Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma

Abstract

Immunotherapy of advanced melanoma with CTLA-4 or PD-1/PD-L1 checkpoint blockade induces in a proportion of patients long durable responses. In contrast, targeting the MAPK-pathway by selective BRAF and MEK inhibitors induces high response rates, but most patients relapse. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. Preclinical data endorsing this hypothesis are accumulating. Inhibition of the PI3K-Akt-mTOR pathway may be a promising treatment option to overcome resistance to MAPK inhibition and for additional combination with immunotherapy. We therefore evaluated to which extent dual targeting of the MAPK and PI3K-Akt-mTOR pathways affects tumor immune infiltrates and whether it synergizes with PD-1 checkpoint blockade in a BRAF^V600E/PTEN^-/--driven melanoma mouse model. Short-term dual BRAF + MEK inhibition enhanced tumor immune infiltration and improved tumor control when combined with PD-1 blockade in a CD8⁺ T cell dependent manner. Additional PI3K inhibition did not impair tumor control or immune cell infiltration and functionality. Analysis of on-treatment samples from melanoma patients treated with BRAF or BRAF + MEK inhibitors indicates that inhibitor-mediated T cell infiltration occurred in all patients early after treatment initiation but was less frequent found in on-treatment biopsies beyond day 15. Our findings provide a rationale for clinical testing of short-term BRAF + MEK inhibition in combination with immune checkpoint blockade, currently implemented at our institutes. Additional PI3K inhibition could be an option for BRAF + MEK inhibitor resistant patients that receive targeted therapy in combination with immune checkpoint blockade.

Keywords: Anti-PD-1; BRAF; MAPK; MEK; PI3K; checkpoint blockade; immunotherapy; mTOR; melanoma; targeted therapy.

Figure 1.

Improved tumor control and lymphocyte infiltration by addition of MEKi and PI3Ki to BRAFi. (A) C57BL/6 mice were injected subcutaneously with 3 × 10⁵ D4M.3A mouse melanoma cells and targeted therapies (14 d) were started after 7 d. BRAFi [B] PLX4720 was provided in chow; MEKi [M] trametinib was dosed by daily oral gavage at 15 µg (on average 0.75 mg/kg); PI3Ki [P] BKM120 daily oral gavage at 400 µg (on average 20 mg/kg); and mTORi [mT] everolimus daily oral gavage at 100 µg (on average 5 mg/kg). Shown are tumor growth curves from mice treated with the indicated combinations (mean ± SEM and n = 9–11). (B) Mean tumor size ± SD at day 28 (no mice removed from experiments) is depicted in a dot plot (Mann–Whitney U-test). (C) D4M.3A tumor-bearing C57BL/6 mice (each group n = 5) were treated for 3 d with MAPK and/or PI3K pathway inhibitors, and tumors were analyzed by immunohistochemistry for CD3⁺ cell infiltration and analyzed by flow cytometry for (D) proportion of IFNγ producing CD8⁺ T cells; (E) PD-L1 expression of CD45⁻ cells; and (F) expression of PD-1 on CD8⁺ T cells.

Figure 2.

BRAFi + MEKi has the strongest short-term synergy with anti-PD1. (A) Tumor-bearing mice were treated as described in Fig. 1 with the indicated small molecules targeting MAPK and/or PI3K pathway for 14 d and concurrently either with anti-PD-1 or isotype mAb (twice weekly 100 µg intraperitoneal). Anti-PD-1 or control antibody was continued beyond day 14. Shown are the tumor sizes of the different treatment groups (mean ± SEM and n = 8–10). (B) Tumor sizes from 2A at day 32 are depicted in a dot plot (mean ± SD) and statistical significance is analyzed comparing isotype versus anti-PD1 treatment (Mann–Whitney U-test). (C) Individual tumor growth curves of mice are plotted per condition and the average of the group is indicated by a bold line.

Figure 3.

Addition of anti-PD1 to MAPK and PI3K pathway inhibition does not alter the infiltration and activation/effector status of CTLs. (A) Tumors from mice treated as described in Fig. 2A (n = 4–5 per group) were analyzed by immunohistochemistry for CD3 infiltration on day 3. The black scale bars equal 3 mm and the high magnification inserts are 400 μm squared. Quantification depicted in (B) In the same setup as A and B, tumors were digested and analyzed by flow cytometry for the percentages of (C) IFNγ producing CD8⁺ T cells (D) granzyme B producing CD8⁺ T cells; and (E) CD44^highCD62L^low expressing CD8⁺ T cells.

Figure 4.

Synergy of targeted therapy with anti-PD-1 is dependent on the presence of CD8⁺ T cells. (A) Tumor growth curves of D4M.3A tumor-bearing C57BL/6 mice treated for 14 d with BRAFi and MEKi combined with isotype or anti-PD-1 as described in Fig. 2A. In addition, mice were treated with twice weekly intraperitoneal isotype mAb, anti-CD4⁺ or anti-CD8⁺ depleting antibodies at 250 µg (mean ± SEM and n = 8–9). (B) Tumor size and mean ± SD at day 28 is depicted in a dot plot (Mann–Whitney U-test).

Figure 5.

Transient infiltration of T cells upon MAPK pathway inhibition. D4M.3A tumor-bearing C57BL/6 mice were treated with BRAFi and/or MEKi. (A) Tumors treated for the respective time were analyzed by immunohistochemistry for CD3⁺ cell infiltration. Representative immunohistochemistry stainings are shown. The black scale bars equal 3 mm and the high magnification inserts are 400 μm squared. (B, C) Automated software quantifications for the proportion of infiltrating CD3⁺ cells are depicted as a fraction of total nuclei (mean indicated by horizontal line). (D, E) Intrapatient pre- and during treatment tumor biopsies from humane melanoma treated with either BRAFi or BRAFi + MEKi. Samples were analyzed by immunohistochemistry for CD8⁺ cell infiltration in a blinded manner. The average CD8⁺ cell count per HPF is plotted and median is indicated by horizontal red line. Patient samples were grouped as early on treatment biopsies (< 15 d of treatment) and late on treatment biopsies (> 15 d).