Search

Background: Despite advances in surgery and pharmacotherapy, there remains significant residual ischemic risk after coronary artery bypass grafting surgery. Methods: In REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl–Intervention Trial), a multicenter, placebo-controlled, double-blind trial, statin-treated patients with controlled low-density lipoprotein cholesterol and mild to moderate hypertriglyceridemia were randomized to 4 g daily of icosapent ethyl or placebo. They experienced a 25% reduction in risk of a primary efficacy end point (composite of cardiovascular death, myocardial infarction, stroke, coronary revascularization, or hospitalization for unstable angina) and a 26% reduction in risk of a key secondary efficacy end point (composite of cardiovascular death, myocardial infarction, or stroke) when compared with placebo. The current analysis reports on the subgroup of patients from the trial with a history of coronary artery bypass grafting. Results: Of the 8179 patients randomized in REDUCE-IT, a total of 1837 (22.5%) had a history of coronary artery bypass grafting, with 897 patients randomized to icosapent ethyl and 940 to placebo. Baseline characteristics were similar between treatment groups. Randomization to icosapent ethyl was associated with a significant reduction in the primary end point (hazard ratio [HR], 0.76 [95% CI, 0.63–0.92]; P =0.004), in the key secondary end point (HR, 0.69 [95% CI, 0.56–0.87]; P =0.001), and in total (first plus subsequent or recurrent) ischemic events (rate ratio, 0.64 [95% CI, 0.50–0.81]; P =0.0002) compared with placebo. This yielded an absolute risk reduction of 6.2% (95% CI, 2.3%–10.2%) in first events, with a number needed to treat of 16 (95% CI, 10–44) during a median follow-up time of 4.8 years. Safety findings were similar to the overall study: beyond an increased rate of atrial fibrillation/flutter requiring hospitalization for at least 24 hours (5.0% vs 3.1%; P =0.03) and a nonsignificant increase in bleeding, occurrences of adverse events were comparable between groups. Conclusions: In REDUCE-IT patients with a history of coronary artery bypass grafting, treatment with icosapent ethyl was associated with significant reductions in first and recurrent ischemic events. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01492361.

Purpose Siblings of individuals with disabilities provide the most long-term care for an individual with disabilities, yet research on their experiences is limited. A majority of previous research focuses on young siblings from a parent’s viewpoint. The purpose of this paper is to investigate the effects of having a sibling with a disability and behaviour described as challenging from adult siblings’ perspectives. Design/methodology/approach Six adult siblings of individuals with intellectual disabilities and behaviour described as challenging were interviewed about their responsibilities pertaining to their sibling, family relationships and the support that had been provided. The study used semi-structured interview methodology based on interview questions from previous research. Findings Siblings described a multifaceted impact on their lives. They attributed aspects of their career choices, personal characteristics and family dynamics to having a sibling with a disability and behaviour that challenges. Siblings stressed the inadequate support that they have received throughout their lives. They are, in a sense, the invisible carers for their sibling but they are perceived by society as just a sibling. Siblings described an optimistic perspective on their lives, even though they expressed the difficulties that they have faced. Research limitations/implications Due to the recruitment process and limited demographic of the participants, the findings may not be generalisable to the general population of siblings of individuals with disabilities. Further research should focus on a broader population. Practical implications This study reinforces the need for more support for siblings of individuals with disabilities in childhood and in adulthood. Originality/value This paper provides perspectives of individuals that have not been fully represented in previous research.

A TPP1 mutation known to cause telomere shortening and bone marrow failure in humans recapitulates telomere loss but results in severe germline defects in mice without impacting murine hematopoiesis., Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

Simple Summary MLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex. Abstract MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

In January 2017, the Colorado Department of Public Health and Environment (CDPHE) identified four epidemiologically linked cases of mumps among persons from a Marshallese community who were members of the same church in the Denver metropolitan area. During 2016-2017, sizable outbreaks of mumps reported in Arkansas, Hawaii, and Washington also affected the Marshallese population (1). CDPHE, the Tri-County Health Department (TCHD), and Denver Public Health collaborated to conduct an outbreak investigation during January-March 2017 using active and passive surveillance that identified 17 confirmed and 30 probable cases. Public health actions included conducting measles-mumps-rubella (MMR) vaccination clinics at local Marshallese churches; these resulted in the vaccination of 126 persons with ≥1 doses of MMR vaccine. Implementation of active surveillance and support from local Marshallese church leaders in promoting vaccination programs likely contributed to interruption of the outbreak.

