Adaptive Regulation of dNTP Homeostasis Confers Osimertinib Resistance in EGFR-Mutant Non-small Cell Lung Carcinoma.

Simple SummaryThe development of oncogene-targeted drugs has radically changed the course of non small cell lung carcinoma (NSCLC) in the advanced stage. Recently, the ADAURA trial demonstrated the efficacy of Osimertinib also in the adjuvant setting of EGFR-mutated NSCLC. This raises the question regarding whether the same paradigm applies also to currently approved drugs directed against non-EGFR NSCLC drivers. Herein we compared actionable genomic alterations in early- and advanced-stage NSCLC in 1961 unselected single-institution cases analyzed by routine molecular diagnostics procedures. Our data add significantly to the currently limited real-world data on actionable mutations in surgically resectable NSCLC. Our finding that distinct NSCLC genomic drivers are mutated at similar frequencies in early- and advanced-stage tumors implies that the relative biological potency of currently actionable NSCLC genomic drivers is conserved throughout clinical evolution and supports the hypothesis that genotype-matched therapies are likely to provide significant benefit in an adjuvant settingThe approval of osimertinib for adjuvant treatment of stage I–II–III EGFR-mutated NSCLC (early stage) represents a paradigm shift, raising the question of whether other genotype-matched therapeutics approved for advanced-stage NSCLC can also provide clinical benefit in the adjuvant setting. However, there is a paucity of real-world data on the prevalence of actionable genomic alterations (GAs) in early-stage NSCLC. We used next-generation sequencing, complemented by immunohistochemistry and fluorescence in situ hybridization, to screen our single-institution cohort of 1961 NSCLC consecutive cases for actionable molecular targets. The prevalence of actionable GAs was comparable in early versus advanced-stage NSCLC, the only exception being KRAS mutations (more frequent in early-stage cases). Consistent with advanced-stage tumors being more aggressive, co-occurrence of TP53 and EGFR GAs as well as copy number gains were less frequent in early-stage tumors. EGFR mutations and high expression of PD-L1 were inversely associated, whereas KRAS mutations and high PD-L1 reactivity showed positive association. Recapitulating advanced-stage tumors, early-stage NSCLC had the highest share of EGFR mutations in lepidic and acinar subtypes. Resected lepidic tumors contained the highest proportion of the KRAS G12C actionable variant. These results, obtained with routine diagnostic technologies in an unselected clinical setting, provide a significant addition of real-world data in early-stage NSCLC.

EGFR (epidermal growth factor receptor) mutant NSCLC (non-small cell lung carcinoma) comprises 35-40% of cases in the Asian NSCLC cohort, compared to 15-20% in the rest of the world. Improved response rates have been observed in terms of PFS (progression-free survival) and ORR (overall response rate) when treated with EGFR TKIs (tyrosine kinase inhibitors). However, resistance eventually ensues regardless of the generation of TKI used. Preclinical studies have reported that PDL1 (programmed death ligand1) is a downstream target of EGFR and is interposed by IL-6/JAK/STAT3 (interleukin-6/Janus kinase/signal transducer and activator of transcription3), NF-κB (nuclear factor kappa beta), and p-ERK1/2/p-c-Jun pathways. Hence, it may potentially be repressed by EGFR TKIs. In this retrospective exploratory analysis, we studied whether PDL1 expression affects efficacy of EGFR TKIs and clinical outcome in patients with untreated metastatic EGFR-mutated lung adenocarcinoma. This single-center retrospective, exploratory analysis was performed between January 2015 and December 2019. Among 1350 cases of NSCLC, 470 were EGFR mutant, of which PDL1 expression testing was done in 193 patients who were included in this study. Median age was 60years (range 24-87years). A total of 116 patients (60.1%) had inframe deletion in exon19, 52 (26.9%) had L858R, and 25 (13%) had uncommon mutations. The number of patients with PDL1 tumour proportion score (TPS) < 1% was 109 (56.5%); 1-49%, 57 (29.5%); and ≥ 50%, 27 (14%). Comparing clinical characteristics among various PDL1 groups, there were no statistically significant correlations obtained. However, patients with PDL1 > 50% were smokers, and showed a trend for higher disease burden at diagnosis. Median PFS of PDL1 < 1% was 10.14months, compared to 9.4months in the PDL1 > 1% group; however, the values did not reach statistical significance. The current study was an exploratory retrospective study; however, the results add to the growing body of evidence that PDL1 expression in EGFR-mutated NSCLC does not have any prognostic significance. Also the efficacy of EGFR TKIs is not influenced by variations in PDL1 TPS.

