KRAS-mutant Lung Cancer Research Articles

IKKβ Kinase Promotes Stemness, Migration, and Invasion in KRAS-Driven Lung Adenocarcinoma Cells.

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase β (IKKβ) to promote lung tumourigenesis, we hypothesized that inhibition of IKKβ would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. IKKβ targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKβ targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKβ is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease.

Abstract 3349: Evolution of the gut Amicrobiome during the pathogenesis of smoking-associated Kras-mutant lung cancer

Abstract Relative to other tumors in smokers, KRAS-mutant lung adenocarcinomas (LUADs) display dismal prognosis warranting the need for early management of this disease. Limiting these advances is our gap in knowledge of events that drive KRAS-mutant LUAD oncogenesis. Host defense systems, such as those elicited by the gut microbiome, were recently shown to influence tumors external to the gastrointestinal tract (e.g. melanomas), thus highlighting the microbiome as an orchestrator of oncogenesis. Yet, gut microbiome changes in early stages of KRAS-mutant LUAD development are not known. We recently reported that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-), in contrast to wild type (WT) littermates, develop LUADs which are accelerated following exposure to tobacco carcinogen (nicotine-specific nitrosamine ketone/NNK). LUADs in the tobacco exposed Gprc5a-/- mouse model exhibit high somatic mutation burdens, driver Kras variants and other co-occurring drivers, features constituting a “perfect storm” for LUAD pathogenesis in smokers. Using this human-relevant model, we performed 16S-Seq of fecal samples collected prior to and at several time points post-NNK exposure during LUAD development. We found significant, some progressive, microbial changes during the pathogenesis of LUAD, including abolishment of phylum Verrucomicrobia, progressive increase in tumor-promoting genera Odoribacter spp., gradual decrease in Akkermansia spp., which was previously shown to be associated with response to PD-1 blockade, as well as reduced abundance of Ruminococcus which was previously reported to be suppressed during colon carcinogenesis. Additionally, and prior to tumor onset, lungs and immune cells of these Gprc5a-/- mice exhibited markedly elevated expression of lipocalin 2 (LCN2), an antimicrobial protein released from host cells during microbiome imbalance and inflammation. We further found that Gprc5a-/- mice with knockout of Lcn2 exhibited increased tumors compared to similarly exposed Gprc5a-/- with intact Lcn2. These effects were accompanied by widespread changes in the gut microbiome including increased abundance of tumor-promoting Alistipes spp. and, conversely, reduced abundance of Lactobacillus spp. Our data show that host defense mediated by LCN2 counteracts Kras-mutant LUAD development by restricting gut microbiome imbalance (i.e., maintaining gut microbiome homeostasis and diversity), suggesting a protective role during Kras-mutant LUAD development. Our overall findings inform on novel pathways implicating antimicrobial host defense mechanisms in the development of smoking-associated Kras-mutant LUAD. Efforts are underway to discern specific microbiome profiles that are likely causally related to smoking-associated Kras-mutant LUAD development as well as to develop and test preclinical microbiome intervention strategies for this malignancy. Citation Format: Warapen Treekitkarnmongkol, Casey Finnicum, Ansam Sinjab, Maya Hassane, Christel Davis, Gareth E. Davies, Kristi L. Hoffman, Junya Fujimoto, Florencia McAllister, Boris Sepesi, Tina Cascone, Robert R. Jenq, Joseph Petrosino, Erik Ehli, Seyed J. Moghaddam, Humam Kadara. Evolution of the gut microbiome during the pathogenesis of smoking-associated Kras-mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3349.

