Trametinib Drives T-cell-Dependent Control of KRAS-Mutated Tumors by Inhibiting Pathological Myelopoiesis

Abstract

Targeted therapies elicit seemingly paradoxical and poorly understood effects on tumor immunity. Here, we show that the MEK inhibitor trametinib abrogates cytokine-driven expansion of monocytic myeloid-derived suppressor cells (mMDSC) from human or mouse myeloid progenitors. MEK inhibition also reduced the production of the mMDSC chemotactic factor osteopontin by tumor cells. Together, these effects reduced mMDSC accumulation in tumor-bearing hosts, limiting the outgrowth of KRas-driven breast tumors, even though trametinib largely failed to directly inhibit tumor cell proliferation. Accordingly, trametinib impeded tumor progression in vivo through a mechanism requiring CD8⁺ T cells, which was paradoxical given the drug's reported ability to inhibit effector lymphocytes. Confirming our observations, adoptive transfer of tumor-derived mMDSC reversed the ability of trametinib to control tumor growth. Overall, our work showed how the effects of trametinib on immune cells could partly explain its effectiveness, distinct from its activity on tumor cells themselves. More broadly, by providing a more incisive view into how MEK inhibitors may act against tumors, our findings expand their potential uses to generally block mMDSC expansion, which occurs widely in cancers to drive their growth and progression. Cancer Res; 76(21); 6253-65. ©2016 AACR.

Figure 1. Trametinib impairs the growth of Brpkp110 tumors

(A) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 3–13. One representative experiment of three, P<0.05, unpaired t test. (B) Brpkp110 cells were cultured for 2 days with trametinib and proliferation was quantified by MTS assay. (C) Mice with Brpkp110 tumors were treated daily with trametinib or vehicle on days 7–9 and tumors were excised on day 10 and stained for Ki-67. Scale bars = 100 μM. (D) Positive Ki-67 as % of total tumor area is shown from two experiments.

Figure 2. Trametinib reduces the accumulation of Ly6C^hi M-MDSCs in tumors

(A–E) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 7–9, harvested on day 10, and analyzed by flow cytometry. (A–D) Percentages of cell populations found in dissociated tumors from two independent experiments. (E) Representative gating for Ly6C^hi and Ly6G⁺ from CD11b⁺MHCII⁻ cells in tumors. (F) CD11b⁺MHCII⁻Ly6C^hi or CD11b⁺MHCII⁻Ly6G⁺ were FACS sorted from advanced Brpkp110 tumor-bearing or naive mice and analyzed by qPCR. Expression normalized to TATA binding protein is shown. (G) CD11b⁺MHCII⁻Ly6C^hi cells were sorted from advanced Brpkp110 tumor-bearing mice and mixed at the indicated ratios with OVA_257-264-peptide-loaded OT-I splenocytes. Proliferation was measured by CellTrace dilution of CD8⁺ cells 3 days later. Representative of two experiments. (H–I). Percentages or total numbers of cell populations from spleens of Brpkp110 tumor-bearing mice from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001, unpaired t test.

Figure 3. Trametinib reduces the accumulation of M-MDSCs and growth of LLC tumors

(A–C)Mice with LLC intraperitoneal tumors were gavaged with trametinib or vehicle on days 7–9, harvested on day 10, and analyzed by flow cytometry. Percentages or total numbers of cell populations found in dissociated tumors (A) and spleens (B–C) from two independent experiments. *P<0.05, unpaired t test. (D) Mice with LLC intraperitoneal tumors were gavaged with trametinib or vehicle on days 3–13. *P<0.001, log-rank. (E) LLC cells were cultured for 2 days with trametinib and proliferation was quantified by MTS assay. (F) Mice with LLC intraperitoneal tumors were gavaged with trametinib or vehicle on day 8 and tumors were harvested 8 hrs later for Western blotting. V = vehicle (2 mice), T = trametinib (2 mice).

