IACS-70654 inhibited the growth of neutrophil-enriched syngeneic mammary tumor models. To examine the effects of CBP/P300 BRD inhibition on TNBC in vivo, we selected 4 immunocompetent mouse models derived from the BALB/c or C57BL/6J background. T6, T12, and 2208L tumors are syngeneic Trp53-null models, which were shown to recapitulate the aggressiveness, heterogeneity, and resistance to standard-of-care therapies in human TNBC (24–26). PyMT-N is a luminal-like subtype derived from MMTV-PyMT tumors, exhibiting stable myeloid cell infiltration and resistance to immune checkpoint blockade (ICB) (7). These models displayed distinct TAN and TAM frequencies. TANs are defined as CD45+CD11b+Ly6G+Ly6Clo/med in flow cytometry and S100A8+ in immunostaining. TAMs are defined as CD45+CD11b+Ly6G–Ly6C–F4/80+ in flow cytometry and F4/80+ in immunostaining. PyMT-N, 2208L, and T6 can be categorized as neutrophil-enriched models, and T12 can be categorized as a macrophage-enriched model (7, 27) (Figure 1, A and B). Tumor pieces (Trp53-null models) or freshly dissociated tumor cells (PyMT-N) were implanted into the mammary fat pad of BALB/c or C57BL/6J mice. When the tumors reached more than 80 mm3 in volume on average, the mice were randomized, and treatment was initiated (Figure 1C). IACS-70654 is a potent and highly selective inhibitor of CBP/P300 BRD (Supplemental Tables 1 and 2 and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.182621DS1). IACS-70654 was orally administered at the dosage of 3.75 mg/kg on a 3-on/2-off regimen for all animal studies, and therefore a total of 6 doses were given in a 7-day experiment (Figure 1C). IACS-70654 treatment was well tolerated and did not lead to significant body weight loss (Supplemental Figure 1B). Surprisingly, IACS-70654 as a single agent resulted in the regression of 2208L tumors, which has the highest TAN frequency (Figure 1, B and D, and Supplemental Figure 1C). Tumor regression is rarely observed with a single-agent treatment in Trp53-null models because they are highly aggressive and resistant to therapies. IACS-70654 reduced the growth of PyMT-N and T6 tumors, which are neutrophil enriched but infiltrated with more TAMs than 2208L tumors (Figure 1, B and D, and Supplemental Figure 1C). The macrophage-enriched T12 model was resistant (Figure 1, B and D, and Supplemental Figure 1C). We also used BrdU to determine the effect of IACS-70654 on tumor cell proliferation. 2208L tumors treated with IACS-70654 showed a reduction in BrdU incorporation and thus were less proliferative (Figure 1E). To test whether tumor inhibition can persist in long-term treatment, we conducted a 27-day treatment study of IACS-70654 with 2208L tumors. IACS-70654 durably inhibited the growth of 2208L tumors, with no signs of resistance (Figure 1F). Histone H3 lysine 27 acetylation (H3K27ac) is the target biomarker for IACS-70654, and reduced H3K27ac was observed after in vitro and in vivo treatments with IACS-70654 (Supplemental Figure 1, D–F). IACS-70654 did not affect the expression of CBP, P300, or the RNA expression of other major epigenetic enzymes (Supplemental Figure 1, E–G). Based on those findings, we hypothesized that the infiltrated myeloid cells might explain the difference in response. Therefore, we next examined the changes in infiltrated myeloid cells after IACS-70654 treatment.

IACS-70654 suppressed the growth of neutrophil-enriched preclinical mouse models of TNBC. (A) Representative images of H&E and immunostaining of all TNBC preclinical mouse models used. F4/80 is a macrophage marker, and S100A8 is a neutrophil marker. Scale bars: 50 μm. (B) Flow cytometry analysis of TANs and TAMs in all TNBC preclinical mouse models used (T6, n = 6; all other models, n = 5). (C) The treatment scheme of IACS-70654 in a 7-day experiment. When the average tumor volume reached 80–250 mm3, the mice were randomized to initiate treatment. Vehicle or IACS-70654 at 3.75 mg/kg was administered orally on a 3-on/2-off schedule. On day 7, mice were euthanized 2 hours after receiving the last treatment. (D) Tumor growth curves of all TNBC models treated with IACS-70654 for 7 days (2208L, n = 6; all other models, n = 5). Two-way ANOVA and Šidák’s multiple-comparison test were used. (E) Left: Representative images of immunostaining of BrdU in 2208L tumor sections treated with vehicle or IACS-70654 for 7 days. Scale bar: 50 μm. Right: Quantification of BrdU staining (n = 15). A 2-tailed, unpaired Student’s t test was used. For B, D, and E, data are presented as mean ± SD. *P < 0.05; ****P < 0.0001. The experiments were conducted 3 times for 2208L and twice for other models. Data from 1 representative experiment are shown. (F) Tumor growth curves (from 1 representative experiment) of 2208L tumors treated with IACS-70654 for 27 days. The experiment was conducted twice.

