Mpox vaccine and infection-driven human immune signatures: an immunological analysis of an observational study.

BACKGROUND: Monkeypox virus has recently infected more than 88 000 people, raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side-effects than previous smallpox vaccines and has shown immunogenicity against monkeypox in animal models. This study aims to elucidate human immune responses to JYNNEOS vaccination compared with mpox-induced immunity. METHODS: Peripheral blood mononuclear cells and sera were obtained from ten individuals vaccinated with one or two doses of JYNNEOS and six individuals diagnosed with monkeypox virus infection. Samples were obtained from seven individuals before vaccination to serve as a baseline. We examined the polyclonal serum (ELISA) and single B-cell (heavy chain gene and transcriptome data) antibody repertoires and T-cell responses (activation-induced marker and intracellular cytokine staining assays) induced by the JYNNEOS vaccine versus monkeypox virus infection. FINDINGS: All participants were men between the ages of 21 and 60 years, except for one woman in the group of mpox-convalescent individuals, and none had previous orthopoxvirus exposure. All mpox cases were mild. Vaccinee samples were collected 6-33 days after the first dose and 5-40 days after the second dose. Mpox-convalescent samples were collected 20-102 days after infection. In vaccine recipients, gene-level plasmablast and antibody responses were negligible and sera displayed moderate binding to recombinant orthopoxviral proteins (A29L, A35R, E8L, A30L, A27L, A33R, B18R, and L1R) and native proteins from the 2022 monkeypox outbreak strain. By contrast, recent monkeypox virus infection (within 20-102 days) induced robust serum antibody responses to monkeypox virus proteins and to native monkeypox virus proteins from a viral isolate obtained during the 2022 outbreak. JYNNEOS vaccine recipients presented robust orthopoxviral CD4+ and CD8+ T-cell responses. INTERPRETATION: Infection with monkeypox virus resulted in robust B-cell and T-cell responses, whereas immunisation with JYNNEOS elicited more robust T-cell responses. These data can help to inform vaccine design and policies for preventing mpox in humans. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), and Icahn School of Medicine.

Mpox vaccine and infection-driven human immune signatures.

Background: Mpox (formerly known as monkeypox) outbreaks outside endemic areas peaked in July 2022, infecting > 85,000 people and raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side effects than previous smallpox vaccines and demonstrated efficacy against mpox infection in humans. Comparing JYNNEOS vaccine- and mpox-induced immunity is imperative to evaluate JYNNEOS' immunogenicity and inform vaccine administration and design. Methods: We examined the polyclonal serum (ELISA) and single B cell (heavy chain gene and transcriptome data) antibody repertoires and T cells (AIM and ICS assays) induced by the JYNNEOS vaccine as well as mpox infection. Findings: Gene-level plasmablast and antibody responses were negligible and JYNNEOS vaccinee sera displayed minimal binding to recombinant mpox proteins and native proteins from the 2022 outbreak strain. In contrast, recent mpox infection (within 20-102 days) induced robust serum antibody responses to A29L, A35R, A33R, B18R, and A30L, and to native mpox proteins, compared to vaccinees. JYNNEOS vaccine recipients presented comparable CD4 and CD8 T cell responses against orthopox peptides to those observed after mpox infection. Interpretation: JYNNEOS immunization does not elicit a robust B cell response, and its immunogenicity may be mediated by T cells. Funding: Research reported in this publication was supported, in part, by the National Cancer Institute of the National Institutes of Health under Award Number U54CA267776, U19AI168631(VS), as well as institutional funds from the Icahn School of Medicine.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

The Combination of Trametinib and Ganitumab is Effective in RAS-Mutated PAX-Fusion Negative Rhabdomyosarcoma Models.

PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Hypomorphic mTOR Downregulates CDK6 and Delays Thymic Pre-T LBL Tumorigenesis.

PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.

T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis.

