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Supplementary Tables 1-2, Figure Legends 1-4 from MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Supplementary Figure 1 from MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Supplementary Figure 3 from MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Supplementary Figure 2 from MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Supplementary Figure 4 from MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Funder: Victorian Cancer Agency, Funder: NIHR Manchester Biomedical Research Centre, Funder: Cancer Research UK, Funder: Cancer Council Tasmania, Funder: Instituto de Salud Carlos III, Funder: Cancer Australia, Funder: NIHR Oxford Biomedical Research Centre, Funder: Fundación Científica de la Asociación Española Contra el Cáncer, Funder: Cancer Council South Australia, Funder: Swedish Cancer Society, Funder: NIHR Cambridge Biomedical Research Centre, Funder: Institut Català de la Salut, Funder: Cancer Council Victoria, Funder: Prostate Cancer Foundation of Australia, Funder: National Institutes of Health, BACKGROUND: Lynch syndrome is a rare familial cancer syndrome caused by pathogenic variants in the mismatch repair genes MLH1, MSH2, MSH6, or PMS2, that cause predisposition to various cancers, predominantly colorectal and endometrial cancer. Data are emerging that pathogenic variants in mismatch repair genes increase the risk of early-onset aggressive prostate cancer. The IMPACT study is prospectively assessing prostate-specific antigen (PSA) screening in men with germline mismatch repair pathogenic variants. Here, we report the usefulness of PSA screening, prostate cancer incidence, and tumour characteristics after the first screening round in men with and without these germline pathogenic variants. METHODS: The IMPACT study is an international, prospective study. Men aged 40-69 years without a previous prostate cancer diagnosis and with a known germline pathogenic variant in the MLH1, MSH2, or MSH6 gene, and age-matched male controls who tested negative for a familial pathogenic variant in these genes were recruited from 34 genetic and urology clinics in eight countries, and underwent a baseline PSA screening. Men who had a PSA level higher than 3·0 ng/mL were offered a transrectal, ultrasound-guided, prostate biopsy and a histopathological analysis was done. All participants are undergoing a minimum of 5 years' annual screening. The primary endpoint was to determine the incidence, stage, and pathology of screening-detected prostate cancer in carriers of pathogenic variants compared with non-carrier controls. We used Fisher's exact test to compare the number of cases, cancer incidence, and positive predictive values of the PSA cutoff and biopsy between carriers and non-carriers and the differences between disease types (ie, cancer vs no cancer, clinically significant cancer vs no cancer). We assessed screening outcomes and tumour characteristics by pathogenic variant status. Here we present results from the first round of PSA screening in the IMPACT study. This study is registered with ClinicalTrials.gov, NCT00261456, and is now closed to accrual. FINDINGS: Between Sept 28, 2012, and March 1, 2020, 828 men were recruited (644 carriers of mismatch repair pathogenic variants [204 carriers of MLH1, 305 carriers of MSH2, and 135 carriers of MSH6] and 184 non-carrier controls [65 non-carriers of MLH1, 76 non-carriers of MSH2, and 43 non-carriers of MSH6]), and in order to boost the sample size for the non-carrier control groups, we randomly selected 134 non-carriers from the BRCA1 and BRCA2 cohort of the IMPACT study, who were included in all three non-carrier cohorts. Men were predominantly of European ancestry (899 [93%] of 953 with available data), with a mean age of 52·8 years (SD 8·3). Within the first screening round, 56 (6%) men had a PSA concentration of more than 3·0 ng/mL and 35 (4%) biopsies were done. The overall incidence of prostate cancer was 1·9% (18 of 962; 95% CI 1·1-2·9). The incidence among MSH2 carriers was 4·3% (13 of 305; 95% CI 2·3-7·2), MSH2 non-carrier controls was 0·5% (one of 210; 0·0-2·6), MSH6 carriers was 3·0% (four of 135; 0·8-7·4), and none were detected among the MLH1 carriers, MLH1 non-carrier controls, and MSH6 non-carrier controls. Prostate cancer incidence, using a PSA threshold of higher than 3·0 ng/mL, was higher in MSH2 carriers than in MSH2 non-carrier controls (4·3% vs 0·5%; p=0·011) and MSH6 carriers than MSH6 non-carrier controls (3·0% vs 0%; p=0·034). The overall positive predictive value of biopsy using a PSA threshold of 3·0 ng/mL was 51·4% (95% CI 34·0-68·6), and the overall positive predictive value of a PSA threshold of 3·0 ng/mL was 32·1% (20·3-46·0). INTERPRETATION: After the