IACS-70654 reduced TANs and immunosuppression in the TIME. To investigate the effects of IACS-70654 on tumor-infiltrated myeloid cells, we analyzed the infiltrated immune cells of the selected models from the 7-day treatment study. IACS-70654 reduced Ly6G expression in TANs from all 4 models and significantly reduced the percentage of TANs in 2208L, PyMT-N, and T12 tumors (Figure 2A and Supplemental Figure 2A). The reduction in TANs was verified with reduced immunostaining of S100A8 in 2208L tumors and was observed within 72 hours after starting treatment (Figure 2B and Supplemental Figure 2B). TANs of neutrophil-enriched models were demonstrated to be immunosuppressive (7). To verify, we applied a published MDSC gene signature to TANs from single-cell RNA sequencing (scRNA-seq) analyses of 2208L tumors treated with IACS-70654 for 7 days (Supplemental Table 3) (5). TANs from 2208L tumors showed higher expression of the MDSC gene signature than the tumor-associated monocytes, and the expression decreased with IACS-70654 treatment (Supplemental Figure 2C). Furthermore, CD84 is an emerging marker used to distinguish PMN-MDSCs from neutrophils, and IACS-70654 substantially reduced CD84+ TANs (PMN-MDSCs) in 2208L tumors (Figure 2C) (4, 5). In contrast, TANs of macrophage-enriched tumors were shown to resemble normal neutrophils (7). Therefore, reduced TANs should not reduce immunosuppression in T12 tumors, and thus this may partially explain why T12 did not respond to IACS-70654. Higher TAM frequency in PyMT-N and T6 might explain why regression was only observed in 2208L tumors. TANs in 2208L and PyMT-N tumors treated with IACS-70654 were also found to have reduced expression of H3K27ac, without changes in RNA expression of genes encoding CBP and P300 (Figure 2D and Supplemental Figure 2D). This result suggested that TANs can be directly reprogrammed by IACS-70654. Moreover, high TAN frequency is accompanied by an accumulation of peripheral blood neutrophils (7, 8). IACS-70654 reduced blood neutrophils in 2208L tumor–bearing mice to a similar level as non–tumor-bearing mice (Figure 2E). We also treated non–tumor-bearing mice with IACS-70654 and observed no significant change in blood neutrophils (Supplemental Figure 2E).

IACS-70654 reduced TANs and reprogrammed myeloid cells in the TIME. (A) Flow cytometry analyses of immune cells in all TNBC models after treatment. Left: Representative contour plots of myeloid (CD45+CD11b+) populations. Middle: Quantification of TANs (T6, n = 6; all other models, n = 5). Right: Median fluorescence intensity (MFI) of Ly6G in TANs relative to the average MFI of the vehicle-treated group (T6 and PyMT-N, n = 6; 2208L and T12, n = 5). (B) Left: Representative images of S100A8 immunostaining on 2208L tumor sections. Scale bar: 50 μm. Right: Quantification of S100A8 staining (n = 12). For A and B, experiments were conducted 3 times for 2208L and twice for other models. Data from 1 representative experiment are shown. (C) Flow cytometry analysis of CD84+ TANs in 2208L tumors after treatment (n = 5). (D) MFI of H3K27ac in TANs relative to the average MFI of the vehicle-treated group in 2208L and PyMT-N tumors after treatment (2208L, n = 5; PyMT-N, n = 6). (E) Flow cytometry analyses of blood neutrophils in 2208L tumor–bearing mice after treatment and non–tumor-bearing BALB/c mice. Ordinary 1-way ANOVA and Tukey’s multiple-comparison test were used (2208L tumor–bearing mice, n = 5; non–tumor-bearing mice, n = 3). (F) UMAP plot of TAM population (monocytes included). (G) Left: Violin plot showing the expression of representative IFN-associated genes across TAM clusters. Right: Fraction of Cluster 5 in TAMs. The fraction values are labeled. (H) Flow cytometry analysis of tumor-associated mMDSCs (Ly6G–Ly6C+CD84+) in 2208L tumors after treatment (vehicle, n = 5; IACS-70654, n = 6). For A–D and H, a 2-tailed, unpaired Student’s t test was used. For A–E and H, data are presented as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001. For A–H, tumors were treated with vehicle or IACS-70654 for 7 days.

