This work evaluated the in vivo performance of an anti-delta-like ligand 3 (DLL3) monoclonal antibody, TDI-Y-010, covalently linked to the MACROPA-derived chelator (mcp) for 225Ac radioimmunotherapy in 2 DLL3-positive neuroendocrine cancer models of the lung and prostate. Methods: Ex vivo biodistribution studies were conducted to evaluate the uptake of [225Ac]Ac-mcp-TDI-Y-010 and determine appropriate therapeutic dosing. On the basis of dosimetry data, 3 doses of [225Ac]Ac-mcp-TDI-Y-010 (9.25, 18.5, and 37.0 kBq) were evaluated in female nude mice bearing Lu149 small cell lung cancer patient-derived xenografts. An additional therapeutic efficacy study was conducted in male nude mice bearing H660 neuroendocrine prostate cancer xenografts, with administered doses of 4.63, 9.25, and 18.5 kBq. Results: In the Lu149 model, the median survival of the [225Ac]Ac-mcp-TDI-Y-010 treatment groups was significantly longer than that of the saline treatment cohorts (P < 0.0001 and P = 0.0002, respectively). In the neuroendocrine prostate cancer model, median survival was significantly longer for mice in the [225Ac]Ac-mcp-TDI-Y-010 groups than in those treated with [225Ac]Ac-mcp-IgG4 (median survival, 37 d; P = 0.002, 0.0001, and 0.0006 for the 4.63-, 9.25-, and 18.5-kBq [225Ac]Ac-mcp-TDI-Y-010 groups, respectively). Hematologic toxicity was transient in both models and comparable across all cohorts. Histopathologic assessment of background organs demonstrated mild to moderate kidney and ovary toxicity in the SC16 group compared with the highest-dose TDI-Y-010 cohort (37.0 kBq). Conclusion: [225Ac]Ac-mcp-TDI-Y-010 exhibited excellent antitumor efficacy with mild and transient hematologic toxicity, supporting its potential as a radioimmunotherapeutic agent for patients with DLL3-expressing neuroendocrine cancers.

Histologic transformation to lung squamous cell carcinoma (LUSC) is an underrecognized mechanism of resistance in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD). Although AKT and MYC activation have been linked to LUSC features, the clinicogenomic determinants of this transformation remain undefined. In this study, we performed comprehensive clinical and multiomic profiling-including genomic, transcriptomic, methylation, and proteomic analyses-of EGFR-mutant tumors that were transforming, adenosquamous (LUAS), or de novo LUSC. Patients with EGFR-mutant LUSC or LUAS had shorter overall survival on first-line osimertinib compared with those with EGFR-mutant LUAD. Transforming tumors were enriched for alterations in the retinoblastoma (Rb) and AKT pathways, particularly cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletions. These alterations were also frequent in EGFR-wild-type LUSC and associated with shorter time-to-osimertinib discontinuation. In genetically engineered human in vivo models, Rb inactivation, in combination with AKT and MYC activation, enhanced the acquisition of LUSC features. Single-cell RNA profiling of such models recapitulated the molecular changes observed in the transforming clinical specimens and identified MET pathway up-regulation during transformation. Combined EGFR and MET inhibition suppressed tumor growth in patient-derived xenograft models of LUSC transformation. Together, these findings highlight Rb pathway inactivation as a promoter of LUSC transformation in EGFR-mutant lung cancer and identify MET signaling as a therapeutic vulnerability that may suppress plasticity in this setting and extend response to targeted therapy.

