RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1
S-H Chen1, X Gong1, Y Zhang1, RD Van Horn1, T Yin1, L Huber1, TF Burke1, J Manro2, PW Iversen2,W Wu1, SV Bhagwat1, RP Beckmann1, RV Tiu3, SG Buchanan1 and S-B Peng1
INTRODUCTION
Oncogenic mutations in KRAS, NRAS and HRAS are among the most frequent genetic alterations and occur in ~ 30% of human cancers.1,2 Among the RAS gene family, KRAS is often mutated in three major cancer types: pancreatic ductal adenocarcinoma (98%), colorectal adenocarcinoma (45%) and lung adenocarci- noma (31%).1 NRAS mutations frequently occur in melanoma (28%), multiple myeloma (20%), thyroid carcinoma (8.5%), color- ectal adenocarcinoma (7.5%) and acute myeloid leukemia (6.7%).1 However, no effective therapeutics targeting RAS mutant cancer has been approved despite more than three decades of efforts by academia and industry.
Among RAF isoforms, BRAF is frequently mutated most commonly at the somatic hot spot Val600, which is substituted by a glutamate side chain in 450% of melanoma.3 BRAF V600E activates the MAPK pathway and functions as a monomer in a RAS-independent manner.3–7 Therefore, among BRAF V600E mutant cancers, BRAF V600E monomer is the primary driver of MAPK signaling, and thus makes it an attractive anti-cancer target.5,8 This led to FDA approval of two BRAF-selective inhibitors vemurafenib and dabrafenib, which showed antitumor activities in BRAF mutant preclinical models,9–11 and significant benefit in melanoma patients.12–14 However, both vemurafenib and dabrafenib induce dimerization of RAF proteins and promote paradoxical pathway activation in BRAF wild-type cells owing to their lack of effective inhibition of RAF dimers.15–18 Therefore, vemurafenib and dabrafenib are considered to be contraindicated for treatment of cancer patients with BRAF wild- type status, including those with KRAS or NRAS mutation. We have developed LY3009120, a pan-RAF inhibitor that is able to effectively inhibit active RAF dimers and is active against tumor cells with BRAF, NRAS or KRAS mutations in vitro and in vivo.19–21
Regulation of the cell cycle in proliferation is controlled by the retinoblastoma (Rb) tumor suppressor protein. Mitogens that stimulate the RAS-MAPK pathway are known to induce expression of D-type cyclins, which activate the Rb kinases CDK4 and CDK6. Activation of cyclin D-CDK4/6 complex promotes phosphorylation and inactivation of Rb, thus releasing E2F transcription factor for G1–S phase transition to promote cell cycle progression.22 The regulation of Rb is frequently disrupted in cancer cells due to the loss of p16INK4A, an inhibitor of CDK4 and CDK6.23 In addition, D-type cyclins and the CDK4/6 axis are frequently overexpressed in broad spectrum of cancers.24,25 This led to the clinical development of several highly selective kinase inhibitors of CDK4/6, including palbociclib (PD0332991), abemaciclib (LY2835219) and ribociclib (LEE011),26 all of which demonstrate potent inhibition of phos- phorylated Rb with relatively good selectivity against other kinases.27–29 Targeting this pathway has been shown to have efficacy in ER-positive metastatic breast cancer patients.
Figure 1. Pan-RAF inhibition by LY3009120 sensitizes KRAS, NRAS or BRAF mutant cancer cells to CDK4/6 inhibition by abemaciclib in vitro.
(a) LY3009120 and abemaciclib combination index (CI) in 328 cell tumor cell lines. Red, yellow and blue colors indicate tumor cells with a KRAS, BRAF or NRAS mutation, respectively. (b) Growth inhibition of Calu-6 cells (KRAS Q61K) by LY3009120 or abemaciclib alone, or combination of LY3009120 with a fixed concentration of abemaciclib (0.31 μM). (c) Growth inhibition of HCT116 cells (KRAS G13D) by LY3009120 or abemaciclib alone, or combination of LY3009120 with a fixed concentration of abemaciclib (0.16 μM). (d) Growth inhibition of A375 (BRAF V600E) cells by LY3009120 or abemaciclib alone, or combination of LY3009120 with a fixed concentration of abemaciclib (0.31 μM). (e) Growth inhibition of SK-Mel30 cells (NRAS Q61K) by LY3009120 or abemaciclib alone, or combination of LY3009120 with a fixed concentration of abemaciclib (0.62 μM). (f) Growth inhibition of HT-29 cells (BRAF V600E) by LY3009120 or abemaciclib alone, or combination of LY3009120 with a fixed concentration of abemaciclib (0.16 μM). Cell proliferation was measured by CellTiter Glo (Promega) with 5 days of treatment.
MAPK signaling controls transcription of cell cycle proteins via activation of AP1 or ETS family of transcription factors, whereas MAPK inhibition resulted in downregulation of cyclin D and inhibition of cell proliferation.30 It has been shown that RAS or BRAF mutations render cancer cells mitogen-independent by constitutive activation of the Cyclin D-CDK4/6 complex. Consistent with this idea, MAPK inhibition can lead to downregulation of cyclin D1 and inhibition of cell proliferation.24,31,32 In many tumor types, including melanoma, lung and pancreatic cancer, aberra- tions in D-cyclin/Rb axis, such as CDKN2A loss or CCND1 amplification, frequently co-occur with mutations affecting the RAS-MAPK pathway.33 Furthermore, studies in preclinical models revealed that driver oncogenes KRAS, NRAS or BRAF co-operated with CDK4 activation for tumor progression.34,35
We hypothesized that combined inhibition of RAF and CDK4/6 might provide synergistic antitumor effects in cancers with a KRAS,NRAS or BRAF mutation. In this study, we found that concurrent targeting of CDK4/6 by abemaciclib and RAF by LY3009120 resulted in synergistic effects on inhibition of proliferation of RAS and BRAF mutant cancer cells in vitro and suppression of tumor growth in vivo. Mechanistically, this combination led to more significant inhibition of Rb phosphorylation and cyclin D1 suppression, which resulted in more robust cell cycle G1 arrest and inhibition of cell proliferation.
