Combined inhibition of PLK1 and NOTCH in melanoma
Statistical analysis
Tumor growth curves were plotted by mean volume (mm\(^3\)) \(\pm\) standard deviation at each measurement occasion. Between-group comparisons were based on the average growth volume, i.e., the area under the growth curve (AUC) divided by the number of weeks (Patten et al. 2022), and tested by the \(t\)-test. The combination index (CI) was calculated to assess the synergy of combined treatments (Chou and Talalay 1984; Chou 2006). Mouse weights and tumor weights were analyzed similarly. P-values \(< 0.05\) were considered statistically significant. All analyses were conducted using R version 4.1.2.
Results
Mouse weights
Mouse weight growth curves in each treatment group (mean \(\pm\) SD) are plotted in Figure 1 by gender and overall.
- Male mice are much heavier than female mice;
- Vehicle group appears to gain weight faster (lower weight at week 0 and higher weight at week 4) compared to other groups.
The average weight gain in 4 weeks (95% confidence interval) and p-values comparing each group to Vehicle are shown in Table 1.
| Group | Avg. increase (95% conf. int.) | P |
|---|---|---|
| Vehicle | 2.1 (1.5, 2.7) | — |
| V5 | 1.4 (1.2, 1.7) | 0.051 |
| V10 | 1 (0.6, 1.5) | 0.006 |
| O30 | 1.4 (0.8, 1.9) | 0.081 |
| O60 | 1.2 (0.7, 1.7) | 0.028 |
| MK-12.5 | 1.4 (0.9, 1.8) | 0.061 |
| MK-25 | 1.1 (0.8, 1.4) | 0.005 |
| V5MK12.5 | 1.1 (0.6, 1.6) | 0.014 |
| V5MK25 | 1.1 (0.6, 1.5) | 0.009 |
| V10MK12.5 | 1 (0.7, 1.3) | 0.004 |
| V10MK25 | 0.9 (0.6, 1.3) | 0.002 |
| O30MK12.5 | 1 (0.7, 1.2) | 0.002 |
| O30MK25 | 1.1 (0.8, 1.3) | 0.004 |
| O60MK12.5 | 1.2 (0.9, 1.6) | 0.017 |
| O60MK25 | 1.2 (0.7, 1.6) | 0.015 |
Weekly mouse weights
The weekly increase in mouse weight is summarized by treatment group with p-values comparing each group to Vehicle in Table 2.
| Group | Wt. increase (95% conf. int.) | P |
|---|---|---|
| Vehicle | 0.9 (0.5, 1.2) | — |
| V5 | 0.6 (0.4, 0.9) | 0.253 |
| V10 | 0.3 (0, 0.6) | 0.025 |
| O30 | 0.7 (0.4, 1.1) | 0.575 |
| O60 | 0.5 (0.2, 0.8) | 0.110 |
| MK-12.5 | 0.6 (0.3, 0.9) | 0.194 |
| MK-25 | 0.3 (0.1, 0.4) | 0.007 |
| V5MK12.5 | 0.5 (0.1, 0.9) | 0.160 |
| V5MK25 | 0.4 (0.2, 0.6) | 0.023 |
| V10MK12.5 | 0.4 (0.1, 0.6) | 0.022 |
| V10MK25 | 0.5 (0.1, 0.8) | 0.110 |
| O30MK12.5 | 0.3 (0, 0.6) | 0.018 |
| O30MK25 | 0.3 (0.1, 0.5) | 0.016 |
| O60MK12.5 | 0.7 (0.5, 1) | 0.482 |
| O60MK25 | 0.7 (0.4, 0.9) | 0.389 |
| Group | Wt. increase (95% conf. int.) | P |
|---|---|---|
| Vehicle | 2 (1.4, 2.6) | — |
| V5 | 1.4 (1, 1.7) | 0.075 |
| V10 | 0.9 (0.5, 1.4) | 0.007 |
| O30 | 1.4 (0.8, 2) | 0.127 |
| O60 | 1.4 (0.9, 2) | 0.149 |
| MK-12.5 | 1.3 (0.8, 1.9) | 0.097 |
| MK-25 | 1.1 (0.8, 1.4) | 0.013 |
| V5MK12.5 | 1 (0.4, 1.5) | 0.013 |
| V5MK25 | 1.1 (0.8, 1.5) | 0.015 |
| V10MK12.5 | 1 (0.7, 1.4) | 0.009 |
| V10MK25 | 0.9 (0.5, 1.2) | 0.003 |
| O30MK12.5 | 1 (0.7, 1.2) | 0.004 |
| O30MK25 | 1.1 (0.9, 1.4) | 0.013 |
