Abstract Richter's Syndrome (RS) arises from chronic lymphocytic leukemia (CLL) and has long been acknowledged as presenting a bleak clinical outlook for CLL patients. New treatment combinations, however, incorporating novel agents suggest the feasibility of achieving deeper and longer-lasting remissions in patients with RS. These include treatment with immune checkpoint blockade (ICB; -PD1 therapy) together with ibrutinib, and combination of the BCL2 inhibitor venetoclax with chemotherapy. These experiences provide encouraging developments for an entity hitherto considered a death sentence for CLL patients. Despite these promising therapeutic developments, little information is available regarding the determinants of clinical response to these therapies. The recent increase in clinical investigation of novel therapeutic agents for RS now provides an opportunity to more consistently collect biospecimens. Moreover, the recent availability of novel sequencing capabilities, analytic tools and animal models (generated by our efforts over this past funding period) now enable us to maximally extract information about the diverse molecular and phenotypic features of RS from primary patient material. We hypothesize that therapeutic response and resistance to RS therapy are shaped by both tumor-intrinsic and -extrinsic features, and that these features underlie the differential sensitivity to novel agents between RS and CLL. Herein, we will undertake in-depth analysis of specimens collected on therapeutic trials, focusing on longitudinal analyses of both RS specimens and their microenvironment, and also functional validation of these findings in a novel animal model. These studies are supported by strong interactions with Projects 1 and 3, and each of the individual Cores. Our aims are thus to: (i) Define the RS-intrinsic features that govern responses to RS therapies. We will evaluate the molecular characteristics of serially collected RS specimens from patients treated with: (i) BCL2 inhibitor-based and (ii) ICB-based therapy, and link these attributes with clinical features to identify markers/pathways predictive of response or resistance to therapy. (ii) Delineate the key components of the RS immune microenvironment impacting therapeutic response. New immunogenomic approaches enable us to gain fresh mechanistic insights directly from analyzing patient samples. We will perform scRNA-seq profiling of marrow or lymph node-infiltrating immune cells from RS responders and nonresponders per therapy (enrolled on the aforementioned trials) to systematically characterize the composition and functional state of cellular populations participating in response or resistance to novel RS therapeutic combinations. Finally, we will: (iii) Model differential therapeutic sensitivity of RS to -PD1 therapy compared to CLL. Using novel genetically engineered mouse models that reflect CLL or RS disease, we will perform in vivo treatment studies and functional/phenotypic a...