Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether ...peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets.
High-resolution fluorescence microscopy is increasingly contributing to our understanding of molecular processes. By utilizing single-molecule intensity information, imaging experiments can be ...rendered quantitative, yielding insights into the stoichiometry and kinetics of the components of a molecular assembly. Here, we describe the experimental and analytical steps needed to study the assembly of clathrin-coated vesicles with single-molecule resolution, using total internal reflection fluorescence microscopy. Many components of the protocol are broadly applicable to the characterization of other molecular processes.
The large majority of variants identified by GWAS are non-coding, motivating detailed characterization of the function of non-coding variants. Experimental methods to assess variants' effect on gene ...expressions in native chromatin context via direct perturbation are low-throughput. Existing high-throughput computational predictors thus have lacked large gold standard sets of regulatory variants for training and validation. Here, we leverage a set of 14,807 putative causal eQTLs in humans obtained through statistical fine-mapping, and we use 6121 features to directly train a predictor of whether a variant modifies nearby gene expression. We call the resulting prediction the expression modifier score (EMS). We validate EMS by comparing its ability to prioritize functional variants with other major scores. We then use EMS as a prior for statistical fine-mapping of eQTLs to identify an additional 20,913 putatively causal eQTLs, and we incorporate EMS into co-localization analysis to identify 310 additional candidate genes across UK Biobank phenotypes.
During cancer therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms in an individual patient
. Previous case reports and small case ...series have suggested that liquid biopsy (specifically, cell-free DNA (cfDNA)) may better capture the heterogeneity of acquired resistance
. However, the effectiveness of cfDNA versus standard single-lesion tumor biopsies has not been directly compared in larger-scale prospective cohorts of patients following progression on targeted therapy. Here, in a prospective cohort of 42 patients with molecularly defined gastrointestinal cancers and acquired resistance to targeted therapy, direct comparison of postprogression cfDNA versus tumor biopsy revealed that cfDNA more frequently identified clinically relevant resistance alterations and multiple resistance mechanisms, detecting resistance alterations not found in the matched tumor biopsy in 78% of cases. Whole-exome sequencing of serial cfDNA, tumor biopsies and rapid autopsy specimens elucidated substantial geographic and evolutionary differences across lesions. Our data suggest that acquired resistance is frequently characterized by profound tumor heterogeneity, and that the emergence of multiple resistance alterations in an individual patient may represent the 'rule' rather than the 'exception'. These findings have profound therapeutic implications and highlight the potential advantages of cfDNA over tissue biopsy in the setting of acquired resistance.
Tens of thousands of genetic variants associated with gene expression (cis-eQTLs) have been discovered in the human population. These eQTLs are active in various tissues and contexts, but the ...molecular mechanisms of eQTL variability are poorly understood, hindering our understanding of genetic regulation across biological contexts. Since many eQTLs are believed to act by altering transcription factor (TF) binding affinity, we hypothesized that analyzing eQTL effect size as a function of TF level may allow discovery of mechanisms of eQTL variability. Using GTEx Consortium eQTL data from 49 tissues, we analyzed the interaction between eQTL effect size and TF level across tissues and across individuals within specific tissues and generated a list of 10,098 TF-eQTL interactions across 2,136 genes that are supported by at least two lines of evidence. These TF-eQTLs were enriched for various TF binding measures, supporting with orthogonal evidence that these eQTLs are regulated by the implicated TFs. We also found that our TF-eQTLs tend to overlap genes with gene-by-environment regulatory effects and to colocalize with GWAS loci, implying that our approach can help to elucidate mechanisms of context-specificity and trait associations. Finally, we highlight an interesting example of IKZF1 TF regulation of an APBB1IP gene eQTL that colocalizes with a GWAS signal for blood cell traits. Together, our findings provide candidate TF mechanisms for a large number of eQTLs and offer a generalizable approach for researchers to discover TF regulators of genetic variant effects in additional QTL datasets.
Clathrin‐mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin‐coated pit (CCP) dynamics led us to propose the existence of a rate‐limiting, regulatory step(s) ...that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this “checkpoint.” Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin‐1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non‐neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin‐1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR‐Cas9n‐mediated knockout and reconstitution studies establish that dynamin‐1 is activated by Akt/GSK3β signaling in H1299 non‐small lung cancer cells. These findings provide direct evidence for an isoform‐specific role for dynamin in regulating CME and reveal a feed‐forward pathway that could link signaling from cell surface receptors to the regulation of CME.
Synopsis
Dynamin‐1, previously thought to be neuron specific, is activated by an Akt/GSK3β signaling cascade in non‐neuronal cells. Dynamin‐1 activation alters the rate and regulation of clathrin‐mediated endocytosis, providing a feed‐forward pathway between endocytosis and signaling.
An endocytic checkpoint monitors the fidelity of clathrin‐coated vesicle (CCV) formation.
Dysregulated clathrin‐mediated endocytosis (CME) alters cell signaling and proliferation.
Dynamin‐1 and dynamin‐2 differentially regulate early stages of CME.
Endogenous dynamin‐1 is activated by an Akt/GSK3β signaling cascade, illustrating a first non‐neuronal role of dynamin‐1.
Activation of dynamin‐1 by Akt and GSK3β in non‐neuronal cells alters the rate and regulation of CME, providing a feed‐forward pathway between signaling and endocytosis.
Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with a 10% annual risk of progression. Various prognostic models exist for risk stratification; however, those are ...based on solely clinical metrics. The discovery of genomic alterations that underlie disease progression to MM could improve current risk models.
We used next-generation sequencing to study 214 patients with SMM. We performed whole-exome sequencing on 166 tumors, including 5 with serial samples, and deep targeted sequencing on 48 tumors.
We observed that most of the genetic alterations necessary for progression have already been acquired by the diagnosis of SMM. Particularly, we found that alterations of the mitogen-activated protein kinase pathway (
and
single nucleotide variants SNVs), the DNA repair pathway (deletion 17p,
, and
SNVs), and
(translocations or copy number variations) were all independent risk factors of progression after accounting for clinical risk staging. We validated these findings in an external SMM cohort by showing that patients who have any of these three features have a higher risk of progressing to MM. Moreover, APOBEC associated mutations were enriched in patients who progressed and were associated with a shorter time to progression in our cohort.
SMM is a genetically mature entity whereby most driver genetic alterations have already occurred, which suggests the existence of a right-skewed model of genetic evolution from monoclonal gammopathy of undetermined significance to MM. We identified and externally validated genomic predictors of progression that could distinguish patients at high risk of progression to MM and, thus, improve on the precision of current clinical models.
Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular ...clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.
Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma ...coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of
-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (
), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous
impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities.
.
Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on ...chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the 'CLL map,' we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.
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