CDK4/6 blockade provides an alternative approach for treatment of mismatch-repair deficient tumors

Abstract

Mismatch repair-deficient (dMMR) tumors show a good response toward immune checkpoint inhibitors (ICI), but developing resistance impairs patients' outcomes. Here, we compared the therapeutic potential of an α-PD-L1 antibody with the CDK4/6 inhibitor abemaciclib in two preclinical mouse models of dMMR cancer, focusing on immune-modulatory effects of either treatment. Abemaciclib monotherapy significantly prolonged overall survival of Mlh1^-/- and Msh2^{loxP/loxP;TgTg(Vil1- cre)} mice (Mlh1^-/-: 14.5 wks vs. 9.0 wks (α-PD-L1), and 3.5 wks (control); Msh2^{loxP/loxP;TgTg(Vil1- cre)}: 11.7 wks vs. 9.6 wks (α-PD-L1), and 2.0 wks (control)). The combination was not superior to either monotherapy. PET/CT imaging revealed individual response profiles, with best clinical responses seen with abemaciclib mono- and combination therapy. Therapeutic effects were accompanied by increasing numbers of tumor-infiltrating CD4⁺/CD8⁺ T-cells and lower numbers of M2-macrophages. Levels of T cell exhaustion markers and regulatory T cell counts declined. Expression analysis identified higher numbers of dendritic cells and neutrophils within tumors together with high expression of DNA damage repair genes as part of the global stress response. In Mlh1^-/- tumors, abemaciclib suppressed the PI3K/Akt pathway and led to induction of Mxd4/Myc. The immune-modulatory potential of abemaciclib renders this compound ideal for dMMR patients not eligible for ICI treatment.

Graphical abstract

Figure 1.

In vitro analysis on Mlh1^−/− tumor cells. (a) Apoptosis/necrosis quantification. Mlh1^−/− A7450 T1 M1 tumor cells were treated with CDKIs for 72 h and apoptosis/necrosis was quantified from Yo-Pro1/PI-stained cells. n = 3 independent experiments, *p < .05; ** p > .01; ***p < .001; two-way ANOVA (Tukey’s multiple comparisons test). (b) Cell cycle analysis after 48 h of treatment with abemaciclib. n = 4 independent experiments, *p < .05; t-test. (c) Colony formation assay after treatment with abemaciclib or dinaciclib. Two experimental conditions were studied: (i) 6 days treatment and direct analysis and (II) 6 days treatment + 6 days re-culture without (w/o) treatment (= reconvalescence). Thereafter, colonies were counted using ImageJ software. n = 3 independent experiments, *p < .05; ***p < .001; ****p < .0001; two-way ANOVA (Tukey’s multiple comparisons test). (d) Detection of ROS (RosBrite), cytoskeleton (Phalloidin) and mitochondria (mitolite) after 48 h of treatment with abemaciclib. Representative images are shown out of n = 3 independent experiments. Read out was done with the ZEISS Elyra 7 Confocal Laser Microscope (Zeiss). Original magnification 400 x. (e) Flow cytometric measurement of CalR-positive cells after 48 h and 72 h of treatment. n = 3–4 independent experiments. (f) HMGB1 secretion after 72 h of treatment. HMGB1 levels were determined from supernatants of MLH1^−/− tumor cells. Control cells were left untreated. Experiments were repeated three times each of them performed in duplicates. *p < .05. (g) Co-culture of tumor and immune cells. Tumor and immune cells were simultaneously treated for 1 × 72 h with abemaciclib, α-PD-L1 antibody or a combination. The effector to target ratio was 1:10. Residual tumor cells were counted by adding fluorescent beads. Read out was done via flow cytometry. Representative dot plots of tumor cells treated with immune cells and drugs are shown. n = 3 independent experiments, *p < .05; **p < .01, ***p < .001; ****p < .0001, One-way ANOVA (Tukey’s multiple comparisons test). (a-g) All data are given as mean + SEM. Doses used in each experiment are as follows: abemaciclib: 1 µM, dinaciclib: 0.1 µM, THZ-1: 0.83 µM; α-PD-L1 antibody: 10 µg/ml.

Figure 2.

Treatment schedule, longitudinalF-FDG PET/CT imaging in vivo and overall survival of Mlh1^−/− and Msh2^{loxP/loxP;TgTg(Vil−cre)} mice. (a) Experimental protocol. Mice with gastrointestinal tumors were conducted to mono- or combination therapy. Abemaciclib: 1x/week, 8 times in total (q1wx8); α-PD-L1: biweekly, 3 times in total (q2wx3); combination: abemaciclib first (=lead-in), followed by α-PD-L1 injection. (b – g) Baseline and follow-up PET/CT imaging of individual mice. (b, c) Data are presented in column (each dot stands for an individual mouse). (D, E, F, G) data are presented as best % change from baseline according to clinical definitions and depicted for each individual mouse either after short-term (day 30) (d, e) or long-term follow-up (~day 50) (f, g); PD – progressive disease (tumor volume > 25% vs. baseline), SD – stable disease (tumor volume similar to initial staging (≤ 25% vs. day 0); PR – partial response (tumor volume 50% lower or more vs. baseline). (h, i) Kaplan-Meier survival curve. Mlh1^−/−: isotype vs. α-PD-L1: p < .05; control vs. abemaciclib: p < .01; control vs. combination: p < .01; ctrl: n = 6, isotype: n = 7, α-PD-L1 n = 7, abemaciclib: n = 6, combination n = 6. Msh2^{loxP/loxP VillinCre:} isotype vs. α-PD-L1: p < .05; control vs. abemaciclib: p < .01; control vs. combination: p < .001. ctrl: n = 10, isotype: n = 7, α-PD-L1 n = 10, abemaciclib: n = 9, combination n = 9. Log-rank analysis (Mantel Cox).

