FOXA1 Mutations Research Articles

Abstract PR012: KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer

Abstract The estrogen receptor (ER) is expressed in ∼70% of breast cancers where it functions as a critical transcription factor that regulates cell growth and tumor progression. ER inhibition is a mainstay treatment for ER-positive (ER+) breast cancer, but despite the clinical benefit of targeting ER signaling, breast cancer remains the second leading cause of cancer-related death in women, with ∼50% of deaths resulting from ER+ tumors. While recurrent and metastatic ER+ tumors are typically resistant to endocrine therapies they continue to rely on ER signaling suggesting that disruption of other chromatin associated complexes that mediate ER-driven gene expression may present an alternative therapeutic strategy. One chromatin modifier important in regulating ER function is the histone acetyltransferase KAT6A. KAT6A is amplified and/or overexpressed in ∼15% of ER+ tumors and a novel KAT6A/B inhibitor has entered clinical trials for advanced breast cancer. However, as with most single agent therapies, compensatory mechanisms may attenuate the effects of KAT6A/B inhibition on ER-driven gene expression. We hypothesized that the dual targeting of distinct chromatin complexes would enhance the effects of KAT6A/B inhibition, by altering chromatin state and transcriptional output. To that end we performed an epigenetic focused CRISPR-Cas9-based functional genetic screen to identify chromatin-associated proteins that enhance KAT6A/B inhibition. CRISPR guides targeting MEN1 (encoding Menin) were among our top hits as sensitizers to treatment with the KAT6A/B inhibitor PF-9363. We confirmed that co-treatment of PF-9363, and the Menin inhibitor, SNDX-5613, had strong synergistic anti-proliferative effects in a panel of ER+ breast cancer cell lines. Mechanistically, our data revealed that KAT6A/B and Menin cooperatively regulate ER-driven gene expression and chromatin accessibility, both through direct effects on ESR1 expression as well as through effects at ER target genes. Specifically, we found that KAT6A and Menin-KMT2A complexes co-localized at promoters of ER target genes and treatment with KAT6A/B or Menin inhibitors displaced KAT6A and Menin/KMT2A from ER target genes, respectively. Yet only the combination effectively displaced both complexes from chromatin, which produced more dramatic changes in ER-directed gene expression than either small molecule alone. Combined displacement of KAT6A and Menin/KMT2A at ER-regulated genes induced loss of an active chromatin landscape characterized by reduction of activating histone modifications and loss of RNA Polymerase II binding to ER target loci. Importantly, the combination of KAT6A/B and Menin inhibition was effective in a panel of ER+ patient-derived organoid models as well as in various models of endocrine resistance, including models with ESR1 mutations, NF1 loss, and FOXA1 mutations. Both KAT6A/B and Menin inhibitors are in Phase I/II clinical trials and have shown manageable toxicity profiles to date. This combination represents a novel potential therapeutic combination for ER+ breast cancer. Citation Format: Sarah Naomi Olsen, Bryn Anderson, Charles Hatton, Rinath Jeselsohn, Myles Brown, Eneda Toska, Scott Armstrong. KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR012.

Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Abstract 2147: Genetic ancestry associations with prostate adenocarcinoma mutational profiles: New Insights from a diverse 5,959-patient real-world cohort

Abstract In prostate cancer, Black men face higher incidence rates, more advanced diagnoses and worse outcomes than White men. These disparities partially stem from environmental, socioeconomic, and healthcare access factors. Recent data highlight racial differences in prostate cancer molecular profiles, with tumors from Black men showing increased SPOP and decreased PIK3CA mutations and amplified androgen receptor signaling and inflammatory pathway activations. Thus, somatic mutation patterns may also contribute to these disparities. We sought to identify associations of mutational profiles in prostate adenocarcinoma (PRAD) with genetic ancestry instead of race and ethnicity categories, analyzing a de-identified cohort of 5,959 patients who underwent the Tempus xT 648-gene NGS tumor profiling test. We used 654 ancestry-informative markers to estimate genetic ancestry by calculating similarity to reference populations for five regions: Africa (AFR), Americas (AMR), East Asia (EAS), Europe (EUR), and South Asia (SAS). Associations between genetic ancestry proportions and somatic variants were assessed for 67 PRAD-related genes using logistic regression models. Analyses included copy number alterations (CNAs), oncogenic gene fusions, small splice and non-synonymous mutations (NS), OncoKB L1/2 & R1, and small missense driver somatic mutations predicted with boostDM. Likelihood ratio tests were employed to compare models with and without ancestry terms. The Benjamini-Hochberg method was utilized to adjust p-values and control the FDR at 5%. We confirmed reported associations in PRAD between AFR ancestry and SPOP NS mutations (OR=1.03 per doubling of AFR ancestry proportion, p=0.03) and decreased AFR with PIK3CA OncoKB and driver mutations (OR=0.94, p=0.02), PTEN CNAs (OR=0.95, p=0.0001), and TMPRSS2-ERG fusions (OR=0.95, p<0.0001). However, we also observed negative associations between AFR and AR CNAs (OR=0.95, p=0.004), EAS and PTEN CNAs (OR=0.96, p=0.01), and EAS and TMPRSS2-ERG fusions (OR=0.97, p=0.02). Additionally, a negative association was found between EAS and TP53 driver NS mutations (OR=0.96, p=0.04), while positive associations with driver mutations in FOXA1 (OR=1.06, p=0.03) and NS mutations in the PI3K/AKT/mTOR pathway genes (OR=1.04, p=0.01) were noted. Lastly, we identified novel positive associations between AMR and driver mutations in PIK3C3A (OR=1.12, p=0.0003) as well as small NS mutations or CNAs in the PI3K/AKT/mTOR pathway genes (OR=1.03, p=0.04). By analyzing a large, diverse real-world cohort and leveraging NGS-inferred genetic ancestry, our study confirms known associations between somatic alterations in PRAD cancer genes and race and ethnicity, while unveiling novel associations in understudied populations of potential significance for understanding disparities in disease outcomes. Citation Format: Brooke Rhead, Yannick Pouliot, Francisco M. De La Vega, David W. Craig, John Carpten. Genetic ancestry associations with prostate adenocarcinoma mutational profiles: New Insights from a diverse 5,959-patient real-world cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2147.