Abstract ALK gene fusions drive oncogenesis in ∼5% of lung adenocarcinomas (LUADs), and ALK tyrosine kinase inhibitors (TKIs) such as lorlatinib have improved outcomes. However, acquired resistance remains a challenge, with >30% of mechanisms unknown. To uncover novel drivers of resistance, we performed multi-omic profiling of clinical samples and PDXs. Genomic analysis of 83 ALK+ LUADs revealed frequent co-occurring alterations, including CDKN2A deletion (34%), TP53 mutation (32%), and MYC amplification (13%). Transcriptomic profiling highlighted enrichment of DNA replication and repair pathways, MYC targets, epithelial-to-mesenchymal transition, and TGF-β signaling in resistant tumors. scRNAseq of matched sensitive and resistant PDXs revealed extensive heterogeneity and diverse resistance pathways, mirroring those in clinical samples. Notably, elevated DNA replication and repair activity strongly correlated with TP53 missense mutations in clinical samples. Analysis of MSK clinical cohort showed that 80% of TP53 mutations in ALK+ LUAD occur in the DNA-binding domain, predominantly missense variants with potential gain-of-function (GOF) properties. The presence of TP53 missense mutations predicted worse prognosis in ALK-fusion LUAD patients treated with TKIs.To assess the role of mutp53 in resistance, we overexpressed R175H and R273H in ALK+ H3122 and H2228 cells. Co-immunoprecipitation and mass spectrometry revealed that mutp53 associates with DNA repair, cell cycle, and chromatin remodeling proteins. Mutp53-expressing cells were refractory to lorlatinib compared to isogenic p53-deficient clones. Overexpression of mutp53 upregulated DNA replication proteins, including ORC and MCM family members, as well as DNA repair proteins such as BRCA1, ATM, CHEK1, and MSH6. Notably, mutp53-expressing cells maintained DNA replication and repair protein levels following lorlatinib-induced DNA damage, unlike p53-deficient counterparts. Immunofluorescence of the DNA damage marker pH2A demonstrated complete repair of lorlatinib-induced lesions in the presence of mutp53, revealing a novel GOF role for these mutants. Furthermore, mutp53 enhanced DNA synthesis by promoting origin firing, thereby facilitating cellular survival under targeted therapy. Similar effects were observed in EGFR-driven LUAD cells, indicating that mutp53 contributes to resistance across oncogene-driven LUADs.To target this axis, we tested the proteasome inhibitor carfilzomib which inhibited mutp53 proteins and promoted the degradation of DNA replication proteins. Carfilzomib synergized with lorlatinib in resistant ALK+ p53 mutant cells and induced robust tumor response in TP53 mutant ALK+ PDX models resistant to lorlatinib, demonstrating therapeutic potential. In summary, missense mutp53 promotes ALK TKI resistance by enhancing DNA replication and repair. Citation Format: Esther Redin, Barbara P. Mello, Yingian A. Zhan, Nicholas Socci, Samuel Tischfield, Alexander Lim, Hong Zhong, Mark Donoghue, Richard Koche, Elisa De Stanchina, Alexander Drilon, Alvaro Quintanal-Villalonga, Charles M. Rudin. Missense mutant p53 regulates DNA replication and damage response to promote resistance to targeted therapies in ALK fusion lung adenocarcinoma [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 7042.

Small cell lung cancer (SCLC) is known for its high metastatic potential, with most patients demonstrating clinically evident metastases in multiple organs at diagnosis. The factors contributing to this exceptional metastatic capacity have not been defined. To bridge this gap, we compare gene expression in SCLC patient samples who never experienced metastasis or relapse throughout their clinical course, versus primary SCLC patient samples from more typical patients who had metastatic disease at diagnosis. This analysis identifies FOXA2 as a transcription factor strongly associated with SCLC metastasis. Subsequent analyses in experimental models demonstrates that FOXA2 induces a fetal neuroendocrine gene expression program and promotes multi-site metastasis. Moreover, we identify ASCL1, a transcription factor known for its initiating role in SCLC tumorigenesis, as a direct binder of the FOXA2 promoter and regulator of FOXA2 expression. Taken together, these data define the ASCL1-FOXA2 axis as a critical driver of multiorgan SCLC metastasis.

Large cell (LCNEC), prostate (NEPC) and small cell lung (SCLC) neuroendocrine carcinomas are highly aggressive malignancies with few therapeutic options. Lurbinectedin, is an FDA-approved DNA-damaging agent used for the treatment of SCLC, although its efficacy remains limited. Our group has found that neuroendocrine (NE) tumors have higher levels of DNA damage repair (DDR) proteins than non-NE tumors, which may contribute to their reduced response to DNA-damaging agents. Exportin 1 (XPO-1), is an export receptor that regulates DDR proteins, and it can be inhibited with selinexor. We have explored the combination of lurbinectedin with selinexor as a potential strategy for treating neuroendocrine tumors. XPO-1 was found significantly upregulated in neuroendocrine carcinomas (NECs) biospecimens compared to normal tissues, and its expression positively correlated with DDR genes such as CHEK1, WEE1 and PARP1. Combination of selinexor and lurbinectedin demonstrated a potent synergistic effect in SCLC, LCNEC and NEPC cell lines and significantly enhanced apoptosis compared to single-agent treatments. RNA sequencing (RNAseq) analysis revealed a pronounced induction of DNA damage, alongside a reduction in DNA repair pathways in double treated cells compared to untreated cells. Western blotting confirmed increased levels of γ-H2AX and cleaved PARP, while showing decreased expression of CHK1, CHK2, and MLH1. Comet assays further demonstrated increase in DNA strand breaks in cells treated with the combination compared to monotherapies. We next evaluated the efficacy of this combination therapy in patient-derived xenograft (PDX) models, including two SCLC, two LCNEC, and one NEPC model. The combination significantly suppressed the tumor growth of three chemoresistant PDXs while delayed tumor relapse in another two naïve models. Furthermore, the dual treatment activated both STING and autophagy pathways, as evidenced by the increased levels of mTOR and LC3B-I/II in vitro. RNA sequencing analysis revealed the upregulation of inflammatory and interferon-γ (IFN-γ)-related pathways, indicating that this therapeutic combination may have an immunomodulatory effect. Consistent with this, the combination of selinexor and lurbinectedin showed strong synergy in a Rb1−/−; Trp53−/−; cMyc+ (NEPC) and a Rb1−/−; Trp53−/−; Rb2−/− (SCLC) triple mutant mouse models, both resistant to either treatment alone. Single-cell RNAseq of these models revealed increased infiltration of immune cells (CD45+ cells), along with enhanced antigen presentation in tumors treated with either lurbinectedin alone or in combination. Furthermore, treatment with lurbinectedin or the combination, enhanced the infiltration of T cells, monocytes, and macrophages into the tumor. In summary, selinexor and lurbinectedin is a promising strategy for targeting NE tumors. Esther Redin, Parvathy Manoj, Juan Qiu, Barbara de Mello, Hong Zhong, Suleyman Vural, Yingqian Zhan, Rodrigo Romero, Nicholas Socci, Richard Koche, Charles Sawyers, Elisa de Stanchina, Charles M Rudin, Alvaro Quintanal-Villalonga. Lurbinectedin synergizes with selinexor by inhibiting DNA damage repair and enhancing immune cell infiltration in neuroendocrine lung and prostate tumors [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 1166.

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.

INTRODUCTION:Small Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC. METHODS:ATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo. RESULTS:SMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib. CONCLUSIONS:This study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.

e20105 Background: 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. Methods: To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model, followed by functional validation in in vivo prostate and lung models of NE transformation, including pharmacologic as well as genetic (isogenic cell line with overexpression or knock of CDC7) validation. Results: Our 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. 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. 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. 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. Conclusions: 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.

We previously reported that activation of p53 by APR-246 reprograms tumor-associated macrophages to overcome immune checkpoint blockade resistance. Here, we demonstrate that APR-246 and its active moiety, methylene quinuclidinone (MQ) can enhance the immunogenicity of tumor cells directly. MQ treatment of murine B16F10 melanoma cells promoted activation of melanoma-specific CD8+ T cells and increased the efficacy of a tumor cell vaccine using MQ-treated cells even when the B16F10 cells lacked p53. We then designed a novel combination of APR-246 with the TLR-4 agonist, monophosphoryl lipid A, and a CD40 agonist to further enhance these immunogenic effects and demonstrated a significant antitumor response. We propose that the immunogenic effect of MQ can be linked to its thiol-reactive alkylating ability as we observed similar immunogenic effects with the broad-spectrum cysteine-reactive compound, iodoacetamide. Our results thus indicate that combination of APR-246 with immunomodulatory agents may elicit effective antitumor immune response irrespective of the tumor's p53 mutation status.

