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Vikram Sharma, Moloy Goswami, Qitao Zhang, Sraboni Chaudhury, Katie Li, Hima Bindu Durumulta, Anthony Andren, Costas A Lyssiotis, Cagri G Besirli, Jason Matthew Lewis Miller, Thomas J Wubben; Promoting PKM2 tetramerization to treat RPE epithelial-to-mesenchymal transition in proliferative vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3051 – F0422.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) represents the greatest risk of failure of retinal detachment repair surgery. No pharmacotherapies have prevented the formation of PVR, so a significant unmet need exists. The epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is critical in PVR pathogenesis. Increased glycolysis is a hallmark of RPE EMT. Pyruvate kinase M2 (PKM2) is a key regulator of glycolysis that exists in different oligomeric states, dimer and tetramer, and has been implicated in glycolytic reprogramming of cells. Small molecule modulators of PKM2, like ML-265, induce tetramerization reducing glycolytic reprogramming. We assessed the therapeutic potential of pharmacologically inducing PKM2 tetramerization in preventing PVR.
To mimic the loss of cell contact observed in the PVR process and stimulate EMT, primary human fetal RPE (hfRPE) were seeded at 10% density. Expression of EMT markers, PKM2, and mediators of glycolytic reprogramming and proliferation were assessed. A continuous, enzyme-coupled assay that measures the depletion of NADH measured PK activity. Targeted metabolomics assessed the impact of ML-265 on the metabolic profile of hfRPE. The therapeutic potential of ML-265 in attenuating RPE EMT and PVR was evaluated in in vitro assays. ML-265 toxicity was assessed via transepithelial electrical resistance (TEER) and cell death markers.
Low seeding density of hfRPE demonstrated a fibroblastic-like PVR phenotype (hfRPE EMT) with induction of EMT markers α-SMA and NCAD. PKM2 but not PKM1 expression was increased. Yet, overall PK activity was decreased and the phosphoenolpyruvate (PEP) to pyruvate ratio was increased in hfRPE EMT suggesting PKM2 is in the low activity, dimeric state. ML-265 treatment increased PK activity >3-fold in hfRPE EMT. ML-265 induced a decrease in glycolytic intermediates and the expression of HIF1A, GLUT1, and PDK1in hfRPE EMT. ML-265 decreased the proliferation of EMT hfRPE cells and the expression of MYC and CCND1 as well as inhibited EMT hfRPE-mediated gel contraction with a reduction in α-SMA expression. Exposure to ML-265 did not reduce TEER or increase annexin or PI staining in differentiated hfRPE.
This study suggests that pharmacologically inducing PKM2 tetramerization may be an innovative therapeutic strategy for PVR.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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