Mantle Cell Lymphoma Tumor Research Articles

SOX11 Cooperates with CCND1 in Mantle Cell Lymphoma Pathogenesis

MCL (Mantle cell lymphoma) is an aggressive and incurable B cell malignancy with a median survival of 5-6 years. Cyclin D1 (CCND1) overexpression is a key diagnostic feature of this disease, observed in more than 90% of MCL tumors. However, murine models over-expressing CCND1 in B cells do not recapitulate the phenotype of MCL. The SOX11 transcription factor is aberrantly expressed in 80-90% of primary MCL. Our published data demonstrated that SOX11 binds and functionally regulates key components in multiple oncogenic pathways in MCL such as WNT and TGFβ pathways. Recent studies have also showed that SOX11 regulates PAX5 and PDGFA to block differentiation and facilitate lymphoma growth. We thus hypothesize that SOX11 expression may contribute directly and functionally cooperate with CCND1 in MCL pathogenesis.To study the role of SOX11 in MCL tumorigenesis in vivo, we have generated a novel SOX11 transgenic mouse model with B cell-specific tissue expression under the E-mu enhancer and an IRES-eGFP tag to monitor the expression of SOX11. The presence of SOX11 can be readily detected in pre-pro-B stage in the bone marrow coincided with the activation of E-mu enhancer and was persistent through all stages of B cells. SOX11 over-expression in our mouse model led to an aberrant oligo-clonal expansion of CD19+/CD5+ B cells. This phenotype was evident in all SOX11 transgenic mice studied (100% penetrance, n= 42 mice) with an average of 7-12 fold increase (p<0.03) of the CD5+ B cell populations as compared to littermate controls starting from 1.5 months. Using Mass Cytometry (CyTOF), we further characterized this B cell population to be CD23-, CD21/35 dim, CD138-, high surface IgM, and variable IgD expression, a naive B cell phenotype consistent with an early precursor stage of human MCL. This MCL phenotype is most prominent in peripheral blood and spleen and, to a much lesser extent, in peritoneal cavity and bone marrow. Transplanting bone marrow from SOX11 transgenic mouse to lethally-irradiated wild type mice successfully transferred the observed phenotypic CD19+/CD5+/CD23- B cell hyperplasia, suggesting that SOX11 overexpression in early B cells drives this MCL phenotype.We next studied the cooperation between CCND1 and SOX11 by crossing SOX11 transgenic mice with a CCND1 transgenic mouse model, which over-expresses CCND1 in a B-cell specific manner under a similar E-mu enhancer. Overexpression of both CCND1 and SOX11 in the double transgenic mice model dramatically enhanced (average 10x, range 6x-30x) the aberrant MCL phenotype (CD19+/CD5+/CD23-) in peripheral blood, spleen, bone marrow, peritoneal cavity and lymph nodes compared to age-matched SOX11 and CCND1 single-transgenic mice.We report here the first direct evidence in vivo that SOX11 expression drives an aberrant expansion of B cells consistent with early human MCL and functionally collaborates with CCND1 in "full blown" MCL pathogenesis, mimicking the commonly observed co-expression of SOX11 and CCND1 in most human MCL tumors. This model captures the underpinning molecular pathogenesis events occurred in the majority of human MCL and overcomes constraints of previous MCL models that develop a phenotype after long latency or with low penetrance, making it a valuable tool for testing anti-MCL therapeutics. We are currently developing small molecule SOX11 inhibitors using SOX11 DNA binding domain models and consensus SOX11 binding nucleotides to screen a large library of compounds to identify new therapeutics for this fatal disease and gain better understanding of the molecular mechanisms of MCL tumorigenesis. DisclosuresNo relevant conflicts of interest to declare.

JQ1, a Selective Bromodomain Inhibitor, Augment the Immunogenicity of Mantle Cell Lymphoma By Influencing the Expression of PD-L1

Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin's B-cell lymphoma and represents 4% to 8% of NHL. New therapeutic strategies are under development to improve the clinical outcome of patients with this disease. Recent studies have highlighted the importance of BRD/acetyl-lysine binding in orchestrating molecular interactions in chromatin biology and regulating gene transcription. The Bromodomain (BRD) is a diverse family of evolutionary conserved protein-interaction modules, which recognizes acetylated lysine residues such as those on the N-terminal tails of histones.We have recently determined the functional consequences of inhibiting Bromodomains in MCL. First, we evaluated the effects of JQ1, a selective small-molecule bromodomain inhibitor upon MCL's immunogenicity and antigen-specific CD4+ T-cell responses. In vitro treatment of MCL cells with increasing concentrations of JQ1 resulted in a decreased expression of the tolerogenic molecule PD-L1 as compared to control cells. Second, we evaluated the capabilities of JQ1-treated MCL cells to present cognate antigen to naïve or anergic antigen-specific CD4+ T-cells. We found that treatment of MCL cells with JQ1 enhances their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells and restoring the responsiveness of tolerant T-cells as determined by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, in vivo studies clearly demonstrated a strong anti-tumor effect in JQ1-treated MCL tumor bearing mice. Mechanistically, we found a decreased STAT3 phosphorylation and c-Myc expression in JQ1-treated MCL cells, which in turn might be responsible for the diminished expression of PDL1 and augmentation of MCL's immunogenicity.Taken together, we have found that MCL cells treated with the Bromodomain specific inhibitor JQ1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and, are capable of restoring the responsiveness of tolerant T-cells. More importantly, JQ1 treatment in vivo inhibits tumor growth in MCL tumor bearing animals. Our studies therefore have unveiled a previously unknown immunological effect of BRD inhibitors and have identified a novel immunotherapeutic approach in MCL. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-160: HDAC9 expression is deregulated in malignant B-cell lymphomas in particular in diffuse large B-cell lymphoma and mantle cell lymphoma

Abstract Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme that, within the hematopoietic system, is preferentially expressed in the B-cell lineage. In our previous works, in order to identify HDAC9 function in the B cell lineage we developed mice that constitutively expressed human HDAC9 from early stages of B-cell development, under the control of the immunoglobulin heavy chain (IgH) enhancer. These mice developed lymphoproliferative disorders, including indolent Marginal Zone Lymphoma (MZL) and more aggressive post-Germinal Center (GC) lymphomas, demonstrating an oncogenic role for HDAC9 in B-cells. In order to examine the relationship between diseases observed in the mouse model and human primary lymphoma, we have examined, using immunohistochemistry (IHC) the expression of full length HDAC9 isoform in a panel of various B-cell malignancies from human tumor samples. The study group included 59 non-Hodgkin lymphomas (NHL), and 3 classical HL. Non-HL consisted of 34 diffuse large B cell lymphoma (DLBCL), 9 follicular lymphoma (FL), 5 marginal zone lymphoma (MZL), 6 mantle cell lymphoma (MCL), and 2 small lymphocytic lymphomas (SLL). HDAC9 expression was assessed by IHC using tissue microarray and/or routine tissue sections. Protein expression was scored as negative (0), low (1), or high (2) depending on the staining signal intensity. Expression of HDAC9 in the nuclei of the tumor cells was compared with that seen in adenocarcinoma cells; if equal or higher, then expression of HDAC9 was considered high and if lower, then expression of HDAC9 was considered low. Five reactive lymph nodes were studied to assess the baseline expression of HDAC9. Rectal adenocarcinomas were used as positive controls. In reactive lymph nodes, HDAC9 was weakly expressed in a subset of germinal center cells, a subset of lymphoid cells in the paracortex as well as in endothelial cells. HDAC9 expression was detected in all subsets of B-cell lymphomas analyzed and in most cases with a level of expression higher than those seen in reactive lymph nodes. DLBCL and MCL tumors had the highest frequency of high HDAC9 expression among the B-cell lymphomas analyzed, 77 and 83% (Fisher's exact test P = 1.0), respectively. No differences in HDAC9 expression were detected in DLBCL of GC and non-GC type. In contrast, most (69%) of the low-grade B cell lymphomas showed no or lower expression of HDAC9 (Fisher's exact test P = 0.004; as compared to DLBCL). Classical HL showed frequently low-expression of HDAC9 in the tumor cells. In summary, HDAC9 is frequently expressed in B-cell lymphomas with the highest level of expression found in the most aggressive lymphomas such as DLBCL and MCL. These findings support the biological role of HDAC9 in the pathobiology of aggressive B cell neoplasms and highlight the need to further study HDAC9 function in these malignancies as well as its importance as a therapeutic target. Citation Format: Elena Cubedo, Veronica Gil, Chae Hwa Kim, German Campuzano-Zuluaga, Nitin Kumar Agarwal, Louise Howell, Kevin R Petrie, Francisco Vega, Arthur Zelent. HDAC9 expression is deregulated in malignant B-cell lymphomas in particular in diffuse large B-cell lymphoma and mantle cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-160. doi:10.1158/1538-7445.AM2015-LB-160

