Class IIa HDAC inhibition reduces breast tumours and metastases through anti-tumour macrophages

Abstract

Although the main focus of immuno-oncology has been manipulating the adaptive immune system, harnessing both the innate and adaptive arms of the immune system might produce superior tumour reduction and elimination. Tumour-associated macrophages often have net pro-tumour effects1, but their embedded location and their untapped potential provide impetus to discover strategies to turn them against tumours. Strategies that deplete (anti-CSF-1 antibodies and CSF-1R inhibition)2,3 or stimulate (agonistic anti-CD40 or inhibitory anti-CD47 antibodies)4,5 tumour-associated macrophages have had some success. We hypothesized that pharmacologic modulation of macrophage phenotype could produce an anti-tumour effect. We previously reported that a first-in-class selective class IIa histone deacetylase (HDAC) inhibitor, TMP195, influenced human monocyte responses to the colony-stimulating factors CSF-1 and CSF-2 in vitro6. Here, we utilize a macrophage-dependent autochthonous mouse model of breast cancer to demonstrate that in vivo TMP195 treatment alters the tumour microenvironment and reduces tumour burden and pulmonary metastases by modulating macrophage phenotypes. TMP195 induces the recruitment and differentiation of highly phagocytic and stimulatory macrophages within tumours. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade in this model significantly enhances the durability of tumour reduction. These data introduce class IIa HDAC inhibition as a means to harness the anti-tumour potential of macrophages to enhance cancer therapy.

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Figure 1: TMP195 modulates macrophages in breast tumours.
Figure 2: TMP195-activated myeloid cells are highly phagocytic and induce vasculature normalization in breast tumours.
Figure 3: TMP195 induces macrophage-dependent reduction in tumour burden and decreases pulmonary metastasis.
Figure 4: TMP195 improves the efficacy of chemotherapy and checkpoint blockade, and induces a durable response.

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Acknowledgements

We thank A. Benowitz and C. Leach for their support and discussions in the preparation of the manuscript. This work was supported by NIH NCI F32CA180733 (J.L.G.), The Friends of Dana-Farber, Dancing for a Cure (J.L.G.), NIH NCI R01CA205967 (A.L.) and a sponsored research agreement from GlaxoSmithKline (A.L., J.L.G.).

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Authors

Contributions

J.L.G., M.A.N. and A.L. conceived the project and designed the experiments. J.L.G., A.S., H.E.P., A.L.P., S.S., S.F.J., J.A.C. and M.A.N. performed the biological experiments and analysed the data. R.T.B. and R.D.C. performed histopathological analysis. S.L. assisted flow cytometry analysis. S.P.D. assisted with statistical analysis of gene expression arrays. J.L.G., S.P.D. and M.A.N. prepared figures and tables. J.L.G., M.A.N. and A.L. wrote and edited the manuscript with help from co-authors. J.L.G., M.L., M.A.N. and A.L. contributed to oversight of and advice on the overall project. J.L.G., M.A.N and A.L. provided overall project leadership.

Corresponding authors

Correspondence to Michael A. Nolan or Anthony Letai.

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Competing interests

This work was supported though a sponsored research agreement with GlaxoSmithKline to A.L. and J.L.G. S.P.D., M.L. and M.A.N. are GlaxoSmithKline employees.

Additional information

Reviewer Information Nature thanks B. Ruffell and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 TMP195 modulates CD11b+ cells in breast tumours.

