Pharmacological PIK3C2B inhibition rescues XLMTM phenotype in mouse models and identifies molecular markers of disease
Andrew Shearer, Melissa L. Brooks, Maxine M. Chen, Thiwanka Samarakoon, John Hsieh, Gramoz Kondakci, Emanuele Perola, Jason Brubaker, Kristina Fetalvero, Stefanie Schalm, Joana Caetano-Lopes
Andrew Shearer, Melissa L. Brooks, Maxine M. Chen, Thiwanka Samarakoon, John Hsieh, Gramoz Kondakci, Emanuele Perola, Jason Brubaker, Kristina Fetalvero, Stefanie Schalm, Joana Caetano-Lopes
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Research Article Development Genetics Muscle biology

Pharmacological PIK3C2B inhibition rescues XLMTM phenotype in mouse models and identifies molecular markers of disease

Abstract

X-linked myotubular myopathy (XLMTM) is a rare genetic disorder that typically presents at birth with progressive muscle weakness and respiratory difficulties and is caused by myotubularin 1 (MTM1) gene mutations. Here, we examine the role of phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2-β (PIK3C2B), a lipid kinase that interacts with MTM1, in XLMTM in various models. We examined the effect of BLU3797, a highly potent, selective, orally bioavailable PIK3C2B inhibitor, on survival, muscle development, myofiber phenotypes, and gene expression in MTM1–/y mice. PIK3C2B-deficient XLMTM animals demonstrated increased survival, restored muscle function, fewer myofibers with centralized nuclei, and normalization of disease-associated molecular markers. BLU3797 alleviated the XLMTM phenotype in a dose-dependent and reversible manner. Loss of functional PIK3C2B in XLMTM mice promoted a more differentiated, adult-like myofiber profile, which was strongly associated with normalization of disease surrogates and a reduction in markers of early muscle development and regeneration. BLU3797 treatment appears to modulate the expression of miRNAs associated with satellite cell activation and myofiber fusion. These findings indicate that PIK3C2B inhibition with BLU3797 effectively reverses the XLMTM disease phenotype by enhancing muscle function and promoting development toward a more mature state.

Authors

Andrew Shearer, Melissa L. Brooks, Maxine M. Chen, Thiwanka Samarakoon, John Hsieh, Gramoz Kondakci, Emanuele Perola, Jason Brubaker, Kristina Fetalvero, Stefanie Schalm, Joana Caetano-Lopes

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Figure 7

PIK3C2B inhibition alters miRNA expression and decreases muscle SFRP1 protein levels.

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PIK3C2B inhibition alters miRNA expression and decreases muscle SFRP1 pr...
(A) Survival of MTM1–/y mice treated with BLU3797 at the indicated number of times following a once every 36 hours dosing schedule. Arrowhead represents start of the study. (B) Clustering of treatment groups by differential miRNA expression. (C) Validation of select miRNAs by quantitative real-time PCR (qRT-PCR). All sample values are normalized to Rnu1a1 expression and relative expression in WT mice treated with BLU3797. (D and E) qRT-PCR analysis of Sfrp1 and Smad1 levels normalized to Gapdh and expression in WT mice treated with BLU3797. (F) SFRP1 protein levels from MTM1–/y mouse muscle treated with and without BLU3797. (G) qRT-PCR of Sfrp1 expression in WT C2C12 cells or deficient for MTM1 with BLU3797 treatment (final 48 hours of 5-day protocol); values normalized to Gapdh and WT untreated cells. (H) Western blot of SMAD1 and total actin in lysates from the muscle of MTM1–/y mice. (I) Quantification of SMAD1 levels shown in H; values are normalized to actin and protein levels in WT mice treated with BLU3797. In box-and-whisker plots, median (line), mean (symbol), 25th–75th quartile range (box), and minimum and maximum (whiskers) are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Comparisons of select miRNAs across BLU3797 dosing regimens were done with an unpaired 2-tailed t test. Comparisons of SFRP1 protein levels were made using Dunnett’s correction.