Primary Mantle Cell Lymphoma Tumor Research Articles

RAC2 Links B-Cell Receptor Signaling and Cell Adhesion in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin B-cell lymphoma. B-cell receptor (BCR) signaling is chronically active in several mature B-cell malignancies including mantle cell lymphoma. Inhibition of the BCR and downstream pathway is highly effective in B-cell neoplasia and can be achieved at the cellular level through BTK inhibition by ibrutinib, for example. Ibrutinib has generated remarkable patients' response in large clinical trials and has been approved by FDA for the treatment of relapsed/refractory MCL. Ibrutinib targets Bruton tyrosine kinase primarily by inhibition of B-cell receptor signaling and cell proliferation, and secondarily by induction of tumor apoptosis. In addition, the drug inhibits the interaction of tumor cells with their microenvironment by disruption of cell adhesion. The effect on cell adhesion appears to be a mechanism shared by many BCR-targeted therapies including inhibitors of LYN (Leuk Res 38, 34, 2014), SYK (Blood, 115, 2578, 2010; Blood 125, 2336, 2015), and PI3K (idelalisib) (NEJM, 370, 997, 2014; Blood, 125, 2306). This class action raised a question regarding how the BCR pathway is connected to the cell adhesion phenotype. We set out to investigate the underlying mechanisms that are largely unknown at the present time.Methods: We used RNA-sequencing to identify pathways that have differential responses to ibrutinib in MCL cell lines that are either intrinsically sensitive or resistant to ibrutinib. We then validate the RNA-Seq findings in sensitive and resistant cell lines via RNA interference and transfection, xenograft mouse models, and human primary MCL samples.Results: We found that sensitivities of six MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. These include sensitive cell line Jeko-1, intermediate cell lines Mino, REC-1 and JVM2, and resistant lines Maver-1 and Granta 519. RNA-sequencing revealed that BCR signaling and cell adhesion transcriptional programs were coordinately down-regulated by ibrutinib in ibrutinib-sensitive cells, but not in ibrutinib-resistant cells. Among genes that are differentially regulated in the sensitive and resistant cell lines, we identified RAC2, a regulator of cell adhesion this is also involved in BCR signaling. To determine whether RAC2 is relevant, we examined RAC2 expression in primary MCL tumors by immunohistochemical staining (IHC). We found that RAC2 was weakly expressed in 7/10 classic MCL tumors and strongly expressed in 3/3 aggressive MCL. In contrast, in tonsilar lymphoid reactive tissues, RAC2 positivity was restricted only to mantle zone and T-zone of the follicles. For further validation, we found that RAC2 expression, at the protein level, was down-regulated by ibrutinib treatment in sensitive, but not resistant, cell lines. More interestingly, using co-immunoprecipitation, we found that RAC2 is physically associated with BLNK, an adaptor of early BCR signaling pathway, in the sensitive cells but not in the resistant cells. To establish that RAC2 is a regulator of MCL cell adhesion in the context of MCL, we demonstrated that siRNA knock-down of RAC2 impaired cell adhesion while exogenous introduction of RAC2 reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib showed slower tumor growth with reduced RAC2 expression by IHC. Lastly, we show that in primary human MCL cells, RAC2 was down-regulated by ibrutinib, and this down-regulation correlated well with cell adhesion impairment following ibrutinib treatment.Conclusions: Our findings suggest the RAC2 mediates the link between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC expression and via disruption of RAC2-BLNK interaction. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development. DisclosuresLu:Portola Pharmacueticals, Inc.: Research Funding. Wang:Portola: Research Funding; Karyopharm: Research Funding.

XPO1 Inhibitor Selinexor Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma Via Nuclear Retention of IκB

