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Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x(L), Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-x(L) and Mcl-1. Expression of Bcl-x(L), Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-x(L) and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatemnt with specific small molecule inhibitors of Bcl-x(L) (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-x(L) induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-x(L) and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-x(L) in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-x(L) as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Biliary tract cancers (BTCs) have poor prognosis and limited therapeutic options. The impact of O

XLSX file 47K, Supplementary table 1: Clinical and biological features of all samples and timepoints used in the study including all genetic alterations found per sample

XLSX file 15K, Supplementary table 2: Primer sequences used for targeted bisulfite-sequencing

This file contains supplementary figures S1-S10.

This excel file contains supplementary tables S1-S7.

PDF file 1498K, Supplemental figures 1-10: Methylation analysis of CLL and healthy donor samples using 450k arrays, WGBS and targeted BS-seq, including technical evaluations of the methods used

This file contains supplementary methods and references.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population.Significance:Rare cancers are difficult to treat; in particular, molecular pathogenesis–oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659

Generanks for differential shRNA effects from GENE-E class comparison analysis

Table S2. Genetic characterization of BL cell lines; Table S4. Genetic aberrations in aggressive B-NHL patients from the MMML consortium; Table S5. Annotation of TP53 mutation and MDM4 gain in cell lines from the Achilles project; Figure S1. RNAi screen identifies common and context-specific essential genes; Figure S2. Genes essential in the context of TP53 mutation; Figure S3. p53 reactivation causes cytotoxic effect; Figure S4. Molecular impact of MDM2 or MDM4 knock-down; Figure S5. Genetic profile in TP53wt and TP53mut aggressive B-NHL patients; Figure S6. Basal MDM4 mRNA in aggressive lymphomas stratified by TP53 mutation and 1q status.

Oncogenic MYC activation promotes proliferation in Burkitt lymphoma, but also induces cell-cycle arrest and apoptosis mediated by p53, a tumor suppressor that is mutated in 40% of Burkitt lymphoma cases. To identify molecular dependencies in Burkitt lymphoma, we performed RNAi-based, loss-of-function screening in eight Burkitt lymphoma cell lines and integrated non-Burkitt lymphoma RNAi screens and genetic data. We identified 76 genes essential to Burkitt lymphoma, including genes associated with hematopoietic cell differentiation (FLI1, BCL11A) or B-cell development and activation (PAX5, CDKN1B, JAK2, CARD11) and found a number of context-specific dependencies including oncogene addiction in cell lines with TCF3/ID3 or MYD88 mutation. The strongest genotype–phenotype association was seen for TP53. MDM4, a negative regulator of TP53, was essential in TP53 wild-type (TP53wt) Burkitt lymphoma cell lines. MDM4 knockdown activated p53, induced cell-cycle arrest, and decreased tumor growth in a xenograft model in a p53-dependent manner. Small molecule inhibition of the MDM4–p53 interaction was effective only in TP53wt Burkitt lymphoma cell lines. Moreover, primary TP53wt Burkitt lymphoma samples frequently acquired gains of chromosome 1q, which includes the MDM4 locus, and showed elevated MDM4 mRNA levels. 1q gain was associated with TP53wt across 789 cancer cell lines and MDM4 was essential in the TP53wt-context in 216 cell lines representing 19 cancer entities from the Achilles Project. Our findings highlight the critical role of p53 as a tumor suppressor in Burkitt lymphoma and identify MDM4 as a functional target of 1q gain in a wide range of cancers that is therapeutically targetable.Significance:Targeting MDM4 to alleviate degradation of p53 can be exploited therapeutically across Burkitt lymphoma and other cancers with wild-type p53 harboring 1q gain, the most frequent copy number alteration in cancer.

