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Despite routine use of DNA-hypomethylating agents (HMAs) in AML/MDS therapy, their mechanisms of action are not yet unraveled. Pleiotropic effects of HMAs include global methylome and transcriptome changes. We asked whether in blasts and T-cells from AML patients HMA-induced in vivo demethylation and remethylation occur randomly or non-randomly, and whether gene demethylation is associated with gene induction. Peripheral blood AML blasts from patients receiving decitabine (20 mg/m2 day 1–5) were serially isolated for methylome analyses (days 0, 8 and 15, n = 28) and methylome-plus-transcriptome analyses (days 0 and 8, n = 23), respectively. T-cells were isolated for methylome analyses (days 0 and 8; n = 16). We noted massive, non-random demethylation at day 8, which was variable between patients. In contrast, T-cells disclosed a thousand-fold lesser, random demethylation, indicating selectivity of the demethylation for the malignant blasts. The integrative analysis of DNA demethylation and transcript induction revealed 87 genes displaying a significant inverse correlation, e.g. the tumor suppressor gene IFI27, whose derepression was validated in two AML cell lines. These results support HMA-induced, non-random early in vivo demethylation events in AML blasts associated with gene induction. Larger patient cohorts are needed to determine whether a demethylation signature may be predictive for response to this treatment.

Supplementary Information: Supplementary Figures 1-11, Supplementary Figure Legends, Supplementary Tables 1-4

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3–1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3–1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group.Significance:These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813

Aberrant DNA methylation plays a pivotal role in tumor development and progression. DNA hypomethylating agents (HMA) constitute a class of drugs which are able to reverse DNA methylation, thereby triggering the re-programming of tumor cells. The first-generation HMA azacitidine and decitabine have now been in standard clinical use for some time, offering a valuable alternative to previous treatments in acute myeloid leukemia and myelodysplastic syndromes, so far particularly in older, medically non-fit patients. However, the longer we use these drugs, the more we are confronted with the (almost inevitable) development of resistance. This review provides insights into the mode of action of HMA, mechanisms of resistance to this treatment, and strategies to overcome HMA resistance including next-generation HMA and HMA-based combination therapies.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3–1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3–1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. Significance: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes. See related commentary by O'Hagan et al., p. 813

Background Repeat elements constitute a large proportion of the human genome and recent evidence indicates that repeat element expression has functional roles in both physiological and pathological states. Specifically for cancer, transcription of endogenous retrotransposons is often suppressed to attenuate an anti-tumor immune response, whereas aberrant expression of heterochromatin-derived satellite RNA has been identified as a tumor driver. These insights demonstrate separate functions for the dysregulation of distinct repeat subclasses in either the attenuation or progression of human solid tumors. For hematopoietic malignancies, such as Acute Myeloid Leukemia (AML), only very few studies on the expression/dysregulation of repeat elements were done. Methods To study the expression of repeat elements in AML, we performed total-RNA sequencing of healthy CD34 + cells and of leukemic blast cells from primary AML patient material. We also developed an integrative bioinformatic approach that can quantify the expression of repeat transcripts from all repeat subclasses (SINE/ALU, LINE, ERV and satellites) in relation to the expression of gene and other non-repeat transcripts (i.e. R/G ratio). This novel approach can be used as an instructive signature for repeat element expression and has been extended to the analysis of poly(A)-RNA sequencing datasets from Blueprint and TCGA consortia that together comprise 120 AML patient samples. Results We identified that repeat element expression is generally down-regulated during hematopoietic differentiation and that relative changes in repeat to gene expression can stratify risk prediction of AML patients and correlate with overall survival probabilities. A high R/G ratio identifies AML patient subgroups with a favorable prognosis, whereas a low R/G ratio is prevalent in AML patient subgroups with a poor prognosis. Conclusions We developed an integrative bioinformatic approach that defines a general model for the analysis of repeat element dysregulation in physiological and pathological development. We find that changes in repeat to gene expression (i.e. R/G ratios) correlate with hematopoietic differentiation and can sub-stratify AML patients into low-risk and high-risk subgroups. Thus, the definition of a R/G ratio can serve as a valuable biomarker for AML and could also provide insights into differential patient response to epigenetic drug treatment.

