Development Of Mantle Cell Lymphoma Research Articles

Disruption of cyclin D1 degradation leads to the development of mantle cell lymphoma

Cyclin D1 has been recognized as an oncogene due to its abnormal upregulation in different types of cancers. Here, we demonstrated that cyclin D1 is SUMOylated, and we identified Itch as a specific E3 ligase recognizing SUMOylated cyclin D1 and mediating SUMO-induced ubiquitination and proteasome degradation of cyclin D1. We generated cyclin D1 mutant mice with mutations in the SUMOylation site, phosphorylation site, or both sites of cyclin D1, and found that double mutant mice developed a Mantle cell lymphoma (MCL)-like phenotype. We showed that arsenic trioxide (ATO) enhances cyclin D1 SUMOylation-mediated degradation through inhibition of cyclin D1 deSUMOylation enzymes, leading to MCL cell apoptosis. Treatment of severe combined immunodeficiency (SCID) mice grafted with MCL cells with ATO resulted in a significant reduction in tumor growth. In this study, we provide novel insights into the mechanisms of MCL tumor development and cyclin D1 regulation and discover a new strategy for MCL treatment.

Development of a clinically relevant chemoresistant mantle cell lymphoma cell culture model.

Mantle cell lymphoma is an aggressive subtype of non-Hodgkin’s lymphoma that claims the lives of tens of thousands of people every year. Although combination chemotherapy treatments such as CHOP have yielded promising outcomes in the clinic, the development of chemoresistance in patients has limited their long-term success. The lack of in vitro chemoresistance models has limited our ability to understand the mechanisms by which cells develop resistance, and thus our ability to develop novel therapeutics to overcome this issue. Here, we describe the development of a clinically relevant chemoresistant mantle cell lymphoma model using the JeKo-1 cell line. This was achieved through a stepwise treatment selection strategy using gradually increasing concentrations of CHOP. We show that resistant JeKo-1 cells display strong recovery and fast proliferation after treatment with an IC50 dose of CHOP. We also found that resistant JeKo-1 cells overexpress three oncogenes implicated in the development of mantle cell lymphoma—Cyclin D1, Mcl-1, and Bcl-2—compared to normal JeKo-1 cells. We anticipate that in vitro models such as this one will enable the discovery of new therapeutic strategies for overcoming chemoresistance and improve clinical outcomes in mantle cell lymphoma patients. Impact statement Mantle cell lymphoma remains one of the deadliest subtypes of non-Hodgkin’s lymphoma, in large part because patients become resistant to frontline chemotherapy. The development of strategies to treat advanced disease will be contingent upon testing in appropriate models. Most in vitro models of resistant mantle cell lymphoma are laboratory grade models that do not recapitulate the low level of chemoresistance typically observed in patients, limiting their utility. This study develops a clinically relevant in vitro model that can be used to establish the mechanisms of resistance and test new therapeutics intended to treat recurrent disease.

Sox11 promotes head and neck cancer progression via the regulation of SDCCAG8

BackgroundSOX11 is a transcription factor that plays an important role in mantle cell lymphoma development. However, its functional role in head and neck squamous cell carcinoma (HNSCC) remains unknown.MethodsProtein expression was measured with Western blotting, immunohistochemistry or quantitative proteomics, and gene expression was measured with quantitative RT-PCR. Functional role of SOX11 in HNSCC was evaluated with MTS/apoptosis, migration, invasion assays and a xenograft model. A SOX11-targeting gene, SDCCAG8, was confirmed with chromatin immunoprecipitation (ChIP), luciferase reporter and rescue assays.ResultsSOX11 was up-regulated in recurrent versus primary HNSCC and in highly invasive versus low invasive HNSCC cell lines. Silencing SOX11 in HNSCC cell lines significantly inhibited the cell proliferation, migration, invasion and resistance to Cisplatin, and vice versa. Quantitative proteomic analysis of SOX11-silencing HNSCC cells revealed a number of differentially expressed proteins, including a down-regulated tumor antigen SDCCAG8. Silencing of SDCCAG8 in HNSCC cells also significantly inhibited the cell proliferation, migration and invasion, and vice versa. ChIP assays demonstrated that endogenous SOX11 strongly bound to Sdccag8 gene promoter in highly invasive HNSCC cells. When over-expressed in low invasive HNSCC cells, wild type SOX11 but not mutant SOX11 induced the promoter activity of Sdccag8 and significantly induced the expression of SDCCAG8. However, exogenous mutant SOX11 abolished the expression of SDCCAG8 in highly invasive HNSCC cells. In addition, the inhibitory effects of SOX11 knockdown were partially rescued by over-expression of SDCCAG8 in HNSCC cells.ConclusionCollectively, our findings indicate SOX11 promotes HNSCC progression via the regulation of SDCCAG8.

