Multiple Kinase Pathways Research Articles

88. Fostamatinib for the Treatment of Hospitalized Patients With COVID-19 Who Required Oxygen Supplementation: Results of a Phase 3 Trial

Abstract Background Severe COVID-19 is linked to hyperactivation of the host immune response involving signaling through multiple spleen tyrosine kinase (SYK) pathways. Fostamatinib is an orally administered potent and selective inhibitor of SYK that has been approved in the US, Canada, Japan, and Europe for the treatment of chronic immune thrombocytopenia (ITP). Methods We conducted a double-blind, randomized, placebo-controlled phase 3 trial of fostamatinib in adults hospitalized for COVID-19 who required oxygen supplementation (NCT04629703). Patients (pts) were randomly assigned 1:1 to receive fostamatinib (150 mg BID administered orally) or placebo for 14 days. All pts additionally received standard of care at their hospital. The primary endpoint was days on oxygen (days 1-29). Results A total of 280 pts underwent randomization (with 141 assigned to fostamatinib and 139 to placebo). The primary endpoint was met; those who received fostamatinib had lower mean days on oxygen than those who received placebo (4.8 vs. 7.6 days, P=0.0136; Table 1). Fostamatinib showed significance or trend towards significance in all secondary endpoints of reducing mortality and morbidity compared to placebo. The mean change in the 8-point ordinal score from baseline to the average of Day 5 to 15 was significantly improved in pts who received fostamatinib vs. placebo (P=0.0092,Table 1). Furthermore, 6 pts were enrolled with a baseline ordinal score of 6 (3 in each group). All patients in the fostamatinib group survived, and all in the placebo group died by Day 30. A significantly higher proportion of pts who received fostamatinib were discharged from the hospital by Day 15 compared to placebo (P=0.0029,Table 1). Significantly more patients were alive and oxygen-free by Day 29 and Day 60 with fostamatinib treatment in comparison to placebo (P=0.0213 and P=0.0271, respectively, Table 1). Treatment-emergent adverse events were consistent with previous studies and were similar between the 2 groups. Conclusion The addition of fostamatinib to standard of care treatment resulted in significantly fewer days on oxygen, an improved 8-point ordinal scale score, and significantly more patients alive and oxygen-free by Day 60 compared to placebo in pts with COVID-19 requiring hospitalization and supplemental oxygen. Disclosures Deepa B. Gotur, MD, FCCP, FCCM, GSK: Advisor/Consultant|GSK: Speaker at ATS|Moderna: Advisor/Consultant Vadim Markovtsov, PhD, Rigel Pharmaceuticals, Inc.: Employee|Rigel Pharmaceuticals, Inc.: Stocks/Bonds Lucy Yan, MD, PhD, Rigel Pharmaceuticals, Inc.: Employee|Rigel Pharmaceuticals, Inc.: Stocks/Bonds Wolfgang Dummer, MD, PhD, Rigel Pharmaceuticals, Inc.: Stocks/Bonds.

Multi-omic subtyping and molecular determinants of neoadjuvant therapeutic regimen for locally advanced HER2-positive and triple-negative breast cancer: Implications of novel therapy.

