Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer which nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

Case report: reinitiating pembrolizumab treatment after small bowel perforation.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have emerged as paradigm shifting treatment options for a number of cancers. Six antibodies targeting the immune checkpoint proteins programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1) or cytotoxic T-lymphocyte associated protein 4 (CTLA4) have been approved. In some cases, response rates have been impressive, but not uniformly so and not consistently; similarly, toxicity to this class of therapeutic is often unpredictable and can be life threatening. Predicting treatment response and toxicity are two main obstacles to truly individualize treatment with ICIs. One of the most severe and life-threatening adverse events is colitis induced colonic perforation, estimated to occur in 1.0 to 1.5% of patients treated with ICIs. An important question to address is, under what circumstances is it appropriate to reinitiate ICI treatment post-bowel perforation? CASE PRESENTATION: The patient is a 62-year-old woman, who presented with stage IV lung cancer. Immunohistochemical staining indicated that 80% of the patient's tumor cells expressed PD-L1. The patient was started on a three-week cycle of pembrolizumab. Subsequent reducing in tumor burden was observed within ten weeks. Initially, pembrolizumab was tolerated fairly well, with the exception of immunotherapy related hypothyroidism. However, the patient experienced a second, more serious immune-related adverse event (irAE), in the form of enteritis, which led to small bowel perforation and necessitated exploratory laparotomy. The concerning part of the small bowel was resected, and a primary anastomosis was created. Based on the pathological and surgical findings, the patient was diagnosed with pembrolizumab-associated small bowel perforation. The patient recovered well from surgery and, considering the patient's remarkable response to treatment, a collective decision was made to reinitiate pembrolizumab on post-operative day twenty-eight. The patient is continuing her immunotherapy with ongoing partial response and is able to continue her full-time job. CONCLUSIONS: This case report highlights the challenges of identifying patients likely to respond to ICIs and those that are likely to experience irAEs and it discusses the impressive work that has been done to start to address these challenges. Lastly, the topic of reinitiating pembrolizumab treatment even after colonic perforation is discussed.

Synthesis of 13-vertex dimetallacarboranes by electrophilic insertion into 12-vertex ruthenacarboranes.

The electrophilic insertion of organometallic species into metallacarboranes was studied in detail for the model compound - the 12-vertex closo-ruthenacarborane anion [Cp*Ru(C2B9H11)]- (1). Reactions of the anion 1 with the 12-electron cationic species [M(ring)]+ (M(ring) = RuCp, RuCp* and Co(C4Me4)) gave the 13-vertex closo-dimetallacarboranes Cp*Ru(C2B9H11)M(ring). Similar reactions of the neutral ruthenacarborane Cp*Ru(Me2S-C2B9H10) produce the cationic dimetallacarboranes [Cp*Ru(Me2S-C2B9H10)M(ring)]+. The symmetrical 13-vertex diruthenacarboranes (C5R5)Ru(R2C2B9H9)Ru(C5R5) can be prepared by the direct reactions of Tl2[7,8-R2-7,8-C2B9H9] (R = H and Me) with two equivalents of [CpRu(MeCN)3]+ or [Cp*RuCl]4. The insertions of the 14-electron cationic species [M(ring)]+ (M(ring) = NiCp, NiCp* and Co(C6Me6)) into 1 gave the 13-vertex dimetallacarboranes Cp*Ru(C2B9H11)M(ring), which have a distorted framework with one open face. The structures of Cp*Ru(C2B9H11)Co(C4Me4) and Cp*Ru(C2B9H11)NiCp were established by X-ray diffraction. Some of the 13-vertex dimetallacarboranes have two electrons less than required by Wade's rules. This violation is explained by the absence of the appropriate pathway for the distortion of the framework.

The First Triple-Decker Complex with a Carbenium Center, [CpCo(µ-C3 B2 Me5 )RuC5 Me4 CH2 ]+ : Synthesis, Reactivity, X-Ray Structure, and Bonding.

The first derivative of the methylium cation with the triple-decker substituent, [CpCo(C3 B2 Me5 )RuC5 Me4 CH2 ]PF6 (2PF6 ), was synthesized from the reaction of the triple-decker complex CpCo(C3 B2 Me5 )RuCp* (1) with the salt of the trityl cation [CPh3 ]+ . The X-ray crystal structure of 2PF6 reveals that the methylium carbon is bound to the ruthenium with Ru-C bond length of 2.259 Šand corresponds to the description of its structure as η6 -fulvene-ruthenium. Reactions of 2PF6 with nucleophiles OH- , Ph3 P, Et3 N led to the corresponding derivatives of 1 in high yields. Aromatic amines PhNEt2 and 4-MeC6 H4 NH2 react with 2PF6 to give the electrophilic aromatic substitution products quantitatively. Chemical reduction of 2PF6 with Zn powder in tetrahydrofuran leads to the formation of the bis(triple-decker) derivative (CpCo(C3 B2 Me5 )RuC5 Me4 CH2 )2 (10) with a CH2 CH2 -bridge. The structures of complexes 4, 7-10 were determined by X-ray diffraction. Density functional calculations support the crystallographically determined geometry of 2 and allow rationalization of some characteristics of its structure, spectroscopy, and reactivity.

Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets.

Aberrant gene expression that drives human cancer can arise from epigenetic dysregulation. Although much attention has focused on altered activity of transcription factors and chromatin-modulating proteins, proteins that act posttranscriptionally can potently affect expression of oncogenic signaling proteins. The RNA-binding proteins (RBP) Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance. Following identification of Musashi as a regulator of progenitor cell identity in Drosophila, the human Musashi proteins were initially linked to control of maintenance of hematopoietic stem cells, then stem cell compartments for additional cell types. More recently, the Musashi proteins were found to be overexpressed and prognostic of outcome in numerous cancer types, including colorectal, lung, and pancreatic cancers; glioblastoma; and several leukemias. MSI1 and MSI2 bind and regulate the mRNA stability and translation of proteins operating in essential oncogenic signaling pathways, including NUMB/Notch, PTEN/mTOR, TGFß/SMAD3, MYC, cMET, and others. On the basis of these activities, MSI proteins maintain cancer stem cell populations and regulate cancer invasion, metastasis, and development of more aggressive cancer phenotypes, including drug resistance. Although RBPs are viewed as difficult therapeutic targets, initial efforts to develop MSI-specific inhibitors are promising, and RNA interference-based approaches to inhibiting these proteins have had promising outcomes in preclinical studies. In the interim, understanding the function of these translational regulators may yield insight into the relationship between mRNA expression and protein expression in tumors, guiding tumor-profiling analysis. This review provides a current overview of Musashi as a cancer driver and novel therapeutic target. Clin Cancer Res; 23(9); 2143-53. ©2017 AACR.

Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer.

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-ß)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-ß/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-ß/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.

Musashi-2 (MSI2) supports TGF-ß signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis.

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-ß receptor 1 (TGFßR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFßRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFßR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

A Novel HSP90 Inhibitor-Drug Conjugate to SN38 Is Highly Effective in Small Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is a highly aggressive disease representing 12% to 13% of total lung cancers, with median survival of <2 years. No targeted therapies have proven effective in SCLC. Although most patients respond initially to cytotoxic chemotherapies, resistance rapidly emerges, response to second-line agents is limited, and dose-limiting toxicities (DLT) are a major issue. This study performs preclinical evaluation of a new compound, STA-8666, in SCLC. EXPERIMENTAL DESIGN: To avoid DLT for useful cytotoxic agents, the recently developed drug STA-8666 combines a chemical moiety targeting active HSP90 (concentrated in tumors) fused via cleavable linker to SN38, the active metabolite of irinotecan. We compare potency and mechanism of action of STA-8666 and irinotecan in vitro and in vivo RESULTS: In two SCLC xenograft and patient-derived xenograft models, STA-8666 was tolerated without side effects up to 150 mg/kg. At this dose, STA-8666 controlled or eliminated established tumors whether used in a first-line setting or in tumors that had progressed following treatment on standard first- and second-line agents for SCLC. At 50 mg/kg, STA-8666 strongly enhanced the action of carboplatin. Pharmacokinetic profiling confirmed durable STA-8666 exposure in tumors compared with irinotecan. STA-8666 induced a more rapid, robust, and stable induction of cell-cycle arrest, expression of signaling proteins associated with DNA damage and cell-cycle checkpoints, and apoptosis in vitro and in vivo, in comparison with irinotecan. CONCLUSIONS: Together, these results strongly support clinical development of STA-8666 for use in the first- or second-line setting for SCLC. Clin Cancer Res; 22(20); 5120-9. ©2016 AACR.

General Route to Cyclobutadiene Rhodium Complexes.

Cyclobutadiene rhodium complexes bear high potential for applications in organometallic synthesis and catalysis. We have found that the cyclobutadiene complexes with substitutionally labile p-xylene ligands [(C4 R4 )Rh(p-xylene)](+) can be synthesized in one step from the commercially available bis(ethylene) complex [{(C2 H4 )2 RhCl}2 ], p-xylene, and internal alkynes. The replacement of p-xylene by various ligands provides a general access to other [(C4 R4 )Rh] compounds, such as [(C4 R4 )RhCl]x , [(C4 R4 )RhL3 ](+) , [(C4 R4 )Rh(C5 H5 )], and [(C4 R4 )Rh(arene)](+) . Complex [(C4 Et4 )Rh(p-xylene)](+) also catalyzes an unusual cycloisomerization of a 1,11-dien-6-yne into a bicyclic diene.

Selective ruthenium labeling of the tryptophan residue in the bee venom Peptide melittin.