Abstract 1742: Ibrutinib plus proteasome or MALT1 inhibitors overcome resistance to BCR antagonists in CARD11 mutant-expressing B-lymphoma cells

Abstract Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with poor prognosis. Ibrutinib, a covalent first in-human Bruton's tyrosine kinase inhibitor, approved for second line treatment for MCL patients, has an overall response rate of nearly 70% in patients with relapsed and refractory MCL (Wang, 2013). However, combination strategies with novel therapies remain an important option for the 30% of non-responding or progressing patients. Caspase recruitment domain-containing protein (CARD)11 is a critical component of NF-κB signaling in B and T cells (Stepensky, 2013). Mutations in the coiled-coil domain of CARD11 can result in constitutive NF-κB activation and resistance to B-cell receptor (BCR) antagonists in ABC-DLBCL (Lenz, 2008) and MCL cell lines (Rahal, 2014). This study investigated the effect of CARD11 mutations on MCL tumor growth, and evaluated combination strategies to overcome resistance to BCR antagonists. Methods: Jeko cells (an ibrutinib-sensitive MCL cell line [IC50 = 0.5 μM]) were co-infected with lentiviral vector pCDH-EF1 expressing either wildtype (WT) or CARD11 mutants (mut2 [L244P] or mut10 [L225LI], Lenz, 2008), generated by site-directed mutagenesis, and with lentiviral vector pGreenPuro expressing an endogenous knockdown small hairpin (sh)RNA targeting the 3′-untranslated region of CARD11. Stable transfectants were compared for their sensitivity to ibrutinib. Pharmacological inhibitors of NF-κB, PI3K/SYK, proteasomes, or apoptosis pathways were also evaluated in combination with ibrutinib. Results: The mutant stable cell lines have similar levels of CARD11 expression, which are comparable to levels of endogenous CARD11. CARD11 shRNA inhibited 70% of endogenous CARD11 expression resulting in cell death. Jeko cells expressing WT or mutant CARD11 were protected from shRNA toxicity, and cells expressing mut10 CARD11 proliferated 40% faster than WT-expressing cells. Mutant cells were less sensitive to ibrutinib (IC50 = 0.8 μM [WT]; IC50 = 1.9 [mut2] or 2.6 μM [mut10]). In mutant CARD11-expressing Jeko cells, ibrutinib inhibited the activity of downstream BCR signaling molecules ERK and AKT, but not NF-κB. Addition of the proteasome inhibitors (carfilzomib or bortezomib) or the MALT1 inhibitor MI2 sensitized CARD11 mutant cells to ibrutinib treatment (IC50 = 2.6 μM [ibrutinib]; IC50 = 0.2 μM [ibrutinib+bortezomib]; IC50 = 0.1 μM [ibrutinib+MI2]). MI2 also facilitated the degradation of CARD11 protein, and synergized with ibrutinib for complete inhibition of the NF-κB pathway. Analysis of the ibrutinib and MI2 combination in murine models is ongoing. Conclusion: This study showed that the combination of ibrutinib with proteasome or MALT1 inhibitors can sensitize CARD11 mutant cells to treatment and may provide a strategy to overcoming resistance to BCR-antagonists in-vivo. Note: This abstract was not presented at the meeting. Citation Format: Ling Xue, Mutiah Apatira, Mint Sirisawad, Betty Chang. Ibrutinib plus proteasome or MALT1 inhibitors overcome resistance to BCR antagonists in CARD11 mutant-expressing B-lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1742. doi:10.1158/1538-7445.AM2015-1742

The STAT3 inhibitor WP1066 synergizes with vorinostat to induce apoptosis of mantle cell lymphoma cells

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) characterized by the translocation t (11; 14) (q13; q32). Drug resistance remains a formidable obstacle to treatment and the median survival for MCL patients is between 3 and 5 years. Thus, there is an urgent need to discover novel approaches to MCL therapy. The signal transducer and activation of transcription 3 (STAT3) has been found to be constitutively activated in several subtypes of MCL cell lines and MCL tumors. WP1066, a small-molecule inhibitor of STAT3, exerted antitumor activity in hematological and solid malignancies by inhibiting key survival and growth signaling pathways. In the present study, we evaluated the antiproliferative and proapoptotic activity of WP1066 combined with pan-histone deacetylase (HDAC) inhibitor vorinostat (SAHA) in a panel of MCL cell lines. In addition, potential mechanisms involved were also explored. The outcome showed that combination of WP1066 with SAHA resulted in synergistic growth inhibition and apoptosis induction in MCL cell lines in vitro. Furthermore, combination of WP1066 with SAHA inhibited the constitutive STAT3 activation and modulated mRNA expressions of anti- and pro-apoptotic genes. Our findings suggest that agents targeting the STAT3 pathway such as WP1066 may be useful therapeutic drugs for MCL when combined with SAHA.

