Michelle DeVeaux

DNA polymerase beta participates in DNA End-joining

Abstract DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol μ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol β), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol β, fewer small deletions are observed. In addition, depletion of Pol β results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol β in functions in aNHEJ and provide mechanistic insight into its role in this process.

Counseling patients with higher-risk MDS regarding survival with azacitidine therapy: are we using realistic estimates?

Azacitidine is the only drug proven to prolong overall survival (OS) in patients with higher-risk myelodysplastic syndromes (HR-MDS) in the large randomized trial AZA001 with a median OS of 24.5 months among azacitidinetreated patients vs. 15.0 months in the conventional care regimens (CCR) arm, with a hazard ratio of 0.58; p= 0.0001. Two-year OS probability was 51% in azacitidine arm compared with 26% in the CCR arm (p < 0.0001). These numbers are often quoted to counsel HR-MDS patients regarding expected benefits with azacitidine therapy. However, several real-life data and registry studies suggest that the median OS benefit with azacitidine is much lower (a median of 13–16 months) than what is suggested by AZA-001 trial. Additionally, in a recent large Surveillance, Epidemiology, and End Results (SEER)-Medicare-linked database study of 532 patients with refractory anemia and excessive blasts (RAEB), the median OS was found to be only 11 months [95% confidence interval (CI), 10–14 months] for azacitidine treated patients in the United States (US). Furthermore, comparing OS for all patients with MDS before the approval of azacitidine and decitabine (2001–2003) and after their approval (2007–2010) using SEER Medicare data shows that the OS for MDS patients has not improved substantially after the approval of the two hypomethylating agents azacitidine and decitabine in 2004 and 2006, respectively, in the US. When analysis was restricted to patients with RAEB, a proxy for HR-MDS, an improvement in OS of only 3 months was observed, which is quite small compared with the initial survival advantage of 9.5 months reported in the AZA-001 trial. Similarly, an analysis of 1000 MDS patients at the Mayo Clinic showed that OS has not significantly improved for patients with MDS over the last two decades. Certainly, the marked difference in median OS between the AZA-001 trial and real-life analyses can be attributed to many factors including the stricter selection of patients in clinical trials compared to registry studies, which often include older and frail patients. To get a more realistic estimate of OS with azacitidine and minimize the effect of selection bias when comparing clinical trial data with reallife data, we pooled OS data from these clinical trials including the landmark AZA-001 trial. First, we conducted a literature search for published prospective clinical trials that had an azacitidine monotherapy arm at the standard approved dose (75 mg/m/day for 7 days) in which OS results were presented in Kaplan–Meier (KM) methodology. Next, we used GetData Graph Digitizer Version 2.26 to digitize the published KM curves of the azacitidine monotherapy arms in these trials. An algorithm developed by Guyot et al. was implemented in the R statistical software to recreate individual patient level data based on the information from each KM curve, number of patients at risk, and number of events. Individual patient level data were pooled for patients receiving azacitidine to produce overall KM estimates and estimates of median OS and 1-, 2and 3-year OS probabilities. We found four published articles that fit the research criteria (Table 1). The baseline characteristics of patients who received azacitidine monotherapy in these trials are relatively comparable in terms of age, Eastern Cooperative Oncology Group Performance Status (ECOG

The use of immunosuppressive therapy in MDS: clinical outcomes and their predictors in a large international patient cohort

Most studies of immunosuppressive therapy (IST) in myelodysplastic syndromes (MDS) are limited by small numbers and their single-center nature, and report conflicting data regarding predictors for response to IST. We examined outcomes associated with IST and predictors of benefit in a large international cohort of patients with MDS. Data were collected from 15 centers in the United States and Europe. Responses, including red blood cell (RBC) transfusion independence (TI), were assessed based on the 2006 MDS International Working Group criteria, and overall survival (OS) was estimated by Kaplan-Meier methods. Logistic regression models estimated odds for response and TI, and Cox Proportional Hazard models estimated hazards ratios for OS. We identified 207 patients with MDS receiving IST, excluding steroid monotherapy. The most common IST regimen was anti-thymocyte globulin (ATG) plus prednisone (43%). Overall response rate (ORR) was 48.8%, including 11.2% (95% confidence interval [CI], 6.5%-18.4%) who achieved a complete remission and 30% (95% CI, 22.3%-39.5%) who achieved RBC TI. Median OS was 47.4 months (95% CI, 37-72.3 months) and was longer for patients who achieved a response or TI. Achievement of RBC TI was associated with a hypocellular bone marrow (cellularity < 20%); horse ATG plus cyclosporine was more effective than rabbit ATG or ATG without cyclosporine. Age, transfusion dependence, presence of paroxysmal nocturnal hemoglobinuria or large granular lymphocyte clones, and HLA DR15 positivity did not predict response to IST. IST leads to objective responses in nearly half the selected patients with the highest rate of RBC TI achieved in patients with hypocellular bone marrows.

Hypomethylating agents in relapsed and refractory AML: outcomes and their predictors in a large international patient cohort

Although hypomethylating agents (HMAs) are frequently used in the frontline treatment of older acute myeloid leukemia (AML) patients, little is known about their effectiveness in relapsed or primary treatment-refractory (RR)-AML. Using an international multicenter retrospective database, we studied the effectiveness of HMAs in RR-AML and evaluated for predictors of response and overall survival (OS). A total of 655 patients from 12 centers received azacitidine (57%) or decitabine (43%), including 290 refractory (44%) and 365 relapsed (56%) patients. Median age at diagnosis was 65 years. Best response to HMAs was complete remission (CR; 11%) or CR with incomplete count recovery (CRi; 5.3%). Additionally, 8.5% experienced hematologic improvement. Median OS was 6.7 months (95% confidence interval, 6.1-7.3). As expected, OS differed significantly by best response, with patients achieving CR and CRi having a median OS of 25.3 and 14.6 months, respectively. In multivariate analysis, the presence of ≤5% circulating blasts and a 10-day schedule of decitabine were associated with improved response rates, whereas the presence of >5% circulating blasts and >20% bone marrow blasts were associated with decreased OS. A significant subset of RR-AML patients (16%) achieved CR/CRi with HMAs and experienced a median OS of 21 months. Outside of a clinical trial, HMAs represent a reasonable therapeutic option for some patients with RR-AML.

DNA Polymerase Beta Germline Variant Confers Cellular Response to Cisplatin Therapy

Resistance to cancer chemotherapies leads to deadly consequences, yet current research focuses only on the roles of somatically acquired mutations in this resistance. The mutational status of the germline is also likely to play a role in the way cells respond to chemotherapy. The carrier status for the POLB rs3136797 germline mutation encoding P242R DNA polymerase beta (Pol β) is associated with poor prognosis for lung cancer, specifically in response to treatment with cisplatin. Here, it is revealed that the P242R mutation is sufficient to promote resistance to cisplatin in human cells and in mouse xenografts. Mechanistically, P242R Pol β acts as a translesion polymerase and prefers to insert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair (NER) pathway, removal of crosslinks, and resistance to cisplatin. In contrast, wild-type (WT) Pol β preferentially inserts the incorrect nucleotide initiating mismatch repair and cell death. Importantly, in a mouse xenograft model, tumors derived from lung cancer cells expressing WT Pol β displayed a slower rate of growth when treated with cisplatin, whereas tumors expressing P242R Pol β had no response to cisplatin. Pol β is critical for mediating crosstalk in response to cisplatin. The current data strongly suggest that the status of Pol β influences cellular responses to crosslinking agents and that Pol β is a promising biomarker to predict responses to specific chemotherapies. Finally, these results highlight that the genetic status of the germline is a critical factor in the response to cancer treatment. Implications: Pol β has prognostic biomarker potential in the treatment of cancer with cisplatin and perhaps other intrastrand crosslinking agents. Mol Cancer Res; 15(3); 269–80. ©2017 AACR.

The effect of iron chelation therapy on overall survival in sickle cell disease and β-thalassemia: A systematic review.

Red blood cell transfusions have become standard of care for the prevention of life‐threatening anemia in patients with β‐thalassemia and sickle cell disease (SCD). However, frequent transfusions can lead to accumulation of iron that can result in liver cirrhosis, diabetes mellitus, arthritis, arrhythmias, cardiomyopathy, heart failure, and hypogonadotropic hypogonadism. Iron chelation therapy has been shown to reduce serum ferritin levels and liver iron content, but limitations of trial design have prevented any demonstration of improved survival. The objective of this systematic review was to investigate the impact of iron chelation therapy on overall and event‐free survival in patients with β‐thalassemia and SCD. Eighteen articles discussing survival in β‐thalassemia and 3 in SCD were identified. Overall iron chelation therapy resulted in better overall survival, especially if it is instituted early and compliance is maintained. Comparative studies did not show any significant differences between available iron chelation agents, although there is evidence that deferiprone is better tolerated than deferoxamine and that compliance is more readily maintained with the newer oral drugs, deferiprone and deferasirox. Iron chelation therapy, particularly the second‐generation oral agents, appears to be associated with improved overall and event‐free survival in transfusion‐dependent patients with β‐thalassemia and patients with SCD.

Performance of the Medical Research Council (MRC) and the Leukemia Research Foundation (LRF) score in predicting survival benefit with hypomethylating agent use in patients with relapsed or refractory acute myeloid leukemia

Maximilian Stahl, Michelle DeVeaux, Pau Montesinos, Rapha€el Itzykson, Ellen K. Ritchie, Mikkael A. Sekeres, John Barnard, Nikolai A. Podoltsev, Andrew Brunner, Rami S. Komrokji, Vijaya R. Bhatt, Aref Al-Kali , Thomas Cluzeau, Valeria Santini, Gail J. Roboz, Pierre Fenaux, Mark Litzow , Amir T. Fathi, Sarah Perreault, Tae Kon Kim, Thomas Prebet , Norbert Vey, Vivek Verma, Ulrich Germing, Juan Bergua, Josefina Serrano, Steven D. Gore and Amer M. Zeidan Department of Internal Medicine, Section of Hematology, Yale University School of Medicine, New Haven, CT, USA; Department of Biostatistics, Yale School of Public Health, New Haven, CT, USA; Department of Medicine, University of Valencia, Hospital Universitario y Polit ecnico La Fe, Valencia, CIBERONC, Instituto III, Madrid, Spain; Department of Hematology/Oncology, Saint-Louis Hospital, University of Paris 7, France; Division of Hematology and Oncology, Weill Cornell Medicine and The New York Presbyterian Hospital, New York, NY, USA; Leukemia Program Cleveland Clinic, Cleveland, OH, USA; Massachusetts General Hospital Cancer Center Harvard Medical School, Boston, MA, USA; Department of Malignant Hematology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA; Division of Hematology/Oncology, University of Nebraska Medical Center, Omaha, NE, USA; Mayo Clinic, Rochester, MN, USA; Cote d’Azur University, Nice Sophia Antipolis University, CHU of Nice, Nice, France; Division of Hematology, University of Florence, Florence, Italy; Department of Pharmacy, Yale New Haven Hospital, New Haven, CT, USA; Department of Hematology, Institut Paoli Calmettes, Marseille, France; Department of Hematology, Oncology and Clinical Immunology, HeinrichHeine-University Duesseldorf, Duesseldorf, Germany; Hospital San Pedro Alc antara, C aceres, Spain; Division of Hematology/ Oncology, University Hospital Reina Sofia, Cordoba, Spain

Allogeneic Hematopoietic Stem Cell Transplantation Following the Use of Hypomethylating Agents among Patients with Relapsed or Refractory AML: Findings from an International Retrospective Study

Patients with primary refractory or relapsed acute myeloid leukemia (RR-AML) have very poor prognosis. Due to limited treatment options, some patients are treated with hypomethylating agents (HMAs) due to their tolerability. Little is known about the role of allogeneic hematopoietic stem cell transplantation (HSCT) following HMA therapy in this setting. We retrospectively analyzed an international cohort of 655 RR-AML patients who received HMA therapy to study patterns and outcomes with HSCT. Only 37 patients (5.6%) patients underwent HSCT after HMA therapy. The conditioning regimen was myeloablative in 57% and nonmyeloablative in 43%. Patients received matched unrelated donor, matched sibling, haploidentical and mismatched unrelated HSCT in 56%, 24%, 16% and 4% of cases, respectively. Acute GvHD and chronic GvHD were observed in 40% and 17% of patients. While the median OS for the entire cohort of patients was 15.3 months (95% CI 9.5 - 21.7 months), OS reached 29.7 months (95% CI 7.01 - not-reached) for patients who achieved a complete remission (CR) to HMA and no intervening therapies between HMA therapy and HSCT. Our study suggests that HMA therapy can effectively bridge some patients with RR-AML to HSCT.