Abstract 1433: FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice

Abstract Androgen receptor (AR) signaling is critical for survival of prostate cancer (PCa) cells, thus making androgen deprivation therapy (ADT; e.g., castration) as the mainstay for treatment. Notably, oncogenic functions of AR rely on chromatin-binding regulatory proteins, which includes a pioneer transcription factor called FOXA1. FOXA1 de-compacts chromatin to enable AR’s binding to the DNA and expression of it's target genes. Recently, our lab found FOXA1 alterations to recur within three distinct structural classes in over 35% of metastatic castration resistant PCa (mCRPC) in Caucasian men. Subsequent studies found FOXA1 mutations to be prevalent in over 40% of primary PCa in Chinese men, positioning FOXA1 as a principal oncogene in this disease. Yet, hitherto, no studies have defined the pathobiology of FOXA1 alterations in mouse models. Here, we have developed the first-in-field transgenic mice with conditional overexpression of FOXA1 mutants in the prostate luminal epithelia. We found truncal FOXA1 Class1 mutants to initiate luminal hyperplasia in a monogenic model, or hyperproliferative prostate adenocarcinoma in a compound Trp53-deficient genetic background. Mechanistically, Class1 mutants concurrently upregulate the AR and mTORC1/2 pathways to drive transformation, with this being the first report of FOXA1-driven PCa formation in mice. In contrast, FOXA1 Class2 mutants—which are acquired in mCRPC—do not drive prostate luminal transformation. Instead, single-cell multi-omics (RNA+ATAC) profiling of Class2-mutant mouse prostate tissue uncovered extensive transcriptional remodeling of epithelial cells into a luminal stem-like cell fate, leading to a dramatic 15-20-fold expansion of the progenitor population vs control tissue. These luminal stem cells are similar to the Club/Hillock cells detected in the human prostate that have been implicated in driving resistance to ADT. Consistently, we found Class2-mutant prostates to show minimal atrophy upon castration, with immunohistological assessment uncovering a higher density of Ki67+ luminal epithelial cells relative to wild type and Class1 tissues. Class2-mutant organoids also showed higher subcutaneous grafting ability in mice in limiting dilution assays. Mechanistically, we found the cistromically-dominant Class2 mutants to pioneer over 40,000 neo-enhancer elements harboring motifs of stemness-associated transcription factors, which in turn instruct the ADT-resistant luminal progenitor gene program. Altogether, findings from our mouse models uncover FOXA1’s versatility as a driver oncogene that, depending on the mutation type, either activates enhancer-wired luminal tumorigenesis (Class1-initiating event) or therapy resistance-associated stemness (Class2-promoting alteration) gene programs in the mouse prostate tissue. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James Matthew George, Rahul Mannan, Sandra E. Carson, Yuping Zhang, Jean Tien, Mustapha Jaber, Jie Luo, Matthew Pang, Rohit Mehra, Xuhong Cao, Fengyun Su, Rui Wang, Marcin Cieslik, Dong Kee Lee, Jianming Xu, Arul M. Chinnaiyan. FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1433.

The opposing effects of Class 1B and Class 2 FOXA1 mutations in prostate cancer.

216 Background: PCa harbors recurrent FOXA1 alterations, yet distinct alteration types may drive divergent consequences. Here, we established several classes of FOXA1 alterations and evaluated their clinical outcomes, molecular correlates, and immunologic landscape in a large cohort of primary and metastatic PCa samples from Caris Life Sciences. Methods: We interrogated the Caris DNA (592-gene/whole exome) and RNA (whole transcriptome) NGS databases. FOXA1 alterations were annotated based on amino acid (a.a.) location and prior functional studies (Table). Real-world overall survival (OS) was determined from insurance claims data using Kaplan-Meier estimates. Gene set enrichment analyses (GSEA) was used to estimate signaling pathway activity. Immune-cell fractions were inferred from bulk RNA-seq using quanTIseq. All comparisons are made relative to FOXA1 WT cases. Results: The overall prevalence of FOXA1 alterations was 16%, with no enrichment in metastatic PCa samples (15.5%). Class 2 mutations were enriched in histologic NEPC, were associated with a classical NEPC mRNA signature, and showed poor survival with ADT (HR 5.6, 95% CI: 2.5–12.5, p<0.001). Conversely, metastatic PCa patients with Class 1B mutations had numerically improved survival with ADT (HR 0.76, 95% CI: 0.50–1.14, p=0.18). All FOXA1 alterations (except Class 2) resulted in increased FOXA1 transcripts, with 1B and 1C having the greatest effects (q<0.0001). Class 0 and 3 alterations had 2–5 fold higher frequency of TMB-high and MSI-high status (q<0.05). Conversely, Class 1B exhibited lower TMB and markedly fewer TP53 mutations (35.7% vs 20.3%, q<0.001) and TMPRSS2-ERG fusions (34.5% vs 4.6%, q<0.001). Class 1B was depleted of AR transcriptional signatures (q=0.01) but enriched of MYC signaling in GSEA analysis (FDR<0.001), and showed reduced expression of B7-H3. On immune-cell deconvolution analysis, Class 1B showed reduced dendritic cells, but increased B cells and neutrophils (DC: 1.96% vs 0.01%, B cell: 4.27% vs 5.29%, neutrophils: 5.05% vs 8.00%, each q<0.001). Conclusions: Different FOXA1 alterations exhibit divergent molecular and clinical features, and should not be interpreted in aggregate. In particular, Class 1B mutations are associated with a unique molecular and immunological landscape with potentially better outcomes to ADT, while Class 2 mutations are associated with NEPC phenotype with inferior ADT sensitivity. [Table: see text]