e12600 Background: Neoadjuvant chemotherapy is the backbone treatment modalities for BC patients for locally advanced breast cancer (BC) improves R0 resection rate. Determinants of differentially therapeutic response to anthracycline or platinum-based neoadjuvant therapy were largely unknown but were urgently needed to maximize patients’ benefit. Methods: A total of 149 local advanced (cT2-3, cN0-2, cM0-1) BC patients (HER2 positive BC, n = 54 and TNBC, n = 95) were retrospectively enrolled in the study, whole-exon sequencing (WES), mRNA sequencing and (phosphor-)proteomics were systematically conducted and comprehensively analyzed. The primary end point of the study was the pathological complete response (pCR) rate. The Cancer Genome Atlas (TCGA) BC dataset and GSE130787 set were used to validate our analysis. Cancer cell line encyclopedia (CCLE) dataset was utilized to screen putative small molecular inhibitors to BC cells with molecular features of resistance. Results: Through integrated analysis of multi-omics data, three subtypes with distinct molecular features were identified: immune-activated (IA) subtype, vesicular transport pathways activation (VTs) subtype, and kinase activation (KA) subtype. Same scenario of molecular subtypes can be repeated in TCGA dataset and prognosis of these subtypes can be differentiate ( p= 0.02). Patients with IA subtype majorly favored cisplatin-based treatment ( p= 0.005). And this phenomenon was confirmed in external dataset (GSE130787, n = 26, HER2-positive BC receiving TCbH treatment). Patients with VTs subtype, with significant enrichment of vesicular transport-related pathways, displayed anthracycline sensitivity. Anthracycline transporter, ABCB1 expression was significantly reduced in VTs subtype at both mRNA ( p < 0.001) and protein ( p < 0.05) levels. CCLE breast cancer cells with the characteristics of VTs subtype and lower ABCB1 expression also displayed the sensitivity of epirubicin ( p = 0.0037). Patient with KA subtype was insensitive to both platinum and anthracycline-based therapy. Multiple kinase pathways, especially, non-canonical TGF-β signaling pathway-related genes were overexpressed (p < 0.05), and significantly correlated with non-pCR ( p= 0.002). Phosphor-proteomic analysis indicated TGFβ1-mediated RhoA/ROCK pathway (Cofilin, RhoA kinase, MYH14 proteins) were highly activated. Eleven drugs were found in CCLE inhibiting growth of BC cell lines with upregulated non-canonical TGF-β pathway genes ( p < 0.05). Conclusions: Three multi-omic subtypes with distinct response to anthracycline or platinum-based therapy were identified. Moreover, activated non-canonical TGF-β pathway may mediate therapeutic resistance, but also indicated therapeutic vulnerabilities of several kinase inhibitors.

Combination of mTOR inhibitor PP242 and AMPK activator metformin exerts enhanced inhibitory effects on colorectal carcinoma cells in vitro by blocking multiple kinase pathways

The second-generation mammalian target of rapamycin (mTOR) inhibitor PP242 has demonstrated limited success in some rapamycin-insensitive tumours. We examined the therapeutic potential of combining PP242 with adenosine 50- monophosphate-activated protein kinase (AMPK) activator metformin, using a panel of colorectal carcinoma (CRC) cell lines. We found that the PP242 and metformin combination enhanced the suppression of CRC cell proliferation, colony formation, and cancer cell apoptosis induction. The effect of this combination was observed on AMPK phosphorylation. Western blotting showed that PP242 inhibited mTORC1 activation, as indicated by the reduced expression of its major substrate p-S6K1 and the partially reduced phosphorylation of eIF4E-binding protein 1 (4E-BP1). The inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT Ser473) was transient and occurred in the first few hours of PP242 treatment; metformin exposure decreased the PP242 activity, counteracting AKT activation. We further demonstrated that this was related to direct AMPK-mediated phosphorylation of IRS-1 at Ser789. Thus, the combination of PP242 and metformin completely blocked the activity of both mTORC1 and mTORC2 kinase. This study suggests that this combination could be a more effective strategy for the treatment of CRC.

Targeting HSF1 as a Therapeutic Strategy for Multiple Mechanisms of EGFR Inhibitor Resistance in EGFR Mutant Non-Small-Cell Lung Cancer.

Simple SummaryWe attempted to identify target proteins and compounds that can be used to overcome EGFR-TKI resistance in NSCLC. To accomplish this, we generated EGFR inhibitor erlotinib-resistant HCC827-ErlR cells and obtained a list of differentially expressed genes. Then, we performed connectivity map analysis and identified heat shock factor 1 (HSF1) as a potential target protein to overcome erlotinib resistance. Using specific HSF1 shRNAs and KRIBB11 (N2-(1H-Indazol-5-yl)-N6-methyl-3-nitropyridine-2,6-diamine), we proved the effectiveness of HSF1 inhibition for overcoming erlotinib resistance in vitro. In addition, we proved the efficacy of emetine in inhibiting HSF1 activity and the tumor growth of erlotinib-resistant PC9-ErlR cells in a mouse model.Although EGFR-TKI treatment of NSCLC (non-small-cell lung cancer) patients often achieves profound initial responses, the efficacy is transient due to acquired resistance. Multiple receptor tyrosine kinase (RTK) pathways contribute to the resistance of NSCLC to first- and third-generation EGFR-TKIs, such as erlotinib and osimertinib. To identify potential targets for overcoming EGFR-TKI resistance, we performed a gene expression signature-based strategy using connectivity map (CMap) analysis. We generated erlotinib-resistant HCC827-ErlR cells, which showed resistance to erlotinib, gefitinib, osimertinib, and doxorubicin. A list of differentially expressed genes (DEGs) in HCC827-ErlR cells was generated and queried using CMap analysis. Analysis of the top 4 compounds from the CMap list suggested HSF1 as a potential target to overcome EGFR-TKI resistance. HSF1 inhibition by using HSF1 shRNAs or KRIBB11 decreased the expression of HSF1 downstream proteins, such as HSP70 and HSP27, and also decreased the expression of HSP90/HSP70/BAG3 client proteins, such as BCL2, MCL1, EGFR, MET, and AXL, causing apoptosis of EGFR-TKI-resistant cancer cells. Finally, we demonstrated the efficacy of the HSF1 inhibitor on PC9-ErlR cells expressing mutant EGFR (T790M) in vivo. Collectively, these findings support a targetable HSF1-(HSP90/HSP70/BAG3)-(BCL2/MCL1/EGFR/MET/AXL) pathway to overcome multiple mechanisms of EGFR-TKI resistance.

