on targeting G4 within BCR genes critical for ABC DLBCL cell survival, CD79A, CD79B, CARD11, and MYD88. We first interrogated the BCR-related genes within the hg19 human reference genome for G-rich DNA using a G4 algorithm and discovered each of the four genes contain G4 forming sequences near promoter regions. These G4 elements formed stable G4 structures as determined by circular dichroism (CD) spectroscopy, the standard for visualizing macromolecule secondary structure formation. Melting curves are also generated from CD spectroscopy to determine the thermal stability of a given structure. The CD79A, CD79B, CARD11, and MYD88 G-rich sequences displayed classic, stable G4 structure spectra consisting of negative minima absorption peaks at 240-265 nm and a positive maximum at 260-295 nm with melting temperatures ranging from 62 to 95 °C. We then developed a high-throughput screening assay based on fluorescence resonance energy transfer (FRET) to identify G4 interactive compounds from the NCI Diversity Set IV library (1584 compounds) that uniquely interact with each of the BCR G4 sequences. This screen used the BCR G4 sequences as molecular bait where the 5´-end and 3´-end of the oligomers were labeled with a FAM- and a TAMRA-fluorophore, respectively, such that G4 formation leads to an increase in fluorescence emission (Figure 1). The initial FRET screen tested compounds at a 1:5 molecular ratio of probe to compound and measured the change in fluorescence relative to probe alone. Overall, the screen resulted in a ~1% "hit" rate for each BCR target, except for CD79B, which yielded a lower percent of interactive compounds (0.3%). Seven compounds, which included ellipticine, quinoline, and daunomycin derivatives, were identified to selectively target the CARD11 (n=3), MYD88 (n=3), or CD79A (n=1) G4s relative to other G4, single-stranded, and double-stranded DNA. Of note, all five compounds found to interact with the CD79B G4 also altered FRET of the other BCR G4 sequences. Subsequent FRET validation and CD analyses where each of the BCR sequences was incubated with increasing concentrations of candidate compounds demonstrated dose-dependent effects on G4 structure formation, particularly stabilization of the CARD11 G4 with compound NCI 9037 that resulted in a 300% FRET increase and an 8 °C shift in melting temperature at a 1:10 ratio. This study identifies DNA G4 as a new class of molecular targets for inhibiting an important oncogenic pathway. Discovery of selective compounds in addition to those with "pan" interaction, suggests the CARD11, MYD88, and CD79A G4 have unique folding patterns whereas the CD79B G4 may exhibit more common structural features. These compounds will be used as molecular tools to provide further insight into the structures and mechanisms in which G4 regulate gene transcription. In establishing a high-throughput screen, we discovered compounds for which preclinical development is ongoing and includes evaluation of the effects on BCR target gene and protein expression, inhibition of downstream BCR signaling, and consequent ABC DLBCL tumor growth and survival. This treatment strategy has high potential for leading to a breakthrough in effectively targeting the constitutively active molecules and greatly impacting the clinical management of patients with BCR-dependent DLBCL. Disclosures No relevant conflicts of interest to declare.

Abstract TP166: Age-Associated Transcriptomic Alterations in Patients With Ischemic Stroke

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Gina Sykes ◽  
Yusra Batool ◽  
Joseph Kamtchum Tatuene ◽  
Sarah Zehnder ◽  
Glen C Jickling
Keyword(s):  
Gene Expression ◽  
Functional Analysis ◽  
Ischemic Stroke ◽  
Immune System ◽  
B Cell ◽  
Differentially Expressed Genes ◽  
Hemorrhagic Transformation ◽  
Differentially Expressed ◽  
P Value ◽  
Humoral Immune

