scholarly journals Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma

Blood ◽  
2012 ◽  
Vol 119 (9) ◽  
pp. 1963-1971 ◽  
Author(s):  
Robert Kridel ◽  
Barbara Meissner ◽  
Sanja Rogic ◽  
Merrill Boyle ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Transcriptome Sequencing ◽  
Cell Lymphoma ◽  
Notch Pathway ◽  
Clinical Samples ◽  
Mantle Cell ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome ◽  
Notch1 Mutations

Abstract Mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma, is characterized by the hallmark translocation t(11;14)(q13;q32) and the resulting overexpression of cyclin D1 (CCND1). Our current knowledge of this disease encompasses frequent secondary cytogenetic aberrations and the recurrent mutation of a handful of genes, such as TP53, ATM, and CCND1. However, these findings insufficiently explain the biologic underpinnings of MCL. Here, we performed whole transcriptome sequencing on a discovery cohort of 18 primary tissue MCL samples and 2 cell lines. We found recurrent mutations in NOTCH1, a finding that we confirmed in an extension cohort of 108 clinical samples and 8 cell lines. In total, 12% of clinical samples and 20% of cell lines harbored somatic NOTCH1 coding sequence mutations that clustered in the PEST domain and predominantly consisted of truncating mutations or small frame-shifting indels. NOTCH1 mutations were associated with poor overall survival (P = .003). Furthermore, we showed that inhibition of the NOTCH pathway reduced proliferation and induced apoptosis in 2 MCL cell lines. In summary, we have identified recurrent NOTCH1 mutations that provide the preclinical rationale for therapeutic inhibition of the NOTCH pathway in a subset of patients with MCL.

Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 436-436 ◽  
Author(s):  
Robert Kridel ◽  
Barbara Meissner ◽  
Sanja Rogic ◽  
Merrill Boyle ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
The Other ◽  
Mantle Cell ◽  
Whole Transcriptome ◽  
Notch1 Mutations

Abstract Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion: We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures: Sehn: Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

scholarly journals Disrupting GRINL1A Association with Cereblon Accelerates IKZF1/3 Degradation and IMiD Killing of Mantle Cell Lymphoma Cells By CDK4/6 Inhibition

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 299-299
Author(s):  
Nicole Zhao ◽  
Maurizio Di Liberto ◽  
Olivier Elemento ◽  
Selina Chen-Kiang ◽  
Xiangao Huang
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Mantle Cell Lymphoma ◽  
Transcriptome Sequencing ◽  
Cell Lymphoma ◽  
G1 Arrest ◽  
Immunomodulatory Drugs ◽  
Mantle Cell ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome

Lenalidomide (Len) and Pomalidomide (Pom) are immunomodulatory drugs (IMiDs) used for the treatment of multiple myeloma (MM) and have efficacy in other hematologic malignancies. Although as single agents, IMiDs rarely achieve complete remission despite a high overall response rate and durability, they potentiate the clinical response to diverse partners including Rituximab in mantle cell lymphoma (MCL). In MM, the expression of cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Binding of IMiDs to CRBN promotes the recruitment of transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) to CRL4CRBN, for ubiquitination-mediated degradation. This leads to loss of IRF4 and ultimately apoptosis. Crystal structure studies have further identified five putative inhibitors of CRBN (GRINL1A, MBOAT7, OTUD7B, C6orf141 and MEIS2). But no information for their endogenous expression or biological function is available, and the mechanism that mediates IMiD sensitivity in MCL is unknown. To determine the mechanism of IMiD action and if any of the putative CRBN inhibitors acts endogenously in MCL, we first demonstrated by whole transcriptome sequencing (WTS), that only POLR2M (encoding GRINL1A) and MBOAT7 mRNAs were expressed along with IKZF1 and IKZF3 in primary human MCL cells and MCL cell lines. Single-cell RNA sequencing (scRNA-seq) analysis of peripheral mononuclear cells (PBMCs) from treatment-naïve MCL patients further demonstrated that POLR2M, but not MBOAT7, was co-expressed with CRBN and IKZF1 in MCL cells, T cells and NK cells. These data suggest that GRINL1A may modulate the CRBN activity in MCL cells. To address this, we found that Len effectively kills the CCMCL1 MCL cell line, despite c-Myc overexpression and loss of p16INK4a, but not the JEKO-1 cell line, which harbors a deletion in TP53. However, Pom kills both MCL cell lines in a time-dependent manner via apoptosis, as indicated by PARP cleavage. Moreover, Pom killing was accelerated and markedly augmented by prior induction of prolonged early G1 arrest (pG1) through selective inhibition of CDK4/6 with palbociclib. GRINL1A and CRBN proteins were coordinately elevated by Pom within 6 hours, unless the MCL cells were already arrested in G1 by palbociclib pretreatment. By contrast, the MBOAT7 protein level remained unchanged by Pom and even decreased in pG1. This implies that GRINL1A and CRBN proteins may be stabilized by association with each other selectively, and this association is opposed by induction of pG1. Indeed, IP-Western blotting confirmed that GRINL1A and CRBN were in the same immune complex in both CCMCL1 and JEKO-1 cells while MBOAT7 was not. Importantly, induction of pG1 reduced the association of GRINL1A with CRBN, greater by one hour of exposure to Pom and more prominently in CCMCL1 cells than in JEKO-1 cells. Consistent with augmented Pom killing by cooperative reduction of the GRINL1A-CRBN complex in pG1, IKZF1 and IKZF3 were completely degraded by 6 hours of Pom exposure in pG1, but significantly less so by Pom alone. Thus, GRINL1A selectively associates with CRBN in MCL cells, and rapid disruption of this association through induction of G1 arrest by CDK4/6 inhibition accelerates Pom killing via IKZF1/3 degradation. In summary, we have demonstrated for the first time that 1) two putative CRBN inhibitors POLR2M and MBOAT7 are expressed in primary human MCL cells by whole transcriptome sequencing; 2) POLR2M, but not MBOAT7, is co-expressed with CRBN and IKZF1 in primary human MCL cells, T cells and NK cells by scRNA-seq; 3) Induction of prolonged early G1 arrest by CDK4/6 inhibition augments Pom killing, even in Len-resistant MCL cells; and 4) GRINL1A, but not MBAOT7, associates with CRBN in MCL cells and induction of G1 arrest cooperates with Pom to reduce this complex, concurrent with accelerated loss of IKZF1/3 and apoptosis despite overexpression of c-Myc, deletion of p16INK4a or TP53 in MCL cells. Our findings shed light on the mechanism of IMiD action in MCL and identify GRINL1A as the first endogenous inhibitor for the CRL4CRBN E3 ligase in MCL cells. As immunomodulatory drugs, Pom and Len act on immune cells in the tumor-microenvironment as well, especially NK cells and T cells. Co-expression of POLR2M with CRBN and IKZF1 in human PBMCs, as we discovered, provides further new insights into controlling the endogenous CRBN inhibitor in IMiD therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 476-487 ◽  
Author(s):  
Mamta Gupta ◽  
Andrea E. Wahner Hendrickson ◽  
Seong Seok Yun ◽  
Jing Jing Han ◽  
Paula A. Schneider ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Clinical Samples ◽  
Lymphoid Malignancies ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

scholarly journals Oncogenic MALT1 Promotes Cell Survival and Mediates Ibrutinib Resistance and Ibrutinib-Venetoclax Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-18
Author(s):  
Vivian Changying Jiang ◽  
Junwei Lian ◽  
Shengjian Huang ◽  
Shaojun Zhang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Mtor Signaling ◽  
Mantle Cell ◽  
Whole Transcriptome Sequencing ◽  
Ibrutinib Resistance ◽  
Whole Transcriptome