Cancer development requires a favorable tissue microenvironment. By deleting Myd88 in keratinocytes or specific bone marrow subpopulations in oncogenic RAS-mediated skin carcinogenesis, we show that IL17 from infiltrating T cells and IκBζ signaling in keratinocytes are essential to produce a permissive microenvironment and tumor formation. Both normal and RAS-transformed keratinocytes respond to tumor promoters by activating canonical NF-κB and IκBζ signaling, releasing specific cytokines and chemokines that attract Th17 cells through MyD88-dependent signaling in T cells. The release of IL17 into the microenvironment elevates IκBζ in normal and RAS-transformed keratinocytes. Activation of IκBζ signaling is required for the expression of specific promoting factors induced by IL17 in normal keratinocytes and constitutively expressed in RAS-initiated keratinocytes. Deletion of Nfkbiz in keratinocytes impairs RAS-mediated benign tumor formation. Transcriptional profiling and gene set enrichment analysis of IκBζ-deficient RAS-initiated keratinocytes indicate that IκBζ signaling is common for RAS transformation of multiple epithelial cancers. Probing The Cancer Genome Atlas datasets using this transcriptional profile indicates that reduction of IκBζ signaling during cancer progression associates with poor prognosis in RAS-driven human cancers. IMPLICATIONS: The paradox that elevation of IκBζ and stimulation of IκBζ signaling through tumor extrinsic factors is required for RAS-mediated benign tumor formation while relative IκBζ expression is reduced in advanced cancers with poor prognosis implies that tumor cells switch from microenvironmental dependency early in carcinogenesis to cell-autonomous pathways during cancer progression.

MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma.

The RAS isoforms are frequently mutated in many types of human cancers, including PAX3/PAX7 fusion-negative rhabdomyosarcoma. Pediatric RMS arises from skeletal muscle progenitor cells that have failed to differentiate normally. The role of mutant RAS in this differentiation blockade is incompletely understood. We demonstrate that oncogenic RAS, acting through the RAF-MEK [mitogen-activated protein kinase (MAPK) kinase]-ERK (extracellular signal-regulated kinase) MAPK effector pathway, inhibits myogenic differentiation in rhabdomyosarcoma by repressing the expression of the prodifferentiation myogenic transcription factor, MYOG. This repression is mediated by ERK2-dependent promoter-proximal stalling of RNA polymerase II at the MYOG locus. Small-molecule screening with a library of mechanistically defined inhibitors showed that RAS-driven RMS is vulnerable to MEK inhibition. MEK inhibition with trametinib leads to the loss of ERK2 at the MYOG promoter and releases the transcriptional stalling of MYOG expression. MYOG subsequently opens chromatin and establishes super-enhancers at genes required for late myogenic differentiation. Furthermore, trametinib, in combination with an inhibitor of IGF1R, potently decreases rhabdomyosarcoma cell viability and slows tumor growth in xenograft models. Therefore, this combination represents a potential therapeutic for RAS-mutated rhabdomyosarcoma.

M7824, a novel bifunctional anti-PD-L1/TGFß Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine.

Tumors evade host immune surveillance through multiple mechanisms, including the generation of a tumor microenvironment that suppresses immune effector function. Secretion of TGFß and upregulation of immune checkpoint programmed cell death ligand-1 (PD-L1) are two main contributors to immune evasion and tumor progression. Here, we examined the efficacy of a first-in-class bifunctional checkpoint inhibitor, the fusion protein M7824, comprising the extracellular domain of human TGFßRII (TGFß Trap) linked to the C-terminus of human anti-PD-L1 heavy chain (αPD-L1). We demonstrate that M7824 reduces plasma TGFß1, binds to PD-L1 in the tumor, and decreases TGFß-induced signaling in the tumor microenvironment in mice. In murine breast and colon carcinoma models, M7824 decreased tumor burden and increased overall survival as compared to targeting TGFß alone. M7824 treatment promoted CD8+ T cell and NK cell activation, and both of these immune populations were required for optimal M7824-mediated tumor control. M7824 was superior to TGFß- or αPD-L1-targeted therapies when in combination with a therapeutic cancer vaccine. These findings demonstrate the value of using M7824 to simultaneously target TGFß and PD-L1/PD-1 immunosuppressive pathways to promote anti-tumor responses and efficacy. The studies also support the potential clinical use of M7824 as a monotherapy or in combination with other immunotherapies, such as therapeutic cancer vaccines, including for patients who have progressed on αPD-L1/αPD-1 checkpoint blockade therapies.

Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues.

To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinase C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatin 1.

Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation.

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivoMol Cancer Ther; 16(9); 2008-21. ©2017 AACR.

Liver Label Retaining Cancer Cells Are Relatively Resistant to the Reported Anti-Cancer Stem Cell Drug Metformin.

