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The key epigenetic regulator DNA methyltransferase 1 (DNMT1) is a scientifically validated target in p53-null chemorefractory cancers like pancreatic ductal adenocarcinoma (PDAC) since DNMT1-depletion effects cancer cell cycle exits by p53-independent epithelialization. DNMT1 can be depleted by the pyrimidine nucleoside analog pro-drugs decitabine (Dec) or 5-azacytidine (5Aza). However, PDAC clinical trials with Dec/5Aza disappointed. In pre-clinical and clinical analyses, we found resistance was caused by configurations of pyrimidine metabolism in PDAC cells that forestall Dec or 5Aza processing into DNMT1-depleting nucleotide: high expression of cytidine deaminase (CDA) that rapidly catabolizes Dec/5Aza; and suppression of deoxycytidine kinase (DCK) and uridine kinase 2 (UCK2) that rate limit Dec/5Aza pro-drug processing respectively. Accordingly, combination of Dec with a CDA clinical inhibitor, tetrahydrouridine (THU), enabled DNMT1-depletion and PDAC cytoreduction in vitro and in Dec/gemcitabine-refractory PDAC pre-clinical in vivo models. We then conducted a pilot clinical trial in 13 patients with chemorefractory PDAC given oral THU ~10 mg/kg/day combined with decitabine ~0.2 mg/kg/day, for 5 consecutive days, then twice weekly. This Phase 2 was based on several PK/PD studies in human subjects showing potent non-cytotoxic DNMT1-targeting in myeloid cells. Yet again, there were no meaningful clinical responses in the patients. A reason for this was a surprising lack of neutropenia, the most sensitive indicator of systemic DNMT1-targeting. Upon measuring plasma CDA enzyme activity, we found a >10-fold increase in patients with metastatic vs resectable PDAC. Thus, CDA activity is increased not only locally but also systemically in metastatic PDAC, suggesting a need for higher THU doses. We have also observed DCK downregulation, necessary for Dec/gemcitabine uptake and processing, as a cause of PDAC resistance to Dec/gemcitabine. To counter this mechanism, we discovered that 5Aza upregulates DCK as an adaptive response to 5Aza-mediated decrease in dCTP, while Dec upregulates UCK2 (that mediates 5Aza uptake) as an adaptive response to Dec mediated reductions in dTTP. Thus, we alternated Dec with 5Aza in an in vivo model of gemcitabine-resistant PDAC, to exploit their mutual cross-priming, together with THU to inhibit CDA: median vehicle control tumor measurements 972 mm3(range 726-1267.5); median THU-Dec/THU-5Aza 16 mm3 (range 0-87.5); P Citation Format: Rita Tohme, Francis Enane, Caroline Schuerger, Xiaorong Gu, Melissa Fishel, John Pink, Daniel Lindner, Davendra Sohal, Yogen Saunthararajah. Advancing non-cytotoxic DNMT1-targeting to treat chemorefractory pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1088.

The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow–derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine–treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98–HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846–57. ©2017 AACR.

Background: A deeper molecular understanding of cancer biology has led to the development of therapies targeting driver mutations. Genomic profiling of tumors is commercially available and has become integrated into many clinical practices as part of a paradigm shift towards personalized care of cancer patients. The current impact of genomic profiling on clinical decision making for patients with advanced breast cancer is not well described. Methods: Patients with metastatic breast cancer (mBC) who had tumors submitted for commercial genomic analysis from 2013-2015 were identified consecutively at two large academic cancer centers with genomic basket trials open for the majority of the collection period. Demographics, tumor pathology, clinical, and treatment histories were extracted through medical chart review as per an IRB approved protocol. Data from genomic analysis reports was extracted including number and type of mutations, FDA approved therapies and clinical trials available. Genomic analysis was determined to have impacted clinical decision making if the next line of therapy was influenced either by accrual to clinical trial, or a decision to prescribe an FDA-approved therapy. The most frequent somatic mutations and their relative frequencies were determined. Results: A total of 82 patients with mBC who had undergone commercially available genomic testing were identified. The median age was 49 (range: 29-70). 42 patients (51%) had ER-positive HER2-negative disease, 33 (40%) had ER-negative HER2-negative disease, 4 (5%) had ER-negative HER2-positive disease and 3 (4%) had ER-positive HER2-positive disease. The median number of lines of therapy received prior to genomic profiling was 2 (range 0-15). Genomic analysis reports suggested that 61 (74%) of these patients had at least one FDA approved medication available for at least one somatic mutation, and 79 (96%) had at least one clinical trial available (39 (46%) in the same state, 11 (13%) in the same institution). Genomic testing influenced management in 8 patients (10%), with 6 patients (7%) experiencing a change in next line of therapy attributable to genomic information. In 74 patients (90%), genomic testing results did not affect clinical decision-making. The most frequently observed somatic mutations included: TP53, PI3KCA, MYC, CCDN1, FGF, ZNF703, GATA3, ARID1A, MCL1, PTEN, MYST3, and BRCA1. Conclusions: Genomic testing did not affect management in the vast majority of mBC patients treated at two major academic cancer centers. Furthermore, the most identified mutated genes found were not targetable. The real world clinical utility of genomic analysis remains limited in breast cancer but may influence clinical decision making in a minority of patients. Citation Format: Kruse ML, Santa-Maria CA, Raska P, Swoboda A, Jain S, Sohal D, Moore H, Budd GT, Abraham J, Montero AJ. Impact of genomic medicine on clinical decision making in patients with advanced breast cancer at two academic medical centers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-24.

