Amy H. Lin

Virtually 100% of melanoma cell lines harbor alterations at the DNA level within CDKN2A, CDKN2B, or one of their downstream targets

The cyclin‐dependent kinase inhibitor 2A (CDKN2A), or p16INK4a, gene on 9p21 is important in the genesis of both familial and sporadic melanoma. Homozygous deletions and intragenic mutations of this gene have been identified in both melanoma cell lines and uncultured tumors, although the frequency of these alterations is higher in the cell lines. A proportion of melanoma cell lines and tumors without deletion/mutation of CDKN2A have also been determined to harbor transcriptionally inactive CDKN2A alleles or carry alterations in other components of the pathway through which p16 INK4a acts on pRb to mediate cell cycle arrest. We sought to determine the frequency of these alternative events (in relationship to those that specifically inactivate CDKN2A) in a panel of 45 melanoma cell lines. Surprisingly, at the DNA level alone, 96% (43/45) of melanoma cell lines examined were found to be deleted/mutated/methylated for CDKN2A (34/45), homozygously deleted for CDKN2As neighbor and homolog CDKN2B (6/45), and/or mutated/amplified for CDK4 (5/45). In two of these 43 cases, homozygous deletions of CDKN2A were detected along with a CDK4 mutation or amplification of the cyclin D1 (CCND1) gene. The latter discoveries were made in two of three cell lines which harbored extremely large (3–6 Mb) homozygous deletions on 9p21; all other homozygous deletions in similarly affected cell lines (N = 23) were confined to a region immediately surrounding the CDKN2A/CDKN2B loci. These results suggest that (1) only melanoma cells with alterations in this pathway can be propagated in culture, and (2) the homozygous deletions on 9p21 in the cell lines, which are also mutated/amplified for CDK4 or CCND1, could serve to target tumor suppressor genes other than CDKN2A. Genes Chromosomes Cancer 22:157–163, 1998.

Receptor-specific targeting mediated by the coexpression of a targeted murine leukemia virus envelope protein and a binding-defective influenza hemagglutinin protein

The entry of retroviral vectors into cells requires two events: binding to a cell surface receptor and the subsequent fusion of viral and cellular membranes. The host range of a vector is therefore determined largely by the receptor specificity of the fusion protein contained in the outer viral envelope. Previous attempts to generate targeted retroviral vectors have included the addition of targeting ligands to the murine leukemia virus envelope protein (MuLV Env). Although such proteins frequently display modified cell-binding characteristics, the interaction with the targeted receptors fails to trigger virus-cell fusion. Here, we report the use of a binding-defective but fusion-competent hemagglutinin (HA) protein to complement the fusion defect in a chimeric MuLV Env targeted to the Flt-3 receptor. Retroviral vectors containing both proteins showed enhanced transduction of cells expressing Flt-3, which was abrogated by preincubating the target cells with soluble Flt-3 ligand. Furthermore, the fusion function of HA was absolutely required. These data demonstrate that it is possible to separate the binding and fusion events of retroviral entry, using two separate proteins, and suggest that varying the binding protein component in this scheme may allow a general strategy for targeting retroviral vectors.

Use of pseudotyped retroviral vectors to analyze the receptor-binding pocket of hemagglutinin from a pathogenic avian influenza A virus (H7 subtype)

The hemagglutinin (HA) protein of influenza virus binds to terminal sialic acid residues present on cell surface glycoproteins and glycolipids. The specific amino acids involved in this interaction have been identified for a H3 subtype HA from the human non-pathogenic virus, A/Aichi/2/68, by both crystallographic and mutagenesis studies. We were interested to examine the receptor-binding pocket of a H7 subtype protein from the avian pathogenic virus A/FPV/Rostock/34. Accordingly, we made amino acid substitutions at six conserved residues (Y88, T126, H174, E181, L185, and G219), suggested by comparison with the receptor-binding pocket of the H3 protein, and analyzed the resulting proteins using pseudotyped retroviral vectors. The use of these vectors enabled us to quantitate both the ability of the mutant HA proteins to bind with receptor-expressing cells, and also to promote virus-cell fusion by measuring vector titer. Using this system, we identified a subset of mutants with impaired receptor-binding activity and a corresponding decrease in titer, but which retained the ability to induce syncytia in low pH cell-cell fusion assays. The most severely affected mutants contained more than one substitution, with the triple mutant Y88F/E181Q/G219K being the most defective. These observations highlight the importance of multiple contact points for the interaction between sialic acid and HA.