| O60MK12.5 | 1.2 (0.9, 1.6) | 0.031 |
| O60MK25 | 1.2 (0.8, 1.6) | 0.035 |
| Group | Wt. increase (95% conf. int.) | P |
|---|---|---|
| Vehicle | 3.3 (2.4, 4.2) | — |
| V5 | 2.4 (2, 2.7) | 0.057 |
| V10 | 1.7 (1, 2.3) | 0.005 |
| O30 | 2 (1.1, 2.8) | 0.030 |
| O60 | 1.9 (1.1, 2.7) | 0.018 |
| MK-12.5 | 2.1 (1.4, 2.8) | 0.040 |
| MK-25 | 1.7 (1.2, 2.2) | 0.003 |
| V5MK12.5 | 1.7 (0.9, 2.4) | 0.007 |
| V5MK25 | 1.6 (0.8, 2.4) | 0.006 |
| V10MK12.5 | 1.6 (1.1, 2.1) | 0.003 |
| V10MK25 | 1.5 (1, 2) | 0.002 |
| O30MK12.5 | 1.6 (1.1, 2) | 0.002 |
| O30MK25 | 1.6 (1.2, 2) | 0.002 |
| O60MK12.5 | 1.8 (1.3, 2.3) | 0.006 |
| O60MK25 | 1.7 (1, 2.4) | 0.008 |
| Group | Wt. increase (95% conf. int.) | P |
|---|---|---|
| Vehicle | 4.2 (3, 5.4) | — |
| V5 | 2.8 (2.5, 3.1) | 0.026 |
| V10 | 2.4 (1.6, 3.2) | 0.014 |
| O30 | 2.9 (1.7, 4) | 0.099 |
| O60 | 2.2 (1.1, 3.2) | 0.012 |
| MK-12.5 | 2.8 (1.9, 3.7) | 0.062 |
| MK-25 | 2.3 (1.5, 3.1) | 0.010 |
| V5MK12.5 | 2.4 (1.3, 3.4) | 0.019 |
| V5MK25 | 2.4 (1, 3.7) | 0.040 |
| V10MK12.5 | 2.2 (1.6, 2.8) | 0.004 |
| V10MK25 | 1.8 (1, 2.6) | 0.001 |
| O30MK12.5 | 2.1 (1.4, 2.7) | 0.003 |
| O30MK25 | 2.4 (1.7, 3.1) | 0.010 |
| O60MK12.5 | 2.4 (1.7, 3.1) | 0.010 |
| O60MK25 | 2.1 (1.1, 3) | 0.006 |
Tumor volumes
For each mouse, the average volume between the two tumors (T1 and T2) is considered.
All groups
Tumor growth curves in each treatment group (mean \(\pm\) SD) are plotted in Figure 2 by gender and overall.
Based on Figure 2 (all mice), we can order the treatments by the following:
- Best: O60MK25 \(\gtrsim\) O60MK12.5
- Good: V10MK25 \(\approx\) O30MK25
- Medium: All other treatments except vehicle
- Worst: Vehicle
The average increase in tumor volume (95% confidence interval) in 4 weeks and p-values comparing each group to Vehicle are shown in Table 3 (all mice). All treatments are significantly different from Vehicle.
| Group | Avg. increase (95% conf. int.) | P |
|---|---|---|
| Vehicle | 198.6 (157.5, 239.7) | — |
| V5 | 120.8 (92.8, 148.9) | 0.003 |
| V10 | 125.8 (97.3, 154.3) | 0.004 |
| O30 | 139.6 (102.8, 176.4) | 0.029 |
| O60 | 126.4 (81.2, 171.7) | 0.017 |
| MK-12.5 | 123.3 (81.6, 164.9) | 0.010 |
| MK-25 | 134 (99.9, 168) | 0.015 |
| V5MK12.5 | 108.7 (77.4, 139.9) | <0.001 |
| V5MK25 | 120.2 (69.2, 171.1) | 0.016 |
| V10MK12.5 | 102.4 (69.4, 135.4) | <0.001 |
| V10MK25 | 86.8 (61.4, 112.2) | <0.001 |
| O30MK12.5 | 112.7 (62.5, 163) | 0.008 |
| O30MK25 | 82.9 (51, 114.8) | <0.001 |
| O60MK12.5 | 60.9 (43.6, 78.2) | <0.001 |
| O60MK25 | 53.5 (29.9, 77) | <0.001 |
Volasertib-only vs Onvansertib-only
Table 4 shows that there are no significance differences between Volasertib-only vs Onvansertib-only treatments.
| Group 1 | Group 2 | P |
|---|---|---|
| V5 | O30 | 0.389 |
| V10 | O30 | 0.529 |
| V5 | O60 | 0.819 |
| V10 | O60 | 0.980 |
Volasertib-only treatments vs combinations
Table 5 shows that only V10MK25 is significantly different (better) than Vo and MK groups alone.
| Group 1 | Group 2 | P |
|---|---|---|
| V5 | V5MK12.5 | 0.530 |
| V5 | V5MK25 | 0.980 |
| V10 | V10MK12.5 | 0.251 |
| V10 | V10MK25 | 0.036 |
| MK-12.5 | V5MK12.5 | 0.544 |
| MK-12.5 | V10MK12.5 | 0.397 |
| MK-25 | V5MK25 | 0.629 |
| MK-25 | V10MK25 | 0.024 |
Onvansertib-only treatments vs combinations
Table 6 shows that O30MK25, O60MK12.5, and O60MK25 are significantly different (better) than the corresponding single treatments.
| Group 1 | Group 2 | P |
|---|---|---|
| O30 | O30MK12.5 | 0.360 |
| O30 | O30MK25 | 0.019 |
| O60 | O60MK12.5 | 0.010 |
| O60 | O60MK25 | 0.006 |
| MK-12.5 | O30MK12.5 | 0.728 |
| MK-12.5 | O60MK12.5 | 0.008 |
| MK-25 | O30MK25 | 0.026 |
| MK-25 | O60MK25 | <0.001 |
Tumor weights
For each mouse, the average weight between the two tumors (T1 and T2) is considered.
Tumor weights in each treatment group (mean \(\pm\) SE (standard error of the mean)) at the end of follow-up are plotted in Figure 3 by gender and overall.
For all mice, the ranking among treatments roughly coincided with that of tumor volume growth:
- The four best groups are: O60MK25, O60MK12.5, V10MK25, and O30MK25;
- Vehicle is the worst group.
The average tumor weight (95% confidence interval) and p-values comparing each group to Vehicle are shown in Table 8.
| Group | Avg. tumor weight (95% conf. int.) | P |
|---|---|---|
| Vehicle | 1.1 (0.7, 1.6) | — |
| V5 | 0.8 (0.6, 1) | 0.125 |
| V10 | 0.7 (0.5, 0.9) | 0.103 |
| O30 | 0.9 (0.6, 1.1) | 0.253 |
| O60 | 0.7 (0.3, 1) | 0.105 |
| MK-12.5 | 0.8 (0.5, 1.1) | 0.222 |
| MK-25 | 0.7 (0.6, 0.9) | 0.114 |
| V5MK12.5 | 0.7 (0.4, 1) | 0.079 |
| V5MK25 | 0.6 (0.3, 0.9) | 0.061 |
| V10MK12.5 | 0.6 (0.4, 0.7) | 0.020 |
| V10MK25 | 0.5 (0.3, 0.6) | 0.010 |
| O30MK12.5 | 0.6 (0.3, 0.9) | 0.061 |
| O30MK25 | 0.5 (0.3, 0.8) | 0.022 |
| O60MK12.5 | 0.4 (0.3, 0.6) | 0.008 |
| O60MK25 | 0.3 (0.1, 0.5) | 0.003 |
Single vs combo treatments
Single vs combo treatments are compared in Table 9 and Table 10.
- There is synergistic effect between V10 and MK25 (both significantly worse than combination);
- There is synergistic effect between O60 and MK25 (both borderline-significantly worse than combinations).
| Group 1 | Group 2 | P |
|---|---|---|
| V5 | V5MK12.5 | 0.577 |
| MK-12.5 | V5MK12.5 | 0.468 |
| V5 | V5MK25 | 0.440 |
| MK-25 | V5MK25 | 0.475 |
| V10 | V10MK12.5 | 0.125 |
| MK-12.5 | V10MK12.5 | 0.125 |
| V10 | V10MK25 | 0.031 |
| MK-25 | V10MK25 | 0.027 |
| Group 1 | Group 2 | P |
|---|---|---|
| O30 | O30MK12.5 | 0.229 |
| MK-12.5 | O30MK12.5 | 0.381 |
| O30 | O30MK25 | 0.052 |
| MK-25 | O30MK25 | 0.150 |
| O60 | O60MK12.5 | 0.185 |
| MK-12.5 | O60MK12.5 | 0.039 |
| O60 | O60MK25 | 0.063 |
| MK-25 | O60MK25 | 0.004 |
References
Chou, Ting-Chao. 2006. “Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies.” Pharmacological Reviews 58 (3): 621–81. https://doi.org/10.1124/pr.58.3.10.
Chou, Ting-Chao, and Paul Talalay. 1984. “Quantitative Analysis of Dose-Effect Relationships: The Combined Effects of Multiple Drugs or Enzyme Inhibitors.” Advances in Enzyme Regulation 22 (January): 27–55. https://doi.org/10.1016/0065-2571(84)90007-4.
Patten, Luke W., Patrick Blatchford, Matthew Strand, and Alexander M. Kaizer. 2022. “Assessing the Performance of Different Outcomes for Tumor Growth Studies with Animal Models.” Animal Models and Experimental Medicine 5 (3): 248–57. https://doi.org/10.1002/ame2.12250.