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Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumor regression but ultimately fail to elicit cures. As a result, there is an intense interest in how to best combine targeted therapies with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumors with the host immune system are inadequate, in part due to the low tumor mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS–driven lung cancer with an elevated tumor mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumor mutational burden and autochthonous tumors remained refractory to immunotherapy. However, establishing clonal cell lines from these tumors enabled the generation of an immunogenic syngeneic transplantation model of KRAS-mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, antitumor immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C -expressing tumors was markedly potentiated by the adaptive immune system, highlighting the importance of using immunocompetent models for evaluating targeted therapies. Overall, this model provides a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer.Significance:This study develops a mouse model of immunogenic KRAS-mutant lung cancer to facilitate the investigation of optimal combinations of targeted therapies with immunotherapies.

Oncogenic KRAS impairs anti-tumour immune responses, but effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identifies mechanisms by which oncogenic KRAS promotes immune evasion, most notably expression of immunosuppressive cyclooxygenase-2 (COX-2) in cancer cells. Oncogenic KRAS was a potent inducer of COX-2 in both mouse and human lung cancer which was suppressed using KRAS inhibitors. COX-2 acting via prostaglandin E2 (PGE2) promotes resistance to immune checkpoint blockade (ICB) in both mouse and human lung adenocarcinoma. Targeting COX-2/PGE2 remodelled the tumour microenvironment by inducing pro-inflammatory polarisation of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX-2 expression contributed to tumour relapse after prolonged KRAS inhibition. We propose testing COX-2/PGE2 pathway inhibitors in combination with KRAS G12C inhibition or ICB in patients with KRAS-mutant lung cancer.SIGNIFICANCEHere we identify cyclooxygenase 2 signalling via prostaglandin E2 as a major mediator of immune evasion that is driven by oncogenic KRAS in both mouse and human lung cancer. COX2/PGE2signalling contributes to immunotherapy and KRAS-targeted therapy resistance which can be overcome by COX2/PGE2pathway inhibitors. This work suggests novel combination therapies for the treatment of KRAS-mutant lung cancer.

Supplementary Data from An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations

Supplementary Table from An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations

Environmental carcinogenic exposures are major contributors to global disease burden yet how they promote cancer is unclear. Over 70 years ago, the concept of tumour promoting agents driving latent clones to expand was first proposed. In support of this model, recent evidence suggests that human tissue contains a patchwork of mutant clones, some of which harbour oncogenic mutations, and many environmental carcinogens lack a clear mutational signature. We hypothesised that the environmental carcinogen

Recently developed KRAS G12C inhibitory drugs are beneficial to lung cancer patients harboring KRAS G12C mutations, but drug resistance frequently develops. Because of the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, these drugs can indirectly affect antitumor immunity, providing a rationale for their combination with immune checkpoint blockade. In this study, we have characterized how KRAS G12C inhibition reverses immunosuppression driven by oncogenic KRAS in a number of preclinical lung cancer models with varying levels of immunogenicity. Mechanistically, KRAS G12C inhibition up-regulates interferon signaling via Myc inhibition, leading to reduced tumor infiltration by immunosuppressive cells, enhanced infiltration and activation of cytotoxic T cells, and increased antigen presentation. However, the combination of KRAS G12C inhibitors with immune checkpoint blockade only provides synergistic benefit in the most immunogenic tumor model. KRAS G12C inhibition fails to sensitize cold tumors to immunotherapy, with implications for the design of clinical trials combining KRAS G12C inhibitors with anti-PD1 drugs.

