Polyploidy Is Necessary for Apoptotic Cell Death in TP53-Mut AML in Response to Polo-like-Kinase 4 (PLK4) Inhibition and Results in Caspase 3 Cleavage

Abstract

Background Approximately twenty percent of patients with newly diagnosed acute myeloid leukemia (AML) have TP53 mutations, associated with complex karyotype and the highest risk of relapse(Döhner et al., 2017; Giacomelli et al., 2018). Although combination therapies, such as Ara-C + anthracyclines and Venetoclax (VEN) + hypomethylating agents (HMA) result in higher remission rates in patients with TP53-mutant AML, the mutant clone is never eliminated and invariably leads to relapse(Short et al., 2021; Takahashi et al., 2016). Novel targets and mechanisms associated with TP53-mut AML need to be identified. Results: We performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations of TP53-mutant or TP53-wild-type (wt) high-risk AML patients before and after treatment. Amongst overexpressed genes, we identified Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis and therefore of polyploidy, as a potential target highly expressed in TP53-mutant AML using differential gene expression analysis of TP53-wt vs TP53-mutant samples collected (TP53-mutant=36, TP53-wt=31, PLK4 LOG2FC=~2, p<0.001). We validated this finding using an independent RNA-seq dataset from the Munich Leukemia Lab of 726 AML samples (TP53-mutant=72, TP53-wt=654, PLK4 LOG2FC=0.48, p<0.001) and investigated the impact of PLK4 expression levels on AML patient outcomes in the context of TP53-status. High levels of PLK4 expression in AML are associated with poor outcomes (Figure 1). Targeting PLK4 by CFI-400945 (25nM, Treadwell Therapeutics, Toronto, Canada) in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 for 72 hours resulted in increased polyploid cells in TP53-mut cells compared to TP53-wt AML (> 2 fold, p< 0.0001). PLK4 inhibition triggered higher levels of cleaved caspase 3 in polyploid TP53-mut polyploid MOLM-13 cells compared to TP53-wt MOLM-13 cells (Figure 2). Polyploidization in TP53-mut MOLM-13 cells 48 hours post treatment with 15nM of PLK4 inhibitor was not reversible and retained higher apoptogenic activity in TP53-mut MOLM-13 cells even after wash out for additional 48 hours. To translate our findings to in vivo models, we transplanted CRISPR-generated TP53-wt, TP53-ko, and TP53-248 mutant MOLM-13 cells (Boettcher et al., 2019) into NSG mice (n=58: untreated n= 28, treated n=30), confirmed successful engraftment, and treated the mice with CFI-400945 (oral gavage 7.5 mg/kg, 5 times/week) for 21 days. Our preliminary data indicates that all treated mice showed improved survival compared to untreated mice. The median survival for treated TP53-wt vs untreated TP53-wt was 17.5 vs 14.5 days (20.7% increase, n=20 (untreated n=10, treated n=10), p=0.00086). The median for treated TP53-R248Q vs untreated TP53-R248Q was 15.0 vs 12.0 days respectively (25% increase, n=19 (untreated n=9, treated n=10), p=0.00039). The median OS for treated TP53-ko vs untreated TP53-ko was 11 vs 8 days respectively (37.5% increase, n=19 (untreated n=9, treated n=10), p<0.0001). Next, we performed a Cox proportional hazard model to investigate the contribution of TP53 genotype and PLK4 inhibition on overall survival. The resulting model (Likelihood ratio test=83.09 on 3 degrees of freedom and p<2.2e-16) showed a higher hazard ratio in recipients with TP53-R248Q and TP53-ko (~4.3, and ~1.2 hazard coefficients respectively) in comparison to TP53-wt (p=2.67e-14, and 0.000708 respectively). Additionally, treated mice had statistically significant lower hazard ratios compared to untreated (hazard coefficients=-2.47277 and p=8.38e-10). The data suggest that PLK4 inhibition is a promising treatment approach for AML with TP53 mutation. We are presently testing different dosing schedules in other aggressive TP53-mut AML models to further ascertain the efficacy of PLK4 inhibition. Conclusion: We established an association between PLK4 overexpression and poor survival outcomes in patients with newly diagnosed AML. TP53-mutant AML express higher levels of PLK4 in comparison to TP53-wt AML and targeting PLK4 induces polyploidy and apoptotic cell death predominantly in TP53-mutant AML. We demonstrate that targeting of PLK4 improves overall survival in TP53-mut and TP53-ko AML cells in vivo. Data suggest that targeting PLK4 represents a potential treatment approach for TP53-mut AML. An early phase, multi-center clinical trial is ongoing with CFI-400945 in AML (NCT04730258, TWT-202). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal