Activation Of Downstream Kinases Research Articles

Abstract 3171: The effect of estrogen and phytoestrogens in Inflammatory breast cancer

Abstract Inflammatory breast cancer is an aggressive and lethal type of breast cancer. Frequently, this cancer is diagnosed in an advanced stage with metastasis. IBC diagnosis is associated with a worse survival rate than other types of breast cancer. The presence of dermal and stromal tumor emboli blocking the lymphatic vessels under the skin is considered a hallmark of IBC and assumed to be responsible for the high metastatic behavior and aggressiveness of IBC disease. To this date, there are no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of alternate estrogen receptors including estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, naturally derived structural analogs to 17β-estradiol (E2), were evaluated as an alternative treatment option using in vitro IBC cell models (SUM149 and SUM190). Given the estrogenic properties of phytoestrogens, the drugs were hypothesized to interact with targets in pathways similar to E2 in non-genomic signaling. Several phytoestrogens including coumestrol (Cou) can induce a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test these hypotheses, IBC cells were treated with E2 or Cou to determine their effects on the activation of downstream kinases by western blot and phospho-kinase array assays. In addition, transcriptional effects upon treatment with Cou were identified using RNA-seq. A dose-response curve in 2D and 3D models was generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decreases migration, proliferation, and tumor emboli formation in IBC cell lines. In addition, IBC cells were treated using monotherapy and combination therapy with phytoestrogens including Cou, genistein (GN), and/or 8-prenylnaringenin (8-PN). Observed reductions of cell viability in IBC cell lines treated with the different phytoestrogens and combinations make them worthy of further characterization as anti-cancer therapies in in vitro and in vivo models. In summary, IBC cells are responsive to E2 making this signaling pathway attractive for the development of innovative therapeutic strategies with phytoestrogens as potential candidates for treatment. Citation Format: Esther A. Peterson-Peguero, Keishla Rodriguez-Martir, Madeline Ganshert, Dalissa Negron-Figueroa, Ayeisha N. Colon-Ortiz. The effect of estrogen and phytoestrogens in Inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3171.

Formyl Peptide Receptor 2-Dependent cPLA2 and 5-LOX Activation Requires a Functional NADPH Oxidase.

Phospholipases (PL) A2 catalyzes the hydrolysis of membrane phospholipids and mostly generates arachidonic acid (AA). The enzyme 5-lipoxygenase (5-LOX) can metabolize AA to obtain inflammatory leukotrienes, whose biosynthesis highly depends on cPLA2 and 5-LOX activities. Formyl Peptide Receptor 2 (FPR2) belongs to a subfamily of class A GPCRs and is considered the most versatile FPRs isoform. Signaling triggered by FPR2 includes the activation of several downstream kinases and NADPH oxidase (NOX)-dependent ROS generation. In a metabolomic analysis we observed a significant increase in AA concentration in FPR2-stimulated lung cancer cell line CaLu-6. We analyzed cPLA2 phosphorylation and observed a time-dependent increase in cPLA2 Ser505 phosphorylation in FPR2-stimulated cells, which was prevented by the MEK inhibitor (PD098059) and the p38MAPK inhibitor (SB203580) and by blocking NOX function. Similarly, we demonstrated that phosphorylation of 5-LOX at Ser271 and Ser663 residues requires FPR2-dependent p38MAPK and ERKs activation. Moreover, we showed that 5-LOX Ser271 phosphorylation depends on a functional NOX expression. Our overall data demonstrate for the first time that FPR2-induced ERK- and p38MAPK-dependent phosphorylation/activation of cPLA2 and 5-LOX requires a functional NADPH oxidase. These findings represent an important step towards future novel therapeutic possibilities aimed at resolving the inflammatory processes underlying many human diseases.

Selective Activation of M1 Muscarinic Receptors Attenuates Human Colon Cancer Cell Proliferation.

M3 muscarinic receptor (M3R) activation stimulates colon cancer cell proliferation, migration, and invasion; M3R expression is augmented in colon cancer and ablating M3R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M3R, M1R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M1R versus M3R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M1R agonists. We detected divergent expression of M1R and M3R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M1R agonists, we found that in contrast to the effects of M3R activation, selective activation of M1R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M1R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M1R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M1R agonist-induced attenuated cell proliferation. Lastly, adding M1R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M1R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.

The histone H2B Arg95 residue efficiently recruits the transcription factor Spt16 to mediate Ste5 expression of the pheromone response pathway

In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.

Modulating retinoid-X-receptor alpha (RXRA) expression sensitizes chronic myeloid leukemia cells to imatinib in vitro and reduces disease burden in vivo.

Introduction: The ligand-activated transcription factors, nuclear hormone receptors (NHRs), remain unexplored in hematological malignancies except for retinoic acid receptor alpha (RARA). Methods: Here we profiled the expression of various NHRs and their coregulators in Chronic myeloid leukemia (CML) cell lines and identified a significant differential expression pattern between inherently imatinib mesylate (IM)-sensitive and resistant cell lines. Results: Retinoid-X-receptor alpha (RXRA) was downregulated in CML cell lines inherently resistant to IM and in primary CML CD34+ cells. Pre-treatment with clinically relevant RXRA ligands improved sensitivity to IM in-vitro in both CML cell lines and primary CML cells. This combination effectively reduced the viability and colony-forming capacity of CML CD34+ cells in-vitro. In-vivo, this combination reduced leukemic burden and prolonged survival. Overexpression (OE) of RXRA inhibited proliferation and improved sensitivity to IM in-vitro. In-vivo, RXRA OE cells showed reduced engraftment of cells in the bone marrow, improved sensitivity to IM, and prolonged survival. Both RXRA OE and ligand treatment markedly reduced BCR::ABL1 downstream kinase activation, activating apoptotic cascades and improving sensitivity to IM. Importantly, RXRA OE also led to the disruption of the oxidative capacity of these cells. Conclusion: Combining IM with clinically available RXRA ligands could form an alternative treatment strategy in CML patients with suboptimal response to IM.

Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands.

The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling. By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells. We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases. Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.

Abstract 2405: Delineating molecular vulnerabilities of ATM mutant prostate cancers

Abstract Background: Mutations in DNA Damage Response (DDR) genes, including Ataxia, Telangiectasia, Mutated (ATM), are common in advanced castration-resistant prostate cancers (CRPCs). Poly (ADP-ribose) polymerase (PARP) inhibitors are approved in DDR mutant CRPCs, but demonstrate limited clinical efficacy in CRPCs with ATM mutations. In this project, we sought to specifically define the impact of ATM loss on DDR pathways in CRPC, with the goal of identifying alternate therapeutic vulnerabilities. Methods: ATM-KO CRPC cell lines were generated via CRISPR-Cas9 mediated knockout. ATM loss and abolishment of downstream ATM kinase activity was confirmed via western blot. We performed an unbiased phospho-proteomic evaluation of DDR pathways in parental and ATM-KO cells after ionizing radiation (IR). Clonogenic survival assays were performed with either an ATR inhibitor (VX970), the selective DNA-PKcs inhibitor (M3814), or combination therapy. Kinetics of DDR protein recruitment and resolution were interrogated with immunofluorescence (IF) staining for γH2ax, 53BP1, MDC1, and Rad51 foci. Results: ATM-KO cells were able to effectively repair DNA damage following IR, as measured by recruitment and resolution of γH2ax, 53BP1, MDC1, and Rad51 foci. Phospho-proteomic studies demonstrated that ATM-KO cells maintain canonical DDR pathways through ATR and DNA-PKcs kinase activation. Treatment of ATM-KO cells with either VX-970 or M3814 only incrementally affected DDR in ATM-KO cells compared to parental controls, as evidenced by clonogenic survival assays and maintenance of DDR foci. Importantly, combination treatment with VX-970 and M3814 prevented downstream DDR foci recruitment and radio-sensitized ATM-KO CRPC to a greater extent than parental controls. This suggested that activity of any of the trinity of kinases is sufficient to mediate DDR, and that blockade of both ATR and DNA-PKcs is required to effectively prevent DDR in ATM-KO CRPC. We then leveraged a RUVBL1 ATPase inhibitor Compound B, which significantly attenuates levels of these kinases in lung cancer cells. We confirmed that Compound B treatment attenuated ATR and DNA-PKcs protein expression and kinase activity in ATM-KO CRPC cells, and demonstrated sensitivity of ATM-KO cells to Compound B. Conclusions: Our data demonstrates that dual targeting of ATR and DNA-PKcs is necessary in ATM-KO CRPC, as either kinase is independently capable of mediating DDR following IR. Our initial studies indicate that the RUVBL1 ATPase inhibitor Compound B may effectively block DDR in ATM-mutant CRPC, and could be utilized as a novel therapeutic strategy in this molecular subtype. Citation Format: Mia Hofstad, Lan Yu, Andrea Woods, Zoi Sychev, Collin Gilbreath, Xiaofang Huo, Ralf Kittler, Justin M. Drake, Ganesh V. Raj. Delineating molecular vulnerabilities of ATM mutant prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2405.

