Project Summary/Abstract Richter’s syndrome (RS) is a critical complication of up to 10% of chronic lymphocytic leukemia (CLL) patients, which develops as an aggressive transformation into a diffuse large B cell histology, and is mostly refractory to existing therapies. RS pathogenesis remains largely unknown and cellular and mouse models for molecular studies are limited. To address this challenge, Dr. ten Hacken has developed novel human-genetics inspired mouse models through CRISPR-Cas9 multiplexed B-cell editing, recapitulating CLL transformation into RS. Already through her preliminary studies, Dr. ten Hacken demonstrated how selected mutational co-occurrences facilitate disease transformation, and are associated to distinct transcriptional changes and therapeutic vulnerabilities—work that is presently near completion. Dr. ten Hacken is now planning to introduce a new set of mutations in genes involved in epigenetic programming of B cells, which were identified as putative RS drivers in recent human genomic analyses. In Aim 1, Dr. ten Hacken will introduce epigenetic drivers in mice to assess the impact of the selected alterations (and their combinations) on CLL transformation. As part of this Aim, Dr. ten Hacken will also assess the transcriptional and genetic faithfulness of these models to human disease. In Aim 2, Dr. ten Hacken will functionally characterize the modeled gene mutations, while dissecting changes in the epigenetic landscape underlying transformation of CLL into RS. Epigenetic dependencies identified through these studies will be cross-compared with human RS datasets and validated in human primary samples with similar genetic make-ups. In Aim 3, Dr. ten Hacken will perform in vitro and in vivo preclinical testing of a panel of agents (comprehensive of chemotherapy and novel targeted agents) in order to design mutation (or co- mutation) specific treatment strategies. To carry out the proposed work, Dr. ten Hacken has enlisted collaborators who are experts in computational biology, systems immunology, mouse pathology, molecular pharmacology, biostatistics, functional genomics and epigenetics. Dr. ten Hacken has outlined a 3-year career development plan that will allow her to foster her personal professional development (including leadership, grant writing, negotiation and communication skills), and to gain additional scientific training in bioinformatics and biostatistics. Dr. ten Hacken’s independent research program will be focused on translational research in hematological malignancies, with the longer-term objective of undertaking clinical correlative research and functional genomic analyses of other lymphoid and myeloid malignancies. Through her proposed work, Dr. ten Hacken anticipates to contribute 2 high-impact manuscripts within the award term. She will present yearly at international conferences, and will be ready for her first R01 submission towards the end of Year 2. The proposed studies are expected to provide cri...