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

9056 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a key treatment for EGFR-mutated non-small-cell lung carcinoma (NSCLC). To date, a biomarker to predict whether NSCLC will exhibit a short- or long-term response to first- or second-generation EGFR-TKIs has not been established for clinical use. Human epidermal growth factor receptor-2 (HER2) aberrations are mechanisms for acquired resistance to EGFR-TKIs; however, their impact on EGFR-TKI therapy outcomes in EGFR-mutant NSCLC has not yet been systematically evaluated. Methods: Patients with advanced NSCLC were prospectively registered from more than 35 institutes (HER2-CS STUDY UMIN 000017003). EGFR mutations or anaplastic lymphoma kinase gene translocations were assessed at each institution using a commercially approved test. HER2 protein expression levels were determined by immunohistochemistry (IHC) using the Ventana I-VIEW PATHWAY anti-HER-2/neu (4B5). The IHC status scoring system applied to gastric cancer was used. Results: Of 1,126 screened patients with NSCLC, 354 (31.8%) had EGFR-mutated tumors, and the HER2 protein statuses were as follows: IHC0 (n = 71, 26%), IHC1+ (n = 148, 53%), IHC2+ (n = 51, 18%), and IHC3+ (n = 7, 3%). The patients’ demographics were almost identical in those with lung tumors harboring EGFR mutations and HER2-IHC2+/3+ (group P) or EGFR mutations and HER2-IHC0/1 (group N). The EGFR-TKI response rates were not different between these groups (Table). However, group P showed significantly shorter time to EGFR-TKI treatment failure than group N (median 19.1 vs. 13.3 months; log rank p = 0.038). Conclusions: These data from a large prospective cohort show that HER2 protein expression in EGFR-mutant NSCLC may have a negative impact on the effect of EGFR-TKIs. A clinical trial of EGFR/HER2-TKIs (e.g., afatinib) is warranted for this population. [Table: see text]

The relationship between epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance, including osimertinib, and programmed cell death-ligand 1 (PD-L1) expression status in EGFR-mutated non-small cell lung carcinoma (NSCLC) remains unclear. We retrospectively analyzed 64 patients with unresectable advanced or metastatic NSCLC carrying EGFR exon 19 deletions (ex19del) or EGFR exon 21 L858R substitutions (L858R) who received osimertinib as the first-line treatment. We compared progression-free survival (PFS) between eligible patients with PD-L1 tumor proportion scores (TPS) ≥20% and PD-L1 TPS <20% using the Kaplan-Meier survival plots with a log-rank test. Multivariate analysis was performed to examine the poor prognostic factors of PFS. The PD-L1 TPS ≥20% group included 22 cases (median [range] age: 70.5 [33-86] years; 10 women [45.5%]; 11 current or ex-smokers [50%]); ECOG performance status (PS) of 0-1/2/3/4 was noted in 16/4/1/1 patients, respectively. The PD-L1 TPS <20% group included 42 patients (median [range] age 73 [43-88] years; 29 women [69%]; 12 current or ex-smokers [28.6%]); ECOG PS of 0-1/2/3/4 was noted in 33/6/3/0 cases, respectively. The median PFS was 9.1 and 28.1 months in the PD-L1 TPS ≥20% and PD-L1 TPS <20% groups, respectively (log-rank p = 0.013). Multivariate analysis revealed that PD-L1 TPS ≥20% was associated with PFS (hazard ratio: 2.35, 95% confidence interval: 1.09-5.08, p = 0.030). PD-L1 TPS ≥20% in patients with EGFR-mutated NSCLC may be associated with early resistance to osimertinib.

