Prolyl Hydroxylase 3 (Phd3) Is a Therapeutic Target in Isocitrate Dehydrogenase 1 (IDH1) Mutated Acute Myeloid Leukemia
Authors: Chaturvedi Anuhar , Araujo Cruz Michelle Maria , Goparaju Ramya , Gabdoulline Razif , Schottmann Renate , Görlich Kerstin , Kloos Arnold , Thol Felicitas , Geffers Robert , Mougiakakos Dimitrios , Carmeliet Peter , Ganser Arnold , Heuser Michael 
Published: October 2019

Abstract
Background: About 40% of IDH1 mutated (IDH1mut) acute myeloid leukemia (AML) patients respond to IDH1 inhibitors with a median duration of response of 8.2 months. A better understanding of the biology of IDH1mut leukemia may further improve the treatment of these patients. IDH1mut produces R-2-hydroxyglutarate (R-2HG), which activates PHD1 and PHD2 but have negligible effects on PHD3. In the present study we assessed whether PHD3 plays a role in the pathogenesis of IDH1 mutated leukemia and can be targeted in a patient-derived xenograft (PDX) model of IDH1 mutated AML.
Methods: Bone marrow cells from Phdwt and Phd3ko mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt) and IDH1mut respectively, were constitutively expressed. The effects on cell proliferation, apoptosis and colony formation were evaluated in vitro, whereas the leukemic potential was evaluated in vivo by transplantation in syngeneic mice. To show that PHD3 is a therapeutic target, either IDH1mut cells from AML patients were transduced with shRNA against PHD3 and transplanted in immunocompromised mice, or leukemic cells from an AML patient with mutated IDH1 were xenografted in immunocompromised mice and treated with the PHD inhibitor molidustat.
Results: In in-vitro functional assays loss of Phd3 specifically impaired proliferation, apoptosis and clonogenic capacity of HoxA9-IDH1mut but not HoxA9-IDH1wt cells. Likewise, in mouse transplantation assays, loss of Phd3 eliminated HoxA9-IDH1mut induced leukemia. However, Phd3 was dispensable to the engraftment and proliferation of HoxA9-IDH1wt cells. Additionally, the IDH1-independent model of MN1-induced leukemia remained unaltered in the absence of Phd3, indicating the specificity of the role of Phd3 in mutant IDH1-induced transformation. To identify molecular pathways that might explain in vitro and in vivo phenotypes gene expression profiling was performed. Immune and stress-response pathways as well as metabolism-related genes were most prominently dysregulated in Phd3ko IDH1-mutant cells. Analysis of dysregulated transcription factors by gene set enrichment analysis revealed a depletion of key oncogenic transcription factors (Myc, Rb, Stk33, and Rps14) in Phd3ko IDH1mut cells compared to Phd3ko IDH1wt cells. To study if IDH1mut signals to Phd3 through R-2HG, we transduced Phd3kocells, with a splice variant of mutant IDH1, which does not produce R-2HG but causes leukemia in mice with similar kinetics as in mice with the full-length IDH1 mutant protein. Interestingly, loss of Phd3 also eliminated leukemia in these mice, which demonstrates that mutant IDH1 signals through Phd3 independently of R-2HG. To study the functional relevance of PHD3 inhibition in patients, cells from an IDH1 mutated AML patient were transduced with an shRNA against PHD3 and were transplanted in immunodeficient NSG mice. Inhibition of PHD3 depleted human AML cells in the IDH1-mutated PDX model. Moreover, the PHD inhibitor molidustat was 50-fold more active in IDH1mut (80 nM) compared to IDH1wt AML patient cells (4000 nM) in colony-forming unit assays. In a xenograft model of IDH1 mutated AML, molidustat significantly prolonged survival compared to control-treated mice (P<.001).
Conclusion: We demonstrate that the leukemogenic activity of the mutant IDH1 protein depends on PHD3 independently of R-2HG. We identified inhibition of PHD3 as a novel therapeutic strategy in IDH1 mutated AML. Since PHD3 can be targeted pharmacologically, combinatorial treatment of PHD3 and IDH1 inhibitors is warranted to improve eradication of leukemic stem cells in IDH1 mutated AML.
#AC and MMAC share first authorship

Disclosures
Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Karyopharm: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.