Synergistic Immunostimulatory Effects and Therapeutic Benefit of 2 Combined Histone Deacetylase and Bromodomain Inhibition in Non-Small Cell 3 Lung Cancer

Author Manuscript Published OnlineFirst on April 13, 2017; DOI: 10.1158/2159-8290.
CD-16-1020
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
1 Title: Synergistic immunostimulatory effects and therapeutic benefit of
2 combined histone deacetylase and bromodomain inhibition in non-small cell
3 lung cancer
5 Authors: Dennis O. Adeegbe*1, Yan Liu*1, Patrick H. Lizotte1,2, Yusuke Kamihara1, Amir R.
6 Aref2, Christina Almonte1, Ruben Dries1, Yuyang Li1%, Shengwu Liu1, Xiaoen Wang1, Tiquella
7 Warner-Hatten1, Jessica Castrillon1, Guo-Cheng Yuan4, Neermala Poudel-Neupane1, Haikuo
8 Zhang1, Jennifer L. Guerriero1, Shiwei Han1, Mark M. Awad1,3, David A. Barbie1,3, Jerome
9 Ritz1, Simon S. Jones5, Peter S. Hammerman1,3, James Bradner1, Steven N. Quayle5, Kwok-Kin
10 Wong6
11 Affiliations:
1
12 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
2
13 Belfer Center for Applied Cancer Science, Dana Farber Cancer Institute, Boston, Massachusetts
3
14 Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston,
15 Massachusetts
4
16 Harvard Chan school of Public Health, Boston, Massachusetts
5
17 Acetylon Pharmaceuticals, Inc., Boston, Massachusetts
6
18 Laura & Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York, New York
19 % Yuyang Li’s current address:
20 Shandong Provincial Hospital, Shandong University, Jinan, China
21 *These authors contributed equally to this work
22
23 Running title: HDAC and bromodomain inhibition as immunotherapeutics in NSCLC
24 Keywords: Histone deacetylase inhibitor, Bromodomain inhibitor, Non-small cell lung cancer, immunomodulatory,
25 immunotherapy.
Downloaded from cancerdiscovery.aacrjournals.org on July 4, 2017. © 2017 American Association for Cancer
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Author Manuscript Published OnlineFirst on April 13, 2017; DOI: 10.1158/2159-8290.CD-16-1020
26
27 Corresponding author: Kwok-Kin Wong, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA
28 02115. Phone: 617-632-6084; Fax: 617-582-7839; E-mail: kwong1@partners.org
29 Conflict of interest disclosure: SSJ and SNQ are both employees and shareholders of Acetylon Pharmaceuticals. All
30 other authors have no competing financial interests.
31
32 Abstract
33 Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an
34 increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now
35 clear that therapeutic agents with potential to impact the tumor immune microenvironment
36 potentiate immune-orchestrated therapeutic benefit. Herein we evaluated the immunoregulatory
37 properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that
38 modulate the epigenome, with a focus on key cell subsets that are engaged in an immune
39 response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective
40 HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T cell
41 activation and improved function of antigen presenting cells. The bromodomain inhibitor JQ1
42 attenuated CD4+Foxp3+ T regulatory cell suppressive function and synergized with ricolinostat
43 to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with
44 lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two
45 epigenetic modifiers that, together, promote T cell-mediated anti-tumor immunity and
46 demonstrate their therapeutic potential for treatment of NSCLC.
47
48 Statement of Significance: Selective inhibition of histone deacetylases and bromodomain
49 proteins modulates tumor-associated immune cells in a manner that favor improved T cell
50 function and reduced inhibitory cellular mechanisms. These effects facilitated robust anti-tumor
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51 responses in tumor-bearing mice demonstrating the therapeutic potential of combining these
52 epigenetic modulators for the treatment of NSCLC.
53 Introduction
54 The tumor microenvironment contains a variety of stromal/supporting cells, including
55 those of hematopoietic origin. Specific cell types such as T lymphocytes, natural killer (NK)
56 cells, and various myeloid cells play diverse roles in shaping the course of the immune response
57 against cancer [1-3]. Of particular importance are antigen-presenting cells (APCs), which
58 process tumor-associated antigens (TAAs) and present them to potential tumor-reactive T cells in
59 a process that leads to T cell priming. Despite their potential to generate an anti-tumor response,
60 T cell antigen-specific responses are often inhibited by immunosuppressive cells such as
61 myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3+ regulatory T cells (Tregs) [4-6].
62 Thus, agents that promote stimulatory immuno-phenotypic and functional changes in APCs and
63 effector T cells or disrupt inhibitory cellular mechanisms may generate a more permissive tumor
64 microenvironment favoring enhanced anti-tumor responses.
65 Histone deacetylases (HDACs) are a family of enzymes that modulate the expression of
66 genes or their products by removing acetyl groups from lysine residues on histone and non-
67 histone proteins. As a consequence, they regulate numerous cellular processes, some of which
68 have been implicated in aspects of tumor development and behavior such as differentiation and
69 cell cycle progression in malignant cells, as well as apoptosis [7-9]. Class I and II HDACs are
70 among the most active regulators of histone and non-histone proteins in cancer [10] and their
71 pharmacologic inhibition is seeing increasing interest for treatment of solid cancers. The
72 contribution of the various classes of the HDAC family to tumor pathogenesis is well
73 documented. Class I HDACs have been reported to contribute to the transformation of epithelial
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74 cells and/or metastasis [11, 12] while members of class II HDACs play critical roles in regulating
75 autophagy, proliferation and differentiation in tumor cells [13-16]. The roles of class III
76 (Sirtuins) and class IV HDAC family members are still emerging although existing reports
77 implicate their involvement in DNA damage response pathways, chromatin regulation, genome
78 integrity, cell survival under stress, tumor metabolism and cell death [17-19]. Given their clear
79 importance in cancer pathogenesis, pharmacologic inhibition of HDACs has become an area of
80 significant interest for cancer therapy.
81 Several studies have evaluated the relative utility of both pan- and isozyme-selective
82 HDAC inhibitors for cancer therapy [20, 21]. Although promising therapeutic outcomes have
83 been demonstrated using pan-HDAC inhibitors, especially when combined with
84 chemotherapeutic drugs, their use is associated with adverse effects and dose-limiting toxicity in
85 patients [22-25]. Thus, isozyme-specific inhibitors may offer improved benefits arising from
86 their selective inhibition of some but not all HDACs. While many translational studies exploring
87 the therapeutic efficacy of HDAC inhibitors have focused on their effects on malignant cells [26-
88 28], multiple studies have highlighted the immunomodulatory activity of HDAC inhibitors on
89 various immune cellular subsets when used alone or in combination with other
90 immunotherapeutic agents [8, 9, 20, 21, 29-33]. Furthermore, HDAC inhibitors have been shown
91 to promote tumor cell recognition by immune cells via induction of MHC constituents and
92 antigen-presentation machinery on tumor cells [34]. Despite these data, the effects of HDAC
93 inhibitors on the cellular constituents of the tumor immune microenvironment remains poorly
94 understood.
95 Bromodomain inhibitors are a second class of agents that alter cellular epigenetic and
96 transcriptional programs through regulating the recognition of acetylated lysine residues by
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97 transcriptional machinery. JQ1 is a well-characterized inhibitor of the bromodomain and
98 extraterminal (BET) family of bromodomain-containing proteins and may indirectly regulate
99 epigenetic footprints by modulating the interactions of histone acetyl transferases (HATs) and
100 HDACs with transcription factors and proteins involved in gene expression [35]. While JQ1, like
101 other BET inhibitors, has demonstrated efficacy in hematologic malignancies, its efficacy in
102 solid cancers has only recently been explored. For example, JQ1 was shown to synergize with
103 the pan-HDAC inhibitor SAHA to suppress tumor growth in models of pancreatic ductal and
104 lung adenocarcinoma [36]. Although the mechanisms governing therapeutic synergy were not
105 fully explored in that report, it was suggested that these agents function to repress MYC
106 expression and inflammatory cascades that regulate IL-6 levels [36]. Thus, the effect of JQ1 on
107 tumor-associated immune cells from this and other studies remains largely undefined.
108 Recent preclinical and clinical data generated with antibodies that target immune
109 checkpoints, namely PD-1/PD-L1 and CTLA-4, demonstrate that anti-tumor immunity can be
110 driven in multiple tumor types by targeting inhibitory interactions among T cells, APCs, and
111 tumor cell populations [37, 38]. While the use of these agents represents a major advance in the
112 treatment of cancer, it remains the case that in most tumor types, only a minority of patients
113 respond and the barriers to response are diverse and poorly characterized. As the durability of
114 anti-tumor immunity is increasingly appreciated to hinge upon immune cell contributions, we
115 sought to identify the immunomodulatory effects of small molecule epigenetic modulators
116 including selective HDAC inhibitors and JQ1. We describe significant dynamic changes with
117 respect to phenotype and function of T cells and monocytes/macrophages in peripheral tissues as
118 well as in the tumor microenvironment by ricolinostat (ACY-1215), an HDAC inhibitor selective
119 for the class II HDAC6. We also describe the Treg-specific effects of JQ1, which serves as the
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120 basis for its use as a partner agent with ricolinostat, a combination that facilitated sustained anti-
121 tumor responses in immunocompetent, genetically engineered mouse models of non-small cell
122 lung cancer (NSCLC).
123
124 Results
125 Ricolinostat reduced the frequency of CD4+FOXP3+ Tregs and promoted transient activation of
126 peripheral and tumor-associated T lymphocytes.
127 HDAC inhibition results in tumor growth arrest in immunocompetent animal models [39-
128 41]. As HDACs are ubiquitously expressed in many tissues, including immune cells, an
129 outstanding issue is whether some of the reported therapeutic benefits of HDAC inhibitors stem
130 from their effects on immune cells. In this regard, we sought to determine the effects of selective
131 inhibition of members of class I and class II HDACs on leukocytes. First, we cultured peripheral
132 blood mononuclear cells (PBMCs) from healthy donors with ricolinostat and compared it with a
133 panel of HDAC inhibitors with varying selectivity for class I HDACs 1/2/3 and class II HDAC6
134 including ACY-241, ACY-738, ACY-775, and tubastatin A. This panel also included entinostat
135 (MS-275), a class I HDAC-selective agent currently being evaluated in clinical trials, in order to
136 assess the relative impact of selective inhibition of class I HDAC enzymes (Supplementary Table
137 1). Notably, viability of cells cultured for 24 hours with these HDAC inhibitors was largely
138 similar (Supplementary Fig. S1A). Furthermore, the proportions (Supplementary Fig. S1B) and
139 phenotype (not shown) of leukocyte subsets, including monocytes, B cells, NK cells,
140 CD4+FOXP3- and CD8+ conventional T cells were not significantly impacted by these drugs.
141 While the percentage of CD4+FOXP3+ Tregs within PBMCs was reduced when cultured with
142 ACY-738 or entinostat, this effect was most striking with ricolinostat or ACY-241 treatment
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143 (Supplementary Fig. S1C and S1D). Interestingly, the highly selective HDAC6 inhibitors, ACY-
144 775 and tubastatin A, did not significantly impact Treg frequency. This finding suggests that
145 inhibition of class I HDAC and class II HDAC6 by compounds such as ricolinostat and ACY-
146 241 may impact Treg frequency more so than single class-selective agents such as entinostat.
147 Next, PBMCs from NSCLC patients (Supplementary Table 2) or bulk single cell
148 suspensions from dissociated freshly resected patient lung tumors were cultured with ricolinostat
149 or entinostat for 24 hours. Flow cytometry analysis of leukocyte subsets within the cell cultures
150 revealed that ricolinostat significantly reduced Treg proportions relative to DMSO control or
151 entinostat (Fig. 1A, p=0.04; 1D, p=0.05, Supplementary Fig. S1E), similar to what was observed
152 for healthy donor PBMCs (Supplementary Fig. S1C and S1D). In parallel, expression of the
153 CD69 activation marker was substantially upregulated on conventional CD8+ and CD4+ T cells
154 in both patient (Fig. 1B, p=0.003; 1C, p=0.01) and healthy donor (Supplementary Fig. S2A and
155 S2B) PBMC cultures in the presence of ricolinostat but not entinostat, suggesting that an
156 increased activation profile is promoted by ricolinostat. In addition, we observed substantial
157 increase in phosphorylated STAT3 levels that accompanied increased CD69 expression in T cells
158 within healthy donor PBMCs exposed to ricolinostat (Supplementary Fig. S2C and S2D).
159 In contrast to patient PBMCs, we found that tumor infiltrating immune cells in the two
160 dimensional (2-D) cultures of NSCLC patient tumors were significantly less viable in the
161 presence of entinostat when compared to ricolinostat (Supplementary Fig. S3), precluding further
162 analysis of entinostat-treated tumor cultures. Although many tumor-infiltrating T cells already
163 exhibited an activated profile based on CD69 positivity ex vivo (data not shown), exposure to
164 ricolinostat led to a modest increase in the frequency of CD69+CD8+ T cells (Fig 1E, p=0.39)
165 and a significant increase in CD69+CD4+ cells (Fig. 1F, p=0.04). This observation suggests that
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166 ricolinostat enhanced activation of T cells in a setting where tumor-relevant antigens were likely
167 present. Furthermore, after removal of drugs from primary cultures, CD8+ T cells from patient
168 PBMCs that were re-stimulated in secondary cultures with PMA and ionomycin showed a higher
169 frequency of IFN-γ positivity (Fig. 1G, p=0.05). (Supplementary Fig. S4A-C), indicating
170 enhanced effector cytokine secretion and cytotoxic capability following exposure to ricolinostat.
171
172 Ricolinostat promotes increased expression of MHC and co-stimulatory molecules on human
173 monocytes and tumor-associated macrophages.
174 Existing reports demonstrate that HDAC6 inhibition can effect functional changes in
175 APCs through a mechanism that involves regulation of inflammatory cytokine production [42].
176 We therefore explored whether ricolinostat alters APC phenotype and function. Specifically, we
177 evaluated CD14+CD11b+ monocytes in the peripheral blood of NSCLC patients and CD14-
178 CD45+CD68+CD11b+ macrophages within disaggregated NSCLC tumors. Upon 24-hour
179 culture with ricolinostat, the CD14+ monocyte fraction significantly up-regulated surface
180 expression of MHC class II molecules (p=0.04), as well as CD86 (p=0.01) but not CD80 (data
181 not shown), phenotypic changes that are associated with increased priming ability of APCs [43].
182 In contrast, entinostat only promoted upregulation of CD86 while not affecting MHC class II
183 expression (Fig. 2A and B). Ricolinostat elicited a similar pattern of increased expression of
184 MHC class II and CD86 on the CD14-CD45+CD68+CD11b+ macrophages within 2-D tumor
185 cultures (Fig. 2C and D) and in PBMCs obtained from healthy donors (Supplementary Fig. S5A
186 and S5B). These findings demonstrate a unique modulatory effect of ricolinostat on human
187 monocytic cells and tumor-infiltrating macrophages, and suggest that ricolinostat promotes
188 phenotypic changes that support enhanced antigen presentation and co-stimulatory capabilities.
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189 Consistent with this notion, ricolinostat-exposed CD14+ monocytes were superior at inducing
190 allogeneic T cell proliferative responses in mixed lymphocyte reactions (Fig. 2E).
191
192 Ricolinostat promotes quantitative and phenotypic changes that facilitate tumor-infiltrating T
193 cell activation and function in a non-small cell lung cancer model.
194 Given the importance of tumor-associated immune cells in shaping the course of tumor
195 progression and anti-tumor immunity, we next investigated whether the phenotypic and
196 functional changes seen in vitro on immune cells upon ricolinostat treatment are recapitulated in
197 vivo, especially in the context of tumor antigens. To address this issue, we examined the effects
198 of ricolinostat on immune cells infiltrating lung adenocarcinomas that spontaneously develop in
199 two fully immunocompetent mouse models: 1) genetically engineered mice (GEM) harboring an
200 activating KrasG12D mutation concurrent with p53 loss (designated KP), or 2) GEM with
201 T790M/L858R EGFR mutations (designated TL), as previously described [44].
202 Mice with established adenocarcinomas (as confirmed by MRI) were treated once daily
203 with ricolinostat for seven days prior to evaluation of dissociated tumors by comprehensive
204 multi-parametric flow cytometry. Under this short-term treatment, we observed modest increases
205 in the frequency of tumor-infiltrating CD8+ T cells and significant decreases in CD4+Foxp3+
206 Treg cells among lymphoid cells (Fig. 3A and B), resulting in a significant elevation of
207 CD8:Treg ratios in treated tumors (Fig. 3C). Such increases in CD8:Treg ratio are often
208 associated with enhanced anti-tumor responses [45]. Although there were no substantial
209 phenotypic changes with respect to inhibitory receptor molecule expression (Supplementary Fig.
210 S6A-D), tumor-infiltrating CD8+ T cells in ricolinostat-treated mice exhibited increased
211 expression of the activation marker CD69 (Fig. 3D) and enhanced secretion of the effector
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212 cytokine IFN-γ following ex vivo stimulation compared with equivalent cells in the vehicle
213 control group (Fig. 3E).
214 Consistent with this finding, single cell RNA-sequencing (RNA-seq) revealed that a
215 number of genes associated with T cell activation and TCR signaling including Cd69, Cd44,
216 Cd247, and Zap70 were up-regulated in tumor-infiltrating T cells from KP mice treated with
217 ricolinostat when compared to equivalent cells derived from the tumors of the vehicle group
218 (Supplementary Fig. S7). Among myeloid cell populations, the proportions of CD11c+CD11blo
219 tumor-associated macrophages (TAMs; Supplementary Fig. 8A) were comparable between the
220 treatment group and vehicle controls (data not shown). However, under this short-term
221 ricolinostat treatment, TAMs exhibited significantly elevated expression of MHC class II as well
222 as co-stimulatory molecule CD86 (Fig. 3F and G, Supplementary Fig. S8B). Consistent with this
223 phenotype, ricolinostat-exposed TAMs analyzed by single cell RNA-seq showed higher
224 expression of key genes associated with MHC class II expression including Cd74 and H2-Aa
225 relative to their counterparts in the control mice (Supplementary Fig. S8C).
226 As increase in acetylation of tubulin is considered to be one measure of pharmacologic
227 inhibition of HDAC6 [42, 46, 47], we observed that ricolinostat treatment led to increased levels
228 of acetyl α-tubulin (lysine 40) in the tumor (supplementary Fig. S9), providing supporting
229 evidence that changes occurring consequent to ricolinostat treatment stem from drug activity in
230 tumor-associated immune cells.
231 Collectively, these findings from in vivo treatment of tumor-bearing mice are consistent
232 with our in vitro observations in PBMCs, and indicate that upon ricolinostat treatment, tumor-
233 infiltrating macrophages underwent changes analogous to more mature APC phenotype that
234 supports a more activated, effector profile of surrounding T cells.
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235
236 BET bromodomain inhibitor JQ1 disrupts Treg signature protein expression and attenuates
237 suppressive function.
238 Based on the positive immuno-dynamic and phenotypic changes mediated by ricolinostat
239 in immune cell subsets, we reasoned that these effects should translate to augmented anti-tumor
240 immune response. In initial efficacy studies, we found that ricolinostat treatment in
241 immunocompetent KP mice provided only marginal therapeutic benefit; that is, minimal
242 inhibition of tumor growth was observed relative to vehicle-treated controls (Supplementary Fig.
243 10). In an attempt to identify a partner agent to augment anti-tumor effect, we focused on JQ1, an
244 inhibitor of the BET family of bromodomain proteins. Our choice of JQ1 was based on our
245 hypothesis that, due to its indirect regulation of epigenetic "reader" proteins, which are key to
246 immune cell biology, JQ1 should also potentiate immunomodulatory effects in the tumor
247 microenvironment [48-50]. To this end, we treated KP mice with JQ1 in order to determine
248 whether it promotes changes in tumor-associated immune cells that are beneficial for tumor
249 immunity. Analysis of lung tumor-infiltrated T cells revealed that CD4+Foxp3+ Tregs showed
250 significant downregulation of Foxp3, CTLA-4, and PD-1 upon JQ1 treatment, whereas, little
251 phenotypic changes were observed in the conventional T cell subsets (Fig. 4A and B,
252 Supplementary Fig. S11A, and S11B). In addition, phospho-STAT5, a bromodomain-dependent
253 target gene [51-53]. was downregulated in tumors of JQ1-treated mice, specifically in Tregs,
254 supporting the notion that JQ1 activity is dominant in Tregs relative to conventional T cells
255 (Supplementary Fig. S12A-D).
256 Furthermore, the phenotypic changes in Tregs noted above was accompanied by
257 decreased suppressive function as Tregs isolated from the tumors of JQ1-treated mice were less
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258 efficient at inhibiting the proliferation of responding effector T cells when compared to their
259 counterparts in vehicle-treated mice (Fig. 4C and D). In contrast, Tregs isolated from the spleens
260 of these tumor-bearing mice were comparable in their inhibitory function (Supplementary Fig.
261 S13) demonstrating that tumor-Tregs were more susceptible to JQ1 effect, possibly due to their
262 unique and heightened expression of PD-1, CTLA-4, and CD69 (Supplementary Fig. S14A and
263 S14B). Consistent with these findings in the GEM, FOXP3+ Tregs present within dissociated
264 tumors from NSCLC patients cultured with JQ1 showed diminished expression levels of FOXP3,
265 CTLA-4, and PD-1 relative to those cultured in the presence of ricolinostat or control DMSO
266 (Supplementary Fig. S15). In summary, these results indicate that JQ1 preferentially modulates
267 Treg phenotype and function in tumor-infiltrating immune cells.
268
269 BET bromodomain inhibitor JQ1 synergizes with ricolinostat to enhance immune-dependent
270 suppression of tumor growth
271 A recent report by Mazur and colleagues showed that JQ1 has an anti-tumor effect in KP
272 mice in part due to downregulation of MYC and induction of apoptosis in tumor cells [36]. The
273 preceding data also demonstrate that JQ1 exhibits a unique effect on tumor-associated Tregs. We
274 therefore hypothesized that these tumor and immune cell-specific JQ1 effects, when combined
275 with the immunomodulatory effects of ricolinostat, should foster a tumor immune
276 microenvironment that favors stronger stimulation of tumor infiltrating T cells and an augmented
277 anti-tumor response. Thus, mice with lung adenocarcinomas were treated with ricolinostat and/or
278 JQ1 in long-term efficacy studies (Supplementary Fig. S16). Immunocompetent GEM with
279 established tumors (150-250mm3) were administered a daily dose of ricolinostat and/or JQ1, and
280 tumor growth kinetics evaluated. In KP mice, ricolinostat or JQ1 monotherapy led to minimal or
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281 moderate delay in tumor growth, respectively, but combination treatment with both agents
282 resulted in significant arrest of tumor growth (Fig. 5A-C). In alignment with these outcomes,
283 vehicle-treated mice harbored large tumors with high grade adenocarcinomas, while single and
284 combination therapy tumors were mostly low grade adenocarcinomas often exhibiting the lowest
285 histopathology grade and increased cellular infiltrates (Supplementary Fig. S17A). Additionally,
286 tumors in the treatment groups particularly the combination, displayed reduced Ki67 and
287 elevated cleaved Caspase-3 expression, signaling reduced proliferation and increased cell death,
288 respectively (Supplementary Fig. S17B and S17C).
289 These anti-tumor effects were largely dependent on CD8+ and CD4+ T cells, because
290 tumor growth arrest was abrogated upon incorporation of CD8- or CD4-depleting antibodies in
291 the combination treatment regimen (Fig. 5B). Furthermore, combination therapy led to longer
292 progression-free survival compared to either agent alone (Fig. 5D), suggesting that JQ1
293 synergizes with ricolinostat to drive T cell-mediated anti-tumor response. Similar to KP mice,
294 the combination was also efficacious in mice bearing mutant EGFR (TL) lung tumors, as well as
295 Lewis lung carcinomas, as evidenced by marked inhibition of tumor growth in each of these
296 models (Supplementary Fig. S18A and S18B). These data suggest that the therapeutic benefits of
297 this combinatorial approach extend beyond lung adenocarcinomas driven by ectopic expression
298 of driver mutations to those arising by spontaneous development.
299 Immune profiling of tumor nodules further revealed that, relative to the single agent
300 treatments, tumor-infiltrating CD8+ T cells were more activated, as evidenced by increased
301 expression of CD69 (Fig. 5E). In addition, these tumor-infiltrating CD8+ T cells demonstrated
302 significantly enhanced secretion of effector cytokine IFN-γ (Fig. 5F) as well as increased
303 degranulation as indicated by CD107ɑ staining (Fig. 5G).
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304 Furthermore, when equivalent numbers of T cells, TAMs, and Epcam+ tumor cells sorted
305 from tumor nodules of ricolinostat and/or JQ1-treated mice were co-cultured together with tumor
306 cell lysate, we found a higher proportion of tumor cell death in the presence of T cells isolated
307 from the tumors of dual agent-treated mice (Fig. 5H). Thus, combination treatment enhanced the
308 tumor killing potential of tumor infiltrating T cells. With respect to the TAMs, ricolinostat
309 treatment, either alone or in combination with JQ1, markedly promoted upregulation of MHC
310 class II molecules and CD86 (Fig. 5I and J).
311
312 Ricolinostat and JQ1 treatment promote quantitative changes in tumor-infiltrating immune cell
313 subsets that signal a re-shaping of tumor-immune cell dynamics.
314 Although we detected very little change in the proportions and phenotypes of T cells
315 under acute (7 days) drug treatments with the exception of Tregs (Fig. 3A-C, Supplementary Fig.
316 S6), we wondered whether the immune cell dynamics change over time especially upon long-
317 term treatment where we observed change in tumor size after prolonged drug exposure. While
318 the proportion of conventional T cells did not change dramatically within the tumors across
319 treatment groups (Fig. 6A), tumors of KP mice treated with JQ1 alone or in combination with
320 ricolinostat exhibited substantially reduced Treg numbers, yielding significantly increased
321 CD8:Treg ratios, an effect that was not evident in peripheral Treg cells (Fig. 6A, B,
322 Supplementary Fig. S19A and S19C). In contrast, CD11c+ TAMs increased with more
323 pronounced clusters of these cells within the tumors of mice treated with JQ1 and/or ricolinostat
324 (Fig. 6C, Supplementary SFig. 19B), demonstrating peripheral reshaping of immune cell
325 dynamics within the tumor in the presence of these drugs. While their frequencies did not differ
326 dramatically, tumor-infiltrating T cells, particularly CD8+ cells, in JQ1- and JQ1/ricolinostat-
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327 treated mice exhibited significantly reduced expression levels of the immune checkpoint
328 receptors PD-1, CTLA-4, TIM-3, and BTLA relative to the vehicle- and ricolinostat-treated
329 groups (Supplementary Fig. S20A-D), indicating a correlation between the expression levels of
330 inhibitory proteins on these T cells and the outcome of tumor growth kinetics.
331 Next, we wondered whether despite the lack of substantial changes in immune cell
332 dynamics under acute drug exposure, other changes were already being imprinted in tumor-
333 immune cell populations. In this regard, we performed gene expression profiling to address this
334 issue and as an approach to elucidate the mechanistic underpinnings for each drug as a sole
335 agent. Our results revealed that TAMs sorted from the tumors of ricolinostat-treated mice, but
336 not JQ1-treated mice, show increased gene transcripts for the MHC class II transactivator Ciita
337 and MHC class II H2-DMb relative to vehicle controls (Supplementary Fig. S21A and S21B).
338 On the other hand, JQ1 but not ricolinostat treatment remarkably led to downregulation of Treg-
339 associated genes and/or genes encoding inhibitory co-receptors including Foxp3, Ctla4, Cd25,
340 Ccr4, Pdcd1, and Btla in sorted tumor T cells (Supplementary Fig. S22A and S22B). This effect
341 is not due to a reduction in Treg numbers within the analyzed T cell samples as these were
342 comparable between ricolinostat and JQ1 treatments, and only slightly lower than the vehicle
343 group (Supplementary Fig. S23). Interestingly, this gene expression pattern was largely absent in
344 ricolinostat-exposed tumor T cells, which displayed a distinct profile with minimal changes in
345 the gene transcripts for the above-mentioned inhibitory receptors (Supplementary Fig. S22A and
346 S22B).
347 Taken together, these findings demonstrate that selective inhibition of HDAC6 by
348 ricolinostat and bromodomain inhibition by JQ1 evoke distinct gene transcriptional landscape
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349 that yield striking immunomodulatory features which, when combined, synergizes to potentiate
350 robust immune-mediated anti-tumor response in our described NSCLC models.
351 Discussion
352 The utility of HDAC inhibitors as anti-cancer agents is gaining traction in oncology in
353 part due to reported cytostatic effects on tumor cells [54]. However, their effects on immune
354 cells recruited to tumors are still not well understood. Such knowledge is critical as cross-talk
355 between immune cells and cancer cells shapes the course of tumor progression. While pan-
356 HDAC inhibitors are currently being evaluated as therapeutic options for a number of cancers,
357 isozyme-selective HDAC inhibitors may possess unique properties. Indeed, we observed that
358 ricolinostat, which is highly selective for HDAC6 but retains some inhibitory activities on class I
359 HDACs as well, has striking immuno-modulatory effects. In the present study, we characterized
360 the effects of ricolinostat on immune cells and evaluated the therapeutic potential of combining
361 this HDAC inhibitor with an inhibitor of BET bromodomain proteins in models of NSCLC.
362 The increased expression of MHC class II and co-stimulatory molecule CD86 that we
363 observed on tumor-associated macrophages (TAMs) upon ricolinostat treatment in lung tumor-
364 bearing mice indicates that ricolinostat possesses immunomodulatory properties that promote
365 phenotypic changes favoring improved delivery of co-stimulatory signals and antigen
366 presentation. Furthermore, the observed increase in CD69 expression on ricolinostat-exposed T
367 cells also suggests that it may facilitate cellular events that lead to T cell activation. Such
368 activating effect may be beneficial for amplification of T cell receptor (TCR)-induced antigen-
369 driven T cell priming, especially in the context of tumor-associated antigens. While they did not
370 focus on T cells, Sotomayor and colleagues also reported that HDAC6 inhibition improved APC
371 function in a manner involving the regulation of STAT3/IL-10 tolerogenic axis in APCs [42],
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372 raising the possibility that selective HDAC6 inhibition may differentially regulate activation and
373 function in multiple cell types.
374 Our results suggest that ricolinostat has the potential to facilitate T cell priming in cancer
375 patients. This notion is supported by the increased activation and enhanced effector function of
376 tumor-infiltrating CD8+ T cells observed in tumor-bearing mice treated with ricolinostat.
377 Although ricolinostat treatment alone did not robustly dampen tumor growth in our NSCLC
378 models, it substantially increased T cell mediated anti-tumor efficacy when coupled with JQ1.
379 Thus, the enhancement of the therapeutic activity of ricolinostat by JQ1 cotreatment suggests
380 that promoting antigen presentation and co-stimulation alone is not sufficient to achieve
381 sustained anti-tumor responses. Rather a significant reduction in inhibitory cellular mechanisms
382 operative within the tumor bed as achieved by JQ1 therapy is fundamental to augmenting the
383 therapeutic benefit of ricolinostat treatment. Nevertheless, the enhanced APC function conferred
384 by ricolinostat warrants further exploration, as does its application in NSCLC treatment in
385 tandem with vaccination strategies.
386 Given that CD4+FOXP3+ T regulatory cells accumulate in many solid cancers [55],
387 agents that target these cells may offer therapeutic benefits. In this regard, we found that JQ1,
388 more than ricolinostat treatment, led to substantial reduction in tumor-infiltrating Treg fractions
389 evidenced by reduced Foxp3 expression level, as well as reduced Treg function. These findings
390 suggest that Treg cells may be more susceptible to epigenetic modifications by bromodomain
391 proteins than their conventional T cell counterparts. Given that FOXP3, CTLA-4, and PD-1 are
392 critical for Treg stability and/or function [56-58], their diminished expression or transcriptional
393 repression may compromise tumor-Treg survival and suppressive function as we observed in
394 lung tumor-bearing, JQ1-treated mice. Although existing reports demonstrate that certain HDAC
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395 inhibitors affect Treg biology [35, 59, 60], and ricolinostat clearly evoked deleterious effects on
396 patient tumor-Tregs in vitro, the more profound anti-Treg effect in our in vivo lung tumor model
397 is attributed to JQ1.
398 Our gene expression profiling provides a snapshot of potential mechanisms of action by
399 these drugs in our NSCLC investigational model: ricolinostat promoted increased transcription
400 of genes that are key components of MHC class II expression in tumor macrophages, molecular
401 changes that support improved antigen presentation and T cell priming [61]. On the other hand,
402 bromodomain inhibition by JQ1 remarkably led to a decrease in transcription of gene signature
403 associated with Treg maintenance/function which significantly overlap with those associated
404 with immune checkpoint co-receptors [62-64]. We postulate that ricolinostat plays a pleiotropic
405 role on immune cells and functions by increasing activation of T cells and promoting APC
406 function while JQ1 contributes to reduction of Treg proportions in addition to disrupting the
407 suppressive function of residual Tregs present in the tumor bed. The net effect is promotion of
408 leukocyte-orchestrated anti-tumor response by way of optimizing antigen delivery, increasing
409 co-stimulatory signals, and lowering inhibitory cellular mechanisms (Fig. 7).
410 As the race continues towards finding effective therapeutic modalities for solid cancers,
411 such as NSCLC, our findings show that the combination of ricolinostat and JQ1 lead to robust
412 immune-mediated anti-tumor effects in lung tumors driven by different mutation genotypes,
413 which is timely and important. This crucial contribution by the immune cells, particularly T
414 cells, is underscored by the abrogation of tumor growth arrest seen under ricolinostat and JQ1
415 therapy upon T cell depletion. In a nutshell, our findings highlight previously unknown
416 immunomodulatory effects of these two classes of drugs in combination and support their
417 therapeutic testing in lung adenocarcinoma. While not the focus of this study, both agents have
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418 direct anti-tumor effects [36, 41, 48, 65]. This raises the exciting possibility that ricolinostat and
419 JQ1 could be utilized in combination as “hybrid” therapies that harness not just direct anti-tumor
420 effects, but also indirect effects via peripheral reshaping of immune cell dynamics and function,
421 converging to foster strong anti-cancer benefits. With JQ1 currently being evaluated in clinical
422 trials for solid cancers, and ricolinostat, which has a good safety profile presently in clinical
423 development, both drugs are promising therapeutic agents that could be combined as epigenetics-
424 based immunotherapeutics for the treatment of lung adenocarcinomas.
425
426 Online Methods
427 Mice
428 Genetically engineered mice harboring Kras+/LSL-G12DTrp53L/L (KP) or EGFRLSL-T790M/L858R (TL)
429 have been previously described [44]. For lung tumor induction, mice received 1 × 106 CFU Cre-
430 encoding adenovirus intranasally at 5–6 weeks of age. Tumor formation (typically 6–8 weeks
431 post-inoculation) and volume were confirmed by MRI and quantified using 3D Slicer software.
432 C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). All mice were
433 maintained under pathogen-free conditions in accordance with institutional guidelines for animal
434 welfare and experiments involving human tissues were conducted under approved, Dana-
435 Farber/Harvard Cancer Center (DF/HCC) IRB protocol 02-180 with informed consent by
436 patients.
437
438 Drugs and reagents
439 Ricolinostat (ACY-1215), ACY-241, ACY-738, and ACY-775 were provided by Acetylon
440 Pharmaceuticals (Boston, MA). Tubastatin was obtained from ApexBio Technology (Houston,
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Research.
441 TX) and Entinostat was obtained from selleckchem (Houston, TX). Ricolinostat was formulated
442 in 10% DMSO in 5% dextrose and intraperitoneally administrated to mice at 50 mg/kg daily.
443 JQ1, kindly provided by James Bradner (Dana Farber Cancer Institute, Boston, MA), was
444 formulated in 10% DMSO in 10% 2-hydroxylpropyl β-cyclodextrin (Sigma-Aldrich, Saint
445 Louis, MO) and intraperitoneally administrated to mice at 25 mg/kg daily. Each drug’s vehicle
446 control represents DMSO diluted into the vehicle agent. Recombinant human cytokines IL-2, IL-
447 4, and GM-CSF were purchased from Peprotech (Rocky Hill, NJ).
448
449 Antibodies
450 In vivo antibodies anti-CD4 (GK1.5), anti-CD8 (53-6.72), and rat IgG (LTF-2) were purchased
451 from Bio X Cell (Lebanon, NH). Mice were first administered two consecutive doses (400
452 μg/mouse) of antibodies at day -2 and day -1 and twice per week thereafter along with
453 ricolinostat and JQ1 combination treatment. Antibodies utilized for flow cytometric analyses are
454 listed in supplementary information.
455
456 Tumor profiling and flow cytometry
457 To generate cell suspensions, tumor nodules were excised from lungs of KP and TL mice, cut
458 into small pieces, and further dissociated in RPMI-1640 buffer containing 5% FBS, 100 IU/ml
459 collagenase type IV (Invitrogen, Carlsbad, CA), and 50 μg/ml DNAse I (Roche, Basel,
460 Switzerland) for 45 min at 37°C. After incubation, cells were treated with red blood cell lysis
461 buffer and filtered through a 70 μm cell strainer. After centrifugation, cell pellets were
462 resuspended in 1X PBS/2% FBS. About 0.5–1 x 106 cells were stained for surface markers in 1X
463 PBS/2% FBS for 15 min at 4°C. Intracellular staining was performed for Foxp3 using the Foxp3
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464 staining kit (ebioscience, Santa Clara, CA) according to manufacturer’s instructions. The
465 Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA) was used for intracellular cytokine
466 staining. Briefly, fixed and permeabilized cells were stained for surface markers, including CD4,
467 CD8, and CD3, followed by intracellular staining with PE-conjugated anti-IFN-γ, or isotype-
468 matched mAbs. In all stained samples, dead cells were excluded using Live/Dead Fixable Dead
469 Cell staining kit (Invitrogen, Carlsbad, CA). Cells were acquired on the LSR Fortessa (BD
470 Biosciences) and analyzed with FlowJo software (Treestar). Gating strategy for FACS analyses
471 are shown in Supplementary Figure 24.
472
473 Cell purifications and in vitro studies
474 PBMCs were isolated by Ficoll gradient from fresh or frozen blood samples obtained from
475 healthy donors and patients. All subjects provided consent and all procedures were followed
476 under IRB-approved institutional protocols. When frozen, PBMCs were thawed and rested in
