TRAIL receptor targeting agents potentiate PARP inhibitor efficacy in pancreatic cancer independently of BRCA2 mutation status

Chemotherapy, the standard treatment for pancreatic ductal adenocarcinoma (PDAC), has only a modest effect on the outcome of patients with late-stage disease. Investigations of the genetic features of PDAC have demonstrated a frequent occurrence of mutations in genes involved in homologous recombination (HR), especially in the breast cancer susceptibility gene 2 (BRCA2). Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is approved as a maintenance treatment for patients with advanced PDAC with germline BRCA1/2 mutations following a platinum-containing first-line regimen. Limitations to the use of PARP inhibitors are represented by the relatively small proportion of patients with mutations in BRCA1/2 genes and the modest capability of these substances of inducing objective response. We have previously shown that pancreatic cancer with BRCA2 mutations exhibits a remarkably enhanced sensitivity towards tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor-stimulating agents. We thus aimed to investigate the effect of combined treatment with PARP inhibitors and TRAIL receptor-stimulating agents in pancreatic cancer and its dependency on the BRCA2 gene status. The respective effects of TRAIL-targeting agents and the PARP inhibitor olaparib or of their combination were assessed in pancreatic cancer cell lines and patient-derived organoids. In addition, BRCA2-knockout and -complementation models were investigated. The effects of these agents on apoptosis, DNA damage, cell cycle, and receptor surface expression were assessed by immunofluorescence, Western blot, and flow cytometry. PARP inhibition and TRAIL synergized to cause cell death in pancreatic cancer cell lines and PDAC organoids. This effect proved independent of BRCA2 gene status in three independent models. Olaparib and TRAIL in combination caused a detectable increase in DNA damage and a concentration-dependent cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also significantly increased the proportion of membrane-bound death receptor 5. Our results provide a preclinical rationale for the combination of PARP inhibitors and TRAIL receptor agonists for the treatment of pancreatic cancer and suggest that the use of PARP inhibitors could be extended to patients without BRCA2 mutations if used in combination with TRAIL agonists

Genome‐wide cooperation of EMT transcription factor ZEB 1 with YAP and AP ‐1 in breast cancer

Invasion, metastasis and therapy resistance are the major cause of cancer-associated deaths, and theEMT-inducing transcription factorZEB1 is a crucial stimulator of these processes. While work onZEB1 has mainly focused on its role as a transcriptional repressor, it can also act as a transcriptional activator. To further understand these two modes of action, we performed a genome-wideZEB1 binding study in triple-negative breast cancer cells. We identifiedZEB1 as a novel interactor of theAP-1 factorsFOSL1 andJUNand show that, together with the Hippo pathway effectorYAP, they form a transactivation complex, predominantly activating tumour-promoting genes, thereby synergising with its function as a repressor of epithelial genes. High expression ofZEB1,YAP,FOSL1 andJUNmarks the aggressive claudin-low subtype of breast cancer, indicating the translational relevance of our findings. Thus, our results link critical tumour-promoting transcription factors:ZEB1,AP-1 and Hippo pathway factors. Disturbing their molecular interaction may provide a promising treatment option for aggressive cancer types

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development

Background: SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517–595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. Results: By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516–584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1–639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. Conclusions: Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination

Supplementary table 2.xlsx

<p><b>Lipidomic data </b> <b></b></p><p>Lipidomic maps were generated for mouse KGC pancreatic tumor organoid lines expressing doxycycline-inducible constitutively-active<b> </b>mutant GNAS (GNAS^R201C). The organoids were analyzed in the presence of doxycycline; Dox(+) or absence of doxycycline; Dox(-). LC-MS experiments for non-polar metabolites were performed using a KCG organoid line in triplicate for the +Dox and -Dox conditions and denoted as Dox(+)-1; Dox(+)-2; Dox(+)-3; Dox(-)-1; Dox(-)-2; Dox(-)-3. LC-MS/MS-based lipidomic experiments and data analyses were performed as described in the reference¹. The data were normalized to the total number of cells in the organoids from replicate wells. Metabolites were quantified by integrating the area under the curve. Lipidation (lipid species detected); Class (class of lipids. see lipid keys in the document)</p><p>Reference: 1) Smulan, L. J.<i>et al.</i>Cholesterol-Independent SREBP-1 Maturation Is Linked to ARF1 Inactivation. <i>Cell reports</i><b>16</b>, 9-18, doi:10.1016/j.celrep.2016.05.086 (2016).</p

Supplementary table 3

<p><b>Polar metabolite data</b></p><p>Polar metabolite profiling data were generated for a mouse KGC pancreatic tumor line expressing doxycycline-inducible constitutively-active<b> </b>mutant GNAS (GNAS^R201C) growing in 2D culture in the presence of doxycycline; Dox(+) or absence of doxycycline; Dox(-). The cells were harvested as 5 biological replicates and flash-frozen. The polar metabolites were extracted with 40:40:20 acetonitrile/methanol/water and replicates are denoted as Dox(+)-1, Dox(+)-2, Dox(+)-3, Dox(+)-4, Dox(+)-5 and Dox(-)-1, Dox(-)-2, Dox(-)-3, Dox(-)-4, Dox(-)-5. Polar metabolites were analyzed by QQQ-LC/MS/MSas described in the reference^1.The ion counts were normalized to the total cell number. Metabolites were quantified by integrating the area under the curve, and then normalized to internal standard values. </p><p> </p><p>1) Louie, S. M.<i>et al.</i>GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity. <i>Cell Chem Biol</i><b>23</b>, 567-578, doi:10.1016/j.chembiol.2016.03.017 (2016). </p