Abstract A37: Implication of targeting YAP1 in KRAS-mutant lung cancer cells

Abstract Lung cancer is the leading cause of cancer death worldwide, and it is estimated that 1.6 million deaths occur per year. Non-small cell lung cancer (NSCLC), mainly consisting of adenocarcinoma and squamous cell carcinoma, accounts for more than 80% of all new lung cancers with poor prognosis. Cytotoxic chemotherapy has improved the prognosis of NSCLC, and new advances in the discovery of oncogenic drivers and specific targeted therapies have brought significant improvement in the outcome of NSCLC patients. KRAS is one of the most frequently mutated oncogenes in human NSCLC. Despite intense efforts to develop drugs targeting the mutant KRAS, no effective therapeutic strategies have been successfully made in the clinical stage. In an attempt to interrogate the therapeutic candidate for the obstacle named KRAS, we performed small-molecule screening by assessing drug sensitivity from cell viability assay. We have previously described the identification of benzimidazole derivatives as selective cytotoxic agents for KRAS-mutant lung cancer cells. Here, we report another compound, verteporfin, a YAP1 inhibitor, showed a significant effect for reducing cell viability in KRAS-mutant lung cancer cells. Further validation assays using 16 cell lines (8 KRAS-mutant and 8 wild-type) strengthened the results of the primary screening. Recent studies have provided the relationship between YAP1 and KRAS for tumorigenesis in various cancers and urged us to explore the YAP1 inhibitor as an archer of this evil oncogene, KRAS. To determine the role of YAP1 in these cascades, we carried out the validation assay to compare the effect of verteporfin and the knockdown of YAP1 on cell viability in KRAS-mutant lung cancer cells. Whereas verteporfin showed the inhibitory effects in KRAS-mutant lung cancer cells in a dose-dependent manner, YAP1 knockdown did not show such inhibitory effect to that extent. The results of Western blotting and immunofluorescence suggested that the effects between verteporfin and YAP1 knockdown showed different manner. These facts led us to formulate the hypothesis that the activity of verteporfin is not only serving as a YAP1 inhibitor but other mechanisms are involved. Thus, we planned to examine the difference between the effects of verteporfin and YAP1 knockdown with RNA sequencing analyses and evaluated the factors surrounding the Hippo pathway or other pathways. As a result, there are many genes that overlap between verteporfin treatment and YAP1 knockdown, and genes related significantly only when treated with verteporfin include oncogenes such as TP53 and ERBB2, suggesting that elements other than YAP1 inhibition may be involved in cancer cell proliferation treated with verteporfin. These analyses pose a challenge to the use of verteporfin as a specific YAP1 inhibitor and will provide additional insights into the pathogenesis and the therapeutic strategy targeting via the Hippo pathway for KRAS-mutant lung cancer cells. Citation Format: Iwao Shimomura, Yusuke Yamamoto, Yuji Tada, Koichiro Tatsumi, Takahiro Ochiya. Implication of targeting YAP1 in KRAS-mutant lung cancer cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A37.

Impaired AKT signaling and lung tumorigenesis by PIERCE1 ablation in KRAS-mutant non-small cell lung cancer

KRAS-mutant non-small cell lung cancer (NSCLC) is a major lung cancer subtype that leads to many cancer-related deaths worldwide. Although numerous studies on KRAS-mutant type NSCLC have been conducted, new oncogenic or tumor suppressive genes need to be detected because a large proportion of NSCLC patients does not respond to currently used therapeutics. Here, we show the tumor-promoting function of a cell cycle-related protein, PIERCE1, in KRAS-mutant NSCLC. Mechanistically, PIERCE1 depletion inhibits cell growth and AKT phosphorylation (pAKT) at S473, which is particularly observed in KRAS-mutant lung cancers. Analyses of AKT-related genes using microarray, immunoblotting, and real-time quantitative PCR indicated that PIERCE1 negatively regulates the gene expression of the AKT suppressor, TRIB3, through the CHOP pathway, which is a key regulatory pathway for TRIB3 expression. Similarly, in vivo analyses of PIERCE1 depletion in the KRAS mutation-related lung cancer mouse models revealed the suppressive effect of PIERCE1 knockout in urethane- and KRASG12D-induced lung tumorigenesis with decreased pAKT levels observed in the tumors. Tissue microarrays of human lung cancers indicated the expression of PIERCE1 in 83% of lung cancers and its correlation with pAKT expression. Thus, we illustrate how PIERCE1 depletion may serve as a therapeutic strategy against KRAS-mutant NSCLC and propose the clinical benefit of PIERCE1.

Integrin-linked kinase (ILK) regulates KRAS, IPP complex and Ras suppressor-1 (RSU1) promoting lung adenocarcinoma progression and poor survival.

Integrin-linked kinase (ILK) forms a heterotrimeric protein complex with PINCH and PARVIN (IPP) in Focal Adhesions (FAs) that acts as a signaling platform between the cell and its microenvironment regulating important cancer-related functions. We aimed to elucidate the role of ILK in lung adenocarcinoma (LUADC) focusing on a possible link with KRAS oncogene. We used immunohistochemistry on human tissue samples and KRAS-driven LUADC in mice, analysis of large scale publicly available RNA sequencing data, ILK overexpression and pharmacological inhibition as well as knockdown of KRAS in lung cancer cells. ILK, PINCH1 and PARVB (IPP) proteins are overexpressed in human LUADC and KRAS-driven LUADC in mice representing poor prognostic indicators. Genes implicated in ILK signaling are significantly enriched in KRAS-driven LUADC. Silencing of KRAS, as well as, overexpression and pharmacological inhibition of ILK in lung cancer cells provide evidence of a two-way association between ILK and KRAS. Upregulation of PINCH, PARVB and Ras suppressor-1 (RSU1) expression was demonstrated in ILK overexpressing lung cancer cells in addition to a significant positive correlation between these factors in tissue samples, while KRAS silencing downregulates IPP and RSU1. Pharmacological inhibition of ILK in KRAS mutant lung cancer cells suppresses cell growth, migration, EMT and increases sensitivityto platinum-based chemotherapy. ILK promotes an aggressive lung cancer phenotype with prognostic and therapeutic value through functions that involve KRAS, IPP complex and RSU1, rendering ILK a promising biomarker and therapeutic target in lung adenocarcinoma.