Figure 3.

Cytokine levels of plasma from Mlh1^−/− and Msh2^{loxP/loxP;TgTg(Vil−cre)} mice. (a) Mlh1^−/− and (b) Msh2.^{loxP/loxP;TgTg(Vil−cre)} Plasma samples were collected before treatment (= baseline) and at the experimental endpoint. Cytokine levels were determined as described in material and methods. Given is the x-fold change of the indicated marker in comparison to day 0 (= baseline). Mlh1^−/−: ctrl: n = 3, isotype: n = 3, α-PD-L1 n = 7, abemaciclib: n = 6, combination n = 5. Msh2^{loxP/loxP VillinCre}: ctrl: n = 4, isotype: n = 7, α-PD-L1 n = 6, abemaciclib: n = 9, combination n = 8. *p < .05; ***p < .001, Kruskal-Wallis test (Dunn’s multiple comparisons test). (a-b) All data are given as mean + SEM.

Figure 4.

Spectral flow cytometry of peripheral blood and spleens from Mlh1^−/− and Msh2^{loxP/loxP;TgTg(Vil−cre)} mice. (a – d) Blood phenotyping. Given is the number of % immune cells before treatment (= baseline) and at the experimental endpoint resulting from 100,000 events measured on a flow cytometer. Mlh1^−/− n = 3–8 mice/group, Msh2^{loxP/loxP;TgTg(Vil−cre)} n = 7–9 mice/group. ^#p < .05 endpoint vs. day 0 α-PD-L1; ⁺p < .05 endpoint vs. day 0 abemaciclib; ⁺⁺p < .01 endpoint vs. day 0 abemaciclib, °°°°p < .0001 endpoint vs. day 0 combination. Kruskal-Wallis test (Dunn’s multiple comparisons test). (e – h) Spleen phenotyping. Given is the number of % immune cells at the experimental endpoint resulting from 100,000 events measured on a flow cytometer. *p < .05; Kruskal-Wallis test (Dunn’s multiple comparisons test). (g, h) tSNE plots showing single T cell subpopulations of spleens from Msh2^{loxP/loxP;TgTg(Vil−cre)} mice. The expression profile of the exhaustion marker CTLA-4 as well as Tregs were illustrated for the treatment and control groups, respectively.

Figure 5.

Immunofluorescence of tumor specimens from Mlh1^−/− and Msh2^{loxP/loxP;TgTg(Vil−cre)} mice. Residual tumor slides were fixed, stained and embedded. Confocal laser scanning microscopy was done on a Zeiss Elyra 7 microscope. The infiltration pattern of T cells, regulatory and tumor-associated macrophages differed between individual treatment groups. In most cases, the differences reached statistical significance. Upper panel: representative images of tumor slides; lower panel: quantitative analysis of tumor-infiltrating immune cells. (a, d) Given is the number of infiltrating CD3⁺CD4⁺ T helper cells, CD3⁺CD8⁺ cytotoxic T cells and IRF5⁺ macrophages counted in 2–3 HPFs/slide with n = 3–10 mice/group. (b, c) The infiltration pattern was semi-quantitatively analyzed using a scoring system. 0 = no; 1 = mild; 2 = moderate; 3 = strong. Each symbol represents one case. *p < .05; **p < .01, Two-way ANOVA (Tukey’s multiple comparisons test).

Figure 5.

(Continued)

Figure 6.

Nanostring gene expression analysis of tumors from Mlh1^−/− and Msh2^{loxP/loxP;TgTg(Vil−cre)} mice. The PanCancer IO 360 Gene Expression Panel was applied. Relative abundances measuring various contrasts between cell types reported for each group. Data result from n = 3 samples/group.

Figure 7.

Expression levels of selected genes and functional immunological analysis. (a-f) Total RNA from tumors (A-F) and spleens (g, h) was reverse transcribed into cDNA and qPCR was done as described in material and methods. All data are given as 2-^ΔΔCT values + SEM. Analysis was done in triplicates with n = 3 mice/group, respectively, * p < .05; *** p < .001; **** p < .0001; # p < .05; ^## p < .01; ^#### p < .0001 vs. control. One-way ANOVA (Tukey’s multiple comparisons test) or Kruskal-Wallis test (Dunn’s multiple comparisons test).

Figure 7.

(Continued)