Exploring redox vulnerabilities in JAK2V617F-positive cellular models

Abstract

Background In Philadelphia chromosome-negative myeloproliferative neoplasm (MPN) models, reactive oxygen species (ROS) are elevated and have been implicated in genomic instability, JAK2/STAT signaling amplification, and disease progression. Although the potential effects of ROS on the MPN phenotype, the effects of ruxolitinib treatment on ROS regulation have been poorly explored. Herein, we have reported the impact of ruxolitinib on redox signaling transcriptional network, and the effects of diphenyleneiodonium (DPI), a pan NOX inhibitor, in JAK2 V617F -driven cellular models. Method Redox signaling-related genes were investigated in SET2 cells upon ruxolitinib treatment by RNA-seq (GEO accession GSE69827). SET2 and HEL cells, which represent JAK2 V617F -positive MPN cellular models with distinct sensitivity to apoptosis induced by ruxolitinib, were used. Cell viability was evaluated by MTT, apoptosis by annexin V/PI and flow cytometry, and cell signaling by quantitative PCR and Western blot. Main results Ruxolitinib impacted on a network composed of redox signaling-related genes, and DUOX1 and DUOX2 were identified as potential modulators of ruxolitinib response. In SET2 and HEL cells, DPI reduced cell viability and, at low doses, it significantly potentiated ruxolitinib-induced apoptosis. In the molecular scenario, DPI inhibited STAT3, STAT5 and S6 ribosomal protein phosphorylation and induced PARP1 cleavage in JAK2 V617F -positive cells. DPI combined with ruxolitinib increased PARP1 cleavage in SET2 cells and potentiated ruxolitinib-reduced STAT3, STAT5 and S6 ribosomal protein in HEL cells. Conclusion Our study reveals a potential adaptation mechanism for resistance against ruxolitinib by transcriptionally reprogramming redox signaling in JAK2 V617F cells and exposes redox vulnerabilities with therapeutic value in MPN cellular models.

Insights on JAK2 Modulation by Potent, Selective, and Cell-Permeable Pseudokinase-Domain Ligands

“…These results indicated that it has an IC 50 below 2 μΜ, which is in accord with literature reports of IC 50 values of 274 nΜ 44 and 30−100 nΜ. 45,46 Compound 1 exhibited the same inhibition patterns for HEL and TF-1 cells, with an estimated IC 50 between 1 and 5 μΜ for HEL cells (Figure S3 B), which is also in accord with the literature. 47 Given 13's cellular activity, its effects in HEL cells and against TF1 cells (expressing only WT JAK2) were examined in more detail.…”

“…Ruxolitinib was tested at only one concentration (2 μΜ), and it showed complete inhibition of STAT-5 phosphorylation for both HEL and TF-1 cells (Figure S3 Α). These results indicated that it has an IC 50 below 2 μΜ, which is in accord with literature reports of IC 50 values of 274 nΜ and 30–100 nΜ. , Compound 1 exhibited the same inhibition patterns for HEL and TF-1 cells, with an estimated IC 50 between 1 and 5 μΜ for HEL cells (Figure S3 B), which is also in accord with the literature …”

“…Product: off-white solid. Yield: 46 Methyl 6-(4-(5-Amino-3-((4-sulfamoylphenyl)amino)-1H-1,2,4triazole-1-carboxamido)phenyl)pyrazine-2-carboxylate (4i). According to General Method D: Methyl 6-(4-((phenoxycarbonyl)amino)phenyl)pyrazine-2-carboxylate (B2) (111.8 mg, 0.32 mmol) was reacted at 110 °C for 2 h (1.9 mL/mmol of anhydrous dioxane, 1 equiv triethylamine).…”

NADPH Oxidase 4: A Potential Therapeutic Target of Malignancy

“…Fulvene-5 could revert adaptive responses of CRC cells from pro-survival to radio-sensitization ( Murley et al, 2018 ). As a pan NOX inhibitor, DPI inhibits phosphorylation of STAT3 and STAT5, and drives PARP1 cleavage in JAK2V617F-positive cells ( Lima et al, 2021 ). DPI can also eliminate ROS production to inhibit tumor progression in ovarian, prostate and lung cancer ( Xia et al, 2007 ; Ma et al, 2021 ).…”

Insights into the Potential Mechanisms of JAK2V617F Somatic Mutation Contributing Distinct Phenotypes in Myeloproliferative Neoplasms

“…Subsequent evidence indicates that IRS2 is associated with JAK2V617F induced malignant transformation and upregulation of IGF1R signaling, further inducing the MPN phenotype [60]. Reactive oxygen species (ROS) signaling also potentially contributes to the MPN phenotype [90]. It was shown in a mouse model that JAK2V617F induced ROS production, which would lead to genomic instability and disease occurrence [91].…”