Longitudinal multi-omics study of palbociclib resistance in HR-positive/HER2-negative metastatic breast cancer.

BACKGROUND: Cyclin-dependent kinase 4/6 inhibitor (CDK4/6) therapy plus endocrine therapy (ET) is an effective treatment for patients with hormone receptor-positive/human epidermal receptor 2-negative metastatic breast cancer (HR+/HER2- MBC); however, resistance is common and poorly understood. A comprehensive genomic and transcriptomic analysis of pretreatment and post-treatment tumors from patients receiving palbociclib plus ET was performed to delineate molecular mechanisms of drug resistance. METHODS: Tissue was collected from 89 patients with HR+/HER2- MBC, including those with recurrent and/or metastatic disease, receiving palbociclib plus an aromatase inhibitor or fulvestrant at Samsung Medical Center and Seoul National University Hospital from 2017 to 2020. Tumor biopsy and blood samples obtained at pretreatment, on-treatment (6 weeks and/or 12 weeks), and post-progression underwent RNA sequencing and whole-exome sequencing. Cox regression analysis was performed to identify the clinical and genomic variables associated with progression-free survival. RESULTS: Novel markers associated with poor prognosis, including genomic scar features caused by homologous repair deficiency (HRD), estrogen response signatures, and four prognostic clusters with distinct molecular features were identified. Tumors with TP53 mutations co-occurring with a unique HRD-high cluster responded poorly to palbociclib plus ET. Comparisons of paired pre- and post-treatment samples revealed that tumors became enriched in APOBEC mutation signatures, and many switched to aggressive molecular subtypes with estrogen-independent characteristics. We identified frequent genomic alterations upon disease progression in RB1, ESR1, PTEN, and KMT2C. CONCLUSIONS: We identified novel molecular features associated with poor prognosis and molecular mechanisms that could be targeted to overcome resistance to CKD4/6 plus ET. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03401359. The trial was posted on 18 January 2018 and registered prospectively.

Predictive biomarkers for 5-fluorouracil and oxaliplatin-based chemotherapy in gastric cancers via profiling of patient-derived xenografts.

Gastric cancer (GC) is commonly treated by chemotherapy using 5-fluorouracil (5-FU) derivatives and platinum combination, but predictive biomarker remains lacking. We develop patient-derived xenografts (PDXs) from 31 GC patients and treat with a combination of 5-FU and oxaliplatin, to determine biomarkers associated with responsiveness. When the PDXs are defined as either responders or non-responders according to tumor volume change after treatment, the responsiveness of PDXs is significantly consistent with the respective clinical outcomes of the patients. An integrative genomic and transcriptomic analysis of PDXs reveals that pathways associated with cell-to-cell and cell-to-extracellular matrix interactions enriched among the non-responders in both cancer cells and the tumor microenvironment (TME). We develop a 30-gene prediction model to determine the responsiveness to 5-FU and oxaliplatin-based chemotherapy and confirm the significant poor survival outcomes among cases classified as non-responder-like in three independent GC cohorts. Our study may inform clinical decision-making when designing treatment strategies.

DNA Damage Repair Inhibitor for Breast Cancer Treatment.

Cancer has been defined as a genetic disorder caused by the accumulation of genetic alterations, which result from various internal and external DNA damage that is left unrepaired. One of the main characteristics of cancer is a partial loss of DNA damage repair (DDR) pathway, resulting in increased DNA damage levels and replication stress. DDR inhibitors have been suggested as a new anticancer strategy, under the concept of synthetic lethality. The poly-(ADP-ribose) polymerase (PARP) inhibitor is the first DDR inhibitor to be used in clinical practice. PARP inhibitors have been tested in patients with BRCA1/2 germline mutations (gBRCA1/2mt) and shown robust clinical benefits in breast cancer with gBRCA1/2mt and serous ovarian cancer patients. The concept of synthetic lethality is not limited to gBRCAmt for PARP inhibitor, and discovering homologous recombination deficiency (HRD) markers beyond BRCA1/2 and identifying best candidates for DDR inhibitors are the active research areas. At the same time, various combinations of DDR inhibitors and other anticancer drugs are being tested in both preclinical and clinical studies. In addition, based on recent evidence of the immune-modulatory effect of PARP inhibitors, the combination of DDR inhibitors and immune checkpoint inhibitors is being actively investigated. Acquired resistance mechanism of DDR inhibitors, as well as defining best candidates and best combinations, would be future research topics for DDR inhibitors. Furthermore, it would also be crucial to establish a clinically relevant standardized method to detect HRD for future clinical use.

