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RAD51 paralogs are key components of the homologous recombination (HR) machinery. Mouse mutants have been reported for four of the canonical RAD51 paralogs, and each of these mutants exhibits embryonic lethality, although at different gestational stages. However, the phenotype of mice deficient in the fifth RAD51 paralog, XRCC3, has not been reported. Here we report that Xrcc3 knockout mice exhibit midgestational lethality, with mild phenotypes beginning at about E8.25 but severe developmental abnormalities evident by E9.0–9.5. The most obvious phenotypes are small size and a failure of the embryo to turn to a fetal position. A knockin mutation at a key ATPase residue in the Walker A box results in embryonic lethality at a similar stage. Death of knockout mice can be delayed a few days for some embryos by homozygous or heterozygous Trp53 mutation, in keeping with an important role for XRCC3 in promoting genome integrity. Given that XRCC3 is a unique member of one of two RAD51 paralog complexes with RAD51C, these results demonstrate that both RAD51 paralog complexes are required for mouse development.

Direct-to-Patient (DTP) Multiple Myeloma (MM) research studies have been launched recently, including PCROWD (NCT02269592), PROMISE (NCT03689595) and the MMRF CureCloud Research Initiative (NCT03657251), aimed at enrolling thousands of individuals from whom comprehensive molecular and immune analyses will be generated from blood specimens and the resulting data aggregated with the correlating clinical information. To support the molecular characterization of liquid biopsies for such DTP efforts, a set of myeloma-specific liquid biopsy approaches were developed. First, a hybrid selection panel was developed that detects somatic variants present in a patient's circulating-free DNA (cfDNA) in 70 commonly altered MM and Clonal Hematopoiesis of Indeterminate Potential (CHIP) genes. For this MM 70-Gene cfDNA Assay, samples are received as blood in a StreckTM tube designed for stabilization of cfDNA and DNA is extracted from buffy coat using magnetic bead-based chemistry. Deep coverage sequencing (80,000x depth) is performed and duplex BAM files generated with UMI alignment and error correction allowing for sensitive detection of clinically relevant variants. Technical validation data on healthy donor cfDNA mixes was generated using samples with a range of cfDNA inputs. This data determined that the assay is capable of achieving >90% sensitivity for detecting somatic events present at 1% variant allele frequency with a specificity of Because one the aims of this effort is to return results to treating physicians, a clinical-grade (CLIA) pipeline was established. For that CLIA pipeline, the variants reported are a subset of all the events detected by the MM 70-Gene Assay. The events detected in the assay are reviewed by experienced molecular pathologists at the Dana Farber Cancer Institute (DFCI) who have developed a customized reporting process. These reports utilize an internally-developed knowledgebase of variant/gene annotations that leverages the DFCI expertise in hematologic malignancies and myeloma specifically. The reports are then provided back through providers to the patient via the CureCloud system for their use in clinical care and trial identification. In order to complement the MM-70 Gene panel with copy number and translocation information, we have been exploring Circulating Multiple Myeloma Cells (CMMCs). Our current approach involves automated capture of CMMCs using ferrofluids coated with MM-selective and discriminating antibodies to immunomagnetically enrich circulating plasma cells. The highly enriched CMMCs fractions generated in such a fashion are then submitted for molecular characterization. At the submission date of this abstract, 163 patients have been fully enrolled into CureCloud from which results will be presented. In summary, we have developed a robust and very sensitive clinical-grade next-gen liquid biopsy sequencing platform allowing for minimally invasive monitoring of MM disease genomics that can be used to complement other more classical approaches and to help support our Direct-To-Patient Initiatives. Especially in this post-COVID19 era, such liquid biopsy-based approaches that avoid clinic visits for the patients and can be performed through at-home mobile phlebotomy are emerging as important new options. Disclosures Kim: LabCorp: Consultancy; Quanterix, Inc: Consultancy; PapGene, Inc: Consultancy. Ghobrial:AbbVie: Consultancy; GNS Healthcare: Consultancy; GlaxoSmithKline: Consultancy; Genentech: Consultancy; Noxxon Pharma: Consultancy; Novartis: Consultancy; Adaptive Biotechnologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Cellectar: Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Direct-to-patient (DTP) multiple myeloma (MM) research studies have been launched recently, including PCROWD (NCT02269592), PROMISE (NCT03689595), and the MMRF CureCloud Research Initiative (NCT03657251), aimed at enrolling thousands of individuals from whom comprehensive molecular and immune analyses will be generated from blood specimens and the resulting data aggregated with the correlating clinical information. To support the molecular characterization of liquid biopsies for such DTP efforts, a myeloma-specific hybrid selection panel was developed that captures 70 commonly altered genes. The assay detects somatic point mutations and indels present in a patient’s circulating-free DNA (cfDNA). For this MM 70-Gene cfDNA Assay, samples are received as blood in a Streck’s tube and DNA is extracted from buffy coat using magnetic bead-based chemistry. Coverage sequencing (80,000x depth) is performed and duplex BAM files are generated with UMI alignment and de-duplication. As will be presented, MM blood specimens present a unique challenge as circulating MM cells are often present at significant levels in the buffy coat blood fraction used as the source of normal genomic DNA. The performance of the 70-Gene cfDNA Assay was thoroughly validated in order to establish the sensitivity, specificity, and reproducibility of the technical approach. First, the reference genomic DNA from unrelated healthy individuals was sequenced in replicate at deep coverage. Next, two cohorts were used, one from Dana-Farber and one from the MMRF CureCloud pilot. For both cohorts, tumor DNA samples from bone marrow aspirates (BMAs) with matched normal DNA from blood were sequenced on an orthogonal platform and compared to results from the MM 70-Gene Assay on cfDNA extracted from the same individuals. The yield of extracted cfDNA ranged from 6 ng to 80 ng, and about two third of cases yielded enough material to attempt sequencing, with failures coming mostly from individuals in remission. As will be presented, there was a very strong correlation between BMA and cfDNA and additional events could actually be detected in the blood that were not seen in the BMAs. Because this MM 70-Gene cfDNA Assay may potentially be used by treating physicians for management of care, a clinical-grade (CLIA) pipeline was established. For that CLIA pipeline, the variants reported are a subset of all the events detected by the MM 70-Gene Assay. The events detected in the assay are reviewed by a Genomic Tumor Board within the appropriate subset of territory predefined for reporting. The territory limitations are defined by the Genomic Tumor Board knowledgebase of actionable genomic territory available. In summary, we have developed a robust and very sensitive clinical-grade next-gen liquid biopsy sequencing platform allowing for less invasive monitoring of MM disease genomics that can be used to complement other more classical approaches and to help support direct-to-patient Initiatives. Citation Format: Mark W. Bustoros, Carrie Cibulskis, Teni Dowdell, Svetlana Gavrilov, Cody Boehner, Jennifer Yesil, Steven E. Labkoff, Shaadi Mehr, Jihye Park, Romanos Sklavenitis Pistofidis, Salomon Manier, Annette S. Kim, Keith L. Ligon, Niall Lennon, Viktor Adalsteinsson, Jane Wilkinson, Irene M. Ghobrial, Daniel Auclair. A novel clinical-grade liquid biopsy platform for multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A38.

Background The dual-specificity T-box/basic helix-loop-helix leucine zipper transcription factor MGA is part of the MAX-interacting network of proteins. In the mouse, MGA is necessary for the survival of the pluripotent epiblast cells of the peri-implantation embryo and a null, gene-trap allele MgaGt results in embryonic lethality shortly after implantation. We have used this allele to document expression of Mga in postimplantation embryos and also investigated a second, hypomorphic gene-trap allele, MgaInv. Results Compound heterozygotes, MgaGt/MgaInv, die prior to midgestation. The extraembryonic portion of the embryos appears to develop relatively normally while the embryonic portion, including the pluripotent cells of the epiblast, is severely retarded by E7.5. Mga expression is initially limited to the pluripotent inner cell mass of the blastocyst and epiblast, but during organogenesis it is widely expressed, notably in the central nervous system and sensory organs, reproductive and excretory systems, heart, somites and limbs. Conclusions Widespread yet specific areas of expression of Mga during organogenesis raise the possibility that the transcription factor may play roles in controlling proliferation and potency in the progenitor cell populations of different organ systems. Documentation of these patterns sets the stage for the investigation of specific progenitor cell types.

Ventral folding morphogenesis, a vital morphogenetic process in amniotes, mediates gut endoderm internalization, linear heart tube formation, ventral body wall closure and encasement of the fetus in extraembryonic membranes. Aberrant ventral folding morphogenesis underlies a number of birth defects, such as gastroschisis and ectopia cordis in human and misplacement of head and heart in mouse. Recent cell lineage-specific mouse mutant analyses identified the Bone Morphogenetic Protein (BMP) pathway and Anterior Visceral Endoderm (AVE) as key regulators of anterior ventral folding morphogenesis. Loss of BMP2 expression solely from embryonic visceral endoderm (EmVE) and the AVE blocks formation of foregut invagination, and simultaneously, aberrantly positions the heart anterior/dorsal to the head, suggesting a mechanistic link between foregut and head/heart morphogenesis.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.

Early mammalian embryonic development is characterized by the existence of pluripotent or multipotent cell populations that are present for limited periods of time during lineage specification and cellular differentiation. In addition to the pluripotent embryonic stem cell lines that can be derived from the epiblast lineage of the inner cell mass of the blastocyst, stem cell lines have now been derived from the trophectoderm layer, the primitive endoderm, and the postimplantation epiblast, as well as from the primordial germ cells. These stem cell lines generally have the developmental potential and characteristics corresponding to their tissue of origin and the conditions for their derivation reflect the intrinsic genetic programs of lineage specification and differentiation. The extensive investigation of mouse embryo-derived stem cell lines has paved the way for therapeutic applications of stem cells. The derivation of pluripotent stem cells from human embryos as well as the development of induced pluripotent stem cell lines from non-embryo sources brings widespread application of stem cell-based therapies closer to reality.

Publisher Summary Regenerative medicine entails a broad range of methodologies that utilize cells or tissues as therapeutic tools to rebuild or repair damaged or diseased structures and restore their function. The source of therapeutic cells can be autologous, allogenic, or xenogenic. If allogenic, then these cells can be harvested without harm or risk of harm or cooperation with harm to another human being or harvested with corresponding harm. Ethical issues abound in experimental medicine but those that arise uniquely in regenerative medicine reflect this latter circumstance, wherein one human life is used as an object to satisfy the needs or desires of another. The developmental potential of embryonic cells and fetal cells and tissues has attracted the compelling interest of investigators. The interest of the political establishment and the media are similarly engaged in part because the ethics of regenerative medicine are so frequently intertwined with the ethics of abortion. The ethics of human embryonic stem cells (hESCs) and of abortion both hinge on the respect, or the absence of respect, accorded to the human organism at a prenatal stage of development.

We report the derivation and characterization of two new human embryonic stem cells (hESC) lines (CU1 and CU2) from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED) 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion ofin vitrofertilization (IVF) embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.