ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
ZBTB7A is a transcription factor with function in hematopoietic lineage fate decisions (reviewed in Lunardi et al., 2013, Blood). Moreover, ZBTB7A has been shown to also be involved in regulation of glycolysis in solid tumors (Liu et al., 2014, Genes Dev.). Recently, we found ZBTB7A frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. What is more, high expression of ZBTB7A correlated with better clinical outcome in cytogenetically normal AML (Hartmann et al., 2016, Nat Commun). The functional role of ZBTB7A and its alterations in myeloid malignancies, however, remains unclear, especially concerning the regulation of leukemia metabolism. To investigate the effect of ZBTB7A mutations in leukemia, we generated K562 ZBTB7A knockout (KO) cells using CRISPR/Cas9, followed by RNA-Seq in KO and control cells. Thereby, we confirmed de-repression of the previously reported ZBTB7A target gene SLC2A3 (glucose transporter 3) as well as upregulation of several other glycolysis related genes (PGM2, PGM3, SLC2A1 and ENO2). ZBTB7A binding to all of these candidate target genes was validated using publicly available ChIP-Seq data from K562 cells (ENCSR000BME). Interestingly, Gene Set Enrichment Analysis revealed deregulation of distinct pathways related to fatty acid metabolism in this model (Figure 1A). KO cells overexpress genes involved in the fatty acid beta oxidation pathway (ACAA2, ACOX1, ACSL1, ACADVL, CPT1A and CPT1B) as well as genes related to other fatty acid metabolism (EPHX2, FADS2 and others) (Figure 1B). We could further validate ACAA2, ACOX1, ACADVL and CPT1A as direct ZBTB7A targets using ChIP-Seq data. Of special interest are CPT1A and CPT1B, which are targetable through Etomoxir treatment. KO cells showed an increased sensitivity to this drug compared to control (IC50= 120.6 and 125.5 µM in KOs vs 228.4 µM in control, p<0.0001). Moreover, analysis of RNA-Seq data from patients with AML t(8;21) revealed a significantly higher expression of EPHX2 (p=0.049), FADS2 (p=0.003) and FASD1 (p=0.021) in patients harboring ZBTB7A mutations (Figure 1C) using a two-tailed unpaired Student's t-test. In order to evaluate our findings on a functional level, we performed metabolic flux assays in K562 ZBTB7A KO vs control cells. Using Seahorse technology (Agilent), we found that KO cells show a modest increase in extracellular acidification rate (ECAR), indicating a higher glycolysis. This effect becomes more obvious after mitochondrial respiratory chain inhibition: 41.20 and 51.66 mpH/min in KO clones vs 34.33 mpH/min in control (p=0.002 and p<0.001, respectively) (Figure 1D). This result suggests that loss of ZBTB7A may confer an advantage to cells in specific microenvironment with low oxygen availability, such as the bone marrow. Moreover, metabolic flux assays also revealed a nearly 50% increase in oxygen consumption rate (OCR) in KO cells after 1h glucose starvation: 140.53 and 148.53 pmol/min in KO clones vs 96.46 pmol/min in control (p<0.001 in both comparisons) (Figure 1E). Since the cells were deprived from glucose, the observed oxygen consumption may arise mainly from glutamate metabolism or fatty acid oxidation. Deprivation from glutamate reduced overall OCR but KO cells still showed increased oxygen consumption compared to control. These results therefore suggest that an increased beta oxidation of fatty acids leads to the higher OCR observed in KO cells. In summary, we have demonstrated that the previously described role of ZBTB7A as a regulator of glycolysis in solid tumors is also relevant in myeloid malignancies. In addition, we identified the beta oxidative pathway and fatty acid synthesis as novel mechanisms underlying the perturbed function of ZBTB7A in tumor metabolism. ZBTB7A downregulation or mutation may lead to an increased energy production providing an advantage to leukemia cells. These findings likely have therapeutic implications, as metabolic inhibitors such as 2-deoxy-d-glucose and Etomoxir may specifically target ZBTB7A deficient malignancies. Figure Disclosures Hiddemann: Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria.
ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood. To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b). To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562. Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation. To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation. With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells. In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. Disclosures Hiddemann: F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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