a, Gating strategy for double-sorting tumour cell suspensions from Fig. 1b. Mononuclear cells gated on the basis of FSC-A versus SSC-A were sequentially gated to select single cells (FSC-H versus FSC-W then SSC-H versus SSC-W). Single cells were then gated to select live CD45+ (7-AAD versus CD45). Live CD45+ cells were then gated to select CD19 cells (CD19 versus CD3). Live/CD45+/CD19 cells were then gated to select either CD11b+ or CD3+ cells (CD11b versus CD3). Tumour suspensions were sorted on a BD Aria II into CD11b+ or CD3+ fractions. These fractions were concentrated by centrifugation and sorted through the same gating strategy a second time to increase the purity of each population. The purity of the double-sorted population was confirmed for each sample before RNA isolation. b, c, Representative purity checks of the double-sorted CD3+ (b) and CD11b+ (c) populations are shown. d, Mean versus expression value plots of Affymetrix transcriptional profiling data. All probe sets are shown, highlights apply only to probe sets with a δ-factor >1.5. ej, Starting with the list of genes most affected by 5-day TMP195 treatment (δ-factor >1.5), we queried their biological processes in the PANTHER GO-Slim gene ontology database and compared that to the distribution of biological processes represented in the genome. e, The genes induced by TMP195 treatment had a significant over- or under-representation of the ontologies illustrated in the pie charts and embedded table of statistics. Unbiased analysis of TMP195-induced differential gene expression through GSEA of all probe sets revealed a significant bias (χ2 P value <0.05) in the distribution of the expression values of five gene sets as highlighted on the volcano plots (y axis, Student’s t-test P value). fj, Probe sets representing genes in both the δ-factor list we selected and the GSEA gene set are labelled with the gene symbol corresponding to that probe set.

Extended Data Figure 2 TMP195 induced recruitment of tumour infiltrating leukocytes.

Tumour-bearing MMTV-PyMT mice were randomly placed into treatment groups and received daily intraperitoneal (i.p.) injections of either vehicle (DMSO) or 50 mg kg−1 of TMP195 for the indicated days. a, b, IHC was performed on tumour sections for the myeloid marker CD11b (a) and the macrophage-specific marker F4/80 (b). Quantitation as percentage of total tissue is shown to the right of each representative section. Vehicle (5 days of DMSO) and 5 day TMP195 quantitation is taken from Figs 1c and 2a for reference. Scale bar, 100 μm. t-test *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. c, Negative controls for the CD40 IHC staining (Fig. 2f) are shown. Both rabbit IgG and no primary antibody controls are shown in TMP195-treated tumours and reveal no background or non-specific positive signal. dp, Whole tumours were processed into single cells and flow cytometry was performed to determine the extent of immune cell infiltration into tumours. Representative graphs are shown from at least 3 independent experiments of 3–5 animals per group. All graphs shown mean and error bars represent s.e.m., t-test *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Figure 3 Characterization of recruited TMP195-activated myeloid cells.

Tumour-bearing MMTV-PyMT mice were randomly placed into treatment groups and received daily i.p. injections of either vehicle (DMSO) or 50 mg kg−1 of TMP195 for 5 days. Whole tumours were processed into single cells and flow cytometry was performed. a, Gating strategy for MHCII+CD11blo versus MHCII+CD11bhi. b, Representative mammary tissue macrophages (MTMs) versus tumor associated macrophages (TAMs) plots of 5 vehicle-treated and 5 TMP195-treated mice with quantitation. Graphs show the results from 5 independent experiments in which there were 3–5 animals per treatment group. ce, To identify pre-existing versus new tumour macrophages, mice were pretreated with dextran labelled with Alexa555, which is ingested by macrophages. Then mice were treated for 5 days with vehicle or TMP195. Mice were injected with dextran labelled with Alexa594 before being euthanized. Whole tumours were processed into single cells and flow cytometry was performed. d, There is an increase in new, but not pre-existing macrophages, in tumours from TMP195-treated animals. Representative graph from two separate experiments with n = 3 per treatment group (unpaired t-test). e, Of note, the new macrophages are MHCII+CD11bhi (mammary tissue macrophages; MTMs). t-test *P < 0.05. f, Mice received one i.p. injection of either vehicle (DMSO) or 50 mg kg−1 of TMP195. The following day mice were i.v. injected with CD11b+ cells labelled with CFSE. Mice were then treated for an additional 5 days with vehicle or TMP195. g, Whole tumours were processed into single cells and flow cytometry was performed. 13 mice from 3 different experiments are shown. There is a significant increase in recruitment of i.v. injected CD11b+CFSE+ monocytes to tumours in TMP195-treated mice. Graphs show the results from 2 independent experiments (unpaired t-test). All graphs show mean and error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Figure 4 TMP195-activated myeloid cells are highly phagocytic, engulf breast tumour cells and are co-stimulatory.

