DNA Methyltransferase Inhibitor Therapy Research Articles

The role of DNA methylation in ovarian cancer chemoresistance: A narrative review.

Ovarian cancer (OC) is the most lethal gynecological cancer. In 2018, it was responsible for over 180,000 deaths worldwide. The high mortality rate is the culmination of a lack of early diagnosis and high rates of chemotherapy resistance, which is synonymous with disease recurrence. Over the last two decades, an increasingly significant role of epigenetic mechanisms, in particular DNA methylation, has emerged. This review will discuss several of the most significant genes whose hypo/hypermethylation profiles are associated with chemoresistance. Aside from functionally elucidating and evaluating these epimutations, this review will discuss recent trials of DNA methyltransferase inhibitors (DNMTi). Finally, we will propose future directions that could enhance the feasibility of utilizing these candidate epimutations as clinical biomarkers. To perform this review, a comprehensive literature search based on our keywords was conducted across the online databases PubMed and Google Scholar for identifying relevant studies published up until August 2022. Epimutations affecting MLH1, MSH2, and Ras-association domain family 1 isoform A (DNA damage repair and apoptosis);ATP-binding cassette subfamily B member 1 and methylation-controlled J (drug export); secreted frizzled-related proteins (Wnt/β-catenin signaling), neurocalcin delta (calcium and G protein-coupled receptor signaling), and zinc finger protein 671all have potential as biomarkers for chemoresistance. However, specific uncertainties relating to these epimutations include histotype-specific differences, intrinsic versus acquired chemoresistance, and the interplay with complete surgical debulking. DNMTi for chemoresistant OC patients has shown some promise; however, issues surrounding their efficacy and dose-limiting toxicities remain; a personalized approach is required to maximize their effectiveness. Establishing a panel of aberrantly methylated chemoresistance-related genes to predict chemoresponsiveness and patients' suitability to DNMTi could significantly reduce OC recurrence, while improving DNMTi therapy viability. To achieve this, a large-scale prospective genome-wide DNA methylation profile study that spans different histotypes, includes paired samples (before and after chemotherapy), and integrates transcriptomic and methylomic analysis, is warranted.

The combination of DNA methyltransferase inhibitor therapy and immunotherapy for acute myeloid leukemia

The combination of DNA methyltransferase inhibitor therapy and immunotherapy for acute myeloid leukemia

Molecular mechanisms of Guadecitabine induced FGFR4 down regulation in alveolar rhabdomyosarcomas

Fibroblast growth factor receptor 4 (FGFR4) aberrant expression and activity have been linked to the pathogenesis of a variety of cancers including rhabdomyosarcomas (RMS). We found that treatment of alveolar rhabdomyosarcoma (aRMS) cells with Guadecitabine (SGI-110), a next-generation DNA methyltransferase inhibitor (DNMTi), resulted in a significant reduction of FGFR4 protein levels, 5 days post treatment. Chromatin immunoprecipitation-sequencing (ChIP-seq) in aRMS cells revealed attenuation of the H3K4 mono-methylation across the FGFR4 super enhancer without changes in tri-methylation of either H3K4 or H3K27. These changes were associated with a significant reduction in FGFR4 transcript levels in treated cells. These decreases in H3K4me1 in the FGFR4 super enhancer were also associated with a 240-fold increase in KDM5B (JARID1B) mRNA levels. Immunoblot and immunofluorescent studies also revealed a significant increase in the KDM5B protein levels after treatment in these cells. KDM5B is the only member of KDM5 (JARID1) family of histone lysine demethylases that catalyzes demethylation of H3K4me1. These data together suggest a pleiotropic effect of DNMTi therapy in aRMS cells, converging to significantly lower FGFR4 protein levels in these cells.

Oral Azacitidine and Cedazuridine Approximate Azacitidine Efficacy in a Murine Model

Background: DNA methyltransferase inhibitors (DNMTi) induce remissions and improve survival for patients with MDS and those with AML unable to receive standard cytotoxic chemotherapy. Accordingly, DNMTi therapy is the backbone of SOC treatment for MDS and AML. Given the inconvenience, pain, and general detriments to QOL with SQ or IV therapy daily for 5-7 days every month with azacitidine (AZA) or decitabine (DAC), many have attempted to provide the therapy orally, but encountered difficulties with this method of administration given rapid first-pass clearance via the enzyme cytidine deaminase (CDA) which is ubiquitous in the gut and liver. Recently, DAC was combined with cedazuridine (CDZ), an oral CDA inhibitor, in a fixed-dose (35mg/100mg) combination tablet (ASTX727) to approximate the pharmacokinetics of IV DAC (Savona et al Lancet Haematology 2019). To determine if a similar strategy might be feasible with AZA, we attempted to increase the bioavailability of oral AZA with CDZ in a murine tumor model. Methods: We measured GI50 in AML cell lines treated with vehicle (DMSO), AZA, and AZA/CDZ combination. Although cancer cell lines produce CDA, total levels are negligible in comparison CDA in the gut and liver. For this reason, we then studied CDZ, AZA, and the AZA/CDZ combination in a systemic model of AML in immunocompromised mice. NSGS mice were sub-lethally irradiated and administered MOLM-13 AML cells via tail vein injection. At day seven post-transplant, engrafted mice were randomized to receive 2.5 mpk i.p. AZA(n=8), 2.5 mpk oral AZA(n=8), 3 mpk CDZ + oral AZA(n=6), or CDZ alone at 30 mpk (n=7). All oral AZA and CDZ was administered via oral gavage daily for 7 consecutive days; i.p. therapy was similarly given for 7 consecutive days. During treatment, the kinetics of MOLM-13 expansion was defined by detection of human AML in the blood as detected by flow cytometry. At approximately three weeks after transplant, CDZ-only treated mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Results: After 72 hour of treatment, no differences were noted in viability of cell lines between AZA and AZA+ CDZ in vitro. In the xenograft model, as expected, i.p. AZA-treated mice had significant decreases in leukemic expansion in the bone marrow and spleen, whereas CDZ alone did not (AZA i.p. vs CDZ, p = 0.004 and <0.001). More importantly, whereas oral AZA alone failed to decrease AML expansion in both the bone marrow and spleen of treated mice (oral AZA vs CDZ., p = 0.677 and 0.249, respectively), the addition of CDZ to oral AZA led to significant decreases in AML in both bone marrow and spleen (Oral AZA + CDZ vs CDZ, p = 0.012 and 0.004, respectively). Likewise, addition of CDZ to oral AZA showed no significant differences in activity against AML compared to traditional AZA i.p. dosing in either bone marrow or splenic tissue (p= 0.204 and 0.224, respectively). Furthermore, in a Kaplan-Meier survival analysis, mice treated with CDZ alone die within 21 days of transplant, but both i.p. AZA and oral AZA + CDZ treated mice had a 50% extended survival with no significant difference in survival between i.p. AZA and oral AZA + CDZ treated mice (p= 0.502). H&E staining revealed no significant bone marrow toxicity in treated mice, suggesting that AZA preferentially effected transplanted MOLM-13 AML cells. Conclusion: Consistent with previous preclinical and clinical studies with ASTX727, the oral AZA approximated AZA anti-tumor activity when co-treated with CDA-inhibitor CDZ. These preliminary data provide rationale for the development of CDZ + oral AZA therapy as a fixed dose combination (ASTX030) in myeloid disease. Clinical trials evaluating ASTX030 are being planned. Figure Disclosures Oganesian: Astex Pharmaceuticals, Inc.: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Savona:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.

DNA methyltransferase inhibitors induce a BRCAness phenotype that sensitizes NSCLC to PARP inhibitor and ionizing radiation

A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC.

DNMT Inhibitors Increase Methylation in the Cancer Genome.

DNA methyltransferase inhibitors (DNMTi) decitabine and azacytidine are approved therapies for myelodysplastic syndrome and acute myeloid leukemia, and their combinations with other anticancer agents are being tested as therapeutic options for multiple solid cancers such as colon, ovarian, and lung cancer. However, the current therapeutic challenges of DNMTis include development of resistance, severe side effects and no or partial treatment responses, as observed in more than half of the patients. Therefore, there is a critical need to better understand the mechanisms of action of these drugs. In order to discover molecular targets of DNMTi therapy, we identified 638 novel CpGs with an increased methylation in response to decitabine treatment in HCT116 cell lines and validated the findings in multiple cancer types (e.g., bladder, ovarian, breast, and lymphoma) cell lines, bone marrow mononuclear cells from primary leukemia patients, as well as peripheral blood mononuclear cells and ascites from platinum resistance epithelial ovarian cancer patients. Azacytidine treatment also increased methylation of these CpGs in colon, ovarian, breast, and lymphoma cancer cell lines. Methylation at 166 identified CpGs strongly correlated (|r|≥ 0.80) with corresponding gene expression in HCT116 cell line. Differences in methylation at some of the identified CpGs and expression changes of the corresponding genes was observed in TCGA colon cancer tissue as compared to adjacent healthy tissue. Our analysis revealed that hypermethylated CpGs are involved in cancer cell proliferation and apoptosis by P53 and olfactory receptor pathways, hence influencing DNMTi responses. In conclusion, we showed hypermethylation of CpGs as a novel mechanism of action for DNMTi agents and identified 638 hypermethylated molecular targets (CpGs) common to decitabine and azacytidine therapy. These novel results suggest that hypermethylation of CpGs should be considered when predicting the DNMTi responses and side effects in cancer patients.