Genomic alterations related to HPV infection status in a cohort of Chinese prostate cancer patients

BackgroundHuman papillomavirus (HPV) has been proposed as a potential pathogenetic organism involved in prostate cancer (PCa), but the association between HPV infection and relevant genomic changes in PCa is poorly understood.MethodsTo evaluate the relationship between HPV genotypes and genomic alterations in PCa, HPV capture sequencing of DNA isolated from 59 Han Chinese PCa patients was performed using an Illumina HiSeq2500. Additionally, whole-exome sequencing of DNA from these 59 PCa tissue samples and matched normal tissues was carried out using the BGI DNBSEQ platform. HPV infection status and genotyping were determined, and the genetic disparities between HPV-positive and HPV-negative PCa were evaluated.ResultsThe presence of the high-risk HPV genome was identified in 16.9% of our cohort, and HPV16 was the most frequent genotype detected. The overall mutational burden in HPV-positive and HPV-negative PCa was similar, with an average of 2.68/Mb versus 2.58/Mb, respectively, in the targeted whole-exome region. HPV-negative tumors showed a mutational spectrum concordant with published PCa analyses with enrichment for mutations in SPOP, FOXA1, and MED12. HPV-positive tumors showed more mutations in KMT2C, KMT2D and ERCC2. Copy number alterations per sample were comparable between the two groups. However, the significantly amplified or deleted regions of the two groups only partially overlapped. We identified amplifications in oncogenes, including FCGR2B and CCND1, and deletions of tumor suppressors, such as CCNC and RB1, only in HPV-negative tumors. HPV-positive tumors showed unique deletions of tumor suppressors such as NTRK1 and JAK1.ConclusionsThe genomic mutational landscape of PCa differs based on HPV infection status. This work adds evidence for the direct involvement of HPV in PCa etiology. Different genomic features render HPV-positive PCa a unique subpopulation that might benefit from virus-targeted therapy.

Abstract PR001: An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and AR therapy resistance

Abstract Tumor mutational burden and heterogeneity fuel resistance to many targeted therapies, which inevitably limits the clinical outcome of patients. The cytosine deaminase APOBEC proteins are major drivers of mutagenesis in human cancer, with over 70% of tumors exhibiting a mutational signature that is impacted by APOBEC activity. Nevertheless, the mechanisms through which tumor cells hijack the powerful APOBEC mutagenesis machinery to promote heterogeneity and therapy resistance remain largely unknown. Through a multi-disciplinary approach integrating bulk and single cell RNA-Seq (scRNA-Seq), whole-genome exome-sequencing (WES), and CRISPR library screening, we identified a long sought-after cell-intrinsic mechanism that prevents APOBEC-driven mutagenesis in non-malignant cells. The loss of this “molecular brake” unleashes APOBEC3B-driven mutagenesis in malignant cells, which then becomes a key mutator and represents the long sought-after molecular source of driver mutations in some frequently mutated genes in cancers, including FOXA1, EP300, and AR. Functional screening identified eight crucial drivers for Androgen Receptor (AR)-targeted therapy resistance in prostate cancer that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. Finally, contrary to conventional understanding, our study reveals that driver mutations in FOXA1 induced by APOBEC3B, not mutations in AR, evolutionarily outcompete other driver mutations and eventually dominate the resistant tumors. Collectively, these results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring targeted therapy resistance and could be the potential therapeutic targets to overcome resistance. Citation Format: Ping Mu, Xiaoling Li, Yunguan Wang. An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and AR therapy resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR001.

Clinical implications of molecular alterations in intraductal carcinoma of the prostate.

