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Federal agencies draft statutes. Indeed, they are often the chief architects of the statutes they administer. Even when federal agencies are not the primary substantive authors, they routinely respond to congressional requests to provide technical assistance in statutory drafting. Yet despite their substantial role in the legislative process, our understanding about how agencies interact with Congress is greatly undertheorized and perhaps even less understood empirically. This Report, which was commissioned by the Administrative Conference of the United States (ACUS), explores the latter role of federal agencies in the legislative process: the provision of technical assistance in statutory drafting.To better understand the technical drafting assistance process, the Author met with agency officials at some twenty executive departments and independent agencies for a total of over sixty hours of interviews. Ten of these agencies agreed to participate on the record: the Departments of Agriculture, Commerce, Homeland Security, Education, Energy, Health and Human Services, Housing and Urban Development, and Labor as well as the Federal Reserve and the Pension Benefit Guaranty Corporation. Subsequent to the interviews, the participating agencies responded to an anonymous, forty-question follow-up survey. The Report presents the key findings from this study, identifies the best practices that certain agencies have developed to address these challenges, and proposes recommendations for ACUS to consider. These recommendations focus both on internal agency practices to improve the technical drafting assistance process and external practices to strengthen agencies’ relationship with Congress in the legislative process.

King v. Burwell is a crucial victory for the Obama Administration and for the future of the Affordable Care Act. It also has important implications for tax law and administration, as explored in the other terrific contributions to this Pepperdine Law Review Symposium. In this Essay, we turn to another tax-related feature of the Chief Justice’s opinion for the Court: It is hard to ignore the fingerprints of a tax lawyer throughout the opinion. This Essay focuses on two instances of a tax lawyer at work.First, in the Chief’s approach to statutory interpretation one sees a tax lawyer as interpreter. As others have observed, the Chief and the Court more generally seem to be embracing a brand of contextualism to depart from the textualism Justice Scalia has imbued in the Court’s statutory interpretation jurisprudence during his tenure. The Chief’s articulation of this interpretive approach in King, however, tracks tax law’s substance-over-form doctrine. In particular, the Chief looks beyond Congress’s formal, textual characterization of the statutory provision to recharacterize the provision based on strong proof of its true nature or substance. Indeed, this is not the first time the Chief has taken an approach that is functionally similar to the substance-over-form doctrine when interpreting the Affordable Care Act. He also seemed to do so when rejecting the constitutional challenge to the statute three Terms prior.Second, as to King’s sweeping administrative law holding, the Chief crafts a new major questions doctrine that could significantly cut back on federal agency lawmaking authority by not applying Chevron deference at all to questions of deep economic or political significance. Yet the Chief seems to develop this doctrine against the backdrop of tax exceptionalism — the notion that general administrative law principles do not apply to tax — and thus this development may have a more limited application to extraordinary circumstances at the intersection of tax and administrative law. Tax exceptionalism may well limit the opinion’s impact on administrative law, and tax law may better inform the Chief’s (and the Court’s) evolution from textualism to contextualism in statutory interpretation. The growing call to abandon the perception that tax is exempt from general principles of administrative law may well be correct. We believe it is. But that does not mean tax law cannot provide us with special insights that can be applied in other legal contexts.

In response to Kevin M. Stack, Interpreting Regulations, 111 Mich. L. Rev. 355 (2012). In Interpreting Regulations, Professor Stack provides the first comprehensive approach to regulatory interpretation and situates this approach within the larger literature on legal interpretation. His theory of regulatory interpretation is simple yet pioneering: “a regulation should be read in light of its purposes, with the regulation’s text and the statement of basis and purpose constituting the privileged interpretive sources.” This Research Note takes a look inside regulatory interpretation to explore the empirical foundation for Professor Stack’s novel approach to regulatory interpretation. In 2013, the author conducted a 195-question survey of 128 federal agency rule drafters at seven executive departments (Agriculture, Commerce, Energy, Homeland Security, Health and Human Services, Housing and Urban Development, and Transportation) and two independent agencies (the Federal Communications Commission and the Federal Reserve). Part I of this Research Note presents the findings from this study for the questions that were designed to assess Professor Stack’s theory of regulatory interpretation from the perspective of the agency officials who draft these statements of basis and purpose. These findings largely support his theory. Part II then takes a step back to explain how the other findings from the study bear on regulatory interpretation.

Claude Steele’s stereotype threat hypothesis posits that when there are negative stereotypes about the intellectual capacity of certain (stigmatised) groups, members of that group suffer aversive consequences; group members who are most strongly identified with the stigmatised domain in question (e.g., intellectual or academic ability) are those most likely to suffer the effects of stereotype threat. In education, it is widely held that personal investment in schooling should lead to more positive outcomes. However, highly‐invested individuals will most keenly experience the negative effects of stigma. Thus those most at risk for withdrawing from school among students of colour (who suffer a stigma of intellectual inferiority) could be those most invested in schooling. This hypothesis was tested by measuring identification with academics among a group of incoming students at a racially diverse inner‐city high school in the Midwest USA. Regardless of race, the students who most strongly identified with aca...

The BAALC/miR-3151 locus on chromosome 8q22 contains both the BAALC gene (for brain and acute leukemia, cytoplasmic) and miR-3151, which is located in intron 1 of BAALC. Older acute myeloid leukemia (AML) patients with high expression of both miR-3151 and the BAALC mRNA transcript have a low survival prognosis, and miR-3151 and BAALC expression is associated with poor survival independently of each other. We found that miR-3151 functioned as the oncogenic driver of the BAALC/miR-3151 locus. Increased production of miR-3151 reduced the apoptosis and chemosensitivity of AML cell lines and increased leukemogenesis in mice. Disruption of the TP53-mediated apoptosis pathway occurred in leukemia cells overexpressing miR-3151 and the miR-3151 bound to the 3′ untranslated region of TP53. In contrast, BAALC alone had only limited oncogenic activity. We found that miR-3151 contains its own regulatory element, thus partly uncoupling miR-3151 expression from that of the BAALC transcript. Both genes were bound and stimulated by a complex of the transcription factors SP1 and nuclear factor κB (SP1/NF-κB). Disruption of SP1/NF-κB binding reduced both miR-3151 and BAALC expression. However, expression of only BAALC, but not miR-3151, was stimulated by the transcription factor RUNX1, suggesting a mechanism for the partly discordant expression of miR-3151 and BAALC observed in AML patients. Similar to the AML cells, in melanoma cell lines, overexpression of miR-3151 reduced the abundance of TP53, and knockdown of miR-3151 increased caspase activity, whereas miR-3151 overexpression reduced caspase activity. Thus, this oncogenic miR-3151 may also have a role in solid tumors.

