KRAS-driven Lung Adenocarcinoma Research Articles

NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis

The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. Invivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile invivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.

A mouse model for the study of anti-tumor T cell responses in Kras-driven lung adenocarcinoma

SummaryKras-driven lung adenocarcinoma (LUAD) is the most common lung cancer. A significant fraction of patients with Kras-driven LUAD respond to immunotherapy, but mechanistic studies of immune responses against LUAD have been limited because of a lack of immunotherapy-responsive models. We report the development of the immunogenic KP × NINJA (inversion inducible joined neoantigen) (KP-NINJA) LUAD model. This model allows temporal uncoupling of antigen and tumor induction, which allows one to wait until after infection-induced inflammation has subsided to induce neoantigen expression by tumors. Neoantigen expression is restricted to EPCAM+ cells in the lung and expression of neoantigen was more consistent between tumors than when neoantigens were encoded on lentiviruses. Moreover, tumors were infiltrated by tumor-specific CD8 T cells. Finally, LUAD cell lines derived from KP-NINJA mice were immunogenic and responded to immune checkpoint therapy (anti-PD1 and anti-CTLA4), providing means for future studies into the immunobiology of therapeutic responses in LUAD.

Abstract 2682: STK11-loss-dependent YAP1-mediated transcriptional activation promotes immune evasion in KRAS-driven lung adenocarcinoma

Abstract Why Serine Threonine Kinase 11 (STK11) loss of function (LoF) correlates with anti-PD1 therapy resistance in the context of KRAS-driven lung adenocarcinomas remains unclear and represents a critical outstanding question in the field. To address this, we knocked out STK11 in KRAS-driven human lung adenocarcinoma cell lines and performed RNAseq to identify STK11-loss-dependent differentially expressed genes. Subsequent pathway enrichment analyses implied activation of the HIPPO/YAP1 signaling network occurs in the absence of STK11. Based on work linking YAP1 with transcriptional regulation of cytokine-mediated inflammation and immune-evasion in colorectal cancer and gastric cancer, we wondered whether YAP1 activation might impact anti-PD1 therapy response in KRAS-driven lung cancers lacking STK11. To evaluate this possibility, we applied a YAP1 transcriptional signature to the differentially expressed genes and performed gene set enrichment analysis (GSEA) revealing a strong positive correlation between STK11 LoF and upregulation of YAP1 target genes, most notably induction of IL-6, IL-8 and IL-32. To validate YAP1-mediated transcriptional activation is downstream of STK11 loss, we reasoned we could rescue STK11-loss-dependent YAP1-mediated cytokine induction by genetic disruption of YAP1, or pharmacologic inhibition of YAP1 with verteporfin. STK11/YAP1 double knock-out cell lines express significantly less IL-6, IL-8 and IL-32 compared with STK11 KO lines. Further, inhibition of YAP1 with verteporfin phenocopied YAP1 genetic disruption in these cell lines, changes mirrored at the level of secreted protein. Finally, we demonstrate YAP1 chromatin occupancy is STK11-dependent. Together these data link STK11 LoF with YAP1-mediated transcriptional activation, specifically the upregulation of immune-evasion promoting cytokines including IL-6, IL-8 and IL-32. We posit these STK11-loss-dependent YAP1-mediated tumor-intrinsic changes could result in anti-PD1 therapy resistance and immune-evasion by promoting recruitment of myeloid-derived suppressor cells, neutrophils and T-regulatory cells to the tumor microenvironment. Further, our results indicate that YAP1 antagonism may represent a valid therapeutic approach to circumvent STK11-loss-dependent transcriptional changes and potentially restore sensitivity to anti-PD1 therapy. Elucidating the mechanism(s) linking STK11 with YAP1 transcriptional activation and evaluating the potential for beneficial therapeutic intervention remains the focus of our continuing work. Citation Format: David Joseph Seward. STK11-loss-dependent YAP1-mediated transcriptional activation promotes immune evasion in KRAS-driven lung adenocarcinoma [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 2682.

