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Supplementary Figures S1-S6 and Tables S1-S4

Supplementary Materials and Methods

Prognostic values of MHC expression

IHC antibodies and cell line information

Supplementary Methods, Figures and Legends

Genetic backgrounds of MHC deficient cases

Genetic analysis in the MHC-I/II double-positive cases

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II–deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of EZH2 mutations in both MHC-I– and MHC-II–negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant Ezh2Y641. Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in EZH2-mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming.Significance:We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin–specific context. Specifically, EZH2 mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination.See related commentary by Velcheti et al., p. 472.This article is highlighted in the In This Issue feature, p. 453

EZH2 mutation data

Gene expression analyses of clinical samples

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma–specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma–associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II–deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma.Significance:We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell–like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.See related commentary by Fisher and Oh, p. 342.This article is highlighted in the In This Issue feature, p. 327

Supplementary Methods, Supplementary Figures 1-19, Supplementary References

MHC expression status in the cohort

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and ‘eat-me’ signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation—a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Integrative analysis in patients with diffuse large B-cell lymphoma uncovers that biallelic mutations on TMEM30A are associated with a favorable outcome and enhanced sensitivity to CD47 blockade.

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma–specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma–associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II–deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. Significance: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell–like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints. See related commentary by Fisher and Oh, p. 342. This article is highlighted in the In This Issue feature, p. 327

Background: With the goal of translating biological discovery into clinical actionability, deciphering crosstalk in the cellular ecosystem of the tumor microenvironment (TME) has emerged as a research focus. Although comparatively little is known about the immune biology of diffuse large B-cell lymphoma (DLBCL), as reflected in clonal selection of specific somatic gene mutations in response to immune system pressure and the specific composition of the TME, PRAME has emerged as a prominent member of the cancer germline antigen/ tumor associated antigen (TAA) family of proteins. It is expressed in various types of cancers, but generally not in normal tissues, apart from male germinal cells, and triggers autologous T-cell mediated immune responses. PRAME is highlighted as a new cancer therapeutic target of T-cell or antibody-based immunotherapies with promising anti-tumor responses in early phase clinical trials and pre-clinical models for several types of cancers. In the context of developing new immunotherapies, targeting TAAs that are presented by major histocompatibility complexes on tumor cells is a promising therapeutic strategy for patients that experience treatment failure. Material and methods: We performed integrative genomic analysis of whole-transcriptome RNAseq, targeted genomic sequencing, and high-resolution copy number analysis in 347 de novo DLBCL tumors from patients uniformly treated with R-CHOP. Findings were correlated with pathological and clinical parameters, as well as TME composition. Using DLBCL-derived cell lines (7 EZH2 mutated and 5 wt) and CRISPR-Cas9 genome editing, we performed in vitro functional studies to characterize cell-intrinsic effects of PRAME knockout. Moreover, we studied EZH2 inhibition in a murine model of Ezh2 mutant lymphoma with a focus on mechanistic links between EZH2 activity and PRAME expression, as well as TME composition (cell-extrinsic effects). Results: We discovered recurrent, and highly focal deletions of 22q11.22 including the PRAME gene, which were associated with poor treatment outcome, independent of pathological and clinical risk factors. To explore PRAME-deletion-associated phenotypes and interaction with the tumor microenvironment (TME), we analyzed corresponding RNAseq, immunohistochemistry (IHC), and flow-cytometry data from our DLBCL cohort and utilized enzyme-linked immunospot (ELISPOT) assays using patient-derived peripheral blood mononuclear cells. PRAME deletions contributed to an immunologically "cold" TME, representing a somatically acquired mechanism to evade anti-tumor T-cell response (cell-extrinsic effect). Using PRAME knock out by CRISPR-Cas9 in vitro, TRAIL-mediated apoptotic signaling was impaired. In addition, PRAME down-modulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. Using proximity ligation assays and co-IP, we demonstrated that PRAME directly interacted with EZH2 as a negative regulator, establishing a link between PRAME deletions and EZH2 mutations with anti-apoptotic signaling in DLBCL (cell-intrinsic effect). An in vivo murine model of Ezh2 mutant lymphoma showed decreased T-cell infiltration in the TME and Ezh2 inhibition induced PRAME restoration as compared to vehicle controls. IHC for CD3, CD4, FOXP3, and GZMB revealed significant increases in various T-cell populations of the TME, including Tregs and cytotoxic T cells. EZH2 inhibition with EPZ-6438 abrogated these dualistic effects leading to increased immune cell infiltration in the tumor microenvironment and acceleration of apoptosis via PRAME restoration. Moreover, restoration of PRAME antigen presentation by EZH2 inhibition resulted in enhancement of PRAME binding using a T-cell receptor mimic PRAME antibody (Pr20), suggesting immunotherapeutic potential. Conclusion: Our findings highlight multiple functions of PRAME during lymphomagenesis. PRAME restoration by EZH2 inhibition provides a preclinical rationale for synergistic therapies combining epigenetic re-programming with PRAME-targeted therapies. Disclosures Dao: Eureka Therapeutics: Consultancy. Melnick:Jubilant: Consultancy; Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding. Scheinberg:Lantheus: Current equity holder in private company; Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Enscyse: Current equity holder in private company; Arvenas: Current equity holder in private company; Pfizer: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Oncopep: Consultancy. Scott:Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding. Steidl:Curis Inc: Consultancy; Roche: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy.

