Promoter-Driven Overexpression in Chromobacterium vaccinii Facilitates Access to FR900359 and Yields Novel Low Abundance Analogs.

Access to the cyclic depsipeptide FR900359 (FR), a selective Gq/11 protein inhibitor of high pharmacological interest and a potential lead molecule for targeted therapy of cancers with oncogenic GNAQ or GNA11 mutations (encoding Gq and G11 respectively), has been challenging ever since its initial discovery more than three decades ago. The recent discovery of Chromobacterium vaccinii as a cultivable FR producer enables the development of approaches leading to a high-yielding, scalable and sustainable biotechnological process for production of FR, thereby removing this bottleneck. Here we characterize different promoters in exchange of the native promoter of the FR assembly line, resulting in an overexpression mutant with significantly increased production of FR. Thereby, the isolation and structure elucidation of novel FR analogs of low abundance is enabled. Further, we explore the antiproliferative activities of fifteen chromodepsins against uveal melanoma cell lines harboring Gq/11 mutations and characterize the major metabolite of FR formed in plasma.

M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth.

Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.

INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.