Tax exceptionalism — the view that tax law does not have to play by the administrative law rules that govern the rest of the regulatory state — has come under attack in recent years. In 2011, the Supreme Court rejected such exceptionalism by holding that judicial review of the Treasury Department’s interpretations of the tax code is subject to the same Chevron deference regime that applies throughout the administrative state. The D.C. Circuit followed suit by rejecting the IRS’s position that its notices are not subject to judicial review under the Administrative Procedure Act (APA). This Article calls for the demise of another instance of tax exceptionalism: the United States Tax Court’s longstanding view that it is not governed by the APA.In addition to presenting the legal case against Tax Court exceptionalism, the Article explores administrative law and tax policy considerations that favor the Tax Court following traditional administrative law, including consistent application of the law, efficient allocation of resources, horizontal and vertical equity, comparative agency expertise, and a proper separation of powers. Moreover, by following administrative law principles that may be more deferential to the IRS in a particular case, the Tax Court can establish a richer dialogue with the IRS to improve agency procedures and decision-making — thus advancing tax policy’s interest in protecting less sophisticated taxpayers while increasing economic efficiency. With a growing circuit conflict as to whether the Tax Court is bound by the APA, the Tax Court should reverse course now before the Supreme Court intervenes to declare the death of tax exceptionalism in yet another area of tax law.

This appendix reproduces a 195-question survey of federal agency rule drafters that covers a variety of topics related to agency rule-drafting and statutory interpretation. The survey is modeled on the path-breaking empirical work Lisa Bressman and Abbe Gluck have conducted on congressional drafting, though it differs in substantial respects. The author administered the survey over a five-month period in 2013 at seven executive departments (Agriculture, Commerce, Energy, Homeland Security, Health and Human Services, Housing and Urban Development, and Transportation) and two independent agencies (Federal Communications Commission and Federal Reserve). Of the 411 officials sent the survey, 128 responded for a 31% response rate. The findings from this survey will be reported in a series of papers, starting with Chevron Inside the Regulatory State: An Empirical Assessment, 83 Fordham L. Rev. 703 (2014) (invited contribution to Chevron at 30 symposium), available at http://ssrn.com/abstract=2483760, and Inside Agency Interpretation, 67 Stan. L. Rev. 999 (2015), available at http://ssrn.com/abstract=2501716.

For three decades, scholars (as well as courts and litigants) have written thousands of articles (and opinions and briefs) concerning the impact of the Chevron deference regime on judicial review of agency statutory interpretation. This should come as no surprise as Chevron is the most-cited administrative-law decision of all time. Little attention, however, has been paid to how Chevron and its progeny have actually shaped statutory interpretation inside the regulatory state. As part of the Fordham Law Review symposium Chevron at 30: Looking Back and Looking Forward, this Essay presents the findings of the first comprehensive empirical investigation into the effect of Chevron and related doctrines on how federal agencies interpret statutes they administer. The Essay draws on a larger, 195-question survey of federal agency rule drafters that covered a variety of topics related to agency statutory interpretation. (The survey is available online at http://ssrn.com/abstract=2481631.) The author administered the survey during a five-month period in 2013 at seven executive departments (Agriculture, Commerce, Energy, Homeland Security, Health and Human Services, Housing and Urban Development, and Transportation) and two independent agencies (Federal Communications Commission and Federal Reserve). Responses were received from 128 agency officials whose primary duties included statutory interpretation and rulemaking (for a 31% response rate). This Essay provides a new and important window into the role of Chevron and its progeny inside the regulatory state. Based on the findings presented in this Essay, it would not be an understatement to conclude that thirty years of Chevron have saturated the federal agency rulemaking process. The rule drafters surveyed overwhelmingly indicated familiarity with and use of Chevron and related doctrines in their statutory interpretation efforts. Moreover, two in five rule drafters surveyed agreed or strongly agreed — and another two in five somewhat agreed — that a federal agency is more aggressive in its interpretive efforts if it is confident that Chevron deference (as opposed to Skidmore deference or de novo review) applies.

CBF-AML is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a "second hit" to initiate leukemogenesis. Although compared with other AML subtypes patients with CBF-AML have a relatively favorable prognosis, still almost 40% of CBF-AML patients experience relapses of their disease. Mutation assessment of 84 leukemia- and/or cancer-associated genes using a customized targeted next-generation sequencing (NGS) approach was done on samples from 177 adults with CBF-AML [t(8;21), n=68; inv(16)/t(16;16), n=109]. We identified frequent mutations in the CCND1 and CCND2 genes as novel molecular alterations in AML with t(8;21). They were detected in 10 (15%) patients with t(8;21), making CCND1/2 mutations the third most frequently detected mutations in t(8;21) AML patients. In contrast, we found a single CCND2 mutation in only 1 (0.9%) patient with inv(16), and CCND1/2 mutations in only 11 of 1,426 non-CBF-AML patients (0.77%). Testing for CCND1 and CCND2 mutations using Sanger sequencing in additional 25 CBF-AML patients with t(8;21), for whom NGS analysis was not possible, identified 3 CCND1/2 mutated patients, thereby confirming the CCND1/2 mutation frequency in t(8;21) AML (15% in the NGS set, and 12% in Sanger sequencing set). All CCND2 mutations cluster around the highly conserved amino acid residue threonine 280 (Thr280), and encode a degradation-resistant CCND2 protein. Curiously, de novo germline CCND2 mutations affecting the identical amino acid residues Thr280 or Pro281 have been recently found to cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH) type 3, an extremely rare neurodegenerative disorder (Mirzaa et al., Nat Genet. 2014;46:510-5). The variant allele fractions of the detected CCND1/2 mutations were variable, suggesting that these mutations can represent both early and later mutational events. Interestingly, three of the 11 CCND1/2 mutated patients had two different CCND1/2 mutations, suggesting a dependence of their leukemia on this mutational event. The evaluation of mutations in other genes found in CBF-AML patients with CCND1/2 mutations did not reveal any statistically significant co-occurring or mutually exclusive mutations. The most frequent mutation co-occurring with CCND1/2 was ASXL2, detected in 4 of the 10 (40%) cases with t(8;21). Patients with CCND1/2 mutations presented with lower percentages of blood (P=0.02) and bone marrow (P=0.05) blasts than patients without these mutations, but did not differ significantly with regard to other pretreatment characteristics. No differences in the achievement of complete remission, disease-free or overall survival were observed between CCND1/2 mutated and CCND1/2 wild-type patients. Mechanistically, we show that Thr280Ala mutated CCND2 significantly increased cell growth (as assessed by TiterGlo assays and cell counts) in all experimental set-ups. Western blotting revealed an increase of retinoblastoma gene phosphorylation, indicating increased downstream signaling. Moreover, both propidium iodide mediated cell cycle analysis and immunofluorescence staining for phospho-histone H3 indicated an increased mitotic rate and a decreased G1 fraction. In summary, recurrent mutations in CCND1/2 are consistent with the currently known mutational landscape of t(8;21) AML, which is dominated by receptor tyrosine kinase, RAS, and chromatin remodeling gene mutations─all of which can result in targeting of CCND1/2. While CCND1 and CCND2 play a crucial role in the regulation of hematopoietic differentiation and cell proliferation, and their differential activation has been implicated in leukemogenesis, frequent mutations in these genes have not previously been reported in AML. The fact that no outcome difference was seen when comparing CCND1/2-mutated versus wild-type patients may be due to the relatively small sample size, or may possibly reflect the common use of cell cycle affecting high-dose cytarabine-based therapy in CBF-AML patients. Thus, the identification of CCND1/2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease. Disclosures Stone: Merck: Consultancy; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy; Celator: Consultancy; Agios: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

In response to Jud Mathews, Deference Lotteries, 91 Texas Law Review 1349 (2013).In Deference Lotteries, Jud Mathews proposes that the deference framework in administrative law be viewed through the game theory lens of a lottery. Such an approach helps us think critically about how varying standards of review may affect the behavior of agencies and courts engaged in the judicial review process. This Response suggests that the lottery lens can also help agencies think more strategically about how to develop and defend interpretations of statutes they administer. Assuming the validity of the lottery framework, the Response suggests a playbook for agencies to win the deference lottery. As the playbook reveals, this lottery is not a win-or-go-home contest. Instead, it is a repeated game -- a dialogue of sorts between courts and agencies -- where agencies have multiple opportunities to play and replay (and win). The predictive effect of tightening or loosening the lottery thus may not be as strong as one would hope.

The Dodd-Frank Wall Street Reform and Consumer Protection Act (Dodd-Frank) has raised the stakes for financial regulation by requiring more than twenty federal agencies to promulgate nearly 400 new rules. Scholars, regulated entities, Congress, courts, and the agencies themselves have all recognized — even before Dodd-Frank — the lack of rigorous cost-benefit analysis in the context of financial rulemaking. The D.C. Circuit has struck down several financial regulations because of inadequate cost-benefit analysis, with three more challenges to be decided this summer. Members of Congress have introduced legislation to address this problem, including a call for the President to intervene to require more exacting economic analysis. Regulated entities and investor protection groups are vigorously debating whether (and how) financial regulators should engage in cost-benefit analysis, as are a variety of policymakers, academics, and commentators. Absent from these debates, however, is a serious discussion of the importance of cost-benefit analysis in promoting good governance and democratic accountability. This Essay seeks to fill that void. The lack of attention to accountability is particularly troubling in the Dodd-Frank context, where most regulators are independent agencies and thus less democratically accountable via presidential oversight. In particular, independent agencies are not required to submit proposed rules and accompanying economic analyses for presidential review. Nor are their high-ranking officials subject to plenary presidential removal authority. Without another means of accountability — e.g., a robust cost-benefit analysis embedded in notice-and-comment rulemaking — independent agencies are more vulnerable to agency capture. This Essay argues that Dodd-Frank regulators should consider more seriously the democratic accountability concerns at play when regulating the financial markets. And those who regulate the regulators (via statutory command, executive order, or judicial review) should pay more attention to the good governance rationales for cost-benefit analysis when deciding whether and how to encourage Dodd-Frank regulators to engage in more rigorous and transparent economic analysis.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

Currently, there is no recommended confirmatory procedure for detecting doping with the anabolic steroid 5 alpha-dihydrotestosterone (DHT) in sportsmen. To develop a method, we determined ratios of hormone concentrations in urine from 120 healthy men and used these to set discrimination limits. These limits were then applied to results for urine specimens from 10 volunteers given DHT percutaneously (125 mg twice daily for 4 days). The ratio of DHT to epitestosterone (EpiT) was chosen as the primary marker of DHT administration, and ratios of 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha-ADIOL) to EpiT, 5 alpha-ADIOL to luteinizing hormone, and 5 alpha-ADIOL to 5 beta-androstane-3 alpha, 17 beta-diol were proposed as secondary markers. To evaluate method suitability, we analyzed 194 samples from sports competitors; results for 193 samples were negative, but the ratios in 1 sample greatly exceeded all the chosen limits. In conclusion, we propose that a test scheme based on our recommendations be considered for implementation in all Olympic drug-testing laboratories.

Background: Over 15 genome-wide association studies (GWASs) have identified a narrow region in chromosome 6q23.3 as a major hematopoietic quantitative trait locus (QTL) for many hematologic traits. The associated phenotypes have yet to be fully explained by genes in the region, including MYB. We noted a newly annotated microRNA within the QTL, miR-3662, and sought to characterize its role in the QTL by exploring its function in normal and malignant hematopoiesis, specifically acute myeloid leukemia (AML). Results: To assess the association between the QTL-defining SNPs and miR-3662 abundance, we genotyped 10 GWAS SNPs in 200 blood samples from non-leukemic individuals. The risk alleles of all 10 SNPs associated with higher miR-3662 abundance. Homozygosity for rs66650371, a three base pair (bp) deletion, showed the strongest association (P To explore the role of miR-3662 in malignant hematopoiesis, we overexpressed miR-3662 in AML cell lines (Kasumi1, KG1a, MV4-11) and primary AML patient blasts (n = 12). Overexpression of miR-3662 reduced cell growth and viability in vitro, and significantly reduced tumor size in a murine xenograft model (scramble vs. miR-3662; n = 9 mice/group). We used targeted RNA sequencing to identify differentially expressed genes between forced miR-3662-expresssing- and control AML patient blasts using a panel of 361 candidate genes (TruSeq RNA platform, Illumina). IKBKB was identified as a promising target, and direct regulation of IKBKB's 3′-UTR by miR-3662 was confirmed via luciferase assay. MiR-3662 overexpression lowered IKBKB mRNA and protein expression in both HPCs and AML patient blasts. As IKBKB promotes activation and nuclear localization of NF-κB, overexpression of miR-3662 led to decreased NF-κB transactivation potential via luciferase assay. Confocal microscopy of HPCs and AML cells, as well as subcellular fraction immunoblots, revealed decreased NF-κB nuclear localization upon miR-3662 overexpression. Re-introduction of IKBKB partly rescued miR-3662's phenotype by increasing nuclear localization of NF-κB, decreasing growth of HPCs, and increasing viability of AML cells. Conclusion: The risk alleles of the 6q23.3 region associated with higher miR-3662 abundance, and miR-3662 abundance was functionally linked to rs66650371. We characterized miR-3662 as an agent in normal and malignant hematopoiesis, and demonstrated that miR-3662 reduces NF-κB activity by targeting IKBKB. Citation Format: Sophia E. Maharry, Christopher J. Walker, Sandya Liyanarachchi, Sujay Mehta, James S. Blachly, Denis C. Guttridge, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML. [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 1117.

