Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance

Abstract

Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8⁺ and CD4⁺ T cells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment.

Keywords: CD3ζ; CDK6; CP: Cancer; CP: Immunology; PTP1B; TCPTP; cyclin D3.

Figure 1.. High CDK6 expression correlates with immunotherapy resistance, and CDK6 ablation in the TME inhibits tumor growth

(A) CDK6 expression was positively correlated with risk of death in 6 of 7 immunotherapy studies for melanoma patients analyzed with the Tumor Immune Dysfunction and Exclusion (TIDE) software. (B) Two clinical studies showed that high CDK6 expression is significantly associated with poor PFS in patients treated with anti-PD1 Abs or ACT. (C and D) Graphs of MC38 tumor weights from CDK6 (C, left) or CDK4 (D, left) KO mice. Tumors were harvested on day 15. Also shown are representative tumors from WT and KO mice (C and D, right). CDK6 KO, n = 10; CDK6 WT, n = 11; CDK4 KO, n = 7; CDK4 WT, n = 5. (E and F) CDK6 ablation in the TME inhibited MC38 tumor growth. Shown are tumor growth curves from individual mice (E) and survival (F) of tumor-bearing mice. n = 8 mice/group. (G and H) CDK6 ablation in the TME inhibited B16F10 melanoma tumor growth. Shown are tumor growth curves (G) and overall survival of tumor-bearing mice (H). CDK6 KO, n = 9; WT, n = 11. (C) and (D) show mean ± SEM, two-tailed Student’s t test. Each dot represents an individual mouse. *p < 0.05, ***p < 0.001; n.s., not significant.

Figure 2.. CDK6 depletion in the TME reshapes the tumor immune microenvironment and increases tumor-infiltrating T cell cytotoxicity

(A–F) Comparison of cell populations of the TME in MC38 tumors derived from CDK6 KO or WT littermate controls. n = 7. (G) Relative amounts of tumor-infiltrating CD8⁺, CD4⁺, and Treg cells from CDK6 KO or WT littermate controls. Treg, CDK6^−/−, n = 6; others, n = 7. (H and I) Comparative analysis of memory T cells among CD8⁺ (H) or CD4⁺ (I) tumor-infiltrating T cells from CDK6 KO or WT littermate controls. Shown are representative flow cytometry plots (right). CDK6 KO, n = 5; WT, n = 6. (J–O) Activities of CD8⁺ (J–L) or CD4⁺ (M–O) tumor-infiltrating T cells isolated from CDK6 KO or WT mice. IFN-γ (J and M), GzmB (K and N), and TNF-α (L and O) were detected with flow cytometry. CDK6 KO, n = 5; WT, n = 7. Mean ± SEM, two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.. Depletion of CD8⁺ or CD4⁺ T cells reverses the host antitumor effects in CDK6 KO mice

(A and D) Schematic timeline for pre-depletion of CD8⁺ (A) or CD4⁺ (D) T cells with anti-CD8 (A) or anti-CD4 (D) Abs in CDK6 KO mice. (B and E) Representative flow cytometry assays showing pre-depletion of CD8⁺ (B) or CD4⁺ (E) T cells. (C and F) Pre-depletion of CD8⁺ (C) or CD4⁺ (F) T cells reversed the host anti-tumor effect in CDK6 KO mice. Shown are MC38 tumor weights (left) and representative tumors (right). n ≥ 5 (C), n ≥ 6 (F). (G) Schematic timeline for NK cell depletion with anti-NK1.1 Abs in CDK6 KO mice. (H and I) Pre-depletion of NK cells did not affect the host anti-tumor effect in CDK6 KO mice. Representative flow cytometry assays show pre-depletion of NK cells (H). MC38 tumor volumes from mice are also shown (I). n ≥ 4. (J) Schematic timeline for adoptive T cell therapy. (K and L) Survival of mice challenged with MC38-OVA (K) or B16F10-OVA (L) receiving an adoptive transfer of CDK6 KO (red lines) or WT (green lines) OT-I CD8⁺ T cells. Tumor-bearing mice that received no therapy served as controls (black lines). n ≥ 9 (K), n ≥ 7 (L). (C), (F), and (I) represent mean ± SEM; 1-way ANOVA (Dunnett test). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. CDK6 inhibition increases tyrosine phosphorylation of proteins functioning in TCR signaling, and cyclin D3/CDK6 phosphorylates/activates PTP1B and TCPTP

