Subset Of Mantle Cell Lymphoma Research Articles

A Multicenter Phase 2 Trial of Zanubrutinib, Obinutuzumab, and Venetoclax (BOVen) in Patients with Treatment-Naïve, TP53-Mutant Mantle Cell Lymphoma

Background: TP53-mutant mantle cell lymphoma (MCL) is associated with poor survival outcomes with chemoimmunotherapy (Eskelund, Blood 2017) with median progression-free survival (PFS) of 0.9 years and median overall survival (OS) of 1.8 years. There is no standard frontline treatment for this high-risk subset. The combination of Bruton's tyrosine kinase (BTK) inhibition and BCL2 inhibition were synergistic and active in relapsed, refractory MCL, including in patients with TP53 mutation (Tam, NEJM 2018). Obinutuzumab, ibrutinib, and venetoclax were well tolerated and efficacious in relapsed and untreated MCL patients (Le Gouill, Blood 2021). We hypothesized that treatment with zanubrutinib (Zanu), Obinutuzumab (Obin), and venetoclax (Ven) (BOVen) would be well-tolerated and efficacious in untreated TP53-mutant MCL. Methods:In this multicenter, investigator-initiated phase 2 trial (NCT03824483), eligible patients had previously untreated MCL with TP53 mutation (of any variant allele frequency) and ECOG PS ≤2, ANC >1, PLT >75, HGB ≥9 (unless if due to MCL). BOVen is administered in 28-day cycles: Zanu 160 mg PO BID starting D1; Obin 1000 mg IV D1 or split D1-2, 8, 15 of C1, D1 of C2-8; Ven ramp up initiated C3D1 (target 400 mg QD). Treatment duration is 2 years at minimum and the primary endpoint is 2-year PFS. Response was assessed using Lugano criteria (Cheson, JCO 2014). PFS and OS were estimated using the Kaplan-Meier method. Peripheral blood minimal residual disease (PB-MRD) assessment was performed using the Adaptive clonoSEQ ® assay. After 24 cycles, Zanu and Ven can be discontinued if MRD undetectable (<10 -6) complete remission (CR) is achieved. With subsequent PB-MRD monitoring, patients can be retreated if clinical progression or MRD detectability (>10 -6). Adverse events (AE) were assessed per CTCAE v5. Results: All 25 planned patients have been enrolled (May 15, 2023 data cut). The median age on study was 65 years (range 29 - 82); 76% were male (19/25); various histologic subtypes were included (15 conventional MCL, 5 non-nodal leukemic, and 5 blastoid variant); 100% stage IV (25/25); by MIPI: 68% high risk (17/25), 28% intermediate (7/25), and 4% low risk MIPI score (1/25); 67% Ki67 ≥30% (14/21); 33% Ki67≥50% (7/21); 100% TP53 mutation, and 48% 17p deletion (12/25). Treatment was overall well-tolerated. The most common treatment-related AEs (≥20%) were predominantly low-grade and manageable, including diarrhea (52%), neutropenia (28%), infusion-related reaction (24%), bruising (20%), COVID-19 infection (20%), nausea (20%), thrombocytopenia (20%), and rash (20%). The grade 3 or higher treatment-related AEs were neutropenia (12%), infusion-related reaction (8%), COVID-19 (8%), diarrhea (4%), transaminitis (4%), thrombocytopenia without bleeding (4%), and rash (4%). Detailed toxicity data will be presented in future. Median follow-up was 16.1 months. There were 5 progressions and 4 deaths on study (2 COVID-related, 1 post-operative aspiration pneumonia, 1 unknown cause), see Figure 1. All deaths occurred in patients who were in ongoing response at time of death. The best overall response rate was 95% (24/25) with 88% (22/25) achieving a CR. At C3 post Zanu-Obin, 68% (n=17/25) achieved PET-CR and post cycle 3, 5 patients converted to a PET-CR (Figure 1). The 1-year PFS and overall survival (OS) were 84% (95% CI: 71%, 100%) and 96% (95% CI: 89%, 100), respectively. The 16-month PFS and OS were 75% (95% CI: 60%, 95%) and 87% (95% CI: 75%, 100%), respectively. The 16-month PFS and OS for patients less than 65 years of age (n=9) were both 100%. Seven patients completed 24 treatment cycles. Of these, 71% (5/7 pts) were in CR and MRD undetectable (treatment discontinued) and 29% (2/7 pts) were in CR and MRD detectable (continued treatment). Outcomes post C24 will be presented in future. At 10 -6 MRD sensitivity level, 7 (28%) of 23 patients had undetectable MRD at C3 and 16 (100%) of 16 patients at C13. Conclusions:BOVen is a well-tolerated, outpatient regimen associated with high response rates and high rates of undetectable MRD in untreated TP53-mutant MCL. The early PFS and OS estimates with BOVen compare favorably with historical outcomes of chemoimmunotherapy in this high-risk subset of MCL. Based on this data, BOVen emerges as a promising treatment option for TP53-mutant MCL and, therefore, the study was expanded to include an additional 25 (total 50) TP53-mutant MCL patients.

The pathologic diagnosis of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell non-Hodgkin lymphoma usually characterized by t(11;14) (q13;q32), or CCND1 translocation and Cyclin D1 over expression. A very small subset of MCL may lack the t(11;14) (q13;q32) translocation and Cyclin D1 over expression, but show alternative translocations involving CCND2 and CCND3, and over expression of SOX11. In general, MCL has been considered a very aggressive and incurable lymphoma and patients with MCL usually have a poor prognosis. However, indolent variants, including in situ mantle cell neoplasm and the recently recognized leukemic non-nodal MCL do exist. In recent years, genome-wide molecular genetic studies have revealed a characteristic MCL genetic profile. This review will focus on the pathologic diagnosis of MCL using the traditional morphological and immunophenotypic strategies combined with cytogenetic characteristics and recently identified molecular profile. Morphological subtypes, immunophenotypic variants, recently recognized indolent variants, as well as MCL risk stratification will also be discussed.

Identify AMPK-Mediated Autophagy and Oxphos As a Novel Metabolic Vulnerability for Targeted Therapy in Mantle Cell Lymphoma

Introduction: Metabolic reprogramming promotes cancer cell growth, metastasis, and therapeutic resistance in a multitude of cancers. Mantle cell lymphoma (MCL) is a rare and aggressive hematopoietic malignancy that exhibits dramatic alterations in the cellular metabolism, especially in advanced stages and at the time of relapse. Unchecked pro-tumor growth signaling and multidrug therapy induce genomic instability and metabolic stress. To sustain tumor survival, MCL cancer cells upregulate multiple essential metabolic pathways that include mitochondria biogenesis, OXPHOS, glutaminolysis, cytoprotective autophagy, and fatty acid β-oxidation. While we recently have demonstrated that OXPHOS is upregulated and mediates resistance to ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, in MCL, the underlying mechanism remains to be elucidated. Cytoprotective autophagy is known to be induced in chronic hypoxic microenvironments such as bone marrow and secondary lymphoid organs, and it in turn contributes to drug resistance and promotes MCL tumor survival. AMPK, a central regulator of cellular metabolism, is activated when MCL cells are challenged with metabolic stress. Consistently, AMPKα1 is highly activated in a subset of MCL at the leukemic phase. AMPK dependence and metabolic reprogramming represent vulnerabilities that could be potentially targeted for MCL treatment. Methods: Primary MCL biopsies, apheresis, or blood specimens, as well as MCL cell lines, were used for metabolic and functional analyses. Metabolomics profiling was carried out using Liquid Chromatography Mass Spectrometry (LC-MS). Mitochondrial membrane potential was determined by staining MCL cells with MitoStatus or TMRE and flow cytometry analysis. OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were determined by Seahorse metabolic flux analysis. Autophagy flux was monitored with tandem mRFP-EGFP-LC3 fluorescence reporter analysis. Other analyses included western blotting, real-time qPCR, and cell viability assay (Cell Titer-Glo). Pharmacological inhibitors were used for the down-modulation of ULK1, AMPK, autophagy, and OXPHOS. Results: Metabolomics profiling of steady-state levels of TCA metabolites showed significant increases in levels of α-KG, glutamate, malate, and fumarate, indicating upregulated glutaminolysis in subsets of MCL that are recurrent or refractory to targeted therapeutics including ibrutinib. Functional analysis illustrates that glutamine deprivation leads to reduced ATP and cell viability in a subset of MCL, while pharmacological inhibition of glutamine metabolism leads to reduced mitochondria membrane potential and increased cellular apoptosis. Seahorse metabolic flux analysis revealed enhanced OXPHOS activity in ibrutinib-resistant MCL cells as indicated by increased OCR. Inhibition of OXPHOS or glutamine metabolism impairs OCR and subsequently induces dose-dependent cellular apoptosis. Further, autophagy flux is increased in the same MCL cells with mRFP-EGFP-LC3 as a reporter. IHC and western blot analysis demonstrated that AMPKα1 is highly activated in a subset of MCL at the leukemic phase, concomitant with an increased NADP:NADPH and AMP:ATP ratio, indicating energy stress. AMPK activation promotes cytoprotective autophagy, and AMPK blockade suppresses autophagy, mitochondria biogenesis, and activity (OCR and ATP production), ultimately inhibiting MCL proliferation under metabolic stress. Conclusions: Our results demonstrate that AMPK activation promotes cytoprotective autophagy, glutaminolysis, and OXPHOS in established MCL, thus representing unique metabolic vulnerabilities that could be potentially targeted for MCL treatment. As AMPKα1 inhibitors are underexplored, it is critical to identify and develop more potent and specific inhibitors of the kinase and define the in vivo functions of its activation in certain advanced cancers. Similarly, pharmacological blockade of the dysregulated autophagy, glutaminolysis, and OXPHOS can be extensively exploited for treatment of MCL patients, especially those at advanced stages of the disease. Disclosures Wang: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; MoreHealth: Consultancy, Equity Ownership; Acerta Pharma: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Guidepoint Global: Consultancy; BioInvent: Consultancy, Research Funding; VelosBio: Research Funding; Loxo Oncology: Research Funding; Celgene: Honoraria, Research Funding; Juno Therapeutics: Research Funding; Aviara: Research Funding; Dava Oncology: Honoraria.