Emerging Therapeutic Implications of STK11 Mutation: Case Series.

STK11 was first recognized as a tumor suppressor gene in the late 1990s based on linkage analysis of patients with Peutz-Jeghers syndrome. STK11 encodes LKB1, an intracellular serine-threonine kinase involved in cellular metabolism, cell polarization, regulation of apoptosis, and DNA damage response. Recurrent somatic loss-of-function mutations occur in multiple cancer types, most notably in 13% of lung adenocarcinomas. Recent reports indicate that KRAS-mutant non-small cell lung cancers harboring co-mutations in STK11 do not respond to PD-1 axis inhibitors. We present three patients with STK11-mutated tumors and discuss the proposed mechanisms by which germline and somatic alterations in STK11 promote carcinogenesis, potential approaches for therapeutic targeting, and the new data on resistance to immune checkpoint inhibitors. KEY POINTS: STK11 is a tumor suppressor gene, and loss-of-function mutations are oncogenic, due at least in part to loss of AMPK regulation of mTOR and HIF-1-α. Clinical trials are under way, offering hope to patients whose STK11-mutated tumors are refractory and/or have progressed on chemotherapeutic regimens. Whether gastrointestinal cancers with STK11 loss of function will show the same outcome and potential refractoriness to immune therapy that were reported for lung cancer is unknown. However, physicians managing such patients should consider the experience in lung cancer, particularly outside the context of a clinical trial. In the CheckMate-057 trial lung tumors harboring co-mutations in KRAS and STK11 had an inferior response to PD-1 axis inhibitors. Coupled with the observation that STK11-mutated tumors were found to have a cold immune microenvironment regardless of KRAS status, the conclusion could extend to KRAS wild-type tumors with STK11 mutation. Current data suggest that the use of PD-1 axis inhibitors may be ill advised in the presence of STK11 mutation.

Synergistic effects of piperlongumine and gemcitabine against KRAS mutant lung cancer.

To determine the combined efficacy of piperlongumine and gemcitabine for treatment of KRAS mutant lung cancer. The cell growth inhibition of piperlongumine, gemcitabine, and piperlongumine plus gemcitabine was measured by Cell Counting Kit‑8 assay and the combination index was calculated. In addition, the combined effects of piperlongumine and gemcitabine on cell apoptosis, reactive oxygen species (ROS) contents, and microtubule-associated protein 1 light chain 3B (LC3B) expression were examined. Piperlongumine increased ROS contents and LC3B-II expression. Following the combined treatment with piperlongumine and 10 mM N-acetyl-L-cysteine (NAC), intracellular ROS and cell viability returned to normal levels, and the expression of LC3B-II decreased to the predose level. Gemcitabine also induced cell apoptosis, increased ROS contents, and LC3B-II expression. The combination of piperlongumine with gemcitabine exhibited a synergetic anticancer activity with the combination index <1. The combined application of gemcitabine and piperlongumine yielded synergistic effects on cell apoptosis, but failed to synergistically increase ROS levels and LC3B-II expression. Combination therapy with piperlongumine and gemcitabine is a promising treatment option for KRAS mutant lung cancer.

FCHO1560−571 peptide, a PKB kinase motif, inhibits tumor progression

BackgroundCell division is regulated by protein kinase B (PKB)-mediated FCH domain only 1 (FCHO1) phosphorylation. MethodsFCHO1560−571, a synthetic water-soluble peptide, was generated from the PKB substrate motif 560PPRRLRSRKVSC571 found in the human FCHO1 protein. ResultsIn this study, we found that in vitro FCHO1560−571 inhibits cell proliferation via PKB/ERK/SMAD4 pathways in KRAS-mutated A549 lung cancer cells. In addition, FCHO1560−571, at effective doses of 15 and 30 mg/kg, significantly suppressed tumor growth and decreased the size and weight of tumors in A549-xenograft mice. ConclusionThese results suggest that the FCHO1560−571 peptide could be a potential therapy for lung cancer.

