https://openworks.mdanderson.org/sumexp22/1020/thumbnail.jp

Andreeff, Michael, MD, PhD

Ayoub, Edward, PhD

Haferlach, Torsten, MD, PhD

Marupudi, Akhil S

Nishida, Yuki, MD, PhD

Walter, Wencke, PhD

English

OpenWorks @ MD Anderson

TARGETING POLO-LIKE KINASE 4 (PLK4) TRIGGERS POLYPLOIDY AND APOPTOSIS IN TP53-MUTANT ACUTE MYELOID LEUKEMIA AND RESULTS IN IMPROVED SURVIVALAkhil Marupudi1,2, Edward Ayoub2, Yuki Nishida2, Wencke Walter3, Torsten Haferlach3, Michael Andreeff2  1Department of Biomedical Sciences, Texas A&M University, 2Department of Leukemia, The University of Texas MD Anderson Cancer Center, 3MLL Munich Leukemia Laboratory Funding: This work was funded by The University of Texas MD Anderson AML Moonshot project. Acknowledgements: Many thanks to Marites Melancon, Chandra Bartholomeusz, and Nancy Strange for their organization of the MD Anderson Cancer Center Summer Experience and Summer Undergraduate Research Program (SURP).  PLK4 inhibition results in higher polyploidy in TP53-mut vs TP53-wt MOLM13A.                                        B.C.                                        D.Figure 4: TP53-wt and TP53-mut MOLM13 cell lines were treated with 25nM CFI-400945 for 72 hours. Ploidy status shown in A) TP53-wt, B) TP53-ko, C) TP53-R248Q, and D) TP53-R273H.Polyploid TP53-mut MOLM13 cells have increased levels of cleaved Caspase-3                Figure 5: Monitoring DNA content with the Click-iT EdU labeling. Pseudocolor plots showing the levels PI and Edu levels in TP53-wt, TP53-KO, and TP53-R234Q MOLM13 cell lines. Left column shows untreated samples. Right column shows samples treated with 25nM CFI-400945 for 48 hours. Color axis represents the levels of cleaved Caspase-3. TP53-mut PDX have worst OSFigure 6: Survival analysis performed on three different PDX models. TP53-WTTP53-KOTP53-R248QUntreated 25nM PLK4i (48hrs)p = 8.9e-04 p = 3.9e-04BULK LSC0246MDACC AML MoonshotTP53−wtTP53−mutTP53−wtTP53−mutp = 3.5e−05234567TP53−wtTP53−mutPLK4 normalized RNA expressionMunich Leukemia LaboratoryBULKPLK4 normalized RNA expressionTreatmentGenotypeTreatedUntreatedTP53−R248QTP53−KOTP53−WT(N=30)(N=28)(N=19)(N=19)(N=20) 0.084reference 3.30373.828reference( 0.038 −   0.19)( 1.654 −   6.59)(24.394 − 223.44)<0.001 ***<0.001 ***<0.001 ***# Events: 58; Global p−value (Log−Rank): 6.653e−18 AIC: 283.82; Concordance Index: 0.89 0.05 0.1 0.5 1 5 10 50 100 500Hazard ratiop = 0.000390.000.250.500.751.000 3 6 9 12 15 18DaysOverall Survival ProbabilityTP53-R248Q Treated TP53-R248Q Untreated10 10 10 10 10 8 19 9 9 9 7 1 0TP53-R248Q UntreatedTP53-R248Q TreatedNumber at riskp = 0.000860.000.250.500.751.000 3 6 9 12 15 18DaysOverall Survival ProbabilityTP53-wt Treated TP53-wt Untreated10 10 10 10 10 10 510 10 10 10 10 5 0TP53-wt UntreatedTP53-wt TreatedNumber at riskp < 0.00010.000.250.500.751.000 3 6 9 12 15 18DaysOverall Survival ProbabilityTP53-ko Treated TP53-ko Untreated10 10 10 10 4 0 09 9 9 3 0 0 0TP53-ko UntreatedTP53-ko TreatedNumber at riskp < 0.00010.000.250.500.751.000 3 6 9 12 15 18 21 24DaysOverall Survival ProbabilityGenotype=TP53−KO Genotype=TP53−R248Q Genotype=TP53−WT9 9 9 9 9 9 0 0 09 9 9 9 9 9 9 1 010 10 10 10 10 10 10 8 1Genotype=TP53−WTGenotype=TP53−R248QGenotype=TP53−KONumber at riskBackground• TP53 mutations in acute myeloid leukemia (AML) are associated with complex karyotype and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018). The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and investigating novel mechanisms is critical to develop more effective therapies.• In order to shed light on the defective p53 signaling pathways underlying TP53 mutant AML, we performed RNA-sequencing (RNA-seq) on bulk mononuclear cells or FACS-sorted leukemic stem cells (LSCs) using samples collected from TP53-mutant or TP53-wt high-risk AML patients. • We identified a key regulator of centriole biogenesis: Polo-like kinase 4 (PLK4) as a potential target highly expressed in TP53-mutant AML samples. • Previous publications showed that PLK4 is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Fischer et al., 2014; Li et al., 2005). Here we show that TP53-mutant AML samples lack the p53-dependent PLK4 repression and have higher levels of PLK4 compared to TP53-wt AML.