Supplementary Table 1. Flow cytometry antibody details:

Supplementary Figure 2. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of representative histograms for the MFI of MHC-I (H2Kb/Kd and H2Db/Dd), MHC-II, PD-L1, CD80 and CD86 are shown.

Supplementary Figure 13. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in LLC tumor. (A) Schema of LLC tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Numbers of cells/mg of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 12. The triple combination increases activation of CD8+ T cells, CD4+ Teffs, and Tregs in CT26 TDLN. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Absolute number of immune cell populations in the TDLN; n=4-5. (C) Phenotypes of T cells from the TDLN; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplemental Figure 11. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while Tregs remain unaffected in LLC TDLN. (A) Schema of LLC tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Image of TDLNs from LLC tumor bearing mice. (C) Gating strategy for all in vivo flow experiments. (D) Absolute number of immune cell populations in the TDLN; n=4-5. (E) Phenotypes of T cells from the TDLN; n=9-10. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 14. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in CT26 tumor. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Tumor weights, numbers of cells/mg, and CD8:Treg ratio of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 5. Intermittent CKI27 allows for immune cell recovery in the spleen, increases frequencies in the TDLN, and inhibits TILs similarly to continuous treatment. (A) Schema of LLC tumor bearing mice treated with vehicle, daily 2mg/kg CKI27, or intermittent 5mg/kg 4on/3off CKI27. Mice were treated in a staggered schedule and all timepoints were harvested on day 23. (B-D) All fold changes were calculated by normalizing to DMSO. (B) Fold changes of absolute number (cells/uL) of spleen immune cell populations. (C) Fold changes of absolute number (cells/uL) of TDLN immune cell populations. (D) Fold changes of tumor weights (mg) and TILs (cells/mg); n=4-5. Data are shown as mean±SEM. Unpaired, nonparametric Mann-Whitney test was used to compare each time point. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 1. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. (A-B) Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of (A) MHC-I (H2Kb/Kd and H2Db/Dd) and MHC-II and (B) PD-L1, CD80 and CD86 surface expression. Median fluorescence intensity (MFI) values were normalized to DMSO and log transformed. Data are shown as mean±SEM.

Supplementary Figure 7. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-stimulatory markers. Representative dot plot data for FACS analysis of co-stimulatory markers expressed by CD8+ T cells.

Supplementary Figure 15. The triple combination favorably alters the genetic profile of immune cells in the TDLN. LLC tumor bearing mice were treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. TLDNs were harvested on day 21 (7 days post treatment). Live CD45+ cells were FACS sorted and processed for sc-RNA sequencing. (A-B) Heatmap showing top genes expressed by each cluster for (A) CD8+ and (B) CD4+ T cells. (C) UMAPs of each treatment groups showing different clusters and annotations. (D) UMAPs of CD8+ and CD4+ T cells from each treatment group showing specific activation genes and proteins.

Supplementary Figure 10. The triple combination reduces tumor growth, is T cell dependent, and protects from re-challenge in LLC and CT26. (A-D) LLC tumor bearing mice were treated with vehicle, isotypes, GITR, CTLA-4, 5mg/kg 4on/3off CKI27, and/or CD8 for 4 weeks and tumor growth was monitored over time. (A) Average tumor growth (volume, mm3) of immunocompetent mice. (B) Average tumor growth (volume, mm3) of immunodeficient mice. (C) Average tumor growth (volume, mm3) of CD8 depleted mice. (D) Average tumor growth (volume, mm3) of mice from (A) that were re-challenged. (E-H) CT26 tumor bearing mice were treated with vehicle, isotypes, GITR, αCTLA-4, 2mg/kg 4on/3off CKI27, and/or αCD8 for 4 weeks and tumor growth was monitored over time. (E) Average tumor growth (volume, mm3) of immunocompetent mice. (F) Average tumor growth (volume, mm3) of immunodeficient mice. (G) Average tumor growth (volume, mm3) of CD8 depleted mice. (H) Average tumor growth (volume, mm3) of mice from (E) that were re-challenged. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 8. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of activation markers. Representative dot plot data for FACS analysis of activation markers expressed by CD8+ T cells.

Supplementary Figure 4. Intermittent CKI27 treatment partially relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr) or washout CKI27 (24hr on, 72hr off); n=2-3. (A) Proliferation fold change of CTVlow CD8+ and CD4+ T cells was calculated by normalizing to DMSO. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 3. MEK inhibition with CKI27 increases HLA and checkpoint ligand expression. (A-B) Human tumor cell lines were treated with DMSO or CKI27 for 72hr and either with or without IFNγ (10ng/mL) for the last 24hr; n=3. FACS analysis of (A) HLA-ABC and HLA-DR and (B) PD-L1, CD80 and CD86 surface expression. MFI values are shown as mean±SEM.

Supplementary Figure 6. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-inhibitory markers. (A) Gating strategy for all T cell activation assays. (B) Representative dot plot data for FACS analysis of co-inhibitory markers expressed by CD8+ T cells.

Supplementary Table 2. Gene list associated with each cluster.

Abstract MEK inhibitors (MEKi) have shown limited success as a treatment for MAPK/ERK pathway–dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression in tumor cells and improved T cell–mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T-cell receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and costimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor-draining lymph nodes and tumors and led to intratumoral regulatory T-cell destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.