5024 Background: Intraductal carcinoma of the prostate (IDC-P) is a recent addition to the 2016 WHO classification system and little is known about the nature of its true incidence and biology. Clinically, it has been observed to be associated with higher grade group tumors and a more aggressive disease course with a high risk of local recurrence and distant metastasis. However, the molecular underpinnings which define IDC-P are not well elucidated. Initial case series with molecular profiling have suggested an enrichment of alterations in DNA damage/repair (DDR), PI3K, WNT and MAPK pathways. We now present comprehensive molecular profiling data from the largest cohort of IDC-P reported to date in order to better characterize this relatively new histologic entity. Methods: Eligible patients included those with radical prostatectomy tumor tissue labeled as IDC-P on testing requisition with available tissue-based DNA and RNA sequencing utilizing a commercially available CLIA-certified assay (Caris Life Sciences). Prostatic tumor specimens were analyzed using NextGen DNA sequencing (NextSeq, 592 genes and NovaSeq, WES). Digitized H&E slides underwent central pathological review by a board certified pathologist (SW) specializing in genitourinary malignancies to confirm presence of intraductal histology according to the 2022 WHO classification. Results: 31 of 44 submitted cases were confirmed to be IDC-P on central review. Median age of patients was 64 yrs (range 44-79). The majority of cases (26/31 - 84%) were Grade Group 5 and all were Grade Group 3 or higher. Overall the most frequently detected alterations were in TP53 (39%), TMPRSS2 fusion (39%), FOXA1 (16%), CDK12 (10%), PIK3CA (10%), SPOP (10%), ATM (7%), AKT1 (7%) and RB1 (5%). The most commonly altered pathways were in cell cycle regulators (50%), DDR ( BRCA1/2, ATM, CDK12) (23%), and PI3K/AKT/MTOR (23%) and WNT (10%). The FOXA1 mutation rate in this cohort was higher compared to the localized prostate cancer cohort from the TCGA (16% - 5/31 vs 6% - 82/1325). In the IDC-P cohort, there were no tumors which demonstrated mismatch-repair deficiency (dMMR) or microsatellite instability and none of the profiled tumors had a high tumor mutational burden (≥10 mutations/megabase). Conclusions: This data suggests that IDC-P harbors targetable molecular alterations in DDR genes ( BRCA1/2 and CDK12) with approved targeted agents (e.g. PARP inhibitors). These findings should underscore the importance of germline and somatic mutation testing when IDC-P is identified. We also found enrichment of oncogenic FOXA1 mutations in IDC-P which are associated with prostate cancer progression, castrate-resistance and an unfavorable prognosis. Further efforts are ongoing to expand this cohort of IDC-P cases and compare them with a matched cohort of pure adenocarcinoma cases and will include analysis of key RNA expression signatures.

Abstract 2048: Mutations in FOXA1 alter chromatin remodeling and cell fate in prostate organoids

Abstract Genomic analysis of targeted patient tumor sequencing identified frequent mutations, 41% in prostate cancer (Li, et al., 2020) in the gene FOXA1, a developmentally important pioneer transcription factor (TF) in mammary and prostate tissues. Previous work by our group and others has shown that these FOXA1 mutations alter global chromatin accessibility and promote growth in prostate cells (Adams, 2019), but the underlying molecular details, including the identity of partner TFs, remain unclear. To address this topic, we generated mouse prostate organoids expressing Foxa1 alleles harboring three distinct classes of mutations: (i) overexpression of WT Foxa1 (reflecting focal gene amplification seen in tumors), (ii) a series of mutants within the Wing2region of the forkhead binding domain (FHBD) and (iii) a mutant bearing a stop codon after the FHBD to represent a series of C-terminal truncation mutants. We performed single nucleus multiome sequencing to obtain gene expression (snRNA-seq) and chromatin accessibility (snATAC-seq) readouts from the same individual nuclei. Whereas each Foxa1 mutant has distinct, often mutant-specific features, several themes emerge. These include alterations in the relative proportion of stem-like (L2) luminal cells vs secretory (L1) luminal cells as well as changes in luminal or basal gene signatures, increased androgen receptor signaling output, and enrichment for motifs of distinct classes of partner TFs. For example, cells expressing the truncation mutant show gain in the accessibility of Gata3 and Pou2f1 TF binding motifs, as well as enhanced numbers of L1-like luminal cells. Functional studies demonstrate that Pou2f1 is specifically required for the pro-luminal phenotype in cells expressing the truncation mutant whereas Gata3 plays a more general pro-luminal role. Correlations in motif accessibility and transcription factor expression across single cells further revealed a composite androgen receptor (AR)-FOXA1 motif enriched in the pro-luminal truncation mutant, while the canonical AR motif was enriched in pro-basal cell mutants. Finally, Foxa1 mutants cooperative with Trp53 and Pten loss in orthotopic prostate tumorigenicity assays, most strikingly manifest by reversion of the basal-like features characteristic of Trp53/Pten loss tumors to Ck8+ luminal adenocarcinoma histology, mirroring that seen in Foxa1-mutant human prostate cancer tumors in mice. Thus, mutant Foxa1 alleles cooperate with canonical prostate cancer tumor suppressors and alter the histologic phenotype of prostate cancers in mice through the activation of basal or luminal lineage differentiation programs. Citation Format: Erik Ladewig, Abbas Nazir, Christina Leslie, Charles Sawyers. Mutations in FOXA1 alter chromatin remodeling and cell fate in prostate organoids [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 2048.