The protein NRAS is part of the NRAS-BRAF-MEK-ERK signaling cascade. Recently, 5 NRAS isoforms were discovered by our group, and it was observed that isoform 5 overexpression in vitro resulted in a more aggressive cell phenotype. NRAS isoform 5 increases phosphorylation of downstream targets (AKT, MEK, and ERK). The structural report is unavailable for NRAS isoform 5. In addition, it was found in the current study that the NRAS isoform 5 does not have GTPase activity due to lack the canonical GTP binding region. Given the importance of this pathway in driving melanoma progression, we studied the structural details of this novel NRAS isoform 5 by two biophysical techniques, NMR and CD spectroscopy. The circular dichroism spectra of NRAS isoform 5 that was C-acetylated and N-amidated was measured using 11%, 14%, 21%, 45%, 56% and 85% trifluoroethanol (TFE) in a phosphate buffer. The secondary structural elements were induced at ∼14-15% TFE. There were no qualitative differences in the CD spectra between 56% and 85% TFE. Therefore 56% deuterated TFE was utilized in the NMR structural studies. Two dimensional (2D) homonuclear and heteronuclear experiments were acquired at 25°C with Bruker Avance III HD 600 and 800 MHz NMR spectrometers (Campus Chemical Instrument Center NMR facility), each equipped with a triple resonance z axis gradient TXI cryoprobe for amino acid chemical shift assignments and structural determination of NRAS isoform 5. The NMR chemical shifts for the protons were assigned using traditional 2D TOCSY, COSY, and NOESY experiments. The 2D proton-carbon HSQC was utilized to facilitate the assignment of the alpha and side chain aliphatic protons. In the absence of TFE, there were no observable NOEs that could represent ordered secondary structures. Other than intra residue and short range sequential NOEs (i-j ≤ 2), only a weak medium range NOE was observed from the aromatic protons of Tyr 4 to the gamma proton of Val 7/8. The secondary structure of NRAS isoform 5 was determined via the same NMR techniques in 56% TFE. The secondary structure was defined by 69 sequential NOEs and 39 medium range NOEs. NOEs characteristic of helical structure were observed in the NOESY spectra. For example, NOEs were measured from H(alpha, i) to H(beta, i+3) for residues Tyr 4, Lys 5, and Leu 6. The tertiary fold was determined by 10 key long-range NOEs. The flexible C terminal is brought in close proximity to the N-terminal helix by 4 NOEs between Val 8/9 and Val 14, and 5 NOEs between Tyr 4 and Try 20 to form a helix-turn-coil structure in presence of TFE. In addition, the proton chemical shifts for the aromatic proton chemical shifts for the tyrosine aromatic ring became distinct. NRAS isoform 5 is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy. This data may be utilized as a starting point to understand the biophysical interactions of this novel NRAS isoform. Citation Format: Joseph Markowitz, Tapas K. Mal, Chunhua Yuan, Nicholas B. Courtney, Mitra Patel, Andrew R. Stiff, James Blachly, Christopher Walker, Ann-Kathrin Eisfeld, Albert de la Chapelle, William E. Carson. 3D structural report of NRAS isoform 5. [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 3093.

In contrast to aberrant MYC-signaling, MYC associated factor X (MAX) mutation is not common in many cancers but occurs in a limited number of specific tissue types, where it may be important. We sequenced MAX in 540 endometrioid endometrial cancer (EEC) DNAs. Mutations were seen 5.37% of samples with two hotspots, H28R (n = 12) and R60Q/W (n = 4 R60Q and n = 1 R60W), which accounted for ∼60% of the mutations seen. MAX mutation was associated with reduced recurrence-free survival (RFS) (P = 0.03; HR [95% CI] = 3.798 [1.10-13.0]) and with advanced stage (P = 0.024) and high tumor grade (P = 0.048). The association with reduced RFS remained significant in multivariable analysis (P = 0.05 HR = 2.40 [1.00-5.74]), strongly suggesting MAX mutation contributes to clinical aggressiveness in EEC. Focusing on the H28R hotspot mutation, we performed whole transcriptome expression profiling using AN3CA endometrial cancer cell lines stably expressing exogenous long and short isoforms of flag-tagged MAX-WT and MAX-H28R. Unsupervised hierarchical clustering and principle component analysis proved that the expression profiles for the MAX-H28R long cells were strikingly different than the MAX-H28R short, MAX-WT and unmodified cells. The MAX:DNA interface contains a predicted hydrogen bond between his28 and a guanine residue in the E-box motif. We hypothesized that the his-arg mutation seen in EECs would alter DNA binding, and assessed altered binding via chromatin immunoprecipitation/qPCR (ChIP). MAX-H28R had higher affinity for the NPM1 and CDK4 promoter regions, both of which include E-boxes that are well-established MYC/MAX targets. EMSAs for the same regions confirmed ChIP results, and showed that the supershifted MAX-WT and MAX-H28R complexes had different motilities, consistent with altered confirmation. We showed that an anti-MYC antibody was not able to supershift the EMSA fragment, indicating MYC is not part of the MAX:DNA complex. Co-IP, however, showed MAX-H28R retains MYC binding ability. Based on these findings, we conclude the differences seen in EMSA were with MAX homodimers. RNA-seq revealed differential gene expression in MAX-H28R mutated tumors, and ChIP showed enhanced MAX-H28R affinity for E-boxes in several of the differentially expressed genes. In vivo, MAX-H28R AN3CA xenografts were markedly hemorrhagic compared to MAX-WT. MECA-32 (pan-endothelial marker) staining proved that the MAX-mutant tumors had significantly more vessels per field (2.4-fold increase, P = 0.002). Work is ongoing to test for paracrine factors elaborated by the H28R-mutant AN3CA cells that might contribute to the vascular phenotype. Taken together these data implicate MAX mutation as a novel driver of endometrial tumorigenesis, contributing to tumor aggressiveness through enhanced tumor vascularity and potentially other mechanisms. Citation Format: Christopher J. Walker, Craig Rush, Paola Dama, Maggie Stein, Reena Shakya, Matthew O’Hern, David G. Mutch, David E. Cohn, Paul J. Goodfellow. MAX mutations in endometrial cancer are associated with poor patient outcome, altered E-box binding, and increased tumor vascularity. [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 2002.

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virus-specific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses ('eHAV') resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between 'enveloped' and 'non-enveloped' viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

The International Olympic Committee-approved drug test to detect testosterone administration is based on the concentration ratio of urinary testosterone to epitestosterone (T/E). Usually the T/E is ∼1, but after administration, the urinary excretion rate of testosterone increases and hence the ratio will be augmented;International Olympic Committee-accredited laboratories report T/E >6. Constant refrigeration of competitors’ samples in transport is logistically difficult; hence, samples are usually transported at ambient temperature and may take several days to reach these laboratories. Preservative is not added because it is reasoned that adulteration with a foreign substance may lead to legal challenges. Concern has been raised about maintaining the integrity of the sample, particularly because possible urinary microbial production of testosterone may cause an adverse finding (1), and several appeals have been made on that basis, e.g., the case of Mrs. Diane Modahl (2)(3), a British athlete who had competed internationally. A wide range of microorganisms can contaminate urine. Thus, testing the general hypothesis that microbial production of testosterone is possible simply by incubating a limited number of untreated urine samples at ambient temperature has not been fruitful. We decided that a better approach was to perform a relatively large-scale study in which urine samples from females were inoculated with a single potential candidate organism. To rationalize the choice of organism, a fresh approach was used that involved searching a comprehensive protein database (SWall; comprising >500 000 entries) to identify putative organisms that are most likely to produce enzymes able to convert steroid precursors to testosterone. Unlike prokaryotes (e.g., Escherichia coli ), many eukaryotic microorganisms are capable of both de novo synthesis of steroids and transformation of steroid substrates. Essential enzymes in the conversion of the precursor steroids, androstenedione (androst-4-ene-3,17-dione) and androstenediol (androst-5-ene-3β,17β-diol), to testosterone in humans are 17β-hydroxysteroid dehydrogenase …

This Article explores the conflicting commands of modern constitutional avoidance (courts must construe ambiguous statutes not only to adopt a constitutional construction but to avoid constructions that raise constitutional questions) and Chevron deference (courts must defer to an agency’s reasonable interpretation of an ambiguous statute it administers). While courts and commentators have suggested that constitutional avoidance trumps Chevron deference (at either step one or two), this Article advocates that modern avoidance should play no role in the review of administrative interpretations of law. Once Congress has empowered an agency to interpret an ambiguous statutory provision, a court cannot simply invalidate the agency’s interpretation and replace it with one the court believes better avoids constitutional questions. Instead, if an agency’s reasonable interpretation raises constitutional questions, a court must determine whether the interpretation is indeed unconstitutional and thus an impermissible interpretation at Chevron step two. This approach constitutes a return to the classical doctrine of constitutional avoidance, and it finds support in the Supreme Court’s decision in National Cable & Telecommunications Ass’n v. Brand X Internet Services. As the Article illustrates in a variety of administrative contexts, this Brand X doctrine of constitutional avoidance is necessary to preserve a proper separation of powers between the courts, the Executive, and Congress. It is also justified under Dean Edward Rubin’s network theory of administrative law.

