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Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency., (© 2022 by The American Society of Hematology.)

The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration., Competing Interests: Declaration of competing interests The authors declare that they have no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Loading controls are necessary for semiquantitative Western blotting to compensate for loading errors. Loading control methods include the reprobing of membranes with an antibody against a constitutively expressed protein or staining the membrane with a total protein stain. We compared the loading control performance of recently released Stain-Free (SF) gels with Sypro Ruby (SR) and reprobing using β-actin. SF gels demonstrated superior performance in that they were faster, required fewer steps and consumables, and allowed the quality of electrophoresis and Western transfer to be assessed before committing to costly and time-consuming Western blots., (Copyright © 2012 Elsevier Inc. All rights reserved.)