Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing...

Intracellular Ca²⁺ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca²⁺ is intracellular BAPTA (BAPTA_i), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTA_i enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca²⁺ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTA_i enhances cell death in B-cell cancers. In this study, we discovered that BAPTA_i alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTA_i provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTA_i diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTA_i. Notably, these effects were also induced by a BAPTA_i analog with low affinity for Ca²⁺. Consequently, our findings uncover PFKFB3 inhibition as an Ca²⁺-independent mechanism through which BAPTA_i impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTA_i are not necessarily related to Ca²⁺ signaling. Our data support the need for a reassessment of the role of Ca²⁺ in cellular processes when findings were based on the use of BAPTA_i.