RESULTS
Pan-RAF inhibition by LY3009120 sensitizes KRAS, NRAS or BRAF mutant cancer cells to CDK4/6 inhibition by abemaciclib in vitro As part of our Lilly internal high-throughput cell line panel screening effort to discover potential combinatorial therapies that sensitize abemaciclib, pan-Raf inhibitor LY3009120 scored among the top sensitizers of RAS and BRAF mutant cancer cells. In a 328 cancer cell screen panel (CCSP), synergistic effects (combination index CIo1) by treatment of LY3009120 and abemaciclib combination were observed for ~ 60% of cells, particularly cells with MAPK pathway alterations (Figure 1a). To further verify these results, representative tumor cells with KRAS, NRAS or BRAF mutation were treated with LY3009120, abemaciclib or their combination. In KRAS mutant Calu-6 and HCT116 cells, either LY3009120 or abemaciclib alone showed a concentration depen- dent inhibition of cell proliferation, and their combination exhibited synergistic inhibitory effects with CI value of 0.31 in Calu-6 cells and 0.22 in HCT116 cells, respectively (Figures 1b and c). Synergistic inhibition of HCT116 cells was also observed in a colony formation assay (Supplementary Figure S1). In NRAS mutant melanoma SK- Mel30 cells, combination of LY3009120 and abemaciclib also showed synergistic inhibition of cell proliferation with CI of 0.36 (Figure 1e). In BRAF mutant melanoma A375 and colorectal HT-29 cells, either LY3009120 or abemaciclib inhibited cell proliferation of these cells, and their combination showed synergistic inhibition with CI value of 0.3 in A375 and 0.36 in HT-29 cells, respectively (Figures 1d and f). Collectively, our in vitro data indicate that the combination of LY3009120 and abemaciclib synergistically inhibits the proliferation of tumor cells with a KRAS, NRAS or BRAF mutation. Supplementary Figure S2 summarized the combination effects of these two agents in different cells.
Figure 2. In vitro activities of LY3009120 and abemaciclib combination in cancer cell screen panel (CCSP) and their association with genetic alterations. (a), absolute IC50 of LY3009120 and abemaciclib combination against 328 tumor cell lines. Red, yellow and blue colors indicate cells with KRAS, BRAF or NRAS mutation, respectively, and gray color indicates cell lines with wild-type (WT) KRAS, NRAS and BRAF. (b) Average IC50 values and statistical analysis between KRAS, NRAS or BRAF mutant cell line group vs WT cell line group. (c), average IC50 values and statistical analysis among KRAS, NRAS or BRAF mutant cell line subgroups vs WT cell line group. (d) Association of genetic alterations with sensitivity or resistance of tumor cells to LY3009120 and abemaciclib combination. Blue color indicates sensitivity, and red color indicated resistance. The bigger average LOD (log of odds) value in the y axis suggests the stronger correlation of the sensitivity (blue) or resistance (red).
Figure 3. Combination of LY3009120 and abemaciclib leads to additive or synergistic antitumor growth activities and tumor growth regression in KRAS, NRAS or BRAF mutant xenograft models. (a) Calu-6 xenografts (n = 7) were treated with LY3009120 (10 mg/kg) twice daily, abemaciclib (20 mg/kg) once daily, or the combination for 21 days and tumor volumes (mean ± s.e.m.) were measured every 3 ~ 6 days.
(b) HCT116 xenografts (n = 7) were treated with LY3009120 (15 mg/kg) twice daily, abemaciclib (20 mg/kg) once daily, or the combination for 21 days and tumor volumes (mean ± s.e.m.) were measured every 3 ~ 7 days. (c), SKMel-30 xenografts (n = 8) were treated with LY3009120 (15 mg/kg) twice daily, abemaciclib (20 mg/kg) once daily, or the combination for 21 days and tumor volumes (mean ± s.e.m.) were measured every 3 ~ 7 days. (d) A375 xenografts (n = 8) were treated with LY3009120 (5 mg/kg) twice daily, abemaciclib (35 mg/kg) once daily, or the combination for 28 days and tumor volumes (mean ± sem) were measured every 3 ~ 5 days. (e) WM-266-4 xenografts (n = 8) were treated with LY3009120 (15 mg/kg) twice daily, abemaciclib (20 mg/kg) once daily, or the combination for 21 days and tumor volumes (mean ± s.e.m.) were measured every 3 ~ 5 days. (f) HT-29 xenografts (n = 7) were treated with LY3009120 (15 mg/kg) twice daily, abemaciclib (20 mg/kg) once daily, or the combination for 21 days and tumor volumes (mean ± s.e.m.) were measured every 3 ~ 4 days. (g) Intermittent combinational dose schedule in HCT116 xenograft model. Mice (n = 5) were treated with Vehicle (black), LY3009120 at 15 mg/kg twice daily (red) or abemaciclib at 20 mg/kg once daily (blue) for 28 days, or their combination in two intermittent dose schedules: continuous dosing of LY3009120 with abemaciclib week on/week off for 28 days (green), or continuous dosing of abemaciclib with LY3009120 week on/week off for 28 days (purple). (h) Summary of Bliss effect analysis of above in vivo studies. The D[T/C]% and mean of expected additive tumor volumes with combination treatment were calculated by Bliss independent method. The actual tumor volumes with combination treatment lower than the expected additive tumor volumes are considered as synergy. *Indicates combination study with abemaciclib dosed intermittently. **Indicates combination study with pan-RAF inhibitor LY3009120 dosed intermittently.
