Metabolic Alterations May Contribute to Cabozantinib Resistance in Acute Myeloid Leukemia Cells with FLT3-ITD

Background: Internal tandem duplication in the juxtamembranal region of FLT3 gene (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML), resulting in constitutive activation of FLT3 signaling pathway. Therefore FLT3 have been proved to be as a useful target for AML treatment. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor (TKI), could selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Recently, cabozantinib was reported to be well tolerated in AML patients with FLT3-ITD and potentially be useful in the treatment of AML with FLT3-ITD. However, it is known that TKI-resistance in AML often cause higher relapse rates and lower survival rates. In order to study the drug resistance mechanism, we established cabozantinib-resistant cell lines from MV4-11 and Molm13 cells, after gradual escalating concentration of cabozantinib incubation, with increasing IC₅₀ from 9.5nM to 1.5μM and from 1.06nM to 473.36nM, respectively. The cabozantinib-resistance cell lines were named MV4-11 XR and Molm-13 XR, respectively.

Aims: To elucidate the mechanism of survival advantage of cabozantinib-resistance of MV4-11-XR and Molm13-XR.

Materials and Methods: The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape recourse and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to predict the biological functions of DEGs. Quantitative PCR (Q-PCR) were used to validate the RNA-seq results. The extracellular acidification rate (ECAR) was measured using Seahorse bio-analyzer. In addition, to investigate the mitochondrial metabolism, analyses of oxygen consumption rate (OCR), glucose uptake, GAPDH activity, lactate production, ATP content were performed.

Results: The gene expression profile in MV4-11 cells and Molm13 cells were used as baselines to establish the up- or down-regulated genes in MV4-11-XR and Molm13-XR cells, respectively. The FPKM were estimated with the selection criteria of q value<0.05 and [log₂ (fold change0)>1 or <1 for significantly differential expression for up- and down-regulation, respectively. We identified a total of 1113 DEGs between the MV4-11 and MV4-11-XR cells, and a total of 1057 DEGs between the Molm13 and Molm13-XR cells. By using KEGG Mapper to interrogate pathways significance, we found that the metabolic pathway was the most significant in both up- and down-regulated DEGs in both resistant cells (MV4-11-XR up: 30 DEGs, down: 52 DEGs; Molm13-XR up: 27 DEGs, down: 58 DEGs). The information suggests that metabolic alterations occur in both drug resistant cell lines. Both MV4-11-XR and Molm13-XR cells showed higher glucose uptake, GAPDH activity, lactate production and ATP content than corresponding parental cells. Consistent with increased lactate export, analysis of glycolytic function showed a significant increase in glycolysis, glycolytic capacity and glycolytic reserve in both resistant cells. In addition, we showed increased basal mitochondrial and ATP-coupled respiration in both resistant cells, compared to their corresponding parental cells. Finally, decreased OCR / ECAR ratios indicated the relatively higher reliance on glycolysis in both resistant cells: 15.79 in Molm13-XR cells compared to 24.93 in Molm-13 cells, and 1.75 in MV4-11-XR cells compared to 9.97 in MV4-11 cells.

Conclusion: Our study highlights that alteration of metabolic pathways may contribute cabozantinib resistance. We suggest that targeting these pathways may be a viable strategy to overcome cabozantinib resistance.