The identification of gene fusions from RNA sequencing data is a routine task in cancer research and precision oncology. However, despite the availability of many computational tools, fusion ...detection remains challenging. Existing methods suffer from poor prediction accuracy and are computationally demanding. We developed Arriba, a novel fusion detection algorithm with high sensitivity and short runtime. When applied to a large collection of published pancreatic cancer samples (
= 803), Arriba identified a variety of driver fusions, many of which affected druggable proteins, including ALK, BRAF, FGFR2, NRG1, NTRK1, NTRK3, RET, and ROS1. The fusions were significantly associated with
wild-type tumors and involved proteins stimulating the MAPK signaling pathway, suggesting that they substitute for activating mutations in
In addition, we confirmed the transforming potential of two novel fusions,
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and
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, in cellular assays. These results show Arriba's utility in both basic cancer research and clinical translation.
Chronic lymphocytic leukemia cells have an altered energy metabolism compared to normal B cells. While there is a growing understanding of the molecular heterogeneity of the disease, the extent of ...metabolic heterogeneity and its relation to molecular heterogeneity has not been systematically studied. Here, we assessed 11 bioenergetic features, primarily reflecting cell oxidative phosphorylation and glycolytic activity, in leukemic cells from 140 chronic lymphocytic leukemia patients using metabolic flux analysis. We examined these bioenergetic features for relationships with molecular profiles (including genetic aberrations, transcriptome and methylome profiles) of the tumors, their
responses to a panel of 63 compounds, and with clinical data. We observed that leukemic cells with mutated immunoglobulin variable heavy-chain show significantly lower glycolytic activity than cells with unmutated immunoglobulin variable heavy-chain. Accordingly, several key glycolytic genes (
and
) were found to be down-regulated in samples harboring mutated immunoglobulin variable heavy-chain. In addition, 8q24 copy number gains, 8p12 deletions, 13q14 deletions and
mutations were identified as determinants of cellular respiration. The metabolic state of leukemic cells was associated with drug sensitivity; in particular, higher glycolytic activity was linked to increased resistance towards several drugs including rotenone, navitoclax, and orlistat. In addition, we found glycolytic capacity and glycolytic reserve to be predictors of overall survival (
<0.05) independently of established genetic predictors. Taken together, our study shows that heterogeneity in the energy metabolism of chronic lymphocytic leukemia cells is influenced by genetic variants and this could be therapeutically exploited in the selection of therapeutic strategies.
Classical hairy cell leukemia (cHCL) is characterized by a near 100% frequency of the BRAFV600E mutation, whereas ∼30% of variant HCLs (vHCLs) have MAP2K1 mutations. However, recurrent genetic ...alterations cooperating with BRAFV600E or MAP2K1 mutations in HCL, as well as those in MAP2K1 wild-type vHCL, are not well defined. We therefore performed deep targeted mutational and copy number analysis of cHCL (n = 53) and vHCL (n = 8). The most common genetic alteration in cHCL apart from BRAFV600E was heterozygous loss of chromosome 7q, the minimally deleted region of which targeted wild-type BRAF, subdividing cHCL into those hemizygous versus heterozygous for the BRAFV600E mutation. In addition to CDKN1B mutations in cHCL, recurrent inactivating mutations in KMT2C (MLL3) were identified in 15% and 25% of cHCLs and vHCLs, respectively. Moreover, 13% of vHCLs harbored predicted activating mutations in CCND3. A change-of-function mutation in the splicing factor U2AF1 was also present in 13% of vHCLs. Genomic analysis of de novo vemurafenib-resistant cHCL identified a novel gain-of-function mutation in IRS1 and losses of NF1 and NF2, each of which contributed to resistance. These data provide further insight into the genetic bases of cHCL and vHCL and mechanisms of RAF inhibitor resistance encountered clinically.
•KMT2C mutations occur in 15% and 25% of patients with cHCL and vHCL, respectively, along with CCND3 and U2AF1 mutations each in 13% of vHCLs.•NF1, NF2, N/KRAS, and IRS1 alterations contribute to clinical resistance to vemurafenib treatment in patients with cHCL.
Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the ...underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by "omics" profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components (e.g., BTK, PI3K, SYK) had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target's position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations.
