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Supplementary Material and Methods

DNA methylation aberrations have been implicated in acquired resistance to platinum drugs in ovarian cancer. In this study, we elucidated an epigenetic signature associated with platinum drug resensitization that may offer utility in predicting the outcomes of patients who are coadministered a DNA methyltransferase inhibitor. The ovarian cancer specimens we analyzed were derived from a recent clinical trial that compared the responses of patients with recurrent platinum-resistant ovarian cancer who received carboplatin plus the DNA methyltransferase inhibitor guadecitabine or a standard-of-care chemotherapy regimen selected by the treating physician. Tumor biopsies or malignant ascites were collected from patients before treatment (day 1, cycle 1) or after treatment (after 2 cycles) for epigenomic and transcriptomic profiling using the Infinium HumanMethylation450 BeadChip (HM450). We defined 94 gene promoters that were hypomethylated significantly by guadecitabine, with 1,659 genes differentially expressed in pretreatment versus posttreatment tumors. Pathway analysis revealed that the experimental regimen significantly altered immune reactivation and DNA repair pathways. Progression-free survival correlated with baseline expression levels of 1,155 genes involved in 25 networks. In functional investigations in ovarian cancer cells, engineered upregulation of certain signature genes silenced by promoter methylation (DOK2, miR-193a, and others) restored platinum drug sensitivity. Overall, our findings illuminate how inhibiting DNA methylation can sensitize ovarian cancer cells to platinum drugs, in large part by altering gene expression patterns related to DNA repair and immune activation, with implications for improving the personalized care and survival outcomes of ovarian cancer patients.Significance: Epigenomic targeting may improve therapeutic outcomes in platinum-resistant and recurrent ovarian cancer in part by effects on DNA repair and antitumor immune responses. Cancer Res; 78(3); 631–44. ©2017 AACR.

Table S3. 77 integrated genes

Figure S1. (A) Predicted cancer/stromal signatures in TCGA and our data. (B) Correlation between NMF and ESTIMATION estimated cancer cell purity in TCGA OV data. (C,D) Predicted relative proportion of cancer/stromal cell in our and TCGA data. Figure S2. Full list of pathways enriched by differentially expressed genes induced by guadecitabine. Figure S3. A-D. Spearman correlation between the beta values of 24 CpG sites within the promoters of 19 genes significantly demethylated in ascites samples and predicted proportions of B cells (A), T cells (B), NK cells (C), and total cells (D). E-H. Spearman correlation between the beta values of 352 CpG sites within the promoters of 94 genes significantly demethylated in tumor samples and predicted proportion of B cells (E), T cells (F), NK cells (G), and total cells (H). Complete sets of correlation coefficients are provided in Supplementary Table 8. X axis corresponds to Spearman correlation coefficients.

Table S1. Genes demethylated by Guadecitabine Table S2. Differentially expressed genes

Table S4. 1155 genes associated with PFS Table S5. 203 guadecitabine deM genes associated with PFS Table S6. 293 guadecitabine upregulated genes associated with PFS

Table S7. 165 cancer and 218 stromal markers

Table S8. deconvolution_results

Purpose: Platinum resistance in ovarian cancer is associated with epigenetic modifications. Hypomethylating agents (HMA) have been studied as carboplatin resensitizing agents in ovarian cancer. This randomized phase II trial compared guadecitabine, a second-generation HMA, and carboplatin (G+C) against second-line chemotherapy in women with measurable or detectable platinum-resistant ovarian cancer. Patients and Methods: Patients received either G+C (guadecitabine 30 mg/m2 s.c. once-daily for 5 days and carboplatin) or treatment of choice (TC; topotecan, pegylated liposomal doxorubicin, paclitaxel, or gemcitabine) in 28-day cycles until progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS); secondary endpoints were RECIST v1.1 and CA-125 response rate, 6-month PFS, and overall survival (OS). Results: Of 100 patients treated, 51 received G+C and 49 received TC, of which 27 crossed over to G+C. The study did not meet its primary endpoint as the median PFS was not statistically different between arms (16.3 weeks vs. 9.1 weeks in the G+C and TC groups, respectively; P = 0.07). However, the 6-month PFS rate was significantly higher in the G+C group (37% vs. 11% in TC group; P = 0.003). The incidence of grade 3 or higher toxicity was similar in G+C and TC groups (51% and 49%, respectively), with neutropenia and leukopenia being more frequent in the G+C group. Conclusions: Although this trial did not show superiority for PFS of G+C versus TC, the 6-month PFS increased in G+C treated patients. Further refinement of this strategy should focus on identification of predictive markers for patient selection.

