Genome-Wide DNA Methylation Analysis Reveals Epigenetic Deregulation of Key Biological Pathways in Therapy-Related Myeloid Neoplasms

Abstract Therapy-related myeloid neoplasms (tMN), including therapy-related myelodysplastic syndromes (tMDS) and therapy-related acute myeloid leukemia (tAML) are associated with resistance to chemotherapy and a more unfavorable prognosis than their de novo counterparts. While many studies have explored the epigenome of de novo MDS, little is known about the epigenetic status of tMN. Recently, we reported that tMN showed distinct mutational profiles whether they arose after an initial solid tumor or a lymphoproliferative disorder, indicating that aside from the type of therapy received it is possible the type of primary neoplasm might also help determine the phenotype of t-MN. Therefore, we sought to perform a comprehensive epigenomic study of tMN that would help improve our current understanding of epigenetic deregulation in this disorder. Using next-generation bisulfite sequencing we analyzed the DNA methylation status at ~3M CpG sites of 15 cases, including 5 de novo MDS (dnMDS), 5 tMN with a prior history of Hodgkin lymphoma (HL-tMN) treated with MOPP/BEACOPP-based regimens, and 5 tMN arising after breast cancer (BC-tMN) treated with combined chemo-radiotherapy. There were no significant differences in median age, median bone marrow blast percentage, or median latency to t-MN for the HL and BC groups. Karyotype mainly consisted of abnormalities of chromosomes 5 or 7, or complex karyotypes in all groups. None of the patients had balanced translocations or abnormalities of the 11q23 locus. We first sought to determine whether tMDS and dnMDS are epigenetically distinct. For this we compared the DNA methylation profiles of the tMDS (n=6) vs. dnMDS (n=5). We identified 1504 statistically significant (FDR<0.1 and mean methylation difference ≥25%) differentially methylated regions (DMRs). Relative to dnMDS, tMDS showed a markedly hypomethylated profile. Pathway analysis using DAVID revealed that these DMRs were significantly enriched for genes in the Wnt signaling (FDR=3.8x10-6) and cadherin pathways (FDR=5.3x10-7), indicating that these key pathways maybe play different roles in these two forms of MDS. Next we asked whether among tMNs the type of neoplasm preceding the development of tMN might also influence the epigenome. To address this we performed a direct comparison of the DNA methylation profiles for the HL-tMN (n=5) vs. BC-tMN (n=5), which identified 457 statistically significant DMRs, the vast majority of which localized to intronic and intergenic regions and were hypomethylated in HL-tMN relative to BC-tMN. Finally, in order to determine the role that epigenetic deregulation may play in different forms of AMLs not arising de novo, we compared the epigenetic profiles of tAML (n=4) to a cohort of AMLs arising after progression from a myeloproliferative neoplasm (MPN) (n=5). Notably, despite their shared features of chemoresistance and poor prognosis, the epigenetic profiles of these two entities were vastly different, with 14,887 statistically significant DMRs detected. These DMRs were depleted at promoter regions (DMRs 4% vs. Background [BG] 12%, Fisher p-value[p]=0.038) and CpG islands (DMRs 9% vs. BG 30%, p=0.0003), but were enriched at regions outside of CpG islands (DMRs 73% vs. BG 54%, p=0.008). Gene ontology analysis showed these DMRs were significantly enriched for genes involved transcriptional regulation (FDR=0.02), nucleotide binding (FDR=0.00025), regulation of RNA metabolic process (FDR=0.02), and protein kinase activity (FDR=0.009). In addition, these DMRs were significantly enriched at enhancer regions (p<0.0001), indicating they may play key regulatory roles. In summary, we have completed the first comprehensive analysis of the epigenome-wide abnormalities associated with tMN. Our findings demonstrate that tMN are epigenetically distinct from dnMDS and that these abnormalities target biological pathways known to play a key role in self-renewal and differentiation of hematopoietic stem and cancer cells, as well in MDS pathogenesis. Moreover, we demonstrate that similar to our findings at the mutational level, epigenetic deregulation in tMN also has distinct profiles strongly correlated with the type of first malignancy preceding the tMN. Finally, AML arising as a progression of MPN or after prior chemotherapy show robust epigenetic differences that clearly distinguish these two entities. Disclosures Voso: Celgene: Consultancy

Transcription and methylation analyses of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