Introduction: Immune system dysregulation occurs with age. This includes an increase in inflammation, and immunosenescence, the inability to efficiently respond to new immune challenges. These changes are evident in various diseases but have yet to be evaluated in a population with ischemic stroke. Age is an important factor in stroke, contributing to stroke risk, outcome and risk of hemorrhagic transformation. This study aimed to assess the changes that occur with age in the leukocyte gene expression of patients with ischemic stroke. Methods: Two cohorts of acute ischemic stroke patients were analyzed; cohort 1 (n=94) and cohort 2 (n=79). RNA was isolated from PAXgene tubes and processed on Affymetrix microarrays. Differentially expressed genes associated with age quartiles were identified by ANCOVA, adjusted for sex and batch. Functional analysis identified age-associated pathways. Differentially expressed genes were compared with previous non-stroke aging studies in whole blood. Results: There were 61 and 442 age-associated genes in cohorts 1 and 2 respectively (FDR-corrected p<0.05, partial correlation coefficient ≥ |0.3|). Nineteen genes, including CR2, CCR6 and CXCR5 , were found in common and decreased with age among both cohorts (max-log10(p value) = 17). Functional analysis of the 61 and 442 genes revealed with advancing age there is a change in the humoral immune system, including antibody production and B cell proliferation. When compared to aging gene expression studies in controls, 52% of age-associated genes in cohort 1 and 31% of cohort 2 age-associated genes overlapped with those found in controls, and 16 of the 19 common genes to both cohorts overlapped in controls (max-log10(p value) = 15). Conclusion: In patients with acute stroke there is a change in leukocyte gene expression with advancing age. Changes included a shift in humoral immune response with a potentially impaired B cell response. While many of the age-associated alterations in gene expression present in stroke are similar to non-stroke controls, these changes warrant further investigation for their impact on stroke outcome and risk.

The PI3K Delta Inhibitor Idelalisib Interferes With Pre-B Cell Receptor Signaling In Acute Lymphoblastic Leukemia (ALL): A New Therapeutic Concept

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2632-2632
Author(s):  
Nathalie Y. Rosin ◽  
Ekaterina Kim ◽  
Stefan Koehrer ◽  
Zhiqiang Wang ◽  
Susan O'Brien ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Differentially Expressed ◽  
B Cell Malignancies ◽  
Bcr Signaling ◽  
Dose Dependent

Abstract Phosphoinositide-3-kinases (PI3K) play an important role in transmitting signals from surface receptors such as the B-cell receptor (BCR), cytokine receptors and receptor tyrosine kinases, that result in survival and growth of normal and malignant B cells. In mature B-cell malignancies such as CLL and indolent B-NHL, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a p110δ-isoform-specific PI3K inhibitor that is highly active in patients with CLL and indolent NHL. In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. RNA expression of the PI3K isoforms α, β, γ, and δ was detected in all B-ALL cell lines. Protein expression was analyzed by immunoblotting. We noted that in vitro responsiveness to idelalisib treatment was associated with protein expression of the δ isoform and presence of the Pre-BCR. We found that treatment of B-ALL cell lines with idelalisib at concentrations between 10 nM and 5 µM inhibited metabolism and growth of B-ALL cells expressing a pre-BCR, whereas only minor effects were observed in Pro-B. To monitor the expression and phosphorylation of proteins (CD72, Akt, Plcγ2, S6, Vav) involved in BCR and PI3K signaling, we prepared protein lysates of B-ALL cell lines. Cells were treated with idelalisib with/without stimulation of the µ-heavy chain of the (Pre-) BCR. We were able to show an increase of pAktSer473 and pS6Ser235/236 after stimulation, as well as a decrease in a dose dependent manner with idelalisib. The decreased amount of Akt phosphorylation was linear with the sensitivity to idelalisib treatment of the cell lines. To investigate intracellular calcium mobilization which occurs in B cells through pre-BCR stimulation, we treated cells with idelalisib and stimulated them with anti-Igµ. A dose dependent decrease in calcium flux was observed in 5 of 6 pre-B cell lines. To examine the effects of idelalisib treatment on the gene expression of pre-B ALL cells, gene expression profiling (GEP) was performed. This revealed down regulation of several genes involved in MAP-Kinase signaling, (Pre-) BCR signaling and Natural Killer cell (NK-cell) mediated cytotoxicity. For the Pre-BCR signaling pathway several genes were differentially expressed, including genes encoding the following proteins, which were found to be down regulated after 3 days of 1 µM idelalisib treatment: BCL-6, CD72, CD79a, Vav, and Plcγ2. To verify the data of the GEP, qPCR analysis was performed. To further investigate the effects of idelalisib on proteins involved in BCR signaling, six Pre-B-ALL cell lines, as well as one mature and one Pro-B cell line were treated with 1 µM idelalisib and the protein expression was quantified after immunoblotting. Most of the proteins that were differentially expressed on genomic levels have also been differentially expressed on proteomic levels and therefore confirmed the effect on Pre-BCR signaling. In summary, these experiments demonstrate inhibition of Pre-BCR signaling on both gene and protein expression levels via idelalisib treatment of Pre-B-ALL cell lines and support the importance of clinical development of the δ isoform specific PI3K inhibitor idelalisib. Disclosures: Burger: Gilead Sciences Inc: Research Funding.

