Abstract Copy-number (CN) amplification is a major mechanism of oncogene activation and a therapeutic target, yet its evolution in human cancers is incompletely understood. We analyzed single-cell whole-genome sequencing (scWGS) data from >80,000 cancer cells in 100 tumors from major cancer types (ovary, breast, lung, and brain) and experimental models to resolve mechanisms and evolution of oncogene amplification. CN distributions across cells revealed two patterns: narrow, uniform peaks from symmetric segregation, consistent with intrachromosomal amplifications (ICamps), and broad, heavy-tailed variation with extreme outliers (>100 copies/cell) indicative of extrachromosomal circular DNA (ecDNA). A probabilistic mixture model of these distributions classified 74 (15%) of 503 amplified regions as ecDNA. These ecDNAs most frequently involved MYC, EGFR, and MDM2, and were enriched in glioblastoma and lung cancers, whereas ovarian and triple-negative breast cancers predominantly showed ICamps. Notably, ICamp events showed significant subclonal specificity, with 227 (53%) of 429 events displaying multiple modes in the CN distribution. These modes were congruent with other genomic features from phylogenetic analysis, suggesting lineage inheritance patterns of symmetric division and clonal expansion. Diversification arose via numeric mechanisms (aneuploidy or genome doubling) and subclone-specific structural variants (e.g., breakage-fusion-bridge cycles or chromothripsis). CN modulation was bidirectional, including loss of amplified derivative chromosome via subclone-specific aneuploidy. Joint analysis of copy-number and structural variants at single-cell resolution uncovered several mechanisms of ecDNA-driven cancer evolution. First, remodeling of ecDNAs through internal rearrangements and recombination between distinct species was often observed, leading to oncogene co-selection. Second, convergent evolution via acquisition of distinct EGFR-containing ecDNAs was observed in a glioblastoma, suggesting the potential role of ecDNA loss in shaping subsequent evolution. Third, scWGS of isogenic cell lines distinguished present ecDNAs from historical ecDNAs that underwent genomic rearrangements resulting in chromosomal re-integration. Finally, we show that comparison between circular genome graph-based prediction versus the CN distribution-based prediction of ecDNAs revealed substantial discrepancy with notable tissue-type specificity. In conclusion, this study reveals interpretable distributions of oncogene amplifications consistent with distinct ICamp and ecDNA generative processes. This approach, refining ecDNA identification beyond standard genome graphs, further elucidates how distinct mechanisms of oncogene amplification diversify cancer cell populations. We suggest these new insights will inform patient selection for emerging ecDNA-directed therapies. Citation Format: Jake June-Koo Lee, Sohrab Salehi, Matthew Myers, Melissa Yao, Seongmin Choi, Duaa Hassan Al-Rawi, Ignacio Vazquez-Garcia, Eliyahu Havasov, Michelle Wu, Jin Lee, Fathema Uddin, Parvathy Manoj, Pedram Razavi, Samuel Aparicio, Natasha Rekhtman, Kenny Kwok Hei Yu, Helena A. Yu, Charles M. Rudin, Andrea Ventura, Andrew William McPherson, Marc Williams, Sohrab Shah. Dynamic evolution of oncogene amplification across 80,000 cancer cell genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3526.