Long-term follow-up of a single institution pilot study of sirolimus, tacrolimus, and short course methotrexate for graft versus host disease prophylaxis in mismatched unrelated donor allogeneic stem cell transplantation

Dear Editor, Allogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment for patients with hematologic malignancies [1]. However, overall survival (OS) is often compromised by treatment-related complications such as graftversus-host disease (GVHD). Recipients of fully HLAmatched sibling donors (MSD) have an acute GVHD rates between 30 and 40% and severe life-threatening acute GVHD may occur in 15% of patients [2, 3]. Many patients who could potentially benefit from allo-SCT do not have a MSD or a matched unrelated donor (MUD) based on highresolution molecular matching techniques [4]. Partially, HLAmismatched unrelated donor (MMUD) transplantation is a viable option for patients lacking a fully matched donor; however, HLA mismatches significantly increase the risk of GVHD and confer an inferior OS [5]. Treatment for acute GVHD is not successful in all patients and may impact long-term survival. Patients with grade III–IV acute GVHD who do not respond to frontline therapy with corticosteroids have only a 30% 2-year overall survival [6]. Pharmacologic immunosuppression is the most commonly applied method for prevention of GVHD and has been traditionally performed using a calcineurin inhibitor in combination with a short course of methotrexate [7, 8]. These regimens effectively prevent severe GVHD in the majority of MSD andMUD transplantation. However, there is no uniformly accepted guideline for GVHD prophylaxis for MMUD transplants. Sirolimus is an immunosuppressant derived from Streptomyces hygroscopicus [9], which is structurally similar to tacrolimus [10]. Sirolimus binds mammalian target of rapamycin (mTOR), that inhibits co-stimulatory pathway (e.g., CD28-AKT) and IL-2 driven pathway [11]. Sirolimus has potent anti-rejection activity in solid organ transplantation [12] and demonstrated activity as therapy of steroid-resistant GVHD [13]. Since tacrolimus and sirolimus have distinct mechanisms of action, combination therapy confers a synergistic effect [14, 15] to inhibit rejection in human organ allografting. Sirolimus does not cause nephrotoxicity and neurotoxicity, and is therefore less likely to cause synergistic adverse effects with a calcineurin inhibitor. The combination of sirolimus, tacrolimus, and low-dose methotrexate was initially tested as GVHD prophylaxis in both matched related and limited numbers of mismatched related transplants, with reported rates of overall acute GVHD of 26% and severe acute GVHD (grades III–IV) of 13% [16]. Here, we aimed to study the activity of sirolimus/ tacrolimus/low-dose methotrexate solely in MMUD transplantation and performed extended follow-up of patients registered in our pilot study. This pilot study enrolled 25 recipients of MMUD allografts recruited at Yale University between 2008 and 2011.Wemonitored the efficacy of a regimen of sirolimus, tacrolimus, and methotrexate as GVHD prophylaxis for MMUD allo-grafting with extended follow-up until 2015. This study was approved by the Institutional Review Board of Yale University and * Stuart Seropian stuart.seropian@yale.edu

Abstract 2492: DNA polymerase beta participates in DNA end-joining

DNA double strand breaks (DSBs) are one of the most deleterious lesions. If left unrepaired, DSBs lead to genomic instability and carcinogenesis. The cells combat DSBs by two classical pathways that include homologous recombination (HR) which requires the sister chromatid for sequence homology, and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred non-canonical NHEJ which has been given many names, such as alternative NHEJ or microhomology-mediated end joining (MMEJ). The enzymatic mechanisms of non-canonical NHEJ are not well defined. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes in the pathway are poorly understood. Recent studies have implicated a role for DNA polymerase theta (Pol θ), an A-family polymerase is essential for non-canonical NHEJ pathway. Emerging evidence indicates X-family polymerases, mammalian DNA polymerases lambda (λ) and mu (μ) and yeast Pol 4 promote DNA end-joining. Our laboratory has recently provided evidence for a role for DNA polymerase beta (Pol β), another X-family polymerase, in V(D)J recombination, a process that requires end-joining. Here, using a recently developed fluorescence based assay that monitors non-canonical NHEJ and HR, we provide evidence that Pol β plays a role in the non-canonical NHEJ process. DNA sequencing at the break point junctions revealed Pol β-depleted cells have fewer small deletions than control cells, but significantly greater numbers of insertions and large deletions. We further demonstrate that Pol β-depleted cells have increased sensitivity to DNA damaging agents that induce double-strand breaks and that there is persistent accumulation of DSBs in these cells. In combination, our results suggest that Pol β is critical for double-strand break repair. Citation Format: Sreerupa Ray, Michelle DeVeaux, Gregory Breuer, Ranjit Bindra, Daniel Zelterman, Joann Sweasy. DNA polymerase beta participates in DNA end-joining [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 2492. doi:10.1158/1538-7445.AM2017-2492