ALBERT

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

Description: Abstract 426 One of the important challenges in screening anti-cancer drugs is the lack of available “primary cultures systems” that is easy to use to screen new compounds or their combinations. The low yield of primary cancer cell cultures is mainly due to suboptimum environment in vitro and inefficient 2-dimensional cell culture conditions. To create an optimum in vitro environment, lymphoma cell lines were grown in 3-dimension model by using a scaffold and the stromal cells derived from neonatal foreskin was used as the feeder component. This 3-dimensional (3D) stromal co-culture generates an in-vitro model that may mimic the conditions/microenvironment of blood cancer cells interacting with stromal compartments. A specific 3D tissue culture scaffold 3D Insert-PS™ (300 μ m in fiber diameter and 400μ m in pore size) significantly enhances the cell proliferation and maintenance of liquid cancer cells in comparison to 2D stromal co-culture control. The combination of the neonatal stroma cells, a novel 3D scaffold, the constant gyration and a frequent nutrient stimulation allows the lymphoma cells to proliferate 10-fold faster than the cells grown in 2D under the same condition. Starting from the 2nd day of 3D cell culture, these lymphoma cells grew to form layers of aggregated clusters and caused disappearance of single cells morphology and phenotype that is typical of cells growing in suspension. The cell aggregates are continuously produced from the 3D scaffold, subsequently dislodge from the scaffold and then remain viable at the bottom of the dish below the scaffold. When the cell clusters are harvested and cultured in 3D condition, the contamination of fibroblasts is over 1,000 fold less than the cell clusters that are generated from 2D environment. In addition, the clusters of cancer cells generated from 3D co-culture using 3D scaffolds contained the fibroblasts contamination that is less than 0.00001% of the total cell count, suggesting that this novel 3D environment can be implicated for the isolation of primary lymphoma/cancer cells from patient's blood or tissue specimen. To investigate this feasibility,

Description: Abstract 1579 Background: There is a growing awareness of the biological heterogeneity of MCL, which likely translates into differences in outcomes. Cytokines play an important role in the pathogenesis of lymphoma. Recent reports suggest that cytokine profiles can identify DLBCL (ASH 2010: Abstract 991) and HL pt (ASH 2011: Abstract 429) subsets with inferior outcomes. A distinct cytokine profile for MCL has not yet been defined. We therefore examined serum cytokines levels in MCL pts to gain insight into which may have biological relevance and their association with clinical outcomes. Methods: Serum samples from MCL pts (57% newly diagnosed, 43% relapsed) were prospectively collected and linked to relevant clinical data from our MCL outcomes database. Cytokine levels were determined quantitatively utilizing a commercially available protein array system. We first used Wilcoxan tests to identify relevant cytokines by comparing the distribution of 507 serum cytokines levels in MCL cases (n=97) to serum cytokines levels from normal controls (n=20). For cytokines that showed significant differences, we report the ratio of median values (cases vs. controls). To maintain a type I error rate of.05 we adjusted for multiple comparisons (Hochberg's method). Using Cox regression model and adjusting for multiplicity, we next examined the association between serum cytokines levels and outcome (PFS) in a MCL cohort uniformly treated with R-HyperCVAD (R-HCVAD). Results: 214 pts with MCL were identified of which 97 pts had available serum samples. Clinical characteristics were as follows: med age 62 yr, med MIPI score of 4, med Ki67 25% (range 5–95%) and median follow up of 35 months. Our analysis identified 22 cytokines with statistically significantly distinct levels in MCL pts (vs. normal serum controls) including (p

Description: Abstract 895 The SOX11 (Sex determining region Y)-box 11 transcription factor is aberrantly expressed in the majority (78-93%) of Mantle Cell Lymphoma (MCL) patients. Functionally, SOX11 represses MCL cell proliferation in vitro. We identify SOX11 mediated repression of Wnt signaling as contributory to the proliferation block in vitro and determine the clinical consequences of SOX11 overexpression in a meta-analysis of 3 large international patient cohorts. We have previously identified SOX11 direct binding target genes using chromatin immunoprecipitation coupled with high resolution, next generation sequencing (ChIP-Seq). Members of the Wnt signaling pathway, which is known to promote proliferation in MCL, were significantly enriched in our ChIP analysis in MCL patients and cell lines. Using a luciferase reporter assay for nuclear beta-catenin activity, we find that overexpression of SOX11 represses Wnt signaling (p