Key Points mRNA decapping gene EDC4 is a novel fusion partner of MLL in AML. Genes functioning in mRNA decapping may compose a distinct group of MLL fusion partners that links MLL function with mRNA decapping in AML.

DNA methyltransferase inhibitors (DNMTi) approved for older AML patients are clinically tested in combination with histone deacetylase inhibitors (HDACi). The mechanism of action of these drugs is still under debate. In colon cancer cells, 5-aza-2'-deoxycytidine (DAC) can downregulate oncogenes and metabolic genes by reversing gene body DNA methylation, thus implicating gene body methylation as a novel drug target. We asked whether DAC-induced gene body demethylation in AML cells is also associated with gene repression, and whether the latter is enhanced by HDACi.Transcriptome analyses revealed that a combined treatment with DAC and the HDACi panobinostat or valproic acid affected significantly more transcripts than the sum of the genes regulated by either treatment alone, demonstrating a quantitative synergistic effect on genome-wide expression in U937 cells. This effect was particularly striking for downregulated genes. Integrative methylome and transcriptome analyses showed that a massive downregulation of genes, including oncogenes (e.g., MYC) and epigenetic modifiers (e.g., KDM2B, SUV39H1) often overexpressed in cancer, was associated predominantly with gene body DNA demethylation and changes in acH3K9/27. These findings have implications for the mechanism of action of combined epigenetic treatments, and for a better understanding of responses in trials where this approach is clinically tested.

An amendment to this paper has been published and can be accessed via the original article.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment

Lysine‐specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site‐specific LSD1 inhibition, we developed an enzyme‐prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1‐NTR+. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2‐nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild‐type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches., Site‐specific targeting: Nitroaromatic prodrugs were activated by the bacterial nitroreductase NfsB (NTR) to selectively inhibit lysine‐specific demethylase 1 (LSD1) in transfected leukaemic cells. The effects of LSD1 inhibition and cytotoxicity of the side products were distinguished by negative controls. This system may be applied in targeting approaches to reach site‐specific LSD1 inhibition.

