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Purpose: Patients with advanced non–small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. Experimental Design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell–mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

IL-6 modulates expression of NK receptor and their ligands in EGFR mutant tumors.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1–knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting.Prevention Relevance:PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

Percentage of CD4+CD44hi

Table 1: Pathology classification criteria. Table 2: Flow cytometry antibodies

In KRAS-mutant lung adenocarcinoma, tumors with LKB1 loss (KL) are highly enriched for concurrent KEAP1 mutations, which activate the KEAP1/NRF2 pathway (KLK). Here, we investigated the biological consequences of these cooccurring alterations and explored whether they conferred specific therapeutic vulnerabilities. Compared with KL tumors, KLK tumors exhibited increased expression of genes involved in glutamine metabolism, the tricarboxylic acid cycle, and the redox homeostasis signature. Using isogenic pairs with knockdown or overexpression of LKB1, KEAP1, and NRF2, we found that LKB1 loss results in increased energetic and redox stress marked by increased levels of intracellular reactive oxygen species and decreased levels of ATP, NADPH/NADP+ ratio, and glutathione. Activation of the KEAP1/NRF2 axis in LKB1-deficient cells enhanced cell survival and played a critical role in the maintenance of energetic and redox homeostasis in a glutamine-dependent manner. LKB1 and the KEAP1/NRF2 pathways cooperatively drove metabolic reprogramming and enhanced sensitivity to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, these findings elucidate the adaptive advantage provided by KEAP1/NRF2 pathway activation in KL tumors and support clinical testing of glutaminase inhibitor in subsets of KRAS-mutant lung adenocarcinoma.Significance:In KRAS-mutant non–small cell lung cancer, LKB1 loss results in enhanced energetic/redox stress, which is tolerated, in part, through cooccurring KEAP1/NRF2–dependent metabolic adaptations, thus enhancing glutamine dependence and vulnerability to glutaminase inhibition.

Supplementary figures

Supplementary tables S1-S6

Purpose:Radiotherapy with or without chemotherapy is a mainstay of treatment for locally advanced non–small cell lung cancer (NSCLC), but no predictive markers are currently available to select patients who will benefit from these therapies. In this study, we investigated the association between alterations in STK11/LKB1, the second most common tumor suppressor in NSCLC, and response to radiotherapy as well as potential therapeutic approaches to improve outcomes.Experimental Design:We conducted a retrospective analysis of 194 patients with stage I–III NSCLC, including 164 stage III patients bearing mutant or wild-type STK11/LKB1 treated with radiotherapy, and assessed locoregional recurrence (LRR), distant metastasis rates, disease-free survival (DFS), and overall survival (OS), and we investigated the causal role of LKB1 in mediating radiotherapy resistance using isogenic pairs of NSCLC cell lines with LKB1 loss or gain.Results:In stage III patients, with 4 years median follow-up, STK11/LKB1 mutations were associated with higher LRR (P = 0.0108), and shorter DFS (HR 2.530, P = 0.0029) and OS (HR 2.198, P = 0.0263). LKB1 loss promoted relative resistance to radiotherapy, which was dependent on the KEAP1/NRF2 pathway for redox homeostasis. Suppression of the KEAP1/NRF2 pathway via KEAP1 expression, or pharmacologic blockade of glutaminase (GLS) 1 sensitized LKB1-deficient tumors to radiotherapy.Conclusions:These data provide evidence that LKB1 loss is associated with LRR and poor clinical outcomes in patients with NSCLC treated with radiotherapy and that targeting the KEAP1/NRF2 pathway or GLS inhibition are potential approaches to radiosensitize LKB1-deficient tumors.

Abbreviations used in the manuscript.

Supplementary bioinformatic analysis and CB-839 effects on in vitro cell proliferation, ROS stress and metabolism.

Deletion or loss of function mutations of the STK11/LKB1 tumor suppressor are associated with primary resistance to immunotherapy in KRAS-mutant lung adenocarcinoma (LUAD) and drive metabolic reprogramming of tumor cells. We observed that LKB1-deficient tumors were resistant to anti-angiogenic therapy in the hypoxic and nutrient-depleted or acidic tumor microenvironment (TME). We determined that MYC which is elevated in LKB1-deficient cells, regulated the expression of the lactate transporter, MCT4. Moreover, knockdown of MYC decreases glycolysis and cell proliferation. Therefore, we hypothesize that metabolic changes in LKB1-deficient tumor cells is at least partially driven by MYC upregulation, and that depletion of MYC or targeting of key enzymes or transporters downstream of MYC such as MCT4 may abrogate lactate reutilization and sensitize LKB1-deficient tumors to anti-angiogenic therapy. To explore the impact of MYC on lactate metabolism in LKB1-deficient NSCLC cells, we performed isotope tracing in KRAS mutant (K) and KRAS mutant LKB1 knockout (KL) syngeneic murine lung cancer cells. 13C3-lactate tracing revealed that isotopologues were significantly enriched in TCA components such as pyruvate, citrate and a-ketoglutarate in KL cells. Meanwhile, lactate-treated KL cells shows less reactive oxygen species (ROS), suggesting that LKB1-deficient tumor cells reutilize lactate as an energy source more efficiently than LKB1-intact cells. MCT4 KO significantly abrogated lactate incorporation into TCA cycle. Similarly, MYC knockdown or MCT4 KO decreased lactate-induced oxygen consumption, but increased ROS levels. In contrast, exhausted T cells were inefficient at lactate reutilization, and high levels of lactate increased ROS in T cells. Hence, LKB1-deficient tumors have a survival advantage over T cells in the lactate-rich TME. We queried scRNAseq data from K, KL and KL MCT4KO syngeneic models and similarly observed that KL tumor cells exhibited elevated hypoxia and angiogenesis gene expression signatures, which was reversed by MCT4 KO. However, ROS detoxification was decreased in T cells from KL tumors but increased in MCT4 KO tumors. Finally, we injected KL murine tumor cells into immunocompetent mice, and randomly treated them with vehicle or the VEGF blocking antibody, DC101. MYC knockdown or MCT4 KO sensitized KL tumors to VEGF inhibition, and significantly increased overall survival. Collectively, our data indicates that in LKB1-deficient tumors, upregulation of MYC promotes tumor cell metabolic reprogramming and that targeting MYC or MCT4 can inhibit lactate reutilization and enhance the efficacy of anti-angiogenic agents. These findings provide insight into the mechanisms driving the aggressive phenotype of KRAS-mutant LKB1-deficient tumors and identify a novel therapeutic strategy for targeting this patient population. Citation Format: Yu Qian, David Molkentine, Chendong Yang, Ana Galan Cobo, Irene Guijarro, Minghao Dang, Alissa Poteete, Peixin Jiang, Ferdinandos Skoulidis, Linghua Wang, Alexandre Reuben, John D. Minna, Ralph J. DeBerardinis, John V. Heymach. MYC mediates enhanced lactate reutilization and resistance to anti-angiogenesis therapy in preclinical models of LKB1-deficient NSCLC [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 271.

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.

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.

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.

NRG1 gene fusions are rare, clinically actionable somatic alterations identified in 0.1% of all tumors. Previous studies have demonstrated that NRG1 fusions signal through ERBB/HER family members and that HER2 inhibition has anti-tumor activity in NRG1 fusion-driven cancers. However, NRG1 can also bind to HER4 in addition to HER3, and the contribution of individual HER family members in tumor cells with NRG1 fusions has not been fully elucidated. To determine the role of individual HER family members in NRG1 fusion positive cancer cells, we engineered Ba/F3 cells to express various HER family members along with NRG1 fusions. Expression of NRG1 fusions in combination with EGFR/HER3, HER2/HER4 or HER4 induced cellular transformation as measured by IL-3 independent growth. These results indicated that in addition to HER2/HER3 signaling, EGFR/HER3, HER4, and to a greater extent, HER2/HER4 signaling, may activate key oncogenic signaling pathways in cells expressing NRG1 fusions. We next assessed whether targeted inhibition of HER family members could inhibit the growth of NRG1 fusion positive cancer cells using our panel of engineered Ba/F3 cells, human tumor cell lines, and xenograft models. Our data indicated that pan-HER tyrosine kinase inhibitors (TKIs), such as poziotinib, were more effective at blocking HER2/HER3, EGFR/HER3 and HER4 signaling in NRG1-fusion expressing cells as compared to TKIs with greater relative specificity for EGFR (erlotinib, lapatinib), HER2 (pyrotinib), EGFR/HER2 (afatinib, dacomitinib, neratinib) or HER2/HER4 (TAS0728,tucatinib). In NRG1 fusion-positive cancer cell lines, poziotinib showed potent anti-tumor cell activity with IC50 values of 0.2 - 0.4 nM. The IC50 values of EGFR/HER2 TKIs were 0.3 - 23 nM, and HER2/HER4 TKIs showed modest activity with IC50 values of 11 - 560 nM. Next, we assessed whether inhibition of HER family dimerization also had activity against tumor cells harboring NRG1 fusions. We observed that pertuzumab, a HER2 antibody which inhibits HER2 dimerization with other HER family members, had improved single agent activity as compared to trastuzumab, a HER2 antibody that inhibits HER2 activation, suggesting crosstalk between HER family members. Moreover, an additive effect was observed when tumor cells were treated with trastuzumab in combination with pertuzumab. Treatment with cetuximab, an antibody that targets EGFR, in combination with trastuzumab and pertuzumab yielded a synergistic effect on tumor cell killing. These data indicate that HER4 and EGFR can play a role in NRG1 fusion-driven signaling through crosstalk with HER2/HER3 and thus, pan-HER/EGFR inhibitors are more effective than EGFR/HER2 or HER2/HER4-selective inhibitors, highlighting the therapeutic potential of targeting multiple members of the HER family in NRG1 fusion driven cancers. Citation Format: Hibiki Udagawa, Monique Nilsson, Junqin He, Alissa Poteete, John Victor Heymach. Role of individual HER family members and pan-HER targeting treatment strategy in NRG1 fusion positive 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 4042.

BackgroundKRAS-mutant non-small cell lung cancers (NSCLC) have exhibited unique response patterns to immunotherapy based on their co-occurring mutations. Patients harboring KRAS & STK11/LKB1 co-mutations (KL) have experienced shorter progression-free and overall survival compared to those with only KRAS mutations (K). Despite their limited responses, KL tumors exhibit a tumor mutational burden comparable to their K counterparts, suggesting the presence of additional mechanisms impairing antigen-specific responses. Accordingly, here we investigated the role of the MHC I antigen processing and presentation pathway in KL tumors.MethodsTCGA lung adenocarcinoma (LUAD) data were investigated for changes in expression of HLA molecules and chaperones involved in antigen processing and presentation. In mice, we performed single cell RNA sequencing of resected LKR13 K and KL tumors to evaluate changes in the tumor microenvironment and intrinsic differences in tumor antigen processing machinery. In vitro experiments were performed using the ovalbumin antigen to evaluate changes in antigen-specific T cell responses.ResultsExpression of HLA-A (pConclusionsKRAS-mutant tumors harboring STK11/LKB1 alterations have an immunosuppressed phenotype and resistance to PD-1/PD-L1 inhibitors. Our findings provide evidence that these alterations are associated with markedly reduced antigen presentation and resistance to T cell killing, responsiveness to IFN-gamma stimulation, and impaired production of T cell chemokines, providing mechanistic insights into this immunosuppressed phenotype that could help guide the development of new therapeutic strategies for enhancing anti-tumor immunity.

Approximately 15% of all patients with non-small cell lung cancer (NSCLC) and nearly 35% of Asian patients with NSCLC harbor activating mutations within the epidermal growth factor receptor (EGFR). Although these patients are initially highly sensitive to first or second generation EGFR tyrosine kinase inhibitors (TKIs) including erlotinib or third-generation inhibitors including osimertinib, EGFR TKI-refractory disease inevitably emerges. While therapeutic strategies to target resistant disease that emerges though secondary EGFR mutations or MET amplification have been developed, there remains a void of therapeutic options for patients where resistance occurs through EGFR-independent mechanism such as epithelial to mesenchymal transition (EMT) or transformation to small cell lung cancer (SCLC). To identify cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches we interrogated RNAseq data from EGFR mutant NSCLC cell lines (HCC827 and HCC4006) and their associated EGFR TKI resistant variants previously shown to have developed resistance through EMT and filtered gene expression data to include only genes which transcribed proteins localized to the cell surface. We identified CD70 as a being highly upregulated in EGFR TKI resistant cells (p = 7.2e-42). Given that CD70 expression is highly restricted and only transiently expressed on immune cells, CD70 was selected as a top candidate cell surface protein for targeting studies. Western blotting and flow cytometry analysis confirmed CD70 protein levels to be highly upregulated in EGFR TKI resistant cells that had undergone EMT but not in cells harboring secondary EGFR mutations or MET amplifications. We also observed CD70 upregulation in osimertinib-treated drug tolerant persister cells, indicating that CD70 upregulation is an early event in the evolution of TKI resistance. Moreover, patient-derived models of acquired EGFR TKI resistance also exhibited CD70 positivity. Our data also indicated that in EGFR mutant NSCLC cells, CD70 could be upregulated through decreased CD70 promoter methylation as well as by the EMT regulators, transforming growth factor-β (TGF-β) and ZEB1, both of which were upregulated in TKI resistant cells. In EGFR TKI resistant cells, CD70 knockdown impaired cell viability and invasiveness, and stimulation of CD70 using the exogenous binding partner CD27 resulted in activation of AKT and MAPK, pathways known to be re-activated with acquired TKI resistance. CD70-targeting approaches including anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting CAR T cell and CAR NK cells showed promising in vitro and in vivo activity against CD70 positive tumor cells and in osimertinib drug-tolerant persister cells. These results identify CD70 as a novel therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits further investigation in the clinic. Citation Format: Monique B. Nilsson, Yan Yang, Sonia Patel, Simon Heeke, Xiuning Le, Thiru Aruguman, Jacqulyne Robichaux, Xiaoxing Yu, Alissa Poteete, Xiaoyang Ren, Lixia Diao, Li Shen, Qi Wang, Fahao Zhang, Leticia Campos Clemente, Luisa Solis Soto, Chunhua Shi, Hai Tran, Jason Bock, Jing Wang, Ignacio I. Wistuba, John D. Minna, John V. Heymach. CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1827.

Genomic alterations that result in loss of function of the tumor suppressor serine/threonine kinase STK11/LKB1 occur in 20-30% of lung adenocarcinomas. We previously observed that STK11/LKB1 mutations are genomic drivers of primary resistance in lung adenocarcinoma. Moreover, LKB1-mutant NSCLCs exhibit higher hypoxia and glycolysis rates, resulting in enhanced production and secretion of lactate. Accordingly, we hypothesize that high production of lactate of LKB1 mutant tumor may contribute to its immunologically cold phenotype and that blockade of the lactate pathway may potentiate the efficacy of immune checkpoint blockade (ICB) therapies. We characterized the immune landscape of LKB1 mutant clinical samples and performed scRNAseq analysis in KRAS mutant (K) and KRAS mutant LKB1 knockout (KL) syngeneic murine models. To evaluate inhibition of lactate metabolism as a therapeutic strategy, we knocked out the lactate transporter SLC16A3/MCT4 and characterized the impact on the tumor microenvironment (TME), and response to ICB. Clinical analysis of LKB1 mutant NSCLC patients from the MD Anderson’s ICON and PROSPECT cohorts suggested that LKB1 mutant tumors showed reduced immune cell infiltration, restricted T cell function, and enhanced M2-like macrophages phenotypes. Moreover, in preclinical models, LKB1 mutant tumors showed enhanced glycolysis and upregulation of MCT4 expression in a variety of human and murine cell lines. Deletion of MCT4 dramatically reduced glycolysis, energy production, and cell proliferation. By scRNAseq, we identified distinct immune subclusters modulated by LKB1 mutation. Hypofunctional T cells and M2-like macrophages were abundant in LKB1 mutant tumors, while these populations were significantly reduced in KL tumors with MCT4 KO. The conditioned medium from KL cells impaired T cell activation and decreased T cell killing, IFNγ production and glycolysis rate. Moreover, conditioned medium from KL cells induced M2-associated genes expression, as well as CD206+ expression in both peritoneal macrophages and Raw264.7 cells. These effects were at least in part MCT-dependent, as medium from MCT4 KO cells induced the opposite effects on T cells and macrophages, and the effects could be reversed by introducing exogenous lactate, suggesting that blockade of lactate transport reactivated T cells and reversed M2 polarization. Importantly, MCT4 KO in LKB1-mutant tumors sensitized tumors to anti-PD1 immunotherapy in syngeneic murine tumors and promoted long-term anti-tumor immunity. Collectively, our data indicate that LKB1 mutant tumors enhanced lactate secretion into the TME and this results in decreased T cell cytotoxic potential as well as higher pro-tumor M2 polarization, leading to resistance to immunotherapy. These data suggest that therapeutic inhibition of MCT4 is a promising strategy to overcome immunotherapy resistance in NSCLC patients harboring LKB1 mutant tumors. Citation Format: Yu Qian, Irene Guijarro, Ana Galan-Cobo, Minghao Dang, Alissa Poteete, Fahao Zhang, Qi Wang, Jing Wang, Edwin Parra, Ferdinandos Skoulidis, Ignacio Wistuba, Svena Verma, Taha Merghoub, Linghua Wang, Jedd Wolchok, Alexandre Reuben, John Heymach. MCT4 blockade reverses lactate-mediated immunosuppression in LKB1-deficient NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2160.

Around 10-15% of non-small cell lung cancer (NSCLC) cases in the United States harbor an activating mutation in epidermal growth factor receptor (EGFR) which are initially highly sensitive to treatment EGFR tyrosine kinase inhibitors (TKI) before eventually developing resistance to these agents. Therefore, there is a current unmet clinical need to identify novel treatment options for this patent population. While anti-PD-1/PD-L1 immunotherapy has been effective for many NSCLC patients, EGFR-mutant tumors have response rates of less than 10%. The mechanisms driving resistance to immunotherapy in EGFR-mutant NSCLC are not well understood. We previously reported that IL-6 is highly upregulated in NSCLC cells with acquired resistance to EGFR-TKIs. Because IL-6 is a pleiotropic cytokine known to impact immune populations within the tumor microenvironment, we hypothesized that IL-6 may drive immunosuppression in EGFR mutant NSCLC. Using genetically engineered mouse models (GEMMs) of EGFR-mutant NSCLC with and without IL-6 knockout, we evaluated the effects of IL-6 on the tumor microenvironment. EGFR-mutant mice with knockout of IL-6 had a significantly increased overall survival compared to those with intact-IL-6. Knockout of IL-6 increased total immune infiltration in these tumors as assessed by flow cytometry. Infiltrating T cells from IL-6 knockout mice displayed a smaller population of T helper 17 cells compared to IL-6-expressing tumors. Knockout of IL-6 also increased the population of infiltrating activated CD8 T cells and a reduced T-regulatory cell population. Due to these changes in T cell activity, we wanted to determine any potential synergistic effects of IL-6 blockade used during anti-PD-1 therapy. We observed that combination treatment extended survival in EGFR-mutant NSCLC mice. Corresponding in vitro T cell cytotoxicity assays confirmed that EGFR-TKI resistant cells treated with IL-6 blocking antibody were more sensitive to T cell-mediated killing. Furthermore, infiltration of NK cells was increased in tumors with knockout of IL-6. Infiltrating NK cells found in these IL-6 knockout mice displayed a more activated phenotype as demonstrated by increased expression of NKG2D. Next, we evaluated the effects of IL-6 from conditioned media collected from human EGFR-mutant and TKI-resistant cells lines on human T and NK cells cultured ex vivo. e determined that IL-6 suppressed expression of activation markers including granzyme B and IFN-y on the surface of both T and NK cells. Blockade of IL-6 increased NK-mediated cytotoxic killing of EGFR-TKI resistant cells in vitro. In conclusion, our data indicates that in EGFR-TKI resistance, upregulated IL-6 suppresses T and NK cell cytotoxic potential and drives immunosuppression. Citation Format: Sonia Patel, Monique Nilsson, Yan Yang, Xiaoxing Yu, Fahao Zhang, Alissa Poteete, Xiaoyang Ren, Li Shen, Jing Wang, Xiuning Le, John Heymach. IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3125.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer with a notably low survival rate. Chemotherapy and more recently its combination with immunotherapy are the standard of care for extensive stage SCLC. Although this combinatorial treatment provides clinical benefit, it can be hampered by the effect of chemotherapy on inducing immunosuppression. Cyclin dependent kinases 4/6 (CDK4/6) phosphorylate retinoblastoma (Rb) protein and promote proliferation of Rb-intact hematopoietic stem progenitor cells (HSPCs) by allowing cells to progress through S phase. Rb loss is a signature mutation in SCLC tumors, making these tumor cells unresponsive to CDK4/6 inhibition. Therefore, we hypothesized that CDK4/6 inhibitors could be utilized to block the proliferation of Rb-intact HSPCs and protected them from chemotherapy without altering the effect of chemotherapy on Rb-deficient SCLC cells. We treated Rb-deficient SCLC cancer cells and Rb-intact NK and T cells with increasing concentrations of cisplatin, etoposide and topotecan with or without the CDK4/6 inhibitor G1T28. Cell proliferation, apoptosis and cell cycle were analyzed using CellTiter-Glo, Annexin V staining, and PI-based cell cycle analysis. In vitro, CDK4/6 inhibition did not alter the impact of chemotherapy on Rb-deficient SCLC cell viability, whereas with Rb-intact NK and T cells, CDK4/6 inhibitor treatment induced cell cycle arrest and prevented apoptosis induced by chemotherapeutic agents. In the TKOTmG Rb-deficient mouse model of SCLC, CDK4/6 inhibition improved the efficacy of cisplatin plus etoposide. In vivo studies using the KP-1 syngeneic mouse models showed that treatment with a CDK4/6 inhibitor improved the efficacy of chemotherapy and immunotherapy individually or chemo-immunotherapy combinations by reducing the tumor growth rate and prolonging animal survival. CDK4/6i treatment prior to chemotherapy preserved white blood cell counts in comparison to chemotherapy alone. Tissue and blood immune marker analysis showed that CDK4/6 inhibitor treatment increased both the circulating and tumor-resident NK cells. Collectively, we demonstrated that CDK4/6 inhibition does not antagonize the therapeutic impact of chemotherapy on Rb-deficient SCLC cells but enhances the efficacy of chemotherapy and immunotherapy in KP-1 syngeneic SCLC mouse model. CDK4/6i also at least partially abrogated the chemotherapy-induced reductions in subpopulatons of immune cells. CDK4/6 inhibition may be considered as a therapeutic strategy for mitigating chemotherapy-induced immunosuppression and enhance antitumor immunity in SCLC patients. Citation Format: Thiruvengadam Arumugam, Irene G. Muñoz, Alissa Poteete, Fahoa Zhang, Monique Nilsson, John S. Yi, Jessica A. Sorrentino, Andrew P. Beelen, John V. Heymach. CDK4/6 inhibition mitigates chemotherapy-induced immunosuppression and enhances the efficacy of chemo- immunotherapy in small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4204.

