CDK4/6 inhibition triggers anti-tumour immunity

Abstract

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.

Conflict of interest statement

Competing Financial Interests

Shom Goel has served as a paid scientific advisor to Eli Lilly, and conducts laboratory research funded by Eli Lilly. Eli Lilly did not fund the present study. Matthew Ellis has performed ad hoc consulting for Novartis, Pfizer, and AstraZeneca, receives royalties for PAM50-based diagnostics including Prosigna, and holds stock in Bioclassifier LLC for PAM50-based diagnostics.

Figures

Extended Data Figure 1. Tumor cell proliferation and expression of cell cycle related genes after CDK4/6 inhibition

a, Immunohistochemistry for Ki-67 in MMTV-rtTA/tetO-HER2 tumors treated for 12d with abemaciclib or vehicle; representative images (scale bar=100 μm) and quantification (n=7 tumors/group). b, Expression of E2F transcription factors, S phase genes, and G2/M genes in MMTV-rtTA/tetO-HER2 tumors treated with abemaciclib for 12d compared to vehicle (vehicle, n=11; abemaciclib, n=12 tumors). c–d, Gene ontology terms with p<0.05 (c) or GSEA terms significantly downregulated (d) by abemaciclib compared to vehicle in MMTV-rtTA/tetO-HER2 tumors (vehicle, n=11; abemaciclib, n=12 tumors). Unpaired two-tailed t-tests (a–b). Error bars, SD. *p<0.05, **p<0.01.

Extended Data Figure 2. CDK4/6 inhibition enhances antigen presentation

a, Antigen processing and presentation gene expression in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). b, Gene expression in cell lines treated with DMSO or palbociclib (7d, n=3). c, β2M/MHC-I flow cytometry in cell lines; grey, FMO control. (For MDA-MB-453, vehicle and abemaciclib, n=2; palbociclib, n=3. For MDA-MB-361, n=3) d, Gene expression in TCGA samples (CCND1 shallow deletion, n=101; CCND1 diploid, n=503; CCND1 gain, n=203; CCND1 amplified, n=153). e, H-2Kb SIINFEKL flow cytometry after 7d abemaciclib or DMSO (B16-OVA, n=9; MMTV-PyMT-S2WTP3-OVA, n=3). f, CD8+ T cell proliferation in response to abemaciclib-pretreated tumor cells (n=3). g, IFNγ and TNFα production in tumor cell/OT-I assay by ELISA (n=3). One-way ANOVA adjusted for multiple comparisons (c–d,f), unpaired two-tailed t-tests (a–b,e,g). Error bars, SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Extended Data Figure 3. Effects of CDK4/6 inhibition on breast cancer cell proliferation and apoptosis in vitro

a, Relative numbers of breast cancer cells cultured in 250 nM (MDA-MB-453) or 500 nM (MDA-MB-361, BT474) abemaciclib or DMSO for 11d, followed by drug withdrawal (arrow). b, Representative SA-β-galactosidase staining of MDA-MB-453 cells (left) and BT474 cells (right) treated with DMSO or abemaciclib (MDA-MB-453, 250 nM; BT474, 500 nM) for 0, 4, and 7 days. c, Western blot of SKBR3, BT474, MDA-MB-453, and MDA-MB-361 cells treated with DMSO, lapatinib, or abemaciclib for 48h. d, Western blot of MDA-MB-453 cells pretreated with DMSO or abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500 nM) for 4h. For western blot source images, see Supplementary Figure 1.

