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Our Laboratory was established in 1994 at Univ. of Pennsylvania. Lab members demonstrated initial competencies by performing cell culture, western blots, immunofluorescence, and flow cytometry showing induction of p53/p21(WAF1) in cells treated with chemotherapy. Years later, our Laboratory of Translational Oncology & Experimental Cancer Therapeutics moved to Penn State Univ., Fox Chase Cancer Center/Temple Univ. and then Brown Univ. By 2020, with desire for inclusiveness (everyone succeeds), scientific rigor/reproducibility mandated by NIH, and as a training and mentoring activity (lab scientists/trainees/students mentoring others at High School level and beyond), we established a process for onboarding and training new cancer researchers. By Fall of 2022, there were 17 current Brown University undergraduate students (10 receiving research credit and 7 not receiving credit), HS students, 7 graduate students (PhD, masters, MD/PhD), and 6 medical students working with collaborating faculty at our laboratory at Brown’s Legorreta Cancer Center. After completion of biosafety training, and required trainings such as by IACUC, new lab members complete basic competencies in cell culture, cell viability, and western blot analysis that include technical, presentation quality output, and quantitative/statistical rigor to satisfy current standards for journal publication. For cell culture this includes pathogen free conditions, authentication, attention to details of routine procedures, documentation of morphology, freezing, thawing, passaging, seeding density, and managing cell populations to not run out of cells. Cell viability assessment includes attention to culture conditions, synergy analysis, data robustness, and presentation, and for western blots attention to quality of blots, protein quantification, loading, labeling, antibody specificity and sensitivity controls, presentation at 2022 standards, conventions for splicing, and issues with reproducibility including biological replicates, and generalizability. Additional and advanced competencies include RT-PCR, long-term colony assays, 3-D cultures (spheroids, organoids), transfection (overexpression, knockdown, CRISPR), co-culture and triculture with immune cells and fibroblasts, cytokine profiling, in vivo studies, in vivo imaging, immunohistochemistry, flow cytometric analysis, single cell techniques, viral infection, circulating tumor cell isolation, blood immune and cytokine analysis, and work with transgenic organoids and inducible cancer predisposing alleles. Modeling the tumor microenvironment, relevance to human cancer and translational directions are emphasized. Shared online lab resources, protocols, practices, videos, and manuscripts are available for lab members. The framework herein may be of interest to others involved in similar training programs. Citation Format: Wafik S. El-Deiry, Andrew George, Francesca Di Cristofano, Praveen Srinivasan, Lindsey Carlsen, Kelsey E. Huntington, Arielle De La Cruz, Leiqing Zhang, Marina Hahn, Shuai Zhao, Attila Seyhan, Bradley D. DeNardo, Aaron W. Maxwell, Dae Hee Kim, Alex Raufi, Hina Khan, Stephanie L. Graff, Don S. Dizon, Christopher Azzoli, Abbas E. Abbas, Roxanne Wood, Rishi R. Lulla, Howard P. Safran, Benedito A. Carneiro, Arunasalam Navaraj, Xiaobing Tian, Shengliang Zhang, Lanlan Zhou. Inclusive basic and advanced translational laboratory research competencies for research in cancer biology and therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4185.

Thoracic therapeutic ionizing radiation is limited by toxicities such as pneumonitis and fibrosis of the lungs. Such limitation restricts therapeutic doses and adversely affects patient quality of life while undergoing and following treatment. ONC201/TIC10 is a small-molecule anti-cancer drug that activates the integrated stress response (ISR) and drives the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. Pegylated recombinant long-acting TRAIL (TLY012) has been shown in preclinical models to induce the reversal of fibrosis and currently has orphan drug status for systemic sclerosis and chronic pancreatitis. We show a similar effect of both TLY012 and ONC201 in vivo in protecting from radiation pneumonitis and fibrosis of the lungs. WT and TRAIL-/- C57Bl/6 mice receiving a single 20 Gy thoracic radiation dose with shielding of other organs and treated with 10 mg/kg of TLY012 twice a week showed a significantly reduced alveolar wall thickness and lessened inflammation compared to controls and DR5-/- mice receiving the same treatment upon histological analysis of the lungs conducted 13 days post-irradiation. WT and TRAIL-/- C57Bl/6 mice treated with 100 mg/kg of ONC201 once a week showed similar effect to a lesser extent. Further analysis in C57Bl/6 WT mice bearing orthotopic mammary fat pad e0771 TNBC tumors similarly receiving a single 20 Gy thoracic radiation dose revealed the same pattern of protection from radiation pneumonitis upon TRAIL-pathway agonism through treatment with TLY012 and ONC201 both in combination and alone, while also showing a significant reduction in tumor burden at the experimental endpoint (day 9 post-irradiation) in the combination treated mice. Further, pulse oximetry readings of the hind paw revealed a notable reduction in oxygen saturation in all mice except those treated with TLY012. Cytokinomic profiling of mouse serum upon sacrifice revealed a significant reduction in CCL22/MDC levels in the TLY012 cohort. Additional post-hoc analysis including immunophenotyping, immunostaining, and bulk RNA analysis through Nanostring nCounter technologies is underway. Altogether, these findings suggest a role for TLY012, ONC201, or broader modulation of the TRAIL/DR5 pathway in mitigating adverse effects and outcomes of therapeutic radiation, and may serve as a foundation for safer use of radiation in the clinic. Citation Format: Jillian R. Strandberg, Anna Louie, Marina Hahn, Praveen Srinivasan, Andrew George, Arielle De La Cruz, Leiqing Zhang, Liz Hernandez Borrero, Kelsey Huntington, Christopher Azzoli, Abbas E. Abbas, Lanlan Zhou, Seulki Lee, Wafik S. El-Deiry. Post exposure suppression of radiation pneumonitis by TRAIL pathway agonists TLY012 and ONC201. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4457.

Pre-treatment FDG-PET/CT scans were analyzed with machine learning to predict progression of lung malignancies and overall survival (OS).A retrospective review across three institutions identified patients with a pre-procedure FDG-PET/CT and an associated malignancy diagnosis. Lesions were manually and automatically segmented, and convolutional neural networks (CNNs) were trained using FDG-PET/CT inputs to predict malignancy progression. Performance was evaluated using area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, and specificity. Image features were extracted from CNNs and by radiomics feature extraction, and random survival forests (RSF) were constructed to predict OS. Concordance index (C-index) and integrated brier score (IBS) were used to evaluate OS prediction.1168 nodules (n=965 patients) were identified. 792 nodules had progression and 376 were progression-free. The most common malignancies were adenocarcinoma (n=740) and squamous cell carcinoma (n=179). For progression risk, the PET+CT ensemble model with manual segmentation (accuracy=0.790, AUC=0.876) performed similarly to the CT only (accuracy=0.723, AUC=0.888) and better compared to the PET only (accuracy=0.664, AUC=0.669) models. For OS prediction with deep learning features, the PET+CT+clinical RSF ensemble model (C-index=0.737) performed similarly to the CT only (C-index=0.730) and better than the PET only (C-index=0.595), and clinical only (C-index=0.595) models. RSF models constructed with radiomics features had comparable performance to those with CNN features.CNNs trained using pre-treatment FDG-PET/CT and extracted performed well in predicting lung malignancy progression and OS. OS prediction performance with CNN features was comparable to a radiomics approach. The prognostic models could inform treatment options and improve patient care.NIH NHLBI training grant (5T35HL094308-12, John Sollee).

Lung cancer is the leading cause of cancer death in both men and women according to the American Cancer Society, with more deaths than colon, breast, and prostate cancer combined. Small cell lung cancer (SCLC) comprises about 10 to 15 percent of all lung cancer diagnoses. Frequent loss of RB1 and mutations in p53 in SCLC make it rather difficult to develop targeted therapies. Many current therapies rely on toxic chemotherapy or immunotherapy to combat the aggressive nature of SCLC; however, even with the advent of new therapies, the median survival rate has remained relatively low at 12 to 16 months. SCLC is also characterized by a high mutation rate, resulting in patients becoming increasingly resistant to treatments. This calls for a novel combination to extend patient life expectancy. ONC201 is an imipridone that is currently in clinical trials for its proapoptotic effects in several cancer types. We propose that combining ONC201 with standard first-line chemotherapeutic agents etoposide and carboplatin will result in a synergistic effect and serve as a novel treatment plan for SCLC patients. SCLC cell lines H1048 and H1105 were treated with the novel triple drug combination ONC201, etoposide, and carboplatin. Cell-Titer Glo cell viability assays were conducted for 72 hours to see the additive and/or synergistic effect of novel triple drug treatment. Western blots were also run to evaluate the expression of proapoptotic protein cleaved PARP or expression of cellular metabolic stress protein ATF4 to explore the synergistic effect of triple drug treatments in SCLC cell lines. Cell viability after the triple drug treatment in both cell lines H1048 and H1105 may indicate a potential synergistic effect. Preliminary western results indicate that the triple drug treatment results in an increase in cPARP or ATF4 expression relative to the vehicle, suggesting a synergistic effect of the triple drug treatment. We are currently exploring the novel combination and potential underlying mechanisms behind the synergistic effects of the triple drug treatment. Future studies will evaluate this triple drug combination treatment in mouse models of SCLC. Citation Format: Young S. Lee, Lanlan Zhou, Christopher Azzoli, Wafik S. El-Deiry. ONC201 in combination with carboplatin and etoposide as a novel triple drug treatment for small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4056.