Shen, Na

Abstract Introduction: Primary myelofibrosis (PMF), a myeloproliferative neoplasm (MPN), features bone marrow fibrosis, splenomegaly, and abnormal blood cell production. Aberrant JAK/STAT signaling, elevated cytokines or hyperactive NFκB are implicated in MPN pathogenesis; however, their interplay remains unclear. JAK inhibitors (JAKi) alleviate PMF symptoms, but they don't fully address disease progression or mitigate cytokine storms. NFκB inhibitors have shown promise in MPN models but are associated with off-target effects and toxicities. This study aims to elucidate the mechanisms of JAKi resistance and develop novel therapeutic strategies targeting hyperactive NFκB in vitro and in vivo, offering potential benefits beyond current JAKi therapies. Methods: To investigate JAKi resistance in PMF, ruxolitinib-resistant HEL cells were generated and NFκB/JAK2/STAT signaling was examined by Western blot. Biomarkers associated with JAKi response were identified in PMF patients treated with BMS-9115437 (Gangat et al, Blood Cancer J, 2023) via Western blot. Molecular mechanisms of MPN pathogenesis were further elucidated using Affymetrix HG-U133Plus2 assay on RNA from treatment-naïve PMF, essential thrombocythemia (ET), polycythemia vera (PV) patients and healthy donors, followed by KEGG and gene set enrichment (GSE) analysis. In vitro, NFκB was inactivated by shRNA, Ixazomib or emetine followed by Western blot for NFκB/JAK2/STAT phosphorylation as well as flow cytometry and clonogenic assays for cell growth. NFκB DNA binding activity was determined by electrophoretic mobility-shift assays (EMSA). The therapeutic and preventive effects of Ixazomib and emetine were evaluated in HEL-engrafted nude mice by assessing tumor formation, development, and underlying mechanisms using Western blot, H&E and IHC staining. Results: Short-term JAKi (BMS-911543, ruxolitinib) exposure altered JAK2 phosphorylation, increasing Tyr1008 phosphorylation and decreasing Tyr221, Tyr570, and STAT5 phosphorylation. Notably, JAK2, STAT3 and STAT5 were phosphorylated in ruxolitinib-resistant HEL cells or after treatment cessation, implying sustained signaling activity. Western blot analysis and clinical data in PMF patients (n=3) receiving BMS-911543 showed no hyperphosphorylation of JAK2 Tyr1008 with diverse responses independent of JAK2V617F status. A lack of correlation between JAK2 and STAT5 expression/phosphorylation suggests that other factors contribute to hyperactive STAT5 in PMF. Symptomatic improvement in a JAK2V617F-positive patient correlated with decreased STAT5 protein expression and phosphorylation, potentially serving as a predictor for JAKi response. Transcriptomic analysis in MPN patients (PMF (n=11), ET (n=6), PV (n=4), HD (n=5)) pinpointed the transcriptomic dissimilarities between PMF and PV or ET, and highlighted the higher NFκB activity in PMF compared to PV or ET. The importance of NFκB in PMF was further evidenced by the network analysis for the overproduction of circulating cytokines in PMF patients (Tefferi et al, JCO, 2011). In vitro studies revealed that NFκB overexpression promotes cell proliferation and inhibits apoptosis through enhanced IL-6/IL-6R expression and JAK2/STAT5 phosphorylation. Inactivating NFκB using shRNA, emetine, or ixazomib reversed these effects. Further, IL-6 treatment increased JAK2 phosphorylation. In vivo studies showed that Ixazomib significantly reduces tumor volume (734 ±133 to 98 ±14 mm3; P <0.001) and weight (582 ±156 to 87 ±22 mg; P =0.0005) compared to controls. Emetine similarly suppressed tumor growth dose-dependently without affecting spleen weight and even prevented tumor formation when administered prior to cell engraftment. Mechanistically, both Ixazomib and emetine inhibited NFκB and JAK2 phosphorylation while increasing cleaved caspase-3 in tumors. Conclusions: We discovered that STAT inactivation independently predicts improved outcomes in PMF and that NFκB hyperactivity holds particular significance for PMF compared to other MPN subtypes. We identified a NFκB-IL-6-JAK2-STAT axis in regulating PMF growth regardless of JAK2V617F status. Targeting NFκB with novel inhibitors Ixazomib and emetine disrupts the NFκB-IL-6-JAK2-STAT axis, suppressing PMF growth and offering potential therapeutic alternatives to overcome current JAKi limitations.