477 10% complete medium (RPMI-1640 supplemented with 10% pooled human serum, 100 μg/ml
478 penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine) at 37ºC for 6 h, after which cells
479 (0.5 x 106) were cultured with DMSO as a control, or 2.5 μM of individual HDAC inhibitors
480 (ricolinostat, ACY-241, ACY-738, ACY-775, tubatstain, and entinostat) as indicated for 24 h.
481 For tumor cell cultures, freshly resected tumors from consented NSCLC patients were
482 dissociated into single cell suspensions and cultured similarly for 72 h in culture media
483 supplemented with 20 IU/ml of IL-2 and 20 ng/ml of GM-CSF. Cells were then washed and
484 subjected to phenotypic analysis by FACS. In some experiments, cultures were maintained in
485 100IU/ml of IL-2 for 48 hours prior to analysis of Treg populations.
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486 For human proliferation assays, T cells were isolated from PBMCs by positive selection using a
487 T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per manufacturer's
488 instructions. Purified T cells (typically ~90% pure) were then labeled with 2.5 μM
489 carboxyfluorescein succinimidyl ester (CFSE) or cell trace violet (CTV) (Life Technologies,
490 Carlsbad, CA at 37°C for 7 min. Cells were washed thrice with RPMI + 10% FBS, counted,
491 and used as responder cells. Purified CD14+ cells isolated from patient PBMCs by positive
492 selection using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were cultured
493 with ricolinostat or entinostat for 24 h, after which cells were washed thrice before co-culture
494 with allogeneic T cells in mixed lymphocyte reactions.
495 For mouse Treg suppression assays, CD4+CD25hi Tregs were sorted from spleen or tumor cell
496 suspensions and cultured at graded numbers with Carboxyfluorescein diacetate succinimidyl
497 ester (CFSE)-labelled CD4+CD25- cells (0.5x105) isolated from the spleen of the same mouse. T
498 cell-depleted, mitomycin C-treated splenocytes (0.25x105) were added as APCs and cultures
499 were stimulated with soluble ɑ-CD3 (clone 2C11, ebioscience) at 0.5µg/ml concentration for 3
500 days.
501 For intracellular cytokine detection assays, immune cells from tumors of treated or control mice
502 were obtained after Ficoll gradient separation as previously described [66]. Cells (1 × 106) were
503 cultured with PMA (50 ng) and ionomycin (500 ng) for 6 h at 37°C. GolgiPlug (BD Pharmingen)
504 and FITC-conjugated CD107ɑ (Biolegend, San Diego, CA), were added for the last 5 h of
505 culture. For equivalent assays utilizing human PBMCs, 0.5 × 106 cells were similarly stimulated
506 before analysis.
507
508 Immunohistochemistry
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509 Tumors were extracted from lungs of tumor-bearing mice and fixed in 10% formalin overnight
510 after which they were embedded in paraffin and sectioned at the Specialized Histopathology core
511 at Harvard Medical School. Prior to immunohistochemical staining, tumor sections were washed
512 twice with Histo-Clear II (National Diagnostics), followed by two washes with 100% ethanol,
513 and subsequent hydration with washes of 90%, 80%, 70%, and 50% ethanol. Tumor sections
514 were heated in 10mM sodium citrate buffer (pH 6.0) for antigen unmasking. After cooling,
515 sections were washed in de-ionized water, then incubated in 3% Hydrogen peroxide for 10
516 minutes at room temperature followed by washes in de-ionized water and 1X PBS. For antigen
517 blocking, sections were incubated in PBS buffer containing 0.5% Tween, 1% BSA plus 5%
518 serum for 1 hour at room temperature. Sections were then stained in block buffer containing
519 primary antibodies: anti-alpha tubulin (acetyl K40), clone EPR16772 at 1;100 (abcam,
520 Cambridge, MA); or Phospho-STAT5 alpha (Tyr694) at 1:200 (Invitrogen, Rockford, Illinois)
521 overnight in a wet chamber at 4°C in the dark. The following day, sections were washed three
522 times in 1X PBS and then stained with secondary biotinylated antibody in PBS blocking buffer
523 for 1 hour at room temperature. Sections were washed three times with 1X PBS and Elite
524 Vectastain ABC Kit (Vector Laboratories) was applied for 30 minutes following the
525 manufacturer’s instructions at room temperature in the dark. Sections were washed with 1X PBS,
526 developed with DAB reagent (Peroxidase Substrate Kit, Vector Laboratories), and
527 counterstained with hematoxylin. Sections were then washed twice with de-ionized water
528 followed by one wash with 1X PBS, and additional washes of increasing ethanol concentration
529 for dehydration followed by incubation in Histo-Clear II. Slides were mounted with VectaMount
530 Permanent Mounting Medium (Vector Laboratories) and covered with glass coverslips (Denville
531 Scientific). After 24 hours, sections were viewed with an Olympus BX43 Trinocular
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532 Microscope. For all IHC quantitations, ten randomly selected fields from at least 4 different
533 tumors in each treatment group were used to quantitate the percent of tissue positive for each
534 marker using ImageJ software. Images were converted to a greyscale red-green-blue (RGB)
535 stack. Positive stain in the greyscale image was quantified at the appropriate threshold as %total
536 image area positive for stain. Quantitation as percent of total tissue is shown where indicated.
537
538 Immunofluorescence
539 Tumors were excised from lungs of tumor-bearing mice, embedded in and immediately snap-
540 frozen in O.C.T. compound (Fisher Healthcare). Frozen fresh tissues were cryosectioned at the
541 Specialized Histopathology core at Harvard Medical School. Sections were fixed for 10 minutes
542 in pre-cooled (-20°C ) acetone, washed three times in ice cold 1X PBS, and blocked in 1X PBS
543 with 10% BSA for 1 hour at room temperature. Sections were then stained with fluorochrome-
544 conjugated antibodies: anti-mouse CD3 Alexa Fluor® 488, clone 17A2 at 1:200, anti-mouse
545 CD11c Alexa Fluor® 594, clone N418 at 1:100, (BioLegend, San Diego, CA) overnight in a wet
546 chamber at 4°C in the dark. Sections were subsequently washed three times in ice cold 1X PBS
547 and counter stained with DAPI (FluorPureTM Grade, Life Technologies) at 0.5ug/ml for 7
548 minutes. Following this, sections were further washed thrice with 1X PBS and mounted with
549 ProlongGold mounting media and coverslips (Corning) and allowed to cure for 24 hr at room
550 temperature in the dark. Sections were imaged using a Nikon Eclipse 80i fluorescence
551 microscope equipped with CoolSNAP CCD camera (Roper Scientific). NIS elements Imaging
552 software was used to create merged images.
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553
554 Single cell RNA sequencing of tumor-infiltrating TAMs and T cells
555 Tumor-associated macrophages (TAMs; CD45+CD11c+CD11blo) and CD45+CD3+CD25lo/- T
556 cells were sorted into 96-well plates containing 50 μl of 1X PBS with recombinant ribonuclease
557 inhibitor (Life Technologies, Carlsbad, CA). Briefly, tumor cell suspensions were incubated with
558 live/dead fixable dye after FcγR blocking and subsequently stained with antibodies against
559 CD45, CD3, CD8, CD11b, and CD11c. The SmartSeq2 libraries were prepared according to the
560 SmartSeq2 protocol [67, 68] with some modifications [69], the details of which are provided in
561 Supplementary information. Correlation between expression levels for genes of interest
562 highlighted in this study is shown in Supplementary Figure 25.
563
564 Statistical analysis
565 Statistical significance was evaluated by Student’s t-test for comparisons between two groups
566 (DMSO/vehicle versus depicted drug) or one-way ANOVA for multi-group comparisons using
567 GraphPad Prism software (La Jolla, CA). P < 0.05 was considered statistically significant (*); P
568 < 0.01 was considered very significant (**); and P < 0.001 was considered highly significant
569 (***).
570
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731
732 Supplementary Information are available in the online version of the paper
733
734 Acknowledgements:
735 We thank Glenn Dranoff for critical reading of the manuscript, Elena Ivanova for technical
736 assistance with immunofluorescence. The data presented in this paper are tabulated in the main
737 paper and in the Supplementary materials. This work was supported by the National Cancer
738 Institute R01 CA195740, CA163896, CA166480, CA122794, and CA140594 to K.K. Wong,
739 CA205150 to P.S. Hammerman, 5RO1CA183560 to J. Ritz, the Thoracic Foundation (K.K.
740 Wong), the Gross-Loh Family Fund for Lung Cancer Research and Susan Spooner Family Lung
741 Cancer Research Fund at Dana-Farber Cancer Institute (K.K. Wong), and research funding from
742 Acetylon Pharmaceuticals.
743
744 Competing financial interests: SSJ and SNQ are both employees and shareholders of Acetylon
745 Pharmaceuticals. All other authors have no competing financial interests.
746 Materials and correspondence: Reprints and permissions information is available at
747 www.nature.com/nm. Correspondence and requests for materials should be addressed
748 to kwong1@partners.org
29
Research.
749 Figure Legends
750
751 Figure 1. Reduction of CD4+FOXP3+ Treg cells and up-regulation of CD69 on T cells in
752 NSCLC patient PBMC and dissociated tumor cultures in the presence of ricolinostat.
753 Peripheral blood mononuclear cells (PBMCs) from NSCLC patients (A,-C, G) or dissociated
754 tumors (D-F) were cultured for 24 or 72 hours, respectively with ricolinostat or entinostat after
755 which the frequency and phenotype of T cell subsets were assessed by FACS. DMSO was used
756 as a control. (A, D) Percent of CD4+FOXP3+ Treg cells in (A) PBMCs cultured for 24 hours or
757 (D) tumors of NSCLC patients cultured for 72-hours with 2.5µm of indicated HDAC inhibitors.
758 (B,C,E,F) Summary (left), and representative histograms (right) of expression levels of CD69 on
759 gated (B, E) CD8+ and (C, F) CD4+ T cells within the PBMC and tumor cultures. (G) Patient
760 PBMCs cultured and treated as described above were washed and re-stimulated with
761 PMA+ionomycin in the presence of golgi plug for 6 hours. Percent of IFN-γ positive
762 CD3+CD8+ T cells as assessed by intracellular cytokine staining. Data represent the mean
763 ±SEM of samples analyzed from 5 patients. * indicates p-value ˂ 0.05, ** indicates p-value
764 ˂0.01, *** indicates p-value ˂0.001.
765
766 Figure 2. Increased expression of MHC class II and CD86 on monocytes/macrophages in

