Stabilization of RUNX1 Induced by O-GlcNAcylation Promotes PDGF-BB-Mediated Resistance to CDK4/6 Inhibitors in Breast Cancer

Abstract

Cyclin-dependent kinases 4 and 6 (CDK4/6) are crucial in regulating cell-cycle progression and cancer development. Targeting CDK4/6 has shown considerable promise in treating various cancers, including breast cancer. Despite significant therapeutic efficacy, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial hurdle in clinical practice. Using a coculture system, cytokine array, and quantitative high-throughput combinatorial screening, we discovered a mechanism by which the Runt-related transcription factor (RUNX) 1-platelet-derived growth factor (PDGF)-BB axis regulates palbociclib resistance in breast cancer cells. Specifically, RUNX1 functioned as a transcription factor to drive expression of PDGFB, leading to resistance to palbociclib by enhancing the Akt pathway and suppressing senescence. Furthermore, in resistant cells, RUNX1 was O-GlcNAcylated at serine 252 by O-GlcNAc transferase, resulting in the stabilization of RUNX1 by preventing ubiquitin-mediated degradation. Inhibition of the RUNX1-PDGF-BB axis by specific inhibitors overcame palbociclib resistance both in vitro and in vivo. Notably, the RUNX1-PDGF-BB axis was upregulated in resistant patient-derived xenograft lines and in patients with breast cancer following treatment with CDK4/6i. These findings not only unveil O-GlcNAcylation-mediated activation of a RUNX1-PDGF-BB pathway as a driver of palbociclib resistance but also provide clinical evidence supporting the repurposing of FDA-approved PDGFR inhibitors as a therapeutic strategy to treat patients with CDK4/6i-resistant breast cancer. Significance: RUNX1-PDGF-BB signaling drives resistance to CDK4/6 inhibition in breast cancer, providing the foundation to develop approaches to target the RUNX1-PDGF-BB axis to overcome CDK4/6 inhibitor resistance in breast cancer patients.

Figure 1.. Identification of PDGF-BB that promotes palbociclib-resistance in ER+ breast cancer cells

(A) MCF-7 WT-GFP cells and MCF-7 PR-RFP cells were monocultured or co-cultured in the presence of palbociclib (2μM) for 8 days. Images were taken on day 0, day 2, day 4, day 6, and day 8. The scale bar represents 10 μm. (B) Viability of MCF-7 WT treated with the indicated amount of conditioned medium (CM) from MCF-7 WT or MCF-7 PR cells for two days, followed by treatment with the indicated concentrations of palbociclib for 3 days. (C-D) Representative image (C) and quantification (D) of cytokine arrays showing the expression levels of the indicated upregulated cytokines in the culture medium of MCF-7 WT and MCF-7 PR cells from three independent experiments. The spots highlighted within the red rectangle indicate cytokines that are upregulated in MCF-7 PR CM compared to MCF-7 WT CM. (E) The schematic illustrates the overlap of upregulated cytokines identified by the cytokine array from MCF-7 PR & MCF-7 WT CM and T-47D PR & T-47D WT CM. (F-G) MCF-7 WT/MCF-7 PR cells (F) and T-47D WT/T-47D PR cells (G) were harvested and then subjected to qPCR to examine the mRNA levels of the indicated genes. (H) Cell viability was assessed in MCF-7 PR cells treated with 1 μg/mL PDGFB neutralizing antibody or IgG, in combination with increasing concentrations of palbociclib, for 3 days. (I) Cell viability was examined in MCF-7 WT cells treated with 100ng/mL recombinant PDGF-BB protein or IgG, in combination with increasing concentrations of palbociclib, for 3 days. (J) Surface plots show the synergy between palbociclib and sunitinib in MCF-7 PR cells (n = 3). (K) MCF-7 PR cells were transfected with the indicated siRNAs for 48 hr, then cell viability was examined with indicated concentrations of palbociclib for 3 days. Data are presented as mean ± SEM. Statistic analysis was performed by GraphPad Prism 9.5.0 using Welch’s t-test for the data shown in (D, F, G), using Multiple t-tests followed by Holm–Sidak correction for the data shown in (B, H, I, K). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; “NS” indicates no significant difference.

Figure 2.. Inhibition PDGF-BB overcomes palbociclib resistance by inhibiting Akt and activating the senescence pathway in breast cancer cells

(A) Schematic of qHTCS to identify Akti (Tricirbine) that can overcome palbociclib resistance. (B) Surface plots showing the synergy between palbociclib and Tricirbine in MCF-7 PR cells (n = 3). (C) GSEA profiling shows the enrichment of the PI3K_Akt_MTOR geneset and E2F geneset in MCF7 PR cells treated with siCon or si-PDGF-BB. NES scores and FDR values were determined by GSEA software. (D) MCF-7 PR cells (top) and T-47D PR cells (bottom) were transfected with indicated siRNA for 48 hr, cells were then harvested and immunoblotted for the indicated proteins. (E-F) MCF-7 PR cells (E) and T-47D PR cells (F) were pretreated with/without SC-79 for 24h, then transfected with the indicated siRNAs for 48h, and cell viability was examined with the indicated concentrations of palbociclib for 3 days. (G-H) MCF-7 PR cells (G) and T-47D PR cells (H) were transfected with indicated siRNA for 24h and then treated with 4 μM Palbociclib for 2 days, then cell lysed were harvested and immunoblotted for indicated proteins. (I) Representative images of SA-β-gal staining in MCF-7 PR cells transfected with the indicated siRNA for 24 hours, followed by treatment with 4 μM Palbociclib for 4 days. The scale bar represents 20 μm. All data are presented as mean ± SEM and analyzed by Multiple t-tests followed by Holm–Sidak correction in GraphPad Prism 9.5.0. **P ≤ 0.01, ***P ≤ 0.001; “NS” indicates no significant difference.

