Response of parental, AfaR, and ErlR models to AURKAi combinations with erlotinib or afatinib. A, Loewe synergy plot showing synergistic interaction between VIC-1911 and afatinib or erlotinib in FaDu and Cal27 parental, ErlR, and AfaR cell lines treated for 72 hours, n = 3. B, Quantification of dead cells from the models indicated treated for 72 hours with drugs indicated. FaDu and derived models were treated with vehicle, VIC-1911 (0.6 μmol/L), afatinib (0.2 μmol/L), erlotinib (1 μmol/L), or combination. Cal27 and derived models were treated with vehicle, VIC-1911 (0.6 μmol/L), afatinib (0.8 μmol/L), erlotinib (2 μmol/L), or combination for 24 hours. C and D, Clonogenic survival assay primary data (C) and corresponding (D) quantification. Cells were treated with the drug concentration indicated in C for 11 days. E, Quantification of annexin-positive cells for Fadu parental and Fadu AfaR treated for 3 days (left) and 5 days (right) with VIC-1911 (600 nmol/L). P values are based on one-way ANOVA followed by the Tukey multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Data are shown as mean ± SEM of three biological replicates.

In vivo response of FaDu parental and FaduAfaR xenografts to VIC-1911 + adavosertib drug combination. A, Mice harboring either FaDu parental or FaDuAfaR xenograft tumors were daily treated with vehicle (n = 8 for parental, n = 5 for AfaR) or VIC-1911 60 mg/kg (n = 8 for parental, n = 5 for AfaR) for 12 days to compare response to monotherapy. B and C, Mice harboring FaDu parental (B) or FaduAfaR (C) xenografts were daily treated with vehicle (n = 7), adavosertib 120 mg/kg (n = 7), VIC-1911 30 mg/kg (n = 8), or combination (n = 8) for 21 days. P values are based on two-way ANOVA followed by the Tukey multiple comparison test. D and E, Representative IHC staining images (D) and quantification (E) of Ki-67 and cleaved caspase 3 in FaDu parental or FaDuAfaR xenograft tumors treated with the indicated drugs. Mice harboring FaDu parental or FaDuAfaR xenografts were daily treated with vehicle (n = 7), adavosertib 120 mg/kg (n = 7), VIC-1911 30 mg/kg (n = 8), or combination (n = 8) for 21 days. Scale bars, 300 μm. Violin plots display median (-) and quartiles (…). P values are based on two-way ANOVA followed by the Tukey multiple comparison test. Only significant comparisons are displayed in the violin plots. P = 0.0332; **, P = 0.0021; ***, P = 0.0002; ****, P ≤ 0.0001.

Response of parental, AfaR, and ErlR models to AURKAi monotherapy. A, Representative Western blot images showing baseline activation and total levels of AURKA. B, Quantification of immunoprecipitation assay probing for phospho-AURKA (T288) and total AURKA protein levels. C, Representative Western blot images of TPX2 and NEDD9. D, Western blot quantification for baseline levels of TPX2 and NEDD9, with antibodies to total protein. E, Dose–response curves for parental, ErlR, and AfaR cell models treated with the indicated dose range of VIC-1911 for 72 hours. F, Fadu/Cal27 parental and derived resistant models were treated with vehicle and VIC-1911 (0.6 μmol/L) for 24 hours. Protein level quantification for phospho-EGFR (y1068) was relative to total EGFR, phospho-AKT (S473) relative to total AKT, phospho-ERK (Y204) relative to total ERK, total AURKA, TPX2, and BimEL relative to the loading control. Bar graphs display data normalized to each parental cell line. All graphs were quantified from at least three independent biological repeats. P values are based on one-way ANOVA followed by Tukey multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Data are shown as mean ± SEM of three biological replicates.

