RNAi Screen Reveals TYK2 as a Novel Therapeutic Gene Target In T-Cell Acute Lymphoblastic Leukemia

Tyrosine kinases are widely implicated in the genesis of hematologic malignancies and tyrosine kinase inhibitors have achieved remarkable clinical outcomes. However, most cancer patients are ineligible for this form of therapy because we lack knowledge of the oncogenes driving the growth of their malignant cells. In particular, 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, and these relapsed patients have a very poor prognosis. Most current T-ALL therapy regimens lack tyrosine kinase inhibitors suggesting that addition of effective kinase inhibitors to T-ALL therapy may improve clinical outcomes. We report that tyrosine kinase 2 (TYK2), signaling through signal transducer and activator of transcription 1 (STAT1), is vital for the maintenance of T-ALL growth and viability, suggesting that incorporation of TYK2 inhibitors into T-ALL therapy regimens may improve patient outcomes.

T-ALL diagnostic specimens were collected with informed consent and neoplastic cells were isolated on a Ficoll gradient. Cells were treated with an siRNA library that individually silences each member of the tyrosine kinase gene family and cultured for 4 days at which time an MTS assay was used to assess cell viability. DNA was isolated from 16 T-ALL cell lines as well as 50 T-ALL patient specimens. TYK2 was sequenced and identified point mutant were cloned and introduced into Ba/F3 cells for assessment of transformative capacity. Transformed Ba/F3 cells were lysed and immunoblotted with antibodies specific for total or phospho-TYK2, STAT1, STAT3, and ERK1/2.

RNAi functional profiling of cells from a T-ALL patient specimen demonstrated dependence on TYK2 for cell viability. 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). Introduction of these point mutants into Ba/F3 cells revealed that 7/8 (all except R425H) transformed these cells to factor-independent growth. Analysis of whole cell extracts from transformed cells revealed 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.

We demonstrate for the first time that the TYK2-STAT1 pathway is constitutively active and required for viability in the setting of T-ALL, sometimes due to gain-of-function point mutations in TYK2. 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. Interestingly, this pathway has previously been associated with tumor suppression, therefore, our findings indicate that activation of this pathway can have varying effects depending on cellular context. 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:Molecular MD: Equity Ownership.