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PURPOSE Although level 1 evidence supports 45-Gy twice-daily radiotherapy as standard for limited-stage small-cell lung cancer, most patients receive higher-dose once-daily regimens in clinical practice. Whether increasing radiotherapy dose improves outcomes remains to be prospectively demonstrated. METHODS This phase III trial, CALGB 30610/RTOG 0538 (ClinicalTrials.gov identifier: NCT00632853 ), was conducted in two stages. In the first stage, patients with limited-stage disease were randomly assigned to receive 45-Gy twice-daily, 70-Gy once-daily, or 61.2-Gy concomitant-boost radiotherapy, starting with either the first or second (of four total) chemotherapy cycles. In the second stage, allocation to the 61.2-Gy arm was discontinued following planned interim toxicity analysis, and the study continued with two remaining arms. The primary end point was overall survival (OS) in the intention-to-treat population. RESULTS Trial accrual opened on March 15, 2008, and closed on December 1, 2019. All patients randomly assigned to 45-Gy twice-daily (n = 313) or 70-Gy once-daily radiotherapy (n = 325) are included in this analysis. After a median follow-up of 4.7 years, OS was not improved on the once-daily arm (hazard ratio for death, 0.94; 95% CI, 0.76 to 1.17; P = .594). Median survival is 28.5 months for twice-daily treatment, and 30.1 months for once-daily treatment, with 5-year OS of 29% and 32%, respectively. Treatment was tolerable, and the frequency of severe adverse events, including esophageal and pulmonary toxicity, was similar on both arms. CONCLUSION Although 45-Gy twice-daily radiotherapy remains the standard of care, this study provides the most robust information available to help guide the choice of thoracic radiotherapy regimen for patients with limited-stage small-cell lung cancer.

Supplementary Figure S1. Talazoparib inhibition of PBMC PARP activity.

Supplementary Methods includes additional details on these topics: tumors eligible for dose escalation phase; tumors eligible for dose expansion phase; patients enrolled using a local laboratory; study design and participants; additional inclusion criteria; full list of exclusion criteria; study treatment; study procedures; pharmacodynamic (PD) analysis; inhibition of PBMC PARP activity.

Supplementary Table S1. Dose-response and exposure-response parameters.

This file contains data showing perturbations in the S phase progression of UMSCC1 cells following prexasertib treatment. This file is linked to main figure 2.

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PDF file - 136KB, Supplementary Table 1. Sumary of the clinicopathological characteristics of the patients with lung adecarcinomas examined for EZH2 and miR-101 expressions. Supplementary Table 2. Association between markers' expression and lung adenocarcinoma patient's clinical and pathological characteristics. Supplementary Table 3. Summary of Multivariate Analysis of Outcome in Lung Adenocarcinoma Patients by Adjuvant Chemoterapy and EZH2, miR-101 and Combination of EZH2/miR-101 Expression.

Purpose: To investigate the mechanisms of regulation and role associated with enhancer of zeste homolog 2 (EZH2) expression in lung cancer cells.Experimental Design: We investigated the mechanisms of EZH2 expression associated with the VEGF/VEGFR-2 pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. In addition, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients' clinical characteristics.Results: In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and hypoxia-inducible factor-1α (HIF1α), and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. In addition, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti–VEGFR-2 drug AZD2171.Conclusion: Our results suggest that the VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF1α, and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy. Clin Cancer Res; 20(14); 3849–61. ©2014 AACR.

Purpose:Primary analysis of VISION showed tepotinib had durable clinical activity in patients with MET exon 14 (METex14) skipping non–small cell lung cancer (NSCLC). We present updated outcomes for clinically relevant subgroups.Patients and Methods:This phase II, open-label, multi-cohort study of 500 mg (450 mg active moiety) tepotinib in patients with METex14 skipping NSCLC assessed efficacy and safety in predefined subgroups according to age, prior therapies (chemotherapy and immune checkpoint inhibitors), and brain metastases. An ad hoc retrospective analysis using Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) criteria assessed intracranial activity.Results:152 patients were evaluable for efficacy (median age: 73.1). Overall, objective response rate (ORR) was 44.7% [95% confidence interval (CI): 36.7–53.0]. Patients aged n = 84) and ≥75 (n = 68) had ORRs of 48.8% (95% CI: 37.7–60.0) and 39.7% (95% CI: 28.0–52.3), respectively. Treatment-naïve (n = 69) versus previously treated (n = 83) patients showed consistent efficacy [ORR (95% CI): 44.9% (32.9–57.4) vs. 44.6% (33.7–55.9); median duration of response (95% CI): 10.8 (6.9–not estimable) vs. 11.1 (9.5–18.5) months]. Of 15 patients analyzed by RANO-BM (12 received prior radiotherapy), 13 achieved intracranial disease control; 5 of 7 patients with measurable brain metastases had partial intracranial responses. Of 255 patients evaluable for safety, 64 (25.1%) experienced grade ≥3 treatment-related adverse events (TRAE), leading to discontinuation in 27 patients (10.6%). Rates of adverse events (AE) were broadly consistent irrespective of prior therapies.Conclusions:Tepotinib showed meaningful activity across subgroups by age, prior therapies, and brain metastases, with a manageable safety profile and few treatment discontinuations.See related commentary by Rosner and Spira, p. 1055

Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy.Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781–97. ©2017 AACR.

Supplementary Table S1.

Supplementary Data from Tepotinib Efficacy and Safety in Patients with MET Exon 14 Skipping NSCLC: Outcomes in Patient Subgroups from the VISION Study with Relevance for Clinical Practice

PDF file - 453KB, Figure 1. EZH2, VEGFR-2 and miR-101 expression in in additional cell lines. Figure 2. Effect of VEGF stimulation in the E2F expression. Figure 3. Effect of Hypoxia in the expression of EZH2,HIF-1alpha and miR-101. Figure 4. Effect of miR-101 overexpression in the expression of EZH2. Figure 5. Effect of treatment with DZNep on lung adenocarcinoma cell viability. Figure 6. Effect of VEGFR-2 overexpression in sensitivity to cisplatin.

Purpose: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC).Experimental Design: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data.Results: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo.Conclusion: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted. Clin Cancer Res; 17(7); 1815–27. ©2011 AACR.

Supplementary Figure Legends 1-3 from Control of HIF-1α Expression by eIF2α Phosphorylation–Mediated Translational Repression

Supplementary Figure 1 from Control of HIF-1α Expression by eIF2α Phosphorylation–Mediated Translational Repression

Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non–small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA–mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor–resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer. Cancer Res; 70(16); 6477–85. ©2010 AACR.

Hypoxia inducible factor 1α (HIF-1α) plays a central role in regulating tumor angiogenesis via its effects on vascular endothelial growth factor (VEGF) transcription, and its expression is regulated through proteasome-mediated degradation. Paradoxically, previous studies have shown that proteasome inhibitors (PI) block tumor angiogensis by reducing VEGF expression, but the mechanisms have not been identified. Here, we report that PIs down-regulated HIF-1α protein levels and blocked HIF-1α transcriptional activity in human prostate cancer cells. PIs induced phosphorylation of the translation initiation factor 2α (eIF2α), which caused general translational repression to inhibit HIF-1α expression. Furthermore, PIs induced HIF-1α accumulation in LNCaP-Pro5 cells depleted of eIF2α via siRNA transfection and in MEFs expressing a phosphorylation-deficient mutant form of eIF2α. Finally, PIs failed to induce eIF2α phosphorylation or translational attenuation in DU145 or 253JB-V cells, and, in these cells, PIs promoted HIF-1α accumulation. Our data established that PIs down-regulated HIF-1α expression in cells that display activation of the unfolded protein response by stimulating phosphorylation of eIF2α and inhibiting HIF-1α translation. [Cancer Res 2009;69(5):1836–43]

Supplementary Figures 1-3, Tables 1-3 from Steroid Receptor Coactivator-3 Expression in Lung Cancer and Its Role in the Regulation of Cancer Cell Survival and Proliferation

Supplementary Figure 2 from Control of HIF-1α Expression by eIF2α Phosphorylation–Mediated Translational Repression

