ID OF MOLECULES THAT SUBSTITUTE FOR ACTIVE NOTCH1 IN T LINEAGE LEUKEMOGENESIS

University Of California, San Francisco, San Francisco CA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. NOTCH1 encodes a transmembrane receptor that is critical for T cell development and is mutated in ~55% of human T lineage acute lymphoblastic leukemia (T-ALL) cases. Activation of the Notch1 protein requires cleavage by the gamma-secretase protease, which releases the Notch1 intracellular domain (NICD) that then translocates to the nucleus and upregulates transcription. Mutated forms of Notch1 are gamma-secretase dependent and several gamma-secretase inhibitors (GSIs) have been developed. We harnessed retroviral insertional mutagenesis (RIM) in Mx1-cre;LSL-KrasG12D mice to generate a large panel of genetically and biochemically diverse T-ALLs. In this model, Notch1 is frequently mutated and NICD can be detected using a specific antibody that recognizes Val 1477 that is exposed only after gamma-secretase cleavage. We have thoroughly interrogated Notch1 dependence in 20 tumor-derived cell lines and found that sensitivity to GSI correlates with the presence of NICD and GSI treatment reduces NCID levels. This work is currently in press at PNAS. Interestingly, we identified 3 cell lines that robustly express a protein recognized by the NICD specific antibody, but are GSI-resistant. This protein does not disappear following GSI exposure. Further analysis revealed that these lines do not express Notch1 mRNA or have upregulation of Notch1 target genes, indicating that Notch1 is not activated. Importantly, we never detect this protein and would like to use mass spectrometry to determine its identity. Future experiments would involve 1) using shRNA to knockdown the protein in T-ALL cells to assess its role in tumorigenesis, and 2) screening human T-ALL patient samples for mutations in the gene that encodes this protein.