MD2 Inhibits Choroidal Neovascularization via Antagonizing TLR4/MD2 Mediated Signaling Pathway

Purpose To explore the pathological mechanism of Toll-like receptor 4 (TLR4) mediating neovascular age-related macular degeneration (nAMD) and the potential role of the TLR4 coreceptor myeloid differentiation protein 2 (MD2). Methods In the study, we inhibited MD2 with the chalcone derivative L2H17 and we utilized a laser-induced choroidal neovascularization (CNV) mouse model and Tert-butyl hydroperoxide (TBHP)-challenged rhesus choroid-retinal endothelial (RF/6A) cells to assess the effect of MD2 blockade on CNV. Results Inhibiting MD2 with L2H17 reduced angiogenesis in CNV mice, and significantly protected against retinal dysfunction. In retina and choroid/retinal pigment epithelium (RPE) tissues, L2H17 reduced phospho-ERK, phospho-P65 but not phospho-P38, phospho-JNK, and reduced the transcriptional levels of IL-6, TNF-α, ICAM-1 but not VCAM-1. L2H17 could protect RF/6A against TBHP-induced inflammation, oxidative stress, and apoptosis, via inhibiting the TLR4/MD2 signaling pathway and the following downstream mitogen-activated protein kinase (MAPK) and nuclear transcription factor-κB (NF-κB) activation. Conclusions Inhibiting MD2 with L2H17 significantly reduced CNV, suppressed inflammation, and oxidative stress by antagonizing TLR4/MD2 pathway in an MD2-dependent manner. MD2 may be a potential therapeutic target and L2H17 may offer an alternative treatment strategy for nAMD.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our invitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

P10.23.B Polycystin-1 induces mechanotransduction pathways in gioblastoma cells under hydrostatic pressure

Abstract Background Increased mechanical load and stiffness of the tumor microenvironment are key features during progression of gliomas. Polycystin-1 (PC1) is a mechanosensitive protein which perceives extracellular mechanical cues. The aim of the study was to investigate PC1 participation in mechanisms of glioblastoma (GBM) progression under a mechanically-induced microenvironment. Material and Methods We generated monolayer cell cultures and tumor spheroids of the T98G glioblastoma cell line. Cells were subjected to continuous hydrostatic pressure (HP), inhibition of PC1 mechanical perception through functional blockage of its extracellular domain, and silencing of PKD1 expression. The level of activation of PC1 mechano-induced downstream kinases (mTOR, ERK, FAK) and transcription factors (YAP, TAZ) was evaluated via Western blot at specific time-points. Functional assays were performed to assess cell proliferation, clone formation, and cell migration. qPCR was employed to quantify the mRNA expression of markers regarding certain traits of cancer cells. Results In monolayer cell cultures, PC1 functional inhibition under HP is associated with mTOR and ERK activation and elevated expression of TAZ. On the other hand, PKD1 knockdown under HP is associated with activation of FAK, but not mTOR or ERK. In tumor spheroids, PC1 inhibition acts in synergy with HP to activate FAK and upregulate YAP, but not ERK or mTOR. PC1 inhibition under HP is associated with compact 3-dimensional spheroid structures. PC1 inhibition acts in synergy with HP to suppress cell proliferation, clone formation, and cell migration in GBM cells. The same synergistic effect is observed on the expression of respective markers of apoptosis, angiogenesis, proliferation, and epithelial-to-mesenchymal transition of GBM cells. Conclusion PC1 seems to be able to sense applied mechanical forces in GBM cells by regulating the activity of downstream molecules and enhance certain traits of tumor promotion. This work was co-financed by Operational Programme “Human Resources Development, Education and Lifelong Learning”, National Strategic Reference Framework (NSRF) 2014-2020, Ministry of Economy and Development, Greece & European Union (European Social Fund (ESF), in the context of the project “Investigation of the role of polycystin proteins in glioma neoplastic cells”; Grant/Award Number: MIS 5047967