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. In order 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 ofin vitroexperiments.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x

Introduction: Thymic epithelial tumors (TETs) are very rare. Thymoma A and AB have a better prognosis than more aggressive thymoma B, thymic carcinoma (TC) and neuroendocrine tumors of the thymus (NET). While previous efforts such as TCGA have mainly characterized thymomas (Radovich et al., Cancer Cell 2018), the molecular landscape of TCs and NETs is still elusive. Patients and Methods: Between 03/2014 and 07/2020, we enrolled 44 TET patients (27 TCs, 11 thymomas, 6 NETs) in a prospective observational study (MASTER) conducted by the National Center for Tumor Diseases (NCT) Heidelberg, NCT Dresden and the German Cancer Consortium (DKTK). MASTER applied whole genome/exome sequencing (WGS, n=22; WES, n=22), transcriptome (n=40) and germline analysis to inform therapy recommendations by a dedicated molecular tumor board (MTB). We systematically gathered follow-up data to evaluate outcome and compared progression-free survival (PFS) of the first treatment according to an MTB recommendation (PFS2) to the last prior systemic treatment (PFS1) in each patient (PFS ratio). Results: Tumor mutational burden (TMB) was low (median=0.99 mutations/Mb, range 0.08-3.48) but higher than in TCGA (p 6 months and a PFS ratio > 1.3. The best outcome was achieved using imatinib in a patient with a KIT mutation (p.W557R). After progression, the MTB recommended ponatinib based on a secondary KIT mutation (p.V654A). The patient was still on ponatinib when the observation period ended. Conclusion: We demonstrate that comprehensive molecular analysis provides clinically relevant information in a subgroup of TET patients. Thymoma, TCs, and NETs present with different molecular characteristics. Distinction between immunologically hot and cold TCs may have value for risk stratification and therapeutic strategies. PARP inhibition could be a potential new treatment option in a small subgroup of TETs. Molecular testing of KIT, germline analysis and genetic counseling should be recommended for all patients with advanced TETs. Citation Format: Lino Möhrmann, Lysann Rostock, Małgorzata Oleś, Arne Jahn, Marie Arlt, Nagarajan Paramasivam, Korinna Jöhrens, Luise Rupp, Marc Schmitz, Daniela Richter, Sebastian Uhrig, Martina Fröhlich, Barbara Hutter, Jennifer Hüllein, Elena E. Wolf, Dorothea Hanf, Laura Gieldon, Simon Kreutzfeldt, Christoph E. Heilig, Veronica Teleanu, Daniel B. Lipka, Andreas Mock, Ivan Jelas, Damian T. Rieke, Marcel Wiesweg, Melanie Boerries, Anna L. Illert, Alexander Desuki, Thomas Kindler, Angela M. Krackhardt, C. Benedikt Westphalen, Heidrun Grosch, Leonidas Apostolidis, Albrecht Stenzinger, Irina A. Kerle, Christoph Heining, Daniel Hübschmann, Evelin Schröck, Stefan Fröhling, Hanno Glimm. Genomics-based personalized oncology of advanced thymic epithelial tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 926.

Chronic Lymphocytic Leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex-vivo drug response data from 217 Chronic Lymphocytic Leukemia (CLL) cases. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to the known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n=547 patients). We find that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL-PD was linked to the activation of mTOR-MYC-oxidative phosphorylation (OXPHOS) through transcriptomic, proteomic and single cell resolution analysis. CLL-PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.

Oncogenic MYC activation promotes proliferation in Burkitt lymphoma, but also induces cell-cycle arrest and apoptosis mediated by p53, a tumor suppressor that is mutated in 40% of Burkitt lymphoma cases. To identify molecular dependencies in Burkitt lymphoma, we performed RNAi-based, loss-of-function screening in eight Burkitt lymphoma cell lines and integrated non-Burkitt lymphoma RNAi screens and genetic data. We identified 76 genes essential to Burkitt lymphoma, including genes associated with hematopoietic cell differentiation (FLI1, BCL11A) or B-cell development and activation (PAX5, CDKN1B, JAK2, CARD11) and found a number of context-specific dependencies including oncogene addiction in cell lines with TCF3/ID3 or MYD88 mutation. The strongest genotype–phenotype association was seen for TP53. MDM4, a negative regulator of TP53, was essential in TP53 wild-type (TP53wt) Burkitt lymphoma cell lines. MDM4 knockdown activated p53, induced cell-cycle arrest, and decreased tumor growth in a xenograft model in a p53-dependent manner. Small molecule inhibition of the MDM4–p53 interaction was effective only in TP53wt Burkitt lymphoma cell lines. Moreover, primary TP53wt Burkitt lymphoma samples frequently acquired gains of chromosome 1q, which includes the MDM4 locus, and showed elevated MDM4 mRNA levels. 1q gain was associated with TP53wt across 789 cancer cell lines and MDM4 was essential in the TP53wt-context in 216 cell lines representing 19 cancer entities from the Achilles Project. Our findings highlight the critical role of p53 as a tumor suppressor in Burkitt lymphoma and identify MDM4 as a functional target of 1q gain in a wide range of cancers that is therapeutically targetable. Significance: Targeting MDM4 to alleviate degradation of p53 can be exploited therapeutically across Burkitt lymphoma and other cancers with wild-type p53 harboring 1q gain, the most frequent copy number alteration in cancer.