Description: Abstract 673 Mantle Cell Lymphoma (MCL) is an aggressive and incurable malignancy arising from naïve B cells (NBC) in the mantle zone of lymph node follicles. Murine models over-expressing Cyclin D1, the putative oncogene implicated in the majority of MCL, do not fully recapitulate the disease phenotype. We therefore hypothesize that there are additional mechanisms contributing to MCL pathogenesis and undertook an integrative approach by studying genome-wide DNA methylation and gene expression in primary MCL to uncover additional genes and pathways involved in MCL pathogenesis. We therefore compared and contrasted the DNA methylation levels of 14,000 gene promoters in MCL patients and normal tonsillar NBC controls using the HELP (HPA II tiny fragment Enrichment by Ligation mediated PCR) assay. All patient samples were obtained prior to any treatment from peripheral blood or apheresis specimens from newly diagnosed patients with histologically confirmed MCL. We found significant hypo-methylation of gene promoters in the MCL patients as compared to normal NBCs. Integrating genomic methylation data from HELP and gene expression data from Affymetrix U133 arrays, we determined a signature of differentially methylated genes with reciprocal changes in mRNA levels. Using pathway analysis and gene ontogeny, we selected genes for validation by choosing loci that were differentially methylated and fulfilling the following characteristics (i) demonstrating a clear methylation state change (from hypomethylated in normal B cells to methylated in MCL or vice-versa) using a threshold of 0.0 on the log ratio scale, (ii) Genes that function as tumor suppressors and were hypermethylated and suppressed in MCLs in our data (iii) Overexpressed/ hypomethylated genes with existing therapeutic options available or in clinical trial (iv) involved in pathways controlling biological processes with known relevance to MCL i.e. cell cycle control, apoptosis. Our panel included four differentially hypermethylated genes CDKN2B, MLF-1, PCDH8, HOXD8 and four differentially hypomethylated genes CD37, HDAC1, NOTCH1 and CDK5. MassArray Epityper analysis confirmed the presence of differential methylation at the promoter region of these genes, which was consistent between MCL patients and cell lines in all 8 genes studied. Remarkably, PCDH8 and CDKN2B have previously been shown to be silenced by methylation at their gene promoters and transfection of PCDH8 and CDKN2B have been shown to reduce tumor growth in breast cancer and MCL cell line models respectively. Based on these data, we hypothesized that the aberrantly hypermethylated and thereby silenced tumor suppressor genes in MCL could be pharmacologically induced by DNA hypomethylating agents and HDAC inhibitors for therapeutic benefit. We therefore next treated MCL cell lines MINO and Z138 with the DNA methyltransferase inhibitor Decitabine alone and in combination with the HDAC inhibitor SAHA. HELP analysis of MINO and Z138 cells treated with hypomethylating doses of decitabine (0.5uM × 3days) showed widespread reversal of aberrant gene promoter hypermethylation. Hypomethylation in these cell lines was accompanied with 3-7 fold increase in mRNA levels of tumor suppressor genes CDKN2B, MLF-1, PCDH8 and HOXD8. Concurrent treatment with SAHA (1 uM x 1 dose) synergized with Decitabine leading to 5-15 fold increase in mRNA of these tumor suppressor genes in Z138 cells. Importantly, treatment with Decitabine and SAHA as single agents decreased MCL cell viability by 60% and 40% respectively and the combination synergised in anti-MCL cytotoxicity with 〉 90% decrease in cell viability. In conclusion, our analysis shows prominent aberrant gene promoter methylation patterns in MCL genome and identifies novel differentially methylated and expressed genes in MCL cell lines and primary MCLs. Furthermore, we demonstrate that reversal of aberrant hypermethylated and silenced genes can be targeted for therapeutic benefit using epigenetic drugs in MCL. We are currently modeling our combination epigenetic drug therapy in primary MCL cells in culture and murine xenograft systems. We expect these results to support the development of clinical trials investigating the prospective use of combination epigenetic therapy in MCL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 557 Mantle cell lymphoma (MCL) is an aggressive B-lymphoid neoplasm with frequent relapses after initial response to standard chemotherapy. Bortezomib is the first proteasome inhibitor with promising clinical efficacy in relapsed MCL, where it achieved durable responses in 30%–50% of patients. The mechanisms contributing to Bortezomib resistance remain poorly understood. Our group and others have identified aberrantly methylated genes in MCL controlling critical cellular processes like cell cycle, transcription and regulation of gene