A lymphatic-absorbed multi-targeted kinase inhibitor for myelofibrosis therapy

Activation of compensatory signaling nodes in cancer often requires combination therapies that are frequently plagued by dose-limiting toxicities. Intestinal lymphatic drug absorption is seldom explored, although reduced toxicity and sustained drug levels would be anticipated to improve systemic bioavailability. A potent orally bioavailable multi-functional kinase inhibitor (LP-182) is described with intrinsic lymphatic partitioning for the combined targeting of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways without observable toxicity. We demonstrate selectivity and therapeutic efficacy through reduction of downstream kinase activation, amelioration of disease phenotypes, and improved survival in animal models of myelofibrosis. Our further characterization of synthetic and physiochemical properties for small molecule lymphatic uptake will support continued advancements in lymphatropic therapy for altering disease trajectories of a myriad of human disease indications.

Abstract 6366: A novel small molecule inhibitor of MAPK and PI3K ameliorates disease phenotypes in myelofibrosis

Abstract Myelofibrosis is a myeloproliferative neoplasm (MPN) caused primarily by mutations in hematopoietic stem and progenitor cells (HSPCs) that activate Janus kinase 2 (JAK2). Central roles for JAK2 signaling in myelofibrosis (MF) have been established, leading to marked extramedullary hematopoiesis, splenomegaly, and bone marrow fibrosis, but the use of JAK inhibitors has shown limited ability to produce durable remissions in most patient populations. Bypass or resistance mechanisms to JAK inhibition have been linked to concomitant activation of compensatory phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways, and while genetic and functional studies have not fully defined the mechanisms for transformation and maintenance of the proliferative state in MF, targeting multiple signaling pathways will be required to alter the disease course and improve therapeutic outcomes. Treatments using kinase inhibitor combinations remain a challenge clinically, and trials have struggled to create positive balance between gains in survival, therapeutic efficacy, and dose-limiting toxicity. A multi-functional kinase inhibitor was developed to deliver consistent and synergistic dosing ratios against multiple targets simultaneously, thereby blocking compensatory oncogenic signaling pathways while minimizing the prospect for adverse effects. Using synthetic medicinal chemistry, we present a potent and selective, orally bioavailable, single-molecule multi-targeted kinase inhibitor (LP-182) against MAPK and PI3K/mTOR signaling pathways. We demonstrate selectivity and therapeutic efficacy through in vitro screening of kinome inhibition, reduction of cell growth and downstream kinase activation, renormalization of immune cell populations, amelioration of splenomegaly and bone marrow fibrosis, and improved survival in the myeloproliferative leukemia oncogene (MPLW515L) mouse model of myelofibrosis. Furthermore, treatment with LP-182 was well tolerated showing no observable pharmacotoxicity, and combination with the JAK inhibitor Ruxolitinib also showed ~35% reduction in spleen size as compared to either treatment alone with a ~60% spleen volume reduction compared to vehicle treatment, in the JAK2+/V617F mutant mouse model. Overall, these data suggest that in vivo bioavailability and distribution of LP-182 to secondary lymphoid tissues attenuates MAPK and PI3K signaling to alleviate disease phenotypes in animal models of MF. Simultaneous targeting of both MAPK and PI3K pathways with LP-182 alone or in combination with JAK inhibition provides the potential for significant new opportunities to improve the clinical outcome of MF patients. Citation Format: Christopher A. Bonham, Youngsoon Jang, Kevin Heist, Amanda Welton, Tanner Robison, Kathryn E. Luker, Gary D. Luker, Winston Y. Lee, Thomas L. Chenevert, Marcian Van Dort, Brian D. Ross. A novel small molecule inhibitor of MAPK and PI3K ameliorates disease phenotypes in myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6366.