Abstract PR06: An in vivo KRAS allelic series reveals distinct phenotypes of common oncogenic variants

Abstract KRAS is the most frequently mutated oncogene in cancer. Tumor sequencing has revealed a complex spectrum of KRAS mutations across different cancer types, yet there is little understanding of how specific KRAS alterations impact tumor in initiation, progression, or therapy response. Using high-fidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRASG12C), pancreas (KRASG12R), and colon (KRASG13D) cancers. Induction of each mutation in the developing mouse pancreas reveals striking quantitative and qualitative differences in the degree of ductal transformation and premalignant progression. Further, using organoid models we show that KRASG13D mutants respond to EGFR inhibition, while the antiproliferative effect of KRASG12C-selective inhibitors can be overcome by upstream EGFR signaling. Together, these new mouse strains provide an ideal method for investigating KRAS biology in vivo and for developing preclinical precision oncology models of KRAS-mutant pancreas, colon, and lung cancers. This abstract is also being presented as Poster A41. Citation Format: Maria Paz Zafra, Direna Alonso-Curbelo, Sukanya Goswami, Emma M Schatoff, Teng Han, John E. Wilkinson, Lukas E. Dow. An in vivo KRAS allelic series reveals distinct phenotypes of common oncogenic variants [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR06.

The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

Identifying additional mitogen-activated protein kinase (MAPK) pathway regulators is invaluable in aiding our understanding of the complex signaling networks that regulate cellular processes, including cell proliferation and survival. Here, using in vitro kinase assays and by expressing WT or kinase-dead MAPK kinase kinase 19 (MAP3K19) in the HEK293T cell line and assessing activation of the extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) signaling pathways, we defined MAP3K19 as a novel regulator of MAPK signaling. We also observed that overexpression of WT MAP3K19 activates both the ERK and JNK pathways in a panel of cancer cell lines. Furthermore, MAP3K19 sustained ERK pathway activation in the presence of inhibitors targeting the RAF proto-oncogene Ser/Thr protein kinase (RAF) and MAPK/ERK kinase, indicating that MAP3K19 activates ERK via a RAF-independent mechanism. Findings from in vitro and in-cell kinase assays demonstrate that MAP3K19 is a kinase that directly phosphorylates both MAPK/ERK kinase (MEK) and MAPK kinase 7 (MKK7). Results from an short-hairpin RNA screen indicated that MAP3K19 is essential for maintaining survival in KRAS-mutant cancers; therefore, we depleted or inhibited MAP3K19 in KRAS-mutant cancer cell lines and observed that this reduces viability and decreases ERK and JNK pathway activation. In summary, our results reveal that MAP3K19 directly activates the ERK and JNK cascades and highlight a role for this kinase in maintaining survival of KRAS-mutant lung cancer cells.

Clinical characteristics and anti-PD-(L)1 treatment outcomes of KRAS-G12C mutant lung cancer compared to other molecular subtypes of KRAS-mutant lung cancer.

9596 Background: KRAS mutations are identified in approximately 30% of NSCLC. There are no FDA approved targeted therapies for patients with KRAS-mutant non-small cell lung cancer (NSCLC) but novel direct inhibitors of KRAS G12C have shown some activity in early phase clinical trials. We hypothesized that patients with KRAS-G12C mutations may have distinct clinical characteristics and responses to systemic therapies compared to patients with non-G12C subtypes. Methods: We identified patients with KRAS-mutant lung cancers who underwent next-generation sequencing with MSK-IMPACT, between January 2014 and December 2018. Baseline characteristics were compared with the Chi-square and Fisher’s exact test for categorical data and Wilcoxon rank-rum test for continuous data. Overall survival was calculated from time of diagnosis of metastatic/recurrent disease to date of death or last follow up, with left truncation to account for time of MSK-IMPACT. Overall survival was compared between groups using the Cox proportional-hazards model. Response evaluations where performed by independent thoracic radiologists according to RECIST 1. and compared between group with the Fisher’s exact test. Results: We identified 1194 patients with KRAS -mutant NSCLC, 772 with recurrent or metastatic disease. Of patients with advanced disease, 46% (352/772) had mutations in KRAS-G12C and 54% harbored non-G12C mutations (15% G12D, 16% G12V, 8% G12A, 4% G13D). Co-mutation patterns were similar with respect to KEAP1 (p=0.9) and STK11 (p=1.0). Patients with non-G12C mutations had a higher proportion of never smokers (10% vs 1.4% p&lt;0.001). The median OS from diagnosis was 13 months for G12C and non-G12C patients (p=0.99). 45% (347/772) received 1L or 2L line treatment with PD-(L)1 inhibitor. RECIST measurements were available for 290/347 cases (84%). ORR with anti-PD-(L)1 treatment was 24% vs 28% in G12C vs non-G12C patients (p=0.5). In patients with PD-L1 50% (n=103), ORR was 39% for G12C vs 58% non-G12C patients (p=0.06). Conclusions: KRAS G12C mutations are present in 12% of patients with NSCLC and represent a relevant subtype of NSCLC given KRAS G12C inhibitors now in clinical development. Baseline characteristics including co-mutation patterns are similar between patients with G12C and non-G12C, except for smoking history. The efficacy of KRAS G12C direct inhibitors will need to be compared to other available therapies for KRAS mutant NSCLC (chemotherapy and PD-(L)1 inhibitors) to identify most effective therapeutic strategy.

Abstract B02: Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer

Abstract Lung cancer is the foremost cause of cancer-related deaths in the U.S. KRAS mutations are one of the most frequent oncogenic aberrations in lung adenocarcinoma patients. Despite harboring identical mutations, the response of distinct tumor cells to therapeutic agents varies due to epigenetic reprogramming of the tumor cells. Epithelial-mesenchymal transition (EMT) contributes to phenotypic heterogeneity and determines the response of tumor cells to therapeutic agents. Kras-mutant NSCLC cells have been shown to acquire EMT as a mechanism to evade therapeutic targeting. We have previously observed that murine and human epithelial lung cancer cells are highly sensitive to MEK inhibition whereas mesenchymal tumor cells remain largely resistant. We aim to identify the mechanisms conferring survival advantage and an intrinsic resistance to MEK inhibition to mesenchymal cells. Using a loss-of-function shRNA dropout screen for targets with FDA-approved drugs, we identified cyclin dependent kinase 4 (CDK4) as an important driver of growth in mesenchymal cells both in vitro and in vivo. Murine NSCLC cells are resistant to cell cycle arrest induced by serum starvation, which corresponds to higher protein levels of CDK4 and cyclin D1 allowing cells to transition from G1 to S phase. After release of cells from arrested state, the mesenchymal cells transition into S phase more rapidly than the epithelial cells, suggesting a higher dependency on CDK4-cyclin D1 complex of mesenchymal tumor cells. An Ex Vivo Tumor (EVT) system to culture lung tumors in a 3D matrix environment that retains tumor cell heterogeneity was utilized to test the therapeutic sensitivity of distinct tumor cell subpopulations. In response to treatment with a CDK4 inhibitor, we observed a depletion of mesenchymal tumor cells within heterogeneous EVTs. In combination with MEK inhibitor treatment, there was a net decrease in size and viability of EVTs. Single-agent treatment with CDK4 inhibitor upregulated pErk in mesenchymal tumor cells, increasing their sensitivity to MEK inhibition and achieving an additive response with combination treatment. Mechanistically, the effects are mediated by FOXM1, a transcriptional regulator of cell cycle. FOXM1 positively regulates CDK4-cyclin D1 activity and is negatively regulated by the miR-200 family, which is preferentially expressed in epithelial cells. Upon induction of miR-200 expression, there was a decrease in FOXM1. CDK4, cyclin D1 and pRB levels follow a similar trend, suggesting the phenotype observed in mesenchymal cells is mediated by FOXM1. Since EMT is modulated by the miR-200/Zeb1 axis, higher levels of the transcriptional repressor Zeb1 and consequently FOXM1 in mesenchymal tumor cells confer a survival advantage and an intrinsic resistance to MEK inhibition. Combinatorial treatment approaches with MEK and CDK4 inhibitors targeting different subpopulations within a tumor are an effective strategy to combat resistant outgrowth of epigenetic subsets in a heterogeneous Kras mutant tumor. Citation Format: Aparna Padhye, B. Leticia Rodriguez, Jared J. Fradette, Christin Ungewiss, Don L. Gibbons. Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B02.

Abstract IA16: The interplay between RAS/MAPK signaling and tumor immunology

Abstract The role of MAPK in regulating the expression of pathways important for immune recognition of tumors such as modulation of the antigen presenting machinery and MHC Class I expression is now well studied preclinically. The clinical experience of the combination of MAPK inhibition with checkpoint inhibition (anti-PD-L1/PD-1) in cancer, however, has been less understood. Here, we will review the clinical data for checkpoint inhibitors and correlation with outcomes in the context of KRAS mutations in lung cancer and melanoma. In addition, the clinical activity of combinations of checkpoint inhibitors with MAPK inhibitors will be reviewed for both inflamed cancers such as melanoma and noninflamed cancers such as colorectal cancer. Citation Format: Priti Hegde. The interplay between RAS/MAPK signaling and tumor immunology [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA16.

Abstract B23: Insight towards therapeutic susceptibility of KRAS mutant cancers from MRTX1257: A prototype selective inhibitor of KRAS G12C

Abstract KRAS G12C is a driver mutation and the most frequent KRAS mutation in lung cancer. The ability to effectively target mutated KRAS has remained elusive despite decades of research. A structure-based drug design discovery program identified mutant-selective, covalent inhibitors of KRAS G12C with low nanomolar cell potency and favorable oral drug properties. MRTX1257 is a research tool compound that demonstrates selective irreversible modification of KRAS G12C and inhibits ERK1/2 phosphorylation with an IC50 value of 1 nM in a H358 cell-based assay. MRTX1257 was evaluated in 3D ultra-low adherent (ULA) viability assays across a panel of KRAS G12C mutated cancer cell lines in vitro and inhibited the growth of 16 out of 17 KRAS G12C-mutant cell lines tested with IC50 values ranging from 0.3 to 62 nM. MRTX1257 was inactive in non-KRAS G12C-mutant cell lines. Oral administration of MRTX1257 to mice bearing H358 xenograft tumors demonstrated a dose-dependent modification of KRAS G12C-mutant protein and inhibition of ERK1/2 and S6 phosphorylation. MRTX1257 also demonstrated dose-dependent KRAS target modification and cytoreductive antitumor efficacy in the KRAS G12C-mutant MIA PaCa-2 xenograft model with durable complete regressions. MRTX1257 was evaluated across an additional large panel of KRAS G12C-mutant subcutaneous cell-derived and patient-derived xenografts and demonstrated broad-spectrum antitumor activity including regressions greater than 30% in 18 out of 23 models tested. A small subset of models, exemplified by the H2122 model, demonstrated rapid initial tumor regression, followed by tumor stasis, suggesting that there may be mechanisms of adaptive tolerance to MRTX1257 treatment. Consistent with drug tolerance kinetics, MAP kinase pathway reactivation was observed as evidenced by downregulation of dual specificity phosphatases (DUSPs) and rebound of pERK and pS6 signaling during continuous treatment. To further elucidate mechanisms of drug tolerance and to identify combination strategies that could address feedback signaling, we tested MRTX1257 together with ~70 rationally selected compounds across a panel of genetically characterized cell lines. Combination treatment with a pan-EGFR family inhibitor more fully inhibited downstream signaling in vitro and led to increased antitumor activity in some models in vivo. A subset of KRAS G12C mutation-positive tumors may exhibit heterogenous molecular characteristics and mechanisms of reactivating KRAS dependent signaling, which is susceptible to combinatorial treatment strategies. Together, these data indicate the therapeutic susceptibility and broad dependence of KRAS G12C mutation-positive tumors on KRAS for tumor cell growth and survival and support the feasibility of developing mutant-selective small molecules. Citation Format: Jill Hallin, Ruth Aranda, Brian R. Baer, David M. Briere, Michael R. Burkhard, Andrew Calinisan, Harrah Chiang, Lars D. Engstrom, Jay B. Fell, John P. Fischer, Lauren Hargis, Matthew A. Marx, Pete Olson, Niranjan Sudhakar, James G. Christensen. Insight towards therapeutic susceptibility of KRAS mutant cancers from MRTX1257: A prototype selective inhibitor of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B23.

Anlotinib Exerts Anti-Cancer Effects on KRAS-Mutated Lung Cancer Cell Through Suppressing the MEK/ERK Pathway.

BackgroundWith a high frequency of 30%, KRAS mutations in patients with non-small cell lung cancer (NSCLC) often lead to their poor response to most anti-cancer therapies. As a multi-target tyrosine kinase inhibitor, Anlotinib shows clinical efficacy against several types of cancer. However, its effects on KRAS mutant NSCLC and the underlying molecular mechanisms remain unclear.Materials and MethodsCell counting Kit-8 assay, colony formation assay, flow cytometry analysis, wound healing scratch assay, Transwell assay and xenograft mouse model were used to evaluate the anti-cancer effects of Anlotinib. The potential molecular mechanisms were determined by immunohistochemistry (IHC) and Western blotting.ResultsAnlotinib inhibited proliferation of KRAS mutant lung cancer cells and induced apoptosis in vitro. In addition, the migration and invasion abilities of these cells were also decreased after treatment with Anlotinib. It significantly suppressed tumor growth in vivo and prolonged the survival of the xenograft-bearing mice, which correlated to lower expression levels of Ki67 in the tumor tissues. Mechanistically, Anlotinib downregulated MEK and ERK as well as their phosphorylated forms in the KRAS mutant lung cancer cells.ConclusionAnlotinib inhibits the growth of KRAS mutant lung cancer cells partly via the suppression of the MEK/ERK pathway. Our findings provide novel insights into treating recalcitrant KRAS mutated NSCLC.

Abstract A44: SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors

Abstract FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate this pathway feedback activation is mediated through mutant KRAS. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huai-Xiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A44.

Abstract IA11: Tissue- and cell-specific functions of Hras, Kras4A, and Kras4B in skin and lung cancer

Abstract Mouse models of cancer have been instrumental in the development of our understanding of the processes that drive multistage carcinogenesis from the initiation stage to metastasis. Tumors induced in mice by exposure to carcinogenic chemicals replicate many of the biologic and genetic features of multistage cancer in human populations. Skin and lung tumors initiated by chemical mutagens generally have carcinogen-specific mutations in Hras or Kras, respectively, but the reasons for this selectivity are unknown. We have used mouse models of skin and lung cancer to address some of the unanswered questions in cancer biology related to the specific functions of Hras, Kras4A, and Kras4B in cancer development in vivo. The KRAS gene, encoding two splice isoforms (KRAS4A and KRAS4B), is mutated in many lethal human tumors and has become a major target for cancer drug discovery. We show that germline deletion of either the major Kras4B or the minor Kras4A isoform in mice confers complete resistance to development of Kras mutant lung cancers. Double heterozygotes that are able to express both isoforms, but in which splicing has been uncoupled, are also tumor resistant, suggesting that coordinated expression of both splice variants is necessary for cancer initiation in vivo. KRAS4A is enriched in human tumor cells with stem cell properties, and the two splice variants show different responses to inducers of hypoxia or ER stress. Our data help to rationalize previous conflicting information on metabolic changes in KRAS mutant tumors and suggest that KRAS4A and 4B may require separate targeting in cancer stem and progenitor cells. Citation Format: Allan Balmain. Tissue- and cell-specific functions of Hras, Kras4A, and Kras4B in skin and lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA11.

Abstract B32: Silencing of oncogenic KRAS by a mutant-favoring short interfering RNA

Abstract Oncogenic mutations in the KRAS gene are well-established drivers of cancer. Promising preclinical strategies including RNA interference (RNAi) have been developed to target oncogenic KRAS function, yet a clinically effective therapy to directly target KRAS remains to be achieved. While genetic knockdown of mutant KRAS (mKRAS) with RNAi is one promising approach, current methods are not mutant-selective, and thus they also decrease normal KRAS levels, raising concerns about normal cell toxicity. Here, we describe a custom short interfering RNA (siRNA) oligonucleotide (D1) designed to preferentially bind the most commonly occurring RAS missense mutations in codons 12 and 13. Our approach designed the siRNA to have three mismatches binding wild-type mRNA, but only two mismatches for the oncogenic mRNAs, thus having binding affinities more favorable for the mutant-specific sequences. Using NIH/3T3 mouse fibroblasts stably expressing human KRAS WT or mutant cDNA sequences and KRAS mutant lung cancer cell lines, we determined that D1 siRNA preferentially reduced mKRAS mRNAs versus the wild-type sequence at the transcriptional and protein levels. Phenotypically, D1 siRNA significantly reversed mKRAS-induced morphologic and growth transformation of NIH/3T3 cells. Furthermore, D1 siRNA significantly impaired the proliferation of several mKRAS lung cancer cells in 2D as well as 3D spheroids embedded in an extracellular matrix. To overcome clinical limitations of delivering the siRNA to lung tumors, we evaluated lipid nanoparticles (LNPs) as carriers of D1 siRNAs in orthotopic mKRAS lung cancer models and observed that Cy5.5-labeled LNPs are highly enriched in lung tumors and metastases. Compared with control siRNA-LNP treated mice, 344SQ (KRAS G12D) tumor-bearing mice injected once with D1 siRNA-LNPs had drastically decreased KRAS protein levels in tumors 72 h later (73% decrease, p=0.001). Using an orthotopically injected luciferase-labeled A549 (KRAS G12S) human lung cancer model, D1 siRNA-LNP treated mice had pronounced reductions in luciferase signal (56% reduction, p=0.045), chest wall metastases (67% reduction, p&amp;lt;0.001) and contralateral lung metastases (73% reduction, p&amp;lt;0.01). Interestingly, D1 siRNA-LNP did not impair tumor growth when applied in the 344SQ mKRAS orthotopic mouse lung model, consistent with the mKRAS-independent growth of this line. This result supports our conclusion that the effects of D1 siRNA-LNP in the A549 model are KRAS specific and highlight the importance of determining RAS-dependency a priori for KRAS-targeting therapeutics. Taken together, our data indicate a novel use of RNAi to specifically target oncogenic KRAS-driven cancers. Citation Format: Bjoern Papke, Amanda E.D. Van Swearingen, Anne Y. Feng, Salma H. Azam, Emily B. Harrison, Runying Yang, Adrienne D. Cox, Channing J. Der, Chad V. Pecot. Silencing of oncogenic KRAS by a mutant-favoring short interfering RNA [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B32.

Abstract IA06: Oncogenic Ras control of the tumor immune microenvironment

Abstract Despite RAS proteins being exceptionally well-validated cancer targets, little progress has been made towards targeting them effectively in the clinic. We have optimized combination targeting of effector pathways downstream of RAS for specific activity on KRAS mutant cells, with combinations targeting MEK, IGF-1R and mTORC1 being highly effective in preclinical models. Replacing MEK with G12C KRAS inhibitor reduces the toxicity of the combination. However, these combinations are not able to eradicate tumors, which recur once treatment is stopped. In order to improve the effectiveness of RAS-targeted therapies, we have investigated the mechanisms whereby RAS signaling pathways can promote local immune suppression in the tumor microenvironment through inducing the expression of immune checkpoint proteins, immune suppressive cytokines, and extracellular enzymes. One such pathway involves the control of mRNA stability of PD-L1 and multiple cytokines by the 3′AU rich element binding protein, tristetraprolin. Another pathway that appears to be strongly upregulated in RAS mutant tumors is signaling by extracellular adenosine, which is strongly immunosuppressive. In order to test potential combinations of targeted therapies and immunotherapies resulting from these studies, we have developed new mouse models for KRAS mutant lung cancer that show more realistic interaction of the immune system with the tumor, which is not seen in existing genetically engineered mouse models used to study RAS mutant cancer due their lack of tumor heterogeneity and mutational burden. This work also involves detailed analysis of the tumor immune microenvironment using imaging mass cytometry and comparison between manipulatable mouse models and human clinical samples from the TRACERx lung cancer study. Citation Format: Julian Downward. Oncogenic Ras control of the tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA06.

Abstract B49: “Triple wild-type” co-mutational profile in early-stage KRAS-mutant lung cancer

Abstract Introduction: Kirsten rat sarcoma viral oncogene (KRAS) is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), occurring in approximately 30% of cases. Recent work suggests KRAS -mutant (KRASm) NSCLC is a microcosm for diverse immune checkpoint inhibitor responses, partly dictated by its co-mutation with TP53 (“KP” group=~1/3 cases, “immune hot”), STK11 (“KL” group=~1/3 cases, “immune cold”) or CDKN2A (“KC” group=~1/3 cases). Here we identify and describe genomic and clinical associations of a further subset of KRASm NSCLC defined by “triple WT” status for common KRASm co-mutations. Method: Sequencing results from 364 early-stage NSCLC cases from the Cancer Research UK TRACERx program (whole-exome sequencing) and the Genomics England 100,000 genome program (whole-genome sequencing) were analyzed for KRASm, associated co-mutations in TP53, STK11 and CDKN2A, copy number changes in the RAS-RAF-MEK-ERK axis, tumor mutational burden (TMB) and mutational signatures. Clinical demographics and factors including tumor size, nodal status and stage were ascertained and assessed for statistical associations with sequencing data using the Mann-Whitney and Fisher’s exact tests. Results: Overall, 143/364 lung cancer cases (39%) were KRASm with 65/143 (45%) tumors identified as “KP,” 26/143 (18%) as “KL,” and 3/143 (2%) as “KC.” There were 49/143 KRASm cases (34%) with no co-mutation in TP53, STK11 or CDKN2A, defined as “triple wild-type” (triple WT). Relative to the KP, KL and KC genotypes, there was a positive association of the KRAS G12D mutation with triple WT status: 10/49 cases, 20%; KP/KL/KC: 6/94 cases, 6%; p=0.022). In the 100,000-genome cohort, TMB was similar for the triple WT and KL groups but significantly lower than that observed for KP (KP: median 10.55 mut/Mb, 95% CI 8.8-15.33; triple WT: median 6.96, 95% CI 4.87-12.53; p=0.036). Despite this lower median TMB, smoking-associated mutational signature 4 was common in triple WT tumors (triple WT: 13/14 pts, 93%; overall 76/114 pts, 67%). In the triple WT group overall, there was a reduction of &amp;gt;50% in cancers with copy number changes of NF1 and NRAS. There was an inverse association between pathologic stage III tumors and KRASm triple WT genotype (p=0.0014). Conclusion: There are a significant proportion of KRASm NSCLC patients whose tumors are triple WT for TP53, STK11 and CDKN2A. Despite their high frequency of smoking-associated mutational signatures, these tumors are characterized by low TMB and are more common in early-stage disease. They also associate more commonly with KRAS G12D. Our observations suggest a discrete KRASm subset that may have implications for stratification in trials of immune checkpoint inhibitors and/or targeted therapeutics of KRASm tumors. Citation Format: Colin R. Lindsay, Pantelis Nicola, Mariam Jamal-Hanjani, Andrew Wallace, Gareth Wilson, George Burghel, Helene Schlecht, Katie Baker, Eleanour Baker, Lynsey Priest, Jane Rogan, Sharzad Moghadam, Mathew Carter, Caroline Dive, Robert G. Bristow, Charles Swanton, William Newman, Fiona Blackhall. “Triple wild-type” co-mutational profile in early-stage KRAS-mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B49.