During July 2016-January 2017, two unrelated measles cases were identified in the Denver, Colorado area after patients traveled to countries with endemic measles transmission. Each case resulted in multiple exposures at health care facilities and public venues, and activated an immediate and complex response by local and state public health agencies, with activities led by the Tri-County Health Department (TCHD), which serves Adams, Arapahoe, and Douglas counties. To track the economic burden associated with investigating and responding to single measles cases, personnel hours and supply costs incurred during each investigation were tracked prospectively. No secondary cases of measles were identified in either investigation. Postexposure prophylaxis (PEP) was administered to 31 contacts involving the first case; no contacts of the second case were eligible for PEP because of a delay in diagnosing measles disease. Public health costs of disease investigation in the first and second case were estimated at $49,769 and $18,423, respectively. Single measles cases prompted coordinated public health action and were costly and resource-intensive for local public health agencies.

Background In this study cull dairy cows from six California dairy herds were sampled seasonally over the course of a year. The objectives were to determine the prevalence of antimicrobial resistant (AMR) Salmonella spp. shed in cull cow feces, and the factors associated with fecal shedding of AMR and multidrug resistant (MDR) Salmonella. Methods Six dairy farms located in the San Joaquin Valley of California were identified and enrolled as a convenience sample. On each dairy, and once during each of the four seasons, 10 cull cows were randomly selected for fecal sampling on the day of their removal from the herd. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous 4 month’s herd management and the specific cattle sampled. Fecal samples were submitted to the California Animal Health and Food Safety laboratory for Salmonella isolation. Antimicrobial resistance was evaluated using broth microdilution method and a gram-negative assay plate following Clinical Laboratory Standards Institute (CLSI) guidelines and breakpoint references. All statistical models were survey adjusted for number of animals on sampling day. Results A total of 62 Salmonella were isolated from 60 of the 239 fecal samples collected. For 12% (95% confidence interval (CI) [3–20]) of fecal samples a multidrug resistant Salmonella was isolated. The survey-weighted results for the two most common drug classes for which isolates were resistant were tetracycline (39%; 95% CI [27–51]) and ampicillin (18%; 95% CI [9–27]). An important finding was the identification of cephalosporin as the third most common drug class for which isolates were resistant, with ceftriaxone (10%; 95% CI [2–17]) being the most common drug associated with resistance in that class. At the cow-level, reason for culling, prior treatment with antimicrobial drugs as the reason for culling was associated with higher odds of isolating an AMR Salmonella isolate. At the herd-level, percent of animals monthly culled on the farm as well as number of milking cows in the herd were associated with isolation of antimicrobial resistant Salmonella in cull cows. Discussion Salmonella isolated from fecal samples from cull cows were resistant to important antimicrobials, such as ceftriaxone. The most common drug classes for which isolates were resistant were tetracyclines and beta-lactams, with ampicillin, ceftriaxone and ceftiofur being the three most common drugs within the latter. Cow and herd level factors were associated with isolating antimicrobial resistant Salmonella that should be further investigated for their potential role in promoting occurrence of AMR Salmonella. Our results also highlight the importance of monitoring dairy cattle sent to slaughter for shedding of Salmonella resistant to medically important antimicrobial drugs.

Rapidly cycling fetal and neonatal hematopoietic stem cells (HSCs) generate a pool of quiescent adult HSCs after establishing hematopoiesis in the bone marrow. We report an essential role for the trithorax group gene absent, small, or homeotic 1-like (Ash1l) at this developmental transition. Emergence and expansion of Ash1l-deficient fetal/neonatal HSCs were preserved; however, in young adult animals, HSCs were profoundly depleted. Ash1l-deficient adult HSCs had markedly decreased quiescence and reduced cyclin-dependent kinase inhibitor 1b/c (Cdkn1b/1c) expression and failed to establish long-term trilineage bone marrow hematopoiesis after transplantation to irradiated recipients. Wild-type HSCs could efficiently engraft when transferred to unirradiated, Ash1l-deficient recipients, indicating increased availability of functional HSC niches in these mice. Ash1l deficiency also decreased expression of multiple Hox genes in hematopoietic progenitors. Ash1l cooperated functionally with mixed-lineage leukemia 1 (Mll1), as combined loss of Ash1l and Mll1, but not isolated Ash1l or Mll1 deficiency, induced overt hematopoietic failure. Our results uncover a trithorax group gene network that controls quiescence, niche occupancy, and self-renewal potential in adult HSCs.

