Restoration of ceramide de novo synthesis by the synthetic retinoid ST1926 as it induces adult T-cell leukemia cell death

Abstract

Ceramide is a potently bioactive cellular lipid with compartmentalized and tightly regulated levels. Distinct metabolic pathways lead to the generation of several ceramide species with distinguishable roles in oncogenesis, cancer therapy, and the stress response. Deregulation of ceramide pathways has emerged as an important mechanism for acquired chemotherapeutic resistance and disease progression. In fact, decreased cellular ceramide seems to be a hallmark of innate hemotherapy resistance. Adult T cell leukemia/lymphoma (ATL) is an aggressive neoplasm that develops following infection with Human T cell Lymphotropic Virus-1 (HTLV-1) where the viral oncogene Tax largely contributes to the pathogenesis and progression of the disease. Given the fact that ATL, similar to most blood cancers, displays a genetically heterogeneous clonal profile with extensive chemotherapy resistance, alternative therapies that could restore lethal levels of ceramide might have potential promise. In this context, retinoids could be used alone or as an adjuvant treatment to trigger a ceramide-centered approach to cancer therapy. We have previously reported that the synthetic retinoid ST1926 is a potent inducer of growth inhibition and massive apoptosis in malignant T cells and that ATL cells are defective in ceramide synthesis. The perturbation of ceramide pathways through which Tax exerts its tumor promoting functions in ATL remains poorly defined. At the mitochondrial level, the intrinsic pathway of apoptosis is tightly regulated by the Bcl-2 family members and orchestrated by the bioactive sphingolipid ceramide.

We investigated the effect of ST1926 on ceramide accumulation in malignant T cells, modulation of ceramide and dihydroceramide species, mechanisms of ceramide production, ceramide synthases (CerS) activity, gene, and protein expression levels, and cell death and ceramide response in a Bcl-2 overexpression leukemia model. HTLV-1 positive and negative cell lines were treated with ST1926 to determine the doseresponse and kinetics of ceramide accumulation. Total cellular ceramide was measured using the diacylglycerol kinase assay. De novo ceramide synthesis was determined by measuring [3H] labelled palmitic acid incorporation into ceramide. Sphingolipidomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS). The activities of CerS(s) and dihydroceramide desaturase enzymes were indirectly measured by labeling cells with the unnatural 17-carbon sphinganine followed by LC-MS measurement of the resulting 17C-backbone products.

ST1926 treatment results in early Tax oncoprotein degradation in HTLV-1 treated cells. ST1926 induces cell death and a dose- and time-dependent accumulation of ceramide in HTLV-1 positive and negative malignant T cells. The kinetics and degree of ceramide production shows an early unique response upon ST1926 treatment versus previous synthetic retinoids such as HPR and CD437. Similarly, Tax degradation is unique to ST1926. Compared to HPR, which leads to the preferential accumulation of dihydroceramide, ST1926 enhances de novo ceramide synthesis via activation of CerS without inhibiting dihydroceramide desaturase, thereby accumulating ceramide rather than the less bioactive dihydroceramide. We showed that ST1926 preferentially induces the activities of a distinct set of CerS(s). Treatment with ST1926 increases gene expression of CerS(s) more prominently in HTLV-1 positive cells. Since ceramide synthesis follows ST1926-induced degradation of Tax, this raises the possibility that Tax is responsible for the ceramide synthesis defect in ATL cells. We detected a delay in cell death response and interruption of ceramide generation in response to ST1926 in Molt-4 cells overexpressing Bcl-2. These results highlight the potential role of ST1926 in inducing ceramide levels, thus lowering the threshold for cell death in malignant T cells.