KRAS Mutant NSCLC Research Articles

Abstract B156: Discovery and characterization of AZ6197, a potent and selective ERK1/2 inhibitor

Abstract The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial positive clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops by reactivation of the RAS/MAPK pathway. Direct targeting of ERK1/2 may provide another therapeutic option in tumors with mutations in BRAF or RAS genes and, importantly, may overcome acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Here we describe the discovery and characterization of compound 35 (Ward et al., J Med Chem 2017 27;60), also known as AZ6197. AZ6197 is a highly potent and selective inhibitor of ERK1 and ERK2, with IC50 of <1nM in biochemical assays. AZ6197 reduces cellular phospho-p90RSK1 levels in BRAF and RAS mutant cancer cell lines and inhibits proliferation of cancer cell lines exhibiting dysregulation of RAS/MAPK pathway signaling, including cell lines with mutations in BRAF, NRAS, or KRAS. AZ6197 is also active in multiple BRAF- and MEK- inhibitor resistant A375 melanoma cell lines, which have acquired a range of mutations that reactivate the RAS/MAPK pathway. In tumor xenograft models, AZ6197 reduces the pharmacodynamic (PD) biomarkers phospho-p90RSK1 and phospho-FRA1 in tumors in line with compound exposure levels, and induces significant tumor regression in the KRAS mutant NSCLC Calu 6 xenograft model. AZ6197 can be combined safely and effectively with the MEK1/2 inhibitor selumetinib in KRAS mutant NSCLC xenograft models, and this simultaneous targeting of MEK and ERK leads to synergistic tumor regressions being observed in multiple xenografts. In summary, AZ6197 is a potent and selective ERK1/2 inhibitor with activity in cell lines and tumor xenograft models with RAS/MAPK pathway activation. AZ6197 also has significant activity in BRAF mutant melanoma cell lines in which resistance to RAF and MEK inhibitors is due to the acquisition of NRAS and MEK1 mutations. Combination treatment of AZ6197 and the MEK1/2 inhibitor selumetinib drives synergistic tumor regression in multiple KRAS mutant xenograft models. Citation Format: Vikki Flemington, Iain Simpson, Emma Davies, David Robinson, Nicola Lindsay, Lyndsey Hanson, Philip Hopcroft, Michael Tonge, Karen Roberts, Richard Ward. Discovery and characterization of AZ6197, a potent and selective ERK1/2 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B156.

Highlights of This Issue

The receptor tyrosine kinase AXL is aberrantly expressed in many tumors, is a potent driver of invasive cell motility and has become increasingly recognized as a broadly relevant mechanism in cancer drug resistance. Here, it is reported that exposure of cancer cells to phosphatidylserine (PS)-expressing apoptotic bodies enhances migration of tumor cells via a PS-AXL signaling axis. These findings suggest that anti-cancer therapies that induce fractional cell killing contribute to the motility of surviving cells in AXL-expressing tumors, hence generating potentially problematic invasive and metastatic behavior.Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyltransferase (NAPRT) are rate-limiting enzymes in the nicotinamide adenine dinucleotide (NAD+) synthesis pathway. In this study, in vitro experiments reveal that chondrosarcoma cells exhibit a dose-dependent decrease in cell viability, 3D collagen invasion and colony formation upon treatment with NAMPT inhibitors. Strikingly, higher methylation of the NAPRT promoter was observed in high-grade versus low-grade chondrosarcomas. Therefore, chondrosarcoma patients, especially those of high histological grade with lower expression and hypermethylation of NAPRT, may benefit clinically from inhibition of the NAD synthesis pathway.Few actionable oncogenic driver mutations have been identified in nasopharyngeal carcinoma (NPC). However, a marked lymphocytic infiltrate and association with viral infection make immunotherapy an intriguing treatment option. Zhang and colleagues perform the first integrated analysis of gene expression, mutation, copy number, and clinical data in NPC, identifying gene expression subtypes characterized by distinct immune and Epstein-Barr virus (EBV) associated gene expression patterns. Prognostic associations with stromal tumor-infiltrating lymphocytes and a proliferation signature were also identified. This study is an important step in understanding how NPC tumor cells and EBV influence the tumor microenvironment (TME), and may help guide immunotherapy strategies in this disease.TWIST1 promotes tumorigenesis through induction of EMT, invasion and metastasis, as well as suppression of oncogene-induced senescence and apoptosis. Additionally, TWIST1 expression is required for oncogene-driven lung cancer, including tumors driven by mutant KRAS. Using a chemical-bioinformatic approach, Yochum and colleagues identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC. Targeting TWIST1 with a small-molecule therapeutic such as harmine and its potential analogues/derivatives, represents a novel treatment strategy in oncogene-driven lung cancer and other solid malignancies in which TWIST1 promotes tumorigenesis.