Non-small-cell lung cancer (NSCLC) is responsible for the majority of cancer-related mortality worldwide. Lung adenocarcinoma (LUAD) is the most common NSCLC subtype. Despite advances in targeted therapies, treatment resistance remains a critical challenge. Ribonucleotide reductase (RNR), a crucial enzyme in deoxyribonucleotide triphosphate (dNTP) biosynthesis, is frequently upregulated in cancer, contributing to genomic instability and poor prognosis in multiple malignancies. However, the role of the RNR complex in driving tumorigenesis is not fully understood in oncogenic-driven LUAD. Transcriptomic analysis of more than 27,000 real-world NSCLC patient samples revealed that RNR subunits (RRM1 and RRM2) are significantly upregulated in TP53 mutated NSCLC and correlated with significantly poor prognosis in multiple oncogene-driven LUAD. Using pharmacologic and genetic approaches to inhibit RNR in LUAD models, we assessed functional consequences through molecular, biochemical, and imaging techniques. RNR inhibition induced appreciable replication stress and triggered DNA damage, leading to cell death in LUAD cells. Notably, we uncovered that RNR suppression preferentially induced ferroptosis, an iron-dependent cell death driven by lipid peroxidation. This represents a previously unrecognized mechanism of RNR-mediated cell death by which mutant LUAD cells can be selectively targeted. Our study establishes RNR inhibition as a potent strategy to selectively induce ferroptosis in oncogenic addicted LUAD, offering a new therapeutic avenue for genetically defined patient subgroups. Targeting nucleotide metabolism could serve as an effective approach to overcome treatment resistance and improve clinical outcomes for patients with high-risk LUAD.

e20721 Background: Pts with NSCLC bearing uncommon EGFR alterations experienced inferior survival with tyrosine kinase inhibitors (TKIs) vs. those with sensitizing exon 19 deletions or L858R in prior studies. While TP53 co-mutations are well-recognized to be associated with poorer outcomes in sensitizing EGFR-mutant NSCLC, no research to our knowledge has examined the contribution of this adverse co-mutation to the inferior survival observed in pts with uncommon EGFR alterations receiving front-line TKIs. Methods: The Flatiron Health-Foundation Medicine clinical-genomic database (~800 sites of care) was used to evaluate pts (N = 108) with advanced NSCLC (Stage IIIB/IV) bearing uncommon EGFR alterations receiving front-line osimertinib or afatinib from 6/2014 – 9/2024. Kaplan-Meier methods (accounted for left truncation) estimated real-world progression-free survival (rwPFS) and overall survival (OS) in months (m). Comparisons by presence of TP53 co-mutations vs. wild-type were assessed using multivariable Cox regression after adjusting for the covariates gender, age, smoking history, performance status, presence of brain and liver metastases, EGFR subtype (e.g. G917X, L861Q, compound), and TKI received. Results: Of the 108 pts, 30 (27.8%) had G719X, 30 (27.8%) had L861Q, 3 (2.8%) had S768I, 3 (2.8%) had E709X, 2 (1.9%) had L747X, and 40 (37.0%) had compound mutations made-up of ˃ 1 EGFR alteration. Seventy-six (70.4%) pts had a TP53 co-mutation, which is higher than the rate reported for pts with sensitizing EGFR-mutant NSCLC in prior studies. Sixty-nine pts (63.9%) received osimertinib and 39 (36.1%) received afatinib as a front-line TKI. Pts with TP53 co-mutations had a shorter median rwPFS (5.9 m vs. 15.7 m, HR 1.9, 95% CI 1.1-3.3, P = 0.017) and OS (11.0 m vs. 24.7 m, HR 2.4, 95% CI 1.4-4.4, P = 0.003) with front-line TKIs as compared to wildtype pts after adjusting for relevant covariates. In our multivariable model, the TKI received (i.e. osimertinib vs. afatinib) did not alter survival. However, having L861Q correlated with a poorer OS (HR 2.5, 95% CI 1.3-4.7, P = 0.006) and having G719X predicted inferior rwPFS (HR 2.5, 95% CI 1.3-4.5, P = 0.004) and OS (HR 2.3, 95% CI 1.2-4.5, P = 0.016) as compared to pts with compound EGFR mutations. Conclusions: This is the first study to demonstrate that the occurrence of a TP53 co-mutation is a significant predictor of inferior rwPFS and OS in pts with NSCLC bearing uncommon EGFR alterations. Although conventional thought is that pts with uncommon EGFR alterations derive less benefit from TKIs, our analysis suggests TP53-wildtype pts experience survival comparable to that of pts with sensitizing EGFR alterations in the real-world. Pts possessing uncommon EGFR alterations with a TP53 co-mutation critically need more effective therapies. Emerging strategies that target mutant p53 may benefit this high-risk subgroup.