Besides neutrophils, IACS-70654 also affected other infiltrated myeloid cells. Even in neutrophil-enriched tumors, TAMs remain the second most abundant immune cell, indicating their importance (7) (Figure 1B). TAMs can have various functions, leading to an antitumor or protumor phenotype (28). To determine transcriptional changes in TAMs, we analyzed TAMs from the scRNA-seq of 2208L tumors treated with vehicle or IACS-70654 for 7 days. Six TAM clusters with distinct expression profiles were identified (Figure 2F, Supplemental Figure 2, F and G, and Supplemental Tables 6–12). Cluster 5 of TAMs highly expressed IFN-response genes and pathways, which are associated with the antitumor phenotype (29) (Figure 2G, Supplemental Figure 2H, Supplemental Table 4, and Supplemental Table 5). IACS-70654 treatment led to an almost 2-fold increase in the fraction of cluster 5 (Figure 2G). Moreover, monocytes are defined as CD45+CD11b+Ly6G–Ly6C+, and those that express CD84 can be considered monocytic myeloid-derived suppressor cells (mMDSCs) (4, 5). IACS-70654 reduced mMDSC infiltration in 2208L and PyMT-N tumors (Figure 2H). Arginase 1 (Arg) is also a marker of immunosuppression, and IACS-70654 reduced Arg+ monocytes/mMDSCs in 2208L tumors (Supplemental Figure 2I). These results suggested that IACS-70654 can reduce immunosuppression and might be an effective therapy for neutrophil-enriched TNBC, which has been correlated with poor patient outcomes and therapy resistance (3, 7). Because IACS-70654 reduced neutrophils in tumors and blood, we further hypothesized that IACS-70654 might inhibit the abnormal neutrophil generation in BM promoted by tumor outgrowth (8–10).

IACS-70654 reprogrammed and reduced the proliferation of neutrophils in BM. To characterize the changes in BM after IACS-70654 treatment, we first performed scRNA-seq analyses on all CD45+ cells collected from BM of non–tumor-bearing WT and 2208L tumor–bearing mice. The 2208L tumor–bearing mice were treated with vehicle or IACS-70654 for 6 days (5 total treatments). As expected, the fraction of neutrophils was increased in 2208L tumors, with a concomitant decrease in the fraction of monocytes and dendritic cell progenitors (pDCs) (Figure 3A). IACS-70654 treatment reduced BM production of neutrophils and restored that of monocytes and pDCs (Figure 3A). This observation was confirmed by flow cytometry analyses coupled with measuring BrdU incorporation (Figure 3B and Supplemental Figure 3A). Reduced H3K27ac was observed in the CD11b+Ly6Glo/medF4/80– neutrophil precursors (Supplemental Figure 3B). Neutrophils were then isolated and clustered to identify subpopulations using pseudotime analysis and previously identified markers such as Itgam and Cxcr2 expression (26, 30, 31) (Figure 3C and Supplemental Figure 3C). Pro-neutrophils (proNeu) and Pre-neutrophils (preNeu) are the proliferative precursors (31). We examined the cell cycle score of the proliferative proNeu/preNeu and found that IACS-70654 decreased the fraction of cells exhibiting a G2/M signature (Figure 3D). Previous studies showed BM neutrophils in tumor-bearing mice are immunosuppressive, especially in those with advanced tumors (7, 32). Among the mature neutrophils, we identified a distinct subpopulation with high expression of IL-1β, an MDSC marker, and this subpopulation was upregulated in 2208L tumor–bearing mice compared with the non–tumor-bearing mice (4) (Figure 3E and Supplemental Figure 3D). BM mature neutrophils in 2208L tumor–bearing mice also expressed a higher MDSC signature than those in non–tumor-bearing mice, and the expression decreased with IACS-70654 treatment (Figure 3, F and G). We further verified that IACS-70654 reduced CD84+ BM neutrophils in 2208L tumor–bearing mice using flow cytometry (Supplemental Figure 