Abstract NG07: Targeting polyamines metabolism in acute myeloid leukemia stem cells
Authors: Rondeau Vincent , Culp-Hill Rachel , O’Brien Cristiana , Berman Jacob , Reisz Julie A. , Yendi Duhan , Ling Tianyi , Janangiri Soheil , Tikhonova Anastasia , D’Alessandro Angelo , Jones Courtney 
Published: April 2023

Abstract
Acute myeloid leukemia (AML) is a devastating hematologic malignancy diagnosed in over 20,000 patients each year in the United States. The five-year survival rate in adult AML is less than 30%, highlighting the need for novel therapies to treat this malignancy. Current therapeutic approaches fail in part due of their inability to fully eradicate the disease-initiating leukemia stem cells (LSCs). Our previous work and the work of many others has demonstrated that LSCs have unique metabolic dependencies compared to the more differentiated AML blasts population. Importantly, these metabolic differences can be targeted to specifically eradicate the LSC population. To date, limited studies have examined the difference in metabolism between human LSCs and their normal counterparts, hematopoietic stem cells (HSCs), at the level of individual metabolites. To address this gap in our knowledge, we performed an unbiased mass spectrometry-based metabolomics analysis hematopoietic stem and progenitor cells (HSPCs) enriched from the bone marrow of 7 healthy donors and LSCs enriched from 18 primary AML patient specimens. This analysis revealed 38 metabolites that were significantly different between LSCs and HSPCs (FDR&lt;0.05) including several amino acids, nucleotides, and TCA cycle intermediates. Pathway analysis revealed that the most significantly enriched pathways between LSCs and HSPCs is arginine biosynthesis (FDR =9.99 × 10−14). To determine if arginine is functionally important for LSCs biology we cultured LSCs and HSCs in arginine depleted media and measured colony forming potential which showed that arginine depletion targeted LSCs without harming HSPCs. Arginine metabolism has been widely studied in cancer, including AML, and shown to be essential for urea cycle function, polyamine synthesis, pyrimidine synthesis, and creatine metabolism. In contrast to preclinical studies, clinical trials evaluating safety and efficacy of arginine-depleting therapies in relapsed AML (NCT02899286, NCT02732184, NCT01910012) have shown limited efficacy. This is likely because cancer cells upregulate arginine biosynthesis upon arginine depletion therapy. Therefore, we hypothesized that targeting metabolic pathways downstream of arginine may have more clinical benefit. To determine what metabolic pathways arginine contributes to in LSCs, we performed tracing analysis using uniformly labeled 13C15N arginine. LSCs were enriched from primary AML specimens and incubated 100µM with L-arginine-13C6,15N4 hydrochloride for 1, 6, 12, and 24 hours. Mass spectrometry, analysis revealed that arginine is metabolized through the urea cycle (ornithine) into polyamines (putrescine, spermidine, and spermine). While polyamine metabolism has been explored in several cancer types, its role in AML LSCs is unknown. Therefore, we sought to examine the importance of polyamines in AML and LSCs by depleting polyamines from cells using DENSpm, a potent inducer of SAT1, which is responsible for spermidine and spermine acetylation, resulting in their cellular export. We observed decreased polyamine concentration and overexpression of SAT1 in AML cell lines following DENSpm treatment. Importantly, DENSpm treatment resulted in decreased viability and colony forming potential of LSCs enriched from 12 primary AML specimens but did not harm normal human HSPCs. To determine if polyamine depletion alters the function of LSC or HSPCs we measured engraftment of primary AML and healthy bone marrow into immune deficient mice, which is considered the gold-standard for assessing stem cell function. DENSpm treatment resulted in a significant decrease in engraftment in 4 primary AML specimens but did not alter engraftment levels of a normal bone marrow specimen. Finally, to determine if targeting polyamine metabolism in AML is a clinically relevant approach, we transplanted mice with primary human AML, treated the mice with 60mg/kg DENSpm for 2 weeks, and measured leukemic burden. Leukemia burden in the bone marrow was significantly decreased upon DENSpm treatment compared to vehicle treated mice. Overall, these data suggest that polyamine metabolism represents a metabolic target in LSCs. Finally, we sought to explore the molecular mechanisms by which polyamine levels are essential for LSCs but not HSPCs. To do this, we performed RNA-sequencing on LSCs and HSPCs treatment with DENSpm for 24 hours and performed gene set enrichment analysis. These data revealed that protein translation gene sets were altered in LSCs but not HSPCs. Interestingly, the polyamine spermidine regulates protein translation through its role as a precursor of hypusine, an essential modification of eIF5A. We determined that polyamine depletion through DENSpm treatment decreased hypusination of eIF5A by western blot, which was rescued by co-treatment with exogenous spermidine. Further, DENSpm treatment resulted in a decrease in protein synthesis which was determine by measuring OP-Puro incorporation. Importantly, protein synthesis was also rescued by co-treatment with spermidine. Overall, these data suggest that DENSpm reduces protein synthesis through decreased hypusination of eIF5A, resulting in effective targeting of human LSCs.
Citation Format: Vincent Rondeau, Rachel Culp-Hill, Cristiana O’Brien, Jacob Berman, Julie A. Reisz, Duhan Yendi, Tianyi Ling, Soheil Janangiri, Anastasia Tikhonova, Angelo D’Alessandro, Courtney Jones. Targeting polyamines metabolism in acute myeloid leukemia stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG07.