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To interrogate signaling pathways activated in mantle cell lymphoma (MCL) in vivo, we contrasted gene expression profiles of 55 tumor samples isolated from blood and lymph nodes from 43 previously untreated patients with active disease. In addition to lymph nodes, MCL often involves blood, bone marrow, and spleen and is incurable for most patients. Recently, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrated important clinical activity in MCL. However, the role of specific signaling pathways in the lymphomagenesis of MCL and the biologic basis for ibrutinib sensitivity of these tumors are unknown. Here, we demonstrate activation of B-cell receptor (BCR) and canonical NF-κB signaling specifically in MCL cells in the lymph node. Quantification of BCR signaling strength, reflected in the expression of BCR regulated genes, identified a subset of patients with inferior survival after cytotoxic therapy. Tumor proliferation was highest in the lymph node and correlated with the degree of BCR activation. A subset of leukemic tumors showed active BCR and NF-κB signaling apparently independent of microenvironmental support. In one of these samples, we identified a novel somatic mutation in RELA (E39Q). This sample was resistant to ibrutinib-mediated inhibition of NF-κB and apoptosis. In addition, we identified germ line variants in genes encoding regulators of the BCR and NF-κB pathway previously implicated in lymphomagenesis. In conclusion, BCR signaling, activated in the lymph node microenvironment in vivo, appears to promote tumor proliferation and survival and may explain the sensitivity of this lymphoma to BTK inhibitors.

Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%-50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylation in MCL patient samples following BZM treatment. Pathway analysis of differentially methylated genes identified molecular mechanisms of cancer as a top canonical pathway enriched among hypomethylated genes in BZM treated samples. Noxa, a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of BZM, was significantly hypomethylated and induced following BZM treatment. Therapeutically, we could demethylate Noxa and induce anti-lymphoma activity using BZM and the DNA methytransferase inhibitor Decitabine (DAC) and their combination in vitro and in vivo in BZM resistant MCL cells. These findings suggest a role for dynamic Noxa methylation for the therapeutic benefit of BZM. Potent and synergistic cytotoxicity between BZM and DAC in vitro and in vivo supports a strategy for using epigenetic priming to overcome BZM resistance in relapsed MCL patients.

Mantle cell lymphoma (MCL) is a malignancy of mature B cells characterized by aberrant expression of cyclin D1 due to the translocation t(11;14). Epigenomic and genomic lesions in pathways regulating B-cell activation, cell cycle progression, protein homeostasis, DNA damage response, cell proliferation and apoptosis contribute to its pathogenesis. While patients typically respond to first-line chemotherapy, relapse is the rule resulting in a median survival of 5-7 years. The PI3K/AKT/mTOR appears as a key pathway in the pathogenesis and can be targeted with small molecules. Most experience is with mTOR inhibitors of the rapamycin class. Second-generation mTOR inhibitors and the PI3K inhibitor CAL-101 are novel options to more effectively target this pathway. Bruton's tyrosine kinase inhibition by PCI-32765 has promising activity and indicates immunoreceptor signaling as a novel therapeutic target. Up to 50% of relapsed patients respond to the proteasome inhibitor bortezomib suggesting that MCL may be particularly sensitive to disruption of protein homeostasis and/or induction of oxidative stress. Recent work has focused on elucidating the mechanism of bortezomib-induced cytotoxicity and the development of second-generation proteasome inhibitors. DNA hypomethylating agents and histone deacetylase inhibitors effect epigenetic de-repression of aberrantly silenced genes. These epigenetic pharmaceuticals and HSP90 inhibitors can synergize with proteasome inhibitors. Finally, BH3 mimetics are emerging as tools to sensitize tumor cells to chemotherapy. Participation in clinical trials offers patients a chance to benefit from these advances and is essential to maintain the momentum of progress. Innovative trial designs may be needed to expedite the clinical development of these targeted agents., (Published by Elsevier Ltd.)