One of the most discussed recent models for cancer development is based on the finding of an activated DNA damage response in early stages of cancer development. We now know that this DNA damage occurs due to replication stress (RS) induced by oncogenes. In mammalian cells, RS is chiefly limited by the action of ATR and Chk1 kinases. In what regards to cancer, several Chk1 inhibitors have been (and are being) tested in clinical trials, so far with modest results. However, we believe that whereas Chk1 (or ATR) inhibitors might fail as generic anti-cancer therapies, they might be much more efficient for the treatment of tumours with high levels of oncogene-induced RS. To address this hypothesis, we have (1) provided genetic proof-of-principle to show that limited ATR levels are indeed particularly toxic for tumours with high levels of RS (Murga et al Nat Struct Mol Biol, 2011), and (2) generated ATR inhibitors which show synthetic lethal interactions with cancer-associated mutations (Toledo et al Nat Struct Mol Biol, 2011). In what regards to the genetics, and with the use of an ATR hypomorphic mouse strain previously developed in our lab (Murga et al Nat Genet, 2009), we now know that limited ATR levels fully prevent the development of Myc-induced lymphomas or pancreatic tumours. Moreover, we also showed that Chk1 inhibitors are very effective for the treatment of Myc-induced lymphomas. In contrast, these inhibitors were largely ineffective in the treatment of Ras-induced pancreatic tumours, which had no detectable evidences of RS. Hence, these data strongly suggest that the use of Chk1 (or ATR) inhibitors would be particularly beneficial for the treatment of tumours harbouring high levels of RS. Importantly, the levels of RS in tumours can be analyzed in tumour biopsies. Hence, we have provided a rationale that could be readily used in the clinic for a more efficient use of ATR and Chk1 inhibitors in cancer chemotherapy. Until recently, no potent inhibitors of ATR existed. One of the limitations for the discovery of ATR inhibitors is that the activity of the kinase is restricted to replicating cells. This hindered cell-based screenings due to the large number of false positives that would derive from an indirect effect of the tested compound on the cell cycle. Overcoming this limitation, we previously developed a cellular system in which ATR activity can be unleashed at will, throughout the cell cycle and in the absence of any actual DNA damage (Toledo et al Genes &amp; Dev 2008). After adapting this system for a High-Throughput Imaging pipeline, and with the help of the Experimental Therapeutics Programme of the CNIO, we have now identified several compounds that can inhibit ATR in the nanomolar range (Toledo et al Nat Struct Mol Biol, 2011). We are now at the early stages of characterizing these inhibitors and their potential uses, and have shared these reagents with many investigators around the world. Besides the published ones, we have now also identified some compounds with good pharmacological properties in vivo, so that we are now ready to test the effect of ATR inhibitors in mouse preclinical models of cancer.

Combined Inhibition of Chk1 and Wee1 As a New Therapeutic Strategy for High-Risk Multiple Myeloma Patients

BackgroundInterleukin-35 (IL-35) is an added member of the IL-12 heterodimeric cytokine family, composed of two subunits: EBI3 and P35 subunits, implicated in tumor immune evasion. This study investigates the expression and immunosuppressive role of IL-35 in epidermal growth factor receptor (EGFR)-mutant non-small cell lung carcinoma (NSCLC), with a focus on its interaction with natural killer (NK) cells.MethodsEighty-two NSCLC tissue samples (47 EGFR-mutant and 35 wild-type) were assessed for IL-35 expression via immunohistochemistry (IHC) targeting EBI3 and P35. ELISA, Western blot, and PCR validated protein and mRNA expression in fresh tissues (n = 14). The degree of NK-cell infiltration was evaluated as the percentage of NKp46-positive cells among CD45-positive cells. Peripheral NK cells were isolated from healthy donors and subjected to IL-35 treatment. Functional assays included CCK8, flow cytometry for CD3-CD56+ cells and NKG2D, ELISA for cytokine secretion, and cytotoxicity assays on NSCLC cell lines. In vivo, H1975 and PC-9 xenograft models with EGFR-sensitive mutations were used to assess the effects of IL-35 on tumor growth and NK-cell infiltration.ResultsIL-35 was significantly overexpressed in EGFR-mutant NSCLC tissues, with strong concordance between EBI3 and P35(r = 0.795, P < 0.0001). High IL-35 expressions associated with larger tumor size (χ2 = 16.140, P = 0.000) and EGFR mutation status (χ2 = 4.843, P = 0.028). IL-35 expression levels were associated with patient prognosis in both the overall population and the EGFR-mutant subgroup (Kaplan–Meier, P < 0.05).IL-35 expression inversely correlated with NKp46- cell density (r = -0.526, P = 0.000). The percentage of NKp46-positive cells among CD45-positive cells differed significantly between mutant and wild-type NSCLC tissues (t=-9.083,P=0.000). IL-35 inhibited NK cell proliferation and function in vitro, reducing CD3-CD56+ cell proportion (F = 101.3, P < 0.0001), NKG2D expression (F = 49.29, P = 0.0002), and cytokine secretion (IFN-γ, F = 252.388, P = 0.000, Perforin, F = 39.372, P = 0.000, Granzyme, F = 1001.822, P = 0.000); Conditioned medium from IL-35-treated NK cells enhanced proliferation, invasion, and migration of NSCLC cell lines. In vivo, IL-35 promoted tumor growth, while IL-35 neutralization reduced tumor size. The proportion of NKp46+ cells among CD45+ cells differed significantly across the control, IL-35, and IL-35 neutralizing antibody groups (One-way ANOVA, P = 0.000). The in vivo results in mice indicated that IL-35 expression remained inversely related to NKP46 expression (r = -0.753, P = 0.000).ConclusionIL-35 is upregulated in EGFR-mutant NSCLC and mediates immune suppression by impairing NK cell activity. Targeting IL-35 may offer a therapeutic avenue to restore NK cell function and enhance anti-tumor immunity.

Immunotherapy with antibodies against B7/CD28 family members, including PD-1, PD-L1, and CTLA-4 has shifted the treatment paradigm for non-small cell lung carcinoma (NSCLC) with improved clinical outcome. HHLA2 is a newly discovered member of the family. By regulating T-cell function, HHLA2 could contribute to tumor immune suppression and thus be a novel target for cancer immunotherapy. There is limited information and critical need to characterize its expression profile and clinical significance in NSCLC. We performed IHC with an HHLA2-specific antibody (clone 566.1) using tissue microarrays constructed from 679 NSCLC tumor tissues, including 392 cases in the discovery set and 287 cases in the validation cohort. We also studied clinicopathologic characteristics of these patients. Overall, HHLA2 was not detected in most of normal lung tissue but expressed in 66% of NSCLC across different subtypes. In particular, EGFR-mutated NSCLC was significantly associated with higher tumor HHLA2 expression in both discovery (EGFR vs. WT: 76% vs. 53%, P = 0.01) and validation cohorts (89% vs. 69%, P = 0.01). In one of the two cohorts, HHLA2 expression was higher in lung adenocarcinoma as compared with squamous and large cell histology, non-Hispanic White versus Hispanics, and tumors with high tumor-infiltrating lymphocyte (TIL) density. In the multivariate analysis, EGFR mutation status and high TIL intensity were independently associated with HHLA2 expression in lung adenocarcinoma. HHLA2 is widely expressed in NSCLC and is associated with EGFR mutation and high TILs in lung adenocarcinoma. It is potentially a novel target for lung cancer immunotherapy. Clin Cancer Res; 23(3); 825-32. ©2016 AACR.

Landscape for the treatment of cancer patients has recently been changed with the arrival of targeted therapies in different malignancies with the drugs targeting specific mutations and genetic alterations such as, EGF-receptor blockers and BRAF inhibitors, and the drugs such as PARP inhibitors which exploit defects in the cancers and cause synthetic lethality. In this thesis, I investigated to understand the mechanism of action of a drug that targets the cell cycle checkpoint regulator, checkpoint kinase 1 (CHK1) and explored its potential therapeutic usage in specific cancer types. Checkpoint kinase 1 inhibitor (CHK1i) as a single-agent treatment is effective in some of the cancer types with high levels of replication stress, including melanoma. However, the mechanism and manner of cell-killings induced by CHK1i single-agent treatment is still poorly understood. To identify the patient population who can benefit from CHK1i single-agent treatment, it is important to understand how single-agent CHK1i induced the cell killing. CHK1i has been investigated in pre-clinical studies and clinical trials, and shown to enhance the efficacy of chemotherapeutic drugs, particularly those that promote replication stress such as gemcitabine. Most of those investigations were based on the previous notion that CHK1i abrogates the G2/M phase checkpoint to cause mitotic catastrophe and results in cancer cell death. However, considering numerous roles of CHK1 playing in cell cycle and DNA damage response pathway, this mechanism alone may not represent the entirety of sensitivity to CHK1i. CHK1 has different roles in S and G2/M phases of cell cycle such as controlling the G2/M checkpoint, stabilising the stalled replication fork, and inhibiting the apoptosis together with other DNA damage repair and cell cycle checkpoint regulators. When one of these other regulators/pathways has defects, inhibiting CHK1 can be synthetically lethal to the cancer cells. Another CHK1 function in S phase is regulating CDC25A; CHK1 triggers the destabilisation of CDC25A upon DNA damage and replication stress. The normal role of CDC25A in S phase progression is activation of CDK2 which is required for progression into and through S phase. CDC25A dysregulation and overexpression has been reported in various cancers and shown to create increased replication stress in the cells. Tumours with high level of replication stress have been suggested to be selectively susceptible to the inhibition of CHK1 and its upstream regulator, ATR. In this thesis, it was firstly shown that S phase cell cycle checkpoint defect is a common feature of CHK1i-hypersensitive melanoma cell lines, and this defect is often associated with failure to degrade CDC25A in response to replication stress. Furthermore, CDC25A over-expression and/or dysregulation contributes to CHK1i sensitivity in hypersensitive melanoma cell lines. Secondly, it was demonstrated that CHK1i induced high level of replication stress via RPA hyper-phosphorylation and subsequently RPA depletion occurred in hypersensitive cell lines resulting in replication catastrophe, while only low level of replication stress was observed in CHK1i-insensitive cell lines. Thirdly, adding low level replication stress by the addition of low-dose (0.2 mM) hydroxyurea (HU) significantly sensitised not only CHK1i-insensitive melanoma cell lines but also the lung cancer cell lines to CHK1 inhibition. Finally, I have developed a three-dimensional (3D) tumoursphere drug-testing platform. Using CHK1i single-agent treatment as a test system, I have demonstrated this system to be more predictive of in vivo CHK1i sensitivity than the traditional 2D model. Taken together, these data suggest that CHK1i single-agent treatment has potential use in cancer with high levels of endogenous replication stress which can be identified by the presence of defective S phase checkpoint, overexpression/impaired degradation of replication stress proteins such as CDC25A or presence of hyper-phosphorylation of RPA. The tumours which are insensitive to CHK1i single-agent can also be sensitised to the drug by inducing replication stress with low-dose HU.