International audienceAcute promyelocytic leukemia is an aggressive malignancy characterized by the accumulation of promyelocytes in the bone marrow. PML/RARA is the primary abnormality implicated in this pathology, but the mechanisms by which this chimeric fusion protein initiates disease are incompletely understood. Identifying PML/RARA targets in vivo is critical for comprehending the road to pathogenesis. Utilizing a novel sorting strategy, we isolated highly purified promyelocyte populations from normal and young preleukemic animals, carried out microarray and methylation profiling analyses, and compared the results from the two groups of animals. Surprisingly, in the absence of secondary lesions, PML/RARA had an overall limited impact on both the transcriptome and methylome. Of interest, we did identify down-regulation of secondary and tertiary granule genes as the first step engaging the myeloid maturation block. Although initially not sufficient to arrest terminal granulopoiesis in vivo, such alterations set the stage for the later, complete differentiation block seen in leukemia. Further, gene set enrichment analysis revealed that PML/RARA promyelocytes exhibit a subtle increase in expression of cell cycle genes, and we show that this leads to both increased proliferation of these cells and expansion of the promyelocyte compartment. Importantly, this proliferation signature was absent from the poorly leukemogenic p50/RARA fusion model, implying a critical role for PML in the altered cell-cycle kinetics and ability to initiate leukemia. Thus, our findings challenge the predominant model in the field and we propose that PML/RARA initiates leukemia by subtly shifting cell fate decisions within the promyelocyte compartment

DRAGONFLY: in situ exploration of Titan's meteorology

International audienceDragonfly is a rotorcraft lander mission currently in a Phase A study under NASA's New Frontiers Program that would take advantage of Titan's dense atmosphere and low gravity to visit a number of surface locations to study how far chemistry can progress in environments that provide key ingredients for life. This mission architecture also permits and demands investigation of Titan's atmosphere. First, Dragonfly is a lander that will spend >2 Earth years on Titan's surface, long enough to observe many diurnal cycles, atmospheric waves, and perhaps even some seasonal change. The DraGMet (Dragonfly Geophysics and Meteorology) instrument package includes measurement of wind speed and direction (using sensors on each of the four rotor pylons, to assure that one or more sensors are upwind of and thus unperturbed by the vehicle), temperature and pressure, and methane humidity. A camera suite will also include panoramic imaging, informing atmospheric optics and possibly cloud motions. Second, through its flight capability, Dragonfly can explore the micrometeorology at a number of different locations with different terrain settings, as well as making repeated vertical profiles of temperature, methane, and hydrogen to constrain mixing in the atmospheric boundary layer at different times of day. Dragonfly will also contribute to atmospheric science in a number of other ways: electric field measurements; seismic observations, which may include an atmospheric component; measurements of surface properties, which include soil moisture; chemical composition of surface deposits, which may contain the products of high-altitude photochemistry; identification of fluvial sediments may inform our understanding of the hydrologic cycle; and measurement of the saltation threshold (using the vehicle's rotors) will improve interpretation of dune morphology and circulation patterns. Dragonfly results will test and improve atmospheric models, feeding forward into a deeper understanding of the local and global Titan climate system

Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis

Distinct DNA Methylation and Expression Profiles Underlie CMML Responsiveness to Decitabine and Uncover Novel Mechanism of Resistance