Extended Data Figure 4. CDK4/6 inhibition increases interferon signaling

a–b, Top ranked GO terms (a) and expression of interferon-responsive transcription factors (b) in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). c, Expression of interferon sensitive genes (ISGs) in MMTV-PyMT-S2WTP3 cells treated with DMSO or abemaciclib (500 nM, 7d) (n=3). d, Expression of ISGs in MDA-MB-361 and MCF7 cells treated with abemaciclib (100 nM), fulvestrant (100 nM), or the combination for 7d (n=3). e, Expression of ISGs in MDA-MB-453, MCF7, and MDA-MB-231 cells treated with abemaciclib or DMSO (7d, n=3). f, Expression of ISGs in PDX 14-07 tumors treated with abemaciclib or vehicle (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). g, phospho- and total STAT1 in cells treated with 500 nM abemaciclib as indicated. h, Confirmation of p16-FLAG overexpression in MDA-MB-453 and BT474 cells (left) and gene expression in these cell lines by qPCR (right) (n=6). i–j, Gene expression changes in MMTV-rtTA/tetO-HER2 tumors from mice treated with vehicle or abemaciclib for 12d (vehicle, n=11; abemaciclib, n=12 tumors/group). Relative expression of interferon-responsive T cell chemoattractants (i); relative expression of ISGs (j). k, Correlation of expression of Stat1 and Nlrc5 with genes involved in antigen processing and presentation in MMTV-rtTA/tetO-HER2 tumors. Blue dots, vehicle-treated tumors; red dots, abemaciclib-treated tumors. (r is Pearson product-moment correlation coefficient). Unpaired two-tailed t-tests (b,d–f,h–j) adjusted for multiple comparisons (c). Error bars, SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For western blot source images, see Supplementary Figure 1.

Extended Data Figure 5. CDK4/6 inhibitors enhance expression of immune-related signatures in breast cancer patients

a–c, NeoPalAna schema (a) Endo. Rx denotes endocrine therapy for breast cancer. Down-regulated GSEA signatures after 2 wks palbociclib treatment (b); Nom p<0.001, FDR q<0.001. Up-regulated GSEA signatures after 2 wks palbociclib treatment (c); Nom p<0.001, FDR q<0.001 (C1D1, n=34; C1D15, n=29; Surgery, n=23).

Extended Data Figure 6. CDK4/6 inhibition mediates Type III interferon production

a, Phospho- and total STAT1 in MDA-MB-453 cells treated with abemaciclib +/− ruxolitinib for 7d. b, Effect of neutralization of IFN-α or IFN-γ on STAT1 mRNA expression (n=2–4). c–d, Impact of neutralization of IFN-α (c) and IFN-γ (d) on phospho-STAT1 and total STAT1 protein in indicated cell lines. e, Expression of type III interferon genes in indicated cell lines treated with abemaciclib for 7d compared to DMSO (n=3). f, Type III interferon production measured by ELISA (7d, n=2). Unpaired two-tailed t-tests (e–f) adjusted for multiple comparisons (b). Error bars, SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For western blot source images, see Supplementary Figure 1.

Extended Data Figure 7. DNMT1 suppression mediates dsRNA response

a, DNMT1 expression after treatment with abemaciclib (n=3). b, DNMT1 protein expression after treatment with abemaciclib. c, DNMT3A expression after treatment with abemaciclib (500 nM) for 7d (n=3, except abemaciclib 24 hrs, n=2). d, DNMT1 expression after abemaciclib or control in MMTV-rtTA/tetO-HER2 tumors, p=0.05, (12d, vehicle, n=11; abemaciclib, n=12 tumors/group). e, RB1 knockdown in MDA-MB-453 cells and its effect on indicated gene expression after 7d abemaciclib (500 nM) (two biological replicates each associated with three technical replicates). f, ERV3-1 methylation. g, ERV expression after abemaciclib or DMSO (7d, n=3). h, Relative dsRNA expression after 7d of abemaciclib compared to DMSO (n=3). i–j, Cytosolic pattern recognition receptors in cells (i) (7d, n=3) or PDX tumors (j) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). k, Western blot of overexpression of DNMT1 in MDA-MB-453 cells and quantification of mRNA expression (n=3). Unpaired two-tailed t-tests (d–e,g–k) adjusted for multiple comparisons (a). Error bars, SD. *p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001. For western blot source images, see Supplementary Figure 1.

Extended Data Figure 8. Abemaciclib induces a ‘senescence-like’ phenotype without evidence of SASP

a, Representative SA-β-galactosidase staining (left) of MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (scale bar=500 μm). Quantification of relative SA-β-galactosidase positive area (right, n=6 tumors/group). b, mRNA expression of SASP factors in MMTV-rtTA/tetO-HER2 tumors treated as in Fig. 1a. Il6 expression determined by qPCR (n=10 tumors/group); Il1a and Il1b by transcriptome analysis (vehicle, n=11; abemaciclib, n=12 tumors/group). c, MDA-MB-453 and BT474 cells treated with DMSO or abemaciclib (500 nM) for 7d, and expression determined by qPCR. d, mRNA expression of IL6 upon doxorubicin-induced senescence (n=3). MDA-MB-453 and BT474 cells were treated with doxorubicin (200 nM) for 24h, and mRNA extracted 3d later for qPCR. Unpaired two-tailed t tests (a,d). Error bars, SD. **p<0.01.

