Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2–restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097–1105 and pCDC25b38–46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097–1105 or pCDC25b38–46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097–1105 was presented on HLA-A2+ melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser1100-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38–46 was narrower, but still evident on HLA-A2+ melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097–1105–specific and pCDC25b38–46–specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2+ cancers. Cancer Res; 74(23); 1–12. ©2014 AACR.

Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2-restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097-1105 and pCDC25b38-46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097-1105 or pCDC25b38-46. Expression of these TCR in human CD8 T-cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097-1105 was presented on HLA-A2+ melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser1100-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38-46 was narrower, but still evident on HLA-A2+ melanoma, breast carcinoma and lymphoblastoid cells. Notably, pIRS-21097-1105-specific and pCDC25b38-46-specific TCR-expressing human CD8 T-cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2+ cancers.

The kinase PRKD2 (protein kinase D) is a crucial regulator of tumor cell-endothelial cell communication in gastrointestinal tumors and glioblastomas, but its mechanistic contributions to malignant development are not understood. Here, we report that the oncogenic chaperone HSP90 binds to and stabilizes PRKD2 in human cancer cells. Pharmacologic inhibition of HSP90 with structurally divergent small molecules currently in clinical development triggered proteasome-dependent degradation of PRKD2, augmenting apoptosis in human cancer cells of various tissue origins. Conversely, ectopic expression of PRKD2 protected cancer cells from the apoptotic effects of HSP90 abrogation, restoring blood vessel formation in two preclinical models of solid tumors. Mechanistic studies revealed that PRKD2 is essential for hypoxia-induced accumulation of hypoxia-inducible factor-1α (HIF1α) and activation of NF-κB in tumor cells. Notably, ectopic expression of PRKD2 was able to partially restore HIF1α and secreted VEGF-A levels in hypoxic cancer cells treated with HSP90 inhibitors. Taken together, our findings indicate that signals from hypoxia and HSP90 pathways are interconnected and funneled by PRKD2 into the NF-κB/VEGF-A signaling axis to promote tumor angiogenesis and tumor growth. Cancer Res; 74(23); 1–12. ©2014 AACR.

The kinase PRKD2 is a crucial regulator of tumor cell-endothelial cell communication in gastrointestinal tumors and glioblastomas, but its mechanistic contributions to malignant development are not understood. Here we report that the oncogenic chaperone HSP90 binds to and stabilizes PRKD2 in human cancer cells. Pharmacologic inhibition of HSP90 with structurally divergent small molecules currently in clinical development triggered proteasome-dependent degradation of PRKD2, augmenting apoptosis in human cancer cells of various tissue origins. Conversely, ectopic expression of PRKD2 protected cancer cells from the apoptotic effects of HSP90 abrogation, restoring blood vessel formation in two preclinical models of solid tumors. Mechanistic studies revealed that PRKD2 is essential for hypoxia-induced accumulation of HIF-1α and activation of NF-κB in tumor cells. Notably, ectopic expression PRKD2 was able to partially restore HIF-1α and secreted VEGF-A levels in hypoxic cancer cells treated with HSP90 inhibitors. Taken together, our findings indicate that signals from hypoxia and HSP90 pathways are interconnected and funneled by PRKD2 into the NF-κB/ VEGF-A signaling axis to promote tumor angiogenesis and tumor growth.

Multiple myeloma (MM) is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, is expressed and active in all eleven MM-derived cell lines examined regardless of the type of cytogenetic abnormality, the mutation state of RAS and FGFR3 genes, or the activation state of ERK and AKT. Our results revealed that PDPK1 is a pivotal regulator of molecules that are essential for myelomagenesis, such as RSK2, AKT, c-MYC, IRF4, or Cyclin Ds, and that PDPK1 inhibition caused the growth inhibition and the induction of apoptosis with the activation of BIM and BAD, and augmented the in vitro cytotoxic effects of anti-myeloma agents in myeloma cells. In the clinical setting, PDPK1 was active in myeloma cells of approximately 90% of symptomatic patients at diagnosis, and the smaller population of MM patients exhibiting myeloma cells without active PDPK1 showed a significantly less frequent proportion of the disease stage III by the International Staging System and a significantly more favorable prognosis, including the longer overall survival period and the longer progression free survival period by bortezomib treatment, than patients with active PDPK1, suggesting that PDPK1 activation accelerates the disease progression and the resistance to treatment in MM. Our study demonstrates that PDPK1 is a potent and an universally targetable signaling mediator in MM regardless of the types of cytogenetic/molecular profiles.