IGSF9-targeted therapy inhibits the progression of acute myeloid leukemia

Abstract

Previously, we reported that targeting immunoglobulin superfamily member 9 (IGSF9) could enhance antitumor T-cell activity and sensitivity to anti-PD-1 immunotherapy, although the detailed mechanism remains unclear. In this study, we find that, similar to the regulation of PD-L1 expression, interferon gamma (IFN-γ) also induces the expression of IGSF9 in acute myeloid leukemia (AML). The small interfering RNA specifically targeting JAK1 and a STAT1 inhibitor blocking IFN-γ signal pathway significantly inhibit the expression of IGSF9 and PD-L1. As a tumor-specific immune checkpoint molecule, IGSF9 plays a significant role in promoting tumor escape. The induction of both PD-L1 and IGSF9 by IFN-γ in the tumor microenvironment explains why IGSF9 is highly expressed in tumors and tumor-infiltrating immune cells. This induction also underpins the strong synergistic effects when combining anti-IGSF9 and anti-PD-1 therapies. Additionally, IGSF9 also mediates the extramedullary infiltration of AML cells, which can be inhibited by depletion of IGSF9 or anti-IGSF9. The binding epitopes of anti-IGSF9 are located within the immunoglobulin G2 and fibronectin type-III-2 domains of IGSF9. Based on these findings, we develop an antibody-drug conjugate (ADC) targeting IGSF9 (anti-IGSF9-linker-DXd). This ADC exhibits 99.7% purity, and primarily exists in monomeric form, demonstrating excellent homogeneity (drug-to-antibody ratio, 8-10) and specificity. Anti-IGSF9-linker-DXd effectively kills IGSF9-positive tumor cells and exhibits a potent bystander effect. In vivo, anti-IGSF9-linker-DXd almost completely eliminates early- and mid-stage tumors and significantly inhibits the progression of advanced tumors. In summary, our findings underscore the potential of IGSF9 as a novel therapeutic target for AML treatment, highlighting its role in disease progression and the efficacy of targeted therapies.

Graphical abstract

Figure 1.

IGSF9 level is detected by flow cytometry. (A-E) IGSF9 levels are detected by flow cytometry in T cells, B cells, and myeloid cells from healthy donors (n = 10), CD33⁺/CD13⁺ leukemia cells (n = 27), CD34⁺/CD117⁺ leukemia stem cells (n = 15), umbilical cord blood (n = 5), and AML cell lines including THP-1, MV4-11, Molm13, and U937 cells. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PBMC, peripheral blood mononuclear cell.

Figure 2.

IGSF9 is induced by IFNγ. (A) Mouse bone marrow cells are differentiated into macrophages. (B) Mouse IGSF9 expression is assessed in bone marrow cells, M0, M1, and M2 macrophages. (C-F) IFN-γ induces the expression of IGSF9 and PD-L1 in THP-1 and MV4-11 cells. APC, allophycocyanin; BMNC, bone marrow mononuclear cells.

Figure 3.

Silencing JAK1 inhibits the expressions of IGSF9 and PD-L1. (A) The siRNA targeting JAK1 blocks the IFN-γ signal pathway, (B-E) leading to reduced levels of IGSF9 and PD-L1 when JAK1 is silenced by siRNA. si, small interfering.

Figure 4.

The characterization of anti–IGSF9-DXd. (A) The basic structure of anti–IGSF9-DXd. (B) The purity of anti–IGSF9-DXd is assessed by size exclusion chromatography, and (C) the DAR values are determined by hydrophobic interaction chromatography. (D,E) The binding activities and serum half-life of anti-IGSF9 and anti–IGSF9-DXd are determined.

Figure 5.

Internalization and cytotoxic assay. (A) The antigen-antibody complexes are endocytosed, and anti–IGSF9-DXd is fluorescently labeled with green, and anti–LAMP-1 is fluorescently labeled with red. (B-E) The different concentrations of anti–IGSF9-DXd are used to treat THP-1-IGSF9-WT, -KO, MV4-11, and U937 cells. (F-H) The cleaved PARP, pCHK1, and pH2A are detected by western blotting in THP-1 and MV4-11 cells. Ab, antibody; Con, concentration.

Figure 6.

Anti–IGSF9-DXd treats the early and mid-stage AML models. About 1 × 10⁶ MV4-11-luc cells are injected into NSG mice (n = 10). Treatment with anti-IGSF9 as a control and anti–IGSF9-DXd is initiated the following day and administered every 5 days thereafter. (A) Disease progression is monitored by bioluminescence imaging. (B) The GFP⁺ cell percentages in bone marrow, spleen and lungs are detected by flow cytometry, and (C) the tumors in all organs are shown by bioluminescence imaging. About 1 × 10⁶ THP-1-luc cells are injected into NSG mice (n = 10), (D) and anti-IGSF9 or anti–IGSF9-DXd are administered on days 7 and 14, and (E) the survival curves are drawn.

Figure 7.

Anti–IGSF9-DXd–treated the advanced AML models. A total of 1 × 10⁶ THP-1-luc cells are injected into NSG mice via the tail vein. (A) At 15 days after injection administration, treatment is initiated using isotype control, anti-IGSF9, DXd, and anti–IGSF9-DXd, and administered every 4 days. (B,C) The bioluminescence imaging is used to detect disease progression, and the survival curve is drawn. (D) H&E staining is used to observe the tumors in lungs and liver, and tissue structure and cell morphology of kidney, heart, and brain.