Sphingosine Kinase Inhibition Enhances Dimerization of Calreticulin at the Cell Surface in Mitoxantrone-Induced...

Agents that induce immunogenic cell death (ICD) alter the cellular localization of calreticulin (CRT), causing it to become cell surface-exposed within the plasma membrane lipid raft microdomain [cell surface-exposed CRT (ectoCRT)] where it serves as a damage associated-molecular pattern that elicits an antitumor immune response. We have identified the sphingolipid metabolic pathway as an integral component of the process of ectoCRT exposure. Inhibition of the sphingosine kinases (SphKs) enhances mitoxantrone-induced production of hallmarks of ICD, including ectoCRT production, with an absolute mean difference of 40 MFI (95% CI: 19-62; P = 0.0014) and 1.3-fold increase of ATP secretion with an absolute mean difference of 87 RLU (95% CI: 55-120; P < 0.0001). Mechanistically, sphingosine kinase inhibition increases mitoxantrone-induced accumulation of ceramide species, including C16:0 ceramide 2.8-fold with an absolute mean difference of 1.390 pmol/nmol Pi (95% CI: 0.798-1.983; P = 0.0023). We further examined the localization of ectoCRT to the lipid raft microdomain and demonstrate that ectoCRT forms disulfide-bridged dimers. Together, our findings suggest that ceramide accumulation impinges on the homeostatic function of the endoplasmic reticulum to induce ectoCRT exposure and that structural alterations of ectoCRT may underlie its immunogenicity. Our findings further suggest that inhibition of the SphKs may represent a means to enhance the therapeutic immunogenic efficacy of ICD-inducing agents while reducing overt toxicity/immunosuppressive effects by allowing for the modification of dosing regimens or directly lowering the dosages of ICD-inducing agents employed in therapeutic regimens. SIGNIFICANCE STATEMENT: This study demonstrates that inhibition of sphingosine kinase enhances the mitoxantrone-induced cell surface exposure of a dimeric form of the normally endoplasmic reticulum resident chaperone calreticulin as part of the process of a unique form of regulated cell death termed immunogenic cell death. Importantly, inhibition of sphingosine kinase may represent a means to enhance the therapeutic efficacy of immunogenic cell death-inducing agents, such as mitoxantrone, while reducing their overt toxicity and immunosuppressive effects, leading to better therapeutic outcomes for patients.