ALBERT

Description: The NOTCH1 gene locusencodes a cell surface receptor which is recurrently mutated in chronic lymphocytic leukemia (CLL), with a rising incidence in higher risk cases. Upon ligand-binding two cleavage events occur. As a consequence the receptor's intracellular domain (NICD) is released and shifts to the nucleus, where it functions as a transcription factor. Since in CLL NOTCH1 mutations mainly affect the protein's PEST-domain, which is involved in NICD degradation, they were assumed to be activating mutations. However, to date this has only been proven for mutations that lead to the loss of the complete PEST-domain or major parts of it, whereas the hotspot mutation in CLL (ΔCT7544-7545; E2515fs) leads to the loss of only 39 amino acids. To prove the activating nature of the CLL hotspot mutation, we developed a cell culture based model using lentiviral transduction of B-cell lymphoma cell lines. A full-length NOTCH1 construct was cloned into a pCCL transfer vector and the CLL hotspot mutation was introduced. The cell lines SU-DHL4, Raji and Daudi were transduced with wild-type NOTCH1 (NOTCH1WT), ΔCT-mutant NOTCH1 (NOTCH1ΔCT) and empty vector. For validation experiments, primary CLL samples were screened for PEST-domain NOTCH1 mutations by targeted next-generation sequencing of exon 34. Activation of NOTCH1 was induced via treatment of cells with the Ca2+ chelator EGTA (1.0 mM, 1 hour). Liberated NICD was semi-quantitatively assessed by Western Blot and image densitometry from whole cell lysates or nuclear protein fractions. In addition, the NICD was visualized by intracellular immunofluorescent staining along with nuclear staining for DAPI and histone H3K27me / histone H3K27ac. Expression of the NOTCH1 target gene HES1 was quantified using TaqMan® RT-PCR analysis. Treatment with EGTA robustly activated NOTCH1 signalling; with the highest levels of NICD seen in the nucleus 2 to 4 hours after activation. Immunofluorescent staining of the NICD in NOTCH1 transduced cells 3 hours after activation revealed a speckled pattern in the nucleus without differences between NICDWT and NICDΔCT. Speckles co-localized with areas of relaxed chromatin, with NICD staining coincident with histone H3K27ac staining and separate from histone H3K27me staining. Co-localization of NICD speckles with areas of relaxed chromatin was confirmed in primary CLL samples where it was again observed independent of NOTCH1 mutation status. In Western Blot analyses, NICDWT and NICDΔCT were distinguishable due to their different molecular weight. Without prior activation, NOTCH1WT transduced cell lines displayed low levels of NICD. In contrast, NOTCH1ΔCT transduced cell lines presented with considerably higher levels of NICDΔCT despite comparable cell surface levels of transduced NOTCH1. After activation of NOTCH1WT and NOTCH1ΔCT with EGTA, the levels of NICDWT and NICDΔCT rose to a similar maximum, but a subsequent time-course showed a slower degradation of the NICDΔCT compared to the NICDWT. The slower degradation of mutant NICD was confirmed in primary CLL samples including samples with truncating mutations of the PEST-domain other than the hotspot mutation E2515fs (Q2440*, S2486*, Q2307*). HES1 is an established NOTCH1 target gene activated by nuclear NICD. Baseline expression levels of HES1 did not differ between NOTCH1WT and NOTCH1ΔCT transduced SU-DHL4 cells. After NOTCH1 activation, HES1 expression was up-regulated ~2.9 fold compared to baseline. Peak expression levels were seen around 3 hours after activation. HES1 expression levels fell more slowly in NOTCH1ΔCT transduced SU-DHL4 cells suggesting longer lasting effects on target genes by NICDΔCT than by NICDWT. In summary, our data demonstrates for the first time that the ΔCT7544-7545 mutant of NOTCH1 maintains transcriptional activity for longer due to slower degradation of its NICD. Our understanding about NICD target genes remains incomplete, but their identification will be crucial to understand the role of NOTCH1 mutations in CLL pathogenesis. Disclosures Tausch: Gilead: Other: Travel support, Speakers Bureau; Celgene: Other: Travel support; Amgen: Other: Travel support. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding. Cragg:Roche: Consultancy, Research Funding; GSK: Research Funding; Bioinvent International: Consultancy, Research Funding; Baxalta: Consultancy; Gilead Sciences: Research Funding.

Description: Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies. The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function. In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced. We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines. Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

Description: Background: Genomically instable (GI) chronic lymphocytic leukemia (CLL) is characterized by frequent alterations in DNA-damage response (DDR) genes (e.g. TP53,ATM) and related pathways. Conversely, pathogenic networks in CLL cases which maintain genomic integrity and operate with a functional DDR remain incompletely described. Methods: Molecular profiling was conducted on CD19 sorted samples derived from patients registered on the CLL8 study (1st-line, FC vs. FCR) for gene expression (GEP)(n=337, Exon 1.0 ST arrays, Affymetrix), copy number aberrations (CNAs) (n=309, SNP Arrays 6.0, Affymetrix) and mutation analyses/signature projections (n=171, whole exome sequencing, Illumina). FISH, IGHV and TP53 mutation analysis was conducted at trial enrolment. Results: Unsupervised consensus clustering (k=2-6) on variably expressed genes (SD〉0.5) was used for class discovery. Two small, but highly differentiated clusters were identified, characterized through NRIP1 and EBF1/tri12. GSEA also segregated the remaining samples into four major clusters showing signatures of inflammation (I) and without inflammation (NI). These clusters were further segregated into GI-CLL clusters with increased "DNA-repair" or clusters with "epithelial-mesenchymal transition"-like signatures (EMT-L). Variability for del(17p)/TP53 mutation was found across clusters (p

Description: Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.