Abstract 1422: Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Treatment most commonly relies on ionizing radiation (IR) and platinum-based DNA damaging agents, but long-term survival is poor and patients tend to suffer chronic side-effects due to the high radiation dose to the surrounding normal tissues. Therefore, new treatments are needed that can be used in combination lower radiation doses. We have recently reported that low, non-cytotoxic doses Poly ADP ribose polymerase inhibitors (PARPi) Talazoparib in combination with DNA methyltransferase inhibitors (DNMTi) Decitabine (DAC) or azacytidine (AZA) significantly increase cytotoxicity in acute myeloid leukemia and breast cancer models in vitro and anti-tumor effects in vivo. Simultaneous administration of both inhibitors result in increased PARP binding in DNA, leading to higher levels of DNA double strand breaks (DSBs), yielding increased cytotoxicity, compared with each agent treatment alone. We first studied the efficacy of Talazoparib and AZA combination therapy in multiple NSCLC cell lines (A549, H358 and H838) in vitro through colony forming assays. Results showed, compared to single agent treatments, combination drug treatment significantly decreased colony formation. Cell viability was also significantly decreased with the drug combination in MTS assays (P<0.05). These results also showed synergistic activity between the two drugs, with a combination index of less than 1 for all tested NSCLC cell lines. Importantly, the drug combination increased PARP trapping in chromatin and DSB formation, as measured by immunofluorescent staining for γH2AX. We next determined whether AZA/Talazoparib in combination with a single dose of radiation (2Gy) had an increased anti-cancer effect compared to each modality alone in in vivo mouse xenografts of NSCLC A549. While the combination of PARPi+DNMTi with radiation treatment decreased tumor growth, compared to PARPi/DNMTi alone or RT alone, no significant enhancement with radiation was observed. We next determined whether low doses of fractionated IR (2Gy 3 fractions) would improve the efficacy of DNTMi and PARPi combination treatment. In vitro studies with colony forming assays of NSCLC cell line A549 show that IR doses in combination with PARPi and DNMTi decrease clonogenicity, compared with non-IR controls. Furthermore, A549 xenografts were treated with the drug combination and then irradiated 1 week later with 2Gy daily for 3 consecutive days. Mice treated with the drug combination followed by IR had significant decreases in tumor volume and survival (P<0.05). This suggests that low doses of PARPi and DNMTi therapy in combination with low dose IR can potentially target NSCLC tumors. This represents a novel treatment approach for NSCLC patients that may reduce chronic side-effects of high dose IR. Citation Format: Christopher Biondi, Daniel Fontaine, Lora Stojanovic, Pratik Nagaria, Rena Lapidus, Eun Yong Choi, Javed Mahmood, Stephen Baylin, Feyruz V. Rassool. Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers [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 1422. doi:10.1158/1538-7445.AM2017-1422

Analysis of Myelodysplastic Syndrome Stem Cells at Single Cell Resolution during DNA Methyltransferase Inhibitor Therapy

Abstract The myelodysplastic syndromes (MDS) arise in and are maintained by hematopoietic stem cells (HSCs). Serial sampling of patients treated with DNA methyltransferase inhibitors (DNMTIs) and lenalidomide has demonstrated that disease HSCs (MDS HSCs) persist at significant levels even in patients achieving complete clinical and cytogenetic responses. As MDS HSCs are the functional unit of clonal selection both during therapy and subsequent disease progression, we hypothesized that the molecular heterogeneity of MDS HSCs may underlie therapeutic resistance. We therefore sought to perform single cell RNA-sequencing (RNA-seq) on MDS HSCs from patients with known responses to therapy, with the intention of identifying novel therapeutic vulnerabilities. To characterize MDS HSC heterogeneity, we FACS-purified HSCs (Lin-CD34+CD38-CD90+CD45RA-) from paired bone marrow (BM) specimens taken from four MDS patients before and after two to four 28-day cycles of the DNMTI decitabine, as well as two patients who were not treated due to stable disease, and two normal age matched controls. Specimens from both responding and non-responding patients were included. We captured and sequenced a total of 869 single cells from 14 samples, sequencing to an average depth of 4.8 million reads. In a subset of samples (n=7) we also performed bulk RNA-seq (average 1500 cells) for comparison. The sequencing data was of high quality, with an average of 80% mapped reads. We confirmed our ability to accurately quantify transcript levels using ERCC spike-in controls, observing a linear correlation between expected concentration and observed FPKM (fragments per kilobase per million). Single cell RNA-seq revealed vast intratumoral heterogeneity in MDS HSCs that was otherwise missed by bulk RNA-seq, as evidenced by the presence of transcripts variably expressed among cells from the same specimen (Fig. 1A). Despite this intratumoral heterogeneity, single cell transcriptomes were able to completely separate individual MDS patients using principal components analysis and hierarchical clustering, consistent with the known heterogeneity of MDS. MDS HSCs further clustered separately from normal age-matched HSCs, with the top 10% of genes contributing to this separation enriched for Gene Ontology (GO) categories including pathways implicated in MDS biology such as "mRNA splicing," "nonsense mediated decay," and "P53 mediated DNA Damage Response" (all P<1e-9). Unsupervised hierarchical clustering of all pre-treatment MDS HSCs revealed clustering of cells from responders separately from non-responders (Fig. 1B). Differential gene expression analysis identified a cluster of genes (FDR<0.01) enriched for GO categories including "translational termination," "SRP dependent co-translational protein targeting to membrane," and "nonsense mediated decay" (all P<1e-9). Notably, this cluster included 60 ribosomal proteins, all of which were decreased in non-responders (t-test, P<1e-16), with responders demonstrating levels of expression closer to but still lower than normal controls (t-test, P<1e-3). Thus, defective ribosomal biogenesis, a hallmark of MDS pathogenesis, may also contribute to therapeutic resistance. Finally, within each sample we measured the spread of gene expression using dispersion (log[variance/mean]) within bins based on expression levels, defining variable genes as those with a dispersion >1.75 at a mean FPKM >2. The highest number of variable genes were in normal HSCs (mean=141), with the next highest in responders prior to treatment (mean=80), and the least number of variable genes in non-responders prior to treatment (mean=9.5). We speculate that the low number of variable genes in non-responders reflects a higher degree of clonal dominance. All post-treatment MDS HSCs demonstrated a relatively low number of variable genes (mean=25), suggesting that therapy induces clonal selection. In sum, our data illustrate the robustness of single cell RNA-seq to define the intrinsic variability of individual MDS HSCs, implicating perturbed ribosomal biogenesis and transcriptional variability as novel predictors of response to therapy. As we expand our data set with additional patients, we expect to identify additional pathways that mediate therapeutic response and resistance, as well as mutations and variably expressed genes that are selected for during therapy and drive disease progression. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