Cell surface integrin α5ß1 clustering negatively regulates receptor tyrosine kinase signaling in colorectal cancer cells via glycogen synthase kinase 3.

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

Identifying Kinase Inhibitors as Potential Adjuvant Chemotherapeutics in Treatment for Ovarian Cancer

In the United States, 22,000 women are diagnosed with ovarian cancer annually resulting in 14,000 deaths. This makes ovarian cancer the fifth leading cause of cancer deaths in women despite its relatively low frequency. Plasma and ascites levels of lysophosphatidic acid (LPA) are elevated in ovarian cancer patients even at early stages of the disease. LPA is a bioactive lipid involved in signal transduction. The increased LPA within the peritoneal cavity causes a cascading effect with the activation of multiple different kinase pathways in ovarian cancer cells such as the Rho‐A/Rock, Ras/MAPK, and the PI3K/Akt pathways. The activation of these kinase pathways through elevated LPA levels are known to prevent apoptosis, increase cell proliferation and metastasis, and decrease sensitivity to platinum‐based therapy. In efforts to increase the overall survival, enhance progression‐free intervals, and to improve the overall quality of life for patients with ovarian cancer, it has been hypothesized that combining traditional chemotherapeutics with kinase inhibitors would slow tumor cell proliferation and decrease disease progression. In these studies, two ovarian adenocarcinoma cell lines SKOV‐3 and CAOV‐3 are used to evaluate cell proliferation to determine IC50 values of common platinum‐based chemotherapeutics, Cisplatin, and Carboplatin, as well are the PARP inhibitors, Olaparib and Rucaparib, independently and in conjunction with of one of four kinase inhibitors; BI‐D1870 (Rsk inhibitor), MK‐2206 (Akt inhibitor), SCH772934 (Erk inhibitor), and Y27632 (Rock inhibitor). We demonstrate that the addition of kinase inhibitors to traditional and modern ovarian cancer chemotherapeutics decreases the IC50 values for these chemotherapeutics thus providing a potential pathway for improved ovarian cancer therapy.

Phosphorylation and Palmitoylation Dynamically Regulate Sodium Hydrogen Exchanger Isoform 1 (NHE1) Activity: Implications in Health and Disease

