SET-2

Ruxolitinib Uptake by BM-Mscs and Antiproliferative Effects on Leukemic Stem Cells

Myelofibrosis (MF) is a chronic myeloproliferative disorder marked by bone marrow (BM) hyperplasia, fibrosis, and splenomegaly. Over 50% of patients carry the JAK2V617F mutation, driving uncontrolled cell proliferation. Ruxolitinib, a JAK1/2 inhibitor, can relieve inflammation in these patients but the crosstalk with BM-MSCs has not been evaluated so far, and MSCs could modulate the distribution of drugs. Marino et al. analyzed in vitro ruxolitinib uptake by MF patient-derived BM-MSCs and tested the impact of drug-treated MSCs and their conditioned media on leukemic cell proliferation.

The antiproliferative activity of CM from BM-MSCs treated with 5 μg/mL ruxolitinib for 24 h was evaluated on leukemic cells. Serial dilutions of ruxolitinib or CM were prepared and 2 × 10⁴ SET-2 cells were added. After 7 days of incubation, cell proliferation was assessed by CCK-8 assay. The IC₅₀ of the CM from BM-MSCs previously treated with ruxolitinib was significantly lower compared to ruxolitinib (57.3 ng/mL vs 74.2 ng/mL), while the IC₁₀ was lower (10.1 ng/mL vs 19 ng/mL) and the IC₉₀ was similar (353.1 ng/mL vs 289.1 ng/mL). This data suggests that the CM of ruxolitinib-treated BM-MSCs from MF patients exert stronger antiproliferative effects compared to ruxolitinib alone and further studies are needed to assess whether this response is mediated by the release of drug-loaded exosomes or by cytokine release (Fig. 1C).

Fig. 1. Ruxolitinib uptake by BM-MSCs and antiproliferative effects on leukemic stem cells (Marino L, Charlier B, et al., 2020).

Identification of DNMT3A^R882H and IDH2^R140Q Neopeptides Using HLA Class I Peptidomics

DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are epigenetic regulators mutated in 7–23% of patients with acute myeloid leukemia (AML) (6). Struckman et al. assessed whether hotspot mutations in these two genes encode immunogenic neoantigens. They transduced five human B-lymphoblastoid cell lines expressing common HLA class I alleles with a minigene construct harboring DNMT3A or IDH1/2 mutations and used mass spectrometry to identify HLA-A01: 01-binding DNMT3A^R882H and HLA-B07:02-binding IDH2^R140Q peptides as candidate neoantigens (Table S2).

They next asked whether DNMT3A^R882H and IDH2^R140Q peptides are HLA class I-presented in AML. They used parallel reaction-monitoring mass spectrometry (PRM-MS) to assess HLA class I peptide eluates from AML cell lines and patient-derived AML samples (Fig. 1D). SET-2 is an AML cell line harboring a natural heterozygous DNMT3A^R882H mutation, and K562-^R140Q is a chronic myelogenous leukemia cell line into which homozygous IDH2^R140Q mutations were introduced using gene editing technology. Sanger sequencing confirmed these mutations in these cell lines. PRM-MS analysis did not detect the 11mer YTDVSNMSHLA peptide in SET-2 transduced with HLA-A01: 01 or HLA-A01:01-positive patient-derived AML cells with a DNMT3A^R882H mutation. YTDVSNMSHLA was detected, however, in SET-2 transduced with HLA-A*01:01 and the full-length DNMT3A^R882H gene (Fig. 2C). YTDVSNMSH, the 9mer counterpart to YTDVSNMSHLA, was not detected in any HLA eluates.

Fig. 2. Surface presentation of DNMT3A and IDH2 neopeptides assessed with HLA class I immunopeptidomics (Struckman NE, de Jong RCM, et al., 2024).