Publications

Abstract Phosphatidylethanolamine‐binding protein 4 (PEBP4), a member of the PEBP family, has been reported to play a pivotal role in tumor progression. However, its role in epithelial‐to‐mesenchymal transition (EMT) in non–small cell lung cancer (NSCLC) cells remains unclear. Here, we investigated the effects and underlying mechanism of PEBP4 in NSCLC EMT. Three human NSCLC cell lines (A549, H1299, and H460) were transfected with pcDNA3.1‐PEBP4 or PEBP4‐targeting small interfering RNA. Then, cell proliferation was analyzed by the MTT assay, and cell migration and invasion were analyzed by the transwell chamber assay. Protein and messenger RNA expression of the related genes and proteins were assessed by Western blot analysis and quantitative real‐time polymerase chain reaction, respectively. Results showed that PEBP4 was highly expressed in the human lung cancer tissues and three human NSCLC cell lines. Pretreatment with pcDNA3.1‐PEBP4 promoted the proliferation, invasion, and migration of NSCLC cells and increased EMT in vitro and lung tumor metastasis in vivo. Whereas knockdown of PEBP4 suppressed NSCLC cell migration, PEBP4, and invasion with prevented EMT. Furthermore, PEBP4 overexpression significantly promoted the transcriptional activity of sonic hedgehog (Shh) signaling in NSCLC cells. Further analysis showed that using cyclopamine to inhibit Shh signaling significantly ameliorated the effect on cell proliferation, invasion, and migration, as well as EMT triggered by PEBP4 overexpression. Together, these results suggest that PEBP4 may promote tumorigenesis in NSCLC by regulating cell proliferation and EMT via activation of the Shh signaling pathway.

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.

218 Background: CRPC is enriched for germline mutations in DNA damage repair genes (gDDRm). BRCA2 mutations (g BRCA2m) associate with poor prognosis from localized PC, but prognostic and predictive value for standard therapy in CRPC is unclear. We reviewed clinical outcome of 390 patients previously tested for gDDRm. Methods: Patient records were reviewed for 372 patients from 3 institutions (Royal Marsden UK, Weill-Cornell NY, University of Washington, WA) with gDDRm status previously published (Pritchard et al, NEJM 2016) and 18 g BRCA1/2m carriers from KConFab consortium (Australia). Baseline characteristics and survival were annotated. Response (PSA50%/RECIST) and PFS (RECIST/PSA progression or start of a new therapy due to clinical progression) were collected for Abiraterone, Enzalutamide and Docetaxel. To account for potential differences between cohorts, a mixed effect model (Weibull distribution) with random intercept per cohort was pursued. Results: dDDRm status was available for n = 390 (60 gDDRm+, including 37 g BRCA2m, and 330 gDDRm-). Overall, 74% and 69% received Docetaxel and Abiraterone/Enzalutamide respectively; 47% gDDRm+ and 34% gDDRm- received PARPi and/or platinum. Median overall survival from CRPC was 3.0 vs 3.2 years in gDDRm+ vs gDDRm- (p = 0.73; g BRCA2m = 3.0 years, p = 0.72). Age and Gleason score at diagnosis were associated with survival from castration-resistance in multivariate analysis. Median PFS on Docetaxel for gDDRm+ (6.8 months; 6.3 for g BRCA2m) and gDDRm- (5.1 months) were not significantly different (p = 0.2). Similarly, RR to Docetaxel was similar for the two groups (61% vs 54% in gDDRm+ vs gDDRm-; 63% g BRCA2m). Median PFS and RR on first Abiraterone/Enzalutamide were similar across groups (PFS: 8.3 months gDDRm+, 8.3 months for gDDRm-; p = 0.9; RR 46% vs 56% respectively) The Interaction of PARPi/platinum therapy among gDDRm+ patients resulted in an aHR for OS from CRPC of 0.59 (95%CI 0.28-1.25; p = 0.17). Conclusions: In this retrospective analysis, CRPC patients with gDDRm still benefited from standard therapies similarly to non-mutation carriers; interpretation of survival data should consider the high proportion treated with PARPi/platinum.