The SLC9 family is made up of nine isoforms of sodium hydrogen exchangers (NHE) which regulate pH by exchanging an intracellular proton for an extracellular sodium ion. Isoform 1 (NHE1) is ubiquitously expressed and plays a vital role in multiple cellular processes in addition to regulating pH such as cellular migration and proliferation, control of cell volume, and stress fiber formation. Additionally, NHE1 is a scaffolding protein that organizes protein complexes to regulate various signaling pathways within the cell. Therefore, it is important to understand the factors contributing to NHE1 regulation as maintenance of these critical cellular functions is vital ensure a healthy cell. Regulation of NHE1 occurs onthe large intracellular C‐terminus where binding partners and posttranslational modifications (PTMs) influence the exchanger in both short‐ and long‐term manners. Phosphorylation and palmitoylation are both dynamic reversible posttranslational modifications (PTMs) that regulate proteins to influence critical cellular processes. Multiple studies have shown phosphorylation and palmitoylation can work together in a barcode to regulate proteins and alter cellular functions. Previous work in our lab has shown NHE1 is regulated by palmitoylation and that inhibition of palmitoylation in cells expressing NHE1 decreases NHE1 activity, as well as NHE1 associated cellular functions such as stress fiber formation and cell migration. NHE1 is regulated by phosphorylation of multiple sites through various kinase pathways. To better understand the relationship between palmitoylation and phosphorylation on NHE1 we used various stimuli known to regulate multiple kinase pathways that impact NHE1 phosphorylation and measured the impact on NHE1 palmitoylation. Using lysophosphatidic acid (LPA) and phorbol 12‐myristate (PMA) to increase NHE1 phosphorylation leads to increased palmitoylation of NHE1, while activation of the NHE1 phosphorylation with insulin leads to decreased NHE1 palmitoylation. Furthermore, we have shown that using these stimuli NHE1 activity increases with NHE1 palmitoylation and this can be attenuated by irreversibly inhibiting palmitoylation using 2‐bromopalmitate (2BP). Using this information, we have begun to understand how the presence or absence of palmitoylation and phosphorylation in a coordinated manner contributes to a barcode that dictates a regulatory outcome and cellular response involving the sodium hydrogen exchanger isoform 1 (NHE1). Future studies will target specific kinases involved in these pathways to further explore the mechanism through which palmitoylation is altered.Support or Funding InformationThis work was supported by the University of North Dakota School of Medicine and Health Sciences and P20 GM103442 (to U.N.D.) from the INBRE program of the National Institute of General Medical Sciences. ND EPSCoRDoctoral Dissertation Assistantship UND0023386

Gas6 negatively regulates the Staphylococcus aureus-induced inflammatory response via TLR signaling in the mouse mammary gland.

Staphylococcus aureus (S. aureus)-induced mastitis is the most frequent, pathogenic, and prevalent infection of the mammary gland. The ligand growth arrest-specific 6 (Gas6) is a secretory protein that binds to and activates Tyro3, Axl, and MerTK receptors. This study explored the role of Gas6 in S. aureus-induced mastitis. Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S. aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways. Moreover, S. aureus also induces Gas6, which then activates the TAM receptor kinase pathway, which is related to the inhibition of TLR2- and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction. Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and messenger RNA expression of SOCS1 and SOCS3. S. aureus-induced MAPK and NF-ĸB p65 phosphorylation were also dependent on Gas6, which negatively regulated the production of Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in S. aureus-treated mammary tissues and mammary epithelial cells. Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism, which inhibits TLR2- and TLR6-mediated MAPK and NF-ĸB signaling by activating TAM receptor kinase (MerTK, Axl, and Tyro3) through the induction of SOCS1/SOCS3 proteins.

Abstract 2188: Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells

Abstract Background & Aims: The new therapy for pancreatic cancer which has the poorest prognosis among malignant tumors is high demand nowadays. The overall 5-year survival rate in all stages of this type of cancer is less than 5%, even with the multimodality therapy, including surgery, chemotherapy and radiotherapy. Although gemcitabine (GEM) is a key drug in the treatment of advanced pancreatic cancer, the therapeutic effect is limited because of drug resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition has shown promise in the treatment of solid malignancies. Src kinases work as a signal switch for multiple molecular signal transduction pathways. The objective of this study is to evaluate an antitumor effect of three kinds of small molecular weight compounds (antimetabolite Gemcitabine, HSP90 inhibitor NVP-AUY922, and Src kinase inhibitor Dasatinib) on tumor growth and chemotaxis in pancreatic cancer cell lines. Methods: Pancreatic cancer cell lines were treated with each of the 3 compounds to examine the effect on proliferation assay, apoptosis assay, Western blotting, and chemotaxis assay. Results: Of these compounds, HSP90 inhibitor inhibited cell proliferation in pancreatic cancer cells best in a dose- and time-dependent manner. Growth inhibitory effect of HSP90 inhibitor for pancreatic cancer cells were highest of the three. Further, HSP90 inhibitor inhibited the growth of gemcitabine sensitive and resistant pancreatic cancer cell lines. With flowcytometry and immunostaining, HSP90 inhibitor had higher apoptosis-inducing effect than gemcitabine. In the chemotaxis assay, HSP90 inhibitor inhibited the migration of pancreatic cancer cell best in the three compounds. In western blotting, HSP90 inhibitor suppressed the expression of multiple receptor tyrosine kinase proteins as well as the signal transduction in PI3K-Akt and RAS-MAPK pathways. Conclusion: In this study, HSP90 inhibitor was the best of 3 small molecular compounds and has proven effective in gemcitabine sensitive and resistant pancreatic cancer. The HSP90 inhibitor is useful as an antitumor drug by inhibiting proliferation and chemotaxis of pancreatic cancer cells through multiple kinase pathways. Citation Format: Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Masakiyo Sakaguchi, Yosuke Katata, Hiroaki Tanioka, Makoto Okawaki, Takeshi Nagasaka, Yoshiyuki Yamaguchi. Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2188.