MiRNA Predictors of Response to DNA Methyltransferase Inhibitors

BackgroundMyelodysplastic syndrome (MDS) is a common disease of the elderly characterized by ineffective maturation of hematopoietic cells manifesting as low blood counts, morphologic atypia, and predisposition to the development of acute leukemia. The only FDA-approved drugs for the treatment of all subytpes of MDS are the DNA methyltransferase inhibitors (DNMTis), azacytidine and decitabine. However, only a minority of patients achieve a hematologic response or better on these agents. We have previously identified a miRNA signature which distinguishes MDS patients from normal controls. We sought to investigate whether miRNA expression might also be predictive of how patients respond to therapy with DNA methyltransferase inhibitors (DNMTis).DesignWe collected paired fresh frozen bone marrow mononuclear cells (BM-MNCs) from 28 patients with MDS both pre and post treatment with DNMTis as well as from 30 normal controls. Following enrichment for miRNAs and other small RNA species using the TruSeq Small RNA sample preparation kit (Illumina, San Diego, CA), miRNAs were sequenced on an Illumina HiSeq 2500. Alignment was performed using Bowtie 2 against reference sequences from mirBASE and differential expression analysis was analyzed using a rank sum score from the following analysis programs: DESeq, edgeR and baySeq. The response to the DNMTis was characterized based upon the following scale: 1 = complete remission, 2 = marrow complete remission, 3 = partial remission, 4 = hematologic improvement, 5 = stable disease, and 6 = progressive disease. Linear regression analysis was conducted using the response score as outcome and miRNA expression value as the predictor.ResultsSeveral differences were found between the control samples and the pre-treatment MDS samples, many of which have been described previously, including hsa-miR-125b, hsa-miR-342, hsa-miR-140, and hsa-miR-150. Not unexpectedly, the expression of numerous miRNAs was significantly altered as a result of treatment with DNMTis. However, there were no significant miRNA expression differences between responders and non-responders in the post-treatment samples and no significant differences between the changes in miRNA expression (pre versus post treatment changes) in responders and non-responders. Interestingly, 5 miRNAs demonstrated significant association between their pre-treatment expression and the degree of response (FDR < 0.05): hsa-miR-125a, hsa-miR-342, hsa-miR-146b, hsa-miR-146a, hsa-miR-423, and hsa-miR-150. These miRNAs share striking overlap in their mRNA targets including key pathway regulators in the p53 pathway as well as numerous growth factors implicated in hematopoietic stem cells as well as hematopoietic stem cell markers.ConclusionThese studies confirm that miRNA expression differences differentiate between MDS and normal controls. The administration of DNMTis results in widespread expression changes regardless of the response to therapy. However, the expression of a small handful of miRNA are positively correlated with the degree of response in MDS pre-treatment samples to DNMTis and therefore may be useful as markers which predict responders to DNMTi therapy in addition to suggesting biological pathways implicated in the mechanism of response. These results await further prospective studies for confirmation. DisclosuresNo relevant conflicts of interest to declare.

