Many of the diverse functions of cilia depend upon tight control of their length. Steady-state length reflects a balance between rates of ciliary assembly and disassembly, two parameters likely ...controlled by a length sensor of unknown identity or mechanism.
A null mutation in Chlamydomonas CNK2, a member of the evolutionarily conserved family of NIMA-related kinases, reveals feedback regulation of assembly and disassembly rates. cnk2-1 mutant cells have a mild long-flagella (lf) phenotype as a consequence of reduced rates of flagellar disassembly. This is in contrast to the strong lf mutant lf4-7, which exhibits an aberrantly high rate of assembly. Cells carrying both mutations have even longer flagella than lf4-7 single mutants do. In addition to their high rate of assembly, lf4-7 mutants have a CNK2-dependent increase in disassembly rate. Finally, cnk2-1 cells have a decreased rate of turnover of flagellar subunits at the tip of the flagellum, demonstrating that the effects on disassembly are compensated by a reduced rate of assembly.
We propose a model wherein CNK2 and LF4 modulate rates of disassembly and assembly respectively in a feedback loop that is activated when flagella exceed optimal length.
•Chlamydomonas cells null for the CNK2 kinase have a mild long-flagella phenotype•cnk2-1 cells are defective in flagellar disassembly and therefore resorption•The cnk2-1 mutation enhances the long-flagella phenotype of lf4-7 mutants•cnk2-1 cells exhibit a compensatory decrease in incorporation of new tubulin
Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (oncovin), and ...prednisone R-CHOP); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.
•KMT2D and TP53 are predominantly mutated in rrDLBCL and remain clonally stable following therapy.•MS4A1 mutations are commonly acquired and undergo clonal expansion following treatment with rituximab-containing therapy.
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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
. 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.
High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard ...chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit–like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.
•Gene expression identifies DHITsig+ DLBCL tumors with BCL2 and MYC translocations detectable by whole-genome sequencing, but not by breakapart FISH.•Additional genetic mechanisms of MYC dysregulation include focal MYC and MIR17HG copy-number gains and PVT1 promoter deletions.
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Diffuse large B-cell lymphoma (DLBCL) is heterogeneous both in clinical outcomes and the underlying disease biology. Over the last 2 decades, several different approaches for dissecting biological ...heterogeneity have emerged. Gene expression profiling (GEP) stratifies DLBCL into 3 broad groups (ABC, GCB, and DZsig/MHG), each with parallels to different normal mature B cell developmental states and prognostic implications. More recently, several different genomic approaches have been developed to categorize DLBCL based on the co-occurrence of tumor somatic mutations, identifying more granular biologically unified subgroups that complement GEP-based approaches. We review the molecular approaches and clinical evidence supporting the stratification of DLBCL patients based on tumor biology. By offering a platform for subtype-guided therapy, these divisions remain a promising avenue for improving patient outcomes, especially in subgroups with inferior outcomes with current standard-of-care therapy.
With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by ...shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2 Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone.
•DHITsig expression extends beyond HGBCL-DH-BCL2 to identify dark zone lymphomas and was thus renamed the “DZsig.”•DZsig refines cell-of-origin classification by identifying patients within GCB-DLBCL ...with inferior outcomes and shorter time to treatment.
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Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)–defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called “double-hit signature” (DHITsig)—a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)–DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the “dark zone signature” (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)–DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.
Aggressive B-cell lymphomas are both molecularly and clinically heterogeneous, creating challenges for systems that attempt to classify them into clinically relevant entities. Alduaij et al advance the field by reporting on the utility of a dark zone signature (DZsig), originally identified in double-hit lymphoma in the diagnosis of aggressive B-cell lymphomas with diffuse large B-cell morphology. The authors show that DZsig has a strong negative prognostic value, and these analyses provide a direction for further refinement of current classifications of aggressive B-cell lymphomas.
Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish ...the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.
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