Melittin is a membrane-active peptide from bee venom with promising antimicrobial and anticancer activity. Herein we report on a simple and selective method for labeling of the tryptophan residue in melittin by the organometallic fragment [(C5 H5 )Ru](+) in aqueous solution and in air. Ruthenium coordination does not disturb the secondary structure of the peptide (as verified by 2D NMR spectroscopy), but changes the pattern of its intermolecular interactions resulting in an 11-fold decrease of hemolytic activity. The high stability of the organometallic conjugate allowed the establishment of the biodistribution of the labeled melittin in mice by inductively coupled plasma MS analysis of ruthenium.

CAS proteins in health and disease: an update.

The CAS family of scaffolding proteins has increasingly attracted scrutiny as important for regulation of cancer-associated signaling. BCAR1 (also known as p130Cas), NEDD9 (HEF1, Cas-L), EFS (Sin), and CASS4 (HEPL) are regulated by and mediate cell attachment, growth factor, and chemokine signaling. Altered expression and activity of CAS proteins are now known to promote metastasis and drug resistance in cancer, influence normal development, and contribute to the pathogenesis of heart and pulmonary disease. In this article, we provide an update on recently published studies describing signals regulating and regulated by CAS proteins, and evidence for biological activity of CAS proteins in normal development, cancer, and other pathological conditions.

Simple synthesis of ruthenium pi complexes of aromatic amino acids and small peptides.

The interaction of [Ru(eta(6)-C(10)H(8))(Cp)](+) (Cp=C(5)H(5)) with aromatic amino acids (L-phenylalanine, L-tyrosine, L-tryptophane, D-phenylglycine, and L-threo-3-phenylserine) under visible-light irradiation gives the corresponding [Ru(eta(6)-amino acid)(Cp)](+) complexes in near-quantitative yield. The reaction proceeds in air at room temperature in water and tolerates the presence of non-aromatic amino acids (except those which are sulfur containing), monosaccharides, and nucleotides. The complex [Ru(eta(6)-C(10)H(8))(Cp)](+) was also used for selective labeling of Tyr and Phe residues of small peptides, namely, angiotensin I and II derivatives.

Extremely efficient alkane oxidation by a new catalytic reagent H(2)O(2)/Os(3)(CO)(12)/pyridine.

Triosmium dodecacarbonyl catalyzes a very efficient oxidation of alkanes by H(2)O(2) in MeCN to afford alkyl hydroperoxides (primary products) as well as alcohols and ketones (aldehydes) at 60 degrees C if pyridine is added in a low concentration. Turnover numbers attain 60,000, and turnover frequencies are up to 24,000 h(-1).

Fusion of a 1,3-diboraruthenocene to form a slipped mu-hexahydrotetraboranaphthalene triple-decker complex with two axial C--H bonds.

Colliding double deckers: Addition of zinc to a reaction mixture of [{Cp*RuCl}(4)]/pentamethyl-2,3-dihydro-1,3-diborole (C(3)B(2)Me(5)H) in THF leads to three known double- and triple-decker complexes of [C(3)B(2)Me(5)](-), and unexpectedly to the slipped triple-decker (see picture) with two fused diborole rings. The endo C--H bonds of two MeC--H groups donate two additional electrons to achieve the stable 34 VE configuration.

Synthesis and rearrangements of aminosubstituted ferra- and ruthenatricarbaboranes.

A room-temperature reaction between the [7-tBuNH-nido-7,8,9-C3B8H10]- anion (1a) and [Cp*RuCl]4 leads to the ruthenatricarbollide [1-Cp*-12-tBuNH-1,2,4,12-RuC3B8H10] (2) (yield 85%). Analogously, the room-temperature photochemical reaction of 1a with [CpFe(C6H6)]PF6 gives the previously reported iron complex [1-Cp-12-tBuNH-1,2,4,12-FeC3B8H10] (3) (yield 82%). Both reactions are associated with extensive polyhedral rearrangement, which occurs under very mild conditions and brings the carbon atoms to positions of maximum separation within the framework. Compounds 2 and 3 were also surprisingly obtained via complexation of the isomeric [8-tBuNH-nido-7,8,9-C3B8H10]- (1b) anion. Complex 2 rearranges further to [1-Cp*-10-tBuNH-1,2,4,10-RuC3B8H10] (4) upon refluxing in xylene (145 degrees C). Density functional theory calculations at the B3LYP/SDD level were used to estimate relative stabilities of these metallacarborane isomers. Compounds 2 and 4, along with the 11-vertex closo compounds [1-Cp*-1,2,3,10-RuC3B7H10] (5) and [1-Cp*-10-tBuNH-1,2,3,10-RuC3B7H9] (6), were also isolated from the reaction between [Cp*RuCl2]2 and 1a in boiling xylene. The structure of 2 was established by an X-ray diffraction study, and the constitution of all compounds was determined unambiguously by multinuclear NMR spectroscopy, mass spectrometry, and elemental analyses.