PURPOSE In head and neck squamous cell carcinoma (HNSCC), HRAS mutation is a new actionable oncogene driver. We aimed to evaluate HRAS mutational variants, comutation profile, and survival outcomes of this molecularly defined population. METHODS We leveraged four deidentified patient data sets with HRAS-mutant HNSCC, MD Anderson Cancer Center, Kura Oncology, Inc trial, Foundation Medicine, and American Association for Cancer Research GENIE v.12. Patient demographic information and clinical courses were extracted, when available, in addition to HRAS mutation type and co-occurring mutations. Survival outcomes were analyzed (Kaplan-Meier method). RESULTS Two hundred forty-nine patients with HRAS-mutant HNSCC were identified from the four data sets. Median age ranged from 55 to 65 years, with a higher frequency in male patients (64%); the majority of HRAS-mutant HNSCC occurred in human papillomavirus–negative HNSCC. HRAS mutation patterns were similar across data sets; G12S was the most common (29%). Treatment responses to tipifarnib were not codon-specific. Compared with wild-type, significantly co-occurring mutations with HRAS were Casp8 (Fisher's exact test, P < .00013), TERT ( P < .0085), and NOTCH1 ( P < .00013). Analysis of clinical courses from the MD Anderson Cancer Center and Kura Oncology, Inc data sets demonstrated poor clinical outcomes with a high rate of recurrence following primary definitive treatment (50%-67% relapse < 6 months) and short disease-free survival (4.0 months; 95% CI, 1.0 to 36.0) and overall survival (OS; 15.0 months; 95% CI, 6.0 to 52.0). Use of tipifarnib in this data set demonstrated improved OS (25.5 months; 95% CI, 18.0 to 48.0). CONCLUSION Oncogenic mutations in HRAS occur in 3%-4% of HNSCC, with G12S being the most frequent. Without targeted therapy, patients with HRAS-mutant HNSCC had poor clinic outcomes; observable trend toward improvement in OS has been noted in cohorts receiving treatments such as tipifarnib. The comutation pattern of HRAS-mutant in HNSCC is distinct, which may provide insight to future therapeutic combination strategies.

BI 1810631 is a human HER2-selective tyrosine kinase inhibitor that covalently binds to both wild-type and mutated HER2 receptors, including exon 20 insertion mutations, whilst sparing EGFR signaling. This phase Ia/Ib, open-label, non-randomized study will determine the safety, maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics, and preliminary efficacy of BI 1810631 in patients with HER2 aberration-positive solid tumors (NCT04886804).In phase Ia, patients with histologically/cytologically confirmed HER2 aberration-positive advanced/metastatic solid tumors will receive BI 1810631 orally twice daily (BID) or once daily (QD) at escalating doses. Starting dose level is 15 mg BID; QD schedule will begin after one dose level above estimated therapeutic dose of BI 1810631 is determined safe by the Dose Escalation Committee. Dose escalation will continue until MTD/recommended phase II dose and preferred phase Ib schedule for each schedule is determined. In phase Ib, patients with HER2 tyrosine kinase domain (TKD) mutation-positive non-small cell lung cancer (NSCLC) who have previously received ≥1 line of systemic therapy will be enrolled initially, with possible inclusion of additional NSCLC cohorts in the future, including untreated patients. The primary endpoints will be MTD based on number of dose-limiting toxicities (DLTs)/number of patients with DLTs (phase Ia) and objective response (phase Ib). Secondary endpoints include PK parameters (phase Ia/Ib); duration of response, disease control, duration of disease control, and progression-free survival (phase Ib).BI 1810631 could be an effective and tolerable EGFR-sparing oral treatment for patients with HER2 mutation-positive NSCLC, including exon 20 insertion mutations.NCT04886804.

