Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia.

We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate Insulin Like Growth Factor 1 Receptor (IGF1R) signaling in leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL in vitro, implicating additional myeloid-mediated signals in leukemia progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion activates downstream focal adhesion kinase (FAK)/ proline-rich tyrosine kinase 2 (PYK2), which are required for myeloid-mediated T-ALL support, partly through activation of IGF1R. Blocking integrin ligands or inhibiting FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated adhesion or FAK/PYK2 also reduces survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate with higher FAK signaling and myeloid gene signatures and are associated with an inferior prognosis in pediatric T-ALL patients. Together, these findings demonstrate that integrin activation and downstream FAK/PYK2 signaling are important mechanisms underlying myeloid-mediated support of T-ALL progression.

Tumor-associated myeloid cells provide critical support for T-ALL.

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells, from the thymic tumor microenvironment support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in 2 distinct mouse models of T-ALL and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of antitumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid-depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

Peptides as surface coatings of nanoparticles that penetrate human cystic fibrosis sputum and uniformly distribute in vivo following pulmonary delivery.

Therapeutic delivery of drug and gene delivery systems have to traverse multiple biological barriers to achieve efficacy. Mucosal administration, such as pulmonary delivery in cystic fibrosis (CF) disease, remains a significant challenge due to concentrated viscoelastic mucus, which prevents drugs and particles from penetrating the mucus barrier. To address this problem, we used combinatorial peptide-presenting phage libraries and next-generation sequencing (NGS) to identify hydrophilic, net-neutral charged peptide coatings that enable penetration through human CF mucus ex vivo with ~600-fold better penetration than control, improve uptake into lung epithelial cells compared to uncoated or PEGylated-nanoparticles, and exhibit enhanced uniform distribution and retention in the mouse lung airways. These peptide coatings address multiple delivery barriers and effectively serve as excellent alternatives to standard PEG surface chemistries to achieve mucus penetration and address some of the challenges encountered using these chemistries. This biomolecule-based strategy can address multiple delivery barriers and hold promise to advance efficacy of therapeutics for diseases like CF.

Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment.

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts as a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) affect their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and quantify their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and spatial ordering impact diffusive transport. The P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited ∼40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to functionalized control particles. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.

Transcriptomic analysis of salt stress responsive genes in Rhazya stricta.

Rhazya stricta is an evergreen shrub that is widely distributed across Western and South Asia, and like many other members of the Apocynaceae produces monoterpene indole alkaloids that have anti-cancer properties. This species is adapted to very harsh desert conditions making it an excellent system for studying tolerance to high temperatures and salinity. RNA-Seq analysis was performed on R. stricta exposed to severe salt stress (500 mM NaCl) across four time intervals (0, 2, 12 and 24 h) to examine mechanisms of salt tolerance. A large number of transcripts including genes encoding tetrapyrroles and pentatricopeptide repeat (PPR) proteins were regulated only after 12 h of stress of seedlings grown in controlled greenhouse conditions. Mechanisms of salt tolerance in R. stricta may involve the upregulation of genes encoding chaperone protein Dnaj6, UDP-glucosyl transferase 85a2, protein transparent testa 12 and respiratory burst oxidase homolog protein b. Many of the highly-expressed genes act on protecting protein folding during salt stress and the production of flavonoids, key secondary metabolites in stress tolerance. Other regulated genes encode enzymes in the porphyrin and chlorophyll metabolic pathway with important roles during plant growth, photosynthesis, hormone signaling and abiotic responses. Heme biosynthesis in R. stricta leaves might add to the level of salt stress tolerance by maintaining appropriate levels of photosynthesis and normal plant growth as well as by the participation in reactive oxygen species (ROS) production under stress. We speculate that the high expression levels of PPR genes may be dependent on expression levels of their targeted editing genes. Although the results of PPR gene family indicated regulation of a large number of transcripts under salt stress, PPR actions were independent of the salt stress because their RNA editing patterns were unchanged.

Gene expression profile and functionality of ESC-derived Lin-ckit+Sca-1+ cells are distinct from Lin-ckit+Sca-1+ cells isolated from fetal liver or bone marrow.

In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.

Small RNA profiling reveals antisense transcription throughout the KSHV genome and novel small RNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus that encodes 12 precursor microRNAs (pre-miRNAs) that give rise to 17 different known approximately 22-nucleotide (nt) effector miRNAs. Like all herpesviruses, KSHV has two modes of infection: (1) a latent mode whereby only a subset of viral genes are expressed and (2) a lytic mode during which the full remaining viral genes are expressed. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. Here, we developed a method to profile small RNAs ( approximately 18-75 nt) from populations of cells undergoing predominantly lytic infection. Using two different next-generation sequencing platforms, we cloned and sequenced both pre-miRNAs and derivative miRNAs. Our analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5' end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. We report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miR-K12-8 and pre-miR-K12-12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. We identify several novel small RNAs of low abundance, including viral miRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. Finally, we observe widespread antisense transcription relative to known coding sequences during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, we observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs).