ABSTRACT
Inflammatory and oncogenic signaling converge in disease evolution of BCR-ABL-negative myeloproliferative neoplasms, clonal hematopoietic stem cell disorders characterized by gain-of-function mutation in JAK2 kinase (JAK2V617F), with highest prevalence in patients with polycythemia vera (PV). Despite the high risk, DNA-damaging inflammatory microenvironment, PV progenitors tend to preserve their genomic stability over decades until their progression to post-PV myelofibrosis/acute myeloid leukemia. Using induced pluripotent stem cells-derived CD34+ progenitor-enriched cultures from JAK2V617F+ PV patient and from JAK2 wild-type healthy control, CRISPR-modified HEL cells and patients' bone marrow sections from different disease stages, we demonstrate that JAK2V617F induces an intrinsic IFNγ- and NF-κB-associated inflammatory program, while suppressing inflammation-evoked DNA damage both in vitro and in vivo. We show that cells with JAK2V617F tightly regulate levels of inflammatory cytokines-induced reactive oxygen species, do not fully activate the ATM/p53/p21waf1 checkpoint and p38/JNK MAPK stress pathway signaling when exposed to inflammatory cytokines, suppress DNA single-strand break repair genes' expression yet overexpress the dual-specificity phosphatase (DUSP) 1. RNAi-mediated knock-down and pharmacological inhibition of DUSP1, involved in p38/JNK deactivation, in HEL cells reveals growth addiction to DUSP1, consistent with enhanced DNA damage response and apoptosis in DUSP1-inhibited parental JAK2V617F+ cells, but not in CRISPR-modified JAK2 wild-type cells. Our results indicate that the JAK2V617F+ PV progenitors utilize DUSP1 activity as a protection mechanism against DNA damage accumulation, promoting their proliferation and survival in the inflammatory microenvironment, identifying DUSP1 as a potential therapeutic target in PV.
Subject(s)
Cell Proliferation , DNA Damage , Dual Specificity Phosphatase 1/genetics , Hematopoietic Stem Cells/pathology , Induced Pluripotent Stem Cells/pathology , Inflammation/metabolism , Janus Kinase 2/genetics , Oxidative Stress , Polycythemia Vera/genetics , Cell Line, Tumor , Cytokines/genetics , Cytokines/metabolism , Humans , Mutation , Reproducibility of Results , STAT1 Transcription Factor/metabolism , Tumor MicroenvironmentABSTRACT
The development of the most common multidrug resistance (MDR) phenotype is associated with aâ¯massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: Sâ¯cells - parental drug-sensitive cells; Râ¯cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; Tâ¯cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is aâ¯ligand of both RARs and nuclear retinoid Xâ¯receptors (RXRs). In aâ¯previous work, we described that the combined treatment of Râ¯cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, Râ¯and Tâ¯cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in Râ¯and Tâ¯cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in Râ¯and Tâ¯cells. Particularly, combined treatment of Râ¯cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of Tâ¯cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.