Clinical activity and safety of the dual pathway inhibitor rigosertib for higher risk myelodysplastic syndromes following DNA methyltransferase inhibitor therapy.

Rigosertib (ON 01910.Na) is an inhibitor of the phosphoinositide 3-kinase and polo-like kinase pathways that induces mitotic arrest and apoptosis in neoplastic cells, while sparing normal cells. Our purpose is to summarize the clinical activity and safety of intravenous (IV) rigosertib delivered by an external ambulatory infusion pump in patients with refractory anemia with excess blasts-1, -2, or, -t myelodysplastic syndromes (MDS) following prior treatment with DNA methyltransferase (DNMT) inhibitors. A total of 39 patients with MDS who fulfilled these criteria were enrolled in four phase 1-2 clinical trials of IV rigosertib. Thirty five (88%) had higher risk disease according to the Revised International Prognostic Scoring System. Median overall survival for this group of 39 patients was 35 weeks. Of 30 evaluable patients with follow-up bone marrow biopsies, 12 (40%) achieved complete (n = 5) or partial (n = 7) bone marrow blast responses. In addition, 15 patients achieved stabilization of bone marrow blasts. One patient with a complete bone marrow response also achieved a complete cytogenetic response. A second patient with stable bone marrow blasts achieved a partial cytogenetic response. Two of the responding patients and three patients with stable disease had hematological improvements. Rigosertib-induced bone marrow blast decreases and stability appeared to be predictive of prolonged survival. IV rigosertib had a favorable safety profile without significant myelosuppression. Most common drug-related toxicities included fatigue, diarrhea, nausea, dysuria, and hematuria. In summary, IV rigosertib is well tolerated and has clinical activity in patients with higher risk MDS following DNMT inhibitor treatment. A multinational pivotal phase 3 randomized clinical trial of rigosertib versus best supportive care for patients with MDS with excess blasts following prior treatment with DNMT inhibitors (ONTIME: ON 01910.Na Trial In Myelodysplastic SyndromE) has recently completed enrollment.

Comprehensive Mutational Profiling In Myelodysplastic Syndromes Treated With Decitabine and Tretinoin

Although DNA methyltransferase Inhibitor (DNMTI) therapy demonstrates efficacy in patients (pts) with Myelodysplastic Syndromes (MDS), to date there are no clinical or genomic markers to select patients most likely to benefit from DNMTI therapy. Two recent retrospective studies have suggested TET2 mutations (muts) are associated with a higher response rate to 5-azacytidine (5-aza) and decitabine (DAC). We investigated the efficacy of DAC (20 mg/m2/day x 5 days) plus tretinoin (ATRA) given on days 10-19 of a 28-day schedule in MDS pts in a phase I/II study. We comprehensively analyzed pretreatment samples from 23 pts (15M,8F; median age 67, range, 44-78) treated on this study for alterations in known oncogenes to identify potential correlations between mut profile and clinical outcomes. The median follow-up was 41 mos (range, 10-66). IPSS cytogenetics (CG) risk categories included Good (n=8), Intermediate (n=5), and High risk (n=8) pts (2 karyotype failures). The overall IPSS risk categories were Int-1 (n=3), Int-2 (n=16), and High (n=4). Of 23 pts, 16 responded, including 2 CRs, 9 mCR+/-HI, 1 PR, and 4 HI.Genomic DNA and total RNA were isolated from all pt samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >584X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The median number of muts detected was 2 (range, 0-6), with 22/23 (96%) pts having at least 1 mut. The number of muts detected for 14 PB samples with ≤ 1% blasts was 2 (range, 0-6) compared to 4 muts (range, 1-5) for 8 pts with > 1% PB or BM blasts (unknown source for 1 sample).The most common mut identified in this cohort was TP53 (35%). Previous studies have shown that complex CGs and mut TP53 are strong poor prognostic indicators. Six pts with complex CGs and either mut or deleted TP53 had a median of 0 (range 0-2) additional muts, whereas pts with non-complex CGs who were wild-type (WT) TP53 (n=12) had a median of 3 (range 1-6) muts in other disease alleles (p=0.006). We identified mutually exclusive recurrent muts in RNA splicing factors including SRSF2 (26%), SF3B1 (17%), and ZRSR2 (9%), and recurrent muts in RUNX1 (22%), IDH1/2 (22%), TET2 (17%), ASXL1 (17%), PTPN11 (13%), CEBPA (13%), STAG2 (9%), and KRAS (9%). Thirteen additional muts were detected in single pts, and we identified novel candidate muts in DNA repair pathways (ATM, ATR, FANCA BRCA2), and in the recently described tumor suppressor FAT3. We also hypothesized that prognostication of pts with normal karyotype (NK) MDS may be aided by molecular stratification, as we and others have shown in AML. Of note, all 5 pts with IDH1/2 muts occurred in pts with NK, and in 4/5 cases they were associated with concomitant SRSF2 muts that were previously shown to be associated with adverse outcomes.We identified TET2 muts in 4 pts who were also ASXL1 WT. Notably, all 4 TET2 mutated/ASXL1 WT pts responded to DAC/ATRA therapy. The other 12 responders included 7 pts with TP53 mut/loss +/- other muts. No other muts were associated with a differential response to DAC/ATRA in this trial cohort. The strong association between TET2 muts and DAC/ATRA response, and more detailed mutational profiling of the 12 TET2 WT pts who responded to DAC/ATRA therapy is being investigated in additional pt samples and will be presented in more detail. Pts with TP53 muts showed a trend toward a worse survival which was not statistically significant; however, no other individual mut was associated with a differential survival in this small cohort. ConclusionsThe use of comprehensive mutational profiling employed in this study allowed us to identify muts in 25 genes in MDS pts, and allowed us to show that the overall mut frequency was higher in pts with a NK and without TP53 muts, thereby providing evidence for alternate disease initiation events and pathogenesis in these two genomically defined MDS subsets. All pts with mutated TET2 responded to DAC/ATRA, suggesting that pts with muts in TET2 are the best candidates for novel therapeutic trials which include DNMTI therapy. Disclosures:Wang: Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc.: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Differential DNA Methylation Predicts Response To Combined Treatment Regimens With a DNA Methyltransferase Inhibitor In Acute Myeloid Leukemia (AML)