Cutting Edge: ROR1/CD19 Receptor Complex Promotes Growth of Mantle Cell Lymphoma Cells Independently of the B Cell Receptor-BTK Signaling Pathway.

Inhibitors of Bruton tyrosine kinase (BTK), a kinase downstream of BCR, display remarkable activity in a subset of mantle cell lymphoma (MCL) patients, but the drug resistance remains a considerable challenge. In this study, we demonstrate that aberrant expression of ROR1 (receptor tyrosine kinase-like orphan receptor 1), seen in a large subset of MCL, results in BCR/BTK-independent signaling and growth of MCL cells. ROR1 forms a functional complex with CD19 to persistently activate the key cell signaling pathways PI3K-AKT and MEK-ERK in the BCR/BTK-independent manner. This study demonstrates that ROR1/CD19 complex effectively substitutes for BCR-BTK signaling to promote activation and growth of MCL cells. Therefore, ROR1 expression and activation may represent a novel mechanism of resistance to inhibition of BCR/BTK signaling in MCL. Our results provide a rationale to screen MCL patients for ROR1 expression and to consider new therapies targeting ROR1 and/or CD19 or their downstream signaling pathways for MCL-expressing ROR1.

Metabolic Profiling Identifies De Novo Nucleotide Synthesis As a Potential Metabolic Vulnerability for Targeted Therapy Against Mantle Cell Lymphoma