3E). In addition to the immunosuppressive neutrophils, a previous study identified IFN-stimulated neutrophils (IFN+ Neu) in blood. Increased IFN+ Neu was correlated with a better response of mouse tumor models and cancer patients to immunotherapy (33). We applied the published gene signature and identified IFN+ Neu in BM neutrophils (Supplemental Figure 3F and Supplemental Table 13). IACS-70654 increased the fraction of IFN+ Neu (Supplemental Figure 3D). Moreover, IACS-70654 reduced the expression of Csf3r, which encodes the receptor for granulocyte colony–stimulating factor (G-CSF), in mature neutrophils (Figure 3H, Supplemental Table 16, and Supplemental Table 18). G-CSF level was upregulated by the 2208L tumor (Supplemental Figure 3G). IACS-70654 might reduce G-CSF signaling, a critical driver for neutrophil production, migration, and immunosuppression (8, 34, 35). IACS-70654 also decreased the RNA expression of genes encoding CCR1 and Ly6G, both of which are critical for neutrophil migration (36, 37) (Supplemental Figure 3, H and I, Supplemental Table 14, Supplemental Table 16, and Supplemental Table 18). In addition, we discovered that BM neutrophils in 2208L tumor–bearing mice downregulated the expression of Thbs1, which encodes thrombospondin-1 (TSP-1), and IACS-70654 restored the expression (Supplemental Figure 3J). The expression of cystatins (e.g., Stfa2, Stfa3, Cstdc5, Cstdc6) and hydrolase/peptidase activity inhibition pathways were also significantly upregulated in BM neutrophils after IACS-70654 treatment (Supplemental Figure 3K and Supplemental Tables 14–19). TSP-1 and cystatins are both inhibitors of neutrophil serine protease that were reported to promote neutrophil release into the blood (38, 39). They can also contribute to the release of C-X-C motif chemokine ligand 2, which promotes neutrophil recruitment, and activates IL-1β (40, 41). These results revealed that IACS-70654 might reduce the proliferation, migration, and protumor activity of BM neutrophils. However, hematopoietic stem and progenitor cells (HSPCs) may also be affected by tumor-derived factors (8, 9). Accordingly, we next investigated the changes in HSPCs after IACS-70654 treatment.

IACS-70654 reprogrammed BM neutrophils. (A) Left: UMAP plot of BM myeloid cells with annotations. Right: Fractions of BM myeloid cells in treated 2208L tumor–bearing and untreated non–tumor-bearing WT BALB/c mice from scRNA-seq analyses. (B) Representative flow cytometry analyses of neutrophils in the BM of treated 2208L tumor–bearing mice (7-day treatment with vehicle or IACS-70654) and untreated non–tumor-bearing WT BALB/c mice (2208L tumor–bearing mice, n = 5; non–tumor-bearing mice, n = 3). Ordinary 1-way ANOVA and Tukey’s multiple-comparison test were used. **P < 0.01; ****P < 0.0001; NS, P > 0.05. Data are presented as mean ± SD. The experiment was conducted twice. (C) Left: Pseudotime analysis of integrated BM neutrophils. The root is circled. Right: UMAP plot of BM neutrophils with annotations. (D) Fractions of pre-neutrophils and pro-neutrophils in G1, G2/M, or S cell cycle stage in the BM of 2208L tumor–bearing mice after treatment from scRNA-seq analyses. (E) Expression distribution of Il1b in integrated BM neutrophils. (F) Expression distribution of the MDSC gene signature in BM neutrophils of treated 2208L tumor–bearing mice and untreated non–tumor-bearing mice (WT). (G) Fraction of IL-1β+ neutrophils that have a high expression level (>0.55) of the MDSC gene signature in treated 2208L tumor–bearing mice and untreated non–tumor-bearing mice (WT). (H) Expression distribution of Csf3r in BM neutrophils of treated 2208L tumor–bearing mice and untreated non–tumor-bearing mice (WT). For A and C–H, 2208L tumor–bearing mice were treated with vehicle or IACS-70654 for 6 days.

