Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid onset of chemoresistance and poor clinical outcomes. Transcriptional heterogeneity among treatment-naïve SCLC tumors underlies four transcriptional subtypes, each with distinct clinical vulnerabilities. Though previously hypothesized to delineate a distinct subtype, expression of YAP1 is largely absent from treatment-naïve, pure SCLC. To characterize relapsed SCLC, circulating tumor DNA, circulating tumor cells, and core needle biopsies from SCLC patients and preclinical models following resistance to standard-of-care therapies were analyzed. In contrast to treatment-naïve SCLC, these analyses reveal an emergent YAP1-positive cell population that coincides with treatment resistance. These YAP1-positive cells exhibit characteristics of drug tolerant persister cells, including senescence, stemness, and plasticity, as YAP1 positive cells largely abandon features characteristic of SCLC to adopt those of large-cell neuroendocrine carcinoma (LCNEC). As a result of this SCLC-like to LCNEC-like evolution, YAP1-positive cells lack several clinically relevant SCLC surface targets (i.e., DLL3, SEZ6), but are enriched for others (i.e., B7-H3, TROP2). We propose a model where YAP1 expressing cells emerge with SCLC treatment resistance and characterize a tenacious subpopulation capable of diverging from the treatment naïve lineage and adopting features to evade therapeutic response.

Abstract Background: Small cell lung cancer (SCLC) exhibits profound epigenetic remodeling and transcriptional plasticity, yet tissue scarcity limits multi-omic profiling. Circulating tumor DNA (ctDNA) methylation analysis via reduced representation bisulfite sequencing (RRBS) or enzymatic methyl-seq (EM-seq) provides a minimally invasive window into tumor biology. However, the relationship between ctDNA methylation features and transcriptomic states such as epithelial-mesenchymal transition (EMT) or neuroendocrine (NE) differentiation remains poorly defined but may serve as means to monitor patient therapeutic response. Methods: We analyzed matched ctDNA methylation RRBS and bulk RNA-seq data from N = 43 SCLC patients collected at baseline. CpG methylation levels were quantified and mapped to gene bodies, promoters, and distal regulatory regions. To capture functional methylation signatures, CpG sites were ranked by their correlation with RNA expression of the corresponding genes and by feature importance predicting expression levels or calculated transcriptomic scores (e.g., EMT, MYC, ASCL1/NEUROD1 subtype indices). High-ranking CpG sites were aggregated into gene-level and pathway-level signatures, followed by GO and KEGG enrichment analyses to identify key regulatory networks. These were further evaluated in SCLC patients treated longitudinally with frontline chemotherapy. Results: Genome-wide ctDNA methylation profiles predicted bulk RNA-seq-derived gene expression as well as predefined score metrics (such as EMT score, NE score) with high concordance (Wilcoxon R2 = 0.63 ± 0.07, p < 0.001). Ranking analysis revealed that predictive CpG sites were enriched in enhancer and promoter regions associated with EMT regulators and NE lineage genes. Functionally, GSEA further linked high NE scores to activation of Hedgehog signaling, and EMT scores to enrichment of G2/M checkpoint pathways, suggesting distinct regulatory programs underlying SCLC phenotypes. Conclusions: Integrative analysis of ctDNA methylation and transcriptomic data reveals that ranked, functionally annotated CpG features can accurately infer gene expression programs and biological states in SCLC. These approaches provide a mechanistic framework to interpret ctDNA methylation signatures and enable noninvasive characterization of tumor subtypes and therapeutic resistance in SCLC patients. Citation Format: Yuanxin Xi, Allison Stewart, Lixia Diao, Qi Wang, Li Shen, Runsheng Wang, Alberto Duarte, Alexa Halliday, Kavya Ramkumar, Robert Cardnell, Bingnan Zhang, Carl M. Gay, Lauren A. Byers, Jing Wang, . Inferring transcriptomic programs from circulating tumor DNA methylation signatures in small cell lung cancer using RRBS and EM-seq [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 4127.

Background: Small cell lung cancer (SCLC) is an aggressive, high-grade neuroendocrine carcinoma (hgNEC) with poor prognosis. Tarlatamab, a DLL3-targeting T cell engager was approved in 2024 for relapsed SCLC, with almost doubled overall survival benefit compared to chemotherapy. However, over half of patients do not respond to tarlatamab and others develop resistance within months. With multiple DLL3-targeting therapies in clinical trials, there are currently no validated predictive biomarkers to identify patients most likely to benefit from tarlatamab. In this study, we evaluate baseline DLL3 IHC level in patients as a predictive biomarker to tarlatamab clinical outcome. Methods: We assembled a cohort of 138 patients within IRB-approved MD Anderson GEMINI database (PA13-0589) with DLL3 expression by CLIA-validated immunohistochemistry (IHC), and those treated with tarlatamab from 7/1/2024 to 3/30/2026. Only those with DLL3 IHC results and treated with tarlatamab were included in this predictive biomarker study. All DLL3 levels were reported as percentage cells with cytoplasmic or surface DLL3 expression, including 24 with additional intensity score and H-score calculation. demographic, clinical, and outcome data were collected. Systemic and intracranial response data were assessed by RECIST and mRANO BM criteria respectively. Longitudinal liquid biopsies were taken from a subset of patients for circulating tumor DNA (ctDNA) and (Cytometry by Time-of-Flight) CyTOF analyses. Results: In the predictive biomarker cohort of 54 patients with SCLC treated with tarlatamab, Median DLL3% level in tarlatamab treated cohort was 75%. Median follow-up time was 9.9 months, median time on tarlatamab treatment (ToT) was 5.8 months. At the data cut-off, 61% (33 out of 54) patients were alive. Kaplan-Meier curve stratified by DLL3 % of <=75% vs. >75 showed significant longer ToT and a trend toward longer median OS (mOS). In DLL3>75% compared to DLL3 <=75%: mToT was 10.1 months vs. 2.9 months (p=0.036), mOS: not reached vs. 9.5 months (p= 0.066). Among 41 patients who completed one cycle of tarlatamab treatment and were eligible for systemic (extra-cranial) response per RECIST criteria; we observed partial response (PR) in 51% (21) patients, stable disease (SD) in 22% (9) patients; and progressive disease (PD) in 27% (11) patients. The median DLL3 % in those with PR vs. PD were 80% vs. 40% (p=0.006); median DLL3% comparing PR and SD were 80% vs. 60% (p=0.051). Among 35 patients evaluable for intracranial response, best objective response prior to any brain radiation were 5 (14%) CR,11(31%) PR, 8 (23%) SD and 11 (31%) PD. 9 received brain radiation (8 SRS, 1 WBRT) during tarlatamab to achieve better intracranial control while systemic control is maintained. DLL3% does not appear to be correlated with intracranial response. Longitudinal ctDNA and CyTOF analyses showed persistent ctDNA positivity, as well as increasing NEUROD1 subtype cells in circulating tumor cells in post-tarlatamab progression samples. We additionally profiled DLL3 expression by IHC in 138 patients with SCLC and found heterogenous DLL3 expression, with Median DLL3% of 70% across different biopsy sites. Conclusions: Our data suggests DLL3 expression in patients with SCLC is heterogeneous and not ubiquitous, with median DLL3 IHC of 70%. DLL3 IHC predicts objective response and long-term outcome with tarlatamab. In addition, dynamic monitoring of ctDNA level and CTCs by CyTOF are suggestive of development of tarlatamab resistance, however further studies with larger cohort of paired longitudinal samples are needed to validate the novel blood-based biomarkers. To our knowledge, this study is the first to validate DLL3 IHC as a predictive biomarker for tarlatamab in SCLC, which leads the way for optimizing treatment selection and combinatorial therapies for the subset of patients less likely to respond to tarlatamab.