Genomic profile of metastatic breast cancer patient-derived xenografts established using percutaneous biopsy.

BACKGROUND: Metastatic breast cancer (mBC) is a complex and life-threatening disease and although it is difficult to cure, patients can benefit from sequential anticancer treatment, including endocrine therapy, targeted therapy and cytotoxic chemotherapy. The patient-derived xenograft (PDX) model is suggested as a practical tool to predict the clinical outcome of this disease as well as to screen novel drugs. This study aimed to establish PDX models in Korean patients and analyze their genomic profiles and utility for translational research. METHODS: Percutaneous core needle biopsy or punch biopsy samples were used for xenotransplantation. Whole exome sequencing and transcriptome analysis were performed to assess the genomic and RNA expression profiles, respectively. Copy number variation and mutational burden were analyzed and compared with other metastatic breast cancer genomic results. Mutational signatures were also analyzed. The antitumor effect of an ATR inhibitor was tested in the relevant PDX model. RESULTS: Of the 151 cases studied, 40 (26%) PDX models were established. Notably, the take rate of all subtypes, including the hormone receptor-positive (HR +) subtype, exceeded 20%. The PDX model had genomic fidelity and copy number variation that represented the pattern of its donor sample. TP53, PIK3CA, ESR1, and GATA3 mutations were frequently found in our samples, with TP53 being the most frequently mutated, and the somatic mutations in these genes strengthened their frequency in the PDX model. The ESR1 mutation, CCND1 amplification, and the APOBEC signature were significant features in our HR + HER2- PDX model. Fulvestrant in combination with palbociclib showed a partial response to the relevant patient's tumor harboring the ESR1 mutation, and CCND1 amplification was found in the PDX model. AZD6738, an ATR inhibitor, delayed tumor growth in a relevant PDX model. CONCLUSIONS: Our PDX model was established using core needle biopsy samples from primary and metastatic tissues. Genomic profiles of the samples reflected their original tissue characteristics and could be used for the interpretation of clinical outcomes.

Homologous repair deficiency score for identifying breast cancers with defective DNA damage response.

Breast cancer (BC) in patients with germline mutations of BRCA1/BRCA2 are associated with benefit from drugs targeting DNA damage response (DDR), but they account for only 5-7% of overall breast cancer. To define the characteristics of these tumors and also to identify tumors without BRCA mutation but with homologous recombination deficiency (HRD) is clinically relevant. To define characteristic features of HRD tumors and analyze the correlations between BRCA1/BRCA2 and BC subtypes, we analyzed 981 breast tumors from the TCGA database using the signature analyzer. The BRCA signature was strongly associated with the HRD score top 10% (score ≥ 57) population. This population showed a high level of mutations in DDR genes, including BRCA1/BRCA2. HRD tumors were associated with high expression levels of BARD1 and BRIP1. Besides, BRCA1/2 mutations were dominantly observed in basal and luminal subtypes, respectively. A comparison of HRD features in BC revealed that BRCA1 exerts a stronger influence inducing HRD features than BRCA2 does. It reveals genetic differences between BRCA1 and BRCA2 and provides a basis for the identification of HRD and other BRCA-associated tumors.

Antitumor effect of a WEE1 inhibitor and potentiation of olaparib sensitivity by DNA damage response modulation in triple-negative breast cancer.

Due to its regulation of CDK1/2 phosphorylation, WEE1 plays essentially roles in the regulations of G2/M checkpoint and DNA damage response (DDR). WEE1 inhibition can increase genomic instability by inducing replication stress and G2/M checkpoint inactivation, which result in increased cellular sensitivity to DNA damaging agents. We considered an increase in genomic instability induced by WEE1 inhibition might be used to augment the effects of drugs targeting DNA repair protein. Typically, PARP inhibitors are effective in germline BRCA 1/2 mutated breast and ovarian cancer, but their applicabilities in triple-negative breast cancer (TNBC) are limited. This study was conducted to investigate the anti-tumor effects of the WEE1 inhibitor, AZD1775, and the mechanism responsible for its potentiation of sensitivity to olaparib (a PARP inhibitor) via the modulation of DDR in TNBC cells. Our results suggest that AZD1775 could be used to broaden the application range of olaparib in TNBC and provide a rationale for a clinical trial of combined olaparib and AZD1775 therapy.

