Targeting of ITK-SYK fusion oncogene through in-silico generated and biologically validated ITK-PH domain modulators

Abstract A subset of human Peripheral T cell Lymphomas, Not Otherwise Specified (PTCL-NOS), harbors a chromosomal translocation of interleukin-2 (IL-2) inducible T cell kinase (ITK) and spleen tyrosine kinase (SYK), giving rise to the composite ITK-SYK protein. ITK, a TEC family member kinase, plays an essential role in TCR and CD28 signaling. SYK signaling pathway is more broadly linked to activation of diverse lymphoid and myeloid cell types. ITK-SYK fusion protein consists of the PH domain of ITK and the SYK kinase domain allowing localization to the signalosome complex at the membrane via the PH domain and constitutive activity in T cells. Given the ubiquitous activity of SYK, it is predicted that targeting the ITK-PH domain of the fusion protein with small molecules can be a precise and less toxic approach to suppress ITK-SYK+PTCL-NOS in vivo. To this end, we performed a computational screen of small molecule libraries to identify candidate ITK-PH domain interacting compounds. A thermal shift assay was used to verify the specificity of binding of candidates to the target protein domain. Cellular assays tested small molecule activity in human and mouse T cells constitutively expressing the ITK-SYK fusion protein. We showed that the first-pass small molecule candidates specific to ITK-PH domain inhibit cytokine production driven by ITK-SYK fusion protein in human and mouse T cells. Our results demonstrate the efficiency of the virtual screening to identify drug candidates with biological activity in lymphocytes. Development of drugs that target ITK-PH domain is a rationale strategy towards first-in-class therapeutics for this subset of PTCL-NOS with very poor prognoses.