Deregulated epigenetic mechanisms have been identified as major components of acute myeloid leukemia (AML) pathogenesis. This improved mechanistic understanding has started to translate into clinics and leads to the development of novel therapeutic options as exemplified by the DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-azaC) and decitabine (DAC). However, biomarkers for response prediction to epigenetic therapy are urgently needed. Recently, we and others demonstrated that in-depth characterization of leukemia-associated DNA-methylation patterns contributes to refinement of the molecular classification and of prognostication in AML. Thus, disease associated methylation patterns might also harbor predictive relevance for identification of patients who will profit from DNMT inhibitor therapy and for support of therapeutic decision making.In order to identify a DNA methylation based response predictor, we applied a two-step strategy and generated genome-wide profiles underlying response and resistance to a combination chemotherapy applied within the AMLSG 12-09 Study (ClinicalTrials.gov Identifier: NCT01180322) comprising the drugs idarubicin and etoposide plus the demethylating agent 5-azaC as induction therapy. By methylated-CpG immune-precipitation and next generation sequencing (MCIp-seq), we generated DNA methylation profiles of responders (n=12) and non-responders (n=23). A supervised empirical Bayes approach for the analysis of sequencing read count data (“edgeR”) was applied to identify differentially methylated regions (DMRs) associated with 5-azaC response.We identified 550 genomic regions (based on 500 bp binning) that exposed highly significant read count differences indicating differential DNA methylation between both patient groups. The GC content distribution within the identified differentially methylated regions (DMRs) was comparable to the entire genome. 14% of the DMRs were located in gene promoter regions, 60% in intragenic and 26% in intergenic regions. Overall, the detected DMRs were considerably enriched in the vicinity of transcriptional start sites and preferentially targeted genes acting as transcriptional regulators (including transcription factors involved in hematopoiesis).Within the set of 550 DMRs, we selected the 40 most significantly discriminating regions and validated them with quantitative DNA methylation data from the Illumina Infinium® HumanMethylation450 Bead Chip. 25% of the selected DMRs were covered by only one probe whereas the majority was covered by up to six probes totaling in 107 probes (CpGs). We detected a good correlation between MCIp-seq und 450k-derived methylation data for each patient (median Spearman's rho = 0.69, 95%-CI [0.32, 0.87]) and could validate 90% of DMRs via quantitative 450k array data. Comprising 95 probes, these validated DMRs were used to create a multivariable signature for therapy response prediction.Through a penalized logistic regression model (“elastic-net”-penalty) applied to the 450k M-values in our discovery sample set, we identified a signature containing 17 probes (CpGs) associated with 12 genes which predicted response perfectly. Four of the identified CpGs were located in promoters, 11 in intragenic and two in intergenic regions. Among the genes targeted by differential methylation in our signature, we found WNT10A, a component of the WNT-beta-catenin-TCF signaling pathway, and PKMYT1. The latter one is a membrane-associated serine/threonine protein kinase which is regulated by polo-like kinase 1. Its inhibition has been reported recently to sensitize for cytarabine-mediated toxicity in vitro. Furthermore, two DMRs associated with the promoters of miRNAs (miR-3154, miR-3186) were contained in the signature.In summary, by genome-wide screening approaches, we identified differentially methylated genes and genomic regions that are associated with response to treatment regimens containing the DNMT inhibitor 5-azaC. At the same time, the predictive DMRs also harbor high potential to be functionally linked to molecular mechanisms and pathways involved in therapy response. By variable selection, we created a minimal signature that accurately predicts response in our discovery sample set. Further validation of this response-signature in independent cohorts of AML cases also comprising patients treated with decitabine are underway. Disclosures:Schlenk:Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Ambit: Honoraria.