scholarly journals PARP14 Is a Novel Therapeutic Target in STAT6 mutant Follicular Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2842-2842 ◽  
Author(s):  
Verena Passerini ◽  
Michael Boesl ◽  
Elisabeth Silkenstedt ◽  
Elisa Osterode ◽  
Michael Heide ◽  
...  
Keyword(s):  
Gene Expression ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Board Of Directors ◽  
Molecular Diagnostics ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Wild Type ◽  
Advisory Committees

Abstract The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood. IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23. We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4. RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype. In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. Disclosures Richter: HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Comparative Gene Expression Profiling of Leukemia Cells in Peripheral Blood and Tissue Compartments Reveals a Prominent Role of the Microenvironment for CLL Cell Proliferation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 355-355 ◽  
Author(s):  
Yair Herishanu ◽  
Patricia Perez-Gelen ◽  
Delong Liu ◽  
Angelique Biancotto ◽  
Berengere Vire ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Lymph Node ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Peripheral Blood ◽  
Expression Profiling ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Gene Set Enrichment Analysis

Abstract Abstract 355 In-vitro studies suggest that chronic lymphocytic leukemia (CLL) cells depend on the tissue microenvironment. Different molecules and cell types have been reported to enhance the proliferation and survival of CLL cells. The presence of CLL cells in three distinct compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), provides a unique opportunity to investigate the effects of the microenvironment on tumor cell biology in-vivo. To this effect, we used gene expression profiling (Affymetrix HU133 plus arrays) to compare purified CLL cells sampled from PB, BM, and/or LN from 24 previously untreated patients. Initially, an unsupervised hierarchical clustering of all samples appeared to be dominated by the effect of the individual patient. However, in 12 patients where all three sites had been sampled, we used a 3-level one-way ANOVA blocked by patients to estimate patient effect and tissue effect. Three principal components of the 36 samples revealed a clear separation of the tumor cells according to their compartment of origin. Furthermore, supervised analysis with a cutoff of >2-fold change and false discovery rate <0.2 identified 151 genes that discriminated between circulating and LN resident CLL cells (n=17), most of which were more highly expressed in LN, and 27 genes that were differentially expressed in BM as compared to PB cells (n=19). Among the genes upregulated in the lymph node many are readily recognized as related to cell proliferation (e.g. Cyclin D2 and c-MYC) or NF-κB signaling. However, to use an observer independent, unbiased discovery tool to query the gene list for the presence of functional gene signatures we used gene set enrichment analysis (GSEA) and identified several gene expression signatures that were preferentially expressed in LN resident cells: a proliferation signature characterized by E2F and c-MYC regulated genes, signatures related to B-cell receptor and NF-kB signaling were prominent (FDR for all <0.02, normalized enrichment scores 1.81-2.15). A gene expression based E2F score was highest in LN, followed by BM and weakest in PB. Increased nuclear accumulation of E2F1 and c-MYC in LN compared to PB CLL cells was confirmed by Western blotting in paired samples. In general these changes were more prominent in the IgVH unmutated CLL subtype as compared to IgVH mutated CLL cases. In particular, the proliferation E2F score was higher in LN biopsies of IgVH unmutated CLL than IgVH mutated CLL (P=0.04). The E2F score was also an excellent predictor of tumor progression measured as progression free survival (PFS) from diagnosis to treatment: patients with a high E2F score had a median PFS of 16.6 months compared to a PFS in excess of 10 years for patients with a low score (P=0.015). The acquired proliferation and activation signatures in CLL cells which are more prominent in LN resident CLL cells than in cells residing in the BM, suggests that the two microenvironment niches are not identical. Possible upstream cascades driving the signature of CLL cells in the tissue appear to be related to NF-kB and B-cell receptor activation. In conclusion: proliferation and cell activation signatures are acquired in the tissue and are more prominent in LN resident CLL cells than in the BM, suggesting that these two microenvironmental niches have different effects on tumor biology. The LN E2F proliferation signature was more prominent in IgVH unmutated CLL cells and correlated with clinical disease progression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals B-Cell Receptor Driven MALT1 Activity Regulates MYC Signaling in Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 611-611
Author(s):  
Beiying Dai ◽  
Michael Grau ◽  
Mélanie Juilland ◽  
Pavel Klener ◽  
Elisabeth Höring ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Advisory Committees ◽  
Mantle Cell ◽  
Bcr Signaling

