The use of of kinase inhibitors for personalized medicine holds great promise for the treatment of patients with hematologic malignancies. In order to accomplish this goal, matching kinase inhibitor efficacy to the underlying causative genetic lesions in all patients will be required. One challenge in this process arises from the observation that kinase mutations are much less frequent than kinase dysregulation, indicating that mutations in non-kinases must result in downstream kinase activation. Hence a functional genomics approach is required to understand the genetic etiology of kinase pathway dependence and to match effective kinase inhibitors with genetic biomarkers. Using this functional genomics approach, we have identified TNK2 as a kinase upon which a chronic neutrophilic leukemia patient sample showed dependence for growth and viability. Deep sequencing also identified that this patient sample possessed a mutation in the GCSF receptor, CSF3R. This mutation leads to an increase in the levels of TNK2 and confers sensitivity to the TNK2/SRC inhibitor, dasatinib.
To understand the underlying tyrosine kinase sensitivities in patients with leukemia we have employed siRNA and inhibitor screens in parallel with deep sequencing of ∼1800 kinase-associated genes. Genetic variants were prioritized for validation based on proximity to functional targets in protein interaction network databases and subsequently confirmed via Sanger sequencing. Oncogenic capacity of these mutations was assessed in an interleukin-3 (IL-3) dependent Ba/F3 murine pro-B cell system. Expression levels of CSF3R and TNK2 were assessed by immunoblot analysis in Ba/F3 and HEK293 cells. A mouse bone marrow colony assay was performed to assess the sensitivity of CSF3R-mutant cells to dasatinib.
Using this functional genomics approach we examined cells from a patient with chronic neutrophilic leukemia using siRNA and kinase inhibitor screens and observed hyper-sensitivity to siRNA directed against the kinase TNK2 and to the TNK2/SRC inhibitor dasatinib. Sequencing of ∼1800 kinase associated genes in this patient sample revealed a one-base pair insertion that results in a premature stop and truncation of the cytoplasmic domain of the GCSF receptor, CSF3R. This CSF3R mutation transformed Ba/F3 cells to IL-3 independence, and induced an upregulation of TNK2. The CSF3R truncation mutation also led to increased formation of colonies compared with wild type in a mouse bone marrow colony assay. Furthermore, colony formation was inhibited by dasatinib, indicating that dasatinib is a potential therapeutic for CSF3R truncation mutations. CSF3R is known to promote neutrophil proliferation and differentiation and similar truncation mutations in CSF3R have been previously found in patients with severe congenital neutropenia, a disease that can transform to acute myeloid leukemia. Published reports of CSF3R mutations in de novo leukemia have been scarce, however, subsequent analysis of sequencing data sets has identified recurrent CSF3R truncation mutations in patients with neutrophilic/myeloid leukemia.
This work identifies CSF3R truncation mutations as a leukemogenic mechanism in patients with de novo myeloid leukemia. CSF3R truncation mutations elevate TNK2 levels and confer sensitivity to the TNK2/SRC inhibitor, dasatinib. CSF3R truncation mutations are therefore promising novel biomarkers for dasatinib treatment in patients with myeloid leukemia.
Off Label Use: Dasatinib, for off-label use in MPN. Fleischman:Incyte: Speakers Bureau. Druker:Bristol-Myers Squibb: Clinical Trial Funding Other.
Asterisk with author names denotes non-ASH members.