Background Study of the geographic distribution of cellular populations and their interaction with malignant cells in non-small cell lung cancer (NSCLC) is essential to understand the roles of cellular populations and potentially design new therapeutic approaches. Material and Methods We studied 225 formalin-fixed, paraffin-embedded tumor tissue samples from patients with stage I-III NSCLC—142 adenocarcinomas and 83 squamous cell carcinomas—placed in tissue microarrays. Twenty-three markers were used, including T-cell, B-cell, immune checkpoint, and myeloid cell markers, placed in five multiplex immunofluorescence panels. We used dimensional data reduction and cellular spatial distribution to identify cell phenotypes and their cellular distribution across panels. In addition, we analyzed associations between cellular spatial distribution patterns and clinicopathologic variables, tumor mutational status, and outcomes. Results Overall, we observed two patterns of cellular distribution—unmixed and mixed—related to T-cells, B-cells, granulocytic cells, and macrophages, and we detected various cellular interactions between malignant and immunosuppressive cells and various associations between cell distribution and clinicopathologic characteristics. Cellular distribution patterns and the distance from various cell phenotypes to malignant cells were associated with recurrence-free survival and overall survival in univariate and multivariable analysis. Conclusions Diverse populations of tumor cells and tumor-associated immune cell phenotypes can be present in NSCLC samples. Spatial distribution is a new tool to better understand the tumor microenvironment and help select therapeutic interventions.

Radiation and platinum-based chemotherapy form the backbone of therapy in HPV-negative head and neck squamous cell carcinoma (HNSCC). We have correlated focal adhesion kinase (FAK/PTK2) expression with radioresistance and worse outcome in these patients. However, the importance of FAK in driving radioresistance and its effects on chemoresistance in these patients remain unclear. We performed an in vivo shRNA screen using targetable libraries to address these questions and identified FAK as an excellent target for both radio- and chemosensitization. Because TP53 is mutated in over 80% of HPV-negative HNSCC, we hypothesized that mutant TP53 may facilitate FAK-mediated therapy resistance. FAK inhibitor increased sensitivity to radiation, increased DNA damage and repressed homologous recombination and non-homologous end joining repair in mutant, but not wild-type, TP53 HPV-negative HNSCC cell lines. Mutant TP53 cisplatin-resistant cell line had increased FAK phosphorylation compared to wild-type, and FAK inhibition partially reversed cisplatin resistance. To validate these findings, we utilized a HNSCC cohort to show that FAK copy number and gene expression were associated with worse disease-free survival in mutant TP53, but not wild-type TP53, HPV-negative HNSCC tumors. Thus, FAK may represent a targetable therapeutic sensitizer linked to a known genomic marker of resistance.