RET-fusion-driven lung adenocarcinomas (LUADs) are a rare and particularly aggressive subtype of lung cancer, often affecting young, non-smoker populations. While selective RET-TKI therapies are initially highly effective, they often fail to provide durable responses, with resistance mechanisms emerging over time. Notably, approximately 40% of patients exhibit resistance mechanisms that remain unidentified. To uncover these unknown mechanisms, we performed a comprehensive multi-omic analysis of clinical samples from patients with RET-fusion lung cancers before and after RET-TKI treatment. RNA sequencing analysis revealed inactivation of RB1 and upregulation of DNA damage repair (DDR) pathways in resistant tumors relative to TKI-sensitive samples. Accordingly, methylome analysis confirmed hypomethylation of E2F targets and DDR pathways, suggesting that their transcriptomic upregulation is mainly driven by epigenetic changes. We hypothesized that DDR pathway upregulation was a result of inhibition of Rb1 during TKI treatment. To validate this, we knocked down Rb1 or overexpressed of E2F, and confirmed upregulation of key DDR genes such as BRCA1, MSH6, and Rad18 in RET cell lines. To validate the role of DDR pathways in resistance, we established isogenic cell lines by overexpressing or knocking out BRCA1, MSH6, and Rad18, and conducted drug-tolerant persister (DTP) assays. Genetic inhibition of each of these DDR proteins significantly reduced the generation of DTPs after high dose treatment with the RET-TKI selpercatinib (4.5 uM) while overexpression increased the number of DTPs. We next evaluated the inhibition of XPO1, an exportin protein linked to DDR pathway activation, as a strategy to target DTPs. Treatment with selinexor, an FDA-approved XPO1 inhibitor, in combination with selpercatinib strongly reduced the number of DTPs in vitro, and overexpression of the DDR genes BRCA1, MSH6, and Rad18 rescued this effect. In vivo experiments using RET-driven patient-derived xenograft (PDX) models confirmed that treatment with combination of selpercatinib with selinexor significantly delayed tumor relapse in TKI-sensitive PDX models (n=4) and resensitized resistant PDX tumors to selpercatinib (n=2). Our research highlights DDR pathways as a critical mechanism of resistance via Rb1 inactivation in TKI-treated RET-driven lung cancers and identifies XPO1 inhibition as a promising therapeutic strategy to overcome this resistance. Finally, we also found upregulation of DDR proteins upon treatment with lorlatinib and osimertinib in ALK and EGFR driven LUAD PDXs, respectively, suggesting that targeting DDR pathways could be an effective strategy against resistance in a pan-TKI setting across other RTK-driven LUADs. Fathema Z. Uddin, Samuel Tischfield, Romel Somwar, Alexander Lim, Hyung Jun Woo, Christina Falcon, Barbara P. Mello, Dennis Kinyua, Harsha Sridhar, Parvathy Manoj, Hong Zhong, Juan Qiu, Elisa de Stanchina, Mark Donoghue, Mark Ladanyi, Esther Redin, Charles M. Rudin, Alvaro Quintanal-Villalonga, Alexander Drilon. DNA damage repair pathways enable a drug-tolerant persister state in RET-fusion NSCLC and precedes TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6382.

Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.

