Whole-Genome Sequencing Identifies Genetic Variances in Culture-Expanded Human Mesenchymal Stem Cells

Culture-expanded human mesenchymal stem cells (MSCs) are increasingly used in clinics, yet full characterization of the genomic compositions of these cells is lacking. We present a whole-genome investigation on the genetic dynamics of cultured MSCs under ex vivo establishment (passage 1 [p1]) and serial expansion (p8 and p13). We detected no significant changes in copy-number alterations (CNAs) and low levels of single-nucleotide changes (SNCs) until p8. Strikingly, a significant number (677) of SNCs were found in p13 MSCs. Using a sensitive Droplet Digital PCR assay, we tested the nonsynonymous SNCs detected by whole-genome sequencing and found that they were preexisting low-frequency mutations in uncultured mononuclear cells (∼0.01%) and early-passage MSCs (0.1%–1% at p1 and p8) but reached 17%–36% in p13. Our data demonstrate that human MSCs maintain a stable genomic composition in the early stages of ex vivo culture but are subject to clonal growth upon extended expansion

Histone Demethylases KDM4A and KDM4C Regulate Differentiation of Embryonic Stem Cells to Endothelial Cells

SummaryUnderstanding epigenetic mechanisms regulating embryonic stem cell (ESC) differentiation to endothelial cells may lead to increased efficiency of generation of vessel wall endothelial cells needed for vascular engineering. Here we demonstrated that the histone demethylases KDM4A and KDM4C played an indispensable but independent role in mediating the expression of fetal liver kinase (Flk)1 and VE-cadherin, respectively, and thereby the transition of mouse ESCs (mESCs) to endothelial cells. KDM4A was shown to bind to histones associated with the Flk1 promoter and KDM4C to bind to histones associated with the VE-cadherin promoter. KDM4A and KDM4C were also both required for capillary tube formation and vasculogenesis in mice. We observed in zebrafish that KDM4A depletion induced more severe vasculogenesis defects than KDM4C depletion, reflecting the early involvement of KDM4A in specifying endothelial cell fate. These findings together demonstrate the essential role of KDM4A and KDM4C in orchestrating mESC differentiation to endothelial cells through the activation of Flk1 and VE-cadherin promoters, respectively

Durvalumab Combined With Pemetrexed-Based Chemotherapy in Trial-Ineligible Patients With Mesothelioma: A Brief Report

Introduction: Chemoimmunotherapy is associated with promising activity in mesothelioma in phase II to III trials. Studies exploring this approach in patients ineligible for clinical trials are lacking. We assembled a cohort of patients receiving pemetrexed-based chemotherapy with durvalumab outside of clinical trials. Methods: Patients with pleural mesothelioma received pemetrexed plus durvalumab or carboplatin plus pemetrexed plus durvalumab via off-label authorization at Massachusetts General Hospital. Response to chemoimmunotherapy was assessed per modified Response Evaluation Criteria in Solid Tumors version 1.1. A retrospective chart review was conducted to assess safety per Common Terminology Criteria for Adverse Events version 5.0. Results: Twelve patients were included in the series. Nine patients were treated with triplet chemoimmunotherapy. Three patients received doublet chemoimmunotherapy because of platinum ineligibility. Concurrent active malignancies and symptomatic cardiac disease were present in three patients (25%) and two patients (17%), respectively. Ten patients had measurable disease at baseline. With the triplet regimen, partial responses were observed in four of the seven (57%) patients with measurable disease. All three patients receiving pemetrexed plus durvalumab had measurable disease and experienced a partial response. Primary progression was not observed with either regimen. Overall, eight patients (75%) remained on treatment for more than 6 months without progression. Five patients developed immune-related adverse events (n = 1 each pyrexia, arthritis, neutropenia, Raynaud’s disease, stomatitis). Three patients discontinued treatment because of toxicity or symptomatic comorbid conditions (n = 1 grade 3 heart failure, n = 1 grade 2 fever + progressive kidney cancer, n = 1 grade 2 fatigue). Conclusions: Antitumor activity of chemoimmunotherapy reported in phase II to III clinical trials is generalizable to the broader patient population with mesothelioma. However, the tolerability of chemoimmunotherapy is impacted by comorbid conditions in real-world patients

Abstract 1360: A mouse model of double heterozygosity for protein kinase A regulatory subunits promotes osteoblastic differentiation of cAMP-induced bone tumors

Abstract Background: Carney Complex (CNC) is a multiple neoplasia syndrome inherited in an autosomal dominant manner and causing various endocrine and bone tumors. The PRKAR1A gene coding for the regulatory subunit type 1A of protein kinase A (PKA) is mutated in CNC patients, but tumorogenesis mechanisms are not well understood. Regulatory subunits suppress activity of catalytic subunits of PKA responsible for cAMP signaling and cell differentiation and maturation. Mice with a deleted prkar1a allele develop a variety of tumors overlapping those of CNC patients. Deletion of a catalytic subunit allele prkaca+/− on the prkar1a+/ background unexpectedly increased the numer and aggressiveness of bone tumors without development of schwannomas or thyroid lesions. Cells from prkar1a+/−prkaca+/ tumors have more type II PKA complexes, indicating that unbalanced expression of type II and type I regulatory subunits may also dysregulates PKA catalytic activity, potentially increasing tumorigenesis. Methods: To explore this hypothesis, single mice with single alleles of prkar1a, prkar2a and prkar2b were crossed to generate heterozygous mice for double heterozygotes prkar1a+/−/prkar2a+/− or prkar1a+/−/prkar2b+/− and double knockouts. The resulting mice of 3,6,9,12,18 month age were phenotyped and compared to prkar1a+/−. Tumor specimens were examined by histology, polarized microscopy and confocal Raman micro-spectroscopy. Results: Mice with both alleles prkar1a+/+ did not develop any tumors. Double heterozygotes developed bone tumors whose onset age was delayed to 9 months in prkar1a+/−/prkar2b+/− mice compared to 3-6 months in the others. However, the mean number of tumors per animal became similar for prkar1a+/− and double heterozygotes by 12 months. Histology of the bone tumors showed abnormal proliferation of a fibroblastoid-like cell with better osteogenic differentiation in the lesions from double heterozygous mice. Polarized microscopy and Raman micro-spectroscopy revealed that bone material growing in tumors was immature and poorly organized in prkar1a+/− mice (disorganized collagen and osteocytes and undermineralized matrix). Prkar2α+/− deletion on the prkar1α+/− background rescued local organization and mineralization but worsened global organization of cortical tumor bone. The prkar1a+/−/prkar2b+/− deletion rescued both global and local organization and mineralization, resulting in formation of mature bone. Conclusion: Prkar1a haploinsufficiency requires both prkar2a and prkar2b for tumorigenesis in most tissues except bone. In bone, however, prkar1a is the major tumor-suppressor gene. Unexpectedly, Prkar2b or prkar2a deficiencies in addition to prkar1a lead to better differentiation of bone tumors, indicating a compensating role for the type II regulatory subunits in bone tumirogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1360. doi:1538-7445.AM2012-1360</jats:p