Introduction: Cancer cells exhibit dramatic alterations in cellular metabolism, such as enhanced de novo nucleotide synthesis, to support cell growth, proliferation and survival. The abundance of the nucleotide pool as well as the level and activity of different rate-limiting enzymes belonging to the nucleotide synthetic pathway limit the maximal proliferative capacity of cells. Maintenance of an adequate pool of deoxyribonucleotide triphosphates is essential for DNA replication and DNA repair, and consequently, the genetic integrity of nuclear and mitochondrial genomes. We and others have demonstrated that mantle cell lymphoma (MCL) undergoes metabolic reprogramming to progress and develop resistance to targeted therapy; however, the contribution of de novo nucleotide synthesis to the development and progression of MCL remains poorly understood. In contrast, oncogenic Myc is demonstrated to be highly upregulated in a subset of MCL. In addition to its pro-glycolysis, pro-biogenesis and pro-tumor growth functions, oncogenic levels of Myc induce the expression of multiple genes involved in the nucleotide biosynthetic pathway (e.g., IMPDH2, CTPS1, and CAD). Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis. The γ-nitrogen amide group of glutamine is an indispensable donor of nitrogen for de novo synthesis of both nucleobases purine and pyrimidine. Here, we hypothesize that a subset of MCL depends on de novo nucleotide synthesis for anabolic cell growth and cancer progression due to aberrant Myc expression and Myc-induced glutaminolysis.Methods: Primary MCL biopsy, apheresis, and blood specimens as well as MCL cell lines were utilized for metabolic and functional analyses. Liquid Chromatography Mass Spectrometry (LC-MS) metabolomics was employed to measure the steady-state level of metabolites. Western-blotting and real-time qPCR were utilized determine protein and gene expression levels. BrdU incorporation and the Cell-Trace Violet Cell Proliferation Assay were employed to assess DNA synthesis and cell proliferation. Cell viability was measured with the Cell Titer-Glo Cell Viability Assay. Pharmacological agents were employed to inhibit either de novo nucleotide synthesis or glutaminolysis.Results: Metabolomics profiling of steady-state levels of intracellular metabolites showed significant increases in N-carbamoyl aspartate/dihydroorotate and 5-phosphoribosyl-1-pyrophosphate (PRPP), which are critical intermediates in de novo pyrimidine and purine synthesis, respectively, as well as CTP, dUTP, dCTP, in a subset of MCL, indicating remarkably upregulated de novo nucleotide synthesis. The protein and mRNA levels of c-Myc and its target genes involved in the metabolism of nucleotides (IMPDH2, CTPS1, CAD) were significantly increased. Inhibition of pyrimidine synthesis with DON (6-diazo-5-oxo-L-norleucine), a CTPS1 inhibitor, dramatically reduced the pool of pyridine nucleotides, leading to remarkable apoptosis and halted cell proliferation of a subset of MCL cell lines. Consistent with c-Myc overexpression, increased glutamine uptake was also observed in a subset of MCL cell lines. Glutamine deprivation or pharmacological inhibition of glutamine metabolism showed a similar effect on the inhibition of pyrimidine synthesis as DON (6-diazo-5-oxo-L-norleucine), manifested by a significant reduction of pyrimidine nucleoside triphosphate levels, a dramatic increase in apoptosis, and retarded cell proliferation of a subset of MCL cell lines.Conclusions: Our preliminary results indicate that de novo nucleotide synthesis is upregulated in a subset of MCL with aberrant c-Myc expression. The expression of genes involved in nucleotide metabolism as well as glutaminolysis is also elevated in these cancer cells. Disruption of de novo nucleotide synthesis or glutaminolysis induces apoptosis and suppresses proliferation of a subset of MCL. Myc does not possess enzymatic activity and is considered “undruggable”; therefore, the inhibition of Myc target genes such as those involved in de novo nucleotide synthesis and glutaminolysis presents a promising alternative approach. Taken together, MCL dependency on de novo nucleotide synthesis may represent a metabolic vulnerability for targeted therapy for MCL. DisclosuresWang:AstraZeneca: Consultancy, Research Funding; Juno: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Dava Oncology: Honoraria; MoreHealth: Consultancy; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Aberrant expression of CD10 and BCL6 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by distinctive histological and molecular features. Aberrant expression of BCL6 and CD10 has been reported occasionally, but the biological features of such cases are largely unknown. This study aimed to define the epidemiological, histological and cytogenetic characteristics of BCL6 and CD10-positive MCLs, also investigating possible biological features. A total of 165 cases of cyclin D1 and t(11;14)(q13;q34)-positive MCLs were studied for CD10 and BCL6 immunohistochemical expression, which was documented in 26 of 165 (15.8%) cases (BCL6 17 of 165; CD10 11 of 165; BCL6 and CD10 co-expression two of 165). CD10-positivity was significantly more frequent in females (63.3%; P < 0.01). Either expression correlated significantly with higher mean proliferation index and higher prevalence of MUM1 positivity (P < 0.05). Fluorescence in-situ hybridization (FISH) for BCL6 (3q27) gene derangements was performed on the BCL6- and CD10-positive cases and 98 matched controls: amplifications were documented more frequently in BCL6-positive than -negative cases (50.0% versus 19.4% of cases) (P < 0.05). The mutational status of the variable immunoglobulin heavy chain genes (IGVH) was investigated by Sanger sequencing: five of the six successfully tested cases (83.3%) showed no somatic hypermutations. Aberrant CD10 and BCL6 expression defines a subset of MCLs with higher mean Ki-67 index and higher prevalence of MUM1 expression. BCL6 protein positivity correlates with cytogenetic aberrations involving the BCL6 gene. Although examined successfully in few cases, the high prevalence of unmutated IGVH genes also points at a pregerminal cell origin for these phenotypically aberrant cases.