Background: B-cell receptor signaling (BCR) plays a central role in mantle cell lymphoma (MCL) and inhibition of BCR signaling through the BTK inhibitor, ibrutinib, has generated remarkable responses in patients. However, approximately one-third of the patients do not respond well to the drug, and disease relapse on ibrutinib is nearly universal. Alternative therapeutic strategies aimed to prevent and overcome ibrutinib resistance are needed. Selinexor is a first-in-class selective inhibitor of nuclear export. It binds and inhibits exportin XPO-1, which mediates the nuclear export of proteins and mRNAs. Inactivation of XPO-1 increases the retention of tumor suppressors in the nuclei to enhance suppression of tumor cell growth. Inactivation of exportin also decreases the the expression of oncogenes of which translation is eIF4E-dependent. Since many of the oncogenes and tumor suppressor genes in MCL also shuttle between nuclei and cytoplasm, we speculate that selinexor would be active in MCL. As selinexor acts on a cellular process rather than a particular molecular target, we thus postulate selinexor may have the potential to act broadly in both ibrutinib-sensitive and -resistant mantle lymphoma cells. In this study, we compared and contrasted the intrinsic cellular response of MCL cells to ibrutinib versus selinexor. We aim to identify molecular mechanisms underlying differential cellular response to these two drugs in order to obtain a deeper understanding of what underlies tumor cells' susceptibility to drug intervention.Methods: We first determined the sensitivity of six MCL cell lines to both ibrutinib and selinexor in a cell-based MTT assay, which measures cellular metabolic activity, and then tested the effects of the drugs on cell growth. Next, we examined the effects of both inhibitors on apoptosis and proliferation to further delineate the differential sensitivity of MCL cells to ibrutinib and selinexor. We performed RNA-seq analyses to identify signaling pathways that underlie differential cellular response to these two drugs. Lastly, we validated the major findings in both cell lines and primary MCL tumors using a variety of assays including Flowsight analysis.Results: We found that selinexor had a broader anti-tumor activity in MCL than ibrutinib. MCL cell lines resistant to ibrutinib were sensitive to selinexor. We showed that selinexor induced apoptosis in addition to causing cell cycle arrest. Further, inhibition of NF-kB gene expression signature, as opposed to BCR signature, was a common feature that underlies the response of MCL cell lines to both selinexor and ibrutinib while unchanged NF-kB signature was associated exclusively with ibrutinib resistance. Using Flowsight, we showed that selinexor increased nuclear retention of IκB that was accompanied by the reduction of NF-kB DNA binding activity. In primary MCL tumor cells, we further demonstrated that the number of cells with IκB nuclear retention was linearly correlated with the degree of cellular apoptosis.Conclusions: Together, our results show that inhibition of NF-κB transcription via IκB nuclear retention is an important action of selinexor in MCL tumor cells. Selinexor is effective MCL cells with intrinsic resistance to ibrutinib and has the potential to prevent and overcome ibrutinib resistance. Our study warrants further clinical investigation of this compound in MCL. DisclosuresKadri:Portola Pharmacueticals, Inc.: Research Funding. Shacham:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment. Lu:Portola Pharmacueticals, Inc.: Research Funding.

Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

S897 VALIDATION OF A NOVEL MECHANISM OF ACTION FOR TREATMENT OF CCR7+ HEMATOLOGICAL MALIGNANCIES WITH FIRST‐IN‐CLASS ANTIBODY CAP‐100

Background:The homeostatic CC‐chemokine receptor 7 (CCR7) controls trafficking of immune cells to the lymph node (LN). Accordingly, CCR7 is highly expressed in hematological malignancies with nodal involvement including chronic lymphocytic leukemia (CLL), several B‐cell non‐Hodgkin lymphomas (NHL) such as mantle cell lymphoma (MCL), and various mature T‐cell neoplasia like T‐cell prolymphocytic leukemia (T‐PLL) and Sézary syndrome (SS). Upon engagement by its ligands, the chemokines CCL19 and CCL21, CCR7 directs tumor cells to LN. In this protective microenvironment, CCR7 contributes to tumor cell survival and proliferation. Indeed, both high CCR7 surface expression levels and high CCR7‐triggered migratory responses correlate with lymphadenopathy, adverse prognostic factors, and shorter patient survival. Therefore, strategies targeting CCR7 could provide a novel therapeutic approach for CCR7+ hematological malignancies.Aims:In this study we aimed to validate CCR7 as a novel target for antibody‐based therapies in CCR7‐expressing blood cancers.Methods:We have generated CAP‐100, the first humanized IgG1 monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand‐mediated signaling, and evaluated the antibody in various in‐vitro and in‐vivo preclinical models.Results:CAP‐100 potently inhibited in vitro migration of primary CLL, MCL, T‐PLL and SS tumor cells. Furthermore, in vivo pre‐clinical studies confirmed that CAP‐100 effectively blocked entry of CCR7‐expressing cells to LNs. CAP‐100 also abrogated survival elicited by CCR7 in CLL, and showed potent antibody‐dependent cell‐mediated cytotoxicity (ADCC) against CLL or CCR7+ T‐lymphomas cells. This Fc‐mediated cell killing activity clearly outperformed anti‐CD20 mAb rituximab and anti‐CD52 mAb alemtuzumab, standards of care in B‐cell and T‐cell lymphomas respectively. In all cases, CAP‐100 activity was independent of prognostic markers for high risk disease. Finally, when given as monotherapy in disseminated B‐NHL and CLL xenograft tumors in SCID mice, CAP‐100 inhibited tumor growth and extended survival significantly. In addition, CAP‐100 strongly inhibited the infiltration into as well as the survival of tumor cells in the LN.Summary/Conclusion:Our results demonstrate that CAP‐100, the first‐in‐class anti‐CCR7 mAb, is a potent antagonist with biological activity in several CCR7+ hematological malignancies, including relapsed/refractory disease. Moreover, these results highlight the relevance of the CCR7‐CCL19/CCL21 pathway as a therapeutic target in these diseases. CAP‐100's unique ability to block migration of tumor cells to the LN (where also inhibits tumor cell survival), in combination with its potent cell killing activity, represents a novel and differentiating mechanism of action. This provides the biological rationale for use of CAP‐100, either as monotherapy or in combination with novel agents. Clinical trials in CLL and CCR7‐expressing NHL will be initiated soon.