Both as monotherapies and in combination, the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax have proven to be efficacious and are now widely used treatment options for mantle cell lymphoma (MCL) patients. However, mono- and dual- resistance frequently develops, necessitating investigation into the mechanisms mediating resistance to these therapies. To investigate the mechanism of ibrutinib resistance, we generated two ibrutinib-resistant cells due to marked BTK knockdown via CRISPR/CAS9 from JeKo-1, which is ibrutinib-sensitive and venetoclax-resistant. To understand the mechanism of venetoclax resistance, we generated three venetoclax-resistant cell lines with acquired resistance via chronic exposure to increasing doses of venetoclax from ibrutinib/venetoclax double sensitive Mino and Rec-1 cells, and ibrutinib-resistant but venetoclax-sensitive Granta519 cells. All these paired cell lines with various resistance to ibrutinib and venetoclax were subject to whole transcriptome sequencing of these paired MCL cell lines. We discovered that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is significantly overexpressed in ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells, especially in cells with BTK knockdown. This was further validated in primary MCL patient cells (n=24). Interestingly, MALT1 overexpression inversely correlates with CARD11 expression and enhances non-canonical NF-κB signaling, suggesting a switch from a highly-dependent BTK-CARD11 mechanism to an independent mechanism in both ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells. Chromosomal translocations of MALT1 are the hallmarks of MALT lymphoma, which result in oncogenic fusion of MALT1 products. MALT1 is constitutively active in driving aggressive ABC-type DLBCL. This indicates MALT1 can be oncogenic when its activity is dysregulated. Therefore, we hypothesized that constitutive MALT1 activity may be responsible for the resistance to ibrutinib and venetoclax in MCL cells. To demonstrate this relationship, MALT1 ablation using genetic manipulation resulted in significant growth defects both in vitro and in vivo. Pharmaceutical approaches using MALT1 inhibitor MI-2 resulted in similar effects on cell survival and using cell viability assays. Whole transcriptome sequencing analysis revealed that MYC, NF-kB, ROS, cell cycle and mTOR signaling are the most significantly downregulated pathways upon MI-2 treatment. Intriguingly, MYC, NF-kB, PI3K-AKT-mTOR and mTOR signaling pathways were reported to be upregulated in ibrutinib-resistant MCL cells compared to sensitive MCL cells. To address this further, proteomics analysis by reverse phase protein array (RPPA) using more than 400 antibodies confirmed that MI-2 significantly downregulated AKT-mTOR signaling. NF-kB modulation, ROS production, AKT-mTOR, and metabolism changes were further confirmed through multiple biochemical approaches. In addition, MI-2 treatment resulted in a dramatic reduction of MALT1 expression, suggesting that MI-2 treatment affected both its scaffold and paracaspase activities. Furthermore, MI-2 treatment resulted in significant inhibition of in vivo tumor growth of ibrutinib-venetoclax dual-resistant MCL subcutaneous xenografts and tumor homing to the spleen and bone marrow in an ibrutinib-venetoclax dual-resistant MCL patient-derived xenograft (PDX) mouse model. In conclusion, we discovered that MALT1, an essential regulator of NF-κB signaling, is hyperactive in ibrutinib-resistant cells and ibrutinib-venetoclax dual-resistant MCL cells, which puts MALT1 forward as a potentially new therapeutic target in ibrutinib and venetoclax-resistant MCL tumors. Genetic depletion or pharmaceutical inhibition of MALT1 resulted in remarkable defects in cell survival and cell proliferation. The MALT1 inhibitor MI-2 proved its in vivo potency by its pro-apoptotic effect and its significant tumor growth inhibition. In conclusion, targeting a hyperactive MALT1 is a promising therapeutic strategy that could lead to clinical implementation of a new treatment strategy meant to overcome ibrutinib and ibrutinib-venetoclax dual resistance in MCL patients by reversing the NF-kB and ROS/mTOR- mediated resistance in these tumors. Disclosures Wang: Targeted Oncology: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; MoreHealth: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses.

scholarly journals Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5171-5179 ◽  
Author(s):  
Pascal Gelebart ◽  
Mona Anand ◽  
Hanan Armanious ◽  
Anthea C. Peters ◽  
Jennifer Dien Bard ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Selective Inhibition ◽  
Canonical Pathway ◽  
Oligonucleotide Arrays ◽  
Downstream Target ◽  
Naturally Occurring ◽  
Inactive Form ◽  
Mantle Cell

Abstract Aberrations of the Wnt canonical pathway (WCP) are known to contribute to the pathogenesis of various types of cancer. We hypothesize that these defects may exist in mantle cell lymphoma (MCL). Both the upstream and downstream aspects of WCP were examined in MCL cell lines and tumors. Using WCP-specific oligonucleotide arrays, we found that MCL highly and consistently expressed Wnt3 and Wnt10. β-catenin, a transcriptional factor that is a downstream target of WCP, is localized to the nucleus and transcriptionally active in all 3 MCL cell lines examined. By immunohistochemistry, 33 (52%) of 64 MCL tumors showed nuclear localization of β-catenin, which significantly correlated with the expression of the phosphorylated/inactive form of GSK3β (p-GSK3β; P = .011, Fisher). GSK3β inactivation is directly linked to WCP stimulation, since addition of recombinant sFRP proteins (a naturally occurring decoy for the Wnt receptors) resulted in a significant decrease in p-GSK3β. Down-regulation of DvL-2 (an upstream signaling protein in WCP) by siRNA or selective inhibition of β-catenin using quercetin significantly decreased cell growth in MCL cell lines. To conclude, WCP is constitutively activated in a subset of MCL and it appears to promote tumorigenesis in MCL.

