Transcriptomic-Based in silico Drug Screening Reveals Dual PI3K/mTOR Inhibitors Overcome Cabozantinib-Resistance of FLT3-ITD AML Cells

Background: Elucidating the mechanisms involved in tyrosine kinase inhibitor (TKI) resistance is critical for developing effective therapies in acute myeloid leukemia (AML). Cabozantinib (CBZ), an oral multi-target TKI, is well tolerated in AML patients and effectively inhibits the FLT3-mutated AML. We previously established CBZ-resistant MV4-11 cells (MV4-11-XR). However, the precise mechanisms of CBZ resistance are still not fully studied.

Aims: To elucidate the molecular mechanism in MV4-11-XR and explore the potential therapeutic approaches to overcome CBZ-resistance.

Materials and Methods: MV4-11-P and MV4-11-XR myeloid leukemia cell lines were used in this study. Cabozantinib (BMS-907351), omipalisib (GSK2126458), and gedatolisib (PF-05212384) were used for experiments. Cell proliferation was measured by Cell Titer 96 AQueous One Solution Cell Proliferation Assay. The protein expression was analyzed by immunoblotting. RNA-seq was performed using an Illumina NextSeq 500 platform. Sanger's sequencing and pyrosequencer were used to characterize the SNVs (single nucleotide variants). FusionCatcher was also used to explore potential fusion events related to resistance. Differentially expressed genes (DEGs) between MV4-11-P and MV4-11-XR were identified by EBseq. A threshold of p ≤ 0.05 was used to select the DEGs. Connectivity map was used to predict small molecules which had potentials to reverse enriched metabolic pathways in MV4-11-XR. The quantitative reverse transcription PCR (qRT-PCR) was performed by QuantStudio 3 Real-Time PCR Systems. The cellular ATP production rate was analyzed by the XFe 24 extracellular flux analyzer. Flow cytometry was used for cell cycle and apoptosis analysis.

Results: We demonstrated that the anti-proliferative effect of CBZ on MV4-11-XR and parental MV4-11 (MV4-11-P). MV4-11-XR was significantly resistant to CBZ with IC₅₀ of 1500 nM compared with MV4-11-P with IC₅₀ of 9.5 nM. Subsequently, we performed RNA-seq to evaluate SNVs, indels, and enriched pathways in MV4-11-XR compared to MV4-11-P. We found 376 SNVs in MV4-11-XR. Of them, we noticed the emergence of FLT3 c.2503G>T (p.D835Y) mutation in MV4-11-XR. Pyrosequencing analysis confirmed that the frequency of mutant allele increased from 5 % in MV4-11-P to 99 % in MV4-11-XR. Immunoblotting confirmed the hyperactivation of FLT3 downstream signaling pathways in MV4-11-XR compared to MV4-11-P. FusionCatcher analysis indicated no novel fusion transcript related to CBZ resistance. Transcriptomic analysis revealed that a total of 1113 (366 upregulated and 725 downregulated) DEGs were detected in MV4-11-XR compared to MV4-11-P. Metascape analysis revealed that those up-regulated DEGs were enriched in "HALLMARK MTORC1 SIGNALING” and "HALLMARK HYPOXIA", indicating increased glycolysis in MV4-11-XR. qRT-PCR validated the overexpression of glycolytic genes including HIF1A, GLUT1, HK2, PKM2, LDHA, and PDK1 in MV4-11-XR. Real-time ATP rates analysis showed that MV4-11-XR had significantly increased rates of glycolytic ATP production. To explore small molecules that have the potential to reverse the metabolic phenotypes and overcome CBZ resistance, we performed a connectivity map analysis and predicted a PI3K inhibitor (wortmannin) and an mTOR inhibitor (sirolimus) as candidates. Therefore, we selected and evaluated the effects of gedatolisib and omipalisib, which have dual PI3K/mTOR inhibitory capacity, on MV4-11-XR. qRT-PCR showed that omipalisib significantly inhibited the expression of glycolytic genes in MV4-11-XR. Further experiments showed that gedatolisib and omipalisib significantly inhibited the proliferation of MV4-11-XR with IC₅₀ of 28.33 nM and 37.56 nM, respectively. More importantly, gedatolisib and omipalisib significantly suppressed PI3K-AKT-mTOR signaling, promoted cell apoptosis, and induced cell cycle arrest in MV4-11-XR.

Summary: In this study, we identified the emergence of FLT3 D835Y mutation and the upregulation of glycolysis in MV4-11-XR. As suggested by connectivity map analysis, we identified PI3K/mTOR dual inhibitor gedatolisib and omipalisib had potent anti-leukemic effects and could reverse the metabolic signature of CBZ-resistant cells. These findings highlight the potentials of PI3K/mTOR dual inhibitors as therapeutic molecules in AML to overcome TKI resistance.

No relevant conflicts of interest to declare.