HPV-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) tumors typically have p53 loss due to the activity of the human papillomavirus (HPV)-encoded E6 protein and the E6-associated protein (HPVE6-AP) which mediate the degradation of wild-type (WT) p53 (p53α). The loss of p53 is thought to be a major contributor to the pathogenesis of HPV+ HNSCC, which comprise approximately 35% of all HNSCC. Currently, standard care for HPV+HNSCC includes radiation and chemotherapy. However long-term toxicity related to these treatments is a concern, and there is a need for newer therapeutic strategies. Previously, we reported that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising of exons 1 to 9β or 9γ, respectively) are degraded by nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. Here, using HPV+HNSCC cell line models, we show that NMD inhibition rescues p53β/γ isoforms and activates p53 pathway. Furthermore, we show that p53β/γ isoforms are more stable compared to p53α in these cells, with reduced vulnerabililty to HPVE6-AP- mediated degradation, and that p53β/γ isoforms contribute to increased expression of p53 transcriptional targets p21 and PUMA following NMD inhibition. Consistent with p53 pathway activation, NMD inhibition enhanced radiosensitivity of HNSCC cells. NMD inhibition attenuated colony forming ability and disrupted cell cycle progression. To evaluate the therapeutic implications of NMD inhibition, we assessed the in vivo growth of HPV+ UMSCC47 tumors. Nude mice were injected with UMSCC47 cells either subcutaneously or orthotopically in the tongue and randomized to receive vehicle or with an NMD inhibitor. In both tumor models, we observed a significant reduction in tumor volume with NMD inhibition as compared to the vehicle-treated animals. To investigate whether NMD inhibition induced the expression of p53β/γ isoforms and activated the p53 pathway in vivo, we collected tumor tissues from animals and evaluated expression of p53 isoforms and transcriptional targets by RT-PCR. We observed increased expression of p53γ, p21, GADD45A and PUMA mRNAs in NMD inhibitor treated UMSCC47 tumors, compared to their respective vehicle treated controls. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in major subgroups of p53-deficient HPV+ HNSCC tumors. Citation Format: Jayanthi Gudikote, Tina Cascone, Alissa Poteete, Piyada Sitthideatphaiboon, Sonia Patel, Yan Yang, Fahao Zhang, Lerong Li, Li Shen, Monique Nilsson, Phillip Jones, Jing Wang, Jean-Christophe Bourdon, Faye M. Johnson, John V. Heymach. Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5733.

The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.