Triple-hit therapeutic approach for triple negative breast cancers using docetaxel nanoparticles, EN1-iPeps and RGD peptides.

Triple negative breast cancers (TNBC) are aggressive malignancies for which chemotherapy is the only treatment option. Many TNBC acquire chemotherapy resistance, notably docetaxel, which has been associated with the overexpression of transcription factors (TFs), such as ENGRAILED1 (EN1). Here, we have developed a tumor delivery system for docetaxel-PGMA-PAA-nanoparticles and interference peptides designed to specifically inhibit EN1 (EN1-iPeps). To promote tumor specific targeting, we functionalized these nanoparticles with EN1-iPeps engineered with RGD sequences. We found that these peptides reduce cell viability and induce apoptosis in TNBC cells with negligible effects on normal cells (EN1-). Moreover, EN1-RGD-iPeps-mediated nanoparticle internalization into breast cancer cells was via integrins and intravenous injection of this nanoformulation increased tumor accumulation. Furthermore, docetaxel nanoparticles functionalized with EN1-RGD-iPeps significantly reduced TNBC growth both in vitro and in vivo without showing toxicity. Our results suggest that this targeted nanoformulation represents a new and safe therapeutic approach for chemoresistant TNBCs.

Csk-binding protein controls red blood cell development via regulation of Lyn tyrosine kinase activity.

Erythropoiesis is controlled principally through erythropoietin (Epo) receptor signaling, which involves Janus kinase 2 (JAK2) and Lyn tyrosine kinase, both of which are important for regulating red blood cell (RBC) development. Negative regulation of Lyn involves C-Src kinase (Csk)-mediated phosphorylation of its C-terminal tyrosine, which is facilitated by the transmembrane adaptor Csk-binding protein (Cbp). Although Cbp has significant functions in controlling Lyn levels and activity in erythroid cells in vitro, its importance to primary erythroid cell development and signaling has remained unclear. To address this, we assessed the consequence of loss of Cbp on the erythroid compartment in vivo and whether Epo-responsive cells isolated from Cbp-knockout mice exhibited altered signaling. Our data show that male Cbp-/- mice display a modest but significant alteration to late erythroid development in bone marrow with evidence of increased erythrocytes in the spleen, whereas female Cbp-/- mice exhibit a moderate elevation in early erythroid progenitors (not seen in male mice) that does not influence the later steps in RBC development. In isolated primary erythroid cells and cell lines generated from Cbp-/- mice, survival signaling through Lyn/Akt/FoxO3 was elevated, resulting in sustained viability during differentiation. The high Akt activity disrupted GAB2/SHP-2 feedback inhibition of Lyn; however, the elevated Lyn activity also increased inhibitory signaling via SHP-1 to restrict the Erk1/2 pathway. Interestingly, whereas loss of Cbp led to mild changes to late RBC development in male mice, this was not apparent in female Cbp-/- mice, possibly due to their elevated estrogen, which is known to facilitate early progenitor self-renewal.

Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA.

Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention. Although efficiencies and specificities of genome editing technologies have improved with the development of TALEs and CRISPR platforms, intracellular delivery of these larger constructs still remains a challenge using existing delivery agents. Viral vectors, including lentiviruses and adeno-associated viruses, as well as some non-viral strategies, such as cationic polymers and liposomes, are limited by packaging capacity, poor delivery, toxicity, and immunogenicity. We report a highly controlled synthetic strategy to engineer a flexible dendritic polymer using click chemistry to overcome the aforementioned delivery challenges associated with genome engineering technologies. Using a systematic approach, we demonstrate that high transfection efficiencies and packaging capacity can be achieved using this non-viral delivery methodology to deliver zinc fingers, TALEs and CRISPR/dCas9 platforms.

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system.

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling.

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.