Rodent models have contributed significantly to the understanding of haematological malignancies. One important model system in this context are patient-derived xenografts (PDX). In the current study, we examined 20 acute leukaemia PDX models for growth behaviour, infiltration in haemopoietic organs and sensitivity towards cytarabine. PDX were injected intratibially (i.t.), intrasplenicaly (i.s.) or subcutaneously (s.c.) into immune compromised mice. For 18/20 models the engraftment capacity was independent of the implantation site. Two models could exclusively be propagated in one or two specific settings. The implantation site did influence tumour growth kinetics as median overall survival differed within one model depending on the injection route. The infiltration pattern was similar in i.t. and i.s. models. In contrast to the s.c. implantation, only one model displayed circulating leukaemic cells outside of the locally growing tumour mass. Cytarabine was active in all four tested models. Nevertheless, the degree of sensitivity was specific for an individual model and implantation site. In summary, all three application routes turned out to be feasible for the propagation of PDX. Nevertheless, the distinct differences between the settings highlight the need for well characterized platforms to ensure the meaningful interpretation of data generated using those powerful tools.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Background: DNA-hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/del(7q) embedded in a complex, monosomal karyotype (MK). We have noted that MK pts with multiple monosomies responded better to DAC treatment than pts with only one monosomal autosome (Blum, Greve and Lübbert, Curr Opin Hematol. 2017). This important characteristic of HMAs differentiates them from cytarabine (AraC, lacking DNMT-inhibitory activity) as shown in randomized studies with DAC (Wierzbowska et al., Am. J. Hematology 2018) and AZA (Döhner et al., Leukemia 2018). The mechanism of action is currently under investigation and may involve preferential gene reactivation on monosomic chromosomes by DAC (Greve et al., ASH Annual Meeting 2017, #2612). In order to address this important difference between DAC and AraC, we compared their antileukemic activity in PDX models representing cytogenetically normal (CN) AML and adverse cytogenetics AML (complex, monosomal karyotypes including del(7q) and 1 or more monosomies). Materials and Methods: PDX models were generated for 5 CN and 3 MK AML pts with a median age of 68 years (range 55-80), a median WBC of 3.0 x 103/µl (range 1.4-139.7) and a median of 42% bone marrow (BM) blasts (range 5-97). 7/8 pts were sampled at diagnosis, i.e. prior to first-line treatment (3 pts received DAC, 3 pts induction and 1 pt upfront allografting); 1/8 pt had had multiple relapses. T-cell depleted peripheral blood or bone marrow cells (3x106 cells/mouse) were injected into NSG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in BM, peripheral blood (PB) and spleen during the course of the experiment and at the end of a study. Leukemic cells were transplanted serially at least 4-5 times to propagate and ensure full leukemic potential of the cells. Mice were treated with low-toxic concentrations of DAC (1 mg/kg/day given intraperitoneally), AraC (15 mg/kg/day given intravenously) or vehicle (PBS) for 5 consecutive days (one cycle). Drug doses were titrated to be equitoxic and as potent as possible without causing side effects. Treatment commenced upon disease onset, as evaluated by FC positivity for human CD33, increased WBC in PB and weight loss. Overall survival served as the read-out. The study was carried out in accordance with the recommendations by the Society of Laboratory Animal Science in an AAALCA accredited animal facility. The animal experiments were approved by the regional council (Regierungspräsidium Freiburg, ref. 35, permit no. G-12/86). Results: We successfully established well-characterized PDX from pts with CN (n = 5), and with MK (n = 3) with a median of 2 autosomal monosomies (range 1-6; of chromosomes 4, 6, 7 [2x], 15, 17 [2x], 18, 21). TP53 (mutational status available for 6/8 pts) was wildtype in 4 CN pts and 1 MK pt, and mutated in 1 MK pt. All models were treated with either DAC, AraC or vehicle. Across both cytogenetic cohorts, DAC treatment significantly prolonged median survival by 36 days when compared to vehicle administration (p = 0.0002, see Fig 1A). Specifically, DAC increased median survival of CN PDX by 33 days (p = 0.0104, range 61-105 days with DAC, compared to 43-65 days with vehicle, Fig 1B), and in MK PDX by 40 days (p = 0.0197; range 72-105 days with DAC, compared to 39-50 days with vehicle, Fig 1C). Notably, disease progression in two DAC-treated PDX (one CN, one MK with 6 monosomies) was delayed to 105 days (Fig 1, marked with ⋆). AraC treatment also resulted in longer median survival across both cohorts compared to vehicle, albeit not as pronounced as with DAC, by an average of 20 days (p = 0.0066). In more detail, AraC increased median survival of CN PDX by 19 days (p = 0.0025, range 53-82 days), and, though not significantly, of MK PDX by 15 days (p = 0.2048, range 46-92 days). Conclusion: In this PDX study recapitulating two different cytogenetic risk groups of AML, DAC showed encouraging activity also in models with complex, monosomal karyotype, even in the presence of 6 autosomal monosomies, and was superior to AraC in extending survival. These data lend further support to the treatment of such adverse genetics AML patients with HMAs. Disclosures No relevant conflicts of interest to declare.

Non-small cell lung cancer (NSCLC) still constitutes the most common cancer-related cause of death worldwide. All efforts to introduce suitable treatment options using chemotherapeutics or targeted therapies have, up to this point, failed to exhibit a substantial effect on the 5-year-survival rate. The involvement of epigenetic alterations in the evolution of different cancers has led to the development of epigenetics-based therapies, mainly targeting DNA methyltransferases (DNMTs) and histone-modifying enzymes. So far, their greatest success stories have been registered in hematologic neoplasias. As the effects of epigenetic single agent treatment of solid tumors have been limited, the investigative focus now lies on combination therapies of epigenetically active agents with conventional chemotherapy, immunotherapy, or kinase inhibitors. This review includes a short overview of the most important preclinical approaches as well as an extensive discussion of clinical trials using epigenetic combination therapies in NSCLC, including ongoing trials. Thus, we are providing an overview of what lies ahead in the field of epigenetic combinatory therapies of NSCLC in the coming years.