(A) GO enrichment analysis of TMT-MS data showed the top-ranked annotation groups among increased tyrosine-phosphorylated proteins in KOPTK1 cells upon treatment with palbociclib. 13 of 105 tyrosine-phosphorylated proteins relate to the TCR signaling pathway. (B) A list of PTPs, derived from TMT-MS data analysis, dephosphorylated at S/TP phosphorylation sites. (C) IB with anti-phospho-S/TP Abs of PTP1B or TCPTP immunoprecipitated from KOPTK1 cells treated with palbociclib (24 h). (D) IB with anti-phospho-S/TP Abs of immunoprecipitated PTP1B or TCPTP from interleukin-2 (IL-2)/CD3-activated splenic CD8⁺ T cells isolated from WT, CDK6 KO (center), or CDK4 KO mice (right) or from WT CD8⁺ T cells treated with vehicle or a CDK6 degrader (BSJ-03–123, 10 μM) for 24 h (left). (E) Phosphatase activities of PTP1B (left) and TCPTP (right) immunoprecipitated from Jurkat cells treated with a CDK4/6 inhibitor (palbociclib,1 μM) or CDK6 degrader (BSJ-03–123, 10 μM) for 24 h. Immunoblotting of PTP1B or TCPTP following immunoprecipitation (IP) shows protein loading of PTPs. (F) IB of recombinant PTP1B or TCPTP protein phosphorylated by cyclin D3/CDK6 following an in vitro kinase assay with anti-phospho-S/TP Abs. The recombinant Rb fragment was a positive control. (G) Phosphatase activities of PTP1B and TCPTP with or without cyclin D3/CDK6 phosphorylation. (H) IB with anti-phospho-S/TP Abs following kinase assays showed that PTP mutants were not or less phosphorylated by cyclin D3/CDK6 compared with the corresponding WT PTP. (I) Normalized phosphatase activities of PTP mutants following kinase assays with cyclin D3/CDK6. (E), (G), and (I) represent mean ± SEM; 1-way ANOVA (Dunnett’s test) (E and G) and two-tailed Student’s t test (I). n = 3 (E, G, and I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.. CDK6 inhibition increases CD3ζ tyrosine phosphorylation mediated by PTP1B and TCPTP

(A–C) Immunoprecipitation using Abs specific to CD3ε, CD3γ, or CD3ζ, followed by immunoblotting using pY99 phosphotyrosine Abs to detect phosphorylation of CD3 subunits in Jurkat cells treated with palbociclib (24 h). (D) Comparative CD3ζ phosphorylation measured with flow cytometry by phospho-Tyr142 Abs in Jurkat cells with and without palbociclib treatment (24 h). n = 2. (E–G) In vitro phosphatase assay showed that CD3ε, CD3γ, and CD3ζ were the substrates of PTP1B and/or TCPTP. (H) In vitro phosphatase assay to detect the phosphatase activity of PTP1B or TCPTP immunoprecipitated from Jurkat cells treated with palbociclib (8 h). The phosphorylation level of PTP1B/TCPTP was detected using p-S/TP Abs. (I) IB using pY99 to detect phosphorylated CD3ζ immunoprecipitated from Jurkat cells treated with PTP1B-IN2 (1 μM, 24 h). (J) Phosphorylation levels of CD3ζ at Tyr142 in CD3/CD28/IL-2-activated splenic CD4⁺ or CD8⁺ T cells from CDK6 KO mice or WT littermates. n = 4. (K) IB of tyrosine phosphorylation of CD3ζ in CD8⁺ T cells from CDK6 KO mice or WT littermates or in WT CD8⁺ T cells treated with a CDK6 degrader for 48 h (L and M) Comparative levels of tyrosine phosphorylated CD3ζ (phospho-CD3ζ [Tyr142] Abs) in OVA peptide-activated CD8⁺ T cells isolated from OT-I mice (L) or primary human T cells derived from peripheral blood mononuclear cells (M) upon treatment with PTP1B-IN2 (1 μM), palbociclib (1 μM), or BSJ-03–123 (5 μM) for 24 h n = 4 (L). Con, n = 6; PTPIB-IN2, n = 7; palbociclib (Palbo), n = 7; CDK6 degrader, n = 6 (M). (N) Phosphorylation levels of CD3ζ at Tyr142 in human T cells upon genetic deletion of CDK6 (shCDK6) or cyclin D3 (shcyclinD3). n = 3. (D), (J), and (L)–(N) represent mean ± SEM; two-tailed Student’s t-test (D and J) and 1-way ANOVA (Dunnett’s test) (L–N); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6.. Cyclin D3/CDK6 depletion or PTP1B/TCPTP inhibition increases cytokine production in T cells