5090 Background: We previously reported that ENZA improved overall survival (OS) after median follow-up times of 34 and 68 months, in comparison with a NSAA, when added to testosterone suppression, with or without concurrent early docetaxel, for mHSPC. We now report outcomes after median follow-up of 98 months. Methods: Participants (pts) with mHSPC were randomly assigned (1:1) from 31MAR2014-24MAR2017 to treatment with ENZA 160 mg or NSAA, in addition to testosterone suppression. Concurrent early docetaxel was used in 45%. OS was the primary endpoint and analysed with the Kaplan-Meier method, log-rank test for p-values, and Cox regression for hazard ratios (HR). Secondary outcomes included deaths due to prostate cancer (PC) versus (vs) other causes. The numbers of pts experiencing specified adverse events (AE) of grade 3-5 are expressed per 100 person-years of study treatment exposure to account for differing treatment durations. Results: After a median follow-up of 98 months, data cut-off 30JUN2024, death was reported in 285/563 (51%) pts assigned ENZA vs 337/562 (60%) assigned NSAA. OS was longer among those assigned ENZA than NSAA (medians 95 vs 70 months; OS at 96 months 50% vs 40%; HR 0.73, 95% CI 0.63 to 0.86; p=0.0001). Clinical PFS also continued to favour ENZA over NSAA (HR 0.49; 95% CI 0.42 to 0.57; p<0.0001). PC accounted for 468 of all 622 deaths, and were less frequent among those assigned ENZA than NSAA (207 vs 261). Deaths due to other causes accounted for a total of 154 deaths, and were similarly frequent among those assigned ENZA vs NSAA (78 vs 76). Mean duration of study treatment was longer for ENZA than NSAA (58 vs 36 months). 185/562 (33%) remain on ENZA with 88% on full dose. G3-5 AE of interest were reported in the following numbers of pts per 100 years of study treatment with ENZA vs NSAA: cardiac disorder 2.2 vs 2.2, nervous system disorder 2.3 vs 2.0, fall 0.70 vs 0.24. Causes of death according to PSA at 7 months are tabulated below. Among those with PSA at 7 months ≤0.2, deaths were due to PC in 29%, and other causes in 13%. Among those with PSA at 7 months >0.2, deaths were due to PC in 60%, and other causes in 13%. Conclusions: Treatment with enzalutamide continues to confer substantial OS benefits at 8 years. These findings highlight long-term safety, toxicities, non-PC causes of death, and survival outcomes of those with and without PSA ≤0.2 at 7 months. ClinicalTrials.gov Identifier NCT02446405. Clinical trial information: NCT02446405 . Landmark analysis by PSA at 7 months ENZA (N=555) NSAA (N=549) ALL (N=1104) Deaths due to prostate cancer, PSA ≤0.2 100/375 (27%) 87/270 (32%) 187/645 (29%) Deaths due to other causes, PSA ≤0.2 54/375 (14%) 33/270 (12%) 87/645 (13%) Deaths due to prostate cancer, PSA >0.2 104/180 (58%) 170/279 (61%) 274/459 (60%) Deaths due to other causes, PSA >0.2 22/180 (12%) 39/279 (14%) 61/459 (13%)

Background The programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) axis plays a central role in suppressing antitumor immunity; axis dysregulation can be used by cancer cells to evade the immune system. Tislelizumab, an investigational monoclonal antibody with high affinity and binding specificity for PD-1, was engineered to minimize binding to FcγR on macrophages to limit antibody-dependent phagocytosis, a potential mechanism of resistance to anti-PD-1 therapy. The aim of this phase IA/IB study was to investigate the safety/tolerability, antitumor effects and optimal dose and schedule of tislelizumab in patients with advanced solid tumors. Methods Patients (aged ≥18 years) enrolled in phase IA received intravenous tislelizumab 0.5, 2, 5 or 10 mg/kg every 2 weeks; 2 or 5 mg/kg administered every 2 weeks or every 3 weeks; or 200 mg every 3 weeks; patients in phase IB received 5 mg/kg every 3 weeks. Primary objectives were to assess tislelizumab’s safety/tolerability profile by adverse event (AE) monitoring and antitumor activity using RECIST V.1.1. PD-L1 expression was assessed retrospectively with the VENTANA PD-L1 (SP263) Assay. Results Between May 2015 and October 2017, 451 patients (n=116, IA; n=335, IB) were enrolled. Fatigue (28%), nausea (25%) and decreased appetite (20%) were the most commonly reported AEs. Most AEs were grade 1–2 severity; anemia (4.9%) was the most common grade 3–4 AE. Treatment-related AEs led to discontinuation in 5.3% of patients. Grade 5 AEs were reported in 14 patients; 2 were considered related to tislelizumab. Pneumonitis (2%) and colitis (1%) were the most common serious tislelizumab-related AEs. As of May 2019, 18% of patients achieved a confirmed objective response in phase IA and 12% in phase IB; median follow-up duration was 13.6 and 7.6 months, respectively. Pharmacokinetics, safety and antitumor activity obtained from both phase IA and IB determined the tislelizumab recommended dose; ultimately, tislelizumab 200 mg intravenous every 3 weeks was the dose and schedule recommended to be taken into subsequent clinical trials. Conclusions Tislelizumab monotherapy demonstrated an acceptable safety/tolerability profile. Durable responses were observed in heavily pretreated patients with advanced solid tumors, supporting the evaluation of tislelizumab 200 mg every 3 weeks, as monotherapy and in combination therapy, for the treatment of solid tumors and hematological malignancies. Trial registration number NCT02407990 .

<div>AbstractPurpose:<p>In this article, we describe a combination chimeric antigen receptor (CAR) T-cell therapy that eradicated the majority of tumors in two immunocompetent murine pancreatic cancer models and a human pancreatic cancer xenograft model.</p>Experimental Design:<p>We used a dual-specific murine CAR T cell that expresses a CAR against the Her2 tumor antigen, and a T-cell receptor (TCR) specific for gp100. As gp100 is also known as pMEL, the dual-specific CAR T cells are thus denoted as CARaMEL cells. A vaccine containing live vaccinia virus coding a gp100 minigene (VV-gp100) was administered to the recipient mice to stimulate CARaMEL cells. The treatment also included the histone deacetylase inhibitor panobinostat (Pano).</p>Results:<p>The combination treatment enabled significant suppression of Her2<sup>+</sup> pancreatic cancers leading to the eradication of the majority of the tumors. Besides inducing cancer cell apoptosis, Pano enhanced CAR T-cell gene accessibility and promoted CAR T-cell differentiation into central memory cells. To test the translational potential of this approach, we established a method to transduce human T cells with an anti-Her2 CAR and a gp100-TCR. The exposure of the human T cells to Pano promoted a T-cell central memory phenotype and the combination treatment of human CARaMEL cells and Pano eradicated human pancreatic cancer xenografts in mice.</p>Conclusions:<p>We propose that patients with pancreatic cancer could be treated using a scheme that contains dual-specific CAR T cells, a vaccine that activates the dual-specific CAR T cells through their TCR, and the administration of Pano.</p></div>

9563 Background: With the evolving treatment landscape in metastatic melanoma, approaches to disease surveillance post resection in stage 3 disease requires reconsideration. We previously reported the outcomes of sub-stage-specific schedules of combined fluorodeoxyglucose-positron emission and computerized tomography (PET/CT) surveillance at high risk of relapse following surgery. The aim of this study was to provide an update on our surveillance protocol with an extended sample size and longer duration of follow-up. Methods: From 2009, patients with AJCC stage 3 melanoma underwent PET/CT scans according to pre-specified schedules based on Bayesian probabilities of sub-stage-specific relapse. Schedules were stage 3A: 6, 18 mo; stage 3B: 6, 12, 18, 24, 36, 48, 60 months; stage 3C: 6, 12, 18, 24, 36 months. Contingency tables were used to evaluate the sensitivity, specificity and predictive values of these schedules. Results: In total, 171 patients (3A: 34; 3B: 93; 3C:44) underwent 553 PET/CT scans with a median follow up of 47 months. Relapses were identified in 65 (38%) patients of which (72%) were asymptomatic at the time of radiologically documented relapse. False positive results occurred in 8%. The positive predictive value (PPV) of an individual scan for diagnosing true recurrence was 77% (64-87%). Negative scans at 6 months had negative predictive values (NPV) between 57% in Stage 3A to 69% in Stage 3B for relapse. Sensitivity and specificity of the overall approach of sub-stage-specific PET/CT surveillance for detecting disease relapse were 70% and 89%, respectively. Evaluable predictive values for detecting disease relapse were: stage 3A: PPV:56%, NPV:76%; 3B: PPV 83%, NPV 86%; stage 3C: PPV 84%, NPV 84%. 32 of 65 patients (49%; 3A: 1; 3B: 7; 3C: 2) underwent resection of relapsed disease and 10 of these patients remained free of disease with a median follow-up of 24 months. Conclusions: Sub-stage-specific PET/CT is effective in detecting asymptomatic recurrence in stage 3 melanoma, and is associated with a high rate of disease resection at relapse.

<p>Supplementary Figure 7. No cytokine storm was observed in WAP- and MMTV-Her2 mice post the ACTIV+Pano treatment.</p>