IntroductionEpigenetic modification is an important mechanism for the regulation of transcription and maintenance of cell identity with cell division. Hence, many histone-modifying enzymes have been identified as crucial for maintaining normal hematopoietic stem cells (HSCs) as well as leukemia-initiating cells (LICs). Disruptor of telomeric silencing 1 (Dot1) is a novel class of histone methyltransferase (HMT) that was first identified in yeast for its ability to dysregulate gene silencing near telomeres. 1-3 Dot1 and its mammalian homolog, Dot1l (Dot1-like), is currently the only identified histone 3 lysine 79 (H3K79) methyltransferase. 4,5 Since its discovery, many studies have shown an essential role for Dot1l and H3K79 methylation in embryonic development, prenatal hematopoiesis, and leukemia. 6,7 Recently, Dot1l has been shown to be specifically required for transformation by Mixed Lineage Leukemia (MLL) fusion proteins. 8 However, the role of Dot1l in normal postnatal hematopoiesis has not been definitely shown.In general, Dot1l and H3K79 methylation is associated with transcriptional activation. [9][10][11] Interestingly, the loss of Dot1l seems to regulate a relatively short list of genes instead of globally downregulating the entire transcriptome. This effect has been particularly noted in the context of embryonic stem cells, suggesting a specific biologic role for Dot1l. 12 Further studies using constitutive Dot1l knockout mouse models provide additional evidence for a role of Dot1l in stem cell biology. Analyses of Dot1l knockout embryonic stem cells and yolk sac cells show that the loss of Dot1l leads to cell cycle defects, chromosomal aberrations, and prenatal hematopoietic abnormalities. 13,14 However, the 2 constitutive Dot1l knockout mouse lines are embryonic lethal by E10.5 and E13.5, precluding the examination of Dot1l loss in postnatal mice and in definitive hematopoiesis.Dot1l is strongly associated with leukemias arising from translocations of the MLL gene, which fuse MLL in frame to more than 60 different translocation partners. 15 Multiple studies show that Dot1l interacts with many of the most common MLL translocation partners, such as AF9, ENL, AF4, and AF10, in a complex promoting transcriptional elongation. [16][17][18][19][20][21] Recent studies also show that the reciprocal translocation protein AF4-MLL can interact with Dot1l while exhibiting different gene expression profiles from conventional MLL translocation proteins. 21,22 For this paper, MLL translocation refers to the fusion of N-terminus of MLL with a translocation partner that can associate with Dot1l. Additional evidence from MLL translocation-containing cell lines and patient samples shows up-regulation of H3K79 methylation at MLL translocation target genes, including HOXA9 and MEIS1, which are critical for leukemogenesis. 19,23 Furthermore, the loss of H3K79 methylation through knockdown of Dot1l leads to reduced expression of these genes. 19,23 However, it is not clear whether Dot1l is required for MLL translocation-m...

Objective-The aim of this study was to better understand how 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 immuno-purified after expression in myeloblastic M1 cells and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative PCR 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 EAP for Elongation Assisting Proteins, 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. Conclusion-The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci.

Background Human papillomvirus (HPV)-16 is associated with an improved prognosis in a subset of patients with head and neck squamous cell carcinoma (HNSCC). Cervical carcinoma models have also demonstrated that HPV oncoproteins, E6 and E7, can induce VEGF and HIF-1 gene expression. The purpose of this study was to 1) determine the presence of high-risk HPV 16 in patients with HNSCC enrolled in a prospective phase II clinical trial, 2) assess the impact of HPV status on treatment response and survival in this select cohort treated with combined modality therapy, and 3) identify the differences between HIF/VEGF expression in HPV-positive and -negative tumors. Experimental Design The effect of HPV status on treatment response and outcome was prospectively evaluated in a single-institution phase II clinical trial. Patients had resectable untreated stage III, IV HNSCC of the oral cavity, oropharynx, hyopharynx, or larynryx, and stage II cancer of the base of tongue, hypopharynx, and larynx. All patients received neoadjuvant chemotherapy with two course of docetaxel (T) 60 mg/m2, then a 96-hour infusion of cisplatin (P) 25mg/m2/d, 5-fluorouracil (F) 700 mg/m2/d, and leucovorin (L) 500 mg/m2/d (TPFL). Those with at least a partial response received a third course. Responding patients then underwent surgery including modified lymph node dissection followed by adjuvant radiation or chemoradiation. Patients who progressed during neoadjuvant chemotherapy proceeded directly to surgery. HPV status was determined by conventional PCR in fresh frozen biopsy samples and Taqman PCR assay on formalin-fixed, paraffin-embedded specimens. HIF-1a and VEGF-A expression were assessed by immunohistochemistry (IHC) and quantitative real-time PCR (RT-PCR). Multivariate Cox proportional hazards regression analysis of time to disease progression or death was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for HPV-positive status. Results Of the 24 evaluable cases, HPV16 DNA was detected in 14 specimens, 13 of which were oropharyngeal tumors. HPV18 was not detected in any of the specimens. Treatment compliance was similar between both groups. There was no difference in either the response rates seen after NCT (85.7% vs. 90%), or the pathologic complete response rate for surgical patients (38.5% vs. 42.9%) for the HPV-positive and –negative tumors, respectively. After a median follow-up time of 52.9 months, there was a trend toward better progression-free (HR 0.15; p = 0.06) and overall survival (HR 0.14; p = 0.10), but this was not statistically significant. There was no difference in the level of VEGF expression at the protein level, however, in a subset of 13 fresh frozen tissue samples, quantitative RT-PCR revealed a statistically significant increase in VEGF mRNA transcript in the HPV-positive tumors (p < 0.01). No difference was seen for HIF-1a expression. Conclusions HPV-positivity portended a better prognosis in patients with oropharyngeal SCC treated with induction chemotherapy and adjuvant radiation in ...

Background: MLL fusion proteins use similar strategy for leukemic transformation through DOT1L recruitment. Results: Ten amino acids in DOT1L were identified as essential for binding and transformation by MLL-AF9. Conclusion: Biochemical and functional results indicate that blocking DOT1L recruitment represents a promising therapeutic strategy for mixed lineage leukemia. Significance: Identified DOT1L peptide will lay a foundation toward discovery of chemical tools able to block DOT1L recruitment.

Giant encephalocele is an uncommon congenital anomaly with very few published reports available in the English literature. Tetralogy of Fallot associated with situs inversus is also infrequently reported. To our knowledge there are no published reports of an association between giant encephalocele and Tetralogy of Fallot. The additional finding of situs inversus results in a rare pathologic triad, not heretofore described.

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Osteoarthritis and osteoporosis are widely prevalent and have far‐reaching public health implications. There is increasing evidence that epigenetics, in particular, histone 3 lysine 79 methyltransferase DOT1L, plays an important role in the cartilage and bone biology. In this study, we evaluated the role of Dot1l in the articular cartilage, growth plate, and trabecular bone utilizing conditional KO mouse models. We generated chondrocyte‐specific constitutive and inducible conditional Dot1l KO mouse lines using Col2a1‐Cre and Acan‐CreER systems. Prenatal deletion of Dot1l in mouse chondrocytes led to perinatal mortality, accelerated ossification, and dysregulation of Col10a1 expression. Postnatal deletion of Dot1l in mouse chondrocytes resulted in trabecular bone loss decreased extracellular matrix production, and disruption of the growth plate. In addition, pharmacological inhibition of DOT1L in a progeria mouse model partially rescued the abnormal osseous phenotype. In conclusion, Dot1l is important in maintaining the growth plate, extracellular matrix production, and trabecular bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.