PDF file - 84K, Percentage of CD4+Foxp3+ Tregs in IgG vs DTA-1 treated tumors

Multiple suppressive mechanisms within the tumor microenvironment (TME) are capable of blunting anti-tumor T cell responses. These include engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8 T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). While immune checkpoint blockade therapies aimed at reversing the dysfunctional state of tumor-associated T cells have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8 T cells to be reinvigorated by, for example, PD-1/PD-L1 blockade. As CD8 T cell exhaustion (or dysfunction) is a major therapeutic challenge, gaps in our understanding of cellular and molecular mechanisms underlying the T cell exhaustion (or dysfunction) program in cancer warrant further study of pathways that program T cells toward exhaustion (or dysfunction). Through comprehensive immune profiling of tumor-infiltrating T lymphocytes (TILs), we found that CD47 expression in CD8 TILs isolated from melanoma patients significantly correlates with expression of several checkpoint inhibitory molecules (e.g., TIM-3, PD-1 and LAG-3). Additionally, our re-analysis of single cell data from melanoma patients revealed that terminally exhausted T cells (Tex) and TCF7hi Tex precursor cells exhibit high levels of CD47 transcripts, suggesting phenotypic association of CD47 with T cell exhaustion. We confirmed our observations in murine B16-F10 melanoma where CD47 expression is significantly upregulated in exhausted CD8 TILs. We also show that CD47 functions as a negative regulator for T cell proliferation and function during T cell priming. To address the role of CD47 during the development of CD8 T cell exhaustion/dysfunction in cancer, we performed adoptive T cell transfer of the naïve-sorted Cd47+/+ (WT) and Cd47+/- (Het) antigen specific Pmel-1 CD8 T cells (but not Cd47-deficient Pmel-1 CD8 T cells as they would be subject to innate immune clearance) into B16 tumor-bearing mice and found that Cd47-Het Pmel-1 CD8 TILs, as compared to the Cd47-WT Pmel-1 CD8 TILs, exhibit less expression of exhaustion-related genes (e.g. Pdcd1, Lag3 and Tox), and increased expression of genes associated with T cell activation and proliferation (e.g. Mki67, Lck, Cd69, Gzma, Gzmk). We further confirmed that thrombospondin-1 (TSP-1), as an extracellular matrix protein and a ligand of CD47, contributes to driving the differentiation of CD8 T cells toward exhaustion. Our data highlight for the first time the potential of extracellular matrix protein TSP-1 in programming CD8 T cell exhaustion in cancer through its interaction with CD47 expressed on CD8 T cells. Citation Format: Chien-Huan Weng, Fadi Samaan, Sadna Budhu, Levi Mangarin, Sébastien Monette, Cailian Liu, Stephane Pourpe, Linda Hamadene, Hong Zhong, Xia Yang, David Schroder, Roberta Zappasodi, Pamela Holland, Jedd D. Wolchok, Taha Merghoub. Potential role of CD47 in T cell exhaustion program [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 6150.

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.

Abstract Canonical p53-activated pathways can influence a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages (TAMs). We examined whether p53 activity in the tumor microenvironment (TME) influences antitumor immunity and show that p53 signaling induced pharmacologically with APR-246 (eprenetapopt) can augment the efficacy of immune checkpoint blockade (ICB) in preclinical models, a strategy that is also being tested in patients (NCT04383938). We first investigated the effects of combining APR-246 with ICB in wildtype C57BL6 (B6) mice bearing syngeneic p53 wildtype MC38 colon cancer and B16 melanoma tumors. The combination of an anti-PD-1 antibody (RMP1-14) with APR-246 in mice significantly delayed tumor growth (p < 0.001) and improved survival of tumor-bearing mice, compared to monotherapies (p < 0.01). To further dissect the effects of APR-246 on myeloid and T cells in the TME, we used a conditional knockout of p53 in CSF1R+myeloid cells (CSF1Rcre/p53fl mice), or T cells (CD8cre/p53fl mice). CSF1Rcre/p53fl had loss of tumor control and worse survival with APR-246+anti-PD-1. CD8cre/p53fl had intact tumor control. To study enhanced p53 activity in the TME, we performed flow cytometry, cytokine multiplex and global transcriptional profiling by RNA seq. We found enhanced p53-activity led to increased infiltration of T cells, increased MHC-II expression in TAMs and downregulation of M2-associated cytokines. This was associated with cellular senescence in TAMs and induction of canonical p53-induced senescence-associated secretory phenotype (SASP). Our preclinical findings informed the development of a phase I/II clinical trial using APR-246 with pembrolizumab for patients with advanced solid tumors (NCT04383938). We studied peripheral blood samples from two of the patients with tumor regression and two patients in whom tumors progressed on therapy. We analyzed peripheral blood mononuclear cells (PBMCs) and serum prior to therapy, and at the beginning of cycle 2 and 5 for the patients with tumor control, and at the end of therapy for patients who had progression. Single cell RNA-seq of PBMCs demonstrated a signature consistent with T cell activation and proliferation, and SASP-associated changes in the myeloid compartment as seen in mice. T cell profiling of PBMCs by flow cytometry demonstrated strong proliferation of T cells in patients with tumor control. Serum cytokine analysis demonstrated robust in IL-12, IFN-gamma and Eotaxin-1 in the two responders, which was not seen in the patients whose tumors progressed. Our study illustrates p53-induced SASP in TAMs as a mechanism to reprogram the TME and augment responses to ICB. Ongoing studies will help determine biomarkers that are predictive of response to APR-246+ICB therapy. Citation Format: Arnab Ghosh, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, Nathan Suek, Aliya Holland, Yu-Jui Ho, Mohsen Abu-Akeel, Luis Felipe Campesato, Levi Mark Bala Mangarin, Cailian Liu, Hong Zhong, Sadna Budhu, Andrew Chow, Roberta Zappasodi, Marcus Ruscetti, Scott W. Lowe, Taha Merghoub, Jedd D. Wolchok. Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy [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 250.

