Fig 2: IRF8 Activity Suppresses Inflammasome Activation in BMDCs and Macrophages(A) Caspase-1 cleavage (hCD4+FLICA+) was quantified by flow cytometry following NLRP3 inflammasome activation (LPS+ATP) in BMDCs that were transduced with retrovirus expressing IRF8, IRF4, or control plasmids. Mean fluorescence intensity (MFI) from four independent experiments is quantified.(B) IL-1β was measured by ELISA in the supernatants of FACS-sorted hCD4+ GM-Macs following NLRP3 inflammasome activation. See also Figure S1.(C) Immunoblot analysis of pro-casp1 (p45) and cleaved casp1 (p20) in the lysates of iBMDMs transduced with lentivirus expressing IRF8, IRF4, or control plasmids and stimulated with NLRP3 inflammasome ligands.(D) IL-1β was measured by ELISA in the supernatants of iBMDMs from (C).(E) qPCR of designated mRNA in the lysates of mock, IRF8, or IRF4 transduced iBMDMs following 4 h of LPS stimulation.See also Figure S4. Error bars indicate SEM; (A, B, D, and E) paired t test; (C) data representative of three to four independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

Fig 3: A selective activator protein‐1 (AP‐1) inhibitor augments the effects of lenalidomide in murine multiple myeloma (MM) models. (A) Luciferase‐expressing MM.1S cells were inoculated subcutaneously into the right thigh of NOD/SCID mice (1 × 106 cells/mouse). When the inoculated tumour became measurable (defined as day 1), we started treatment with vehicle alone (0.9% NaCl, twice a week), 20 mg/kg T‐5224 (twice a week), 10 mg/kg lenalidomide (twice a week), or the combination of T‐5224 and lenalidomide (20 mg/kg T‐5224 and 10 mg/kg lenalidomide, twice a week) intraperitoneally for two weeks. Left panel: Representative photographs of mice on day 1 and day 25 (original magnification: 2×). Right panel: Quantitative data of ex vivo bioluminescence imaging on day 1 and day 25 expressed in photons/s (photons/s). The mean ± S.D. (bars) is shown. p‐Values were determined by one‐way ANOVA with Student–Newman–Keuls multiple comparison test. (B) Left panel: Representative photographs of tumours resected on day 25 (original magnification: ×2). Right panel: The mean ± S.D. (bars) of tumour weight. *p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (C) Upper panel: Tumor sections were prepared from mice and stained with hematoxylin and eosin (HE). Lower panel: Serial tumour sections were immunostained for IRF4 (green) and CD138 (red), followed by counterstaining with DAPI (blue). Merged images are shown at the bottom. The data are representative of several independent experiments. (D) We transplanted luciferase‐expressing KMS26 cells (1 × 106 cells/mouse) into BALB/c mice via tail‐vein injection. After confirming the engraftment of transplanted cells on day 14 via ex vivo luciferase assays, we assigned recipients to four groups (n = 5), which were standardized on the basis of total luciferase activity, and started the treatments with vehicle alone (0.9% NaCl, three times a week), 20 mg/kg T‐5224 (three times a week), 10 mg/kg lenalidomide (three times a week), or the combination of T‐5224 and lenalidomide (20 mg/kg T‐5224 and 10 mg/kg lenalidomide, three times a week) intraperitoneally for 3 weeks. p‐Values and hazard ratios (95% confidential interval) were calculated by log‐rank test between the combined treatment group (red) and the lenalidomide‐alone (green) or T‐5224‐alone treatment group (blue). No significant differences were observed between the vehicle‐treated group (Control) and the lenalidomide‐alone or T‐5224 alone‐treatment group.

Fig 4: Direct interaction of IKZF1 and c‐FOS in multiple myeloma (MM) cells. (A) Left panel: Nuclear extracts from MM.1S and KMS12‐BM cells were immunoprecipitated with rabbit anti‐IKZF1 antibody or isotype‐matched immunoglobulin (IgG). The immunoprecipitates were analyzed by immunoblotting with specific antibodies against IKZF1, IKZF3, c‐FOS, c‐JUN, IRF4 and rabbit IgG. Input: Immunoblotting of nuclear extracts fractioned before immunoprecipitation. Right panel: The same experiments were carried out with c‐FOS immunoprecipitates. (B) The binding of the IKZF1 complex to oligonucleotides containing an IKZF consensus motif was measured by sandwich immunoassay and is shown as the relative activity against the data obtained in the absence of blocking antibodies. Antibody perturbation was carried out with isotype‐matched immunoglobulin (Control), an anti‐IKZF1 antibody, an anti‐c‐FOS antibody and a combination of the two antibodies. p < .05 by Student's t‐test against Control (n = 5). (C) HEK293T cells were transfected with an empty vector (Mock) or expression vectors carrying HA‐tagged full‐length IKZF1 protein, the exon 1‐exon 4 fragment, the exon 5‐exon 6 fragment, or the exon 7 fragment of IKZF1 together with a FLAG‐tagged c‐FOS expression vector. Nuclear extracts were isolated 24 h after transfection and immunoprecipitated with an anti‐HA antibody, followed by immunoblotting with antibodies against HA tag (IKZF1), FLAG tag (c‐FOS) or rabbit immunoglobulin (IgG). Red arrows denote the positions of the transfected IKZF1 fragments. (D) Structure‐based prediction of IKZF1‐c‐FOS interactions using the AlphaFold2 program.

Fig 5: Global gene expression analysis of response to tirabrutinib treatment in a TMD-8 xenograft model.Microarray analysis was conducted in the TMD8 xenograft model after 21 days of tirabrutinib administration. (A) Volcano plot for differential gene expression between the vehicle and tirabrutinib 10 mg/kg group with Welch’s t test. (B) Heat map of top altered gene group including 59 upregulated probes and 87 downregulated probes (fold change, >4 or <0.25; P < 0.00001) in the vehicle, tirabrutinib 3 mg/kg, and 10 mg/kg groups. (C) Gene set enrichment analysis (GSEA) plots involved in IRF4, MYC, and mTORC1 signaling in the tirabrutinib 10 mg/kg group versus vehicle group.