Prognostic Role of Androgen Receptor Expression in Surgically Resected Early Breast Cancer Patients.

PURPOSE: Endocrine therapy is a standard treatment for hormone receptor-positive breast cancer, which accounts for 60%-75% of all breast cancer. Hormone receptor positivity is a prognostic and predictive biomarker in breast cancer. Approximately 50%-80% of breast cancer is also positive for androgen receptor (AR), but the prognostic and predictive value of AR expression in breast cancer is controversial. Here, we investigated AR expression and its prognostic value in patients with surgically resected breast cancer in Korea. METHODS: We retrospectively reviewed the medical records of patients who had surgically resected breast cancer to collect AR expression data and other clinicopathological data. The optimal cut-off for AR positivity was determined using a receiver operating characteristic curve analysis. RESULTS: We reviewed 957 patients with surgically resected breast cancer from June 2012 to April 2013. The median follow-up was 62 months, and relapse events occurred in 101 (10.6%) patients. Unlike the cut-off value of 1% or 10% in previous reports, 35% was determined to be best for predicting relapse-free survival (RFS) in this study. At the cut-off value of 35%, 654 (68.4%) patients were AR-positive. AR expression was more prevalent in luminal A (87.6%) and luminal B (73.1%) types than in human epidermal growth factor receptor 2-positive (56.2%) or triple-negative (20.6%) types. AR expression of ≥ 35% was significantly related to longer RFS in a multivariate analysis (hazard ratio, 0.430; 95% confidence interval, 0.260-0.709; p = 0.001). CONCLUSION: We propose a cut-off value of 35% to best predict RFS in patients with surgically resected breast cancer. AR expression was positive in 68.4% of patients, and AR positivity was found to be an independent prognostic factor for longer RFS.

TDP1 and TOP1 Modulation in Olaparib-Resistant Cancer Determines the Efficacy of Subsequent Chemotherapy.

The aim of this study was to elucidate the carryover effect of olaparib to subsequent chemotherapy and its underlying mechanisms. We generated olaparib-resistant SNU-484, SNU-601, SNU-668, and KATO-III gastric cancer cell lines and confirmed their resistance by cell viability and colony forming assays. Notably, olaparib-resistant cell lines displayed cross-resistance to cisplatin except for KATO-III. Inversely, olaparib-resistant SNU-484, SNU-668, and KATO-III were more sensitive to irinotecan than their parental cells. However, sensitivity to paclitaxel remained unaltered. There were compensatory changes in the ATM/ATR axis and p-Chk1/2 protein expression. ERCC1 was also induced in olaparib-resistant SNU-484, SNU-601, and SNU-668, which showed cross-resistance to cisplatin. Olaparib-resistant cells showed tyrosyl-DNA phosphodiesterase 1 (TDP1) downregulation with higher topoisomerase 1 (TOP1) activity, which is a target of irinotecan. These changes of TOP1 and TDP1 in olaparib-resistant cells was confirmed as the underlying mechanism for increased irinotecan sensitivity through manipulated gene expression of TOP1 and TDP1 by specific plasmid transfection and siRNA. The patient-derived xenograft model established from the patient who acquired resistance to olaparib with BRCA2 mutation showed increased sensitivity in irinotecan. In conclusion, the carryover effects of olaparib to improve antitumor effect of subsequent irinotecan were demonstrated. These effects should be considered when determining the subsequent therapy with olaparib.

PARP Inhibitors as Therapeutics: Beyond Modulation of PARylation.

Poly (ADP-ribose) polymerase (PARP) 1 is an essential molecule in DNA damage response by sensing DNA damage and docking DNA repair proteins on the damaged DNA site through a type of posttranslational modification, poly (ADP-Ribosyl)ation (PARylation). PARP inhibitors, which inhibit PARylation through competitively binding to NAD+ binding site of PARP1 and PARP2, have improved clinical benefits for BRCA mutated tumors, leading to their accelerated clinical application. However, the antitumor activities of PARP inhibitors in clinical development are different, due to PARP trapping activity beyond blocking PARylation reactions. In this review, we comprehensively address the current state of knowledge regarding the mechanisms of action of PARP inhibitors. We will also discuss the different effects of PARP inhibitors in combination with cytotoxic chemotherapeutic agents regarding the mechanism of regulating PARylation.

Immune recurrence score using 7 immunoregulatory protein expressions can predict recurrence in stage I-III breast cancer patients.

BACKGROUND: Immune cells in the tumour microenvironment play an essential role in tumorigenesis. This study aimed to evaluate the immunoregulatory protein expression of breast cancer and reveal their prognostic role. METHODS: Expression of 10 immune markers (PD-1/PD-L1/PD-L2/IDO/TIM-3/OX40/OX40L/B7-H2/ B7-H3/B7-H4) with known/possible clinical relevance was identified in stromal tumour-infiltrating lymphocytes or tumour tissue of stage I-III breast cancer patients. RESULTS: A total of 392 patients, including 271(69.1%) luminal A, 36(9.2%) luminal B, 32(8.2%) HER2-positive and 53(13.5%) triple negative disease, were included. Expression of PD-1 and PD-L1 was higher in HER2-positive and triple negative disease. By contrast, expression of TIM-3, OX40 and OX40L were higher in luminal disease. We devised an immune recurrence score (IRS) using seven markers with prognostic value (B7-H2/B7-H3/B7-H4/OX40/OX40L/PD-L1/PD-L2). Patients were classified as high-risk (7.9%), intermediate-risk (67.6%), or low-risk (24.5%). In the multivariate analysis, IRS low-risk (adjusted HR 0.14, p = 0.001) and intermediate-risk (adjusted HR 0.32, p = 0.002) had significantly lower risk of recurrence compared with high-risk. The prognostic role of IRS was maintained in both luminal A and non-luminal A patients. CONCLUSIONS: This study identified immunoregulatory protein expression of breast cancer patients using 10 immune markers. In addition, we devised an IRS which may predict recurrence in stage I-III breast cancer patients.

Prognostic role of body mass index is different according to menopausal status and tumor subtype in breast cancer patients.

PURPOSE: Although controversial, obesity and underweight may have a negative impact on breast cancer outcome. However, the relationship between body mass index (BMI) and breast cancer outcomes according to tumor subtype and menopausal status remains unclear. METHODS: This study investigated the association between BMI and breast cancer outcome in stage I-III breast cancer patients. The relationships were further evaluated according to tumor subtype and menopausal status. RESULTS: A total of 5919 patients, 3475 (58.7%) hormone receptor (HR)(+) human epidermal growth factor receptor 2 (HER2)(-), 608 (10.3%) HR(+)HER2(+), 621 (10.5%) HR(-)HER2(+), and 1079 (18.2%) HR(-)HER2(-) were included. Underweight and obesity had a negative impact on relapse-free survival but did not affect overall survival. Importantly, the prognostic role of BMI was different according to tumor subtype and menopausal status. In HR(+)HER2(-) patients, underweight was associated with poor relapse-free survival and overall survival in pre-menopausal women. In contrast, obesity had negative impact on relapse-free survival and overall survival in HR(+)HER2(-) post-menopausal patients. Underweight may have a negative prognostic role in HR(+)HER2(+) patients. However, BMI did not impact the outcome of HR(-)HER2(+) and HR(-)HER2(-) patients. CONCLUSIONS: The impact of BMI on breast cancer outcome was dependent on tumor subtype and menopausal status. In HR(+)HER2(-) patients, underweight and obesity had a negative prognostic role in pre-menopausal and post-menopausal women, respectively. These findings in Asian population should be further evaluated and compared in Western population.

Prognostic effects of abnormal DNA damage response protein expression in breast cancer.

PURPOSE: We aimed to explore the expression of DNA damage response machinery proteins and their integrated prognostic value in different subgroups of breast cancer. METHODS: Expression of NBS1, BRCA1, BRCA2, ATM, and p53 was determined by immunohistochemistry in 419 surgically resected breast tumors. RESULTS: Loss of NBS1, BRCA1, ATM, and abnormal p53 expression was significantly associated with lower disease-free survival rates. Abnormal DNA damage response protein expression, defined as loss of any one of NBS1, BRCA1, ATM, and/or abnormal p53 expression, was observed in 258 of 399 evaluable cases (64.7%) and was significantly associated with higher tumor grade, larger tumor size, and ER-negative, and/or PR-negative status. Most patients with luminal B (86.1%), HER2-enriched (94.4%), and triple-negative (86.8%) breast cancers had abnormal DNA damage response protein expression. In contrast, abnormal DNA damage response protein expression was found in only 53.8% of luminal A tumors. Abnormal DNA damage response protein expression was associated with significantly lower 5-year disease-free survival rates in all patients (95.6% vs. 84.8%, p = 0.001), as well as in the luminal A subgroup (97.4% vs. 89.0%, p = 0.011). In multivariate analysis, abnormal DNA damage response protein expression remained an independent predictor of shorter disease-free survival for luminal A subtype (hazard ratio 3.14, 95% confidence interval 1.16-8.47; p = 0.024). CONCLUSION: Abnormal DNA damage response protein expression is found in most luminal B and HER2-enriched breast cancers as frequently as in triple-negative breast cancer. In the luminal A subtype, abnormal DNA damage response protein expression is an independent prognostic marker.