MA 05.02 STK11/LKB1 Loss of Function Genomic Alterations Predict Primary Resistance to PD-1/PD-L1 Axis Blockade in KRAS-Mutant NSCLC

MA 05.02 STK11/LKB1 Loss of Function Genomic Alterations Predict Primary Resistance to PD-1/PD-L1 Axis Blockade in KRAS-Mutant NSCLC

Selumetinib for the treatment of non-small cell lung cancer

ABSTRACTIntroduction: KRAS is the most frequently mutated oncogene in NSCLC, occurring in around a third of patients. However, this largest genomically defined subgroup of lung cancer patients seem to remain ‘undruggable’, with any effective targeted therapy approved at the moment. The prognostic and predictive power and thus the clinical utility of KRAS oncogenic mutations in lung cancer are highly debated issues, not supportive of KRAS testing in clinical practice of NSCLC therapy.Areas covered: A phase II trial in KRAS-mutant NSCLC had shown significant improvements in PFS and ORR in patients treated with selumetinib plus docetaxel compared to docetaxel alone. Disappointing data emerged from the next phase III trial in which the addition of selumetinib to docetaxel in patients with advanced KRAS mutant lung cancer did not improve survival or show clinical benefit.Expert opinion: Promising strategies against this common mutation are under evaluation in clinical trials. Combination therapies represent a potential approach for overcoming this complex pathway and potentiating the activity of other antitumor agents, by simultaneous inhibition of the RAS–RAF–MEK–MAPK pathway. Identifying predictive biomarkers, and delineating de novo and acquired resistance mechanisms are essential for future clinical development of MEK inhibitors.

Abstract CT046: Phase I dose escalation study of the CDK4/6 inhibitor palbociclib in combination with the MEK inhibitor PD-0325901 in patients with RAS mutant solid tumors

Abstract Background: Both MEK and CDK4/6 have been investigated as therapeutic targets in preclinical models of RAS mutant solid tumors. Multiple mechanisms contribute to synergism including the development of MAP kinase-dependence in RAS mutant cells exposed to CDK4/6 inhibition, leading to increased apoptosis, as well as enhanced induction of senescence with combinatorial vs. monotherapy treatment. We conducted a Phase 1 study of combined palbociclib and PD-0325901 in patients with RAS mutant solid tumors to assess safety, tolerability, and MTD, as well as pharmacokinetic parameters, preliminary efficacy and effects on mutant RAS allelic burden in plasma. Methods: Patients with RAS mutant solid tumors were enrolled utilizing a 3 + 3 design. Palbociclib and PD-0325901 were given orally once and twice daily, respectively for three of every four weeks. The maximum planned administered doses were 125 mg palbociclib daily and 8 mg PD-025901 twice daily. Pharmacokinetic parameters were measured on cycle 1 day 21 and plasma samples to measure RAS allelic burden were serially collected. Results: Twenty-five patients (17 with KRAS mutant NSCLC) who received at least one dose of each study drug were enrolled over 5 dose levels including (palbociclib/PD-0325901) 75/2, 75/4, 100/4, 125/4 and 125/8. The maximum administered dose of 125 mg palbociclib daily and 8 mg twice daily PD-0325901 was tolerable. One DLT of pneumonitis occurred at the 100/4 dose level. The most frequent (>10%) drug-related toxicities were leukopenia (72%), anemia (72%), thrombocytopenia (72%), neutropenia (64%), acneiform rash (64%), diarrhea (52%), fatigue (44%), lower extremity edema (32%), vomiting (28%), nausea (28%), oral mucositis (24%), increased AST (20%), increased creatinine (12%), epistaxis (12%), and blurred vision (12%). The median number of cycles completed was 3 (range: 1 - 28+). Across all patients, 1 patient (4%) with KRAS mutant NSCLC achieved partial response and 18 (72%) had stable disease as the best response. Eleven patients were progression-free > 3 months, and 6 were progression-free > 6 months, including 5 with KRAS mutant NSCLC, two of whom received prior immune checkpoint blockade. Among patients with KRAS mutant NSCLC, clinical benefit was seen among those with tumors harboring KRAS mutation alone, as well as those with tumors demonstrating concomitant loss of TP53 or CDKN2AB. A dose-dependent decrease in plasma RAS allelic burden was observed across dose levels. Conclusions: Administration of combined palbociclib and PD-0325901 was tolerable and produced promising progression-free survival among patients with KRAS mutant NSCLC. Additional dose levels utilizing continuous MEK inhibition are being evaluated. Citation Format: Geoffrey I. Shapiro, John Hilton, Leena Gandi, Nicole Chau, James Cleary, Andrew Wolanski, Adrienne Anderson, Brian Beardslee, Faith Hassinger, Ketki Bhushan, Elizabeth Downey, Joseph Gibson, Solida Pruitt-Thompson, Alona Muzikansky, Suzanne Barry, Nora Feeney, Cloud Paweletz, Geoffrey Oxnard, Jeffrey Supko, Pasi Jänne, Kwok-Kin Wong, Bruce Johnson. Phase I dose escalation study of the CDK4/6 inhibitor palbociclib in combination with the MEK inhibitor PD-0325901 in patients with RAS mutant solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT046. doi:10.1158/1538-7445.AM2017-CT046

Abstract 627: NLRC5 co-mutations are associated with impaired antigen presentation and immune exclusion in KRAS-mutant lung adenocarcinoma