Background: The therapeutic effect of DNA-hypomethylating agents (HMAs) in AML/MDS is discussed to be via its effects on aberrant gene silencing by reactivation (e.g. through promoter hypomethylation). While this has been broadly studied in cell line models, only very few studies have addressed the global effects of HMAs in primary blasts serially isolated from AML patients (pts) undergoing HMA treatment (Claus et al., Leuk. Res. 2013, Klco et al., Blood 2013, Welch et al., N. Engl. J. Med. 2016). We therefore conducted prospective serial methylome and transcriptome analyses on AML blasts from pts of the DECIDER trial (NCT00867672), hypothesizing that both random and non-random effects of the HMA may be observed in vivo. Patients, Materials and Methods: Of a total of 200 newly diagnosed AML pts included into the DECIDER randomized phase II trial (Decitabine/DAC treatment, 20 mg/m2 intravenous 1-hour infusion over 5 days, with add-on drugs Valproic acid and/or ATRA added at day 6; Grishina et al., BMC Cancer 2015), serially obtained peripheral blood (pb) samples from a total of 28 pts yielded sufficient numbers of purified blasts at 3 timepoints (days 0, 8 and 15 from DAC treatment start) to allow a "triplet analysis" of these matched samples. Baseline pt characteristics: median WBC 11,900/µl (range 1,200 - 53,800), median pb blasts 37.5% (range 1% - 93%). Blasts were sorted using anti-CD34, CD117 MACS microbeads, respectively (median purity >90%). Methylomes were obtained using Infinium Human Methylation 450 BeadChip arrays (Illumina). For expression analyses, GeneChip Human Gene 2.0 ST arrays were used. A linear-model based approach was used to identify the differentially methylated CpGs and expressed genes post vs. prior to treatment. Results: To address in vivo methylation changes occurring at day 8 and 15 from DAC treatment start, complete "triplets" of DNA preparations (thus from purified pb blasts of all 3 time points) were interrogated. Significant hypomethylation at day 8 (Δβ Conclusions: In our study, a subset of genes was specifically targeted by hypomethylation in all pts, arguing against a completely random effect of this treatment on the methylome. Only a limited number of hypomethylating events was associated with gene reactivation. A better understanding of hypo- and remethylation kinetics in vivo may aid in schedule optimization of HMA therapy. Correlative studies on hypomethylation kinetics and treatment outcome in the DECIDER trial are therefore ongoing, as well as serial methylome analyses in the EORTC trial AML21 ("inDACtion vs. induction", NCT02172872). Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Döhner:Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria. Lübbert:Cheplapharm: Other: Study drug; Janssen: Honoraria, Research Funding; TEVA: Other: Study drug; Celgene: Other: Travel Support.

The aim of this study was to determine the influence of the engraftment site on the tumor biology and drug sensitivity of a panel of hematological patient derived xenografts (PDX). PDX cells (3x10e6 cells/mouse) were injected intratibialy (i.t.), intrasplenal (i.s.) or subcutaneously (s.c.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice. Tumor engraftment was determined by flow cytometry (FC) in bone marrow (BM), peripheral blood (PB) and spleen during the course of engraftment and at the end of a study. Overall survival (OS) served as an additional read-out. In 3 models sensitivity towards cytarabine (Cy) was evaluated. Our group has established 18 PDX of acute leukemia (16 AML, 1 ALL, 1 APL). 16/17 lines engrafted when injected i.t., 10/12 developed tumors after i.s. implantation and 13/14 established tumors post s.c. cell injection. Thus, the overall engraftment capacity was for most of the models not depending on the injection site. Nevertheless, some models could only be propagated in a specific setting: LEXFAM 2713, grew exclusively i.t. or i.s., whereas LEXFAM 2824 could be propagated solely when injected s.c.. The implantation site did influence tumor growth rate: Mean OS ranged from 151.4 (±25.21) days for i.t. to 89.2 (±16.82) days for s.c. propagation. I.s. transplanted mice had to be sacrificed after 91.9 (±17.33) days. The dissemination pattern of individual lines was affected by the injection site. In general, infiltration of the hematopoietic organs was higher when cells were engrafted i.t. or i.s.. Nevertheless, also s.c. implanted, AML cells infiltrated murine PB, spleen and BM, although not consistently and to a much lower extent. Interestingly, the expression pattern of the 6 investigated surface markers (CD45, CD3, CD34, CD33, CD38 & HLA-ABC) was not influences by the application route. Every model depicted its distinct expression pattern irrespective of the application route. Cy was highly active in 1 AML (LEXFAM 2531) and 1 ALL (LEXFAL 2665) model. OS was significantly prolonged in the s.c. as well as in the disseminated setting (p< 0.003, Log-rank (Mantel-Cox) test). Another AML line (LEXFAM 2734) depicted a less pronounced sensitivity towards Cy (p< 0.007, Log-rank (Mantel-Cox) test) both growing s.c. or i.t.. Thus, drug sensitivity was not influenced by the injection site of the leukemic cells. Of note, the PDX drug responses mimicked the responsiveness of the respective donor patient. Taken together the leukemia PDX panel represents the molecular diversity of the disease and mirrors sensitivity towards standard of care. Our recent careful investigation of the subcutaneous approach in these models highlights their suitability for this type of studies. This enhances the value of the platform as it combines the possibilities of a mid-throughput screening enabled by the subcutaneous approach with the general advantages of a patient-derived leukemia model. Citation Format: Julia B. Schüler, Eva Oswald, Gabriele Greve, Dorothee Lenhard, Kerstin Klingner, Milena Pantic, Michael Luebbert. Influence of the injection site on the dissemination pattern and drug sensitivity of patient derived leukemia cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4821. doi:10.1158/1538-7445.AM2017-4821

