Identification of Burkitt lymphoma vulnerabilities using RNAi

Oncogenic activation of MYC drives cell proliferation in Burkitt lymphoma (BL), but also evokes stress signals that have to be counterbalanced to escape apoptosis. Pro-survival signals from tonic B cell receptor and PI3K signaling are essential, but additional mutations are required for malignant transformation. TP53 mutations were identified in 43% of BL patients. Mutations in the conserved MYC box I that abrogate activation of pro-apoptotic Bcl2-family member Bim were present in 17% of BL patients. Notably, MYC box I and TP53 mutations occurred independently and accounted for 54% of cases, suggesting that alternative failsafe mechanisms may be inactivated in BL. To study alternative mechanisms of transformation in the absence of TP53 mutations, we analyzed the pattern of recurrent genetic aberrations in BL for associations with TP53 status. We observed an overrepresentation of chromosome 1q gains in TP53 wild-type BL patients, which was not observed in diffuse large B cell lymphoma (DLBCL) patients. Minimally gained regions comprised 1q21-q23 and 1q32 and amplified regions displayed a gene dosage effect as shown by gene expression analysis. To identify genes essential for p53 wild-type BL cells we performed a RNAi loss-of-function screen in a panel of genetically defined cell lines. We used a pooled shRNA library targeting 5,000 genes in key signaling pathways across the genome. Our data was probed against published RNAi screens across cancer entities and showed a high overlap of common essential and non-essential genes. p53 wild-type BL cell lines showed a strong and specific dependence on the p53 inhibitor MDM4. Depletion of MDM4 resulted in an upregulation of p53 target genes and induced cell cycle arrest. In a mouse xenograft model, MDM4 knock-down significantly reduced tumor growth. These effects were p53 dependent as confirmed in an isogenic p53 knock-out cell line. MDM4 is located within the minimally gained region 1q32 associated with p53 wild-type BL patients and might therefore contribute to BL pathogenesis by inactivation of the p53 pathway. Our data suggest that reactivation of p53 in patients lacking TP53 mutation, e.g. by specific MDM4 inhibition, is a promising therapeutic approach. Re-analysis of published RNAi screening data revealed p53-specific sensitivity of MDM4 knock-down across cancer cell lines, suggesting a broader application for MDM4 inhibitors. Our data set on essential genes in Burkitt lymphoma proved to be a valuable resource for identification of genotype-specific vulnerabilities. This analysis could be extended by integration of published RNAi screening data in non-lymphoid cell lines to identify interesting potential entity-specific vulnerabilities

Subgroup-specific gene expression profiles and mixed epistasis in chronic lymphocytic leukemia

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 non-additive 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

Drug-perturbation-based stratification of blood cancer

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.Peer reviewe

aRgus: Multilevel visualization of non-synonymous single nucleotide variants & advanced pathogenicity score modeling for genetic vulnerability assessment

The widespread use of high-throughput sequencing techniques is leading to a rapidly increasing number of disease-associated variants of unknown significance and candidate genes. Integration of knowledge concerning their genetic, protein as well as functional and conservational aspects is necessary for an exhaustive assessment of their relevance and for prioritization of further clinical and functional studies investigating their role in human disease. To collect the necessary information, a multitude of different databases has to be accessed and data extraction from the original sources commonly is not user-friendly and requires advanced bioinformatics skills. This leads to a decreased data accessibility for a relevant number of potential users such as clinicians, geneticist, and clinical researchers. Here, we present aRgus (https://argus.urz.uni-heidelberg.de/), a standalone webtool for simple extraction and intuitive visualization of multi-layered gene, protein, variant, and variant effect prediction data. aRgus provides interactive exploitation of these data within seconds for any known gene of the human genome. In contrast to existing online platforms for compilation of variant data, aRgus complements visualization of chromosomal exon-intron structure and protein domain annotation with ClinVar and gnomAD variant distributions as well as position-specific variant effect prediction score modeling. aRgus thereby enables timely assessment of protein regions vulnerable to variation with single amino acid resolution and provides numerous applications in variant and protein domain interpretation as well as in the design of in vitro experiments

Evolution of DNA Methylation Is Linked to Genetic Aberrations in Chronic Lymphocytic Leukemia

ABSTRACT Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefi ned. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintain-ing low methylation heterogeneity and up to 10 % of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and inde-pendent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL. SIGNIFICANCE: Epigenetic alterations are pervasive in cancer and continually develop during disease progression; however, the mechanisms that promote changes in the tumor epigenome at large are cur-rently undefi ned. The current work provides insight into the coevolution of genetic and epigenetic aber-rations and highlights the infl uential role of genetic aberrations in the selection of novel methylatio

TRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma

Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function (GOF) 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 (RNAi) screen in a Burkitt's lymphoma (BL) cell line model. We identified TRRAP, a constituent of several histone acetyltransferase (HAT) complexes, as a critical positive regulator of both mutp53 and wild-type p53 (wtp53) levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, while TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through CRISPR-Cas9 screening, we identified a 109 amino acid region in the N-terminal HEAT repeat region of TRRAP which 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 HDACs1/2/3, 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 Protein Landscape of Chronic Lymphocytic Leukemia (CLL)

Many functional consequences of mutations on tumor phenotypes in chronic lymphocytic leukemia (CLL) are unknown. This may be in part due to a scarcity of information on the proteome of CLL. We profiled the proteome of 117 CLL patient samples with data-independent acquisition mass spectrometry (DIA-MS) and integrated the results with genomic, transcriptomic, ex vivo drug response and clinical outcome data. We found trisomy 12, IGHV mutational status, mutated SF3B1, trisomy 19, del(17)(p13), del(11)(q22.3), mutated DDX3X, and MED12 to influence protein expression (FDR < 5%). Trisomy 12 and IGHV status were the major determinants of protein expression variation in CLL as shown by principal component analysis (1055 and 542 differentially expressed proteins, FDR=5%). Gene set enrichment analyses of CLL with trisomy 12 implicated BCR/PI3K/AKT signaling as a tumor driver. These findings were supported by analyses of protein abundance buffering and protein complex formation, which identified limited protein abundance buffering and an upregulated protein complex involved in BCR, AKT, MAPK and PI3K signaling in trisomy 12 CLL. A survey of proteins associated with trisomy 12/IGHV-independent drug response linked STAT2 protein expression with response to kinase inhibitors including BTK and MEK inhibitors. STAT2 was upregulated in U-CLL, trisomy 12 CLL and required for chemokine/cytokine signaling (interferon response). This study highlights the importance of protein abundance data as a non-redundant layer of information in tumor biology, and provides a protein expression reference map for CLL

Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia

Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefined. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintaining low methylation heterogeneity and up to 10% of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and independent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL

Targeted resequencing for analysis of clonal composition of recurrent gene mutations in chronic lymphocytic leukaemia

Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were most frequently mutated (16·3%, 16·9%, 10·7%). We found evidence for subclonal mutations in 67·5% of CLL cases with mutations of cancer consensus genes. We observed selection of subclones and found initial evidence for convergent mutations in CLL. Our data suggest that assessment of (sub)clonal structure may need to be integrated into analysis of the mutational profile in CL