Citations

Abstract DNA methylation at cytosine position 5 (5mC) is a genetic modification that regulates gene expression and is critical for cancer initiation and development. The methylcytosine dioxygenase TET2 participates in active DNA demethylation by converting 5mC to 5-hydroxymethylcytosine (5hmC) and its subsequent intermediates thereby regulating gene expression. TET2 gene is frequently mutated in hematological disorders, including 15-25% of myelodysplastic syndrome (MDS) cases. Loss of TET2 function leads to DNA hypermethylation and subsequent dysregulated gene expression in hematopoietic stem cells, and has been considered as an initial step of myeloid malignant transformation including MDS and acute myeloid leukemia (AML). While enzymatic activity of TET2 is well studied, little is known about post-translational modifications (PTMs) that regulate its activity in hematopoietic cells. Lysine acetylation, one of the most crucial PTMs, occurs in a variety of proteins and modulates protein-protein or protein-nucleic acids interactions. Here, we first observed that ectopic expression of histone acetyltransferase EP300 or CREB binding protein (CBP) increased the endogenous TET2 acetylation in 293T cells. Mass spectrometry analysis of TET2 identified several conserved acetylated lysine residues located on the exterior surface of its catalytic domain (CD), which is predicted to be essential for the interaction of TET2 with DNA. Modeling of TET2/DNA interaction using x-ray crystal structure (PDB code: 4NM6) indicates acetylated TET2 interferes with DNA binding thereby impairing the catalytic activity of the enzyme. Like other acetylated proteins, TET2 modification is reversible, since ectopic expression of the protein deacetylase Sirtuin1 (SIRT1) significantly decreases acetylation of TET2. A domain mapping analysis revealed that SIRT1 preferentially interacts with TET2 CD, further supporting the regulatory role of SIRT1 on TET2. To explore the role of SIRT1 mediated deacetylaion of TET2 in more physiological condition, SIRT1 knockdown (KD) MDS-L cells as well as SIRT1 knockout (KO) murine hematopoietic c-kit+ cells were used. In MDS-L cells derived from a MDS patient, SIRT1 KD promoted hyperacetylation of endogenous TET2 which is associated with 50% decrease in 5hmC levels compared to control (p=0.03, n=3). Similarly, SIRT1 KO murine c-Kit+ cells showed TET2 hyperacetylation and 62% decrease in 5hmC compared to wildtype (WT) counterpart (p=0.009, n=5). Phenotypically, the transforming ability of SIRT1 shRNA transduced MDS-L cells was evaluated in vitro through serial replating assay. In contrast to first plating which showed no difference between two groups, the number of colonies derived from SIRT1 KD cells was 2.39 fold higher than that from the control shRNA transduced group after two rounds of replating (p=0.0002, n=3). Importantly, we tested the in-vivo effect of SIRT1 KD on MDS-L cells engrafted in immunodeficient NSGS mice. SIRT1 KD significantly enhanced MDS-L cell engraftment compared to control shRNA (50±5% of human CD45+ cells in SIRT1 KD group vs. 19±4% human CD45+ cells in control group, p=0.003, n=6). Interestingly, increased transformation capacity of SIRT1 KD MDS-L cells was associated with decreased expression of TET2 regulated tumor suppressor genes. For example, expression levels of Mtss1 and Dusp6 were significantly decreased in SIRT1 KD MDS-L cells compared to control shRNA counterpart (Mtss1, 49±5% decrease compared to control, p=0.005; Dusp6, 25±2% decrease compared to control, p=0.01). Similar results were observed in SIRT1 KO cells. The validation of physical binding of TET2 to its targets is ongoing. Moreover, the effect of an SIRT1 allosteric activator-SRT1720 in MDS-L cells is similar to that of Vitamin C, a known TET2 activator. Specifically, SRT1720 significantly increased Mtss1 expression in MDSL cells (p=0.0005, n=3), indicating that SIRT1 agonist may lead to activation of TET2 downstream tumor suppressor genes. In summary, our data demonstrate that TET2 activity can be functionally modified by acetylation and may be enhanced in MDS patients that do not harbor loss-of-function mutations through the SIRT1 mediated deacetylation. These results support further exploration of molecular mechanisms inducing TET2 acetylation, and evaluation of SIRT1 activation as a potential therapeutic approach in MDS. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3701 Background: Salvage chemotherapy and autologous stem cell transplantation (ASCT) in the management of relapsed or refractory Hodgkin's lymphoma (HL) results in prolonged disease-free survival in only around 50% of patients (pts). Median survival following relapse from ASCT is estimated at 2.4 years, with poor outcomes in those experiencing relapse or progressive disease within one year from ASCT (Horning 2008 et al). Recently, a pivotal multicenter Phase 2 trial investigated the efficacy of brentuximab vedotin (BV) in pts with recurrent HL following ASCT. The study demonstrated an overall response rate of 75% and median duration of response of 6.7 months. Furthermore, 34% of patients achieved a complete response (CR), with a median progression free survival of 29 months (mos). Whether treatment with BV renders an OS benefit in pts with relapsed or refractory disease after ASCT remains to be determined. Objective: 1) To compare OS in pts with relapsed HL after receiving ASCT in a cohort of 102 HL pts treated with BV, with 756 pts from 6 international centers before the introduction of BV. 2) To evaluate predictors of durable CR in pts treated with BV. Methods: Kaplan-Meier method was used to depict time-to-event outcomes, including OS. The Chi-square test was used to evaluate the association between two categorical variables and log-rank test to compare time-to-event endpoints among pt groups. Based on the univariate proportional hazard model, we examined whether certain variables (sex, stage at diagnosis, number of prior treatments, and time to CR) were predictors of CR duration in pts receiving BV. Results: In the non-randomized comparison between those with relapsed-refractory HL following ASCT who received BV and those who did not, pts were well matched by age, with a relatively higher percentage of females treated on the pivotal study. Dates of progression from ASCT ranged from 1996–2009 in the BV group and 1981–2003 in the international cohort. Median follow-up after ASCT for the censored observations of pts who received BV and those in the international cohort was 49.4 and 58.8 mos. The difference in median OS was statistically significant (p<0.0001) between those receiving BV and those not exposed to the drug (91.49 vs. 27.99 mos). Improvement in OS was irrespective of time to relapse from ASCT, despite previous studies noting a higher risk of recurrence in pts relapsing within 12 mos of ASCT (Sureda 2005 et al). Neither age nor sex was found to impact OS. In the subgroup analysis of pts achieving a CR with BV, univariate analysis of duration of CR and PFS uncovered no significant factors, while only stage of disease at initial diagnosis had a significant effect on OS (P=0.0004). Disease status characterized as relapsed vs. refractory had borderline significance with a positive impact on OS (P=0.0581). No significant associations were noted between the variables and duration of response post BV stratified into periods of less or greater than 12 mos. However, among those pts who experienced a duration of CR greater than 12 mos, 81.8% (n=18) had an initial CR after receiving ≤ 4 doses of BV, compared with 58.3% entering CR after receiving > 4 doses. Conclusions: While the impact of BV on OS can only be determined through a randomized clinical trial, our analysis indicated that treatment with BV in pts with relapsed/refractory HL following ASCT is associated with prolonged OS when compared with historical control patients. Those who achieve a rapid CR with BV may also have a longer duration of response. A randomized clinical trial is ongoing to evaluate prospectively the efficacy of BV following ASCT. Disclosures: Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau. Gopal:Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Ansell:Seattle Genetics, Inc.: Research Funding; Celgene: Consultancy. Savage:Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Ramchandren:Seattle Genetics, Inc.: Speakers Bureau. Forero-Torres:Seattle Genetics: Research Funding. Moskowitz:Seattle Genetics: Speakers Bureau. Connors:Seattle Genetics, Inc.: Research Funding. de Vos:Seattle Genetics: Speakers Bureau. Engert:Takeda: Honoraria, Research Funding; Millennium: Honoraria, Research Funding. Illidge:Seattle Genetics, Inc.: Consultancy, Research Funding; Millennium/Takeda: Consultancy, Honoraria. Morschhauser:Takeda: Honoraria. Younes:Seattle Genetics: Honoraria.

