Hunting for dark halo substructure using submilliarcsecond-scale observations of macrolensed radio jets

Dark halo substructure may reveal itself through secondary, small-scale gravitational lensing effects on light sources that are macrolensed by a foreground galaxy. Here, we explore the prospects of using Very Long Baseline Interferometry (VLBI) observations of multiply-imaged quasar jets to search for submilliarcsecond-scale image distortions produced by various forms of dark substructures in the 1e3-1e8 Msolar mass range. We present lensing simulations relevant for the angular resolutions attainable with the existing European VLBI Network (EVN), the global VLBI array, and an upcoming observing mode in which the Atacama Large Millimeter Array (ALMA) is connected to the global VLBI array. While observations of this type would not be sensitive to standard cold dark matter subhalos, they can be used to detect more compact forms of halo substructure predicted in alternative structure formation scenarios. By mapping ~5 strongly lensed systems, it should be possible to detect or robustly rule out primordial black holes in the 1e3-1e6 Msolar mass range if they constitute >1% percent of the dark matter in these lenses. Ultracompact minihalos are harder to detect using this technique, but 1e6-1e8 Msolar ultracompact minihalos could in principle be detected if they constitute >10% of the dark matter.Comment: 13 pages, 8 figures; v.2 accepted for publication in MNRA

Preclinical Modeling of ACVR1-Dependent Hepcidin Production and Anemia By Momelotinib

Abstract Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Elevated levels of hepcidin in MF patients suggest that deregulated iron homeostasis is a driver of anemia in MF (Pardanani et al., Am J Hematol, 2013). A phase 2 study in MF patients demonstrated that momelotinib (MMB) treatment resulted in improvement of anemia (Pardanani et al., Blood, 2013, ASH Suppl). We have demonstrated that in addition to inhibiting JAK1/2, MMB also inhibits the bone morphogenetic protein receptor kinase, activin A receptor type 1 (ACVR1), a key regulator of hepcidin production in hepatocytes (Asshoff et al., Blood, 2015, ASH Suppl). This work supports a model by which the improvement of anemia observed in MF patients with MMB treatment results from the inhibition of ACVR1-mediated hepcidin expression in the liver, increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis. Here we demonstrate that MMB treatment in anemic rats resulted in a reduction of hepcidin and amelioration of anemia; whereas ruxolitinib dosing affected neither hepcidin nor anemia. Short-term MMB oral treatment in anemic rats resulted in a dose responsive inhibition of serum hepcidin induction with complete suppression at 25 mg/kg QD, 82% inhibition at 10 mg/kg QD, and 55% inhibition at 5 mg/kg QD (Table 1). Interestingly, only the two higher doses show significant time above EC50 levels, whereas the 5 mg/kg dose (which most closely approximates MMB human clinical exposures) only shows significant time above EC20. This suggests that systemic drug concentrations alone may not accurately predict activity in the model as 5 mg/kg demonstrated a 55% reduction in serum hepcidin. In a rat quantitative whole body autoradiography tissue distribution study, MMB levels have been demonstrated to be 3-4 fold higher in the liver relative to blood (Figure 1). Thus, the significant inhibiton of hepcidin levels in animals dosed with 5 mg/kg MMB may be due to higher concentrations in the liver. MMB concentrations would be expected to be higher in the liver relative to blood in humans as well. Our results provide preclinical evidence for a direct role of MMB in regulating iron homeostasis through the ACVR1/hepcidin axis. Evaluation of the activity of MMB on the ACVR1/hepcidin axis is being done in ongoing MMB clinical studies in patients with MF. Table 1 MMB exposure levels and % inhibition of serum hepcidin induction in ACD rats. Human (healthy volunteer) and ACD rat exposure levels for MMB. Table 1. MMB exposure levels and % inhibition of serum hepcidin induction in ACD rats. Human (healthy volunteer) and ACD rat exposure levels for MMB. Figure 1 Tissue distribution of radioactivity in rats following a single oral dose of [14C]-MMB. LOQ is 4, 159 ng-eq/g. 24h rat blood levels were below LOQ. Figure 1. Tissue distribution of radioactivity in rats following a single oral dose of [14C]-MMB. LOQ is 4, 159 ng-eq/g. 24h rat blood levels were below LOQ. Disclosures Warr: Gilead Sciences: Employment, Equity Ownership. Zheng:Gilead Sciences: Employment, Equity Ownership. Sharma:Gilead Sciences: Employment, Equity Ownership. Maciejewski:Gilead Sciences: Employment, Equity Ownership. Theurl:Gilead Sciences: Research Funding. Whitney:Gilead Sciences: Employment, Equity Ownership. </jats:sec

