Evaluating dose-limiting toxicities of MDM2 inhibitors in patients with solid organ and hematologic malignancies: A systematic review of the literature.

INTRODUCTION: Mouse double minute 2 protein (MDM2), a negative regulator of the p53 tumour suppressor gene, is frequently amplified in malignancies. MDM2 antagonists have shown efficacy in treating malignancies with MDM2 overexpression and can overcome chemoresistance in acute myeloid leukemia. We systematically evaluated the safety profile of MDM2 inhibitors in the treatment of solid organ and hematologic malignancies. MATERIALS AND METHODS: We searched Medline and EMBASE from January 1947 to November 2018 for prospective clinical studies, in English or French, investigating any MDM2 inhibitor in pediatric or adult cancers, and reporting dose and toxicity outcomes. Primary outcome was dose-limiting toxicity (DLT) and secondary outcome was death. RESULTS: The search yielded 493 non-duplicate citations. Eighteen studies of 10 inhibitors met inclusion criteria (total N = 1005 patients). Two-thirds of included studies did not define DLTs and the reporting of toxicities was highly variable. The most commonly reported DLTs were cytopenias, gastrointestinal toxicity, metabolic disturbances, fatigue and cardiovascular toxicity; there was one death attributed to treatment toxicity. CONCLUSION: MDM2 antagonists have been studied in a variety of malignancies with toxicities similar to other commonly used chemotherapy agents and may represent a safe adjuvant treatment for further study in in acute leukemia.

Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

OBJECTIVE: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. DESIGN: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. RESULTS: This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. CONCLUSION: This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies.

Critical components of the pluripotency network are targets for the p300/CBP interacting protein (p/CIP) in embryonic stem cells.

p/CIP, also known as steroid receptor coactivator 3 (SRC-3)/Nuclear Receptor Coactivator 3 (NCoA3), is a transcriptional coactivator that binds liganded nuclear hormone receptors, as well as other transcription factors, and facilitates transcription through direct recruitment of accessory factors. We have found that p/CIP is highly expressed in undifferentiated mouse embryonic stem cells (mESCs) and is downregulated during differentiation. siRNA-mediated knockdown of p/CIP decreased transcript levels of Nanog, but not Oct4 or Sox2. Microarray expression analysis showed that Klf4, Tbx3, and Dax-1 are significantly downregulated in mESCs when p/CIP is knocked down. Subsequent chromatin immunoprecipitation (ChIP) analysis demonstrated that Tbx3, Klf4, and Dax-1 are direct transcriptional targets of p/CIP. Using the piggyBac transposition system, a mouse ESC line that expresses Flag-p/CIP in a doxycycline-dependent manner was generated. p/CIP overexpression increased the level of target genes and promoted the formation of undifferentiated colonies. Collectively, these results indicate that p/CIP contributes to the maintenance of ESC pluripotency through direct regulation of essential pluripotency genes. To better understand the mechanism by which p/CIP functions in ESC pluripotency, we integrated our ChIP and transcriptome data with published protein-protein interaction and promoter occupancy data to draft a p/CIP gene regulatory network. The p/CIP gene regulatory network identifies various feed-forward modules including one in which p/CIP activates members of the extended pluripotency network, demonstrating that p/CIP is a component of this extended network.

Gene-trap mutagenesis: past, present and beyond.

Although at least 35,000 human genes have been sequenced and mapped, adequate expression or functional information is available for only approximately 15% of them. Gene-trap mutagenesis is a technique that randomly generates loss-of-function mutations and reports the expression of many mouse genes. At present, several large-scale, gene-trap screens are being carried out with various new vectors, which aim to generate a public resource of mutagenized embryonic stem (ES) cells. This resource now includes more than 8,000 mutagenized ES-cell lines, which are freely available, making it an appropriate time to evaluate the recent advances in this area of genomic technology and the technical hurdles it has yet to overcome.

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death.

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration.

Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility, and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kgamma were generated. We show that PI3Kgamma controls thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kgamma-deficient neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) agonists and chemotactic agents. PI3Kgamma links GPCR stimulation to the formation of phosphatidylinositol 3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated kinases 1 and 2. Thus, PI3Kgamma regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.

Gene trapping of two novel genes, Hzf and Hhl, expressed in hematopoietic cells.

Using an expression gene trapping strategy, we have identified and characterized two novel hematopoietic genes, Hzf and Hhl. Embryonic stem (ES) cells containing a gene trap vector insertion were cultured on OP9 stromal cells to induce hematopoietic differentiation and screened for lacZ reporter gene expression. Two ES clones displaying lacZ expression within hematopoietic cells in vitro were used to generate mice containing the gene trap integrations. Paralleling this in vitro expression pattern, both Hzf and Hhl were expressed in a tissue-specific manner during hematopoietic development in vivo. Hzf encodes a novel protein containing three C(2)H(2)-type zinc fingers predominantly expressed in megakaryocytes and CFU-GEMM. Hhl encodes a novel protein containing a putative phosphotyrosine binding (PTB) domain expressed in megakaryocytes, CFU-GEMM and BFU-E. These results demonstrate the utility of expression trapping to identify novel hematopoietic genes. Future studies of Hzf and Hhl should provide valuable information on the role these genes play during megakaryocytopoiesis.

Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells.

Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.

Expression trapping: identification of novel genes expressed in hematopoietic and endothelial lineages by gene trapping in ES cells.

We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZ reporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressed lacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) null mice.

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A-deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.

Mouse embryos lacking the receptor tyrosine kinase, Flk1, die without mature endothelial and hematopoietic cells. To investigate the role of Flk1 during vasculogenesis and hematopoiesis, we examined the developmental potential of Flk1-/- embryonic stem cells in chimeras. We show that Flk1 is required cell autonomously for endothelial development. Furthermore, Flk1-/- cells do not contribute to primitive hematopoiesis in chimeric yolk sacs or definitive hematopoiesis in adult chimeras and chimeric fetal livers. We also demonstrate that cells lacking Flk1 are unable to reach the correct location to form blood islands, suggesting that Flk1 is involved in the movement of cells from the posterior primitive streak to the yolk sac and, possibly, to the intraembryonic sites of early hematopoiesis.

Blood island formation in attached cultures of murine embryonic stem cells.

Differentiation of murine embryonic stem cells in suspension culture results in the formation of cystic embryoid bodies that develop blood islands. In this study pre-cystic embryoid bodies were attached to a substratum, and the program of differentiation was monitored. The attached ES cell cultures formed blood islands on a cell layer that migrated out from the center of attachment and beneath a mesothelial-like cell layer. Morphological and in situ marker analysis showed benzidine-positive hematopoietic cells surrounded by vascular endothelial cells that expressed PECAM and took up DiI-Ac-LDL. Waves of morphological differentiation were evident, suggesting a graded response to differentiation signals. Electron microscopy of the blood islands showed that they were similar to blood islands of cystic embryoid bodies and mouse yolk sacs, and cell-cell junctions were evident among the blood island cells. RNA expression analysis was consistent with the presence of hematopoietic precursor cells of several lineages and a primitive vascular endothelium in the cultures. Thus a program of vascular and hematopoietic development can be elaborated in attached ES cell cultures, and these blood islands are accessible to experimental manipulation.

Cloning and developmental expression analysis of the murine c-mer tyrosine kinase.

We have cloned the putative mouse homologue of the human c-mer receptor tyrosine kinase proto-oncogene. Comparison of the mouse and human c-mer amino acid sequences reveals an overall identity of 88%. Similar to the human, the extracellular region of the murine c-mer protein possesses two amino terminal immunoglobulin-like domains and two membrane proximal fibronectin type III domains, which places it in the Axl family of tyrosine kinases. Our analysis of the Axl family identifies eight different regions of amino acid consensus that have residues characteristic of this and no other tyrosine kinase family; six of the eight are within tyrosine kinase subdomains. The homology within the Axl family is highest between c-mer and c-eyk, the chicken proto-oncogene of the tumor virus gene product v-eyk. Northern analysis of adult tissues suggests that the mouse c-mer, although expressed in many tissues, has an expression pattern unique among Axl family members. In normal adult hematopoietic cells c-mer seems to be expressed predominantly if not exclusively in the monocytic lineage. Mouse c-mer is expressed during most, if not all, stages of embryological development beginning in the morula and blastocyst and progressing through the yolk sac and fetal liver stages. This early and consistent expression of c-mer was confirmed during in vitro differentiation of embryonic stem cells. The embryonic expression profile of c-mer suggests that this tyrosine kinase may play an important function in the developing mouse.

Evidence that drug-resistant alloreactive T cells may contribute to human graft rejection.

The objective of our study was to determine whether resistance to immunosuppressive drugs by transplant recipient's T cells could contribute to continued graft rejection, in spite of immunosuppressive therapy. The T cell lines used in this series of experiments were originally established from T cells that had infiltrated kidney or liver grafts and initiated rejections in patients receiving immunosuppressive drugs, including the purine analogue azathioprine (AZ). We have used a proliferation assay and the Strauss-Albertini test to analyze the T cell lines. Both assays use 6-thioguanine (6-TG), an amino derivative of AZ, as the selective agent to measure the resistance to AZ. Although the frequency of 6-TG-resistant variants in the majority of T cell lines closely resembled the frequency found in peripheral blood lymphocytes of controls, we identified four cell lines that demonstrated virtually complete resistance to the purine analogue 6-TG in both assays. These observations indicate that a more effective immunosuppressive treatment may be achieved by using a combination of drugs.

The Computerized Laboratory Notebook concept for genetic toxicology experimentation and testing.

We describe a microcomputer system utilizing the Computerized Laboratory Notebook (CLN) concept developed in our laboratory for the purpose of automating the Battery of Leukocyte Tests (BLT). The BLT was designed to evaluate blood specimens for toxic, immunotoxic, and genotoxic effects after in vivo exposure to putative mutagens. A system was developed with the advantages of low cost, limited spatial requirements, ease of use for personnel inexperienced with computers, and applicability to specific testing yet flexibility for experimentation. This system eliminates cumbersome record keeping and repetitive analysis inherent in genetic toxicology bioassays. Statistical analysis of the vast quantity of data produced by the BLT would not be feasible without a central database. Our central database is maintained by an integrated package which we have adapted to develop the CLN. The clonal assay of lymphocyte mutagenesis (CALM) section of the CLN is demonstrated. PC-Slaves expand the microcomputer to multiple workstations so that our computerized notebook can be used next to a hood while other work is done in an office and instrument room simultaneously. Communication with peripheral instruments is an indispensable part of many laboratory operations, and we present a representative program, written to acquire and analyze CALM data, for communicating with both a liquid scintillation counter and an ELISA plate reader. In conclusion we discuss how our computer system could easily be adapted to the needs of other laboratories.