ALBERT

Description: Arrhenius plots are often used to determine energy barriers and prefactors of thermally activated processes. The precision of thus determined values depends crucially on the precision of the temperature measurement at the sample surface. We line out a procedure to determine the absolute temperature of a metal sample in a cryogenic scanning tunneling microscope between 5 K and 50 K with sub-Kelvin precision. We demonstrate the dependence of prefactor and diffusion energy on this calibration for diffusion of CO on Cu(111) and on Ag(100) measured in the temperature range from 30 K to 38 K and 19 K to 23 K, respectively.

Description: In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids.

Description: Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that 15 human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene, NOTCH2NL, demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution.

Description: FL is the most common indolent nodal lymphoma and considered incurable for the majority of patients. Virtually all patients eventually develop resistant disease, and up to 45% histologic transformation (tFL) with poor outcome. Alterations associated with progression to tFL include increased copy number variations (e.g. CDKN2A deletions, MYC amplification), acquisition of additional translocations (e.g. MYC or BCL6), and higher mutation load (including TP53). However the underlying molecular mechanisms promoting this genomic instability remain elusive. Interestingly, histologic transformation occurs at a remarkably constant rate of ~3% per year implying a rather stochastic process. We previously described convergent evolution for disruptive ARID1A mutations in FLs of a donor and the recipient patient following an allogeneic bone marrow transplantation (Weigert, Canc Discovery 2012). The independent acquisition of ARID1Aloss in two separate hosts after transplantation of an identical FL ancestor clone suggested (i) a later acquired event in the molecular ontogeny and (ii) functional relevance. ARID1A promotes the formation of SWI/SNF nucleosome remodeling complexes containing BRG1 or BRM, which catalyze disruption of DNA-histone contacts, thereby controlling chromatin condensation and DNA accessibility. Nucleosome-remodelling has been linked to DNA damage response, including nucleotide and base excision repair, homologous recombination and non-homologous end-joining (NHEJ). We thus hypothesized that ARID1Ahaplodeficiency might contribute to genetic and genomic instability in FL. In a cohort of 305 FL specimens, we identified 44 ARID1A mutations in 43 patients (14%). The majority of these mutations were disruptive (66%; 19 nonsense and 10 frame shift mutations), 6 cases harbored splice site mutations. MutSigCV analysis (Lawrence, Nature 2013) indicated that ARID1A was significantly mutated in FL, i.e. more often than expected by chance given background mutation processes. Consistent with our hypothesis, the number of non-synonymous mutations in 104 targeted genes was higher in ARID1A mutated FLs (5.6+0.34, mean+sem) compared to ARID1A wild-type (wt) FLs (4.7+0.12; p=0.0099), and similar compared to 17 FLs with TP53 mutations (5.6+0.12). Of note, ARID1A and TP53 mutations were mutually exclusive. For functional studies, we confirmed deleterious ARID1A alterations in 6 lymphoma cell lines (Namalwa, Karpas422, SUPHD-1, SU-DHL5, WSU-FSCCL, U-OH1). Immunoblotting showed decreased ARID1A protein expression in all 6 lines compared to 5 ARID1A wt lymphomas. Tet-induced re-expression of ARID1A in two tested ARID1A mutant cell lines (Namalwa and SUP-HD1) reduced cell proliferation compared to tet-induced control (LacZ), but had no effect in ARID1A wt OCI-Ly1. As a proof-of-principle experiment we assayed the impact of ARID1A loss on DNA double strand break (DSB) repair efficiency in genetically well defined mouse embryo fibroblasts (MEFs), avoiding uncontrolled bias introduced by the genetic and genomic complexity in lymphoma cell lines. Early passage MEFs from either wt or conditional ARID1A knock-out mice (ARID1Af/f; Gao, PNAS 2008) were transiently transfected or retrovirally transduced with self-excising Cre-recombinase (Silver&Livingston, Mol Cell 2001) or control, and irradiated with 2 Gy after 36 hrs. Cre-induced ARID1A loss was confirmed by Western blot. To assay NHEJ, the predominate DSB-repair pathway used by cells in G1 phase, we stained cells for γH2A.X and 53BP1. In 3 independent and blinded experiments, evaluation of 〉100 cells per condition demonstrated significantly more double-positive cells (〉6 foci/cell) in ARID1A-/- MEFs (43+14%) compared to both ARID1Af/fMEFs (9+1%) and Cre-exposed wt MEFs (15+5%) at 16 hrs post irradiation, whereas there was no significant difference for ARID1Af/for wt MEFs with or without Cre at baseline (all 50%). This data indicates that ARID1A loss slows the repair kinetics of NHEJ and delays the clearance of DSB. We conclude that ARID1A is recurrently and significantly mutated in FL. Most mutations are disruptive leading to functionally relevant ARID1A protein haplodeficiency. ARID1A loss impairs NHEJ, which might sensitize tumors to DNA damaging agents and irradiation, but also contribute to genetic and genomic instability promoting histologic transformation. Disclosures No relevant conflicts of interest to declare.