Sodium Hydrogen Exchanger Isoform 1 (NHE1) Palmitoylation and Phosphorylation Barcoding: Implications on Regulation and Function

The sodium hydrogen exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by exchanging an intracellular proton for an extracellular sodium ion. Multiple cellular processes are associated with NHE1 activity including coordinated cell migration, cellular proliferation, and control of cell volume. NHE1 also functions as a membrane anchoring and scaffolding protein resulting in the organization of protein complexes, formation of stress fibers, and regulation of signaling pathways within the cell. The large C‐terminus (amino acids 503–815) of NHE1 extends intracellularly and functions as the major regulatory domain of the exchanger where binding partners and posttranslational modifications (PTM) influence the exchanger in both a short‐ and long‐term manner. Elucidating mechanisms of NHE1 regulation by posttranslational modifications has been focused on contributions from the many phosphorylation sites on functional cellular outcomes. Previously, we have shown NHE1 is also regulated by palmitoylation, a reversible lipid modification in which a 16‐carbon fatty acid is covalently linked to a cysteine residue via a thioester bond and have demonstrated that inhibition of palmitoylation in cells expressing NHE1 decreases NHE1 activity, as well as stress fiber formation and cell migration, two cellular functions that are regulated through NHE1 phosphorylation. Multiple studies demonstrating a relationship between phosphorylation and palmitoylation on protein regulation have been reported with our lab recently demonstrating a reciprocal relationship between phosphorylation and palmitoylation in regulation of the dopamine transporter. The goal of this study was to begin to understand how the presence or absence of palmitoylation and phosphorylation in a coordinated manner contributes to a barcode that dictates a regulatory outcome and cellular response involving NHE1. We utilized various stimuli including insulin, reduced serum, and lysophosphatidic acid (LPA), all of which are known to impact NHE1 phosphorylation and regulation through multiple kinase pathways. We show that increasing phosphorylation with phorbol 12‐myristate (PMA) and LPA results in increased palmitoylation of NHE1, while treatment with insulin which also increases NHE1 phosphorylation, results in decreased NHE1 palmitoylation. Using this information, we have begun studies to inhibit specific kinases involved in these pathways to further explore the mechanism through which palmitoylation is altered. Together, this data supports the idea of a barcode hypothesis where phosphorylation and palmitoylation and other PTMs work in concert to regulate protein function including NHE1. Future studies will identify the specific palmitoylation sites and their role in the barcode hypothesis pertaining to NHE1.Support or Funding InformationThis work was supported by the University of North Dakota School of Medicine and Health Sciences and P20 GM103442 (to U.N.D.) from the INBRE program of the National Institute of General Medical Sciences and the ND EPSCoR Doctoral Dissertation AssistantshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Cl−in signaling and ion transport by IRBIT‐mediated recruitment of multiple kinase and phosphatase pathways

IRBIT is a multifunctional protein that also controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters, including NBCe1‐B, CFTR and Slc26a6, by unknown mechanisms. With the ubiquitous NBCe1‐B, IRBIT interacts with the autoinhibitory domain (AID), exposing two cryptic GXXXP motifs to confer regulation by Cl−in. Here, we report that IRBIT‐dependent combinatorial dephosphorylation of S232/S233/S235 controls NBCe1‐B close and open states by determining autoinhibition and regulation by Cl−in. The pS233/pS235 conformer is fully active, no longer activated by IRBIT or inhibited by Cl−in. The pS232/pS235 conformer is similar to wild‐type NBCe1‐B, while the pS232/pS233 conformer is partially active, but retains inhibition by Cl−in. IRBIT then recruits the kinases SPAK and CaMKII to control phosphorylation of S65 and S12, respectively, which determine inhibition by Cl−in at the 32GXXXP36 and 194GXXXP198 motifs. Recruitment by IRBIT of PP1 and calcineurin dephosphorylates S65 and S12, respectively, and relieves inhibition by Cl−in. These findings reveal how multiple kinase/phosphatase pathways use multiple phosphorylation sites to fine tune a transport function, which has important implications for epithelial fluid and HCO3− secretion.Support or Funding InformationAll the work was supported by NIH fundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Artesunate Represses the Growth and Metastasis of Pancreatic Cancer Cells but Upregulates the Expression Levels of Proangiogenic Genes Through Inhibition of Protein Phosphatase 2A

Background: Artesunate (ART) has been found to exhibit remarkable anti-tumor activity. This study is aimed at investigating the significance and mechanisms of ART against pancreatic cancer cells in vitro and in vivo. Methods: MTT and pancreatic xenografts model were used to evaluate cell proliferation of pancreatic cancers. Transwell invasion and wound healing assay were used for cell invasion and migration. RNA sequencing was used to determine gene expression. PP2A activity assay was performed to confirm the inhibition of PP2A activity by ART. Molecular docking analysis was performed to predict the structure of the enzyme bond to the compound. Findings: ART markedly inhibited cell proliferation and the growth of pancreatic xenografts. ART arrested the cell cycle at G2/M phase, induced autophagy, and triggered extrinsic and intrinsic apoptotic pathways. Furthermore, ART significantly attenuated migration, invasion, and epithelial-mesenchymal transition of pancreatic cancer cells. ART exerted pro-angiogenesis potential in vivo. RNA sequencing analyses revealed that ART upregulates several pro-vascularization genes, and the downregulates a protein phosphatase 2A (PP2A) catalytic subunit (PP2Ac), which inhibition can lead to activating multiple pro-angiogenic kinase pathways. Overexpression of PP2A Y307F (constitutive active form) attenuated the upregulation of pro-vascularization genes upon ART treatment. A PP2A activity assay further confirmed a direct inhibition of PP2A activity by ART. Interpretation: Our study indicated that PP2A is a direct target of ART, thereby suggesting that ART can be a newly identified PP2A inhibitor. Funding Statement: This work was supported by the National Natural Science Foundation of China (grant numbers 81873587, 81472296, 81602091, 81874454, 81501970), the National Natural Science Foundation of Jiangsu Province (grant number BK20141162), the Six Major Talent Peak Project of Jiangsu Province (grant number 2015-WSN-022), the Project of Invigorating Health Care through Science, Technology and Education, Jiangsu Provincial Medical Youth Talent (grant number QNRC2016709), the Project of Jiangsu Provincial Commission of Health and Family Planning (grant number H201518), the Special Projects for Diagnosis and Treatment of Clinical Special Diseases in Suzhou (grant number LCZX201713), and the Science and Education for Health Foundation of Suzhou for Youth (grant number kjxw2015003). Declaration of Interests: The authors claim no conflicts of interest regarding the study or the manuscript. Ethics Approval Statement: Mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and received humane care according to the guidelines of Soochow University Institutional Animal Care and Treatment Committee.

Phase II study of sitravatinib in combination with nivolumab in patients with advanced or metastatic urothelial carcinoma (UC) after checkpoint inhibitor therapy (CIT).

TPS498 Background: Combination therapy with agents targeting molecular and cellular mechanisms of CIT resistance is a rational approach to restoring CIT efficacy in patients (pts) with immunotherapy-resistant UC. Sitravatinib is a tyrosine kinase inhibitor (TKI) that targets multiple closely-related receptor tyrosine kinase (RTK) pathways, including the split RTKs VEGFR-2 and KIT, as well as the TAM (TYRO3, AXL, and MER) RTKs. Inhibition of the split RTKs enhances antitumor activity by reduction of immunosuppressive regulatory T cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME). Inhibition of TAM RTKs further restores a more immune-supportive TME by depletion of MDSCs and repolarization of tumor-associated macrophages to the M1 phenotype associated with secretion of pro-inflammatory cytokines. Given these pleiotropic immune-activating effects, the combination of sitravatinib with nivolumab is a rational approach to restoring or enhancing CIT clinical activity in pts with immunotherapy-resistant UC. This combination has been shown to be safe and tolerable in an ongoing Phase 2 study in pts with metastatic non-small cell lung cancer. Methods: This open-label Phase 2 study evaluates tolerability and clinical activity of sitravatinib in combination with nivolumab in pts with advanced or metastatic UC who experienced disease progression on or after CIT as the most recent systemic therapy. Enrollment is stratified by prior receipt of platinum-based chemotherapy. If the initial 2 cohorts are of high interest for efficacy, the protocol allows for addition of new cohorts to include pts previously treated with selected immunotherapies (anti-CTLA-4, anti-OX40 or anti–CD137 therapy) or who are CIT-naïve. The primary objective is Objective Response Rate. A Predictive Probability Design will be applied allowing for expansion to 40 pts per cohort. Sitravatinib is administered orally daily in continuous 28-day cycles at 120 mg; nivolumab intravenously at 240 mg every 2 weeks or 480 mg every 4 weeks. Status: The study is open for enrollment and recruitment is ongoing. Clinical trial information: NCT03606174.

Discovery of Pyrido[3',2':5,6]thiopyrano[4,3-d]pyrimidine-Based Antiproliferative Multikinase Inhibitors.

Protein kinases dysregulation is extremely common in cancer cells, and the development of new agents able to simultaneously target multiple kinase pathways involved in angiogenesis and tumor growth may offer several advantages in the treatment of cancer. Herein we report the discovery of new pyridothiopyranopyrimidine derivatives (2-4) showing high potencies in VEGFR-2 KDR inhibition as well as antiproliferative effect on a panel of human tumor cell lines. Investigation on the selectivity profile of the representative 2-anilino-substituted compounds 3b, 3i, and 3j revealed a multiplicity of kinase targets that should account for the potent antiproliferative effect produced by these pyridothiopyranopyrimidine derivatives.

Regulation of Yeast-to-Hyphae Transition in Yarrowia lipolytica.

The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae We confirmed that the Y. lipolyticamsn2 (Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1 or Ylswi6 homologs decreased hyphal growth and that deletion of either Ylmbp1 or Ylswi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1 and Ylnik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1 Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCE Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in Yarrowia lipolytica We confirmed that the transcription factor Ylmsn2 is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1 and Ylnik1 as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest that Y. lipolytica transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.

Modulation of Cl- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl- in Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl- in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl- in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3 - secretion.

Epidermal growth factor receptor and ligand family expression and activity in glioblastoma.

Epidermal growth factor family of receptor tyrosine kinases (ERBB) family cell surface receptors, including epidermal growth factor receptor (EGFR/ERBB1), are phosphorylated upon binding by various EGF family ligands and signal via multiple kinase pathways. EGFR signaling is enhanced because of mutational activation of EGFR in almost half of glioblastomas, the most common malignant primary brain tumor. Therapeutic targeting of EGFR in glioblastoma has remained largely unsuccessful. Here, we profiled nine long-term (LTC) and five glioma-initiating (GIC) cell lines for expression and activation of ERBB family receptors and expression of their ligands. Receptors and ligands were abundantly expressed, with patterns overall similar to glioblastoma expression profiles invivo as deposited in The Cancer Genome Atlas database. No differences between LTC and GIC emerged. Irrespective of ligand or receptor expression, neither an EGFR antibody, erbitux, nor an EGFR tyrosine kinase inhibitor, gefitinib, were particularly active against LTC or GIC at clinically relevant concentrations. Self-renewal capacity of GIC was severely compromised by epidermal growth factor (EGF) withdrawal, but rescued by transforming growth factor alpha (TGF-α), although not by neuregulin-1 (NRG-1). Subcellular fractionation indicated high levels of nuclear phosphorylated EGFR in all LTC and GIC. In LN-229 cells, pERBB2 and pERBB3 were also detected in the nucleus. Nuclear pERBB2 was less sensitive, whereas pERBB3 was induced, in response to gefitinib. This study provides an extensive characterization of human glioma cell models, including stem-like models, with regard to ERBB receptor/ligand expression and signaling. Redundant signaling involving multiple ERBB family ligands and receptors may contribute to the challenges of developing more effective EGFR-targeted therapies for glioblastoma.