ROR1-targeted delivery of OSU-2S, a nonimmunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle-cell lymphoma in vitro and in vivo

Mantle-cell lymphoma (MCL) remains incurable despite numerous therapeutic advances. OSU-2S, a novel nonimmunosuppressive FTY720 (Fingolimod) derivative, exhibits potent cytotoxicity in MCL cell lines and primary cells. OSU-2S increased the surface expression of CD74, a therapeutic antibody target in MCL cells. OSU-2S, in combination with anti-CD74 antibody milatuzumab, enhanced cytotoxicity in MCL. Moreover, MCL tumor antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeted immunonanoparticle-carrying OSU-2S (2A2-OSU-2S-ILP)-mediated selective cytotoxicity of MCL in vitro, as well as activity in a xenografted mouse model of MCL in vivo. The newly developed OSU-2S delivery using ROR1-directed immunonanoparticles provide selective targeting of OSU-2S to MCL and other ROR1(+) malignancies, sparing normal B cells.

HDAC9 Expression Is Deregulated in Malignant B-Cell Lymphomas in Particular in Diffuse Large B Cell Lymphoma and Mantle Cell Lymphoma

Aberrant epigenetics leading to changes in chromatin structure and patterns of gene expression is an important factor in cancer pathogenesis. Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme that, within the haematopoietic system, is preferentially expressed in the B-cell lineage. Mice that constitutively express human HDAC9 from early stages of B-cell development, under the control of the immunoglobulin heavy chain (IgH) enhancer, develop lymphoproliferative disorders, including germinal center (GC) and post-GC lymphomas, demonstrating an oncogenic role for HDAC9 in B-cells and highlighting its importance as a therapeutic target. In order to examine the relationship between disease observed in the mouse model and human primary lymphoma, we have examined, using immunohistochemistry (IHC) the expression of full length HDAC9 isoform in a panel of various B-cell malignancies.The study group included 59 non-Hodgkin lymphomas (NHL), and 3 classical HL. Non-HL consisted of 34 diffuse large B cell lymphoma (DLBCL), 9 follicular lymphoma (FL), 5 marginal zone lymphoma (MZL), 6 mantle cell lymphoma (MCL), and 2 small lymphocytic lymphomas (SLL). HDAC9 expression was assessed by IHC using tissue microarray and/or routine tissue sections. Protein expression was scored as negative (0), low (1), or high (2) depending on the staining signal intensity. Expression of HDAC9 in the nuclei of the tumor cells was compared with that seen in adenocarcinoma cells; if equal or higher, then expression of HDAC9 was considered high and if lower, then expression of HDAC9 was considered low. Five reactive lymph nodes were studied to assess the baseline expression of HDAC9. Rectal adenocarcinomas were used as positive controls. In reactive lymph nodes, HDAC9 was weakly expressed in a subset of germinal center cells, a subset of lymphoid cells in the paracortex as well as in endothelial cells. HDAC9 expression was detected in all subsets of B-cell lymphomas analyzed and in most cases with a level of expression higer than those seen in reactive lymph nodes. DLBCL and MCL tumors have the highest frequency of high HDAC9 expression among the B-cell lymphomas analyzed, 77 and 83% (Fisher’s exact test P=1,0), respectively. No differences in HDAC9 expression were detected in DLBCL of GC and non-GC type. In contrast, most (69%) of the low-grade B cell lymphomas show no or lower expression of HDAC9 (Fisher’s exact test P=0.004; as compared to DLBCL). Classical HL showed frequently low-expression of HDAC9 in the tumor cells. In summary, HDAC9 is frequently expressed in B-cell lymphomas with the highest level of expression found in the most aggressive lymphomas such as DLBCL and MCL. These findings support the biological role of HDAC9 in the pathobiology of aggressive B cell neoplasms. DisclosuresNo relevant conflicts of interest to declare.

