Francesca Milletti

Cell-penetrating peptides: classes, origin, and current landscape.

With more than ten new FDA approvals since 2001, peptides are emerging as an important therapeutic alternative to small molecules. However, unlike small molecules, peptides on the market today are limited to extracellular targets. By contrast, cell-penetrating peptides (CPPs) can target intracellular proteins and also carry other cargoes (e.g. other peptides, small molecules or proteins) into the cell, thus offering great potential as future therapeutics. In this review I present a classification scheme for CPPs based on their physical-chemical properties and origin, and I provide a general framework for understanding and discovering new CPPs.

Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design

Cell-penetrating peptides (CPPs) enhance the cellular uptake of membrane-impermeable molecules. Most CPPs are highly cationic, potentially increasing the risk of toxic side effects and leading to accumulation in organs such as the liver. As a consequence, there is an unmet need for less cationic CPPs. However, design principles for effective CPPs are still missing. Here, we demonstrate a design principle based on a classification of peptides according to accumulated side-chain polarity and hydrophobicity. We show that in comparison to randomly selected peptides, CPPs cover a distinct parameter space. We designed peptides of only six to nine amino acids with a maximum of three positive charges covering this property space. All peptides were tested for cellular uptake and subcellular distribution. Following an initial round of screening we enriched the collection with short and hydrophobic peptides and introduced d-amino acid substitutions and lactam bridges which increased cell uptake, in particular for long-term incubation. Using a GFP complementation assay, for the most active peptides we demonstrate cytosolic delivery of a biologically active cargo peptide.

Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as a fusion protein with mCherry.

Abstract 4708: Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines

Small cell lung cancer (SCLC), which comprises 15% of lung neoplasms, is an aggressive malignancy with limited treatment options. Survival in refractory settings is typically less than one year, exemplifying the need for more effective therapeutics. Recent published results suggest that epigenetic modulation mediated by Lysine Specific Demethylase-1 (LSD1) inhibition can impact this disease setting (Mohammed et al., Cell, 2015 28(1):57-69). RG6016 is a potent and selective inhibitor of LSD1 that promotes growth inhibition in vitro and in vivo SCLC xenograft models. Here we describe the identification of a gene expression profile associated with neuroendocrine lineages that predicts responses to RG6016 in SCLC cell lines. RG6016 was screened on a panel of 19 SCLC cell lines; potent sub-nanomolar to nanomolar activity was exhibited on a subset of these cell lines. Growth inhibitory responses were greater in classic compared to variant SCLC cell lines (p-value 0.0055). 9 of the 11 classic SCLC cell lines tested were responsive to growth inhibitory effects of RG6016, while 7 of the 8 variant cell lines were insensitive to RG6016 treatment. Differential gene expression analysis comparing classic to variant cell lines ranked neuroendocrine lineage-associated markers, such as ASCL1, amongst the highest differentially expressed genes (adj. p-value = 2.6 × 10-23). ASCL1 is a transcription factor required for proper development of pulmonary neuroendocrine cells, and was recently identified as essential for the survival of a majority of small cell lung cancers (Augustyn et al., Proc Natl Acad Sci USA 2014 111(41):14788-93). Two other established neuroendocrine markers, DDC and GRP, ranked within the top ten differentially expressed genes. We also found that a subset of insensitive SCLC cell lines harbor c-MYC amplifications, suggesting a potential pathway for resistance to LSD1 inhibition. An RG6016 response gene expression pattern that highly correlates with growth inhibition was defined using baseline expression levels of neuroendocrine lineage transcripts and c-MYC amplification. In vitro activity also correlated with in vivo responses; RG60106 treatment inhibited xenograft growth of response signature positive cell line NCI-H510A, while the response signature negative NCI-H526 xenografts were refractory to RG6016 exposure. Similar gene expression patterns were observed within SCLC cell lines and a panel of SCLC patient samples, suggesting that use of the RG6016 response gene signature may increase the likelihood of identifying patients who will clinically benefit from LSD1- based therapies. Citation Format: Francesca Milletti, Wei-Yi Cheng, Tamara Maes, Serena Lunardi, Mark DeMario, William E. Pierceall, Fiona Mack. Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines. [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 4708.

Regulatory T-cell genes drive altered immune microenvironment in adult solid cancers and allow for immune contextual patient subtyping.

Background: The tumor microenvironment is an important factor in cancer immunotherapy response. To further understand how a tumor affects the local immune system, we analyzed immune gene expression differences between matching normal and tumor tissue. Methods: We analyzed public and new gene expression data from solid cancers and isolated immune cell populations. We also determined the correlation between CD8, FoxP3 IHC, and our gene signatures. Results: We observed that regulatory T cells (Tregs) were one of the main drivers of immune gene expression differences between normal and tumor tissue. A tumor-specific CD8 signature was slightly lower in tumor tissue compared with normal of most (12 of 16) cancers, whereas a Treg signature was higher in tumor tissue of all cancers except liver. Clustering by Treg signature found two groups in colorectal cancer datasets. The high Treg cluster had more samples that were consensus molecular subtype 1/4, right-sided, and microsatellite-instable, compared with the low Treg cluster. Finally, we found that the correlation between signature and IHC was low in our small dataset, but samples in the high Treg cluster had significantly more CD8+ and FoxP3+ cells compared with the low Treg cluster. Conclusions: Treg gene expression is highly indicative of the overall tumor immune environment. Impact: In comparison with the consensus molecular subtype and microsatellite status, the Treg signature identifies more colorectal tumors with high immune activation that may benefit from cancer immunotherapy. Cancer Epidemiol Biomarkers Prev; 27(1); 103–12. ©2017 AACR.

Quantifying the probability of clinical trial success from scientific articles.

We sought to analyze how the number and quality of publications predict clinical trial success for a set of gene-disease associations. Limiting the scope of our analysis to genes in the protein kinase family and to oncology indications, we extracted gene-disease relationships from more than 12 million article titles and abstracts published between 1992 and 2012. We integrated these data with clinical trial information for FDA-approved kinase inhibitors and kinase inhibitors that failed owing to lack of efficacy. We found that, up until the year when a compound enters clinical trials, the cumulative number of publications about a gene-disease relationship corresponding to the compounds mechanism of action is, at the median, 30 for approved compounds but only four for failed compounds.