Extended Data Figure 9. Impact of CDK4/6 inhibition on immune cell populations and Treg biology

a, Flow cytometric analysis of immune cell populations in MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (vehicle, n=15; abemaciclib, n=17 tumors/group). b, Peripheral blood Tregs in MMTV-rtTA/tetO-HER2 mice (12d, n=4 mice/group). c–d, Tregs (CD4+ FoxP3+) quantified by flow cytometry of MMTV-PyMT tumors (c, vehicle, n=18; abemaciclib, n=16 tumors/group) and CT-26 tumors (d, n=12 tumors/group) treated as indicated for 12d. e, Tregs in lymph nodes of tumor-free mice (12d, one-way ANOVA corrected for multiple comparisons, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). f, Treg:CD8 ratio in the spleens and lymph nodes (LN) of tumor-free FVB mice (12d, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). g, Plasma autoantibodies in tumor-free and tumor-bearing mice treated with vehicle or abemaciclib for 12d (tumor-free vehicle, n=8; tumor-free abemaciclib, n=7; tumor-bearing vehicle, n=7; tumor-bearing abemaciclib, n=6 mice/group). h–l, Tumor-free FVB mice treated with abemaciclib or vehicle for 12d. Thymic mass (h). Thymic cell populations were quantified by flow cytometry. CD4+CD8+ double positive (DP) thymocytes (i); CD4+ single positive (SP) thymocytes (j); CD8+ SP thymocytes (k); CD4+FoxP3+ regulatory T cells (l). (vehicles and palbociclib, n=5; abemaciclib, n=4 mice/group). m, Effect of abemaciclib or DMSO on ex vivo differentiation of CD4+CD25− T cells into Tregs in the presence of TGF-β for 72hr (n=4). n, Effect of DMSO or abemaciclib treatment for 72hr on Treg apoptosis measured by Annexin V staining (n=2). o, Quantification of immunofluorescent staining of MMTV-rtTA/tetO-HER2 tumors (n=7 tumors/group). p–q, Dnmt1 (p) and Cdkn1a (q) in T cells from tumor-free mice (12d, two-way ANOVA corrected for multiple comparisons, n=7 mice/group [pooled]). Unpaired two-tailed t-tests (b–d), one-way ANOVA corrected for multiple comparisons (e–f,h–l). Error bars, SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Extended Data Figure 10. Role of adaptive immunity in response to abemaciclib and combination with immunotherapy

a, Relative change in volume of MMTV-rtTA/tetO-HER2 tumors implanted into Foxn1nu mice and then treated with vehicle (n=11 tumors) or abemaciclib (n=9 tumors) (one way ANOVA). b, Immunohistochemistry of tumors in (a) for Ki-67. Representative images (left), and quantification of percent of Ki-67+ cells (right) (vehicle, n=7; abemaciclib, n=10/group, scale bar=100 μm). c, Flow cytometric analysis of absolute number (left) and percentage (right) of CD8+ T cells in peripheral blood after administration of anti-CD8 or isotype control antibodies (isotype, n=6; anti-CD8, n=4 mice/group). d–g, Expression of inhibitory co-receptors on intratumoral CD8+ T cells in MMTV-rtTA/tetO-HER2 tumors after 6d of treatment with abemaciclib (n=6 tumors) or vehicle (n=5 tumors). PD-1 cell-surface expression (d); representative flow cytometry plots (left), quantification (right). Representative flow cytometry plots for CTLA-4 (e) and LAG3 (f). g, Quantification of (e) and (f). h, Quantification of number of inhibitory receptors per cell on intratumoral CD8+ T cells in MMTV-PyMT tumors after treating mice with abemaciclib or vehicle (12d, n=18 tumors/group). i, Ifng in MMTV-rtTA/tetO-HER2 tumors (12d, Mann-Whitney test, n=10 tumors/group). j, Experimental schema for Fig. 4j. k, Quantification of mean change in tumor volume at d19 (top) and maximal reduction in tumor volume (bottom) of curves in Fig. 4j. l, Spider plots of CT-26 tumor growth with indicated treatments (n=4/group, one experiment). m, Kaplan-Meier curves of incidence of CT-26 tumor formation after rechallenge. n, Schematic. Unpaired two-tailed t-tests (b–c), two-way ANOVA corrected for multiple comparisons (d,g). Error bars, SD; except (a), SEM. *p<0.05, ***p<0.001, ****p<0.0001. For source data, see Supplementary Table 2.

