An Enhanced Sensitivity DNA Sequencing Protocol for the Detection in AML of Measurable Residual Disease (MRD) Applicable for All Mutations

Abstract Background: One of the more important prognostic factors used to predict the outcome in acute myeloid leukemia (AML) is the persistence of leukemic cells after treatment. The reliable measurement of residual disease (MRD) offers many other clinical uses besides. An assay that was facile, affordable, and applicable to the broadest group of patients would find immediate favor. Next-generation sequencing (NGS) combined with various enrichment methods allow high sequencing depth on restricted targets. For instance, by enriching for 50% of the genomic diversity of that input DNA was preserved, i.e., >15,000 unique genomes were captured and sequenced from an initial 30,000 genomes resulting in a LOD of 0.1% with high precision as a mutation present at 0.1% in 15,000 genomes could be observed ~15 times. As shown in Table 1B, this method scales (non-linearly) as input DNA is increased; samples containing 500ng of DNA (~150,000 genomes) reproducibly permit an LOD of 0.01%, a sensitivity far exceeding any other non-allele specific method for measuring residual disease. In a real world application, we followed 3 AML patients from diagnosis to clinical CR. We tracked all mutations present at diagnosis using both IDT hybrid capture (HC) and the ThermoFisher (TF) method. One patient had molecular evidence of residual disease (VAF3%) that was detected and similarly quantitated by both HC and TF, in the second patient mutation were present at 1% by HC and 3% by TF . The TF value is the reliable one as it is derived from multiple independent templates. Finally the third patient was in molecular remission by HC but had a mutation allele detected by the TF with a frequency of 0.07%. We have extended these studies to include more than a dozen patients followed through relapse with similar results. These pilot studies provide clear evidence that standard sequencing methods cannot reliably promise LODs below 1%; further, the ThermoFisher method enhances the LOD at least 20-fold in an assay that can be applied to the great majority of AML patients. Table 1. Table 1. Disclosures Rienhoff: Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Natsoulis:Imago BioSciences, Inc.: Consultancy, Equity Ownership. Jones:Imago BioSciences, Inc.: Employment, Equity Ownership. Peppe:Imago BioSciences, Inc.: Employment, Equity Ownership. Cao:Thermo Fisher Scientific: Employment. Hanif:Thermo Fisher Scientific: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding

The Small Molecule Img-98, a Potent and Selective Inhibitor of the Lysine Demethylase Lsd-1, Effectively Augments the Pro-Differentiation Effects of ATRA in a Pre-Clinical Model of AML