Background: Translocations involving the MLL gene located on chromosome 11q23 are usually found in de novo acute myeloid leukemia (AML) and generally confer a dismal prognosis unless the AF9 gene is involved (Döhner et al., Blood 2010;115:453-74). MLL can be fused to multiple different genes, resulting in the large and growing "MLL recombinome" (Meyer et al., Leukemia 2013;27:2165-76). Thus far, only two genes encoding proteins that are part of the mRNA decapping protein complex (i.e., DCP1A, DCPS) have been described as rarely being fused to MLL. Here, we describe an AML with an indolent disease course arising from myelodysplastic syndrome (MDS) that disclosed a unique MLL fusion with another component of the mRNA decapping complex, i.e., EDC4. Patient and Methods: Briefly, a 55 year-old female patient presented with an MDS [timepoint (t) -1] that within 10 months progressed to an AML with 2% blood and 40% bone marrow myeloblasts (t0). The patient refused treatment beyond supportive care. Six months later, a marked blast expansion of 80% was detectable in the blood (t1). The patient received 5 cycles of decitabine (t2, cycle 5), followed by 3 months of hydroxyurea (t3). Samples were depleted from CD3+ cells via MACS; CD3+ cells served as germline control. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), and the SureSelect Human All Exon v5 kit (Agilent Technologies) was used for exome capturing from gDNA. Next generation sequencing was performed on an Illumina Hiseq 2500. The analyses were performed as previously described (Kataoka et al. Nature Genetics 2015;47:1304-15, Becker et al. Blood 2014;123:1883-6). NOD scid gamma mice were used as hosts for patient derived tumor xenografts (PDX). Results: Standard metaphase cytogenetics at the diagnosis of AML (t0) revealed a previously undescribed translocation involving the MLL gene, i.e., t(11;16)(q23;q22), as the sole cytogenetic abnormality. The unknown fusion partner on chromosome 16 was identified by RNA-sequencing as the EDC4 gene (a.k.a. Ge-1), which encodes a key scaffold protein of the mRNA decapping complex; the fusion was confirmed by PCR on cDNA. The translocation led to the in-frame fusion of MLL exon 13 to EDC4 exon 6 which was linked by 19 nucleotides from EDC4 intron 5. The predicted amino acid sequence of the linker was ALNTLLR. Further analyses including exome sequencing on the samples collected over the disease course demonstrated STAG2 as a potential founder mutation that was already present during the MDS (t-1) and persisted throughout the disease course at variant allele frequencies (VAFs) of approximately 45-50%. At the time of transformation to AML (t0), the MLL-EDC4 and two RAS mutations (KRAS p.G13D, NRAS p.G12C) were detectable. Towards the terminal phase (t3), the RAS mutations disappeared and a clone that acquired a mutation in the FLT3 tyrosine kinase domain (TKD; p.D835V; VAF 43%) expanded. Primary blasts from the patient engrafted in NOD scid gamma mice and established a stable PDX serial transplantation mouse model used for drug testing. Conclusions: This report provides the first demonstration of an MLL-EDC4 in-frame fusion, with potential cooperativity with a founder mutation in the STAG2 splicing factor gene during the transformation of MDS to AML and the additional acquisition of a FLT3-TKD mutation during disease progression. RNA sequencing proved to be a very feasible approach to identify novel fusion partners of known oncogenes such as MLL. Disclosures Becker: BMS: Honoraria; Novartis: Honoraria. Kataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Schüler:Oncotest GmbH: Employment. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid.