Despite the extensive catalogue of recurrent mutations in chronic lymphocytic leukaemia (CLL), the diverse molecular driving events and the resulting range of disease phenotypes remain incompletely understood. To study the molecular heterogeneity of CLL, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major independent axes of gene expression variation: the first one aligned with the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, with the three-group stratification of CLL by global DNA methylation pattern, and affected biological functions including B- and T-cell receptor signaling. The second one aligned with trisomy 12 status and affected chemokine signaling. Furthermore, we searched for differentially expressed genes associated with gene mutations and copy-number aberrations and detected strong signatures for TP53, BRAF and SF3B1, as well as for del(11)(q22.3), del(17)(p13) and del(13)(q14) beyond the dosage effect. We discovered strong non-additive effects (i.e., genetic interactions, or 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. Our study reveals previously underappreciated gene expression signatures for (epi)genomic variants in CLL and the presence of epistasis between them. The findings will serve as a reference for a functional resolution of CLL molecular heterogeneity.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. Significance: Rare cancers are difficult to treat; in particular, molecular pathogenesis–oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials. See related commentary by Eggermont et al., p. 2677. This article is highlighted in the In This Issue feature, p. 2659

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 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 (false discovery rate [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 B-cell receptor (BCR)/phosphatidylinositol 3-kinase (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 Bruton tyrosine kinase and mitogen-activated protein kinase kinase (MEK) inhibitors. STAT2 was upregulated in unmutated IGHV CLL and trisomy 12 CLL and required for chemokine/cytokine signaling (interferon response). This study highlights the importance of protein abundance data as a nonredundant layer of information in tumor biology and provides a protein expression reference map for CLL.

Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL.

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.

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.

Myxoid liposarcomas (MLS), malignant tumors of adipocyte origin,are driven by theFUS-DDIT3fusion gene encoding an aberrant tran-scription factor. The mechanisms whereby FUS-DDIT3mediatessarcomagenesis are incompletely understood, and strategies toselectively target MLS cells remain elusive. Here we show, using anunbiased functional genomic approach, that FUS-DDIT3-expressingmesenchymal stem cells and MLS cell lines are dependent on YAP1,atranscriptional co-activator and central effector of the Hippo path-way involved in tissue growth and tumorigenesis, and that increasedYAP1activity is a hallmark of human MLS. Mechanistically, FUS-DDIT3promotes YAP1expression, nuclear localization, and transcrip-tional activity and physically associates with YAP1in the nucleus ofMLS cells. Pharmacologic inhibition of YAP1activity impairs thegrowth of MLS cellsin vitroandin vivo. These findings identifyoveractive YAP1signaling as unifying feature of MLS developmentthat could represent a novel target for therapeutic intervention., publishedVersion

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.

Within primary colorectal cancer (CRC) a subfraction of all tumor-initiating cells (TIC) drives long-term progression in serial xenotransplantation. It has been postulated that efficient maintenance of TIC activity in vitro requires serum-free spheroid culture conditions that support a stem-like state of CRC cells. To address whether tumorigenicity is indeed tightly linked to such a stem-like state in spheroids, we transferred TIC-enriched spheroid cultures to serum-containing adherent conditions that should favor their differentiation. Under these conditions, primary CRC cells did no longer grow as spheroids but formed an adherent cell layer, up-regulated colon epithelial differentiation markers, and down-regulated TIC-associated markers. Strikingly, upon xenotransplantation cells cultured under either condition equally efficient formed serially transplantable tumors. Clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in contributing clone numbers, indicating that phenotypic differentiation does not select for few individual clones adapted to unfavorable culture conditions. Our results reveal that CRC TIC can be propagated under conditions previously thought to induce their elimination. This phenotypic plasticity allows addressing primary human CRC TIC properties in experimental settings based on adherent cell growth.

