Cytotoxic T lymphocytes (CTL) and natural killer cells (NK)-mediated elimination of tumor cells is mostly dependent on Granzyme B apoptotic pathway, which is regulated by the wild type (wt) p53 protein. Because TP53 inactivating mutations, frequently found in human tumors, could interfere with Granzyme B-mediated cell death, the use of small molecules developed to reactivate wtp53 function in p53-mutated tumor cells could optimize their lysis by CTL or NK cells. Here, we show that the pharmalogical reactivation of a wt-like p53 function in p53-mutated breast cancer cells using the small molecule CP-31398 increases their sensitivity to NK-mediated lysis. This potentiation is dependent on p53-mediated induction of autophagy via the sestrin-AMPK-mTOR pathway and the ULK axis. This CP31398-induced autophagy sequestrates in autophagosomes several anti-apoptotic proteins, including Bcl-XL and XIAP, facilitating Granzyme B-mediated mitochondrial outer membrane permeabilization, caspase-3 activation and Granzyme B- or NK cell-induced apoptosis. Together, our results define a new way to increase cytotoxic lymphocyte-mediated lysis of p53-mutated breast cancer cell, through a p53-dependent autophagy induction, with potential applications in combined immunotherapeutic approaches.

Aberrant Receptor Tyrosine Kinase (RTK) signaling allows cancer cells to modulate survival, proliferation and death, leading to tumorigenesis and chemoresistance. In leukemia, the RTK FMS-Related Tyrosine Kinase 4 (FLT4) (also known as VEGFR-3, Vascular Endothelial Growth Factor- 3) is deregulated and correlates with cancer progression. However, the underlying consequences of its deregulation remain to be determined. Moreover, chemotherapy treatment requires that cancer cells retain a wild type p53 (wt) in order to respond to DNA damage by tumor suppressing activities, i.e. apoptosis. p53 activity is predominantly limited by its two major negative regulators, MDM2 and MDMX, which inactivate p53 by promoting its degradation and/or cytoplasmic localization. In this study, we have shown that activation of FLT4 by either overexpression or binding of its ligand, VEGF-C, leads to an increase in MDM2/MDMX stability, inactivation of p53 and resistance to DNA damaging therapies. Through immunoprecipitation and mass spectrometry analysis, we observed that FLT4 induced phosphorylation of MDMX at Ser-314, a consensus sequence of CDK4/6. Our data revealed that phosphorylation of MDMX on Ser-314 increases the stability of MDMX, which subsequently affects MDM2 and p53 degradation and could be reversed by the CDK4/6 inhibitor Palbociclib. More importantly, leukemic cells treated with Palbociclib were more susceptible to DNA damaging induction of apoptosis and had reduced cell proliferation. Altogether, our research proposes an innovative way to reactivate p53 in pediatric leukemia through the pharmacological inhibition of FLT4 signaling, which could serve as a potential treatment option for this disease.