expression. We therefore hypothesized that a subset of aberrantly methylated genes may be contributing to Bortezomib resistance. Using a massively parallel sequencing approach, HELP-tagging, we analyzed genomic methylation in MCL patients treated in a Phase II trial at the NIH. This high-resolution technique covers about 1.8 million CpGs in the human genome including gene promoters, gene bodies and CpG shores and is more sensitive and yields greater coverage of the genome as compared to array-based platforms. Unsupervised analysis of genome-wide promoter methylation of Bortezomib sensitive patients (having more than 50% reduction in absolute lymphocyte count after one cycle of Bortezomib) as compared to resistant patients revealed that methylation patterns could independently distinguish between these two groups. Similarly, three-dimensional Principal Component Analysis also clustered Bortezomib resistant patients distinctly from Bortezomib sensitive patients, confirming genomic differences in methylation between these patients. We further analyzed the differentially methylated genes using pathway analysis software, gene ontology and gene set enrichment analysis. We found that majority of differentially methylated genes were relevant to cancer biology involving metabolic pathways, regulation of transcription, cell cycle and programmed cell death. Comparing methylation profiles of patient samples taken before and 96 hours after Bortezomib treatment showed a striking genome-wide hypomethylation following Bortezomib. Western blotting of MCL cell lines showed a significant reduction in DNMT1 levels following Bortezomib treatment, which may help us understand the post-treatment hypomethylation seen in patients. NOXA1 is a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of Bortezomib. The NOXA1 gene promoter was differentially methylated in Bortezomib-sensitive MCL patients. NOXA1 was also significantly hypomethylated to a greater extent 96 hours following Bortezomib treatment in Bortezomib sensitive patients. NOXA1 hypomethylation was associated with induction of NOXA1 mRNA and protein expression in sensitive patients alone. These findings suggest a possible role for dynamic NOXA1 methylation and expression as biomarkers for Bortezomib sensitivity. Treatment of MCL cell lines Z138 and MINO with demethylating agents Decitabine also induced NOXA1 hypomethylation and increased NOXA1 protein expression. Low dose (0.1-0.5μM) Decitabine pretreatment potentiated low (IC25) dose Bortezomib causing more than 80–90% cell kill in MINO and Z138 cells. The combination of Decitabine and Bortezomib led to increased NOXA1 hypomethylation and protein expression as compared to the individual drugs. Depletion of NOXA1 by specific shRNA rescued 40–50% of the cells from the cytotoxicity of this combination. Tumor progression and chemoresistance are commonly associated with the overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1). ABT-263 is a small molecular inhibitor of the Bcl-2 family of proteins, excepting Mcl-1, which can be neutralized by an increase in NOXA1. Decitabine treatment also potently enhanced the cytotoxicity of ABT-263 in MCL cell lines. Our data suggest that inducing hypomethylation of NOXA1 promoter can be a novel therapeutic strategy for overcoming Bortezomib resistance in MCL, and that genomic methylation patterns and specifically NOXA1 hypermethylation may be useful biomarkers for Bortezomib response. More broadly, these data demonstrate that high-resolution genomic methylation profiling can identify genes and signatures with important prognostic and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 585 SOX11 belongs to the high-mobility group of transcription factors that bind to DNA and induce large conformational changes that facilitate the binding of other transcription factors. Genome-wide methylation analysis of Mantle cell lymphoma (MCL) patients using Nimblegen arrays identified SOX11 as hypomethylated and overexpressed in comparison to purified naïve B cells, which are the cells from which MCLs originate. Direct sequencing of SOX11 in MCL cell lines and patient samples failed to identify any activating mutations in SOX11, strengthening the role of aberrant promoter hypomethylation in MCL as the cause of its overexpression. SOX11 expression has been previously compared between Non-Hodgkin's lymphomas and found to be specific for MCL. The function and direct binding targets of SOX11 are unknown. To understand the functional role of SOX11 in MCL, we used high-resolution whole-genome chromatin immunoprecipitation sequencing (ChIP-Seq) to identify the target genes directly bound by SOX11. As a model, genomic DNA from the Granta-519 MCL cell line was immunoprecipitated by SOX11 antibody and IgG and genomic enrichment sites were analyzed by Illumina sequencing. Using stringent statistical criteria (p-value