Description: The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood. IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23. We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4. RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype. In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. Disclosures Richter: HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Description: Follicular lymphoma (FL) is among the most common lymphomas worldwide and considered incurable for the majority of patients who present with advanced disease. FL is a clinically and molecularly heterogeneous disease. We have recently established a clinicogenetic risk model (m7-FLIPI) that integrates the mutation status of 7 genes, including ARID1A, into a predictive algorithm for improved risk stratification for failure-free survival in patients receiving first-line immunochemotherapy (Pastore et al., Lancet Oncology 2015). When adjusted for FLIPI and ECOG performance status, the presence of ARID1A mutations correlated with longer failure-free survival (HR 2.85, 95% confidence interval (CI) 1.12-7.27; p=0.049) in patients receiving first-line R-CHOP. The underlying molecular mechanism for better treatment outcome of patients with ARID1A mutated FL is unclear. ARID1A is a member of SWI/SNF nucleosome remodeling complex. Evolving evidence indicates that the SWI/SNF complex functions as a tumor suppressor and is implicated in DNA damage repair. ARID1Ais the most commonly mutated SWI/SNF complex member in FL: in our series, we identified 44 ARID1A mutations in 304 FL cases (14%). These mutations were primarily heterozygous and disruptive (66%), another 6 cases harbored splice site mutations. Primary patient samples and lymphoma cell lines that harbored ARID1A mutations (e.g., SU-DHL5, WSU-FSCCL, Namalwa) were had lower ARID1A protein expression as compared to ARID1A wild type (wt) t(14;18)-positive lymphoma cell lines (OCI-Ly1, -Ly8, DB). Non-homologous end joining (NHEJ) has been reported to be the predominant pathway of DNA double strand break (DSB) repair in FL (Koues et al., Immunity 2015) and is indispensible for successful VDJ rearrangement during early B-cell maturation.To analyze the impact of Arid1a disruption on B-cell maturation in vivo, we used a conditional Arid1a knock-out mouse model (Arid1af/f; Gao, PNAS 2008). 8-10 week old Mx1-Cre+Arid1af/f mice and control (Mx1-Cre-Arid1af/f) mice were treated with poly(IC). Bone marrow (BM) were harvested from these animals 14 days later. Conditionally deletion of Arid1a (n=3) resulted in severely impaired B-cell maturation in BM cells affecting Hardy fractions B/C though F. E.g., flow cytometric analysis showed that the frequency of fraction C (B220+ CD43+ HSA+ BP-1+) was greatly reduced in BM from Mx1-Cre+Arid1af/f vs in the control group (0.1 % vs 1.2 %, n=3), consistent with the inability to successfully complete VDJ recombination. As a proof-of-principle experiment we assayed the impact of Arid1a loss on DSB repair efficiency using mouse embryo fibroblasts (MEFs) from either wt or conditional Arid1a knock-out mice. MEFs were irradiated with 2 Gy 36 hrs after retroviral transduction with Cre and stained for γH2A.X and 53BP1 to assay for NHEJ. In three independent and blinded experiments, evaluation of 〉100 cells per condition demonstrated significantly more double-positive cells (〉6 foci/cell) in Arid1a-/- MEFs (43+/-14%) compared to both Arid1af/f MEFs (9+/-1%) and Cre-exposed wt MEFs (15+/-5%) at 16 hrs post irradiation, whereas there was no significant difference for Arid1af/f or wt MEFs with or without Cre at baseline (all 50%). ARID1A haplodeficient lymphoma cell lines (SU-DHL5, WSU-FSCCL, Namalwa) were 10-100 fold more sensitive to the DSB inducing doxorubicin treatment compared to ARID1A wt lymphoma cell lines (OCI-Ly1, -Ly8, DB). Tet-induced shRNA knock-down (via pTRIPZ vectors) of endogenous ARID1A in two t(14;18)-positive lymphoma cell lines with wt ARID1A (OCI-Ly1 and OCI-Ly8) doubled the number of NHEJ foci by 53-BP1 staining in two independent experiments. Furthermore, shRNA knock-down of ARID1A resulted in increased sensitivity to doxorubicine (IC50

