Synergistic effects of PRIMA-1Met (APR-246) and 5-azacitidine in TP53-mutated myelodysplastic syndromes and acute myeloid leukemia

Abstract

Myelodysplastic syndromes and acute myeloid leukemia with TP53 mutations are characterized by frequent relapses, poor or short responses, and poor survival with the currently available therapies including chemotherapy and 5-azacitidine (AZA). PRIMA-1^Met(APR-246,APR) is a methylated derivative of PRIMA-1, which induces apoptosis in human tumor cells through restoration of the transcriptional transactivation function of mutant p53. Here we show that low doses of APR on its own or in combination with AZA reactivate the p53 pathway and induce an apoptosis program. Functionally, we demonstrate that APR exerts these activities on its own and that it synergizes with AZA in TP53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) cell lines and in TP53-mutated primary cells from MDS/AML patients. Low doses of APR on its own or in combination with AZA also show significant efficacy in vivo Lastly, using transcriptomic analysis, we found that the APR + AZA synergy was mediated by downregulation of the FLT3 pathway in drug-treated cells. Activation of the FLT3 pathway by FLT3 ligand reversed the inhibition of cell proliferation by APR + AZA. These data suggest that TP53-mutated MDS/AML may be better targeted by the addition of APR-246 to conventional treatments.

Figure 1.

PRIMA-1^Met (APR-246, APR) is active on TP53-mutated acute myeloid leukemia (AML) cell lines and the combination of APR and azacitidine (AZA) is synergistic. (A) Relative proliferation with increasing APR concentrations (10- to 10⁻⁵ M) in SKM1, K562, KG1a, THP-1, and HL60 cell lines from day-0 to day-3. (B) Dose-response curves of APR for the five cell lines. (C) Dose-response curves of AZA for the five cell lines, and (D) at a low APR concentration (IC₁₀). (E) The proliferation rate relative to the untreated control at day 3 with IC₅₀ AZA with or without IC₁₀ APR in the five cell lines indicated above. ^*P<0.05, ^**P<0.01.

Figure 2.

The combination of PRIMA-1^Met (APR-246, APR) and azacitidine (AZA) promotes G0/G1 arrest and apoptosis in various TP53-mutated acute myeloid leukemia (AML) cell lines. (Left) Percentage of Annexin V-positive cells at day 3 post treatment with IC₁₀ APR, IC₅₀ AZA, or the combination of these two drugs at these same concentrations. (Right) Proportion of cells in G0/G1, S, or G2/M phase 24 hours after treatment with IC₁₀ APR, IC₅₀ AZA or the combination of these two drugs at these same concentrations. (A) SKM1, (B) K562, (C) KG1a, (D) THP-1, and (E) HL60 cell lines. ^*P<0.05, ^**P<0.01.

Figure 3.

Effects of PRIMA-1^Met (APR-246, APR) on primary cells from TP53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) patients and healthy donors. The median numbers of (A) myeloid and (B) erythroid colonies relative to the untreated control for 34 bone marrow samples from MDS/AML patients treated with APR, azacitidine (AZA), or the combination APR + AZA in semi-solid medium (methylcellulose). Relative numbers of (C) myeloid and (D) erythroid colonies according to the TP53 status (WT: wild-type) treated with APR or the combination of APR + AZA. Median numbers of (E) myeloid and (F) erythroid colonies relative to untreated control for 3 CD34⁺ cells from healthy donors treated with APR, AZA or the combination APR + AZA. ^*P<0.05, ^****P<0.0001.

Figure 4.

Effects of PRIMA-1^Met (APR-246, APR) on primary cells from TP53 wild-type or mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) patients and healthy donors. (Left) Relative proliferation of CD34⁺ cells treated with 1 mM APR, 1 mM AZA or the combination APR + AZA at these concentrations. (Right) Percentages of Annexin V-positive cells at day 3 post treatment with 1 mM APR, 1 mM of AZA or the combination APR + AZA at these concentrations. (A) TP53-mutated MDS/AML samples (n=3), (B) Wild-type TP53 MDS/AML samples (n=3), and (C) healthy donors (n=3). ^*P<0.05.

Figure 5.

In vivo efficacy of PRIMA-1^Met (APR-246, APR) and azacitidine (AZA) in a xenotransplantation model. (A) Disease development monitored by luciferase activity and bioluminescence imaging. Images of untreated mice at day 14 (day 1 of treatment) and treated mice at day 18 (day 5 of treatment) following injection of SKM1-Luc cells. (B) The tumor volume (p/sec/cm²/sr) during and after treatment with PBS, APR, AZA, or the APR + AZA combination (treatment days are indicated by the solid arrows). The drug treatments were started when the tumor volume had reached 10⁶ p/sec/cm²/sr. ^*P<0.05.

Figure 6.

Changes in gene expression induced by treatment with PRIMA-1^Met (APR-246, APR) + azacitidine (AZA). (A) A Venn diagram representing the number of deregulated genes with APR on its own, AZA on its own, or the combination of APR + AZA. (B) Gene enrichment plots and associated heatmaps showing reactivation of the p53 pathway and induction of an apoptosis program in SKM1 cells treated with IC₁₀ APR compared to untreated cells. (C) Gene enrichment plots and associated heatmaps showing reactivation of the p53 pathway and induction of an apoptosis program in SKM1 cells treated with the combination of APR IC₁₀ + AZA IC₅₀ (APR + AZA) compared to AZA on its own (AZA). (D) RT-qPCR expression of the main p53 targets (CDKN1A and BAX) and pro-apoptotic factors (CASP1 and FAS) in SKM1 cells treated with APR compared to untreated cells. (E) RT-qPCR expression of the main p53 targets (CDKN1A and BAX) and pro-apoptotic factors (CASP1 and FAS) in SKM1 cells treated with the combination of APR + AZA versus AZA on its own. ^***P<0.001. NES: Normalized Enrichment Score; FDR: False Discovery Rate.

Figure 7.

The antiproliferative effect of PRIMA-1^Met (APR-246, APR) + azacitidine (AZA) combination is related to downregulation of the FLT3 pathway. (A) Gene enrichment plots and heatmaps representing the FLT3 activated pathway (Valk, FLT3-ITD) in APR versus untreated SKM1 cells and APR + AZA versus AZA-treated cells. (B) Real-time quantitative polymerase chain reaction analysis of gene expression by FLT3 and FLT3-L with APR, AZA, or APR + AZA relative to the untreated control. (C) Absolute numbers of untreated and APR + AZA-treated SKM1 cells exposed to increasing concentrations of FLT3-L. (D) Proliferation of SKM1 cells treated with APR, AZA, or the combination APR + AZA relative to controls, without or with 10 ng/mL FLT3 ligand (FLT3-L). (E) Proportion of Annexin V-positive SKM1 cells at day 3 following treatment with APR, AZA, or the combination of APR + AZA, with or without FLT3L. ^*P<0.05, ^**P<0.01, ^***P<0.001.