Protemic identification of Germline Proteins in Caenorhabditis elegans

Sexual reproduction involves fusion of 2 haploid gametes to form diploid offspring with genetic contributions from both parents. Gamete formation represents a unique developmental program involving the action of numerous germline-specific proteins. In an attempt to identify novel proteins involved in reproduction and embryonic development, we have carried out a proteomic characterization of the process in Caenorhabditis elegans. To identify candidate proteins, we used 2D gel electrophoresis (2DGE) to compare protein abundance in nucleus-enriched extracts from wild-type C. elegans, and in extracts from mutant worms with greatly reduced gonads (glp-4(bn2) worms reared at 25°C); 84 proteins whose abundance correlated with germline presence were identified. To validate candidates, we used feeding RNAi to deplete candidate proteins, and looked for reduction in fertility and/or germline cytological defects. Of 20 candidates so screened for involvement in fertility, depletion of 13 (65%) caused a significant reduction in fertility, and 6 (30%) resulted in sterility (\u3c5 % of wild-type fertility). Five of the 13 proteins with demonstrated roles in fertility have not previously been implicated in germline function. The high frequency of defects observed after RNAi depletion of candidate proteins suggests that this approach is effective at identifying germline proteins, thus contributing to our understanding of this complex organ

Rapid generation of human B-cell lymphomas via combined expression of Myc and Bcl2 and their use as a preclinical model for biological therapies

Although numerous mouse models of B-cell malignancy have been developed via the enforced expression of defined oncogenic lesions, the feasibility of generating lineage-defined human B-cell malignancies using mice reconstituted with modified human hematopoietic stem cells (HSCs) remains unclear. In fact, whether human cells can be transformed as readily as murine cells by simple oncogene combinations is a subject of considerable debate. Here, we describe the development of humanized mouse model of MYC/BCL2-driven ‘double-hit’ lymphoma. By engrafting human HSCs transduced with the oncogene combination into immunodeficient mice, we generate a fatal B malignancy with complete penetrance. This humanized-MYC/BCL2-model (hMB) accurately recapitulates the histopathological and clinical aspects of steroid-, chemotherapy- and rituximab-resistant human ‘double-hit’ lymphomas that involve the MYC and BCL2 loci. Notably, this model can serve as a platform for the evaluation of antibody-based therapeutics. As a proof of principle, we used this model to show that the anti-CD52 antibody alemtuzumab effectively eliminates lymphoma cells from the spleen, liver and peripheral blood, but not from the brain. The hMB humanized mouse model underscores the synergy of MYC and BCL2 in ‘double-hit’ lymphomas in human patients. Additionally, our findings highlight the utility of humanized mouse models in interrogating therapeutic approaches, particularly human-specific monoclonal antibodies.Kathy and Curt Marble Cancer Research FundSingapore-MIT Alliance for Research and TechnologyNational Institutes of Health (U.S.) (Grant R01-CA128803)Virginia and Daniel K. Ludwig Graduate FellowshipNational Institute of General Medical Sciences (U.S.) (Medical Scientist Training Program Grant T32GM007753)MIT School of Science (Cancer Research Fellowship

Current findings for recurring mutations in acute myeloid leukemia

The development of acute myeloid leukemia (AML) is a multistep process that requires at least two genetic abnormalities for the development of the disease. The identification of genetic mutations in AML has greatly advanced our understanding of leukemogenesis. Recently, the use of novel technologies, such as massively parallel DNA sequencing or high-resolution single-nucleotide polymorphism arrays, has allowed the identification of several novel recurrent gene mutations in AML. The aim of this review is to summarize the current findings for the identification of these gene mutations (Dnmt, TET2, IDH1/2, NPM1, ASXL1, etc.), most of which are frequently found in cytogenetically normal AML. The cooperative interactions of these molecular aberrations and their interactions with class I/II mutations are presented. The prognostic and predictive significances of these aberrations are also reviewed

Diverse roles of GSK-3: Tumor promoter-tumor suppressor, target in cancer therapy.

