Page 84 - Haematologica - Vol. 105 n. 6 - June 2020

N. Maslah et al.
The combination of APR and AZA synergistically reduces the proliferation of myeloid cell lines
AZA is considered to be a first-line treatment for most patients with high-risk MDS, but its effect is relatively limited in MDS patients who have TP53 mutations. After we evaluated the activity of AZA on TP53-mutated AML lines (IC50 ranging from 0.54x10-6 M for SKM1 to 4.5x10-6 M for HL60 cells) (Figure 1C), we investigated whether its efficacy could be enhanced by combination with APR. The addition of low doses of APR (IC10) (Online Supplementary Table S1) to increasing doses of AZA reduced the proliferation of the five cell lines compared to AZA on its own, with the exception of HL60 cells (Figure 1D and E). SKM1 cells were the most sensitive to the com- bination, as the IC50 of AZA in these cells was significantly reduced in the presence of low doses of APR. This syner- gistic effect was confirmed at different concentrations of each drug by calculating the combination index (CI < 1) for all of the tested cell lines, but not at low concentrations of both drugs (i.e. both drugs at their IC10) (Online Supplementary Table S1).
The combination of APR and AZA promotes G0/G1 cell cycle arrest and apoptosis
To elucidate the mechanisms underlying the antiprolifer- ative effect of the combination compared with each drug on their own, we performed apoptosis and cell cycle studies using low dose APR (i.e. at the IC10) with a standard dose of AZA (i.e. at the IC50). Other associations are shown in the Online Supplementary Figures S2-S4. Under these conditions, APR alone did not induce apoptosis, while AZA on its own induced low levels of apoptosis in some of the cell lines. The combination of APR with AZA significantly increased apoptosis compared to the individual drugs in every cell line that was tested. This effect was more pronounced in SKM1 cells (39% apoptotic cells with APR + AZA vs. 19% with AZA alone, P<0.01) (Figure 2A-E).
We then analyzed the cell cycle distribution after treat- ment with IC10 APR, IC50 AZA, or the combination of these two drugs at these concentrations. At this low con- centration, APR on its own did not affect the cell cycle dis- tribution of the various cell lines tested, while AZA tended to increase the proportion of cells in the G0/G1 phase. However, SKM1 cells treated with the combination of AZA and APR underwent cell cycle arrest in G0/G1 as early as 24 hours after exposure (83% of the cells were in G0/G1 phase following APR + AZA treatment vs. 62% for APR-treated cells, P<0.01). In parallel, the proportion of cells in the S and G2/M phases was significantly reduced (Figure 2A). Similar results, although less pronounced, were observed in the other cell lines (Figure 2B-E), while various combinations of these drugs confirmed the observed synergism between the two drugs in terms of cell cycle arrest and the induction of apoptosis (Online Supplementary Figures S2-S4). Altogether, although the effect is more pronounced on apoptosis than on cell cycle, these results suggest that the addition of APR to AZA in various TP53-mutated cell lines potentiates the AZA antiproliferative effect by increasing G0/G1 arrest and a pro-apoptotic effect.
APR is active on primary cells of TP53-mutated MDS/AML samples
We then investigated the effect of these drugs on pri- mary cells from MDS/AML patients. Of the 34 patients
included in our study, 15 patients had mutations in the TP53 gene (10 had a complex karyotype, 2 had an isolated 5q deletion, and 14 had deletion of the other TP53 allele). All mutations were located in the DNA-binding domain hotspots (DBD) (Table 1).
We performed clonogenic assays in semi-solid medium using the previously determined IC50 for SKM1 cells. AZA on its own at 3 μM induced only a modest reduction (30%) of myeloid and erythroid colony growth, while APR at 0.5 μM significantly inhibited colony growth by approximately 60% (Figure 3A and B).
We then sought to determine whether there was a tar- geted effect on cells from patients with mutated TP53. When compared to AZA on its own, the drug combina- tion always had a greater inhibitory effect on the growth of myeloid or erythroid progenitor colonies, irrespective of the TP53 genotype of the patients (Online Supplementary Figure S5). However, when compared to APR on its own, the APR + AZA combination significantly reduced the number of myeloid and erythroid colonies formed by pro- genitors derived from patients with mutated TP53, while this was not observed with wild-type TP53 patients (Figure 3C and D). This indicates that the drug combina- tion may be more beneficial to patients with TP53 muta- tions. However, the molecular analysis of the residual colonies from four patients with mutated TP53, irrespec- tive of the treatment conditions, only revealed mutant TP53 colonies (Table 2 and data not shown), thus making it impossible to conclude that cells with mutated p53 are tar- geted specifically. To further study the specificity of the combination, we performed liquid cultures of CD34+ cells isolated from TP53-mutated or wild-type MDS/AML patients and studied proliferation and apoptosis when treated by APR and/or AZA. A significant efficacy of the combination compared to drugs given alone was observed in TP53-mutated patients only (Figure 4A-D). Accordingly, CD34+ cells isolated from healthy blood donors did not show increased sensitivity to the combination compared to isolated drugs (Figure 4E and F).
In vivo efficacy of APR and AZA in a xenotransplantation model
In order to evaluate the in vivo antiproliferative effect of APR and AZA, we developed a xenograft model of SKM1- Luciferase cells in NSG mice that allowed us to use biolu- minescence to measure tumor volumes before and after APR and AZA treatment. Intravenous injections of 107 cells yielded highly reproducible tumor engraftment and growth over time (100% of the mice had engraftment) (Figure 5A). The mice were treated with the drugs as soon as the tumor bioluminescence signal reached 106 p/sec/cm2/sr.
Treatment with low doses of APR in combination with AZA resulted in pronounced inhibition of disease progres- sion as early as four days after starting the drug treatment compared to APR or AZA treatment on their own (median tumor volume: 91.1x106 p/sec/cm2/sr in untreated mice, 64.4x106 p/sec/cm2/sr with APR on its own, 103.2x106 p/sec/cm2/sr with AZA on its own vs. 18.9x106 p/sec/cm2/sr mm3 in the APR + AZA group; P<0.05) (Figure 5A). There was still a beneficial impact of the drug combination after eight days of treatment (Figure 5B). These results confirmed in vivo the efficacy of the combi- nation of a low dose of APR with standard AZA doses that we had previously shown in vitro.
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