Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression. Cancer Res; 75(15); 1–12. ©2015 AACR.

The optimal T-cell attributes for adoptive cancer immunotherapy are unclear. Recent clinical trials of ex vivo–expanded tumor-infiltrating lymphocytes indicated that differentiated T effector cells can elicit durable antitumor responses in some patients with cancer, with their antitumor activity tightly correlated with their persistence in the host. Thus, there is great interest in the definition of intrinsic biomarkers that can predict the conversion of short-lived tumor antigen–specific T effector cells into long-lived T memory cells. Long-term persistence of ex vivo–expanded tumor-specific CD8+ T effector clones has been reported in refractory metastatic melanoma patients after adoptive T-cell transfer. By using highly homogeneous clone populations from these preparations, we performed a comparative transcriptional profiling to define preinfusion molecular attributes that can be ascribed to an effector-to-memory transition. Through this route, we discovered that preinfusion T-cell clones that expressed the IL7 receptor (IL7R) and c-myc were more likely to persist longer after adoptive transfer to patients. The predictive value of these two biomarkers was strengthened by using IL7R protein, IL7-induced pSTAT5, and c-myc mRNA expression to prospectively identify human tumor-specific T effector clones capable of engraftment into immunodeficient mice. Overall, our findings reveal IL7R and c-myc expression as intrinsic biomarkers that can predict the fate of CD8+ T effector cells after adoptive transfer. Cancer Res; 75(16); 1–11. ©2015 AACR.

The optimal T cell attributes for adoptive cancer immunotherapy are unclear. Recent clinical trials of ex vivo expanded tumor-infiltrating lymphocytes indicated that differentiated T effector can elicit durable anti-tumor responses in some cancer patients, with their anti-tumor activity tightly correlated with their persistence in the host. Thus, there is great interest in the definition of intrinsic biomarkers that can predict the conversion of short-lived tumor antigen-specific T effector cells into long-lived T memory cells. Long-term persistence of ex vivo expanded tumor specific CD8+ T effector clones has been reported in refractory metastatic melanoma patients after adoptive T cell transfer. By utilizing highly homogeneous clone populations from these preparatoins, we performed a comparative transcriptional profiling to define pre-infusion molecular attributes that can be ascribed to an effector-to-memory transition. Though this route, we discovered that pre-infusion T cell clones which expressed c-MYC and the IL-7 receptor (IL-7R) were more likely to persist longer after adoptive transfer to patients. The predictive value of these two biomarkers was strengthened by utilizing IL-7R protein, IL-7 induced pSTAT5, and c-myc mRNA expression to prospectively identify human tumor-specific T effector clones capable of engraftment into immunodeficient mice. Overall, our findings reveal IL-7R and c-MYC expression as intrinsic biomarkers that can predict the fate of effector CD8+ T cells after adoptive transfer.

Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single nucleotide polymorphism (SNP) associations reside within CASC15, a long non-coding RNA that we define as a tumor suppressor at 6p22. Low level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.

Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-RafS338) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPKS308), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEFC-Raf−/− or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPKS308, this drug–target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-RafS338 and pDAPKS308 translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPKS308 to DAPK. PP2A then dissociated from the C-Raf–DAPK complex and induced profound cancer cell death. Increased pDAPKS308 modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPKS308 may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. Cancer Res; 75(17); 1–15. ©2015 AACR.