Thioridazine requires calcium influx to induce MLL-AF6–rearranged AML cell death

Abstract

In pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug’s safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials.

Drug Repurposing for Targeting Acute Leukemia With KMT2A (MLL)—Gene Rearrangements

“…These three proteins are known to form a complex associated with cytoskeletal filaments ( Bode et al, 2008 ). Therefore, the authors investigated the hypothesis that thioridazine treatment may induce cytoskeletal changes in the MLL-AF6 expressing cells and confirmed that morphologic changes and accumulation of large F-actin aggregates were induced upon exposure to thioridazine ( Tregnago et al, 2020 ). The treatment with thioridazine caused an influx of external Ca 2+ possibly via the cytoskeletal rearrangement caused by the drug.…”

“…In a high-throughput drug screening of 1,280 compounds, 104 were identified as active against MLL-AF6 rearranged cell lines, with 20 of them showing activity specifically in MLL- rearranged cells and 10 exclusively on the t (6;11)-rearranged cells ( Tregnago et al, 2020 ). One of those hits was thioridazine, an anti-psychotic agent primarily used for schizophrenia.…”

“…The treatment with thioridazine caused an influx of external Ca 2+ possibly via the cytoskeletal rearrangement caused by the drug. This Ca 2+ overload led to reactive oxygen species (ROS) overproduction that was detrimental for the t (6;11) AML cells ( Tregnago et al, 2020 ). Intriguingly, another class of drugs that leads to cellular and mitochondrial ROS accumulation in MLL -rearranged cells are the artemisinins, a family of antimalarial compounds.…”

The long non-coding RNA CDK6-AS1 overexpression impacts on acute myeloid leukemia differentiation and mitochondrial dynamics

“…Primary AML cells were cultured in RPMI1640 10% FBS with the addition of recombinant human (rh) cytokines: interleukin3 (rhIL-3; 20 ng/mL), rhIL-6; 20 ng/mL, rhSCF; 50 ng/mL, rhTPO; 50 ng/mL and rhFLT-3 ligand (50 ng/mL) ( Tregnago et al., 2020 ). All cell cultures were maintained at 37°C, 5% CO 2 .…”

Inhibition of Autophagy Enhances the Antitumor Effect of Thioridazine in Acute Lymphoblastic Leukemia Cells

“…Chlorpromazine suppressed the growth of primary AML cells and AML cells with mutated tyrosine kinase receptor [ 11 ]. Yet, in AML cells, TR induced cell death promoting cytoskeletal remodeling of blast cells selectively in variant t (6;11) AML cells involving Ca 2+ overload, ROS production, and mitochondrial dysfunction [ 12 ]. Although several mechanisms have been proposed to explain its cytotoxicity, the molecular mechanisms underlying TR-induced apoptosis has not been completely understood, especially in acute lymphoblastic leukemia.…”