Page 12 - Haematologica - Vol. 105 n. 6 - June 2020

Editorials
drug combinations that can simultaneously target the undifferentiated leukemic cell populations as well as the more mature myeloid lineages (especially monocytes) may represent a powerful way to prioritize the most promising drug combinations for pre-clinical study and for clinical development. Clearly, this report demonstrates just the beginning of utility of this exciting drug screening platform.
References
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3. Majumder MM, Leppa AM, Hellesoy M, et al. Multi-parametric sin-
gle cell evaluation defines distinct drug responses in healthy hemato- logical cells that are retained in corresponding malignant cell types. Haematologica. 2020;105(6):1527-1538.
4. Pemovska T, Kontro M, Yadav B, et al. Individualized systems med- icine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discov. 2013;3(12):1416-1429.
5. Lagadinou ED, Sach A, Callahan K, et al. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell. 2013;12(3):329-341.
6. Kuusanmaki H, Leppa AM, Polonen P, et al. Phenotype-based drug screening reveals association between venetoclax response and dif- ferentiation stage in acute myeloid leukemia. Haematologica. 2020;105(3):708-720.
7. Pei S, Pollyea DA, Gustafson A, et al. Monocytic Subclones Confer Resistance to Venetoclax-Based Therapy in Patients with Acute Myeloid Leukemia. Cancer Discov. 2020;10(4):536-551.
To target the untargetable: elucidation of synergy of APR-246 and azacitidine in TP53 mutant myelodysplastic syndromes and acute myeloid leukemia
David A. Sallman
Department of Malignant Hematology, H. Lee Moffitt Cancer Center, Tampa, FL, USA E-mail: DAVID A. SALLMAN - david.sallman@moffitt.org
doi:10.3324/haematol.2020.249060
Mutations of the tumor suppressor gene TP53 rep- resent a common mutation in myeloid malignan- cies, occurring in 10-20% of patients with de novo myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with profound negative impact on out- comes and a median overall survival (OS) of 6-12 months.1- 3 Critically, the clonal burden of TP53, that is the variant allele frequency (VAF) and/or allelic state of TP53, is inti- mately tied with the clinical trajectory of these patients and is a robust, independent predictor of survival.4-7 Given the poor OS and lack of therapeutic options for TP53 mutant MDS/AML patients, a number of novel agents are being investigated in this patient group.8 Of these, APR- 246 has evoked considerable excitement based on its robust clinical efficacy in combination with azacitidine in TP53 mutant MDS/AML patients.9,10 In this issue of Haematologica,11 Maslah et al. describe compelling preclini- cal synergy of APR-246 in combination with azacitidine in TP53 mutated MDS and AML and, more importantly, identify a novel molecular mechanism underlying the observed synergy.
Recent elegant work has definitively identified that TP53 missense mutations in myeloid malignancies result in a dominant-negative effect without evidence of neo- morphic gain-of-function activities, ultimately leading to a selection advantage when exposed to DNA damage.12 Thus, restoring wild-type function in TP53 mutant clones would be of profound beneficial impact. APR-246, a methylated PRIMA-1 analog, is a novel, first-in-class, small molecule that selectively induces apoptosis in TP53 mutant cancer cells. Mechanistically, APR-246 is sponta- neously converted into the active species methylene quin- uclidinone (MQ), which is able to covalently bind to cys- teine residues in mutant p53 thereby producing thermo-
dynamic stabilization of the protein and shifting equilibri- um toward a functional conformation.13,14 APR-246 monotherapy was originally investigated in a phase I trial including AML patients with clinical activity and correla- tive data identifying activation of p53-dependent path- ways.15,16
Maslah et al. identified in TP53 mutant cell lines, in vivo models, and primary patient samples that the combina- tion of APR-246 and azacitidine results in a synergistic pro-apoptotic effect as well as a dramatic reduction in cell proliferation via cell cycle arrest (Figure 1). As the majority of TP53 mutations are missense and located in the DNA binding domain, synergy experiments were performed with the SKM1 cell line, which harbors a homozygous hotspot mutation of TP53 (p. R248Q), and thus is an appropriate representation of clinical disease.17 Combination therapy of APR-246 and azacitidine resulted in a doubling of apoptotic cells versus azacitidine alone as well as 83% of cells undergoing cell cycle arrest in G0/G1. This synergistic effect was confirmed in a xenotransplan- tation model where combination therapy resulted in a pronounced inhibition of disease progression which occurred early and was durable. Subsequently, the authors interrogated differential gene expression profiles of SKM1 cells treated with either drug alone versus the combination of APR-246 and azacitidine. As expected, Gene Set Enrichment Analysis (GSEA) and DAVID analyses of APR- 246 treated cells showed robust induction of p53-target genes including CDKN1A, CASP1, BAX and FAS, which was confirmed by reverse transcription real-time quantita- tive polymerase chain reaction (RT-qPCR), resulting in activation of an early apoptotic program. Furthermore, GSEA analysis of “synergistic only” genes (i.e. genes dif- ferentially expressed only with combination treatment)
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