MAPK pathway mutations and D-cyclin activation predict sensitivity, whereas PI3K pathway or Rb mutations predict resistance to combinational inhibition of LY3009120 and abemaciclib We have genetically characterized cells in CCSP by whole genome and RNA sequencing as described.21 As demonstrated in Figure 2a, cancer cells with a KRAS, NRAS or BRAF mutation indicated are generally more sensitive, whereas KRAS, NRAS and BRAF wild-type cells are less sensitive or resistant to combinational treatment of LY3009120 and abemaciclib. These two groups showed statistically significant difference in sensitivity to this combination with a P-value of 2.15E-8 (Figure 2b). When we separated cancer cells of MAPK mutation into subgroups with KRAS, NRAS or BRAF mutation, respectively, all three subgroups are significantly more sensitive to the combination treatment when they are compared with KRAS, NRAS and BRAF wild-type tumor cells (Figure 2c). Further analysis revealed that, in addition to KRAS, NRAS and BRAF mutation, cancer cells with D-cyclin activation or APC mutation are associated with sensitivity to the combination (Figure 2d, Supplementary Figure 3). In contrast, PI3K pathway mutations, such as PTEN, PIK3CA and PIK3R1, and Rb mutations are associated with resistance of cancer cells to this combination (Figure 2d, Supplementary Figure 3).
These sensitivity and resistance profiles could guide us to further enrich patients who are more likely to be responsive to this combination treatment.Combination of LY3009120 and abemaciclib leads to additive or synergistic antitumor growth activities and tumor growth regression in KRAS, NRAS or BRAF mutant xenograft models We next evaluated whether combined RAF and CDK4/6 inhibition by LY3009120 and abemaciclib is synergistic in vivo. In the KRAS mutant Calu-6 lung tumor xenograft model, single agent of LY3009120 at 15 mg/kg inhibited tumor growth by 96%, whereas single agent of abemaciclib at 20 mg/kg showed 89.2% tumor growth inhibition. However, their combination resulted in a significant tumor regression (−87.1%) (Figure 3a), which scores as synergistic using Bliss effect analysis (Figure 3h). In the KRAS mutant HCT116 colorectal tumor xenograft model (Figure 3b), a synergistic regression in tumor size (−25.4%) was also observed by the combination, whereas single-agent LY3009120 and abemaci- clib showed tumor growth inhibition of 33.2% and 71.5%, respectively (Figures 3b and h). In NRAS-mutant SK-Mel30 melanoma model (Figure 3c), LY3009120 and abemaciclib combination also exhibited superior antitumor activity to single agent alone, and an additive effect was observed. In BRAF V600E mutant A375 melanoma model, single agent LY3009120 at 5 mg/kg or abemaciclib at 35 mg/kg significantly inhibited tumor growth (95.3% and 31%, respectively), whereas their combination led to − 88.6% tumor growth regression (Figure 3d), which is synergistic by Bliss effect analysis. Similarly in BRAF V600E mutant WM-266-4 melanoma model, combination of LY3009120 and abemaciclib exhibited tumor growth regression and additive antitumor growth activities (Figures 3e and h). Finally, in BRAF V600E mutant HT-29 colorectal tumor model, combination of LY3009120 and abemaciclib also achieved additive antitumor growth effect (Figure 3f). It is noteworthy that the combination of LY3009120 and abemaciclib at these doses was generally tolerated, and minimal to moderate body weight loss was observed in some of these studies (Supplementary Figures S4A-F). Considering that concurrent inhibition of MAPK pathway and CDK4/6 may encounter tolerability issue in clinic, we have conducted a combination study with an intermittent dose schedule in KRAS mutant HCT116 model. As revealed in Figure 3g, when we dosed one drug either LY3009120 or abemaciclib continuously and the other drug 1 week on and 1 week off, the combinational effects were also observed. The efficacy and synergy between intermittent and continuous dosing of both drugs were similar (Figures 3b, g and h). The animal body weight change data of intermittent dose scheduled were shown in Supplementary Figure 4G). Overall, these in vivo data suggest that combined inhibition of pan-RAF and CDK4/6 signaling has additive to synergistic antitumor activities in tumor models with KRAS, NRAS or BRAF mutations.