Determination of the ratio of testosterone to epitestosterone (T/E) in urine is used to detect testosterone administration in athletes, with a ratio > 6 considered as evidence of an offense. We show that administration of ketoconazole, which inhibits testosterone biosynthesis, may be useful for differentiating between an athlete who is using testosterone and one who naturally gives a ratio > 6. In a control subject pretreated with testosterone, ketoconazole caused the ratio to increase; conversely, it caused a decrease in the ratio in an athlete under investigation. Repeated administration of ketoconazole to two normal men caused a decrease in the ratio due to a large decrease in the urinary excretion rate of testosterone relative to epitestosterone. Stimulation with human chorionic gonadotropin exacerbated the differences in excretion rates. A single administration of ketoconazole to six normal men caused the T/E ratios to decrease significantly within 8 h, a suitable time scale for use in a dynamic test.

The current drastic escalation in obesity may be contributing to the exponential rise in drugs used for image enhancement. Drugs such as anabolic-androgenic steroids (AAS) are perceived as a viable method of achieving a perfect physique. They are also the most widely abused drugs in sport. The Internet has encouraged the abuse of expensive drugs, particularly human growth hormone (hGH), resulting in increased importation for personal use. The substantial increase in this market has opened up avenues for counterfeiting, estimated as a multi-million pound business. The acute adverse effects from contaminated vials may result in a variety of pathologies including communicable diseases. In 2007, in the UK, a series of intramuscular abscesses, requiring surgical treatment, led us to study samples obtained from the underground market. The analysis of 38 parenteral samples and 19 oral samples of tablets was performed by a World Anti-Doping Agency (WADA) accredited laboratory, in an attempt to establish the extent of available counterfeit products. Fifty-three per cent (20) of the injectable AAS esters and 21% (4) of the oral tablets were counterfeit. Culture and sensitivity revealed the presence of skin commensal organisms, which may have contributed to the development of the abscesses. Users of AAS and hGH for sport, including bodybuilding, are currently risking their health because of counterfeit and poorly controlled products.

This paper examines the 225 banks that failed between February 2, 2007, and April 23, 2010, comparing them to a random sample of banks that had not failed as of April 23, 2010. We performed regression and discriminant analysis on quarterly call report data for one year, two years, three years, and four years prior to bank failure to determine whether the failure could have been predicted. Our model is statistically significant at the 1% level and predicts bank failures with 88.2% accuracy one year prior to failure, 78.6% two years prior to failure, 71.4% three years prior to failure, and 66.0% four years prior to failure.

Dihydrotestosterone (DHT) can be used by an athlete as an anabolic steroid to evade the current International Olympic Committee approved drug tests. To investigate the possibility of a method for its detection, the heptanoate ester of DHT was administered to two male subjects (150 mg i.m.). Urine samples, collected before and after the injection, were subjected to enzymatic hydrolysis and the excretion rates of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and testosterone (T) were determined by radioimmunoassay. Relative changes in the excretion of DHT, 3 alpha-diol, 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol), T and epitestosterone (17 alpha hydroxyandrost-4-en-3-one; Epi-T) were determined by gas chromatography-mass spectrometry (GC-MS). Following administration of DHT, the urinary excretion rates of DHT, 3 alpha-diol and 3 beta-diol increased when compared to those of T, Epi-T, 5 beta-diol and luteinizing hormone (LH). Concentrations of DHT in the plasma increased whereas those of T, LH and follicle stimulating hormone decreased. The changes following such modest doses of DHT suggest that these ratios of urinary hormones may be used for the detection of doping with DHT.

[1] Present-day shortcomings in the representation of upper tropospheric ice clouds in general circulation models (GCMs) lead to errors in weather and climate forecasts as well as account for a source of uncertainty in climate change projections. An ongoing challenge in rectifying these shortcomings has been the availability of adequate, high-quality, global observations targeting ice clouds and related precipitating hydrometeors. In addition, the inadequacy of the modeled physics and the often disjointed nature between model representation and the characteristics of the retrieved/observed values have hampered GCM development and validation efforts from making effective use of the measurements that have been available. Thus, even though parameterizations in GCMs accounting for cloud ice processes have, in some cases, become more sophisticated in recent years, this development has largely occurred independently of the global-scale measurements. With the relatively recent addition of satellite-derived products from Aura/Microwave Limb Sounder (MLS) and CloudSat, there are now considerably more resources with new and unique capabilities to evaluate GCMs. In this article, we illustrate the shortcomings evident in model representations of cloud ice through a comparison of the simulations assessed in the Intergovernmental Panel on Climate Change Fourth Assessment Report, briefly discuss the range of global observational resources that are available, and describe the essential components of the model parameterizations that characterize their "cloud" ice and related fields. Using this information as background, we (1) discuss some of the main considerations and cautions that must be taken into account in making model-data comparisons related to cloud ice, (2) illustrate present progress and uncertainties in applying satellite cloud ice (namely from MLS and CloudSat) to model diagnosis, (3) show some indications of model improvements, and finally (4) discuss a number of remaining questions and suggestions for pathways forward.

19-Norandrosterone (19-NA) is the principal urinary metabolite of the anabolic steroid nandrolone and its prohormones. The administration of these 19-nor androgens is prohibited in sport by the World Anti-Doping Agency (WADA) but, even so, adverse findings for 19-NA continue to be commonly reported. Little is known about the urinary concentrations of 19-NA that can occur in women who are not using anabolic steroids, including those using oral contraceptives containing the 19-nor progestogen norethisterone. In 2004, WADA lowered the reporting threshold for 19-NA for females from 5 to 2 ng/mL. The lack of any substantial data on 19-NA excretion in women prompted this large-scale investigation. In this investigation, single untimed urines collected from 1202 female volunteers, 38 of whom were taking norethisterone containing contraceptives, were analysed for 19-NA. None of the women was a competitive athlete and pregnancy had been excluded by a urinary test for human chorionic gonadotropin (hCG). Only one sample exceeded the 19-NA reporting threshold having a concentration of 4.1 ng/mL. This sample was from a user of a norethisterone-containing contraceptive.

viral infections transmitted to infants: from mechanisms to prevention and care Meeting

This chapter introduces the United Nations global e-government readiness reports, for which we serve as primary researchers, with particular focus on Latin America. The UN reports provide useful composite scores and rankings on 191 countries worldwide with respect to e-government, e-readiness, and e-participation, as well as highlighting best practices that have been identified throughout the world. In particular, the UN reports provide meaningful insights into the development of e-government in Latin America—facilitating comparative analysis between regions, as well as country comparisons within Latin America.Basing itself on the UN report, the chapter provides a comparative analysis of e-government in Latin America—both intra- and interregional comparisons—with the main objective of elevating e-government literature to a more quantitatively rigorous and sophisticated level. This chapter aims at introducing the UN report, outlining its main findings, and reporting how Latin America compares with the world in e-government readiness and development. It first outlines the background of the UN report, followed by the research methodology. Then it discusses the findings relevant to Latin America, with a brief conclusion on topics for future research and discussion.