The Jak1/Jak2 Inhibitor Momelotinib Inhibits Alk2, Decreases Hepcidin Production and Ameliorates Anemia of Chronic Disease (ACD) in Rodents

Abstract Many patients with myelofibrosis (MF) develop anemia and are, or become, dependent on frequent red blood cell transfusions [1]. Results from the phase 2 studies for the treatment of myelofibrosis (MF) with the Jak1/2 inhibitor momelotinib (MMB) demonstrated that MMB treatment provided an anemia benefit, in addition to providing a durable spleen response and improvement in constitutional symptoms in MF patients [2]. MMB's anemia benefit was an unexpected outcome as erythropoietin (EPO)-mediated JAK2 signaling is essential for erythropoiesis [3, 4]. Systemic iron homeostasis is controlled by the peptide hormone hepcidin produced by the liver. Hepcidin reduces iron export from duodenal enterocytes and splenic and hepatic macrophages by binding to and down-regulating the iron exporter ferroportin [5-7]. In chronic disease, there is a significant increase in hepcidin levels which can result in severe anemia; this pathologic condition is termed anemia of chronic disease (ACD). Recently, MF patients have been shown to have elevated serum hepcidin levels and this increase is associated with inferior overall survival in these patients [8]. This study aimed to determine whether MMB's clinical anemia benefit is driven by direct activity of MMB on the hepcidin pathway. We assayed MMB inhibitory activity on the BMP-receptor kinase pathway (the central driver of hepcidin transcription in hepatocytes) and assessed the activity of MMB in a rodent model of anemia of chronic disease (ACD). We demonstrate that MMB inhibits BMP6-induced in vitro production of hepcidin in cultured hepatocytes (HepG2 cells) with an EC50 = 651 +/- 203 nM (n=3). This inhibitory activity is mediated by direct suppresion of the BMP-receptor kinase Alk2 as MMB inhibits Alk2 enzymatic activity with an IC50 = 8.4 +/- 1.5 nM (n=3). Ruxolitinib has no activity on either Alk2 or the BMP-receptor kinase pathway. To understand whether MMB could modulate hepcidin levels in vivo and ameliorate anemia in vivo we assessed the effect of MMB in a peptidoglycan-polysaccharide fragment (PG-APS)-induced rat ACD model. Treatment with clinically relevant exposure levels of MMB for 3 days resulted in a dose dependent reduction in both liver RNA and serum protein hepcidin levels and caused an increase in serum iron. Furthermore, long-term treatment with MMB for 21 days increased the numbers and percent of reticulocytes and mature red blood cells in the bone marrow and increased Hgb and hematocrit to normal levels in the blood. Our data suggest that MMB's clinical anemia benefit results from inhibition of ALK2-mediated expression of hepcidin in the liver, which results in increased release of iron from sequestered cellular stores and enhanced erythropoiesis. A phase 2 translational study in anemic subjects with MPNs is scheduled to confirm this mechanism. The Alk2-mediated activity on iron metabolism through hepcidin could prove beneficial in a number of additional indications, and facilitate the combination of MMB with myelosuppressive agents. 1. Tefferi, A., et al., One thousand patients with primary myelofibrosis: the mayo clinic experience. Mayo Clin Proc, 2012. 87 (1): p. 25-33. 2. Pardanani, A., et al., Update On The Long-Term Efficacy and Safety Of Momelotinib, a JAK1 and JAK2 Inhibitor, For The Treatment Of Myelofibrosis. Vol. 122. 2013. 108-108. 3. Verstovsek, S., et al., Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med, 2010. 363 (12): p. 1117-27. 4. Verstovsek, S., et al., A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012. 366 (9): p. 799-807. 5. Nemeth, E., et al., Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 2004. 306 (5704): p. 2090-3. 6. Theurl, I., et al., Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease. Blood, 2006. 107 (10): p. 4142-8. 7. Theurl, I., et al., Autocrine formation of hepcidin induces iron retention in human monocytes. Blood, 2008. 111 (4): p. 2392-9. 8. Pardanani, A., et al., Associations and prognostic interactions between circulating levels of hepcidin, ferritin and inflammatory cytokines in primary myelofibrosis. Am J Hematol, 2013. 88 (4): p. 312-6. Disclosures Warr: Gilead Science: Employment. Maciejewski:Gilead Science: Employment. Fowles:Gilead Science: Employment. Whitney:Gilead Sciences: Employment. Theurl:Gilead Science: Research Funding. </jats:sec

Abstract C38: Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia

Abstract Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients' cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients' cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.</jats:p

New Allosteric Inhibitors of Mutant IDH1 in Acute Myeloid Leukemia

Abstract Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival". Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers. Disclosures Gao: GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment. </jats:sec