Adaptation to TKI Treatment Reactivates ERK Signaling in Tyrosine Kinase-Driven Leukemias and Other Malignancies.

FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors (TKI) have been tested extensively to limited benefit in acute myeloid leukemia (AML). We hypothesized that FLT3/internal tandem duplication (ITD) leukemia cells exhibit mechanisms of intrinsic signaling adaptation to TKI treatment that are associated with an incomplete response. Here, we identified reactivation of ERK signaling within hours following treatment of FLT3/ITD AML cells with selective inhibitors of FLT3. When these cells were treated with inhibitors of both FLT3 and MEK in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared with either drug alone. ERK reactivation was also observed following inhibition of other tyrosine kinase-driven cancer cells, including EGFR-mutant lung cancer, HER2-amplified breast cancer, and BCR-ABL leukemia. These studies reveal an adaptive feedback mechanism in tyrosine kinase-driven cancers associated with reactivation of ERK signaling in response to targeted inhibition. Cancer Res; 77(20); 5554-63. ©2017 AACR.

All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo.

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)(+) LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD(+) cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD(+) cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD(+) LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD(+) patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drug's apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD(+) LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.

Identification of novel small molecule modulators of K2P18.1 two-pore potassium channel.

Two-pore domain potassium (K2P) channels are responsible for background potassium (K+) current, which is crucial for the maintenance of resting membrane potential. K2P18.1, also called TWIK-related spinal cord K+ channel (TRESK) or KCNK18, is thought to be a major contributor to background K+ currents, particularly in sensory neurons where it is abundantly expressed. Despite its critical role and potential therapeutic implication, pharmacological tools for probing K2P18.1 activity remain unavailable. Here, we report a high-throughput screen against a collection of bioactive compounds that yielded 26 inhibitors and 8 activators of K2P18.1 channel activity with more than 10-fold selectivity over the homologous channel K2P9.1. Among these modulators, the antihistamine loratadine inhibited K2P18.1 activity with IC50 of 0.49±0.23 µM and is considerably more potent than existing K2P18.1 inhibitors. Importantly, the inhibition by loratadine remains equally efficacious upon potentiation of K2P18.1 by calcium signaling. Furthermore, the loratadine effect is dependent on transmembrane residues F145 and F352, providing orthogonal evidence that the inhibition is caused by a direct compound-channel interaction. This study reveals new pharmacological modulators of K2P18.1 activity useful in dissecting native K2P18.1 function.