Expression of STAT3 and its phosphorylated forms in mantle cell lymphoma cell lines and tumours

2002 ◽  
Vol 199 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Raymond Lai ◽  
George Z Rassidakis ◽  
L Jeffrey Medeiros ◽  
Vasiliki Leventaki ◽  
Micheal Keating ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Lymphoma Cell ◽  
Mantle Cell Lymphoma Cell ◽  
Mantle Cell

scholarly journals BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Antitumour Activity ◽  
Single Agent ◽  
Treatment Strategies ◽  
Bet Inhibitor ◽  
Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

scholarly journals Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

2018 ◽  
Vol 2 (16) ◽  
pp. 2039-2051 ◽  
Author(s):  
Jimmy Lee ◽  
Liang Leo Zhang ◽  
Wenjun Wu ◽  
Hui Guo ◽  
Yan Li ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Intrinsic Resistance ◽  
Mantle Cell ◽  
Myc Gene ◽  
Bona Fide ◽  
Ibrutinib Resistance ◽  
Induced Apoptosis

Abstract The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

scholarly journals PRMT5 Inhibition Drives Therapeutic Vulnerability to BCL-2 Inhibition with Venetoclax and Provides Rationale for Combination Therapy in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 302-302 ◽  
Author(s):  
Fiona Brown ◽  
Yang Zhang ◽  
Claire Hinterschied ◽  
Alexander Prouty ◽  
Shelby Sloan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Cell Lymphoma ◽  
Targeted Agents ◽  
Mantle Cell ◽  
Anti Tumor Activity

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50&lt;10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50&gt;1uM). All lines reached an IC50 &lt;1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores &gt; 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Analysis of mRNAs and ncRNAs and Prediction Competing Endogenous RNA Networks in Colorectal Cancer Chemo-Resistance by Whole-Transcriptome Sequencing

2020 ◽  
Author(s):  
Fei Yao ◽  
Chuanren Zhou ◽  
Qiyou Huang ◽  
Xiaoying Huang ◽  
Jie Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cell Lines ◽  
Transcriptome Sequencing ◽  
Regulatory Networks ◽  
Parental Cell ◽  
Resistant Cell ◽  
Competing Endogenous Rna ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome

Abstract Background: Chemo-resistance is a major clinical obstacle to the treatment of colorectal cancer (CRC), mRNAs and non-coding RNAs (ncRNAs) have been reported to modulate the development of chemo-resistance. However, the profiles of mRNAs and ncRNAs as well as competing endogenous RNA (ceRNA) networks in CRC chemo-resistance are still unclear, and whether different drug resistance of CRC have the same mechanisms also needs to be explored. This study aims to uncover the expression of mRNAs and ncRNAs in parental cell lines and different chemo-resistant cell lines, and construct ceRNA regulatory networks by whole-transcriptome sequencing.Methods: The expression of mRNAs and ncRNAs in parental cell lines and drug-resistant cell lines were identified by whole-transcriptome sequencing and bioinformatics methods.Results: A total of 1779 mRNAs, 64 miRNAs, 11 circRNAs and 295 lncRNAs were common differentially expressed in two different chemo-resistant cell lines when compared with the control. In addition, 5,767 lncRNA-miRNA-mRNA relationship pairs and 47 circRNA-miRNA-mRNA pathways were constructed according to ceRNA regulatory rules, in which AC109322.2-hsa-miR-371a-5p-BTNL3 and hsacirc_027876-hsa-miR-582-3p-FREM1 were identified as the most potential ceRNA networks involved in drug resistance to CRC. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of two ceRNA regulatory networks showed that the TNF signaling pathway may be crucial in the process of CRC drug resistance.Conclusions: A large number of mRNAs and ncRNAs in chemo-resistant cell lines were different expressed, which may play pivotal roles in development of drug resistance through the ceRNA regulatory network. This study may improve our understanding of the underlying mechanisms and provide a promising therapeutic strategy for CRC chemo-resistance.

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