8538 Background: Class I BRAF mutant ( BRAF mut) non–small cell lung cancer (NSCLC) is biologically heterogeneous, occurring in both smokers and never-smokers with disparate benefits from immunotherapy (IO). Genomically defined tobacco-induced damage may better identify biologically and clinically distinct subsets than self-reported smoking history. We evaluated COSMIC mutational signature-SBS4 as a genomic surrogate of smoking exposure and examined its association with molecular features, tumor microenvironment (TME) and outcomes in BRAF mut NSCLC. Methods: Retrospective review of 33,217 NSCLC specimens that underwent whole exome and whole transcriptome sequencing at Caris Life Sciences. Mutation profiles of specimens were deconvolved using the COSMIC SBS4 signature to estimate tobacco-associated mutational exposure (filter: total mutation count>=200, Nfiltered=26448; BRAF mut = 276). TME was estimated using QuanTIseq method. Overall survival (OS) and survival on IO (IO-OS) were obtained from insurance claims and calculated from date of tumor biopsy (for OS) or initiation of IO (for IO-OS) to last contact using Kaplan-Meier estimates and Cox proportional hazards models. Statistical significance was determined by Fishers Exact, chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons ( P <0.05). Results: Among 6,405 patients with smoking history and SBS4 data, SBS4+ (SBS4>0) was strongly associated with smoking (OR 11.2, P <0.001). SBS4+ tumors exhibited elevated TMB (mean:13 vs 9 mut/Mb, and TMB-High [>=10 mut/Mb], P <0.05, Table). SBS4+ BRAF mut tumors had a lower prevalence of mutations in SETD2 (OR 0.33), PIK3CA (OR 0.26) and SMAD4 (OR 0.25, all P <0.05). Regardless of SBS4 status, BRAF mut tumors were more often PD-L1+ (OR 3.2, TPS>=1), while mutations in STK11, KEAP1 , and SMARCA4 were less frequent (OR 0.07-0.37, all P <0.05). Evaluation of the TME revealed that SBS4+ BRAF mut were enriched for regulatory T cells (vs. SBS4-,1.44 fold, P <0.05). In metastatic disease, BRAF mut showed improved OS (HR 0.8[0.66-0.97], P =0.03) and IO-OS (HR 0.8[0.66-0.99], P =0.04) compared to WT. The OS benefit was preserved in the SBS4+ tumors (HR 0.64[0.43-0.95], P =0.03), but not in SBS4- tumors. No difference in IO-OS was observed in SBS4+ subgroups likely due to small size. Conclusions: SBS4 identifies biologically distinct subsets in class I BRAF mut NSCLC, with differences in mutational landscape, TMB, and TME. SBS4+ tumors show a survival benefit over WT disease, whereas SBS4- tumors do not. These findings support SBS4 as a genomic marker of smoking-related biology and a potential tool to refine therapeutic decision-making between targeted therapy and IO in NSCLC and potentially other smoking-associated cancers. Smoking signature in BRAF mut NSCLC (% prevalence and OS). Characteristics SBS4 + SBS4 - SETD2 20 43 PIK3CA 6 19 SMAD4 3 12 TMB-high 41 18 OS (months) 29.9 vs. 11.9 ( P =0.03) 16.0 vs. 10.9 ( P =0.47)

Cell cycle and signaling changes induced by treatment with VIC-1911 and adavosertib in parental, AfaR, and ErlR HNSCC models.

