Targeting Homologous Recombination in Notch-Driven <i>C</i>. <i>elegans</i> Stem Cell and Human Tumors

<div><p>Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ <i>glp-1(ar202)</i>, a temperature-sensitive gain-of-function <i>C</i>. <i>elegans</i> NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, <i>glp-1(ar202)</i> is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.</p></div

RNAi of DDR orthologs detects genes that enhance <i>ar202</i> tumor radiosensitivity.

<p>N* represents number of animals examined</p><p>RNAi of DDR orthologs detects genes that enhance <i>ar202</i> tumor radiosensitivity.</p

Adenoviral Transduction of Human Acid Sphingomyelinase into Neo-Angiogenic Endothelium Radiosensitizes Tumor Cure

<div><p>These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, <i>PPE1(3x)</i>, and a hypoxia-inducible dual-binding <i>HIF-2α-Ets-1</i> enhancer element upstream of the <i>asmase</i> gene, inserted into a replication-deficient adenovirus yielding the vector <i>Ad5H2E-PPE1(3x)-ASMase</i>. This vector confers ASMase over-expression in cycling angiogenic endothelium <i>in vitro</i> and within tumors <i>in vivo</i>, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with <i>Ad5H2E-PPE1(3x)-ASMase</i> markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, <i>Ad5H2E-PPE1(3x)-ASMase</i> treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.</p></div

Response of <i>C</i>. <i>elegans</i> germline tumors to ionizing radiation.

<p>(A) Time course of germ cell accumulation in wild-type and <i>glp-1(ar202)</i> hermaphrodites. Worms were stained with DAPI at the indicated times after egg laying and imaged (20x magnification). Data (mean±s.e.m) represent number of germ nuclei⁄gonad in a minimum of 10 gonad arms. (B) Representative images of germline tumors in adult <i>glp-1(ar202)</i> post radiation. Worms were irradiated at the L2-L3 stage (30h after egg laying) and DAPI stained at 40h post irradiation. (C) Stage sensitivity of germline tumors to ionizing radiation. <i>glp-1(ar202)</i> were irradiated at the L2-L3 or late L4 stage, and quantified as in (A). All experiments were performed at 25°C as described in Methods.</p

Knockdown of HDR genes results in abnormal morphology in <i>C</i>. <i>elegans</i> oocytes.

<p>15–25 animals were examined per group at each dose</p><p>Knockdown of HDR genes results in abnormal morphology in <i>C</i>. <i>elegans</i> oocytes.</p

Infection with <i>Ad5H2E-PPE1(3x)-GFP</i> confers GFP expression specific to endothelial cells.

<p>(<b>A</b>) Schematic representation of gene therapy vectors used to overexpress the GFP reporter or human ASMase. (<b>B</b>) Primary cultures of bovine and human endothelial cells (BAEC, HUVEC and HCAEC) and non-endothelial cells (HeLa) were infected with <i>Ad5H2E-PPE1(3x)-GFP</i>. GFP expression was measured in live cells following detachment 72 h post-infection by flow cytometry.</p

Expression of ASMase in endothelium of MCA/129 fibrosarcomas implanted in <i>asmase^−/−</i> mice restores radiation-induced endothelial apoptosis and tumor cure.

<p>MCA/129 fibrosarcoma cells (10⁶, resuspended in PBS) were injected intra-dermally into the right hind limb of <i>asmase^−/−</i> mice (A, B, C) and tumor volume (based on caliper measurements) was calculated daily according to the formula by Kim et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069025#pone.0069025-Kim3" target="_blank">[54]</a>. At 90–130 mm³, 1×10¹⁰ PFU of <i>Ad5Empty</i> or <i>Ad5H2E-PPE1(3x)-ASMase</i> was administered intravenously. 5 days post viral administration tumors were locally irradiated with 15 Gy or left untreated. (A) Representative cross sections of MCA/129 fibrosarcoma excised from unirradiated animals (upper panel) and at 6 hours post 15 Gy (lower panel), and co-stained for an endothelial-specific marker (Meca-32, blue) and apoptosis (TUNEL; brown). (B) Quantitation of the effect of <i>Ad5H2E-PPE1(3x)-ASMase</i> treatment on radiation-induced endothelial cell apoptosis. Data (mean ± SEM) represent TUNEL-positive endothelial cells quantified from 20 fields/tumor (400× magnification) and 2 tumors per group. (C) Impact of treatment with <i>Ad5Empty</i> (gray lines) or <i>Ad5H2E-PPE1(3x)-ASMase</i> (black lines) followed by 15 Gy on MCA/129 fibrosarcoma response. N equals number of animals per group. Tumors were measured daily up to 40 days and twice weekly thereafter. (D) Lack of impact of <i>Ad5H2E-PPE1(3x)-ASMase</i> on small intestinal radiation sensitivity. Mice, pre-treated for 5 days with <i>Ad5Empty</i> or <i>Ad5H2E-PPE1(3x)-ASMase,</i> were subjected to 15 Gy (left) or 8–14 Gy (right) total body irradiation known to cause GI damage. Mice were sacrificed at 4 h (left) for endothelial apoptosis measurement or after 3.5 days (right) for Crypt Microcolony Assay as standardized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069025#pone.0069025-Rotolo1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069025#pone.0069025-Withers1" target="_blank">[27]</a>. Endothelial apoptosis was identified by microscopic co-detection of TUNEL (brown) and MECA-32 (blue) staining. Data (mean ± SEM) were compiled from 2 mice each, analyzing apoptotic cells in the lamina propria of 20 intact crypt-villus units per mouse. Crypt survival curves were calculated using 2 mice per dose analyzing 5–10 crypt-villus units per mouse by least square regression analysis, with a modification of the FIT software program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069025#pone.0069025-Albright1" target="_blank">[55]</a>. The program fits curves by iteratively weighted least squares to each set of dose–survival data, estimates covariates of survival curve parameters and corresponding confidence regions, and plots the survival curve.</p