The t(2;12)(p12;p13), leading to the juxtaposition of the cyclin D2 CCND2 gene with the immunoglobulin kappa locus (IGK), defines a small subset of mantle cell lymphomas lacking cyclin D1 expression.

The t(2;12)(p12;p13), leading to the juxtaposition of the cyclin D2 CCND2 gene with the immunoglobulin kappa locus (IGK), defines a small subset of mantle cell lymphomas lacking cyclin D1 expression.

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

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. DisclosuresTrneny: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.

Genetic heterogeneity in primary and relapsed mantle cell lymphomas: Impact of recurrent CARD11 mutations

The genetic mechanisms underlying disease progression, relapse and therapy resistance in mantle cell lymphoma (MCL) remain largely unknown. Whole-exome sequencing was performed in 27 MCL samples from 13 patients, representing the largest analyzed series of consecutive biopsies obtained at diagnosis and/or relapse for this type of lymphoma. Eighteen genes were found to be recurrently mutated in these samples, including known (ATM, MEF2B and MLL2) and novel mutation targets (S1PR1 and CARD11). CARD11, a scaffold protein required for B-cell receptor (BCR)-induced NF-κB activation, was subsequently screened in an additional 173 MCL samples and mutations were observed in 5.5% of cases. Based on in vitro cell line-based experiments, overexpression of CARD11 mutants were demonstrated to confer resistance to the BCR-inhibitor ibrutinib and NF-κB-inhibitor lenalidomide. Genetic alterations acquired in the relapse samples were found to be largely non-recurrent, in line with the branched evolutionary pattern of clonal evolution observed in most cases. In summary, this study highlights the genetic heterogeneity in MCL, in particular at relapse, and provides for the first time genetic evidence of BCR/NF-κB activation in a subset of MCL.

SOX11 is a biomarker for cyclin D1-negative mantle cell lymphoma.

Cyclin D1 (CCND1) protein overexpression and/or the t(11;14)(q13;q32) translocation are the pathognomonic hallmarks of mantle cell lymphoma (MCL). However, there have been cases that lacked both t(11;14) and cyclin D1 protein but still had a gene expression profile suggesting a diagnosis of MCL. SOX11 expression was detected in most cyclin D1- negative MCL and can serve as a specific biomarker for the diagnosis of this subset of MCL. Lack of SOX11 expression in MCL was associated with an indolent subset and favorable prognosis.

The EMT activator ZEB1 promotes tumor growth and determines differential response to chemotherapy in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (β-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by β-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and anti-apoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.

Genome‐wide methylation analyses identify a subset of mantle cell lymphoma with a high number of methylated CpGs and aggressive clinicopathological features

