The high rate of recurrence and treatment-related mortalities in pediatric acute myeloid leukemia (AML) under- scores the critical need to develop targeted therapies. As one of the most frequently mutated genes in cytoge- netically normal AML, nucleophosmin (NPM1) represents a promising target. Normally a nucleolar protein, mu- tation of NPM1 leads to mislocalization in the cytoplasm. NPM1 has multiple functions that safeguard from cancer, while the mutant form is associated with increased chemosensitivity. The applicant previously reported that NPM1 activates the pro-apoptotic protease and tumor suppressor, caspase-2 in the nucleolus. Preliminary data from the applicant?s laboratory indicates rather than inducing apoptosis, the nucleolar NPM1-dependent caspase-2-activating complex impedes cell division and may even facilitate cell survival. In contrast, apoptosis in NPM1 mutant AML cells is dependent on caspase-2. This suggests that a caspase that has long been con- sidered to be pro-apoptotic has both pro-apoptotic and pro-survival functions depending on where NPM1 is localized in the cell. The central hypothesis is that NPM1-mediated regulation of caspase-2 is a critical node in the regulation of AML cell death and survival, providing a key determinant of chemosensitivity. This hypothesis will be tested by pursuing three specific aims: 1) Determine how NPM1 activates caspase-2 to modulate sensi- tivity to apoptosis and cell cycle arrest in AML; 2) Determine how altering the localization of caspase-2 activation impacts resistance to chemotherapy in AML; and 3) Determine the therapeutic potential of targeting the caspase- 2-NPM1 pathway in AML. Under the first aim, CRISPR/Cas9 will be used to delete caspase-2 to determine the effects of blocking caspase-2 on DNA damage-induced apoptosis and cell cycle arrest in the NPM1 wild type and mutant subtypes. Under the second aim, cells will be engineered to switch the localization of caspase-2 activation from the nucleolus to the cytoplasm or from the cytoplasm to the nucleolus. The impacts of chemo- sensitivity and resistance will be measured. Under the third aim, the impact of inhibiting this pathway on leukemia engraftment, progression and chemosensitivity of AML xenografts will be measured. These studies will provide important mechanistic insights into the role of NPM1 in modulating AML chemosensitivity. Ultimately, these stud- ies are expected to identify targets in this pathway that reduce leukemia burden, providing candidates for rational drug design for AML therapies.
Nucleophosmin is one of the most frequently mutated genes in AML and the mutant form is associated with increased sensitivity. The proposed research is relevant to public health because it will determine how activation of the pro-apoptotic caspase-2 by nucleophosmin leads to cell death or survival depending on where in the cell it is activated and if these functions can be reversed by modulating the localization of caspase-2 activation. Thus, the proposed research is relevant to part of the mission of the National Cancer Institute (NCI) that pertains to stimulating research in the interplay between cell death pathways in nave and drug resistant cancers.