Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome. Cancer Res; 75(19); 1–11. ©2015 AACR.

Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. Additionally, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome.

Glioblastoma (GBM) is the most common form of primary brain tumor in adults that is essentially incurable. Despite aggressive treatment regimens centered on radiotherapy, tumor recurrence is inevitable and thought to be driven by GBM stem-like cells (GSCs) that are highly radioresistant. DNA damage response pathways are key determinants of radiosensitivity, but the extent to which these overlapping and parallel signaling components contribute to GSC radioresistance is unclear. Using a panel of primary patient-derived GBM cell lines and clonogenic survival assays, we confirmed that GSCs were significantly more radioresistant than paired tumor bulk populations. DNA damage response targets ATM, ATR, CHK1, and PARP-1 were upregulated in GSCs, and CHK1 was preferentially activated following ionizing radiation. Consequently, GSCs exhibited rapid G2/M cell cycle checkpoint activation and enhanced DNA repair. Inhibition of CHK1 or ATR successfully abrogated G2/M checkpoint function,
leading to increased mitotic catastrophe, but modestly increased radiation sensitivity. Conversely, ATM inhibition exerted dual effects on cell cycle checkpoint regulation and DNA repair that were associated with greater radiosensitizing effects than inhibition of CHK1, ATR or PARP alone. Combined PARP and ATR inhibition resulted in profound radiosensitization of GSCs that was greater than in bulk populations and also exceeded the effect of ATM inhibition. These data demonstrate that multiple, parallel DNA damage signaling pathways contribute to GSC radioresistance and that combined inhibition of cell cycle checkpoint and DNA repair targets provides the most effective means to overcoming therapy resistance.

Castration-resistant prostate cancers (CRPC) that arise after the failure of androgen-blocking therapies cause most of the deaths from prostate cancer, intensifying the need to fully understand CRPC pathophysiology. In this study, we characterized the transcriptomic differences between untreated prostate cancer and locally recurrent CRPC. Here, we report the identification of 145 previously unannotated intergenic long noncoding RNA transcripts (lncRNA) or isoforms that are associated with prostate cancer or CRPC. Of the one third of these transcripts that were specific for CRPC, we defined a novel lncRNA termed PCAT5 as a regulatory target for the transcription factor ERG, which is activated in approximately 50% of human prostate cancer. Genome-wide expression analysis of a PCAT5-positive prostate cancer after PCAT5 silencing highlighted alterations in cell proliferation pathways. Strikingly, an in vitro validation of these alterations revealed a complex integrated phenotype affecting cell growth, migration, invasion, colony-forming potential, and apoptosis. Our findings reveal a key molecular determinant of differences between prostate cancer and CRPC at the level of the transcriptome. Furthermore, they establish PCAT5 as a novel oncogenic lncRNA in ERG-positive prostate cancers, with implications for defining CRPC biomarkers and new therapeutic interventions. Cancer Res; 75(19); 1–6. ©2015 AACR.

Castration-resistant prostate cancer (CRPC) that arise after the failure of androgen blocking therapies cause most of the deaths from prostate cancer (PC), intensifying the need to fully understand CRPC pathophysiology. In this study, we characterized the transcriptomic differences between untreated PC and locally recurrent CRPC. Here we report the identification of 145 previously unannotated intergenic long non-coding RNA transcripts (lncRNA) or isoforms that are associated with PC or CRPC. Of the one-third of these transcripts that were specific for CRPC, we defined a novel lncRNA termed PCAT5 as a regulatory target for the transcription factor ERG, which is activated in ~50% of human PC. Genome-wide expression analysis of a PCAT5-positive PC after PCAT5 silencing highlighted alterations in cell proliferation pathways. Strikingly, an in vitro validation of these alterations revealed a complex integrated phenotype affecting cell growth, migration, invasion, colony-forming potential and apoptosis. Our findings reveal a key molecular determinant of differences between PC and CRPC at the level of the transcriptome. Further, they establish PCAT5 as a novel oncogenic lncRNA in ERG-positive prostate cancers, with implications for defining CRPC biomarkers and new therapeutic interventions.