Abstract Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor prognosis. Recent studies revealed the importance of constitutive B-cell receptor (BCR) signaling in maintaining survival of a subset of MCLs. MALT1 is an essential component of the CARD11-BCL10-MALT1 (CBM) complex that links BCR signaling to the nuclear factor kappa-B (NF-κB) pathway. Additionally, MALT1 functions as a protease that cleaves various substrates to promote proliferation and survival. However, its role in the molecular pathogenesis of MCL is unknown. To elucidate the functional role of MALT1 in the biology of MCL, we determined its proteolytic activity in primary MCL cells and in MCL derived cell lines. A large fraction of MCLs displayed constitutive activity of MALT1. This MALT1 activity is driven by constitutive BCR signaling, as we were able to show that RNA interference-mediated knockdown of central components of the BCR cascade abolished MALT1 activity. To gain insights into the functional significance of MALT1 in MCL, we knocked down its expression by different MALT1 shRNAs. Transduction of these shRNAs induced cytotoxicity in models that are characterized by constitutive MALT1 activity, whereas no effect on survival was observable in MCLs without MALT1 activation. To determine if this MALT1 addiction translates into an in vivosetting, we knocked down MALT1 in mouse MCL models and detected a significant inhibition of tumor growth. This indicates that MALT1-activated MCLs are dependent on the function of MALT1. These results were confirmed as pharmacologic inhibition of MALT1 significantly reduced cell viability in MALT1-activated MCLs, implying that MALT1 inhibition might represent a promising therapeutic strategy for MCL patients. To understand which biologic processes are regulated by MALT1 in MCL, we profiled gene expression changes at different time points following MALT1 inhibition. An unbiased gene set enrichment analysis identified various previously described MYC gene expression signatures to be among the top downregulated signatures, suggesting that MALT1 regulates MYC expression. MYC downregulation following MALT1 inhibition or MALT1 knockdown was confirmed at the protein level and various analyses revealed that MALT1 regulates MYC expression posttranslationally by preventing its proteasomal degradation. These results were further confirmed in primary mouse splenocytes, indicating that this novel molecular mechanism of regulating MYC expression is not restricted to MCL. To confirm that MYC is indeed expressed in primary MCLs, we determined MYC expression in 234 primary MCL samples by immunohistochemistry. These analyses revealed that 75 samples (32.1%) displayed an intermediate and 55 samples (23.5%) a high MYC positivity, suggesting that MYC is expressed in a substantial number of primary MCLs. As common alterations such as MYC high-levelamplifications and translocations determined by FISH occurred extremely rarely in our primary MCL samples (1% of samples), it is conceivable that BCR-driven MALT1 signaling is the predominant mechanism of MYC upregulation in MCL. In summary, we report for the first time that a substantial fraction of MCLs is addicted to constitutive MALT1 signaling. Thus, MCLs can be divided based on their MALT1 activation status into two distinct subgroups. We further identified a novel regulatory mechanism of MYC expression by MALT1. Thus, our study provides a strong mechanistic rationale to investigate the therapeutic efficacy in targeting the MALT1-MYC axis in MCL patients. Disclosures Trneny: Janssen Research & Development: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreyling:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau.

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