Targeted Delivery of Epidermal Growth Factor to the Human Placenta to Treat Fetal Growth Restriction.

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50–100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery; however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

First-in-Class Oral Small Molecule Against the Tyrosine Kinase ROR1 (KAN0439834) Induced Significant Apoptosis of Chronic Lymphocytic Leukemia Cells

Background: ROR1 is a transmembrane protein of the receptor tyrosine kinase (RTK) family normally expressed during embryogenesis, but repressed in most normal adult tissues. ROR1 is of importance for cell proliferation, survival, differentiation, metabolism, and polarity. In chronic lymphocytic leukemia (CLL), ROR1 is upregulated and constitutively phosphorylated. Inhibition of ROR1 by siRNA and monoclonal antibodies (mAbs) abrogated downstream kinase activities as well as induced apoptosis of CLL cells. KAN0439834, a small molecule inhibitor (535 Da) was synthesized with the aim to bind to the TK domain of ROR1 and inhibit phosphorylation.Aim: In the present study we investigated the effects of KAN0439834, on survival of CLL cells and signaling, and in vivo effects in NOD-SCID mice xenografted with human CLL cells.Methods: MTT and Annexin V/PI assays were used to analyse cytotoxicity of KAN0439834. Fresh CLL cells were also co-cultured with the human stromal cell line HS-5 (ROR1 negative) and incubated in vitro with KAN0439834 and apoptosis measured (Annexin V/PI). CLL cells were also cultured with KAN0439834 (50-250 nM) for 2 h and recombinant Wnt5a (25-50 µg/mL) was added. The cells were further incubated for 30 min before lysate preparation and cytotoxicity assayed after 24 h. Proximity ligation assay was used to investigate the effects of KAN0439834 on ROR1/LRP6 heterodimerization. Phosphorylated RTKs in CLL cells and the effects of KAN0439834 on phosphorylation of RTKs were analysed by the Human-phospho-RTK array and WB. Finally, in two mice experiments, fresh CLL cells were grafted into NOD-SCID mice and oral KAN0439834 treatment was started 7 days after transplantation and continued for 7 days in the first study and 14 days in the second study.Results: KAN0439834 effectively induced apoptosis of CLL cells (EC50=250 nM) with a 60-fold selectivity for CLL cells compared to normal PBMC. The molecule induced significant apoptosis of CLL cells from all tumor compartments. Importantly, KAN0439834 was significantly more effective than an anti-ROR1 mAb (against the external part of ROR1) in inducing apoptosis of CLL cells. When CLL and HS-5 cells were co-cultured, HS-5 cells could partially prevent induction of apoptosis of CLL cells at low concentrations of KAN0439834, while at higher concentrations of KAN0439834 the presence of stromal cells had no effect. Wnt5a increased phosphorylation of ROR1 in a dose dependent manner. KAN0439834 dephosphorylated both ROR1 and SRC as well as inhibited Wnt5a-induced ROR1 phosphorylation in this system. KAN0439834 dephosphorylated ROR1, LRP6 and associated downstream signaling molecules as GSK3β, PKCδ, MAPK, PI3K, AKT, mTOR, and CREB. KAN0439834 was highly specific for inhibition of ROR1 phosphorylation as compared to other kinase inhibitors in clinical use for cancer treatment. KAN0439834 could be administered in a dose-schedule to mice giving a plasma conc. of > 600 nM for at least 6 h which according to in vitro pharmacokinetic analyses should be sufficient to induce irreversible apoptosis of CLL cells. Oral administration of KAN0439834 to NOD-SCID mice significantly inhibited growth of xenografted human CLL cells. No major side effects were observed in the KAN0439834 treated mice. Immunohistochemistry staining of spleens from the two studies showed a significant decrease of CLL cells in mice treated with KAN0439834 but not in controls.Conclusions: KAN0439834 is the first generation of a novel class of ROR1-inhibiting small molecule drugs developed by phenotypic screening using CLL cells. The small molecule was much more effective in inducing CLL apoptosis than the anti-ROR1 antibody. KAN0439834 inactivated various signaling pathways in the leukemic cells suggested to be associated with ROR1 signaling. The development of new anti-cancer drugs with other mechanisms of action than those clinically available when existing drugs fail is warranted to improve the prognosis of CLL. KAN0439834 may not only be potent in CLL but also in other ROR1 expressing tumors; such studies are in progress. Our results support the further development of ROR1 inhibitors as a new therapeutic principle. DisclosuresHansson:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janssen-Cilag: Honoraria, Research Funding. Vågberg:Kancera AB: Employment. Byström:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Schultz:Kancera AB: Employment. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment. Österborg:Abbvie: Honoraria; Pfizer: Honoraria; Celgene: Research Funding; Gilead: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding. Moshfegh:Kancera AB: Employment.

