Tacedinaline (CI-994), a class I HDAC inhibitor, targets intrinsic tumor growth and leptomeningeal dissemination in MYC-driven medulloblastoma while making them susceptible to anti-CD47-induced macrophage phagocytosis via NF-kB-TGM2 driven tumor inflammation

Abstract

Background: While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway.

Methods: We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models.

Results: CI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment ('eat-me') signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice.

Conclusion: Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Keywords: Brain Neoplasms; Cytotoxicity, Immunologic; Immunotherapy; Macrophages; Phagocytosis.

Figure 1

Drug screening of distinct malignant brain tumor entities reveals the significant vulnerability of MYC-driven medulloblastoma towards HDAC inhibition in vitro. (A) Heatmap representing the activity of 78 epigenetic modulators across 36 brain tumor cell lines derived from medulloblastoma (MB, n=14), glioblastoma (GBM, n=11), and atypical teratoid/rhabdoid tumors (AT/RT, n=11). Inhibitor response was subdivided into three groups. Group 1 consists of inhibitors that showed a lower median IC₅₀ for MYC-driven MB compared with the remaining entities. Group 2 inhibitors were largely inactive across the entities, and group 3 inhibitors were less active in the MYC-driven MB cell lines compared with the other entities. Inhibitors are sorted according to their respective p value when comparing the IC₅₀ values of MYC-driven MB against the IC₅₀ values of all other cell lines (unpaired t-test). (B) The screened epigenetic library is composed of various inhibitor classes targeting different epigenetic modulators (the inner circle). Out of the 78 inhibitors screened, 13 drugs were significantly more active in MYC-driven MB (outer layer). Nine of those selective inhibitors are HDAC inhibitors (HDACi), three compounds inhibit DNA methyltransferases, and one drug is an aurora kinase inhibitor. (C) A focused re-screen of selected HDACi in brain tumor cell lines identified CI-994 as a selective inhibitor for MYC-driven MB. The heatmap represents the IC₅₀ values (µM) of 20 HDACi in 29 cell lines. Inhibitors are sorted according to the respective p value derived from comparing IC₅₀ values of the MYC-driven MB against the IC₅₀ values of all other tested cell lines (unpaired t-test). (D) The response of the HDACi was highly correlated between the primary screen (Epigenetic library, x-axis) and the secondary screen (HDACi library, y-axis). The graph displays the log IC₅₀ (nM) values of the 15 overlapping HDACi in the 29 cell lines included on both screens. (E) Mean dose-response profile and mean IC₅₀ (µM) of CI-994 for each entity/subgroup. (F) The validation screen of CI-994 confirmed the selective response in MYC-driven MB. The values shown represent mean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001. HDAC, histone deacetylases.

Figure 2

CI-994 induces apoptosis and downregulates MYC expression in MYC-driven medulloblastoma cell lines. (A) Representative FACS analysis of MED8A and D425 MED cells treated either with DMSO (control), 5 or 7.5 µM of CI-994 for 48 hours, and bar graphs displaying the mean of n=4 replicates. CI-994 treatment resulted in a dose-dependent increase in mean early apoptotic and late apoptotic cells on treatment in both cell lines. (B) For MYC expression analyses MED8A and D425 MED cells were treated with 2.5, 5, or 7.5 µM of CI-994 for 48 hours. MYC mRNA expression values were normalized to housekeeping controls, and expression was calculated relative to DMSO control. Representative Western blots for MYC and ACTIN loading controls for MED8A and D425 MED cells following treatment with 5 or 7.5 µM CI-994 treatment for 48 hours. (C) MYC overexpressing UW-228–3 cells (UW-228–3 MYC OE) were more sensitive toward CI-994 treatment than the respective isogenic control cells with low endogenous MYC expression (UW-228–3 ctrl). Values shown represent mean±SD of 3–4 replicates per condition. *, p<0.05; **, p<0.01; ***, p<0.001. DMSO, dimethyl sulfoxide; FACS. flow cytometry; mRNA, messenger RNA.

Figure 3

CI-994 inhibits tumor growth and reduces leptomeningeal dissemination of MYC-driven medulloblastoma in vivo. (A) Treatment schedule for the evaluation of in vivo efficacy of CI-994 in MYC-amplified medulloblastoma. (B) Bioluminescence imaging and luminescence signal quantification of MED8A or D425 MED (C) xenograft from CI-994 treated and control mice. CI-994 treatment reduced tumor growth significantly compared with control mice (MED8A p=0.0015; D425 MED p=0.0344). (D) CI-994 significantly increased survival in both models of MYC-amplified medulloblastoma (MED8A p=0.0026; D425 MED p=0.01). (E) The formation of spinal metastases was significantly reduced in CI-994 treated mice compared with control mice, as determined by quantification of bioluminescence intensity along the spine (MED8A p=0.005; D425 MED p=0.009).