Chemoresistance contributes to the majority of deaths in women with ovarian cancer (OC). Altered DNA repair and metabolic signaling is implicated in mediating therapeutic resistance. DNA damage checkpoint kinase 1 (CHK1) integrates cell cycle and DNA repair in replicating cells, and its inhibition causes replication stress, repair deficiency and cell cycle dysregulation. We observed elevated Poly-ADP-ribosylation (PAR) of proteins (PARylation) and subsequent decrease in cellular NAD+ levels in OC cells treated with the CHK1 inhibitor prexasertib, indicating activation of NAD+ dependent DNA repair enzymes poly-ADP-ribose polymerases (PARP1/2). While multiple PARP inhibitors are in clinical use in treating OC, tumor resistance to these drugs is highly imminent. We reasoned that inhibition of dePARylation by targeting Poly (ADP-ribose) glycohydrolase (PARG) would disrupt metabolic and DNA repair crosstalk to overcome chemoresistance. Although PARG inhibition (PARGi) trapped PARylation of the proteins and activated CHK1, it did not cause any significant OC cell death. However, OC cells deficient in CHK1 were hypersensitive to PARGi, suggesting a role for metabolic and DNA repair crosstalk in protection of OC cells. Correspondingly, OC cells treated with a combination of CHK1 and PARG inhibitors exhibited excessive replication stress-mediated DNA lesions, cell cycle dysregulation, and mitotic catastrophe compared to individual drugs. Interestingly, increased PARylation observed in combination treatment resulted in depletion of NAD+ levels. These decreased NAD+ levels were also paralleled with reduced aldehyde dehydrogenase (ALDH) activity, which requires NAD+ to maintain cancer stem cells. Furthermore, prexasertib and PARGi combinations exhibited synergistic cell death in OC cells, including an isogenic chemoresistant cell line and 3D organoid models of primary patient-derived OC cell lines. Collectively, our data highlight a novel crosstalk between metabolism and DNA repair involving replication stress and NAD+-dependent PARylation, and suggest a novel combination therapy of CHK1 and PARG inhibitors to overcome chemoresistance in OC.

MicroRNA abundance as a particular biomarker for precisely identifying cancer metastases has emerged in recent years. The expression levels of miRNA are analyzed to get insights into cancer tissue detection and subtypes. Similar to other cancer types, the miRNA shows high levels of target mRNA dysregulation in association with non-small cell lung carcinoma (NSCLC). Among many promising cancer biomarkers for NSCLC, miR-7-5p has shown significant downregulation in the NSCLC tissues and targets proto-oncogenes like PAK2 and NOVA2. The expression levels of different proto-oncogenes targeting the miR-7-5p in NSCLC showed that the EGFR-mutated NSCLC has an experimental validation. The target validation of the miR-7-5p could be analyzed using SPR (Surface plasmon resonance) based sensors at a single nanoparticle level, such as Au nanocube, due to its high specificity and accountability. Despite being an accountable tool for cancer diagnosis, miRNA-based biomarkers sometimes cause poor diagnostic specificity and reproducibility due to their heterogenicity and immunogenicity in cancer detection. To overcome these shortcomings, the biomarkers need to be validated according to recent clinical studies.

Lung cancer is responsible for high morbidity and mortality worldwide. In general, lung cancer can be divided into two major types, including small cell lung carcinoma (SCLC) and the more common non-small cell lung carcinoma (NSCLC). Molecular events underlying lung cancer development, growth, and progression remain complex. In addition to a variety of genetic aberrations, alterations in cellular metabolism have been implicated. Epidermal growth factor receptor (EGFR) is a cell surface protein that is frequently mutated in NSCLC. In this review, we discuss the effects of EGFR mutants on cell proliferative and survival signals, as well as metabolic reprogramming, in NSCLC. We also discuss the use and mechanisms of action of tyrosine kinase inhibitors (TKIs) that target EGFR-mutants and mediate their inhibitory effects by inducing cell death. Development of resistance to EGFR-TKIs is a problem in the clinic. We further discuss the approaches that are used to overcome this resistance, including the development of fourth-generation EGFR-TKIs. Immunotherapy is not very effective in EGFR-mutant NSCLC. We also discuss possible underlying mechanisms for the inadequate response of EGFR-mutant tumors to immunotherapeutics. Given that mutant EGFR transduces survival signals, and affects cellular metabolism, a better understanding of the crosstalk between mutant EGFR-mediated signals and metabolic reprogramming is expected to facilitate the development of newer personalized therapeutics to manage lung cancer.

DNA damaging anticancer agents activate checkpoints that stop the cell cycle to prevent additional DNA replication &amp; mitosis until damaged region has been repaired. One of the important regulatory proteins activated in DNA damage response pathway is Checkpoint kinase 1 (Chk1). Therefore, Chk1 inhibitors have emerged as promising new therapeutics for cancer. In order to find novel Chk1 inhibitor, we have conducted a structure based virtual screening. Several small molecules have shown 75 - 100 % inhibition of Chk1 in the binding affinity assay. Combined with the results of kinase assay with ATP &amp; mechanism based cell assay, hit candidates has been led. Lung cancer is the leading cause of cancer related deaths in Korea. About 85 % of lung cancer is non-small cell lung carcinoma (NSCLC) worldwide. Chemotherapy for NSCLC is often regarded ineffective. To test the feasibility of Chk1 inhibitor to sensitize antimetabolite in NSCLC, growth inhibition induced by gemcitabine plus MK8776 was assessed in A549 &amp; H1129 cells. These cells are insensitive to the MK8776 when treated as a single agent (IC50 &amp;gt;10 μM). In contrast, MK8776 reduced the concentration of gemcitabine required to inhibit H1299 or A540 cell growth by almost 3-30 fold or 6-15 fold, respectively, depending on the concentration used. These results would imply the potential clinical utility of Chk1 inhibitor as an adjuvant for antimetabolite such as gemcitabine for NSCLC. Further studies to evaluate the effectiveness of Chk1 inhibitor including new molecules would be warranted in additional preclinical and clinical settings. [The chemical library used in this study was kindly provided by Korea Chemical Bank &amp; KIST. This study was supported by a grant (2015R1D1A1A01057014) from the National Research Foundation of Korea.] Citation Format: Injae Chung. New chk1 inhibitor development &amp; the feasibility of use of chk1 inhibitor with antimetabolite for NSCLC [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 6. doi:10.1158/1538-7445.AM2017-6

Simple SummaryLung cancer is one of the most frequently diagnosed malignant diseases in Poland. Presented real-world data might help in making a clinical decision on cancer treatment and provide an argument for the fast availability of breakthrough therapy. The REFLECT study is one of the largest real-world studies dedicated to patients with non-small-cell lung carcinoma (NSCLC). The results of presented descriptive analysis of the Polish population of the REFLECT study highlight the need for effective treatments and diagnostics of patients with advanced NSCLC with EGFR mutations.Non-small-cell lung cancer (NSCLC) represents 85% of new cases of lung cancer. Over the past two decades, treatment of patients with NSCLC has evolved from the empiric use of chemotherapy to more advanced targeted therapy dedicated to patients with an epidermal growth factor receptor (EGFR) mutation. The multinational REFLECT study analyzed treatment patterns, outcomes, and testing practices among patients with EGFR-mutated advanced NSCLC receiving first-line EGFR tyrosine kinase inhibitor (TKI) therapy across Europe and Israel. The aim of this study is to describe the Polish population of patients from the REFLECT study, focusing on treatment patterns and T790M mutation testing practice. A descriptive, retrospective, non-interventional, medical record-based analysis was performed on the Polish population of patients with locally advanced or metastatic NSCLC with EGFR mutations from the REFLECT study (NCT04031898). A medical chart review with data collection was conducted from May to December 2019.The study involved 110 patients. Afatinib was used as the first-line EGFR-TKI therapy in 45 (40.9%) patients, erlotinib in 41 (37.3%), and gefitinib in 24 (21.8%) patients. The first-line EGFR-TKI therapy was discontinued in 90 (81.8%) patients. The median progression-free survival (PFS) on first-line EGFR-TKI therapy was 12.9 months (95% CI 10.3–15.4). A total of 54 patients started second-line therapy, of whom osimertinib was administered to 31 (57.4%). Among 85 patients progressing on first-line EGFR-TKI therapy, 58 (68.2%) were tested for the T790M mutation. Positive results for the T790M mutation were obtained from 31 (53.4%) tested patients, all of whom received osimertinib in the next lines of therapy. The median overall survival (OS) from the start of first-line EGFR-TKI therapy was 26.2 months (95% CI 18.0–29.7). Among patients with brain metastases, the median OS from the first diagnosis of brain metastases was 15.5 months (95% CI 9.9–18.0). The results of the Polish population from the REFLECT study highlight the need for effective treatment of patients with advanced EGFR-mutated NSCLC. Nearly one-third of patients with disease progression after first-line EGFR-TKI therapy were not tested for the T790M mutation and did not have the opportunity to receive effective treatment. The presence of brain metastases was a negative prognostic factor.