Modern cancer care has seen an increase in the use of targeted therapies and the number of FDA-approved biomarkers. To take advantage of these options, many have turned to commercially available comprehensive genome profiling (CGP) services, like Foundation Medicine's FoundationOne assay. FoundationOne (FO) offers a list of clinically relevant genomic alterations and biomarkers, as well as a list of clinical trials, on-label and off-label therapies that may benefit the patient, that is, therapeutic associations. We performed a retrospective cohort study to assess the clinical benefit of therapy associations from FO reports. We collected demographics, therapy records, and clinical outcomes for 1004 patients who had a history of advanced cancer in the Cleveland Clinic system and had received a FO report between 2012 and 2017. FO therapy associations were considered as “applied” if a patient received an order for said therapy for the first time after their report date. We classified patients as follows: No Associated Therapy (NAT) if the patient did not receive any recommendations, Therapy Applied (TA) if at least one associated therapy was ordered after report date, and Therapy Not Applied (TNA) if none of the associated therapies were applied. We evaluated differences in demographic and clinical features among the 3 groups using Chi-squared and Kruskal-Wallis tests where appropriate. We performed survival analysis using univariate and multivariate COX Proportional Hazards regression models, and Log-Rank tests on Kaplan Meier Curves with overall survival (OS) as our metric. Our cohort demographics were 55% male, 85% white, 92% non-Hispanic, and a median age at report of 60 (IQR: 51-69). The most common diagnoses were Lung Adenocarcinoma (14%), Glioblastoma (8%), Colon Adenocarcinoma (8%), and Breast Cancer NOS (4%). Most of our patients belonged to the TNA class (64%), 21% were NAT, and 15% were TA. There was no statistical significance in the demographic distribution among the therapy classes. In the pan-cancer analysis, we found no statistically significant difference in OS among the therapy groups or any of the adjusted covariates except for metastasis status. Similarly, when evaluating the top cancer diagnoses individually, we found no significant differences in OS. The data in our study indicates that the application of FO therapy associations is not correlated with a statistically significant difference in OS for advanced cancer patients. This suggests that larger studies should be performed to better understand how CGP services provide clinical benefits to patients, and how we can maximize these benefits in the real-world community setting. Citation Format: Jean Rene Clemenceau, Sung Hak Lee, Alex Milinovich, Jian Jin, Nathan Pennell, Davendra Sohal, Tae Hyun Hwang. Analysis of the clinical benefit of comprehensive genome profiling derived therapeutic associations in advanced cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3638.

Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations. Cancer Res; 73(3); 1076–85. ©2012 AACR.