Prospective serial evaluation of 2-hydroxyglutarate, during treatment of newly diagnosed acute myeloid leukemia, to assess disease activity and therapeutic response

Mutations of genes encoding isocitrate dehydrogenase (IDH1 and IDH2) have been recently described in acute myeloid leukemia (AML). Serum and myeloblast samples from patients with IDH-mutant AML contain high levels of the metabolite 2-hydroxyglutarate (2-HG), a product of the altered IDH protein. In this prospective study, we sought to determine whether 2-HG can potentially serve as a noninvasive biomarker of disease burden through serial measurements in patients receiving conventional therapy for newly diagnosed AML. Our data demonstrate that serum, urine, marrow aspirate, and myeloblast 2-HG levels are significantly higher in IDH-mutant patients, with a correlation between baseline serum and urine 2-HG levels. Serum and urine 2-HG, along with IDH1/2-mutant allele burden in marrow, decreased with response to treatment. 2-HG decrease was more rapid with induction chemotherapy compared with DNA-methyltransferase inhibitor therapy. Our data suggest that serum or urine 2-HG may serve as noninvasive biomarkers of disease activity for IDH-mutant AML.

Clinical Activity of DNA Methyltransferase Inhibitors In Therapy-Related Myelodysplastic Syndromes: A Retrospective Study

AbstractAbstract 1891Therapy-related myelodysplastic syndrome (tMDS) is a poor-risk subtype of MDS with no standard treatment options, and yet patients (pts) with tMDS are often excluded from trials where they would have the opportunity to benefit from novel treatment approaches. DNA methyltransferase inhibitors are a treatment option for tMDS, and are being evaluated as a bridge to stem cell transplant in these often heavily pre-treated patients to avoid the organ toxicity of intensive chemotherapy. However, the response rate of tMDS to DNA methyltransferase inhibitor (DNMTI) therapy is unknown. In this retrospective study, adult patients tMDS were culled from a fully annotated, IRB-approved database of all MDS patients who received either decitabine (DAC) on both the 3-day and 5-day schedules or 5-azacytidine (5-aza) at our institution from 4/8/2002 to 6/18/2010. Patients who received interrupted therapy were only analyzed for response to their initial course of therapy. Patients who received sequential DNMTI therapy (i.e., DAC followed by 5-aza or 5-aza followed by DAC) were included, but response to only their initial therapy was assessed. Responses were determined using the modified International Working Group criteria (Cheson BD, et al, 2006). Of the 35 patients initially identified with tMDS who received DNMTI therapy, 4 were deemed inevaluable for response due to marrow involvement with the primary malignancy (n= 1), missing records (n=2), or delivery of < 1 full cycle of therapy (n=1). The 31 evaluable pts included 14 males and 17 females with a median age of 65 years (range 25–85). Therapy for the primary malignancy included chemotherapy alone (n=13), chemotherapy plus radiotherapy (n=14), radioactive iodine and radiotherapy (n=2), radioactive iodine and chemotherapy (n=2), and autologous stem cell transplant (n=3). Prior to DNMTI therapy, the MDS FAB subtypes were as follows: RA, n= 6; RARS, n= 3; RAEB, n= 19; RAEBt, n=2; CMMoL, n=1. Pre-DNMTI therapy included lenalidomide (n=4) and alloSCT (n=1). Of the 31 evaluable patients, 20 received DAC, including 7 pts who received tretinoin with DAC in a clinical trial, and 11 received 5-aza. DAC recipients received a median of 2 cycles of therapy (range, 1–12) and 5-aza recipients received a median of 5 cycles (range, 1–9). Best responses were as follows: CR, n=1; Marrow CR plus HI, n=6 (3 trilineage HI, 1 HI-P+ HI-N, 2 HI-P); Stable Disease, n=6; Progressive Disease, n=6; Failures (death during 1st cycle or before response evaluation), n=3. Rate of CR + mCR was 22% (n=7). Additional patients had inevaluable (aparticulate) marrows, or refused follow-up marrow studies, but showed signs of stable (n= 3), improved, (n=2; HI-P, HI-P+HI-N), or progressive cytopenias (n= 3). Median time to best response was 1.5 cycles (range 1–6). Fifty-eight percent (n=18) of 31 pts achieved stable disease or better responses (including 4 pts with stable cytopenias or HI with inevaluable marrow response). Four patients proceeded directly to transplant after DNMTI therapy. Two subsequently died from relapsed disease after transplant, while 1 pt is lost to follow-up and 1 pt is without evidence of MDS 2.5 years after transplant. Nine pts had persistence of their primary malignancy during DNMTI therapy, and 5/9 pts required interruption or cessation of their DNMTI therapy because of progressive primary malignancy. 24/31 pts died from complications of MDS (n=5) or subsequent AML (11), complications of MDS/AML with likely contribution from their primary malignancy (n=4), infection during DNMTI nadir (n=2), GVHD post AlloSCT (n=1), or unknown reasons (n=1). Living pts (n=7; median follow-up from start of DNMTI therapy = 12.5 months, range 4.1 – 35.1 months) include 5 who are not transplant candidates. In conclusion, DNMTI therapy produced modest clinical benefit in our tMDS cohort. In some patients, persistence of the primary malignancy interfered with our ability to deliver optimal DNMTI therapy and to assess response. Although DNMTIs represent a potential therapeutic option for tMDS, treatment of a larger cohort is required to clarify the response rate of these agents in tMDS. Disclosures:Klimek:Celgene: Consultancy.

Prior Therapy with DNA Methyltransferase Inhibitors (DNMTI) Predicts for Lower Remission Rates and Worse Survival In Secondary Acute Myeloid Leukemia Patients (sAML) Receiving Remission Induction Therapy

AbstractAbstract 4033 Background:DNMTIs are the most commonly used disease modifying agents for myelodysplastic syndromes (MDS), particularly in patients (pts) with higher-risk MDS. Many will subsequently develop acute myeloid leukemia (AML) and will be treated with cytarabine-based remission induction therapy. The clinical impact of prior DNMTI therapy on the effectiveness of AML induction therapy is unknown for pts with AML arising secondary to MDS (sAML). Methods:We reviewed all pts treated with cytarabine-based induction chemotherapy for sAML arising from MDS at Cleveland Clinic between 2002 and 2009, years during which DNMTI therapy may have been used for MDS. Data on demographics, cytogenetic risk classification (per CALGB 8461), and disease duration were collected for MDS and for AML diagnoses. Response to DNMTI therapy (using International Working Group (IWG) 2006 criteria), complete response (CR) to induction chemotherapy (using IWG 2003 AML criteria), and overall survival (OS) were analyzed; comparisons between groups were performed using univariate and regression analyses, controlling for age, gender, white blood cell count, cytogenetics, reinduction, disease duration, and International Prognostic Scoring System (IPSS) score. Results:Of 330 AML pts treated with remission induction chemotherapy over the 7-year period, 27 had an antecedent MDS; 14 were treated with DNMTI prior to AML transformation, 13 were not. For these sAML pts, mean age was 67.5 years (range, 39–85), 11 (41%) were female, AML cytogenetic risk groups were intermediate (13), poor (10), and unknown (4), while IPSS cytogenetic risk groups were good (12), intermediate (7), poor (3), and unknown (5). Only 2 pts (7%) had a chromosome 7 abnormality. By MDS WHO classification, pts had RA/RARS (4), RCMD (6), RAEB-1 (6), RAEB-2 (9), and CMML-1 (2). Pts had MDS a median of 1.76 years (range .33-6.0). Compared to pts not treated with DNMTI, pts who received a DNMTI for MDS were younger (63 vs. 72 years, p=.013) but duration from MDS diagnosis to AML diagnosis was similar between groups (1.6 years vs 1.9 years, p=.60), as were other baseline factors identified above. Among sAML pts, reinduction rate for persistent leukemia was qualitatively higher in the DNMTI group (36% vs 8%), with a trend towards significance (p=.08). CR to AML induction for the sAML pts was 41% (compared to 52% for all AML patients ≥60 years who were induced), but was significantly worse in the DNMTI group (21% vs 62% for non-DNMTI, p=.034), and by log rank there was a trend toward worse OS in those treated with a DNMTI (.5 years vs 1.1 years, p=.11, similar to all AML patients ≥60 years, at .41 years). When compared to sAML without prior DNMTI, by logistic regression analyses, prior use of a DNMTI led to a worse CR rate to induction (hazard ratio .026, p=.047), and using Cox regression, prior use of a DNMTI led to worse OS (hazard ratio 22.95, p=.004). Within the MDS group treated with a DNMTI, achievement of complete or partial response by IWG MDS criteria was significantly correlated with CR to induction chemotherapy for AML (Pearson correlation coefficient .61, p=.021) but not with OS (Pearson correlation coefficient .40, p=.15). Conclusions:Induction therapy in sAML pts previously treated with DNMTI for MDS is less effective and associated with worse OS when compared to sAML pts not treated with DNMTI, controlling for baseline factors including MDS duration and IPSS score. However, within this poor-risk group, clinical response to DNMTI therapy is correlated with improved CR to AML induction therapy. Treatment alternatives to induction chemotherapy for AML after prior DNMTI therapy should be investigated. Disclosures:Mohan:Celgene: Research Funding; Eisai: Research Funding. Maciejewski:Celgene: Research Funding; Eisai: Research Funding. Sekeres:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. [Display omitted]