Characterization of erlotinib-resistant (ErlR) and afatinib-resistant (AfaR) HNSCC cell models.

Signaling changes induced by treatment with VIC-1911 and adavosertib in parental, AfaR, and ErlR HNSCC models. A–E, Representative Western blots and quantification for phospho-CDK1 (Y15) and total-CDK1 (A), γH2AX (S139; B), TPX2 (C), total AURKA (D), and NEDD9 (E) in FaDu parental, ErlR, and AfaR cell models treated with DMSO, VIC-1911 (0.25 μmol/L), adavosertib (0.5 μmol/L), or combination for 24 hours. A and B share the same loading control, as they were derived from the same gel. Bar graphs display data normalized to the DMSO of each cell line. P values are based on one-way ANOVA followed by the Tukey multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Data are shown as mean ± SEM of three biological replicates.

Importance:For patients with advanced non-small cell lung cancer (NSCLC) and programmed cell death 1 ligand 1 (PD-L1) expression of 50% or higher, programmed cell death 1 protein or PD-L1 (PD-[L]1) inhibitor monotherapy is commonly used as first-line therapy; however, whether adding chemotherapy improves outcomes in this population remains unknown. Objective:To compare overall survival (OS) and progression-free survival (PFS) associated with PD-(L)1 inhibitor monotherapy vs chemoimmunotherapy in treatment-naive patients with advanced NSCLC and high PD-L1 expression. Data Sources:PubMed, Embase, and major oncology conference proceedings were searched for phase 3 randomized clinical trials (RCTs) published before August 3, 2025. Study Selection:Eligible studies were phase 3 RCTs that enrolled patients with untreated advanced NSCLC, evaluated PD-(L)1 inhibitor monotherapy or chemoimmunotherapy vs chemotherapy alone, and reported outcomes in patients with high PD-L1 expression. Data Extraction and Synthesis:Hazard ratios (HRs) for OS and PFS were extracted from published studies and synthesized using inverse variance methods. Additional analyses included meta-regression, network meta-analysis, and reconstructed individual patient data from published Kaplan-Meier curves. Main Outcomes and Measures:Primary outcome was OS; secondary outcome was PFS. Results:Among 24 trials including 5546 patients with PD-L1-high NSCLC, 16 evaluated chemoimmunotherapy and 8 PD-(L)1 inhibitor monotherapy. Compared with chemotherapy, survival was improved by both chemoimmunotherapy (OS: HR, 0.63 [95% CI, 0.56-0.72]; P < .001; PFS: HR, 0.44 [95% CI, 0.39-0.49]; P < .001) and PD-(L)1 inhibitor monotherapy (OS: HR, 0.74 [95% CI, 0.69-0.80]; P < .001; PFS: HR, 0.70 [95% CI, 0.65-0.76]; P < .001). Tests for subgroup differences suggested improved benefit with chemoimmunotherapy compared to PD-(L)1 inhibitor monotherapy (OS: χ21 = 4.1; P = .04; I2 = 75.8%; PFS: χ21 = 48.1; P < .001; I2 = 97.9%), consistent with meta-regression analyses (OS: HR, 0.85 [95% CI, 0.72-1.00]; P = .048; PFS: HR, 0.61 [95% CI, 0.50-0.75]; P < .001) and network meta-analyses (OS: HR, 0.85 [95% CI, 0.73-0.99]; PFS: HR, 0.61 [95% CI, 0.50-0.75]). In the reconstructed individual patient data analysis, median OS was longer with chemoimmunotherapy (n = 704 patients) compared to PD-(L)1 inhibitor monotherapy (n = 1706 patients) (29.2 months [95% CI, 25.2-35.4] vs 19.8 months [95% CI, 18.3-21.7]; HR, 0.74 [95% CI, 0.66-0.82]; P < .001). Similarly, median PFS was significantly longer with chemoimmunotherapy (n = 701 patients) compared to PD-(L)1 inhibitor monotherapy (n = 1706 patients) (11.3 months [95% CI, 10.3-13.5] vs 6.8 months [95% CI, 6.2-7.1]; HR, 0.67 [95% CI, 0.60-0.75]; P < .001). Conclusions and Relevance:In this meta-analysis of phase 3 RCTs, chemoimmunotherapy was associated with significantly improved OS and PFS compared with PD-(L)1 inhibitor monotherapy in patients with advanced NSCLC and high PD-L1 expression. Prospective trials are needed to confirm these findings.

