Myeloid Cells That Impair Immunotherapy Are Restored in Melanomas with Acquired Resistance to BRAF Inhibitors

Abstract

Acquired resistance to BRAFV600E inhibitors (BRAFi) in melanoma remains a common clinical obstacle, as is the case for any targeted drug therapy that can be developed given the plastic nature of cancers. Although there has been significant focus on the cancer cell-intrinsic properties of BRAFi resistance, the impact of BRAFi resistance on host immunity has not been explored. Here we provide preclinical evidence that resistance to BRAFi in an autochthonous mouse model of melanoma is associated with restoration of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, initially reduced by BRAFi treatment. In contrast to restoration of MDSCs, levels of T regulatory cells remained reduced in BRAFi-resistant tumors. Accordingly, tumor gene expression signatures specific for myeloid cell chemotaxis and homeostasis reappeared in BRAFi-resistant tumors. Notably, MDSC restoration relied upon MAPK pathway reactivation and downstream production of the myeloid attractant CCL2 in BRAFi-resistant melanoma cells. Strikingly, although combination checkpoint blockade (anti-CTLA-4 + anti-PD-1) was ineffective against BRAFi-resistant melanomas, the addition of MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist) prevented outgrowth of BRAFi-resistant tumors. Our results illustrate how extrinsic pathways of immunosuppression elaborated by melanoma cells dominate the tumor microenvironment and highlight the need to target extrinsic as well as intrinsic mechanisms of drug resistance. Cancer Res; 77(7); 1599-610. ©2017 AACR.

Conflict of interest statement

Potential Conflicts of Interest: None

©2017 American Association for Cancer Research.

Figures

Figure 1. Braf/Pten tumors acquire resistance to BRAF-inhibitors

(A) Mice bearing autochthonous Braf/Pten melanomas induced in syngeneic skin grafts (see Methods) received BRAFi (PLX4720) containing diet, and tumor growth was monitored. Data were pooled from 3 independent experiments each involving 4 or 5 mice (14 total). Dotted vertical line represents mean onset of resistance (105 days), calculated based an expanded cohort of 56 total mice. (B) Western blot was performed to detect phospho-ERK1/2 and total-ERK1/2 in lysates from tumors established and treated as in (A), for the indicated numbers of days. Lanes represent tumors from individual mice (4 per group). (C) Viability assay was used to determine EC50 of the BRAF-inhibitor vemurafenib against BPR versus BP melanoma cell lines (derived from BRAFi-resistant and sensitive Braf/Pten tumors, respectively). Symbols represent mean +/− standard deviation (SD) of 4 replicate wells per point. (D) BPR and BP tumors were grown on contralateral flanks of C57BL/6 mice, and BRAFi (PLX4720 in chow) was administered as indicated (treatment period shaded in grey). Symbols and error bars represent mean +/− SEM of 5 mice per group. Statistical significance was calculated by 2-way ANOVA. Data in B, C, and D are representative of at least 2 independent experiments with similar results.

Figure 2. CD8 T cell and Treg proportions are restored to normal in BRAFi-resistant melanomas

Braf/Pten melanomas were induced as in Fig. 1A, and mice received BRAFi-containing diet for the indicated times. Tumors were analyzed for proportion of (A) CD8+ T cells, pre-gated on live CD45+ cells and (B) FoxP3+ (Treg) and FoxP3− (Th) cells pre-gated on live CD45+CD3+CD4+ cells. Absolute numbers of each cell type (per gram tumor weight) are depicted in lower panels. (C) Ratios of intratumoral CD8 T cells to Treg cells were calculated for each sample. Data were pooled from two independent experiments each involving 4–5 mice per group; 30d samples taken on day 27 or 30, and 120d samples were taken on day 117 or 128, relative to starting BRAFi. All points represent individual mice and horizontal lines depict means; statistical significance was calculated by unpaired 2-tailed t-test.

Figure 3. Myeloid derived suppressor cell populations are restored in BRAFi-resistant melanomas

Braf/Pten melanomas were induced as in Fig. 1A, and mice received BRAFi-containing diet for the indicated times. Tumors were analyzed for (A) Proportion and total number of CD11b+Gr-1+ cells, pre-gated on live CD45+ cells, and (B) ratio of CD8 T cells to CD11b+Gr-1+ cells. Samples were the same as those used in Fig. 2; significance was calculated by t-test. (C) Proportions of intratumoral CD11b+Gr-1+ cells were analyzed over time; data were pooled from 3 experiments, and significance was calculated by 1-way ANOVA (* P<0.05, ** P<0.01, ** P<0.001, *** P<0.0001). (D) Untreated and BRAFi-resistant tumors (129 days BRAFi) were analyzed for expression of Ly6G and Ly6C (gated on live CD45+CD11b+ cells) to differentiate g-MDSC and m-MDSC subsets. (A–D) Data points represent individual mice, and horizontal lines depict means. (E) In vitro assay was conducted using purified CD11b+ cells from untreated or BRAFi-resistant tumors (129 days BRAFi) as suppressors of IFN-γ production by activated splenocytes. Points represent individual wells; CD11b+ cells in each group were pooled from 2 tumors. Data in each panel were combined from two independent experiments each involving 4–5 mice/group; mice in A and B are the same as those shown in Fig. 2.

Figure 4. Evolution of the tumor immune gene signature during the acquisition of BRAFi resistance

Braf/Pten tumors (generated as in Fig. 2) were analyzed by whole genome microarray, with SAM used to identify significantly differentially expressed genes (FDR ≤ 10%) comparing tumors from untreated mice to those that had been treated with BRAFi for 4 days (A), 60 days (B), or 120 days (C). Color bar refers to median-centered log2 fold change. In A and B, soluble immune mediators are highlighted, as well as candidate genes involved in tumor metabolic processes. In C, all significantly altered genes are listed. (D, E), g:Profiler tool was used for functional enrichment analysis to identify over-representation of Gene Ontology terms (biological processes) downregulated on d60 relative to untreated, and analyzed across all time points. Enriched gene sets included Myeloid Cell Homeostasis (D) and Myeloid Leukocyte Migration (E). Significance was calculated by Kruskal-Wallis test followed by Dunn’s multiple comparisons test, with **** denoting P<0.0001.

Figure 5. MEKi reduces intratumoral MDSC accumulation and suppresses the growth of BRAFi-resistant melanomas

(A) qRT-PCR assessment of chemokine gene expression in BPR cells treated with 300 nM MEKi (PD0325901) or vehicle for 48h. Bars represent mean ±SEM of 3 wells. Statistical significance was calculated by unpaired t test. (B) BPR tumor-bearing mice receiving BRAFi treatment starting on d9 were co-treated with MEKi (versus vehicle) on days 16, 19, and 21. Spleen was analyzed by flow cytometry on day 22; absolute numbers of myeloid cells (CD11b+) and sub-populations (CD11b+Ly6CHigh, CD11b+Ly6GHigh, or CD11b+Ly6G−Ly6C−) were calculated and normalized per spleen. (C–D) Tumors taken from mice treated as in panel B were analyzed by flow cytometry for (C) total myeloid (CD11b+), and non-myeloid (CD11bneg) cells, and (D) total m-MDSCs (CD11b+Ly6CHigh) versus g-MDSCs (CD11b+Ly6GHigh); normalized per gram tumor weight. Points represent individual mice, horizontal lines depict means, and statistical significance was calculated by t test. Each panel depicts representative data of 2–3 independent experiments.

Figure 6. The CCL2/CCR2 axis mediates the accumulation of monocytic MDSCs in BRAFi-resistant melanomas

(A–B) Braf/Pten tumors, induced as in Fig. 2 and treated with BRAFi for the indicated times, underwent (A) qRT-PCR analysis to detect whole tumor CCL2 gene expression, and (B) Luminex to determine CCL2 protein concentration. (C) qRT-PCR analysis of CCL2 expression in BP (sensitive) and BPR (resistant) melanoma cells in vitro +/− vemurafenib treatment for 48h. (D) Flow cytometric analysis of CCR2 expression on CD11b+ Ly6CHigh versus Ly6GHigh and Ly6G−Ly6C− subsets in transplanted BPR tumors. (E) Transwell assessment of migration of CD11b+Ly6CHigh (left) or CD11b+Ly6GHigh (right) MDSC subsets towards BPR tumor cell-conditioned medium (CM) +/− anti-CCL2-blocking antibody. (F) The migration of CD11b+Ly6CHigh (top) or CD11b+Ly6GHigh (bottom) MDSC subsets toward CCL2 was assessed by a transwell assay. Recombinant mouse CCL2 was added to 0.5% FBS-enriched DMEM:F12 in the bottom. Splenocytes derived from BPR-bearing mice were added to the top chamber and allowed to migrate for 18 hours before analyzing by flow cytometry. (G–H) Flow cytometric analysis of CD11b+ MDSC subsets in BPR tumors growing on (G) wild-type versus CCR2−/− mice, or (H) wild-type mice treated with anti-CCL2 blocking antibody (vs. isotype control) thrice weekly starting on day 0. Both experiments (G–H) were analyzed on day 11, with flow pre-gated on live CD45+ cells. Data in (H) are pooled from two identical experiments. In all experiments, symbols represent individual mice, squares represent individual wells, horizontal lines depict means, and statistical significance was calculated by unpaired t-test (except in D, which used paired t-test). Each experiment was conducted at least twice with similar results.

Figure 7. MDSC depletion/blockade synergizes with checkpoint blockade immunotherapy to prevent the development of BRAFi resistance

Tumor growth in autochthonous Braf/Pten tumor-bearing mice receiving ongoing long-term treatment with BRAFi and various immunotherapeutic interventions; (A) Mice were treated +/− dual checkpoint blockade (anti-PD-1 + anti-CTLA-4 mAbs); data are pooled from two experiments each involving 3 mice/group. Checkpoint blockade was initiated on day 50 relative to BRAFi-initiation, and continued as described in Methods. (B) BPR tumor growth in wild-type mice treated with 90ug CCR2 antagonist every 2 days, beginning on day 0 (left), or treated with 200ug anti-GR-1 antibody on day 0 and day 3 versus isotype (right). (C) Mice were treated +/− MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist SC-202525), or dual checkpoint blockade + MDSC depletion/blockade. All treatments were initiated on day 85 and continued weekly as described in Methods. Dual checkpoint blockade (starting day 52) + MDSC depletion/blockade (starting day 89) was shown to be equally as effective in a second experiment. Tumor growth curves depict individual mice. (D) Onset of resistance in all mice studied receiving long-term BRAFi and immunotherapeutic interventions (n=18 mice in four combined experiments for no treatment, n=6 mice in a single experiment for MDSC depletion/blockade, n=10 mice in three experiments for dual checkpoint blockade, n=10 mice in two experiments for MDSC depletion/blockade + dual checkpoint blockade). Statistical significance was calculated by Mantel-Cox log rank test.