Dhanoop Manikoth Ayyathan

A-lamins, encoded by the LMNA gene, are major structural components of the nuclear lamina coordinating essential cellular processes. Mutations in the LMNA gene and/or alterations in its expression levels have been linked to a distinct subset of human disorders, collectively known as laminopathies, and to cancer. Mechanisms regulating A-lamins are mostly obscure. Here, we identified E3 ubiquitin ligase Smurf2 as a physiological regulator of lamin A and its disease-associated mutant form progerin (LAΔ50), whose expression underlies the development of Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging syndrome. We show that Smurf2 directly binds, ubiquitinates, and negatively regulates the expression of lamin A and progerin in Smurf2 dose- and E3 ligase-dependent manners. Overexpression of catalytically active Smurf2 promotes the autophagic-lysosomal breakdown of lamin A and progerin, whereas Smurf2 depletion increases lamin A levels. Remarkably, acute overexpression of Smurf2 in progeria fibroblasts was able to significantly reduce the nuclear deformability. Furthermore, we demonstrate that the reciprocal relationship between Smurf2 and A-lamins is preserved in different types of mouse and human normal and cancer tissues. These findings establish Smurf2 as an essential regulator of lamin A and progerin and lay a foundation for evaluating the efficiency of progerin clearance by Smurf2 in HGPS, and targeting of the Smurf2-lamin A axis in age-related diseases such as cancer.

Mutant isocitrate dehydrogenase 1 (IDH1) and IDH2 block the differentiation of acute myeloid leukemia (AML) cells through production of R-2-hydroxyglutarate (R-2-HG). IDH inhibitors can induce differentiation of AML cells by lowering R-2-HG but have limited clinical efficacy as single agents. Here, we performed a genome-wide CRISPR knockout screen in an Idh1-mutated hematopoietic progenitor cell line to identify genes that increased the differentiation response to ivosidenib, an IDH1 inhibitor. The screen identified C-type lectin member 5a (Clec5a), which encodes a spleen tyrosine kinase (SYK)-coupled surface receptor, as one of the top hits. Knockout of Clec5a and Syk rendered cells more sensitive to ivosidenib-induced differentiation through a reduction in STAT5-dependent expression of stemness-related genes, including genes in the homeobox (HOX) family. Importantly, direct inhibition of STAT5 activity was sufficient to increase the differentiation response to IDH inhibitors in primary human IDH1- and IDH2-mutated AML cells, including those harboring mutations in receptor tyrosine kinase (RTK) and MAPK genes that have been linked to drug resistance. In patient-derived xenograft models of IDH1-mutated AML, combination treatment with ivosidenib and the STAT5 inhibitor pimozide was superior to each agent alone in inducing differentiation in leukemic cells without compromising normal hematopoiesis. These findings demonstrate that STAT5 is a critical mediator of resistance to IDH inhibitors and provide the rationale for combining STAT5 and IDH inhibitors in the treatment of IDH-mutated AML. SIGNIFICANCE: A CRISPR knockout screen identifies a mechanism of resistance to IDH inhibitors in AML involving activated STAT5 signaling, suggesting a potential strategy to improve the clinical efficacy of IDH inhibitors.