B016 - Inhibition of HDAC6 and ROCK Kinase Impairs Macrophage Migration and Proliferation

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Author Block: M. M. Kloc¹, M. Halasa², A. Wawruszak³, K. Kapusta⁴, B. Walker⁵, R. M. Ghobrial⁶, ¹THMRI, Houston, TX, ²Transplant Immunology, Houston Methodist Research Institute, Houston, TX, ³U of Lublin, Lublin, Poland, ⁴Tougaloo College, Tougaloo, MS, ⁵UT Health, Houston, TX, ⁶Houston Methodist Hospital, Houston, TX
*Purpose: Macrophage-driven chronic (long-term) rejection of organ allografts remains an unsolved challenge in clinical transplantation. Chronic rejection is mostly mediated by macrophages. The resident and recruited macrophages potentiate fibrosis by interacting with fibroblasts and secreting profibrotic factors, ultimately leading to organ rejection. Macrophage infiltration of the allograft depends on the reorganization of their cytoskeleton, which consists of actin filaments, intermediate filaments, and microtubules. The dynamics of actin filaments, which influence macrophage motility, phenotype, and inflammatory properties, are controlled by Rho-associated coiled-coil containing protein kinases (ROCK 1 and 2). ROCKs are the downstream effectors of small GTPase RhoA, which is reciprocally regulated by the mTOR pathway. Indeed, our previous research showed that pharmacological inhibition of ROCKs (in combination with an mTOR inhibitor) prevents macrophage infiltration into the grafts and thus counteracts chronic rejection of rat cardiac transplants. Similarly, to ROCKs, another cytosolic protein, histone deacetylase 6 (HDAC6), is essential for macrophage infiltration during inflammation. HDAC6 is involved in cell motility and filopodia formation by regulating microtubule and cortactin acetylation levels. Filopodia are indispensable for macrophage movement, serving as sensory and mechanical guides for the interaction with the substrate.
*Methods: Here, we show, for the first time, that simultaneous inhibition of HDAC6 and ROCK kinase impairs macrophage migration and proliferation.
*Results: Macrophage treatment with the FDA-approved, selective inhibitors Ricolinostat (HDAC6i) or Rezurock (ROCKi) altered macrophage phenotype, reduced filopodia, and induced G1/S cell cycle arrest and early apoptosis. These changes were caused by dysregulation of the mTORC2 complex, leading to downregulation of Akt phosphorylation and, eventually, affecting its targets. Another protein important for filopodia formation is Src kinase. Our research showed that Rezurock alone and in combination with Ricolinostat significantly decreased Src activity by downregulating phosphorylation of tyrosine 416 and upregulating phosphorylation of tyrosine 527, with the strongest effect observed with combined drug treatment.
*Conclusions: Our research confirms that combined treatment significantly alters macrophage properties and could thus be an effective strategy for reducing fibrosis in post-transplantation patients and other, fibrotic-related diseases.