Comprehensive molecular profiling can be successfully applied to guide targeted treatment in cancer patients, an approach commonly referred to as precision oncology. Over the past years, several clinical trials that employed subgenomic molecular profiling have demonstrated that molecularly informed decision-making across tumor entities is associated with improved clinical outcome in approximately one third of patients. To investigate the feasibility and clinical relevance of comprehensive genomic analysis, i.e. whole-exome/genome sequencing (WES/WGS) and RNA sequencing (RNA-seq), in younger adults with advanced-stage cancer across all histologies and patients with rare tumors, we established MASTER (Molecularly Aided Stratification for Tumor Eradication Research) - a prospective, multicenter precision oncology platform - at NCT Heidelberg/Dresden in 2013, which was extended to the German Cancer Consortium (DKTK) in 2016. Based on a standardized workflow, we have analyzed more than 1,700 poor-prognosis (median overall survival, 12 months) patients with advanced, heavily pretreated (median number of prior therapies, n=2) malignancies representing a broad spectrum of rare histopathologic entities. We here report the actionable findings and clinical outcomes for the first 1,311 patients discussed in cross-institutional molecular tumor board (MTB) conferences. Each MTB recommendation was based on the individual molecular profile and specific predictive molecular biomarkers identified by WES/WGS and RNA-seq. In addition to DNA alterations (single-nucleotide variants, small insertions/deletions, copy number alterations), we also used alterations identified by RNA-seq (gene fusions, aberrant gene expression) to support clinical decision-making. We categorized therapy recommendations into seven different intervention baskets and assigned evidence levels to each recommendation according to a dedicated NCT/DKTK classification system, which addresses the complexity of evaluating predictive molecular biomarkers in clinical routine. MTB recommendations were implemented in one third of cases, and overall response and disease control rates on molecularly guided treatment were improved compared to prior systemic therapies, which translated into a progression-free survival ratio of greater than 1.3 in a significant proportion of patients. Furthermore, comprehensive genomic profiling in combination with histopathologic reevaluation allowed reclassification of approximately 4% of cases, in particular soft-tissue sarcomas not otherwise specified and carcinomas of unknown primary site. This prospective study demonstrates that comprehensive molecular profiling based on WES/WGS and RNA-seq in a multiinstitutional clinical setting creates meaningful therapeutic opportunities for patients with rare cancers. Our data demonstrate the added benefit of germline and RNA analysis, providing a rationale for their routine clinical implementation. Current and future activities of the MASTER network are focused on the standardization of variant classification and evidence levels in MTB conferences, the implementation of molecularly stratified basket trials, and the integration of additional layers of patient characterization. Citation Format: Peter Horak, Christoph Heining, Andreas Mock, Simon Kreutzfeldt, Andreas Lassmann, Lino Möhrmann, Jennifer Hüllein, Dorothea Hanf, Arne Jahn, Leo Ruhnke, Laura Gieldon, Christoph E. Heilig, Veronica Teleanu, Martina Fröhlich, Sebastian Uhrig, Katja Beck, Daniela Richter, Stephan Wolf, Katrin Pfütze, Christina Geörg, Bettina Meissburger, Frederick Klauschen, Ulrich Keilholz, Sebastian Ochsenreither, Gunnar Folprecht, Jens Siveke, Sebastian Bauer, Thomas Kindler, Christian Brandts, Melanie Boerries, Anna L. Illert, Nikolas von Bubnoff, Karsten Spiekermann, Philipp J. Jost, Klaus Schulze-Osthoff, Michael Bitzer, Peter Schirmacher, Christof von Kalle, Richard F. Schlenk, Barbara Klink, Barbara Hutter, Daniel Hübschmann, Albrecht Stenzinger, Wilko Weichert, Evelin Schröck, Benedikt Brors, Hanno Glimm, Stefan Fröhling, German Cancer Consortium (DKTK). Comprehensive genomic analysis of rare cancers: Results of the MASTER precision oncology trial of the German Cancer Consortium [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 821.