Ovarian cancer (OC) cells frequently metastasize to the omentum, and adipocytes play a significant role in ovarian tumor progression. Therapeutic interventions targeting aberrant DNA methylation in ovarian tumors have shown promise in the clinic, but the effects of epigenetic therapy on the tumor microenvironment are understudied. Here, we examined the effect of adipocytes on OC cell behavior in culture and impact of targeting DNA methylation in adipocytes on OC metastasis. The presence of adipocytes increased OC cell migration and invasion, and proximal and direct coculture of adipocytes increased OC proliferation alone or after treatment with carboplatin. Treatment of adipocytes with hypomethylating agent guadecitabine decreased migration and invasion of OC cells toward adipocytes. Subcellular protein fractionation of adipocytes treated with guadecitabine revealed decreased DNA methyltransferase 1 (DNMT1) levels even in the presence of DNA synthesis inhibitor, aphidicolin. Methyl-Capture- and RNA-sequencing analysis of guadecitabine-treated adipocytes revealed derepression of tumor-suppressor genes and epithelial–mesenchymal transition inhibitors. SUSD2, a secreted tumor suppressor downregulated by promoter CpG island methylation in adipocytes, was upregulated after guadecitabine treatment, and recombinant SUSD2 decreased OC cell migration and invasion. Integrated analysis of the methylomic and transcriptomic data identified pathways associated with inhibition of matrix metalloproteases and fatty acid α-oxidation, suggesting a possible mechanism of how epigenetic therapy of adipocytes decreases metastasis. In conclusion, the effect of DNMT inhibitor on fully differentiated adipocytes suggests that hypomethylating agents may affect the tumor microenvironment to decrease cancer cell metastasis. Implications: Epigenetic targeting of tumor microenvironment can affect metastatic behavior of ovarian cancer cells. Mol Cancer Res; 16(8); 1226–40. ©2018 AACR.

Purpose: PARP inhibitors (PARPi) are primarily effective against BRCA1/2-mutated breast and ovarian cancers, but resistance due to reversion of mutated BRCA1/2 and other mechanisms is common. Based on previous reports demonstrating a functional role for DNMT1 in DNA repair and our previous studies demonstrating an ability of DNA methyltransferase inhibitor (DNMTi) to resensitize tumors to primary therapies, we hypothesized that combining a DNMTi with PARPi would sensitize PARPi-resistant breast and ovarian cancers to PARPi therapy, independent of BRCA status. Experimental Design: Breast and ovarian cancer cell lines (BRCA-wild-type/mutant) were treated with PARPi talazoparib and DNMTi guadecitabine. Effects on cell survival, ROS accumulation, and cAMP levels were examined. In vivo, mice bearing either BRCA-proficient breast or ovarian cancer cells were treated with talazoparib and guadecitabine, alone or in combination. Tumor progression, gene expression, and overall survival were analyzed. Results: Combination of guadecitabine and talazoparib synergized to enhance PARPi efficacy, irrespective of BRCA mutation status. Coadministration of guadecitabine with talazoparib increased accumulation of ROS, promoted PARP activation, and further sensitized, in a cAMP/PKA-dependent manner, breast and ovarian cancer cells to PARPi. In addition, DNMTi enhanced PARP “trapping” by talazoparib. Guadecitabine plus talazoparib decreased xenograft tumor growth and increased overall survival in BRCA-proficient high-grade serous ovarian and triple-negative breast cancer models. Conclusions: The novel combination of the next-generation DNMTi guadecitabine and the first-in-class PARPi talazoparib inhibited breast and ovarian cancers harboring either wild-type– or mutant-BRCA, supporting further clinical exploration of this drug combination in PARPi-resistant cancers. Clin Cancer Res; 24(13); 3163–75. ©2018 AACR.

Background: The RAS-RAF-MEK-ERK pathway is commonly upregulated in human cancers. This is an open-label Phase 1 study of ASTX029, a dual-mechanism extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, in subjects with relapsed/refractory solid tumors (NCT03520075). Methods: The primary objectives are to identify a maximum tolerated dose and/or recommended Phase 2 dose. ASTX029 was administered orally daily of 21-day cycles as powder-in-bottle (PiB, Cohort 1/10mg) and tablet formulation (beginning with Cohort 6/80 mg) under fed conditions, and as tablet formulation under fasting conditions (beginning with Cohort 8/40 mg). Dose escalation occurred according to a “3+3 design” based on dose-limiting toxicity (DLT) events. Disease response was evaluated according to RECIST v1.1 and exploratory indicators, including tumor variant allele frequency changes detected by cell-free DNA (cfDNA) quantitation. Results: 56 subjects were treated with at least one dose of ASTX029 in Phase 1A (dose escalation). Of 46 subjects with data, 35 (76%) had any RAS mutations and 4 (9%) had BRAF mutations; 1 subject had both. At the 200 mg dose level (Cohort 5, PiB/fed), one of six evaluable subjects developed a DLT (grade 3 maculopapular rash). At the 280 mg dose level (Cohort 12, tablet/fasting), two subjects experienced grade 2 central serous retinopathy adverse events (CSR AEs) within a few days of dosing. These were the only CSR AEs noted and one event was declared a DLT. Both subjects recovered to baseline within days of dose interruption. One cohort level below this dose was expanded (Cohort 11/200 mg, tablet/fasting); this dose level was deemed safe (without a DLT or grade ≥2 visual AE in 7 subjects) and was selected for Phase 1B dose expansion. Mean pharmacokinetic (PK) exposure was 151% of target exposure, which is defined as the level expected to have biological activity based on animal studies. The most frequent grade ≥2 AEs assessed as drug-related included nausea (4 subjects, grade 2) and transaminitis (4 subjects: 3 grade 2, 1 grade 3). The grade 3 transaminitis occurred in a subject with metastatic sarcoma involving the liver. There was one serious AE of malaise considered related to study drug. Two subjects, one with KRAS-G12A and BRAF-D549N non-small cell lung cancer (120 mg) and one with KRAS-G12D metastatic pancreatic cancer (200 mg), achieved partial responses (cycle 15/ongoing and cycle 3/ongoing, respectively). In 2 subjects with stable disease as the best response, longitudinal cfDNA sequencing showed a decrease of tumor variant allele frequencies after 2 cycles of ASTX029, followed by a return to baseline levels before disease progression. The most common reason for ASTX029 discontinuation was disease progression. Conclusions: This Phase 1A study of the ERK1/2 inhibitor ASTX029 has identified a dose level of 200 mg daily of a 21-day cycle for investigation in the Phase 1B portion of the study. Pharmacokinetic and pharmacodynamic data suggest target exposures are achieved with preliminary clinical activity. Citation Format: Patricia LoRusso, Drew Rasco, Geoffrey Shapiro, Filip Janku, Alain Mita, Nilofer Azad, Marcia Toguchi, Chris Hindley, Shannon Bradley, Danna Chan, Harold Keer, Kim-Hien Dao, Ryan J. Sullivan, Alexander Spira. A first-in-human, Phase 1 study of ASTX029, a dual-mechanism inhibitor of ERK1/2, in relapsed/refractory solid tumors [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 CT108.

Whilst mitogen-activated protein kinase (MAPK) pathway inhibitors are approved therapies in mutant-BRAF driven cancers and have demonstrated a high response rate in the clinic, the duration of response is often short-lived. Approaches targeting the immune system have elicited durable responses and led to the approval of checkpoint inhibitors such as the anti-PD1 therapy, pembrolizumab, in indications where MAPK pathway activation is often observed, such as melanoma. Activation of the MAPK pathway has been associated with an immunosuppressive tumor microenvironment (TME). Further, preclinical studies have demonstrated that in addition to inhibiting MAPK activity in tumor cells, MAPK pathway inhibitors such as the BRAFV600E inhibitor dabrafenib or MEK inhibitor trametinib have promoted a more proinflammatory TME leading to upregulated antigen presentation on tumor cells, increased CD8+ T cell infiltration and tumor cell killing. Similar preclinical results were recently reported for a KRASG12C inhibitor, AMG 510, where treatment of in vivo models led to an increase in tumor infiltrating lymphocytes, macrophages, dendritic cells and an increase in active immune gene signatures leading to tumor immunity. We have recently described the discovery of ASTX029, which is currently undergoing clinical development in advanced solid tumors (NCT03520075). ASTX029 is a dual-mechanism ERK1/2 (ERK) inhibitor, inhibiting both the catalytic activity and phosphorylation of ERK, and shows potent inhibition of MAPK-activated tumor growth in preclinical models. Previous studies have demonstrated a difference in regulation of genes involved in response to type I interferon following treatment with dual-mechanism compared to catalytic ERK inhibitors. We investigated whether treatment with ASTX029 modulates antigen presentation and the TME in MAPK-activated tumors. Following treatment of cell lines in vitro, we observed ASTX029-dependent changes consistent with increased antigen presentation, including an increase in cell surface expression of MHC class I and the increase in gene expression of tumor-specific antigens gp100 and MART-1 following treatment of melanoma cells. We have previously demonstrated that ASTX029 has good bioavailability following oral dosing in mice. To further investigate the effect of ASTX029 on the TME, syngeneic tumors grown in immunocompetent mice dosed with ASTX029 were characterized in terms of immune cell composition of the TME and gene expression. Our results were consistent with increased immune activation, including increased interferon signaling and a change in the immune cell composition of the TME. These data demonstrate that treatment with ASTX029 leads to modulation of the TME and we hypothesize that to optimize therapeutic activity, ASTX029 could be partnered either with an immunomodulatory or tumor-directed agent. Citation Format: Chris Hindley, Andrea Biondo, Kim-Hien Dao, Matthew Davis, Lynsey Fazal, Roberta Ferraldeschi, Keisha Hearn, Vanessa Martins, Harpreet Saini, Martin Sims, Nicola Wallis, Nicola Wilsher, Harold Keer, John Lyons, Joanne Munck. Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated tumor models [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 1661.