Abstract Treatment-naïve small cell lung cancer (SCLC) is highly sensitive to frontline chemotherapy; however relapsed SCLC acquires broad chemoresistance that renders conventional second line agents like topotecan, a topoisomerase inhibitor (TOP1i), largely ineffective. Recent clinical trials with surface targeting strategies such as antibody drug conjugates (ADCs) with TOP1i-based payloads have demonstrated strong responses in relapsed small SCLC. Identifying biomarkers of response and resistance to these agents will be important to optimize the effectiveness of this new therapeutic class. We previously showed that non-neuroendocrine SCLC tumors express low levels of DLL3 and SEZ6, but high levels of TACSTD2 (TROP2) and ERBB2 (HER2). In this study, we examined the activities of different HER2- and TROP2-directed ADCs with TOP1i- and non-TOP1i-based payloads in SCLC cell lines. While target expression did not correlate with ADC sensitivity, SCLC cell lines with high SLFN11 levels showed greater sensitivity to TOP1i-based HER2- and TROP2-ADCs and their payloads. SLFN11 knockdown confirmed that SLFN11 loss reduces ADC sensitivity. Notably, combination with an ATR inhibitor sensitized resistant SLFN11-low SCLC cell lines to TOP1i-based ADCs. In contrast, sensitivity to a non-TOP1i-based HER2 ADC was not associated with SLFN11 expression. High cMYC and ABCB1 (P-glycoprotein) levels were also associated with resistance to TOP1i-based ADC, but not to non-TOP1i ADCs. Mechanistically, TOP1i-based HER2- and TROP2-ADCs induced significant DNA damage and apoptosis. Increases in PDL-1 expression, STING-chemokine CCL5, along with extracellular ATP were also detected, indicating induction of immunogenic responses and supporting combinations with immunotherapy. Overall, these results identify SLFN11 as a clinical biomarker for patient selection and provide preclinical support for combinations with ATR inhibitor or using alternate non-TOP1i-based ADCs to overcome resistance to TOP1i-based HER2- and TROP2-ADCs. Citation Format: Kavya Ramkumar, C. Allison Stewart, Shui-Ping So, Runsheng Wang, Azusa Tanimoto, Alberto Duarte Jr., Jenna H. Gray, Li Shen, Lixia Diao, Yuanxin Xi, Qi Wang, Alejandra G. Serrano, Luisa M. Solis Soto, Jing Wang, Bingnan Zhang, Carl M. Gay, Lauren A. Byers. Predictive biomarkers and combination strategies to overcome resistance to HER2- and TROP2-directed antibody drug conjugates with topoisomerase I inhibitor payloads in SCLC [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 4544.

Abstract High-grade neuroendocrine carcinomas (hgNECs) are aggressive malignancies that arise, most commonly, from the aerodigestive tracts, though, more rarely from other sites. Frustratingly, hgNECs are, initially, exquisitely sensitive to chemotherapy and/or radiation, but these responses are short-lived and inevitable relapses occur. Recent therapeutic developments have focused on hgNEC-associated cell surface antigens which, when targeted with an antibody, can serve as beacons for delivery of cytotoxic or immunologic payloads. T-cell engagers (TCEs) and/or antibody-drug conjugates (ADCs) against hgNEC antigens, including DLL3 and SEZ6, have demonstrated unprecedented responses in relapsed hgNECs. In some cases, such as small cell lung cancer (SCLC), the expression of these antigens is nearly ubiquitous and sensitivity to surface-targeting therapies is contingent primarily on payload. However, in other hgNECs, neuroendocrine (NE) features, including DLL3 and SEZ6 expression, are bimodal, which necessitates strategies for patient selection and alternative antigens for NE-low hgNECs. DLL3 levels have been evaluated by CLIA-validated immunohistochemistry in >340 patients at MDACC, including those diagnosed with rare extrapulmonary hgNECs (i.e., Merkel cell carcinoma, small cell of the breast, thyroid, etc.) for off-label use of tarlatamab (DLL3-targeting TCE). We utilized expression data from public hgNEC cohorts, as well as our own patient and preclinical model cohorts to characterize the relationship between known cell surface target expression (i.e., DLL3, etc.), various established biomarkers, including NE status and SLFN11, and sensitivity to surface targeting therapies. NE status defined two major subsets of hgNECs. NE-high specimens expressed high DLL3 and SEZ6 and were responsive to therapies targeting these antigens. In contrast, the NE-low subsets expressed high TROP2 and HER2. Surprisingly, target expression was not the dominant predictive biomarker for ADCs targeting these antigens as, instead, biomarkers of payload sensitivity (e.g. SLFN11 for topoisomerase I inhibitor payloads, P=0.01) offered superior insight into efficacy. Surface-targeting therapies represent a new paradigm for hgNEC therapeutics and while DLL3- and SEZ6-targeting therapies are showing promise across all hgNECs, their impact will be limited outside SCLC without patient selection. Utilizing biomarkers, including those for target expression, inflammatory potential, and payload sensitivity, will be critical to optimize drug selection for patients with these malignancies. Citation Format: C. Allison Stewart, Kavya Ramkumar, Azusa Tanimoto, Runsheng Wang, Alberto Duarte, Angelo Chen, Alejandra Serrano, Yuanxin Xi, Lixia Diao, Qi Wang, Li Shen, Alexa Halliday, Shui Ping So, Luisa Solis Soto, Jing Wang, Bingnan Zhang, Lauren A. Byers, Carl M. Gay. Tumor cell surface targeting of high-grade neuroendocrine carcinomas [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 4550.

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by robust responsiveness to frontline chemotherapy and immunotherapy followed by rapid resistance and poor survival. Recently, cell-surface targeting drugs, such as T-cell engagers (tarlatamab) and antibody-drug conjugates (ADCs), have demonstrated significant responses in relapsed SCLC patients. Paradoxically, the efficacy of TROP2-ADCs and HER2-ADCs both have failed to correlate with the level of target expression in both SCLC and breast cancer patients, yielding difficulty in predicting the patient population which would receive benefit from these treatments. Treatment efficacy across a range of expression may be due to tumor heterogeneity and the delivery of the payload to a cell presenting the target resulting in a bystander effect on cells with low or no expression. SCLC cell lines and dissociated PDX tumors were evaluated for development of spheroids, which better mimic a solid tumor than 2D cell culture. The growth of these spheroids and PDX organoids were evaluated following treatment with HER2-ADC (trastuzumab deruxtecan), TROP2-ADC (sacituzumab govitecan), tarlatamab, activated T-cells, and combinations of these. Effect of complete siRNA knockdown of the surface target on the efficacy of targeted ADCs was also determined. HER2- and TROP2-ADCs slowed growth of the spheroids as compared to the control. Additionally, the combination of TROP2-ADC, tarlatamab, and T-cells caused contraction of the spheroids. Notably, the tarlatamab and T-cells arm demonstrated what is likely a pseudo-progression sometimes seen in patients as T-cells infiltrate and begin to kill tumor cells. Knockdown of ERBB2 and TACSTD2 demonstrated no demonstrable difference in ADC sensitivity. In conclusion, HER2- and TROP2-ADCs demonstrated efficacy in these models and may be used in combinatorial approaches in the future. Additionally, co-cultured cell line spheroids could provide a better model of tumor heterogeneity. Lastly, HER2- and TROP2- levels are not predictive biomarkers for ADC sensitivity, suggesting that other biomarkers should be explored to identify responsive patient populations. Citation Format: Jenna Gray, Kavya Ramkumar, C. Allison Stewart, Runsheng Wang, Alberto Duarte, Azusa Tanimoto, Robert J. Cardnell, Lauren Averett Byers, Carl M. Gay. HER2- and TROP2-antibody-drug conjugates in small cell lung cancer cell lines and 3D culture: Efficacy and role of target expression [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 4546.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid onset of chemoresistance and poor clinical outcomes. Following decades of, at best, modest clinical advances, the recent FDA approval of tarlatamab, a DLL3 targeting bispecific T-cell engager (BiTE), alongside unprecedented response rates observed with multiple antibody-drug conjugates (ADCs), have ushered in a paradigm shift towards surface targeting strategies in relapsed SCLC patients. These same agents are demonstrating similar efficacy in more rare high-grade neuroendocrine carcinomas, both pulmonary and extrapulmonary; however, they are being largely tested in unselected populations. While providing much-needed optimism for SCLC patients, resistance, both de novo and acquired, is common and must be better characterized to maximize the potential of these new therapeutic classes. We hypothesize that combinatorial targeting of multiple surface proteins using distinct strategies (i.e., BiTEs, ADCs, etc.) represents a novel way to overcome intratumoral heterogeneity common to relapsed SCLC and enhance antitumor immunity engendered by ADC payloads (i.e., topoisomerase 1 [TOP1] inhibitor). To better define the surfaceome of relapsed SCLC, we performed surfaceome mass spectrometry analysis of SCLC cell lines, naïve and relapsed patient derived xenografts (PDXs), and PDXs treated with frontline chemotherapy until relapse occurred, and identified a number of novel and known surface proteins (i.e., TROP2, HER2, B7H3). Surface targeting strategies against HER2, TROP2, and DLL3 are effective in preclinical models (i.e., cell lines and xenograft models) resistant to other common SCLC therapies (i.e., platinum chemotherapy). In particular, ADCs with TOP1 inhibitor payloads were more effective in models with high SLFN11 and target levels, suggesting that sensitivity requires both surface target expression and SLFN11 positivity for greatest response. Notably, single-cell transcriptional profiling of relapsed patient biopsies revealed mutually exclusive expression of surface genes in distinct cell populations, including senescent, drug tolerant persister cells (DTPCs), representing an unique opportunity to target heterogeneous populations. We show that combination targeting against different surface proteins (e.g., DLL3, TROP2, HER2) using both immune (i.e., chimeric antigen receptor T-cells, BiTEs) and payload-based modalities (i.e., ADCs) was more effective than single-agent targeting in resistant, neuroendocrine-low models. Therefore, intratumoral heterogeneity associated with relapsed SCLC, which limits efficacy of single-agent surface targeting strategies, may be exploited with combinatorial therapies to target resistant cell populations, including DTPCs, using payload and immune-based methods. C. Allison Stewart, Kavya Ramkumar, Runsheng Wang, Yan Yang, Bingnan Zhang, Yuanxin Xi, Lixia Diao, Qi Wang, Alberto Duarte, Ping Li, Azusa Tanimoto, Alejandra G. Serrano, Jody Vykoukal, Mukulika Bose, Loukia G. Karacosta, Luisa Solis Soto, Samir Hanash, Jing Wang, John V. Heymach, Lauren Averett Byers, Carl M. Gay. Combination surface targeting strategies in relapsed small cell lung cancer (SCLC) to overcome intratumoral heterogeneity associated with treatment 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 2928.