Recently developed KRAS

The recent approval of KRASG12C mutant-specific inhibitors has transformed the clinical practice of lung cancer patients harboring KRASG12C mutations. However, clinical data show that resistance develops rapidly after initial responses, suggesting that combination therapies will be needed. Ideally, these combinations should not only overcome adaptive or acquired resistance but also maintain or even enhance the positive immunomodulatory effects that KRASG12C inhibitors (G12Ci) have in the tumor microenvironment (TME). Most of the G12Ci being tested in clinical trials target GDP-bound KRAS (OFF state), which makes them vulnerable to upstream pathway reactivation. In this study we use a covalent tri-complex KRASG12C(ON) inhibitor, RM-029, which targets KRASG12C in the active state. Treatment with RM-029 still resulted in some degree of adaptive RAS pathway reactivation, which can be blocked using the SHP2 inhibitor RMC-4550. In vivo, this combination enhanced tumor regressions and improved overall survival in two different KRASG12C lung cancer models. In an immunogenic KRASG12C lung cancer model both KRASG12C(ON) and SHP2 inhibitors remodel the TME and are able to drive durable responses, which are enhanced when both compounds are combined. However, in this anti-PD1 sensitive model the combination of RM-029 with anti-PD1 generated more complete responses than the combination of RM-029 and RMC-4550. In an immune-excluded anti-PD1 resistant model, KRASG12C(ON) and SHP2 inhibitors also promoted a profound remodelling of the TME, with increased infiltration and activation of T cells accompanied by a reduction of tumor-promoting myeloid cells. Importantly, only the combination of both KRASG12C(ON) and SHP2 inhibitors sensitized these immune-excluded tumors to anti-PD1 blockade, resulting in durable responses and immune memory. Our preclinical results show that RM-029 as single agent or in combination with RMC-4550 and/or anti-PD1 can induce anti-tumor immunity and generate complete responses, especially in immunogenic models. To study if this immunological response could also target G12Ci-resistant subpopulations within the tumor, we used our immunogenic cell line (KPAR) to generate reporter-traced G12Ci sensitive (KPARG12C) and resistant (KPARG12D) cells that can be monitored over time via in vivo luciferase imaging and end point flow cytometry. We find evidence of bystander immune mediated killing of G12Ci-resistant cells in response to the different treatment combinations. Overall, our preclinical results demonstrate the potential of the combination of KRASG12C(ON) inhibitors with SHP2 and/or immune checkpoint blockade not only by targeting KRAS-driven proliferation in tumor cells but by stimulating anti-tumor immunity to target both G12Ci sensitive and resistant cells. Citation Format: Panayiotis Anastasiou, Mona Tomaschko, Jesse Boumelha, Sareena Rana, Christopher Moore, Miriam Molina-Arcas, Julian Downward. Combining KRASG12C(ON) inhibition with SHP2 and immune checkpoint blockade to enhance anti-tumor immunity and overcome development of resistance in 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 5733.

The recent development and approval of KRASG12C inhibitors promises to change profoundly the clinical management of lung cancer patients harbouring KRASG12C mutations. However, early clinical data indicate that acquired drug resistance can frequently develop after the initial response. Due to the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, targeted KRASG12C inhibition can indirectly affect anti-tumour immunity. This has served as a rationale for combination with immune checkpoint blockade, showing therapeutic benefit in certain immunogenic pre-clinical tumour models. In this study, we characterised how KRASG12C inhibition reverses immune suppression driven by oncogenic KRAS in a number of pre-clinical lung cancer models with varying levels of immunogenicity. Mechanistically, KRASG12C inhibition upregulates interferon pathway gene expression via inhibition of Myc and, in tumours, leads to reduced infiltration of immunosuppressive cells, increased interferon responses and antigen presentation, and also enhanced infiltration and activation of cytotoxic T cells. However, the combination of KRASG12C inhibitors with immune checkpoint blockade only provides synergistic benefit in the most immunogenic tumour model, with KRASG12C inhibition failing to sensitize cold tumours to immunotherapy. In immunogenic tumours, complete responses to KRASG12C inhibition requires tumour cell autonomous interferon γ signaling. Our data have important implications for the design of clinical trials combining KRASG12C inhibitors with anti-PD-1 drugs and suggest that additional combination strategies will be needed for immunotherapy refractory patients. One sentence summary KRASG12C inhibition mobilizes anti-tumour immunity in immunogenic lung cancer models through derepressing interferon signaling via repression of Myc.