Background: Therapeutic targeting of the PD-1/PDL-1 axis significantly increases the survival of melanoma (MM) and non-small cell lung cancer (NSCLC) patients. Despite this unprecedented efficacy, a sizeable proportion of MM and NSCLC patients fails to benefit from therapy due to primary or secondary resistance to treatment. In this scenario, deepening the knowledge on mechanism(s) underlying treatment failure allows to design novel, mechanism-based, therapeutic approaches to overcome resistance to anti-PD-1/PDL-1 therapy. Along this line, we have extensively characterized the immunomodulatory properties of DNA hypomethylating agents (DHA) in different human malignances. Exposure of neoplastic cells to DHA efficiently improved antigen-restricted and -unrestricted T cell recognition of cancer cells in vitro through the upregulation/induction of the expression of epigenetically regulated tumor associated antigens, HLA class I and/or accessory/co-stimulatory molecules by neoplastic cells. Supporting these ex vivo data, combining the systemic administration of DHA (ie, decitabine or guadecitabine) with antibodies to different immune checkpoints significantly reduced tumor growth of murine syngeneic grafts, compared to single-agent therapy. Supporting these pre-clinical findings, our phase Ib NIBIT-M4 study has been the first clinical trial demonstrating that systemic administration of guadecitabine followed by CTLA-4 blockade with ipilimumab is safe and tolerable in MM patients, shows a promising anti-tumor, and induces a significant modulation of immune related pathways in tumor samples (Di Giacomo AM, Clin Cancer Res 2019). Prompted by these results and to further explore the efficacy of DHA combined with immune-checkpoints blockade we have designed and activated the NIBIT-ML1 trial. This study aims at investigating the efficacy of guadecitabine combined with ipilimumab and nivolumab in reverting the resistance to PD-1/PDL-1 therapy of MM and NSCLC patients. Methods: The NIBIT-ML1 is a randomized, phase II study designed according to the two stages optimal design by Simon, in unresectable Stage III or Stage IV MM (Cohort A) or NSCLC (Cohort B) patients who failed therapy with anti-PD-1/PDL-1 as last treatment. Primary objective of the study is immune (i)-ORR according to iRECIST criteria. Secondary objectives include safety, DCR, PFS, median OS, and survival rate at 1 and 2-years. Extensive cellular and molecular immune correlates will also be explored. Following a safety run-in phase in 6 subjects per Cohort, eligible patients will be randomized to receive: guadecitabine plus ipilimumab and nivolumab (ARM A) or ipilimumab and nivolumab (ARM B). Sample size will range from 6 to 92 patients per Cohort. The first patient first visit is foreseen by March 2020 (NCT04250246) Citation Format: Anna Maria Di Giacomo, Luana Calabrò, Riccardo Danielli, Monica Valente, Elisabetta Gambale, Sandra Coral, Giovanni Amato, Harold Keer, Diana Giannarelli, Mohammad Azab, Andrea Anichini, Alessia Covre, Michele Maio. A randomized, multi-center, phase II study of nivolumab combined with ipilimumab and guadecitabine or nivolumab combined with ipilimumab in melanoma and NSCLC patients resistant to anti-PD-1/-PD-L1: The NIBIT-ML1 Study [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 CT270.

Epithelial ovarian cancer (OC) is the most lethal gynecologic malignancy. The majority of advanced stage patients develop uniformly fatal resistance to standard platinum-based treatments. Epigenetic changes, particularly DNA methylation aberrations have been implicated in acquired resistance to platinum in OC. The goal of the current study was to identify methylation changes associated with the development of acquired resistance to platinum-based chemotherapy. We hypothesized that OC tumors harboring an aberrant DNA methylome could be reversed by DNMTi and re-sensitized to platinum treatment. To achieve our objective, we generated and compared tumor DNA methylation profiles from patients with recurrent platinum-resistant OC treated on a clinical trial with a DNA methyl transferase inhibitor, guadecitabine, before (n=65) and after (n=19) treatment (NCT01696032), and patients with primary (platinum-naïve) OC (n=20). Human ovarian surface epithelial cells (HOSE) were used as controls (n=5). We examined methylation levels across the genome using the Infinium Human Methylation 450 Bead Chip (Illumina). By comparing the methylome of platinum naive and platinum resistant ovarian tumors, normalized to HOSE, we identified 444 CpG island-containing gene promoters that became hypermethylated in resistant tumors. Of those, 45 gene promoters were hypermethylated at an FDR0.1. Among those significantly hypermethylated genes, acquired promoter CpG island methylation of Ras Association Domain Family 1A (RASSF1A; a tumor suppressor gene in multiple cancer types) was observed. Of the 444 hypermethylated promoters in tumor resistant samples, 93 CpG islands became hypomethylated after guadecitabine treatment, suggesting that these promoters may be associated with reversal of resistance. Of those were the IFNA8 promoter, a gene known to be associated with anti-tumor effects in ovarian and other solid malignancies, and DEFB124, which is involved in the innate immune response. Pathways associated with immune reactivation were highly enriched by guadecitabine treatment, including IL-12 signaling and production of macrophages and MAPK and apoptosis signaling. Together, these results support that aberrant DNA methylation affecting gene networks associate with immune response plays a role in platinum resistant development and could be a therapeutic target. Citation Format: Fang Fang, Horacio Cardenas, Guanglong Jiang, Susan M. Perkins, Chi Zhang, Harold Keer, Yunlong Liu, Daniela Matei, Kenneth Nephew. Epigenomic signatures of acquired platinum resistance in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2997.

Background: We report dose escalation results from a phase 1/2 trial of ASTX660 (NCT02503423). This non-peptidomimetic, dual XIAP and cIAP antagonist inhibits tumor cell lines at nanomolar concentrations and is active against xenografts. Methods: Study ASTX660-01, an open-label, multi-center, dose-escalation study in subjects with advanced cancers and lymphomas, used a 3+3 dose escalation design. Study drug was administered orally once daily in 28 day cycles of alternating 7 days on, then 7 days off therapy. Dose escalation was planned from a starting fixed 15 mg dose until the maximum tolerated dose (MTD) was reached. Endpoints included safety, MTD, recommended phase 2 dose (RP2D), pharmacokinetics, pharmacodynamics, and antitumor effects. Results: Forty-five subjects received at least 1 dose of ASTX660 (range 15 mg - 270 mg). Mean age was 62 years (range 36-77); 60% of subjects were female. Median duration of study therapy was 2 cycles (range 1-8). Four dose-limiting toxicities (DLTs) were observed: 3 at 270 mg QD (grade 3/4 lipase increased), and 1 at 210 mg QD (grade 3 lipase increased). These lipasemia DLTs were all asymptomatic. Most declined after dose interruption and did not recur after dose reduction. The MTD was 210 mg QD, and the RP2D was 180 mg QD. Common adverse events (AEs) included fatigue, lipasemia, and vomiting (29%); nausea and lymphopenia (27%); anemia (24%); rash and edema (20%); ALT increase and pruritus (18%); AST increase (16%); appetite decrease and diarrhea (13%); and amylasemia and hyponatremia (11%). Lipasemia and amylasemia appeared to be dose-related. Most AEs were CTCAE grades 1-2 and manageable with supportive treatment. Median time to peak plasma concentration (tmax) was 1 hour. ASTX660 t ½ was 15.3 hours at the RP2D. Day 1 and 7 AUC0-24h plasma exposures after 180 mg ASTX660 were 1450 (67%) and 2080 (62%) ng*hr/mL (geometric mean [CV]), respectively. Modest accumulation (1.4 fold) at Day 7 vs Day 1 was observed for ASTX660 AUC0-24h exposures but not for Cmax. Human RP2D exposure levels reached the biologically active exposure range seen in preclinical models. ASTX660 at all dose levels suppressed cIAP1 in peripheral blood mononuclear cells (PBMCs). At 180 mg cIAP1 suppression was sustained beyond the off-therapy week. A clinical response in cutaneous T cell lymphoma was observed at the 180-mg dose level. Conclusions: In phase 1, ASTX660 caused manageable toxicities, achieved target study drug exposures, and demonstrated both biologic and clinical activity at the RP2D. Enrollment to phase 2 expansion cohorts in lymphoma and other selected cancers is ongoing. Citation Format: Monica Mita, Patricia LoRusso, Michael S. Gordon, Aram Oganesian, Xiaoping Zhang, Roberta Ferraldeschi, Simone Jueliger, Harold N. Keer, Purvee Kumar, Chihche Lin, Edwin P. Rock, Alain Mita, Anthony W. Tolcher. Phase 1 study of IAP inhibitor ASTX660 in adults with advanced cancers and lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A091.

