RB expression confers sensitivity to CDK4/6 inhibitor–mediated radiosensitization across breast cancer subtypes

Standard radiation therapy (RT) does not reliably provide locoregional control for women with multinode-positive breast cancer and triple-negative breast cancer (TNBC). We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor–positive (ER+) cells, but also of TNBC that expresses retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB WT TNBC (n = 4, radiation enhancement ratio [rER]: 1.49–2.22) but failed to radiosensitize RB-null TNBC (n = 3, rER: 0.84–1.00). RB expression predicted response to CDK4/6i + RT (R2 = 0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88–1.13) after RB1 knockdown in isogenic and nonisogenic models. CDK4/6i suppressed homologous recombination (HR) in RB WT cells but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB reexpression. Radiosensitization was independent of nonhomologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult-to-control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

: Abemaciclib and ribociclib radiosensitize TNBC with wild-type RB1 Cell viability was measured in each cell line 72 hours after treatment with either abemaciclib (green) or ribociclib (blue) in RB wild type MDA-MB-231 (A) and CAL-120 (B) cells to calculate IC 50 values. MCF10A cells (C) were treated with palbociclib alone (open circles) or palbociclib + RT (filled squares). Clonogenic survival assays were performed in MDA-MB-231 (D-E) and CAL-120 (F-G) cells with varying doses of either ribociclib or abemaciclib and a one-hour drug pretreatment. RB null BT-549 cells were treated with palbociclib + RT (H). MDA-MB-231 cells were treated with palbociclib 6 hours after RT, instead of a one hour drug pretreatment (I). SF 2 Gy graphs represent the mean of three independent experiments, and for each experiment a one-way ANOVA with Dunnett's post hoc test was used to compare combination-treated groups to the control group treated with RT alone (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

inhibition impairs HR in TNBC in vitro
Two stable BT-549 HR reporter clones were pretreated ± 500nM CDK4/6 inhibitor one hour before SceI-induction of dsDNA breaks (A,B). After a 1-hour pretreatment with ± 1μM palbociclib and ± 4 Gy radiation, coverslips were stained for RAD51 foci 6 hours and 16 hours after radiation in RB wild type SUM-159 cells (C). T-tests were performed between paired radiation and combination treated groups at each timepoint, correcting for multiple comparisons. Western blots were used to assess RAD51 protein expression (D). Representative images of RAD51 foci (red) 6 hours post radiation are shown in MDA-MB-231 cells (E) and CAL-851 cells (F).

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A.   Figure S4: RB1 knockout decreases CDK4/6 inhibitor potency in ER+ and TNBC cell lines Cell viability was measured 72 hours after treatment with either palbociclib (grey), ribociclib (blue), or abemaciclib (green) in Cas9-expressing control cell lines (black circles) or RB1 CRISPR knockout cells (colored squares). Dose-response curves were generated for each drug in CAL-120 Clonogenic survival assays were performed in CAL-120 (A) and T47D (B) RB1 CRISPR cells along with MDA-MB-231 (C) and MCF-7 (D) Cas9 control cells to quantify radiosensitization and calculate radiation enhancement ratios (rER). Western blots were used to confirm successful knockout of RB in CAL-120 and T47D cells (E). rER were compared between parental CAL-120 cells and CAL-120 RB1 CRISPR knockout cells (F). Overexpression of RB was performed in MDA-MB-468 and BT-549 cells in order to assess radiosensitivity in clonogenic survival assays (G-I). All clonogenics represent the pooled results of 3 independent replicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001). Palbociclib-resistant MDA-MB-231 cells were generated through continuous culture in drug-containing media (A) and 72 hour cell viability was used to assess acquired resistance to palbociclib, ribociclib, or abemaciclib. Clonogenic survival assays were performed to quantify the rER for PalboR cells and western blots were used to assess RB expression compared to parental MDA-MB-231 cells (B). Radiosensitization was assessed after transient overexpression of GFP-RB and pretreatment ± palbociclib (C).
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Supplemental Figure S9: CDK4/6 inhibitor-mediated radiosensitization of TNBC in vivo
Time to tumor doubling is shown for MDA-MB-231 parental (RB wild type, A) xenografts treated with palbociclib, and a log-rank (Mantel-Cox) test was used to compare survival curves. Weights are shown for mice with parental (B), Cas9 control (E), and RB1 CRISPR (F) xenografts throughout the study. Ki67 staining (imaged at 40x) was used to assess proliferation of tumor cells in mice treated with short term palbociclib and/or RT (C,D). A one way ANOVA with Tukey's post hoc test was used to compare Ki67 staining across treatment groups. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001).