5-HT1A receptor-mediated activation of neuroendocrine responses and multiple protein kinase pathways in the peripubertal rat hypothalamus

Increasing evidence suggests that multiple factors can produce effects on the immature brain that are distinct and more long-lasting than those produced in adults. The hypothalamic paraventricular nucleus (PVN) is a region integral to the hypothalamic-pituitary-adrenal axis and is affected by anxiety, depression, and drugs used to treat these disorders, yet receptor signaling mechanisms operative in hypothalamus prior to maturation remain to be elucidated. In peripubertal male rats, systemic injection of the selective serotonin 1A (5-HT1A) receptor agonist (+)8-OH-DPAT (0.2 mg/kg) markedly elevated plasma levels of oxytocin and adrenocorticotropic hormone (ACTH) at 5 and 15 min post-injection. The 5-HT1A receptor selectivity was demonstrated by the ability of the 5-HT1A receptor selective antagonist WAY100635 to completely block both oxytocin and ACTH responses at 5 min, with some recovery of the ACTH response at 15 min. At 15 min post-injection, (+)8-OH-DPAT also increased levels of phosphorylated extracellular signal-regulated kinase (pERK) and phosphorylated protein kinase B (pAkt) in the PVN. As previously observed in adults, (+)8-OH-DPAT reduced levels of pERK in hippocampus. WAY100635 also completely blocked (+)8-OH-DPAT-mediated elevations in hypothalamic pERK and pAkt and the reductions in hippocampal pERK, demonstrating 5-HT1A receptor selectivity of both kinase responses. This study provides the first demonstration of functional 5-HT1A receptor-mediated ERK and Akt signaling pathways in the immature hypothalamus, activated by a dose of (+)8-OH-DPAT that concomitantly stimulates neuroendocrine responses. This information is fundamental to identifying potential signaling pathways targeted by biased agonists in the development of safe and effective treatment strategies in children and adolescents.

SH2B3 aberrations enriched in iAMP21 B lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) represents the most common childhood malignancy. Although survival for pediatric B-ALL has approached 90%, variability in outcome among and within cytogenetically defined subgroups persists. While G-banding and fluorescence in situ hybridization (FISH) have been used to characterize leukemic clones, there is added value of chromosomal microarray and next generation sequencing in screening genome-wide for copy number aberrations, copy neutral loss of heterozygosity and nucleotide variations. Evaluation of novel genetic aberrations can provide information about the biologic mechanisms of cytogenetically defined subgroups associated with poor prognosis, explain heterogeneity in patient outcome and identify novel targets for therapeutic intervention. The high risk B-ALL intrachromosomal amplification of chromosome 21, (iAMP21), subtype is characterized by amplification of a region of chromosome 21 that typically encompasses the RUNX1 gene and is associated with poor prognosis. Analysis of chromosomal microarray and FISH data revealed that deletions of SH2B3, encoding a negative regulator of multiple tyrosine kinase and cytokine signaling pathways, are enriched among leukemias harboring iAMP21. Enrichment of SH2B3 aberrations in the iAMP21 subtype may indicate that loss of SH2B3 contributes to disease progression and raises the possibility that these leukemias may be sensitive to tyrosine kinase inhibitors.

Heat shock factor 1 confers resistance to lapatinib in ERBB2-positive breast cancer cells

Despite success of ERBB2-targeted therapies such as lapatinib, resistance remains a major clinical concern. Multiple compensatory receptor tyrosine kinase (RTK) pathways are known to contribute to lapatinib resistance. The heterogeneity of these adaptive responses is a significant hurdle for finding most effective combinatorial treatments. The goal of this study was to identify a unifying molecular mechanism whose targeting could help prevent and/or overcome lapatinib resistance. Using the MMTV-ERBB2;mutant p53 (R175H) in vivo mouse model of ERBB2-positive breast cancer, together with mouse and human cell lines, we compared lapatinib-resistant vs. lapatinib-sensitive tumor cells biochemically and by kinome arrays and evaluated their viability in response to a variety of compounds affecting heat shock response. We found that multiple adaptive RTKs are activated in lapatinib-resistant cells in vivo, some of which have been previously described (Axl, MET) and some were novel (PDGFRα, PDGFRβ, VEGFR1, MUSK, NFGR). Strikingly, all lapatinib-resistant cells show chronically activated HSF1 and its transcriptional targets, heat shock proteins (HSPs), and, as a result, superior tolerance to proteotoxic stress. Importantly, lapatinib-resistant tumors and cells retained sensitivity to Hsp90 and HSF1 inhibitors, both in vitro and in vivo, thus providing a unifying and actionable therapeutic node. Indeed, HSF1 inhibition simultaneously downregulated ERBB2, adaptive RTKs and mutant p53, and its combination with lapatinib prevented development of lapatinib resistance in vitro. Thus, the kinome adaptation in lapatinib-resistant ERBB2-positive breast cancer cells is governed, at least in part, by HSF1-mediated heat shock pathway, providing a novel potential intervention strategy to combat resistance.