(A–D) Bar graphs show a comparison of IFN-γ (A and C) and GzmB (B and D) production of T cells treated with 1 μM PTPIB-IN2, 1 μM Palbo, or 10 μM BSJ-03–123 for 24 h. In (D), 2 μM PTPIB-IN2 was applied for 16 h. CD8⁺ T cells were isolated from OT-I mice (A and B) or human T cells (C and D). n = 4 (A), n = 4 (B), n ≥ 5 (C), n ≥ 3 (D). (E and F) Normalized IFN-γ (E) or GzmB (F) production in human T cells in which cyclin D3 or CDK6 was knocked down. (G) The population of memory T cells differentiated from T cells upon treatment with PTP1B-IN2 (0.5 μM), Palbo (0.5 μM), or BSJ-03–123 (5 μM) for 2 days in the presence of IL-15. (H–J) Bar graphs show a comparison of phospho-Tyr142 of CD3ζ (H), TNF-α (I), and GzmB (J) of T cells treated with inhibitors for LCK kinase (10 μM PP2) or PTPs (1 μM PTPIB-IN2) for 4 h (K–P) Bar graphs show a comparison of IFN-γ (K and N) and GzmB (L and O) production and phospho-Tyr142 of CD3ζ levels (M and P) of PTP knockdown (K–M) or A-mutant expressing (N–P) T cells treated with 1 μM Palbo or 5 μM BSJ-03–123 for 24 h n ≥ 2 (E–P). Mean ± SEM, 1-way ANOVA (Dunnett’s test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7. Targeting the CDK6-PTPs axis enhances the efficacy of T cell killing and adoptive T cell immunotherapy

. (A) In vitro tumor killing assays showed that targeting the CDK6-PTPs axis increased splenic CD8⁺ T cell killing capacity against OVA-expressing B16F10 cells. The control groups were without OT-I CD8⁺ T cells. n = 3. (B) CAR T tumor killing assay was applied to examine the cytotoxicity of human T cells targeting Raji cells expressing GFP upon compound treatments. n ≥ 3. For (A) and (B), 1 μM PTP1B-IN2, 1 μM Palbo, or 10 μM BSJ-03–123 was applied. (C) Graph showing the mean of B16F10-OVA tumor volumes in C57BL/6-Ly5.1 mice receiving CD8⁺ T cells from OT-I mice. n ≥ 5. (D) Activities of tumor-infiltrating OT-I CD8⁺ T cells (top) and analysis of memory T cells among tumor-infiltrating OT-I T cells from C57BL/6-Ly5.1 mice (bottom). Ly5.2⁺ T cells were quantified in this experiment. n ≥ 5. (E and F) Bioluminescence images (E) and overall survival graph (F) of tumor progression in Raji tumor-bearing mice with/without receiving CAR T cells under treatment with BSJ-03–123 (50 mg/kg) or PTP1B-IN2 (5 mg/kg). Shown are representative bioluminescence images (E, top) and quantification of the regions of interest (ROIs) (E, bottom). n = 11/group. (G) Memory T cell analysis of CAR T cells from Raji tumor-bearing mice receiving treatment with PTP1B-IN2 or BSJ-03–123 for 2 weeks. n = 4/group. (H) Overall survival (left) and tumor-free survival (right) of mice challenged with B16F10 melanoma cells with/without receiving pmel CD8⁺ T cells. (I) Schematic depicting the presented study model. Created with BioRender. (A), (B), (D), and (G) represent mean ± SEM, 1-way ANOVA (Dunnett’s test); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.