The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow (BM) stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of BM stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, can potentially affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of BM stroma in MDS. To accomplish this aim, adherent non-hematopoietic cells were isolated from MDS patients and controls by immunomagnetic CD45 negative selection and then used for whole genome methylation studies using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR) . Global epigenetic profiling revealed that MDS stroma (n=7) was epigenetically distinct from normal BM stroma (n=3) (ANOVA, P In subsequent studies, we profiled stroma from another set of MDS patients treated with the DNMT inhibitor, 5-Azacytidine (5-Aza) (n=4). In contrast to untreated MDS patients, the 5-Aza treated MDS patients and healthy controls (p = NS) were epigenetically similar. These 5-Aza exposed stroma cells did not demonstrate increased cytosine methylation thus suggesting that these cells were also targeted by DNMT depletion by the drug. Finally, the ability of MDS and control stromal cells to support the growth of healthy CD34+ cells was tested by in-vitro colony formation assay. MDS stromal cell co-culture led to significant reduction in erythroid and myeloid colony formation, demonstrating the MDS stroma contributes to ineffective hematopoiesis seen in this disease. Strikingly, pretreatment of MDS stromal cells with 5-aza increased the colony formation seen in co-cultures, demonstrating again that DNMT inhibitors exert effects on the microenvironment. Thus our results reveal for the first time that MDS is characterized by widespread aberrant epigenetic changes in the BM microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, contributing in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire BM, including the microenvironment to improve our understanding of the pathophysiology of MDS and therapy. Citation Format: Tushar D. Bhagat, Matthias Bartenstein, Yiting Yu, A. Mario Marcondes, Davendra Sohal, John Greally, H Joachim Deeg, Amit Verma. Myelodysplasia bone marrow stromal cells have widespread aberrant hypermethylation that is targeted by treatment with DNMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1503. doi:10.1158/1538-7445.AM2013-1503

The microenvironment where a tumor originates plays an important role in its initiation, growth, progression and metastatic capability. Since in most cancers the microenvironment is not derived from the malignant clone and does not contain oncogenic mutations, it is likely that the tumor microenvironment is reprogrammed epigenetically to support the growth of the tumor. To test this hypothesis, Fibroblasts associated with primary pancreatic adenocarcinomas (N=7) & healthy fibroblast controls (N=4) were analyzed for genome wide alterations in DNA cytosine methylation by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR). Methylome profiling revealed widespread aberrant cytosine methylation in Pancreatic cancer associated fibroblasts (CAFs) with demethylation of many important gene promoters as the predominant epigenetic event. In addition to loss of methylation, aberrant hypermethylation of GALNTS, JAZF1, MTCH1, SP8, GRB14 was also seen in CAFs. In addition to epigenetic differences, pancreatic CAFs were also found to have widespread transcriptmoic differences as seen by parallel gene expression profiling. Next, we analyzed tumor mediated reprogramming of microenvironment at a higher resolution by a Massive parallel sequencing based methylation analysis (HELP-Tagging) on CAFs differentiated from mesenchymal stem cells (MSCs) in the presence of pancreatic cancer cell conditioned medium. HELP-tagging showed widespread epigenetic reprogramming with 11,100 hypomethylated and 1709 hypermethylated loci. Comparison of in-vitro reprogramed loci with the aberrantly methylated loci from primary CAFs showed a core set of 140 loci that were commonly differentially methylated in both samples. The chemokine receptor CXCR4 was observed to overexpressed & demethylated in both cohorts & was found to be expressed on the surface of primary pancreatic CAFs by immunohistochemistry. Functional studies demonstrated that co-culture of pancreatic cancer cells with CAFs (from MSCs) led to significant increase in malignant cell invasion when compared to co-culture with naïve MSCs. This increased invasion was abrogated by blockade of CXCR4 by AMD-3100 and by knockdown of CXCR4 by siRNAs in orthotopically derived CAFs; demonstrating a critical role for this receptor in regulating tumor promoting abilities of the microenvironment. Thus our results reveal for the first time that pancreatic CAFs are characterized by widespread epigenomic reprogramming that includes loss of methylation at many important loci. Validation of an aberrantly demethylated target, CXCR4, shows that inhibition of this receptor can abrogate the ability of CAFs in promoting cancer cell invasion. These results also provide a preclinical rationale for the use of clinically relevant CXCR4 antagonist AMD-3100 (plerixafor) in pancreatic cancer. Citation Format: Tushar D. Bhagat, Yanique Rattigan, Brijesh Patel, Strepell Mirte, Yiting Yu, Davendra Sohal, Matthias Bartenstein, Orsolya Giricz, Shanisha AK Gordon, Nishanth Vallumsetla, Rahul Polineni, Meher Walia, Paraic Kenny, John Greally, Debabrata Banerjee, Anirban Maitra, Amit Verma. Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2013-1504