Mantle cell lymphoma (MCL) is a B-cell neoplasm with an aggressive clinical behavior characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. To clarify the potential contribution of altered DNA methylation in the development and/or progression of MCL, we performed genome-wide methylation profiling of a large cohort of primary MCL tumors (n = 132), MCL cell lines (n = 6) and normal lymphoid tissue samples (n = 31), using the Infinium HumanMethylation27 BeadChip. DNA methylation was compared to gene expression, chromosomal alterations and clinicopathological parameters. Primary MCL displayed a heterogeneous methylation pattern dominated by DNA hypomethylation when compared to normal lymphoid samples. A total of 454 hypermethylated and 875 hypomethylated genes were identified as differentially methylated in at least 10% of primary MCL. Annotation analysis of hypermethylated genes recognized WNT pathway inhibitors and several tumor suppressor genes as frequently methylated, and a substantial fraction of these genes (22%) showed a significant downregulation of their transcriptional levels. Furthermore, we identified a subset of tumors with extensive CpG methylation that had an increased proliferation signature, higher number of chromosomal alterations and poor prognosis. Our results suggest that a subset of MCL displays a dysregulation of DNA methylation characterized by the accumulation of CpG hypermethylation highly associated with increased proliferation that may influence the clinical behavior of the tumors.

Variability in morphology and cell proliferation in sequential biopsies of mantle cell lymphoma at diagnosis and relapse: clinical correlation and insights into disease progression

The variable morphology of mantle cell lymphoma (MCL) is assumed to reflect progression from an early form with classical cytology and mantle zone or nodular growth to a later, more aggressive variant of the disease with blastoid cytology and a diffuse growth pattern. However, studies of sequential biopsies of MCL are very limited, and we therefore undertook to carry out such a study. We analysed a cohort of 47 MCLs at primary diagnosis and relapse for cytology, growth pattern, and Ki67 index, and correlated the findings with outcome. In the majority of cases, the mantle zone growth pattern was lost, but it had been reacquired in a small subset of MCLs at relapse. Twenty-two per cent of MCLs with classical/small cell cytology acquired blastoid features during the course of the disease. However, 50% of MCLs with blastoid cytology at primary diagnosis recurred as a classical variant. The Ki67 index increased over time, and was associated with prognosis in the primary and the relapse biopsy specimens. This is the first study to show, in a large cohort of MCLs, that the Ki67 index increases over time in MCL. Assessment of the Ki67 index remains a useful prognostic tool if assessment is performed in the relapse situation.

Molecular Subsets of Mantle Cell Lymphoma Defined by the IGHV Mutational Status and SOX11 Expression Have Distinct Biologic and Clinical Features

Mantle cell lymphoma (MCL) is a heterogeneous disease with most patients following an aggressive clinical course, whereas others having an indolent behavior. We conducted an integrative and multidisciplinary analysis of 177 MCL to determine whether the immunogenetic features of the clonotypic B-cell receptors (BcR) may identify different subsets of tumors. Truly unmutated (100% identity) IGHV genes were found in 24% cases, 40% were minimally/borderline mutated (99.9%-97%), 19% significantly mutated (96.9%-95%), and 17% hypermutated (<95%). Tumors with high or low mutational load used different IGHV genes, and their gene expression profiles were also different for several gene pathways. A gene set enrichment analysis showed that MCL with high and low IGHV mutations were enriched in memory and naive B-cell signatures, respectively. Furthermore, the highly mutated tumors had less genomic complexity, were preferentially SOX11-negative, and showed more frequent nonnodal disease. The best cut-off of germline identity of IGHV genes to predict survival was 97%. Patients with high and low mutational load had significant different outcome with 5-year overall survival (OS) of 59% and 40%, respectively (P = 0.004). Nodal presentation and SOX11 expression also predicted for poor OS. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. In conclusion, these observations suggest the idea that MCL with mutated IGHV, SOX11-negativity, and nonnodal presentation correspond to a subtype of the disease with more indolent behavior.

Increased tumor cell proliferation in mantle cell lymphoma is associated with elevated insulin-like growth factor 2 mRNA-binding protein 3 expression