Purpose High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression. Patients and Methods We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data. Results We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non–light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive–transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested. Conclusion We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.

Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas. DLBCL presents as an aggressive disease requiring immediate treatment. Although significant improvement in outcome has been achieved, ~40% of patients still experience treatment failure. Here, we characterized the recurrent genetic alterations and transcriptomic signatures in diagnostic biopsies from a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP. This analysis revealed bi-allelic loss of function mutations of TMEM30A that were associated with favorable treatment outcome. TMEM30A is a chaperone protein, involved in maintaining the asymmetric distribution of phosphatidylethanolamine and phosphatidylserine, an integral component of the plasma membrane and “eat-me” signal recognized by macrophages. Using TMEM30A knockout systems by CRISPR genome editing techniques, we have functionally characterized this loss-of-function mutation in representative human and mouse DLBCL cell line models. We have discovered that TMEM30A loss is associated with increased B-cell signaling following antigen stimulation, including a two-fold increase in the diffusion rate of B-cell receptor (BCR) clustering, using high resolution Single Particle Tracking (SPT) technology. In addition, we have measured three-fold increase in chemotherapeutic drug accumulation in both knockout cell lines and randomly selected patient biopsies with TMEM30A biallelic loss. This observation was validated in a xenograft mouse model, which presented improved survival and limited tumor growth following vincristine treatment in mice injected with TMEM30A null DLBCL cell lines compared with native cell lines. This phenotype explains the improved prognosis observed in DLBCL patients following R-CHOP treatment. Furthermore, we have observed over two fold higher numbers of tumor-associated macrophages in B-cell lymphoma syngeneic mouse models with Tmem30a loss-of-function, prior to any form of treatment, suggesting the existence of “hot” and primed tumors. Our data highlight a multi-faceted role for TMEM30A and plasma membrane physiology in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Characterization of these mechanisms will address a missing link in the cancer field as related insights in lymphoma will outline therapeutic approaches that can be extended to cancer therapy in general. Citation Format: Shannon Healy, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W. Woolcock, Elena Vigano, Libin Abraham, Michael Gold, Adele Telenius, Pedro Farinha, Graham Slack, Susana Ben-Neriah, Daniel Lai, Allen W. Zhang, Sohrab Salehi, Hennady P. Shulha, Derek S. Chiu, Sara Mostafavi, Alina S. Gerrie, Diego Villa, Laurie H. Sehn, Kerry J. J. Savage, Andrew J. J. Mungall, Andrew P. Weng, Marcel Bally, Ryan D. Morin, Gabriela V. Cohen Freue, Joseph M. Connors, Marco A. Marra, Sohrab P. Shah, Randy D. Gascoyne1, David W. Scott, Christian Steidl, Ulrich Steidl. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3480.

The clinical significance of

Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Recognizing biological heterogeneity in diffuse large B-cell lymphoma (DLBCL), significant effort has been made to define distinct molecular subgroups of prognostic importance which harbor potentially targetable biology. Reflecting this, in the recent revision of the WHO classification, DLBCL was divided into cell-of-origin molecular subtypes and a new entity was defined - high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH). ~8% of tumors with DLBCL morphology are HGBL-DH/TH and all HGBL-DH/TH with BCL2 translocations (HGBL-DH/TH-BCL2) are of the GCB molecular subtype. To explore specific biology operating in HGBL-DH/TH-BCL2, we analyzed RNAseq data from 157 de novo GCB DLBCL tumors (25 being HGBL-DH/TH-BCL2) with the aim of defining gene expression signatures that distinguish such cases from other GCB-DLBCLs. We identified 104 genes that were most significantly differentially expressed between HGBL-DH/TH-BCL2 and other GCB-DLBCLs, defined as having a 95% confidence interval of the Importance Score that did not cross 0. A model constructed from the expression of these genes clustered 42 tumors into one group ("double-hit signature" positive - DHITsig pos), and 115 tumors into the DHITsig neg group. 22 tumors were HGBL-DH/TH-BCL2 within the DHITsig pos group compared with only 3 tumors in the DHITsig neg group. We next assessed the clinical impact of the DHITsig within a uniformly R-CHOP treated cohort of de novo GCB-DLBCL drawn from a population-based registry, which included the discovery cases. The DHITsig pos group had significantly inferior outcomes for time to progression (TTP) and overall survival (OS) (P < 0.001 and P = 0.01, respectively) similar to ABC-DLBCL (Figs A, B). Notably, the non-HGBL-DH/TH-BCL2 cases sharing the DHITsig showed the same poor prognosis as the HGBL-DH/TH-BCL2 cases. A multivariate Cox model of TTP revealed that DHITsig remained prognostic, independent of IPI and MYC/BCL2 dual protein expression (HR = 3.1 [1.5 - 6.4], P = 0.002). We then applied this gene expression model to GCB-DLBCL in an independent dataset (n = 262 GCB-DLBCLs; Reddy et al,Cell 2017). Validating the prognostic significance, the DHITsig pos group had significantly inferior OS compared with other GCB-DLBCLs (P < 0.001) similar to ABC-DLBCL (Fig C). We then sought to determine whether differentially expressed genes, according to DHITsig, could inform on the biology of the DHITsig pos group. Gene set enrichment analysis (GSEA) strongly suggested a germinal centre dark-zone (DZ) cell-of-origin for the DHITsig pos tumors with significant enrichment of DZ and light-zone (LZ) gene signatures (Victora et al, Blood 2012) in DHITsig pos and neg tumors, respectively (FDR = 0.002 and < 0.001). Furthermore, the DHITsig pos group had up-regulation of pathways related to mitochondrial metabolism and RNA synthesis (both FDR < 0.001). We separately identified mutations associated with DHITsig pos cases within GCB-DLBCL. In addition to the expected enrichment of MYC and BCL2 mutations, chromatin modifiers EZH2 and CREBBP, as well as RFX7 and DDX3X (mutated in Burkitt lymphoma), were more frequently mutated in DHITsig pos tumors. In contrast, mutations of TNFAIP3, MYD88 and IRF4, more typical of ABC-DLBCLs, were more prevalent in DHITsig neg tumors. To enable application to FFPE biopsies, the DLBCL90 NanoString assay was developed by translating the DHIT gene expression signature into a 30-gene module that was then added to the Lymph2Cx assay. The DLBCL90 assay was applied to 171 DLBCL tumors (including 156 from the discovery cohort), yielding 26% DHITsig pos, 64% DHITsig neg, and 10% unclassified, with a frank misclassification rate of 3% against the RNAseq comparator. The prognostic significance of the groups was maintained (Fig D). Importantly, the DHITsig neg group had a disease specific survival of 91% at 5 years. To validate the association between the DHITsig and HGBL-DH/TH-BCL2 tumors, the DLBCL90 assay was applied to 113 transformed follicular lymphoma tumors. Within the DHITsig pos group, 19/34 tumors were HGBL-DH/TH-BCL2 compared with 0/58 in the DHITsig neg group. In conclusion, we have identified a clinically and biologically distinct subgroup of GCB-DLBCL tumors that are defined by the HGBL-DH/TH-BCL2 gene signature. The translation to an assay applicable to FFPE allows exploration of its utility to guide patient management within the context of clinical trials. Figure. Figure. Disclosures Sehn: Merck: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Steidl:Nanostring: Patents & Royalties: patent holding; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy. Connors:Cephalon: Research Funding; Amgen: Research Funding; F Hoffmann-La Roche: Research Funding; Roche Canada: Research Funding; Bristol Myers-Squibb: Research Funding; Janssen: Research Funding; Bayer Healthcare: Research Funding; Takeda: Research Funding; NanoString Technologies: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Seattle Genetics: Honoraria, Research Funding; Merck: Research Funding; Genentech: Research Funding; Lilly: Research Funding. Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Roche: Research Funding; Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding.

Collapsible soils such as loess are particular types of problematic soils, whose collapse potential have caused severe damages to the buildings and structures. Loess is susceptible to large and sudden volume reduction upon wetting in constant normal stress or upon increasing normal stress in constant water content or a combination of these two stress paths. In this research both of these collapse induced paths: wetting and stress paths are investigated on undisturbed specimens of a Loess to observe the effect of various parameters on the collapse behavior of the tested soil., Seven controlled matric suction unsaturated oedometer tests have been conducted on undisturbed specimens taken from loessial soil of Gorgan, a city in Golestan province in Iran to study the collapse mechanisms of this type of soils. Two identical sets of unsaturated oedometer apparatuses designed and developed at Sharif University of Technology, with the capability of controlling matric suction have been utilized. In these tests, the matric suction, vertical net stress, volumetric water content, and vertical deformation of the samples have been controlled and recorded. Tests have been performed under two separate stress paths: constant matric suction with varying vertical net stress (loading and unloading) and constant vertical net stress with varying matric suction (wetting and drying). Obtained results show the high collapse potential of the tested soil and significant effects of stress path, matric suction, and vertical net stress on the collapse behavior of the tested soil.

Background A variety of DNA binding proteins are involved in regulating and shaping the packing of chromatin. They aid the formation of loops in the DNA that function to isolate different structural domains. A recent experimental technique, Hi-C, provides a method for determining the frequency of such looping between all distant parts of the genome. Given that the binding locations of many chromatin associated proteins have also been measured, it has been possible to make estimates for their influence on the long-range interactions as measured by Hi-C. However, a challenge in this analysis is the predominance of non-specific contacts that mask out the specific interactions of interest. Results We show that transforming the Hi-C contact frequencies into free energies gives a natural method for separating out the distance dependent non-specific interactions. In particular we apply Principal Component Analysis (PCA) to the transformed free energy matrix to identify the dominant modes of interaction. PCA identifies systematic effects as well as high frequency spatial noise in the Hi-C data which can be filtered out. Thus it can be used as a data driven approach for normalizing Hi-C data. We assess this PCA based normalization approach, along with several other normalization schemes, by fitting the transformed Hi-C data using a pairwise interaction model that takes as input the known locations of bound chromatin factors. The result of fitting is a set of predictions for the coupling energies between the various chromatin factors and their effect on the energetics of looping. We show that the quality of the fit can be used as a means to determine how much PCA filtering should be applied to the Hi-C data. Conclusions We find that the different normalizations of the Hi-C data vary in the quality of fit to the pairwise interaction model. PCA filtering can improve the fit, and the predicted coupling energies lead to biologically meaningful insights for how various chromatin bound factors influence the stability of DNA loops in chromatin. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0584-2) contains supplementary material, which is available to authorized users.

Myocyte enhancer factor 2B (MEF2B) is a transcription factor with mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL). To provide insight into the regulatory network of MEF2B, in this study, we analyse global gene expression and DNA-binding patterns. We find that candidate MEF2B direct target genes include RHOB, RHOD, CDH13, ITGA5 and CAV1, and that indirect target genes of MEF2B include MYC, TGFB1, CARD11, MEF2C, NDRG1 and FN1. MEF2B overexpression increases HEK293A cell migration and epithelial–mesenchymal transition, and decreases DLBCL cell chemotaxis. K4E, Y69H and D83V MEF2B mutations decrease the capacity of MEF2B to activate transcription and decrease its' effects on cell migration. The K4E and D83V mutations decrease MEF2B DNA binding. In conclusion, our map of the MEF2B regulome connects MEF2B to drivers of oncogenesis., Mutations in the transcription factor MEF2B are found in diffuse large B-cell lymphoma. In this study, the authors map the DNA-binding sites of the transcription factor in cells in vitro and find that the mutations decrease the ability of MEF2B to activate transcription.

Many proteins are observed to localize to specific subcellular regions within bacteria. Recent experiments have shown that proteins that have self-interactions that lead them to aggregate tend to localize to the poles. Theoretical modeling of the localization of aggregating protein within bacterial cell geometries shows that aggregates can spontaneously localize to the pole due to nucleoid occlusion. The resulting polar localization, whether it be to a single pole or to both was shown to depend on the rate of protein addition. Motivated by these predictions we selected a set of genes from Escherichia coli, whose protein products have been reported to localize when tagged with green fluorescent protein (GFP), and explored the dynamics of their localization. We induced protein expression from each gene at different rates and found that in all cases unipolar patterning is favored at low rates of expression whereas bipolar is favored at higher rates of expression. Our findings are consistent with the predictions of the model, suggesting that localization may be due to aggregation plus nucleoid occlusion. When we expressed GFP by itself under the same conditions, no localization was observed. These experiments highlight the potential importance of protein aggregation, nucleoid occlusion and rate of protein expression in driving polar localization of functional proteins in bacteria.