Mice were treated for 5 days with vehicle or 50 mg kg−1 of TMP195. Whole tumours were processed into single cells. a, Flow cytometry was performed and representative flow cytometry plots are shown indicating intracellular EPCAM signal inside F4/80+ macrophages, which corresponds with Fig. 2c. bd, CD11b+ cells were isolated from tumours and cytospun onto glass slides. Immunofluorescence was performed to identify phagocytosed breast tumour cells (corresponding with Fig. 2d). e, Representative flow cytometry plots of CD40+CD11b+ monocytes, and quantitation. f, g, In vitro TMP195 treatment enhances the co-stimulatory activity of monocytes differentiated in IL-4/GM-CSF. Human monocytes purified from peripheral blood were differentiated with IL-4 and GM-CSF for 5 days in the presence of 300 nM TMP195 or 0.1% DMSO as a control. f, FACS analysis of CD80 and CD86 shows an increase in the proportion of cells expressing the co-stimulatory molecule CD86. g, Following the 5-day differentiation, monocytes were used cells as antigen-presenting cells in a polyclonal T-cell proliferation assay (10 CFSE-labelled naive CD4+ T cells per 1 differentiated monocyte), T cells display a higher degree of proliferation (Division Index, FlowJo, Treestar Inc.) when co-cultured with monocytes differentiated in 300 nM TMP195 compared to the DMSO control monocytes. Data are representative of three independent experiments, each with two unique blood donors per experiment. All graphs show mean and error bars represent s.e.m. t-test **P < 0.01.

Extended Data Figure 5 TMP195 is not directly cytotoxic.

Tumour-bearing MMTV-PyMT mice were randomly placed into treatment groups. Mice received daily i.p. injections of either vehicle (DMSO) for 5 days or 50 mg kg−1 of TMP195 for 1, 3, or 5 days. a, b, IHC was performed on tumour sections for a marker of vasculature, CD34 (a), a marker of proliferation, Ki67 (b), and a marker of apoptosis, cleaved caspase 3 (CC3) (d). Quantitation as a percentage of total tissue is shown to the right of each representative section. Vehicle (5 days of DMSO) and 5 day TMP195 quantitation is taken from Fig. 2h for reference. For IHC representative quantitation and images are shown from two independent experiments with at least 3 mice per group. Scale bar, 100 μm. t-test *P < 0.05, **P < 0.01, ***P < 0.001 and ***P < 0.0001. c, Whole-tissue lysate was used to generate lysates for immunoblotting analysis using markers of apoptosis, parp and cleaved caspase 3. Each lane represents an individual tumour. For gel source data, see Supplementary Fig. 1. e, Human or mouse breast tumour cells were plated and treated with increasing concentrations of TMP195 (0, 0.1, 1, 10 μM), an inactive isomer, TMP058 (ref. 6; 0, 0.1, 1, 10 μM), staurosporine (0, 1, 10, 100 ng ml−1) or etoposide (0, 10, 50, 100 μM), for 48 h. CellTiter-Glo was used to assess cell viability. Error bars represent the average of three independent experiments. All graphs show mean and error bars represent s.e.m. An unpaired t-test was performed for all statistical values.

Extended Data Figure 6 TMP195 induces reduction in tumour burden and decreases pulmonary metastasis.

a, Treatment regimen of three independent experiments testing single-agent efficacy of TMP195. b, Mice with total tumour burden between 150–800 mm3 were treated with DMSO (n = 12) or 50 mg kg−1 of TMP195 (n = 13) for 13 days. Tumour volume was measured and plotted as total tumour burden ± s.e.m. c, The mice whose tumours started between 150–400 mm3 were kept on therapy to day 24 (DMSO n = 5; TMP195 n = 6). d, At day 24, their lungs were removed and H&E staining was performed to assess pulmonary metastasis. The number of metastatic lesions per lung section is quantitated and the mean is shown ± s.e.m. t-test ***P < 0.0001. e, f, Two additional independent experiments were performed to test single-agent efficacy of TMP195.