Abstract Neuroendocrine (NE) transformation occurs as a mechanism of resistance to targeted therapy in up to 14% and 30% of EGFR-mutant lung and AR-dependent prostate adenocarcinomas, respectively, leading to poor prognosis. Even if we know the tumor population at high risk of transformation (TP53/RB1-mutated), no therapies to prevent NE relapse are currently available. To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model. This screen identified CDC7, involved in DNA replication and DNA damage response, as a potential therapeutic target in this setting. Proteogenomic analyses revealed CDC7 upregulation in lung and prostate clinical samples undergoing NE transformation, detected already in pre-transformation adenocarcinomas. These results indicated that CDC7 expression is induced already at early steps of transformation. Consistently, TP53/RB1-mutated lung and prostate adenocarcinomas exhibited higher CDC7 expression than their double wild-type counterparts. Combined ChIP-seq and promoter reporter assays indicated that CDC7 expression was directly regulated by TP53 and RB1 inactivation. Importantly, TP53/RB1-inactivation induced sensitivity to the CDC7 inhibitor simurosertib, unraveling a therapeutic vulnerability in tumors at high risk of NE transformation. Thus, we tested the combination of simurosertib with targeted therapy in vivo in different lung and prostate patient-derived models of NE transformation, namely TP53/RB1-knock out adenocarcinomas known to undergo NE transformation on targeted therapy. In these, simurosertib was able to suppress NE transformation and dramatically delay tumor relapse. Trajectory analysis on single-cell transcriptomic data for such models revealed a NE transformation transcriptional program occurring already in the untreated tumors before transformation. Remarkably, simurosertib treatment reverted this transcriptomic state and induced a reversion to the original adenocarcinoma transcriptomic profile. CDC7 inhibition led to increased proteasomal activity and degradation of MYC, a stemness transcription factor involved in NE transformation. Ectopic overexpression of MYCT58A, a proteasome degradation-resistant MYC isoform, rescued the NE phenotype in these transformation models, suggesting that CDC7 inhibition-induced MYC degradation is the mechanism by which NE transformation is prevented. In sum, CDC7 inhibition may suppress, or at least dramatically delay NE transformation in patients with lung and prostate adenocarcinomas at high risk of transformation, by inducing MYC proteasomal degradation. The clinical availability of CDC7 inhibitors, currently in phase II clinical trials after demonstrating tolerability and preliminary efficacy, will allow rapid translation of these results into the clinics. Citation Format: Alvaro Quintanal-Villalonga, Fathema Uddin, Kenta Kawasaki, Esther Redin, Vidushi Durani, Amin Sabet, Wouter Karthaus, Yingqian A. Zhan, Samir Zaidi, Moniquetta Shaffer, Harsha Sridhar, Juan Qiu, Parvathy Manoj, Elisa De Stanchina, Michael C. Haffner, Charles L. Sawyers, Charles M. Rudin. CDC7 inhibition prevents neuroendocrine transformation in the lung and prostate through MYC degradation [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 2004.

Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8+ T cells. Here, we comprehensively profiled CD39 expression in human lung cancer. CD39 expression enriched for CD8+ T cells with features of exhaustion, tumor reactivity, and clonal expansion. Flow cytometry of 440 lung cancer biospecimens revealed weak association between CD39+ CD8+ T cells and tumoral features, such as programmed death-ligand 1 (PD-L1), tumor mutation burden, and driver mutations. Immune checkpoint blockade (ICB), but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8+ T cells. Higher baseline frequency of CD39+ CD8+ T cells conferred improved clinical outcomes from ICB therapy. Furthermore, a gene signature of CD39+ CD8+ T cells predicted benefit from ICB, but not chemotherapy, in a phase III clinical trial of non-small cell lung cancer. These findings highlight CD39 as a proxy of tumor-reactive CD8+ T cells in human lung cancer.

Supplemental figures and figure legends.

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced res-olution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoanti-gen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+ SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tu-mor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.

Lineage plasticity contributes to therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon drives neuroendocrine (NE) and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is lower than that of either LUAD or de novo LUSC patients. To date, little is known about the molecular effectors enhancing lineage plasticity and driving histological transdifferentiation due to the paucity of well annotated pre- and post-transdifferentiation clinical samples amenable for molecular analyses. Currently no specific therapies for LUSC or NE transdifferentiation prevention are available for patients at high risk of transformation. We performed multi-omic profiling of transdifferentiating clinical samples, as well as control never-transformed LUAD and de novo LUSC and small cell carcinomas, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Our data suggest that histological transdifferentiation is driven by epigenetic -rather than mutational- events, and indicate that transdifferentiated tumors retain molecular features of their previous LUAD state. Integrative analysis revealed biological pathways dysregulated specifically for distinct histological outcomes, including downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses revealed commonly dysregulated pathways for transdifferentiated tumors, including marked downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in EGFR-mutant patient-derived xenograft transdifferentiation models. These results identify common and histology-specific drivers and dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to constrain lineage plasticity and prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Jacklynn Egger, Juan Qiu, Elisa de Stanchina, Natasha Rekhtman, Brian Houck-Loomis, Richard P. Koche, Helena A. Yu, Triparna Sen, Charles M. Rudin. AKT pathway as a therapeutic target to constrain lineage plasticity leading to histological transdifferentiation [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 658.

