A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre‐clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain‐dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.
The primary aim of this study was to evaluate the antitumor efficacy of the bromodomain inhibitor JQ1 in pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (tumorgraft) models. A secondary aim of the study was to evaluate whether JQ1 decreases expression of the oncogene c-Myc in PDAC tumors, as has been reported for other tumor types. We used five PDAC tumorgraft models that retain specific characteristics of tumors of origin to evaluate the antitumor efficacy of JQ1. Tumor-bearing mice were treated with JQ1 (50 mg/kg daily for 21 or 28 days). Expression analyses were performed with tumors harvested from host mice after treatment with JQ1 or vehicle control. An nCounter PanCancer Pathways Panel (NanoString Technologies) of 230 cancer-related genes was used to identify gene products affected by JQ1. Quantitative RT-PCR, immunohistochemistry and immunoblots were carried out to confirm that changes in RNA expression reflected changes in protein expression. JQ1 inhibited the growth of all five tumorgraft models (P<0.05), each of which harbors a KRAS mutation; but induced no consistent change in expression of c-Myc protein. Expression profiling identified CDC25B, a regulator of cell cycle progression, as one of the three RNA species (TIMP3, LMO2 and CDC25B) downregulated by JQ1 (P<0.05). Inhibition of tumor progression was more closely related to decreased expression of nuclear CDC25B than to changes in c-Myc expression. JQ1 and other agents that inhibit the function of proteins with bromodomains merit further investigation for treating PDAC tumors. Work is ongoing in our laboratory to identify effective drug combinations that include JQ1.
Background DNA repair deficiency accumulates DNA damage and sensitizes tumor cells to PARP inhibitors (PARPi). Based on our observation that the BET inhibitor JQ1 increases levels of DNA damage, we evaluated the efficacy of JQ1 + the PARPi olaparib in preclinical models of pancreatic ductal adenocarcinoma (PDAC). We also addressed the mechanism by which JQ1 increased DNA damage. Methods The effect of JQ1 + olaparib on in vivo tumor growth was assessed with patient-derived xenograft (PDX) models of PDAC. Changes in protein expression were detected by immunohistochemistry and immunoblot. In vitro growth inhibition and mechanistic studies were done using alamarBlue, qRT-PCR, immunoblot, immunofluorescence, ChIP, and shRNA knockdown assays. Findings Tumors exposed in vivo to JQ1 had higher levels of the DNA damage marker γH2AX than tumors exposed to vehicle only. Increases in γH2AX was concomitant with decreased expression of DNA repair proteins Ku80 and RAD51. JQ1 + olaparib inhibited the growth of PDX tumors greater than either drug alone. Mechanistically, ChIP assays demonstrated that JQ1 decreased the association of BRD4 and BRD2 with promoter loci of Ku80 and RAD51, and shRNA data showed that expression of Ku80 and RAD51 was BRD4- and BRD2-dependent in PDAC cell lines. Interpretation The data are consistent with the hypothesis that JQ1 confers a repair deficient phenotype and the consequent accumulation of DNA damage sensitizes PDAC cells to PARPi. Combinations of BET inhibitors with PARPi may provide a novel strategy for treating PDAC. Fund NIH grants R01CA208272 and R21CA205501; UAB CMB T32 predoctoral training grant.
Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer. Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types. Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing. Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models. We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation. Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.
Cholangiocarcinoma (CCA) is a fatal disease with a 5-year survival of <30%. For a majority of patients, chemotherapy is the only therapeutic option, and virtually all patients relapse. Gemcitabine is the first-line agent for treatment of CCA. Patients treated with gemcitabine monotherapy survive ∼8 months. Combining this agent with cisplatin increases survival by ∼3 months, but neither regimen produces durable remissions. The molecular etiology of this disease is poorly understood. To facilitate molecular characterization and development of effective therapies for CCA, we established a panel of patient-derived xenograft (PDX) models of CCA. We used two of these models to investigate the antitumor efficacy and mechanism of action of the bromodomain inhibitor JQ1, an agent that has not been evaluated for the treatment of CCA. The data show that JQ1 suppressed the growth of the CCA PDX model CCA2 and demonstrate that growth suppression was concomitant with inhibition of c-Myc protein expression. A second model (CCA1) was JQ1-insensitive, with tumor progression and c-Myc expression unaffected by exposure to this agent. Also selective to CCA2 tumors, JQ1 induced DNA damage and apoptosis and downregulated multiple c-Myc transcriptional targets that regulate cell-cycle progression and DNA repair. These findings suggest that c-Myc inhibition and several of its transcriptional targets may contribute to the mechanism of action of JQ1 in this tumor type. We conclude that BET inhibitors such as JQ1 warrant further investigation for the treatment of CCA. .
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