Deep PIM kinase substrate profiling reveals new rational co-therapeutic strategies for acute myeloid leukemia

Provirus integration site for Moloney murine leukemia virus (Pim) family serine/threonine kinases perform pro-tumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of Pim kinase functions is currently lacking. Multiple small molecule Pim kinase inhibitors are currently being evaluated as co-therapeutics in Cancer patients. To further illuminate Pim kinase functions in Cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative Pim substrates nominated RNA splicing and rRNA processing as PIM-regulated cellular processes. Pim inhibition elicited reproducible splicing changes in PIM-inhibitor-responsive acute myeloid leukemia (AML) cell lines. Pim inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. Pim inhibition also altered rRNA processing, and Pim inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic co-therapeutic strategies. This approach may expand the co-therapeutic armamentarium to overcome kinase-inhibitor resistant disease that limits durable responses in malignant disease.