Project Summary/ Abstract Peripheral T cell lymphoma (PTCL) represents a group of aggressive blood cancers derived from mature T cells, and remains as a high unmet clinical need with poor prognosis and lack of standards of care and effective treatment. Recent exome sequencing in PTCL patients has unveiled a frequent co-occurrence of mutations in an epigenetic modifier (TET2) and a small GTPase (RHOA). This discovery heralds the advent of a molecular era in the dissection of novel pathogenic mechanisms underlying T cell lymphoma. The PI has shown that genetic depletion of murine Tet2 alone in blood cells causes biased differentiation of hematopoietic stem and progenitor cells (HSPCs) toward the myeloid lineage, but is insufficient to cause lymphoid neoplasms. A second hit, such as RHOA-G17V frequently found in PTCL, is required to promote full-blown malignancies. To meet the immediate need for animal models of PTCL, The PI has generated a genetically modified rodent model mimicking the PTCL-associated genotype with genetic lesions in both TET2 and RHOA. This transgenic mouse model is well suited to study PTCL because it developed T cell lymphoma in peripheral lymphoid organs and recapitulated hallmark phenotypes as seen in PTCL patients. These exciting findings laid a strong scientific foundation to hypothesize that: co-existing TET2 and RHOA mutations in CD4 T cells contribute to the pathogenesis of PTCLs by (i) impairing DNA hydroxymethylation and gene transcription to predispose T cells for pre-malignant status (Aim 1; with a mechanistic emphasis on R-loop accumulation and aberrant RNA polymerase II pausing in key genes involved in phosphoinositide metabolism), and (ii) disrupting the Rho GTPase signaling to abnormally activate pro-oncogenic pathways for malignant transformation (Aim 2; with a prioritized focus on the PI3K/Akt signaling). The team has presented compelling evidence in pilot studies that lends strong support to the central hypotheses and the feasibility of our approach. Technical innovations include a unique mouse model that reflects the PTCL-associated genotype and disease hallmarks, as well as a set of novel molecular tools tailored for precise epigenome mapping/editing and optogenetic control of small GTPase signaling. These tools allow the team to overcome a major impediment to studies of epigenotype- phenotype causal relations in pre-malignant and malignant T cells. This research is also conceptually innovative as it introduces a previously underappreciated dimension for studying the epigenetic regulatory mechanisms, as well as aberrant GTPase signaling, that drive lymphomagenesis. The proposed studies will likely illuminate how somatic mutations in epigenetic and GTPase signaling pathways cooperatively contribute to the initiation, transformation and progression of lymphoma. From a translational perspective, the findings may reveal novel molecular targets and pathways for therapies against lymphoma.