Dwight V. Nissley

Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low Frequency

ABSTRACT We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.

Low Frequency Nonnucleoside Reverse-Transcriptase Inhibitor-Resistant Variants Contribute to Failure of Efavirenz-Containing Regimens in Treatment-Experienced Patients

BACKGROUND The contribution of low frequency drug-resistant human immunodeficiency virus type 1 (HIV-1) variants to failure of antiretroviral therapy is not well defined in treatment-experienced patients. We sought to detect minor nonnucleoside reverse-transcriptase inhibitor (NNRTI)-resistant variants at the initiation of multidrug efavirenz-containing therapy in both NNRTI-naive and NNRTI-experienced patients and to determine their association with virologic response. METHODS Plasma samples at entry and at time of virologic failure from patients enrolled in the AIDS Clinical Trials Group study 398 were analyzed by standard genotype, single-genome sequencing and allele-specific polymerase chain reaction (K103N and Y181C) to detect and quantify minor NNRTI-resistant variants. RESULTS Minor populations of NNRTI-resistant variants that were missed by standard genotype were detected more often at study entry in NNRTI-experienced patients than NNRTI-naive patients by both single-genome sequencing (8 of 12 vs 3 of 15; P = .022) and allele-specific polymerase chain reaction (11% Y181C, 5 of 22 vs 3 of 72, respectively; P = .016). K103N variants at frequencies 11% were associated with inferior HIV-1 RNA response to efavirenz-containing therapy between entry and week 24 (change in HIV-1 RNA level, +0.5 vs -1.1 log(10) copies/mL; P < .001). CONCLUSIONS Minor NNRTI-resistant variants were more prevalent in NNRTI-experienced patients and were associated with reduced virologic response to efavirenz-containing multidrug regimens.

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

Significance Despite the significant progress made in the last few years toward targeting phosphodiesterase-δ (PDEδ) for KRAS (Kirsten rat sarcoma isoform)-driven cancers, there is no structural information available on posttranslationally modified KRAS4b in complex with PDEδ. The KRAS4b–PDEδ structure reported here provides the structural details of the protein–protein interaction interface and the atomic details of the hypervariable region of KRAS4b. Structural comparison of the two crystal forms allowed identification of a 5-aa-long sequence motif in KRAS4b that could allow PDEδ to bind to both farnesylated and geranylgeranylated KRAS4b. Structural insights obtained from this study could be used to guide the development of improved and more specific inhibitors of the KRAS4b–PDEδ complex. Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated–methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated–methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated–methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated–methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein–protein interaction interface in KRAS4b–PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5–10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

Towards Better Precision Medicine: PacBio Single-Molecule Long Reads Resolve the Interpretation of HIV Drug Resistant Mutation Profiles at Explicit Quasispecies (Haplotype) Level.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient’s HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients’ HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.

Comparative proteomics of a model MCF10A-KRas G12V cell line reveals a distinct molecular signature of the KRas G12V cell surface

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry–based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.

Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.

We have developed and validated an amplification-free method for generating DNA sequencing libraries from very low amounts of input DNA (500 picograms – 20 nanograms) for singlemolecule sequencing on the Pacific Biosciences (PacBio) RS II sequencer. The common challenge of high input requirements for single-molecule sequencing is overcome by using a carrier DNA in conjunction with optimized sequencing preparation conditions and re-use of the MagBead-bound complex. Here we describe how this method can be used to produce sequencing yields comparable to those generated from standard input amounts, but by using 1000-fold less starting material.

Direct molecular dissection of tumor parenchyma from tumor stroma in tumor xenograft using mass spectrometry-based glycoproteomics

The most widely used cancer animal model is the human-murine tumor xenograft. Unbiased molecular dissection of tumor parenchyma versus stroma in human-murine xenografts is critical for elucidating dysregulated protein networks/pathways and developing therapeutics that may target these two functionally codependent compartments. Although antibody-reliant technologies (e.g., immunohistochemistry, imaging mass cytometry) are capable of distinguishing tumor-proper versus stromal proteins, the breadth or extent of targets is limited. Here, we report an antibody-free targeted cross-species glycoproteomic (TCSG) approach that enables direct dissection of human tumor parenchyma from murine tumor stroma at the molecular/protein level in tumor xenografts at a selectivity rate presently unattainable by other means. This approach was used to segment/dissect and obtain the protein complement phenotype of the tumor stroma and parenchyma of the metastatic human lung adenocarcinoma A549 xenograft, with no need for tissue microdissection prior to mass-spectrometry analysis. An extensive molecular map of the tumor proper and the associated microenvironment was generated along with the top functional N-glycosylated protein networks enriched in each compartment. Importantly, immunohistochemistry-based cross-validation of selected parenchymal and stromal targets applied on human tissue samples of lung adenocarcinoma and normal adjacent tissue is indicative of a noteworthy translational capacity for this unique approach that may facilitate identifications of novel targets for next generation antibody therapies and development of real time preclinical tumor models.

Abstract 3894: A xenograft mouse model coupled with in-depth cell surface proteome analysis facilitates further elucidation of K-Ras driven tumorigenesis in lung carcinoma

The purpose of this study was to develop a proteomic approach for analysis of cell surface proteins in tissue specimens and applied it on a K-Ras driven mouse model of metastatic lung carcinoma. Metastatic disease is the leading cause of lung cancer-related mortality in the United States. In spite of continuous efforts, effective treatments targeting oncogenic K-Ras in lung and other K-Ras driven malignancies are slow to develop. To expand the treatment options for lung cancer and facilitate better understanding of metastasis new targets need to be identified and characterized at the surface of cancer cells, preferably in their native tissue microenvironment. Towards this goal we developed a mass-spectrometry (MS)-based glyco-proteomic approach targeting specifically cell surface proteins in tissue specimens and applied it on a xenograft lung cancer mouse model. We used A549 cells expressing endogenous K-RasG12S to induce lung tumor xenografts in BALB/c mice via tail vein injection. A comparative surface glyco-proteomics of cultured A549 cells, dissected tumor tissue (TT), adjacent tumor (AT) tissue and normal mouse lung tissue (NT) obtained from saline-injected age-matched littermates yielded high enrichment (i.e., ≥80%) of surface proteins. It resulted in extensive catalogue/map of more than 400 glycoproteins identified on tumor cell surface. More than 50% of proteins identified on the surface of A549 cultured cells were unambiguously identified in mouse TT, featuring proteins and pathways implicated in metastasis, invadopodia formation and cancer cell migration. These markers also provide insight regarding species determination of tissue origin (i.e., human vs. mouse) differentiation of tumor parenchyma from stroma, and regulation of tumor immune response. Citation Format: Xiaoying Ye, Gordon Whiteley, Dwight Nissley, Frank McCormick, Josip Blonder. A xenograft mouse model coupled with in-depth cell surface proteome analysis facilitates further elucidation of K-Ras driven tumorigenesis in lung carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3894.

Abstract 1131: Uncovering oncogenic KRAS allele specific phenotypes using an isogenic MEF cell line system

The RAS Initiative at the Frederick National Laboratory has generated an isogenic mouse embryonic fibroblast (MEFs) cell line panel containing single transgene alleles using the RAS-less MEF system developed by Matthias Drosten and Mariano Barbacid (CNIO). These MEFs are H, N and K-RAS null, growth is dependent on exogenous RAS or MAPK pathway activation. The panel includes: KRAS 4A WT, KRAS 4B WT, KRAS 4B G12C, G12D, G12V, G13D, Q61L, Q61R, HRAS WT, NRAS WT or BRAF V600E. The workflow to generate the lines requires cre-lox removal of endogenous KRAS and the cells arrest in the G1 phase of the cycle. Proliferation is resumed through the delivery of the transgene to the cells using lentiviral transduction. Clonal cell lines are derived from the initial pools and are thoroughly characterized including confirmation of endogenous KRAS gene removal, identification of the transgene insertion site(s), calculation of proliferation rates and doubling times, analysis of signaling pathways, response to tool compounds and exome sequenced to exclude lines with mutations in onco-relevant genes. This panel is a unique resource that can be used to ask allele and isoform specific questions (gene expression profiles, GTP-loading, etc.) and screen for RAS inhibitors as well as determine novel compound specificity. This panel is available to the academic RAS research community. Citation Format: Nicole Fer, Brian Smith, Leslie Garvey, William Burgan, Katie Powell, Kanika Sharma, Andrew Waters, Xiaolin Wu, Dan Soppet, Robert Stephens, Dwight Nissley, Matthew Holderfield, Rachel K. Bagni. Uncovering oncogenic KRAS allele specific phenotypes using an isogenic MEF cell line system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1131.