Abstract Myelodysplastic syndromes (MDS) and the related disorder chronic myelomonocytic leukemia (CMML) are characterized by abnormal DNA hypermethylation and the DNA methyltransferase inhibitors (DMTis) azacytidine and decitabine (DAC) are frequently used as frontline therapy in these patients. However, DMTis are ineffective for ~50% of the patients who must still undergo treatment for at least 6 months before they can be deemed resistant. Therefore, it is of critical importance to identify baseline molecular differences associated with DMTi sensitivity that can help (i) to improve patient risk-stratification at diagnosis and (ii) identify the underlying mechanisms of resistance to these agents. Previous efforts to identify baseline DNA methylation differences at promoter regions between DMTi responders and non-responders have not been successful, so we hypothesized that any potential differences would be located distally from promoter regions. For this purpose we studied 40 CMML patients at diagnosis, all of whom had been uniformly treated with DAC 20 mg/m2/day x 5 days as frontline therapy. After 6 cycles of therapy patients were classified as responders (n=19, hematological improvement or better), or non-responders (n=21, stable or progressive disease). Mutational analysis showed no significant differences in the frequency of mutations in TET2, ASXL1, DNMT3A, RUNX1, TP53, JAK2, KIT, KRAS, EZH2, IDH1/2 and spliceosome genes. Using Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) we analyzed the baseline methylation status at ~3M CpG sites across the genome of 39/40 CMML patients. We identified 158 statistically significant differentially methylated regions (DMRs) (FDR<0.1 and methylation difference ≥25%) between the two groups. DAC-sensitive patients displayed both regions of higher methylation as well as regions with lower methylation compared to DAC-resistant patients. As predicted, DMRs were depleted at promoters (DMRs 9% vs. Background [BG] 21%, p-value: 3.4×10-5) and CpG islands (DMRs 8% vs. BG 25%, p-value: 1.5×10-8). Further analysis showed that hypermethylated DMRs were enriched at intronic regions (Hyper DMRs 58% vs. BG 33%, p-value: 3.7×10-6) while hypomethylated DMRs were enriched at intergenic regions (Hypo DMRs 49% vs. BG 38%, p-value: 2.6×10-2). Moreover, hypermethylated DMRs were significantly enriched for enhancer regions, and in particular, enhancers located within gene bodies (hyper DMRs 38% vs. BG 18%, p-value: 2.3×10-5). KEGG pathway analysis showed a significant enrichment of DMRs in the MAPK signaling pathway (FDR<0.01). Next, using a support vector machine algorithm with 10-fold cross validation we were able to develop a classifier capable of predicting response to DAC with high level of accuracy (ROC AUC: 0.99) based solely on the DNA methylation status at diagnosis of 17 genomic regions. Three different random splits of the cohort into training and test sets achieved correct predictions for 85.7%, 89.47%, and 100% of cases, respectively, demonstrating the accuracy and potential utility of such a classifier. Finally, RNA-seq analysis identified 53 differentially expressed genes between responders (n=8) and non-responders (n=6) at diagnosis. Genes implicated in cell cycle and DNA replication were overexpressed in responders. By contrast, very few genes were overexpressed at the time of diagnosis in primary resistant patients. Among these were CXCL4 and CXCL7 which, given their reported contributions to cell cycle arrest and chemoresistance, were tested for their functional roles in DAC resistance. Pre-treatment of normal CD34+ cells for 72 h with 10nM DAC significantly reduced colony formation (p<0.05) but the addition of 50ng/mL of CXCL4 and CXCL7 restored colony formation to that of untreated cells. Moreover, treatment of primary CMML cells with 10 nM DAC for 72h significantly reduced viability of these cells, while concomitant treatment with 50ng/mL of CXCL4 and CXCL7 was sufficient to abrogate this effect. Taken together, our findings demonstrate that (i) specific DNA methylation profiles targeting non-promoter regulatory regions are associated with DAC sensitivity, (ii) these differences can be harnessed for the development of clinical biomarkers predictive of response and (iii) we identified a novel mechanism of resistance to DAC mediated through two chemokines that are exclusively overexpressed in non-responders. Disclosures No relevant conflicts of interest to declare

Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34(+) and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance

Science Goals and Objectives for the Dragonfly Titan Rotorcraft Relocatable Lander

International audienceNASA's Dragonfly mission will send a rotorcraft lander to the surface of Titan in the mid-2030s. Dragonflyʼs science themes include investigation of Titan's prebiotic chemistry, habitability, and potential chemical biosignatures from both water-based "life as we know it" (as might occur in the interior mantle ocean, potential cryovolcanic flows, and/or impact melt deposits) and potential "life, but not as we know it" that might use liquid hydrocarbons as a solvent (within Titan's lakes, seas, and/or aquifers). Consideration of both of these solvents simultaneously led to our initial landing site in Titan's equatorial dunes and interdunes to sample organic sediments and water ice, respectively. Ultimately, Dragonflyʼs traverse target is the 80 km diameter Selk Crater, at 7°N, where we seek previously liquid water that has mixed with surface organics. Our science goals include determining how far prebiotic chemistry has progressed on Titan and what molecules and elements might be available for such chemistry. We will also determine the role of Titan's tropical deserts in the global methane cycle. We will investigate the processes and processing rates that modify Titan's surface geology and constrain how and where organics and liquid water can mix on and within Titan. Importantly, we will search for chemical biosignatures indicative of past or extant biological processes. As such, Dragonfly, along with Perseverance, is the first NASA mission to explicitly incorporate the search for signs of life into its mission goals since the Viking landers in 1976