Dissecting regulatory microRNA-mRNA networks at the single-cell level in T-cell large granular lymphocytic leukemia (T-LGLL)

• Applicant: Dr. Natali Pflug
• Subject Area: Hematology, Oncology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497803382

T-cell large granular lymphocytic leukemia (T-LGLL) is a rare malignant chronic lym-phoproliferative disease. It is characterized by a clonal expansion of cytotoxic, often auto-immune reactive, mature T-cells. Gain-of-function mutations of the JAK/STAT signaling pathway, predominantly of STAT3, represent the molecular hall-mark of a fraction of T-LGLL cases. However, as such genomic aberrations are only detected in a subset of cases, our molecular and mechanistic disease concept of T-LGLL is yet incomplete. Diagnosis and management of T-LGLL is a challenge for even the largest academic centers. Treatment options in T-LGLL are limited and non-curative only. We further lack reliable predictive and prognostic markers. Up to now, gene-regulatory networks, particularly changes of the miR-ome have not been addressed in T-LGLL. Moreover, genomic studies at the single-cell level are of utmost importance, as T-LGLL samples represent an often oligo-clonal composition of reactive pre-malignant expansions coexisting with fully transformed (sub)clones. Studying miR regulatory networks at the single-cell level addresses these voids and will lead to a deeper understanding of the underlying pathogenetic mechanisms, aiming at the design of tailored treatment strategies. In this project, we will continuously feed our comprehensive database and corresponding biobank of T-LGLL patients (Aim 1). Using this unique collection of T-LGLL samples, we will study global profiles of miR and mRNA expression as well as miR-target gene circuits of T-LGLL cases that are being generated by bulk miR and mRNA sequencing of T-LGLL cells and corresponding T-cell controls of healthy donors as part of the supplementing EU JAKSTAT-TARGET consortium(Aim 2). Furthermore, we will investigate the clonal hierarchies of candidate miRs and their regulatory networks identified in Aim 2, by per-forming miR-mRNA co-sequencing of immunophenotypically pre-sorted single cells from T-LGLL patients as well as healthy donors (Aim 3). We will further align the identified hierarchies of miR-ome/transcriptome networks with immunophenotypic subpopulations and clinico-pathological information of these patients, enabling a better basal un-derstanding of T-LGLL’s complex (sub)clonal composition. Finally, we will establish a model of clonal trajectories, by applying this half-cell sequencing approach to sequentially collected samples from individual patients (Aim 4). For this, we selected T-LGLL patients with spontaneous changes in disease characteristics and patients with (-out) treatment responses. Within this proposal, we apply for funding to finance additional sequencing studies at the single-cell level (Aim 3 and Aim 4). Respective complementary work (Aim 1 and Aim 2) is funded via the German CLL Study Group (GCLLSG) and by the EU/BMBF.

• DFG Programme: Research Grants