Extended Data Figure 7 Two week TMP195 treatment results in the biased distribution of select cell-type signatures.

a, Volcano plot of Affymetrix gene expression in RNA isolated from whole tumours (n = 3 mice per treatment group) demonstrates an overall lack of differential gene expression in these samples. bg, However, analysis for biased enrichment of the ImmGen cell-type signature gene sets identifies only 5 populations of cells as significantly (χ2 P value < 0.05) affected by TMP195 treatment as listed in (b) and illustrated in the volcano plots (cg). For a visual point of reference, the unaffected natural killer gene signature is highlighted in volcano plot h. Volcano plot y axes are Student’s t-test P values.

Extended Data Figure 8 Macrophages are required for efficacy of TMP195.

Tumour-bearing MMTV-PyMT mice were randomly placed into treatment groups. Mice received daily i.p. injections of either vehicle (DMSO) or 50 mg kg−1 of TMP195 in combination with (a, b, h, i) a myeloid depleting antibody (anti-CD11b) or (cg) a macrophage depleting antibody (anti-CSF-1). a, Tumour volumes were measured and plotted as average total tumour burden ± s.e.m. b, Tumours were removed from animals at the end of the CD11b depletion experiment and IHC was used to confirm the depletion of myeloid cells in the tumour tissue. cg, Corresponding with Fig. 3c, at the end of the experiment, tumours were removed from animals and flow cytometry was used to confirm depletion of macrophages in the tumour tissue. f, MHCII+CD11bhi but not MHCII+CD11blo macrophage populations were significantly depleted in response to the anti-CSF-1 depletion strategy. h, i, Corresponding with a, at the end of the experiment, tumours were removed from animals and IHC was used to assess cell death (h) and cellular proliferation (i). All bar graphs shown mean and error bars represent s.e.m. (unpaired t-test). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data

Extended Data Figure 9 CD8+ but not CD4+ T cells are required for optimal TMP195 efficacy.

a, b, Wild-type FVB/N (a) and immunodeficient athymic nude (b) mice were implanted with tumour pieces from MMTV-PyMT transgenic mice and treated daily for 16 days or 20 days, respectively, with vehicle (DMSO) or 50 mg kg−1 of TMP195. Athymic nude mice were also treated with paclitaxel (PTX) as a positive control. Tumour burden (a) or relative tumour burden (b) is shown for each treatment group. Relative tumour burden is the volume of the tumour at day 0 of treatment divided by the tumour volume at indicated day post treatment. c, Tumour-bearing MMTV-PyMT mice with similar total tumour burden were randomly placed into treatment groups and received daily i.p. injections of either vehicle or 50 mg kg−1 of TMP195 in combination with IgG, anti-CD8, anti-CD4 or anti-IFNγ for 6 days (corresponding with Fig. 3d). At the end of the experiment tumours were removed and single-cell suspensions were subjected to flow cytometry to confirm depletion of T cells in the tumour. d, e, Tumour-bearing MMTV-PyMT mice with similar total tumour burden were randomly placed into treatment groups and received daily i.p. injections of either vehicle or 50 mg kg−1 of TMP195 in combination with IgG or anti-CD8 for 14 days. e, CD8+ T-cell depletion was confirmed by flow cytometry. f, Mice treated with DMSO or TMP195 in combination with isotype or neutralizing anti-IFNγ antibody for 5 days and their tumours were collected. IHC was performed to identify the extent of vasculature organization. Graphs in b and e show mean and error bars represent s.e.m., (unpaired t-test) *P < 0.05 and **P < 0.01. Source data

Extended Data Figure 10 Representation of MMTV-PyMT breast tumours with and without TMP195 therapy.

Breast tumours in MMTV-PyMT transgenic mice contain leaky vasculature and monocytes and pro-tumour macrophages that suppress the function of CD8 T cells (left). Upon treatment with TMP195 (right side), tumour macrophages become activated, expressing CD40, and are highly phagocytic (engulfment of tumour cells depicted). CD8+ T cells become granzyme B+ indicating their ability to kill tumour cells. Tumour vasculature becomes more organized and less leaky. Additionally, there is a reduction in tumour volume.

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Guerriero, J., Sotayo, A., Ponichtera, H. et al. Class IIa HDAC inhibition reduces breast tumours and metastases through anti-tumour macrophages. Nature 543, 428–432 (2017). https://doi.org/10.1038/nature21409

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