Oncogenic KRAS mutations drive tumourigenesis in 30% of non-small cell lung cancer (NSCLC). Despite much effort, targeted therapies that aim to directly inhibit signalling pathways downstream of KRAS have limited clinical benefits for NSCLC patients, but the recent approval of PD-1/PD-L1 antibodies has led to striking durable responses. However, only a fraction of patients respond and therefore a deeper understanding of the mechanisms that drive immune evasion are required in order to broaden the clinical efficacy of immunotherapy. Increasing evidence suggests that oncogenic signalling pathways greatly influence the tumour immune landscape to impair anti-tumour immune responses. We therefore aim to understand the mechanisms by which KRAS signalling mediates immune evasion in lung cancer. Current mouse models of KRAS-mutant lung cancer are poorly immunogenic, limiting investigations into tumour-immune interactions. To overcome this, we generated a novel transplantable KRAS-mutant lung cancer model, KPAR1.3, which triggers spontaneous anti-tumour immune responses and is sensitive to immune checkpoint blockade. To identify mechanisms of immune evasion we carried out an in vivo pooled CRISPR-Cas9 screen targeting 240 KRAS-regulated genes using this novel immunogenic model. This identified the prostaglandin synthase COX-2 as a mediator of immune evasion and its expression was driven by KRAS in multiple models of mouse and human lung cancer. Loss of COX-2 promoted a pro-inflammatory tumour microenvironment and sensitised tumours to both innate and adaptive anti-tumour immune responses. Furthermore, therapeutic COX inhibition extended the survival of tumour-bearing mice and synergised with PD-1 blockade. Together these data suggest that targeting KRAS-driven mechanisms of immune evasion could broaden the clinical efficacy of immunotherapy in KRAS-mutant NSCLC. Since the approval of immunotherapy, a novel class of mutant-specific KRAS-G12C inhibitors have recently been approved for the treatment of KRAS-mutant NSCLC, however acquired drug resistance is common. We are therefore also currently investigating whether the inhibition of the COX-2/PGE2 axis enhances the therapeutic benefit of KRAS-G12C inhibitors. Citation Format: Jesse Boumelha, Pablo Romero-Clavijo, Sophie de Carné, Miriam Molina-Arcas, Julian Downward. In vivo CRISPR screening identifies mediators of immune evasion in KRAS-mutant lung cancer [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 1352.

Introduction: B cells constitute a major part of the lung tumour microenvironment where they are organised in tertiary lymphoid structures (TLS) that resemble antibody-producing germinal centres. The presence of TLS has been associated with improved patient outcome and response to immunotherapy, though the mechanisms remain unclear. Similarly, the contribution of B cells and TLS to anti-tumour immunity in non-small cell lung cancer (NSCLC) remains largely unexplored. Methods: We evaluated TRACERx and TCGA NSCLC cohorts to compare B cell and TLS signatures as well as BCR sequences from human adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Using a novel immunogenic mouse model of LUAD, we applied lineage tracing, 3D IF, and single-cell sequencing to characterise the evolution of lung germinal centre and antibody responses over tumour progression and immunotherapy treatment. We validated immunotherapy observations in a cohort of melanoma patients with pre- and post-treatment samples. Results: We found that B cell and TLS gene set scores were significantly higher in LUAD compared to LUSC. We examined B cell signals in specific mutational backgrounds, and observed an increase in BCR CDR3 diversity and IgG class switch in the context of clonal TP53 mutation. We therefore investigated the mechanistic basis of B cells in LUAD using an immunogenic mouse model on a KRAS/TP53 background. We found that TLS organisation was triggered by CXCL13 and that exogenous CXCL13 treatment increased B cell infiltration and overall survival. Using serum transfer experiments, we demonstrated that tumour-binding class-switched antibodies played an anti-tumour role and increased survival. We found that immunotherapy increased germinal centre B cell responses and antibody quantity and quality. Using single-cell BCR sequencing of immunotherapy-treated mice, we showed clonal expansion of B cells reactive to re-expressed endogenous retroviral antigens. Furthermore, we found that KRAS G12C but not MEK inhibition boosted B cell and antibody responses. Finally, combined treatment with CXCL13 and checkpoint blockade improved survival, suggesting that boosting local B cell responses can improve immunotherapy outcome. In checkpoint blockade-treated melanoma patients, we found that BCR diversity and the number of unique antigen specificities was elevated in responders post-therapy. Non-responders showed a loss of expanded clones, suggesting that both maintenance of expanded clones and recruitment of new clones associate with successful response. Conclusions: Our work describes a novel role for B cells in the lung tumour microenvironment and establish that TLS directly contribute to anti-tumour immunity by producing class-switched protective antibodies. We demonstrate that selective boosting of anti-tumour B cells shows therapeutic potential in combination with targeted and immunotherapy. Citation Format: Katey Enfield, Kevin Ng, Jesse Boumelha, Kevin Litchfield, Oriol Pich, TRACERx Consortium, Charles Swanton, George Kassiotis, Julian Downward. Immunotherapy boosts B cell responses in lung adenocarcinoma [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 1390.

Oncogenic mutations in KRAS are frequent in non-small cell lung cancer and have been associated with poor prognosis. The recent development and approval of KRASG12C mutant-specific inhibitors could change the clinical practice of lung cancer patients harboring KRASG12C mutations. However, early clinical data indicate the development of acquired resistance after initial responses. All of the KRASG12C inhibitors being tested in clinical trials to date target GDP-bound KRAS (OFF state), which makes them vulnerable to upstream pathway reactivation, as this will increase KRAS in a GTP-bound (ON) state and therefore reduce drug efficacy. In this study we use a covalent tri-complex KRASG12C(ON) inhibitor which targets KRASG12C in the active state. In vitro, the KRASG12C(ON) inhibitor RM-029 exhibited higher efficacy than the KRASG12C(OFF) inhibitor MRTX849 (adagrasib). However, treatment with RM-029 also showed adaptive RAS pathway reactivation after 24 hours of treatment, which can be blocked using the SHP2 inhibitor RMC-4550, suggesting that pathway reactivation is mediated by signaling from RTKs to RAS proteins. In vivo, KRASG12C(ON) treatment led to strong regression of KRASG12C tumors and addition of a SHP2 inhibitor significantly increased anti-tumor responses. Single treatments with KRASG12C(ON) or SHP2 inhibitors led to complete responses in some animals and immunological memory in an immune responsive KRASG12C lung tumor model, with all tumors showing complete responses when the inhibitors were combined. Consistent with this finding, both KRASG12C(ON) and SHP2 inhibition resulted in a profound remodeling of the lung tumor microenvironment (TME), with increased infiltration and activation of T cells accompanied by a reduction of tumor-promoting myeloid cells. Interestingly, similar changes were obtained in an immune evasive KRASG12C lung cancer model. Besides acting upstream RAS, SHP2 has also specific roles in immune cells, including regulation of T cell function. As comparison, responses to SOS1 inhibition, which acts only upstream RAS, have also been analyzed. Overall, our preclinical results show that the anti-tumor activity of the combination of KRASG12C(ON) with SHP2 inhibition is the result of different mechanisms. First, a KRASG12C(ON) inhibitor potently inhibits oncogenic RAS and addition of a SHP2 inhibitor blocks RAS pathway reactivation increasing tumor responses. Second, both inhibitors have beneficial effects in the TME which can be potentiated when combined. Finally, addition of a SHP2 inhibitor could potentially overcome intrinsic resistant populations by directly targeting cells resistant to G12C inhibition and/or by increasing anti-tumor immunity. Citation Format: Panayiotis Anastasiou, Edurne Mugarza, Jesse Boumelha, Sareena Rana, Christopher Moore, Miriam Molina-Arcas, Julian Downward. Combination of KRASG12C(ON) and SHP2 inhibitors overcomes adaptive resistance and enhances anti-tumor immunity [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 4029.

Oncogenic KRAS mutations drive tumorigenesis in 30% of non-small cell lung cancer (NSCLC). Despite much effort, targeted therapies that aim to directly inhibit signaling pathways downstream of KRAS have limited clinical benefits for NSCLC patients, but the recent approval of PD-1/PD-L1 antibodies has led to striking durable responses. However, only a fraction of patients respond and therefore a deeper understanding of the mechanisms that drive immune evasion are required in order to broaden the clinical efficacy of immunotherapy. Increasing evidence suggests that oncogenic signaling pathways greatly influence the tumor immune landscape to impair anti-tumor immune responses. We therefore aim to understand the mechanisms by which KRAS signaling mediates immune evasion in lung cancer. Current mouse models of KRAS-mutant lung cancer are poorly immunogenic, limiting investigations into tumor-immune interactions. To overcome this, we generated a novel transplantable KRAS-mutant lung cancer model, KPAR1.3, which triggers spontaneous anti-tumor immune responses and is sensitive to immune checkpoint blockade. To identify mechanisms of immune evasion we carried out an in vivo pooled CRISPR-Cas9 screen targeting 240 KRAS-regulated genes using this novel immunogenic model. This identified a number of genes that increased sensitivity or caused resistance to anti-tumor immune responses. As an alternative approach we utilized the recently developed class of mutant-specific KRAS-G12C inhibitors to assess the impact of inhibiting KRAS signaling on anti-tumor immune responses in KPAR1.3 tumors. KRAS-inhibition stimulated adaptive immunity in vivo, which contributed to the response of KPAR1.3 tumors in immune-competent mice. Together these data suggest that targeting KRAS, or KRAS-driven mechanisms of immune evasion, could broaden the clinical efficacy of immunotherapy in KRAS-mutant NSCLC. Citation Format: Jesse Boumelha, Sophie de Carné, Pablo Romero, Miriam Moliona, Julian Downward. Uncovering mechanisms of immune evasion in a novel immunogenic model of KRAS-mutant lung cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P026.

