In cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset, and progression. The cancer epigenome is composed of the epigenetic patterns present in ...the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations.
We developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation.
Methyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level.
Methyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.
A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different ...cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.
Deregulated transcriptional control caused by aberrant DNA methylation and/or histone modifications is a hallmark of cancer cells. In chronic lymphocytic leukemia (CLL), the most common adult ...leukemia, the epigenetic ‘landscape’ has added a new layer of complexity to our understanding of this clinically and biologically heterogeneous disease. Early studies identified aberrant DNA methylation, often based on single gene promoter analysis with both biological and clinical impact. Subsequent genome-wide profiling studies revealed differential DNA methylation between CLLs and controls and in prognostics subgroups of the disease. From these studies, it became apparent that DNA methylation in regions outside of promoters, such as enhancers, is important for the regulation of coding genes as well as for the regulation of non-coding RNAs. Although DNA methylation profiles are reportedly stable over time and in relation to therapy, a higher epigenetic heterogeneity or ‘burden’ is seen in more aggressive CLL subgroups, albeit as non-recurrent ‘passenger’ events. More recently, DNA methylation profiles in CLL analyzed in relation to differentiating normal B-cell populations revealed that the majority of the CLL epigenome reflects the epigenomes present in the cell of origin and that only a small fraction of the epigenetic alterations represents truly CLL-specific changes. Furthermore, CLL patients can be grouped into at least three clinically relevant epigenetic subgroups, potentially originating from different cells at various stages of differentiation and associated with distinct outcomes. In this review, we summarize the current understanding of the DNA methylome in CLL, the role of histone modifying enzymes, highlight insights derived from animal models and attempts made to target epigenetic regulators in CLL along with the future directions of this rapidly advancing field.
Abstract
Advancements in state-of-the-art molecular profiling techniques has resulted in better understanding of pediatric cancers and their drivers. Conversely, it also became apparent that ...pediatric cancers are much more heterogeneous than previously thought. Many new types and subtypes of pediatric cancers have been identified with distinct molecular and clinical characteristics. However, for most newly recognized entities there is no specific treatment available yet. The ITCC-P4 consortium is a collaboration between many academic centers across Europe and several pharmaceutical companies involved in preclinical testing, with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers and to use them for in vivo testing of novel mechanism-of-action based treatments. Currently, 340 models are fully established, including 87 brain and 253 non-brain tumor models, together representing different tumor types both from primary (113) and relapsed (92)/metastatic disease (42). 252 of these models have been fully molecularly characterized, representing 18 pediatric cancer entities and 43 different subtypes. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number, transcriptome analysis and methylation profiling we have observed that the molecular profile of most PDX models closely mimics their original tumors. Clonal evolution of somatic variants was only observed in some PDX-tumor pairs or so between disease states. Somatic copy number variant analysis highlights specific alterations for instance MYB, MYC, MYCN, NTRK3, PTEN loss differently distributed between PDX-patient tumor pairs in high-grade gliomas. Overall, our results show that we have established >250 PDX models of solid pediatric cancers, that well represents the disease spectrum and that is currently being used for in vivo testing of standard of care drugs and targeted small molecules. Treatment responses will be directly linked to molecular data to identify potential biomarkers for prioritization or deprioritization of individual, patient-specific specific drugs.
Normal B cells undergo extensive epigenetic programming during normal differentiation and distinct B cell differentiation stages represent unique DNA methylation patterns. Chronic Lymphocytic ...Leukemia (CLL) originates from rapidly differentiating B cells and their DNA methylation signature is stably propagated in CLL. Consequently, CLL methylome data can be used to infer the putative cell-of-origin (COO) for each individual CLL case. We define the COO of CLL as the cell that has acquired a first oncogenic hit and which will initiate tumorigenic growth if one or more additional hits have been acquired. This means that two factors contribute to the epigenetic profile of CLL cells: first, the epigenetic profile of the founder B cell at the time of malignant transformation and second, CLL-specific epigenetic alterations that are acquired during leukemogenesis and progression of the disease. Previous studies using peripheral blood CD19+ B cells as a reference for aberrant methylation calls completely neglected the massive epigenetic programming that occurs during normal B cell differentiation. Thus, novel strategies aiming at identifying truly CLL-specific methylation changes considering the highly dynamic methylome during normal B cell differentiation were urgently needed.
Here we outline a new analytical framework to delineate CLL-specific DNA methylation. We demonstrate how this approach can be applied to detect epigenetically deregulated transcripts in CLL. Firstly, we modeled the epigenome dynamics occurring during normal B cell differentiation using linear regression. The DNA methylomes of CLL cells were then precisely positioned onto the normal B cell differentiation trajectory to define the closest normal B cell methylome for every CLL patient, the COO. The epigenome of the COO then served as a reference for aberrant DNA methylation calls. We dissected two categories of CLL-specific methylation events: those occurring at sites undergoing epigenetic programming during B cell differentiation and those that normally do not change during B cell differentiation. The first group was further subdivided into class A and B, displaying exaggerated methylation loss or gain, respectively, and class C showing both hyper- and hypomethylation relative to the normal differentiation. The second group was classified into class D displaying hypo- and class E showing hypermethylation. Overall, only 1.6% of the CpG-sites (7,248 CpGs) represented on the Illumina 450k array were affected by disease-specific methylation programming, mostly hypomethylation (6,680 CpGs).
Next, the molecular programs underlying the CLL-specific methylation patterns were investigated. We tested enrichment of chromatin states and of transcription factor binding sites (TFBS) as identified in an immortalized B cell line (GM12878). This indicated that disease-specific methylation events target transcriptionally relevant cis-regulatory elements in CLL (enhancers, weak and poised promoters and insulator regions). In line with this, CLL-specific differentially methylated regions affected TFBS associated with signaling pathways known to be important in normal B-cell differentiation (i.e. BATF, EBF1). We also observed altered methylation at CTCF binding sites suggesting their involvement in CLL pathogenesis.
In the present work, we dissected CLL methylomes to distinguish between normal B cell differentiation-associated methylation patterns and CLL-specific methylation events. We showed that this approach is indispensable to identify key pathogenic events driving CLL pathogenesis. The relevance of our approach was demonstrated by contrasting the number of epigenetically deregulated miRNAs and protein-coding genes to those determined with a classic analysis using CD19+ B cells as controls. This highlights the extent of overcalling of CLL-specific methylation patterns in previous studies (~30-fold for protein-coding genes and ~10-fold for miRNAs) and stresses the importance to consider normal differentiation trajectories for the identification of aberrant DNA methylation events. Here we propose 11 protein-coding genes (e.g. DOK2, CLLU1) and 4 miRNAs (e.g. miR-486, miR-195) as being epigenetically deregulated in CLL. Our analytical approach provides a general framework for the identification of disease-specific epigenomic changes that should be applicable to other cancers in the future.
Küppers:the Takeda Advisory Board: Membership on an entity's Board of Directors or advisory committees. Stilgenbauer:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
•NTRK gene fusions are targetable oncogenic drivers independent of tumor type.•Selective TRK inhibitor larotrectinib is active in tumors with NTRK gene fusions.•A diverse array of methods can be used ...to detect NTRK gene fusions.•The most common testing approach was RNA-based next-generation sequencing.•54 different NTRK fusion partners identified in 225 patients with TRK fusion cancer.
NTRK gene fusions are targetable oncogenic drivers independent of tumor type. Prevalence varies from highly recurrent in certain rare tumors to <1% in common cancers. The selective TRK inhibitor larotrectinib was shown to be highly active in adult and pediatric patients with tumors harboring NTRK gene fusions.
We examined the techniques used by local sites to detect tumor NTRK gene fusions in patients enrolled in clinical trials of larotrectinib. We also report the characteristics of the detected fusions in different tumor types.
The analysis included 225 patients with 19 different tumor types. Testing methods used were next-generation sequencing (NGS) in 196 of 225 tumors (87%); this was RNA-based in 96 (43%); DNA-based in 53 (24%); DNA/RNA-based in 46 (20%) and unknown in 1 (<1%); FISH in 14 (6%) and PCR-based in 12 (5%). NanoString, Sanger sequencing and chromosome microarray were each utilized once (<1%). Fifty-four different fusion partners were identified, 39 (72%) of which were unique occurrences.
The most common local testing approach was RNA-based NGS. Many different NTRK gene fusions were identified with most occurring at low frequency. This supports the need for validated and appropriate testing methodologies that work agnostic of fusion partners.
