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Acute myeloid leukemia

Creatine kinase pathway inhibition alters GSK3 and WNT signaling in EVI1-positive AML

Leukemia volume 33, pages 800–804 (2019)Cite this article

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Over the past decade, several inhibitors of key metabolic dependencies have been shown to be active in AML (Acute Myeloid Leukemia), suggesting that targeting cellular metabolism is a promising therapeutic strategy for this disease. Specific inhibitors of the TCA cycle isoenzymes IDH1 and 2, mutated in approximately 20% of AML cases, abrogate the myeloid differentiation block and restore normal granulocytic/neutrophilic differentiation of IDH1/2-mutated AML blasts [1, 2]. Moreover, lonidamine and 2-deoxy-D-glucose, two small molecules that inhibit the first rate-limiting step of glycolysis, have been demonstated to have potent anti-leukemic activities in AML cells in preclinial studies and are now being tested in early phase clinical trials [3, 4]. While the inhibition of metabolic enzymes alters metabolite steady-state levels, it also impacts the activity of other downstream signaling pathways. For instance, glycolysis inhibition by lonidamine is associated with activation of the MEK/ERK pathway, which in the context of AML, could counteract the anti-leukemic effect of glycolysis inhibition [3]. This example suggests that it is essential to identify both the favorable effects and the potential liabilities of metabolic perturbations on downstream signaling pathways in order to (i) prevent potential resistance mechanisms to inhibition of the metabolic targets and (ii) nominate combination therapies that may synergize with metabolic inhibitors.

Next, we performed single-sample Gene Set Enrichment Analysis (ssGSEA) in two large human primary patient AML gene expression datasets Wouters et al. (n = 526, GSE14468) and Tomasson et al. (n = 304, GSE10358). In both datasets, a significant correlation was observed between the transcriptional signatures of GSK3 inhibition and CK pathway inhibition (Fig. 1e, f and S1A). In line with these results, the level of GSK3 phosphorylation at the inhibitory serine sites of the two GSK3 isoforms (S21 on GSK3A and S9 on GSK3B) was strongly enhanced upon cyclocreatine treatment, confirming the inhibition of GSK3 in EVI1-positive AML cell lines and primary patient samples (Fig. 1g, h). Interestingly, the basal level of S21/S9 GSK3 phosphorylation was higher in Kasumi-3, compared to the other EVI1-positive cell lines, suggesting less constitutive activation of GSK3 at baseline. Despite this high basal level of phosphorylation, GSK3 was further phosphorylated upon cyclocreatine treatment in Kasumi-3. Intracellular glycogen accumulation, which was previously reported as a direct phenotypic consequence of GSK3 inhibition [6], was also observed both by Periodic Acid-Schiff positive staining and glycogen dosage in cyclocreatine-treated AML cell lines and primary patient samples (Figs. S1C, 1I and 1J). Moreover, we previously reported that cyclocreatine treatment induced cell cycle arrest in AML cells [5], and the cyclin-dependent kinase inhibitor p27Kip1 is a critical downstream mediator of the cell cycle arrest associated with GSK3 inhibition [7]. Consistent with these reports, cyclocreatine treatment increased p27Kip1 levels in the EVI1-positive AML cell line TF-1 (Figure S1D).

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Acknowledgements

We thank Dr. Kevin Shannon for advice and discussions. We thank the Saint-Louis Hospital’s Tumor Biobank (Daniela Geromin, Carole Albuquerque) for managing patient AML samples. This research was supported with grants from the US National Cancer Institute (NCI) (NIH R35 CA210030; KS), the Children’s Leukemia Research Foundation (CLRF), and the Bridge Project, a collaboration between the Koch Institute for Integrative Cancer Research at MIT and the Dana-Farber–Harvard Cancer Center (DF–HCC) (KS and MTH). KS is a Leukemia and Lymphoma Society Scholar. AP is a recipient of support from the ERC Starting Grant (H2020), the ATIP–AVENIR and LNCC French research programs, the EHA research grant for a Non-Clinical Advanced fellow, and is supported by the St. Louis Association for leukemia research. LB is an MD-PhD candidate of “Ecole de l’INSERM Liliane Bettencourt” and a recipient of Philippe Foundation and GPM fellowships.

Author contributions

LB developed the study, established conditions for in vivo and in vitro experiments, acquired and analyzed the data, and wrote the manuscript. GA performed statistical analysis, biostatistics, and computational analysis of the RNAseq data and mined publicly available databases. AS performed in vitro experiments. ER, JS, and RI provided patient samples. OH and MTH designed experiments and helped interpreting data. AP and KS supervised the study, wrote and revised the manuscript, designed the in vitro and in vivo experiments and analyzed the data, provided material and funding support for the study.

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  1. These authors contributed equally: Kimberly Stegmaier, Alexandre Puissant.

Authors and Affiliations

  1. Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Lina Benajiba, Gabriela Alexe & Kimberly Stegmaier

  2. The Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA, USA

    Lina Benajiba, Gabriela Alexe & Kimberly Stegmaier

  3. INSERM U1163 and CNRS 8254, Imagine Institute, Université Paris Saclay, Paris, France

    Lina Benajiba & Olivier Hermine

  4. Bioinformatics Graduate Program, Boston University, Boston, MA, USA

    Gabriela Alexe

  5. INSERM UMR 944, Institut Universitaire d’Hématologie, Hôpital St. Louis, Paris, France

    Angela Su, Raphael Itzykson & Alexandre Puissant

  6. Département d’Hématologie, Hôpital Saint-Louis, Assistance Publique – Hopitaux de Paris, Paris, France

    Emmanuel Raffoux, Jean Soulier & Raphael Itzykson

  7. Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Michael T. Hemann

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Correspondence to Kimberly Stegmaier or Alexandre Puissant.

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Benajiba, L., Alexe, G., Su, A. et al. Creatine kinase pathway inhibition alters GSK3 and WNT signaling in EVI1-positive AML. Leukemia 33, 800–804 (2019). https://doi.org/10.1038/s41375-018-0291-x

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