Figure 4. Combined inhibition of RAF by LY3009120 and CDK4/6 by abemaciclib, palbociclib or siRNA knockdown results in superior inhibition of phospho-Rb and suppression of cyclin D1 in vitro. (a) LY3009120 and abemaciclib combination. Cells were treated with DMSO, LY3009120 (Calu-6: 0.05 μM, HCT116: 1 μM, SKMel-30: 0.1 μM, A375: 0.01 μM), abemaciclib (Calu-6: 5 μM, HCT116: 5 μM, SKMel-30: 0.5 μM, A375: 1 μM) or the combination for 48 h, and the cell lysates were analyzed by western blotting using the indicated antibodies. (b) LY3009120 and palbociclib combination. Cells were treated with DMSO, LY3009120 (Calu-6: 0.5 μM, HCT116: 1 μM, SKMel-30: 0.1 μM, A375: 0.01 μM), CDK4/6 inhibitor palbociclib (Calu-6: 5 μM, HCT116: 5 μM, SKMel-30: 2.5 μM, A375: 1 μM), or the combination for 48 h, and the cell lysates were analyzed by western blotting using the indicated antibodies. (c) Combination of LY3009120 and CDK4/6 siRNA knockdown. Cells were transfected with control or CDK4 and six siRNA. After 24 h, the cells were treated with DMSO or LY3009120 (Calu-6: 0.5 μM, HCT116: 1 μM, SKMel-30: 0.1 μM, A375: 0.05 μM) for 48 h, and cell lysates were analyzed by western blotting using the indicated antibodies.
Combined inhibition of RAF by LY3009120 and CDK4/6 by abemaciclib, palbociclib or siRNA knockdown results in superior inhibition of phospho-Rb and suppression of cyclin D1 in vitro.To elucidate the molecular mechanism underlying the synergistic effects of pan-RAF and CDK4/6 inhibition, we conducted western blot analysis of many key cell signaling components, including phospho-MEK, phospho-RB and cyclin D1 in tumor cells treated with LY3001920 or CDK4/6 inhibitor (abemaciclib or palbociclib) alone, or their combination. As shown in Figure 4a, treatment of KRAS mutant Calu-6 cells with LY3009120 (0.5 μM) or abemaciclib (5 μM) alone for 48 h resulted in a partial inhibition of phospho-RB at both Ser780 and Ser807/811 sites. However, combination treatment led to nearly complete inhibition of phospho-RB at both sites. In addition, inhibition of CDK4/6 by abemaciclib induced a significant increase of cyclin D1. Addition of LY3009120 com- pletely prevented the cyclin D1 elevation induced by abemaciclib treatment, and also reduced cyclin D1 to levels below those observed in untreated cells (Figure 4a). As expected, treatments of LY3009120 alone or LY3009120 plus abemaciclib led to similar and nearly complete inhibition of phospho-MEK, while abemaciclib alone had a minimal effect on phospho-MEK. Similarly, in KRAS mutant HCT116 cells, LY3009120 (1 μM) or abemaciclib (5 μM) alone only caused partial phospho-RB inhibition, and abemaciclib also induced significant accumulation of cyclin D1. When LY3001920 was combined with abemaciclib, a more robust inhibition of phospho-RB and suppression of cyclin D1 were also observed (Figure 4a). To evaluate if these molecular changes were also occurring in NRAS or BRAF V600E mutant cells, we tested SK- Mel30, a NRAS mutant melanoma cell line and A375, a BRAF V600E mutant melanoma cell line. As revealed in Figure 4a, treatment of single agent LY3009120 or abemaciclib partially inhibited phospho-RB. Combination treatment led to nearly complete inhibition of phospho-RB at both sites. Again, abemaciclib alone induced significant accumulation of cyclin D1, and the combina- tion of LY3009120 and abemaciclib completely prevented cyclin D1 elevation. These results confirmed that these important molecular changes mediated by LY3001920 and abemaciclib combination are consistent in tumor cells with KRAS, NRAS or BRAF mutation. To further validate the molecular synergy of abemaciclib and LY3009120 was due to CDK4/6 inhibition and rule out any possible off target effect of abemaciclib, we also tested LY3009120 in combination with palbociclib, another chemically unrelated CDK4/6 inhibitor. As shown in Figure 4b, palbociclib alone indeed partially inhibited phospho-RB and induced significant upregula- tion of cyclin D1, and LY3009120 and palbociclib combination resulted in more significant inhibition of phospho-RB, and suppressed cyclin D1 increase induced by palbociclib in all the same four cell lines tested.
Consistent with pharmacological inhibition of CDK4/6 by abemaciclib and palbociclib, similar results were also observed by siRNA knockdown of CDK4/6. CDK4/6 depletion by siRNA led to partial inhibition of phospho-RB and a significant increase of cyclin D1, whereas it had no effect on phospho-MEK (Figure 4c). When CDK4/6 siRNA knockdown was combined with LY3009120, a more significant inhibition of phospho-Rb and suppression of cyclin D1 were observed.
Combined inhibition of RAF by LY3009120 and CDK4/6 by abemaciclib enhances cell cycle G0/G1 arrest
For KRAS mutant Calu-6 cells in the growth conditions described in this study, the cells displayed a ratio of 14.72%, 53.83% and 31.45% at G0/G1, S and G2/M phases, respectively. Treatment of LY3009120 led to a moderate increase of G0/G1 cells to 29.84%, whereas treatment of abemaciclib led to a significant increase of G0/G1 cells to 68.7%. The increase of G0/G1 phase was accompanied by a decrease S and G2/M phases. When these cells were treated with LY3001920 and abemaciclib combination, an enhanced G0/G1 population, up to 76.74%, and accompanying decreased S (17.91%) and G2/M (5.34%) phases were observed (Figure 5a). The more significant cell G0/G1 arrest is consistent with more significant phospho-Rb inhibition described in Figure 4. Similarly, in KRAS mutant HCT116 cells, treatment of LY3009120 or abemaciclib alone led to the increase of the G0/G1 cell population to 60.89% and 65.5%, respectively, compared with 34.95% in cells treated with dimethyl sulfoxide control (Figure 5b). Again, treatment of LY3009120 and abemaciclib combination further increased G0/G1 cell population to 76.13% (Figure 5b). In NRAS mutant SK-Mel30 and BRAF V600E mutant A375 cells, treatment with LY3009120 or abemaciclib alone, or their combination, demonstrated similar cell cycle G0/G1 arrest with combination treatment showing more significant G0/G1 arrest (Supplementary Figure S5A and B). The proportions of cells at various cell cycle stages inferred from the data for Calu-6, HCT116, SK-Mel30 and A375 cells treated by LY3009120, abemaciclib or their combina- tion are summarized in Figure 5c. Overall, treatment of either LY3009120 or abemaciclib alone induces an increase of the G0/G1 cell population, and combination of both further enhances G0/G1 arrest of these cells.
