Mechanisms of Resistance and Therapeutic Targets in T-LGL Leukemia

ABSTRACT T-cell large granular lymphocytic leukemia (T-LGLL) is an incurable, underdiagnosed proliferation of clonal CD8+ cytotoxic T-lymphocytes that results in severe neutropenia and anemia, with resultant recurring infections, transfusion dependence, and death. There are no FDA-approved therapies for T-LGLL, and current immune- suppressive based therapies have marginal efficacy. Even if a response is attained with current therapies, patients shift between immune-suppressive agents, are subject to the adverse effects of these drugs, and for those with any effect, indefinite therapy is required. Urgent investigation into the pathogenesis of T-LGLL and development of rational, targeted therapies are needed. T-LGLL is a cytokine-dependent disease, driven by interleukin-15 (IL-15) which has been identified as the ‘master switch’ crucial to induce and potentiate T-LGLL. IL-15 induces pathogenesis in T-LGLL through up-regulation of STAT3, with resultant decrease in Fas/Fas- Ligand mediated apoptosis with resultant T-LGLL proliferation and cytopenias. We evaluated BNZ-1, a γc-inhibiting peptide that blocks IL-15, in patients with T-LGLL in only the second major, multicenter trial completed in T-LGLL. Clinical responses were observed in 20% of patients, but in vivo data revealed that nearly all patients had dramatic apoptosis of T-LGLL cells 24 hours post-BNZ-1, though apoptosis persisted only in responding patients at day 29. These results (in revision, Blood), provide in vivo proof that BNZ-1 induces T- LGLL cell apoptosis, and that T-LGLL cells are dependent on IL-15 in vivo in patients. To evaluate the mechanisms of resistance to IL-15 deprivation induced cell death, we performed single-cell RNA sequencing (scRNAseq) on samples from a non-responder. We identified the emergence of unique T-LGLL sub-populations on day 29, with corresponding up-regulation of anti-apoptotic pathways PI3K and NF-kB; implicating up- regulation of these alternate pathways in T-LGLL as key mechanisms of resistance of T-LGLL to IL-15 deprivation. Yet, validation in a larger sample set is needed to confirm these findings and identify therapeutic targets. To address these translational and clinical gaps, we will perform scRNAseq and single-cell T-cell receptor sequencing (scTCRseq) on remaining clinically annotated samples from 3 responders and 3 non- responders from the BNZ-1 trial to test our hypothesis that resistance to IL-15 deprivation with BNZ-1 is caused by expansion of resistant T-LGLL populations with upregulation of anti-apoptotic pathways (e.g. NF-kB, PI3K). In Aim 1, we will perform scRNAseq at serial timepoints (baseline, 24 hours post-BNZ-1, 29 days post BNZ-1), to identify key anti-apoptotic pathways, genes, and therapeutic targets in resistant populations. In Aim 2, we will apply scTCRseq to evaluate T-LGLL clonal populations (clonotypes), and determine the impact of IL-15 deprivation on gene expression and anti-apoptotic pathways in these groups. Upon completion, we will gain crucial mechanistic insights on the critical genes and anti-apoptotic pathways in T-LGLL that lead to resistance to IL-15 deprivation and identify therapeutic targets for a future NCI R01 proposal and BNZ-1 combinatorial trial.