Abstract Purpose: The heterogeneity of androgen receptor (AR)-activity (AR-A) is well-characterized in heavily treated metastatic castration-resistant prostate cancer (mCRPC). However, the diversity and clinical implications of AR-A in treatment-naïve primary prostate cancer is largely unknown. We sought to characterize AR-A in localized prostate cancer and understand its molecular and clinical implications. Experimental Design: Genome-wide expression profiles from prostatectomy or biopsy samples from 19,470 patients were used, all with independent pathology review. This was comprised of prospective discovery (n = 5,239) and validation (n = 12,728) cohorts, six retrospective institutional cohorts with long-term clinical outcomes data (n = 1,170), and The Cancer Genome Atlas (n = 333). Results: A low AR-active subclass was identified, which comprised 9%–11% of each cohort, and was characterized by increased immune signaling, neuroendocrine expression, and decreased DNA repair. These tumors were predominantly ERG and basal subtype. Low AR-active tumors had significantly more rapid development of recurrence or metastatic disease across cohorts, which was maintained on multivariable analysis [HR, 2.61; 95% confidence interval (CI), 1.22–5.60; P = 0.014]. Low AR-active tumors were predicted to be more sensitive to PARP inhibition, platinum chemotherapy, and radiotherapy, and less sensitive to docetaxel and androgen-deprivation therapy. This was validated clinically, in that low AR-active tumors were less sensitive to androgen-deprivation therapy (OR, 0.41; 95% CI, 0.21–0.80; P = 0.008). Conclusions: Leveraging large-scale transcriptomic data allowed the identification of an aggressive subtype of treatment-naïve primary prostate cancer that harbors molecular features more analogous to mCRPC. This suggests that a preexisting subgroup of patients may have tumors that are predisposed to fail multiple current standard-of-care therapies and warrant dedicated therapeutic investigation.

