A molecularly engineered lectin destroys EGFR and inhibits the growth of non-small cell lung cancer.

Survival rates for non-small cell lung cancer (NSCLC) remain low despite the advent of novel therapeutics. Tyrosine kinase inhibitors (TKIs) targeting mutant epidermal growth factor receptor (EGFR) in NSCLC have significantly improved mortality but are plagued with challenges--they can only be used in the small fraction of patients who have susceptible driver mutations, and resistance inevitably develops. Aberrant glycosylation on the surface of cancer cells is an attractive therapeutic target as these abnormal glycosylation patterns are typically specific to cancer cells and are not present on healthy cells. H84T BanLec (H84T), a lectin previously engineered by our group to separate its antiviral activity from its mitogenicity, exhibits precision binding of high mannose, an abnormal glycan present on the surface of many cancer cells, including NSCLC. Here, we show that H84T binds to and inhibits the growth of diverse NSCLC cell lines by inducing lysosomal degradation of EGFR and leading to cancer cell death through autophagy. This is a mechanism distinct from EGFR TKIs and is independent of EGFR mutation status; H84T inhibited proliferation of both cell lines expressing wild type EGFR and those expressing mutant EGFR that is resistant to all TKIs. Further, H84T binds strongly to multiple and diverse clinical samples of both pulmonary adenocarcinoma and squamous cell carcinoma. H84T is thus a promising potential therapeutic in NSCLC, with the ability to circumvent the challenges currently faced by EGFR TKIs.

Disruptin, a cell-penetrating peptide degrader of EGFR: Cell-Penetrating Peptide in Cancer Therapy.

Disruptin is a cell-permeable decoy peptide designed to destabilize activated EGFR, both by inhibiting Hsp90 chaperoning and dissociating the active asymmetric EGFR dimer, which leads to an increase in engagement of activated EGFR with the proteolytic degradation machinery and subsequent loss from the cells. Disruptin is an N-terminally biotinylated nonadecapeptide, with 8 amino acids from the αC-helix-ß4 sheet loop of EGFR (S767-C774) fused to a TAT undecapeptide. The S767-R775 loop is at the interface with juxtamembrane domains in the active EGFR dimers and is a binding site for Hsp90. Cellular studies in EGFR-activated tumor cells demonstrated that Disruptin causes the disappearance of EGFR protein from cells over a few hours, a growth inhibitory effect, similar but more effective than the EGFR kinase inhibition. Interestingly, cells without activated EGFR remained unaffected. In vivo studies showed that Disruptin slowed the growth of small tumors. Larger tumors responded to intratumoral injections but did not respond to systemic administration at tolerated doses. Investigation of these results revealed that systemic administration of Disruptin has acute toxicities, mainly related to its TAT peptide moiety. Therefore, we conclude that although the efficacy of both in vitro and in vivo intratumoral injection of Disruptin supports the therapeutic strategy of blocking activated EGFR dimerization, Disruptin is not suitable for further development. These studies also highlight the importance of the chosen models and drug-delivery methods for such investigations.

Transcriptomic Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Identifies a Targetable ALDH-Positive Subset of Highly Tumorigenic Cancer Stem-like Cells.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Low-Dose Hsp90 Inhibitor Selectively Radiosensitizes HNSCC and Pancreatic Xenografts.

PURPOSE: Treatment approaches using Hsp90 inhibitors at their maximum tolerated doses (MTDs) have not produced selective tumor toxicity. Inhibition of Hsp90 activity causes degradation of client proteins including those involved in recognizing and repairing DNA lesions. We hypothesized that if DNA repair proteins were degraded by concentrations of an Hsp90 inhibitor below those required to cause nonspecific cytotoxicity, significant tumor-selective radiosensitization might be achieved. EXPERIMENTAL DESIGN: Tandem mass tagged-mass spectrometry was performed to determine the effect of a subcytotoxic concentration of the Hsp90 inhibitor, AT13387 (onalespib), on global protein abundance. The effect of AT13387 on in vitro radiosensitization was assessed using a clonogenic assay. Pharmacokinetics profiling was performed in mice bearing xenografts. Finally, the effect of low-dose AT13387 on the radiosensitization of three tumor models was assessed. RESULTS: A subcytotoxic concentration of AT13387 reduced levels of DNA repair proteins, without affecting the majority of Hsp90 clients. The pharmacokinetics study using one-third of the MTD showed 40-fold higher levels of AT13387 in tumors compared with plasma. This low dose enhanced Hsp70 expression in peripheral blood mononuclear cells (PBMCs), which is a biomarker of Hsp90 inhibition. Low dose monotherapy was ineffective, but when combined with radiotherapy, produced significant tumor growth inhibition. CONCLUSIONS: This study shows that a significant therapeutic ratio can be achieved by a low dose of Hsp90 inhibitor in combination with radiotherapy. Hsp90 inhibition, even at a low dose, can be monitored by measuring Hsp70 expression in PBMCs in human studies.

Enhancing the Radiation Response in KRAS Mutant Colorectal Cancers Using the c-Met Inhibitor Crizotinib.

INTRODUCTION: C-Met plays important roles in treatment resistance, tumor invasion, and metastasis. In this study, we used a small molecule inhibitor of c-Met, crizotinib, in cetuximab-resistant, mutant KRAS-driven colorectal cancer cell lines and assessed radiosensitization. MATERIALS AND METHODS: A tissue microarray containing colorectal tumors was used to study the relationship between KRAS mutations and c-Met expression. For in vivo studies, we used the KRAS mutant cell lines HCT116, DLD1, and LoVo. Colony formation assays were performed to assess the effects of crizotinib and cetuximab. Immunoblot analysis was used to determine the effect of crizotinib on c-Met and downstream pathways and DNA damage response. We then selected noncytotoxic doses of crizotinib to assess clonogenic survival with radiation. To study potential mechanisms of radiosensitization, cell cycle analysis was performed using flow cytometry. RESULTS: Analysis of the tissue microarray revealed that KRAS mutant tumors had active c-Met signaling. KRAS mutant cell lines LoVo, HCT116, and DLD1 were resistant to cetuximab but sensitive to crizotinib. Pretreatment with crizotinib for 24 hours radiosensitized LoVo, DLD1, and HCT116 cell lines with enhancement ratios of 1.54, 1.23, and 1.30, respectively. Immunoblot analysis showed that crizotinib blocked radiation-induced c-Met phosphorylation and attenuated downstream signaling pathways. Cell cycle analysis revealed minimal G1 arrest with crizotinib. Additionally, crizotinib completely blocked HGF induced cell migration. CONCLUSIONS: Inhibition of c-Met with crizotinib effectively sensitizes cetuximab-resistant KRAS mutant colorectal cancer cell lines to radiation. Crizotinib has the potential to improve outcomes in locally advanced rectal cancer patients undergoing chemoradiation.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90ß in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90ß, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90ß in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90ß have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ßß homodimers and no GR•Hsp90αß heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αß heterodimers. The formation of GR•Hsp90αß heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αß heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors.

Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

Zinc-Oxide Nanoparticles Exhibit Genotoxic, Clastogenic, Cytotoxic and Actin Depolymerization Effects by Inducing Oxidative Stress Responses in Macrophages and Adult Mice.

Zinc oxide nanoparticles (ZnO-NPs) have wide biological applications, which have raised serious concerns about their impact on the health and environment. Although, various studies have shown ZnO-NP toxicity on different cells underin vitroconditions, sufficient information is lacking regarding toxicity and underlying mechanisms underin vivoconditions. In this work, we investigated genotoxic, clastogenic, and cytotoxic effects of ZnO-NPs on macrophages and in adult mice. ZnO-NP-treated mice showed signs of toxicity such as loss in body weight, passive behavior and reduced survival. Further mechanistic studies revealed that administration of higher dose caused severe DNA damage in peripheral blood and bone marrow cells as evident by the formation of COMET tail, micronuclei, chromosomal fragmentation, and phosphorylation of H2A histone family member X. Moreover, ZnO-NPs inhibited DNA repair mechanism by downregulating the expression offen-1andpolBproteins. Histopathological examinations showed severe inflammation and damage to liver, lungs, and kidneys. Cell viability and wound healing assays revealed that ZnO-NPs killed macrophages in a dose-dependent manner, caused severe wounds and inhibited cellular migration by irreversible actin depolymerization and degradation. Reduction in the viability of macrophages was due to the arrest of the cell cycle at the G0/G1 phase, inhibition of superoxide dismutase and catalase and eventually reactive oxygen species. Furthermore, treatment with an antioxidant drug N-acetyl cysteine significantly reduced the ZnO-NP induced genotoxicity bothin vitroandin vivo Altogether, this study gives detailed pathological insights of ZnO-NP that impair cellular functions, thus will enable to arbitrate their biological applications.

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.

Topical application of zinc oxide nanoparticles reduces bacterial skin infection in mice and exhibits antibacterial activity by inducing oxidative stress response and cell membrane disintegration in macrophages.

Here we studied immunological and antibacterial mechanisms of zinc oxide nanoparticles (ZnO-NPs) against human pathogens. ZnO-NPs showed more activity against Staphylococcus aureus and least against Mycobacterium bovis-BCG. However, BCG killing was significantly increased in synergy with antituberculous-drug rifampicin. Antibacterial mechanistic studies showed that ZnO-NPs disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity and down-regulate the transcription of oxidative stress-resistance genes in bacteria. ZnO-NP treatment also augmented the intracellular bacterial killing by inducing reactive oxygen species production and co-localization with Mycobacterium smegmatis-GFP in macrophages. Moreover, ZnO-NPs disrupted biofilm formation and inhibited hemolysis by hemolysin toxin producing S. aureus. Intradermal administration of ZnO-NPs significantly reduced the skin infection, bacterial load and inflammation in mice, and also improved infected skin architecture. We envision that this study offers novel insights into antimicrobial actions of ZnO-NPs and also demonstrates ZnO-NPs as a novel class of topical anti-infective agent for the treatment of skin infections. FROM THE CLINICAL EDITOR: This in-depth study demonstrates properties of ZnO nanoparticles in infection prevention and treatment in several skin infection models, dissecting the potential mechanisms of action of these nanoparticles and paving the way to human applications.

Mutations in subunit interface and B-cell epitopes improve antileukemic activities of Escherichia coli asparaginase-II: evaluation of immunogenicity in mice.

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.

Cationic antimicrobial peptides and biogenic silver nanoparticles kill mycobacteria without eliciting DNA damage and cytotoxicity in mouse macrophages.

With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.