The Tumor Microenvironment in Mantle Cell Lymphoma (MCL): Novel Targets to Overcome Chemo-Resistance in MCL

▪Mantle Cell Lymphoma (MCL) is a relatively uncommon, aggressive form of B-cell non-Hodgkin’s lymphoma (NHL), defining approximately 5-8% of adult NHL in the United States. Although individual survival times have increased in recent years, prognosis remains among the poorest of NHL, with a median survival of 5 to 7 years. Despite an initial response to chemotherapy, MCL cells remain residually in the marrow and digestive tract, making MCL prone to recur leading to short survival prognosis. The characteristic re-emergence of MCL is likely related to drug resistance generated from the tumor microenvironment (TME). In particular, stromal cells found in bone marrow and secondary lymphoid sites promote the movement and proliferation and possibly drug resistance from cross-talk between stromal and lymphoma cells. While studies show a correlation between stromal cells and tumor survival in various forms of NHL, the mechanisms are poorly understood and its role in MCL has not extensively explored. Based on our experimental studies on an extensive spectrum of representative patient samples, we have shown that in vitro culturing of primary leukemic phase MCL cells (15 cases) usually leads to the spontaneous formation of activated CD68+ monocytic macrophages and stromal cells. The MCL tumor cells tended to form rosettes, clustering around and adhering to these cultured lymphoma-associated macrophages (LAM). The LAM formed in culture coexisted with rosetted MCL tumor cells for up to 4 months. During this period, a small subset of MCL cells showed limited mitotic figures, suggesting that these cells were proliferating and possibly becoming incipient immortalized MCL cells. When these cells were weaned from the adherent LAM, they began to expand autonomously and proliferate as an autonomous cell line. Using this methodology, we have developed 3 MCL cell lines (PF-1, PF-2, and PF-3) from 15 leukemic phase MCL cases. PF-1 MCL cell line represents classic typical MCL cells, while PF-2 and PF-3 MCL cell lines represent the in situ indolent MCL cells that are CD200 positive. When these cells were xeno-transplanted into SCID mice, lymphoma cell uptakes were disseminated throughout the lymphatic system, including the spleen, lymph nodes, and GI tract, representing excellent in vivo models for MCL. Next, we delineate the biologic functions of LAM in the MCL microenvironment milieu. Our data showed that when we initially removed the accessory cells (monocytes, etc.) from the MCL primary culture, no LAM were formed and the purified MCL cells died out in <2-3 weeks in culture, suggesting that the LAM developed in culture are biologically and pathologically functional in providing growth/survival signals to the tumor cells. We then examined several therapeutic agents that have shown efficacy against relapsed-MCL in the clinic, including the proteasome inhibitor carfilzomib (CFZ) and the BTK inhibitor ibrutintib (IB), on targeting LAM. Our results showed that both CFZ and IB, at very low drug concentrations could affectively eliminate LAM, followed by spontaneous apoptosis of the MCL cells. We have also developed a unique mesenchymal stromal cell line (PF-MSC), derived from a leukemic phase MCL patient. These cells grow in culture as spindle-shaped morphologies and can enhance the growth of MCL cells as well as protect MCL cells from chemotherapy. However, PF-MSC cells did not protect MCL cells from CFZ or IB therapies. Our findings clearly indicate that MCL TME is a critical mediator for growth/survival and chemoresistance mechanisms in the pathophysiology of MCL. Therapeutically targeting these MCL-associated macrophages/stromal cells with CFZ and/or IB should lead to better therapeutic strategies for refractory MCL patients. DisclosuresPham:Onyx/Amgen: Research Funding.

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.

Diagnostic Tumor Mirna Profiling Predicts Molecular Relapse in Mantle Cell Lymphoma Patients Prospectively Followed for Minimal Residual Disease. Results from the Nordic MCL2-3 Trials

