Direct in vivo evidence for B-cell receptor and NF-KB activation in mantle cell lymphoma: role of the lymph node microenvironment and activating mutations. [Affymetrix]
ABSTRACT: We provide direct in vivo evidence for activation of the BCR and canonical NF-KB pathways in MCL that, in the absence of activating mutations, is dependent on the lymph node microenvironment. This finding provides a mechanistic explanation for the surprising efficacy of ibrutinib for the treatment of this type of lymphoma. Mutations in components of the BCR and NF-KB pathways are associated with cell-autonomous signaling and resistance to ibrutinib. Lymph node biopsies and peripheral blood samples were obtained from 55 patients with previously untreated MCL. The samples are distributed into 3 groups: peripheral blood (purified CD19, N=17), unpurified lymph node biopsy (mixed tissue biopsy, N=34), and purified lymph node (purified CD19 Tumor, N=4).
Project description:We provide direct in vivo evidence for activation of the BCR and canonical NF-KB pathways in MCL that, in the absence of activating mutations, is dependent on the lymph node microenvironment. This finding provides a mechanistic explanation for the surprising efficacy of ibrutinib for the treatment of this type of lymphoma. Mutations in components of the BCR and NF-KB pathways are associated with cell-autonomous signaling and resistance to ibrutinib. Lymph node biopsies and peripheral blood samples were obtained from patients with previously untreated MCL.
Project description:Ibrutinib,a novel Bruton'styrosine kinase inhibitor, demonstrated high response rates in B-cell lymphomas but a growing number of ibrutinib treated patients relapse with resistance, fulminant progression and accelerated mortality. Using chemical proteomics and a high-throughput ex vivo assay in a reconstructed tumor microenvironment (TME), we determined the molecular basis for ibrutinib activity and mechanism of acquired ibrutinib resistance. Reciprocal activation of PI3K-AKT-mTOR and integrin β1 signaling were identified as a signaling hub of kinome for ibrutinib resistance, resulting in enforced TME-lymphoma interactions, promoting mantle cell lymphoma (MCL) growth and drug resistance. Combinatorial disruption of BCR signaling and ibrutinib resistance associated pathways led to release of MCL cells from TME, reversal of drugresistance and enhanced anti-MCL activity in murine and patient-derived xenograft models. This study integrated TME-mediated de-novo and acquired drug resistance mechanisms and provides the rationale for novel combination therapeutic strategy against MCL and other B cell malignancies.
Project description:B cell receptor (BCR) signaling has emerged as a therapeutic target in B cell lymphomas, but the precise deployment of inhibitors to target oncogenic BCR signaling requires detailed knowledge of the signaling cascades that the BCR triggers in individual tumors. Here, we have used CRISPR-Cas9 screens to investigate whether the ABC and GCB molecular subtypes of diffuse large B cell lymphoma (DLBCL) utilize distinct BCR signaling modes to sustain their proliferation and survival. Constitutive germinal center (GC) BCR signaling in GCB DLBCLs requires the BCR, CD19 and SYK engaging PI(3) kinase for survival. In ABC DLBCLs with oncogenic mutations in the BCR and MYD88, a novel BCR-TLR9-MYD88 signaling supercomplex is assembled on endolysosomal membranes that engages NF-kB. Our data explain why this subset of ABC DLBCL tumors respond frequently to ibrutinib, an inhibitor of BCR-dependent NF- kB activation, while GCB DLBCLs are insensitive, and thus provide a roadmap for the rational development of BCR pathway inhibitors in molecular subtypes of DLBCL. Overall design: Knockdown of TLR9 (C4) was tested in ABC DLBCL cell lines (HBL1 n=2 and TMD8 n=2) for 1 and 2 days. Knockdown of TLR9 (D7) was tested in ABC DLBCL cell lines (HBL1 n=2 and TMD8 n=2) for 1 and 2 days.
Project description:To determine the global transcriptome changes in mantle cell lymphoma cells following treatment with the BET bromodomain antagonist, JQ1 Mantle Cell Lymphoma (MCL) cells exhibit increased B cell receptor and NFkB activities. The BET protein BRD4 is essential for the transcriptional activity of NFkB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and CDK4/6, inhibits the nuclear RelA levels and the expression of NFκB target genes including Bruton’s Tyrosine Kinase (BTK) in MCL cells. While lowering the levels of the anti-apoptotic BCL2 family proteins, BA treatment induces the pro-apoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Co-treatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared to each agent alone, co-treatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells which overexpress CDK6, BCL2, Bcl-xL, XIAP and AKT, but lack ibrutinib resistance-conferring BTK mutation. Co-treatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL. MO2058 cells treated with vehicle, 250 nM or 1000 nM JQ1 for 8 hours. Samples were acquired and analyzed in duplicate.
