p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5′-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.M utations in the TP53 tumor suppressor gene are the most frequent genetic alterations in human cancer and commonly compromise the gene's tumor suppressor activity. p53-knockout mice succumb to tumors very early in life, arguing that the loss of function associated with p53 mutations is sufficient on its own to explain the high mutation frequency observed in patients with cancer (1). However, in striking contrast to mutations in other tumor suppressor genes, the vast majority of TP53 gene alterations in patients with cancer neither ablate p53 expression nor produce unstable or truncated proteins. Instead, p53 mutations are mostly missense mutations resulting in expression of mutant p53 (mutp53) proteins with only single-amino acid substitutions that accumulate to steady-state levels greatly exceeding those of wild-type p53 (wtp53) in normal tissues. Immunohistochemical positivity for p53 is therefore considered a diagnostic marker for the presence of a TP53 mutation (2). The high prevalence of missense mutations suggests a selective advantage during cancer progression, so it was hypothesized early on in p53 research that p53 mutations are neomorphic and endow the mutp53 protein with novel oncogenic functions that actively promote cancer progression and therapy resistance (2). These oncogenic properties are generally referred to as the mutp53 gain of function (GOF).Over the years, substantial experimental evidence for mutp53 GOF has accumulated (3-5). For example, mice expressing cancer-associated p53 hot spot mutations from the endogenous Trp53 gene locus are remarkably different from p53-deficient mice: tumorigenesis is accelerated, and the spectrum of tumors is shifted toward carcinomas and more meta...

Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene ()-rearranged ALL were established in NOD.Cg-Prkdc Il2rg/SzJ (NSG) mice. Antibody CD19-DE was efficient in prolonging the survival of NSG mice in a minimal residual disease (MRD) model. The majority of surviving mice remained polymerase chain reaction (PCR)-MRD negative after treatment. When antibody therapy was initiated in overt leukemia, antibody CD19-DE was still efficient in prolonging survival of xenografted mice in comparison with nontreated control animals, but the effects were less pronounced than in the MRD setting. Importantly, the combination of antibody CD19-DE and cytoreduction by chemotherapy (dexamethasone, vincristine, PEG-asparaginase) resulted in significantly improved survival rates in xenografted mice. Antibody CD19-DE treatment was also efficient in a randomized phase 2-like PDX trial using 13 -rearranged BCP-ALL samples. Macrophage depletion by liposomal clodronate resulted in a reversal of the beneficial effects of CD19-DE, suggesting an important role for macrophages as effector cells. In support of this finding, CD19-DE was found to enhance phagocytosis of patient-derived ALL blasts by human macrophages in vitro. Thus, Fc-engineered CD19 antibodies may represent a promising treatment option for infants and children with-rearranged BCP-ALL who have a poor outcome when treated with chemotherapy only.

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