Abstract Acute Promyelocytic Leukemia (APL) is a cytogenetically unique subtype of acute myeloid leukemia (AML), characterized by the presence of the t(15;17)-associated PML-RARA fusion gene. This disease is curable in most patients with all-trans-retinoic acid (ATRA) based therapies, which effectively differentiate malignant promyelocytes. In patients with non-APL AML, most patients with die from their disease and ATRA has little activity. Therefore, research strategies that seek to extend the efficacy of ATRA-based treatment in AML are key avenues of investigation. From our previous studies, an epigenetic analysis of primary AML samples revealed that relative to normal CD33+ cells, loss of RARα2 expression in AML is associated with a reduction in H3K4me2 on the RARA2 promoter (a modification that is associated with transcriptional activation). The mono- and di-methyl lysine demethylase LSD1 (KDM1A) is highly expressed in patients with AML, and its overexpression has been implicated in various other tumors. Based on these data we correctly predicted that the use of small-molecule inhibitors targeting LSD1 (LSD1i) could result in epigenetic reprogramming that enhanced or facilitated the execution of the ATRA-induced differentiation program in AML cells. In the current study, we characterized a range of small molecule inhibitors of LSD-1. All the agents tested (RN-1, GSKi, SP2509, TCP, IMG-98 and OG-L002) led to inhibition of LSD-1 in a biochemical assay with varying degrees of potency. From this study, we further characterized the anti-tumor effects of IMG-98 alone and in combination with ATRA. IMG-98 is a novel LSD1 inhibitor relative to drugs of this class with comparatively different specificity, potency, pharmacokinetics, and metabolism. Its greater heavy atom count and chemical complexity contribute to these properties. By fluorine nuclear magnetic resonance (fNMR) and florescent spectrophotometry, the molecule rapidly reacts irreversibly with the FAD co-factor of LSD1 and this polypeptide is necessary to catalyze the reaction. Thermal stability shifts show the inactivated form of the enzyme becomes much more stable suggesting significant structural changes. Treatment with IMG-98 promoted the expression of the cell surface marker CD11b, associated with a differentiated immunophenotype, in both AML cell lines and primary patient material. IMG-98 produced a potent anti-proliferative effect across a range of AML cell lines and also led to growth inhibition of AML blast colony forming ability. In combination studies with ATRA, IMG-98 re-sensitized AML cells to ATRA by reactivating ATRA driven differentiation programs. Post-differentiation apoptosis was more significant for combined therapy (ATRA + IMG-98) than with either agent alone. Heatmap display of unsupervised hierarchical clustering of genes in AML cell lines differentially expressed in response to treatment with combinations of ATRA, IMG-98 or the combination, confirmed that ATRA combined with IMG-98 enhanced the expression of a subset of genes associated with the myeloid differentiation program. Updated studies on mechanisms underpinning mode of action of IMG-98 in this model will be presented. Taken together, these data demonstrate that ATRA combined with pharmacological inhibition of LSD1, may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. A closely related molecule to IMG-98 is currently being optimized in late preclinical development, and clinical trials with this compound are anticipated to start in 2016. Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Disclosures Rienhoff: Imago: Employment

Chemical modulation of Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) induces wide-scale biological and epigenomic changes

Background: Schistosoma mansoni, a parasitic worm species responsible for the neglected tropical disease schistosomiasis, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic and epigenetic processes. As inhibition of S. mansoni epigenetic machinery components impairs key transitions throughout the parasite’s digenetic lifecycle, a greater understanding of how epi-drugs affect molecular processes in schistosomes could lead to the development of new anthelmintics. Methods: In vitro whole organism assays were used to assess the anti-schistosomal activity of 39 Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors on different parasite life cycle stages. Moreover, tissue-specific stains and genomic analysis shed light on the effect of these small molecules on the parasite biology. Results: Amongst this collection of small molecules, compound 33 was the most potent in reducing ex vivo viabilities of schistosomula, juveniles, miracidia and adults. At its sub-lethal concentration to adults (3.13 µM), compound 33 also significantly impacted oviposition, ovarian as well as vitellarian architecture and gonadal/neoblast stem cell proliferation. ATAC-seq analysis of adults demonstrated that compound 33 significantly affected chromatin structure (intragenic regions > intergenic regions), especially in genes differentially expressed in cell populations (e.g., germinal stem cells, hes2+ stem cell progeny, S1 cells and late female germinal cells) associated with these ex vivo phenotypes. KEGG analyses further highlighted that chromatin structure of genes associated with sugar metabolism as well as TGF-beta and Wnt signalling were also significantly perturbed by compound 33 treatment. Conclusions: This work confirms the importance of histone methylation in S. mansoni lifecycle transitions, suggesting that evaluation of LSD1 - targeting epi-drugs may facilitate the search for next-generation anti-schistosomal drugs. The ability of compound 33 to modulate chromatin structure as well as inhibit parasite survival, oviposition and stem cell proliferation warrants further investigations of this compound and its epigenetic target SmLSD1

Safety, Tolerability and Dose Response of FBS0701, a Novel Iron Chelator for Treatment of Transfusional Iron Overload: Results of a 24-Week Multicenter, International Phase 2 Study

53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH)/Symposium on the Basic Science of Hemostasis and Thrombosis -- DEC 10-13, 2011 -- San Diego, CAWOS: 000299597100691Amer Soc Hematol (ASH

A public resource facilitating clinical use of genomes

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review boardapproved "open consent" process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10).We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain - we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.close555