first screening round, carriers of MSH2 and MSH6 pathogenic variants had a higher incidence of prostate cancer compared with age-matched non-carrier controls. These findings support the use of targeted PSA screening in these men to identify those with clinically significant prostate cancer. Further annual screening rounds will need to confirm these findings. FUNDING: Cancer Research UK, The Ronald and Rita McAulay Foundation, the National Institute for Health Research support to Biomedical Research Centres (The Institute of Cancer Research and Royal Marsden NHS Foundation Trust; Oxford; Manchester and the Cambridge Clinical Research Centre), Mr and Mrs Jack Baker, the Cancer Council of Tasmania, Cancer Australia, Prostate Cancer Foundation of Australia, Cancer Council of Victoria, Cancer Council of South Australia, the Victorian Cancer Agency, Cancer Australia, Prostate Cancer Foundation of Australia, Asociación Española Contra el Cáncer (AECC), the Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional (FEDER), the Institut Català de la Salut, Autonomous Government of Catalonia, Fundação para a Ciência e a Tecnologia, National Institutes of Health National Cancer Institute, Swedish Cancer Society, General Hospital in Malmö Foundation for Combating Cancer.

Background: Prostate cancer (PCa) is a clinically and molecularly heterogeneous disease, with differences in incidence and mortality among and between racial groups, which grade and stage only partially predict. Prostate cancer patient-derived xenografts (PCPDXs) are essential for studying PCa biology and testing new therapeutics in models that we expect to be reflective of the clinical setting. To date, PCPDXs have been difficult to establish due to lack of solid tumor content and poor uptake rates in mice. In the present study, we established and characterized the first PCPDX from a highly aggressive and metastatic tumor sample from a patient of African ancestry. Methods: We collected the PCa sample from the patient at the time of surgery after initiating stage-specific standard-of-care treatment. Tissue was minced and implanted into the kidney capsule of 8- to 10-week-old SCID mice. Once the explant was established, tissue was collected and formalin-fixed, paraffin-embedded for histologic evaluation. An initial section was stained using hematoxylin and eosin and another section was stained by PIN4 (p63, CKBE12, racemase) for immunohistochemical (IHC) analysis to confirm diagnosis of PCa. To obtain genetically estimated indicators of race, we performed ancestral genotyping. We evaluated response to chemical castration with 10mg/kg enzalutamide administered 5 days per week by oral gavage. Tumor growth was measured with calipers every day. Results: We established a PCPDX from a core prostate sample taken from a patient diagnosed with adenocarcinoma of the prostate (Gleason 10), metastatic to lymph nodes and penis undergoing a pelvic exenteration. Ancestral genotyping estimated 90% African ancestry. IHC staining showed this model to be highly tumorigenic and PSA negative. Passaged tumors took in 15 out of 15 SCID mice and reached 10mm3 within 3 weeks. This model does not show a significant decrease in growth in response to treatment with the androgen receptor inhibitor, enzalutamide. Further characterization using IHC, RNA and whole-exome sequencing, castration, drug treatment, and assays for metastatic potential are currently under way. Conclusions: Establishing this PCPDX provides a unique model of metastatic, androgen-independent PCa in a patient of African ancestry. Prior to this study, there had yet to be a PCPDX model derived from a patient of African ancestry. We established this PCPDX from a Gleason 10 PCa, supporting previous data of success in grafting this type of aggressive PCa into a mouse. It has a very high take rate and growth rate relative to other PCa models. Such a model will enable interrogation of PCa from a patient of African ancestry. Once a larger panel of PCPDXs from racially diverse patients is established, we will be able to achieve a more complete characterization of this disease and use such models to develop new biomarkers and therapeutic agents. Ultimately, these tools will improve outcomes for all men with aggressive PCa and reduce PCa disparities for patients of African ancestry. Citation Format: Brendon M. Patierno, Wayne Glover, Wen-Chi Foo, Jason A. Somarelli, Kathryn E. Ware, Lingfan Xu, Yanjing Li, Xufeng Chen, Daniel J. George, Rick A. Kittles, Andrew J. Armstrong, Shannon J. McCall, Jiaoti Huang, Jennifer A. Freedman, Steven R. Patierno, David S. Hsu. Characterization of a metastatic prostate cancer xenograft derived from a patient of African ancestry [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A114.