Small cell lung cancer (SCLC) is a highly aggressive malignancy characterized by significant replication stress and DNA damage. Recently an "inflamed" subset of SCLC has been identified, showing high expression of genes associated with Stimulator of Interferon Genes (STING)-induced T-cell attracting chemokines, such as CCL5 and CXCL10. The addition of DNA damage repair inhibitors (DDRis) to immunotherapy has been shown to enhance immune responses through STING-dependent activation pathways.This study focuses on the role of DNA-PK inhibition (DNA-PKi), a key regulator of DDR, in the activation of multiple cytosolic DNA/RNA sensors (STING, cGAS, MAVS, IFI16) to elucidate the molecular mechanisms linking DNA damage to immune responses.Treatment-naïve SCLC patients (n=10) and patients undergoing chemotherapy treatment (n=10) were enrolled. Blood samples were collected at baseline and after chemo-immunotherapy to analyze the expression of cGAS, STING, MAVS, and IFI16. Comprehensive transcriptomic analysis, real-time PCR and protein analysis were performed. STING, MAVS and cGAS knockdown and subcellular localisation analysis were also reported.SCLC tumor tissues (n=81) with higher STING-related chemokines (CCL5/CXCL10) exhibited increased expression of genes involved in MAVS pathway (IFITH1, DDX60), suggesting a simultaneous activation of STING and MAVS. DNA-PKi enhanced STING and MAVS more effectively than other DDRis in SCLC patients-derived PBMCs post-cisplatin compared to naïve patients, indicating its unique role in promoting immune responses. No significant changes in cGAS expression were observed in immune cells from SCLC patients, suggesting that the upregulation of STING mediated by DNA-PK inhibition is independent of cGAS, as knockdown of cGAS did not influence the upregulation of STING and p-IRF3 following DNA-PKi treatment. DNA-PKi treatment resulted in novel mitochondrial co-localization and physical interaction between STING and MAVS in PBMCs, leading to improved functional activation via nuclear translocation of p-IRF3.In conclusion, the study posits that DNA-PK inhibition facilitates a non-canonical interaction between STING and MAVS, thereby enhancing the innate immune response against SCLC. Caterina De Rosa, Luisa Amato, Concetta Tuccillo, Francesca Iommelli, Viviana De Rosa, Virginia Tirino, Federica Papaccio, Kavya Ramkumar, Qi Wang, Jing Wang, Alberto Servetto, Floriana Morgillo, Lauren Averett Byers, Fortunato Ciardiello, Carminia Maria Della Corte. STING-MAVS interaction as novel immune mechanism of DNA-PK-inhibitor in SCLC [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 6158.

Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer with limited treatment options. Patients often respond well to initial chemo-immunotherapy but relapse quickly, necessitating new strategies to enhance immune responsiveness. Recent research explores combining DNA-damaging therapies with immunotherapy to activate the STING pathway and improve the antitumor immune response. The addition of DNA Damage Repair (DDR) inhibitors, such as DNA-PKcs inhibitors, after chemotherapy has shown promise in activating innate immune sensors and enhancing CD8+ T cell and NK cell pathways in SCLC models. This approach could potentially reshape the tumor microenvironment and sustain an antitumor immune response, offering a maintenance strategy for SCLC treatment.

Supplementary Figure 2 shows polyploidy and apoptosis are inhibited by high BCL2 levels

Supplementary table shows AZD2811 sensitivity, transcriptomic and proteomic profiles of SCLC cell lines

Supplementary Figure 3 shows the combination of AZD2811 and BCL2 inhibitor enhances apoptosis and cell death.

Supplementary Figure 1 shows BCL2 is a strong biomarker of resistance to AURK inhibition in SCLC.

INTRODUCTION:Wnt/β-catenin signaling pathway activation contributes to tumorigenesis and chemo-resistance in SCLC, yet clinical attempts to target this pathway have been unsuccessful. TRAF2 and NCK-interacting protein kinase (TNIK), an essential nuclear activator of Wnt/β-catenin target genes, has not yet been validated as a viable therapeutic target in SCLC. Here, we validated that TNIK inhibition is a promising approach for personalized anticancer therapy in SCLC. METHODS:We correlated the IC50 values of a TNIK inhibitor, NCB-0846, with proteomic profiling (reverse phase protein array) data across 28 SCLC cell lines. Cytokine array analysis was performed to quantify changes in 105 cytokines after TNIK inhibitor treatment. RESULTS:We identified c-MYC expression as a top candidate marker of TNIK inhibition response. In xenograft models of c-MYChigh SCLC, TNIK inhibition led to suppression of tumor growth and a decrease in c-MYC expression. In the clinically aggressive POU2F3 expressing subtype of SCLC, the TNIK inhibitor demonstrated antitumor effect by decreasing SOX9 in addition to c-MYC. Furthermore, TNIK inhibition suppressed the production of the immunosuppressive chemokine CCL2 by attenuating its transcription factor FOXK1 in c-MYChigh SCLC cells. Combination of TNIK inhibition and an anti-PD-L1 antibody resulted in greater efficacy and reduced infiltration of immunosuppressive cells compared with each monotherapy in immunocompetent SCLC in vivo models. CONCLUSIONS:TNIK inhibition is more effective in c-MYChigh SCLC, acting through down-regulation of c-MYC levels. It also decreases the production of CCL2, supporting the rationale for combination therapy with immune checkpoint inhibitors in c-MYChigh SCLC.

