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Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li–Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. Significance: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li–Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are “basal” p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027

Background Single-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Classification of bulk tumors by their individual cellular constituents has also created new opportunities to generate single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous promise of this technology, recent evidence studying epithelial tissues and diverse carcinomas suggests the methods used for tissue processing, cell disaggregation, and preservation can significantly bias gene expression and alter the observed cell types. To determine whether sarcomas – tumors of mesenchymal origin – are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma (OS), Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins. Results We systematically assessed dissociation methods across different sarcoma subtypes. We compared gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from ES, DSRCT, and OS PDXs. We detected warm dissociation artifacts in single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of the dissociation method. In addition, we showed that dissociation method biases could be computationally corrected. Conclusions We highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by the dissociation method for various sarcoma subtypes. This work is the first to characterize how the dissociation methods used for sc/snRNA-seq may affect the interpretation of the molecular features in sarcoma PDXs.

Pancreatic ductal adenocarcinoma (PDAC) is considered ‘non-immunogenic’ with trials demonstrating its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and anti-tumor immunity, characterized by modulating T cell subsets with anti-tumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the presence of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor, targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC. Significance: To improve the efficacy of immunotherapy in PDAC, there is a great need to understand the PDAC TIL landscape. This study represents a reference of PDAC TIL subpopulations and their relationships and provides a foundation upon which to base future immunotherapeutic efforts. This article is highlighted in the In This Issue feature, p. 2221

Gliomas are recalcitrant brain tumors. Differential tumor immune reactivity contributes to survival advantage of isocitrate dehydrogenase-mutant (IDHmut) over wild-type (IDHwt) gliomas. Despite this correlative pattern of immunity and survival, only a limited view of a highly complex immune contexture across IDH mutation classified gliomas is known. Herein, we present an unprecedented view of myeloid and lymphoid cell type diversity by single cell RNA sequencing and spectral cytometry-based interrogation of tumor-associated leukocytes from fifty-five IDH stratified primary and recurrent human gliomas and six non-glioma brains. Our analyses revealed twenty-two myeloid and lymphoid cell types within and across glioma subtypes. Glioma severity in relapsed IDHwt correlated with microglial attrition concomitant with a continuum of invading monocyte-derived microglia-like and macrophages amongst other infiltrating conventional T and NK lymphocytes and unconventional mucosa associated invariant T (MAIT) cells. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). As tissue macrophages exhibit multifaceted polarization in response to microenvironmental cues, we clarify the existence of microglia/macrophage functional states beyond M1/M2 paradigms exemplified by the presence of palmitic-, oleic- acid, and glucocorticoid responsive polarized states. Immune related gene ontology analysis identified enriched antigen presentation and phagocytosis gene modules in distinct microglia-like clusters. Importantly, the phagocytic immunomodulator; Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) was upregulated in these microglia-like cells. Contrary to tumor promoting role of TREM2 myeloid cells in non-brain cancers, we identify TREM2 mediated anti-glioma axis as a regulator of antigen presentation Accelerated glioma growth was observed in Trem2 deficient mice implanted with CT2A glioma cells affirming the anti-glioma role of TREM2+ myeloid cells. In addition to providing an advanced landscape of glioma-specific immune contexture for immunotherapy applications, our reverse translational investigations discover TREM2 as a novel immunotherapy target for brain malignancies. Citation Format: Pravesh Gupta, Minghao Dang, Shivangi Oberai, Mekenzie Peshoff, Nancy Milam, Aml Ahmed, Krishna Bojja, Tuan M. Tran, Kathryn Cox, Huma Shehwana, Carlos Kamiya Matsuoka, Jianzhuo Li, Joy Gumin, Alicia Goldman, Sameer A. Seth, Atul Maheshwari, Frederick F. Lang, Nicholas E. Navin, Amy B. Heimberger, Karen Clise Dwyer, Linghua Wang, Krishna P. Bhat. Immunophenotyping of human brain tumors reveals myeloid cell mediated anti-glioma axis [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 669.

Prostate cancer is the most commonly diagnosed cancer among men and a major cause of cancer-related deaths in the modern world. Metastatic castration-resistant prostate cancer (mCRPC) is the most lethal form and has complex and heterogeneous genomic patterns. Despite the recent development of life-prolonging therapies, mCRPC remains incurable since most patients develop resistance over time. A better understanding of the evolution of resistance to therapy in mCRPC is urgently needed. A major logistical challenge is that collecting serial tissue biopsies from the prostate and metastatic sites such as bone is not feasible. To overcome this issue, we developed a minimally invasive liquid biopsy approach to study the genomic basis of resistance through circulating tumor DNA (ctDNA) from longitudinally collected plasma samples. To best account for interpatient tumor heterogeneity and treatment history, we selected 60 mCRPC patients who were serially treated with life-prolonging second generation androgen signaling inhibitors (abiraterone, enzalutamide, or the combination) and taxane-based chemotherapy, and prospectively collected longitudinal blood samples (2 to 10 per patient). ctDNA was isolated from each blood plasma sample for sparse whole-genome sequencing and exome sequencing at 200x coverage depth. Somatic copy number alterations (CNA) and exome mutations were profiled and the resulting data was used to reconstruct the clonal substructure and infer clonal evolutionary dynamics across the treatment timepoints. The mutation signatures and the CNA signatures were also characterized. One of the CNA signatures identified the patients who carry CDK12 mutations through genome-wide focal tandem duplications in ctDNA. The subclones resistant to second-generation androgen signaling inhibitors and taxane-based chemotherapy were also identified, along with their associated genomic aberrations affecting AR, RB1, and PTEN. Furthermore, by analyzing the evolutionary dynamics patterns, we identified three distinct genomic responses to therapy: intrinsic resistance, clonal progression, and clonal responders, which correlated with overall survival outcomes. In summary, our data show that clonal evolutionary dynamics in mCRPC can be tracked over time and in response to treatment with minimally invasive liquid biopsy methods. Our results provide insights into the genomic evolution of life-prolonging therapy resistance in mCRPC and identified candidate resistance biomarkers for subsequent validation. Citation Format: Yuehui Zhao, Naveen Ramesh, Emi Sei, Min Hu, Shanshan Bai, Patricia Troncoso, Paul Corn, Ana Aparicio, Christopher J. Logothetis, Nicholas E. Navin, Amado J. Zurita. Investigating longitudinal therapeutic resistance in serially treated metastatic castration resistant prostate cancer by liquid biopsy genome sequencing [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 3791.

