MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia.

OBJECTIVE: The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia. MATERIALS AND METHODS: The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation. RESULTS: Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C-terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented. CONCLUSIONS: The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci.

Commentary: IDSA guidelines for improving the teaching of preclinical medical microbiology and infectious diseases.

Preclinical microbiology and infectious diseases courses too often primarily depend on PowerPoint lectures and notes, combined with multiple-choice tests, as their primary teaching tools. This strategy sets low expectations for students, encouraging short-term memory and discouraging understanding and long-term memory. These methods also fail to stimulate active participation, collaborative learning, and two-way communication with the professor, and they do not respect the students' diverse talents and ways of learning. The Infectious Diseases Society of America Preclinical Curriculum Committee proposes a new approach that emphasizes active learning and understanding and that addresses all of these failures. It consists of five components: (1) "Just-in-time" teaching that requires students to e-mail the answers to two general questions as well as any areas of misunderstanding to the instructor several hours before each lecture, (2) peer instruction or large-group sessions consisting of student teams of four who electronically answer a conceptual question before each major section of the lecture, (3) teaching from edited textbooks and Internet sources, (4) small-group discussions that emphasize pathogenesis and differential diagnosis, and (5) essay questions that encourage and test understanding in addition to recognition. A national consensus on factual content is proposed, with the goals of reducing information overload and minimizing requirements for excessive memorization. These strategies promise to enhance learning and rekindle interest in the field of infectious diseases. Other subspecialty organizations should create similar teaching guidelines that will encourage future medical students to bring a richer understanding of clinical and basic science to the bedside.

A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification.

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases.

Acting on a broad spectrum of extracellular, intracellular, and membrane-associated substrates, the matrix metalloproteinases (MMPs) are critical to the biological processes of organisms; when aberrantly expressed, many pathological conditions may be born or exacerbated. The prospect of MMP inhibition for therapeutic benefit in cancer, cardiovascular disease, and stroke is reviewed here. MMP inhibitor (MMPI) development constitutes an important branch of research in both academic and industrial settings and advances our knowledge on the structure-function relationship of MMPs. Targeting MMPs in disease treatment is complicated by the fact that MMPs are indispensable for normal development and physiology and by their multi-functionality, possible functional redundancy or contradiction, and context-dependent expression and activity. This complexity was revealed by previous efforts to inhibit MMP activity in the treatment of cancer patients that yielded unsatisfactory results. This review focuses on MMPI development since the late 90s, in terms of natural products and their derivatives, and synthetic compounds of low molecular mass incorporating specific zinc-binding groups (ZBGs). A few polyphenols and flavonoids that exhibit MMPI activities may have chemopreventive and neuro- and cardiovascular-protective effects. A new generation of potent and selective MMPIs with novel ZBGs and inhibition mechanisms have been designed, synthesized, and tested. Although only one collagenase inhibitor (Periostat, doxycycline hyclate) has been approved by the Food and Drug Administration as a drug for the treatment of periodontal disease, new hope is emerging in the form of natural and synthetic MMPIs for the prevention and treatment of stroke, cardiovascular disease, cancer, and other medical conditions.