Jerry C. Cheng

Expression profile of CREB knockdown in myeloid leukemia cells.

BackgroundThe cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.MethodsTo understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA.ResultsBy combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA.ConclusionWe have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle.

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.

Severe lactic acidosis in a 14-year-old female with metastatic undifferentiated carcinoma of unknown primary.

A 14-year-old girl was found to have a large, non-tender breast mass with anemia and thrombocytopenia. The diagnosis of an undifferentiated carcinoma of unknown primary was made after open breast biopsy of the mass with negative immunohistochemical studies for breast malignancies. Further evaluation showed extensive metastatic disease affecting the bone marrow, ribs, liver, and brain with magnetic resonance imaging evidence of carcinomatous meningitis. Despite 2 months of chemotherapy and intensive supportive care, the patient died of severe lactic acidosis and disseminated intravascular coagulation after exaggerated menstrual bleeding. The association of severe lactic acidosis and undifferentiated carcinoma of unknown primary in an adolescent has not been previously described.

Potential role of CREB as a prognostic marker in acute myeloid leukemia

The cAMP response element binding protein (CREB) is a leucine zipper transcription factor that regulates genes responsible for cell proliferation, differentiation and survival. CREB is overexpressed in the bone marrow from most patients with acute leukemia. Overexpression of CREB occurs both at the protein and at the transcript levels and is associated with gene amplification in leukemic blast cells. Higher levels of CREB correlate with a less favorable prognosis in a small cohort of adult patients with acute myeloid leukemia. In one study, patients whose bone marrow over-expresses CREB had an increased risk of relapse and decreased event-free survival. Mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome. These findings suggest that CREB plays an important role in the pathogenesis of acute leukemia and is a potential biomarker of disease.

The emerging role of RNA interference in the design of novel therapeutics in oncology.

RNA interference (RNAi) has emerged as the tool of choice for studying gene function. Dubbed the “breakthrough of the year” in 2002 by the journal Science, RNAi is a naturally occurring host defense mechanism that mediates the sequence-specific degradation of target mRNA transcripts and their protein products. The specificity of RNAi makes it an ideal tool for targeted therapeutics against unique fusion oncogene sequences. RNAi may also be effective against viral-mediated oncogenesis and has the potential to enhance tumor sensitivities to existing chemotherapy. The current interest in the success of RNAi-based therapies will depend on the delivery systems that protect the silencing apparatus from endogenous nucleases, sustain tissue-specific expression of the small-interfering RNAs, and prevent the activation of a destructive non-specific host immune response.

Report on the Workshop “New Technologies in Stem Cell Research,” Society for Pediatric Research, San Francisco, California, April 29, 2006

RNA interference (RNAi) is a powerful tool with which to study gene function, especially in stem cells. Small interfering RNAs (siRNAs) can effectively be introduced either with a vehicle or through viral vectors to transiently or stably inhibit the expression of a particular gene target. Much is known about the optimization of siRNAs and method of delivery in mammalian cells. In this review, we discuss design considerations for siRNAs, methods of delivery, optimization of siRNAs, applications to study genes in stem cells, therapeutic applications, and remaining hurdles. With recent advances in RNAi, it is likely that application of this technology will increase in the future. Correspondence: Kathleen M. Sakamoto, M.D., Ph.D., Division of Hematology-Oncology, Mattel Children’s Hospital at UCLA, 10833 Le Conte Avenue, Los Angeles, California, 90095-1752. Telephone: 310-794-7007; Fax: 310-206-8089; e-mail: kms@ucla.edu Received June 28, 2006; accepted for publication January 5, 2007; first published online in STEM CELLS EXPRESS January 25, 2007. ©AlphaMed Press 1066-5099/2007/

The role of CREB as a proto-oncogene in hematopoiesis and in acute myeloid leukemia

30.00/0 doi: 10.1634/stemcells.2006-0397 MEETING REPORT STEM CELLS 2007;25:1070–1088 www.StemCells.com