Combined inhibition of RAF by LY3009120 and CDK4/6 by abemaciclib results in superior inhibition of phospho-Rb and suppression of cyclin D1 in xenograft models
To determine whether the molecular changes observed in vitro may explain the antitumor effects of the LY3009120 and abemaciclib combination in vivo, we treated animals bearing KRAS mutant HCT116 xenograft tumors with LY3009120 or abemaciclib alone, or their combination. Three hours after the last dose from a 3 day treatment regimen, the tumors were collected and the tumor lysates were prepared for cell signaling analysis. As shown in Figure 6a, LY3009120 single agent treatment caused significant reduction of phospho-MEK and phospho-ERK. Abemaciclib treatment significantly inhibited phospho-Rb at both S780 and S807/S811 sites, and also induced an increase of cyclin D1. Consistent with in vitro results described in Figure 4, the combination of LY3009120 and abemaciclib exhibited enhanced inhibition of phospho-Rb and suppression of cyclin D1, as well as inhibition of phospho-MEK and phospho-ERK. The quantification of western blots revealed that the difference of phospho-Rb inhibition by abemaciclib single agent and combination groups was statistically significant. Abemaciclib treatment alone signifi- cantly enhanced the cyclin D1 level, whereas combination treatment suppressed the cyclin D1 increase (Figure 6b). Similar molecular changes were observed in the NRAS mutant SK-Mel30 xenograft model. LY3009120 inhibited phospho-MEK and phos- pho-ERK, and abemaciclib inhibited phospho-Rb partially and induced an increase of cyclin D1. Combination of these two enhanced phospho-Rb inhibition and caused suppression of cyclin D1 (Figures 6c and d).
Figure 5. Combined inhibition of RAF by LY3009120 and CDK4/6 by abemaciclib enhances cell cycle G0/G1 arrest. (a) Cell cycle analysis of Calu-6 cells treated with DMSO, LY3009120 (0.05 μM), abemaciclib (0.5 μM) or the combination for 24 h and subjected to PI staining and subsequent flow cytometry analysis for cell cycle distribution. Dead cells are indicated as debris. Representative histograms were shown from at least two independent experiments. (b) Cell cycle analysis of HCT116 cells treated with DMSO, LY3009120 (0.1 μM), abemaciclib (0.5 μM) or the combination for 24 h and subjected to PI staining and subsequent flow cytometry analysis for cell cycle distribution. Dead cells are indicated as debris. Representative histograms were shown from at least two independent experiments. (c) Bar graphs show percent cells in G1/G0, S and G2/M phase as determined by PI staining of Calu-6 after the treatment described in a. (c) Bar graphs show percent cells in G1/G0 (red color), S (green color) and G2/M (blue color) phases as determined by PI staining in Calu-6, HCT116, SK-Mel30 and A375 cells.
The role of cyclin D1 in phospho-Rb inhibition mediated by abemaciclib and molecular mechanism of synergy by co-targeting RAF and CDK4/6
To account for effects observed on the Rb pathway in vitro and in vivo, we hypothesized that the failure of single-agent abemaciclib to completely inhibit Rb phosphorylation in KRAS, NRAS or BRAF mutant cells was owing to the increase of cyclin D1. To test this hypothesis, we depleted the cyclin D1 using shRNA in KRAS mutant HCT116 and BRAF mutant A375 cells. As demon- strated in Figures 7a and b, cyclin D1 shRNA knockdown led to decrease of cyclin D1 protein levels, and had a minimal effect on phospho-Rb. Again, abemaciclib treatment demonstrated a partial inhibition of phospho-Rb and an increase of cyclin D1 in cells without cyclin D1 knockdown. However in cells with cyclin D1 knockdown, abemaciclib treatment demonstrated more robust and nearly complete inhibition of phospho-Rb in both cells, suggesting that cyclin D1 upregulation contributes to the incomplete phospho-Rb inhibition by abemaciclib.