Abstract 93: An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and therapy resistance

Abstract Tumor mutational burden and heterogeneity fuel resistance to many targeted therapies, which inevitably limits the clinical outcome of patients. The cytosine deaminase APOBEC proteins are major drivers of mutagenesis in human cancer, with over 70% of tumors exhibiting a mutational signature that is impacted by APOBEC activity. Nevertheless, the mechanisms through which tumor cells hijack the powerful APOBEC mutagenesis machinery to promote heterogeneity and therapy resistance remain largely unknown. Through a multi-disciplinary approach integrating bulk and single cell RNA-Seq (scRNA-Seq), whole-genome exome-sequencing (WES), and CRISPR library screening, we identified a long sought-after cell-intrinsic mechanism that prevents APOBEC-driven mutagenesis in non-malignant cells. The loss of this “molecular brake”, unleashes APOBEC3B-driven mutagenesis in malignant cells, which then becomes a key mutator and represents the long sought-after molecular source of driver mutations in some frequently mutated genes in cancers, including FOXA1, EP300, and AR. Functional screening identified eight crucial drivers for Androgen Receptor (AR)-targeted therapy resistance in prostate cancer that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3 and AR. Finally, contrary to conventional understanding, our study reveals that driver mutations in FOXA1 induced by APOBEC3B, not mutations in AR, evolutionarily outcompete other driver mutations and eventually dominate the resistant tumors. Collectively, these results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring targeted therapy resistance and could be the potential therapeutic targets to overcome resistance. Citation Format: Ping Mu, Xiaoling Li, Yunguan Wang, Tao Wang, Su Deng. An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and therapy resistance [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 93.

PD10-11 DISTINCT CLINICAL GENOMIC VARIATIONS AMONG MEN WITH LOCALIZED VS METASTATIC PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD10-11 DISTINCT CLINICAL GENOMIC VARIATIONS AMONG MEN WITH LOCALIZED VS METASTATIC PROSTATE CANCER Ahmed El-shafie, Mohammed Al-Toubat, Allison H. Feibus, SeyedBehzad Jazayeri, Soroush Bazargani, Devon Thomas, Mark Bandyk, and K.C. Balaji Ahmed El-shafieAhmed El-shafie More articles by this author , Mohammed Al-ToubatMohammed Al-Toubat More articles by this author , Allison H. FeibusAllison H. Feibus More articles by this author , SeyedBehzad JazayeriSeyedBehzad Jazayeri More articles by this author , Soroush BazarganiSoroush Bazargani More articles by this author , Devon ThomasDevon Thomas More articles by this author , Mark BandykMark Bandyk More articles by this author , and K.C. BalajiK.C. Balaji More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003250.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Our objective was to identify unique genetic mutations associated with metastatic prostate cancer (PCa) by comparing next generation sequencing (NGS) data between men with localized and metastatic disease METHODS: We queried the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (GENIE) registry for men diagnosed with PCa. Patients were categorized into localized (m0) and metastatic (m1) groups. The difference in the frequency of genetic mutations between the two groups and the prognostic significance of the differentially expressed genes were analysed using SPSS V24. We included frequency rates of >5% and p values <0.05 were considered statistically significant to maintain over 95% true positive detection rate. RESULTS: A total of 1,027 patients, with localized (n=459) and metastatic PCa (n=568) were included in the study. Patients were mostly non-Hispanic White men (89%) with a median age of 61 years (IQR 56-66 years). 421 (41%) of our cohort had Gleason grade group (GG) 2 disease, 277 (27%) had GG3 disease, and 267 (26%) had GG4 or GG5 disease. We identified alterations in 89 genes in our analyses. Patients with M1PCa had significantly higher genomic aberrations in TMPRSS2 (31.51% vs 11.14%, p<0.001); ERG fusion (23.59% vs 13.13%, p<0.001); PTEN loss (25.7% vs 16.16%, p<0.001); TP53 mutation (38.73% vs 12.23%, p<0.001); FOXA1 (17.43% vs 6.33%, p<0.001); KMT2C (9.51% vs 6.11%, p=0.02) (Figure 1). We then isolated the signalling pathways most likely to be affected by these mutations, and found that the androgen receptor was most often impacted (Figure 2). Patients with alterations in all 6 of the genes listed above had the worst progression-free survival (p = <0.01, log rank test) (Figure 1). CONCLUSIONS: In M1PCa, patients had significantly higher frequency of TMPRSS2, ERG, PTEN loss, TP53, FOXA1, and KMT2C somatic gene mutations compared to samples from patients with m0PCa. Additionally, patients who had mutations in all 6 of these genes harboured more aggressive cancer and poorer outcomes. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e330 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ahmed El-shafie More articles by this author Mohammed Al-Toubat More articles by this author Allison H. Feibus More articles by this author SeyedBehzad Jazayeri More articles by this author Soroush Bazargani More articles by this author Devon Thomas More articles by this author Mark Bandyk More articles by this author K.C. Balaji More articles by this author Expand All Advertisement PDF downloadLoading ...

Determination of TMPRSS2-ERG, SPOP, FOXA1, and IDH1 prostate cancer molecular subtypes in Colombian patients and their possible implications for prognosis.

Prostate cancer (PCa) is one of cancer with of the highest incidence and mortality worldwide. Current disease prognostic markers do not differentiate aggressive from indolent PCa with sufficient certainty, and characterization by molecular subtypes has been sought to allow a better classification. TMPRSS2-ERG, SPOP, FOXA1, and IDH1 molecular subtypes have been described, but the association of these subtypes with prognosis in PCa is unclear; their frequency in Colombian patients is also unknown. Formalin-fixed and paraffin-embedded samples of radical prostatectomy from 112 patients with PCa were used. The TMPRSS2-ERG subtype was assessed with fluorescent in situ hybridization. The mutations in SPOP, FOXA1, and IDH1 in hot-spot regions were evaluated using Sanger sequencing. Fusion was detected in 71 patients (63.4%). No statistically significant differences were found between the state of fusion and the variables analyzed. In the 41 fusion-negative cases (36.6%), two patients (4.9%) had missense mutations in SPOP (p.F102C and p.F133L), representing a 1.8% of the overall cohort. The low frequency of this subtype in Colombians could be explained by the reported variability in the frequency of these mutations according to the population (5%-20%). No mutations were found in FOXA1 in the cases analyzed. The synonym SNP rs11554137 IDH1105GGT was found in tumor tissue but not in the normal tissue in one case. A larger cohort of Colombian PCa patients is needed for future studies to validate these findings and gain a better understanding of the molecular profile of this cancer in our population and if there are any differences by Colombian regions.

