Previous studies showed that most cases of ALK ؉ anaplastic large-cell lymphoma (ALK ؉ ALCL) do not express SHP1, a tyrosine phosphatase and an important negative regulator for cellular signaling pathways such as that of JAK/STAT. To fully assess the biologic significance of loss of SHP1 in ALK ؉ ALCL, we transfected SHP1 plasmids into 2 SHP1 ؊ , ALK ؉ ALCL cell lines, Karpas 299 and SU-DHL-1. After 24 hours of transfection, pJAK3 and pSTAT3 were decreased, and these changes correlated with downregulation of STAT3 downstream targets including cyclin D3, mcl-1, and bcl-2. Expression of SHP1 in these 2 cell lines also resulted in marked decreases in the protein levels of JAK3 and NPM-ALK, and these effects were reversible by proteosome inhibitor MG132. Conversely, when SHP1 expression in SUP-M2 (a SHP1 ؉ ALK ؉ ALCL cell line) was inhibited using siRNA, pSTAT3, pJAK3, JAK3, and NPM-ALK were all up-regulated. Coimmunoprecipitation studies showed that SHP1 was physically associated with JAK3 and NPM-

M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK þ ALCL) is characterized by constitutive activation of the Janus kinase (JAK)3/signal transducers and activators of transcription 3 (STAT3) signaling pathway. SHP1, a tyrosine phosphatase that negatively regulates JAK/STAT, is frequently absent in ALK þ ALCL owing to gene methylation. To test the hypothesis that loss of SHP1 contributes to JAK3/ STAT3 activation in ALK þ ALCL cells, we induced SHP1 expression using 5-aza-2 0 -deoxycytidine (5-AZA), an inhibitor of DNA methyltransferase, in ALK þ ALCL cell lines, and correlated with changes in the JAK3/STAT3 pathway. 5-AZA gradually restored SHP1 expression in Karpas 299 and SU-DHL-1 cells over 5 days. The initially low level of SHP1 expression did not result in significant changes to the expression or tyrosine phosphorylation of JAK3 and STAT3. However, higher levels of SHP1 seen subsequently correlated with substantial decreases in JAK3 and pJAK3, followed by pSTAT3 (but not STAT3). Importantly, the decrease in JAK3 was abrogated by MG132, a proteasome inhibitor. 5-AZA induced no significant increase in apoptosis but it sensitized ALCL cells to doxorubicin-induced apoptosis. Our findings support the concept that loss of SHP1 contributes to the constitutive activation of JAK3/STAT3 in ALK þ ALCL cells. SHP1 appears to downregulate JAK3 by two mechanisms: tyrosine dephosphorylation and increased degradation via the proteasome pathway.

Summary JSI‐124 (cucurbitacin I) has been recently described as a specific inhibitor of signal transducer and activator of transcription‐3 (STAT3). As STAT3 activation is pathogenetically important in anaplastic lymphoma kinase‐positive anaplastic large cell lymphoma (ALK+ ALCL), we investigated whether JSI‐124 can mediate significant inhibitory effects in this cell type. In two ALK+ ALCL cell lines (Karpas 299 and SU‐DHL‐1), JSI‐124 significantly reduced the number of viable cells to 50% of that of negative controls at a dose of 5–10 μmol/l at 24 h and 1–1·25 μmol/l at 48 h. This decrease in viability was associated with apoptosis, as confirmed by the increase in the subG0/1 fraction, poly(ADP‐ribose)polymerase cleavage and expression of active caspase 3. JSI‐124 decreased the phosphorylated‐STAT3 and ‐Janus kinase‐3 (JAK3) levels in a dose‐dependent fashion, and these changes were coupled with significant decreases in several STAT3 downstream targets, including mcl‐1, bcl‐2, bcl‐xL and cyclin D3. Interestingly, JSI‐124 also dramatically decreased the protein levels of JAK3 and nucleophosmin (NPM)‐ALK, and these effects were reversible by MG132. Our data support that JSI‐124 is a potentially useful therapeutic agent for ALK+ ALCL. In addition to its role as a tyrosine kinase inhibitor, JSI‐124 appears to be involved in regulating proteosome degradation for proteins such as JAK3 and NPM‐ALK.