Cyclin D1 is known to be transcriptionally regulated by MAPK signaling in some cells,31 suggest that the cyclin D1 suppression by LY3009120 may be due to transcriptional inhibition of cyclin D1. To investigate this possibility, we conducted quantitative PCR analysis of cyclin D1 in KRAS mutant Calu-6 and HCT116 cells, BRAF mutant A375 and NRAS mutant SK-Mel30 cells after treatment of LY3009120. As revealed in Figures 7c-f, LY3009120 treatment indeed dramatically reduced mRNA levels of cyclin D1 in these four cell lines. As positive controls of transcriptional inhibition by LY3009120, DUSP4 and DUSP6 were also significantly inhibited by LY3009120.Based on all these data, we proposed the following molecular mechanism of the synergy promoted by combined inhibition of RAF and CDK4/6 (Figure 7g). Inhibition of CDK4/6 arrests the tumor cells in G0/G1 phase of the cell cycle and leads the accumulation of cyclin D1 levels and subsequently incomplete inhibition of Rb phosphorylation by a CDK4/6 kinase inhibitor. Cyclin D1 is a downstream target transcriptionally controlled b MAPK signaling. Inhibition of MAPK by LY3009120 suppresses the cyclin D1 expression. Therefore, combination of RAF and CDK4/6 inhibitors suppresses cyclin D1 expression and cooperatively leads to more complete inhibition of Rb phosphorylation, and subse- quently more robust G0/G1 cell cycle arrest and inhibition of cell proliferation.
Figure 6. Combined inhibition of RAF by LY3009120 and CDK4/6 by abemaciclib results in superior inhibition of phospho-Rb and suppression of cyclin D1 in vivo in xenograft models. (a) Western blot analysis of tumor lysates from KRAS mutant HCT116 rat xenograft tumors. The animals were treated with vehicle, LY3009120 (15 mg/kg, twice daily), abemaciclib (20 mg/kg) or their combination for 3 days. The western blot analysis was conducted with indicated antibodies as described under ‘Materials and Methods’. (b) Quantification of western blot images from panel a and statistical analysis. (c) Western blot analysis of tumor lysates from NRAS mutant SK-Mel30 rat xenograft tumors. The animals were treated with vehicle, LY3009120 (15 mg/kg, twice daily), abemaciclib (20 mg/kg) or their combination for 3 days. (d) Quantification of western blot images from a and statistical analysis.
DISCUSSION
LY3009120 is a pan-RAF and RAF dimer inhibitor advanced to clinical studies, and shown to inhibit KRAS, NRAS or BRAF mutant cell proliferation in preclinical models.20 Abemaciclib is a CDK4/6- selective inhibitor in clinical development for breast cancer, NSCLC and other cancer types.36 The goal of this study is to evaluate if combination of abemaciclib and LY3009120 will provide synergis- tic or additive effects on cancers with KRAS, NRAS or BRAF mutation. Indeed, we have found that concurrent inhibition of CDK4/6 by abemaciclib and RAF by LY3009120 resulted in synergistic effects on proliferation of RAS or BRAF mutant cancer cells in vitro and tumor growth in vivo. Mechanistically, combined RAF and CDK4/6 inhibition led to more significant inhibition of Rb phosphorylation and cyclin D1 suppression, which subsequently resulted in a more robust cell cycle G0/G1 arrest and inhibition of cell proliferation and tumor growth.
Rb is a tumor suppressor and a key negative regulator of cell cycle entry in proliferation and frequently inactivated through phosphorylation by activation of cyclin D-CDK4/6 complex in many cancers. The elevated CDK4/6 activity promotes tumor growth by compromising tumor suppressor mechanisms includ- ing senescence and apoptosis.22,23 D-cyclin kinase inhibitors lead to inhibition of Rb phosphorylation and cell cycle arrest.27–29 In accordance with these studies, we observed the inhibition of Rb phosphorylation by abemaciclib, palbociclib or siRNA directed against CDK4/6 in KRAS, NRAS or BRAF mutant cancer cells.
However, the inhibition of phospho-Rb by CDK4/6 inhibitor alone was incomplete, and there was a concomitant increase of cyclin D1. When CDK4/6 inhibitor was combined with a pan-RAF inhibitor, a more significant inhibition of phospho-Rb and cyclin D1 suppression were observed in vitro and in vivo. The cooperative inhibition further translated into the enhancement of G0/G1 arrest in cell cycle and synergistic tumor growth inhibition in vivo. Cyclin D1 is an oncogene across many tumor types and its expression is highly upregulated via the constitutive activation of RAS-MAPK pathway at the transcription level.30,33 Indeed, the increase of cyclin D1 expression induced by CDK4/6 inhibition was signifi- cantly suppressed in the presence of pan-RAF inhibitor LY3009120. This is consistent with previous reports that MAPK inhibition by BRAF inhibitors decrease cyclin D1 expression and consequently blocked the cell cycle entry.30,37 We also further confirmed that cyclin D1 is transcriptionally inhibited by LY3009120 in multiple cells, and observed that cyclin D1 knockdown significantly enhanced the phospho-Rb inhibition by abemaciclib. Taken altogether, cyclin D1 has an important role in Rb regulation, and it appears that a high level of cyclin D1 expression protests phospho-Rb from complete inhibition by CDK4/6 kinase inhibitors. However, inhibition of MAPK and phospho-RB and suppression of cyclin D1 observed in this study may not be the only molecular mechanisms of this combinational synergy. Further studies to investigate additional pathways engaged in synergy or resistance to this combination are ongoing in our laboratory.