Overexpression of human ASMase via <i>Ad5H2E-PPE1(3x)-ASMase</i> increases lysosomal and secretory ASMase activity, radiosensitizes BAEC, and attenuates bFGF protection against radiation-induced apoptosis.

<p>(A) Cellular homogenates and serum-free conditioned media were harvested from BAEC transduced with <i>Ad5Empty</i> or <i>Ad5H2E-PPE1(3x)-ASMase</i> and assayed for ASMase activity using [¹⁴C-methylcholine]sphingomyelin as substrate in the presence of 1 mM EDTA (cellular homogenates) or 0.1 mM Zn²⁺ (conditioned media). (B) BAEC transduced with <i>Ad5Empty</i> or <i>Ad5H2E-PPE1(3x)-ASMase</i> were pre-treated with 1 ng/ml bFGF 15 minutes prior to 10 Gy irradiation. Apoptosis was assessed at various time points after IR by morphologic analysis following <i>bis</i>-benzimide staining. Data (mean ± SEM) are collated from 3 experiments performed in triplicate in which 400 nuclei were analyzed per sample.</p

IR induces CRP formation on the surface of purified tumor endothelium.

<p>(<b>A</b>) Representative image of the clustering of ceramide into platforms on the outer leaflet of the plasma membrane of purified tumor endothelial cells at 1 min post 15 Gy. For these studies, irradiated cells were stained with Texas Red-labeled anti-ceramide antibody, and platforms identified as in Materials and Methods. Images are representative of over 30% of cells from 3 experiments in which over 100 cells were analyzed. (<b>B</b>) Time-dependent generation of platforms in tumor endothelial cells. CRPs in tumor endothelial cells irradiated <i>ex vivo</i> with 15 Gy were quantified as in Materials and Methods. Data (mean ± 95% CI) are collated from 3 experiments in which 200 cells were analyzed per point.</p

Tumor cells in <i>glp-1(ar202)</i> arrest in G2 phase following radiation exposure, and are apoptosis resistant.

<p>(A) Worms were irradiated at L4 stage and after 12h stained with DAPI. Representative germline tumors are outlined. (B) Relative nuclear DNA content of distal germ nuclei in unirradiated (0Gy) or irradiated (480Gy) worms. A total of 207 nuclei from 5 unirradiated worms, and 147 nuclei from 5 irradiated worms were scored. (*<i>p</i><0.05; **<i>p</i><0.01 relative to non-irradiated control). (C) Wild-type and <i>glp-1(ar202)</i> unirradiated or irradiated germline are stained with anti-phospho-Tyr15-CDK-1 antiserum (red) and DAPI (blue) as in Methods. White bar indicates border of proliferative zone. Asterisk indicates position of distal end. Scale bar is 20 μm. (D) Comparison of radiation-induced germ cell apoptosis in wild type and <i>glp(ar202)</i>. Wild type and mutant worms were synchronized at 25°C and irradiated with 240Gy at the L4 stage. Germline apoptosis was scored in one gonad loop per worm. Incidence of germ cell death was quantified by dividing number of apoptotic germ cells by total germ cells. Data (mean±s.e.m) are from 10–12 worms⁄group. (E) Inactivation of apoptosis does not alter <i>ar202</i> response to radiation. <i>glp-1(ar202)</i> and <i>glp-1(ar202);ced-3(n717)</i> double mutant worms were irradiated at the L4 stage. Data (mean±s.e.m) are from 9–12 worms⁄group. Note the line of “<i>glp-1</i> 480Gy” is hidden behind the line of “<i>glp-1;ced-3</i> 480Gy”.</p