Abstract 2706: Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response

Abstract Our work demonstrates RNAscope allows simultaneous quantitative assessment of multiple transcripts in situ using formalin fixed paraffin embedded (FFPE) human non-small cell lung cancer (NSCLC) biopsies and suggests this methodology could be used as a rapid surrogate biomarker for genotype/transcriptome profiling stemming for Serine Threonine Kinase 11 (STK11) loss of function (LoF). Recent clinical studies have correlated anti-PD1 therapy resistance in NSCLC with somatic disruption of STK11. Historically, rapid assessment of STK11 status using immunohistochemistry (IHC) has been a challenge. Our work in human NSCLC cell lines suggests that STK11 loss correlates with changes in tumor intrinsic cytokine expression. Several recent studies have suggested such changes may drive immune evasion. We therefore wondered whether cytokine expression profiles from primary human NSCLC biopsies might be used as a reliable biomarker to predict anti-PD1 therapy response. Here we present our findings using multiplexed RNAscope probes on KRAS-driven primary human NSCLC biopsies with and without STK11 loss. This initial work focuses on cytokines identified using our cell culture models as a proof of principle approach. Using CRISPR-Cas9 we engineered paired STK11 KO cell lines derived from 3 independent KRAS-driven human lung adenocarcinomas (NCI-H441, NCI-H1792, NCI-H2009). RNAseq and differential gene expression analysis revealed STK11-loss-dependent increases in IL-6, IL-8 and IL-32 in all three lines. We then created FFPE cell blocks using these cell lines and validated the RNAseq expression changes with multiplexed RNAscope probes in situ. Next, we informatically searched the molecular pathology archives at the UVM medical center and identified primary KRAS-driven human NSCLC biopsies with and without predicted STK11 loss. We then performed multiplexed RNAscope analysis on these samples to quantify IL-6, IL-8 and IL-32 gene expression as a function of tumor genotype in vivo. Our data suggest RNAscope represents a valid methodology to exploit quantitative differential gene expression as a reliable and reproducible surrogate biomarker of somatic STK11 disruption in NSCLC. Our future studies aim to establish whether genotype-dependent cytokine profiles predict anti-PD1 therapy response in KRAS-driven lung adenocarcinomas. Citation Format: Hailey M. Sarausky, Sean M. Lenahan, David Joseph Seward. Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response [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 2706.

Abstract 2466: Stochastic loss of GLUL expression correlates with STK11-loss-dependent glutamine addiction and may impact anti-PD1 therapy resistance in NSCLC

Abstract Our work aims to delineate the molecular interactions linking somatic disruption of Serine-Threonine Kinase 11 (STK11) with anti-PD1 therapy resistance in KRAS-driven lung adenocarcinoma (LUAD). Data presented here suggests glutamine synthetase (GLUL) may modulate this phenotype. Immune checkpoint inhibitors (ICIs), including anti-PD1 monoclonal antibody therapy, represent a promising therapeutic option for non-small cell lung cancer (NSCLC) patients. Unfortunately, efficient use of these drugs is hampered by an inability to accurately predict which patients will respond. Clinical observations have correlated anti-PD1 therapy resistance in KRAS-driven LUAD with somatic STK11 disruption, but the mechanism(s) responsible remains unknown. To investigate this connection we knocked out STK11 in three different human KRAS-driven LUAD cell lines and performed RNA-seq analysis to identify STK11-loss-dependent differentially expressed genes. Intriguingly, GLUL expression was reproducibly lost in a subset of KRAS-driven/STK11 null clones across two different human lung adenocarcinoma cell lines. GLUL is an enzyme that converts glutamate to glutamine. Glutamine is a critical metabolite supplying carbon and nitrogen necessary for amino acid, nucleotide, and lipid synthesis. Additionally, glutamine plays an important role in redox homeostasis, chromatin organization, and intra-tumoral signaling. Glutamine dependency is a common phenotype across many solid tumor types, and has previously been linked with STK11-deficiency in the context of polycystic kidney disease. Our current work suggests loss of GLUL expression correlates with an altered tumor-intrinsic response to nutrient stress in KRAS-driven LUAD culminating in immune evasion and anti-PD1 therapy resistance. Ongoing experiments aimed to define the relationship between STK11 loss and GLUL expression suggest this phenomenon is stochastic in nature and may provide tumor cells with a selective advantage. In conclusion, understanding the biology underlying the molecular connection between STK11 and GLUL may reveal reliable biomarkers for anti-PD1 therapy response and suggest a method for restoring sensitivity to current therapies. Citation Format: Sean M. Lenahan, Hailey M. Sarausky, David Joseph Seward. Stochastic loss of GLUL expression correlates with STK11-loss-dependent glutamine addiction and may impact anti-PD1 therapy resistance in NSCLC [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 2466.