Abstract Background: We recently reported that co-occurring genetic events constitute major determinants of the molecular diversity of KRAS-mutant lung adenocarcinoma (LUAC) (Skoulidis et al., Cancer Discovery, 2015). However, comprehensive evaluation of the functional impact of KRAS co-mutations on key cancer hallmarks is thus far lacking. Here, we find that inactivating mutations in NLRC5, a major transactivator of MHC class I molecules, are significantly enriched in KRAS-mutant LUAC and examine the impact of NLRC5 loss on the composition of the tumor immune microenvironment. Methods: Our cohorts consist of 513 LUACs from the TCGA (145 KRAS-mutant), 152 chemotherapy-naïve surgically resected LUAC from the PROSPECT cohort, 20 platinum-refractory KRAS-mutant LUAC from the BATTLE-2 clinical trial, as well as a panel of 31 KRAS-mutant NSCLC cell lines. Analysis of immune cell sub-population was performed using automated IF-based enumeration. Antigen presentation score was defined as the geometric mean mRNA expression of HLA-A, HLA-B, HLA-C and β2M. Results: In an unbiased analysis for genes significantly co-mutated with KRAS in LUAC (TCGA cohort) we identified NLRC5 (NLR family, CARD domain containing 5), encoding a recently discovered major transactivator of MHC class I genes (~11% of KRAS-mutant LUAC, odds ratio 2.99, P=0.0197).The spectrum of NLRC5 somatic mutations includes several nonsense and frameshift mutations, as well as missense mutations, many of which are predicted to abrogate normal NLRC5 function. In the TCGA cohort, KRAS/NLRC5 co-mutated tumors exhibited lower antigen presentation score compared to KRAS-mutant NLRC5 wild-type tumors (P=0.0369, t-test). Among KRAS-mutant LUAC from the TCGA, PROSPECT, BATTLE-2 cohorts expression of NLRC5 mRNA correlated tightly with the expression of core antigen presentation pathway components including HLA-A, HLA-B, HLA-C, β2M, TAP1, TAP2, PSMB8 and PSMB9 [in BATTLE-2: HLA-A r=0.7616, P=9.572e-05, HLA-B r=0.834, P=4.884e-06, HLA-C r=0.8029, P=2.036e-05, TAP1 r=0.8189, P=1.009e-05 ]. Similar results were obtained in a panel of 31 KRAS-mutant NSCLC cell line. Thus, both mutational and non-mutational mechanisms can account for NLRC5 inactivation. Finally, in a tumor microarray encompassing surgically resected, chemotherapy naïve LUAC (PROSPECT), NLRC5-low tumors (lower tertile for NLRC5 mRNA expression, N=34), exhibited reduced density of infiltrating CD3+ (P<0.0001, Mann-Whitney U test), CD8+(P<0.0001, Mann-Whitney U test) as well as PD-1+ cells (P<0.0001, Mann-Whitney U test) and PD-L1 Histo-score (P=0.0036, Mann-Whitney U test) compared to NLRC5-high tumors (N=41). Conclusions: Co-mutations in NLRC5, are enriched in KRAS-mutant LUAC and are associated with immune exclusion. KRAS co-mutations can shape the tumor immune micro-environment and may therefore predict for response - or lack thereof- to immunotherapy. Citation Format: Ferdinandos Skoulidis, Taghreed Hirz, Xiang Dong Lee, Jaime Rodriguez Canales, Edwin R. Parra, Pan Tong, Carmen Behrens, Vassiliki A. Papadimitrakopoulou, Jing Wang, Ignacio Wistuba, John V. Heymach. NLRC5 co-mutations are associated with impaired antigen presentation and immune exclusion in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2017-627

Therapeutic Targeting of Nuclear Export Inhibition in Lung Cancer

Intracellular compartmentalization and trafficking of molecules plays a critical role in complex and essential cellular processes. In lung cancer and other malignancies, aberrant nucleocytoplasmic transport of tumor suppressor proteins and cell cycle regulators results in tumorigenesis and inactivation of apoptosis. Pharmacologic agents targeting this process, termed selective inhibitors of nuclear export (SINE), have demonstrated antitumor efficacy in preclinical models and human clinical trials. Exportin-1 (XPO1), which serves as the sole exporter of several tumor suppressor proteins and cell cycle regulators, including retinoblastoma, adenomatous polyposis coli, p53, p73, p21, p27, forkhead box O, signal transducer and activator of transcription 3, inhibitor of κB, topoisomerase II, and protease activated receptor 4—is the principal focus of development of SINE. The most extensively studied of the SINE to date, the exportin-1 inhibitor selinexor (KPT-330 [Karyopharm Therapeutics, Inc., Newton Centre, MA]), has demonstrated single-agent anticancer activity and synergistic effects in combination regimens against multiple cancer types, with principal toxicities of low-grade cytopenias and gastrointestinal effects. SINE may have particular relevance in KRAS-driven tumors, for which this treatment strategy demonstrates significant synthetic lethality. A multicenter phase 1/2 clinical trial of selinexor in previously treated advanced KRAS-mutant NSCLC is under way.

Results from the biomarker-driven basket trial of RO5126766 (CH5127566), a potent RAF/MEK inhibitor, in RAS- or RAF-mutated malignancies including multiple myeloma.