Differential expression analysis table deseq2

Acetate serves as an alternative carbon source in nutrient-limited tumors, yet its role in supporting nucleotide biosynthesis remains poorly understood. Here, we identify the mitochondrial enzyme ACSS1 as a key metabolic driver in mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). ACSS1 is frequently overexpressed and catalyzes the conversion of acetate to mitochondrial acetyl-CoA, sustaining oxidative metabolism and biosynthesis under nutrient stress. Genetic silencing of ACSS1 impairs mitochondrial respiration and disrupts acetate incorporation into acetyl-CoA, TCA cycle intermediates, glutamate, and aspartate, while markedly reducing 13C-acetate labeling of dihydroorotate and orotate, intermediates in de novo pyrimidine synthesis. Untargeted metabolomics reveal enrichment of pyrimidine biosynthesis pathways in ACSS1-high cells. Notably, acetate or uridine supplementation rescues the growth of ACSS1-deficient cells, confirming a functional link between acetate metabolism and nucleotide synthesis. Importantly, in vivo studies using two different MCL xenografts demonstrate that ACSS1 knockdown profoundly suppresses tumor growth, indicating that ACSS1 is required not only for metabolic adaptation of lymphoma cells in vitro but also in vivo. Collectively, our results uncover an ACSS1-dependent mitochondrial acetate-pyrimidine axis that sustains lymphoma growth and represents a previously unrecognized therapeutic vulnerability.

3121 Background: HGNECs, including small-cell (SC) and large-cell (LCNEC), are aggressive malignancies frequently misclassified due to subtle morphology and inconsistent use of neuroendocrine (NE) immunohistochemistry in routine pathology workflows. It is unclear if tumors with transcriptomic signatures reflecting HGNEC-like biology have inferior outcomes with conventional treatment. Leveraging a large real-world dataset, this study aims to identify transcriptomically defined HGNEC to improve upon morphology-based classification. Methods: 49,144 specimens underwent DNA and RNA sequencing at Caris Life Sciences. Using a previously reported framework, we developed a weighted, z-scored based HGNEC activity score (HAS) from 10,137 pathologically defined HG- and LG- NEC specimens and applied it to characterize NSCLC samples (without any pathologist assigned HGNEC diagnosis) further stratified by histology (n=39,007). Overall survival (OS) was obtained from insurance claims data and calculated from specimen biopsy to last contact using Kaplan-Meier and Cox proportional hazards estimates. Results: To identify a high-confidence HGNEC-like subpopulation, a HAS threshold (z-score>=3) was applied, classifying 0.2% squamous (sq) and 2.3% non-squamous (nsq) as HGNEC-like; this analysis is focused in nsq. Patient demographics, including age, gender, race, ethnicity, and smoking history, were similar between the HGNEC-like and non-HGNEC-like nsq cohorts. However, biopsies from lymph nodes (26 vs 11%) and brain (9.1 vs 5.6%) were more frequent, and lung (39 vs 56%) less frequent in HGNEC-like tumors (all p-adj<0.05). HGNEC-like tumors were associated with poor OS (HR:1.18, 95% CI:1.07–1.31, p<0.001). HGNEC-like tumors were more frequently PD-L1 negative (64% vs 47%), and were enriched for mutations in TP53 , PTEN and RB1 (OR: 1.7-14.1), as well as amplifications in MYC and CCNE (OR: 2.2-3.5), with lower odds of KRAS and EGFR mutations (OR:0.4-0.5, p-adj<0.05). Consistent with a highly proliferative phenotype, HGNEC-like tumors were enriched for MKI67 expression (1.4-fold) and higher cell-cycle and replication stress scores (1.5-fold) (all p-adj<0.05) . Gene set enrichment analysis revealed significant activation of neurodevelopmental programs (NES: 2.0-3.0, all p-adj<0.05) and negative enrichment of immune pathways (e.g antigen presentation) (NES: -3.0 to -1.5, all p-adj<0.05). Finally, transcriptomic expression of potentially actionable SCLC-directed targets, including DLL3 (10-fold), SEZ6 (3.7-fold), and SSTR2 (10-fold), were all higher in HGNEC-like tumors (all p-adj<0.05). Conclusions: We identify a transcriptomically defined HGNEC-like subset of NSCLC with inferior survival and targetable NEC biology, supporting transcriptomic stratification to inform clinical trial design and selection of SCLC-directed therapies.