Aspirin-Triggered Resolvin D1 Reduces Proliferation and the Neutrophil to Lymphocyte Ratio in a Mutant KRAS-Driven Lung Adenocarcinoma Model.

Simple SummaryAspirin-triggered resolvin D1 (AT-RvD1) is biosynthesised by leukocytes as a mechanism to resolve inflammation during infection and/or injury. Emerging studies reveal that AT-RvD1 also has anti-cancer properties associated with stimulating macrophage-mediated clearance of tumour debris. No study to date has investigated how AT-RvD1 influences the neutrophil to lymphocyte ratio (NLR) in lung cancer, an established marker of poor prognosis. The biosynthesis of AT-RvD1 is dependent on the ALOX5 gene, and we reveal that ALOX5 mRNA expression was markedly reduced in lung adenocarcinoma tumours. We next utilised an oncogenic KrasG12D lung adenocarcinoma mouse model to investigate the efficacy of AT-RvD1 in vivo. We show for the first time that AT-RvD1 reduces tumour growth in the lungs of KrasG12D mice and alters the immune landscape in tumours by reducing the NLR.Tumour-associated neutrophils (TANs) can support tumour growth by suppressing cytotoxic lymphocytes. AT-RvD1 is an eicosanoid that can antagonise neutrophil trafficking instigated by ALX/FPR2 ligands such as serum amyloid A (SAA). We aimed to establish whether SAA and ALOX5 expression associates with TANs and investigate the immunomodulatory actions of AT-RvD1 in vivo. MPO-positive neutrophils were quantified in tumour blocks from lung adenocarcinoma (n = 48) and control tissue (n = 20) by IHC. Tumour expression of SAA and ALOX5 were analysed by RTqPCR and an oncogenic KrasG12D lung adenocarcinoma mouse model was used to investigate the in vivo efficacy of AT-RvD1 treatment. ALOX5 expression was markedly reduced in lung adenocarcinoma tumours. The SAA/ALOX5 ratio strongly correlated with TANs and was significantly increased in tumours harbouring an oncogenic KRAS mutation. AT-RvD1 treatment reduced tumour growth in KrasG12D mice, which was accompanied by suppressed cellular proliferation within parenchymal lesions. In addition, AT-RvD1 significantly reduced the neutrophil to lymphocyte ratio (NLR), an established prognostic marker of poor survival in adenocarcinoma. This study identifies a novel molecular signature whereby elevated levels of SAA relative to ALOX5 favour accumulation of TANs. Furthermore, the ALOX5/5-LO enzymatic product, AT-RvD1, markedly reduced the NLR and suppressed tumour growth in KrasG12D mice.

A Novel Mouse Model for the Study of Anti-Tumor T Cell Responses in Kras Driven Lung Adenocarcinoma

Kras-driven lung adenocarcinoma (LUAD) is one of the most common forms of lung cancer. A significant fraction of patients with Kras-driven LUAD respond to immunotherapy, but mechanistic studies of immune responses against LUAD have been limited because of a lack of immunotherapy-responsive models. The Kras-Lox-STOP-Lox-G12D p53 flox/flox (KP) model faithfully recapitulates human LUAD but does not elicit tumor-specific T cell responses because developing tumors lack neoantigens. In vivo lentiviral transduction has been used to express neoantigens in KP LUADs but this strategy has limitations. We report here the development of the immunogenic KP x NINJA (iNversion INducible Joined NeoAntigen) (KP-NINJA) LUAD model. This model allows for temporal uncoupling of antigen and tumor induction, which allows one to wait until after adeno/lentiviral infection-induced inflammation has subsided to induce neoantigen expression by tumors. Neoantigen expression is restricted to EPCAM+ epithelial cells, with no neoantigen expression in CD45+ immune cells in the lung. Analysis of neoantigen expression by FACS and immunofluorescence revealed that expression was more consistent between neoantigen+ tumors (~2x variance) than lentiviral-programmed neoantigen+ tumors (>80x variance). Moreover, tumors were infiltrated by tumor-specific CD8 T cells. Finally, LUAD cell lines derived from KP-NINJA mice were immunogenic and responded to immune checkpoint therapy with anti-PD-1 and anti-CTLA4, providing means for future studies into the immunobiology of therapeutic responses in LUAD.

Genetic Determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo.

In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2-the strongest drivers of growth in a KRAS-driven model-reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment. SIGNIFICANCE: By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease.This article is highlighted in the In This Issue feature, p. 1601.

TRIM58 is a Prognostic Biomarker Remodeling the Tumor Microenvironment in KRAS -Driven Lung Adenocarcinoma

Aim: To comprehensively analyze the expression profiles of ubiquitin-related genes (URGs) and determinepotential biomarkers in KRAS-driven lung adenocarcinoma (LUAD). Materials & methods: Differential expression analyses were performed between KRAS-wild and KRAS-mutant LUAD samples from The Cancer Genome Atlasdatabase, and 34 URGs were screened out. ESTIMATE and CIBERSORT methods were used to calculate the ratio of immune and stromal components. Results & conclusion: TRIM58 was positively correlated with abundances of M2 macrophages and resting mast cellsand negatively correlated with follicular helper T-cellabundances in KRAS-driven LUAD. TRIM58was a potential prognosis-associated indicator for tumor microenvironment modulation and playeda key role in TME-specific AS landscapes alterations in KRAS-driven LUAD.

Myc linked to dysregulation of cholesterol transport and storage in nonsmall cell lung cancer

Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC.

HDAC10 Regulates Cancer Stem-Like Cell Properties in KRAS-Driven Lung Adenocarcinoma.

Activation of oncogenic KRAS is the most common driving event in lung adenocarcinoma development. Despite the existing rationale for targeting activated KRAS and its downstream effectors, the failure of clinical trials to date indicates that the mechanism of KRAS-driven malignancy remains poorly understood. Here we report that histone deacetylase 10 (HDAC10) might function as a putative tumor suppressor in mice carrying a spontaneously activated oncogenic Kras allele. Hdac10 deletion accelerated KRAS-driven early-onset lung adenocarcinomas, increased macrophage infiltration in the tumor microenvironment, and shortened survival time in mice. Highly tumorigenic and stem-like lung adenocarcinoma cells were increased in Hdac10-deleted tumors compared with Hdac10 wild-type tumors. HDAC10 regulated the stem-like properties of KRAS-expressing tumor cells by targeting SOX9. Expression of SOX9 was significantly increased in Hdac10-deleted tumor cells and depletion of SOX9 in Hdac10 knockout (KO) lung adenocarcinoma cells inhibited growth of tumorspheres. The genes associated with TGFβ pathway were enriched in Hdac10 KO tumor cells, and activation of TGFβ signaling contributed to SOX9 induction in Hdac10 KO lung adenocarcinoma cells. Overall, our study evaluates the functions and mechanisms of action of HDAC10 in lung carcinogenesis that will inform the rationale for targeting its related regulatory signaling as an anticancer strategy. SIGNIFICANCE: These findings linking HDAC10 and lung tumorigenesis identify potential novel strategies for targeting HDAC10 as a treatment for lung cancer.

Abstract 4053: Mucin 5ac as a potential therapeutic target for KRAS-driven lung adenocarcinoma

Abstract Lung cancer continues to be the leading cause of cancer death in the United States, with more than 140,000 deaths per year. One of the main types of lung cancer is non-small cell lung cancer with lung adenocarcinoma (LUAD) being the most prominent subtype and is mainly due to oncogenic mutations like KRAS, which offers limited and unsuccessful treatments. The mucus barrier produced by mucins, particularly Mucin 5ac (Muc5ac) which is mainly expressed by lung epithelial cells, is essential for airway homeostasis. However, its overproduction can promote respiratory diseases such as chronic obstructive pulmonary disease (COPD), which is known to increase the risk of lung cancer by 3-10 fold. We have previously shown that Muc5ac is commonly expressed in patients with KRAS-driven LUAD and is associated with poor prognosis. Additionally, we found that lack of Muc5ac (genetic deletion) in mice with LUAD either induced chemically by urethane or genetically engineered with mutant KRAS presented with significantly reduced tumor burden. Therefore, we sought a translational approach by inhibiting Muc5ac in our KRAS-driven lung cancer mutant mouse model using an antisense oligonucleotide (ASO) against Muc5ac and hypothesized that the administration of this drug would result in decreased Muc5ac mRNA expression and reduced tumor burden. This was tested by intrapharyngeal administration of either ASO (5mg/kg) or vehicle (saline) twice a week to a cohort of six-week-old KRASG12D mutant mice for a total duration of eight weeks. Our results showed a 5-fold decrease in Muc5ac mRNA expression and a 42% reduction in overall tumor burden when compared to age and sex matched control mice. Additionally, analysis of bronchoalveolar lavage fluid showed a predominance of macrophages with significant downregulation of the protumorigenic M2 type macrophage polarization markers Fizz1 and Arg1 as well as significant upregulation of TNF-a, which suggests a mechanism mediated by direct tumor cytotoxic activity or apoptosis associated with a reduction in macrophage mediated immunosuppressive tumor microenvironment. In conclusion, our study suggests that administration of Muc5ac ASO effectively inhibits mucin production and ameliorates tumor progression, which ultimately could provide insight in both treatments and prevention against KRAS-driven LUAD. Citation Format: Walter V. Velasco Torrez, Shanshan Deng, Marco A. Ramos-Castaneda, Segundo W. Del Aguila-Soto, Seyed Moghaddam. Mucin 5ac as a potential therapeutic target for KRAS-driven lung adenocarcinoma [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 4053.

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.

Targeting platelets for improved outcome in KRAS-driven lung adenocarcinoma.

Elevated platelet count is associated with poor survival in certain solid cancers, including lung cancer. In addition, experimental transplantation of cancer cell lines has uncovered a role for platelets in blood-borne metastasis. These studies, however, do not account for heterogeneity between lung cancer subtypes. Subsequently, the role of platelets in the major subtypes of non-small cell lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SqCC)) is not fully understood. We utilised an autochthonous KrasLSL-G12D/+;p53flox/flox mouse model of lung ADC together with genetic models of thrombocytopenia to interrogate the role of platelets in lung cancer growth and progression. While thrombocytopenia failed to impact primary tumour growth, in experimental metastatic models however, thrombocytopenic mice displayed significantly extended survival. Utilising a novel thrombocytopenic immunocompromised mouse, the importance of platelets in metastatic dissemination was confirmed with human KRAS-mutant ADC cell lines. Finally, retrospective analysis of a NSCLC patient cohort revealed thrombocytosis was predictive of poor survival in ADC patients with metastatic disease. Interestingly, this association was not apparent in SqCC patients. Overall, these data highlight the possibility of patient stratification using thrombocytosis as a biomarker, and indicates opportunities for potential novel treatment strategies that combine anti-platelet and lung cancer therapies.

Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis1-3. We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis4. To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation5, as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.

