Polyamines Are Required for AML LSC Function through Their Role in Regulating eIF5A Dependent Protein Synthesis

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Many studies have demonstrated that LSCs have unique metabolic properties compared to AML blasts and normal hematopoietic stem and progenitor cells (HSPCs) (Jones et al., 2021). In this study we sought to quantify metabolite levels in LSCs compared to HSPCs using an unbiased metabolites screen. To examine metabolite levels in enriched LSCs and HSPCs we used mass spectrometry-based metabolomics analysis on enriched LSCs from 18 AML patients and HSPCs from 5 normal bone marrow (BM) specimens. Pathway analysis showed that arginine metabolism and biosynthesis were the most significantly enriched pathways in LSCs compared to HSPCs.

Clinical studies have shown limited efficacy of targeting arginine levels using arginine degrading enzymes; therefore, we sought to target pathways downstream of arginine. To identify the pathways that arginine metabolism supports in LSCs, we used stable isotope labeled tracing analysis which showed arginine was metabolized through the urea cycle into polyamines in LSCs. Interestingly, the polyamine spermidine was the most significantly enriched metabolite in LSCs compared to HSPCs in our metabolomics analysis. Moreover, expression of SAT1, the enzyme catalyzing the export of polyamines from the cells, was decreased in AML compared to normal BM. Based on these observations, we sought to determine if the polyamine pathway represents a metabolic vulnerability that can be exploited to target LSCs. Polyamines are cationic compounds which have been previously shown to be important for the growth of various cancers but their role in AML LSCs has yet to be determined. To investigate if polyamines are essential for LSC survival and function, we treated LSCs and HSPCs with N¹,N¹¹-diethylnorspermine (DENSpm), a polyamine analog causing polyamine depletion through increased SAT1 expression and subsequent cellular export. DENSpm treatment decreased the viability of LSCs in 10 out of 12 of patients tested but had no impact on HSPCs viability. Further, treatment with DENSpm resulted in decreased engraftment potential of 5 AML specimens but did not alter the engraftment of HSPCs from 3 normal BM specimens, suggesting that targeting polyamines represents a promising approach to kill LSCs while sparing HSPCs.

We then investigated the mechanisms by which polyamines are essential in LSCs. Using RNA-sequencing analysis on enriched LSCs from 5 AML patients and HSPCs from 3 normal BM specimens we observed an enrichment in myeloid differentiation signatures in DENSpm-treated LSCs but not in DENSpm-treated HSPCs. In line with this, expression of myeloid markers CD11b and CD15 were increased in patient-derived xenograft models engrafted with DENSpm-treated LSCs. As we did not observe such dysregulation in mice engrafted with DENSpm-treated HSPCs, these data suggest polyamine depletion promotes differentiation of LSCs but not HSPCs.

Our transcriptomic analysis also unveiled decreased expression of genes involved in protein synthesis in DENSpm-treated LSCs but not in normal HSPCs. Protein synthesis and its regulation are critical for AML survival (Chen et al., 2019; Messling et al., 2022) but the role of polyamines in protein synthesis in LSCs has not been explored. Polyamines stimulate translation notably by serving as precursors for the hypusination of eukaryotic translation initiation factor 5A (eIF5A) (Park et al., 2010). Accordingly, DENSpm led to decreased hypusination of eIF5A in AML cells. This was associated with reduced protein synthesis level in AML cell lines and LSCs but not in normal HSPCs. Strikingly, eIF5A hypusination, protein synthesis and viability were rescued upon co-treatment with spermidine. Moreover, knockdown of eIF5A using siRNA resulted in impaired colony forming potential of AML and altered engraftment in patient-derived xenograft models. Altogether, these results suggest that DENSpm alters AML cell and LSC viability and function at least in part through decreased eIF5A-mediated protein translation.

Altogether, our data demonstrates that depletion of polyamines decreased LSC function while sparing normal HSPCs. Mechanistically, our data suggest that LSC targeting is accomplished through decreased eIF5A-mediated synthesis of proteins crucial for LSCs.