High-grade neuroendocrine carcinomas of the lung (hgNECs), which include small cell lung cancer (SCLC) and pulmonary large-cell neuroendocrine carcinoma (LCNEC), are characterized by poor prognoses, limited treatment options and rapid emergence of resistance. While recent surfaceome-directed approaches targeting DLL3, SEZ6, and TROP2 using chimeric antigen receptor T cells (CAR-Ts), bispecific T cell engagers (BiTEs) and antibody drug conjugates (ADCs) show considerable promise in hgNECs, novel strategies to target the heterogenous resistant disease are needed. AXL, a TAM family receptor tyrosine kinase, is known to mediate resistance to chemotherapy, radiation and targeted therapies in SCLC and other cancers, through its roles in epithelial to mesenchymal transition (EMT), DNA damage repair and replication stress tolerance. We hypothesize that targeting cell surface AXL would provide an effective therapeutic strategy in hgNECs, especially for relapsed disease. We analyzed the transcriptomic (bulk and single-cell RNA sequencing) and proteomic (immunohistochemistry) expression profiles of AXL in treatment-naïve and relapsed SCLC, LCNEC and other extrapulmonary hgNECs patient tumors. High levels of AXL expression were seen in distinct tumor subsets. Notably, AXL-high tumors exhibited molecular signatures associated with resistance (higher EMT scores, lower replication stress and cisplatin response). To evaluate AXL as a viable surface target in hgNECs, we generated a preclinical CAR T cell against AXL. Anti-AXL CAR T cells were effective at inducing cytotoxicity in AXL-expressing SCLC and LCNEC cell lines. Similarly, an anti-AXL ADC also showed selective and potent cytotoxicity in AXL-positive models. These findings establish AXL as both a valuable biomarker and a promising target for the drug-resistant hgNECs, supporting the development of AXL-directed therapeutics such as anti-AXL CAR T cells and anti-AXL ADCs. Kavya Ramkumar, C. Allison Stewart, Yan Yang, Bingnan Zhang, Qi Wang, Yuanxin Xi, Runsheng Wang, Alejandra G. Serrano, Luisa M. Solis Soto, Jing Wang, John V. Heymach, Carl M. Gay, Lauren A. Byers. AXL-directed surface targeting approaches in recalcitrant pulmonary high-grade neuroendocrine carcinomas [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 2931.

KRAS mutations frequently co-occur with alterations in STK11/LKB1 and/or KEAP1, defining an aggressive subset of lung cancers resistant to immuno- and chemotherapy. While LKB1 loss is associated with vulnerability to DNA damage response-based therapies, the impact of KEAP1 alterations remains unknown. We demonstrate that KEAP1-NRF2 pathway drives a compensatory modulation of ATR-CHK1 signaling, enhancing vulnerability to ATR inhibitors (ATRi), particularly in the setting of increased replication stress associated with LKB1 loss. ATRi shows enhanced anti-tumor activity in LKB1 and/or KEAP1-deficient non-small cell lung cancer (NSCLC) models and synergizes with gemcitabine. ATRi also enhances antitumor immunity and mitigates the immunosuppressed phenotype of LKB1/KEAP1-deficient tumors. In the HUDSON trial, LKB1/KEAP1-deficient NSCLC patients demonstrate enhanced benefits to the ATRi ceralasertib plus durvalumab. These findings suggest that alterations in the KEAP1-NRF2 pathway and/or LKB1 are associated with enhanced sensitivity to ATRi and could serve as biomarkers for predicting response to ATRi combination regimens.

Supplementary Figure 4 shows the combination of AZD2811NP with venetoclax is well tolerated in mice and less efficacious in SCLC models with low BCL2 expression.