Figure 3.. RUNX1 transactivates PDGF-BB in palbociclib-resistant breast cancer cells

(A) Venn diagram showing the overlap of transcription-regulated genes with PDGF-BB from PathwayNet, transcription factors targetting the promoter region of PDGF-BB by JARSPR database, and genes upregulated from RNA-seq analysis in MCF-7 PR cells. (B) MCF-7 WT & PR cells (left) and T-47D WT & PR cells (right) were harvested and then subjected to qPCR and immunoblotting to examine the expression of the indicated genes and proteins. (C) ChIP-qPCR was employed to examine the binding of RUNX1 to the promoter region of the PDGFB. (D) Luciferase reporter assay was performed to examine the transcription activities of RUNX1 in MCF-7 PR cells. Renilla luciferase plasmid was used as an internal control. (E) MCF-7 PR cells were transfected with the indicated siRNAs for 48 hr, then cells were harvested and then subjected to qPCR and immunoblotting to examine the expression of indicated genes. (F) MCF-7 PR cells were transfected with the indicated siRNAs for 48 hr, then cell viability was examined with indicated concentrations of palbociclib for 3 days. (G-H) MCF-7 PR cells (G) and T-47D PR cells (H) were transfected with indicated siRNA for 24h, and then incubated with/without recombinant PDGF-BB (100ng/mL) for 48h, cells were then harvested and immunoblotted for the indicated proteins. (I-J) MCF-7 PR cells (I) and T-47D PR cells (J) were transfected with indicated siRNA for 24 hrs, followed by incubation with or without recombinant PDGF-BB (100 ng/mL) in combination with the indicated concentrations of palbociclib for 3 days. (K) Surface plots show the synergy between palbociclib and Ro5-3335 in MCF-7 PR cells. (L-M) Representative images (L) and growth curves (M) of MCF-7 PR xenograft tumors with the indicated treatments for 3 weeks (n = 5 mice/group). Palbociclib and RUNX1 inhibitor Ro5-3335 were administered intraperitoneally every 2 days for 3 consecutive weeks at a dose of 25 mg/kg and 10 mg/kg, respectively. The data in (M) are presented as mean ± SD, whereas data in (B-F, I-J) are shown as mean ± SEM. Statistic analysis was performed by GraphPad Prism 9.5.0 using Welch’s t-test for the data shown in (B-E), using Multiple t-tests followed by Holm–Sidak correction for the data shown in (F, I, J, M). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4.. O-GlcNAcylation of RUNX1 by OGT stabilizes RUNX1 by preventing STUB1-mediated ubiquitination

(A) MCF-7 WT cells were transfected with the indicated plasmids for 48 hr before being harvested for co-IP assay. HA-IPs were immunoblotted for indicated proteins. (B) MCF-7 PR cells were transfected with the indicated siRNAs and plasmids for 48 hr before being harvested. FLAG-IPs were immunoblotted for the indicated proteins. (C-D) MCF-7 PR cells and T-47D PR cells were transfected with the indicated siRNAs for 48 hr before being harvested. Cell lysates were then subjected to immunoblotting for the indicated proteins and qPCR to examine mRNA levels of the indicated genes. (E) MCF-7 WT cells were transfected with the indicated plasmids for 48 hr before being harvested. Cell lysates were then subjected to immunoblotting for the indicated proteins. (F-G) MCF-7 PR cells were transfected with indicated siRNA for 48 hr, followed by treatment with CHX (100μg/mL) for the indicated time points, and then subjected to immunoblotting for the indicated proteins and quantification of the protein levels. (H) HEK293T cells were transfected with the indicated siRNAs and plasmids for 48 hr before being harvested. HA-IPs were immunoblotted for the indicated proteins. (I) HEK293T cells were transfected with the indicated plasmids for 48 hr before being harvested. HA-IPs were immunoblotted for the indicated proteins. (J) HEK293T cells were transfected with the indicated siRNAs and plasmids for 48 hr before being harvested. HA-IPs were immunoblotted for the indicated proteins. (K) HEK293T cells were transfected with the indicated plasmids for 48 hr before being harvested. FLAG-IPs were immunoblotted for the indicated proteins. The data in (D) are presented as mean ± SEM, whereas data in (G) are shown as mean ± SD. Statistic analysis was performed by GraphPad Prism 9.5.0 using Welch’s t-test for the data shown in (D), using Multiple t-tests followed by Holm–Sidak correction for the data shown in (G). *P ≤ 0.05, **P ≤ 0.01; “NS” indicates no significant difference.