Mantle cell lymphoma is an aggressive, non-curable B-cell lymphoma, characterized by the translocation t(11;14)(q13;q32) involving CCND1 and a high number of additional genetic alterations. Chromosomal gains of 7p are frequent in mantle cell lymphoma, with insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3 aka IMP3) being the most upregulated gene in this region. IGF2BP3 is a member of the IGF II mRNA-BP family, and increased IGF2BP3 expression is associated with an aggressive behavior in many malignant tumors. We here analyze selected genes related to IGF signaling in gene expression and genomic array data of 8 mantle cell lymphoma cell lines and 12 primary mantle cell lymphomas and study IGF2BP3 protein expression in 172 well-characterized primary mantle cell lymphomas by immunohistochemistry. The majority of mantle cell lymphoma cell lines and primary cases showed elevated IGF2BP3 mRNA expression and a subset also expressed the IGF1 and IGF2 receptors. On the protein level, 66 of 172 primary mantle cell lymphomas showed IGF2BP3 expression in >50% of tumor cells, and strong IGF2BP3 protein expression was highly associated with increased proliferation as measured by the Ki-67 index, but not with overall survival of mantle cell lymphoma patients. Only a subset of mantle cell lymphomas with marked IGF2BP3 expression had an underlying chromosomal gain in 7p, suggesting that additional mechanisms are involved in the upregulation of IGF2BP3 in mantle cell lymphoma. In seven paired mantle cell lymphoma samples, IGF2BP3 protein expression remained constant between primary diagnosis and relapse. Increased IGF2BP3 expression and, potentially, enhanced IGF signaling may contribute proproliferative stimuli in the evolution of mantle cell lymphoma tumor cells.

PIM2 inhibition as a rational therapeutic approach in B-cell lymphoma

AbstractPIM serine/threonine kinases are overexpressed, translocated, or amplified in multiple B-cell lymphoma types. We have explored the frequency and relevance of PIM expression in different B-cell lymphoma types and investigated whether PIM inhibition could be a rational therapeutic approach. Increased expression of PIM2 was detected in subsets of mantle cell lymphoma, diffuse large B-cell lymphoma (DLBLC), follicular lymphoma, marginal zone lymphoma-mucosa–associated lymphoid tissue type, chronic lymphocytic leukemia, and nodal marginal zone lymphoma cases. Increased PIM2 protein expression was associated with an aggressive clinical course in activated B-like-DLBCL patients. Pharmacologic and genetic inhibition of PIM2 revealed p4E-BP1(Thr37/46) and p4E-BP1(Ser65) as molecular biomarkers characteristic of PIM2 activity and indicated the involvement of PIM2 kinase in regulating mammalian target of rapamycin complex 1. The simultaneous genetic inhibition of all 3 PIM kinases induced changes in apoptosis and cell cycle. In conclusion, we show that PIM2 kinase inhibition is a rational approach in DLBCL treatment, identify appropriate biomarkers for pharmacodynamic studies, and provide a new marker for patient stratification.

Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

BackgroundMantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance.Methodology/Principal FindingsTo identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n = 38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n = 25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9, HOXA9, AHR, NR2F2, and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients.ConclusionsWe have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.

Hypermutation in mantle cell lymphoma does not indicate a clinical or biological subentity

Mantle cell lymphoma is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy-chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear whether the mutational status is stable over time within a given case, whether hypermutation might be influenced by therapy and how it is related to other relevant biological features of mantle cell lymphoma. In this study, we analyzed 23 typical mantle cell lymphoma cases with respect to mutational status and compared the results with clinicopathological and genetic data to determine whether the presence of mutation indicates a subentity with clinical or pathological relevance. We found somatic hypermutation in 26% of our cases and, interestingly, one case showed ongoing somatic hypermutation. In tumor cells of both mutated and unmutated cases, we found a preferential usage of VH3-21 (23%) and VH4-34 (19%). No significant correlations were found between mutation status and the other morphological and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status in mantle cell lymphoma is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance.

Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma

AbstractAberrations 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.

The subcellular Sox11 distribution pattern identifies subsets of mantle cell lymphoma: correlation to overall survival

Gene expression analysis demonstrated high expression of the neuronal transcription factor SOX11 in mantle cell lymphoma (MCL). In contrast to follicular lymphoma, small lymphocytic lymphoma and reactive lymphoid tissue, most MCLs tested (48/53 patients) expressed sox11 protein in the nucleus. Therefore nuclear sox11 expression represents a new tumour marker for a subset of MCL. However, 5/53 MCL cases expressed sox11 only in the cytoplasm; these MCL patients had a shorter survival compared to MCL with nuclear sox11 expression. These results suggest sox11 expression as a new diagnostic marker in MCL that could be related to the clinical and biological behaviour of MCL.