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Targeting mutant p53 in ALL
TP53-mutated leukemias are sensitive to APR-246 but not to genotoxic therapy
In response to genotoxic agents and stress, wildtype p53 suppresses cellular viability and proliferation. However, dysfunctional, mutant p53 fails to mediate tumor-suppres- sive functions such as induction of cell death. Therefore, we analyzed cell death in TP53mut and TP53wt ALL pri- mografts (TP53mut n=4, TP53wt n=4) and cell lines (TP53mut n=2, TP53wt n=4) in response to increasing concentrations of the DNA-damaging agent doxorubicin, a standard genotoxic drug regularly used in ALL treatment protocols, and to APR-246. All TP53mut primografts and cell lines showed, as expected, insensitivity to doxorubicin indicated by significantly higher IC50 values, in contrast to doxorubicin-sensitive TP53wt leukemias (Figure 1C, D; Online Supplementary Table S2A, B). An opposite effect was observed upon exposure to APR-246 with high sensitivity and cell death induction in all TP53mut leukemias, but low APR-246 sensitivity in TP53wt ALL (Figure 1E, F; Online Supplementary Table S2C, D). Interestingly, diagno- sis- (TP53mut-2, -3, -4) or relapse-derived (TP53mut-1) pri- mograft samples did not show differences in APR-246 or doxorubicin sensitivity.
Activation of the p53 pathway results in apoptosis induction. Along with cell death, APR-246 led to annexin- V/propidium iodide positivity and caspase-3 activation indicating apoptosis induction in TP53mut ALL. In con- trast, apoptosis was induced by doxorubicin but not APR- 246 in TP53wt cells (Figure 2 and Online Supplementary Figure S1).
Thus, all identified TP53mut leukemias carried missense mutations in the DNA-binding domain, showed accumu- lation of p53 indicative of dysfunctional mutant p53, resistance to the genotoxic agent doxorubicin, and were highly sensitive to APR-246-induced apoptosis.
APR-246 restores p53’s wildtype conformation reactivating tumor suppressive functions
We further addressed the mode of action of APR-246 in
TP53mut ALL and examined the conformation of p53 and activation of the pathway in response to APR-246. Using a p53 wildtype conformation-specific antibody (PAb1620), larger amounts of p53 with wildtype confor- mation were immunoprecipitated from lysates of TP53mut ALL cells exposed to APR-246, indicating recon- stitution of p53 wildtype conformation in TP53mut ALL by APR-246 (Figure 3A). However, this effect was not observed in TP53wt leukemia cells (Online Supplementary Figure S2). Next, we assessed expression of the p53 tran- scriptional targets PUMA (P53-Upregulated Modulator of Apoptosis), p21 (Cyclin Dependent Kinase Inhibitor 1A, CDKN1), and NOXA upon APR-246 or doxorubicin treat- ment in TP53mut (KOPN-8, RS4;11) and TP53wt (NALM- 6, UoCB-6) ALL lines. In TP53mut ALL, APR-246 led to induction of all p53 targets (Figure 3B, C and Online Supplementary Figure S3). In contrast, an opposite picture of activation of p53 transcriptional targets in TP53wt but not in TP53mut leukemias was observed upon incubation with doxorubicin (Figure 3D, E and Online Supplementary Figure S3). Thus, APR-246 induces restoration of mutant p53 to wildtype conformation, transcriptional target expression, and apoptosis in TP53mut ALL.
Induction of oxidative stress contributes to APR-246-mediated cellular death
Induction of oxidative stress has been described as a sec- ond activity of APR-246 in different cancers.30-32 APR-246 was reported to interfere with different regulators of the cellular redox system, such as thioredoxin reductase, thioredoxin and glutathione, and with the transcription factor NRF2, leading to induction of reactive oxygen species (ROS).28,30-33,44-47 Given the activity of APR-246 in TP53mut ALL, we addressed whether TP53mut and TP53wt ALL display distinct sensitivities in response to ROS generation. Upon treatment with 3-morpholinosyd- nonimine, a spontaneous generator of reactive oxygen and nitrogen species, and the oxidant tert-butyl hydroxyper- oxide, increased ROS levels were observed in both
Table 1. TP53 mutations in acute lymphoblastic leukemia cell lines and primograft samples.
Sample
TP53mut-1 TP53mut-2
TP53mut-3 TP53mut-4 RS4;11 KOPN-8
Exon
exon 6 exon 7
exon 8
exon 8
exon 5
exon 7
exon 7
Mut. (bp)
c.637C>T c.743G>A
c.844C>G
c.818G>A
c.524G>A
c.761T>C
c.743G>A
Mut. (aa) Region Mut. type
p.R213X DBD stop p.R248Q missense
p.R282G DBD missense
p.R273H DBD missense
p.R175H DBD missense
p.I254T DBD missense
p.R248Q DBD missense
del (17p)
-
del del - - -
del (17p)
del
Genotype
heterozygous
hemizygous hemizygous homozygous heterozygous heterozygous
Genotype
hemizygous
Number of chr.s.
47
47 44 46 47 46
Diploidy
diploid
diploid diploid diploid
Mut: mutation; bp: base pair; aa: amino acid; del: deletion; DBD,: DNA-binding domain; Chr.s: chromosomes.
Table 2. TP53 mutations in primary samples from patients with acute lymphoblastic leukemia.
Sample
Patient-1
Patient-2
Patient-3
Patient-4
Exon
exon 4
exon 7
exon 7
exon 7
Mut. (bp)
c.375G>A
c.743G>A
c.743G>A
c.733G>C
Mut. (aa) Region Mut. type
p.T125T DBD splice site
p.R248Q DBD missense - heterozygous
p.R248Q DBD missense - heterozygous
p.G245R DBD missense - heterozygous
Mut: mutation; bp: base pair; aa,: amino acid; del: deletion; DBD: DNA-binding domain.
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