Inhibitory Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer

Breast cancer is the second leading cause of death in females in the United States, being Inflammatory Breast Cancer (IBC) the most aggressive subtype. Usually, this cancer is diagnosed in an advanced stage with metastasis. Experts in IBC pathogenesis think that IBC rapid metastasis is induces by tumor emboli formation, a hallmark of IBC. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease to develop effective targeted therapeutics. To this date, there is no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Importantly, several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of an alternate isoform of estrogen receptor alpha-66, estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, like coumestrol (Cou), are natural compounds found in plants, specifically soybeans or clover sprouts. Also, it is a polyphenolic compound that is structurally similar to estradiol (E2) and several studies have shown that coumestrol induces a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test this hypothesis, IBC cells were treated with E2 or Cou, and activation of downstream kinases was analyzed by Western blot and using a Human Phospho-Kinase Array. A dose-response curve in 2D and 3D models were generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decrease migration, proliferation, and tumor emboli formation in IBC cell line. In summary, IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies show a potential anticancer activity in IBC cell lines.

The Structure and Ubiquitin Binding Properties of TRAF RING Heterodimers

Tumour necrosis factor (TNF) receptor associated factor (TRAF) family members share a common domain architecture, but play non-redundant physiological roles in cell signalling. At the N terminus, most TRAFs have a RING domain, followed by a series of Zinc finger (ZF) domains. The RING domain of TRAF6 dimerizes, and the RING homodimer together with the first ZF assembles ubiquitin chains that form a platform which facilitates activation of downstream kinases. The RING dimer interface is conserved amongst TRAF proteins, suggesting that functional heterodimers could be possible. Here we report the structure of the TRAF5-TRAF6 RING heterodimer, which accounts for the stability of the heterodimer as well as its ability to assemble ubiquitin chains. We also show that the RING domain of TRAF6 heterodimerizes with TRAF3 and TRAF2, and demonstrate that the linker helix and first ZF of TRAF2 can cooperate with TRAF6 to promote chain assembly. Collectively our results suggest that TRAF RING homo- and hetero-dimers have the potential to bridge interaction of nearby TRAF trimers and modulate TRAF-mediated signalling.

Tripartite Motif-Containing 44 is Involved in the Tumorigenesis of Laryngeal Squamous Cell Carcinoma, and its Expression is Downregulated by Nuciferine.

Tripartite motif-containing 44 (TRIM44) was reported to be involved in the tumorigenesis of several tumors, but its function in laryngeal squamous cell carcinoma has not been investigated yet. In the present study, we aimed to elucidate the function of TRIM44 in laryngeal squamous cell carcinoma, and identify the compounds which could inhibit TRIM44 expression. Our results showed that TRIM44 was upregulated in tumor tissues and cell lines of laryngeal squamous cell carcinoma. Knockdown of TRIM44 significantly inhibited cell growth of laryngeal squamous cell carcinoma by suppressing TLR4, phosphorylated AKT and phosphorylated NF-κB p65 expression in vitro. Moreover, TRIM44 knockdown inhibited tumor growth in nude mice, which further suggested that TRIM44 exerted oncogenic activity in laryngeal squamous cell carcinoma. Interestingly, it was found that nuciferine significantly inhibited the mRNA levels of TRIM44 after screening a small natural compound library. Our further studies showed nuciferine markedly downregulated the protein levels of TRIM44 and its substrate TLR4 in a concentration-dependent manner in laryngeal squamous cell carcinoma cells. Moreover, the activation of downstream kinases of TLR4 such as AKT signaling pathway was also inhibited by nuciferine. Additionally, nuciferine markedly inhibited cell survival of laryngeal squamous cell carcinoma in a concentration-dependent manner. In contrast, TRIM44 overexpression significantly reduced the cytotoxicity of nuciferine in laryngeal squamous cell carcinoma cells. In conclusion, this study indicated that inhibiting TRIM44 would be a useful strategy for the treatment of laryngeal squamous cell carcinoma, and nuciferine could be a potential chemical applicated in the therapy of laryngeal squamous cell carcinoma.

Phosphorylation of the RB C-terminus regulates condensin II release from chromatin

The retinoblastoma tumor suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate and render RB inactive in late G1/S, promoting entry into S phase. Recently, monophosphorylated RB species were described to have distinct cell-cycle-independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output. We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK and RB S838/T841 phosphorylation. This signaling pathway disrupts RB and condensin II interaction with chromatin. Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin.

Non-Canonical WNT5A Signaling Through RYK Contributes to Aggressive Phenotype of the Rheumatoid Fibroblast-Like Synoviocytes

We hypothesized that WNT5A could contribute to the enhanced migration and invasiveness of rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), which is one of the incompletely understood aspects of the RA FLS aggressive phenotype. This hypothesis is based on the previous evidence of a WNT5A role in both, RA and cell migration. Migration and invasion of RA FLS were assessed after incubation with recombinant Wnt5a (rWnt5a) or silencing of the endogenous WNT5A expression. The expression of WNT5A, WNT receptors, cytokines, chemokines, and metalloproteinases was quantified with RT-PCR. The WNT pathway was explored with gene silencing, antibody and pharmacological inhibition followed by migration assays and phosphoprotein western blots. Here, we reported that rWnt5a promoted migration and invasion of RA FLS, whereas knockdown of the endogenous WNT5A reduced them. These effects were specific to the RA FLS since they were not observed in FLS from osteoarthritis (OA) patients. Also, rWnt5a induced the expression of IL6, IL8, CCL2, CXCL5, MMP1, MMP3, MMP9, and MMP13 from baseline or potentiating the TNF induction, WNT5A signaling required the RYK receptor and was mediated through the WNT/Ca2+ and the ROCK pathway. These pathways involved the RYK and ROCK dependent activation of the p38, ERK, AKT, and GSK3β kinases, but not the activation of JNK. Together these findings indicate that WNT5A contributes to the enhanced migration and invasiveness of RA FLS through RYK and the specific activation of ROCK and downstream kinases.

Regorafenib suppresses epidermal growth factor receptor signaling-modulated progression of colorectal cancer

Active epidermal growth factor receptors (EGFR) signaling mediates the progression of colorectal cancer (CRC) through activation of downstream kinases and transcription factors. The increased expression of EGFR was associated with worse prognosis in patients with metastatic CRC (mCRC). Regorafenib, the oral kinase inhibitor approved for the treatment of mCRC, has been shown to reduce activation of downstream kinases of EGFR signal pathway in hepatocellular carcinoma and osteosarcoma. However, whether EGFR inactivation was participates in regorafenib-inhibited progression of CRC still remaining ambiguous. The major purpose of present study was to verify effect of regorafenib on EGFR signaling-mediated progression of CRC. Here, we investigated the effect of regorafenib or erlotinib (EGFR inhibitor) on tumor cell growth, invasion ability, apoptotic, and EGFR signal transduction in CRC in vitro and in vivo. Our results indicated regorafenib reduced EGF-induced EGFR and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. Both regorafenib and erlotinib significantly reduced cell invasion ability, activation of protein kinase C-δ (PKCδ), protein kinase B (AKT), extracellular signal-regulated kinases (ERK), and NF-κB. Regorafenib can trigger the inhibition of tumor cell growth and the induction of apoptosis through extrinsic/intrinsic apoptosis pathways. In addition, the expression of NF-κB-mediated proteins involved in tumor progression was also suppressed by regorafenib treatment. Taken together, regorafenib acts as a inhibitor of EGFR signaling that attenuated the activation of EGFR and EGFR related downstream signaling cascades in CRC. Our results suggested that the suppression of EGFR signaling was associated with regorafenib-inhibited progression of CRC.