PROMISE: a real-world clinical-genomic database to address knowledge gaps in prostate cancer.

PURPOSE: Prostate cancer is a heterogeneous disease with variable clinical outcomes. Despite numerous recent approvals of novel therapies, castration-resistant prostate cancer remains lethal. A "real-world" clinical-genomic database is urgently needed to enhance our characterization of advanced prostate cancer and further enable precision oncology. METHODS: The Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort (PROMISE) is a consortium whose aims are to establish a repository of de-identified clinical and genomic patient data that are linked to patient outcomes. The consortium structure includes a (1) bio-informatics committee to standardize genomic data and provide quality control, (2) biostatistics committee to independently perform statistical analyses, (3) executive committee to review and select proposals of relevant questions for the consortium to address, (4) diversity/inclusion committee to address important clinical questions pertaining to racial disparities, and (5) patient advocacy committee to understand patient perspectives to improve patients' quality of care. RESULTS: The PROMISE consortium was formed by 16 academic institutions in early 2020 and a secure RedCap database was created. The first patient record was entered into the database in April 2020 and over 1000 records have been entered as of early 2021. Data entry is proceeding as planned with the goal to have over 2500 patient records by the end of 2021. CONCLUSIONS: The PROMISE consortium provides a powerful clinical-genomic platform to interrogate and address data gaps that have arisen with increased genomic testing in the clinical management of prostate cancer. The dataset incorporates data from patient populations that are often underrepresented in clinical trials, generates new hypotheses to direct further research, and addresses important clinical questions that are otherwise difficult to investigate in prospective studies.

Systemic vs. intrathecal central nervous system prophylaxis in primary adrenal/renal diffuse large b-cell LYMPHOMA: A multi-institution retrospective analysis and systematic review.

Primary adrenal lymphoma (PAL) and primary renal lymphoma (PRL) are rare extranodal lymphomas, predominantly of diffuse large B-cell lymphoma subtype. Primary adrenal and renal lymphomas (PARL) exhibit a high predilection for the central nervous system (CNS). Therefore, current guidelines support the use of CNS prophylaxis in PARL, particularly in cases of high-risk Central Nervous System International Prognostic Index (CNS-IPI). However, the route of administration (i.e. systemic vs. intrathecal chemotherapy) has not been clearly elucidated. With this in mind, we initiated an international collaboration and literature review to analyze 50 patient cases, 20 of which received CNS prophylaxis. Based on our analysis, we conclude that PARL may indicate a need for CNS chemo-prophylaxis in the form of systemic high-dose methotrexate (HD-MTX) over intrathecal methotrexate (IT-MTX), although IT-MTX may still have utility in certain cases.

Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of Munc18.

Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion protein)). However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion. As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro within a preformed Munc18a.syntaxin 1a heterodimer complex and that this results in the disassembly of the complex. Using site-directed mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr574. Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine. The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses. Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness.

Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings.

The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A. Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.

Characterization and distribution of SNARE proteins at neuroendocrine nerve endings.

Substantial evidence now exists to support a defined complex of interacting proteins, comprised of soluble, vesicle and plasma membrane components, as the core of a general membrane fusion mechanism. Specializations to the general secretory model occur based on cell-specific differences in Ca2+ regulation, secretory organelle types and secretory dynamics. The variation in secretory properties may also result, in part, from isoform diversity and selective-pairing of the molecular components of the core complex. The present report attempts to identify the SNARE proteins found in isolated peptidergic nerve endings of the rat neurohypophysis. The results demonstrate the presence of synaptosomal-associated protein of 25 kD, syntaxin and synaptobrevin as membrane-associated proteins in these nerve endings. Furthermore, we have utilized sucrose density gradient subcellular fractionation and immunoprecipitation protocols to investigate the synaptobrevin isotypes present on secretory granules and to probe using electrophysiological methods their functional relationship to secretion. Secretory granules were found to contain only the synaptobrevin 2 isoform, although the nerve endings themselves were found to possess in addition, synaptobrevin 1 and the closely related protein cellubrevin. Analysis of the secretory characteristics of single nerve endings using membrane capacitance measurements together with Botulinum B toxin dialysis demonstrated the critical importance of synaptobrevin 2 to both the rapid exocytotic release and a slower secretory process, that perhaps includes secretory granule recruitment and priming, in these peptidergic nerve endings.