767 NSCLC patient PBMC and dissociated tumor cultures in the presence of ricolinostat.
768 PBMCs from NSCLC patients (A, B) or dissociated tumors (C-D) were cultured for 24 or 72
769 hours, respectively with ricolinostat or entinostat (2.5µm) after which the phenotype of
770 CD14+CD11b+ monocytes or CD45+CD68+CD11b+ tumor macrophages were assessed by
771 FACS. DMSO was used as a control. Summary (left) or representative histograms (right) for the
772 expression levels of (A, C) HLA-DR and (B, D) co-stimulatory molecule CD86 on gated CD3-
773 CD14+ monocytes from (A, B) PBMCs or (C, D) CD14-CD45+CD68+CD11b+ macrophages in
774 dissociated tumors after culture with indicated HDAC inhibitors. (E) Purified CD14+ cells from
775 patient PBMCs that had been cultured with ricolinostat or entinostat for 24 hours were incubated
776 with cell trace violet (CTV)-labelled purified T cells from allogeneic donor PBMCs for 6 days in
777 the presence of 20 IU/ml of recombinant human IL-2. The percent proliferation by the responder
778 T cells was determined by CTV dilution in response to stimulation by CD14+ cells. Data
30
Research.
779 represent the mean ±SEM of 5 patients (A-D) or 2 independent experiments (E). * indicates p-
780 value ˂ 0.05, ** indicates p-value ˂0.01.
781
782 Figure 3. Ricolinostat promotes phenotypic and functional changes in tumor-infiltrating T
783 cell subsets and macrophages that are consistent with immune activation.
784 Single cell suspensions of lung tumor nodules of KP and TL mice treated with ricolinostat or
785 vehicle for 7-days were stained and then subjected to FACS analysis to assess proportions,
786 phenotype and function of CD45+ immune cell subsets. The single cell suspensions were also
787 stimulated ex-vivo for intracellular cytokine production by T cells. Proportion of indicated
788 lymphoid cell subsets in (A) KP or (B) TL tumors and (C) ratio of CD8 to CD4+Foxp3+ Treg
789 cells in tumors of KP and TL mice. Proportion of (D) tumor-infiltrating CD8+ T cells expressing
790 CD69 activation marker and (E) producing IFN-γ after ex-vivo stimulation. (F) The expression
791 levels of MHC class II and (G) CD86 co-stimulatory molecules on tumor-associated
792 CD11c+CD11blo macrophages in vehicle and ricolinostat-treated KP and TL mice. Data are
793 mean ±SEM of 5-9 mice per group. * indicates p-value ˂ 0.05, *** indicates p-value ˂0.001.
794
795 Figure 4. BET bromodomain inhibitor JQ1 disrupts signature protein expression and
796 attenuates suppressive function of Tregs within lung tumors of treated mice.
797 Single cell suspensions generated from lung tumor nodules excised from KP mice treated with
798 JQ1 or ricolinostat for 1 week were subjected to FACS analysis. Mice that received vehicle
799 served as controls. (A) Representative histograms and (B) Summary of expression levels of
800 Foxp3, CTLA-4, PD-1, and CD69 on tumor-infiltrating CD4+Foxp3+ Treg cells. CD4+CD25hi
801 Treg cells sorted from the tumors of vehicle or JQ1-treated KP mice were co-cultured with
802 CFSE-labeled CD4+CD25- T cells isolated from the spleen of the same mouse. Cells were
803 stimulated with ɑ-CD3 in the presence of T-depleted splenocytes as APCs for 3 days. (C)
804 Representative CFSE profiles of proliferating T cells and (D) Summary of percent of cells
805 proliferating in the presence of tumor (left) or splenic (right) Tregs at indicated Treg: T cell
806 ratios. Data in (B) and (D) are mean ±SEM of 5-6 mice per group. * indicates p-value ˂ 0.05.
807
808 Figure 5. JQ1 synergizes with ricolinostat to promote anti-tumor immunity.
31
Research.
809 KP mice with tumor burdens of approximately 200-400 mm3 were injected I.P. once daily with
810 ricolinostat alone, JQ1 alone, or the combination of the two drugs for 5-6 weeks with or without
811 depleting antibodies against CD4 or CD8. Tumor growth was monitored weekly by MRI. (A)
812 Tumor growth kinetics (B) Change in tumor size after 2 weeks of treatment with indicated drugs
813 and antibodies. (C) Representative tumor MRI of mice treated with single agents or combination
814 as indicated. (D) Progression-free survival of KP mice in each treatment condition. For immune
815 profiling, cohorts of KP mice were euthanized after 4-6 weeks of treatment and tumor cell
816 suspensions were subjected to FACS analysis. (E) Percent of CD8+ T cells that expressed CD69
817 within tumor-infiltrating CD45+ leukocytes. (F) Immune cells isolated from tumors of mice
818 treated with indicated drugs were stimulated ex-vivo for 6 hours in the presence of golgi plug.
819 Percent of CD8+ T cells within tumor-infiltrating leukocytes that secreted IFN-γ or (G)
820 expressed CD107ɑ based on mean fluorescent intensity. (H) CD25-CD3+ T cells, CD45-Epcam+
821 tumor cells, and CD11bloCD11c+ TAMs were sorted from tumors of treated mice and equivalent
822 numbers of each population were co-cultured for 2 d in the presence of tumor cell lysates. Graph
823 represents percent of dead Epcam+ tumor cells as determined by populations that stained
824 positive for viability dye. (I) Summary of expression levels of MHC class II and (J) CD86 on
825 TAMs in the tumors of treated mice. Control mice received vehicle and Rat IgG isotype in the
826 depletion studies. Data are mean ±SEM of 6-8 mice per group. * indicates p-value ˂ 0.05, **
827 indicates p-value ˂0.01, *** indicates p-value ˂0.001.
828
829 Figure 6. Dynamics of immune cells infiltrating the tumors of ricolinostat and/or JQ1-
830 treated KP mice.
831 Cell suspensions generated from tumor nodules of KP mice that were treated with vehicle,
832 ricolinostat or JQ1 for 5-6 weeks were subjected to FACS analysis to assess proportions of
833 CD45+ T lymphocyte subsets. (A) Proportion of CD4+, CD8+ conventional T cells, or
834 CD4+Foxp3+ Tregs and (B) ratio of CD8 to CD4+Foxp3+ Treg cells within the tumors. Frozen
835 sections of fresh tumor nodules from these treated KP mice were stained for TAMs (CD11c+;
836 red) and T cells (CD3+; green), and counter stained with DAPI (nuclei; blue). (C) Representative
837 immunofluorescent staining of tumor sections from mice treated as indicated. Images were
838 captured on a Nikon Eclipse 80i fluorescence microscope equipped with CoolSNAP CCD
839 camera and merged images created with NIS elements imaging software. Scale bar; 25µm.
32
Research.
840 * indicates p-value ˂ 0.05, ** indicates p-value ˂0.01, *** indicates p-value ˂0.001.
841
842 Figure 7. Proposed mechanism of action of ricolinostat and JQ1 in GEMM of NSCLC. In
843 treatment-naive lung tumors that develop in GEMM of NSCLC, inhibitory cellular mechanisms
844 such as Tregs outweigh (co)-stimulatory signals resulting in impaired T cell function.
845 Ricolinostat promoted increased MHC and co-stimulatory molecules which favors enhanced
846 antigen presentation while JQ1 led to a reduction in Treg cell numbers and function, effects that
847 facilitate T cell activation and function. The net result is a co-operative immunotherapeutic effect
848 orchestrated by both agents to favor enhanced T cell function, promoting robust anti-tumor
849 immunity.
33
Research.
Figures
Research.
C
B
A
% CD4+CD69+ % CD8+CD69+ % CD 4+FO XP3+
0
10
20
30
40
50
0
10
20
30
0
2
4
6
8
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Patient PBMC
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F
E
% CD4+CD69+ % CD8+CD69+ % CD 4+FO XP3+
0
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60
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Research.
ic
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Isotype
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Ricolinostat
Figure 1
Figure 2
Patient PBMC Patient Tumor

