TWS119

Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However, how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8(+) T cells towards stem cell-like memory (T(SCM)) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here, we evaluated if T(SCM) can be obtained from human mature CD8(+) T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3β (GSK-3β), key inhibitor of the Wnt pathway. Human CD8(+) T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L(+)CD45RA(+) cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T(SCM) cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T(SCM) CD8(+) T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T(SCM) subset in human CD8(+) T cells from TIL and the periphery, which are relevant for ACT.

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways. The Wnt-beta-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation, but its role in the generation and maintenance of memory T cells is unknown. We found that induction of Wnt-beta-catenin signaling by inhibitors of glycogen sythase kinase-3beta or the Wnt protein family member Wnt3a arrested CD8(+) T cell development into effector cells. By blocking T cell differentiation, Wnt signaling promoted the generation of CD44(low)CD62L(high)Sca-1(high)CD122(high)Bcl-2(high) self-renewing multipotent CD8(+) memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of 'stemness' in mature memory CD8(+) T cells and have major implications for the design of new vaccination strategies and adoptive immunotherapies.

It is well known that hepatic stellate cells (HSC) develop into cells, which are thought to contribute to liver fibrogenesis. Recent data suggest that HSC are progenitor cells with the capacity to differentiate into cells of endothelial and hepatocyte lineages. The present study shows that beta-catenin-dependent canonical Wnt signaling is active in freshly isolated HSC of rats. Mimicking of the canonical Wnt pathway in cultured HSC by TWS119, an inhibitor of the glycogen synthase kinase 3beta, led to reduced beta-catenin phosphorylation, induced nuclear translocation of beta-catenin, elevated glutamine synthetase production, impeded synthesis of alpha-smooth muscle actin and Wnt5a, but promoted the expression of glial fibrillary acidic protein, Wnt10b, and paired-like homeodomain transcription factor 2c. In addition, canonical Wnt signaling lowered DNA synthesis and hindered HSC from entering the cell cycle. The findings demonstrate that beta-catenin-dependent Wnt signaling maintains the quiescent state of HSC and, similar to stem and progenitor cells, influences their developmental fate.

In an attempt to better understand and control the processes that regulate stem cell fate, we have set out to identify small molecules that induce neuronal differentiation in embryonic stem cells (ESCs). A high-throughput phenotypic cell-based screen of kinase-directed combinatorial libraries led to the discovery of TWS119, a 4,6-disubstituted pyrrolopyrimidine that can induce neurogenesis in murine ESCs. The target of TWS119 was shown to be glycogen synthase kinase-3beta (GSK-3beta) by both affinity-based and biochemical methods. This study provides evidence that GSK-3beta is involved in the induction of mammalian neurogenesis in ESCs. This and such other molecules are likely to provide insights into the molecular mechanisms that control stem cell fate, and may ultimately be useful to in vivo stem cell biology and therapy.