Figure 4. Trametinib selectively reduces the differentiation of Ly6C⁺ MDSCs from murine bone marrow

(A–B) MDSCs were differentiated from mouse bone marrow with IL-6 and GM-CSF in the presence of vehicle or trametinib for 4 days. Shown are representative gating from all live cells (A) and total number of cells (B). n=4, *P<0.05, Mann-Whitney test. (C–D) MDSCs were differentiated from mouse bone marrow with Brpkp110 conditioned medium (50%) in the presence of vehicle or trametinib for 4 days. Shown are representative plots (C) and total number of cells (D). n=4, *P<0.05, Mann-Whitney test. (E) MDSCs differentiated with Brpkp110 conditioned medium were added at the indicated ratios to mouse splenocytes activated with anti-CD3 and anti-CD28 and cultured for 3 days. (F) MDSCs were differentiated as in (C) and analyzed by Western blot on days 2 and 4 of culture. (G–H) MDSCs differentiated as in (C) were analyzed for AnnexinV and ZombieYellow. Shown are representative plots from day 4 (G) and total cell numbers from days 1, 2, and 4 (H). n=4, *P<0.05 for Z⁻AV⁻, Mann-Whitney test. (I–J) MDSCs differentiated with Brpkp110 conditioned medium for 4 days were washed and plated in fresh, unconditioned media for 4 hours with vehicle or trametinib. Shown are gating (I) and plots (J) of total cells. n=4. V=vehicle, T=trametinib 200 nM.

Figure 5. Trametinib reduces the expansion of CD14⁺ MDSCs from human bone marrow

Dissociated human bone marrow was cultured in GM-CSF and IL-6 with vehicle or trametinib for four days and analyzed by flow cytometry. (A) Gating analysis for one representative donor out of seven is shown. (B) Expression of CD11b, CD33, and VNN2 is shown for indicated cell populations. (C) Proportions within HLA-DR⁻SSC^hi cells and total numbers for indicated cell populations normalized to vehicle are shown. Data combined from seven individual donors. *P<0.05, one sample t-test with respect to 100%.

Figure 6. Full efficacy of trametinib requires CD8⁺ T cells

(A) Tumors from Brpkp110 tumor-bearing mice treated daily with vehicle or trametinib on days 7–9 were dissociated on day 10, stimulated with PMA/Ionomycin for 5 hrs, and stained for intracellular IFN-γ. (B) Tumors from Brpkp110 tumor-bearing mice treated daily with vehicle or trametinib on days 3–12 were dissociated on day 13, stimulated with PMA/Ionomycin for 5 hrs, and stained for intracellular IFN- γ. (C–D) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 3–13. Anti-CD8α (C), anti-NK1.1 (D), or control IgG was administered on day 3 and 10. Combined results from two similar experiments. (E) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 3–9. MDSCs differentiated from bone marrow with Brpkp110-conditioned media were injected i.v. on days 4, 7, and 9. Combined results from two similar experiments. *P<0.05, **P<0.01, ***P<0.001, unpaired t test.

Figure 7. Osteopontin chemoattracts MDSCs and is reduced by trametinib treatment of tumor cells

(A) LC-MS/MS data of cytokines found in supernatants of Brpkp110 cells cultured for 40 hrs in vehicle or 200 nM trametinib. Y axis=MS count (abundance) in vehicle supernatants. X axis=fold change. Positive values=(trametinib/vehicle), negative values = −(vehicle/trametinib). (B) Osteopontin concentration measured from supernatants of Brpkp110 cells cultured overnight in the indicated conditions. (C) Osteopontin concentration from plasma samples collected from Brpkp110-bearing mice (or naïve tumor-free mice) gavaged daily with trametinib on days 7–9, and harvested on day 10. (D) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G⁺ and Ly6G⁻ populations. Pre- and post-sort cell populations were analyzed for Ly6G and Ly6C expression by flow cytometry. (E–F) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G⁺ and Ly6G⁻ populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis). (G) Osteopontin concentration measured from intratumoral fluid collected from four separate Brpkp110 tumors. *P<0.05, **P<0.01, ***P<0.001, unpaired t test.