Purpose: Epigenetic changes, particularly in DNA methylation, have been implicated in acquired resistance to platinum in ovarian cancer (OC). The goal of the current study was to analyze and integrate global RNA expression and DNA methylation profiles of platinum resistant tumors compared to untreated, platinum-sensitive ovarian tumors, as well as to measure genomic and epigenomic changes induced by guadecitabine (SGI-110) in tumors. Methods: An ongoing phase I/II multi-institutional clinical trial uses the novel DNA methyltransferase (DNMT) inhibitor guadecitabine to re-sensitize recurrent platinum resistant OC to carboplatin. Patients enrolled in this trial had recurrent platinum resistant OC. Tumor biopsies were collected at baseline and after two cycles of guadecitabine administered daily for 5 days at a low (30mg/m2) dose (28 days per cycle). RNA and DNA were extracted from 48 and 57 baseline tumors and analyzed for next generation sequencing approaches to interrogate transcriptomes (RNA-seq) and methylomes (Infinium Human Methylation450 (HM450) arrays), respectively. Differential gene expression and DNA methylation profiles were generated and used for Ingenuity Pathway Analysis (IPA) to identify the top altered pathways in response to guadecitabine. Expression of DNMTs was examined by real-time RT-PCR and immunohistochemistry. LINE1 methylation and promoter methylation of selected genes (MAGE-A2, MAGE-A3, MAGE-A11, NY-ESO, RASSF1, MLH1, and HOXA11) were quantified by pyrosequencing before and after guadecitabine treatment (n=12 paired samples). Results: Analysis of a limited number of paired samples before and after treatment (n=8) revealed significant changes in global gene expression profiles induced by guadecitabine, with 960 altered genes representing immunopathway enrichment including cytokine production in macrophages and T helper cells by IL-17A and IL-17F, granulocyte /agranulocyte adhesion and inflammation, IL-8 signaling, p38 MAPK signaling, cAMP-mediated signaling, and innate immunity. Epigenetic profiling using HM450 revealed extensive methylation changes when comparing recurrent platinum resistant ovarian tumors (n=42) to primary, untreated ovarian cancer specimens analyzed as part of the TCGA project (n=10). Six hundred and four promoters were significantly differentially methylated (adjusted p0.2), among which, 498 and 106 were hypermethylated or hypomethylated respectively in recurrent platinum resistant ovarian tumors. IPA analysis of baseline tumor transcriptome and methylome demonstrated significant enrichment in a wide range of pathways associated with cancer, stem cells, inflammation and the immune system. DNMT1, 3A, and 3B mRNA levels in the tumors were highly variable (n=19). Analysis of a limited number of paired samples (n=7) revealed no significant changes in global methylation or in DNMT expression levels induced by treatment with guadecitabine (adjusted p>0.05). However, the DNMT inhibitor induced significant methylome alterations in selected patients. Significant hypomethylation of MAGE-A3 and MAGE–A11 promoters (p Conclusions: These data suggest that treatment with the DNMT inhibitor guadecitabine induces a reactivation of immune responses in human OC. Correlations between methylation changes and expression profiles are being explored. Citation Format: Fang Fang, Horacio Cardenas, Dave Miller, Aaron Buechlein, Qing Yu, Yunlong Liu, Guanglong Jiang, Pietro Taverna, Harold Keer, Doug Rusch, Daniela Matei, Kenneth P. Nephew. Epigenome and genome alterations in platinum resistant ovarian tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A70.

Background: HCC is the sixth most common cancer and the third most common cause of cancer death worldwide. Sorafenib treatment improves survival in advanced disease, but no therapy has demonstrated significant activity after progression on sorafenib. Increased methylation of genes implicated in tumorigenesis has been described in HCC and has been associated with outcome and etiology. We evaluated the therapeutic and biologic effects of SGI-110, a hypomethylating agent in patients with HCC. SGI-110, a dinucleotide of decitabine and deoxyguanosine, increases decitabine exposure by protecting it from deamination due to slow release on subcutaneous (SC) injection. PK and PD results of an open-label, phase 2 study in patients with HCC are presented here. Methods: Adults with histologically confirmed, advanced-stage HCC who had received sorafenib, had evidence of disease progression, and ECOG PS 0-1 were enrolled. SGI-110 (SC) was given on D1-5 of a 28-day cycle. Blood samples were taken for PK/PD analysis and, when possible, paired tumor biopsies were taken for analysis of global DNA (LINE-1) and gene-specific methylation and gene expression. Patients were imaged every 8 weeks and allowed to continue treatment after radiologic but not clinical progression. End points include disease control rate (DCR) at 16 weeks, overall response rate, progression-free survival, and overall survival. Results: 50 HCC patients (43M/7F; median age 60 years [range 32-82]; ECOG PS 0/1: 21/29) were enrolled. The initial dose of SGI-110 was 60 mg/m2 (4 patients treated), but due to grade 4 neutropenia, the dose was decreased to 45 mg/m2 for subsequent patients. SGI 110 was well tolerated at 45 mg/m2; myelosuppression was the major adverse event. Full PK was available from 16 patients (3F/13M). The PK profile for SGI-110 after 45 mg/m2 showed protracted conversion to deliver active metabolite decitabine with exposure window lasting beyond 8-hr and mean(SD) decitabine AUC exposures of 94(22) ng*hr/mL that were comparable to those achieved in AML/MDS after 60 mg/m2. Potent LINE-1 demethylation was observed in blood (-35.6%, n = 27) and in tumor (-12.9%, n = 10) DNA; significant demethylation (-27.4%, n = 6) was also observed on promoter of tumor suppressor gene MZB1 which is frequently hypermethylated and silenced in HCC. Conclusions: SGI-110 was well tolerated at a dose of 45 mg/m2 administered SC on D1-5 of a 28-day cycle. PK was consistent with that seen in another solid tumor study and provided more persistent decitabine exposures compared with PK in hematologic malignancies. The PD changes in blood and tumor LINE-1 and MZB-1 methylation are promising and consistent with the desired biologic effect of SGI-110. Analysis for clinical efficacy is ongoing. Citation Format: Anthony El-Khoueiry, Mary F. Mulcahy, Tanios Bekaii-Saab, Richard Kim, Crystal Denlinger, Rakesh Goel, Shweta Gupta, Simone Jueliger, Aram Oganesian, Harold Keer, John Nemunaitis. Pharmacodynamic (PD) and pharmacokinetic (PK) results of the second-generation hypomethylating agent, SGI-110, in patients with hepatocellular carcinoma (HCC) after progression on sorafenib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2947. doi:10.1158/1538-7445.AM2015-2947

Background: Epigenetic changes have been implicated in acquired resistance to platinum. SGI-110 is a second generation SQ HMA with improved pharmaceutical properties compared to decitabine. Here we report the clinical results and pharmacodynamic analyses of the phase 1 study of SGI-110 in combination with carboplatin in patients with recurrent platinum resistant high-grade serous, epithelial ovarian cancer (EOC), primary peritoneal carcinoma (PPC) or fallopian tube (FT) cancer. Methods: SGI-110 was administered SQ QD x 5 followed by carboplatin IV on Day 8 of a 28-day cycle. Patients were required to have either measurable disease according to RECIST v1.1 or detectable disease (modified Rustin) with clinical response assessed using the applicable criteria. Safety assessments were graded using CTCAE v4. Results: Twenty patients (18 EOC, 1 PPC, 1 FT) were enrolled and treated in the phase 1 portion of the trial. Median age was 55.8 years (38-72); ECOG PS of 0/1/2 was 10/10/0, respectively. Median number of prior regimens was 7 (1-9). The starting doses were SGI-110 45 mg/m2 SQ QD x 5 and carboplatin AUC5 in the first cohort of 6 patients. Four DLTs of myelosuppression (neutropenia and thrombocytopenia) in the first cohort led to dose reduction to SGI-110 30 mg/m2 and carboplatin AUC4 with granulocyte-CSF permitted at the discretion of the physician. No DLTS were observed in 14 patients and this dose was recommended for the subsequent phase 2 study. Grade 3/4 AEs regardless of relationship to the combination ≥ 10% included anemia, leukopenia, neutropenia, thrombocytopenia, nausea, vomiting, constipation, small intestinal obstruction, infusion related reaction and pulmonary embolism. Three PRs and 9 SDs as best response were observed in 20 patients for an overall response rate and clinical benefit rate of 15% and 60%, respectively. All PRs and 3 SDs were accompanied by CA-125 decrease. LINE-1 hypomethylation, a marker of global DNA methylation, was recorded in PBMCs with SGI-110 30 mg/m2 (avg: -19.5%, n=14) and 45 mg/m2 (avg: -17.4%, n=6). Gene specific methylation of RASSF1A and BRCA-1 measured by pyrosequencing was significantly decreased at C2D8 compared to baseline in paired tumor biopsies/ascites (n=9). Gene re-expression measured by quantitative RT-PCR was observed in tumor biopsies. Conclusions: Priming treatment with SGI-110 prior to carboplatin induced clinical responses in a heavily-pretreated platinum resistant ovarian cancer population with expected and manageable safety profile. Potent LINE-1 demethylation and demethylation and re-expression of silenced tumor genes were recorded. The phase 2 portion of the trial is currently ongoing with patients randomized to either the RP2D dose combination or a physician choice of 1 of 4 treatment options (topotecan; liposomal doxorubicin; weekly paclitaxel; or weekly gemcitabine). Citation Format: Gini Fleming, Sharad Ghamande, Yvonne Lin, Angeles Alvarez Secord, John Nemunaitis, Merry-Jennifer Markham, Kenneth Nephew, Fang Fang, Shweta Gupta, Sue Naim, Gavin Choy, Simone Jueliger, Pietro Taverna, Yong Hao, Harold Keer, Mohammad Azab, Daniela Matei. Clinical epigenetic resensitization of platinum-resistant, recurrent ovarian cancer patients with SGI-110, a novel, second-generation, subcutaneously administered hypomethylating agent (HMA). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2320. doi:10.1158/1538-7445.AM2014-2320

Endometrial cancer is the most common gynecological cancer in industrialized countries, with an estimated incidence of approximately 42,000 cases in the United States in 2009. The majority of patients with endometrial cancer are cured with hysterectomy; however, for patients with non-resectable or recurrent disease, there is no curative treatment. A number of publications have recently reported the identification of somatic mutations in the fibroblast growth factor receptor 2 (FGFR2) in 15-16% of endometrial carcinomas. The most common FGFR2 mutation observed was S252W (∼7 % of all cases), which is identical to the germline activating mutation in FGFR2 seen in Apert Syndrome, a congenital human skeletal disorder. Structure-function studies of S252W FGFR2 demonstrate that the mutant receptor has altered ligand specificity and enhanced affinity for multiple FGFs, compared to wild-type FGFR2. FP-1039 is a soluble fusion protein consisting of the extracellular domain of human fibroblast growth factor receptor 1 (FGFR1) isoform α-IIIc linked to the Fc region of human immunoglobulin G1 (IgG1). FP-1039 acts as a ligand “trap” that sequesters multiple fibroblast growth factor (FGF) family ligands, blocking their ability to bind to and activate multiple FGF receptors. FP-1039 is currently being investigated in a single agent Phase I dose-escalation study in patients with advanced solid malignancies (Tolcher et al., AACR-NCI-EORTC 2009). We have examined the in vitro and in vivo sensitivity of FGFR2 S252W bearing mutant (MFE-280 and MFE-319) and wild-type (w.t.; HEC-1-B) human endometrial carcinoma cell lines to FP-1039. Addition of FP-1039 to MFE-280 and MFE-319 cells in tissue culture resulted in a 49% and 14% reduction, respectively, in cell proliferation as determined by 3H thymidine incorporation. FP-1039 had no effect on HEC-1-B cell proliferation in vitro. FP-1039 treatment of mice bearing HEC-1-B and MFE-319 xenografts resulted in a 22% and 23% decrease (p= Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2597.