IACS-70654 induced transcriptional changes in HSPCs to reduce abnormal myelopoiesis. To determine whether IACS-70654 can reprogram the abnormal myelopoiesis induced by the neutrophil-enriched tumor, we performed scRNA-seq on HSPCs in BM of 2208L tumor–bearing mice treated with vehicle or IACS-70654 and non–tumor-bearing mice. The dataset was filtered to contain only HSPCs involved in myelopoiesis and annotated using classic HSPC markers, as described previously (9, 42). Two distinct populations of granulocyte-monocyte progenitors (GMPs) and common myeloid progenitors (CMPs) were found (Figure 4A and Supplemental Figure 4A). Based on clustering and trajectory analyses, GMP-1 and CMP-1 were determined to be involved in myelopoiesis (Figure 4A). 2208L tumors upregulated the fraction of GMP-1, monocyte progenitors (MPs), and proNeu, whereas IACS-70654 treatment reduced those progenitors, indicating inhibition of myelopoiesis (Figure 4B). We also examined the expression of a published gene signature that was shown to predict the differentiation of HSPCs toward neutrophils (43) (Supplemental Table 20). HSPCs, especially CMP-1 and multipotent progenitors (MPPs), showed higher expression of the gene signature in 2208L tumor–bearing mice than those in non–tumor-bearing WT mice, and the expression decreased with IACS-70654 treatment (Figure 4C, Supplemental Figure 4B, and Supplemental Tables 23–26). In cluster 4, a CMP-1 cluster, IACS-70654 led to the downregulation of myeloid differentiation and activation pathways (Figure 4D, Supplemental Table 21, and Supplemental Table 22). In addition, IACS-70654 decreased the expression of Prtn3 and Ms4a3 in CMP-1 (Figure 4, E and F, and Supplemental Table 23). The expression of these genes has been associated with the differentiation and proliferation of myeloid progenitor cells and found to be increased in the granulocytes from the peripheral blood of TNBC patients (9, 44, 45). These changes imply that IACS-70654 might reduce the differentiation of HSPCs into neutrophils. Moreover, in CMP-1, MPPs, and hematopoietic stem cells (HSCs), IACS-70654 induced the expression of Malat1, a long noncoding RNA shown to inhibit differentiation of early HSPCs (46) (Figure 4G, Supplemental Table 23, Supplemental Table 25, and Supplemental Table 26). In HSCs and MPPs, IACS-70654 also increased the expression of Txnip, which can keep HSCs in an undifferentiated state and reduce their mobility (47) (Supplemental Figure 4C, Supplemental Table 25, and Supplemental Table 26). Furthermore, in the peripheral blood of 2208L tumor–bearing mice, IACS-70654 reduced the level of IL-3, a cytokine known to induce HSC and myeloid differentiation (48, 49) (Supplemental Figure 4D). These observations suggest that IACS-70654 treatment might retain the early HSPCs in an undifferentiated state, explaining the accumulation of those progenitors (Figure 4B). Taken together, these results suggested that IACS-70654 reduced the differentiation of early HPSCs to reduce the overproduction of GMP-1 and thus neutrophils. Besides neutrophils, IACS-70654 may also elicit effects on 2208L tumor cells. Thus, we next investigated the effects of IACS-70654 in tumor cells.

IACS-70654 reprogrammed abnormal myelopoiesis induced by the neutrophil-enriched tumor. (A) UMAP plot with annotations (left) and pseudotime analysis (right) of integrated HSPC clusters. (B) Fraction of myeloid progenitor cells in HSPCs of treated 2208L tumor–bearing mice and untreated non–tumor-bearing WT mice. (C) Expression distribution of neutrophil differentiation signature from scRNA-seq analyses of HSPCs in treated 2208L tumor–bearing mice and untreated non–tumor-bearing WT mice. (D) GO pathway enrichment analysis of the significantly downregulated genes in cluster 4 (annotated as CMP-1) of HSPCs of 2208L tumor–bearing mice treated with IACS-70654 versus vehicle. Biological Process (BP) gene sets from the GO database were used. The top 8 pathways are listed. (E) Violin plots showing the RNA expression of Prtn3 in CMP-1 and MPPs of treated 2208L tumor–bearing mice and untreated non–tumor-bearing WT mice. (F) Violin plot showing the RNA expression of Ms4a3 in CMP-1 of treated 2208L tumor–bearing mice and untreated non–tumor-bearing WT mice. (G) Violin plots showing the RNA expression of Malat1 in CMP-1, MPPs, and HSCs from treated 2208L tumor–bearing mice and untreated non–tumor-bearing WT mice. For A–G, 2208L tumor–bearing mice were treated with vehicle or IACS-70654 for 6 days.

IACS-70654 induced both an IFN response and antigen presentation in tumor cells. To investigate how IACS-70654 impacts tumor cells, we first analyzed the tumor cell cluster from scRNA-seq of 2208L tumors treated with vehicle or IACS-70654 for 7 days (Supplemental Figure 5, A and B). IACS-70654 induced the expression of MHCI components (e.g., H2-D1, H2-K1, H2-Q4) (Figure 5, A–C, Supplemental Table 27, and Supplemental Table 28). Using flow cytometry, we confirmed that IACS-70654 treatment induced the protein expression of MHCI on the surface of 2208L tumor cells in vivo (Figure 5D). Higher expression of MHCI component genes such as B2M and HLA-C and the antigen processing and presentation pathway is associated with pathological complete response (pCR) after paclitaxel and pembrolizumab (ICB) treatment in TNBC patients (Figure 5E, Supplemental Figure 5D, and Supplemental Table 29) (50, 51). Besides MHCI, IACS-70654 also induced the expression of genes associated with IFN-β– and virus-response pathways (Figure 5, A–C, Supplemental Table 27, and Supplemental Table 28). Using a cytokine/chemokine array, we detected an elevated level of IFN-β in 2208L tumors treated with IACS-70654 compared with those treated with vehicle (Figure 5E). IACS-70654 also downregulated genes involved in the regulation of inflammation and inhibition of cytokine production, such as Cd200 and Cebpb that have been shown to induce immunosuppression in the TIME (52, 53) (Figure 5A, Supplemental Figure 5C, and Supplemental Table 27). These results demonstrated that IACS-70654 induced both MHCI expression and an IFN response in 2208L tumor cells, suggesting induced antigen presentation and stimulation of the immune response. Therefore, we next investigated the effects of IACS-70654 on tumor-infiltrated lymphocytes and the response of neutrophil-enriched tumors to ICB.

IACS-70654 induced an IFN-associated response and MHCI expression in 2208L tumor cells. (A) Volcano plot showing –log10(P value) versus log2(fold change) in RNA expression in 2208L tumor cells treated in vivo with IACS-70654 versus those treated with vehicle. Genes demonstrating significant changes are represented by red dots. Genes associated with IFN response, antigen representation, and immunosuppression are labeled. (B) GO pathway enrichment analysis of the significantly upregulated genes in 2208L tumor cells treated in vivo with IACS-70654 versus those treated with vehicle. BP gene sets were used. The top 10 pathways are listed. (C) Violin plots showing RNA expression of representative MHCI components (B2m and H2-D1) and IFN response genes (Bst2 and Isg15) in 2208L tumor cells treated in vivo. (D) Flow cytometry analysis of MHCI expression in 2208L tumor cells treated in vivo. Left: Representative histograms of MHCI expression in 2208L tumor cells (CD45–TER119–CD31–EpCAM+). Right: MFI of MHCI in 2208L tumor cells (vehicle arm, n = 5; IACS-70654 arm, n = 6). (E) Violin plots showing RNA expression (mean ± 2 SD) of representative MHCI components B2M (meta P = –0.0373) and HLA-C (meta P = –0.006612) in human TNBC patients who did (responders) or did not (non-responders) achieve pathological complete response (pCR) after treatment of paclitaxel and pembrolizumab (anti–PD-1). Data were retrieved from published studies (50, 51). (F) Quantification of IFN-β level in 2208L tumor homogenate by cytokine/chemokine array (n = 3). For D and F, a 2-tailed, unpaired Student’s t test was used. *P < 0.05; ***P < 0.001. Data are presented as mean ± SD. For A–D and F, 2208L tumors were treated with vehicle or IACS-70654 for 7 days.

IACS-70654 activated cytotoxic T cells and improved response to ICB. Neutrophil-enriched TNBC models such as 2208L usually have low lymphocyte infiltration and complete resistance to ICB (7). After a 7-day treatment with IACS-70654, 2208L tumors showed a significantly higher level of cytotoxic T cell (CTL, defined as CD3+CD8+) infiltration (Figure 6A). This finding was confirmed using immunostaining, and CTLs were observed in the tumor center instead of the stroma after IACS-70654 treatment (Figure 6B). To determine whether CTLs play a critical role in tumor growth inhibition by IACS-70654, 2208L tumor–bearing mice were treated with an anti-CD8 antibody 24 hours before starting IACS-70654 treatment and then throughout the experiment. Successful depletion of tumor-infiltrated CTLs was confirmed by flow cytometry (Supplemental Figure 6A). CTL depletion attenuated the effects of IACS-70654 and suggested that CTLs were important in both the immediate tumor regression and the durable inhibition of tumor growth (Figure 6C). Moreover, in 2208L tumors, IACS-70654 induced higher levels of CXCL10, a chemokine contributing to CTL recruitment and associated with better efficacy of ICB (Figure 6D) (54). IACS-70654 did not increase programmed cell death protein 1+ (PD-1+) CTLs, but increased PD-1+ regulatory T cells (Tregs, defined as CD4+FoxP3+), implying a potential benefit of combining IACS-70654 with anti–PD-1 (Figure 6E and Supplemental Figure 6B). Thus, we tested the efficacy of IACS-70654 in combination with docetaxel (DTX) and anti–PD-1, which partially mimics the current standard-of-care therapy for TNBC (Figure 6F). DTX is known to cause adverse effects in breast cancer patients, and lowering the dose is commonly used to mitigate adverse effects (55, 56). Therefore, in this study, DTX was administered at 10 mg/kg, which is half of the clinically relevant dose. The combination treatment was well tolerated and showed no signs of toxicity (Supplemental Figure 6C). DTX in combination with anti–PD-1 failed to inhibit tumor growth, and all tumors treated with vehicle or DTX in combination with anti–PD-1 reached the ethical endpoint within 25 days (Figure 6G and Supplemental Figure 6D). IACS-70654 in combination with DTX and anti–PD-1, similarly to IACS-70654 alone, durably inhibited the tumor growth (Supplemental Figure 6D). To determine whether the combination treatment has durable long-term effects on the response of 2208L tumors to anti–PD-1, we stopped IACS-70654 and DTX treatment on day 27, continuing only anti–PD-1 treatment (Figure 6G). Compared with IACS-70654 alone, IACS-70654 in combination with DTX and anti–PD-1 significantly delayed the regrowth of 2208L tumors (Figure 6G). Furthermore, immunostaining of the tumors at the endpoint revealed that TANs again accumulated in the tumors treated with IACS-70654, indicating immunosuppression (Figure 6H). However, tumors treated with IACS-70654 in combination with DTX and anti–PD-1 were infiltrated with significantly fewer TANs, which might explain the delayed recurrence (Figure 6H). In summary, IACS-70654 inhibited the growth of 2208L tumors in a CTL-dependent manner and can potentially improve the response of neutrophil-enriched TNBC to standard-of-care therapies. Although we demonstrated the efficacy of IACS-70654 treatment in primary tumors of neutrophil-enriched TNBC models, the clinically unmet need is to treat metastasis. Accordingly, we next investigated the effects of IACS-70654 in the metastatic setting.

IACS-70654 induced a CTL-dependent response and improved response to ICB. (A) Flow cytometry analysis of CTLs in 2208L tumors treated for 7 days (n = 6). (B) Left: Representative images of CD8 immunostaining on sections of 2208L tumors treated for 7 days. Scale bar: 50 μm. Right: Quantification of CD8 staining (n = 12). For A and B, experiments were conducted 3 times. Data from 1 representative experiment are shown. (C) Log2(fold change) of 2208L tumor volume (mean ± SD). Anti-CD8 was administered 24 hours before starting IACS-70654 treatment. Two-way ANOVA and Šidák’s multiple-comparison test were used (n = 5). (D) Quantification of CXCL10 level in 2208L tumor homogenate by cytokine/chemokine array (n = 3). Tumors were treated for 7 days. (E) Flow cytometry analysis of Tregs in 2208L tumors treated for 18 days (n = 5). For A, B, D, and E, a 2-tailed, unpaired Student’s t test was used. (F) Treatment schemes of IACS-70654 in combination with anti–PD-1 and DTX. DTX was administered at half of the clinically equivalent dose. After 27 days of treatment, only anti–PD-1 treatment was administered until all tumors reached the ethical endpoint (≥1500mm3). (G) Kaplan-Meier survival curves of 2208L tumor–bearing mice (vehicle and DTX + anti–PD-1 arms, n = 4; IACS-70654 and combination arms, n = 5). Log-rank test with Bonferroni’s correction was used. *P < 0.05. The experiment was conducted twice. Data from 1 representative experiment are shown. (H) Quantification of S100A8 immunostaining. Ordinary 1-way ANOVA and Šidák’s multiple-comparison test were used (vehicle and DTX + anti–PD-1 arms, n = 9; IACS-70654 and combination arms, n = 12). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. For A–E and H, data are presented as mean ± SD.