In the study of social media in sub-national politics, offline-bounded methods offer an attractive alternative to the current emphasis on content search. When cases are selected according to content, analysis suffers from nebulous content boundaries, reliance on samples instead of populations, missing relationship data, and a lack of distinction between members of different sets and groups. Sub-national political entities, in contrast, typically have clear and substantive boundaries set by formal membership rules in the offline environment. These features make questions of adoption and interconnection more feasible to address. This chapter makes use of the Twitter list feature and data-mining capabilities of R to understand social media adoption, in-group interaction, and between-group intersection among legislators, lobbyists, and business groups in the American state of Maine. We find that Twitter adopters are distinguished from non-adopters to varying degrees according to offline characteristics. Although rates of Twitter adoption are similar for all three groups, only Maine legislators appear to form an internal community of Twitter interaction. Maine lobbyists and business groups, by contrast, maintain political connection through communication with outsiders.

Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic α cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of α cell mass and glucagon secretion. Using mice with tissue-specific deletion of the mTORC1 regulator Raptor in α cells (αRaptorKO), we showed that mTORC1 signaling is dispensable for α cell development, but essential for α cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in αRaptorKO mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In αRaptorKO mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in KATP channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for α cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling α cell-mass maintenance.

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.

This paper briefly considers the recent dramatic reductions in the underlying costs and market prices of solar photovoltaic (PV) systems, and their implications for decision-makers. In many cases, current PV costs and the associated market and technological shifts witnessed in the industry have not been fully noted by decision-makers. The perception persists that PV is prohibitively expensive, and still has not reached ‘competitiveness’. The authors find that the commonly used analytical comparators for PV vis a vis other power generation options may add further confusion. In order to help dispel existing misconceptions, some level of transparency is provided on the assumptions, inputs and parameters in calculations relating to the economics of PV. The paper is aimed at informing policy makers, utility decision-makers, investors and advisory services, in particular in high-growth developing countries, as they weigh the suite of power generation options available to them.

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangement of the MLL1 gene generates fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome-wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. We have mapped the binding site to a short segment of 10 amino acids in DOT1L and shown that DOT1L mutants lacking these residues did not support transformation by MLL-AF9. We hypothesized that by targeting the AF9-DOT1L protein-protein interactions (PPIs), we would selectively kill MLL-AF9 cells without effecting DOT1L role in normal hematopoiesis. Using established DOT1Lf/f MLL-AF9 with reintroduced WT-DOT1L, DOT1L missing 10aa AF9-binding domain (D10), DOT1L with a point mutation in the AF9-binding domain (I867A) and enzymatically inactive DOT1L (RCR), we were able to demonstrate that by disrupting the AF9-DOT1L PPIs, although we can inhibit leukemogenesis similarly to enzymatic inhibition, this interaction is not essential for normal hematopoiesis. Based on our initial studies to map the DOT1L interaction site and in conjunction with utilizing reported NMR structures of the AF9-DOT1L complex, we investigated the nature of the interactions and the minimum length of the peptide. Using different natural and unnatural amino acids, we successfully designed a 7mer peptide with KD of 10 nM and 25 nM against AF9 and ENL, respectively, showing similar potency as the originally identified and validated 10mer peptide. These results lay the groundwork for further optimization of the 7mer peptide towards developing DOT1L peptidomimetics with improved potency and cellular activity, to further validate the PPIs between DOT1L and MLL-fusion proteins as a potential therapeutic target for MLL rearranged leukemia. Citation Format: Sierrah Marie Grigsby, Jennifer Chase, Bridget Waas, Ann Friedman, Lei Du, Aihong Yao, James Ropa, Justin Serio, Andrew Muntean, Ivan Maillard, Haying Sun, Zaneta Nikolovska-Coleska. Towards peptidomimetics to target DOT1L recruitment in MLL-AF9 leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1380.

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.

The use of rodent models to evaluate efficacy during testing is accompanied by significant economic and regulatory hurdles which compound the costs of screening for promising drug candidates. Vasopermeation Enhancement Agents (VEAs) are a new class of biologics that are designed to increase the uptake of cancer therapeutics at the tumor site by modifying vascular permeability in the tumor to increase the therapeutic index of co-administered drugs. To evaluate the efficacy of a panel of VEA clinical candidates, we compared the rodent Miles assay to an equivalent assay in the ex ovo chicken embryo model. Both model systems identified the same candidate (PVL 10) as the most active promoter of vasopermeation in non-tumor tissues. An ex ovo chicken embryo system was utilized to test each candidate VEA in two human tumor models at a range of concentrations. Vasopermeation activity due to VEA was dependent on tumor type, with HEp3 tumors displaying higher levels of vasopermeation than MDA-MB-435. One candidate (PVL 10) proved optimal for HEp3 tumors and another (PVL 2) for MDA-MB-435. The use of the ex ovo chicken embryo model provides a rapid and less costly alternative to the use of rodent models for preclinical screening of drug candidates.

