Strategies for Alleviating Tyrosine Kinase Resistance in Acute Myeloid Leukemia By Modulating p53 Signaling Pathway with Small Molecules

Background:FLT3-ITD can be found in up to 30% of acute myeloid leukemia (AML) patients and confers a poor prognosis. While midostaurin and gilteritinib are FDA-approved for these high-risk patients, they only result in a temporary response without transplantation, suggesting the importance of identifying the acquired resistance. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor, was selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Subsequently, we established cabozantinib-resistant human leukemic MV4-11 derivative (MV4-11-XR) and Molm13 derivative (Molm13-XR) after gradual escalating concentration of cabozantinib incubation from parental MV4-11 (MV4-11-P) and Molm13 (Molm13-P) cells, respectively. Subsequently, cell cytotoxicity assay showed these two cabozantinib-resistant cells were also resistant to quizartinib, sorafenib and gilteritinib. RNA-seq analysis revealed that MV4-11-XR and Molm13-XR cells shared 87 identical differentially down-regulated genes compared to their respective parental cells. Further Metascape for gene enriched pathway analysis demonstrated the most affected was p53 signaling pathway.

Aim: To investigate whether modulation with p53 activator RITA or MDM2 inhibitor RG7388 could alleviate the drug resistance and their underlying molecular mechanism.

Materials and Methods: Human leukemia MV4-11 and Molm13 cells were used in this study. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape enrichment pathway analyses were preformed to predict the biological functions of DEGs. Cabozantinib, RITA, and RG7388 (Idasanutlin) were used. MTS assay was performed to the effects of various drug on cell growth. Western blotting, flow cytometry and quantitative real-time PCR were applied to elucidate potential molecular mechanism. Fluorescent leukemic cell xenografts in zebrafish and high content imaging system were used for investigating the efficacy of various drugs in vivo.

Results: The gene expression profile in MV4-11-P and Molm13-P cells was used as a baseline to establish the up- and down-regulated genes in MV4-11-XR and Molm13-XR cells. The FPKM were estimated with the selection criteria of p value <0.05 and [log₂ (fold change)] <1 for significantly differential expression for downregulation. We identified a total of 747 DEGs between the MV4-11-P and -XR cells and 407 DEGs between the Molm13-P and -XR cells. Among them, 87 DEGs were shared by these two cohorts. Metascape enrichment analysis revealed that those down-regulated DEGs were enriched in p53-signaling pathway, in line with decreased expression of P53-associated genes, BBC3, CCNG2, CKDN1A, ZMAT3, confirmed by using q-RT-PCR. IGV (Integrative Genomics Viewer) analysis showed that the variant allele frequency of R248W progressed from 57% in MV4-11-P to 100% in MV4-11-XR cells, a similar phenomenon also appeared in P72R (65% to 98%). Microsatellite analysis confirmed the loss of heterozygosity in the TP53 gene of MV4-11-XR. On the other hand, there were not TP53 variants in both Molm13-P and Molm13-XR cells, but increased level of MDM2 in Molm13-XR cells was noted. Further study revealed that p53 activator RITA with 77.6nM could reduce IC₅₀ of cabozantinib from 1026nM to 310.7nM, coupled with increased gene expression and protein levels of BBC3 (PUMA), CCNG2 (cyclin G2), CDKN1A (p21) and ZMAT3. In addition, RITA had synergistic effect with cabozantinib on cell cycle arrest on G2/M phase, increased apoptosis and decreased colony formation and proliferation. On the other hand, MDM2 inhibitor RG7388 reduced the IC₅₀ of cabozantinib from 517nM to 101nM with only 50.6 nM in Molm13-XR cells. Similarly, RG7388 could increase those P53-associated gene expression and protein levels, and had synergistic effect with cabozantinib on cell cycle arrest on G0/G1 phase, increased apoptosis and decreased colony formation and proliferation. In addition, in vivo zebrafish experiment demonstrated that the combination of RG7388 and cabozantinib had a synergistic and significant inhibitory effect compared with single use.

Conclusion: These findings suggested that reactivation of p53 is a potential therapeutic option to alleviate drug resistance in FLT3-mutated AML, provided that AML patients are tested for p53 dysfunction, including TP53 mutational status and downregulation.

No relevant conflicts of interest to declare.