Deregulation of protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression. eIF5A1 and its highly related isoform eIF5A2 are translation initiation factors that have been implicated in a range of human malignancies, but how they control cancer development and disease progression is still poorly understood. Here, we investigated how eIF5A proteins regulate pancreatic ductal adenocarcinoma (PDAC) pathogenesis. eIF5A proteins are the only known proteins regulated by a distinct posttranslational modification termed hypusination, which is catalyzed by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The highly selective nature of the hypusine modification and its amenability to pharmacologic inhibition make eIF5A proteins attractive therapeutic targets. We found that the expression and hypusination of eIF5A proteins are upregulated in human PDAC tissues and in premalignant pancreatic intraepithelial neoplasia tissues isolated from Pdx-1-Cre: LSL-KRASG12D mice. Knockdown of eIF5A proteins in PDAC cells inhibited their growth in vitro and orthotopic tumor growth in vivo, whereas amplification of eIF5A proteins increased PDAC cell growth and tumor formation in mice. Small-molecule inhibitors of DHPS and DOHH both suppressed eIF5A hypusination, preventing PDAC cell growth. Interestingly, we found that eIF5A proteins regulate PDAC cell growth by modulating the expression of PEAK1, a nonreceptor tyrosine kinase essential for PDAC cell growth and therapy resistance. Our findings suggest that eIF5A proteins utilize PEAK1 as a downstream effector to drive PDAC pathogenesis and that pharmacologic inhibition of the eIF5A–hypusine–PEAK1 axis may provide a novel therapeutic strategy to combat this deadly disease. Cancer Res; 74(22); 1–11. ©2014 AACR.

Deregulation of protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression. eIF5A1, and its highly related isoform eIF5A2, are translation initiation factors that have been implicated in a range of human malignancies, but how they control cancer development and disease progression is still poorly understood. Here, we investigated how eIF5A proteins regulate pancreatic ductal adenocarcinoma (PDAC) pathogenesis. eIF5A proteins are the only known proteins regulated by a distinct posttranslational modification termed hypusination, which is catalyzed by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The highly selective nature of the hypusine modification and its amenability to pharmacological inhibition make eIF5A proteins attractive therapeutic targets. We found that the expression and hypusination of eIF5A proteins are upregulated in human PDAC tissues and in premalignant pancreatic intraepithelial neoplasia (PanIN) tissues isolated from Pdx-1-Cre: LSL-KRASG12D mice. Knockdown of eIF5A proteins in PDAC cells inhibited their growth in vitro and orthotopic tumor growth in vivo, whereas amplification of eIF5A proteins increased PDAC cell growth and tumor formation in mice. Small molecule inhibitors of DHPS and DOHH both suppressed eIF5A hypusination, preventing PDAC cell growth. Interestingly, we found that eIF5A proteins regulate PDAC cell growth by modulating the expression of PEAK1, a non-receptor tyrosine kinase essential for PDAC cell growth and therapy resistance. Our findings suggest that eIF5A proteins utilize PEAK1 as a downstream effector to drive PDAC pathogenesis, and that pharmacological inhibition of the eIF5A-hypusine-PEAK1 axis may provide a novel therapeutic strategy to combat this deadly disease.

Plasticity in cancer stem–like cells (CSC) may provide a key basis for cancer heterogeneity and therapeutic response. In this study, we assessed the effect of combining a drug that abrogates CSC properties with standard-of-care therapy in a Ewing sarcoma family tumor (ESFT). Emergence of CSC in this setting has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. In the present work, primary ESFT from four patients containing CD133+ CSC subpopulations ranging from 3% to 17% of total tumor cells were subjected to treatment with enoxacin, doxorubicin, or both drugs. Primary ESFT CSC and bulk tumor cells displayed divergent responses to standard-of-care chemotherapy and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin, which enhances miRNA maturation by stimulating TARBP2 function, induced apoptosis but only in ESFT spheres. In combination, the two drugs markedly depleted CSCs and strongly reduced primary ESFTs in xenograft assays. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low-toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation. Cancer Res; 74(22); 1–13. ©2014 AACR.

Plasticity in cancer stem-like cells (CSC) may provide a key basis for cancer heterogeneity and therapeutic response. In this study, we assessed the effect of combining a drug that abrogates CSC properties with standard-of-care therapy in a Ewing sarcoma family tumor (ESFT). Emergence of CSC in this setting has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. In the present work, primary ESFT from four patients containing CD133+ CSC subpopulations ranging from 3-17% of total tumor cells were subjected to treatment with enoxacin, doxorubicin or both drugs. Primary ESFT CSC and bulk tumor cells displayed divergent responses to standard-of-care chemotherapy and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin, which enhances miRNA maturation by stimulating TARBP2 function, induced apoptosis but only in ESFT spheres. In combination, the two drugs markedly depleted CSC and strongly reduced primary ESFT in xenograft assays. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation.

Cerenkov radiation generated by positron-emitting radionuclides can be exploited for a molecular imaging technique known as Cerenkov luminescence imaging (CLI). Data have been limited, however, on the use of medium- to high-energy β-emitting radionuclides of interest for cancer imaging and treatment. We assessed the use of CLI as an adjunct to determine localization of radioimmunoconjugates to hematolymphoid tissues. Radiolabeled 177Lu- or 90Y-anti-CD45 antibody (Ab; DOTA-30F11) was administered by tail vein injection to athymic mice bearing disseminated murine myeloid leukemia, with CLI images acquired at times afterward. Gamma counting of individual organs showed preferential uptake in CD45+ tissues with significant retention of radiolabeled Ab in sites of leukemia (spleen and bone marrow). This result was confirmed in CLI images with 1.35 × 105 ± 2.2 × 104 p/s/cm2/sr and 3.45 × 103 ± 7.0 × 102 p/s/cm2/sr for 90Y-DOTA-30F11 and 177Lu-DOTA-30F11, respectively, compared with undetectable signal for both radionuclides using the nonbinding control Ab. Results showed that CLI allows for in vivo visualization of localized β-emissions. Pixel intensity variability resulted from differences in absorbed doses of the associated energies of the β-emitting radionuclide. Overall, our findings offer a preclinical proof of concept for the use of CLI techniques in tandem with currently available clinical diagnostic tools. Cancer Res; 74(20); 1–9. ©2014 AACR.