Description: The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. Here, we provide biochemical, structural, functional and clinical evidence that aberrant Cathepsin S (CTSS) activity induces a supportive immune microenvironment in FL. By targeted DNA sequencing of 305 diagnostic FL biopsies, we identified somatic mutations of CTSS in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). A subset of CTSS Y132 mutations (N=5) occurred at lower variant allele frequencies (5-10%), indicating subclonality. Another 13% of FL had CTSS amplifications (37/286). CTSS Y132 mutations and CTSS amplifications were mutually exclusive. In a cohort of 51 FL, CTSS amplifications were associated with higher CTSS expression (P=0.05). Of note, a subset of FL without CTSS amplifications also had higher CTSS expression, suggesting additional mechanisms of transcriptional dysregulation. CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells. CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS). We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). Single-cell derived Y132D mutant clones showed 〉3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had 〉3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001). We purified pro-CTSS WT and Y132D and assayed their in vitro autocatalytic cleavage over time. The time required to convert 50% of pro-CTSS decreased from 17 minutes for WT to 11 minutes for Y132D (N=3, P=0.04). In contrast, purified active CTSS WT and Y132 had similar in vitro cleavage activities. Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which may facilitate intramolecular cleavage. By mass spectrometry we could indeed detect novel intermediate-sized CTSS fragments. Thus, Y132D does not increase the activity of the mature enzyme but is a gain-of-function mutation by accelerating the conversion from pro-CTSS to catalytically active CTSS. CD74 (invariant chain) is a physiologic CTSS substrate that plays critical roles in the assembly, trafficking and stabilization of peptide-free MHC-II. CTSS cleaves CD74, thereby allowing binding and presentation of antigens on MHC-II to antigen specific CD4+ T-cells. We could show that CTSS Y132D enhanced CD74 cleavage in Karpas422 cells. We then tested the impact of CTSS on antigen specific CD4+ T-cell activation in co-culture assays. CTSS knock-out lymphoma cells were broadly incapable of activating CD4+ T-cells. Overexpression of CTSS WT activated CD4+ T-cells more efficiently compared to empty vector control. CTSS Y132 had the highest capacity to stimulate antigen specific CD4+ T-cell responses. Furthermore, in primary FL biopsies (N=51) CTSS Y132 mutations had gene expression profiles linked with antigen-processing and chemokine perturbation, including CXCL13, a B-cell chemoattractant produced by activated CD4+ T-follicular helper cells. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012) and overall survival (P=0.041). In summary, we propose that aberrant CTSS activity - even if only present in a FL subclone - can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment via pro-inflammatory and chemotactic cytokines and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Hiddemann:Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Vector Therapeutics: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bayer: Consultancy. Kridel:Gilead Sciences: Research Funding. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.