Background During 2016, an unusually high number (>5,000) of mumps cases were reported in the United States. On January 20, 2017, we identified a mumps outbreak in the Denver metropolitan area among a Marshallese community. We performed active surveillance to assess outbreak magnitude and guide implementation of control measures. Methods On January 22, local and state health departments initiated active case surveillance by using a church-based community roster. Each household was contacted by telephone ≥3 times to identify mumps cases, according to the 2012 CDC/Council of State and Territorial Epidemiologists case definition, and risk factors (e.g., household size). Measles, mumps, and rubella (MMR) vaccination status was reviewed in the Colorado Immunization Information System (CIIS). Four church-based vaccination clinics were held to bring participants up-to-date for MMR vaccination. Targeted messaging about mumps, MMR vaccine, and vaccination clinics was distributed through social media, churches, and Marshallese-language radio. Results Of the 21 households on the church roster, 17 were successfully contacted, 13 of which (76%) provided data for 85 persons (median household size: 6 persons; range: 5–12). Through household interviews and laboratory reporting, we identified 47 mumps cases (17 confirmed; 30 probable). Median patient age was 20 years (range: 3 months–44 years), 24 (51%) were male, and 34 (72%) reported no or unknown prior mumps vaccination and had no MMR vaccination documented in CIIS. During vaccination clinics, 118 (80%) of 148 presenting Marshallese persons were eligible for and received MMR vaccine; of those vaccinated, median age was 21 years (range: 1–55 years) and 104 (88%) had no prior MMR vaccine documentation. Conclusion Active surveillance, facilitated through culturally appropriate communication with church leaders, helped identify cases, disseminate materials, and promote MMR vaccination. Household interviews provided timely data to define outbreak magnitude and need for urgent MMR vaccination. Disclosures All authors: No reported disclosures. sdsdsdsd

Mixed lineage leukemia (MLL) is an aggressive form of leukemia where the MLL gene is translocated and fused to more than 80 different nuclear, cytoplasmic and membrane proteins. MLL-AF9 is one of the most common MLL-fusions. This fusion partner is known to recruit several multiprotein complexes including the AEP, SEC and DOT1L complexes leading to transcriptional activation. In particular, the recruitment of Disruptor of telomeric silencing 1-like (DOT1L), a H3K79 histone methyltransferase, is essential for leukemogenesis by multiple MLL fusion proteins. We mapped the binding site to a 10 amino acid segment (865 - 874) on DOT1L where AF9 binds. Peptides derived from this region that show dose-dependent disruption of this interaction. We also demonstrated that DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. Encouraged by these results, we generated both genetic and chemical tools to elucidate the role of DOT1L recruitment to the MLL fusion partners and the mechanism of leukemogenic inhibition by disrupting the protein-protein interaction (PPI) between MLL-AF9 and DOT1L. Fl/Fl DOT1L MLL-AF9 Cre+ cell lines were generated with different constructs of DOT1L. These constructs consisted of DOT1L mutants lacking the 10 amino acid binding site, a I867A point mutant known to block DOT1L binding, and an enzymatic mutant known to yield a catalytically inactive protein. As control cell lines, MLL-AF6, a MLL-fusion containing a cytoplasmic protein, and E2A-HLF, a non-DOT1L dependent fusion, were generated to demonstrate the specific effects of generated DOT1L mutant constructs. Both DOT1L PPI mutants impaired the transformation by MLL-AF9 and induced cell death by inducing apoptosis and cell cycle arrest similarly to enzymatic inhibition. These results established a foundation for discovering small-molecule inhibitors that disrupting the AF9-DOT1L as potential disease-specific therapies that target chromatin modifications in this highly aggressive leukemia. A high throughput screening was conducted identifying several different chemical classes of small molecules that bind to the AF9 C-terminal hydrophobic binding site and disrupt the PPI between DOT1L and MLL-AF9 fusion protein. Identified small molecule inhibitors were validated with series of biochemical, functional and cell-based assays. Validated compounds selectively inhibit the growth of the DOT1L dependent murine cells and induce cell death in a similar manner to the genetic approach. The small molecules also showed specificity in killing human MLL-fusion cell lines in comparison to non-MLL fusion leukemia. These results show that blocking the recruitment of DOT1L by AF9 using both genetic and chemical tools eliminate MLL-AF9 mediated immortalization emphasizing an essential function for this interaction in leukemogenesis and warrant further development of the identified small-molecule inhibitors. Citation Format: Sierrah M. Grigsby, James Ropa, Justin Serio, Chenxi Shen, Jennifer Chase, Ivan Maillard, Andrew Muntean, Zaneta Nikolovska-Coleska. Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2017-5489

SummaryChromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.