Background Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin‘s lymphoma (NHL) with a variable but often aggressive clinical course. The majority of MCL patients ≤65 years will experience clinical relapse during a 10-year period (Geisler et al. Br J Haematol 2012). Risk-stratification is therefore of great importance in order to identify patients who are eligible for novel or alternative treatment regimens. Minimal residual disease (MRD) monitoring can predict clinical progression in MCL and guide pre-emptive treatment with rituximab as single agent. It has thus far not been possible to predict post-treatment molecular relapse (MRD-positivity). Although some recent studies have shown that aberrant miRNA expression delineate pathogenic molecular pathways and predict survival in MCL patients, miRNA profiling has not been performed in the context of MRD and molecular relapse.Aims We assessed MCL miRNA expression, in a large, prospective, uniformly treated patient cohort followed with molecular markers for MRD to determine if miRNAs could predict molecular relapse.Methods Diagnostic MCL tumor samples from 114 patients in the Nordic MCL2 and MCL3 clinical trials were retrieved. All patients had confirmed CyclinD1 overexpression and 96 patients had Ki-67 expression measurement for use in the MIPI-B predictive score. All patients received almost identical induction treatment with six alternating cycles of maxi-CHOP and high-dose Ara-C in combination with rituximab. Patients in remission were consolidated with high dose chemotherapy followed by autologous stem cell transplantation. All patients had a molecular marker (PCR detectable t(11;14) or clonal IgH rearrangement) for MRD. The median follow-up was 6.4 years for the MCL2 cohort and 3.7 years for the MCL3 cohort. 19 miRNAs, previously found to have prognostic significance in MCL (Husby et al EHA 2014), were measured by qRT-PCR and analyzed in concordance with MRD-data. The main endpoint was first event of molecular relapse (MRD-positive sample).Results Of the 114 patients in the study, 71 (62%) patients became MRD-positive in the follow-up period. Of the 19 examined miRNAs, 10 miRNAs showed unique qPCR melting curves and were analyzed with respect to MRD. Four miRNAs (miR-92a, miR-3687, miR-486-5p and miR-185-5p) were significantly up-regulated in patients who had molecular relapse (t-test; respectively p = 0.010, p = 0.019, p = 0.048, p = 0.043). However miR-18b, previously identified as prognostic marker regarding survival, was not significantly overexpressed. We hereafter investigated if a newly derived prognostic score, the MIPI-B-miR, which incorporates miR-18b with the MIPI-B, could predict molecular relapse. The MIPI-B-miR high-risk patients had significantly shorter time to first molecular relapse than MIPI-B high-risk patients (Figure 1). Functional studies of these aberrantly expressed miRs are ongoing.Conclusion Patients with molecular relapse had significantly increased levels of miR-92a, miR-3687, miR-486-5p and miR-185-5p, and MIPI-B-miR improved MRD prediction compared to MIPI-B. Aberrant miRNA profiles may be able to predict molecular relapse, and may already at diagnosis identify patients eligible to anti-CD20 antibody maintenance or alternative regimens. However validation in other cohorts is needed. [Display omitted] DisclosuresKolstad:Nordic Nanovector: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Räty:GlaxoSmithKline Ltd: Honoraria; Roche Ltd: Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria.

SNS01-T Modulation of eIF5A Inhibits B-cell Cancer Progression and Synergizes With Bortezomib and Lenalidomide

The high rates of recurrence and low median survival in many B-cell cancers highlight a need for new targeted therapeutic modalities. In dividing cells, eukaryotic translation initiation factor 5A (eIF5A) is hypusinated and involved in regulation of protein synthesis and proliferation, whereas the non-hypusinated form of eIF5A is a potent inducer of cell death in malignant cells. Here, we demonstrate the potential of modulating eIF5A expression as a novel approach to treating B-cell cancers. SNS01-T is a nonviral polyethylenimine-based nanoparticle, designed to induce apoptosis selectively in B-cell cancers by small interfering RNA–mediated suppression of hypusinated eIF5A and plasmid-based overexpression of a non-hypusinable eIF5A mutant. In this study, we show that SNS01-T is preferentially taken up by malignant B cells, inhibits tumor growth in multiple animal models of B-cell cancers without damaging normal tissues, and synergizes with the current therapies bortezomib and lenalidomide to inhibit tumor progression. The results collectively demonstrate the potential of SNS01-T as a novel therapeutic for treatment of a diverse range of B-cell malignancies.

A Unique Composite Follicular Lymphoma and Mantle Cell Lymphoma With a Mixed Cell Pattern and Aggressive Course

There are a limited number of reports of composite follicular lymphoma (FL) and mantle cell lymphoma (MCL) in the literature, and all previous cases report that FL and MCL components are separated, even within a single lymph node. Here we report a case of a patient with a distinctive composite FL and MCL with a mixed cell pattern. A 34-year-old man presented with left supraclavicular lymphadenopathy for one month. The lymph node contained closely packed nodules of lymphocytes. Immunostaining and fluorescence in situ hybridization demonstrated the FL nature of the Bcl-2- and CD10-positive tumor cells, as well as the scattered cyclin D1-positive MCL tumor cells in the nodules. Double immunohistochemical staining showed an unusual mixed pattern of both types of tumor cells. The patient received a regimen of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy and achieved partial remission following four cycles of chemotherapy but relapsed 1 month after the last treatment and died of meninges involvement 8 months after the first presentation. To our knowledge, this is the first report of composite FL and MCL with a mixed pattern. A mixture of grade IIIb FL and MCL may explain the poor prognosis of the patient.