Purpose: Proteasome inhibition disrupts protein homeostasis and induces apoptosis. Up to 50% of patients with relapsed mantle cell lymphoma (MCL) respond to bortezomib. We used gene expression profiling to investigate the connection between proteasome inhibition, cellular response, and clinical efficacy., Experimental Design: We assessed transcriptional changes in primary tumor cells from five patients during treatment with bortezomib in vivo, and in 10 MCL cell lines exposed to bortezomib in vitro, on Affymetrix microarrays. Key findings were confirmed by western blotting., Results: MCL cell lines exposed to bortezomib in vitro showed upregulation of endoplasmic reticulum and oxidative stress response pathways. Gene expression changes were strongest in bortezomib-sensitive cells and these cells were also more sensitive to oxidative stress induced by H2O2. Purified tumor cells obtained at several timepoints during bortezomib treatment in 5 previously untreated patients with leukemic MCL showed strong activation of the antioxidant response controlled by NRF2. Unexpectedly, activation of this homeostatic program was significantly stronger in tumors with the best clinical response. Consistent with its proapoptotic function, we found upregulation of NOXA in circulating tumor cells of responding patients. In resistant cells, gene expression changes in response to bortezomib were limited and upregulation of NOXA was absent. Interestingly, at baseline, bortezomib-resistant cells displayed a relatively higher expression of the NRF2 gene-expression signature than sensitive cells (P < 0.001)., Conclusion: Bortezomib triggers an oxidative stress response in vitro and in vivo. High cellular antioxidant capacity contributes to bortezomib resistance., (©2011 AACR.)

Mantle cell lymphoma (MCL) is a malignancy of mature B cells characterized by the translocation t(11;14) that leads to aberrant expression of cyclin D1. Response to first-line chemotherapy is good, but most patients relapse, resulting in a median survival of 5 to 7 years. The important PI3K/AKT/mTOR pathway can be targeted with small molecules. mTOR inhibitors have clinical activity and temsirolimus has been approved in Europe. Second-generation mTOR inhibitors and the PI3K inhibitor CAL-101 offer additional means to target the pathway. Promising results with the BTK inhibitor PCI-32765 suggest that B-cell receptor signaling could play a role. For unknown reasons, MCL appears to be particularly sensitive to disruption of protein homeostasis. The proteasome inhibitor bortezomib achieves responses in up to 50% of relapsed patients. Much work has been done in elucidating the mechanism of its cytotoxicity, its incorporation into combination therapies, and the development of second-generation proteasome inhibitors. Deacetylase and HSP90 inhibitors are also promising classes of drugs that can synergize with proteasome inhibitors. Finally, BH3 mimetics are emerging as tools to sensitize tumor cells to chemotherapy. Participation in clinical trials offers patients an immediate chance to benefit from these advances and is essential to maintain the momentum of progress., (Published by Elsevier Inc.)

Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Mantle cell lymphoma (MCL) is a mostly incurable malignancy arising from naive B cells (NBCs) in the mantle zone of lymph nodes. We analyzed genomewide methylation in MCL patients with the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay and found significant aberrancy in promoter methylation patterns compared with normal NBCs. Using biologic and statistical criteria, we further identified 4 hypermethylated genes CDKN2B, MLF-1, PCDH8, and HOXD8 and 4 hypomethylated genes CD37, HDAC1, NOTCH1, and CDK5 when aberrant methylation was associated with inverse changes in mRNA levels. Immunohistochemical analysis of an independent cohort of MCL patient samples confirmed CD37 surface expression in 93% of patients, validating its selection as a target for MCL therapy. Treatment of MCL cell lines with a small modular immunopharmaceutical (CD37-SMIP) resulted in significant loss of viability in cell lines with intense surface CD37 expression. Treatment of MCL cell lines with the DNA methyltransferase inhibitor decitabine resulted in reversal of aberrant hypermethylation and synergized with the histone deacetylase inhibitor suberoylanilide hydroxamic acid in induction of the hypermethylated genes and anti-MCL cytotoxicity. Our data show prominent and aberrant promoter methylation in MCL and suggest that differentially methylated genes can be targeted for therapeutic benefit in MCL.

Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC(50) 5.9 nM) and three relatively bortezomib resistant cell lines (median IC(50) 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-kappaB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.