25(OH) Vitamin D₃ Affects Acute Myeloid Leukemia Cell Proliferation

We recently demonstrated that acute myeloid leukemia (AML) patients with insufficient serum 25(OH) vitamin D₃ levels had a worse outcome following intensive induction therapy. However, the mechanisms by which 25(OH) vitamin D₃ and its metabolites exert these effects are poorly understood. We utilized in vitro systems with orthogonal genomic and computational approaches to delineate leukemia-specific vitamin D molecular targets that may contribute to its activities. To achieve this goal, we systematically investigated vitamin D anti-proliferative effects using a genomically-annotated panel of human leukemia cell lines, including lines with genetic alterations that are commonly observed in patients. Since the genetic diversity of these cell lines reflects many of the alterations found in AML patients, we hypothesized that they collectively represent a model system to replicate candidate anti-leukemia drug effects. Specifically, we cultured 40 leukemia cell lines with and without 25(OH) vitamin D₃ in a dose- and time-dependent manner for 72 hours and used high-throughput (HT) cell-based assays to simultaneously measure multiple modalities/ parameters related to cell proliferation and cytotoxicity (i.e. combining several HT assay chemistries to simultaneously assess cell number, viability, cytotoxicity and caspase activation events). Six AML cell lines (THP-1, MV4;11, MOLM-6, MOLM-13, EOL-1, JK-1) that responded to 25(OH) vitamin D₃ treatment were identified and selected for multi-parametric RNA-Seq studies (NB 200nM Vitamin D treatment or vehicle control for 24 hours). Total RNA was extracted and analyzed by the Illumina HiSeq 2500 sequencing platform and mRNA, microRNA and ncRNAs were unambiguously quantitated. Utilizing gene expression profiles and in silico computational modeling of canonical or putative vitamin D receptor elements (VDRE) transcription factor promoter binding sites, we (1) identified a conserved/core gene transcriptional signature and (2) subsequently developed a vitamin D – genetic inference model. Gene enrichment analysis of the inferred network model identified genes involved in abnormal hematopoiesis, proliferation and differentiation, interleukin secretion, leukocyte migration, and bone marrow cell morphology and/or development. Notably, our data identified several 25(OH) vitamin D₃ transcriptional regulatory cascades that converge on ETS2, JUND, NCOA4, RXR4, WT1, and ZFP30 transcription factors. Furthermore, we also demonstrate temporal and spatial regulation of protein kinases CAMK1 and FGFR, cytokine and growth factors (cAMP, CCL2, IL-8 and TNF), and cell differentiation markers (CD14, CD97, FCGR2A, IL3RG, IL7R, ITGAM, THBD). In summary, these data suggest that 25(OH) vitamin D₃ exerts both direct anti-proliferative effects on leukemia cells and direct/indirect leukemia inhibitory microenvironment reprograming activities and better clarify the complex molecular mechanisms underlying the therapeutic activity of 25(OH) vitamin D₃ in AML.