Unraveling Metabolic Dependencies and Molecular Mechanisms in CNS ALL: The Role of MiR-181a and VEGF

Acute lymphoblastic leukemia (ALL) of the central nervous system (CNS) is characterized by leukemia cells residing in the nutrient-low and oxygen-poor leptomeningeal compartment. Therapy directed to the CNS is essential for effective disease management and long-term survival of pediatric ALL patients. However, ALL treatment regimens are associated with neurotoxicities and long-term sequelae contributing to a reduced quality of life for leukemia survivors. To advance the development of novel and less toxic CNS-directed therapies, it is important to understand the molecular pathways contributing to the manifestation of leukemia cells in the CNS.

Recently, we identified vascular endothelial growth factor A (VEGF) as a regulator for CNS ALL aiding leukemia cell transmigration into and adaptation to the oxygen- and nutrient-low CNS niche. MiR-181a regulates genes involved in energy metabolism including reprogramming of glucose metabolism, ß-oxidation, and lipid synthesis. Moreover, miR-181a was reported to be a biomarker for CNS ALL and its expression was positively associated with VEGF levels in cerebrospinal fluid of ALL patients. Interestingly, miR-181a has been demonstrated to regulate VEGF expression in other cancers.

In this study, we evaluated the role and association of miR-181a and VEGF in CNS ALL using a xenotransplantation model and performing in vitro experiments modeling the CNS niche by nutrient deprivation.

First, we assessed the expression of miR-181a and VEGF in 22 human ALL patient-derived xenograft (PDX) samples isolated from CNS, spleen, and bone marrow (BM). In line with previous data, we identified a significant upregulation of VEGF expression in CNS- compared to BM- and spleen-derived ALL cells. Moreover, we observed a significant correlation between miR-181a and VEGF expression in PDX ALL samples being concordant with previous data in cerebrospinal fluid of ALL patients. To further investigate this association, we transfected ALL cell lines with miR-181a mimics resulting in increased levels of miR-181a and simulated the CNS microenvironment through nutrient deprivation (FCS starvation). Upon increased miR-181a, the transmigration of ALL cell lines was not affected in vitro. However, we identified increased VEGF secretion upon high miR-181a levels, and, importantly, this effect was only observed in nutrient-low culture conditions resembling the CNS microenvironment. Next, we analyzed protein expression levels and phosphorylation status of genes, which have been previously linked to miR-181a-VEGF signaling in chondrosarcoma (SRCIN1, pSRC). However, in PDX ALL samples obtained from different organ compartments and in cell lines harboring increased miR-181a levels by mimic transfection, the expression and phosphorylation were not altered independent of organ compartment and nutrient content. This suggests that in ALL the positive association between miR-181a and VEGF is mediated by different molecular mechanisms.

Given that miR-181 controls metabolic regulators, we investigated whether ALL cells residing in the CNS are characterized by specific metabolic programs compared to those derived from the BM. Therefore, we performed functional analyses (Seahorse XF Mito Fuel Flex Test) on using different metabolites (glutamine, glucose, long-chain fatty acids) for energy production on ALL cells isolated from CNS and BM (n=4 PDX samples). The results revealed a high flexibility in switching metabolic pathways among the ALL cells suggesting that leukemia cells possess the ability to adapt to environments with low nutrient availability, enabling their survival in nutrient-deprived compartments.

In sum, we identified that miR-181a levels and VEGF expression are positively correlated in PDX ALL samples and that VEGF secretion is regulated by miR-181a exclusively in culture conditions simulating the CNS niche. Moreover, leukemia cells demonstrate a significant adaptability in using various metabolites depending on their availability at the site of ALL manifestation. These findings highlight the microenvironmental influence on miR-181a and VEGF expression in contributing to CNS ALL features in vivo. Although previously identified miR-181a target genes are not affected in ALL, it is crucial to identify genes interposed to VEGF secretion and to investigate the miRNA-VEGF signaling pathway to establish novel CNS-directed drugs.