Blockade of Type I Interferon (IFN) Production by Retroviral Replicating Vectors and Reduced Tumor Cell Responses to IFN Likely Contribute To Tumor Selectivity

ABSTRACT We developed a Moloney mouse leukemia virus (MLV)-based retroviral replicating vector (RRV), Toca 511, which has displayed tumor specificity in resected brain tumor material and blood in clinical trials. Here, we investigated the interaction between Toca 511 and human host cells, and we show that RRVs do not induce type I interferon (IFN) responses in cultured human tumor cells or cultured human primary cells. However, exogenous type I IFN inhibited RRV replication in tumor cells and induced IFN-regulated genes, albeit at a lower level than in primary cells. Unexpectedly, RRVs did not induce IFN-α production upon incubation in vitro with human plasmacytoid dendritic cells (pDCs), whereas lentivirus vector and heat-treated RRVs did. Coincubation of RRVs with heat-treated RRVs or with lentivirus vector suppressed IFN-α production in pDCs, suggesting that native RRV has a dominant inhibitory effect on type I IFN induction. This effect is sensitive to trypsin treatment. In addition, heat treatment inactivated that activity but exposed an immune-stimulatory activity. The immune-stimulating component is sensitive to deglycosidases, trypsin, and phospholipase C treatment. Experiments with retroviral nonreplicating vectors and virus-like particles demonstrated that the immunosuppressive activity is not associated with the amphotropic envelope or the glyco-Gag protein. In summary, our data provide evidence that RRVs do not directly trigger type I IFN responses in IFN-responsive tumor cells. Moreover, RRVs appear to carry a heat-labile component that actively suppresses activation of cellular innate immune responses in pDCs. Inhibition of IFN induction by RRVs and the reduced response to IFN should facilitate tumor-specific infection in vivo. IMPORTANCE RRVs have a convincing preference for replicating in tumor cells in animal models, and we observed similar preferences in the initial treatment of human glioblastoma patients. This study investigates the basis for the interaction between RRV and human host cells (tumor versus nontumor) in vitro. We found that RRVs do not trigger an IFN-α/β response in tumor cells, but the cells are capable of responding to type I IFNs and of producing them when stimulated with known agonists. Surprisingly, the data show that RRVs can actively inhibit induction of cellular innate immunity and that this inhibitory activity is heat labile and trypsin sensitive and not attributable to the envelope protein. These data partially explain the observed in vivo tumor specificity.

Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro

Tumor cells express a number of immunosuppressive molecules that can suppress anti-tumor immune responses. Efficient delivery of small interfering RNAs to treat a wide range of diseases including cancers remains a challenge. Retroviral replicating vectors (RRV) can be used to stably and selectively introduce genetic material into cancer cells. Here, we designed RRV to express shRNA (RRV-shPDL1) or microRNA30-derived shRNA (RRV-miRPDL1) using Pol II or Pol III promoters to downregulate PDL1 in human cancer cells. We also designed RRV expressing cytosine deaminase (yCD2) and miRPDL1 for potential combinatorial therapy. Among various configurations tested, we showed that RRV-miRPDL1 vectors with Pol II or Pol III promoter replicated efficiently and exhibited sustained downregulation of PDL1 protein expression by more than 75% in human cancer cell lines with high expression of PDL1. Immunologic effects of RRV-miRPDL1 were assessed by a trans-suppression lymphocyte assay. In vitro data showed downregulation of PDL1+ tumor cells restored activation of CD8+ T cells and bio-equivalency compared to anti-PDL1 antibody treatment. These results suggest RRV-miRPDL1 may be an alternative therapeutic approach to enhance anti-tumor immunity by overcoming PDL1-induced immune suppression from within cancer cells and this approach may also be applicable to other cancer targets.

Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene

We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (As) in the J-K bifurcation domain sometimes serially added As. Therefore, vectors with 4-12 As in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 As gave roughly comparable levels, 4 and 8 As were within about 4-5-fold of the 6 As, whereas 10 and 12 As were marked lower. In terms of stability, after 10 infection cycles, expansion of As appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 As appeared completely stable; 6, 7, and particularly 8 As showed some level of expansion in the A bulge; 10 and 12 As underwent both expansion and transgene deletion. The strong relative translational activity of the 5 As in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.

Participant–Informant Relationships Affect Quality of Life Ratings in Incipient and Clinical Alzheimer Disease

OBJECTIVE Clinical trials in incipient and clinical Alzheimer disease (AD) often include informant-reported outcomes. Whereas informant reports in AD dementia may be modulated by the nature of participant-informant relationships, whether informant type affects reporting at earlier disease stages is less certain. We sought to determine the effects of participant-informant relationships on informant assessments of quality of life (QOL), functional abilities, and behavioral symptoms in individuals with normal cognition (NC), mild cognitive impairment (MCI), and mild-to-moderate AD dementia. DESIGN Cross-sectional. SETTING Easton Center for Alzheimer Disease Research at the University of California, Los Angeles. PARTICIPANTS A total of 399 individuals who met criteria for NC (N = 100), MCI [amnestic (N = 125) and nonamnestic (N = 61)], and AD (N = 113). Participants were subdivided into groups based on informant-participant relationships (spouse versus other). MEASUREMENTS We examined informant effects on the Quality of Life-Alzheimers Disease (QOL-AD) scale, the Functional Activities Questionnaire (FAQ), and the Neuropsychiatric Inventory (NPI). RESULTS After adjustments for demographic and cognitive factors, spouse informants reported higher participant QOL in the amnestic MCI and AD groups than did other informants. No informant effects were seen on QOL-AD ratings in the nonamnestic MCI or NC groups or on the FAQ or NPI in the MCI and AD groups. CONCLUSIONS Participant-informant relationships may modulate informant responses on subjective measures such as the QOL-AD in both incipient and clinical AD. Clinical trials that use informant measures may need to address these effects.