Isotype
Isotype
DMSO
Ricolinostat DMSO
Ricolinostat
A Entinostat C ns
40000
* 5000
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% of Max
4000
H L A -D R
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H L A -D R
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Figure 3
Figure 4
A
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JQ1
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B
15000 5000 * 25000
* 4000
*
C T LA -4 M F I
Foxp3 M FI
CD 69 M FI
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Counts
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treated
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Treg
Counts
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(JQ1-
treated
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D
100 Tumor 100 Spleen
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Research.
Figure 5
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50 K P _ R ic o lin o s ta t + J Q 1 +  - C D 4 60
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K P _ R ic o lin o s ta t + J Q 1 +  - C D 8
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Figure 6
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5 R ic o lin o s t a t+ JQ 1 5
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Research.
Figure 7
Research.
Synergistic Immunostimulatory Effects and Therapeutic Benefit

of Combined Histone Deacetylase and Bromodomain Inhibition
in Non-small Cell Lung Cancer
Dennis Adeegbe, Yan Liu, Patrick H Lizotte, et al.
Cancer Discov Published OnlineFirst April 13, 2017.
Updated version Access the most recent version of this article at:
doi:10.1158/2159-8290.CD-16-1020
Supplementary Access the most recent supplemental material at:

Material http://cancerdiscovery.aacrjournals.org/content/suppl/2017/04/13/2159-8290.CD-16-1020.DC1
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Research.

Synergistic Immunostimulatory Effects and Therapeutic Benefit of 2 Combined Histone Deacetylase and Bromodomain Inhibition in Non-Small Cell 3 Lung Cancer

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Author Manuscript Published OnlineFirst on April 13, 2017; DOI: 10.1158/2159-8290.

1 Title: Synergistic immunostimulatory effects and therapeutic benefit of

2 combined histone deacetylase and bromodomain inhibition in non-small cell

7 Warner-Hatten1, Jessica Castrillon1, Guo-Cheng Yuan4, Neermala Poudel-Neupane1, Haikuo

19 % Yuyang Li’s current address:

20 Shandong Provincial Hospital, Shandong University, Jinan, China

21 *These authors contributed equally to this work

28 02115. Phone: 617-632-6084; Fax: 617-582-7839; E-mail: kwong1@partners.org

30 other authors have no competing financial interests.

34 increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now

36 potentiate immune-orchestrated therapeutic benefit. Herein we evaluated the immunoregulatory

45 epigenetic modifiers that, together, promote T cell-mediated anti-tumor immunity and

46 demonstrate their therapeutic potential for treatment of NSCLC.

48 Statement of Significance: Selective inhibition of histone deacetylases and bromodomain

51 responses in tumor-bearing mice demonstrating the therapeutic potential of combining these

52 epigenetic modulators for the treatment of NSCLC.

54 The tumor microenvironment contains a variety of stromal/supporting cells, including

60 T cell antigen-specific responses are often inhibited by immunosuppressive cells such as

64 microenvironment favoring enhanced anti-tumor responses.

79 importance in cancer pathogenesis, pharmacologic inhibition of HDACs has become an area of

80 significant interest for cancer therapy.

83 been demonstrated using pan-HDAC inhibitors, especially when combined with

96 transcriptional programs through regulating the recognition of acetylated lysine residues by

97 transcriptional machinery. JQ1 is a well-characterized inhibitor of the bromodomain and

98 extraterminal (BET) family of bromodomain-containing proteins and may indirectly regulate

122 lung cancer (NSCLC).

126 peripheral and tumor-associated T lymphocytes.

156 increased activation profile is promoted by ricolinostat. In addition, we observed substantial

173 monocytes and tumor-associated macrophages.

178 CD45+CD68+CD11b+ macrophages within disaggregated NSCLC tumors. Upon 24-hour

201 T790M/L858R EGFR mutations (designated TL), as previously described [44].

210 S6A-D), tumor-infiltrating CD8+ T cells in ricolinostat-treated mice exhibited increased

213 control group (Fig. 3E).

226 As increase in acetylation of tubulin is considered to be one measure of pharmacologic

230 tumor-associated immune cells.

234 supports a more activated, effector profile of surrounding T cells.

237 suppressive function.

252 Supplementary Fig. S11A, and S11B). In addition, phospho-STAT5, a bromodomain-dependent

255 (Supplementary Fig. S12A-D).

267 Treg phenotype and function in tumor-infiltrating immune cells.

270 suppression of tumor growth

288 respectively (Supplementary Fig. S17B and S17C).

298 of driver mutations to those arising by spontaneous development.

303 degranulation as indicated by CD107ɑ staining (Fig. 5G).

310 class II molecules and CD86 (Fig. 5I and J).

313 subsets that signal a re-shaping of tumor-immune cell dynamics.

350 robust immune-mediated anti-tumor response in our described NSCLC models.

366 presentation. Furthermore, the observed increase in CD69 expression on ricolinostat-exposed T

373 function in multiple cell types.

385 tandem with vaccination strategies.

397 is attributed to JQ1.

408 leukocyte-orchestrated anti-tumor response by way of optimizing antigen delivery, increasing

424 based immunotherapeutics for the treatment of lung adenocarcinomas.

426 Online Methods

428 Genetically engineered mice harboring Kras+/LSL-G12DTrp53L/L (KP) or EGFRLSL-T790M/L858R (TL)

438 Drugs and reagents

444 formulated in 10% DMSO in 10% 2-hydroxylpropyl β-cyclodextrin (Sigma-Aldrich, Saint

454 listed in supplementary information.

456 Tumor profiling and flow cytometry

471 are shown in Supplementary Figure 24.

473 Cell purifications and in vitro studies

485 100IU/ml of IL-2 for 48 hours prior to analysis of Treg populations.