Mutationally Activated TYK2 From T-ALL Specimens Exhibits Tranformative Capacity in Cell Lines and Primary Cell Models and T-Lineage Expansion in Mice

First-line therapy for T-cell acute lymphoblastic leukemia (T-ALL) fails in 25% of children and more than 50% of adults despite improvements in clinical outcomes due to intensification of therapy. Development of new and improved therapeutic regimens will require 1) new gene targets relevant to the pathogenesis of T-ALL, 2) targeted therapeutic agents with activity against those gene targets, and 3) effective models for pre-clinical development of these gene targets and therapies. Current in vivo models of T-ALL recapitulate derangements in the Notch signaling pathway but modeling alternative pathways of T-ALL leukemogenesis are lacking. We report that tyrosine kinase 2 (TYK2) is vital for the maintenance of T-ALL growth and viability, and we have developed a mouse model based on an activating mutation of TYK2 found in a T-ALL specimen. This mouse model will be useful for further exploration of the biological role of TYK2 in T-ALL pathogenesis as well as pre-clinical evaluation of TYK2 inhibitors as components of T-ALL therapy regimens. Methods: Sequence analysis of TYK2 in T-ALL cell lines and patient samples revealed a diversity of TYK2 point mutations. These mutations were located just outside of or within the FERM domain (V15A, G36D, G36R, S47N, R425H), the pseudokinase domain (V731I), and the kinase domain (E957D, and R1027H). Identified point mutant were cloned and introduced into Ba/F3 cells for assessment of transformative capacity. The point mutant E957D was ectopically expressed via retroviral transduction in primary murine bone marrow hematopoietic progenitor cells and co-cultured on OP9-DL1 stromal cell layers to assay the effect of this mutation on in vitro T-lineage development and expansion. A murine transduction/transplantation model was developed to investigate the effect of TYK2 E957D in vivo. Results: Introduction of the point mutants into Ba/F3 cells revealed that 7/8 (all except R425H) transformed cells to factor-independent growth. Transformed cells displayed constitutive phosphorylation of TYK2, STAT1, STAT3, and ERK1/2. Treatment of transformed cells with JAK Inhibitor I revealed extreme sensitivity of cells to this TYK2 kinase inhibitor as well as reduction of TYK2 AND STAT1 phosphorylation. Ectopic expression of TYK2 E957D enhanced in vitro T cell development and subsequent expansion of mature T cells on OP9-DL1 stromal cell layers as compared to TYK2 WT or empty vector. Fifty percent of mice transplanted with bone marrow ectopically expressing TYK2 E957D died at 7 months post-transplant with evidence of hematologic malignancy characterized by massive expansion of the thymus and infiltration of the bone marrow with a monomorphic population of cells. In comparison, none of the mice transplanted with wild-type TYK2 were adversely affected. Conclusions: We demonstrate that mutations in TYK2 in T-ALL can confer constitutive activity to the TYK2 signaling pathway, which can play a functional role in the pathogenesis of T-ALL. Ectopic expression of TYK2 E957D leads to enhanced expansion of T cells and mice expressing this point mutant in the hematopoietic compartment die from hematologic malignancy with grossly enlarged thymuses after a long period of latency. This model could complement the arsenal of currently available T-ALL models and may represent and the latency of disease may allow a search for cooperating elements leading to transformation. Small-molecule kinase inhibitors with activity against TYK2 reduce the growth of these TYK2-dependent cells. Hence, we have defined a new, oncogenic signaling pathway in T-ALL cells that can be readily modulated by kinase inhibitors. Cumulatively, these data offer new therapeutic options for T-ALL patients while simultaneously providing novel insights into the biology of T-cells and the TYK2-STAT1 signaling cascade.

Druker:Novartis:; Bristol-Myers Squibb:; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Cylene: Consultancy.