Inhibition of lymphoma vascularization and dissemination by estrogen receptor β agonists

AbstractMost lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell–derived lymphoma is repressed following activation of estrogen receptor β (ERβ, ESR2). By using ERβ-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERβ agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERβ reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERβ inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERβ activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERβ is expressed in primary MCL tissue. These results suggest that targeting ERβ with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

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.

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.

SOX11 Regulates Angiogenesis In Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive tumor but recent studies have identified a subtype of this lymphoma with an indolent clinical behavior and long survival of the patients even without chemotherapy that may correspond to a different clinical and biological subtype of the disease. These indolent MCLs differ from conventional MCLs in a particular gene signature that lacks the expression of some transcription factors of the High Mobility Group (HMG). SOX11 is one of the best discriminatory genes between these conventional and indolent MCL tumors. We have recently demonstrated the oncogenic implication of SOX11 expression in the aggressive behavior of MCL as SOX11-knockdown derived tumors display a significant reduction on tumor growth compared to SOX11-control tumors in subcutaneous MCL xenograft experiments (Vegliante MC, Palomero J et al. Blood. 2013; 121:2175-85). To uncover SOX11 regulated genes and transcriptional programs in MCL, we performed an integrative analysis coupling data from human genome-wide promoter analysis by SOX11 ChIP-chip experiments and gene expression profiling (GEP) upon SOX11 silencing in MCL cell lines. In these studies we initially identified the role of SOX11 in blocking the terminal B-cell differentiation by the direct positive regulation of PAX5. However, how SOX11 may enhance tumor growth is still not well known.To further characterize the potential oncogenic mechanisms regulated by SOX11, we have integrated our ChIP-chip data with further analyses including GEP derived from the xenografted SOX11-positive and silenced tumors. The gene ontology term analysis of the genome-wide promoter study revealed “blood vessel development” as one of the most significant biological processes overrepresented among the SOX11-bound genes. Concordantly, a gene set enrichment analysis (GSEA) of the differentially expressed genes in the xenografted tumors showed a significant higher enrichment of gene signatures related to tumor angiogenesis in the SOX11-positive tumors. To validate these results in vivo, we first investigated protein extracts of these tumors using an angiogenesis proteome profiler antibody array and discovered a significant higher expression of 20 pro-angiogenic factors in the SOX11-positive tumors. We have then investigated the angiogenic development in tissue sections from four Z-138 SOX11-positive and nine SOX11-silenced xenografted tumors. The microvascular density area recognized by CD31 staining was significantly larger in SOX11-positive than knockdown tumors (90%±µ2m vs 15%±µ2m, respectively, p<1x10-4). SOX11-silenced tumors had larger necrotic areas than positive tumors (20%±µ2m vs 1%±µ2m, respectively, p<1x10-4), suggesting that vascular paucity could contribute to the significant smaller volume of these tumors.To determine whether angiogenic development was also differentially represented in primary human MCL we have studied the GEP of 16 SOX11-positive and 22 SOX11-negative MCL. A GSEA revealed that SOX11 expressing tumors were enriched in signatures related to tumor vasculature, angiogenesis and vasculature development. Concordantly, the CD34 staining of an independent series of primary SOX11-positive (n=8) and negative (n=9) human MCL showed higher microvascular density in SOX11 expressing tumors (30%±µ2m vs 5%±µ2m, respectively, p<1x10-4).In conclusion, these findings indicate that SOX11 promotes angiogenesis in experimental and primary human MCL and this may be an oncogenic mechanism contributing to the aggressiveness of these tumors. Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiling Reveals The Lymph Node Microenvironment As a Niche For BCR Engagement, NFκB Pathway Activation, and Tumor Proliferation In Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are both mature-looking CD5+ B-cell malignancies that invade the peripheral blood (PB), bone marrow (BM), and secondary lymphoid organs. Both are incurable with the exception of transplant. However, in many regards MCL and CLL appear to be different: MCL is aggressive and treated at diagnosis, while CLL is observed and only treated for symptomatic disease. MCL is characterized by the t(11;14)(q13;q32) resulting in cyclin D1 overexpression, anomalies in cell cycle and DNA repair, and recently by the aberrant expression of SOX11. CLL by contrast, lacks a characteristic mutation or translocation but has been recognized for decades to have a bias for certain IGHV genes, indicating that antigen-BCR interaction is a key pathogenic pathway. More recently, MCL also showed a significant IGHVbias and that many MCL cells, like CLL, express virtually identical BCRs, so called “stereotyped BCRs”, that are predicted to recognize distinct antigens (Hadzidimitriou, Blood 2011).Comparing purified CLL cells isolated concomitantly from the PB, BM, and lymph nodes (LN), we showed that LN resident cells activates SYK and upregulate BCR-target genes (Herishanu, Blood 2011). This indicates that antigenic signaling continues throughout the disease course and that the BCR is engaged primarily in the LN. Here we used gene expression profiling (Affymetrix HU133 plus) to interrogate whether MCL, similar to CLL, displays changes in tumor biology in different anatomic compartments. We collected a total of 100 PB and LN samples from previously untreated CLL and MCL patients. The samples fall into 5 groups: MCL-PB (purified CD19, n=13), MCL-LN (biopsy, n=29), CLL-PB (purified CD19, n=24), CLL-LN (biopsy, n=17), and CLL-LNT (purified CD19, n=17).An unsupervised hierarchical clustering of all samples grouped them according to tissue derivation and disease entity into MCL-LN, CLL-LN, MCL-PB, CLL-purified tumor (either PB or LNT). The previously defined MCL-LN signature (Rosenwald, Cancer Cell 2003) distinguished MCL (PB and LN) from each of the CLL groups (2-fold change, p<0.0001). Conversely a CLL specific gene signature (Jelinek, Mol Cancer Res 2003) was preferentially expressed in the 3 CLL groups than in MCL groups (3-fold change, p<0.001). There was no statistically significant difference in the expression of these signatures between PB or LN samples of the same disease entity.To study the effect of the microenvironment (ME) on tumor cells, we compared a set of three well-defined gene signatures (BCR, NFκB, and MCL-proliferation) across our samples spectrum. An individual signature score was generated for each of the five studied groups, and compared to each other using one-way ANOVA.All three signatures scored higher in LN samples (MCL or CLL) relative to PB (average 2-fold increase, p<0.01-0.0001). These scores were comparable between MCL-LN, CLL-LN, and CLL-LNT (p=ns), suggesting that first, these signatures reflect effects on resident tumor cells, and second, this effect is similar between MCL and CLL. The proliferation signature used is linked to aggressive disease in MCL (Rosenwald et al, Cancer Cell 2003), and unexpectedly scored equally across all LN groups, taking into consideration the indolent nature of CLL. This was validated using the E2F proliferation signature in CLL (Herishanu, Blood 2011). Both proliferation scores were not different between MCL-PB and CLL-PB. However, there was some variability in BCR and NFκB scores within the MCL-PB group: A subset with high BCR and NFκB scores (comparable to LN), and another with low scores (comparable to CLL-PB). The two subsets were well separated suggesting that the BCR signaling may contribute significantly to NFκB activation.Activation of MCL tumor cells in LN was confirmed by flow cytometry showing an increase in the activation markers (CD38, CD69, and CD80) and a decrease in the chemokine receptor CXCR4 in LN resident compared to circulating cells in paired samples (n=3).These data suggest that MCL, like CLL, relies on the LN ME for BCR engagement, cell activation and proliferation, and that both may be critically dependent on BCR signaling. This conclusion is underscored by the equally dramatic clinical responses achieved with the BTK inhibitor ibrutinib.Supported by the Intramural Research Program of NHLBI and NCI, NIH. We thank our patients for donating samples to make this research possible. Disclosures:No relevant conflicts of interest to declare.

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.

The E3 ubiquitin ligase UBR5 is recurrently mutated in mantle cell lymphoma

AbstractWe have recently reported the application of RNAseq to mantle cell lymphoma (MCL) transcriptomes revealing recurrent mutations in NOTCH1. Here we describe the targeted resequencing of 18 genes mutated in this discovery cohort using a larger cohort of MCL tumors. In addition to frequent mutations in ATM, CCND1, TP53, and NOTCH1, mutations were also observed recurrently in MEF2B, TRAF2, and TET2. Interestingly, the third most frequently mutated gene was UBR5, a gene encoding a 2799aa protein, with multiple functions, including E3 ligase activity based on a conserved cysteine residue at the C-terminus. Nonsynonymous mutations were detected in 18% (18/102) of tumors, with 61% of the mutations resulting in frameshifts in, or around, exon 58, predicted to result in the loss of this conserved cysteine residue. The recurrence and clustering of deleterious mutations implicate UBR5 mutations as a critical pathogenic event in a subgroup of MCL.