Altered metabolism has long been recognized as a signature finding of malignant cells with changes in glucose handling termed the ‘Warburg effect’. Now recognized as an adaptation of metabolic pathways to meet the distinctive demands of malignant cells, metabolism represents a potential area of opportunity for developing novel therapeutics. We established a system by which a wide spectrum of metabolic pathways could be examined for differential activity in tumor cells and their normal counterparts in the context of acute Myeloid Leukemia (AML).
We studied metabolism using a biased shRNA library comprising 1000 shRNAs on a well-defined population of leukemia stem or initiating cells (LICs) in a model of AML driven by the MLL-AF9 oncogene (Krivtsov, 2006) and performed identical assays on the normal counterparts of LICs called normal granulocyte monocyte precursors (N-GMPs). The shRNAs chosen, targeted canonical ‘rate limiting enzymes’ as well as those enzymes which were found to be differentially expressed in LICs and N-GMPs by gene expression profiling. Both LICs and N-GMPs were maintained in the presence of murine bone marrow mesenchymal cells in order to mimic the native micro-environment in which these cells grow. To maximize physiologic relevance we used primary mouse LICs, N-GMPs and bone marrow mesenchymal cells, instead of cell lines, which may have distinct metabolic dependencies secondary to their adaption to culture conditions.
Our screen has identified Aldehyde Dehydrogenase 3a2 (Aldh3a2 or Fatty Aldehyde Dehydrogenase, FALDH) as a key target gene. Aldh3a2 depletion in LICs with three independent validated shRNAs resulted in improvement in survival of transplanted mice (p value of 0.0004 to 0.0018) and significantly decreased growth of cells in methylcellulose soft agar assays and in co-culture with primary bone marrow stroma. In a knock-out mouse model of Aldh3a2, normal hematopoiesis as assessed by immunophenotype seems unperturbed.
Using Tumorscape, the Broad Institute's database on gene copy number variation in tumors, we showed that Alh3a2 is significantly focally amplified (Q value =1.39−5) across the entire dataset of 3,131 tumors, including hematologic malignancies and is located within a focal peak region containing 11 other genes (chromosome 17:19117656–19932585). We have also probed a gene expression dataset of 436 samples obtained from two different multicenter clinical trials: the AMLSG HD98a trial (patients < 60 years of age) and the AMLSG HD98b trial (patients > 60 years of age). Analysis revealed significant overexpression of Aldh3a2 in MLL-AF9 samples compared to all other AML subtypes (p< 0.0006 by ANOVA). Patients in the highest quartile of Aldh3a2 expression had a significantly prolonged overall survival compared to those in all other quartiles (p=0.038). Whether this survival association is due to increased dependency on Aldh3a2 and sensitivity to chemotherapy is unclear, but an issue for future experiments.
We have observed increased apoptosis, decreased cycling and altered ROS in L-GMPs with knock-down of Aldh3a2. Metabolomic experiments to identify the precise metabolites which are responsible for the differential sensitivity of LICs and normal hematopoietic cells to Aldh3a2 depletion are ongoing.
Our work is aimed at confirming the metabolic dependency of leukemia on Aldh3a2 and dissecting the biology of this enzyme in the context of this disease. We hope this will lead to the development of novel therapeutic strategies and provide the groundwork for the further study of leukemia metabolism.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.