Glycogen synthase kinase-3 (GSK-3) is a critical enzyme which participates in a complex array of important cellular processes and is often involved in various human diseases. It was first characterized in rat skeletal muscle as a serine/threonine (S/T) kinase that phosphorylated and inactivated glycogen synthase (GS). GS is the last enzyme in glycogen biosynthesis . Thus the initially identified role of GSK-3 was in metabolism. However, as we will soon see, GSK-3 has many diverse functions

Targeting the leukemic stem cell: the Holy Grail of leukemia therapy

Since the discovery of leukemic stem cells (LSCs) over a decade ago, many of their critical biological properties have been elucidated, including their distinct replicative properties, cell surface phenotypes, their increased resistance to chemotherapeutic drugs and the involvement of growth-promoting chromosomal translocations. Of particular importance is their ability to transfer malignancy to non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Furthermore, numerous studies demonstrate that acute myeloid leukemia arises from mutations at the level of stem cell, and chronic myeloid leukemia is also a stem cell disease. In this review, we will evaluate the main characteristics of LSCs elucidated in several well-documented leukemias. In addition, we will discuss points of therapeutic intervention. Promising therapeutic approaches include the targeting of key signal transduction pathways (for example, PI3K, Rac and Wnt) with small-molecule inhibitors and specific cell surface molecules (for example, CD33, CD44 and CD123), with effective cytotoxic antibodies. Also, statins, which are already widely therapeutically used for a variety of diseases, show potential in targeting LSCs. In addition, drugs that inhibit ATP-binding cassette transporter proteins are being extensively studied, as they are important in drug resistance-a frequent characteristic of LSCs. Although the specific targeting of LSCs is a relatively new field, it is a highly promising battleground that may reveal the Holy Grail of cancer therapy

Multifaceted roles of GSK-3 and Wnt/β-catenin in hematopoiesis and leukemogenesis: Opportunities for therapeutic intervention

Glycogen synthase kinase-3 (GSK-3) is well documented to participate in a complex array of critical cellular processes. It was initially identified in rat skeletal muscle as a serine/threonine kinase that phosphorylated and inactivated glycogen synthase. This versatile protein is involved in numerous signaling pathways that influence metabolism, embryogenesis, differentiation, migration, cell cycle progression and survival. Recently, GSK-3 has been implicated in leukemia stem cell pathophysiology and may be an appropriate target for its eradication. In this review, we will discuss the roles that GSK-3 plays in hematopoiesis and leukemogenesis as how this pivotal kinase can interact with multiple signaling pathways such as: Wnt/β-catenin, phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR), Ras/Raf/MEK/extracellular signal-regulated kinase (ERK), Notch and others. Moreover, we will discuss how targeting GSK-3 and these other pathways can improve leukemia therapy and may overcome therapeutic resistance. In summary, GSK-3 is a crucial regulatory kinase interacting with multiple pathways to control various physiological processes, as well as leukemia stem cells, leukemia progression and therapeutic resistance. GSK-3 and Wnt are clearly intriguing therapeutic targets. © 2014 Macmillan Publishers Limited

Targeting the leukemic stem cell: the Holy Grail of leukemia therapy

Since the discovery of leukemic stem cells (LSCs) over a decade ago, many of their critical biological properties have been elucidated, including their distinct replicative properties, cell surface phenotypes, their increased resistance to chemotherapeutic drugs and the involvement of growth-promoting chromosomal translocations. Of particular importance is their ability to transfer malignancy to non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Furthermore, numerous studies demonstrate that acute myeloid leukemia arises from mutations at the level of stem cell, and chronic myeloid leukemia is also a stem cell disease. In this review, we will evaluate the main characteristics of LSCs elucidated in several well-documented leukemias. In addition, we will discuss points of therapeutic intervention. Promising therapeutic approaches include the targeting of key signal transduction pathways (for example, PI3K, Rac and Wnt) with small-molecule inhibitors and specific cell surface molecules (for example, CD33, CD44 and CD123), with effective cytotoxic antibodies. Also, statins, which are already widely therapeutically used for a variety of diseases, show potential in targeting LSCs. In addition, drugs that inhibit ATP-binding cassette transporter proteins are being extensively studied, as they are important in drug resistance-a frequent characteristic of LSCs. Although the specific targeting of LSCs is a relatively new field, it is a highly promising battleground that may reveal the Holy Grail of cancer therapy