Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

We previously reported that mast cell α-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. α-Chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1’s α-chymase-mediated cleavage products reveals hydrolysis at Phe12-Cys13 and Phe23-Val24 in loop 1 and Phe101-Val102 and Trp105-Asn106 in loop 3 of the NH2-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to α-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, α-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with α-chymase increases gel B activity 2- to 5-fold, suggesting that α-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of α-chymase blocks degranulation-induced TIMP-1 processing (absent in α-chymase-deficient HMC-1 cells). Purified α-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell α-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.

Proteolytic shedding of cell surface proteins by transmembrane metalloproteinases of the a disintegrin and metalloproteinase (ADAM) 1 family releases receptors, adhesion molecules, and growth factors that participate in a variety of disease pathways (1). Although many ADAMs participate in ectodomain shedding, murine phenotypes resulting from inactivation of tumor necrosis factor ␣-converting enzyme (TACE; ADAM-17) predicted its broad participation in the release of membrane-anchored proteins, including TNF-␣, transforming growth factor-␣, p55 and p75 TNF receptors, type II interleukin-1 receptor, VCAM-1, fractalkine, and amyloid precursor protein (2-7). Shedding mediated by TACE occurs at either low, constitutive levels or in a regulated fashion induced by phorbol stimulation (8), which may be differentiated using cells derived from TACE null mice (5). TACE expression occurs in a ubiquitous fashion in human tissues (6, 9), but the full complement of susceptible substrates, the cell specificity of TACEmediated surface protein shedding, and its relevance in homeostatic or pathophysiologic mechanisms remain unclear.Receptor shedding may program biological responses by diminishing surface ligand binding sites and solubilizing receptor extracellular domains (ectodomains) whose competitive binding to free ligand may antagonize transduction of incoming signals to the cell (10, 11). Serum levels of c-Kit (CD117) ectodomain increase in infiltrative mast cell disorders and correlate with clinical severity, suggesting that receptor shedding occurs as a response of activated mast cells (12,13). c-Kit is a 145-kDa glycosylated transmembrane protein whose extracellular domain contains Ig-like domains that bind c-Kit ligand (KL; stem cell factor) to initiate signaling that programs mast cell proliferation, differentiation, migration, and survival (14 -16). Soluble c-Kit isolated from human serum or supernatants of murine bone marrow-derived mast cells migrates at 98 -100 kDa, which suggests that focalized hydrolysis occurs in the extracellular juxtamembrane region at critical sequences in the fifth . In addition to cleavage at preferred sites, shedding may also depend on variations in the stalk length, which is defined as the distance between the scissile bond and the transmembrane domain, due to mutations in the juxtamembrane region (11,21,22). Divalent metal chelators attenuate release of c-Kit ectodomain from bone marrow-derived mast cells, thus identifying metalloproteinases as

Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell-dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo, and ex vivo. FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro, reduced tumor growth and increased survival in AML mouse models in vivo. Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo, including elimination of FLT3positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.

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