A hallmark of small cell lung cancer (SCLC) is its recalcitrance to therapy. While most SCLCs respond to frontline therapy, resistance inevitably develops. Identifying phenotypes potentiating chemoresistance and immune evasion is a crucial unmet need. Previous reports have linked upregulation of the DNA damage response (DDR) machinery to chemoresistance and immune evasion across cancers. However, it is unknown if SCLCs exhibit distinct DDR phenotypes. To study SCLC DDR phenotypes, we developed a new DDR gene analysis method and applied it to SCLC clinical samples, in vitro, and in vivo model systems. We then investigated how DDR regulation is associated with SCLC biology, chemotherapy response, and tumor evolution following therapy. Using multi-omic profiling, we demonstrate that SCLC tumors cluster into three DDR phenotypes with unique molecular features. Hallmarks of these DDR clusters include differential expression of DNA repair genes, increased replication stress, and heightened G2/M cell cycle arrest. SCLCs with elevated DDR phenotypes exhibit increased neuroendocrine features and decreased “inflamed” biomarkers, both within and across SCLC subtypes. Clinical analyses demonstrated treatment naive DDR status was associated with different responses to frontline chemotherapy. Using longitudinal liquid biopsies, we found that DDR Intermediate and High tumors exhibited subtype switching and coincident emergence of heterogenous phenotypes following frontline treatment. We establish that SCLC can be classified into one of three distinct, clinically relevant DDR clusters. Our data demonstrates that DDR status plays a key role in shaping SCLC phenotypes and may be associated with different chemotherapy responses and patterns of tumor evolution. Future work targeting DDR specific phenotypes will be instrumental in improving patient outcomes.

Given the aggressive nature of small cell lung cancer (SCLC) and the frequent relapse after initial chemotherapy, this work aims to identify novel approaches to enhance the immune response. DNA-dependent protein kinase (DNA-PK) is a targetable enzyme involved in non-homologous end joining (NHEJ) and antiviral responses. DNA-PK plays a role during cGAS/STING dependent and independent viral DNA sensing pathway. Thus, we investigated the role of DNA-PK inhibition (DNA-PKi) as a maintenance strategy following cisplatin treatment in SCLC patients, focusing on its ability to enhance antitumor immune responses. We included different models of SCLC, in particular SCLC patient-derived peripheral blood mononuclear cells (PBMCs), SCLC cell lines and cocultures of PBMCs with SCLC cells and SCLC tumor spheroids. SCLC cells were treated in vitro with low-dose cisplatin (0.5 µM), and blood was collected from SCLC patients on day 3 of cisplatin treatment. SCLC cells and PBMCs were subjected to transcriptomic sequencing and gene expression profiling to assess the effect of DNA-PKi on PBMC subsets (CD8+ T cells, NK cells and monocytes). Gene set enrichment analysis (GSEA) of RNA-seq data revealed that cisplatin treatment enriched several DDR pathways and upregulated immune-related pathways in both SCLC cell lines and patient-derived PBMCs. Sequential treatment with DNA-PKi after cisplatin significantly activated CD8+ T-cell and NK-cell pathways, as well as enhancing interferon-related pathways and gene signatures associated with viral infections. In vitro treatment with DNA-PKi resulted in increased expression of pro-inflammatory markers in PBMCs, promoting M1 polarization of macrophages (TNF-α, IL-6 and CXCL10) while inhibiting M2 polarization (IL-10, IL4 and CCL22). This shift towards an anti-tumor phenotype was confirmed by enhanced NK cell-mediated cytotoxicity against SCLC cell lines. In addition, combination therapy with DNA-PKi and anti-PD-L1 resulted in increased immune cell infiltration into tumor spheroids, demonstrating a synergistic effect on anti-tumor immunity. In conclusion, our data suggests that DNA-PKi may improve immune responsiveness and potentially prolong antitumor effects in SCLC patients. Future investigations are warranted to further elucidate these mechanisms and to evaluate the clinical applicability of DNA-PKi in combination with existing therapies for SCLC. Luisa Amato, Caterina De Rosa, Concetta Tuccillo, Francesca Iommelli, Viviana De Rosa, Virginia Tirino, Federica Papaccio, Kavya Ramkumar, Qi Wang, Jing Wang, Alberto Servetto, Floriana Morgillo, Lauren Averett Byers, Fortunato Ciardiello, Carminia Maria Della Corte. Efficacy of DNA-PK inhibitor as maintenance strategy after cisplatin induction in SCLC [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 2895.