Somatic mutations reveal complex metastatic seeding from multifocal primary prostate cancer.

Primary prostate cancer shows a striking intraorgan molecular heterogeneity, with multiple spatially separated malignant foci in the majority of patients. Metastatic prostate cancer, however, typically reveals more homogenous molecular profiles, suggesting a monoclonal origin of the metastatic lesions. Longitudinal mutational spectra, comparing multiple primary lesions with metastases from the same patients remain poorly defined. We have here analyzed somatic mutations in multisampled, spatio-temporal biobanked lesions (38 samples from primary foci and 1 sample from each of 8 metastases from seven prostate cancer patients) applying a custom-designed panel targeting 68 prostate cancer relevant genes. The metastatic samples were taken at time of primary surgery and up to 7 years later, and sampling included circulating tumor DNA in plasma or solid metastatic tissue samples. A total of 282 somatic mutations were detected, with a range of 0 to 25 mutations per sample. Although seven samples had solely private mutations, the remaining 39 samples had both private and shared mutations. Seventy-four percent of mutations in metastases were not found in any primary samples, and vice versa, 96% of mutations in primary cancers were not found in any metastatic samples. However, for three patients, shared mutations were found suggesting the focus of origin, including mutations in AKT1, FOXA1, HOXB13, RB1 and TP53. In conclusion, the spatio-temporal heterogeneous nature of multifocal disease is emphasized in our study, and underlines the importance of testing a recent sample in genomics-based precision medicine for metastatic prostate cancer.

Genomic alterations and evolution in patients with prostate cancer with histologic evidence of neuroendocrine differentiation.

5029 Background: The incidence of transformation to neuroendocrine prostate cancer (NEPC) has increased in castration resistant prostate cancer (CRPC) in parallel with treatment advances inhibiting androgen receptor signaling. The current understanding is that this occurs in ̃10-20% of CRPC cases. Missing is a determination of the timing of molecular events that drive the process. Methods: Under an IRB-approved protocol, retrospective annotation of all MSK-reviewed pathology reports was conducted for 1447 prostate cancer patients with MSK-IMPACT sequencing data. For patients with pathologically confirmed NEPC, the date of the first sample with unequivocally reported NEPC (described as “neuroendocrine carcinoma” or as having “neuroendocrine differentiation” or “neuroendocrine features”) was recorded. Patients with early signs of histologic transformation not specifically reported as NEPC (double negative prostate cancer or rare/focal staining for NE markers) were analyzed separately. Sequencing results were described by castration-status at collection (CRPC vs castration-sensitive) and, if applicable, the relationship to NEPC diagnosis (i.e. pre- vs post-NEPC). Genomic enrichment analysis was used to identify differentially altered genes between groups. Results: In total, 95 (6.6%) patients had pathologically confirmed NEPC during their disease course, from whom 150 samples with sequencing results were available: including 18 patients with matched pre- and post-NEPC samples. CRPC samples from patients with NEPC (n = 70) were significantly enriched for RB1 alterations (50% vs 12%, p &lt; 10-10, q &lt; 10-10). AR alterations were significantly enriched in CRPC samples from patients without NEPC (n = 380) (63% vs 21%, p &lt; 10-10, q &lt; 1.27*10-8). Further, alterations in numerous genes including TP53, AMER1, ARID5B, YAP1, SOX2, and NKX2.1 were enriched in NEPC patients at the 95% confidence interval (CI) without correction for repeat testing. Matched pre- and post-NEPC samples demonstrated that TP53 alterations in post-NEPC samples are detected in the majority of pre-NEPC samples (8 of 10 patients), but RB1 alterations in post-NEPC samples are detected in a minority of pre-NEPC samples (1 of 8 patients). 54 (3.7%) patients had evidence of early histologic transformation. CRPC samples from these patients (n = 29) were enriched for mutations in RB1, MAP2K2, MUTYH, and CTNNB1 at the 95% CI without correction. FOXA1 mutations were enriched in patients without transformation. Conclusions: RB1, consistent with previous findings, is enriched in NEPC. The inability to detect RB1 alterations in pre-NEPC samples supports divergent evolution, although technical limits of tissue panel sequencing make it difficult to rule out the presence of sub-clonal alterations. Further study of additional genes which contribute to histologic transformation and the development of NEPC is warranted.

Detection of disease-causing mutations in prostate cancer by NGS sequencing.