XPO1 Inhibitor Selinexor Overcomes Intrinsic Ibrutinib Resistance in Mantle Cell Lymphoma via Nuclear Retention of IκB.

Inhibition of B-cell receptor (BCR) signaling through the BTK inhibitor, ibrutinib, has generated a remarkable response in mantle cell lymphoma (MCL). However, approximately one third of patients do not respond well to the drug, and disease relapse on ibrutinib is nearly universal. Alternative therapeutic strategies aimed to prevent and overcome ibrutinib resistance are needed. We compared and contrasted the effects of selinexor, a selective inhibitor of nuclear export, with ibrutinib in six MCL cell lines that display differential intrinsic sensitivity to ibrutinib. We found that selinexor had a broader antitumor activity in MCL than ibrutinib. MCL cell lines resistant to ibrutinib remained sensitive to selinexor. We showed that selinexor induced apoptosis/cell-cycle arrest and XPO-1 knockdown also retarded cell growth. Furthermore, downregulation of the NFκB gene signature, as opposed to BCR signature, was a common feature that underlies the response of MCL to both selinexor and ibrutinib. Meanwhile, unaltered NFκB was associated with ibrutinib resistance. Mechnistically, selinexor induced nuclear retention of IκB that was accompanied by the reduction of DNA-binding activity of NFκB, suggesting that NFκB is trapped in an inhibitory complex. Coimmunoprecipitation confirmed that p65 of NFκB and IκB were physically associated. In primary MCL tumors, we further demonstrated that the number of cells with IκB nuclear retention was linearly correlated with the degree of apoptosis. Our data highlight the role of NFκB pathway in drug response to ibrutinib and selinexor and show the potential of using selinexor to prevent and overcome intrinsic ibrutinib resistance through NFκB inhibition.

Inhibition of B-Cell Receptor Signaling Disrupts Cell Adhesion in Mantle Cell Lymphoma Via RAC2

Background: B-cell receptor (BCR) signaling pathway is recognized as a crucial pathway for the pathogenesis of neoplastic B-cells. Inhibition of the BCR signaling and the downstream pathway is highly effective in B-cell malignancy through Bruton tyrosine kinase inhibition by ibrutinib. In addition to cell proliferation inhibition, ibrutinib disrupts cell adhesion between tumor and its microenvironment through unknown molecular mechanisms, resulting in peripheral lymphocytosis with accompanying lymphadenopathy reduction in patients who receive ibrutinib.Methods and materials: In an effort to elucidate the link between BCR signaling and cell adhesion phenotype, we first characterized ibrutinib sensitive and resistant mantle cell lymphoma (MCL) cell lines. We measured cell proliferation and cell growth, and correlated ibrutinib sensitivity with cell adhesion disruption. We then used RNA-sequencing to identify differential pathways between sensitive or resistant cell lines in response to ibrutinib treatment. We validated RNA-Seq findings using cell lines, as well as animal models and human primary MCL tumor tissues and cells.Results: We found that intrinsic sensitivities of MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion gene signatures were simultaneously down-regulated by ibrutinib in ibrutinib-sensitive but not ibrutinib-resistant cell lines. Among the differentially expressed genes in the BCR gene signature, we identified and validated that RAC2, a regulator of cell adhesion, was down-regulated at both RNA and protein levels by ibrutinib only in ibrutinib-sensitive cells. Physical association of RAC2 with BLNK, an early BCR pathway adaptor, was disrupted by ibrutinib uniquely in sensitive cells. RAC2 knockdown with siRNA impaired cell adhesion while RAC2 over-expression rescued ibrutinib-induced reduction in cell adhesion. In a xenograft mouse model, mice treated with ibrutinib demonstrated tumor growth retardation along with down-regulation in RAC2 protein expression. Using immunohistochemical staining, we demonstrated that RAC2 was expressed in ~65% primary MCL tumor tissues with majority of RAC2-positive tumors characterized as being the more aggressive subtypes. Finally, primary MCL cells treated with ibrutinib demonstrated reduced RAC2 that is accompanied by cell adhesion impairment.Conclusions: Our findings uncover a novel cross-talk between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC2. Our study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and new drug development. DisclosuresWang:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; AstraZeneca: Consultancy, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Honoraria, Research Funding.