Understanding the molecular and phenotypic heterogeneity of cancer is a prerequisite for effective treatment. For chronic lymphocytic leukemia (CLL), recurrent genetic driver events have been ...extensively cataloged, but this does not suffice to explain the disease's diverse course. Here, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major, orthogonal axes of gene expression variation: the first one represented the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, the three-group stratification of CLL by global DNA methylation. The second axis aligned with trisomy 12 status and affected chemokine, MAPK and mTOR signaling. We discovered nonadditive effects (epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion, suggesting that molecular understanding of disease heterogeneity requires studying such genetic events not only individually but in combination. We detected strong differentially expressed gene signatures associated with major gene mutations and copy-number aberrations including SF3B1, BRAF and TP53, as well as del(17)(p13), del(13)(q14) and del(11)(q22.3) beyond dosage effect. Our study reveals previously underappreciated gene expression signatures for the major molecular subtypes in CLL and the presence of epistasis between them.
The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We ...analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care.
•Low doses of the BRAF inhibitor vemurafenib are highly effective in refractory hairy cell leukemia.•Abrogation of BRAF V600E–induced signaling was consistently seen with 240 mg of vemurafenib twice daily.
Summary
Mutations in the N‐terminus of MED12 protein occur at high frequency in uterine leiomyomas and breast fibroepithelial tumours, and are frequently found in chronic lymphocytic leukaemia (CLL). ...MED12 mutations have been previously linked to aberrant Cyclin C‐CDK8 kinase activity, but the exact oncogenic function in CLL is unknown. Here, we characterized MED12 mutations in CLL and identified recurrent mutations in 13 out of 188 CLL patients (6·9%), which clustered in the N‐terminus. MED12 mutations were associated with unmutated IGHV (P = 0·024). Protein analysis of NOTCH1 in primary CLL samples revealed increased levels of NOTCH1 intracellular domain (NICD), the active form of NOTCH1, in the context of MED12 mutations. We found evidence that NICD is the target of Cyclin C‐CDK8 kinase using a specific CDK8 inhibitor. In line with these findings, MED12 mutations were mutually exclusive to mutations in NOTCH1 in CLL, based on a meta‐analysis of 1429 CLL patients (P = 0·011). Our results suggest that MED12 mutations may contribute to CLL pathogenesis by activating NOTCH signalling.
Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To ...discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.
•The HAT complex member TRRAP is vital for maintaining high p53 levels by shielding it against the natural p53 degradation machinery.•Acetylation-modifying complexes regulate p53 protein stability, which may provide a basis for therapeutic targeting of mutant p53.
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The natural course of HCV infection remains controversial. The German HCV (1b)‐contaminated anti‐D cohort provides an ideal population to investigate the natural course of HCV infection in a large, ...homogenous cohort of young women from the date of HCV inoculation. Our previous follow‐up studies at 20 and 25 years after infection suggested slow fibrosis progression rates in this unique cohort. The aim of our prospective, community‐based, multicenter study was to reevaluate the liver disease progression in 718 patients of the original anti‐D cohort at 35 years after infection. Patients with self‐limited HCV infection (n = 189) were compared to those who failed to eliminate the virus spontaneously (n = 529), comprising patients who were treatment naïve (n = 197) or achieved a sustained virological response (SVR; n = 149), respectively, failed to clear the virus (non‐SVR; n = 183) after antiviral therapy. In the overall cohort, 9.3% of patients showed clinical signs of liver cirrhosis at 35 years after infection. Liver disease progression largely depended on HCV infection status. The highest proportion of patients with clinical signs of end‐stage liver disease was observed in the non‐SVR group (15.3%), whereas decreased cirrhosis rates were detected in the SVR group (6%) and in patients with self‐limited HCV infection (1.1%; P = 6.2 × 10−6). Overall survival was significantly enhanced after SVR, compared to treatment‐naïve patients or non‐SVR (P = 0.027). Conclusion: The present study provides further evidence for a mild, but significant, disease progression at 35 years after infection in the German HCV (1b)‐contaminated anti‐D cohort. Patients with self‐limited HCV infection or SVR after antiviral treatment were protected from progressive liver disease and showed the best clinical long‐term outcome. (Hepatology 2014;58:49–57)
Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications ...but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes.
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