Hematopoietic stem and progenitor cells maintain blood homeostasis by giving rise to all mature blood cells and lymphocytes. A recent study identified the trithorax group gene ash1l as a critical regulator of quiescence and self-renewal potential in adult hematopoietic stem cells [1]. Ash1l encodes a large protein with histone methyltransferase activity that cooperates with the leukemia-associated gene Mll1 to regulate hematopoiesis in mice. Epigenetic modifiers have essential functions in normal hematopoiesis and are frequently dysregulated in hematological malignancies. Trithorax group (TrxG) genes were discovered in Drosophila melanogaster for their role in inducing normal body patterning by positively regulating Homeobox (Hox) gene expression [2]. Hox genes also play critical roles in mammalian development and in specific adult tissues, including the hematopoietic system. Although multiple members of the TrxG gene network interact genetically in flies [3], much less is known about their crosstalk and cooperativity in mammals. Mixed Lineage Leukemia 1 (MLL1) is the mammalian homolog of fly trithorax. MLL1 was originally discovered as a recurrent translocation partner in acute leukemias (reviewed in [4]). MLL1 maintains expression of HOX genes during development and regulates the function of normal hematopoietic stem cells. MLL1-driven leukemias are characterized by upregulated expression of HOXA cluster genes. Endogenous MLL1 contains a Su(var)3-9, Enhancer-of-zeste and Trithorax (SET) domain with H3K4 histone methyltransferase (HMT) activity that is lost in MLL1 fusion proteins [4]. MLL1 functions as part of a multiprotein complex that includes RBP4, WDR5, ASH2L, and the cofactor menin. In contrast, MLL1 fusion proteins consist of the N-terminal domain of MLL1 fused to an oncogenic partner. This complex also associates with the cofactor menin, but lacks intrinsic H3K4 HMT activity, which can be provided by endogenous MLL1 encoded by the non-rearranged allele [4]. In addition, the H3K79 histone methyltransferase DOT1L is recruited to the oncogenic complex at least in part through interactions with the MLL1 fusion partners AF10, AF4, AF9 or ENL [4]. Multiple research groups are focusing on targeting members of this complex in leukemia. Careful consideration must be given to disrupting oncogenic transformation while maintaining normal hematopoiesis, for example by targeting regulatory features of the complex that are unique or dominant in malignant cells. Besides MLL1, other TrxG members are poorly characterized in mammals. The TrxG gene ash1 (absent, small, or homeotic discs 1) was discovered in flies and shown to interact genetically with other TrxG genes [3]. Its mammalian homolog Ash1-like (Ash1l) encodes a large protein that contains a SET domain with in vitro H3K36 histone methyltransferase activity [5]. We recently discovered that Ash1l is a critical regulator of adult hematopoietic stem cells (HSCs) [1]. Ash1l-deficient mice generate normal numbers of fetal and neonatal long-term HSCs, but these cells become rapidly depleted in young adult mice. Phenotypically defined Ash1l-deficient HSCs display decreased quiescence and fail to give rise to long-term tri-lineage hematopoietic output after transplantation to irradiated recipients, indicating profoundly defective function. Moreover, Ash1l-deficient HSCs compete poorly for niche space, as evidenced by potent engraftment of wild-type HSCs in Ash1l-deficient recipients even in the absence of irradiation. Despite these defects, Ash1l-deficient mice maintain steady-state hematopoiesis and display enhanced self-renewal of progenitors downstream of HSCs. As observed for Mll1 in normal and malignant hematopoiesis, Ash1l regulates expression of Hoxa cluster genes [1]. Individual deficiency of Ash1l or Mll1 results in decreased but not abolished Hoxa expression [1, 6]. Functionally, inactivation of both Mll1 and Ash1l but not either gene alone leads to rapid hematopoietic failure [1]. Altogether, this is the first in vivo demonstration of cooperativity between mammalian TrxG proteins. More work is needed to define the critical pathways operating downstream of Ash1l and Mll1 in HSCs and the molecular mechanisms of their cooperative effects. As the field moves forward in targeting TrxG proteins in leukemia, careful consideration should be given to understanding how these proteins function during steady-state hematopoiesis in order to exploit critical structural and functional differences. For example, recent work showed that small molecular inhibitors of the menin/MLL1 interaction have substantial in vivo therapeutic activity in mouse models of MLL1-driven leukemia, but minimal negative impact on normal hematopoiesis [7]. Menin is an MLL1 cofactor that is essential for its recruitment to a subset of target genes [6] and is part of the MLL1 complex in normal hematopoiesis and leukemia. Combined deficiency of Men1 and Ash1l in mice results in bone marrow hypocellularity and HSC loss [1]. Thus, the modest impact of Men1 loss alone or menin/MLL1 disruption in normal hematopoiesis may result from compensatory activity of the TrxG gene Ash1l. It is also possible that individual molecular pathways differentially regulate recruitment of MLL1 and MLL1 fusion proteins to target genes, opening a therapeutic window. Systematic studies of TrxG gene networks in normal and malignant hematopoiesis could point to the Achilles' heel of MLL1-driven leukemia.