Abstract Purpose: Receptor tyrosine kinases (RTKs) are frequently deregulated in leukemia, yet the biological consequences of this deregulation remain elusive. The mechanisms underlying aberrant methylation, a hallmark of leukemia, are not fully understood. Here we investigated the role of RTKs in methylation abnormalities and characterized the hypomethylating activities of RTK inhibitors. Experimental Design: Whether and how RTKs regulate expression of DNA methyltransferases (DNMTs), tumor suppressor genes (TSGs) as well as global and gene-specific DNA methylation were examined. The pharmacologic activities and mechanisms of actions of RTK inhibitors in vitro, ex vivo, in mice, and in nilotinib-treated leukemia patients were determined. Results: Upregulation of RTKs paralleled DNMT overexpression in leukemia cell lines and patient blasts. Knockdown of RTKs disrupted, whereas enforced expression increased DNMT expression and DNA methylation. Treatment with the RTK inhibitor, nilotinib, resulted in a reduction of Sp1-dependent DNMT1 expression, the diminution of global DNA methylation, and the upregulation of the p15INK4B gene through promoter hypomethylation in AML cell lines and patient blasts. This led to disruption of AML cell clonogenicity and promotion of cellular apoptosis without obvious changes in cell cycle. Importantly, nilotinib administration in mice and human patients with AML impaired expression of DNMTs followed by DNA hypomethylation, TSG re-expression, and leukemia regression. Conclusions: Our findings demonstrate RTKs as novel regulators of DNMT-dependent DNA methylation and define DNA methylation status in AML cells as a pharmacodynamic marker for their response to RTK-based therapy, providing new therapeutic avenues for RTK inhibitors in overcoming epigenetic abnormalities in leukemia. Clin Cancer Res; 23(20); 6254–66. ©2017 AACR.

Abstract Nilotinib has been approved to treat newly diagnosed, imatinib-resistant or -intolerant leukemia, but the mechanisms underlying its antileukemia actions remain elusive. Here, we report that nilotinib blocks AML growth through its DNA hypomethylating activity. We showed that, in addition to inducing a reduction of KIT and FLT3 protein expression and phosphorylation, nilotinib downregulated DNMT1, 3a and 3b in Kasumi-1, MV4-11 and C1498 cells. Since DNMT1 is maintenance and de novo DNMT, we focused on DNMT1 and proved that nilotinib impaired DNMT1 expression via Sp1 dysfunction, since the luciferase activity driven by DNMT1 promoter containing Sp1 binding sites was decreased by 2.6 fold by nilotinib, while increased by 10.6 fold after Sp1 overexpression. Electrophoretic mobility shift assay disclosed that nilotinib abolished Sp1 binding on DNMT1 promoter. Dotblot using 5mC antibody revealed global DNA hypomethylation and qPCR showed p15INK4B upregulation in Kasumi-1 (3.4 fold) or MV4-11 (5.2 fold). Bisulfite sequencing found a decrease of DNA methylation on p15INK4B promoter from 100% to 89% in Kasumi-1 or 13% to 0% in MV4-11. The functional assays showed a decrease of colony formation in Kasumi-1 (60%) or MV4-11 (58%), and an increase of cell apoptosis in Kasumi-1 (6.9 fold) or MV4-11 (3.2 fold). We verified these findings in AML patient blasts (n = 3) by showing that nilotinib treatment led to the dephosphorylation of KIT, the downregulation of DNMT1, 3a, 3b and Sp1, the reduction of global DNA methylation and the re-expression of p15INK4B (4.4 fold) via promoter demethylation (from 14% to 6%), which was followed by suppressed colony formation (77%), promoted cell apoptosis (1.84 fold) and increased caspase-3 and caspase-8. To test if nilotinib induces AML regression via DNA hypomethylation in vivo, 0.1×106 C1498 cells were injected into C57BL/6 mice (n = 3 mice/group) via the tail vein. The leukemic mice were treated with 5, 10 or 15 mg/kg of nilotinib in PEG400 plus saline via intraperitoneal injection twice a week for a total of 6 doses. No toxicity was observed for the tested drug dosage and schedule, because of no evident change of mouse body weight. Compared to the control group, nilotinib-treated mice had a decrease in spleen size. H&E staining showed that the control mice exhibited increased infiltration of AML cells into the spleen, lung and liver of recipients. The bone marrow (BM) histopathology identified a higher degree of mature post-mitotic cells containing metamyelocytes, bands and segmented neutrophils in nilotinib-treated mice than control. DNMT1 was downregulated (39%), p15INK4B was upregulated and global DNA methylation was reduced (60%) in BM cells from nilotinib-treated mice. Thus, our findings unveil DNA methylation machinery as an innovative target behind nilotinib's anticancer activities, and provide a new therapeutic avenue for nilotinib in overcoming aberrant epigenetics in AML. Note: This abstract was not presented at the meeting. Citation Format: Na Shen, Fei Yan, Jiuxia Pang, Naseema Gangat, Mark R. Litzow, Aref Al-Kali, Shujun Liu. Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2015-2945