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.

Many efforts are made to treat acute leukemia effectively, but still; new treatment options are urgently needed. In the past, mainly chemically induced and cell line-derived mouse models were used to evaluate the efficacy of new drugs. The high failure rate of 96% in early clinical development illustrates the urgent need for more predictive preclinical models as a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. Transplantable patient-derived xenograft (PDX) models of leukemia offer a strong preclinical tool for drug screening and biomarker development as they represent the complex clinical tumor heterogeneity and molecular diversity of human cancer. Between 2011 and 2015 we collected 91 samples of peripheral blood (PB) or bone marrow (BM) from patients diagnosed with acute myeloid or lymphoblastic leukemia (AML, ALL) and injected them intratibially (i.t.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice (n=1-5/patient sample). Infiltration of human leukemic cells was determined by flow cytometry in murine PB, BM and spleen. If more than 5% of hCD45+, hCD33+, hCD34+, hCD38+, and/or HLA-ABC+ cells were detectable in one sample, it was classified as engrafted. Results were confirmed by histo-pathological examination. FISH analyses confirmed the cytogenetical concordance with the donor patient where available. 8 models were treated with cytarabine and results were compared to patient´s outcome and treatment experiments using cell line derived models of AML. Whole exome sequencing analyses of the transplantable models are underway for a deeper characterization of the respective leukemic clone which adapted to the murine microenvironment. Specimens of 44 female and 47 male patients (median age: 59 years, median BM infiltration: 39%) were collected. The donor patient cohort covered a broad range of different molecular subtypes: amongst others 5 MLL-rearrangements, 1 MLL-Deletion, 2 t(8;21) translocations, 20 cytogenetically normal and 7 complex karyotypes were included. Up to now 12 PDX models were in passage (P) 5 and higher, where P1 represents first implantation of human cells into the murine BM. 15 PDX models grew in P3 - P4, 21 in P2, 36 in P1 and 7 samples showed no engraftment. Engraftment capacity of the leukemic cells did not correlate significantly with any of the patient characteristics. BM engraftment ranged from 30% - 80%. Spleen and PB depicted 5 - 30% of leukemic cells. Infiltration rate in different organs and immuno-phenotypic characteristics of the human cells were specific for a defined model and preserved during serial transplantation. Take rates within one mouse cohort in serial transplantation were ≥98% for all transplantable PDX, similar to cell line derived models thereby qualifying the PDX approach as a suitable preclinical platform. Overall survival (OS) in P1 ranged from 52 to > 310 days (d). Models which could be serially transplanted showed model specific median OS ranging from 32 - 150 d. Comparable cell line derived models depicted median OS of 13 - 45 d. Of note, cell line derived models (KG-1, NOMO-1, MOLM-13, THP-1, MV4-11 or HL60, n ≥ 10 mice/cell line) induced hind limb paresis in all recipient mice. These symptoms could not be observed in PDX and most important are not part of the clinical picture. Cytarabine induced a significantly prolonged OS in 8/8 tested PDX models. Respective donor patients showed hematologic response under cytarabine based therapy highlighting the excellent predictivity of the in vivo platform. In contrast none of the investigated cell line derived models showed sensitivity towards cytarabine, although representing similar subtypes of AML as the investigated PDX models. Taken together a constantly expanding panel of well characterized AML/ALL PDX covering a broad range of different subtypes of the disease is available for drug development and tumorbiology studies. The comparison with cell line derived in vivo models revealed significant advantages of the PDX approach as the latter represents the molecular diversity more in detail, mimics the clinical signs of leukemia more realistic and most important mirrors sensitivity towards standard of care in a direct comparison with the donor patient´s clinical outcome. Therefore, we strongly believe that the AML/ALL PDX platform is a robust and predictive tool to address translational challenges in oncology research. Disclosures Oswald: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Schueler:Oncotest GmbH: Employment.

BACKGROUND: We have previously demonstrated that the Y-specific ampliconic fertility genes DAZ (deleted in azoospermia) and CDY (chromodomain protein Y) varied with respect to copy number and position among chimpanzees (Pan troglodytes). In comparison, seven Y-chromosomal lineages of the bonobo (Pan paniscus), the chimpanzee's closest living relative, showed no variation. We extend our earlier comparative investigation to include an analysis of the intraspecific variation of these genes in gorillas (Gorilla gorilla) and orangutans (Pongo pygmaeus), and examine the resulting patterns in the light of the species' markedly different social and mating behaviors. METHODOLOGY/PRINCIPAL FINDINGS: Fluorescence in situ hybridization analysis (FISH) of DAZ and CDY in 12 Y-chromosomal lineages of western lowland gorilla (G. gorilla gorilla) and a single lineage of the eastern lowland gorilla (G. beringei graueri) showed no variation among lineages. Similar findings were noted for the 10 Y-chromosomal lineages examined in the Bornean orangutan (Pongo pygmaeus), and 11 Y-chromosomal lineages of the Sumatran orangutan (P. abelii). We validated the contrasting DAZ and CDY patterns using quantitative real-time polymerase chain reaction (qPCR) in chimpanzee and bonobo. CONCLUSION/SIGNIFICANCE: High intraspecific variation in copy number and position of the DAZ and CDY genes is seen only in the chimpanzee. We hypothesize that this is best explained by sperm competition that results in the variant DAZ and CDY haplotypes detected in this species. In contrast, bonobos, gorillas and orangutans-species that are not subject to sperm competition-showed no intraspecific variation in DAZ and CDY suggesting that monoandry in gorillas, and preferential female mate choice in bonobos and orangutans, probably permitted the fixation of a single Y variant in each taxon. These data support the notion that the evolutionary history of a primate Y chromosome is not simply encrypted in its DNA sequences, but is also shaped by the social and behavioral circumstances under which the specific species has evolved.

The generation of functional and reproducible in vivo models is a prerequisite for a better understanding of hematological malignancies and for the development of improved therapeutic strategies. Here, we report the establishment of transplantable PDX models of acute myeloid and lymphoid leukemia (AML, ALL) growing subcutaneously (s.c.) as well as in a disseminated fashion in immunocompromised mice. Bone marrow (BM) and peripheral blood (PB) cells from 25 AML/ALL patients were injected intratibially into NSG or NOG mice (n = 1-9/patient) and in later passages also s.c. in nude mice (n = 1-3/model). Tumor growth was monitored via determination of overall survival, flow cytometry (verification of patient-derived leukemic clones by hCD34, hCD33, hCD38, hHLA-ABC, hCD3, hCD45, mCD45), and, where applicable, by caliper measurement. Engraftment is defined as ≥5% hCD45+ cells in the BM and/or ≥1% hCD45+ cells in PB. Transplantable models (higher or equal passage 2) were tested with the respective standard of care drugs (SoC) mainly Cytarabine and Dexamethasone. Bone marrow engraftment in passage 1 was observed for 11 out of 25 leukemia models. Four of them could be established as disseminated models in serial passage. No engraftment was detected for nine models during the observation period of at least 95 days while observation for five models is still ongoing. The four established models were further characterized by fluorescence-in-situ-hybridization (FISH) to confirm patient origin. Treatment of mice bearing the models with the SoC drugs cytarabine and dexamethasone significantly prolonged overall survival. Antitumoral activity was consistent with clinical response of the donor patient. One T-ALL and two AMLs were additionally successfully propagated following s.c. injection in nude mice. They showed stable growth behavior and displayed a similar chemo-sensitivity pattern as their disseminated growing counterparts. In conclusion, we have established a panel of transplantable PDX models of ALL and AML displaying stable and reproducible growth patterns suitable for in vivo efficacy studies. Our results confirm that PDX models of hematological malignancies replicate important clinical characteristics of the disease and are valuable tools for preclinical drug testing. Citation Format: Eva Oswald, Kerstin Klingner, Benedikt Hammerich, Gabriele Greve, Dorothee Lenhard, Milena Pantic, Heinz-Herbert Fiebig, Michael Luebbert, Julia B. Schüler. Patient-derived models of human acute myeloid and lymphoid leukemia in immunocompromised mice for preclinical drug development. [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 4210. doi:10.1158/1538-7445.AM2015-4210