Many proteins are observed to localize to the poles within bacterial cells. Some bacteria show unipolar localization, yet under different conditions bipolar patterns can emerge. One mechanism for spontaneous polar localization has been shown to involve the combination of protein aggregation and nucleoid occlusion. Whether the different observed patterns represent global energy minima for the cellular system remains to be determined. In this paper we show that for a model consisting only of protein aggregation along with an excluded volume effect due to the DNA polymer, that unipolar patterns are the global energy ground state regardless of protein concentration and DNA density. We extend the model to allow for proteins to be added to the cellular volume at a constant rate and show that bipolar (or multi-foci) patterns emerge as the result of the system being kinetically trapped in a local energy minimum. Lastly we also consider the situation of a growing cell that starts with a pre-existing aggregate at one of the poles and determine conditions under which either unipolar or bipolar patterns can exist at the point when it is ready to divide. This work sheds new interpretations on recently published experimental data and suggests experiments to test whether such a mechanism can drive patterning in bacteria.

Diffuse large B cell lymphoma (DLBCL) comprises two distinct molecular subtypes: germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. The pathogenesis of ABC-DLBCL is characterized by two processes - the activation of NF-KB and a block in terminal B-cell differentiation. However, in GCB-DLBCL only a few biologically relevant pathways have been identified, which has hampered the development of targeted therapies with specific efficacy in this subtype. Recurrent genetic alterations involved in PI3K-AKT signaling pathway have been identified in the patients with solid cancers, and dramatic responses have been observed to PI3K inhibitors in clinical trials. The prevalence and clinical significances of genetic alterations in PI3K-AKT pathway have not been well studied in DLBCL. Previous studies have reported that loss of PTEN expression was observed in almost half of GCB-DLBCL cases but were largely absent in the ABC-DLBCL. In addition, functional studies have recently shown that the inactivation of Gα13 signaling pathway genes may also activate AKT in germinal center driven lymphoma, raising the possibility that additional genes within these pathways are affected in GCB-DLBCL. Herein, we identified genetic alterations involved in the PI3K-AKT pathway and evaluated their clinical impact. We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 61 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of phospho-AKT (pAKT) was performed on tissue microarrays (n=332). Cell-of-origin (COO) was assigned by gene expression (Lymph2Cx assay) in 323 cases - 183 GCB, 104 ABC and 36 unclassifiable. GISTIC analysis revealed several COO-specific peaks with copy number changes. Among them, focal 10q23.3 deletion including PTEN was detected in GCB-DLBCL (q-value=1.7e-7), but not in ABC-DLBCL. We also identified two focal amplification peaks in GCB-DLBCL containing microRNAs MIR17HG (13q31.1;q=1.01e-24) and MIR21 (17q23.3; q=1.45e-6), which are known to down-regulate PTEN and result in activation of PI3K-AKT signaling. Furthermore, we detected recurrent INPP4B deletion (4q21.23) in GCB-DLBCL (q= 0.004) only. Of note, the lipid phosphatase INPP4B has been shown to play a role as a tumor suppressor that controls the levels of PI3K lipid products leading to AKT activation and metastasis of some solid cancers. This is consistent with the observation that PTEN and INPP4B deletions were individually associated with increased pAKT protein expression in our cohort (p=0.015 and p With respect to mutations, GNA13, P2RY8 and ARHGEF1 were more frequently mutated in the GCB than ABC subtype (26% vs 0.6%; p Based on these genetic data, we found that the PI3K-AKT signaling pathway was more commonly altered in GCB-DLBCL (64% (114/177)) than in ABC-DLBCL (23% (23/98) p In conclusion, genetic alterations in the PI3K-AKT pathway were frequently observed in GCB-DLBCL and were associated with activation of the pathway and inferior outcomes. These results suggested that AKT and mTOR inhibitors might be beneficial in a greater proportion of GCB-DLBCL than expected. Fig 1. The distribution of genetic alterations of PI3K-AKT pathway and pAKT protein expression. Fig 1. The distribution of genetic alterations of PI3K-AKT pathway and pAKT protein expression. KM curves according to pAKT protein expression (Fig 2) and PTEN and INPP4B deletions (Fig 3). KM curves according to pAKT protein expression (Fig 2) and PTEN and INPP4B deletions (Fig 3). Disclosures Sehn: roche/genentech: Consultancy, Honoraria; amgen: Consultancy, Honoraria; seattle genetics: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; TG therapeutics: Consultancy, Honoraria; celgene: Consultancy, Honoraria; lundbeck: Consultancy, Honoraria; janssen: Consultancy, Honoraria. Connors:F Hoffmann-La Roche: Research Funding; Millennium Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; NanoString Technologies: Research Funding. Scott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Asymmetric cell division in C.Crescentus relies on differentially localizing certain proteins to the poles where they bind to the scaffolding protein PopZ that displays a bipolar pattern. On the other hand, expressing PopZ in E. coli favours unipolar patterning. Additionally, the aggregation of misfolded proteins as well as plasmids in bacteria also display unipolar and biopolar patterns, similar to that of PopZ. These different systems have led to the hypothesis that chromosome free regions + biomolecular aggregation can be sufficient to drive localization in bacterial cells. We have performed Monte-Carlo simulations to show that the entropic force provided by the self-avoiding chromosome confined in the cylindrical geometry of the cell and the energy gained from aggregation results in phase separation between proteins and the polymer. We fully explore the phase space showing how patterning depends on protein concentration, chromosome density, cell shape and aggregation strength. The exploration results in a rich phase diagram of patterns which the observed systems can be fit into. Additionally, the dynamics of pattern formation depends somewhat on the rate at which proteins are added to the system. When proteins are added very slowly to the cell, the unipolar phase dominates, whereas at faster rates the bipolar phase dominates and at yet faster still rate, aggregation at nonpolar locations occurs. Such a rate dependency may explain differences in the observed patterns reported for PopZ induced expression in E. coli cells. Lastly, we show how such a localization process may aid the segregation of other cellular components such as the replication origin which anchors to a pole. We find that adding extra interactions does not destabilize the polar patterning, nor is it sufficient to drive the localization of the origin and indeed other localization and stabilization mechanisms are required.