LMAN1 and MCFD2 encode the components of a mammalian cargo-receptor that facilitates the ER-to-Golgi transport of coagulation factors V (FV) and VIII (FVIII) for secretion to the plasma. Mutations in LMAN1 or MCFD2 result in a rare bleeding disorder known as combined deficiency of coagulation factors V and VIII (F5F8D), characterized by FV and FVIII levels that are ~10% of normal. No other clinical phenotypes are known in human patients. Lman1 null mice have ~50% of normal FV and FVIII levels and exhibit a partially penetrant, perinatal lethality, suggesting a critical role for yet unknown LMAN1 secretory cargo(s). To further characterize the function of the LMAN1/MCFD2 complex and identify new cargos, we generated several murine models of F5F8D, including ubiquitous null Lman1 (Lman1-/-) and Mcfd2 (Mcfd2-/-) mice maintained on a C57BL/6J genetic background. Adult Lman1-/- mice were mildly thrombocytopenic, exhibiting a 25% decrease in platelet count relative to wild-type (WT) mice (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004), but no other CBC abnormalities. In contrast, Lman1 heterozygous and Mcfd2-/- mice exhibited normal platelet counts. To further explore the role of LMAN1 in megakaryocyte/platelet development or survival, bone marrow (BM) histology and platelet transmission electron microscopy were performed. Lman1-/-mice had no platelet morphologic abnormalities by light or transmission electron microscopy, as well as normal number and morphology of BM megakaryocytes. Hematopoietic stem cells and megakaryocyte progenitors were indistinguishable between WT and Lman1-/- mice by flow cytometry. In order to determine whether the thrombocytopenia phenotype results from LMAN1 deficiency specifically in the hematopoietic compartment, mice with tissue-specific knockout of Lman1 in hematopoietic and endothelial cells (Tie2-Cre) were generated. Platelet counts of mice with LMAN1 deficiency restricted to the hematopoietic compartment were indistinguishable from those in WT controls. In contrast, hepatocyte-specific (Alb-Cre) Lman1 deficiency, resulted in significant thrombocytopenia relative to WT controls (p < 0.017), with platelet counts comparable to those observed in ubiquitous Lman1 null mice. Since thrombopoietin (TPO) is a major hepatocyte-derived regulator of platelet synthesis, plasma TPO levels were measured by ELISA in ubiquitous Lman1 and Mcfd2 null mice. Plasma TPO levels in Lman1-/- mice were ~56% lower than those in WT levels (128.7 x 103 vs. 229.5 x 103 pg/mL, p < 0.0025). However, TPO levels were not reduced in Mcfd2-/- mice (p > 0.17). TPO mRNA expression in the liver of Lman1-/-mice was indistinguishable from livers of WT littermate controls. In conclusion, global LMAN1-deficient and hepatocyte-specific LMAN1 deficient mice exhibit thrombocytopenia, a phenotype not previously reported in F5F8D patients. Lman1-/- mice, but not Mcfd2-/- mice, exhibit low plasma TPO levels, suggesting that TPO may be a novel LMAN1-dependent secretory cargo. These results raise the possibility that F5F8D patients with LMAN1 mutations may have mild thrombocytopenia, previously unappreciated as a result of the small number of F5F8D patients and the wide range of clinically normal platelet counts. Disclosures No relevant conflicts of interest to declare.

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression. Citation Format: Sierrah Grigsby, Jennifer Chase, James Ropa, Justin Serio, Chenxi Shen, Martha Larsen, Preston Donover, Melvin Reichman, Andrew Muntean, Ivan Maillard, Zaneta Nikolovska-Coleska. Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3803.

Jennifer Chase University of California, Irvine Arlene Stein. 2001. The Stranger Next Door: The Story of a Small Community's Battle Over Sex, Faith, and Civil Rights. Boston: Beacon Press. In The Stranger Next Door, Arlene Stein uses activism in rural Oregon around gay and lesbian civil rights to examine questions of strangeness and intimacy in a town referred to as Timbertown. Stein recounts how, following a failed statewide effort to pass a measure preempting civil rights protection for gays and lesbians, the Oregon Citizens Alliance advocated at the level of local politics across rural Oregon for similar amendments to local bylaws. The statewide initiative had passed in rural Oregon, indicating a receptive audience for the OCA's argument regarding the need to protect against government protection and promotion of homosexuality. Stein's organizing question is why such amendments find broad support in rural Oregon, where gays and lesbians are nearly invisible. Secondly, she explores how a defense of legal protection could be launched given this invisibility, and given the meanings attached to rights protection for gays and lesbians. Stein uses these questions to theorize the nature of group membership, the uses and impact of media on public debate, and to contribute to the literature on conservative Christians in the US. These additional problems are naturally interwoven throughout the text, demonstrating the usefulness of sexuality for addressing complex issues. This usefulness is all the more clear due to Stein's ability to link local, state and national contexts. She has written a personable text that deals cogently with complex issues. Stein's investigation centers on the conservative Christian Timbertowners who lead the campaign to pass a local ordinance against civil rights protection for gays and lesbians. She constructs them as dedicated to an ethos of self-reliance and hard work, arguing that they use these ideals to cope with economic uncertainty and Timbertown's changing social composition. Sexuality thus acts as a symbolic issue around which emotions, charged by a host of anxieties, gather for expression in rhetoric and political action. Treating sexuality as a symbol has a long history in sexuality studies; social scientists since Mary McIntosh's "Homosexual Role"1 have noted how attaching a stigmatizing label onto a segment of the population acts to police the boundaries between what is and is not acceptable. In this case, Stein's contribution is to see how such stigmatization works when the demonized Other is, quite literally, hard to see. What does it mean when heterosexual sisters living together are accused of being lesbians, and a lesbian mother is mistaken as a heterosexual woman? That is, what happens when the labelling being performed is simply wrong? Stein hypothesizes two responses to these questions. First, despite their relative invisbility, gays and lesbians still represent the "most difference" in rural Oregon, where few people of color live. Secondly, the potential for mistakes in identification actually adds urgency to delineating the line between "us" and "them." When social and economic change increased the importance of social boundaries in Timbertown, the existence of gays and lesbians among "normal" Oregonians became a source of great anxiety because they could not be reliably identified. In a broader sense, The Stranger Next Door is also an investigation of the collision between traditional, conservative American values embodied by small town and rural residents and big city, global cultures represented by gays and lesbians. As a liberal, Jewish, lesbian academic, Stein's interest is piqued by the fundamentally different understandings her conservative Christian informants have of the value of multiculturalism and diversity. …

To allow appropriate allocation of prevention and care funding, HIV/AIDS surveillance data must include risk factor information, currently available for less than 70% of cases reported in the United States. The authors evaluated an intervention consisting of provider training and materials to improve risk factor reporting. Facilities were matched prior to randomization to intervention or control, and generalized linear mixed models were used to test for an intervention effect. Twenty-one percent of cases from intervention facilities and 33.4% from control facilities (p = .09) were reported without any risk factor information. The pre—post difference (20.7% for intervention and 36.0% for control) was not significant among HIV cases (p = .11) nor among AIDS cases (p = .12; 21.3% for intervention and 31.1% for control). The methods the authors’ evaluated may need to be combined with other approaches and/or alternative classification schemes to significantly reduce the percentage of cases reported to surveillance without risk factor information.

Congenital Dyserythropoietic Anemia type II (CDAII) is a disease of ineffective erythropoiesis characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroid precursors. CDAII is an autosomal recessive disease resulting from mutations in SEC23B. SEC23 is a core component of COPII vesicles, which transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi. Despite identification of the underlying genetic defect, the molecular mechanism by which SEC23B deficiency produces the unique CDAII phenotype remains unknown. We previously reported that mice homozygous for a Sec23b null allele die perinatally, exhibiting massive pancreatic degeneration, precluding evaluation of the adult erythroid compartment. To examine the impact of SEC23B deficiency on adult murine hematopoiesis, we generated mice with erythroid specific and pan-hematopoietic deficiency for SEC23B by crossing a second, conditional Sec23b allele (Sec23bfl), in which exons 5 and 6 are flanked by loxP sites, to an EpoR-Cre and Vav1-Cre, respectively. These mice did not exhibit anemia or any other CDAII characteristic. Similarly, mice transplanted with SEC23B-deficient fetal liver cells harvested from E17.5 embryos also failed to recapitulate any features of the CDAII phenotype. We next generated mice deficient in SEC23B exclusively in the pancreas by crossing the Sec23bfl allele to either p48-Cre or Pdx1-Cre. These mice exhibit a phenotype indistinguishable from mice with germline deletion of Sec23b, indicating that loss of pancreatic SEC23B is sufficient to explain the perinatal-lethality of global SEC23B deficiency in mice. The mammalian genome contains two paralogs for SEC23, SEC23A and SEC23B. These paralogs are highly identical at the amino acid level (~85%). We examined the relative expression of SEC23B/SEC23A in WT tissues from both humans and mice. This ratio is higher in human BM compared to pancreas, while it was higher in mouse pancreas compared to BM. In order to determine if SEC23A can rescue the phenotype of SEC23B deficient mice when expressed under the regulatory control of Sec23b, we genetically engineered the Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23a-b) in mouse embryonic stem cells via recombinase mediated cassette exchange. A heterozygous Sec23a-b intercross yielded the expected number of mice homozygous for the Sec23a-b allele. These mice exhibited normal survival, development, and fertility. Pancreas tissues dissected from Sec23a-b/a-b mice had normal weights, were histologically indistinguishable from WT controls, and did not exhibit dilated ER by transmission electron microscopy. Western blot analysis confirmed the absence of SEC23B in pancreata of Sec23a-b/a-b mice, with high levels of SEC23A expression. These data demonstrate that the SEC23A and SEC23B proteins overlap significantly (or completely) at the level of protein function, and suggest that the distinct phenotypes of human and mouse SEC23 deficiency are the result of an evolutionary shift in the tissue-specific gene expression programs of SEC23A and SEC23B. These findings also suggest that therapies that increase the expression of either SEC23 paralog in erythroid cells might be effective in CDAII. Disclosures No relevant conflicts of interest to declare.