Background: There is an unmet need for effective TKIs against HER2 mutations in solid tumors, particularly in NSCLC. BI 1810631 is a HER2-selective TKI that covalently binds to both wild-type and mutated HER2 receptors, including exon 20 insertions, whilst sparing EGFR signaling. This ongoing Phase Ia/Ib, open-label, non-randomized study aims to determine the safety, MTD, PK, pharmacodynamics and preliminary efficacy of BI 1810631 in pts with HER2 aberration-positive solid tumors (NCT04886804). Here, we present interim results of Phase Ia. Materials and Methods: In Phase Ia, pts with HER2 aberration-positive (overexpression, gene amplification, somatic mutation, or gene rearrangements) advanced/unresectable/metastatic solid tumors refractory/unsuitable for standard therapy were enrolled. Pts received escalating doses of BI 1810631 BID (starting dose: 15 mg) or BI 1810631 QD (starting dose: 60 mg). Phase Ib will initially include 30 pts with advanced HER2 tyrosine kinase domain mutation-positive, pre-treated NSCLC. Additional cohorts may be included in the future. Primary endpoints: MTD based on number of DLTs; number of pts with DLTs (Phase Ia); objective response (Phase Ib). Secondary endpoints: number of pts with DLTs throughout entire treatment period and PK parameters (Phase Ia/Ib); duration of response, disease control, duration of disease control, and PFS (Phase Ib). Results: As of 21 December 2022, 34 pts had been treated in the US, The Netherlands, Japan, and China. Pts had NSCLC (n = 21), colorectal cancer (n = 3), or other tumors (n = 10). Most pts had a pathological HER2 mutation (n = 25). Pts received BI 1810631 15, 30, 60, 100, 150 mg BID (n = 3/3/4/4/3) or 60, 120, 180, 240 mg QD (n = 5/4/5/3). Median number of cycles was 4 (range 1-14). Treatment is ongoing in 23 pts. To date, three DLTs have been observed (grade 2 edema [60 mg BID], grade 3 anemia [60 mg QD], grade 3 elevated ALT [180 mg QD]). The MTD has not been reached with either schedule. Treatment-related adverse events (TRAEs) were reported in 23 pts (68%). The most common TRAEs were diarrhea (n = 9), anemia (n = 5), increased alkaline phosphatase, increased creatinine, increased ALT, hypoalbuminemia (all n = 4), hypocalcemia, elevated AST, dry skin, increased GGT (all n = 3). Three pts had grade 3 TRAEs (anemia/increased GGT [n = 1], increased ALT [n = 2]). In 29 pts evaluable for response the ORR (regardless of confirmation) was 34% (n = 10, all PRs; NSCLC: n = 8; esophagus, cholangiocarcinoma: n = 1). The DCR was 90%. In 19 NSCLC pts evaluable for response, the ORR was 42% and the DCR was 95%. Updated data will be presented at the meeting. Conclusions: These preliminary data indicate that BI 1810631 is well tolerated and shows encouraging anti-tumor activity in pts with HER2 aberration-positive solid tumors. Recruitment into Phase Ia is ongoing. Citation Format: John Heymach, Frans Opdam, Minal Barve, Hai-Yan Tu, Yi-Long Wu, Neil Gibson, Behbood Sadrolhefazi, Josep Serra, Kiyotaka Yoh, Noboru Yamamoto. Updated data from the Phase I Beamion Lung 1 trial of the HER2 tyrosine kinase inhibitor (TKI), BI 1810631, as monotherapy in patients (pts) with advanced/metastatic solid tumors with HER2 aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT203.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Targeted agents, such as EGFR tyrosine kinase inhibitors (TKIs) and KRAS G12C inhibitors, have provided clinical benefits for patients and can yield significant tumor shrinkage. Inevitably, however, there remain residual tumors that harbor drug-tolerant persister cells (DTPCs) not killed by initial treatment, leading to emerging of drug-resistant cells (DRCs) and tumor recurrence. The identification of effective treatment strategies to target DTPCs is a critical and unmet need. We hypothesized that DTPCs can be targeted using chimeric antigen receptor (CAR)-based cellular therapy approaches directed at cell surface proteins expressed on DTPCs and that the combination of such approaches with targeted agents will more effectively delay the emergence of fully drug-resistant cells than targeted agents alone. To this end, we first performed luciferase-based killing assays and observed that EGFR-targeting CAR-T and CAR-NK cells displayed potent cytotoxicity against DTPCs of osimertinib-treated EGFR mutant NSCLC cells. Moreover, osimertinib DTPCs showed increased sensitivity to EGFR CAR-T and CAR-NK cells compared to their parental cells, likely due to the upregulation of EGFR on the cell surface following osimertinib treatment. In a patient-derived xenograft model of HER2YVMA mutant lung cancer, we observed that HER2 CAR-NK cells showed enhanced antitumor activity in mice treated with the HER2 inhibitor, poziotinib, for two weeks, compared to CAR-NK cells only, poziotinib only, or mice treated upfront with HER2 CAR-NK cells plus poziotinib. These in vitro and in vivo data support that CAR-based cellular therapy can effectively eliminate DTPCs. To identify additional candidate cell surface proteins expressed on DTPCs that could be targeted by CAR-based cell therapy approaches, we interrogated RNA expression from our panel of DTPCs as well as public datasets containing DTPCs of targeted agents. Likewise, we investigated the expression of cell surface proteins using datasets of parental and DRCs to identify the common candidate targets that could be exploited on both DTPCs and DRCs. Our analysis identified CD70 as being consistently upregulated in both EGFR TKI-resistant cells and DTPCs of osimertinib-treated EGFR mutant NSCLC. Moreover, we observed CD70 upregulation in DTPCs and DRCs of other targeted agents, including KRAS, ALK, and RET inhibitors, suggesting that CD70 may be a common target in DTPCs across oncogenic drivers. Furthermore, we determined that CD70-targeting CAR-T and CAR-NK cells showed promising in vitro activity against the DTPCs of osimertinib-treated EGFR mutant NSCLC. These results demonstrate CAR-based cellular therapy as an effective approach to target DTPCs and identify CD70 as a novel therapeutic target for combatting DTPCs in NSCLC. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Xiaoxing Yu, Alissa Poteete, Qian Huang, Simon Heeke, John Heymach. CD70-targeting CAR-T and CAR-NK cells demonstrate potent activity against NSCLC drug-tolerant persister cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 891.

Background: Recently, neoadjuvant immunotherapy plus chemotherapy has been approved for treatment of resectable non-small cell lung carcinoma (NSCLC). Defining the immune landscape of these tumors and its spatial distribution will help to understand lung cancer biology. Here, we analyzed the distribution of immune-related biomarkers in tumor-defined regions and its associations with clinicopathological variables in resected lung adenocarcinomas using high-plex profiling approaches. Methodology: Thirty-three FFPE tumor tissues from surgically resected treatment-naïve lung adenocarcinoma stage I/II were used to construct a tissue microarray from the MD Anderson ICON cohort. We used three 1-mm core per patient [2 from central tumor (CT), and 1 from invasive margin (IM)] and performed the GeoMx Digital Spatial Profiling protein protocol to assess 49 immune biomarkers. Pancytokeratin (panCK; epithelial), CD45 (immune) and SYTO 13 (nuclear) were utilized as morphology biomarkers. Regions of interests were placed in cores containing tumor, and segmented in ‘”Tumor (Tu)” (PanCK+) and the “tumor microenvironment (TME)” (PanCK-). Digital counts were normalized using background correction. Statistical analysis was performed using linear mixed model. A p value equal or less than 0.05 was considered significant. Results: We first compared the relative counts of immune biomarkers in the TME in CT and IM. IM had higher CD3, CD8, CD45RO, as well as CD163 and STING (P ranges 0.006 to 0.035), while CT had higher PD1 (P 0.025). Then we analyzed differential biomarker expression by sex and smoking status. Females had higher counts of Immune related biomarkers: CD45, CD3, CD20, immune checkpoints: PD-L1, VISTA, CTLA4, LAG3, and ICOS, and myeloid: CD68, CD11c, CD163, and B2M (P ranges 0.0005 to 0.046). Smokers had higher counts of CD66b (P 0.007) and B2M (P 0.039) while never smokers had higher counts of HLA-DR, CD34, FoxP3, OX40L, Tim-3, and B7-H3 (P ranges 0.001 to 0.049). Finally, we analyzed Tu segments. IM had higher CD66b, VISTA, CD163, OX40L, HLA-DR, GZMB, STING, and CD8 (P ranges 0.007 to 0.045) than CT. Female patients had higher CD45, CD68, CD11c and CD163 (P ranges 0.008 to 0.045), and males had higher SMA (P 0.005). Smokers had higher CD66b (P 0.007), B2-microglobulin (P 0.043), and never smokers had higher HLA-DR, STING, CD34, CD44, FoxP3 and CD25 (P ranges 0.006 to 0.043). Conclusions: In this study, biomarker analysis of treatment-naïve adenocarcinoma in CT areas and IM indicates a higher immune response in the IM and presence of inhibitory signaling inside the tumor. Our data also showed that tumors from females have higher immune response than tumors from males, which is concordant with previous studies. Distinct profiling by smoking status was also observed. Further analysis of gene expression analysis of this set is ongoing. Citation Format: Sharia D. Hernandez, Wei Lu, Alejandra G. Serrano, Claudio J. Arrechedera, Beatriz Sanchez-Espiridion, Nejla Ozirmak, Max Molina, Larisa Kostousov, Sean Barnes, Khaja Khan, Ximing Tang, Junya Fujimoto, Edwin R. Parra, Gabriela Raso, Stephanie T. Schmidt, Carmen Behrens, John Heymach, Jianjun Zhang, Ken Chen, Boris Sepesi, Tina Cascone, Don Gibbons, Ignacio I. Wistuba, Cara Haymaker, Luisa M. Solis. Spatial profiling of immune biomarkers in resected treatment-naïve early stage lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4460.

Introduction: An improved understanding of early lung carcinogenesis may facilitate development of novel diagnostic, screening, and prevention strategies to decrease lung cancer mortality. We have previously led a series of studies on the molecular and immune landscape of lung adenocarcinoma (LUAD) precursors. However, resected human specimens only provide molecular snapshots. Here, we sought to establish and characterize human-relevant murine lung precancer models to depict molecular evolution during early lung carcinogenesis and to provide novel insights for lung cancer interception. Methods: We have established 3 genetically engineered mouse models (GEMMs) (K: KrasLSL-G12D/+, KP: KrasLSL-G12D/+/Trp53R172H/+, KL: KrasLSL-G12D/+/Lkb1fl/fl) and 2 carcinogen-induced models (CITMs) (UWT: Urethane in wild type mice, URG: Urethane in Rag2−/− mice). Lung tissues were serially collected at multiple time points after induction and subjected to whole exome sequencing (WES), single cell RNA sequencing (scRNA-seq), spatial transcriptomics (Visium), and imaging mass cytometry (IMC). Results: Pathological review of specimens from 252 mice revealed normal lung, precancers and LUAD at different timepoints mirroring the evolution of human LUAD. Overall, the tumor burden was significantly higher in GEMMs than CITMs (p Conclusion: We have established 5 murine carcinogen-dependent and -independent precancer models, morphologically resembling human LUAD and its precursors. Although progressive immunosuppression along with progression of lung precancers is universal across all models, the evolution patterns and the molecular/immune features underlying immunosuppression vary in different models, particularly between CITMs and GEMMs. These models may be valuable assets for studying early lung carcinogenesis and lung cancer interception. Citation Format: Bo Zhu, Jian-Rong Li, Hong Chen, Pingjun Chen, Junya Fujimoto, Yanhua Tian, Muhammad Aminu, Chenyang Li, Lingzhi Hong, Alexandre Reuben, Edwin Roger Parra Cuentas, Ou Shi, Monique Nilsson, Alissa Poteete, Shawna Hubert, Khaja Khan, Wei Lu, Daniel Kraushaar, Xingzhi Song, Jianhua Zhang, Don Gibbons, Luisa Solis Soto, Ignacio Wistuba, Jia Wu, John Heymach, Chao Cheng, Jianjun Zhang. Single cell and spatial profiling reveal molecular and immune evolution from precancers to invasive lung adenocarcinomas in genetic and carcinogen-induced mouse models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6513.

Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with limited treatment options. Most SCLC are “immune-cold” and respond poorly to immunotherapy. Targeting SCLC cell surfaceome with cellular therapies have shown promising efficacy and tolerability in DLL3-targeting CAR T (AMG119) and Bispecific T-cell engager (tarlatamab). Recent work has defined subtypes of SCLC based on master transcription factors ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and an immune cell-rich “inflamed” subtype(SCLC-I). Notably, SCLC-A and SCLC-N express neuroendocrine (NE) features while SCLC-P and SCLC-I are non-neuroendocrine (non-NE). We hypothesize that SCLC may have distinct surfaceome expressions based on their subtypes, which can be successfully targeted with cellular therapies such as CAR T. Methods: Transcriptional profiling of patient tumors identified several putative cell surfaceome targets in SCLC. We confirmed their expression level by Western blot and flow cytometry in SCLC cell lines stratified by subtypes. Top targets were selected based on abundant expression in SCLC and restricted normal tissue expression. We then constructed third-generation CAR T-cells against the top targets and tested their efficacy in cancer cell/CAR T co-culture models and in vivo. Results: We validated cell surface expression of top targets DLL3, CD56 and GD2; and generated CAR Ts against these targets to test cytotoxicity across a large panel of SCLC cell lines. Specifically, DLL3 is present predominantly in SCLC-A and SCLC-N (NE) subtypes. CAR T against DLL3 showed robust cytotoxicity in vitro against DLL3-expressing cell lines (5 out of 6 SCLC-A/N cell lines, versus 1 out of 6 SCLC-P/I cell lines; median cytotoxicity 94% vs. 43%, p=0.0082, Mann-Whitney U test), as well as promising in vivo activity. We also confirmed CD56 (NCAM1) was broadly expressed on the cell surface in SCLC A/P/N subtypes, and 8 out of 9 cell lines with high CD56 expression demonstrated over 50% cytotoxicity in co-culture models. GD2 was found to be enriched specifically in SCLC-N subtype of SCLC. As expected, GD2-targeting CAR T had higher cytotoxicity in SCLC-N subtypes of SCLC compared to other subtypes (median cytotoxicity 91% vs 22%, p=0.047). Moreover, given that novel therapeutics are often tested in the relapsed setting, we tested target expressions in SCLC cell lines after treatment with cisplatin and etoposide (standard frontline chemotherapy for SCLC) to see if target levels change. We found that surface target expressions increased instead of decreased in most cell lines, suggesting that they may be suitable targets in the relapsed setting also. Conclusion: This study demonstrated efficacy of CAR Ts targeting several cell surfaceome targets enriched in certain subtypes of SCLC. The data supports further investigation of a subtype-specific personalized treatment of SCLC using CAR T therapies against surfaceome targets. Citation Format: Bingnan Zhang, Yan Yang, C.Allison Stewart, Kavya Ramkumar, Runsheng Wang, Robert Cardnell, Lixia Diao, Qi Wang, Jing Wang, Lauren Byers, Carl Gay, John Heymach. Subtype-specific targeting of cell surfaceome with CAR T therapies in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3196.