LncRNA MALAT1 promotes development of mantle cell lymphoma by associating with EZH2

BackgroundMantle cell lymphoma (MCL) is considered an aggressive subtype of non-Hodgkin’s lymphoma with variable treatment responses. There is an urgent need to identify novel markers with prognostic and therapeutic value for MCL. Long non-coding RNAs (lncRNAs) have emerged as key regulators in cancers, including MCL. Metastasis-associated lung adenocarcinoma transcript 1(MALAT1), a lncRNA located at pathognomonic translocation site of t (11; 14) of MCL. MALAT1 is known to be overexpressed in solid tumors and hematologic malignancies. However, the pathological role and clinical relevance of MALAT1 in MCL are not completely understood.MethodsWe quantified MALAT1 in MCL samples (40) and CD19+ B cells by quantitative real time polymerase chain reaction (qRT-PCR) and correlated levels with clinical outcome. We silenced MALAT1 in MCL cell lines and analyzed cells in tumorigenic assays and formation of transcription complexes.ResultsWe found that the expression of MALAT1 was elevated in human MCL tumors and cell lines as compared to normal controls, and the elevated levels of MALAT1 correlated with higher MCL international prognostic index (MIPI) and reduced overall survival. MCL with knockdown of MALAT1 showed impaired cell proliferation, facilitated apoptosis and produced fewer clonogenic foci. The increased expression of p21 and p27 upon MALAT1 knockdown was regulated by enhancer of zeste homolog 2 (EZH2). Moreover, decreased phosphorylation of EZH2 at T350 attenuated the binding to MALAT1.ConclusionsOur findings illuminate the oncogenic role of MALAT1, which may serve as a novel biomarker and as a therapeutic target in MCL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1100-9) contains supplementary material, which is available to authorized users.

A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma

The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1-driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1-expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G(1)-S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2's antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1-expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1-expressing tumors.

Evidence of Long Latency Periods Prior to Development of Mantle Cell Lymphoma

AbstractAbstract 323Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by treatment failures and short survival. At presentation, MCL is often widely disseminated. Occasionally, early lymphoma can be seen as in situ lesions involving the mantle zones of secondary follicles. The primary genetic lesion involved in the pathogenesis of the disease is the t(11;14)(q13;q32)/(IGH/CCND1) which leads to the overexpression of cyclin D1, a cell cycle regulatory protein that is not normally expressed in B-cells. However, MCL frequently has numerous additional chromosomal alterations making it one of the most genetically unstable lymphomas. Thus, while the IGH/CCND1 rearrangement is the critical initiating event, it appears that additional alterations are necessary to develop MCL. However, little is known about the latency period required to accumulate sufficient lesions to develop MCL. Recently, it has been appreciated that the initiating chromosomal events in other B-cell malignancies may precede the development of overt disease by many years. In particular, the defining chromosomal events in childhood ALL may be found in the heel sticks of children at birth, even if leukemia does not develop until a number of years later. We recently reported a unique case of mantle cell lymphoma arising simultaneously in the donor and recipient 12 years following allogeneic bone marrow transplantation for chronic myelogenous leukemia(J. Clin. Oncol., in press). This suggested that MCL may exhibit long latency periods prior to the development of frank lymphoma. In order to determine whether in situ MCL may be identified in patients with MCL, we identified 7 patients who were diagnosed with mantle cell lymphoma and who had previous pathological material available that contained lymphoid tissue and that was unrelated to the subsequent MCL. Remarkably, all 7 specimens showed evidence of in situ mantle cell lymphoma, as judged by cyclin D1 over-expression localized to lymphoid cells. Pathological specimens ranged from 2.1–15.5 years prior to the diagnosis of MCL. In all 7 specimens, cyclin D1 positive collections of lymphocytes were identified, 5 with distinct homing to mantle zones, one with follicular colonization and mantle homing, and 1 with a diffuse distribution. FISH studies were undertaken to identify cyclin D1 chromosomal translocations, and evidence of chromosomal translocations could be identified in 2/3 cases evaluated whereas no signals were seen in adjacent uninvolved lymphoid tissue. Taken together, the data presented strongly suggest that a long latency period following the initiating IGH/CCND1 translocation may occur in MCL with patients harboring in situ lesions for years prior to the development of clinical disease. These findings shed interesting new light on the natural history of MCL.Pre-lymphoma locationPattern of in situ MCLLatency (yrs)Lymphoma locationCCND1 FISH in pre-lymphoma specimenObturator node from radical prostatectomyCyclinD1+ focal colonized follicles, scattered cells in mantles7.4Sigmoid colon biopsyNDDuodenal serosal lymph node (perforated benign gastric ulcer)Diffuse scattered CyclinD1+ cells2.1Sigmoid colon biopsyNDRight neck subcutaneous lymph node (neurofibroma excision)CyclinD1+ cells in follicular mantles3.2Bone marrow biopsyNDPeriesophageal lymph nodes from esophagectomySeveral CyclinD1+ colonized primary follicles8.0Right cervical lymph nodeNDAxillary lymph node from mastectomyCyclinD1+ cells in follicular mantles15.5Mesenteric lymph node<5% positive signalsMesenteric lymph node dissection for colon cancerCyclinD1+ cells in follicular mantles10.6Base of tongue lesionPositiveCervical lymph node involved with T cell lymphomaCyclinD1+ cells in follicular mantles3.1Right axillary lymph nodePositive Disclosures:No relevant conflicts of interest to declare.

Characterization of Secondary Alterations in Mantle Cell Lymphomas Using Matrix-/Array CGH.

Overexpression of Cyclin D1 due to the chromosomal translocation t(11;14)(q13;q32) alone is not sufficient to induce the development of mantle cell lymphoma (MCL). Therefore, secondary genetic alterations seem to be necessary for the malignant transformation and may determine the clinical course of the disease. In order to define chromosomal imbalances more precisely, we analyzed six t(11;14)-positive mantle cell lymphoma cell lines (Granta-519, NCEB1, JeKo-1, Rec-1, SP-53 and HBL-2) by means of matrix-/array-based comparative genomic hybridization (array-CGH), a new molecular tool that allows the genome-wide screening of chromosomal imbalances. The microarray used contains 6251 individual BAC/PAC clones. Clones were selected to reach a genome-wide resolution of 1 Mb and an even higher resolution of up to 100kb in recurrently aberrant regions of MCL. In addition, regions containing known tumor suppressor genes and oncogenes were covered by a high density of clones. The hybridization of tumor DNA against normal control DNA and the subsequent washing steps were carried out using the automated slide processor Lucidea (Amersham-Pharmacia Biotech). Based on the normalized fluorescence ratios computed as log2 values, we were able to detect amplifications as well as single gains and losses. The most frequent alterations were losses in 9p21.3, 2p11.2, 22q11.22, 1p21.1-p31.2, 13q14, 11q22, 6q21, 6q27, 17p13 and gains in 7p14.1, 7q11.2, 2q37.1, 8q24, 12q13 and 18q21. These results are in agreement with the recently published data from deLeeuw et al. (HMG Advance Access June 30, 2004). Up to now, losses of 2p11.22 (all cell lines except for JeKo-1) and 22q11.22 (all cell lines except for Rec-1) have not been known as recurrent aberrations of MCL. Selected chromosomal losses (p16, p53) and the amplification of BCL2 in Granta-519 were confirmed by means of fluorescence in situ hybridization using commercially available probes (Abbott Diagnostics, Wiesbaden, Germany). Matrix-/array CGH analyses enabled us to further delineate important regions of gain and loss like 13q14. In conclusion, the cell lines are excellent model systems which will facilitate the identification and characterization of novel genes which play important roles in the pathobiology of MCL.

Cell cycle alterations in the blastoid variant of mantle cell lymphoma (MCL-BV) as detected by gene expression profiling of mantle cell lymphoma (MCL) and MCL-BV.

