All-Trans Retinoic Acid (ATRA) Enhances the Antileukemic Activity of Decitabine By Increasing Proteasomal Degradation of MYC in a p53-Independent Manner

Background

TP53 mutations (MUT) constitute the worst prognostic marker in virtually all cancers, including acute myeloid leukemia (AML). As TP53 MUT increase with age (Jahn et al., Leukemia 2023), they affect mostly elderly patients (pts) who are often unfit for standard, aggressive chemotherapy. Hypomethylating agents (HMAs) can induce remissions also in these pts, providing an important first-line treatment option, even though these remissions are not durable. A randomized phase II trial (“DECIDER”) showed that the addition of ATRA to decitabine (DAC) significantly improves overall survival in newly diagnosed elderly AML pts, by increasing both the response rate and its duration (Lübbert et al., J Clin Oncol. 2020). Recent post hoc analyses revealed that half of the TP53 MUT pts responding to DAC+ATRA lived for >3 years (Thomas, Rehman et al., 1^st revision). The mechanistic explanation of this phenomenon still requires elucidation.

Methods

We employed a MOLM13 isogenic model of TP53+/+, R175H/-, M237I/-, R248Q/-, R273H/-, R282W/-, and TP53-/- clones (Boettcher et al., Science 2019) treated with 100nM DAC by 3 daily pulses, 250nM ATRA on day 4, and studied for proliferation, viability, cell cycle (EdU, Ki67), differentiation (CD11b) at 24h, 48h and 72h post ATRA. RNA-sequencing was also performed on TP53+/+ and TP53-/- at all 3 time points, with results files used for gene set enrichment analyses (GSEA) on ‘MSigDB 50 Hallmark pathways’.

Results

DAC+ATRA significantly reduced proliferation in TP53+/+ (88% by DAC+ATRA from 73% by DAC relative to DMSO), R175H/- (40% from 30%), M237I/- (64% from 46%), R248Q/- (61% from 51%), R273H/- (40% from 30%), R282W/- (66% from 36%), and TP53-/- (48% from 24%) clones respectively, with both an increase in G₁ phase (except R175H/-) and reduction in viability at 48h. Increased differentiation (CD11b by flow cytometry) was noted in TP53+/+ (84% by DAC+ATRA from 75% by DAC relative to DMSO), R175H/- (40% from 24%), M237I/- (45% from 28%), R248Q/-(29% from 22%), R273H/- (73% from 33%), R282W/- (13% from 11%), and TP53-/- (20% from 5%) clones respectively, at 72h.

The global transcriptomic profile of DAC+ATRA treated TP53+/+ and TP53-/- cells was distinct from single-agent treatments at all 3 timepoints, as the majority of DEGs were unique to the combination. Enrichment analysis (GSEA) shows that DAC+ATRA activates the TP53 pathway for antileukemic activity, as most proliferation pathways (e.g. hallmark G₂M checkpoint, hallmark E2F targets) were depleted in a p53-dependent manner. Interestingly, among the proliferation-associated pathways, hallmark MYC target genes along with MYC transcript, was depleted in both clones, hence pointing towards MYC as an important downstream target for DAC+ATRA efficacy observed, independent of p53, in our model.

Regarding MYC proteins levels, MYC was depleted in TP53+/+ (by 48% from 20% by DAC), R175H/- (55% from 31%), M237I/- (25% from 18%), R248Q/- (65% from 27%), R273H/- (32% from 25%), R282W/- (65% from 45%), and TP53-/- (25% from 15%) clones respectively, as early as 24 hours post ATRA, before any significant effects of ATRA on cell cycle inhibition (Ki67) were noted. By immunofluorescence, DAC+ATRA significantly increased pMYC^T58 and global K48 ubiquitination in both TP53 +/+ and TP53-/- cells compared to DAC, ATRA and DMSO, pointing toward increased general proteasomal degradation. A high overlap of pMYC^T58 foci with K48 ubiquitination was observed. Validation of higher K48 ubiquitination on MYC upon DAC+ATRA treatment is ongoing (proximity ligation assay), along with rescue experiments of MYC overexpression in DAC+ATRA treated cells.

Conclusions

The addition of ATRA to DAC enhances proteasomal degradation of MYC, resulting in reduced proliferation, enhanced apoptosis and increased differentiation in the TP53 isogenic model, with notable differences between the mutated clones studied. These findings will be extended to primary AML blasts isolated from pts treated on the DECIDER trial. As the interest in retinoids as treatment option in AML and MDS has recently been revived, our results support the repurposing of retinoids such as ATRA also for treatment of TP53 MUT AML pts. This is pursued in an ongoing phase III, placebo-controlled trial (DECIDER-2, accruing newly diagnosed AML pts with both TP53 WT and TP53 MUT genotypes) with a DAC+venetoclax backbone and an ATRA vs. placebo randomization.

Boettcher:Astellas: Consultancy; Servier: Consultancy; Pfizer: Consultancy. Lübbert:Syros Pharmaceuticals: Honoraria; Cheplapharm: Research Funding; AbbVie: Honoraria; Janssen: Research Funding; Otsuka: Honoraria.