Abstract IA05: Dimerization is critical for the functions of wild-type and mutant KRAS

Abstract Background: Mutations in KRAS are among the most frequent RAS alterations in human cancers and the prevalent driver event in lung adenocarcinoma (LUAD). There are no effective targeted therapies for KRAS-driven LUAD and chemotherapy remains the standard of care. Small-molecule inhibitors of the MAPK pathway, one of the prominent downstream KRAS mediators, show minimal clinical activity either as single agents or in combination with chemotherapy. Recent data demonstrated that loss of wild-type KRAS enhances tumor fitness in KRAS mutant cancer cells while concomitantly increasing sensitivity to MEK inhibition. We hypothesized that dimerization/oligomerization between KRAS proteins could be a key regulator for lung adenocarcinoma biology and determinant of treatment response. Methods: To study the role of KRAS dimerization, we selected and characterized a dimerization-impaired mutant KRAS (KRASD154Q). KRAS-driven murine and human LUAD cell lines lacking the wild-type KRAS allele were reconstituted with either KRASWT or KRASD154Q to evaluate their impact on tumor progression and response to MEK inhibition. In parallel, we generated an inducible system to force either wild-type/mutant or mutant/mutant KRAS dimerization by taking advantage of the rapamycin-analog mediated heterodimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB). The rapamycin-analog AP21967 induces FKBP/FRB dimerization, hence allowing a tunable control of wild-type/mutant versus mutant/mutant KRAS dimers formation. Results: We demonstrated, using FRET (Fluorescence Resonance Energy Transfer) assays, that KRASD154Q mutant disrupts KRAS dimerization without affecting membrane localization or GTPase activity. Introduction of KRASWT into LUAD cells that solely express mutant KRAS decreased their growth both in vitro and in vivo. In the presence of KRASWT both the IC50 and drug concentration required to inhibit pERK1/2 by MEK inhibitors increased in vivo and in vitro. These findings were not observed in the presence of the dimerization-impaired wild-type KRAS protein (KRASD154Q). Similarly, forced dimerization between wild-type/mutant KRAS resulted in impaired cell growth as compared to forced mutant/mutant KRAS dimerization. Interestingly, introduction of the D154Q in cis with an oncogenic KRAS mutation (G12C or G12D), in the absence of the wild-type KRAS allele led to a significant reduction of growth both in vitro and in vivo without impacting GTPase activity. Mechanistically, impaired mutant KRAS dimerization resulted in inefficient CRAF/BRAF heterodimers formation. Conclusions: Dimerization of wild-type KRAS with mutant KRAS results in growth inhibition and changes the therapeutic index for MEK inhibitors. Mutant-mutant KRAS dimerization is critical for the full oncogenic properties of mutant KRAS. Collectively, these observations suggest that strategies designed to interfere with KRAS dimerization should be evaluated as a therapeutic approach in KRAS-mutant cancers. Citation Format: Chiara Ambrogio. Dimerization is critical for the functions of wild-type and mutant KRAS [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 IA05.

Abstract A20: An essential role for Argonaute 2 in mouse models of KRAS driven cancers

Abstract In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing that is required for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed the genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D driven pancreatic cancer. Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in survival of KRASG12D mutant mice deficient in AGO2 expression. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis; however rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Since PanIN development requires EGFR and is not AGO2 dependent, we probed the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS (using co-IP and PLA analyses). In wild-type RAS expressing cells, grown in media containing serum, RAS-AGO2 co-localization was limited to the intracellular regions of the cells, which dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular basal levels. Using phosphorylation-deficient AGO2 mutants, we demonstrate that the disruption of wild type-RAS-AGO2 interaction is due to AGO2Y393 phosphorylation, a target of EGFR. Interestingly, the mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild-type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. Taken together, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. In the lung cancer mouse model, we also observed a similar dependence of AGO2 in KRAS-driven lung adenocarcinoma. Along with related abstracts detailing the mechanisms of OIS mediated by AGO2 (Ronald Siebenaler) and evidence of direct interaction between oncogenic KRAS and AGO2 with an affinity of 200nM (Jessica Waninger), we present our latest studies related to the KRAS-AGO2 interaction. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Xiao-Ming Wang, Kristin M. Juckette, Alice Xu, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Lisha Wang, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS driven cancers [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 A20.

Abstract B29: SHANK3 in oncogenic RAS signaling

Abstract RAS proteins play a direct causal role in human cancers, with activating mutations in RAS occurring in ∼30% of tumors. Despite comprehensive efforts, efficient targeting of RAS has not yet reached the clinic. Here, we report SHANK3, a synaptic scaffolding protein and a regulator of integrin activity, as a novel tumor suppressor targeting RAS proto-oncogenes, especially mutant KRAS. We previously found the N-terminal part of SHANK3 as an unexpected RAS-association domain with high affinity for active RAS proteins. Now we show that the direct binding of SHANK3 to active RAS abrogates the MAPK/ERK signaling and thereby inhibits the RAS-stimulated transformation and slows the growth of RAS-dependent tumor cells. SHANK3 expression is downregulated in RAS-driven human cancers, and high expression of SHANK3 correlates with improved patient survival in KRAS-driven pancreatic and lung adenocarcinoma. These data support that targeting the MAPK/ERK pathway by SHANK3 represents a novel promising therapeutic approach for RAS-driven cancers. Citation Format: Johanna Lilja, Guillaume Jcaquement, Itziar Martinez D. Posada, Petra Laasola, Ella-Maria Vesilahti, Mitro Miihkinen, Siiri Salomaa, Hans-Juergen Kreienkamp, Igor Barsukov, Daniel Abankwa, Johanna Ivaska. SHANK3 in oncogenic RAS signaling [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 B29.

IA02 Insights into KRAS Biology to Identify Potential Therapeutic Strategies

Mutations in KRAS are among the most frequent RAS alterations in human cancers and the prevalent driver event in lung adenocarcinoma (LUAD). There are still no effective targeted therapies for KRAS-driven LUAD, although specific KRAS G12C inhibitors are showing very promising results in clinical trials. Small-molecule inhibitors of the MAPK pathway, one of the prominent downstream KRAS mediators, showed minimal clinical activity either as single agents or in combination with chemotherapy. We observed that loss of wild-type KRAS enhances tumor fitness in KRAS mutant cancer cells while concomitantly increasing sensitivity to MEK inhibition. Given the challenges of reanalyzing prior clinical trials, future clinical studies of targeted inhibitors should evaluate and/or stratify patients based on the relative expression of wild-type and mutant KRAS alleles to determine their correlation with treatment outcome. We also showed that dimerization/oligomerization between KRAS proteins is a key regulator for lung adenocarcinoma biology and determinant of treatment response. We generated an inducible system to force either wild-type/mutant or mutant/mutant KRAS dimerization, which showed that forced dimerization between wild-type/mutant KRAS resulted in impaired cell growth as compared to forced mutant/mutant KRAS dimerization. Loss of wild-type KRAS enhances tumor fitness in KRAS mutant cancer cells while concomitantly increasing sensitivity to MEK inhibition. Dimerization of wild-type KRAS with mutant KRAS results in growth inhibition and changes the therapeutic index for MEK inhibitors. Mutant-mutant KRAS dimerization is critical for the full oncogenic properties of mutant KRAS. Collectively these observations suggest that strategies designed to interfere with KRAS dimerization should be evaluated as a therapeutic approach in KRAS mutant cancers.

An LKB1-SIK Axis Suppresses Lung Tumor Growth and Controls Differentiation.

The kinase LKB1 is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of LKB1 substrates in vivo, we used CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic KRAS-driven lung adenocarcinoma. We demonstrate that members of the SIK family are critical for constraining tumor development. Histologic and gene-expression similarities between LKB1- and SIK-deficient tumors suggest that SIKs and LKB1 operate within the same axis. Furthermore, a gene-expression signature reflecting SIK deficiency is enriched in LKB1-mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key LKB1-SIK tumor-suppressive axis and underscore the need to redirect efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression. SIGNIFICANCE: Uncovering the effectors of frequently altered tumor suppressor genes is critical for understanding the fundamental driving forces of cancer growth. Our identification of the SIK family of kinases as effectors of LKB1-mediated tumor suppression will refocus future mechanistic studies and may lead to new avenues for genotype-specific therapeutic interventions.This article is highlighted in the In This Issue feature, p. 1469.