Description: Abstract 1608 Poster Board I-634 Cytosine arabinoside (Ara-C) is the mainstay of treatment for acute non lymphoblastic leukaemia (ANLL). Currently there is no rapid, inexpensive test to assess patient sensitivity to Ara-C prior to treatment. We have previously reported a bioluminescent 8-hour assay which assesses Ara-CTP levels in KG-1a and THP-1 cell lines (Smith et al., Blood 2005; 106(11): p695a). Here we present results of this assay using six further ANLL cell lines, 45 patients with ANLL, 2 patients with Ph+ acute lymphoblastic leukaemia (ALL) and 12 with B-CLL. Over 30% of patients with ANLL fail to respond to Ara-C. Potential causes of resistance include lack of hENT-1 transporter activity, over expression of cytidine deaminase (cdd) and over expression of Y-family DNA polymerases. The 8-hour assay was initially tested on eight ANLL cell lines with known response to Ara-C (CCRF-CEM, K562, MV-4-11, HEL, KG-1a, HL-60, THP-1, Mo7e) and this correlated with a commercially available 3-day cytotoxicity assay (CellTiterGlo‘, Promega). ANLL patient samples were sourced at presentation from patients with blast burdens 〉80%. The distribution of FAB sub-types was M4 (26%), M2 (20%), secondary AML (20%), M0 (14%), M1 (6%), M3 (6%), JAK2+ (3%) and Ph+ ALL (5%). Patient ages ranged from 24 to 94 (median 67.5 years) and included 25 peripheral blood and 22 bone marrow samples. The Ara-C concentration used in vitro was 25 μM which is equivalent to 2g/m2 in the clinical setting. The luminescence cut-off for sensitivity was an increase in light production of more than 10%. The 8-hour luminescence assay showed 100% correlation with the 3-day CellTiterGlo” test results, both indicating that 51% of patients were Ara-C sensitive and the remainder resistant. In the 32 patients for whom clinical outcome data was available, assay results correlated with clinical outcome in 30 patients (p

Description: Abstract 3638 Although the vast majority of patients with Hodgkin Lymphoma (HL) do well with standard frontline therapy, about 20% will continue to relapse and have poor clinical outcome, even with high dose therapy and autologous stem cell transplant. Recent progress in the understanding of HL has led to an increasing awareness of the importance of the microenvironment and the cross-talk with cytokines and chemokines (JCO, Steidl et.al. 2011). However, given a low number of HL tumor cells present in lymph node biopsies, it is difficult to develop predictive biomarkers criteria for clinical outcome solely based on the characteristics of Reed Sternberg cells. To identify potential HL biomarkers, a bioinformatics approach was used to derive 151 genes that are potentially related to lymphomagenesis and outcome. These genes were screened against a library of non-tissue (mostly effusions) derived 9 HL cell lines that originated from refractory/relapsing HL patients. Two genes, Fibroblast Growth Factor-2 (FGF2/bFGF) and Syndecan-1 (SDC1/CD138), were found consistently overexpressed across all HL cell lines when compared to normal purified B-cells from PBL. We then tested a panel of lymphoma tissues including Hodgkin and non-Hodgkin subtypes by immunohistochemistry. FGF2 and SDC1 again were found overexpressed only in HL against 148 other lymphoma tissue samples (p