2506 Background: RO5126766 is a potent RAF and MEK inhibitor with activity in xenografts models of RAS and RAF-mutated cancers. We present data from the RAS/RAF-mutated advanced solid tumor cohort and the initial results for the multiple myeloma (MM) cohort. Methods: Patients with KRAS, NRAS or BRAF-mutant tumours were treated with RO5127566 using a novel schedule:4mg twice weekly in 4-week cycles. For MM patients, it was given 3 weeks out of 4 and co-administration of weekly dexamethasone was authorised. Response assessment was completed using RECIST 1.1 criteria for solid tumours and the International Myeloma Working Group (IMWG) criteria were used for MM. Results: A total of 20 patients with solid tumours (10 NSCLC, 5 gynaecological cancers and 5 miscellaneous cancers) and 1 MM patients were evaluable. Among the 10 KRAS-mutant NSCLC patients, tumour regression was seen in 6/10 (60 %), of which 3/10 (30 %) were partial responses. Two of these patients had maintained response for over 1 year and one patient is still on study after 30 cycles. Of the gynaecological cancers, 3/5 patients (60%) achieved a partial response ( KRAS-mutant endometrial and ovarian cancer and BRAF-mutant ovarian). Of these patients, 1 of the KRAS mutants had received 2 previous lines of MEK inhibitors and the BRAF mutant had previously received a BRAF inhibitor. In the miscellaneous group, 4 patients with colorectal cancer (2 BRAF and 2 NRAS) and 1 patient with NRAS-mutant melanoma were treated and none responded. Two patients with MM have been treated so far (1 KRAS, 1 KRAS+NRAS). The one evaluable patient has had an IMWG partial response (PR) after 1 cycle (FLC-λ from 324 mg/L to 161mg/L, ratio 0.03 to 0.08) without concomittant dexamethasone. This patient was previously treated with an immunomodulatory drug, a proteasome inhibitor and two ASCTs. Conclusions: RO5126766 has shown exciting preliminary activity across a wide range of RAS- and RAF-mutated malignancies, with significant response rates in lung and gynaecological cancers. To our knowledge, the PR seen in our MM patient represents one of the first responses to a single-agent RAF/MEK inhibitor in multiple myeloma in a trial context. Clinical trial information: NCT02407509.

Highlights of This Issue

Although the use of Hsp90 inhibitors had emerged as a promising therapeutic approach for the “undruggable” KRAS, these agents failed in the clinic due to the development of rapid resistance. Chatterjee and colleagues set out to understand the mechanism(s) of acquired resistance to the Hsp90 inhibitor ganetespib, in order to develop rational Hsp90 inhibitor combinations to test in KRAS mutant NSCLC. They identified p90RSK as a critical targetable node in the ganetespib resistance mechanism and provided the preclinical rationale for the use of novel Hsp90 inhibitor combinations with ERK or PI3K/mTOR inhibitors that can be tested in the clinic.Hairy and Enhancer-of-split related with YRPW motif (Hey) transcription factors are highly expressed in a variety of cancers. However, few articles cover the important roles of Hey in tumor progression and avenues for cancer therapy through targeting Hey factors. Here, Liu and his colleagues discussed the mechanisms governing Hey factors in tumorigenesis, outlined successful agents reversing Hey expression and provided strategies to target Hey directly. Hey factors-targeted therapeutics may provide promising approaches for cancer treatment and help to improve clinical outcomes.Antibodies blocking the PD-1/PD-L1 interaction have been shown to enhance anti-tumor immunity in patients with diverse cancers. This report describes the preclinical characterization of REGN2810, a novel, fully human high-affinity anti-PD-1 antibody. REGN2810 enhances human primary T cell responses in vitro, and inhibits the growth of syngeneic tumors in mice genetically engineered to express a chimeric human/mouse PD-1 receptor from the mouse locus. PD-1 humanized mice allow for the first time to evaluate in vivo the preclinical activity of a clinical PD-1 blocking antibody. These preclinical results validate REGN2810 as a promising candidate for cancer immunotherapy.Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Husain and colleagues analyzed cfDNA in effusions from individuals with metastatic cancer and discovered copy number variations and somatic mutations within the effusate. The results indicate that cfDNA from ascites and pleural effusions may provide additional information complementing tumor and plasma cell-free DNA molecular characterization, and a context for important insights into clonal dynamic change within primary tumor and metastatic deposits.

STK11 Loss Stabilizes MCL1 and Confers Targetable Drug Resistance in KRAS Mutant NSCLC

The combined KRAS/STK11 (serine‐threonine kinase 11, LKB1) mutated genotype is prevalent and predictive of poor clinical outcome in NSCLC as tumors are drug resistant. Here, we demonstrate that the bromodomain and extraterminal inhibitor (BETi) JQ1 can induce apoptosis in KRASm NSCLC by targeting the pro‐apoptotic BIM anti‐apoptotic BCL2/BCL‐XL axis, but not in KRASm/STK11m due to additional elevated MCL1. Specifically, loss of STK11 stabilizes anti‐apoptotic MCL1 protein via activation of the PI3K‐mTOR signaling axis. In NSCLC cell line models, we have demonstrated that overexpressing myristoylated‐PI3K increased MCL1 expression whereas the PI3K/mTOR inhibitor BEZ235 reduced MCL1 in a dose, time, and proteasome‐dependent, yet relatively GSK3β‐independent, manner. Furthermore, MCL1 stability also attenuates the antitumor efficacy of the direct BCL2/BCL‐XL inhibitor, ABT‐263 (Navitoclax) in KRASm/STK11m cells. However, combination of BETi or ABT‐263 with MCL1 protein reduction by siRNA or BEZ235, or direct MCL1 inhibition by the BH3 mimetic, A1210477, could sensitize resistant cells to apoptosis while no monotherapy was effective. Therefore, STK11 mutation or aberrant PI3K/mTOR pathway activations emerge as biomarkers of BET‐ or BCL2/BCL‐XL inhibitor resistance, suggesting patient selection for combined MCL1 inhibition.Support or Funding InformationThis work is supported by research Scholar grant RSG‐14‐229‐01 from the American Cancer Society and United Against Lung Cancer Legacy Program (Lung Cancer Research Foundation). This work is also supported by research fellowship from Sumitomo life social welfare services foundation (E.K), MINECO (SAF2010‐21769), ISCIII (RD12/0036/0045) and Generalitat Valenciana (ACOMP/2013/156 and ACIF/2013/239) (J.C.), and National Institutes of Health grants CA140594, CA122794, CA163896, CA166480 (K.K.W)

P2.03b-031 Impact of PD-L1 Status on Clinical Response in SELECT-1: Selumetinib + Docetaxel in KRASm Advanced NSCLC: Topic: Biomarkers

Anti-PD-1/PD-L1 immunotherapy has delivered clinical benefit for patients with NSCLC, and PD-L1 has emerged as a predictive biomarker. In the Phase III SELECT-1 trial (NCT01933932), selumetinib (AZD6244, ARRY-142886), an oral, potent and selective, allosteric MEK1/2 inhibitor with a short half-life, plus second-line docetaxel did not provide clinical benefit for patients with KRAS-mutant (KRASm) NSCLC compared with placebo plus docetaxel (PBO+DOC). Although no incremental benefit was observed, it is important to evaluate biomarkers, such as PD-L1, to understand more about the biology of patients with KRASm NSCLC. In total, 510 patients with a prospectively, centrally confirmed KRAS mutation (cobas® KRAS Mutation Test, Roche Molecular Systems) were randomized 1:1 to selumetinib 75 mg BID, plus docetaxel 75 mg/m2 q21d (SEL+DOC), or PBO+DOC. Evaluations included progression-free survival (PFS) by investigator assessment (RECIST 1.1; primary endpoint), and overall survival (OS). Association of tumor PD-L1 status with clinical responses was assessed as an exploratory objective. PD-L1 status was centrally determined using the PD-L1 IHC 28-8 pharmDx test (Dako) for all patients with sufficient tumor sample. Samples with a pre-specified cut-off of ≥5% tumor cell staining were considered PD-L1 positive. SEL+DOC did not improve PFS or OS compared with PBO+DOC. PD-L1 status was determined for 385 (75%) patients: 224 (58%) samples were PD-L1 <5%, and 161 (42%) samples were PD-L1 ≥5%; the remaining 125 patients had unknown PD-L1 status due to insufficient tumor sample. Subgroups were balanced across treatments. PD-L1 subgroup analysis of PFS and OS is presented below.Tabled 1SubgroupEvents (%) in SEL+DOC groupEvents (%) in PBO+DOC groupHR (95% CI)PFSPD-L1 <5%94/112 (84%)101/112 (90%)0.89 (0.67, 1.18)PD-L1 ≥5%65/79 (82%)71/82 (87%)0.70 (0.50, 0.99)PD-L1 unknown59/63 (94%)57/62 (92%)1.24 (0.86, 1.79)OSPD-L1 <5%73/112 (65%)74/112 (66%)0.94 (0.68, 1.30)PD-L1 ≥5%55/79 (70%)58/82 (71%)0.89 (0.61, 1.28)PD-L1 unknown48/63 (76%)38/62 (61%)1.57 (1.02, 2.41) Open table in a new tab Prevalence of PD-L1 positive status in this KRASm cohort was similar to that reported for a pan-NSCLC cohort (Borghaei, NEJM 2015). No significant PFS or OS differences were observed between treatments in either PD-L1 positive or negative tumors. Additional biomarker analyses are planned for different KRAS codon mutations, and LKB1 and TP53 status.

P3.02b-024 Dynamics of EGFR Mutational Load in Urine and Plasma Correlates with Treatment Response in Advanced NSCLC: Topic: EGFR Biomarkers

NSCLC is a heterogeneous and dynamic disease where testing for key mutations is essential. With the emergence of clonal resistance, obtaining serial biopsies to assist in the real-time treatment decision-making has proven challenging. Molecular assessments of circulating tumor (ct)DNA has been previously shown feasible utilizing blood specimens. Here we additionally investigated the utility of serial urine ctDNA analysis in NSCLC. This is a prospective observational study of patients with non-squamous, tissue-confirmed EGFR, KRAS or ALK mutant NSCLC preparing to receive a systemic treatment regimen. Urine and blood specimens were collected at baseline, on treatment and at progression for ctDNA analyses. The primary endpoints were correlation between ctDNA and tumor-based molecular results, and measurable change in ctDNA with response by RECIST v1.1. Blood and urine samples were sent to Trovagene for DNA extraction and mutation enrichment NGS. Of the 34 patients enrolled thus far, interim blinded analysis of EGFR activating mutations (L858R, exon 19 deletions) was conducted in 20 patients with EGFR-positive tumors. Of 20 patients, 17 (85%) had detectable concordant EGFR mutation in pre-treatment urine and/or plasma. Of 11 patients with matched serial ctDNA samples, detectable EGFR mutation signal was observed in pre-treatment urine and/or plasma of 9 patients. These 9 patients received first to sixth line treatment with single EGFR TKI (n=3), combination TKIs (n=3), chemotherapy (n=1), immune checkpoint inhibitors alone (n=1) or in combination with TKI (n=1). In 9 of 9 patients, changes in ctDNA levels from baseline to cycle 2 day 1 on therapy correlated with best response to treatment: a 100% decrease in urine and plasma EGFR mutation levels was observed in 6 of 6 patients with partial response (PR, n=3) or stable disease (SD, n=3), while less than a 90% decrease or an increase in urine and plasma EGFR levels was observed in patients with progressive disease (PD, n=3). Mutation enrichment NGS testing by urine and plasma ctDNA correctly identified EGFR activating mutations in 85% of patients. Monitoring EGFR levels in urine/plasma enabled accurate assessment of response in advance of radiographic evaluation and regardless of therapy type in 100% of patients, with cut-point of a 90% decrease in EGFR load discriminating between patients with disease control (PR or SD) and patients with progressive disease. With continued enrollment, our study aims to further investigate clinical utility of urine and plasma ctDNA for early detection of resistance and discontinuation of inefficient therapy.

397PD KRAS mutation-induced upregulation of PD-L1 mediates immune escape in lung adenocarcinoma

Background Programmed death-ligand 1 (PD-L1) expression is associated with EGFR mutation and EML4-ALK rearrangement. Whether PD-L1 is regulated by other driver mutations like KRAS in lung adenocarcinoma and what is the molecular mechanism remain largely unknown. Methods In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by KRAS mutation. Relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by KRAS mutation and immune reactivation by ERK inhibitor and/or PD-1 blocking in tumor cells and T cells co-culture system. Results We found that PD-L1 expression was correlated with KRAS mutation in the cell lines and human tissue of lung adenocarcinoma. PD-L1 was up-regulated by KRAS mutation through p-ERK but not p-AKT signaling.We also found that KRAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3-positive T cells which was reversed by pembrolizumab or ERK-specific inhibitor. Blocking PD-1 by anti-PD-1 antibody or PD-L1 by ERK inhibitor could re-activate the anti-tumor immunity of T cells and decrease the survival rates of KRAS-mutant NSCLC cells in co-culture system in vitro. However, we did not observe the synergistic effect of pembrolizumab and ERK inhibitor together on killing of tumor cells in co-culture system. Conclusions Our study demonstrates that KRAS mutation induces PD-L1 expression through p-ERK signaling in lung adenocarcinoma. Therapy targeting PD-1/PD-L1 axis provides a promising treatment option for KRAS-mutant lung adenocarcinoma. Legal entity responsible for the study Sun Yat-sen University Cancer Center Funding Sun Yat-sen University Cancer Center Disclosure All authors have declared no conflicts of interest.

A Phase 1/1b Study Evaluating Trametinib Plus Docetaxel or Pemetrexed in Patients With Advanced Non–Small Cell Lung Cancer

ObjectivesThis two-part study evaluated trametinib, a MEK1/2 inhibitor, in combination with anticancer agents. Inhibition of MEK, a downstream effector of KRAS, demonstrated preclinical synergy with chemotherapy in KRAS-mutant NSCLC cell lines. Part 1 of this study identified recommended phase 2 doses of trametinib combinations. Part 2, reported herein, evaluated the safety, tolerability, pharmacokinetics, and efficacy of trametinib combinations in patients with NSCLC with and without KRAS mutations. MethodsPhase 1b evaluated trametinib plus docetaxel with growth factor support (trametinib, 2.0 mg once daily, and docetaxel, 75 mg/m2 every 3 weeks) or pemetrexed (trametinib, 1.5 mg once daily, and pemetrexed, 500 mg/m2 every 3 weeks). Eligibility criteria for the expansion cohorts included metastatic NSCLC with measurable disease, known KRAS mutation status, Eastern Cooperative Oncology Group performance status of 1 or lower, and no more than two prior regimens. ResultsThe primary end point of overall response rate (ORR) was met for both combinations. A confirmed partial response (PR) was observed in 10 of the 47 patients with NSCLC who received trametinib plus docetaxel (21%). The ORR was 18% (four PRs in 22 patients) in those with KRAS wild-type NSCLC versus 24% (six PRs in 25 patients) in those with KRAS-mutant NSCLC. Of the 42 patients with NSCLC treated with trametinib plus pemetrexed, six (14%) had a PR; the ORR was 17% (four of 23) in patients with KRAS-mutated NSCLC versus 11% (two of 19) in KRAS wild-type NSCLC. Adverse events—most commonly diarrhea, nausea, and fatigue—were manageable. ConclusionsTrametinib-plus-chemotherapy combinations were tolerable. Clinical activity exceeding the ORRs previously reported with docetaxel or pemetrexed alone in KRAS-mutated NSCLC and meeting prespecified criteria was observed.