The spatial patterning of proteins in bacteria plays an important role in many processes, from cell division to chemotaxis. In the asymmetrically dividing bacteria Caulobacter crescentus, a scaffolding protein, PopZ, localizes to both poles and aids the differential patterning of proteins between mother and daughter cells during division. Polar patterning of misfolded proteins in Escherechia coli has also been shown, and likely plays an important role in cellular ageing. Recent experiments on both of the above systems suggest that the presence of chromosome free regions along with protein multimerization may be a mechanism for driving the polar localization of proteins. We have developed a simple physical model for protein localization using only these two driving mechanisms. Our model reproduces all the observed patterns of PopZ and misfolded protein localization - from diffuse, unipolar, and bipolar patterns and can also account for the observed patterns in a variety of mutants. The model also suggests new experiments to further test the role of the chromosome in driving protein patterning, and whether such a mechanism is responsible for helping to drive the differentiation of the cell poles., Author Summary A key process in biology is the self-assembly of biomolecules into highly organized structures. This spontaneous assembly can give rise to complex spatial patterns that help give spatial order to the cellular environment. In many bacteria, the patterning of proteins to the cell poles allows the bacteria to differentiate one end of the cell from the other. What mechanisms can lead to the spontaneous organization of proteins to the cell poles? Prior work has shown that such patterning can emerge from interactions between proteins and the cell membrane. In this paper we use computational modeling to show that a novel patterning mechanism involving only the presence of the bacterial chromosome and a self-associating protein is sufficient to generate polar patterning in bacteria. This model explains recent experiments on polar patterning in C. crescentus and misfolded protein aggregation in E. coli and provides predictions about how this mechanism could spontaneously lead to asymmetric patterning of the poles.

We report on a novel stochastic analysis of seismic time series for the Earth's vertical velocity, by using methods originally developed for complex hierarchical systems, and in particular for turbulent flows. Analysis of the fluctuations of the detrended increments of the series reveals a pronounced change of the shapes of the probability density functions (PDF) of the series' increments. Before and close to an earthquake the shape of the PDF and the long-range correlation in the increments both manifest significant changes. For a moderate or large-size earthquake the typical time at which the PDF undergoes the transition from a Gaussian to a non-Gaussian is about 5-10 hours. Thus, the transition represents a new precursor for detecting such earthquakes., Comment: 12 pages, 5 figures

Summary Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.