Abstract A04: Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy
Authors: Xie Stephanie Z. , Laurenti Elisa , Ferrari Robin , Dick John E. 
Published: May 2016

Metabolic alterations are a cancer hallmark that are typically evaluated in bulk tissues. However most normal tissues and cancers are hierarchically organized and the metabolic requirements of both normal and cancer stem cells are poorly understood, particularly beyond energy metabolism. By analyzing a comprehensive transcriptional roadmap of human hematopoiesis, and comparing this to a leukemia stem cell (LSC) signature developed from 84 human acute myeloid leukemia (AML) samples, we found that: (1) several metabolic pathways, specifically in bioactive lipids, distinguish normal hematopoietic stem cells (HSC) from progenitors; and (2) while LSC are similar to HSC, specific metabolic pathways are more comparable to those of normal progenitors. We defined a lipid stem signature of 24 lipid genes, including sphingolipid genes, whose expression is higher in HSC than progenitors. Interestingly, sphingosine-1-phosphate (S1P) is known to play a role in HSC egress, and ceramide vs S1P levels serve as a rheostat to regulate cell growth and survival. To determine if sphingolipids play a functional role in the primitive hematopoietic compartment, we altered sphingolipid signaling by plating sorted populations of HSC or granulocyte-myeloid progenitors (GMP) in methylcellulose containing myriocin, which inhibits the first step of de novo sphingolipid synthesis, or FTY720, a S1P mimetic. Myriocin decreased GMP colony output but did not affect CFC derived from HSC. By contrast FTY720 affected HSC-derived CFC but not those from GMP, suggesting differential sensitivity to sphingolipid pathway inhibition between stem and progenitor cells. In vitro treatment of lineage depleted cord blood (Lin- CB) with myriocin for 8 days limited only myeloid differentiation compared to control treated cells. In contrast, FTY720 treatment reduced levels of immunophenotypic stem cells, erythroid and myeloid cells, as would be expected with inhibition of S1P proliferative signaling. However, in vitro FTY720-treated Lin- CB cells exhibit 16-week engraftment capacity comparable to that of controlled-treated cells, suggesting that any effects on HSC function are reversible after drug withdrawal. Furthermore, in vivo treatment with FTY720 in mice with established CB grafts did not decrease engraftment.

Our lipid stem signature is enriched in LSC gene expression profiles from our 84 AML cohort by GSEA analysis, suggesting that differences in lipid metabolism may also exist in LSC vs. non-LSC. To determine if sphingolipids play a functional role in AML biology, we transplanted mice with peripheral blood cells from 13 AML patients, including those with therapy resistant and relapsed disease, and treated engrafted mice with myriocin or FTY720. We observed heterogeneous responses in our cohort, with reduction of leukemic burden in 3 and 5 samples following treatment with FTY720 and myriocin, respectively. Remarkably, serial transplantation of FTY720 responders into untreated secondary mice at limiting dilution demonstrated decreased LSC frequency in FTY720-treated primary mice compared to vehicle-treated controls, whereas myriocin responders showed no alteration of LSC frequency. These results suggest that FTY720 but not myriocin treatment affects LSC number and/or function. To stratify responders from nonresponders, we performed transcriptional analysis of untreated patient samples and compared these data to transcriptional signatures generated from the normal and AML hierarchy. Bioinformatic analysis demonstrated that FTY720 responders had an enriched LSC signature compared to nonresponders. In contrast, myriocin responders exhibited a strong GMP signature compared to nonresponders. Thus, normal human hematopoietic stem and progenitor cells display a variable dependence on sphingolipid biology that is also distinct between LSC and HSC, pointing to targeting of bioactive sphingolipids as a novel therapeutic strategy in AML to eradicate LSC while sparing HSC.

Citation Format: Stephanie Z. Xie, Elisa Laurenti, Robin Ferrari, John E. Dick. Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A04.