In addition to acting as a cellular energy source, ATP can also act as a damage-associated molecular pattern in both animals and plants. Stomata are leaf pores that control gas exchange and, therefore, impact critical functions such as photosynthesis, drought tolerance, and also are the preferred entry point for pathogens. Here we show the addition of ATP leads to the rapid closure of leaf stomata and enhanced resistance to the bacterial pathogen Psuedomonas syringae. This response is mediated by ATP recognition by the receptor DORN1, followed by direct phosphorylation of the NADPH oxidase RBOHD, resulting in elevated production of reactive oxygen species and stomatal closure. Mutation of DORN1 phosphorylation sites on RBOHD eliminates the ability of ATP to induce stomatal closure. The data implicate purinergic signaling via DORN1 in the control of stomatal aperture with important implications for the control of plant photosynthesis, water homeostasis, pathogen resistance, and ultimately yield.

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.

The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca²⁺-imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.

724 Background: RMC-9805 is a potent, oral, RAS(ON) G12D-selective, covalent, tri-complex inhibitor targeting the active, GTP-bound state of oncogenic RAS G12D isoforms. Despite low response rates to current standard of care (SOC), no RAS-targeted therapy is approved for G12D PDAC. Methods: In this Phase 1 study (NCT06040541), patients with previously treated, advanced KRAS G12D solid tumors received escalating RMC-9805 doses (150-1200 mg once daily [QD] or 300-600 mg twice daily [BID]). Treatment cycles were every 3 wks. Antitumor activity was assessed every 6 wks the first 24 wks then every 9 wks. Additional patients were enrolled at RMC-9805 doses that cleared the dose-limiting toxicity (DLT) evaluation to further characterize pharmacokinetics, safety, antitumor activity, and biomarkers. Plasma for ctDNA analysis was collected at baseline (BL; cycle 1, day 1 [C1D1] or screening), and on treatment (OT; C2D1 or C3D1). Results: As of Sept 2, 2024, 179 patients with KRAS G12D solid tumors (most having PDAC; n=104 [58%]) received 5 escalating dose levels of RMC-9805 (150-1200 mg daily). No DLTs or Grade 4 or 5 treatment-related adverse events (TRAEs) were reported, and the maximum tolerated dose (MTD) was not reached. Among patients who received a candidate recommended phase 2 dose (RP2D) of 1200 mg daily (1200 mg QD [n=60] or 600 mg BID [n=39]), the most common (≥10% of patients) TRAEs were nausea (27%), diarrhea (20%), vomiting (15%), and rash (10%), all of which were Grade 1 or 2 severity. One Grade 3 TRAE (ALT elevation) was observed in a patient with PDAC and a history of ALT elevation, biliary stenting, and liver metastasis. No patients treated with 1200 mg daily discontinued treatment due to TRAEs and 4% of patients had dose reductions due to TRAEs. In patients with PDAC receiving RMC-9805 daily at 1200 mg QD (n=20) or 600 mg BID (n=20) who enrolled at least 14 wks prior to data cutoff, the objective response rate (confirmed response or pending confirmation) was 30%, and the disease control rate was 80%. Of 28 patients who had KRAS G12D mutations detected in ctDNA at BL and were evaluable for OT assessment, 86% had an OT decrease >50% from BL of detectable KRAS G12D mutations, and 39% had a 100% OT clearance. Updated data will be presented. Conclusions: Oral RMC-9805 showed encouraging initial antitumor activity with early and deep reductions in KRAS G12D ctDNA in patients with KRAS G12D PDAC. Tolerability was favorable relative to SOC chemotherapy for PDAC and manageable. This overall safety profile and antitumor activity support continued evaluation as monotherapy in patients with KRAS G12D PDAC, and in combination with chemotherapy and targeted therapies, including the RAS(ON) multi-selective inhibitor RMC-6236. Clinical trial information: NCT06040541 .

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5^S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.