Introduction: All-trans retinoic acid (ATRA, RA) has powerful activity in acute promyelocytic leukemia (APL); its efficacy in non-APL acute myeloid leukemia (AML) is still unclear, but may be enhanced by epigenetic drugs such as azanucleoside DNMT inhibitors (Blagitko-Dorfs et al. PLoS ONE 2013). In a randomized phase II study (DECIDER trial, NCT00867672) the addition of RA to decitabine (DAC) in newly diagnosed non-fit older AML patients resulted in a clinically meaningful extension of survival. We hypothesize that the add-on of RA to DAC results in cooperative transcriptome changes (possibly associated with demethylation) which may, at least in part, explain these clinical results. Materials and Methods: U937 cells were treated in triplicates with daily pulses of 200 nM DAC, 1 µM RA was administered after 48 hours (hr). Cells were harvested after 72 and 120 hr of treatment. Proliferation and apoptosis were determined by acridine orange / propidium iodide staining. For the colony formation assay (CFA), 100 cells were seeded after 120 hr treatment in methylcellulose, colonies were counted after 21 days. Stranded cDNA libraries were sequenced on an Illumina HiSeq2000 with 60 million 125 bp paired-end reads. Read quality was checked with FastQC (per base Q-score >38), alignment to the GencodeCompV24 reference genome with RNAStar, generation of count matrices with ht-seq and differential expression testing with DeSeq2. Transcript changes were considered significant with an adjusted FDR p-value < 0.01. GO analysis was performed with Metascape. Methylomes were generated using Infinium Human Methylation 450K BeadChip arrays and differential methylation data was obtained using the RnBeads package for R (Assenov et al. Nature Methods 2014). Results: In suspension culture, RA alone had no effect on the proliferation of U937 cells, DAC alone reduced proliferation by 74 %, the DAC+RA combination by 81 % (with viability always ≥ 90 %). RA alone also had no effect on colony growth, DAC reduced colony formation by 85 %, and DAC+RA decreased colony numbers by 96 %. To determine transcriptional add-on effects of DAC+RA, transcriptomes of cells treated for 72 hr were analyzed by RNA-Seq. RA alone had a modest effect on transcription, whereas DAC alone induced / downregulated 2538 and 620 transcripts, DAC+RA induced / downregulated 2848 and 1173 transcripts, respectively (Table 1, Fig. 1). 29.6 % (1192 transcripts) of all DAC+RA-altered transcripts were uniquely regulated by the combination and not by DAC or RA alone. GO analysis of the DAC+ATRA alone upregulated genes (664 transcripts) revealed a significant enrichment for immune system and cellular decay terms, downregulated genes were enriched for transcription and cell cycle terms. Of the transcripts already up- or downregulated by DAC or RA alone, 55.5 % of were further enhanced by DAC+RA. Among those, the retinoic acid receptors RARA, RARB and the retinoic acid response element (RARE)-containing tumor suppressor HIC-1 (Hassan et al. Cell Reports 2017) were induced 8.6-, 8.3- and 179.8-fold, respectively, compared to 2.4- and 5.3-fold (RARA and RARB, DAC alone) and 26.5-fold (HIC-1, RA alone) (validated by qRT-PCR). Among the most regulated uniquely induced transcripts by DAC+RA, the RA-regulating cytochrome P450 member CYP26A1 (regulated by multiple RAREs) was upregulated 347.3-fold. In order to further investigate this add-on effect of RA on DAC-induced transcriptome changes, DNA methylation was analyzed (72 and 120 hr). As expected, DAC significantly reduced CpG methylation in gene bodies and promoters, without further demethylation upon RA add-on after 72 hr (after 120 hr, a trend towards further demethylation was noted with DAC+ATRA). Conclusion: In vitro, the combination of DAC with RA resulted in enhanced growth inhibition and reduced colony formation compared to DAC alone. Transcriptome analysis revealed a high number of uniquely regulated transcripts, and an enhancement of regulation by the combination treatment. As expected, this also included genes with RAREs. The enrichment for genes involved in immune response (e.g. multiple interferon-response genes) encourages the combination of DNMTi-based therapies with immunotherapeutic agents. Therefore, further transcriptome analyses of transposable elements, when reactivated, may trigger an interferon-mediated immune reaction, are currently ongoing. Disclosures Lübbert: TEVA: Other: Study drug; Celgene: Other: Travel Support; Cheplapharm: Other: Study drug; Janssen: Honoraria, Research Funding.