SOX11 promotes tumor protective microenvironment interactions through CXCR4 and FAK regulation in mantle cell lymphoma

AbstractSOX11 overexpression in mantle cell lymphoma (MCL) has been associated with more aggressive behavior and worse outcome. However, SOX11 oncogenic pathways driving MCL tumor progression are poorly understood. Here, we demonstrate that SOX11 binds to regulatory regions of 2 important genes for microenvironment signals in cancer: (C-X-C motif) chemokine receptor 4 (CXCR4) and PTK2 (encoding for focal adhesion kinase [FAK]). Moreover, SOX11+ xenograft and human primary MCL tumors overexpress cell migration and stromal stimulation gene signatures compared with their SOX11− counterparts. We show that SOX11 directly upregulates CXCR4 and FAK expression, activating PI3K/AKT and ERK1/2 FAK-downstream pathways in MCL. Concordantly, SOX11+ MCL cells have higher cell migration, transmigration through endothelial cells, adhesion to stromal cells, and cell proliferation and display an increased resistance to conventional drug therapies compared with SOX11− MCL cells. Specific FAK inhibition blocks downstream PI3K/AKT- and ERK1/2-mediated phosphorylation. Additionally, specific FAK and PI3K inhibitors reduce SOX11-enhanced MCL cell migration and stromal interactions and revert cell adhesion–mediated drug resistance (CAM-DR) to the same levels as SOX11− MCL cells. In intravenous MCL xenograft models, SOX11+ MCL cells display higher cell migration, invasion, and growth compared with SOX11-knockdown cells, and specific FAK and CXCR4 inhibitors impair SOX11-enhanced MCL engraftment in bone marrow. Overall, our results suggest that SOX11 promotes MCL homing and invasion and increases CAM-DR through the direct regulation of CXCR4 and FAK expression and FAK/PI3K/AKT pathway activation, contributing to a more aggressive phenotype. Inhibition of this pathway may represent an efficient strategy to overcome stromal-mediated chemotherapy refractoriness in aggressive MCL.

CCND1 Mutations Increase Protein Stability and Promote Ibrutinib Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) represents 6-8% of non-Hodgkin's lymphoma with a median survival of 3-5 years. CyclinD1 (CCND1) chromosomal translocation t(11;14) is the hallmark of this neoplasm, which leads to its deregulated expression. Recent knock down studies of CCND1 in MCL have shown that its role is not only limited to promote cell cycle progression but also for maintaining genomic stability. Knock down of CCND1 results in increased DNA damage in MCL cells and makes them more susceptible to chemotherapy. However, CCND1 is not only prone to frequent translocation but also to somatic mutations. Genomic studies have identified recurring somatic mutations in the coding region of CCND1 in approximately 30% of MCL cases. The hot spot of these mutations is concentrated in exon 1 of CCND1. However, the functional consequence of these mutations is not known. We generated the most common CCND1 mutations (E36K, Y44D or C47S) by site directed mutagenesis and overexpressed these mutants in JEKO-1, UPN-1 or Z-138 MCL cell lines. Here, we show that these single mutations, as compared to the wild type (WT), increased CCND1 protein levels in MCL cell lines and primary tumors. Mechanistically, mutations Y44D or C47S increase the stability of CCND1 protein through attenuation of threonine-286 phosphorylation, which is a prerequisite for export of CCND1 from the nucleus to the cytoplasm, followed by proteolysis through the ubiquitin-proteasome pathway. But the two mutants have stronger affinity to interact with the threonine-286 phosphorylating enzyme glycogen synthase kinase 3 beta ,whereas interaction with cyclin-dependent kinase 4 remains unaffected. Furthermore we observed that these two mutants are preferentially localized in the nucleus compared to WT, as determined by cell fractionation and immunofluorescence assays. In order to understand the relevance of stable CCND1 protein in MCL, we studied the response of WT or mutant CCND1-expressing MCL cell lines to ibrutinib, an FDA- approved Bruton tyrosine kinase inhibitor used for MCL treatment. We observed that forced expression of WT or mutant CCND1 increased the resistance of JEKO-1 cells to ibrutinib. The Y44D mutation sustained the resistance to ibrutinib at supraphysiologic concentrations (5-10 μM). Furthermore, primary MCL tumors with CCND1 mutations expressed a more stable protein and were resistant to ibrutinib than WT cases. These findings uncover new mechanisms for MCL cells to increase CCND1 protein level through somatic mutations, which may provide a survival advantage to the lymphoma cells in developing resistance against the emerging ibrutinib therapy. DisclosuresPillai:Trillium Therapeutics: Research Funding. Pham:Vyripharm Biopharmaceuticals: Research Funding.

The Effects of PI3K-δ/γ Inhibitor, Duvelisib, in Mantle Cell Lymphoma in Vitro and in Patient-Derived Xenograft Studies