Oncogenic MYC activation promotes cellular proliferation in Burkitt lymphoma (BL), but also induces cell cycle arrest and apoptosis mediated by TP53, a tumor suppressor gene that is mutated in 40% of BL cases. To identify therapeutic targets in BL, we investigated molecular dependencies in BL cell lines using RNAi-based, loss-of-function screening. By integrating genotypic and RNAi data, we identified a number of genotype-specific dependencies including the dependence of TCF3/ID3 mutant cell lines on TCF3 and of MYD88 mutant cell lines on TLR signaling. TP53 wild-type (TP53wt) BL were dependent on MDM4, a negative regulator of TP53. In BL cell lines, MDM4 knockdown induced cell cycle arrest and decreased tumor growth in a xenograft model in a p53-dependent manner, while small molecule inhibition of the MDM4-p53 interaction restored p53 activity resulting in cell cycle arrest. Consistent with the pathogenic effect of MDM4 upregulation in BL, we found that TP53wt BL samples were enriched for gain of chromosome 1q which includes the MDM4 locus. 1q gain was also enriched across non-BL cancer cell lines (n=789) without TP53 mutation (23% in TP53wt and 12% in TP53mut, pOur findings show that in TP53wt BL, MDM4-mediated inhibition of p53 is a mechanism to evade cell cycle arrest. The data highlight the critical role of p53 as a tumor suppressor in BL, and identifies MDM4 as a key functional target of 1q gain in a wide range of cancers, which is therapeutically targetable.

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.

Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma.

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.

Myxoid liposarcomas (MLS) account for 20% of malignant adipocytic tumors and are characterized by a high rate of local recurrence and development of distant metastases in approximately 40% of patients. Most MLS are driven by the FUS-DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS-DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. In this study, we employed genome-scale RNA interference (RNAi) screening to uncover that human mesenchymal stem cells engineered to express FUS-DDIT3 and MLS cell lines are dependent on YAP1, a transcriptional co-activator and central effector of the Hippo pathway involved in tissue growth and tumorigenesis. Analysis of a large cohort of primary MLS specimens (n=223) revealed that nuclear YAP1 expression was significantly more prevalent in MLS compared to other liposarcoma subtypes. In support of the concept that increased YAP1-mediated transcriptional activity represents an essential feature of MLS development, RNAi-based YAP1 depletion in cultured MLS cells resulted in suppression of cell viability, cell cycle arrest, cellular senescence, and induction of apoptosis accompanied by decreased YAP1 target gene expression, and YAP1-positive primary MLS tumors showed strong expression of YAP1 downstream effectors such as FOXM1 and PLK1. Mechanistically, FUS-DDIT3 promotes YAP1 transcription, nuclear localization, and transcriptional activity and physically associates with YAP1 in the nucleus of MLS cells, pointing to the coordinate establishment of gene expression programs that promote MLS tumorigenesis. Consistent with the hypothesis that a YAP1-directed therapeutic approach could represent a rational strategy to selectively target FUS-DDIT3-expressing MLS cells, pharmacologic inhibition of YAP1 activity with verteporfin suppressed cell viability and YAP1 target gene expression in MLS cell lines, and the growth-inhibitory effects of YAP1 knockdown or verteporfin treatment could be recapitulated in MLS cell line-based xenograft models. Collectively, our data identify dependence on aberrant YAP1 activity as specific liability of FUS-DDIT3-expressing MLS cells, and provide preclinical evidence that YAP1-mediated signal transduction represents a candidate target for therapeutic intervention that warrants further investigation. Citation Format: Marcel Trautmann, Ya-Yun Cheng, Patrizia Jensen, Ninel Azoitei, Ines Brunner, Jennifer Hüllein, Mikolaj Slabicki, Ilka Isfort, Magdalene Cyra, Eva Wardelmann, Sebastian Huss, Bianca Altvater, Claudia Rossig, Susanne Hafner, Thomas Simmet, Anders Ståhlberg, Pierre Åman, Thorsten Zenz, Undine Lange, Thomas Kindler, Claudia Scholl, Wolfgang Hartmann, Stefan Fröhling. Requirement for YAP1 signaling in myxoid liposarcoma [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 3437.

Burkitt's lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments.

Purpose: Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells. Experimental Design: CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726. Results: Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4–induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4–activated (“resistant”) CLL cells was achieved with higher concentrations (IC50: 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC50: 56 μg/mL). Conclusion: We thus show that A771726 overcomes CD40L/IL-4–mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells. Clin Cancer Res; 18(2); 417–31. ©2011 AACR.

Background: Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. However, a systems-level understanding of how the microenvironment and the genetic and molecular alterations of the tumor interact with each other and contribute to drug resistance is lacking. Methods: To address this unmet need, we established an automated microscopy-based phenotyping platform that uses co-culture conditions mimicking the bone marrow environment. We cultured primary tumor cells from more than 100 leukemia patients (CLL, AML, MCL, T-PLL, HCL) with and without bone marrow stroma cell support in DMEM and 10% human serum and treated each condition with 57 drugs in 3 concentrations. After 72h of incubation, 22 000 images per patient were acquired and processed by our custom made image analysis pipeline. Our set-up allows us to increase sensitivity far beyond simple viability testing, as it reads out additional cell type specific features such as cell morphology, autophagy and cell-cell interactions. Results: Quality assessment revealed that in contrast to mono-culture conditions, assay plate edge effects can be avoided under stable stroma cell co-culture conditions. Correlation of replicated patient samples were comparable between mono- and co-cultures (R2>0.75). In the absence of their native microenvironment, primary leukemia cells undergo spontaneous apoptosis ex-vivo. Viability at culture start was always >90% and dropped to a median of 51% (viability range: 17%-90%) after 72h in mono-cultures. Among CLL samples spontaneous apoptosis was not dependent on either IGHV mutation status or any major cytogenetic risk group. Bone marrow stroma cell co-culture conditions protected tumor cells from spontaneous apoptosis (p=8.2e-6, paired t-test). Patient samples with a high degree of spontaneous apoptosis benefited most from co-culture conditions (p=7.2e-10, Pearson correlation). To model interactions of stroma cell conditions and drug-induced apoptosis we established the following linear model: Viability ~ drug-effect + culture-model + drug-effect:culture-model. While activity of some drugs was significantly altered under co-culture conditions, we could also identify drugs with similar activity in mono- and co-cultures. For instance, the activity of common chemotherapeutics (fludarabine: p=0.002 at 0.6µM, cytarabine: p=0.001 at 1.5µM, ANOVA) or bromodomain inhibitors (I-BET-762: p=5.9e-5 at 4.5µM, JQ1: p=1.5e-8 at 1.5µM, ANOVA) was significantly reduced under co-culture conditions. In contrast, PI3K inhibitors idelalisib and duvelisib had a similar activity in mono-culture and stroma co-culture conditions and might represent a starting point to overcome stroma cell mediated drug resistance. In CLL, we identified IGHV mutation status and trisomy 12 as important determinants of response to kinase inhibitors. We confirmed these findings in stroma cell co-cultures, e.g. a better activity of B-cell receptor inhibitors in trisomy 12 and IGHV unmutated CLL. A systematic comparison of ex-vivo drug response pattern in mono- and co-cultures across 171 drug conditions will be presented. Conclusion: Our results suggest that high throughput co-culture drug testing can be robustly performed and provide an unprecedented understanding of how the stroma cell microenvironment and the genetic make-up of tumor cells contribute to drug resistance and sensitivity. Figure: Over 2 million microscopy images were acquired and analysed to assess drug resistance and sensitivity in a co-culture model of primary leukemia and bone marrow stroma cells. blue= Hoechst33342, green=Calcein AM, red=lysosomal dye NIR Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B (p27). Targeted deep sequencing of CDKN1B in a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1B mutations.

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

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. 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 currently undefined. The current work provides insight into the coevolution of genetic and epigenetic aberrations and highlights the influential role of genetic aberrations in the selection of novel methylation patterns. Cancer Discov; 4(3); 348–61. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 259

Sensitive identification of mutations in genes related to the pathogenesis of cancer is a prerequisite for risk-stratified therapies. Next-generation sequencing (NGS) in lymphoma has revealed genetic heterogeneity which makes clinical translation challenging. We established a 454-based targeted resequencing platform for robust high-throughput sequencing from limited material of patients with lymphoma. Hotspot mutations in the most frequently mutated cancer consensus genes were amplified in a two-step multiplex-polymerase chain reation (PCR) which was optimized for homogeneous coverage of all regions of interest. We show that targeted resequencing based on NGS technologies allows highly sensitive detection of mutations and assessment of clone size. The application of this or similar techniques will help the development of genotype-specific treatment approaches in lymphoma.

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 CLL.

Introduction: The impact of mutations and pathway deregulation for drug sensitivity is only partly understood. Here we systematically investigated heterogeneity of drug response and genetic lesions in leukemia and lymphoma using primary tumor cells in order to identify pathway dependencies which can be exploited by rational treatment approaches. Methods: Primary leukemia / lymphoma cells obtained from peripheral blood were characterized with an ex vivo high-throughput drug screening platform in two steps: In an initial screen 2221 different compounds were tested for cytotoxicity on a limited cohort (n=20). 67 promising compounds targeting different pathways were selected for further validation on a larger cohort (n=111; 97 chronic lymphocytic leukemia (CLL), 5 T-prolymphocytic leukemia (T-PLL), 6 non-CLL B-Non-Hodgkin-Lymphoma (B-NHL), mononuclear cells of 3 healthy donors). Heat inactivated human serum was supplemented to mimic micro-environmental conditions. Cell viability was assessed by quantification of ATP (CellTiterGlo®) 48 and 72 hours after drug application. To understand heterogeneous pathway dependencies, drug sensitivity was regressed on genetic profiles. Genetic characterization was performed by FISH, targeted sequencing of recurrent aberrations (BRAF, MYD88, NOTCH1, SF3B1, TP53) as well as whole exome sequencing. Results: We initially focused on the impact of compounds interfering with or dependent on the p53 pathway. Nutlin-3 and fludarabine induced cell death more efficiently in cells with wild-type (n=80) than in CLL with mutated p53 (n=17; Nutlin-3 10µM: 51±18 vs 80±18; fludarabine 10µM: 40±36 vs 67±23 [% viability of untreated control], both p Citation Format: Leopold Sellner, Malgorzata Oles, Mikolaj Slabicki, Carolin Blume, Jennifer Hüllein, Tatjana Stolz, Marina Lukas, Martin Sill, Christopher C. Oakes, Sascha Dietrich, Olaf Merkel, Anna Jauch, Manfred Hensel, Davide Rossi, Katja Zirlik, Jan Dürig, Ingo Ringshausen, Marco Herling, Martina Seiffert, Peter Dreger, Christof von Kalle, Anthony D. Ho, Hanno Glimm, Wolfgang Huber, Thorsten Zenz. Systematic mapping of drug sensitivity in hematological malignancies identifies vulnerability of chronic lymphocytic leukemia with mutant p53. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5557. doi:10.1158/1538-7445.AM2014-5557

Evolution and resulting tumor heterogeneity is currently under investigation for many malignancies since it may explain resistance of tumors to therapies. Pronounced intra-tumor genetic variation has been recently appreciated for solid tumors and leukemias, including chronic lymphocytic leukemia (CLL). Heterogeneous epigenetic alterations, such as DNA methylation, have the potential to add complexity to the leukemic cell population. Studies of the CLL methylome have revealed an abundance of genomic loci that display altered DNA methylation states, including methylation marks showing high prognostic significance. Despite the ubiquity of these epigenetic alterations, the mechanisms and impact of changes to the tumor epigenome in CLL are currently undefined. Here, we have used Illumina 450k arrays and next-generation sequencing to evaluate intra-tumor heterogeneity and evolution of DNA methylation and genetic aberrations in 80 cases of CLL, with 30 cases evaluated at two or more time points. CLL cases exhibit vast inter-patient differences in intra-tumor methylation heterogeneity. Genetically clonal cases maintain low methylation heterogeneity, resulting in up to 10% of total CpGs existing in a monoallelically-methylated state throughout the tumor cell population. Cases with high levels of methylation heterogeneity display a significantly shorter treatment-free time window preceding first therapy (median difference 11 vs. 49 months, P This study articulates the novel finding of epigenetic and genetic coevolution in leukemia and highlights the dominant role of genetic aberrations in the selection of developing methylation patterns. As epigenetics plays a key role in determining cellular phenotypes, we propose that parallel alterations to the genome and epigenome endow expanding subclonal leukemic populations with novel attributes which contribute to acquired therapy resistance. This work also advocates a benefit of monitoring DNA methylation heterogeneity and evolution during CLL disease course. Disclosures: Kipps: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stilgenbauer:Roche: Consultancy, Research Funding, Travel grants Other; Mundipharma: Consultancy, Research Funding.