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.

Sequence comparison of Francisella tularensis LVS, LVS-G and LVS-R.

Francisella tularensis is a gram-negative organism found in many regions of the world. F. tularensis can cause a fatal, febrile illness, although these natural tularemia infections are rare in the United States. However, the development of F. tularensis as a potential weapon of bioterrorism during the Cold War spurred the development of a live attenuated vaccine, LVS, from F. tularensis subsp. holarctica in the 1960s. Two colony morphology variants, LVS-G and LVS-R, were generated from parental LVS by plate passage and by acridine orange mutagenesis, respectively. In vaccinated mice, LVS-G and LVS-R exhibit altered immunogenicity and protective capacities. While the exact nature of the mutations in these strains was unknown, previous studies indicated that both had altered lipopolysaccharide structures. To better understand the impact of these mutations on LVS' immunogenicity, we sequenced the genomes of LVS-G and LVS-R as well as our parental laboratory stock of LVS, originally obtained from ATCC, and compared these to the F. tularensis subsp. holarctica LVS genome currently deposited in GenBank. The results indicate that the genomic sequence of ATCC LVS is nearly identical to that of the human LVS vaccine. Furthermore, a limited number of genomic mutations likely account for the phenotypes of LVS-G and LVS-R.

Distinct Patterns of Expression of Transcription Factors in Response to Interferonß and Interferonλ1.

After viral infection, type I and III interferons (IFNs) are coexpressed by respiratory epithelial cells (RECs) and activate the ISGF3 transcription factor (TF) complex to induce expression of a cell-specific set of interferon-stimulated genes (ISGs). Type I and III IFNs share a canonical signaling pathway, suggesting that they are redundant. Animal and in vitro models, however, have shown that they are not redundant. Because TFs dictate cellular phenotype and function, we hypothesized that focusing on TF-ISG will reveal critical combinatorial and nonredundant functions of type I or III IFN. We treated BEAS-2B human RECs with increasing doses of IFNß or IFNλ1 and measured expression of TF-ISG. ISGs were expressed in a dose-dependent manner with a nonlinear jump at intermediate doses. At subsaturating combinations of IFNß and IFNλ1, many ISGs were expressed in a pattern that we modeled with a cubic equation that mathematically defines this threshold effect. Uniquely, IFNß alone induced early and transient IRF1 transcript and protein expression, while IFNλ1 alone induced IRF1 protein expression at low levels that were sustained through 24 h. In combination, saturating doses of these 2 IFNs together enhanced and sustained IRF1 expression. We conclude that the cubic model quantitates combinatorial effects of IFNß and IFNλ1 and that IRF1 may mediate nonredundancy of type I or III IFN in RECs.