Gene mutations may affect the fate of many tumors including prostate cancer (PCa); therefore, the research of specific mutations associated with tumor outcomes might help the urologist to identify the best therapy for PCa patients such as surgical resection, adjuvant therapy or active surveillance. Genomic DNA (gDNA) was extracted from 48 paraffin‐embedded PCa samples and normal paired tissues. Next, gDNA was amplified and analyzed by next‐generation sequencing (NGS) using a specific gene panel for PCa. Raw data were refined to exclude false‐positive mutations; thus, variants with coverage and frequency lower than 100× and 5%, respectively were removed. Mutation significance was processed by Genomic Evolutionary Rate Profiling, ClinVar, and Varsome tools. Most of 3000 mutations (80%) were single nucleotide variants and the remaining 20% indels. After raw data elaboration, 312 variants were selected. Most mutated genes were KMT2D (26.45%), FOXA1 (16.13%), ATM (15.81%), ZFHX3 (9.35%), TP53 (8.06%), and APC (5.48%). Hot spot mutations in FOXA1, ATM, ZFHX3, SPOP, and MED12 were also found. Truncating mutations of ATM, lesions lying in hot spot regions of SPOP and FOXA1 as well as mutations of TP53 correlated with poor prognosis. Importantly, we have also found some germline mutations associated with hereditary cancer‐predisposing syndrome. gDNA sequencing of 48 cancer tissues by NGS allowed to detect new tumor variants as well as confirmed lesions in genes linked to prostate cancer. Overall, somatic and germline mutations linked to good/poor prognosis could represent new prognostic tools to improve the management of PCa patients.

Comparative ctDNA analyses of African-American and Caucasian patients with CRPC.

24 Background: Somatic genetic analyses have indicated genetic distinctions in AA as compared to C patients. In the Mahal et al. study (1) evaluating a broad spectrum of pts with tissue based assays, FOXA1 mutations were more frequent in AA men and TP53 mutations were less frequent in AA men as compared to C men. In a separate analysis by Khashab et al. (2) conducted in prostate cancer pts receiving androgen deprivation therapy, using both tissue and ctDNA assays, the authors reported AR, TP53, SPOP, and BRCA2 were more frequently mutated in AA men as compared to C men. Herein we assessed the Guardant 360 platform in assessing ctDNA differences in AA and C men, all of whom had CRPC at the time assays were performed. Methods: Guardant 360 was used to analyze ctDNA with a cut-off of &gt;0.5% for allelic fractions for ascertaining the presence or absence of pathogenic mutations and various amplifications. Lower allelic fractions were not analyzed given these may represent less relevant mutations. Depending on the timing of the assays (2015-2021), 70-83 genes were analyzed. All pts had CRPC and all patients were treated at Tulane Cancer Center. Chi Square analyses were used to determine statistical differences. AR, BRCA2, and TP53 were assessed but SPOP and FOXA1 were not assessable in the Guardant ctDNA assay. Both mutations and amplifications were evaluated. Results: Among men with CRPC, a total of 48 AA men and 179 C men were analyzed using ctDNA. Clear distinctions were found in the alteration reported in APC, TP53, and CDK12. TP53 was less frequently mutated and other genes were more frequently altered in the AA men. Conclusions: Using Guardant ctDNA assays in men with CRPC, clear distinctions were found in AA men as compared to C men. It is unclear why these results differ from that reported by others, however distinctions in both the assays and the populations are notable.[Table: see text]

Germline BRCA2, ATM and CHEK2 alterations shape somatic mutation landscapes in prostate cancer.

148 Background: Prostate cancer (PCa) is a highly heritable malignancy. Germline mutations in homologous recombination repair (HRR) genes, especially in in ATM, BRCA2 and CHEK2, are common in men with PCa, but how these influence somatic mutations is unknown. Characterizing the somatic landscape of tumors from germline HRR-altered PCa patients may provide insights into further therapeutic strategies and identify individuals most likely to benefit from existing therapies. Methods: We retrospectively identified 118 men with germline HRR-altered PCa from Johns Hopkins with available somatic mutation data (g ATM, n = 30; g BRCA2, n = 58; g CHEK2, n = 30). In these patients, somatic NGS analysis were done primarily using prostatic (rather than metastatic) tissues. The proportion of patients in each group with somatic biallelic inactivation was determined. We then compared the somatic mutation landscapes of the 3 groups, focusing on key PCa-relevant genes including AR, TMPRSS2-ERG, PTEN, TP53, BRCA2, ATM, CDK12, APC, FOXA1, AKT1, PIK3CA, and KDM6A among others. Somatic mutation data from primary prostate cancer specimens (n = 3965) found in pooled studies (n = 23) from cBioPortal were included as a reference standard for comparison. Results: Biallelic somatic inactivation was detected in 59% (34/58) of g BRCA2-altered, 47% (14/30) of g ATM-altered, and 0% (0/30) of g CHEK2-altered patients. In all 118 germline HRR-altered individuals, the most common somatic mutations were in BRCA2 (31%), FOXA1 (26%), TMPRSS2-ERG (20%), TP53 (16%), PTEN (15%), and ATM (15%). g BRCA2-altered patients had a higher prevalence of somatic mutations in AR (9% vs 3% in cBioPortal, P &lt; 0.05) and FOXA1 (29% vs 13% in cBioPortal, P &gt; 0.05), suggesting a greater dependency on AR signaling, but a lower prevalence of TMPRSS2-ERG fusions (17% vs 33% in cBioPortal, P &lt; 0.05). g ATM-altered patients had a strikingly low prevalence of somatic TP53 mutations (3% vs 25% in cBioPortal, P &lt; 0.01), indicating mutual exclusivity between these genes. g CHEK2-altered patients had a higher prevalence of somatic CDK12 mutations (10% vs 0% in gATM/gBRCA2 patients, P &lt; 0.05), implying an interaction between CHEK2 and CDK12. No differences were seen between the three groups with respect to alterations in the PI3K/PTEN pathway, WNT pathway, or chromatin-related genes. Conclusions: Biallelic inactivation was frequently observed in PCa patients harboring g ATM and g BRCA2 mutations, but not in those with g CHEK2 mutations. g BRCA2-altered patients were enriched for somatic AR (and FOXA1) mutations and were depleted for TMPRSS2-ERG fusions. g ATM-altered patients were depleted for somatic TP53 mutations, while g CHEK2-altered patients were enriched for somatic CDK12 mutations. Thus, germline mutations appear to impose selective pressures that influence somatic mutation landscapes in PCa.