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.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells 1 . By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis 1 . Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8 + T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (T reg ) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of T reg cells is dependent on T reg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with T reg cell function in the presence of glucose.

Background CD4 and CD8 T cells are genetically and functionally distinct cell subsets of the adaptive immune system that play pivotal roles in immune surveillance and disease control. During development in the thymus, transcription factors ThPOK and Runx3 regulate the differentiation and maturation of these two lineages into single positive T cells that enter the periphery with mutually exclusive expression of either the CD4 or CD8 co-receptor.1–2 Despite our expectation that these two cell fates are fixed, mature CD4+CD8+ double positive (DP) T cells have been described in the context of numerous immunological responses, including cancer, but their molecular and functional properties and therapeutic relevance remain controversial and largely unknown.3–5 Methods Our lab has identified and characterized a heterogenous DP T cell population in murine and human melanoma tumors comprised of CD4 and CD8 T cells re-expressing the opposite co-receptor and a parallel uptake in the opposite cell type’s phenotype and function. Using CD4 (Trp1) and CD8 (Pmel) transgenic TCR T cells specific to B16 melanoma antigens gp75 and gp100 respectively, we demonstrate the re-expression of the opposite co-receptor following adoptive T cell transfer in B16 melanoma tumor bearing mice. Results Specifically, up to 50% of transferred CD4 Trp1 T cells will re-express CD8 to become a DP T cell in the tumor microenvironment. Further, these CD4 derived DP T cells upregulate CD8 lineage regulator Runx3 and cytolytic genes Gzmb, Gzmk, and Prf1 to become potent cytotoxic T cells. Alternatively, a subset of CD8 Pmel T cells differentiate into DP T cells characterized by the increased expression of CD4, ThPOK, and regulatory marker FoxP3 (figure 1). In addition, we utilized 10x single cell and ATAC sequencing to further characterize these divergent DP T cell populations among open repertoire T cells isolated from murine and human melanoma tumors. Conclusions Our findings highlight the capability of single positive T cells to differentiate in response to antigen and local stimuli into novel T cell subsets with polyfunctional characteristics. The resulting cell subsets will potentially affect the tumor microenvironment in distinct ways. Our studies may inform therapeutic approaches to identify antigen specific T cells as well as innovative signaling pathways to target when genetically engineering T cells to optimize cytotoxic function in the setting of adoptive cell therapy. Ethics Approval The human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #06-107 References Ellmeier W, Haust L & Tschismarov R. Transcriptional control of CD4 and CD8 coreceptor expression during T cell development. Cell Mol Life Sci 2013;70:4537–4553. Luckey MA, et al. The transcription factor ThPOK suppresses Runx3 and imposes CD4+ lineage fate by inducing the SOCS suppressors of cytokine signaling. Nature Immunology 2014; 15, 638–645. Bohner P, et al. Double positive CD4(+)CD8(+) T Cells are enriched in urological cancers and favor T Helper-2 polarization. Front Immunol 2019; 10, 622. Nascimbeni M, Shin E-C, Chiriboga L, Kleiner DE & Rehermann B. Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood 2004;104:478–486. Nishida K, et al. Clinical importance of the expression of CD4+CD8+ T cells in renal cell carcinoma. Int Immunol 2020;32:347–357.

Over the past decade, blockade of the immune checkpoints CTLA-4, PD-1, and PD-L1 has been demonstrated to significantly extend survival of cancer patients across multiple tumor types, including metastatic melanoma, formally proving that immunotherapy is a viable option for the treatment of cancer. These successes have paved the way for the development of additional immune-modulatory antibodies, blocking alternative inhibitory receptors, or engaging costimulatory receptors such as the TNF receptor family member GITR. However, the clinical experience accumulated thus far with checkpoint blockade has clearly shown that only a limited fraction of patients achieve durable clinical benefit with these treatments. This highlights the need to deepen our understanding of the molecular mechanisms underlying response and resistance to immunotherapy and design more personalized and rational combinations based on these therapies. Immune-regulatory mechanisms are one of the major barriers limiting efficacy of immunotherapy. CTLA-4 blockade and GITR costimulation are two immunotherapeutic strategies known to interfere with conventional immunosuppressive regulatory T cells (Tregs). We thus investigated the effects of CTLA-4 blockade and GITR costimulation on suppressive T cells in in vivo mouse melanoma models resistant to these therapies with the aim to clarify the molecular mechanisms underlying refractoriness and provide the rationale to develop more effective therapeutic combinations. To understand the clinical relevance of these findings, we explored the same effects in cancer patients treated with CTLA-4 blockade or GITR costimulation. We found that CTLA-4 blockade, while counteracting conventional Tregs, promotes the expansion of a subset of CD4+Foxp3-T cells expressing high levels of PD-1 (4PD1hi), which constitute a new immunosuppressive T-cell population with T-follicular-helper-like features. Importantly, we observed that anti-CTLA-4 increases the frequency of circulating 4PD1hi in a dose-dependent manner. In contrast, PD-1 blockade decreases 4PD1hi in function of its clinical activity, underscoring the relevance of this cell subset as a pharmacodynamic and prognostic biomarker of checkpoint blockade. These findings indicate that optimizing checkpoint blockade doses and combination regimens so as to keep 4PD1hi in the right balance may favor a positive outcome. In mouse melanoma models of response (early tumors) and refractoriness (advanced tumors) to GITR agonism, we found that anti-GITR efficiently reduces Tregs and increases effector:Treg ratios in both curative and refractory treatment conditions. However, T-cell activation and cytotoxic functions are favored selectively in the presence of low tumor burden. Counteracting exhaustion with PD-1 blockade in combination with GITR agonism restored responsiveness of advanced tumors and CD8+ T-cell functionality. Aligned with the effects of anti-GITR in mice, we found that the agonist anti-human GITR antibody TRX518 decreases Tregs in peripheral blood and tumor to similar extents in patients treated in the first-in-human single-dose monotherapy trial (NCT01239134). However, coincident downregulation of Tregs in the peripheral blood and at the tumor site upon TRX518 was not sufficient to achieve substantial clinical responses in this population of advanced solid cancer patients. This suggests that Treg elimination from advanced tumors may not be sufficient to activate cytotoxic T-cell responses unless the T-cell exhaustion process is concurrently blocked. Based on these preclinical and clinical observations, we have started to explore anti-GITR in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574). Taken as a whole, these findings illustrate the value of conventional and nonconventional immune-suppressive T cells as biomarkers of biologic and therapeutic activity of immunotherapy in melanoma and other tumor types. In addition, these results indicate that inhibition of immune-regulatory mechanisms, such as immunosuppressive T cells, may need to be associated with strategies able to positively activate T-cell responses to achieve significant clinical benefit. Citation Format: Roberta Zappasodi, Sadna Budhu, Cynthia Sirard, Jingjing Qi, Cailian Liu, Yanyun Li, Yasin Senbabaoglu, Sasikanth Manne, Billel Gasmi, Hong Zhong, Xia Yang, Moshen Abu-Akeel, David Schaer, Alexander Huang, Walter Newman, Philip Wong, Katherine S. Panageas, Michael A. Postow, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Matthew D. Hellmann, E. John Wherry, Taha Merghoub, Jedd D. Wolchok. Overcoming immune resistance with rationally designed combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA04.