Capitalizing on paradoxical activation of the mitogen-activated protein kinase pathway for treatment of Imatinib-resistant mast cell leukemia.

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation." In the present work we re-analyzed the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KIT V560G, D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSAKIT D816V and in KIT D816V hematopoietic progenitors obtained from patient-derived induced pluripotent stem cells (iPS cells) and systemic mastocytosis patient samples. In conclusion, mutated KIT-driven Imatinib resistance and possible TKI-induced paradoxical activation can be efficiently overcome by a low concentration Ponatinib and Trametinib co-treatment, potentially reducing the negative side effects associated with MCL therapy.

Infliction of proteotoxic stresses by impairment of the unfolded protein response or proteasomal inhibition as a therapeutic strategy for mast cell leukemia.

The intensity and duration of endoplasmic reticulum (ER) stress converts the unfolded protein response (UPR) from an adaptive into a terminal response. The first regulates homeostasis, the latter triggers apoptosis. Cells that rapidly proliferate and possess developed secretory capabilities, such as leukemia cells, depend on an efficiently operating UPR to maintain proteostasis. Activation of terminal UPR by either blockade of adaptive UPR or exaggeration of ER stress has been explored as a novel approach in cancer therapy. For mast cell leukemia (MCL) the efficacy of both approaches, by utilizing the KITV560G,D816V-positive MCL cell line HMC-1.2, was investigated. We show that HMC-1.2 cells display a tonic activation of the IRE1α arm of the UPR, which constitutively generates spliced XBP1. Inhibition of IRE1α by different types of inhibitors (MKC-8866, STF-083010, and KIRA6) suppressed proliferation at concentrations needed for blockade of IRE1α-mediated XBP1 splicing. At higher concentrations, these inhibitors triggered an apoptotic response. Blocking the proteasome by bortezomib, which confers an exaggerated UPR, resulted in a marked cytotoxic response. Bortezomib treatment also caused activation of the kinase JNK, which played a pro-proliferative and anti-apoptotic role. Hence, the combination of bortezomib with a JNK inhibitor synergized to induce cell death. In summary, the UPR can be addressed as an effective therapeutic target against KITD816V-positive MCL.