• Gap of knowledge: The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and there are no effective treatments against TP53-mut AML.• We hypothesized that targeting PLK4 will triger mitotic defects, and activate apoptosis in TP53-mut leukemia cells, making it a potential treatment approach for TP53-mut AML. Materials and MethodsRNA sequencing datasets:MD Anderson AML Moonshot RNA seq dataset:   Total = 44 AML samples as following:   TP53-mut samples = 19  (bulk = 12, LSC = 7)   TP53-wt samples = 25 (bulk =14, LSC =11)Munich Leukemia Laboratory (MLL) RNA-seq dataset:   Total = 726 AML samples as following:   TP53-mut samples = 72, TP53-wt samples = 654.Cell lines: MOLM13 cell lines from Dr. S. BoettcherStatistical analysis: Variables were compared using the Wilcoxon rank-sum test for pairwise comparisons. The Kaplan–Meier method was used to estimate the probability of OS, and compared by the log-rank test. Univariate and multivariate Cox proportional hazards models were used to assess the association between TP53 status and PLK4 expression. Analyses were performed using R version 4.0.3.++++++ +++++++++++++++++++++++++++++++++++++++++++++++++ ++++++ ++ +++ + ++ ++++++++++++++ +++++++++++++++++++++ +++++++++ +++++ ++++ ++++++ +++ + ++ ++ + ++ +p < 0.00010.000.250.500.751.000 1 2 3 4 5 6YearsOverall Survival Probability+ +TP53-mut TP53-wt76 15 5 2 1 1 1 0 0 0 0 0 0 0650 318 238 191 163 149 117 98 66 37 22 11 4 3TP53-wtTP53-mutNumber at risk+++++++++++++++++++++++++++++++++++++++++++++++++ ++++++ ++ + + + + + +++++++++++++++ ++++++++p = 0.050.000.250.500.751.000 1 2 3 4 5 6YearsOverall Survival Probability+ +PLK4 others PLK4 high692 322 237 190 161 148 11634 11 6 3 3 2 2PLK4 highPLK4 othersNumber at riskResultsTP53-mut and PLK4 overexpression are associated with poor overall survival (OS) in AML A.                                                                                            B.Figure 1: Survival analysis performed based on A) TP53 status. B) PLK4 expression levels.PLK4 is overexpressed in primary samples from TP53-mut AML patientsA.                                              B. Figure 2: RNA-seq datasets show a significant increase in PLK4 expression in TP53-mut AML. A) MDACC Moonshot (bulk n=26, LSC n=18). B) Munich Leukemia Laboratory (bulk n = 726).PLK4 is overexpressed in TP53-mut AML MOLM13 cell linesA.B. Figure 3: Western blot showing p53 (fig A) and PLK4 (fig B) protein levels in TP53-wt and TP53-mut MOLM13 cell linesPLK4i leads to improved survival outcomes in TP53-wt, TP53-mut, and TP53-ko AML modelsA. B.C.D.Figure 7: OS in untreated & treated A) TP53-wt, B) TP53-R248Q, and C) TP53-ko PDX models. D) Hazard ratio plotted based on treatement & genotype.Conclusions• TP53-mutant AML has an overexpression of PLK4, which is a central regulator for centriole duplication.• Targeting PLK4 results in increased levels of poly-ploidy in TP53-mut AML vs TP53-wt AML.• PLK4 inhibition upregulates cleaved Caspase-3 in polyploid cells, and results in significantly higher apoptosis in TP53-mut MOLM13 cell lines in compar-ison to TP53-wt MOLM13 cell line. • A clinical trial is ongoing testing the efficacy of PLK4 inhibition (CFI-400945) in AML (ID: NCT04730258, TWT-202).TP53-wt (bulk) TP53-mut (bulk)TP53-wt (LSC) TP53-mut (LSC)14 12 11 7654 72MDACC AMLMoonshotMunichLeukemia LabEdUPI

Targeting Polo-Like Kinase 4 (PLK4) Triggers Polyploidy and Apoptosis in TP53-mutant Acute Myeloid Leukemia and Results in Improved Survival

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1020&context=sumexp22

Targeting Polo-Like Kinase 4 (PLK4) Triggers Polyploidy and Apoptosis in TP53-mutant Acute Myeloid Leukemia and Results in Improved Survival

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