IACS-70654 inhibited the growth of established lung metastases. From the scRNA-seq analyses of 2208L tumor cells, we observed that IACS-70654 downregulated the expression of genes associated with migration and negative regulation of cell adhesion pathways (Supplemental Figure 5C and Supplemental Table 28). More importantly, many of the downregulated genes (Fn1, Hspb1, Postn, Mia, Fgfr1, Serpine2) have been associated with tumor metastasis (57–62) (Figure 7A, Supplemental Figure 7A, and Supplemental Table 27). We also observed a reduction in fibroblast growth factor receptor 1 (FGFR1) protein expression in 2208L primary tumors treated with IACS-70654 (Supplemental Figure 7B). Next, we used an experimental metastasis model to test the effects of IACS-70654 on established lung metastases. To generate lung metastases, 100,000 cells dissociated from a 2208L tumor were injected into the tail vein of each WT BALB/c mouse (Figure 7B). Because IACS-70654 was found to affect the immune response, tumor cells were not labeled, since fluorescent reporters may introduce neoantigens (63). We collected the lungs from 2 mice before starting treatment to ensure the successful establishment of lung metastases. The mice were randomized and treated with vehicle or IACS-70654 for 23 days until most vehicle-treated mice reached the ethical endpoint (Figure 7B). We observed that the lung metastatic burden of IACS-70654–treated mice decreased compared with vehicle-treated mice (Figure 7C). The metastatic burden was quantified by counting the metastases using serial sectioning and a size-based scoring system to calculate a metastasis score for each lung section (Figure 7D). The metastasis score of IACS-70654–treated mice was significantly lower than that of vehicle-treated mice (Figure 7D). In the plasma of 2208L lung metastasis–bearing mice, we observed an abnormal upregulation of CCL19, which has been associated with breast cancer metastasis, and the expression level decreased with IACS-70654 treatment (64) (Supplemental Figure 7C). Moreover, neutrophils have been shown to promote metastasis, and 2208L retained its neutrophil-enriched signature in the lung (3) (Figure 7E). IACS-70654 reduced the number of infiltrated neutrophils in 2208L lung metastases (Figure 7E). IACS-70654 also reduced Ly6G expression in the circulating neutrophils of 2208L lung metastasis–bearing mice (Supplemental Figure 7D). Thus, since IACS-70654 inhibited established lung metastasis in these preclinical models, it may potentially provide a therapeutic alternative for the treatment of metastatic neutrophil-enriched TNBC.

IACS-70654 reduced the growth of established 2208L lung metastases. (A) Violin plots showing RNA expression of representative genes that might be involved in migration and metastasis in 2208L tumor cells treated in vivo. Adjusted P < 0.01 for all genes. (B) Experimental design for studying the effects of IACS-70654 on established lung metastases. Dissociated 2208L tumor cells (100,000) were injected into the tail vein of each mouse. The mice were treated for 23 days. One mouse from the vehicle group had to be euthanized on day 22 due to poor body condition. (C) Representative images from H&E staining of lungs with 2208L metastases from mice after treatment. Scale bars: 5 mm. (D) Quantifications of lung metastases after treatment. Serial sectioning was performed to collect a total of 10 sections for each sample (vehicle, n = 40; IACS-70654, n = 50). Metastases with sizes of <1 mm, 1–3 mm, 3–5 mm, and >5 mm were assigned scores of 1, 2, 3, and 4, respectively. A 2-tailed, unpaired Student’s t test was used. Data are presented as mean ± SD. (E) Left: Representative images of S100A8 immunostaining on 2208L lung metastasis sections. Scale bar: 50 μm. Right: Quantification of S100A8 staining (n = 12). A 2-tailed, unpaired Student’s t test was used. ***P < 0.001; ****P < 0.0001. Data are presented as mean ± SD.