Metabolic Stability in Liver Microsomes

Signaling consequences in Cal27 parental, AfaR, or ErlR cells of treatment with VIC-1911 alone or in combination with erlotinib or afatinib

Characterization of baselines and biological response to AURKA inhibition in parental, AfaR, and ErlR HNSCC cell lines.

Importance Distinguishing primary lung squamous cell carcinoma (SCC) from squamous metastases to the lung is a clinical challenge due to histopathologic similarities. Accurate diagnosis is essential to guide treatment decisions. Objective To assess the utility of an artificial intelligence (AI) approach that includes evaluation of key orthogonal evidence in distinguishing primary lung SCCs from metastatic tumors of other tissue origins. Design, Setting, and Participants This cross-sectional study used GPSai, a tissue-of-origin AI model run automatically on each sample submitted for molecular profiling, to flag potential misdiagnoses among research-eligible cases submitted as lung SCC. Molecularly profiled cases within the Caris Life Sciences clinicogenomic database from January 1, 2024, to January 31, 2025, were queried. All cases were reviewed by board-certified pathologists. Main Outcomes and Measures The primary outcome was the rate of misdiagnosis among presumed lung SCCs confirmed by pathologist review and orthogonal evidence, which included clinical history and clinical findings, GATA3 and uroplakin II immunohistochemistry for urothelial carcinoma, UV variant signature for cutaneous SCC, CD5 and CD117 (c-KIT) immunohistochemistry for thymic carcinoma, and human papillomavirus positivity for orogenital SCC (eg, head and neck, cervical). Results Through a combination of AI and orthogonal evidence, 123 (3.1%) misdiagnoses were confirmed among 3958 cases submitted as presumed lung SCC (patients misdiagnosed: median [range] age, 71 [39 to >89]; 76.4% male). The cohort included 50 cutaneous SCCs (40.7%), 33 orogenital SCCs (26.8%) (including 25 head and neck [75.8%]), 20 urothelial carcinomas (16.3%), 15 thymic carcinomas (12.2%), 4 NUT carcinomas (3.3%), and 1 prostate SCC (0.8%). Ninety-two of the 123 patients (74.8%) had clinical history or findings consistent with the new diagnosis. Eighty-eight cases (71.5%) had differences in guideline-preferred first-line systemic therapies following the diagnosis change. Conclusions and Relevance In this cross-sectional study of patients diagnosed with lung SCC, a meaningful number of patients experienced misdiagnosis, which was identified using a multipronged AI-assisted approach. Diagnosis changes prompted by AI and orthogonal evidence may assist clinicians in prognostication and therapy selection.