Figure 1. CDK4/6 inhibitors induce tumor regression and increase antigen presentation

a, Impact of abemaciclib treatment on MMTV-rtTA/tetO-HER2 tumor volume (two-way ANOVA, vehicle, n=17; abemaciclib, n=22 tumors). b–d, In vivo experimental schema depicted in (b) (vehicle, n=11; abemaciclib, n=12 tumors). Gene ontology terms with p<0.05 (c) and expression of antigen presentation genes (d) are shown. e–f, Antigen presentation gene expression in cells (e) (7d, n=3) and PDX tumors (f) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors) after abemaciclib treatment. g, CD8+ T cell proliferation in response to abemaciclib-pretreated B16-OVA cells (OT-I + anti-IgG1, n=6; other conditions, n=3; one-way ANOVA adjusted for multiple comparisons) Unpaired two-tailed t-tests (d–f). Error bars SD; except (a), SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For source data, see Supplementary Table 2.

Figure 2. CDK4/6 inhibition stimulates interferon signaling

a–b, Top ranked GO terms in abemaciclib-treated tumor cells (a) (7d, n=3) or PDX tumors (b) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors). c–d, Interferon-responsive gene expression from samples in (a) and (b). e–f, Upregulated GO terms (e) and expression of interferon-responsive transcription factors (f) in abemaciclib-treated MMTV-rtTA/tetO-HER2 tumors (12d, vehicle, n=11; abemaciclib, n=12 tumors). g, MMTV-rtTA/tetO-HER2 tumor STAT1 staining (12d, scale bar=100 μm, n=21). h, Upregulated GSEA signatures after 12wk of palbociclib in NeoAnaPal trial. (C1D1, n=34; surgery, n=23). Unpaired two-tailed tests (c, d, f); Mann-Whitney test (h); Error bars, SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For all GSEA, nom p<0.001, FDR q<0.001.

Figure 3. CDK4/6 inhibitors suppress DNMT1, inducing viral mimicry

a, Tumor cell type III interferon secretion measured by ELISA (7d). b, Impact of Type III IFN neutralization on phospho- and total STAT1 protein (7d). c–d, DNMT1 expression after abemaciclib or control in cells (b) (7d, n=3) or PDX tumors (d) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors). e–f, ERV3-1 expression in samples from (c) and (d). g, dsRNA levels in abemaciclib-treated tumor cells (7d, n=2). h, Effect of DNMT1 overexpression on gene expression in MDA-MB-453 cells after 7d abemaciclib (50nM) (n=3). Abemaciclib at 500nM in vitro, unless noted, except MCF-7, 250nM. Unpaired two-tailed t-tests (a, c–h). Error bars, SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For western blot source images, see Supplementary Figure 1.

Figure 4. CDK4/6 inhibition modulates the immune milieu

a–c, Intratumoral T cells (a), Tregs (b), and Treg:CD8 ratios (c) in MMTV-rtTA/tetO-HER2 tumors (12d treatment, vehicle, n=15; abemaciclib, n=17 tumors). d, Tregs in spleens of tumor-free mice (12d, vehicle and palbociclib, n=8; abemaciclib, n=7 mice). e, T cell proliferation in vitro. f, Ki-67/FoxP3 staining of MMTV-rtTA/tetO-HER2 tumors (12d, scale bar=50 μm, n=7). g, MMTV-rtTA/tetO-HER2 tumor volume after treatment with abemaciclib +−/ anti-CD8 neutralizing antibody (n=17 tumors). h, Inhibitory co-receptors on MMTV-rtTA/tetO-HER2 intratumoral CD8+ T cells (6d, vehicle, n=5; abemaciclib, n=6 tumors). i, Number of inhibitory receptors per cell on CD8+ T cells from (h). j, MMTV-rtTA/tetO-HER2 tumor volume after treatment with abemaciclib +/− anti-PDL1 (control and anti-PD-L1, n=18; abemaciclib and combination, n=19 tumors). Unpaired two-tailed t-tests (a–c,f), one-way ANOVA corrected for multiple comparisons (d, j), two-way ANOVA (g) corrected for multiple comparisons (e,h). Error bars, SD; except (g,j), SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For source data, see Supplementary Table 2.