Aim: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor in vivo. Materials & methods: We grew HPV16+, patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs. Results & conclusion: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9. These studies provide proof of principle for the hypothesis that CRISPR/Cas has the potential to be used to selectively treat HPV-induced tumors in humans.

Background Standard of care treatment for colorectal cancer liver metastasis consists of a cytotoxic chemotherapy in combination with a targeted agent. Clinical trials have guided the use of these combinatory therapies, but it remains unclear what the optimal combinations of cytotoxic chemotherapy with a targeted agent are. Methods Using a genomic based approach, gene expression profiling was obtained from tumor samples of patient with colorectal cancer liver metastasis who received an oxaliplatin based therapy. Early passaged colorectal cancer liver metastasis cell lines and patient derived xenografts of colorectal cancer liver metastasis were then treated with oxaliplatin and a mTOR inhibitor. Results Gene set enrichment analysis revealed that the mTOR pathway was activated in patients receiving oxaliplatin based therapy. Treatment of early passaged colorectal cancer lines and patient derived xenografts with oxaliplatin resulted in activation of the mTOR pathway. Combination therapy with oxaliplatin and a mTOR inhibitor resulted in a synergistic effect both in vitro and in vivo. Conclusion Our findings suggest a genomic based approach can be used to identify optimal combinations of cytotoxic chemotherapy with a targeted agent and that these observations can be validated both in vitro and in vivo using patient derived colorectal cancer cell lines and patient derived xenografts prior to clinical use.

Background: Prostate cancer (PCa) is a clinically and molecularly heterogeneous disease, with differences in incidence and mortality among and between racial groups, which are only partially predicted by grade and stage. Age-adjusted incidence and mortality rates for PCa among African American (AA) men are 1.6- and 2.4-fold greater, respectively, than among white men. Preclinical models of AA PCa, including cell lines and xenografts, are severely lacking. This work addresses the urgent need to generate such models for study of the biology of AA PCa and for testing of novel therapeutic agents in models that are expected to be reflective of the clinical setting. Methods: To generate AA PCa patient-derived primary cell lines, we have used the fibroblast feeder cell system established at Georgetown University. In this method, human tumor cells are cocultivated with irradiated mouse fibroblasts in medium with addition of ROCK inhibitor. For generation of AA PCa patient-derived xenografts, we have collected sections of human prostate tumors, minced, suspended, and implanted in the renal capsule of immunodeficient mice. Tumor specimens have been collected and formalin-fixed, paraffin-embedded for histologic evaluation. Sections of such blocks have been stained using hematoxylin and eosin. DNA from the tumor specimens has been used to perform ancestral genotyping. Results: We have established eight AA PCa patient-derived primary cell lines and two AA PCa patient-derived xenografts. The AA PCa patient-derived primary cell lines have grown throughout the feeder layer and directly onto the plastic of the flasks. Currently, these lines are growing without addition of fibroblast feeder cells. The two AA PCa patient-derived xenografts have been passaged beyond three generations and key histologic features have been found to be consistent between the patient tumor and the patient-derived explant. In addition, African ancestry of the patient has been confirmed by ancestral genotyping. Further studies to more fully characterize the AA PCa patient-derived primary cell lines and xenografts at the pathologic, biologic, and molecular levels are currently under way. Procurement of individual AA PCa patient tissues continues for establishment of additional AA PCa patient-derived primary cell lines and xenografts. Conclusions: Strategies designed to maximize PCa specimen availability and tumor content enable the establishment of AA PCa patient-derived primary cell lines and xenografts. Such preclinical models of AA PCa will enable a more rigorous interrogation of the molecular mechanisms underlying AA PCa and aid in the development of new biomarkers and therapeutic agents for AA PCa. Citation Format: Brendon Patierno, Wayne Glover, Thomas Ribar, Rick Kittles, Wen-Chi Foo, Shannon McCall, Jiaoti Huang, Daniel George, Jennifer Freedman, Steven Patierno, Kris Wood, David Hsu. Establishment of African American prostate cancer patient-derived primary cell lines and xenografts [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A79.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E–binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy. [Cancer Res 2009;69(19):7803–10]

160 Background: Radium-223 is a targeted alpha therapy that improves survival in men with mCRPC. The biologic basis for radium-223 efficacy is not completely understood. We hypothesized that PC osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to the intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment. Methods: We conducted a pharmacodynamic study of radium-223 in men with bone predominant mCRPC to investigate genomic and phenotypic alterations in circulating tumor cells (CTCs), ctDNA, and metastases. Prior to radium and 3 and 6 months after radium, liquid and metastatic biopsies were collected, including CTCs for phenotypic characterization and CTC/ctDNA genomic analysis. The primary objective was to describe the prevalence of CTC bone alkaline phosphatase (BAP) over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. Results: We enrolled 20 men with heavily pre-treated symptomatic bone predominant mCRPC and treated with radium-223 over a median of 6 doses. 55% had elevated serum BAP. PFS was 5.5 mo; OS was 13.3 mo; 10% had unfavorable (≥5) to favorable ( < 5) Cellsearch CTC conversion; 5% had ≥30% PSA decline. We found evidence of persistent CTC BAP expression in both EpCAM+ and EpCAM- CTCs in the majority of men over time during radium treatment. We identified genomic gain of key osteomimicry regions in CTC DNA, including loci for BAP, osteopontin, and OB-cadherin. Radium-223 uptake was observed in tumor to a greater degree than surrounding normal bone. CTC DNA and matched ctDNA and CTC cultures suggested persistence of CTCs with aggressive genomic alterations such as AR, FOXA1, and MYC gain and PTEN and RB1 loss. We established multiple CTC cultures and one CTC PDX; cells exhibited evidence of epithelial plasticity with BAP expression. Conclusions: Osteomimicry may contribute to the uptake of Radium-223 within bone metastases and may thereby enhance the therapeutic benefit of radium-223. We found genomic and phenotypic evidence of osteomimicry in CTCs and CTC cultures from men with mCRPC. Clinical trial information: NCT02204943.

Introduction: Metastatic prostate cancer patients often become resistant to standard treatments such as androgen ablation therapy and docetaxel treatment and novel treatments are needed. Loss of PTEN and subsequent activation of the PI3K pathway is common in patients with advanced prostate cancer. However, use of mTOR inhibitors such as rapamycin analogues as single agents has had limited clinical impact and the identification of a potent therapeutic strategy for PI3K inhibition remains a critical question. Experimental procedures: We have used gene expression data generated from metastatic tumors collected from patients with advanced prostate cancer both before and after treatment with RAD001 along with in vitro experiments in established prostate cancer cell lines to determine the impact of TORC1 inhibition, PI3K kinase inhibition, and dual inhibition on PI3K pathway signaling and cancer cell kinetics. Summary: Treatment with RAD001 decreased tumor levels of pS6 but failed to result in a single clinical response. Gene set enrichment analysis of metastatic castration-resistant prostate cancer biopsies prior to and following treatment with RAD001 was associated with increased PI3K activity following treatment. Analysis of the activity of the downstream components of the PI3K/AKT pathway following RAD001 treatment showed reduced pS6 levels, had minimal impact on p4EBP1 levels, and increased phosphorylation of AKT1. Cytotoxic synergy is observed when RAD001 is combined with the PI3K inhibitor, LY294002 and dual PI3K/mTOR kinase inhibitors. These combinations result in more profound reduction of TORC1 activity resulting in decreased cell proliferation and increased autophagy but a continued persistence of pAKT. Reverse phase proteomics supports increased AKT activity in an androgen-receptor positive prostate cancer cell line treated with the combination as multiple AKT targets experience increased phosphorylation. Conclusion: TORC1 inhibition results in an upregulation of PI3K activity and therapeutic combinations that inhibit both TORC1 and PI3K demonstrate cytotoxic synergy. The signaling manifestations of these combinations are a profound inhibition of TORC1 activity together with persistent phosphorylation and activity of AKT. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C61.