Modulating Metabolic Reprogramming By Phosphoglycerate Dehydrogenase (PHGDH) Inhibitors in Omipalisib-Refractory AML

Background: Numerous reports indicated the significance of the PI3K/AKT/mTOR signaling pathway in AML. Up to now, there is no FDA-approved PI3K/AKT/mTOR inhibitors available for patients with acute myeloid leukemia (AML). Recently, we demonstrated that PI3K/mTOR dual inhibitor omipalisib had a potent anti-leukemic effect, but it could not completely eliminate leukemia cells in a subcutaneous murine model. To explore the refractory features of omipalisib, we established several OCI-AML3 sublines refractory to omipalisib by an in vivo selection procedure. The OCI-AML3-R cell line was the most refractory one and showed cross-resistance to another PI3K/mTOR inhibitor dactolisib and multi-tyrosine kinase inhibitor dasatinib, in addition to omipalisib. Several genes associated with multidrug-resistance, ABCC1, ABCC2, ABCB1, ABCG2, MDR1, and MRP1, did not express significant difference between OCI-AML3 and OCI-AML3-R.

Aims: To investigate the molecular mechanisms of OCI-AML3-R using transcriptomics and metabolomics analysis, and to explore potential therapeutic strategies from both in vitro and in vivo aspects.

Methods: OCI-AML3 and OCI-AML3-R leukemia cell lines were used in this study. MTS assay was performed to assess cell viability and proliferation. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NovaSeq 6000). Metabolomic analysis was conducted by the C-SCOPE service using capillary electrophoresis mass spectrometry CE-TOFMS and CE-QqQMS with Agilent 6460 TripleQuad LC/MS (HMT, Yamagata, Japan). Q-RT-PCR was used for mRNA quantification. Immunoblotting were used to clarify the variations of total- and phosphor-proteins. Flow cytometry was used for apoptosis and mitochondrial ROS content analysis. OCI-AML3-R xenograft models in nude mice were used for scrutinizing efficacy of PHGDH inhibitor in vivo. The protocol for the xenograft experiments was approved by the IACUC, National Taiwan University.

Results: A total of 1946 DEGs between OCI-AML3 and OCI-AML3-R cells were identified with the selection criteria of p< 0.05 and [log ₂ (fold change)]> 1 or <1, of which 477 and 1469 DEGs were up- and down-regulated, respectively. g:Profiler was used for functional enrichment analysis and revealed that NFkB signaling-related pathways ( p< 0.001), cell cycle-related pathways ( p< 0.001), and nucleotide metabolic pathways ( p< 0.001) were enriched in OCI-AML3-R. Q-RT-PCR confirmed that a series of genes related to pentose phosphate pathway (PPP; TKT and 6PGD), serine synthesis pathway (SSP; PHGDH and PSAT1), purine synthesis and folate/methionine cycle ( PAICS, SHMT2, MTHFD2, and MTHFD1L) were increased in OCI-AML3-R cells. Among them, PHGDH and PSAT1 increased the most, 4.75x and 11.38x, respectively. A total of 103 metabolites were screened out by metabolomic analysis, 58 of which showed significant differences between OCI-AML3 and OCI-AML3-R cells ( p< 0.05). Metabolite enrichment analysis also revealed that the purine metabolism, urea cycle, glycolysis/gluconeogenesis, and several amino acid metabolism pathways were enriched in OCI-AML3-R cells. A comprehensive overview of metabolites in glycolysis and TCA cycle in OCI-AML3-R cells revealed significantly increased levels of PRPP and serine, as well as steady-state levels of glycine, folate, and several purine metabolites. These results indicated that a reprogramming of glucose metabolism towards the PPP and SSP in OCI-AML3-R cells was noted. Based on these results, we proposed targeting the PPP and SSP as a therapeutic approach for omipalisib-refractory AML. The PHGDH inhibitors, WQ-2101 and NCT-503, showed strong anti-leukemic effects in OCI-AML3-R with an IC ₅₀ of 26.17 and 16.81 μM, respectively, while the G6PD inhibitor 6-AN had limited effect. The PHGDH inhibitors increased mitochondrial ROS levels and induced apoptosis in OCI-AML3-R. Additionally, in vivo experiment showed that treatment with NCT-503 significantly prolonged mouse survival and inhibited tumor growth in a dose-dependent manner without adverse effects on body weight.

Summary/Conclusion: Transcriptomic and metabolomic analyses revealed that OCI-AML3-R upregulated PPP and SSP, leading to increased nucleotide synthesis, which contribute cell growth and survival. Targeting PHGDH could significantly suppress omipalisib-refractory in AML and may have implications for future clinical trials.