Therapeutic KRASG12C inhibitors may substantially improve KRAS-mutant cancer patient care, considering the encouraging clinical data emerging from ongoing clinical trials. Since oncogene-targeting therapies often lead to resistance, the development of combinatorial therapies will be of utmost importance to achieve long-lasting tumour control. Given the known immunosuppressive role of KRAS, clinical trials investigating the combination of KRASG12C inhibitors with immune checkpoint blockade are currently underway for NSCLC patients. Here, we show that KRASG12C inhibition using MRTX1257 alleviates KRAS-driven immune suppression, through a myriad of mechanisms including the augmentation of tumour cell-intrinsic interferon responses. Despite harbouring a large number of somatic mutations, orthotopic 3LL (KRASG12CDNRAS) tumours are able to evade anti-tumour immune responses. Combining multicolour flow cytometry, RNA sequencing and imaging mass cytometry, we observed significantly increased T cell activation and infiltration after MRTX1257 treatment in addition to enhanced antigen presentation cells and gene sets, and reduced myeloid cells, creating an immune permissive microenvironment in these lung tumours. Despite these profound changes, combination of KRASG12C inhibitors and anti-PD-(L)1 antibodies fail to show synergism in our model, in contrast with Amgen’s published data using an immunogenic model of colorectal cancer. This data suggests that a pre-existing anti-tumour immunity may be crucial for clinical responses to KRAS inhibitors and immune checkpoint blockade combinations. Henceforth, ongoing clinical trials investigating this approach will likely only benefit a subset of patients and further research will be needed to examine novel therapeutic approaches and combinations for refractory patients. Citation Format: Edurne Mugarza, Febe Van Maldegem, Jesse Boumelha, Chris Moore, Sareena Rana, Miriam Llorian Sopena, Phil East, Miriam Molina Arcas, Julian Downward. Therapeutic KRASG12C inhibition alleviates KRAS-driven immunosuppression [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P111.

Oncogenic KRAS mutations drive tumourigenesis in 30% of non-small cell lung cancer (NSCLC). Despite much effort, targeted therapies that aim to directly inhibit signalling pathways downstream of KRAS have limited clinical benefits for NSCLC patients, but the recent approval of PD-1/PD-L1 antibodies has led to striking durable responses. However, only a fraction of patients respond and therefore a deeper understanding of the mechanisms that drive immune evasion are required in order to broaden the clinical efficacy of immunotherapy. Increasing evidence suggests that oncogenic signalling pathways greatly influence the tumour immune landscape to impair anti-tumour immune responses. We therefore aim to understand the mechanisms by which KRAS signalling mediates immune evasion in lung cancer. Current mouse models of KRAS-mutant lung cancer are poorly immunogenic, limiting investigations into tumour-immune interactions. To overcome this, we generated a novel transplantable KRAS-mutant lung cancer model, KPAR1.3, which triggers spontaneous anti-tumour immune responses and is sensitive to immune checkpoint blockade. To identify mechanisms of immune evasion we carried out an in vivo pooled CRISPR-Cas9 screen targeting 240 KRAS-regulated genes using this novel immunogenic model. This identified a number of genes that increased sensitivity or caused resistance to anti-tumour immune responses. As an alternative approach we utilised the recently developed class of mutant-specific KRAS-G12C inhibitors to assess the impact of inhibiting KRAS signalling on anti-tumour immune responses in KPAR1.3 tumours. KRAS-inhibition stimulated adaptive immunity in vivo, which contributed to the response of KPAR1.3 tumours in immune-competent mice. Together these data suggest that targeting KRAS, or KRAS-driven mechanisms of immune evasion, could broaden the clinical efficacy of immunotherapy in KRAS-mutant NSCLC. Citation Format: Jesse Boumelha, Sophie de Carné, Edurne Mugarza, Pablo Romero-Clavijo, Miriam Molina-Arcas, Julian Downward. Uncovering mechanisms of immune evasion in a novel immunogenic model of KRAS-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1820.