The Rorɣ/SREBP2 Pathway Is a Master Regulator of Cholesterol Metabolism and Serves As Potential Therapeutic Target in T(4;11) Leukemia

Abstract

Chromosomal translocations involving the KMT2A gene (KMT2Ar leukemia) occur in nearly 5 to 10% of acute leukemia and are associated with an aggressive course of disease and poor prognosis. The most common translocation partner of the KMT2A gene is the AF4 gene inducing a t(4;11) leukemia. It is known that a variety of cancer cells change their cholesterol homeostasis among other metabolic reprogramming leading to increased proliferation and survival. Therefore, dysregulated cholesterol metabolism promotes not only tumorigenesis and chemotherapy resistance, but also constitutes a new hallmark of cancer. The importance of understanding the connection between metabolic changes and leukemia development is necessary to identify potential targets and to develop new therapeutic strategies. By using our recently established CRISPR/Cas9-generated t(4;11) model, we identified the sterol regulatory element-binding protein 2 (SREBP2) as key transcription factor being highly upregulated. RAR-related orphan receptor gamma (RORɣ) has been reported to be a possible upstream regulator of SREBP2 but less is known about its function in the metabolic development of KMT2Ar leukemia and its role as therapeutic target. SREBF2 gene expression, measured via RT-qPCR, showed a strong upregulation in leukemic blasts of KMT2Ar patients, cell lines (SEM, RS4;11, MV4-11) and in our t(4;11) model. Furthermore, we found that the significant upregulation of SREBP2 in KMT2Ar leukemia correlates with poor patient survival (gent2, bloodspot.eu). To test whether RORɣ plays a role in hematopoiesis and leukemogenesis we examined intranuclear protein expression via flow cytometry. Cells of our t(4;11) leukemia model generally showed a stable RORɣ expression around 75%, while protein expression in CD34+ hematopoietic stem and progenitor cells (HSPCs) decreased upon differentiation in myeloid culture. We detected anti-leukemic effects of the SREBP2 inhibitor fatostatin (FS), HMGCR inhibitor atorvastatin (ATV) and the newly developed RORɣ antagonist XY018. All compounds induced leukemic cell death in a dose- and time-dependent manner while significantly changing cholesterol-target gene expression (HMGCR, HMGCS1, LDLR, ABCA1, SQLE, MSMO1, FDFT1, SC5D). Strikingly, the strongest effect was observed after XY018 treatment. Moreover, FS and ATV also affected cell growth of healthy HSPCs while anti-proliferative effects of XY018 were restricted to t(4;11). In addition to reduced cell viability and increased apoptosis upon RORɣ inhibition, we observed significant changes in the cell cycle. Genome-wide transcriptomic analysis by RNA-Seq identified SREBF2 as upstream regulator of these effects. Ongoing studies will address the molecular mechanism by which RORɣ and its downstream target SREBP2 regulate t(4;11) leukemogenesis. Combinatorial treatment studies of t(4;11) leukemia cells with XY018 showed promising additive effects (CI=0.90) together with the chemotherapeutic cytarabine and synergistic effects (CI=0.44) were achieved by adding ATV. In summary, our studies showed that t(4;11) leukemia cells exhibit an activated SREBP2-mediated cholesterol metabolism strongly linked to the nuclear receptor RORɣ. Moreover, we demonstrated that this metabolic reprogramming could serve as a therapeutic target with co-treatment of both cholesterol-modulating agents and common cytostatic drugs to improve the poor outcome of KMT2Ar leukemia patients.