C. Glenn Begley

Drug development: Raise standards for preclinical cancer research.

C. Glenn Begley and Lee M. Ellis propose how methods, publications and incentives must change if patients are to benefit.

Thrombopoietic effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with advanced cancer.

BACKGROUND Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is a potent stimulator of megakaryocyte colony formation and platelet production. It is likely to be useful in the management of severe thrombocytopenia. To determine its clinical activity and safety, we gave it to patients with advanced cancer before chemotherapy. METHODS Patients were randomly assigned to receive either PEG-rHuMGDF or placebo in a three to one ratio. PEG-rHuMGDF was given at a dose of 0.03, 0.1, 0.3, or 1.0 microgram/kg body weight. The study drug or placebo were administered daily by subcutaneous injection for up to 10 days or until a target platelet count was reached. FINDINGS 17 patients, median age 59 years, received either PEG-rHuMGDF (13 patients) or placebo (four patients). PEG-rHuMGDF produced a dose-dependent increase in platelet counts. Patients given placebo. 0.03, and 0.1 microgram/kg of PEG-rHuMGDF had median increases in platelet counts of 16%, 12%, and 39%. Those receiving 0.3 and 1.0 microgram/kg of PEG-rHuMGDF had an increase in blood platelets of between 51% and 584%. Platelets rose from day 6 of PEG-rHuMGDF administration and continued to rise after stopping the drug. The platelet count peaked between days 12 and 18 and remained above 450 x 10(9)/L for up to 21 days. There were no alterations in white-blood-cell count or haematocrit, and low toxicity. Platelets taken from patients during PEG-rHuMGDF administration and at the time of peak platelet count were morphologically and functionally normal. INTERPRETATION The potency with which PEG-rHuMGDF stimulates platelet production and its low toxicity indicate that this is likely to be a useful agent for the management of thrombocytopenia.

The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12

Gene targeting studies have shown that the transcription factor SCL is critically important for embryonic hematopoiesis, but the early lethality of SCL null mice has precluded the genetic analysis of its function in the adult. We have now generated a conditional knockout of SCL by using Cre/Lox technology and an IFN-inducible Cre transgenic mouse. Deletion of SCL in adult mice perturbed megakaryopoiesis and erythropoiesis with the loss of early progenitor cells in both lineages. This led to a blunted response to the hematopoietic stress induced by polyinosinic-polycytidylic acid, with a persistently low platelet count and hematocrit compared with controls. In contrast, progenitors of granulocyte and macrophage lineages were not affected, even in the setting of stress. Immature progenitor cells (day 12 colony-forming unit spleen) with multilineage capacity were still present in the SCL null bone marrow, but these progenitors had lost the capacity to generate erythroid and megakaryocyte cells, and colonies were composed of only myeloid cells. These results suggest that SCL is critical for megakaryopoiesis and erythropoiesis, but is dispensable for production of myeloid cells during adult hematopoiesis.

Expression and function of erythropoietin receptors in tumors: implications for the use of erythropoiesis-stimulating agents in cancer patients.

Safety concerns surrounding the use of recombinant human erythropoietin (Epo) to treat anemia in cancer patients were raised after 2 recent clinical studies reported a worse survival outcome in patients who received epoetin α or epoetin β compared with patients who received placebo. Although those findings contrasted with previous clinical studies, which demonstrated no difference in survival for cancer patients who received erythropoiesis‐stimulating agents (ESAs), some investigators have suggested a potential role for ESAs in promoting tumor growth through 1) stimulation of Epo receptors (EpoR) expressed in tumors, 2) stimulation and formation of tumor vessels, and/or 3) enhanced tumor oxygenation. The first and second hypotheses appeared to be supported by some EpoR expression and ESA in vitro studies. However, these conclusions have been challenged because of poor specificity of EpoR‐detection methodologies, conflicting data from different groups, and the lack of correlation between in vitro data and in vivo findings in animal tumor models. For this report, the authors reviewed the biology of EpoR in erythropoiesis and compared and contrasted the reported findings on the role of ESAs and EpoR in tumors. Cancer 2007.

Reproducibility: Six red flags for suspect work

C. Glenn Begley explains how to recognize the preclinical papers in which the data won’t stand up. of a policy that promotes rapid, open access to observing data, following the protocols developed in the International Polar Year. Frameworks for helping to plan and coordinate long-term observing activities across the scientific community and other sectors need to be established. The community-based observing networks from the International Polar Year, which focus on variables related to local environmental threats or benefits, are a good start. But to be accessible to others, these data should be entered into wider networks such as those of the WMO. Similar to the practice of joint resource management, the scientific community, stakeholders and decision-makers all need to be included in governance from the outset to help ensure relevance and efficiency. Opportunities remain for the private sector to contribute to such collaborative networks. Offering up commercial vessels or infrastructure as platforms for scientific observations, sharing data and engaging the research community in the planning stages of industry observing programmes would go a long way towards establishing a ‘network of networks’. Last month, I was fortunate to be out in a small boat off Toksook Bay in Alaska with ice experts and hunters from the Yup’ik people. We were surrounded by jagged, fast-moving chunks of ice that, to me, seemed hostile. To my companions, it was all in a day’s work. I recalled a sentiment I had heard from a marine-mammal expert in Barrow, more than 1,000 kilometres farther north, where the ice is now unstable. He stated that the key to adapting to increasingly dynamic ice is to learn from those to the south, such as in Toksook Bay. The charge to the scientific community is to help to create a foundation for such mutual learning to occur. ■

The scl +18/19 stem cell enhancer is not required for hematopoiesis: identification of a 5' bifunctional hematopoietic-endothelial enhancer bound by Fli-1 and Elf-1.

ABSTRACT Analysis of cis-regulatory elements is central to understanding the genomic program for development. The scl/tal-1 transcription factor is essential for lineage commitment to blood cell formation and previous studies identified an scl enhancer (the +18/19 element) which was sufficient to target the vast majority of hematopoietic stem cells, together with hematopoietic progenitors and endothelium. Moreover, expression of scl under control of the +18/19 enhancer rescued blood progenitor formation in scl−/− embryos. However, here we demonstrate by using a knockout approach that, within the endogenous scl locus, the +18/19 enhancer is not necessary for the initiation of scl transcription or for the formation of hematopoietic cells. These results led to the identification of a bifunctional 5′ enhancer (−3.8 element), which targets expression to hematopoietic progenitors and endothelium, contains conserved critical Ets sites, and is bound by Ets family transcription factors, including Fli-1 and Elf-1. These data demonstrate that two geographically distinct but functionally related enhancers regulate scl transcription in hematopoietic progenitors and endothelial cells and suggest that enhancers with dual hematopoietic-endothelial activity may represent a general strategy for regulating blood and endothelial development.

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus Mix gene

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired‐like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41‐42. Northern analysis and RT‐PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole‐mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid‐ and late‐streak embryos. In early bud‐stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold‐stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Endophilin-1: a multifunctional protein.

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.

Multilineage mobilization of peripheral blood progenitor cells in humans following administration of PEG‐rHuMGDF

The most important physiological regulator of megakaryocytopoiesis is the ligand for the c‐mpl receptor (thrombopoietin/megakaryocyte growth and development factor, MGDF). We examined the effect of pegylated‐recombinant human MGDF (PEG‐rHuMGDF): patients received PEG‐rHuMGDF at doses of 0.03, 0.1, 0.3 or 1.0 μg/kg/d or placebo for 10 d maximum in a double‐blinded randomized study. There was a dose‐dependent elevation in circulating platelet counts but no alteration in erythrocyte or total leucocyte counts. The number of bone marrow megakaryocytes was increased approximately 2‐fold. The frequency of bone marrow progenitor cells was not altered. In contrast, both to the bone marrow results and to published pre‐clinical data, there was a dose‐dependent mobilization into the blood of progenitor cells of multiple cell lineages. Increased levels of Meg‐CFC (maximum increase 30‐fold), day 7 and day 14 GM‐CFC and BFU‐E were demonstrated at doses of 0.3 and 1.0 μg/kg/d PEG‐rHuMGDF. At 0.1 μg/kg/d, mobilization of Meg‐CFC alone occurred in two‐thirds of patients. Maximum blood levels of progenitor cells occurred at day 12. Thus, administration of PEG‐rHuMGDF to humans resulted in mobilization of progenitor cells of multiple lineages despite its ‘lineage‐specific’ activity on mature cell development.

Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch.

Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage‐colony‐stimulating factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems.