Targeting Salt-Inducible Kinase 3 As a Therapeutic Approach for Acute Myeloid Leukemia
Authors: Lin Shan , Tarumoto Yusuke , Wang Jinhua , Milazzo Joseph , Xu Yali , Gray Nathanael , Vakoc Christopher , Stegmaier Kimberly 
Published: November 2019

Acute myeloid leukemia (AML) remains a devastating illness, with a clear need for the development of novel anti-leukemic therapy. CRISPR-Cas9 functional genomic screens have revealed hundreds of genetic vulnerabilities in AML cells, which can be leveraged to nominate clinically actionable targets. Our previous CRISPR screens in AML cell lines identified Salt-Inducible Kinase 3 (SIK3) as an essential gene for a subset of AML cells with high expression of the oncogenic transcription factor MEF2C, such as AMLs harboring Mixed Lineage Leukemia (MLL)-fusion proteins. Mechanistically, we have shown that the maintenance of MEF2C transcriptional output requires SIK3, which phosphorylates histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C, and thus prevents HDAC4 from nuclear entry and from suppressing MEF2C activity.
SIKs 1-3 are serine/threonine kinases belonging to the adenosine monophosphate-activated protein kinase (AMPK) family. The physiological function of SIKs has been investigated in the regulation of gluconeogenesis, myogenesis and osteogenesis, yet the critical role of SIK3 in AML growth is just emerging. Thus, the evaluation of its translational potential in relevant pre-clinic models, such as patient-derived-xenografts (PDXs), is necessary. To do so, we have established a novel MLL-AF9 PDX model tractable for CRISPR genome editing via lentivirally expressing Cas9 and serial transplantation in immunodeficient mice. The PDX model maintains stable Cas9 activity; knockout of SIK3 hampered leukemia growth in vivo, demonstrating the utility of this model and the functional significance of SIK3 in AML.
In parallel, we tested the anti-leukemic effect of the tool compound YKL-05-099, which inhibits the SIK kinase family and has a suitable bioavailability in mice. YKL-05-099 treatment inhibited the growth of AML cell lines in vitro via inducing cell cycle arrest and apoptosis, and AML cells with high MEF2C protein expression tended to be more sensitive to YKL-05-099. YKL-05-099 was well-tolerated by mice, and after daily administration for a month, no obvious toxicities or physiological abnormalities were observed. Normal hematopoiesis was also sustained, as indicated by complete blood counts and lineage flow cytometry analysis. By contrast, a two-week treatment regimen successfully attenuated disease progression in vivo and extended animal survival in both an MLL-AF9 mouse AML and PDX models. Importantly, we have confirmed the action of the compound is via on-target inhibition of the SIK3-HDAC4-MEF2C axis. Western-blot analysis revealed that YKL-05-099 treatment led to rapid dephosphorylation and nuclear accumulation of HDAC4 in AML cells. Accordingly, similar to SIK3 knockout, ChIP-seq analysis showed that YKL-05-099 selectively reduced histone H3 lysine 27 acetylation at a small number of genomic locations, which are enriched for MEF2C occupancy sites. This finding was supported by RNA-seq analysis as well, which showed that YKL-05-099 treatment suppressed a similar transcriptional program as observed following genetic targeting of SIK3 or MEF2C. Additionally, either overexpressing a SIK3 gatekeeper mutant or deleting HDAC4 in MLL-fusion AML cells can attenuate their sensitivity to YKL-05-099.
High MEF2C expression in AML is associated with poor response to conventional chemotherapy. Our findings validate SIK3 inhibition as a possible therapeutic approach in MEF2C-positive AML and provide a rationale for developing improved drug-like inhibitors of SIK3, with enhanced specificity and bioactivity, for definitive pre-clinical investigation and for future studies in patients with AML.

Disclosures
Lin: Leukemia & Lymphoma Society: Research Funding. Tarumoto:Lauri Strauss Leukemia Foundation: Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Vakoc:Boehringer-Ingelheim: Research Funding; KSQ Therapeutics: Consultancy. Stegmaier:Rigel Pharmaceuticals: Consultancy; Novartis: Research Funding.