Activating ERBB2 (HER2) mutations occur across multiple malignancies, and although mutational hotpots vary by disease, HER2 mutations most frequently occur in the tyrosine kinase domain. Among lung cancer patients, the most common HER2 mutation is the exon 20 insertion mutations, Y772dupYVMA, which accounts for over 30% of all HER2 mutations in lung cancer. Recent clinical testing of trastuzumab deruxtecan (T-DXd), a HER2 antibody-drug conjugate (ADC), against HER2 mutant non-small cell lung cancer (NSCLC) demonstrated a confirmed objective response of 55%, a median duration of response of 9.3 months, and a median overall survival of 17.8 months, which ultimately led to the recent approval of T-DXd for HER2 mutant NSCLC. Acquired resistance to targeted agents remains a major clinical challenge, and HER2-mutant patients that initially respond to T-DXd will eventually develop progressive disease. Thus, understanding mechanism of T-DXd resistance and identifying targeted agents that remain effective in cells with acquired T-DXd resistance is of great clinical importance. We generated Ba/F3 cells expressing the HER2 YVMA mutation and assessed sensitivity to HER2 targeting agents by Cell Titer Glo assay. HER2 YVMA cells were sensitive to the EGFR/HER2 tyrosine kinase inhibitor (TKI) poziotinib and the HER2 ADC T-DXd. To generate HER2 ADC resistant cells, we cultured Ba/F3 HER2 YVMA cells in T-DXd continuously for 8 weeks at which point cells resumed rapid proliferation. Cell Titer Glo assay confirmed that Ba/F3 HER2 YVMA cells were sensitive to T-DXd with an IC50 of 195 μg/ml, whereas Ba/F3 HER2 YVMA T-DXd-resistant cells had an IC50 of greater than 1000 μg/ml. Notably, T-DXd resistant cells remained highly sensitive to the HER2 TKI poziotinib with an IC50 of 14 nM and 7.5 nM for T-DXd-resistant and parental cells, respectively. Moreover, T-DXd-resistant cells demonstrated reduced sensitivity to the topoisomerase inhibitor topotecan with an IC50 of 200 nM whereas parental cells had an IC50 of 66 nM, suggesting that acquired T-DXd resistance may be mediated by loss of sensitivity to the ADC payload. Collectively, these data demonstrate that HER2 TKIs such as poziotinib may retain anti-tumor cell activity in HER2 mutant tumor cells with acquired resistance to HER2 ADCs. Citation Format: Monique B. Nilsson, Alissa Poteete, Hibiki Udagawa, Xiaoxing Yu, Junqin He, Xiuning Le, John Heymach. Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 383.