RKI-1447, a ROCK Inhibitor, Targets SRSF2 Mutated Leukemia by Disrupting Cell Mitosis and Nuclear Morphology

Abstract

Spliceosome machinery mutations are common early mutations in myeloid malignancies, however effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 Mut primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation, that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447 treated SRSF2 Mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.

Note: Funding Information: This work by the authors is supported through National Natural Science Foundation of China (81861148029, 81730006, 81890990), National Key R&D Program of China (2020YFE0203000, 2021YFA1100900), CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-040), CAMS Fundamental Research Funds for Central Research Institutes (3332021093), the EU horizon 2020 grant project MAMLE ID: 714731, LLS and rising tide foundation Grant ID: RTF6005-19, ISF-NSFC 2427/18, ISF-IPMP-Israel Precision Medicine Program 3165/19, ISF 1123/21, BIRAX 713023, the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, awarded to LIS. LIS is an incumbent of the Ruth and Louis Leland career development chair. N.K. is an incumbent of the Applebaum Foundation Research Fellow Chair. This research was also supported by the Sagol Institute for Longevity Research, the Barry and Eleanore Reznik Family Cancer Research Fund, Steven B. Rubenstein Research Fund for Leukemia and Other Blood Disorders, the Swiss Society Institute for Cancer Prevention Research at the Weizmann Institute and the Applebaum Foundation. Work in the Papapetrou lab was supported by US National Institutes of Health (NIH) grants R01HL137219 and R01CA225231, the Leukemia and Lymphoma Society and the Edward P Evans Foundation.

Declaration of Interests: Authors declare that they have no competing interests.

Ethical Approval Statement: For human samples, primary peripheral blood samples were obtained with informed consent from AML patients through the Leukemia Tissue Bank at Princess Margaret Cancer Centre in accordance with regulated procedures approved by the Research Ethics Board of the University Health Network (REB 01-0573-C). All patients provided written informed consent for the usage of their samples for research purposes and for the usage of their clinical and biological data.

For murine studies, mice were derived germ-free and housed at the Weizmann Institute of Science animal facility under institutional animal care and use committee approval (11790319-2).

Keywords: SRSF2, Cytoskeleton, ROCK, Nuclear morphology