BACKGROUND & AIMS: Recently, we reported that liver Label Retaining Cancer Cells (LRCC) can initiate tumors with only 10 cells and are relatively resistant to the targeted drug Sorafenib, a standard of practice in advanced hepatocellular carcinoma (HCC). LRCC are the only cancer stem cells (CSC) isolated alive according to a stem cell fundamental function, asymmetric cell division. Metformin has been reported to preferentially target many other types of CSC of different organs, including liver. It's important to know if LRCC, a novel class of CSC, are relatively resistant to metformin, unlike other types of CSC. As metformin inhibits the Sorafenib-Target-Protein (STP) PI3K, and LRCC are newly described CSC, we undertook this study to test the effects of Metformin on Sorafenib-treated HCC and HCC-derived-LRCC. METHODS: We tested various STP levels and phosphorylation status, associated genes' expression, proliferation, viability, toxicity, and apoptosis profiles, before and after treatment with Sorafenib with/without Metformin. RESULTS: Metformin enhances the effects of Sorafenib on HCC, and significantly decreased viability/proliferation of HCC cells. This insulin-independent effect was associated with inhibition of multiple STPs (PKC, ERK, JNK and AKT). However, Metformin increased the relative proportion of LRCCs. Comparing LRCC vs. non-LRCC, this effect was associated with improved toxicity and apoptosis profiles, down-regulation of cell death genes and up-regulation of cell proliferation and survival genes in LRCC. Concomitantly, Metformin up-regulated pluripotency, Wnt, Notch and SHH pathways genes in LRCC vs. non-LRCC. CONCLUSIONS: Metformin and Sorafenib have enhanced anti-cancer effects. However, in contradistinction to reports on other types of CSC, Metformin is less effective against HCC-derived-CSC LRCC. Our results suggest that combining Metformin with Sorafenib may be able to repress the bulk of tumor cells, but as with other anti-cancer drugs, may leave LRCC behind leading to cancer recurrence. Therefore, liver LRCC, unlike other types of CSC, are relatively resistant to the reported anti-cancer stem cell drug metformin. This is the first report that there is a type of CSC that is not relatively resistant to the CSC-targeting drug. Our findings suggest that a drug targeting LRCC may be critically needed to target CSC and prevent cancer recurrence. These may significantly contribute to the understanding of Metformin's anti-cancer effects and the development of novel drugs targeting the relatively resistant LRCC.

RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage-induced cell senescence.

The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin ß-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage.

Pharmacodynamic markers and clinical results from the phase 2 study of the SMAC mimetic birinapant in women with relapsed platinum-resistant or -refractory epithelial ovarian cancer.

BACKGROUND: Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. It was hypothesized that blocking IAPs with birinapant would increase tumor cell death and result in objective responses for women with platinum-refractory and -resistant ovarian cancer. METHODS: In this phase 2, Cancer Therapy Evaluation Program-sponsored study, patients received birinapant at 47 mg/m(2) on days 1, 8, and 15 of 28-day cycles. Pharmacokinetics were obtained during cycle 1. Plasma, peripheral blood mononuclear cells (PBMCs), and percutaneous tumor biopsy samples were collected before cycle 1 and after 6 weeks. The primary endpoint was an objective response or progression-free survival lasting greater than 6 months in a mini-max design. RESULTS: Eleven patients received birinapant; after this, accrual was terminated for lack of a clinical benefit. Birinapant was well tolerated, with predominantly grade 2 adverse events and 1 case of grade 3 lymphopenia. Pretreatment biopsy samples and PBMCs were collected; paired posttreatment biopsy samples and PBMCs were collected from 7 and 10 patients, respectively. There was consistent downregulation of cellular inhibitor of apoptosis protein 1 in tumors (P = .016) and PBMCs (P < .01). Procaspase 3 also decreased in tumors (P = .031) and PBMCs (P < .01); cleaved caspase 3 colocalized with H2A histone family member X (γ-H2AX) in tumors after birinapant exposure. Peripheral T and B cells decreased significantly after treatment, but natural killer cells did not (P = .04, P = .05, and P = .43, respectively). CONCLUSIONS: Birinapant shows consistent target suppression in vivo without single-agent antitumor activity in this small population. Single-agent pharmacodynamics are necessary to understand the drug's mechanism of action and set the stage for rational combination therapy. Preclinical studies are ongoing to identify optimal synergistic combinations for future clinical trials.

A Phase II Trial of AZD6244 (Selumetinib, ARRY-142886), an Oral MEK1/2 Inhibitor, in Relapsed/Refractory Multiple Myeloma.

PURPOSE: AZD6244 is a MEK1/2 inhibitor with significant preclinical activity in multiple myeloma cells. This phase II study used a two-stage Simon design to determine the AZD6244 response rate in patients with relapsed or refractory multiple myeloma. EXPERIMENTAL DESIGN: AZD6244 (75 mg) was administered orally, twice a day, continuously for 28-day cycles. Response was evaluated after three cycles. RESULTS: Thirty-six patients received therapy. The median age was 65 years (range: 43-81) and the median number of prior therapies was 5 (range: 2-11). The most common grade 3 and 4 toxicities included anemia, neutropenia, thrombocytopenia, diarrhea, and fatigue. Three deaths occurred possibly related to AZD6244 (2 due to sepsis, 1 due to acute kidney injury). After AZD6244 discontinuation, three additional deaths occurred due to disease progression. The response rate (CR + PR) was 5.6% with a mean duration of response of 4.95 months and median progression-free survival time of 3.52 months. One patient had a very good partial response (VGPR), 1 patient had a partial response, 17 patients had stable disease, 13 patients had progressive disease, and 4 patients could not be assessed for response. Pharmacodynamic studies revealed variable effects on bone marrow CD138(+) cell MEK1/2 and ERK1/2 phosphorylation. The best clinical response, a prolonged VGPR, occurred in a patient with an MMSET translocation. CONCLUSIONS: Single-agent AZD6244 was tolerable and had minimal activity in this heavily pretreated population.

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics.

There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.

Molecular systems pharmacology: isoelectric focusing signature of protein kinase Cδ provides an integrated measure of its modulation in response to ligands.

Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure-activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development.

Capillary isoelectric-focusing immunoassays to study dynamic oncoprotein phosphorylation and drug response to targeted therapies in non-small cell lung cancer.

Developing proteomic biomarkers is valuable for evaluating therapeutic effects of drugs and generating better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, lack of assay reproducibility, accuracy, and sensitivity. A novel capillary isoelectricfocusing (IEF) immunoassay system (NanoPro) was used to study the dynamic phosphorylation status of signaling molecules in non-small cell lung cancer (NSCLC) cells treated with EGFR tyrosine kinase and MEK inhibitors. NanoPro showed the same dynamic ERK phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation in NanoPro system enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously. With NanoPro, we identified a specific on-target mitogen-activated protein/extracellular signal-regulated kinase (MEK) response pattern to MEK inhibitor PD325901, which was not detectable by Western blot analysis. We also revealed a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGF receptor inhibitor erlotinib, and distinguished erlotinib-sensitive cells from intrinsic as well as acquired resistant cells to erlotinib. Moreover, NanoPro could differentiate human ERK1 isoforms from the mouse isoforms based on their isoelectric point differences and showed that erlotinib effectively inhibited ERK phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. With 8 µg of tumor aspirates, we precisely quantified the response of 18 signaling molecules to erlotinib and MEK1 inhibitor treatments in an NSCLC patient. NanoPro's higher sensitivity, better resolution of protein phosphorylation status, and reduced tissue requirement warrant NanoPro's investigation for future drug development and evaluation of drug effects of targeted therapies.

Overlapping substrate and inhibitor specificity of human and murine ABCG2.

ABCG2 (also known as breast cancer resistance protein) is an ATP-binding cassette (ABC) transporter localized to the plasma membrane where it mediates the efflux of xenobiotics, including potential therapeutics. Studies investigating Abcg2 function at the blood-brain barrier in mouse models are often compared with human ABCG2 function. It is critical to understand the nature of species differences between mouse and human ABCG2, since extrapolations are made from murine data to humans. Two independent drug-selected cell line pairs expressing human or mouse ABCG2 were compared for efflux of fluorescent substrates using flow cytometry. To this end, we developed and characterized a new mouse Abcg2-expressing subline that demonstrated efflux of known fluorescent ABCG2 substrates and increased resistance to mitoxantrone, which is reduced in the presence of the ABCG2 inhibitor Ko143. Our results indicate that the substrate specificity of human and mouse ABCG2 is very similar. We identified a new human and mouse ABCG2 substrate, a porphyrin analog, purpurin-18 (Pp-18), which is not a substrate for P-glycoprotein or multidrug resistance protein 1. The ability of inhibitors to block efflux activity of ABCG2 was assessed using Pp-18. Inhibitors also demonstrated similar effects on human and mouse ABCG2. Chrysin, benzoflavone, and cyclosporin A inhibited Pp-18 efflux in both human and mouse ABCG2. The similarity of the substrate and inhibitor specificity of human and mouse ABCG2 supports interpretation of mouse models in understanding the clinical, pharmacological, and physiologic roles of ABCG2.

Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.

Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities.

Label-retaining liver cancer cells are relatively resistant to sorafenib.

OBJECTIVE: The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label-retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). The objective was to test whether LRCC are resistant to sorafenib. METHODS: We tested human HCC derived LRCC and non-LRCC before and after treatment with sorafenib. RESULTS: LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non-LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless-type MMTV-integration-site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non-uniform activation of specific isoforms of the sorafenib target proteins extracellular-signal-regulated kinases and v-akt-murine-thymoma-viral-oncogene homologue (AKT) in LRCC but not in non-LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. CONCLUSIONS: Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.