Background: Mantle cell lymphoma (MCL) is an incurable subtype of B-cell lymphoma. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton’s tyrosine kinase (BTK) was approved by the FDA for the treatment of MCL in patients previously treated. In our prior multicenter Phase 2 clinical trial, the overall response rate in relapsed/refractory MCL was 68%, with a median progression-free survival (PFS) of 13.9 months. However, the majority of MCL patients treated with ibrutinib relapsed; in these relapsed patients, the one-year survival rate was only 22%. Therefore, there exists an urgent need for additional novel targeted therapies to improve the mortality rate in these patients. In this study, we assessed the in vitro and in vivo effects of duvelisib, a PI3K-δ,-γ inhibitor, in MCL.Methods: The PI3K/AKT/mTOR and other cell survival signaling pathways were investigated by RNASeq and reverse phase protein array (RPPA) in ibrutinib-sensitive and -resistant MCL samples. The expression of PI3K isoforms, α, β, γ, and δ was tested in 11 MCL cell lines, patient and patient-derived xenograft (PDX) MCL cells by western blot analysis. We then investigated the growth inhibition and apoptosis of duvelisib (IPI-145, Infinity Pharmaceuticals, Inc.) in MCL cells by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and Annexin V-binding assay (BD Biosciences). We established a primary MCL-bearing PDX model and passaged the primary MCL tumor to next generations. Mice were administrated with 50 mg/kg duvelisib daily by oral gavage. Tumor burden and survival time were investigated in the MCL-PDX model.Results: We found that the PI3K/AKT/mTOR signaling pathway was activated in both primary and acquired ibrutinib-resistant MCL cell lines and PDX MCL cells. We immunoblotted PI3K isoforms, α, β, γ, and δ in 11 MCL cell lines and the result demonstrated that both ibrutinib-sensitive and ibrutinib-resistant MCL cells dominantly expressed PI3K-δ and -γ. Next, we tested the effects of duvelisib on these MCL cells. Duvelisib had effects on the growth inhibition and apoptosis in both ibrutinib-sensitive and ibrutinib-resistant MCL cells as good as the PI3K-δ inhibitor, idelalisib (Cal-101, GS-1101). The PI3K-δ isoform could play a very important role in PI3K-mediated signals in MCL. We then investigated the effects of duvelisib in vivo through our established MCL-bearing PDX mouse models. These models are created by inoculating the primary tumor cells from MCL patients into a human fetal bone chip implanted into NSG mice to provide a microenvironment that reconstitutes the human environment. MCL tumor mass was then passaged to next generations for therapeutic investigation of duvelisib. Mice were treated with 50 mg/kg duvelisib daily by oral gavage. Our data demonstrated that duvelisib significantly inhibited tumor growth and prolonged survival of MCL-PDX mice.Conclusion: Duvelisib, an oral dual inhibitor of PI3K-δ,-γ, inhibits MCL growth both in vitro and in PDX mice. These preclinical results suggests duvelisib may be effective in the treatment of patients with relapsed/refractory MCL. DisclosuresNo relevant conflicts of interest to declare.

CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

Prosurvival and Chemoprotective Effects of Tumor-Associated Macrophages Reversed By Anti-CSF-1R Monoclonal Antibody in B-Cell Lymphomas

The results of gene expression profiling (GEP) and immunohistochemical studies indicate that survival is worsened by macrophages (MΦ) in the tumor microenvironment of various B-cell lymphomas including follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Tumor-associated macrophages (TAMs) are known to be different from other types of MΦ, but the effects of TAMs that worsen prognosis in B-cell lymphoma are essentially unknown, as are the mechanisms of these effects. Here, we determined the phenotype and effects of TAMs on tumor survival, proliferation, and drug resistance in B-cell lymphomas and evaluated strategies to reverse their effects.As compared to peripheral blood monocytes (Mo) from normal donors (ND), Mo from FL patients were differentiated less into M1 MΦ (defined as CD68+CD163loCD206loCD86hi) by culture with CSF-1 for 5 days followed by IFN-g + LPS for 2 days more. In contrast, Mo from FL patients and ND were differentiated similarly into M2 MΦ (defined as CD68+CD163hiCD206hiCD86lo) by culture with CSF-1 followed by IL-4. Consistent with this, MΦ gene signatures from FL tumors were more similar to previously-described signatures of M2 rather than M1 MΦ (Martinez et al, J Immunol, 2006, 177(10):7303-11).In co-culture, primary FL tumor cells and lymphoma cell lines (including RL, a transformed FL cell line; Granta 519, a mantle cell lymphoma (MCL) cell line; and Raji, a Burkitt lymphoma cell line) induced differentiation of Mo into MΦ. Differentiation could be prevented by CS4 monoclonal antibody (mAb), a fully human IgG1 anti-human CSF-1R mAb (ImClone/Eli Lilly), but not isotype control Ab. Elevated levels of CSF-1 in culture supernatants after addition of CS4 mAb and real-time PCR of tumor cells suggested secretion of CSF-1 by lymphoma cells. Spontaneous apoptosis of primary FL and MCL tumor cells, determined by Annexin V and propidium iodide staining, was significantly reduced by co-culture with ND Mo (p<0.01), whether pre-differentiated into MΦ with CSF-1 or not, but this protection could be reversed by CS4 mAb. Mo and/or pre-differentiated MΦ protected primary FL and MCL tumor cells from cytotoxic effects of doxorubicin and/or bendamustine (p<0.01), but CS4 mAb reversed this effect. To assess effects of MΦ on proliferation, lymphoma cell lines (RL, Granta 519, and Raji) were CFSE-labeled prior to co-culture with Mo and doxorubicin, and proliferation assessed by CFSE dilution by flow cytometry in the presence or absence of CS4 or isotype control mAbs. MΦ promoted proliferation of all three cell lines, but this effect could be reversed by CS4 mAb.To further understand the mechanism by which MΦ promote tumor survival and growth, we performed phosflow analysis and found increased phosphorylation of STAT3 in co-cultured lymphoma cells. Consistent with this, we observed a correlation between an 11-gene STAT3 activation signature, described by Huang et al in DLBCL tumors (J Clin Oncol, 2013, 52.8414), and a MΦ gene signature in whole genome GEP studies of 191 FL tumors (Pearson correlation co-efficient=0.396, p<0.001).In conclusion, our results suggest that Mo from FL patients are predisposed to differentiate into an M2-like MΦ state. The interaction between lymphoma cells and Mo/MΦ is reciprocal: a change in Mo (MΦ differentiation) induced by interaction with lymphoma tumor cells leads to a change in the tumor cells (promotion of survival, proliferation, and chemoresistance). More importantly, our results demonstrate that targeting TAMs using CS4, an anti-CSF-1R mAb, can be an effective strategy to overcome the adverse effects of TAMs and reverse chemoresistance. Further studies are needed to determine whether STAT3 activation contributes to the protumor effects of TAMs. This may provide novel insights into the molecular mechanisms related to TAMs and lymphoma cells and offers additional targets for therapeutic development. In the long term, strategies targeting TAMs is especially appealing, as they should be able to be combined with existing therapies including chemotherapy, other immunotherapy, and targeted therapy, potentially improving their efficacy without increasing toxicity for FL, DLBCL, and other B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome.Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013).Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components.Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting.These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures:Rosenblatt:Seattle Genetics, Inc.: Research Funding.

Integrative Whole Transcriptome and Exome Sequencing Reveals Reprogramming Of Mantle Cell Lymphoma In Prolonged Early G1 Arrest For Clinical Response To Selective Inhibition Of CDK4 In Combination Therapy

CDK4 and CDK6, which drive cell cycle entry and progression through G1 in the presence of cyclin D, are overexpressed at a high frequency in human cancers. Targeting CDK4 with the first selective and potent CDK4/CDK6 inhibitor, palbociclib (PD 0332991), has recently achieved unprecedented clinical efficacy in both hematologic malignancies and solid tumors. Most notably, palbociclib more than tripled the progression free survival of breast cancer patients treated with letrozole. In mantle cell lymphoma (MCL), CDK4 overexpression and aberrant cyclin D1 expression leads to unrestrained cycling and proliferation that underlies disease progression. In the first phase I single-agent palbociclib clinical trial in recurrent MCL, inhibition of CDK4 by palbociclib alone resulted in a durable clinical response with tumor regression in some MCL patients, including one complete response and two partial response.However, the fundamental mechanism for differential clinical response to selective targeting of CDK4/CDK6 remains obscure. To address this question, we have developed a novel strategy that both inhibits proliferation of cancer cells and reprograms them for cytotoxic killing by reversible inhibition of CDK4/CDK6. We have shown that: 1) inhibition of CDK4/6 with palbociclib leads to early G1 arrest that is dependent on Rb, the substrate for CDK4/CDK6; 2) prolonged early G1 arrest (pG1) reprograms cancer cells for killing by diverse agents; 3) pG1 sensitization is exacerbated in synchronous S phase entry (pG1-S) upon palbociclib withdrawal. Further, pG1 sensitization appears to stem from restricted expression of genes scheduled for early G1 only, which is exacerbated in pG1-S due to incomplete restoration of cell cycle-coupled gene expression.To advance targeting CDK4 in MCL, we have implemented this strategy combining palbociclib with bortezomib at a reduced dose (1.0 mg /m2) in a phase I clinical trial (Pa-Btz) in recurrent MCL. palbociclib was administered to MCL patients on days 1-12 of a 21-day cycle to induce pG1; bortezomib was given on days 8 and 11 in pG1 and on days 15 and 18 in pG1-S. Pa-Btz was well tolerated and appeared to have a palbociclib dose-dependent durable clinical activity, with only one of 6 patients progressed at the optimal dose combination.We investigated the genes that mediate pG1 reprogramming by integrative whole exome sequencing (WES) and whole transcriptome sequencing (WTS). The dynamic changes in cell cycle-coupled gene expression were determined within individual patients in primary MCL tumor cells isolated from serial lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) in conjunction with immunohistochemistry (IHC). palbociclib inhibited CDK4 and induced pG1 in all patients initially, regardless of the clinical response, mutations in p53 or ATM, cyclin D1 3'UTR deletion, or other patient-specific deletions, amplifications and mutations. No mutations were detected in CDK4, which was expressed in primary MCL cells over CDK6. Induction of pG1 maintained the expression of cell cycle genes programmed for early G1 (CDK4, cyclin D and Rb), and prevented the expression of those scheduled for late G1 (cyclin A), S phase (Ki 67, TK) and G2/M (CDK1, cyclin B), and this was completely reversible upon release of the early G1 block.However, induction of pG1 also led to an imbalance in the expression of other cellular genes due to restricted expression of only genes programmed for early G1. Among the 868 genes that were suppressed in pG1 (not programmed for early G1) in MCL tumor of clinically-responding patients (N=4, EdgeR, FDR 0.05), 9 were conversely activated in the non-responding patients (N=4, EdgeR, FDR 0.05). These genes are involved in redox stress, metabolism and cell migration, suggesting a potential role of cell cycle-coupled metabolic imbalance in differential clinical response to targeting CDK4/6.Thus, selective inhibition of CDK4 led to Rb-dependent pG1 in tumor cells of all MCL patients despite a multitude of genomic aberrations. Integrative WES and WTS analysis of serial tumor biopsies revealed that pG1 reprograms MCL cells by inducing an imbalance in gene expression that is associated with the clinical response to the Pa-Btz therapy. Defining the functions of the candidate genes identified in the context of clinical response should shed light on the mechanism for therapeutic targeting of CDK4/CDK6 and advance genome-based patient stratification. Disclosures:Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor. Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau. Leonard:millennium: Consultancy.

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.

Induction of prolonged early G1 arrest by CDK4/CDK6 inhibition reprograms lymphoma cells for durable PI3Kδ inhibition through PIK3IP1

Phosphatidylinositol-3-kinase (PI3K) signaling is constitutive in most human cancers. Selective inhibition of PI3Kδ (p110δ) by GS-1101 has emerged as a promising therapy in chronic lymphocytic leukemia and indolent lymphomas. In aggressive non-Hodgkin lymphomas such as mantle cell lymphoma (MCL), however, efficacy has been observed, but the extent and duration of tumor control is modest. To determine if tumor killing by GS-1101 is cell cycle-dependent, we show in primary MCL cells by whole-transcriptome sequencing that, despite aberrant expression and recurrent mutations in Cyclin D1, mutations are rare in coding regions of CDK4, RB1 and other genes that control G1-S cell cycle progression or PI3K/AKT signaling. PI3Kδ is the predominant PI3K catalytic subunit expressed, and inhibition by GS-1101 transiently inhibits AKT phosphorylation but not proliferation in MCL cells. Induction of prolonged early G1-arrest (pG1) by selective inhibition of CDK4/CDK6 with PD 0332991 amplifies and sustains PI3Kδ inhibition, which leads to robust apoptosis. Accordingly, inhibition of PI3Kδ induces apoptosis of primary MCL tumor cells once they have ceased to cycle ex vivo, and this killing is enhanced by PD 0332991 inhibition of CDK4/CDK6. PIK3IP1, a negative PI3K regulator, appears to mediate pG1 sensitization to PI3K inhibition; it is markedly reduced in MCL tumor cells compared with normal peripheral B cells, profoundly induced in pG1 and required for pG1 sensitization to GS-1101. Thus, the magnitude and duration of PI3K inhibition and tumor killing by GS-1101 is pG1-dependent, suggesting induction of pG1 by CDK4/CDK6 inhibition as a strategy to sensitize proliferating lymphoma cells to PI3K inhibition.

Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell Lymphoma with the Translation Inhibitor Silvestrol

During cell-cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression. Mantle cell lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL. Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nmol/L silvestrol at 16 hours, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As showed in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P < 0.0001) prolonged survival in a MCL xenograft model without detectable toxicity. These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies.

Activity of Rituximab and Ofatumumab Against Mantle Cell Lymphoma (MCL) in Vitro in MCL Cell Lines and Ex Vivo in Tumor Cells Derived From Patient Tumor Samples

Abstract 4972Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that frequently presents with advanced stage disease. The addition of rituximab, a monoclonal anti-CD20 antibody, to high dose chemotherapy regimens often followed by stem cell transplant has improved outcomes, but survival still remains low at 3–5 years. Novel agents are needed to improve outcomes in MCL. Ofatumumab is a fully human anti-CD20 monoclonal antibody directed against a novel epitope on the CD20 antigen. Ofatumumab has been shown to be more potent than rituximab against B-NHL cells in pre-clinical investigations. Ofatumumab is FDA approved for the treatment of CLL that is fludarabine and alemtuzumab refractory or with bulky disease resistant to fludarabine and is being investigated in clinical trials in NHL. In order to characterize the activity of ofatumumab against MCL, we performed pre-clinical investigations into the activity of ofatumumab against MCL cell lines and primary MCL tumor cells derived from patient tumor samples (n=2). Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in the MCL cell lines Mino, Jeko, Rec-1 and Z-138 to demonstrate sensitivity to rituximab and ofatumumab. Lymphoma cells were labeled with 51Cr prior to incubation with rituximab or ofatumumab at 10ug/mL plus human serum or effector cells (efector:target ratio of 20:1). 51Cr-release was measured and the percentage of lysis was calculated. Patient tumor cells were isolated from tumor biopsy samples by MACS sorting (negative selection). Patient tumor cells were incubated with ofatumumab or rituximab at 10ug/mL in the presence of human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Means were compared using a t-test. Expression of CD20 and the complement inhibitory proteins (CIPs) CD55 and CD59 in MCL cell lines were determined by flow cytometry and compared to the rituximab-sensitive cell line Raji and the rituximab-resistant cell line Raji 4RH. Surface density of CD20, CD55 and CD59 were determined by Imagestream analysis. Western blot was performed to measure total CD20 protein expression. Ofatumumab induced significantly higher levels of cell lysis compared to rituximab in CDC assays of all MCL cell lines tested (Mino: 65.9% vs 0.5%; JeKo 43.9% vs 13.3%; REC-1 25.4% vs 4.7%; Z-138: 56.4% vs 0.65%; all p-values <0.05). The ADCC assays showed a similar degree of lysis with ofatumumab when compared to rituximab in all cell lines tested. In primary tumor cells, ofatumumab and rituximab demonstrated similar levels of decreased cell viability following 48 hours of antibody exposure. MCL cell lines demonstrated similar expression of surface and total CD20 when compared to the rituximab-sensitive B-NHL Raji cell line. CIP expression was increased in all MCL cell lines compared to Raji cells and was similar to the rituximab-resistant Raji 4RH cell line. Our data suggest ofatumumab is more potent than rituximab against MCL cells in vitro and retains CDC activity despite high expression levels of CIPs. This increased activity was not seen in patient tumor samples; however we were limited by the number of available patient samples. In vivo experiments investigating the activity of ofatumumab in a SCID mouse MCL xenograft model and investigations into the activity of ofatumumab in MCL cells in combination with cytotoxic agents and novel small molecule inhibitors are ongoing. Disclosures:Czuczman:Genmab: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding.

Targeting the Cyclin D - CDK4/6 - Rb Axis in Mantle Cell Lymphoma with the Novel Translation Inhibitor Silvestrol,

Abstract 3498During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit.A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease.Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P<0.0001] without detectable toxicity. Together, these data demonstrate that the translation inhibitor silvestrol has promising in vitro and in vivo activity in MCL preclinical models. Furthermore, as the cyclin D/CDK/Rb axis is disrupted in most tumor types, this strategy may be broadly effective in other cancers as well. Disclosures:No relevant conflicts of interest to declare.

Pathway discovery in mantle cell lymphoma by integrated analysis of high-resolution gene expression and copy number profiling

AbstractThe genome of mantle cell lymphoma (MCL) is, in addition to the translocation t(11;14), characterized by a high number of secondary chromosomal gains and losses that probably account for the various survival times of MCL patients. We investigated 77 primary MCL tumors with available clinical information using high-resolution RNA expression and genomic profiling and applied our recently developed gene expression and dosage integrator algorithm to identify novel genes and pathways that may be of relevance for the pathobiology of MCL. We show that copy number neutral loss of heterozygosity is common in MCL and targets regions that are frequently affected by deletions. The molecular consequences of genomic copy number changes appear complex, even in genomic loci with identified tumor suppressors, such as the region 9p21 containing the CDKN2A locus. Moreover, the deregulation of novel genes, such as CUL4A, ING1, and MCPH1, may affect the 2 crucial pathogenetic mechanisms in MCL, the disturbance of the proliferation, and DNA damage response pathways. Deregulation of the Hippo pathway may have a pathogenetic role in MCL because decreased expression of its members MOBKL2A, MOBKL2B, and LATS2 was associated with inferior outcome, including an independent validation series of 32 MCLs.

PRDM1 is required for mantle cell lymphoma response to bortezomib.

Mantle cell lymphoma (MCL) is an aggressive form of B-cell lymphoma with a poor disease-free survival rate. The proteasome inhibitor bortezomib is approved for the treatment of relapsed and refractory MCL and has efficacy in about 30% of patients. However, the precise mechanism of action of bortezomib is not well understood. This report establishes a requirement for the transcription repressor PR domain zinc finger protein 1 (PRDM1, Blimp1) in the response to bortezomib. Bortezomib rapidly induces transcription of PRDM1 as part of the apoptotic response in both cell lines and primary MCL tumor cells. Knockdown of PRDM1 blocks activation of NOXA and inhibits apoptosis, whereas ectopic expression of PRDM1 alone leads to apoptosis in MCL. Two novel direct targets of PRDM1 were identified in MCL cells: MKI67 (Ki67) and proliferating cell nuclear antigen (PCNA). Both MKI67 and PCNA are required for proliferation and survival. Chromatin immunoprecipitation and knockdown studies reveal that specific repression of MKI67 and PCNA is mediated by PRDM1 in response to bortezomib. Furthermore, promoter studies and mutation/deletion analysis show that PRDM1 functions through specific sites in the PCNA proximal promoter and an MKI67 distal upstream repression domain. Together, these findings establish PRDM1 as a key mediator of bortezomib activity in MCL.