CWR22-RV1

Cytotoxicity and Uptake of GZMB-MU2

Prostate cancer is a slowly growing malignancy that expresses high levels of PSMA. Protein toxins exert their cytotoxicity in a proliferation-independent manner and are non-tumor specific. In order to evaluate the potential for selective delivery, Rogers et al. conjugated a urea-based PSMA-inhibitor to two cytotoxic proteins, human Granzyme B (GZMB) and a cysteine-containing fragment of the pseudomonas exotoxin A gene (PE35). The complex GZMB-MU2 was tested for cytotoxicity against PSMA expressing prostate cancer cell lines (PIP-PC3, LNCaP, CWR22Rv1) and control (Flu-PC3) cells. GZMB-MU2 demonstrated no impact on cell proliferation, morphology or survival when tested at concentrations up to 300 nM (Fig. 1B). To investigate whether the lack of toxicity was due to poor internalization, fluorescein-tagged GZMB-MU2 was incubated with PIP-PC3 and Flu-PC3 cells for 2 h. Confocal microscopy demonstrated punctate fluorescein staining in PIP-PC3 cells localized near the nucleus and membrane, but not in Flu-PC3 cells (Fig. 1C). Treatment with endosome-disrupting agents (HIV TAT peptides, chloroquine) did not enhance cytotoxicity, although chloroquine alone produced significant toxicity. Low cytosolic delivery or SERPIN-mediated inactivation of GZMB may account for lack of apoptosis.

In Vitro RNA-Seq Data Highlights JAZF1 as a Modulator of Response to Polyamine-Targeted Therapies

Metabolic dysregulation plays a critical role in diseases like prostate cancer, where targeting specific pathways (e.g., polyamine biosynthesis and methionine cycle) has shown therapeutic potential. However, the master metabolic transcriptional regulators (MMTRs) controlling these pathways and their impact on treatment response remain unclear. Rosario et al. aimed to identify novel MMTRs influencing prostate cancer's sensitivity to polyamine- and methionine-targeted therapies, focusing on JAZF1 as a key regulator.

A gene signature of treatment-naïve prostate tumors was used to predict intrinsic nonresponse to polyamine-targeted therapy, and this nonresponse was associated with the dysregulation of metabolic pathways (e.g., downregulation of polyamine biosynthesis). Patient-derived ex vivo samples and TCGA tumor data were interrogated to identify 82 DEGs that stratified responders and nonresponders, and metabolic profiling of tumors from each group identified the downregulation of polyamine synthesis in resistant tumors. To identify key MMTRs of treatment sensitivity, they intersected MMTRs of the 82 response-related genes with those found to regulate adaptive responses to therapy. As shown previously, BENSpm + MTDIA treatment of prostate cancer cell lines (LNCaP, C4-2, CWR22-RV1) results in progressive cytotoxicity over 4-12 days. To study adaptive mechanisms to therapy before cell death (8-12 day timepoints), they treated RV1 and C4-2 cells with vehicle, single agents, or combination therapy for 1h, 24h, and 48h/96h (Fig. 2A). RNA-seq showed a cyclical transcriptional response to therapy with initial dysregulation (1h), transcriptional stabilization (24h), and point of irreversible dysregulation (48h/96h; Fig. 2B). MMTR analysis of DEGs showed JAZF1 to be a shared regulator of polyamine/methionine pathways as well as response to therapy with binding motifs in ODC1, MAT2A, PAOX, and SAT1 (Fig. 2C-E).