Development and Repurposing of Small-Molecule Kinase Inhibitors to Target Novel Leukemogenic TNK2 Mutations

BACKGROUND:

A tremendous amount of information now exists detailing the genetic alterations present in leukemia cells. However, our understanding of the functional significance of many of these genetic events remains incomplete. One major challenge involves the identification of pharmacologically targetable mutations for the design and implementation of targeted therapy strategies. To this end, we are using an algorithm called HitWalker (Bottomly, et al. 2013), which can prioritize gene mutations based on functional data, thus revealing the pharmacologic vulnerabilities of leukemia cells from individual patients. This analysis revealed oncogenic mutations in the tyrosine kinase, TNK2 (aka ACK1), which can be targeted with existing small-molecule inhibitors.

METHODS:

Kinase inhibitor screens were run on primary patient samples, and the operationally important kinases underlying drug sensitivity patterns were predicted based on an algorithm that harnesses the known efficacy of each drug against the kinome (algorithm described in Tyner, et al. 2013). The results of the drug screens were integrated with high throughput sequencing data using the HitWalker algorithm such that mutated genes were ranked according to closest associations with kinases implicated by the functional screening data. Oncogenicity of TNK2 mutations was tested by Ba/F3 cytokine independent growth assays. To develop TNK2 inhibitors, we searched our in-house database for inhibitors that exhibited potent and selective binding to TNK2. The kinase selectivity of the compounds was evaluated by screening against a diverse panel of 241 kinases using a chemical proteomic approach, KiNativ.

RESULTS:

The integrated functional genomic analysis revealed TNK2 mutations in AML and CMML leukemia samples, which ranked highly as potential therapeutic targets. The TNK2 point mutations exhibited transformative capacity, and transformed cells exhibited sensitivity to the multi-kinase inhibitor dasatinib, which antagonizes TNK2 kinase activity. In addition, we observed sensitivity to novel TNK2 inhibitors, XMD8-87 and XMD16-5, which possess greater specificity for TNK2. XMD8-87 in particular demonstrated a high degree of selectivity for TNK2, and is more potent than the previously reported TNK2 inhibitor, AIM-100.

CONCLUSION:

Here we prioritized TNK2 mutations as important functional targets using the HitWalker algorithm. This highlights the utility of integrating functional and genomic data to identify actionable genetic lesions. Given the large number of mutations present in a wide variety of tumors, the ability to prioritize genetic lesions greatly reduces the time and resources necessary to validate candidate mutations. Furthermore, this study highlights the utility of drug screening data for understanding the underlying vulnerabilities of leukemia cells and their accompanying gene mutations. In addition, we newly describe two kinase inhibitors that exhibit greater selectivity for TNK2 compared with dasatinib. These compounds represent exciting new lead candidates for further development of clinically applicable, selective TNK2 inhibitors.