Project description:We have developed a transgenic mouse model for Epstein-Barr virus-associated Burkitt's lymphoma. Transgenic mice that express LMP2A and MYC in B cells develop spontaneous lymph node tumors at different rates. Tumor onset occurs at approximately 40-60 days in LMP2A/λ-MYC mice and at 100-200 days in λ-MYC mice. This study compared total gene expression in the tumor cells from each genotype, as well as in B cells isolated from 3 week old mice prior to tumor onset. Comparison of total gene expression in tumor cells from LMP2A/λ-MYC and λ-MYC transgenic mice identified a short list of differentially expressed genes. Comparison of gene expression in B cells from the spleens of 3 week old mice, in contrast, identified a 10-fold increase in the number of differentially expressed genes. B cells from the spleens of 3 week old LMP2A/λ-MYC (n=5), λ-MYC (n=5), LMP2A (n=3), and WT (n=3) transgenic mice were purified using magnetic-activated cell sorting (MACS) beads purchased from Miltenyi for CD19 positive selection. Total RNA was isolated from these purified B cells, as well as from cervical lymph node tumor cells that developed in LMP2A/λ-MYC (n=7) and λ-MYC (n=5) transgenic mice using RNeasy RNA extraction kit from QIAGEN. Cervical lymph node tumor cells were >90% positive for the B cell marker B220, and were not further purified. Total gene expression was assessed in each sample, along with heart and brain reference samples using MouseRef-8 v2.0 Expression Bead Chips purchased from Illumina. GeneSpring analysis software was used to analyze the expression data and identify significantly differentially expressed genes (fold change >1.5-fold and false discovery rate< 0.05).
Project description:MCL cell lines were treated with DMSO or 5uM AFN700 for 20hrs This experiment is designed to see if NFKB-target genes are downregulated by inhibition of IKKB in MCL cell lines that are insensitive to ibrutinib (BTK inhibitor) or sotrastaurin (PKC inhibitor) MCL cells were seeded in 6well dishes and treated for 20hrs with DMSO or 5uM AFN700
Project description:These experiments were designed as a benchmark tool for deconvolution methods. 5 immune cell populations were sorted from 3 healthy donors' peripheral bloods. Peripheral Blood Mononuclear Cells (PBCMs) and PolymorphoNuclear Cells (PMN) were separated using gradient centrifugation. T cells (DAPI-/CD3+/CD14-/CD19-/CD56-), monocytes (DAPI-/CD3-/CD14+/CD19-/CD56-), B cells (DAPI-/CD3-/CD14-/CD19+/CD56-) and NK cells (DAPI-/CD3-/CD14-/CD19-/CD56+) were FACS-sorted from PBMCs and neutrophils (DAPI-/CD66b+/CD19-/CD3-/CD56-/CD14-) were sorted from PMNs. RNA was extracted from the purified cell population, as well as from the HCT116 colon cancer cell line. RNAs from pure populations were then mixed in various proportions. RNA from HCT116 cells and FACS-sorted T cells (DAPI-/CD3+/CD14-/CD19-/CD56-), monocytes (DAPI-/CD3-/CD14+/CD19-/CD56-), B cells (DAPI-/CD3-/CD14-/CD19+/CD56-), NK cells (DAPI-/CD3-/CD14-/CD19-/CD56+), and neutrophils (DAPI-/CD66b+/CD19-/CD3-/CD56-/CD14-) were mixed in various proportions.
Project description:Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of BCR signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 (CBM) complex that transfers BCR signaling to the NF-kB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls a MYC-driven gene expression network predominantly through increased MYC protein stability. Thus our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1 induced MYC regulation is not restricted to MCL, but represents a common mechanism of MYC regulation. MYC itself is pivotal for MCL survival as its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy in targeting the MALT1-MYC axis in MCL patients. Overall design: Gene expression in Mino and Rec cells induced by treatment with 50 µM z-VRPR-fmk was compared with that induced by treatment with DMSO.
Project description:Ibrutinib (Ibr), an orally administered covalent inhibitor of Bruton’s kinase, has generated remarkable responses in CLL patients including those with an unfavorable cytogenetic profile. Once patients develop resistance to ibrutinib, the outcome is poor with few treatment options. To further understand the genomic mechanisms underlying Ibr resistance, we’ve performed genome wide copy number analysis of serial samples collected from nine ibrutinib-relapsed patients. Deletions of 18p often coincide with del(17p)/TP53 mutations, predispose patients to relapse. Overall design: Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from diagnostic bone marrow, peripheral blood or lymph node samples. Copy number analysis of Affymetrix CytoScan or OncoScan SNP arrays was performed on a total of 26 samples collected from 9 patients included in the study. Please note that OncoScan arrays produce 2 CEL files generated from 2 separate chips that are subsequently converted and combined into one OSCHP file.