Introduction: Patients with luminal A and B early-stage ER+/HER2- breast cancer (BrCa) are uniformly treated with adjuvant endocrine therapy (ET) (±chemotherapy). A better understanding of drivers of ET resistance is required as subsequent lethal metastatic disease remains a major clinical problem. Therefore, there is an unmet clinical need to identify biomarkers that select low- and high-risk patients who may benefit from de-escalation of current treatments or alternative therapeutic interventions. In the present study, we hypothesized that while luminal A and B tumors both arise from normal hormone-responsive cells, they are transcriptionally distinct, hence allowing the identification of unique gene signatures that can predict outcomes in each subtype. Methods: Tumors from 10 early-stage ER+/HER2- BrCa patients were subjected to single-cell RNA-sequencing (scRNA-seq) analysis (10X Genomics); 6/10 tumors were classified as luminal A and 4/10 as luminal B based on combined PAM50 and immunohistochemical classification (Ki67 cut-off=20%). We performed a direct transcriptional comparison between luminal A and B tumors, using well-established signatures and unbiased differential gene expression analysis. To identify unique luminal A and B tumor-specific genes, we compared the gene expression profile of each luminal subtype with 10 non-neoplastic breast tissues. A predictive model (LASSO) was applied to select genes with the highest frequency using a training dataset. This resulted in 5- and 4-gene signatures for luminal A and B, respectively, which were used to calculate risk scores that divided each subtype into low- and high-risk groups. The prognostic value of the above signatures was validated in an independent dataset. Results: The integrated scRNA-seq analysis of luminal A and B tumors revealed transcriptionally distinct tumor cell clusters while tumor microenvironment (TME) clusters were well intermixed. Luminal B tumors had higher cell cycle and BrCa-specific scores, low ER pathway-gene expression scores, and increased 8q amplifications. IFNγ, OXPHOS, p53, hypoxia and MYC targets were the most upregulated pathways in the luminal B subtype. The TME of luminal B tumors was comprised of lower CD4+ and CD8+ T cell but higher Treg levels. Comparison with normal breast tissues revealed that early-stage ER+ BrCa arises from hormone-responsive epithelial cells and provided a number of tumor-specific genes that were used to generate prognostic signatures. These signatures were capable of differentiating high- from low-risk patients within each subtype and predicting survival outcomes in two large-scale training and validation cohorts. Conclusions: We developed a novel prognostic tool that can be used to determine duration of adjuvant ET and/or new therapeutic strategies for high-risk luminal A and B patients in the early-stage ER+/HER2- setting. Citation Format: Sofia Mastoraki, Jerome Lin, Xiayu Rao, Sophie R. Liu, Harsh Batra, Maria G. Raso, Edwin R. Parra Cuentas, Akshara S. Raghavendra, Komal S. Rasaputra, Min Yi, Jing Wang, Aysegul Sahin, Debasish Tripathy, Kelly K. Hunt, Nicholas E. Navin, Khandan Keyomarsi. Single-cell transcriptomic analysis of HR+/HER2- breast cancer identifies gene signatures that predict outcomes of luminal A and B subtypes [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 515.

Castration-resistant prostate cancer (CRPC) is a lethal disease. A subset of these patients present with atypical clinical characteristics and aggressive disease behavior. These patients, classified as Aggressive Variant Prostate Cancer (AVPC), may derive benefit from platinum-based chemotherapy. However, the identification of AVPC patients is often challenging due to heterogeneity in clinical presentations and potentially in biological drivers, which may not be fully reflected in a biopsy obtained from a single tumor site. To address these challenges, we developed an unbiased genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the simultaneous detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied this method to plasma specimens obtained from 160 CRPC patients with and without AVPC participating in a randomized trial of cabazitaxel alone or in combination with carboplatin. We were able to successfully isolate ctDNA (>2 ng) for genomic profiling using PEGASUS in 91/160 (57%) CRPC patients. Among these 91 CRPC patients, we detected common prostate cancer mutations as well as genomic copy number changes in genes such as RB1, PTEN and TP53 and several other genes associated with DNA repair. One of the most salient features that distinguished the patients’ outcomes was whether they had diploid or aneuploid genomes detected by whole genome profiling of the ctDNA. Patients with aneuploid ctDNA had worse progression-free and overall survival. Overall, our data shows the feasibility of performing whole-genome and exome profiling of ctDNA in CRPC patients to characterize the molecular features associated with distinct clinical presentations. Candidate markers associated to benefit from platinum-based chemotherapy are being evaluated. These results pave the way for future clinical applications in biomarker discovery to assist treatment decisions in prostate cancer patients. Citation Format: Naveen Ramesh, Emi Sei, Pei Ching Tsai, Christopher Logothetis, Paul Corn, Ana Aparicio, Amado J. Zurita, Nicholas E. Navin. Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 446.