Figure 4

CI-994 treatment induces the expression of NF-κB pathway genes in MYC-driven medulloblastoma. (A–C) MED8A, D425 MED, and D341 MED treated with CI-994 or vehicle control were analyzed by RNA sequencing. The non-parametric supervised analysis identified 130 upregulated and 43 downregulated genes with a fold change ±2 and p≤0.05, which were analyzed by ingenuity pathway analysis (IPA). (A) Table showing top 20 out of 44 canonical pathways with activation z-score ≥2 and ranked by p value. (B) Table of top 10 upstream regulators identified by IPA with an activation score ≥1.8 and p≤0.05. (C/D) Unsupervised hierarchical clustering of genes regulated by NF-κB complex. (E) MED8A and D425 MED cells were treated with 2.5, 5, or 7.5 µM of CI-994 for 48 hours. TGM2 mRNA expression values are normalized to housekeeping controls, and expression is calculated relative to DMSO control. The values shown represent the mean±SD of three replicates per condition. (F) Western blots for TGM2 and Actin loading controls following CI-994 treatment for 24 and 48 hours. *, p<0.05; **, p<0.01; ***, p<0.001. DMSO, dimethyl sulfoxide; mRNA, messenger RNA; NF-kB, nuclear factor-kB; TGM2, transglutaminase 2.

Figure 5

CI-994 induces a TGM2-dependent pro-inflammatory cytokine response from tumor cells and an increase in infiltration of pro-inflammatory macrophages within tumors: (A) Essay for secretion of CCL1, interleukin-1B, and TNF-α was carried out by ELISA. Significant increase (***p<0.001: ordinary one-way analysis of variance) in the secretion of all three cytokines was observed in MB002, D425MED, MED8A, and D283MED cell lines on treatment with 5 µM CI-994. We also observed a significant drop in secretion of all three cytokines on inhibition of TGM2 using two different inhibitors, ZDON and ERW1041E. (B–C) Representative data from In vivo analysis of macrophage infiltration of macrophages in NSG mice with D425MED (B) or MED8A (C) cells xenografted into the cerebellum. Tumors were harvested and analyzed for total macrophage infiltration as identified being GFP– (tumors cells) and CD11b+. Increase in pro-inflammatory macrophages (CD45+CD11b+CD80+CD64+) is seen in CI-994 treated animals as compared with DMSO control. (D) Quantitative presentation of flow cytometry analysis from B and C. significant increase in CD11b+ macrophages (**p<0.01; n=5) and CD80+CD64+ (*p<0.05; n=5) was observed in D425MED cells whereas higher infiltration was observed in CD11b+ (***p<0.001; n=5) and CD80+CD64+ (**p<0.01; n=5 each). Each data point is an individual tumor-bearing mouse. *p<0.05; **p<0.01; ***p<0.001. DMSO, dimethyl sulfoxide; TGM2, transglutaminase 2; TNF, tumor necrosis factor.

Figure 6

CI-994 sensitizes MYC-driven medulloblastoma cells to anti-CD47 phagocytosis checkpoint immunotherapy: (A) Tumor inflammation on CI-994 treatment was evaluated by an ELISA-based assay for IFN-γ secretion. A significant increase (**p<0.05: non-parametric t-test) is seen in MB002, D425, and D283 cell lines on treatment at 2.5 μM CI-994. (B) An increase in expression of surface calreticulin is seen in MB002 (left panel) and D425 (right panel) as assayed by flow cytometry. (C) An increase in the relative surface calreticulin expression per cell was assayed by measuring the MFI for four different cell lines. (D) Levels of HMGB1 were assayed by a standard colorimetric ELISA. (E) D425 cells were incubated with human peripheral blood mononuclear cells-derived macrophages and tumor cells pretreated with either IgG control, CI-994, anti-CD47 mAb (B6H12), or a combination of CI-994+ anti-CD47 mAb. Phagocytosis was assayed using flow cytometry. (F) Survival analysis of D425 tumor-bearing mice treated with either control, CI-994, anti-CD47, or a combination of anti-CD47 and CI-994.(p=0.0002 using the log-rank (Mantel-Cox) test with a HR of 3.669). DMSO, dimethyl sulfoxide; IFN, interferon; mAb, monoclonal antibody; MFI, mean fluorescence intensity.