Overexpression of cyclin D1 is necessary but by itself insufficient for the development of mantle cell lymphoma (MCL). To identify pathways in the pathogenesis of MCL and the blastoid variant (MLC-BV), we compared the gene-expression profiles of microdissected normal mantle cells, MCL, and MCL-BV by oligonucleotide microarrays and quantitative reverse transcriptase PCR (QRT-PCR). We identified and confirmed the overexpression of several genes in MCL-BV that are involved in the cell cycle control at the G1/S and G2/M checkpoints or inhibit apoptotic cell death. The highly expressed cyclin dependent kinase 4 (CDK4) is a cell cycle kinase that associates with cyclin D1 for the progression through the G1/S checkpoint, whereas overexpression of cdc28 protein kinase 1 (CKS1) blocks the inhibition of the cyclin D1/CDK4 complex by the CDK inhibitor p27/Kip1. Other highly expressed genes in MCL-BV that promote the cells through the G1/S-checkpoint include the oncogenes B-Myb, PIM1, and PIM2, and passage through the G2/M-checkpoint is enhanced by high levels of cdc25B. Furthermore, two highly expressed genes that inhibit apoptosis are defender against cell death (DAD1) and RSK1. In summary, our microarray and QRT-PCR analyses identified several candidate genes whose expression increased when comparing normal follicular mantles with MCL and MCLBV, suggesting a potential pathogenic role in the evolution of MCL-BV.

Somatic hypermutation and V(H) gene usage in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is considered to derive from naïve, pregerminal center (GC) CD5+ B-cells. However, the cell of origin has been questioned in recent studies that showed somatic hypermutations in the immunoglobulin (Ig) variable heavy chain (V(H)) genes in subsets of MCL. To clarify this issue, we analyzed the IgV(H) genes for the presence of somatic hypermutations in 51 MCL cases. Twenty percent of the MCL cases displayed somatically mutated V(H) genes (defined as >2% mutated), whereas 80% showed unmutated V(H) genes. This finding suggests that MCL is a genetically heterogeneous disease, with the majority of cases originating from unmutated pre-GC B-cells and a subset deriving from more mature B-cells which have been exposed to the GC environment and have undergone somatic hypermutation. A biased V(H) gene usage has been demonstrated in several B-cell malignancies; however, this has not yet been investigated in MCL, although V(H)4-34 overusage has been indicated by small studies. Interestingly, we found a restricted usage of three individual V(H) genes in our MCL material; V(H)4-34 (22%), V(H)3-21 (16%) and V(H)5-51 (12%). This novel finding of preferential V(H) gene usage in half of the MCL cases may suggest an antigen driven process occurring in B-cells expressing specific VH genes, thus implicating that Ig specificity could be involved in mantle cell lymphoma development.

Molecular mechanisms of lymphomagenesis through transcriptional disregulation by chromosome translocation.

Chromosome translation plays an important role for lymphomagenesis. Transcriptional disregulation type of chromosome translocation involves a majority of B-cell lymphoma. These include BCL1/cyclin D1 translocation in mantle cell lymphoma, BCL2 in follicular lymphoma and BCL6 in diffuse large B-cell lymphoma. It is known that the transcriptional disregulation type causes aberrant gene expression at the stages where those genes are down-regulated in normal counterpart cells. Normal B-cells at mantle zone stage down-regulate the expression of BCL1 gene and become resting in cell cycle. However, BCL1 expression from translocated allele with immunoglobulin gene is not down-regulated at the mantle zone stage, preventing cells from entering in resting state, and puts cells in cell cycle, leading to development of mantle cell lymphoma. BCL2 expression from altered allele also keeps its expression at the germinal center stage where normal counterpart cells down-regulate BCL2 expression, and makes cells to resist apoptosis, leading to development of follicular lymphoma. The same scenario can apply to diffuse large B-cell lymphoma with BCL6 translocation; i.e. aberrant BCL6 expression at the post germinal center stage takes place where normal counterpart cells down-regulate BCL6 expression. Although a strong association of specific translocations with specific disease types is found, these translocations by themselves are not sufficient for malignant transformation. The factors other than chromosome translocations will be discussed.