High-throughput neural stem cell-based drug screening identifies S6K1 inhibition as a selective vulnerability in SHH-medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current treatments have increased overall survival but can lead to devastating side effects and late complications in survivors, emphasizing the need for new, improved targeted therapies that specifically eliminate tumor cells while sparing the normally developing brain. METHODS: Here, we used a SHH-MB model based on a patient-derived neuroepithelial stem (NES) cell system for an unbiased high-throughput screen with a library of 172 compounds with known targets. Compounds were evaluated in both healthy neural stem cells and tumor cells derived from the same patient. Based on the difference of cell viability and drug sensitivity score between normal cells and tumor cells, hit compounds were selected and further validated in vitro and in vivo. RESULTS: We identified PF4708671 (S6K1 inhibitor) as a potential agent that selectively targets Sonic Hedgehog (SHH) driven MB tumor cells while sparing neural stem cells and differentiated neurons. Subsequent validation studies confirmed that PF4708671 inhibited the growth of SHH-MB tumor cells both in vitro and in vivo, and that knockdown of S6K1 resulted in reduced tumor formation. CONCLUSIONS: Overall, our results suggest that inhibition of S6K1 specifically affects tumor growth, whereas it has less effect on non-tumor cells. Our data also show that the NES cell platform can be used to identify potentially effective new therapies and targets for SHH-MB.

Extended cleavage specificities of human granzymes A and K, two closely related enzymes with conserved but still poorly defined functions in T and NK cell-mediated immunity.

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTL) and natural killer cells (NK). Granzyme A is the most abundant of the different granzymes (gzms) expressed by these two cell types. Gzms A and K are found in all jawed vertebrates and are the most well conserved of all hematopoietic serine proteases. Their potential functions have been studied extensively for many years, however, without clear conclusions. Gzm A was for many years thought to serve as a key component in the defense against viral infection by the induction of apoptosis in virus-infected cells, similar to gzm B. However, later studies have questioned this role and instead indicated that gzm A may act as a potent inducer of inflammatory cytokines and chemokines. Gzms A and K form clearly separate branches in a phylogenetic tree indicating separate functions. Transcriptional analyses presented here demonstrate the presence of gzm A and K transcripts in both CD4+ and CD8+ T cells. To enable screening for their primary biological targets we have made a detailed analysis of their extended cleavage specificities. Phage display analysis of the cleavage specificity of the recombinant enzymes showed that both gzms A and K are strict tryptases with high selectivity for Arg over Lys in the P1 position. The major differences in the specificities of these two enzymes are located N-terminally of the cleavage site, where gzm A prefers small amino acids such as Gly in the P3 position and shows a relatively relaxed selectivity in the P2 position. In contrast, gzm K prefers large amino acids such as Phe, Tyr, and Trp in both the P2 and P3 positions and does not tolerate negatively charged residues in the P2 position. This major distinction in extended specificities is likely reflected also in preferred in vivo targets of these two enzymes. This information can now be utilized for high-precision screening of primary targets for gzms A and K in search of their highly conserved but still poorly defined functions in vertebrate immunity.

Extended cleavage specificity of a Chinese alligator granzyme B homologue, a strict Glu-ase in contrast to the mammalian Asp-ases.

Granzyme B (GzmB) is primarily expressed by mammalian cytotoxic T cells and serves as one of the key components in the defense against viral infection by the induction of apoptosis in virus infected cells. By direct cell to cell contact and delivery into target cells by perforin, cytotoxic T cells activate apoptosis through the action of GzmB by both caspase-dependent and -independent pathways. In search for early ancestors of GzmB we have in the current study identified and characterized a GzmB homologue from a reptile, the Chinese alligator. This enzyme is encoded from the same locus as the mammalian counterparts, the chymase locus. Phage display analysis of the cleavage specificity of the recombinant alligator enzyme (named MCP1A-like) shows that it is a relatively strict Glu-ase, with strong preference for glutamic acid in the P1 position of a substrate. The majority of mammalian GzmB:s are, in marked contrast to the alligator enzyme, relatively strict Asp-ases. The alligator enzyme also showed strong preference for Ala, Pro and Gly in the P2 position and Val in the P3 position indicating that it has a narrow specificity, similar to the mammalian counterparts. Analysis of the three amino acids forming the substrate binding pocket (S1 pocket) in three amphibian homologues to MCP1A-like, from the frogs Xenopus laevis and Xenopus tropicalis, shows that these amphibian enzymes have similar substrate binding pocket as their mammalian counterparts. This finding, together with the apparent lack of GzmB homologs in fish, indicates that the ancestor of GzmB did appear with the amphibians at the base of tetrapod evolution. This study is a first step in a larger effort to understand the evolutionary processes involved in shaping anti-viral immunity in non-mammalian vertebrates.

Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity.

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.