In cancers with RAS-MAPK pathway mutations, it is believed that combined inhibition of both RAF kinases and D-cyclin- dependent kinases could provide an effective treatment strategy. For example, BRAF mutations are present in 80% of early non- transformed stage of melanoma such as melanocytic nevi,38,39 whereas interestingly a majority of these BRAF mutant nevi undergo senescence and rarely transform into melanoma,suggesting that activation of MAPK pathways is not always sufficient for malignancies.38 Studies show that loss of tumor suppressor p16 function and subsequent CDK4 activation are critical events that reverse the senescence phenotype and induce transformation from benign nevi to melanoma.40,41 Furthermore, preclinical studies in melanoma reveal that mutually exclusive driver oncogenes such as NRAS and BRAF cooperate with CDK4 activation for malignant transformation of melanocytes.34,35 In genomic sequencing analysis, 76 out of 121 melanoma patients (63%) harbored BRAF V600 mutations and 72% of them carried additional genetic aberrations in the cell cycle regulatory proteins such as CDKN2A, TP53 and CCDN1.33 Constitutive activation of CDK4/6 due to the deletion of CDKN2A or CCND amplifications frequently co-occurred with BRAF mutations. Although clinical efficacy of selective CDK4/6 inhibitors in melanoma patients is yet to be verified, preclinical data indicate that CDK4/6 inhibition is efficacious against BRAF V600E melanomas,32,42 suggesting that combined inhibition of RAF and CDK4/6 could be an effective therapeutic strategy against BRAF mutant melanoma. In this study, treatment of single agent LY3009120 or abemaciclib was demonstrated to inhibit tumor growth of BRAF V600E melanoma, whereas combination of the two agents resulted in more robust tumor regression, suggesting that melanoma patients with upfront combination of RAF and CDK4/6 inhibitors may exhibit more durable response. In BRAF V600E colorectal cancers, it has been reported that o5% of patients were responsive to single agent BRAF inhibitor such as vemurafenib and rapidly developed resistance via RAS reactivation.43,44 In xenograft model, we have also demonstrated that CDK4/6 inhibition sensitized the colorectal tumors with BRAF V600E mutation to RAF inhibition and resulted in superior efficacy as compared with inhibition of either CDK4/6 or RAF alone, providing insight that combination of RAF and CDK4/6 inhibitors could be a potential treatment option for BRAF V600E colorectal cancer patients in the clinic.
Figure 7. The role of cyclin D1 in phospho-RB inhibition mediated by abemaciclib and molecular mechanism of synergy by co-targeting RAF and CDK4/6. (a, b), Effects of cyclin D1 shRNA knockdown on abemaciclib-mediated inhibition of phospho-RB in KRAS mutant HCT116 (a) and BRAF mutant A375 (b) cells. (c–f) LY3009120 transcriptionally inhibits expression of cyclin D1, DUSP4 and DUSP6 in KRAS mutant Calu-6 (c) and HCT116 (d), NRAS mutant SK-Mel30 (e) and BRAF mutant A375 (f) cells. The cells were treated with 1 μM LY3009120 for 24 h, and the quantitative PCR were conducted as described under ‘Materials and Methods’. (g). The proposed molecular mechanism of synergy by
co-targeting RAF and CDK4/6.
Like BRAF, oncogenic RAS mutations often lead to senescence owing to activation of INK4 family inhibitors of D-cyclin-dependent kinases,23 and CDK4 activity has been shown to be essential in a KRAS mutant mouse lung cancer models.31 This may explain why RAS mutations often co-occur with CDKN2A mutation or altera- tions of cyclin D/CDK4/6 signaling in cancers of pancreas, colon and lung. There is currently no effective therapy against RAS mutant cancers. The role of the RAF-MAPK pathway in mediating tumor growth signals from RAS is contextual and not fully understood. It was shown that the RAF proteins function as dimers in RAS mutant tumors.20,45 BRAF-selective inhibitors vemurafenib and dabrafenib induced dimerization of RAF proteins and promoted paradoxical pathway activation in Ras mutant tumor cells.15,20 Those BRAF-selective inhibitors are thus contraindicated for treatment of cancer patients with KRAS or NRAS mutation. However, inhibition of RAF dimers overcomes the paradoxical activation and shows promise in RAS mutant cancer.20 These data in aggregate suggest that combined inhibition of RAF dimers together with CDK4/6 may be particularly effective in RAS mutant cancers. Indeed, in KRAS mutant Calu-6 lung cancer xenograft model, tumor growth was significantly inhibited by LY3009120 or abemaciclib alone, and their combination resulted in significant tumor regression. This synergistic tumor growth inhibition was also observed in KRAS mutant HCT116 colorectal cancer model described in this study. Similar additive and synergistic antitumor activities were also observed in NRAS mutant SK-Mel30 model and other RAS mutant cancer models. In support of our findings, it has been found that the combination of MEK1/2 and CDK4/6 inhibitors resulted in synergistic efficacy in transgenic mice model of NRAS mutant melanoma.46 Preliminary data from clinical studies revealed that combining a MEK1/2 inhibitor with a CDK4/6 inhibitor showed promising antitumor activity in patients with abemaciclib-mediated cyclin D1 upregulation. Therefore, combi- nation of RAF and CDK4/6 inhibitors cooperatively leads to more robust inhibition of Rb phosphorylation, and subsequently more robust G0/G1 cell cycle arrest and inhibition of cell proliferation. The synergistic antitumor activities observed and the molecular mechanism revealed in this study provide a rationale for combination inhibition of CDK4/6 and MAPK inhibitors for treatment of cancer patients with KRAS, NRAS or BRAF mutation.
MATERIALS AND METHODS
Cells, antibodies and reagents
All the cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), grown and maintained as described.20 Characteriza- tion of the cell lines was conducted by a third party vendor (RADIL, Columbia, MO, USA), which included profiling by PCR for contamination by various microorganisms of bacterial and viral origin, and no contamination was detected. The cells were also verified to be of human origin without mammalian inter-species contamination. The alleles for nine different genetic markers were used to determine that the banked cells matched the genetic profile that has been previously reported. The key genetic alterations of cell lines used in this study were summarized in Supplementary Table S1. Antibodies for phospho-RB/S780 (#558385) and phospho-RB/S807/S811 (#558389) were from BD Biosciences (San Jose, CA, USA). Antibodies for cyclin D1(#2978), CDK4 (#2790), CDK6 (#3136), phospho-MEK (#9154), phospho-ERK (#9101) and actin (#4970L) were from Cell Signaling (Danvers, MA, USA). Pan-RAF inhibitor LY3009120, CDK4/6 dual inhibitor abemaciclib (LY2835219) and palbociclib (PD0332991) were synthesized by Eli Lilly and Company, Indianapolis, IN, USA. All siRNAs were obtained from Dharmacon (OnTargetPlus SiRNA). SiRNA transfections were performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Grand Island, NY, USA) according to manufacturer’s instructions.
Proliferation assay and synergy analysis Cells (5 × 103) were plated in 96 well plates (BD Biosciences) one day before the treatment. Cells were typically treated with indicated inhibitors for 5 days or two doubling time, and the cell proliferation was analyzed using CellTiter Glo (Promega, Madison, WI, USA) according to manufacturer’s instructions and a luminescence plate reader (Victor, Perkin Elmer, Waltham, MA, USA). The CI was calculated using the Loewe method, where synergy was defined as Combination Index (CI) o0.5, additivity as CI = 0.5–2.0, and antagonism as CI 42. Four-parameter logistic curves were fit to the single-agent data using the equation, Y = b + (t – b)/(1 + c h), NRAS mutant melanoma.47
Although the combination of abemaciclib and LY3009120 is well tolerated in preclinical models, the tolerability of this able to achieve combination synergy similar to continuous dosing schedule in KRAS mutant HCT116 model, suggesting that an intermittent dose schedule can be considered for this combina- tion in clinic if the tolerability becomes an issue. In addition, based on our analysis, some of the patient tailoring biomarkers, such as exclusion of patients with PI3K pathway or Rb mutation, could further define the patients who likely respond to this combination. As a future direction, there is also an opportunity to use RNAseq data to generate additional predictors of sensitivity to this combination.
In conclusion, we have consistently observed the synergistic or additive antitumor activities of abemaciclib and LY3009120 combination in tumor cells with KRAS, NRAS or BRAF mutation. Molecular analysis revealed that the combination leads to more complete inhibition of phospho-RB and suppression of cyclin D1 in addition to inhibition of phospho-MEK and phospho-ERK. Inhibition of CDK4/6 by abemaciclib or palbociclib alone leads to upregulation of cyclin D1 and only partial inhibition of phospho-Rb. However, MAPK inhibition by LY3009120 is able to transcriptionally block cyclin D1 expression, and suppress range of CI values was summarized via 10th and 90th percentiles. Where synergy was observed for a subset of combination conditions, the CI values were summarized with a median on that subset. The subsets were defined by a range of concentration ratios between the two compounds, and in one case also by a range of percent inhibition.
In vivo xenograft studies
All xenograft studies were performed with 7–8-week-old female NIH nude rats (Taconic Biosciences, Hudson, NY, USA) as previously described.20 The animals were randomized based on weight and tumor volume with group size of typically six to eight. The principal investigator was blinded to the group randomization and allocation during the experiments. In vivo combination synergy analysis was done using the method of Bliss Independence. A primary analysis day was specified for each study, usually at or near the last day of treatment. On that day, the expected additive response (EAR) tumor volume for the combination group was defined, per the Bliss method, as EAR = (V1*V2)/VC, where V1 and V2 are the mean tumor volumes in the single-agent groups, and VC is the mean volume in the control group. Next, the control group tumor volume change from baseline was defined as ΔV = VC − V0. Then an additive range around the EAR was defined, via upper and lower limits, as EARU = min(2 * EAR, EAR + 0.15 * ΔV and EARL = max(EAR/2, EAR – 0.15 * ΔV). If the observed mean combination volume on that day was larger than the mean volume of either single agent, the combination was called antagonistic. If it was smaller than that but larger than EARU, the combination was called less than additive. If it was between EARU and EARL, then the combination was called additive; otherwise, the combination was called synergistic.
Preparation of cell or tumor lysates and western blotting The cells were typically treated by LY3009120 or abemaciclib alone, or their combination for 48 h before lysis for western blot analysis. Cell and tumor lysate preparations and western blot analysis were performed as described previously.20,48
Quantitative PCR
HCT116, Calu-6, SKMel-30 and A375 cells were treated by 1 μM LY3009120 or dimethyl sulfoxide for 24 h. RNA samples were prepared using RNeasy Mini Kit from Qiagen (Germantown, MD, USA, #74104) per manufacturer’s instruction. SuperScript III First-Strand Synthesis System (Invitrogen, #18080-051) was used for cDNA synthesis. Quantitative PCR was performed using Thermo Scientific (Grand Island, NY, USA) ABsolute Blue qPCR Mixes (#AB4139A) with the following cycles: 50 °C for 2 min, then 95 ° C for 15 min, and followed by 95 °C for 15 s and 60 °C for 60 s for 40 cycles. The difference of cycle time (delta CT, or dCT) was used for calibration and quantification.
Flow cytometry
Cell cycle analysis was performed by flow cytometry as described previously.21,48
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
All authors are employees of Eli Lilly and Company at the time that the work was done. All research described herein was funded by Eli Lilly and Company.
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc).