WEE1 promotes endometriosis via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Endometriosis, the presence of active endometrial tissue outside the lining membrane of the uterine cavity, is a common disease in women of childbearing age. The ectopic endometrium has some characteristics of tumor tissue, including invasive and migratory abilities. In addition, endometriosis is associated with inflammation and reduced cellular apoptosis. METHODS: Western blot analysis, qPCR, immunohistochemistry, immunofluorescence microscopy, Transwell assay, wound healing assay, and TUNEL staining. RESULTS: Interleukin-1ß (IL-1ß) induced WEE1 expression in endometrial stromal cells (ESCs), suggesting that WEE1 may be upregulated during the endometriosis-induced inflammatory response. Overexpression of WEE1 in cultured ESCs promoted ESC migration while inhibiting apoptosis, whereas WEE1 knockdown reduced ESC migration while promoting apoptosis. Inhibition of WEE1 attenuates fibrosis in ESCs and female C57BL/6 J mice. This pro-fibrotic effect of WEE1 was significantly decreased by treatment with the Wnt/ß-catenin inhibitor XAV939, suggesting that WEE1 acts via the Wnt/ß-catenin signaling pathway. CONCLUSION: Our study demonstrates that WEE1 promotes ESC migration and fibrosis via the Wnt/ß-catenin signaling pathway. Thus, WEE1 may serve as a potential therapeutic target for the treatment of endometriosis.

Overexpressed MPS-1 contributes to endometrioma development through the NF-κB signaling pathway.

BACKGROUND: Endometriosis is a benign gynecological disease that shares some characteristics with malignant tumors and affects approximately 10% of women of reproductive age. Endometrioma refers to endometriosis that appears in the ovary. Metallopanstimulin-1 (MPS-1) is a component of the 40S subunit of ribosomes that has extra-ribosomal functions that contribute to the development of diseases. This study aimed to explore the expression pattern and role of MPS-1 in endometrioma development. METHODS: Quantitative real time polymerase chain reaction, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay were used to determine the expression of MPS-1 in patients with endometrioma. Following the successful knockdown of MPS-1 by siRNA, CCK-8 assays, flow cytometry, and transwell assays were performed to detect ectopic endometrial stromal cells (EcESCs) proliferation, the rate of apoptosis, and cell cycle, migration, and invasion, respectively. Western blotting was used to explore the effect of MPS-1 knockdown on protein levels in the NF-κB signaling pathway. RESULTS: The expression of MPS-1 was significantly higher in endometrioma and the serum of endometrioma patients than in the patients without endometriosis. In addition, the downregulation of MPS-1 expression inhibited EcESCs proliferation, migration, and invasion. This downregulation led to the arrest of the EcESCs cycle in the G0/G1 phase and apoptosis and depressed the NF-κB signaling pathway. CONCLUSION: MPS-1 can regulate EcESCs proliferation, motility, invasion, apoptosis, and cell cycle via the NF-κB signaling pathway in endometrioma. This may contribute to the formation or development of endometriotic foci. This study suggests the potential role of MPS-1 in the pathogenesis of endometriosis and enabled further research into the use of MPS-1 in the clinical diagnosis of endometrioma.

A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening.

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases.

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

Overexpression of PKMYT1 Facilitates Tumor Development and Is Correlated with Poor Prognosis in Clear Cell Renal Cell Carcinoma.

BACKGROUND Protein kinase membrane-associated tyrosine/threonine (PKMYT1) has been found in many tumors, but its association with clear cell renal cell carcinoma (ccRCC) remains unclear. MATERIAL AND METHODS PKMYT1 expression in ccRCC was examined in the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Tumor Immune Estimation Resource databases. The correlation between PKMYT1 expression and clinicopathological parameters was explored via the chi-square test. Receiver operating characteristic curves were used to estimate the diagnostic performance of PKMYT1. Kaplan-Meier curves, a Cox model, nomogram, time-dependent receiver operating characteristic curves, and decision curve analysis (DCA) were used to evaluate the prognostic value and clinical utility of PKMYT1. Genes coexpressed with PKMYT1 in ccRCC were identified based on TCGA, the gene expression profiling interactive, and cBioPortal. Gene Set Enrichment Analysis revealed biological pathways associated with PKMYT1 in ccRCC. RESULTS Weighted gene coexpression network analysis identified PKMYT1 as one of the genes most significantly correlated with progression of histological grade. PKMYT1 was significantly upregulated in ccRCC compared with normal tissue (P<0.001), with a trend toward differentiating between individuals with ccRCC and those who were healthy (area under the curve=0.942). High PKMYT1 expression was correlated with unsatisfactory survival (hazard ratio=1.67, P=0.001), indicating that it is a risk factor for ccRCC. A nomogram incorporating PKMYT1 level was created and showed a clinical net benefit. PKMYT1 was strongly positively correlated with the anti-silencing function of 1B histone chaperone (ASF1B) gene in ccRCC. CONCLUSIONS PKMYT1 is upregulated in ccRCC and its presence indicates poor prognosis, making it a potential therapeutic target for ccRCC.

Negative Regulation of Tec Kinase Alleviates LPS-Induced Acute Kidney Injury in Mice via theTLR4/NF-κB Signaling Pathway.

Tec kinase is an important mediator in inflammatory immune response that enhances the activity of neutrophils and macrophages. However, information on its function in lipopolysaccharide- (LPS-) induced acute kidney injury (AKI) is limited. This study is aimed at determining whether Tec kinase was a regulator in AKI. An AKI model in mice was successfully established using intraperitoneal LPS. Results showed that the serum levels of creatinine (Cr), blood urea nitrogen (BUN), and cystatin-C (Cys-C) increased after intraperitoneal LPS injection. Renal tissue sustained significantly severe injury as measured by pathological scores. Pretreatment with LFM-A13 improved the function of the kidney in mice and decreased the renal injury score. Enzyme-linked immunosorbent assay showed that LFM-A13 significantly reduced the release of IL-1ß and TNF-α in mice exposed to LPS. LFM-A13 can evidently abrogate the expression of Tec protein, MyD88, TLR4, NF-κB p65, and Tec's phosphorylated protein as determined by Western blot. Immunohistochemistry analysis revealed that LFM-A13 markedly downregulated the expression of Tec kinase in renal tubular epithelial cells. In vitro, Tec kinase protein was expressed highly in NRK-52E cells after LPS exposure. Tec-siRNA also decreased IL-1ß and TNF-α production and obviously abolished phospho-p65 and phospho-IκBα expression in NRK-52E cell stimulated by LPS; however, Tec-siRNA increased the IκBα level. Altogether, these data suggested that Tec kinase can be a modulating protein in AKI through TLR4/NF-κB activation.

Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.

CDC-like kinase 3 (CLK3) is a dual specificity kinase that functions on substrates containing serine/threonine and tyrosine. But its role in human cancer remains unknown. Herein, we demonstrated that CLK3 was significantly up-regulated in cholangiocarcinoma (CCA) and identified a recurrent Q607R somatic substitution that represented a gain-of-function mutation in the CLK3 kinase domain. Gene ontology term enrichment suggested that high CLK3 expression in CCA patients mainly was associated with nucleotide metabolism reprogramming, which was further confirmed by comparing metabolic profiling of CCA cells. CLK3 directly phosphorylated USP13 at Y708, which promoted its binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and activating the transcription of purine metabolic genes. Notably, the CCA-associated CLK3-Q607R mutant induced USP13-Y708 phosphorylation and enhanced the activity of c-Myc. In turn, c-Myc transcriptionally up-regulated CLK3. Finally, we identified tacrine hydrochloride as a potential drug to inhibit aberrant CLK3-induced CCA. These findings demonstrate that CLK3 plays a crucial role in CCA purine metabolism, suggesting a potential therapeutic utility.

Overexpression of miR-26a-5p Suppresses Tau Phosphorylation and Aß Accumulation in the Alzheimer's Disease Mice by Targeting DYRK1A.

OBJECTIVE: It is reported that miR-26a-5p could regulate neuronal development, but its underlying mechanisms in Alzheimer's disease (AD) progression is unclear. METHODS: APP (swe)/PS1 (ΔE9) transgenic mice served as AD mice. Morris water maze test was used to measure the spatial learning and memory ability of mice. The expressions of miR-26a-5p, DYRK1A, phosphorylated-Tau, Aß40, and Aß42 were detected. The relationship between miR- 26a-5p and DYRK1A was explored using dual luciferase reporter assay. The effects of miR-26a- 5p on AD mice was determined. RESULTS: AD mice walked a lot of wrong ways to find the platform area and the latency time to reach the platform was longer. There was low expression of MiR-26a-5p in AD mice. Overexpression of miR-26a-5p inhibited Tau phosphorylation and Aß accumulation. MiR-26a-5p negatively regulated DYRK1A via targeting its 3'UTR. In vivo, increased miR-26a-5p down-regulated Aß40, Aß42, p-APP and p-Tau levels in AD mice through decreasing DYRK1A. Meanwhile, the swimming path and the latency time, to reach the platform, was shorten after enhancing miR-26a-5p expression. CONCLUSION: Overexpression of miR-26a-5p could repress Tau phosphorylation and Aß accumulation via down-regulating DYRK1A level in AD mice.

TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination.

Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in radiosensitization of basal-like BC are critically needed. Using patients' tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown; however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.

Effects of prenatal cigarette smoke exposure on BDNF, PACAP, microglia and gliosis expression in the young male mouse brainstem.

Cigarette smoke exposure during pregnancy into infancy affects brain growth and development in both short and long term (into adulthood). Using a mouse model of pre- into post- natal cigarette smoke exposure (SE), we aimed to determine the effects on brain derived neurotrophic factor (BDNF) and its receptor TrkB, neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor PAC1, and astrocyte (GFAP) and microglia (Iba-1) immunohistochemical expression, in seven nuclei of the medulla and the facial (FAC) nucleus of the pons. Male pups of dams exposed to two cigarettes (nicotine <1.2 mg, CO <15 mg) twice daily for six weeks prior to mating, during gestation and lactation (n = 5; SE), were compared to pups exposed to air under the same condition (n = 5; SHAM) at postnatal day 20. Expression changes were only evident for BDNF, TrkB and PAC1 and included decreased BDNF in the hypoglossal (XII) nucleus and nucleus of the solitary tract (NTS), increased TrkB in XII but decreased TrkB in the FAC, and increased PAC1 in 4 nuclei of the medulla including the NTS. These results suggest that the effect of SE on the brainstem are region and marker selective, affecting regions of respiratory control (XII and NTS), and restricted to the BDNF system and PAC1, with no effect on activation states of astrocytes or microglia.

The Kinome of Human Alveolar Type II and Basal Cells, and Its Reprogramming in Lung Cancer.

The discovery of mutant tyrosine kinases as oncogenic drivers of lung adenocarcinomas has changed the basic understanding of lung cancer development and therapy. Yet, expressed kinases (kinome) in lung cancer progenitor cells, as well as whether kinase expression and the overall kinome changes or is reprogrammed upon transformation, is incompletely understood. We hypothesized that the kinome differs between lung cancer progenitor cells, alveolar type II cells (ATII), and basal cells (BC) and that their respective kinomes undergo distinct lineage-specific reprogramming to adenocarcinomas and squamous cell carcinomas upon transformation. We performed RNA sequencing on freshly isolated human ATII, BC, and lung cancer cell lines to define the kinome in nontransformed cells and transformed cells. Our studies identified a unique kinome for ATII and BC and changes in their kinome upon transformation to their respective carcinomas.

Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1-KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parental Mdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-known modulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatments may not be applicable at all stages of CLI-mediated HCC.-Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

A study detection of the ROS1 gene fusion by FISH and ROS1 protein expression by IHC methods in patients with ovarian malignant or borderline serous tumors.

OBJECTIVE: ROS1 is an orphan receptor protein tyrosine kinase which is supposed to undergo genetic rearrangement in carcinogenesis. In the current study, we aimed to investigate the frequency and clinicopathologic features associated with ROS1 gene fusion and ROS1 protein expression in patients with ovarian serous carcinoma or serous borderline tumors. MATERIALS AND METHODS: Tissue samples of 102 patients with high or low grade serous carcinoma and borderline serous tumors were selected randomly from the archives of Department of Gyneco-pathology, and analyzed for ROS1 gene expression. (Fluorescence in situ hybridization (FISH) method was used to assess ROS1 gene rearrangement, while ROS1 protein expression was analyzed using immunohistochemistry. RESULTS: The study consisted of 94 cases of high-grade serous carcinoma (92.1%), 2 cases of low-grade serous carcinoma (%2) and 6 cases of serous borderline tumor (5.9%). ROS1 gene rearrangement analysis revealed that 4 patients (3.9%) were FISH-positive; whereas the immunohistochemical analysis yielded only 1 patient (0.9%) exhibiting faint positive expression of ROS1 protein. Given the low incidences of ROS1 gene rearrangement and protein expression, their relationships with clinicopathologic parameters could not be statistically analyzed. CONCLUSION: Although rare, patients with ovarian serous carcinoma or serous borderline tumor may exhibit ROS1 gene rearrangement and ROS1 protein expression. Further large-scale studies are necessary to explore the clinicopathologic significance of ROS1 gene expression in ovarian serous carcinoma.

Electroporation-mediated delivery of FER gene enhances innate immune response and improves survival in a murine model of pneumonia.

Previously, we reported that electroporation-mediated (EP) delivery of the FER gene improved survival in a combined trauma-pneumonia model. The mechanism of this protective effect is unknown. In this paper, we performed a pneumonia model in C57/BL6 mice with 500 CFU of Klebsiella pneumoniae. After inoculation, a plasmid encoding human FER was delivered by EP into the lung (PNA/pFER-EP). Survival of FER-treated vs. controls (PNA; PNA/EP-pcDNA) was recorded. In parallel cohorts, bronchial alveolar lavage (BAL) and lung were harvested at 24 and 72 h with markers of infection measured. FER-EP-treated animals reduced bacterial counts and had better 5-day survival compared to controls (80 vs. 20 vs. 25%; p < 0.05). Pre-treatment resulted in 100% survival. With FER, inflammatory monocytes were quickly recruited into BAL. These cells had increased surface expression for Toll-receptor 2 and 4, and increased phagocytic and myeloperoxidase activity at 24 h. Samples from FER electroporated animals had increased phosphorylation of STAT transcription factors, varied gene expression of IL1ß, TNFα, Nrf2, Nlrp3, Cxcl2, HSP90 and increased cytokine production of TNF-α, CCL-2, KC, IFN-γ, and IL-1RA. In a follow-up experiment, using Methicillin-resistant Staphylococcus aureus (MRSA) similar bacterial reduction effects were obtained with FER gene delivery. We conclude that FER overexpression improves survival through STAT activation enhancing innate immunity and accelerating bacterial clearance in the lung. This constitutes a novel mechanism of inflammatory regulation with therapeutic potential in the setting of hospital-acquired pneumonia.

Targeting ALK in pediatric RMS does not induce antitumor activity in vivo.

PURPOSE: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. METHODS: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. RESULTS: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. CONCLUSIONS: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

High Expression of ABL2 Suppresses Apoptosis in Gastric Cancer.

BACKGROUND: Diseases associated with Abelson-related gene (also called ABL2) include leukemia; furthermore, previous researches have studied the expressions and functions of ABL2 in different types of malignancies and found that it plays an important role in almost all kinds of cancers. AIMS: Nevertheless, the mechanism of ABL2 in gastric cancer (GC) remains vague. METHODS: In the present study, the level of ABL2 in human GC tissues was detected by immunohistochemistry. Also, the GC cell lines MGC-803 and BGC-823 were selected to stably knock down and overexpress the level of ABL2 by corresponding lentiviral vectors. Puromycin was used to maintain the stable low expression of ABL2 MGC-803 cells compared with control cells; what is more, the high expression of ABL2 BGC-823 cells was also obtained. Based on it, we detected the proteins associated with apoptosis, such as Bcl-2 family and caspase family by western blotting. RESULTS: The most appropriate concentration of puromycin to kill GC cells is 1 µg/mL; then, we obtained the corresponding stable cell lines. Furthermore, we found that high level of ABL2 in BGC-823 cells increased the expression of Bcl-XL, total PARP, and caspase3, while decreased the level of cleaved caspase3 and cleaved caspase9. Consistent results are received in MGC-803 cells. In addition, ABL2 overexpression led to the protein related with Ras/Erk and PI3K/AKT signaling pathway increased; also, we found that the major proteins play a significant role in it. CONCLUSION: All the data showed that high expression of ABL2 suppresses apoptosis through Ras/Erk and PI3K/AKT signaling pathway in GC cell lines.

Molecular Testing Guideline for the Selection of Patients With Lung Cancer for Treatment With Targeted Tyrosine Kinase Inhibitors: American Society of Clinical Oncology Endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology Clinical Practice Guideline Update.

Purpose In response to advances in the field, the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) recently updated their recommendations for molecular testing for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors. ASCO has a policy and set of procedures for endorsing clinical practice guidelines that have been developed by other professional organizations. Methods The molecular testing guideline was reviewed for developmental rigor by methodologists. Then an ASCO Expert Panel reviewed the content and the recommendations. Results The ASCO Expert Panel determined that the recommendations from the CAP/IASLC/AMP molecular testing guideline are clear, thorough, and based upon the most relevant scientific evidence. ASCO endorsed the guideline with minor modifications. Recommendations This update clarifies that any sample with adequate cellularity and preservation may be tested and that analytical methods must be able to detect mutation in a sample with as little as 20% cancer cells. It strongly recommends against evaluating epidermal growth factor receptor (EGFR) expression by immunohistochemistry for selection of patients for EGFR-targeted therapy. New for 2018 are recommendations for stand-alone ROS1 testing with additional confirmation testing in all patients with advanced lung adenocarcinoma, and RET, ERBB2 (HER2), KRAS, and MET testing as part of larger panels. ASCO also recommends stand-alone BRAF testing in patients with advanced lung adenocarcinoma. Recommendations are also provided for testing methods for lung cancers that have a nonadenocarcinoma non-small-cell component, for patients with targetable mutations who have relapsed on targeted therapy, and for testing the presence of circulating cell-free DNA. Additional information is available at www.asco.org/thoracic-cancer-guidelines and www.asco.org/guidelineswiki .

Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation.

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of leukemia-related deaths. Mutations in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent findings in this heterogeneous disease. The major types of mutations in FLT3 can be categorized as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs not only occur in the juxtamembrane region as originally described, but also in the kinase domain. Although the juxtamembrane ITDs have been well characterized, the tyrosine kinase domain ITDs have not yet been thoroughly studied due to their recent discovery. For this reason, we compared ITD mutations in the juxtamembrane domain with those in the tyrosine kinase domain, as well as with the most common activating point mutation in the tyrosine kinase domain, D835Y. The purpose of this study was to understand whether it is the nature of the mutation or the location of the mutation that plays the main role in leukemogenesis. The various FLT3 mutants were expressed in the murine pro-B cell line Ba/F3 and examined for their capacity to form colonies in semisolid medium. The size and number of colonies formed by Ba/F3 cells expressing either the internal tandem duplication within juxtamembrane domain of the receptor (JMD-ITD) or the tyrosine kinase domain (TKD)-ITD were indistinguishable, while Ba/F3 cells expressing D835Y/FLT3 failed to form colonies. Cell proliferation and cell survival was also significantly higher in TKD-ITD expressing cells, compared to cells expressing D835Y/FLT3. Furthermore, TKD-ITD is capable of inducing phosphorylation of STAT5, while D835Y/FLT3 fails to induce tyrosine phosphorylation of STAT5. Other signal transduction pathways such as the RAS/ERK and the PI3K/AKT pathways were activated to the same level in TKD-ITD cells as compared to D835Y/FLT3 expressing cells. Taken together, our data suggest that TKD-ITD displays similar oncogenic potential to the JMD-ITD but a higher oncogenic potential than the D835Y point mutation.

Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia.

Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3-internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq-based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD-positive (FLT3-ITD+) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects.

MiR-503 enhances the radiosensitivity of laryngeal carcinoma cells via the inhibition of WEE1.

Enhancing the sensitivity of laryngeal cells to radiation is crucial for improving the efficacy of laryngeal carcinoma. MicroRNAs are known to play a major role in regulating cellular radiosensitivity. This study was designed to explore the effect and the molecular basis of miR-503 in the radiosensitivity of laryngeal carcinoma cells. Quantitative real-time polymerase chain reaction analysis showed that miR-503 expression was decreased in human laryngeal carcinoma cell lines Hep-2 and TU212, and the downregulation of miR-503 was also observed after irradiation. Upregulation of miR-503 by pre-miR-503 transfection restrained proliferation, promoted progression of Hep-2 and TU212 cells through the cell cycle after irradiation, and sensitized cells to radiation. Dual-Luciferase Reporter Assay verified a direct interaction between miR-503 and the WEE1 messenger RNA 3'-untranslated region. The overexpression of miR-503 significantly decreased WEE1 expression at the messenger RNA and protein levels, whereas the inhibition of miR-503 upregulated the expression of WEE1. WEE1 knockdown by WEE1 small interfering RNA apparently abrogated the inhibitory effect of anti-miR-503 on radiosensitivity. In conclusion, miR-503 could function as an enhancer of radiation responses in laryngeal carcinoma cells by inhibiting WEE1, which may be a potential novel radiosensitizing strategy for laryngeal carcinoma.