Mitochondrial Kv1.3 Channels as Target for Treatment of Multiple Myeloma.

Despite several new developments in the treatment of multiple myeloma, all available therapies are only palliative without curative potential and all patients ultimately relapse. Thus, novel therapeutic options are urgently required to prolong survival of or to even cure myeloma. Here, we show that multiple myeloma cells express the potassium channel Kv1.3 in their mitochondria. The mitochondrial Kv1.3 inhibitors PAPTP and PCARBTP are efficient against two tested human multiple myeloma cell lines (L-363 and RPMI-8226) and against ex vivo cultured, patient-derived myeloma cells, while healthy bone marrow cells are spared from toxicity. Cell death after treatment with PAPTP and PCARBTP occurs via the mitochondrial apoptotic pathway. In addition, we identify up-regulation of the multidrug resistance pump MDR-1 as the main potential resistance mechanism. Combination with ABT-199 (venetoclax), an inhibitor of Bcl2, has a synergistic effect, suggesting that mitochondrial Kv1.3 inhibitors could potentially be used as combination partner to venetoclax, even in the treatment of t(11;14) negative multiple myeloma, which represent the major part of cases and are rather resistant to venetoclax alone. We thus identify mitochondrial Kv1.3 channels as druggable targets against multiple myeloma.

Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro.

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

Regulation of Arthritis Severity by the Acid Sphingomyelinase.

BACKGROUND/AIMS: Rheumatoid arthritis is a chronic autoimmune disease hallmarked by inflammation in synovial joints. Treatment is hampered by the lack of a cure and current disease-modifying drugs are associated with potentially severe toxicities. METHODS: We investigated arthritis severity by measuring joint swelling and pro-inflammatory cytokine production in a murine experimental model of inflammatory arthritis (antigen-induced arthritis). We analyzed acid sphingomyelinase knock-out mice and wild-type littermates, as well as mice treated with the pharmacological acid sphingomyelinase inhibitor amitriptyline. RESULTS: Genetic ablation or pharmacological inhibition of acid sphingomyelinase reduced joint swelling and levels of pro-inflammatory cytokines in the arthritic joint. CONCLUSION: We identified acid sphingomyelinase as a novel druggable target in rheumatoid arthritis. Functional inhibitors of acid sphingomyelinase have been clinically used for decades, are well tolerated and suitable for long-term treatment. They would be immediately available for clinical development as a novel rheumatoid arthritis therapy.

Melanoma cell metastasis via P-selectin-mediated activation of acid sphingomyelinase in platelets.

Metastatic dissemination of cancer cells is one of the hallmarks of malignancy and accounts for approximately 90 % of human cancer deaths. Within the blood vasculature, tumor cells may aggregate with platelets to form clots, adhere to and spread onto endothelial cells, and finally extravasate to form metastatic colonies. We have previously shown that sphingolipids play a central role in the interaction of tumor cells with platelets; this interaction is a prerequisite for hematogenous tumor metastasis in at least some tumor models. Here we show that the interaction between melanoma cells and platelets results in rapid and transient activation and secretion of acid sphingomyelinase (Asm) in WT but not in P-selectin-deficient platelets. Stimulation of P-selectin resulted in activation of p38 MAPK, and inhibition of p38 MAPK in platelets prevented the secretion of Asm after interaction with tumor cells. Intravenous injection of melanoma cells into WT mice resulted in multiple lung metastases, while in P-selectin-deficient mice pulmonary tumor metastasis and trapping of tumor cells in the lung was significantly reduced. Pre-incubation of tumor cells with recombinant ASM restored trapping of B16F10 melanoma cells in the lung in P-selectin-deficient mice. These findings indicate a novel pathway in tumor metastasis, i.e., tumor cell mediated activation of P-selectin in platelets, followed by activation and secretion of Asm and in turn release of ceramide and tumor metastasis. The data suggest that p38 MAPK acts downstream from P-selectin and is necessary for the secretion of Asm.

Role of Acid Sphingomyelinase-Induced Signaling in Melanoma Cells for Hematogenous Tumor Metastasis.

BACKGROUND: Hematogenous metastasis of malignant tumor cells is a multistep process that requires release of tumor cells from the local tumor mass, interaction of the tumor cells with platelets in the blood, and adhesion of either the activated tumor cells or the complexes of platelets and tumor cells to the endothelial cells of the target organ. We have previously shown that the interaction of melanoma cells with platelets results in the release of acid sphingomyelinase (Asm) from activated platelets. Secreted platelet-derived Asm acts on malignant tumor cells to cluster and activate integrins; such clustering and activation are necessary for tumor cell adhesion to endothelial cells and for metastasis. METHODS: We examined the response of tumor cells to treatment with extracellular sphingomyelinase or co-incubation with wild-type and Asm-deficient platelets. We determined the phosphorylation and activation of several intracellular signaling molecules, in particular p38 kinase (p38K), phospholipase Cx03B3; (PLCx03B3;), ezrin, and extracellular signal-regulated kinases. RESULTS: Incubation of B16F10 melanoma cells with Asm activates p38 MAP kinase (p38K), phospholipase Cx03B3; (PLCx03B3;), ezrin, and extracellular signal-regulated kinases. Co-incubation of B16F10 melanoma cells with wild-type or Asm-deficient platelets showed that the phosphorylation/activation of p38K is dependent on Asm. Pharmacological blockade of p38K prevents activation of ß1 integrin and adhesion in vitro. Most importantly, inhibition of p38K activity in B16F10 melanoma cells prevents tumor cell adhesion and metastasis to the lung in vivo, a finding indicating the importance of p38K for metastasis. CONCLUSIONS: Asm, secreted from activated platelets after tumor cell-platelet contact, induces p38K phosphorylation in tumor cells. This in turn stimulates ß1 integrin activation that is necessary for adhesion and subsequent metastasis of tumor cells. Thus, inhibition of p38K might be a novel target to prevent tumor metastasis.

Regulation of hematogenous tumor metastasis by acid sphingomyelinase.

Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1(-/-) mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of α5ß1 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing ß1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis.

TNFR1-induced sphingomyelinase activation modulates TCR signaling by impairing store-operated Ca2+ influx.

Tumor necrosis factor alpha (TNF-alpha) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF-alpha alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down-regulation of the intracellular calcium ([Ca(2+)](i)) signal in Jurkat T cells after just 30 min exposure to TNF-alpha, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca(2+)](i) was also observed in human CD4+ T lymphocytes. The change in Ca(2+) signal was related to a decrease in the plasma membrane Ca(2+) influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca(2+) influx. The role of TNF-alpha-induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF-alpha led to a time-dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca(2+) response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca(2+) responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF-alpha-induced inhibition of the Ca(2+) influx in an ASM-deficient murine T cell line (OT-II(+/+)ASM(-/-)). This potent ability of TNF-alpha to rapidly modulate the TCR Ca(2+) signal via TNFR1-induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.

Stimulation of erythrocyte ceramide formation by platelet-activating factor.

Osmotic erythrocyte shrinkage leads to activation of cation channels with subsequent Ca2+ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Ca2+ and ceramide then activate a scramblase leading to breakdown of phosphatidylserine asymmetry of the cell membrane. The mediators accounting for activation of erythrocyte sphingomyelinase and phosphatidylserine exposure remained elusive. The study demonstrates that platelet-activating factor (PAF) is released from erythrocytes upon hyperosmotic cell shrinkage. The experiments further disclose the presence of PAF receptors in erythrocytes and show that PAF stimulates the breakdown of sphingomyelin and the release of ceramide from erythrocytes at isotonic conditions. PAF further triggers cell shrinkage (decrease of forward scatter) and phosphatidylserine exposure (annexin binding) of erythrocytes. The stimulation of annexin-binding is blunted by a genetic knockout of PAF receptors, by the PAF receptor antagonist ABT491 or by inhibition of sphingomyelinase with urea. In conclusion, PAF activates an erythrocyte sphingomyelinase and the then formed ceramide leads to the activation of scramblase with subsequent phosphatidylserine exposure.