Therapies for recurrent or advanced endometrioid endometrial cancer (EEC) are ineffective, and no targeted therapies, or genetic markers to identify high-risk patients exist. There is an urgent need to identify factors contributing to aggressiveness. We performed whole exome sequencing of 8 early stage endometrial cancers that subsequently recurred. Two chromosome 16q tumor suppressor genes, CTCF and ZFHX3, were identified as candidates associated with recurrence. The endometrial Cancer Genome Atlas proved that both of these genes are frequently mutated in EEC. To further explore the relationship between CTCF and ZFHX3 mutations and ECC tumorigenesis/aggressiveness, targeted deep sequencing of both genes was performed on 541 EEC tumors. We relied on microsatellite typing and a novel Bayesian method to assess copy number alterations (CNAs). The Bayesian method's use of deep sequencing data to predict CNAs showed 97% concordance with microsatellite typing. The overall mutation rate (SNVs and small indels) was 24% and 18% for CTCF and ZFHX3, respectively. The majority of mutations were loss of function (stop, frameshift or splice site), with CTCF at 89% and ZFHX3 at 59% loss of function defects. There was a significant excess of mutational co-occurrence in both genes (P = 0.003). Deletion/CNA was also common: 79 of 497 informative tumors had CNAs involving both genes (16%). Taken together, the high rate of co-mutation and co-deletion suggests selective advantage for inactivation of both tumor suppressors. Patients with tumors having mutation or CNA of both CTCF and ZFHX3 had significantly shorter recurrence-free survival (RFS) compared to wt (P = 0.007, HR = 2.4, 95%CI = 1.3-4.4). Mutation/loss of both genes was significantly associated with features portending aggressive disease: high grade (P60 years, P = 0.02). There was no association with stage. Although our ability to determine the contributions of CTCF and ZFHX3 individually was limited by sample size and their frequent co-occurrence, we assessed them separately as independent prognostic markers. It is noteworthy that mutation/CNA of ZFHX3 significantly correlated with shorter RFS (P = 0.003, HR = 2.4, 95%CI = 1.4-4.0), whereas no such association was seen with CTCF. In a multivariable analysis including grade, stage, LVSI and therapy, ZFHX3 mutation/CNA remained an independent prognostic marker (P = 0.04). We are currently validating these results in a larger independent cohort. Here we describe emerging markers that could be useful for identifying high-risk patients. Our data point to what may be a complex interplay between the ZFHX3 and CTCF tumor suppressors in EEC tumorigenesis. Citation Format: Christopher J. Walker, Mario A. Miranda, Matthew O'Hern, Kevin Coombes, Ralf Bundschuh, David G. Mutch, Paul J. Goodfellow. Loss of function of two closely linked tumor suppressors contributes to biologic aggressiveness in endometrial cancers: Co-mutation of CTCF and ZFHX3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5300. doi:10.1158/1538-7445.AM2015-5300

Background: Overcoming the block of differentiation is a major effort in Acute Myeloid Leukemia (AML) research. We visited the major quantitative trait locus (QTL) for hematopoietic differentiation on chromosome 6q23 to identify genes involved in hematopoietic development that are possibly deregulated during leukemogenesis. We noticed a novel microRNA within the QTL: miR-3662. Aims: The goal was to elucidate a possible causal involvement of miR-3662 in the association of the 6q23 QTL with hematopoietic differentiation and/or leukemogenesis. Results: Monitoring of miR-3662 expression during cytokine stimulated differentiation of CD34+ hematopoietic progenitor cells (HPCs) revealed increasing miR-3662 abundance during differentiation, with the highest expression found in terminally differentiated cells. In contrast, miR-3662 had very low expression in AML cell lines (KG1a, MV4-11, OCI-AML3, THP1) and primary AML patient blasts (n=12). We stably introduced miR-3662 into HPCs of 3 non-leukemic donors, and differentiated the cells into erythroid, megakaryocytic and granulocytic lineages. Forced expression of miR-3662 led to significant increase of colony formation, most pronounced in the erythroid lineage. In contrast, targeted knock-down of miR-3662 significantly reduced colony formation (Figure 1). To test if the observed downregulation of miR-3662 in leukemic cells is favorable to leukemic cells, we stably introduced miR-3662 in AML cell lines (MV4-11, KG1a) and AML patient blasts (n=12). Increasing the abundance of miR-3662 led to reduction of cell viability, increased cell death and reduced colony formation. To find the downstream targets of miR-3662, we performed a comprehensive targeted RNA sequencing approach with a panel of 361 genes (TruSeq platform, Illumina; followed by Ingenuity Pathway analysis), using RNA from two AML patient blasts infected with miR-3662 vs. scramble. miR-3662 was associated with >20% reduction in the mRNA expression of 34 genes. Among these, 6 genes harbored predicted miR-3662 binding sites in their 3′-UTRs, qualifying as potential direct miR-3662 targets. Of these, the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) had the highest affinity score for miR-3662, and was thus chosen as the primary candidate. Determination of endogenous miR-3662 and IKBKB expression in primary AML patient blasts (n=12) showed a significant association of higher miR-3662 with lower IKBKB abundance. qPCR and Western blotting confirmed miR-3662 mediated downregulation of IKBKB in HPCs, KG1a and MV4-11 cells and primary patient blasts. Because IKBKB leads to reduced phosphorylation of the inhibitor in the inhibitor/NF-ĸB complex (IKBa), it limits NF-ĸB's nuclear localization and consequent transcriptional activation. Increasing miR-3662 should thus decrease NF-ĸB's nuclear localization. Western blotting showed reduced phosphorylation of IKBa and confocal imaging showed less NF-ĸB in the nucleus in miR-3662 expressing MV4-11 and HPC cells (Figure 2). Luciferase assay validated a direct interaction of miR-3662 with the 3′-UTR of IKBKB. Finally, we attempted to gain insights into the upstream regulation of miR-3662. We identified a possible transcription start site for miR-3662 (TSS-3662), whose activation potential was validated by both luciferase assays and enrichment of RNA polymerase II (Pol II), histone H3 methylated Lys (H3K4), and total histone H3 CHIP assays of TSS-3662. Testing of predicted activating transcription factors showed GATA1 and CEBPA to have an activating potential (as shown by luciferase assays) and binding affinity (as shown by electrophoretic mobility shift assays) to TSS-3662. Overexpression of GATA1 and CEBPA significantly increased miR-3662's abundance. Conclusion: We have identified miR-3662, located in the major hematopoietic QTL on chromosome 6q23, as a novel regulator of hematopoietic differentiation that acts by regulating the NF-ĸB pathway. Reduced abundance of miR-3662 in leukemic cells potentially contributes to the de-differentiated phenotype of leukemic cells and to increased cell growth by increasing nuclear localization of NF-ĸB. Figure 1. Effect of miR-3662 overexpression and knock-down on colony formation of HPCs. Figure 1. Effect of miR-3662 overexpression and knock-down on colony formation of HPCs. Figure 2. Cellular localization of NFĸB in miR-3662 vs. scramble-infected HPCs. Figure 2. Cellular localization of NFĸB in miR-3662 vs. scramble-infected HPCs. Disclosures Walker: Karyopharm Therapeutics Inc.: Research Funding.

Endometrial cancer is the most common gynecologic malignancy in the US. At present, therapies for recurrent or advanced stage disease are largely ineffective. Understanding the genetic and epigenetic factors that contribute to biologic aggressiveness holds promise for new treatment strategies. CTCF, a master regulator of gene expression, was identified as a significantly mutated gene in endometrial cancers by TCGA. Our group identified a hot spot mutation in CTCF in microsatellite instability positive (MSI) endometrial cancers not reported by TCGA. It is noteworthy that among cancer types analyzed to date, EEC is the only malignancy in which CTCF mutation is a frequent defect. CTCF has thousands of binding sites throughout the human genome and is capable of direct activation of genes, enhancer binding and far-reaching chromatin modification, and controls expression of numerous critical regulators of cellular growth/survival (e.g. c-myc, p53, p19/ARF, p16/INK4A, BRCA1, p27, E2F1). However, the role CTCF plays in EEC is largely unknown. Our analysis of a large cohort of EEC offers further insights into CTCF's role in EEC. Targeted ultra-deep sequencing of 540 primary ECC samples (966X mean coverage) identified 97 different variants (excluding SNPs, low quality variant calls, and synonymous variants) in 136 samples (25.2%). Mutations were more common in tumors with MSI than tumors with intact DNA mismatch repair (40% and 14% respectively p Because we saw frequent loss of function mutations we undertook studies to determine if CTCF deletion is also a feature of EECs. Q-PCR for 149 samples revealed 8 cases with heterozygous CTCF deletion (5%), and in one tumor a 1741bp homozygous deletion (chr16: 67643579-67645330). Two patients with CTCF deletion recurred and both had short RFIs (1 SD below the mean). Investigation of the effects of CTCF mutation on gene expression in primary tumors is ongoing. Taken together, our analyses serve to contextualize the effects of CTCF mutations in EEC and implicate loss of CTCF in biologic aggressiveness. Citation Format: Christopher J. Walker, Mario A. Miranda, David G. Mutch, Paul Goodfellow. CCCTC-binding factor (CTCF) mutation in endometrioid endometrial carcinoma (EEC): Master epimutator contributing to biologic aggressiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2014-1540

Dendritic cells (DCs), which are potent antigen-presenting cells (APCs), are used as adjuvants for the treatment of cancer and infectious diseases in human and nonhuman primates, with documented clinical efficacy. The hepatitis C virus (HCV)-chimpanzee model is the best available model for testing the immunotherapeutic effects of DCs in the setting of a chronic infection, as chimpanzees develop a persistent infection resembling that seen in humans. However, several reports have suggested that DCs derived from chronically infected individuals or nonhuman primates are functionally compromised. As a prelude to clinical studies, we evaluated whether functionally mature DCs could be generated in chimpanzee plasma by good manufacturing practice using CD14+mononuclear precursors from chronically infected chimpanzees. DCs generated in a medium with HCV-negative plasma and treated with a defined cocktail of cytokines or a CD40 ligand trimer matured fully, as measured by the induction of CD83 expression and the upregulation of costimulatory molecules. Furthermore, the expression of CCR7 was induced, suggesting an acquisition of migration capacity. Mature DCs were capable of stimulating allogeneic T cells, antigen-specific memory CD4+T cells, and HCV-specific CD8+-T-cell clones. In all cases, there was no evidence of HCV infection in DCs. Furthermore, these DCs maintained their phenotype and APC function after cryopreservation. Finally, no discernible differences were noted between DCs derived from HCV-infected and uninfected chimpanzees. In summary, precursor cells from HCV-infected chimpanzees are fully capable of differentiating into functional, mature DCs, which can now be reproducibly prepared for investigations of their immunotherapeutic potential in the setting of chronic HCV infection.

Abstract 35 As tyrosine kinase inhibitors (TKIs) do not induce long-term response in blast crisis chronic myeloid leukemia (CML-BC) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and are unable to kill quiescent Ph+ hematopoietic stem cells, alternative therapies targeting dysregulated pathways in BCR-ABL1+ acute leukemias are needed. CRM1, a karyopherin aberrantly overexpressed in several cancers, controls the nuclear export of proteins (e.g. ABL1, SET, p53, p21, FOXO and RB) that regulate normal and malignant hematopoietic cell survival, self-renewal and proliferation. Here we show that enhanced CRM1 expression also occurs in Ph+ leukemias and the clinical stage small molecule inhibitor of CRM1, KPT-330, has a detrimental effect on malignant but not normal hematopoietic progenitors. Specifically, a substantial upregulation (≥ 65% increase) in the expression levels of CRM1 was detected by immunoblot in CD34+ progenitors from bone marrow (BM) of leukemic primary samples (n=3), when compared to CD34+ progenitors from healthy (n=3) donors. Interestingly, CRM1 protein levels in CML CD34+ cells were markedly but not totally reduced by treatment with the ABL1 kinase inhibitor Imatinib (1μM, 72h), suggesting that BCR-ABL1-driven pathways are not the only factor contributing to CRM1 upregulation. Additionally, treatment of primary CML cells with KPT-330 (1μM, 72h) resulted in a 75% reduction in BCR-ABL1 kinase activity, consistent with an interrelationship between BCR-ABL1 and CRM1 activities. Finally, the ability of BCR-ABL1 to induce CRM1 expression was demonstrated upon ectopic BCR-ABL1 expression in myeloid 32Dcl3 precursor cells in which CRM1 protein levels became ∼10-fold higher. To determine the therapeutic relevance of CRM1 inhibition, cell survival was assessed in MACS-purified CD34+ progenitors. Treatment with KPT-330 resulted in ≥80% induction of apoptosis in Ph+ (n=5) CML (2μM, 72hr) and B-ALL (500nM, 72h) CD34+ cells. Conversely, KPT-330 exerted only a minimal effect on the survival of CD34+ progenitors from healthy donor BM (15–30% Annexin V positive, KPT-330 0.5–2μM, 72h). Consistent with the existence of BCR-ABL1-independent signaling leading to increased CRM1 expression, marked apoptosis was also observed in KPT-330-treated CD34+ progenitors isolated from a Ph-negative B-ALL patient. Thus, CRM1 inhibitor-based therapies might also benefit BCR-ABL1-negative leukemias. To formally determine the effects of KPT-330 on leukemic cell survival, methylcellulose-based clonogenic assays were performed. KPT-330 treatment (1μM) resulted in almost total suppression (97% reduction) of the clonogenic potential of leukemic but not normal (30% reduction) CD34+ progenitors. Because of evidence that CRM1 directly interacts with the protein phosphatase 2A (PP2A) inhibitor SET, and that CRM1 inhibition alters trafficking of hnRNP A1, a direct regulator of the SET-PP2A interplay in Ph+ leukemias, it is conceivable that the anti-leukemic activity of KPT-330 is, at least in part, mediated by PP2A activation. Indeed, KPT-330 treatment (1μM, 48hr) of BCR-ABL1+ cell lines resulted in full restoration of PP2A activity, comparable to levels observed in BCR-ABL1-negative cells. Accordingly, KPT-330 treatment (1μM, 12h) of 32Dcl3BCR-ABL1 cells caused a nuclear accumulation (4-fold increase) and cytoplasmic decrease (95% lower) of SET protein levels, as indicated by both confocal microscopy and immunoblots of subcellular fractions. Moreover, KPT-330-treated cells showed altered hnRNP A1 cellular distribution and overall downregulation. However, unexpectedly, hnRNP A1 protein levels were proportionally higher in the cytoplasm and lower in the nucleus, suggesting that the deleterious effect of CRM1 inhibition on hnRNP A1 might not depend on direct inhibition of hnRNP-A1 nuclear export. Because KPT-330 displays favorable ADME properties and has been shown to alleviate leukemic burden using in vivo models of different cancers, we are currently testing the anti-leukemic activity of this compound in a mouse model of CML-BC. We do expect a profound inhibition of leukemogenesis, and all treated mice are currently alive with no signs of toxicity. Thus, CRM1 inhibition by KPT-330 represents a potential new therapeutic avenue which could easily be exploited in malignancies like CML-BC and Ph+ ALL that show a dismal outcome to current available therapies. Disclosures: Walker: Karyopharm Therapeutics Inc: Research Funding. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Perrotti:Karyopharm Therapeutics Inc: Research Funding.

Tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis (BC) chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Increased expression/activity of nucleocytoplasmic-shuttling heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, are critical for the expression of factors (e.g. SET/PP2A, C/EBPβ/miR-328, and c-Myc) regulating proliferation and survival of Ph+ progenitors. Because the karyopherin CRM1 controls nuclear export of hnRNPs, we assessed the therapeutic potential of CRM1 inhibitors in CML-BC and Ph+ ALL models. Thus, myeloid 32D-p210BCR-ABL1 and lymphoid Baf3-p190BCR-ABL1 progenitors were exposed to the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays revealed that KPT-185 and KPT-207 decreased cell viability by ∼80% at concentrations ranging from 150-350 nM. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70-100% viable cells) by KPT-SINE. As expected, treatment (1 μM; 48h) with these inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. As a result, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization, which is important for regulation of the PP2A inhibitor SET and, therefore, for BCR-ABL1 leukemogenesis, we assessed whether KPT-207 and KPT-185 negatively regulate PP2A activity. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3839. doi:1538-7445.AM2012-3839

Abstract 3758 Despite the remarkable response in chronic phase CML, tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis CML (CML-BC) and Ph+ ALL, and are unable to kill quiescent Ph+ hematopoietic stem cells. We reported that CML disease progression is characterized by a BCR-ABL1 dose- and kinase-dependent increase in the expression/activity of nucleocytoplasmic-shuttling of heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, which are essential post-transcriptional and translational modulators of critical regulators of cell proliferation, survival and differentiation of CD34+ CML-BC and/or CD34+/CD19+ Ph+ ALL progenitors including SET/PP2A, E2F3, Bcl-xL, C/EBPa, miR-328, and c-Myc. The karyopherin (a class of proteins involved in regulating nuclear import/export) chromosome region maintenance 1 (CRM1) controls the nuclear export of several hnRNPs, and because growth/survival of CML-BC and Ph+ ALL progenitors requires the aberrant cytoplasmic activity of hnRNPs A1, E2 and/or K, we have explored the therapeutic potential of CRM1 inhibitors in p210 and p190 BCR-ABL1+ cell line models of CML-BC and Ph+ ALL, respectively. Thus, the cytokine-dependent myeloid 32Dcl3 and lymphoid BaF3, and the derived cytokine-independent 32D-p210BCR-ABL1 and Baf3-p190BCR-ABL1 mouse progenitors were exposed for 48 hr to different concentrations (0–10 μM) of the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays (n=3) revealed that KPT-185 and KPT-207 decreased viability ∼80% in 32D-p210BCR-ABL1 cells (KPT-185≥207) at concentrations ranging from 150–350 nM. Similar results were obtained in Baf3-p190BCR-ABL1 cells as the EC50 was 300nM for KPT-185 and KPT-207. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70–100% viable cells) at concentrations (150–350 nM) of KPT-185 and KPT-207 that impair survival of BCR-ABL1+ cells. Mechanistically, we found that KPT-SINE CRM1 inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. Indeed, in KPT-207-treated (1 μM; 48h) BCR-ABL1+ cells hnRNP A1, E2 and K accumulated in the cytoplasm. Likewise, KPT-185 treatment (1 μM; 48h) resulted in hnRNP A1 being sequestered in the nucleus whereas hnRNP E2 distribution was not altered and, unexpectedly, levels of hnRNP K expression were markedly decreased in both subcellular compartments. Interestingly, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization in addition to impairing BCR-ABL1 expression/activity, and suppression of PP2A tumor suppressor activity is essential for both p210 and p190 BCR-ABL1-driven leukemogenesis, it is highly plausible that KPT-207 and KPT-185 negatively regulate the BCR-ABL1-dependent and hnRNP A1-mediated induction of the PP2A inhibitor SET thereby rescuing PP2A phosphatase activity, which in turn decreases BCR-ABL1 expression and kinase activity, and triggers in vitro and in vivo apoptosis of primary CML-BC and Ph+ ALL progenitors. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Disclosures: Shacham: Karyopharm: Equity Ownership. Kauffman:Karyopharm: Equity Ownership.

Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assays; Western blot, confocal microscopy and FACS analyses. Results: We observed increased BCR-ABL1 expression with impaired kinase activity in quiescent CML HSCs, in which BCR-ABL1 per se is required for induction of JAK2 that subsequently activated β-catenin and inhibited PP2A. In fact, PP2A was suppressed in CML but not normal CD34+/CD38−/CD90+ HSCs. FTY720 and/or its non-immunosuppressive (S)-FTY720-OMe derivative markedly reduced survival and self-renewal of CML but not normal quiescent HSCs through BCR-ABL1 kinase-independent and PP2A-mediated JAK2 and β-catenin inhibition. Importantly, FTY720 also strongly diminished BCR-ABL1+ LT-HSC frequency in serial BM transplantation assays. Conclusions: The pharmacologic targeting of the newly-identified BCR-ABL1 kinase-independent JAK2/β-catenin interplay in quiescent HSCs with FTY720 and its derivatives, might lead to cessation of lifelong patient dependence on TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2011-LB-109

Abstract 775 We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2). Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients. To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells. Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min). Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures: Verstovsek: Incyte Corporation: Research Funding.