XPO1 May Be Therapeutically Targetable in KRAS-Mutant NSCLC

Abstract XPO1 inhibition selectively targets KRAS-mutant NSCLC cells by suppressing NFκB signaling.

LBA47_PR - Selumetinib in combination with docetaxel as second-line treatment for patients with KRAS-mutant advanced NSCLC: Results from the phase III SELECT-1 trial

LBA47_PR - Selumetinib in combination with docetaxel as second-line treatment for patients with KRAS-mutant advanced NSCLC: Results from the phase III SELECT-1 trial

Abstract LB-214: FASN inhibitor TVB-2640 shows pharmacodynamic effect and evidence of clinical activity in KRAS-mutant NSCLC patients in a phase I study

Abstract TVB-2640 is an oral, first-in-class, selective and reversible inhibitor of fatty acid synthase (FASN) in Phase 1 testing in solid tumor patients (study 3V2640-CLIN-002). FASN is a central mediator of neoplastic lipogenesis and uniquely catalyzes the production of palmitate, a key signaling molecule and the building block for long chain fatty acids. Tumor cells frequently increase FASN expression compared to normal cells to support an increased dependence on de novo lipogenesis to produce palmitate, phospholipids, lipid second messengers, membranes and lipid rafts for oncogenic signaling. High FASN correlates with poor prognosis in several tumor types including NSCLC. Palmitoylation, the reversible attachment of palmitate to proteins regulates membrane interactions and dynamically alters protein trafficking and function. RAS proteins require palmitoylation for plasma membrane localization and signaling, and KRAS4A mutated at G12 specifically requires palmitoylation for oncogenic activity. FASN inhibitors may therefore provide a novel approach to target KRAS which to date has eluded many drug development approaches. In vitro viability assays showed that KRAS mutant NSCLC cell lines show greater sensitivity to FASN inhibitors than KRAS WT. Furthermore gene expression analysis of TCGA NSCLC tumors and PDX models showed that KRAS mutant cases are associated with a higher incidence of lipogenic features than KRAS WT. The ongoing Phase 1 study includes 2 NSCLC expansion cohorts; TVB-2640 as monotherapy or in combination with paclitaxel. Several biomarker approaches are ongoing to investigate pharmacodynamic activity and patient enrichment strategies. Metabolomic profiling of serum from 12 NSCLC patients showed increased serum malonyl carnitine and decreased palmitate- derived lipids after 8 or 15 days of TVB-2640 treatment. These changes are consistent with FASN inhibition, and also observed in preclinical models. A novel non-invasive approach using Sebutape patches to collect forehead sebum showed inhibition of de novo lipogenesis in 7/7 patients including 2 NSCLC. Decreases in wax esters, saturated and monounsaturated triglycerides were observed, indicating that these lipids are not restored by diet. To date, 16 NSCLC patients are considered evaluable for clinical activity; 6 have KRAS mutant tumors, 2 have wild type KRAS and 8 are unknown. Of the 6 known KRAS mutant patients, 3 are in the monotherapy cohort and all 3 have stable disease for 20, &amp;gt;25 and &amp;gt;37 weeks respectively. The remaining 3 KRAS mutant patients are on combination therapy; one achieved a partial response and remains on therapy (&amp;gt;22 weeks), 1 had stable disease (24 weeks), and 1 progressed. This study shows that TVB-2640 1) inhibits palmitate production and lipogenesis, and 2) shows evidence of clinical activity in KRAS mutant NSCLC patients. Additional biomarker and clinical analyses are ongoing in NSCLC and other tumor types. Citation Format: Marie O’Farrell, Tim Heuer, Katharine Grimmer, Richard Crowley, Joanna Waszczuk, Marina Fridlib, Richard Ventura, Claudia Rubio, Julie Lai, Doug Buckley, William McCulloch, George Kemble. FASN inhibitor TVB-2640 shows pharmacodynamic effect and evidence of clinical activity in KRAS-mutant NSCLC patients in a phase I study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-214.

Abstract 1869: Dislodgement of K-Ras from plasma membranes, induction of apoptosis and tumor regression by PCAIs, a novel class of polyisoprenylated small molecules

Abstract Although mutation-induced dysregulation of Ras signaling constitutes the biochemical change that drives some of the most difficult-to-manage cancers, directly targeting the constitutively active mutant Ras GTPases has not resulted in clinically useful drugs. Therefore, modulating Ras activity for targeted treatment of cancer remains an urgent healthcare need. In the current study, we investigated a novel class of compounds, the polyisoprenylated cysteinyl amide inhibitors (PCAIs), for their anticancer molecular mechanisms using the NSCLC cell panel with K-Ras and/or other mutant genes. Treatment of the lung cancer cells with PCAIs, NSL-RD-035, NSL-BA-036, NSL-BA-040, NSL-BA-055 and NSL-BA-040 resulted in concentration-dependent cell death in both K-Ras mutant (A549 and NCI-H1573), N-Ras mutant (NCI-H1299) and other (NCI-H661, NCI-H460, NCI-H1975, NCI-H1563) NSCLC cells. The PCAIs at sub- to low micromolar 1.0 -10 μM concentrations induced the degeneration of 3D spheroid cultures, inhibited, clonogenic cell growth, and induced marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 (p&amp;lt;0.001). NSL-BA-040 at 5 μM prominently dislodged YFP-tagged K-Ras, but not H-Ras and N-Ras from the plasma membranes of transfected A549 and NCI-H661 cells. NSL-RD-035 attenuated the tumor growth of A549 cells in xenografted athymic mice in vivo. Taken together, PCAIs likely suppress NSCLC cell growth by disrupting growth signaling through the dislodgement of K-Ras the plasma membrane. Citation Format: FELIX AMISSAH, ELIZABETH NTANTIE, ROSEMARY A. POKU, AUGUSTINE T. NKEMBO, OLUFISAYO O. SALAKO, HERNAN FLORES-ROZAS, NAZARIUS S. LAMANGO. Dislodgement of K-Ras from plasma membranes, induction of apoptosis and tumor regression by PCAIs, a novel class of polyisoprenylated small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1869.

Abstract 3865: Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer

Abstract PP2A is a phosphatase tumor suppressor that is dysregulated and deactivated in lung cancer. It is one of the most abundant cellular proteins and regulates the activity of numerous kinases Where achievable, restoration of PP2A function inhibits cancer progression, and notably, by the inhibition of the downstream effectors of the oncogenic kinases that initiate and drive cancer progression. In this study, we determined PP2A inactivation in human lung cancer with specific molecular genotypes and we ascertained the biological and functional consequences of PP2A reactivation. In assessing a lung cancer TMA, we identified that PP2A inactivation was correlated with poor survival and was significantly higher in patients with Kras mutations. In order to understand the therapeutic potential of restoration of PP2A activity in KRAS mutant lung cancer, our lab developed a series of small molecule activators of PP2A (SMAPs) through reverse engineering of tricyclic neuroleptic drugs. SMAP treatment of lung cancer cell lines resulted in an induction of apoptosis and decreased cell viability. Structural and biophysical studies have identified the site of drug binding and mechanism for PP2A activation by this small molecule series. Additionally, cell lines harboring drug-binding mutations were resistant to SMAP therapy as compared to wild type PP2A and EGFP control. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines revealed ERK signaling as a commonly perturbed pathway in drug treated cell lines. Given the marked dephosphorylation of ERK upon treatment of cell lines with SMAPs, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation for the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in a blunted apoptotic response to SMAP treatment. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Furthermore, combination of SMAPs with kinase inhibitors further decreased tumor growth in vivo. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of KRAS-mutant NSCLC. Citation Format: Jaya Sangodkar, Rita Tohme, Janna Kiselar, Sudeh Izadmehr, Divya Hoon, Sahar Mazhar, Abbey Perl, Danica Wiredja, Daniela Schlatzer, Shen Yao, David Kastrinsky, Neelesh Sharma, David Brautigan, Mark Chance, Alain Borczuk, Michael Ohlmeyer, Yiannis Ioannou, Goutham Narla. Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3865.

Kras mutations increase telomerase activity and targeting telomerase is a promising therapeutic strategy for Kras-mutant NSCLC.

As shortened telomeres inhibit tumor formation and prolong life span in a KrasG12D mouse lung cancer model, we investigated the implications of telomerase in Kras-mutant NSCLC. We found that Kras mutations increased TERT (telomerase reverse transcriptase) mRNA expression and telomerase activity and telomere length in both immortalized bronchial epithelial cells (BEAS-2B) and lung adenocarcinoma cells (Calu-3). MEK inhibition led to reduced TERT expression and telomerase activity. Furthermore, telomerase inhibitor BIBR1532 shortened telomere length and inhibited mutant Kras-induced long-term proliferation, colony formation and migration capabilities of BEAS-2B and Calu-3 cells. Importantly, BIBR1532 sensitized oncogenic Kras expressing Calu-3 cells to chemotherapeutic agents. The Calu-3-KrasG12D xenograft mouse model confirmed that BIBR1532 enhanced the antitumor efficacy of paclitaxel in vivo. In addition, higher TERT expression was seen in Kras-mutant NSCLC than that with wild-type Kras. Our data suggest that Kras mutations increase telomerase activity and telomere length by activating the RAS/MEK pathway, which contributes to an aggressive phenotype of NSCLC. Kras mutations-induced lung tumorigenesis and chemoresistance are attenuated by telomerase inhibition. Targeting telomerase/telomere may be a promising therapeutic strategy for patients with Kras-mutant NSCLC.