Analysis of BRCA Germline Mutations in Chinese Prostate Cancer Patients.

Recent studies have indicated that prostate cancer (PCa) with BRCA2 mutations is more aggressive. However, these reports mostly focused on Caucasus populations, and large-scale studies on BRCA mutations in Chinese PCa populations remain limited. Herein, we screened, from multiple centers in China, a total of 172 patients with PCa carrying BRCA1/2 germline mutations. The variant distribution and type, associated somatic variant, and frequency of the BRCA germline variants in these patients were analyzed retrospectively. We found that Chinese patients with PCa carrying BRCA1/2 germline mutations were diagnosed at an earlier age, i.e., 67 years (range, 34–89 years), and most had metastatic castration-resistant PCa (mCRPC) (54.65%, 94/172). The top three BRCA variants were frameshift, missense, and splicing variants. The overall pathogenic rates of the BRCA1 and BRCA2 variants were 17.46% (11/63) and 56.55% (82/145), respectively. Among the somatic mutations associated with BRCA2 germline mutations, the highest frequency was for FOXA1 (circulating tumor DNA [ctDNA] sequencing, 7.4%; tissue samples, 52%) and NCOR2 mutations (ctDNA sequencing, 7.4%; tissue samples, 24%); TP53 was the dominant somatic mutation associated with BRCA1 germline mutations (ctDNA sequencing, 25%; tissue samples, 17%). Ultimately, in Chinese patients, PCa with BRCA1/2 germline mutations tends to be more aggressive. Compared with BRCA1, BRCA2 has a higher frequency of germline pathogenic mutations. FOXA1, NCOR2, and TP53 somatic mutations associated with higher BRCA1/2 germline pathogenic mutations. Our description of BRCA germline mutations in the Chinese PCa patients provides more reference data for the precise diagnosis and treatment of Chinese PCa patients.

Differences in Prostate Cancer Genomes by Self-reported Race: Contributions of Genetic Ancestry, Modifiable Cancer Risk Factors, and Clinical Factors.

Black men die from prostate cancer twice as often as White men, a disparity likely due to inherited genetics, modifiable cancer risk factors, and healthcare access. It is incompletely understood how and why tumor genomes differ by self-reported race and genetic ancestry. Among 2,069 men with prostate cancer (1,841 self-reported White, 63 Asian, 165 Black) with access to clinical-grade sequencing at the same cancer center, prevalence of tumor and germline alterations was assessed in cancer driver genes reported to have different alteration prevalence by race. Clinical characteristics such as prostate-specific antigen and age at diagnosis as well as cancer stage at sample procurement differed by self-reported race. However, most genomic differences persisted when adjusting for clinical characteristics. Tumors from Black men harbored fewer PTEN mutations and more AR alterations than those from White men. Tumors from Asian men had more FOXA1 mutations and more ZFHX3 alterations than White men. Despite fewer TP53 mutations, tumors from Black men had more aneuploidy, particularly chromosome arm 8q gains, an adverse prognostic factor. Genetic ancestry was associated with similar tumor alterations as self-reported race, but also with modifiable cancer risk factors. Community-level average income was associated with chr8q gains after adjusting for race and ancestry. Tumor genomics differed by race even after accounting for clinical characteristics. Equalizing access to care may not fully eliminate such differences. Therapies for alterations more common in racial minorities are needed. Tumor genomic differences should not be assumed to be entirely due to germline genetics.

A deep imputation and inference framework for estimating personalized and race-specific causal effects of genomic alterations on PSA.

Prostate Specific Antigen (PSA) level in the serum is one of the most widely used markers in monitoring prostate cancer (PCa) progression, treatment response, and disease relapse. Although significant efforts have been taken to analyze various socioeconomic and cultural factors that contribute to the racial disparities in PCa, limited research has been performed to quantitatively understand how and to what extent molecular alterations may impact differential PSA levels present at varied tumor status between African-American and European-American men. Moreover, missing values among patients add another layer of difficulty in precisely inferring their outcomes. In light of these issues, we propose a data-driven, deep learning-based imputation and inference framework (DIIF). DIIF seamlessly encapsulates two modules: an imputation module driven by a regularized deep autoencoder for imputing critical missing information and an inference module in which two deep variational autoencoders are coupled with a graphical inference model to quantify the personalized and race-specific causal effects. Large-scale empirical studies on the independent sub-cohorts of The Cancer Genome Atlas (TCGA) PCa patients demonstrate the effectiveness of DIIF. We further found that somatic mutations in TP53, ATM, PTEN, FOXA1, and PIK3CA are statistically significant genomic factors that may explain the racial disparities in different PCa features characterized by PSA.