Figure 5.. O-GlcNAcylation of RUNX1 at Serine 252 is required for its stability and palbociclib-resistance

(A) HEK293T cells expressing RUNX1 or its O-GlcNAc mutants from plasmids were harvested 48 hr after transfection, followed by co-IP. HA-IPs were then immunoblotted for the indicated proteins. (B) HA-RUNX1 and Myc-OGT were co-expressed in HEK293T cells, then HA-RUNX1 was purified and analyzed by nanoUPLC-MS/MS to identify O-GlcNAc sites with the annotated mass spectrum (S252 O-GlcNAcylation is illustrated; matched b, y, c, z fragments are highlighted; the modified residue is showed in red; m, methione oxidation). (C) The RUNX1 S252 site is conserved across various species. (D-E) HEK293T cells were transfected with the indicated siRNAs and plasmids for 48 hr before being harvested. HA-IPs were immunoblotted for the indicated proteins. (F-G) MCF-7 WT cells and T-47D WT cells were transfected with RUNX1 WT or RUNX1 S252A mutant plasmids for 48 hr, followed by treatment with CHX (50μg/mL) for the indicated time points, and then subjected to immunoblotting for the indicated proteins and quantification of the protein levels. (H) HEK293T cells were transfected with the indicated plasmids for 48 hr before being harvested. HA-IPs were immunoblotted for the indicated proteins. (I) MCF-7 PR cells were transfected with shRUNX1 and selected with 2ug/ml puromycin for 2 weeks. Cells were then transfected with RUNX1-WT or RUNX1-S252A plasmids, followed by examination of viability after treatment with the indicated concentrations of palbociclib for 3 days. (J) Representative images (left) and growth curves (right) of MCF-7 PR xenograft tumors with the indicated treatments for 3 weeks (n = 5 mice/group). Palbociclib was administered intraperitoneally every 2 days for 3 consecutive weeks at a dose of 25 mg/kg. The data in (G, J) are presented as mean ± SD, whereas data in (I) are shown as mean ± SEM. Statistic analysis was performed using Multiple t-tests followed by Holm–Sidak correction in GraphPad Prism 9.5.0. *P ≤ 0.05, **P ≤ 0.01.

Figure 6.. Palbociclib treatment activates the RUNX1-PDGF-BB axis both in vitro and in vivo

(A-B) MCF-7 WT cells were treated with various doses of palbociclib for 4 days (A) or with 1μM palbociclib for different durations (B). The cells were then lysed, and qPCR was performed to analyze the mRNA levels of the indicated genes. (C) MCF-7 WT cells were collected after the indicated treatments for 4 days and then subjected to immunoblotting for the indicated proteins. (D) MCF-7 WT cell culture medium was collected after the indicated treatment for 4 days for examination of secreted PDGF-BB levels by ELISA. (E) A schematic presentation of the experiments presented in F-H. (F) Representative images of MCF-7 WT xenograft tumors with intraperitoneal palbociclib treatment every 2 days for 3 consecutive weeks at a dose of 10mg/kg (n = 7 mice/group). (G-H) MCF-7 WT xenograft tumor tissues were collected after the indicated treatments for 3 weeks, and then subjected to immunostaining (G) and immunoblotting for the indicated proteins (H). (I) Western Bloting shows increased PDGF-BB protein levels in MCF-7 WT cells with overexpression of OGT, RUNX1, or OGT and RUNX1 together. (J) Cell viability of the cells treated in (I). Data are presented as mean ± SEM and analyzed by Multiple t-tests followed by Holm-Sidak correction in GraphPad Prism 9.5.0. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; “NS” indicates no significant difference.

Figure 7.. RUNX1-PDGF-BB axis is upregulated in breast cancer patients treated with palbociclib

(A) Schematic of NeoPalAna Trial for ER⁺ breast cancer patients. (B) GSEA profiling showing the enrichment of the Cytokine_Receptor_Interaction geneset in the Surgery group vs. C1D1 group and Surgery group vs. C1D15 group; (C) The enrichment of the RUNX1-PDGF-BB signature geneset in the Surgery group vs. C1D15 group. NES scores and FDR values were determined by GSEA software. (D) Palbociclib-resistant and sensitive breast cancer PDX lines were collected and subjected to Western blotting. (E) Quantification results of the indicated proteins. (F) Palbociclib-resistant and sensitive breast cancer PDX lines were collected and subjected to qPCR to examine the mRNA levels of the indicated genes. (G-H) Surface plots to exhibit the synergy between palbociclib and Ro5-3335 (G) as well as palbociclib and sunitinib (H) in palbociclib-resistant organoid cells derived from the PDX line WHIM81 (n = 3). (I) Schematic representation of how O-GlcNAcylation of RUNX1 regulates PDGF-BB signaling and palbociclib resistance in breast cancer cells. See text for details. The data in (E) are presented as mean ± SD, whereas data in (F) are shown as mean ± SEM. Statistic analysis was performed using Welch’s t-test in GraphPad Prism 9.5.0. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.