Pan-Pim Kinase Inhibitor AZD1208 Suppresses Tumor Growth and Synergistically Interacts with Akt Inhibition in Gastric Cancer Cells.

PURPOSE: Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells. MATERIALS AND METHODS: The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis. RESULTS: AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. CONCLUSION: Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.

Androgen Receptor Inhibitor Enhances the Antitumor Effect of PARP Inhibitor in Breast Cancer Cells by Modulating DNA Damage Response.

The androgen receptor (AR) is expressed in 60%-70% of breast cancers regardless of estrogen receptor status, and has been proposed as a therapeutic target in breast cancers that retain AR. In this study, the authors aimed to investigate a new treatment strategy using a novel AR inhibitor AZD3514 in breast cancer. AZD3514 alone had a minimal antiproliferative effect on most breast cancer cell lines irrespective of AR expression level, but it downregulated the expressions of DNA damage response (DDR) molecules, including ATM and chk2, which resulted in the accumulation of damaged DNA in some breast cancer cells. Furthermore, AZD3514 enhanced cellular sensitivity to a PARP inhibitor olaparib by blocking the DDR pathway in breast cancer cells. Furthermore, the downregulation of NKX3.1 expression in MDA-MB-468 cells by AZD3514 occurred in parallel with the suppression of ATM-chk2 axis activation, and the suppression of NKX3.1 by AZD3514 was found to result from AZD3514-induced TOPORS upregulation and a resultant increase in NKX3.1 degradation. The study shows posttranslational regulation of NKX3.1 via TOPORS upregulation by AZD3514-induced ATM inactivation-increased olaparib sensitivity in AR-positive and TOPORS-expressing breast cancer cells, and suggests the antitumor effect of AZD3514/olaparib cotreatment is caused by compromised DDR activity in breast cancer cell lines and in a xenograft model. These results provide a rationale for future clinical trials of olaparib/AR inhibitor combination treatment in breast cancer.

Cyclin E overexpression confers resistance to the CDK4/6 specific inhibitor palbociclib in gastric cancer cells.

Palbociclib is a specific inhibitor of CDK4/6 and has been shown to provide a survival benefit in hormone receptor-positive advanced breast cancer. TCGA database reported that about half of gastric cancers exhibit abnormalities in cell-cycle-related molecules, suggesting that gastric cancer is a good candidate for palbociclib treatment; however, the antitumor effects and predictive markers of palbociclib in gastric cancer remain incompletely described. Herein, the effect and predictive markers of palbociclib on gastric cancer cells were investigated. Our results reveal that palbociclib showed anti-proliferative effects by inducing G1 phase cell-cycle arrest and cellular senescence in some gastric cancer cells. Basal protein expression level of cyclin E showed an inverse correlation of cancer cell sensitivity to palbociclib. In addition, palbociclib enhanced the antitumor effect of 5-FU in vitro and in vivo by modulating thymidine synthase expression. These results suggest that cyclin E protein expression determines the anti-proliferative effect of palbociclib, and palbociclib acts synergistically with 5-FU in gastric cancer. These findings provide a rationale for future clinical trials of palbociclib and 5-FU combination-based chemotherapy in gastric cancer.

Profiling of protein-protein interactions via single-molecule techniques predicts the dependence of cancers on growth-factor receptors.

The accumulation of genetic and epigenetic alterations in cancer cells rewires cellular signalling pathways through changes in the patterns of protein-protein interactions (PPIs). Understanding these patterns may facilitate the design of tailored cancer therapies. Here, we show that single-molecule pull-down and co-immunoprecipitation techniques can be used to characterize signalling complexes of the human epidermal growth-factor receptor (HER) family in specific cancers. By analysing cancer-specific signalling phenotypes, including post-translational modifications and PPIs with downstream interactions, we found that activating mutations of the epidermal growth-factor receptor (EGFR) gene led to the formation of large protein complexes surrounding mutant EGFR proteins and to a reduction in the dependency of mutant EGFR signalling on phosphotyrosine residues, and that the strength of HER-family PPIs is correlated with the strength of the dependence of breast and lung adenocarcinoma cells on HER-family signalling pathways. Furthermore, using co-immunoprecipitation profiling to screen for EGFR-dependent cancers, we identified non-small-cell lung cancers that respond to an EGFR-targeted inhibitor. Our approach might help predict responses to targeted cancer therapies, particularly for cancers that lack actionable genomic mutations.

EGFR or HER2 inhibition modulates the tumor microenvironment by suppression of PD-L1 and cytokines release.

BACKGROUND: Characteristics of tumor microenvironment have been suggested as predictive markers of anti-EGFR or anti-HER2 treatment response. However, the effect of EGFR/HER2 signal blockade on the tumor immune microenvironment is unclear. METHODS: EGFR/HER2 pathway signaling and PD-L1 expression in gastric cancer cell lines were screened by western blot analysis. PD-L1 and HER2 expressions in 251 resected gastric tumors were determined by immunohistochemistry, and changes in EFGR, HER2, and PD-L1 expression in paired specimens between pre- and post-chemotherapy were evaluated. PD-L1 expression in HER2-amplified cell lines was evaluated by western blotting, fluorescence-activated cell sorting, reverse transcription, and real-time quantitative PCR analyses before and after afatinib, lapatinib, pictilisib and trametinib treatment. Changes in cytokines were evaluated by reverse transcription, real-time quantitative PCR, and enzyme-linked immunosorbent assay after EGFR/HER2 inhibition. RESULTS: Cell lines with pEGFR or pHER2 overexpression showed higher PD-L1 expression. In resected gastric tumors, HER2 expression was significantly associated with PD-L1 expression (p=0.030). PD-L1 overexpression accompanied by increased HER2 expression was identified in a post-chemotherapy specimen from a patient with an initial HER2/PD-L1-negative tumor. In HER2-overexpressing cell lines, PD-L1 expression was decreased in a dose- and time-dependent manner after afatinib and lapatinib treatment. PI3K pathway inhibition by pictilisib, but not MEK pathway inhibition by trametinib, resulted in PD-L1 suppression. After lapatinib treatment, the release of CCL2, CCL21, VEGF and CXCL1 decreased in a dose-dependent manner. CONCLUSIONS: Inhibition of the EGFR/HER2 signaling pathway, particularly of downstream PI3K activity, suppressed PD-L1 and release of cytokines, suggesting that EGFR/HER2 inhibition may create a more favorable milieu for tumor immunotherapy.

AZD6738, A Novel Oral Inhibitor of ATR, Induces Synthetic Lethality with ATM Deficiency in Gastric Cancer Cells.

Ataxia telangiectasia and Rad3-related (ATR) can be considered an attractive target for cancer treatment due to its deleterious effect on cancer cells harboring a homologous recombination defect. The aim of this study was to investigate the potential use of the ATR inhibitor, AZD6738, to treat gastric cancer.In SNU-601 cells with dysfunctional ATM, AZD6738 treatment led to an accumulation of DNA damage due to dysfunctional RAD51 foci formation, S phase arrest, and caspase 3-dependent apoptosis. In contrast, SNU-484 cells with functional ATM were not sensitive to AZD6738. Inhibition of ATM in SNU-484 cells enhanced AZD6738 sensitivity to a level comparable with that observed in SNU-601 cells, showing that activation of the ATM-Chk2 signaling pathway attenuates AZD6738 sensitivity. In addition, decreased HDAC1 expression was found to be associated with ATM inactivation in SNU-601 cells, demonstrating the interaction between HDAC1 and ATM can affect sensitivity to AZD6738. Furthermore, in an in vivo tumor xenograft mouse model, AZD6738 significantly suppressed tumor growth and increased apoptosis.These findings suggest synthetic lethality between ATR inhibition and ATM deficiency in gastric cancer cells. Further clinical studies on the interaction between AZD 6738 and ATM deficiency are warranted to develop novel treatment strategies for gastric cancer. Mol Cancer Ther; 16(4); 566-77. ©2017 AACR.

Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis.

PURPOSE: KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. MATERIALS AND METHODS: The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. RESULTS: KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. CONCLUSION: KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC50 values of nine cell lines were less than 1 µmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment.