The prognosis for patients presenting with metastatic pediatric RMS and for those patients with ARMS harboring the PAX3/FOXO1a gene product remains poor. This renewal application explores developmental therapeutic approaches specifically for application to pediatric RMS. Five of eleven RMS cell lines are sensitive to the cytotoxic ligand TRAIL. FOXO1a, when transduced, selectively induces caspase-3 activation and apoptosis in TRAIL-resistant ARMS, and sensitizes Rh30 ARMS cells to TRAIL. The hypothesis is that these events are mediated by selective regulation via FADD, Bim or caspase-3, and may involve activation of p38 MAPK, DR5 or TRAIL. The overall goal is to elucidate downstream targets of FOXO1a that may selectively regulate apoptosis in TRAIL-resistant ARMS and may be exploitable therapeutically. SphK, which inhibits the conversion of proapoptotic Sph to SIP (involved in transformation and proliferation), is elevated in all RMS cell lines. Sph, and the SphK inhibitor, DHSph, induce apoptosis in all cell lines, independent of TRAIL, FADD, caspase-8, Bcl-2 or Bcl-xL, in contrast to Cer, which induces apoptosis in a FADD- and caspase-8-independent manner, but is inhibited by Bcl-2 or Bcl-xL. Sph and DHSph also potentiate TRAIL-induced apoptosis. The hypothesis is that Sph- or DHSph-induced apoptosis are regulated by INK activation, phosphorylation of Bcl-2, or the orphan nuclear receptor Nur77. The overall goal is to elucidate the role of SphK as a therapeutic target in both ERMS and ARMS. In addition to TRAIL, the cytolytic antibody HGS-ETR2 (Human Genome Sciences;anti-DR5) but not HGS-ETR1 (anti-DR4) is active in pediatric RMS cell lines, and DR5 is expressed at high levels. HGS-ETR2 demonstrates 1) activity superior to TRAIL, 2) formation of a large death-inducing signaling complex, 3) reduced activation of NF-KB, and 4) exquisite sensitivity in TRAIL-resistant JR1 ERMS cells. The hypothesis is that HGS-ETR2 targets a broader spectrum of RMS than TRAIL due to differences in DR5 binding or reduced influence of survival signaling pathways, and has superior activity over TRAIL either alone, in FOXO1a-transduced ARMS, or in combination with DHSph. The overall goal is to develop therapeutic approaches for both ERMS and ARMS that exploit the TRAIL signaling pathway. The long-term objectives are to develop highly effective therapy for metastatic RMS from understanding specific signaling pathways involved in the regulation of cell death, the identification of new molecular targets, and developing new therapeutic strategies based upon specific molecular characteristics.
