Ryan Morin Lab

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets^1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

ABC-DLBCLs have unfavorable outcomes and chronic activation of CBM signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutation abrogated a novel motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutation enhanced BCL10 filament formation; forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes.

Formalin fixation of paraffin-embedded tissue samples is a well-established method for preserving tissue and is routinely used in clinical settings. Although formalin-fixed, paraffin-embedded (FFPE) tissues are deemed crucial for research and clinical applications, the fixation process results in molecular damage to nucleic acids, thus confounding their use in genome sequence analysis. Methods to improve genomic data quality from FFPE tissues have emerged, but there remains significant room for improvement. Here, we use whole-genome sequencing (WGS) data from matched Fresh Frozen (FF) and FFPE tissue samples to optimize a sensitive and precise FFPE single nucleotide variant (SNV) calling approach. We present methods to reduce the prevalence of false-positive SNVs by applying combinatorial techniques to five publicly available variant callers. We also introduce FFPolish, a novel variant classification method that efficiently classifies FFPE-specific false-positive variants. Our combinatorial and statistical techniques improve precision and F1 scores compared to the results of publicly available tools when tested individually.

Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.

To gain insight into pre-contact Coast Salish fishing practices, we used new palaeogenetic analytical techniques to assign sex identifications to salmonid bones from four archaeological sites in Burrard Inlet (Tsleil-Waut), British Columbia, Canada, dating between about 2300-1000 BP (ca. 400 BCE-CE 1200). Our results indicate that male chum salmon (Oncorhynchus keta) were preferentially targeted at two of the four sampled archaeological sites. Because a single male salmon can mate with several females, selectively harvesting male salmon can increase a fishery's maximum sustainable harvest. We suggest such selective harvesting of visually distinctive male spawning chum salmon was a common practice, most effectively undertaken at wooden weirs spanning small salmon rivers and streams. We argue that this selective harvesting of males is indicative of an ancient and probably geographically widespread practice for ensuring sustainable salmon populations. The archaeological data presented here confirms earlier ethnographic accounts describing the selective harvest of male salmon.

In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in D. melanogaster and S. cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sub-lethal, stress response pathway that is tailored to an organism's environmental norms. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse, and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species, highlights the physiological significance of this protein conformation and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events.

The expression of BCL6 in B cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase involved in the degradation of BCL6 and is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of Burkitt lymphoma (BL) patients. FBXO11 mutations impaired BCL6 degradation and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of either one or two copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B cell lymphoma onset, providing experimental evidence that FBXO11 is a haplo-insufficient oncosuppressor in B cell lymphoma. In WT and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degrader or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches such as BCL6 degraders and direct MYC inhibition could be exploited as a targeted therapy for BL.

The term diffuse large B-cell lymphoma (DLBCL) includes a heterogeneous collection of biologically distinct tumours. This heterogeneity currently presents a barrier to the successful deployment of novel, biologically targeted therapies. Molecular profiling studies have recently proposed new molecular classification systems. These have the potential to resolve the biological heterogeneity of DLBCL into manageable subgroups of tumours that rely on shared oncogenic programmes. In many cases these biological programmes straddle the boundaries of our existing systems for classifying B-cell lymphomas. Here we review the findings from these major molecular profiling studies with a specific focus on those that propose new genetic subgroups of DLBCL. We highlight the areas of consensus and discordance between these studies and discuss the implications for current clinical practice and for clinical trials. Finally, we address the outstanding challenges and solutions to the introduction of genomic subtyping and precision medicine in DLBCL.

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP.

Tazemetostat represents the first epigenetic therapy approved for the treatment of follicular lymphoma (FL). It inhibits the activity of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase, the first of a multitude of epigenetic regulators that have been identified as recurrently mutated in FL and germinal center diffuse large B-cell lymphoma. In this review, we discuss the initial discovery and ongoing exploration of the functional role of EZH2 mutations in lymphomagenesis. We also explore the path from the preclinical development of tazemetostat to its approval for the treatment of relapsed FL, and potential future therapeutic applications. We discuss the clinical data that led to the approval of tazemetostat and ongoing research into the function of EZH2 and of tazemetostat in lymphomas that derive from the germinal center, which could increase the applicability of this drug in the future.

This study uses ancient DNA analysis to identify the species of salmonids from a number of pre-contact Coast Salish settlements in Burrard Inlet, Canada dating from about 390 BCE to CE 1600. Our results indicate that chum salmon (Oncorhynchus keta) dominates all Burrard Inlet zooarchaeological assemblages through time, followed distantly by pink salmon (Oncorhynchus gorbuscha), sockeye (Oncorhynchus nerka), coho (Oncorhynchus kisutch), and Chinook (Oncorhynchus tshawytscha), indicative of very stable local fisheries. These results indicate that the four well-sampled sites appear to have been occupied during the fall and winter and perhaps during the spring.

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