SEC23B mutations in humans result in the autosomal recessive disease Congenital Dyserythropoietic Anemia type-II (CDAII). CDAII is characterized by moderate anemia in increased bone marrow (BM) bi/multi-nucleated erythroblasts. Despite the identification of the genetic defect underlying CDAII, the pathophysiology of this disease remains unknown. SEC23A and SEC23B are paralogous components of the coat protein complex II (COPII)-coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum to the Golgi apparatus. We generated SEC23B-deficient mice and demonstrated that these animals die perinatally exhibiting massive pancreatic degeneration (Tao et. al PNAS). To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, we harvested fetal liver cells (FLC), which contain hematopoietic stem cells, from SEC23B-deficient or wild-type (WT) control E17.5 embryos and transplanted them into lethally irradiated C57BL/6J mice. Recipients of SEC23B-deficient FLC did not exhibit anemia or any other CDAII characteristic (Khoriaty et. al, Mol Cell Biol), and SEC23B deficient FLC competed effectively with WT FLC at reconstituting hematopoiesis when transplanted into lethally irradiated recipient mice (Khoriaty et. al, Mol Cell Biol). A Sec23bfl conditional-allele was also generated. Mice with hematopoietic-specific SEC23B-deficiency, generated by crossing Vav1-Cre into Sec23bfl mice, also exhibited normal hematopoiesis. In contrast, mice with pancreas-specific Sec23b deficiency generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre resulted in a phenotype indistinguishable from complete SEC23B-deficiency, demonstrating that loss of pancreatic Sec23b expression is sufficient to explain the perinatal lethality of SEC23B-deficient mice. To investigate different phenotypes of SEC23B deficiency in humans and mice, the SEC23B/SEC23A expression ratio was examined in murine and human tissues. This ratio is higher in mouse pancreas (12.7) compared to BM (2.6), whereas it is higher in human BM (7.8) relative to pancreas (5.5). Taken together with the high degree of amino-acid identity between SEC23A and SEC23B (~85%), these data suggest that the tissue-specific functions of SEC23A and SEC23B have shifted during evolution between humans and mice. To determine if Sec23a can rescue the lethality of SEC23B-deficient mice, we have engineered Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23A-B) via recombinase-mediated cassette exchange. A heterozygous Sec23A-B intercross is in progress. Rescue of the SEC23B deficient phenotype by SEC23A protein expressed under control of Sec23b regulatory sequences would suggest that increasing the expression of either paralog in erythroid cells might be effective in the treatment of CDAII. Disclosures No relevant conflicts of interest to declare.

Smooth muscle cell (SMC) proliferation is an important component of restenosis in response to injury after balloon angioplasty. Inhibition of proliferation in vivo can limit neointima hyperplasia in animal models of restenosis. Ribozymes against c- myb mRNA have been shown to be effective inhibitors of SMC proliferation in vitro. The effectiveness of adenovirus as a gene therapy vector in animal models of restenosis is well documented. In order to test the utility of ribozymes to inhibit SMC proliferation by a gene therapy approach, recombinant adenovirus expressing ribozymes against c- myb mRNA was generated and tested both in vitro and in vivo. This adenovirus ribozyme vector is shown to inhibit SMC proliferation in culture and neointima formation in a rat carotid artery balloon injury model of restenosis.