Small cell lung cancer (SCLC) is an aggressive disease characterized by early metastasis and exceptional lethality, comprising 13% of all lung cancer cases. With few treatment options, typically resulting in only transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Here, we performed an in vitro CRISPR screen in SCLC cell lines from all major SCLC subtypes, including short-term cultured cells from patient-derived xenografts (PDXs), to identify potential therapeutic targets to enhance sensitivity to chemotherapy. Candidate hits were validated genetically and pharmacologically with in vitro synergy assays, in vivo clonal competition assays and pharmacologic assessments in PDX models. Signaling pathways were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Our CRISPR screen revealed the nuclear exporter exportin 1 (encoded by the XPO1 gene) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor or hematological malignancy, and demonstrated consistently high protein expression by IHC in clinical TMAs. A potent and selective exportin 1 inhibitor, selinexor, is approved for use in hematological malignancies. Combination of selinexor with cisplatin or irinotecan demonstrated synergy in vitro and efficacy in vivo in an array of chemonäive and chemoresistant SCLC PDXs, including all major SCLC subtypes. The combinations were well tolerated in mice. The chemo-sensitizing effects of selinexor were associated with suppression of chemotherapy-induced AKT activation. In conclusion, exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively, and these effects are independent of the SCLC subtype. These results provide preclinical rationale for the combination of selinexor with cisplatin or irinotecan in naïve and relapsed SCLC. The clinical availability of selinexor will allow rapid clinical translation of these results in a disease setting with extremely limited therapeutic options. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yuan Hao, Andrew Chow, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Juan Qiu, Elisa de Stanchina, Richard P. Koche, Triparna Sen, John T. Poirier, Charles M. Rudin. Exportin 1 inhibition as a therapeutic strategy for small cell lung cancer [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 3594.

ABSTRACT The repertoire of tumor-infiltrating lymphocytes (TILs) can be vast, and many of these TILs are not endowed with tumor reactivity. While a number of reports have shown that tumor-reactive TILs express CD39, few reports have demonstrated that conversely, CD39 can be leveraged to serve as a proxy of tumor-reactive CD8 T cells. Using single-cell CITE/RNA/TCRseq, we show that CD39 + CD8 T cells in human lung cancers demonstrate transcriptional and proteomic features of exhaustion, tumor reactivity, and clonal expansion. Moreover, TCR cloning revealed that CD39 enriched for tumor-reactive CD8 T cell clones. Flow cytometry of 440 lung cancer specimens revealed that CD39 level on CD8 T cells is only weakly correlated with tumoral features that currently guide lung cancer therapy, such as histology, driver mutation, PD-L1 and tumor mutation burden. PD-1 axis blockade, but not cytotoxic chemotherapy, increased intratumoral CD39 + CD8 T cells. CD39 correlated with PD-1 expression on CD8 T cells and high pre-treatment/early-on-treatment levels were associated with improved clinical outcomes, but not immune-related adverse events, from immune checkpoint blockade therapy. This comprehensive profiling of the clinical, pathological and molecular features highlights the utility of CD39 as a proxy for tumor-reactive CD8 T cells in human lung cancer.

INTRODUCTION:SCLC is a highly aggressive neuroendocrine tumor that is characterized by early acquired therapeutic resistance and modest benefit from immune checkpoint blockade (ICB). Repression of the major histocompatibility complex class I (MHC-I) represents a key mechanism driving resistance to T cell-based immunotherapies. METHODS:We evaluated the role of the lysine-specific demethylase 1 (LSD1) as a determinant of MHC-I expression, functional antigen presentation, and immune activation in SCLC in vitro and in vivo through evaluation of both human SCLC cell lines and immunocompetent mouse models. RESULTS:We found that targeted inhibition of LSD1 in SCLC restores MHC-I cell surface expression and transcriptionally activates genes encoding the antigen presentation pathway. LSD1 inhibition further activates interferon signaling, induces tumor-intrinsic immunogenicity, and sensitizes SCLC cells to MHC-I-restricted T cell cytolysis. Combination of LSD1 inhibitor with ICB augments the antitumor immune response in refractory SCLC models. Together, these data define a role for LSD1 as a potent regulator of MHC-I antigen presentation and provide rationale for combinatory use of LSD1 inhibitors with ICB to improve therapeutic response in SCLC. CONCLUSIONS:Epigenetic silencing of MHC-I in SCLC contributes to its poor response to ICB. Our study identifies a previously uncharacterized role for LSD1 as a regulator of MHC-I antigen presentation in SCLC. LSD1 inhibition enables MHC-I-restricted T cell cytolysis, induces immune activation, and augments the antitumor immune response to ICB in SCLC.

CD39 has emerged as a marker of tumor-reactive CD8 T cells. Using single-cell CITE/RNA/TCRseq, we show that CD39+ CD8 T cells in human lung cancers demonstrate transcriptional and proteomic features of exhaustion, tumor reactivity, and clonal expansion. Flow cytometry of 440 lung cancer specimens revealed that CD39 level on CD8 T cells is only weakly correlated with tumoral features, such as histology, driver mutation, PD-L1 and tumor mutation burden. CD8 T cells reactive against EGFR driver mutations preferentially expressed CD39, suggesting that CD39 can be leveraged to identify neoantigen-reactive CD8 T cells. PD-1 axis blockade, but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8 T cells. CD39 correlated with PD-1 expression on CD8 T cells and high pre-treatment/early-on-treatment levels were associated with improved clinical outcomes from immune checkpoint blockade. This extensive and comprehensive profiling of the clinical, pathological and molecular features highlights the utility of CD39 as a proxy for tumor-reactive CD8 T cells in human lung cancer.

This standardized protocol describes the preparation of PCR amplified and purified samples from human cell lines passaged and collected from CRISPR screening. High-quality samples can be used to perform next-generation sequencing (NGS) to uncover changes in sgRNA abundance from the timepoint at which library-transduced cells are selected to the timepoint when the screen is ended. Here, we describe proper calculation methods for library representation and show how to overcome potential issues often encountered by researchers. For complete information on the use and execution of this protocol, please refer to Wohlhieter et al. (2020).

Abstract Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUAD) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre/posttransformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre/posttransformation samples, supports that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in the PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacologic inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer. Significance: The difficulty in collection of transformation samples has precluded the performance of molecular analyses, and thus little is known about the lineage plasticity mechanisms leading to LUAD-to-SCLC transformation. Here, we describe biological pathways dysregulated upon transformation and identify potential predictors and potential therapeutic vulnerabilities of NE transformation in the lung. See related commentary by Meador and Lovly, p. 2962. This article is highlighted in the In This Issue feature, p. 2945

Immune checkpoint blockade (ICB) has been a remarkable clinical advance for cancer; however, the majority of patients do not respond to ICB therapy. We show that metastatic disease in the pleural and peritoneal cavities is associated with poor clinical outcomes after ICB therapy. Cavity-resident macrophages express high levels of Tim-4, a receptor for phosphatidylserine (PS), and this is associated with reduced numbers of CD8+ T cells with tumor-reactive features in pleural effusions and peritoneal ascites from patients with cancer. We mechanistically demonstrate that viable and cytotoxic anti-tumor CD8+ T cells upregulate PS and this renders them susceptible to sequestration away from tumor targets and proliferation suppression by Tim-4+ macrophages. Tim-4 blockade abrogates this sequestration and proliferation suppression and enhances anti-tumor efficacy in models of anti-PD-1 therapy and adoptive T cell therapy in mice. Thus, Tim-4+ cavity-resident macrophages limit the efficacy of immunotherapies in these microenvironments.

Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in multiple cancers. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations. The incidence of transdifferentiation into squamous carcinoma in EGFR mutant tumors, the setting where this histologic shift has been most extensively described, occurs in up to 9% of cases relapsed on osimertinib and has been associated to poor prognosis.

ABSTRACTPaired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of ‘large’ and ‘dual-expanded’ clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.