Abstract
Thanks to state-of-the-art molecular profiling techniques we by now have a much better understanding of pediatric cancers and what is driving them. On the other hand, we have also realized ...that pediatric cancers are much more heterogeneous than previously thought. Many new types and subtypes of pediatric cancers have been identified with distinct molecular and clinical characteristics. However, for many if not most of these new types and subtypes there is no specific treatment available, yet. In order to develop specific treatment protocols and to increase survival rates for pediatric cancer patients further, both at diagnosis and relapse/metastasis, we need a large collection of well-characterized preclinical models representing all the different types and subtypes. These models can be used for preclinical drug testing to prioritize the pediatric development of anticancer drugs that would be best targeting pediatric tumor biology. The ITCC-P4 consortium, which is a collaboration between many academic centers across Europe, several companies involved in in vivo preclinical testing, and ten pharmaceutical companies, started in 2017 with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers and to use them for in vivo testing of novel mechanism-of-action based treatments. Currently, 340 models have been fully established, including 87 brain tumor models and 253 non-brain tumor models, together representing many different tumor types both from primary and relapsed/metastatic disease. Out of these 340 models, 252 have been fully molecularly characterized, most of them together with their matching original tumors, and almost of all these models are currently being subjected to in vivo testing using three standard of care drugs and six novel mechanism-of-action based drugs. In this presentation, an update on the current status of the ITCC-P4 platform and the data we collectively have generated thus far will be presented.
Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ...ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking may be a more widespread consequence of translocations where no oncofusion product was identified, including e.g. t(1;3) or t(4;12) AML.Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking may be a more widespread consequence of translocations where no oncofusion product was identified, including e.g. t(1;3) or t(4;12) AML.
Abstract Cancer remains the main cause of disease-related death in childhood. Pediatric tumors are characterized by a low mutational burden and high intertumoral heterogeneity, with multiple subtypes ...compared to their adult counterparts. The lack of access to many innovative therapies remains one of the main challenges in the pediatric oncology, especially for the 25% of patients who experience relapses. In this context, the need for the development of a well characterized collection of pediatric models, to provide large scale preclinical testing, is capital for the subsequent identification and prioritization of promising novel therapeutic options. The EU funded “Innovative Therapies for Children with Cancer-Pediatric Preclinical Proof-of-Concept Platform” (ITCC-P4) consortium is a unique public-private collaborative project consisting of academic and industrial partners that aimed at establishing a collection of >400 patient-derived xenograft (PDX) models representing the most common high-risk pediatric cancers. The project involved various aspects of model development including the thorough molecular and pharmacological characterization. XenTech’s participation was focused on the development and preclinical in vivo drug testing of Ewing sarcoma (n=17), hepatoblastoma (n=10), rhabdoid tumors (n=6), synovial sarcoma (n=2), rhabdomyosarcoma (n=2) and other tumors (n=6), as part of overall cohort. PDXs were obtained by transplantation of post-surgery tumor specimens, either by grafting tumor fragments into the interscapular region or subcutaneously in the right flank of nude, NOD-Scid or NOD-Scid gamma mice. Tumor xenografts were amplified by serial transplantation, and tissue samples were retained at early passages for molecular characterization. Fragments from established PDX models where frozen to generate a revivable ITCC-P4 PDX collection. Then, proof-of-concept drug testing was conducted, in a single mouse trial format: each tumor type (n=X PDX models) was treated with a dedicated panel of Standard-of-Care (SoC;n=3) and novel targeted therapies (n=6), or combinations of 2 or 3 novel targeted therapies; for each PDX model n=1 mouse being included per treatment. All molecular and drug-testing data obtained by the different partners are being centralized in the R2 repository (https://r2.amc.nl), providing a powerful tool for data integration, visualization and interpretation of the results. A unique collection of well characterized pediatric PDX models derived from the most relevant pediatric tumor types was enabled by a strong public-private collaborative project. This large cohort is now available for preclinical testing of novel therapeutic agents within a non-for-profit spinoff company, ITCC-P4 gGmbH (www.itccp4.com), offering new perspectives to the identification of promising treatment options for children with cancer. Citation Format: Emilie Indersie, Sophie Branchereau, Brice Fresneau, Christophe Chardot, Didier Surdez, Alexandra Saint-Charles, Maria Eugénia Marques da Costa, Ángel M. Carcaboso, Katia Scotlandi, Massimo Moro, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Louis Chesler, Chris Jones, Beat Schäfer, Marco Wachtel, Johannes Gojo, Walter Berger, Christina Guttke, Maureen Hattersley, Frédéric Colland, Ashley Strougo, Dennis Gürgen, Jens Hoffmann, Julia Schueler, Pablo M. Aviles, María José Guillén, Aniello Federico, Apurva Gopisetty, Justyna Anna Wierzbinska, Andreas Schlicker, Sara Colombetti, Olaf Heidenreich, Fatima Iradier, Nicole Huebener, Natalie Jäger, Jan Koster, Marcel Kool, Gudrun Schleiermacher, Jan J. Molenaar, Birgit Geoerger, David J. Shields, Hubert N. Caron, Louis F. Stancato, Stefan M. Pfister, Gilles Vassal, Eva-Maria Rief, Olivier Déas. ITCC-P4, a preclinical proof-of-concept drug testing platform as a tool for pharmacological screening in pediatric tumor models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5469.
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