Anti-Inflammatory Response to Anti-Folate and Vitamin D Analog Combination in Large Granular Lymphocyte Leukemia

Abstract

Large granular lymphocyte (LGL) leukemia is a rare chronic hematological disorder characterized by clonal expansion of circulating cytotoxic T or natural killer (NK) cells. The disease is associated with a number of comorbidities including neutropenia, anemia, and autoimmune diseases. There is no standard of care or curative therapy for LGL leukemia. Instead, it is treated when symptoms arise with a variety of immunosuppressive therapies, chief among them, the anti-folate, methotrexate (MTX). However, a significant proportion of patients remain refractory to treatment, and little characterization has been done regarding MTX's mechanism in vitro. Thus, there is a need to identify new therapeutic approaches such as stronger anti-folates or combination of MTX with existing therapies. Pralatrexate (PDX) is a more potent analog of MTX, not yet utilized in LGL leukemia. It is approved for treatment of relapsed or refractory peripheral T cell lymphomas and may offer a promising new avenue for therapy. Vitamin D is a natural supplement that we have shown to impact key LGL leukemia signaling pathways in vitro by decreasing cytokine levels and STAT1 and STAT3 signaling, all of which are typically elevated in LGL leukemia. Treatment with vitamin D also increases levels of vitamin D receptor (VDR), a known transcriptional controller of reduced folate carrier (RFC), the method of cellular entry for both MTX and PDX. Therefore, we hypothesized that treatment with EB1089, a synthetic vitamin D analog, would sensitize leukemic LGLs to anti-folates, PDX and MTX, by leading to a VDR-mediated increase in RFC thus allowing increased MTX/PDX uptake and therapeutic efficacy. In this study, single MTX treatment of LGL leukemia model cell lines (NKL and TL-1) demonstrated strong decreases in viability at 72h time points (EC50 NKL 10.5nM, TL-1 15.4nM). Single agent PDX effects on viability were even stronger (4-11 fold lower EC50) (Figure A). Key LGL leukemia signaling pathways like STAT phosphorylation were lowered as early as 24h after high dose treatment with MTX (10 µM) in TL-1 cells, and VDR was also lowered by MTX single high dose treatment. When MTX or PDX was given in combination with EB1089, critical inflammatory signaling, such as phosphorylation of STAT1 and STAT3, was attenuated, while VDR was stabilized from anti-folate mediated decrease. Dose dependent changes to STAT3 regulation, RFC, VDR, and PTPN2, a phosphatase specific to STAT1 and STAT3, occurred as early as 6h post combinatorial treatment with a range of MTX dosages (1, 10, 100 nM) in the NK cell line model. A similar anti-inflammatory response in PTPN2, STAT1, and STAT3 levels was observed at 24h following co-treatment in TL-1 cells (Figure B). Additionally, treatment with a combination of EB1089 and PDX (0.1, 1, 10 nM; 10% of the MTX doses) led to anti-inflammatory response with decreases in STAT1 activation and increases in RFC in the TL-1 cell line. In short term assays, there were no measurable changes in anti-folate mediated cell killing when the drugs were combined across cell lines and different dosages of the combination. Taken together, these data suggest a strong single agent potency for PDX and an anti-inflammatory benefit to MTX/PDX and EB1089 combination. Continued mechanistic study of the anti-folate/vitamin D receptor agonist pairing as well as further investigation into PDX's impact on LGLs will be crucial to follow up investigations.