High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance.

Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.

Estrogen-dependent control and cell-to-cell variability of transcriptional bursting.

Cellular decision-making and environmental adaptation are dependent upon a heterogeneous response of gene expression to external cues. Heterogeneity arises in transcription from random switching between transcriptionally active and inactive states, resulting in bursts of RNA synthesis. Furthermore, the cellular state influences the competency of transcription, thereby globally affecting gene expression in a cell-specific manner. We determined how external stimuli interplay with cellular state to modulate the kinetics of bursting. To this end, single-cell dynamics of nascent transcripts were monitored at the endogenous estrogen-responsive GREB1 locus. Stochastic modeling of gene expression implicated a two-state promoter model in which the estrogen stimulus modulates the frequency of transcriptional bursting. The cellular state affects transcriptional dynamics by altering initiation and elongation kinetics and acts globally, as GREB1 alleles in the same cell correlate in their transcriptional output. Our results suggest that cellular state strongly affects the first step of the central dogma of gene expression, to promote heterogeneity in the transcriptional output of isogenic cells.

PET of HER2-positive pulmonary metastases with 18F-ZHER2:342 affibody in a murine model of breast cancer: comparison with 18F-FDG.

UNLABELLED: Targeted therapies often depend on the expression of the target present in the tumor. This expression can be difficult to ascertain in widespread metastases. (18)F-FDG PET/CT, although sensitive, is nonspecific for particular tumor markers. Here, we compare the use of a human epidermal growth factor receptor 2 (HER2)-specific (18)F-Z(HER2)(:342)-Affibody and (18)F-FDG in HER2-expressing pulmonary metastases in a murine model of breast cancer. METHODS: The lung metastasis model was established by intravenous injection of MDA-MB-231(HER2)-Luc human breast cancer cells into the tail vein. Bioluminescence imaging was used to evaluate metastasis progression. Uptake of (18)F-Z(HER2)(:342)-Affibody and (18)F-FDG was confirmed by coregistration of the PET images with MR and CT images. At the end of the study, the presence of neoplastic cells and HER2 expression in lung tissues, and distribution of the tracer, were assessed ex vivo by immunohistochemistry and autoradiography. RESULTS: (18)F-Z(HER2)(:342)-Affibody successfully targeted HER2-positive lesions in the lung and allowed detection of metastases as early as 9 wk after injection of cells. In contrast, (18)F-FDG uptake was often masked by surrounding inflammatory changes and was nonspecific for HER2 expression. HER2 expression at a cellular level correlated well with tracer uptake on autoradiography. CONCLUSION: (18)F-Z(HER2)(:342)-Affibody is a promising tracer for evaluation of HER2 status of breast cancer metastases and is more specific for detecting HER2-positive lesions than (18)F-FDG.

HER2-affitoxin: a potent therapeutic agent for the treatment of HER2-overexpressing tumors.

PURPOSE: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. EXPERIMENTAL DESIGN: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. RESULTS: In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. CONCLUSIONS: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies.

HER2- and EGFR-specific affiprobes: novel recombinant optical probes for cell imaging.

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell-surface receptors/tyrosine kinases. EGFR- and HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683-696). We have created photostable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/ receptors.