AbstractThe deubiquitinase (DUB) ubiquitin C‐terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small‐molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine‐based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Précis: Over 10 years in a tertiary care setting, glaucoma drainage devices (GDDs) have not increased as a reason for keratoplasty. Purpose: The purpose of this study was to determine whether the reasons for keratoplasty have changed between 10 years in a tertiary care setting, with special attention to the rate of GDDs as a reason for keratoplasty. Methods: Patients aged 18 years or above who underwent keratoplasty at Mayo Clinic, Rochester, MN from 2005 to 2006 and 2015 to 2016 were studied. All reasons for keratoplasty performed in the study time period are assessed, including patients who previously had a GDD implanted in the same eye. After analyzing the reasons for keratoplasty, we assess whether the reasons for keratoplasty have changed between 2005 to 2006 and 2015 to 2016 in association with the increasing placement of GDDs. Results: The number of keratoplasty procedures performed in the 2 time periods increased by 62% from 163 (2005 to 2006) to 264 (2015 to 2016), whereas GDD placement increased by 164% from 80 GDD (2005 to 2006) to 211 GDD (2015 to 2016). Although the performance of keratoplasty increased between the 2 points in time, the frequency of each cause for keratoplasty did not change significantly. The majority of keratoplasties were performed due to corneal disease, and GDDs made up a small portion of reasons for keratoplasty (2005 to 2006, 4.29%; 2015 to 2016, 5.68%). Conclusions: The frequency of GDDs as a reason for keratoplasty has not changed significantly between 10 years in this tertiary care setting. Patients with GDDs who later required keratoplasty had associated features including multiple surgical procedures and comorbid infection, pseudophakic bullous keratopathy, Fuchs dystrophy, pseudoexfoliation, uveitis, and congenital glaucoma.

Abstract Resistance to chemotherapy remains a major obstacle to improving outcomes of cancer treatment. Recent data suggest intrinsic functions of immune checkpoint proteins, including PD-L1, that may be distinct from their immunoregulatory roles. We found that colorectal cancer (CRC) cells with mutant BRAF(V600E) or KRASalleles show increased PD-L1 transcripts and expression of cell surface PD-L1 protein compared to those with wild-type copies. Furthermore, ectopic expression of BRAFV600E or mutant KRAS induced PD-L1 whose upregulation was attenuated by inhibition of RAS-RAF-MEK-ERK signaling. Combined knockdown of MEK/ERK effectors c-JUN and YAP markedly reduced PD-L1. Knockout of PD-L1 in RKO cells resulted in a reduction in chemotherapy-induced DNA double strand breaks (pH2AX) and caspase-3 cleavage compared to parental cells. Results were confirmed in PD-L1 knockout MC38 murine CRC cells where re-expression of PD-L1, but not its deletion mutants, was shown to induced pH2AX and apoptosis. In cells with knockout of PD-L1, reductions in p-AKT and the BH3-only proteins BIM and BIK were observed that could be restored by re-expression of PD-L1. Treatment of cells with an anti-PD-L1 antibody reduced p-AKT, BIM and BIK, and also attenuated chemotherapy-induced apoptosis. Re-expression of BIM in PD-L1 knockout cells restored apoptosis. Murine tumor xenografts generated from cells with knockout of PD-L1 displayed resistance to oxaliplatin-induced tumor regression compared to tumors derived from parental cells. Using TCGA datasets, human CRCs with high vs low PD-L1 mRNA levels were significantly associated with better survival. In summary, we identified a non-immune function of tumor cell-intrinsic PD-L1 that upregulates BH3-only proteins BIM and BIK to promote apoptosis, thereby suggesting its potential as a predictive biomarker. Citation Format: Daofu Feng, Krishnendu Pal, Debabrata Mukhopadhyay, Xin Liu, Haidong Dong, Shengbing Huang, Frank A. Sinicrope. Tumor intrinsic PD-L1 is mediated by BRAFV600E and can regulate drug-induced apoptosis in human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 705.

AbstractBackgroundMolecular studies have tried to address the unmet need for prognostic biomarkers in prostate cancer (PCa). Some gene expression tests improve upon clinical factors for prediction of outcomes, but additional tools for accurate prediction of tumor aggressiveness are needed.MethodsBased on a previously published panel of 23 gene transcripts that distinguished patients with metastatic progression, we constructed a prediction model using independent training and testing datasets. Using the validated messenger RNAs and Gleason score (GS), we performed model selection in the training set to define a final locked model to classify patients who developed metastatic‐lethal events from those who remained recurrence‐free. In an independent testing dataset, we compared our locked model to established clinical prognostic factors and utilized Kaplan‐Meier curves and receiver operating characteristic analyses to evaluate the model's performance.ResultsThirteen of 23 previously identified gene transcripts that stratified patients with aggressive PCa were validated in the training dataset. These biomarkers plus GS were used to develop a four‐gene (CST2, FBLN1, TNFRSF19, and ZNF704) transcript (4GT) score that was significantly higher in patients who progressed to metastatic‐lethal events compared to those without recurrence in the testing dataset (P = 5.7 × 10−11). The 4GT score provided higher prediction accuracy (area under the ROC curve [AUC] = 0.76; 95% confidence interval [CI] = 0.69‐0.83; partial area under the ROC curve [pAUC] = 0.008) than GS alone (AUC = 0.63; 95% CI = 0.56‐0.70; pAUC = 0.002), and it improved risk stratification in subgroups defined by a combination of clinicopathological features (ie, Cancer of the Prostate Risk Assessment‐Surgery).ConclusionOur validated 4GT score has prognostic value for metastatic‐lethal progression in men treated for localized PCa and warrants further evaluation for its clinical utility.

Abstract Background: Alzheimer’s disease (AD) is an epidemic with tremendous public health impacts because there are currently no disease-modifying therapeutics. Randomized controlled trials (RCTs) for prevention of AD dementia often use clinical endpoints that take years to manifest (e.g., cognition) or surrogate endpoints that are costly or invasive (e.g., magnetic resonance imaging [MRI]). Blood biomarkers represent a clinically applicable alternative surrogate endpoint for RCTs that would be both cost-effective and minimally invasive, but little is known about their value as surrogate endpoints for treatment responses in the prevention of AD dementia. Methods: The objective of this study is to investigate blood neuropathological, neurodegenerative, and neurotrophic biomarkers as surrogate endpoints for treatment responses to 3 interventions in older adults with amnestic mild cognitive impairment (aMCI, a prodromal stage of AD): aerobic exercise; cognitive training; and combined aerobic exercise and cognitive training (ACT). We chose these three sets of biomarkers for their unique mechanistic associations with AD pathology, neurodegeneration and neurogenesis. This study is built on the ACT Trial (1R01AG055469), a single-blinded, multi-site, 2×2 factorial Phase II RCT that examines the synergistic effects of a 6-month ACT intervention on cognition and MRI biomarkers (AD-signature cortical thickness and hippocampal volume) (n=128). In this ACT Trial Blood Biomarkers Study, we will enroll 120 ACT Trial aMCI participants and measure blood biomarkers at baseline, 3, 6, 12 and 18 months. The goals are to: (1) Determine the effect of interventions on blood biomarkers over 6 months; (2) Evaluate blood biomarkers as surrogate endpoints for predicting cognitive responses to interventions over 18 months; and (3; exploratory) Examine blood biomarkers as surrogate endpoints for predicting brain MRI biomarker responses to interventions over 18 months. Discussion: This study aims to identify new blood biomarkers that can track cognitive decline or AD-related brain atrophy among aMCI patients subjected to a regimen of aerobic exercise and cognitive training. Findings from this study will drive the further use of blood biomarkers in developing effective prevention and treatment strategies for AD dementia. Trial registration number: This study is an ancillary study of the ACT Trial which was registered with ClinicalTrials.gov, NCT03313895.

Abstract Purpose: Protein kinases are known to play a prominent role in oncogenic progression across multiple cancer subtypes, yet their role in prostate cancer progression remains underexplored. The purpose of this study was to identify kinases that drive prostate cancer progression. Experimental Design: To discover kinases that drive prostate cancer progression, we investigated the association between gene expression of all known kinases and long-term clinical outcomes in tumor samples from 545 patients with high-risk disease. We evaluated the impact of genetic and pharmacologic inhibition of the most significant kinase associated with metastatic progression in vitro and in vivo. Results: DNA-dependent protein kinase (DNAPK) was identified as the most significant kinase associated with metastatic progression in high-risk prostate cancer. Inhibition of DNAPK suppressed the growth of both AR-dependent and AR-independent prostate cancer cells. Gene set enrichment analysis nominated Wnt as the top pathway associated with DNAPK. We found that DNAPK interacts with the Wnt transcription factor LEF1 and is critical for LEF1-mediated transcription. Conclusions: Our data show that DNAPK drives prostate cancer progression through transcriptional regulation of Wnt signaling and is an attractive therapeutic target in aggressive prostate cancer.