Therapeutic Synergy from Combined Inhibition of the SERCA Channel and MAPK Signaling Pathway in MAPK-Dependent Leukemia
Authors: Wang Meng , Eskiocak Ugur , Agathokleous Michalis , Yuan Stacy , Crawley Charles , Morrison Sean J. 
Published: October 2019

Abstract
Background
The generation and maintenance of electrochemical gradients by ion transporters are vital in almost all cellular processes. Ion transporters are often dysregulated in cancer cells and play important roles in cancer cell function. For example, aberrant expression of voltage gated potassium channels Kv10.1 (Eag1) and Kv11.1, (hERG) have been associated with increased proliferation and worse prognosis in hematological malignancies. Targeting ion transporters may thus offer a novel therapeutic strategy against cancer. To this end, we identified ion transporters that when inhibited, result in selective toxicity in hematological malignancies with a focus on acute myeloid leukaemia (AML).
Method and Results
Ion transporter inhibitors exhibiting selective activity against leukaemia/lymphoma cells were identified from screening compound libraries targeting ion transporters (total 126 inhibitors) against 7 leukaemia/lymphoma cell lines (HL-60, Kasumi-1, HuT-78, BC-2, U266B1, K562 and Raji). Eight unique compounds were selected (Antibiotic A-23187; Niguldipine; Ouabain; Penitrem A; SKF-96365; Tetrandrine; Thapsigargin; Triamterene). The most potent compound was thapsigargin, exhibiting toxicity at nano-molar concentrations against leukemia cells (IC50 1.8 nM - 8.4 nM) whilst sparing human umbilical cord blood cells (IC50 20 nM). In AML cell lines dependent on activated MAPK signaling pathway (HL-60, Kasumi-1), the potency of thapsigargin was enhanced when combined with MEK inhibitor trametinib (thapsigargin IC50 0.7 nM - 2.6 nM in presence of trametinib), with significantly increased apoptosis as measured by both caspase 3 activation and Annexin-V surface binding. The therapeutic activity of thapsigargin likely acted through the inhibition of sarco-endoreticulum calcium ATPase (SERCA) channel as similar efficacy against the AML cells could be reproduced by treatment with trametinib combined with other SERCA inhibitors (CPA and BHQ).
SERCA is ubiquitously expressed on the endoplasmic reticulum (ER) and functions to sequester cytoplasmic calcium into the ER. SERCA inhibition by thapsigargin is known to trigger ER stress and to activate the unfolded protein response (UPR), which we postulated might underlie the mechanism of activity from combined trametinib/thapsigargin treatment. Compared to human umbilical cord blood cells, inhibition of the MAPK pathway in AML cell lines by trametinib reduced the expression of GRP-78, a major chaperone protein acting both as an initiator and effector of UPR. When trametinib was combined with thapsigargin, the downregulation of GRP-78 was associated with increased level of CHOP, a pro-apoptotic effector downstream of UPR activation. Taken together, these data suggested the downregulation of GRP-78 by trametinib in the AML cells potentially reduces cellular capacity to adapt to the ER stress induced by thapsigargin, thus triggering UPR mediated apoptosis pathway. Indeed, the addition of the chemical chaperone tauro-ursodeoxycholic acid (TUDCA) rescued for the loss of GRP-78 and the toxicity from combined trametinib and thapsigargin treatment.
Conclusion
Inhibition of SERCA channel either alone or in combination with MAPK pathway inhibition was selectively toxic in MAPK-dependent AML cell lines. The combination-induced toxicity resulted from a deficiency in GRP-78 chaperone protein that sensitized the cells to ER stress mediated apoptosis. The combined inhibition of SERCA channel and MAPK signaling may thus offer a novel therapeutic opportunity in MAPK-dependent leukemia.

Disclosures
No relevant conflicts of interest to declare.