Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

The function of wild-type (wt) p53 in acute myeloid Leukemia (AML) is suppressed by MDM2, MDM4 and XPO-1 (Andreeff et al, Exp Hematol, 2016). We propose that wt p53 protein misfolding and cytosolic localization are contributing to its inactivation in AML. Immunofluorescence staining with OpalR TSA amplification demonstrated that p53 is localized both in the nucleus and in the cytosol of AML cells with prominent para-nuclear accumulation. We show here that misfolded wt p53 is localized mainly in the cytoplasm of AML cells, similar to what we reported for mutant (mt) p53 previously (Zeng et al, Blood, 2016). p53 misfolding promotes its aggregation which was recently reported as a novel mechanism promoting loss of its anti-tumor functions (Xu et al, Nat Chem Biol, 2011; Soragni et al, Cancer Cell, 2016). A pro-aggregating segment in the p53 DNA binding domain is exposed when p53 is misfolded. We showed that ReACp53, a cell permeable peptide designed to inhibit the aggregation of this segment, induced apoptosis in ovarian cancers bearing mt p53 (Soragni et al, Cancer Cell, 2016). We also reported that wt p53 AML cells responded to ReACp53 treatment (Zeng et al, Blood, 2016). ReACp53 eliminated misfolded p53, promoted its mitochondrial translocation and induced rapid apoptosis, suggesting that cytoplasmic misfolded wt p53 is a novel target in AML. MDM2 promotes p53 degradation, and inhibitors of MDM2 such as Nutlin derivatives are currently in trials for AML. These molecules inhibit p53 proteasomal degradation and result in p53-mediated apoptosis, as we demonstrated pre-clinically and in a Phase I trial of RG7112 in AML (Andreeff et al, Clin Cancer Res, 2015). p53 aggregation is initiated by protein misfolding, and progresses with increasing accumulation of misfolded p53. While p53 degradation is promoted by MDM2, binding of MDM2 to p53 causes p53 to misfold (Sasaki et al, J Biol Chem, 2007). This raises concerns about induction of p53 misfolding and consequent aggregation in tumors treated with MDM2 inhibitors, which could diminish therapeutic efficacy. We observed that levels of total and misfolded p53 and protein aggregation as identified by Proteostat positivity were MDM2 inhibitors dose- and time-dependent in wt p53 AML cells. This supports the hypothesis that MDM2 inhibition can cause not only p53 misfolding but also aggregation. Consequently, we show that adding a p53-aggregation inhibitor such as ReACp53 to an MDM2 inhibitor to limit p53 misfolding and aggregation results in increased cytotoxic activity in wt p53 AML. Co-aggregation of mt p53 with p63/p73 proteins carrying similar pro-aggregating segments has been reported (Xu et al, Nat Chem Biol, 2011). Next, we tested whether coaggregation could be an additional factor sequestering and inactivating wt p53. High levels of ΔNp73α, a tumor-promoting isoform of p73, can antagonize p53 function possibly through hetero-tetramer formation (Coutandin et al, Cell Death Differ, 2009), resulting in chemoresistance (Kazushi et al, Subcell Biochem, 2014). We hypothesize that upregulated ΔNp73α could constrain wt p53 through protein co-aggregation causing inactivation. Increased levels of misfolded p53 and protein aggregation were detected in both ΔNp73α-overexpressing HEK293T and MOLM13 (M13) cells. ΔNp73α-overexpressing M13 cells were resistant to MDM2 inhibitor-induced apoptosis compared to controls but sensitive to ReACp53. Treatment with Nutlin-derivatives (RG7388 or DS3032b) did not alter ΔNp73α levels but caused dose- and time-dependent increases in total and misfolded p53 and protein aggregation. HEK293T and M13 cells overexpressing ΔNp73α had higher levels of misfolded and aggregated p53, which we interpret as ΔNp73α providing a "seed" to accelerate p53 co-aggregation due to MDM2 inhibition. This suggests that ΔNp73α-overexpression conferred resistance to MDM2-mediated apoptosis that could be overcome by inhibition of p53 aggregation. Thus, combination of Nutlin derivatives and ReACp53 treatment exerted enhanced cytotoxicity in both cells lines. In conclusion, our data supports cytoplasmic, misfolded wt p53 as a novel target in AML and offers a rationale to combine therapeutic approaches supplementing MDM2 inhibition with p53 aggregation-targeting molecules to increase effectiveness. The model of wt p53 aggregation and coaggregation induced by MDM2 inhibition may apply to other cancer types. Disclosures Andreeff: Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Ishizawa:Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed.