Enhanced cytotoxicity of a Pseudomonas Exotoxin A based immunotoxin against prostate cancer by addition of the endosomal escape enhancer SO1861.

Immunotoxins consist of an antibody or antibody fragment that binds to a specific cell surface structure and a cytotoxic domain that kills the cell after cytosolic uptake. Pseudomonas Exotoxin A (PE) based immunotoxins directed against a variety of tumor entities have successfully entered the clinic. PE possesses a KDEL-like motif (REDLK) that enables the toxin to travel from sorting endosomes via the KDEL-receptor pathway to the endoplasmic reticulum (ER), from where it is transported into the cytosol. There, it ADP-ribosylates the eukaryotic elongation factor 2, resulting in ribosome inhibition and finally apoptosis. One major problem of immunotoxins is their lysosomal degradation causing the need for much more immunotoxin molecules than finally required for induction of cell death. The resulting dose limitations and substantially increased side effects require new strategies to achieve improved cytosolic uptake. Here we generated an immunotoxin consisting of a humanized single chain variable fragment (scFv) targeting the prostate specific membrane antigen (PSMA) and the de-immunized PE variant PE24mut. This immunotoxin, hD7-1(VL-VH)-PE24mut, showed high and specific cytotoxicity in PSMA-expressing prostate cancer cells. We deleted the REDLK sequence to prevent transport to the ER and achieve endosomal entrapment. The cytotoxicity of this immunotoxin, hD7-1(VL-VH)-PE24mutΔREDLK, was greatly reduced. To restore activity, we added the endosomal escape enhancer SO1861 and observed an up to 190,000-fold enhanced cytotoxicity corresponding to a 57-fold enhancement compared to the initial immunotoxin with the REDLK sequence. A biodistribution study with different routes of administration clearly showed that the subcutaneous injection of hD7-1(VL-VH)-PE24mutΔREDLK in mice resulted in the highest tumor uptake. Treatment of mice bearing prostate tumors with a combination of hD7-1(VL-VH)-PE24mutΔREDLK plus SO1861 resulted in inhibition of tumor growth and enhanced overall survival compared to the monotherapies. The endosomal entrapment of non-toxic anti-PSMA immunotoxins followed by enhanced endosomal escape by SO1861 provides new therapeutic options in the future management of prostate cancer.

Calpain-mediated cleavage generates a ZBTB18 N-terminal product that regulates HIF1A signaling and glioblastoma metabolism.

Proteolytic cleavage is an important post-translational mechanism to increase protein variability and functionality. In cancer, this process can be deregulated to shut off tumor-suppressive functions. Here, we report that in glioblastoma (GBM), the tumor suppressor ZBTB18 is targeted for protein cleavage by the intracellular protease calpain. The N-terminal (Nte) ZBTB18 cleaved fragment localizes to the cytoplasm and thus, is unable to exert the gene expression repressive function of the uncleaved protein. Mass spectrometry (MS) analysis indicates that the Nte ZBTB18 short form (SF) interacts with C-terminal (Cte) binding proteins 1 and 2 (CTBP1/2), which appear to be involved in HIF1A signaling activation. In fact, we show that the new ZBTB18 product activates HIF1A-regulated genes, which in turn lead to increased lipid uptake, lipid droplets (LD) accumulation, and enhanced metabolic activity. We propose that calpain-mediated ZBTB18 cleavage represents a new mechanism to counteract ZBTB18 tumor suppression and increase tumor-promoting functions in GBM cells.

An Anti-PSMA Immunotoxin Reduces Mcl-1 and Bcl2A1 and Specifically Induces in Combination with the BAD-Like BH3 Mimetic ABT-737 Apoptosis in Prostate Cancer Cells.

Background: Upregulation of anti-apoptotic Bcl-2 proteins in advanced prostate cancer leads to therapeutic resistance by prevention of cell death. New therapeutic approaches aim to target the Bcl-2 proteins for the restoration of apoptosis. Methods: The immunotoxin hD7-1(VL-VH)-PE40 specifically binds to the prostate specific membrane antigen (PSMA) on prostate cancer cells and inhibits protein biosynthesis. It was tested with respect to its effects on the expression of anti-apoptotic Bcl-2 proteins. Combination with the BAD-like mimetic ABT-737 was examined on prostate cancer cells and 3D spheroids and in view of tumor growth and survival in the prostate cancer SCID mouse xenograft model. Results: The immunotoxin led to a specific inhibition of Mcl-1 and Bcl2A1 expression in PSMA expressing target cells. Its combination with ABT-737, which inhibits Bcl-2, Bcl-xl, and Bcl-w, led to an induction of the intrinsic apoptotic pathway and to a synergistic cytotoxicity in prostate cancer cells and 3D spheroids. Furthermore, combination therapy led to a significantly prolonged survival of mice bearing prostate cancer xenografts based on an inhibition of tumor growth. Conclusion: The combination therapy of anti-PSMA immunotoxin plus ABT-737 represents the first tumor-specific therapeutic approach on the level of Bcl-2 proteins for the induction of apoptosis in prostate cancer.

c-Jun-N-terminal phosphorylation regulates DNMT1 expression and genome wide methylation in gliomas.

High-grade gliomas (HGG) are the most common brain tumors, with an average survival time of 14 months. A glioma-CpG island methylator phenotype (G-CIMP), associated with better clinical outcome, has been described in low and high-grade gliomas. Mutation of IDH1 is known to drive the G-CIMP status. In some cases, however, the hypermethylation phenotype is independent of IDH1 mutation, suggesting the involvement of other mechanisms. Here, we demonstrate that DNMT1 expression is higher in low-grade gliomas compared to glioblastomas and correlates with phosphorylated c-Jun. We show that phospho-c-Jun binds to the DNMT1 promoter and causes DNA hypermethylation. Phospho-c-Jun activation by Anisomycin treatment in primary glioblastoma-derived cells attenuates the aggressive features of mesenchymal glioblastomas and leads to promoter methylation and downregulation of key mesenchymal genes (CD44, MMP9 and CHI3L1). Our findings suggest that phospho-c-Jun activates an important regulatory mechanism to control DNMT1 expression and regulate global DNA methylation in Glioblastoma.

Epigenetic Regulation of ZBTB18 Promotes Glioblastoma Progression.

Glioblastoma (GBM) comprises distinct subtypes characterized by their molecular profile. Mesenchymal identity in GBM has been associated with a comparatively unfavorable prognosis, primarily due to inherent resistance of these tumors to current therapies. The identification of molecular determinants of mesenchymal transformation could potentially allow for the discovery of new therapeutic targets. Zinc Finger and BTB Domain Containing 18 (ZBTB18/ZNF238/RP58) is a zinc finger transcriptional repressor with a crucial role in brain development and neuronal differentiation. Here, ZBTB18 is primarily silenced in the mesenchymal subtype of GBM through aberrant promoter methylation. Loss of ZBTB18 contributes to the aggressive phenotype of glioblastoma through regulation of poor prognosis-associated signatures. Restitution of ZBTB18 expression reverses the phenotype and impairs tumor-forming ability. These results indicate that ZBTB18 functions as a tumor suppressor in GBM through the regulation of genes associated with phenotypically aggressive properties.Implications: This study characterizes the role of the putative tumor suppressor ZBTB18 and its regulation by promoter hypermethylation, which appears to be a common mechanism to silence ZBTB18 in the mesenchymal subtype of GBM and provides a new mechanistic opportunity to specifically target this tumor subclass. Mol Cancer Res; 15(8); 998-1011. ©2017 AACR.

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression.

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.