Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib
Authors: Raspe ES , Coulonval K , Pita J , Paternot S , Rothé F , Larsimont D , Van Laere S , Piccart M , Ignatiadis M , Sotiriou C , Roger PP 
Published: March 2017

Abstract
Although the specific CDK4/6 inhibitor PD0332991 (Palbociclib) was recently approved by the FDA to treat advanced ER+ breast tumors, there is yet no reliable sensitivity prediction tool. Cyclin D-CDK4/6 are the first CDK complexes to be activated in G1 phase in response to oncogenic pathways. They phosphorylate and inactivate the central cell cycle/tumor suppressor pRb. CDK4 activity requires its binding to a cyclin D (CCND1-3 genes) with which INK4 CDK4 inhibitors such as p16 (CDKN2A-D genes) compete. Although the assembly of the CDK4-cyclin D complexes was considered to be the main level of CDK4 activity control, we have shown that the activating T172-phosphorylation of CDK4 is actually the central rate-limiting event that initiates the cell cycle decision and signals the presence of active CDK4.
Here, using 2D-gel electrophoresis to separate the modified forms of CDK4, we found in breast cancer cell lines that only the CDK4 T172-phosphorylation correlates with the sensitivity to PD0332991. The only exception was in the rare case of combined CCNE1 amplification and CDKN2A loss wherein combination of PD0332991 with a CDK2 inhibitor is required to block entry in the cell cycle. Additionally, three types of CDK4 modification profile were identified by 2D-gel electrophoresis in 56 breast tumors. In the first profile, the phosphorylated CDK4 was undetectable as in normal breast samples despite a high KI67 index. In the second and third profiles, the CDK4 phosphorylation was detectable and its intensity was either above or below 90% of the intensity of a second yet unidentified form of CDK4, respectively. The proportions of these profiles differ among breast tumors according to their clinic-pathological characteristics, molecular subtypes and risk. Finally, we identified a 11-gene expression signature that faithfully predicted the CDK4 modification profiles of breast tumors and cell lines (concordance rates of 84% and 100% in the 56 analyzed breast tumor samples or cell lines respectively). All three CDK4 modification profiles were evaluated in a merged independent dataset of 4034 published gene expression profiles. In these 4034 patients, 70% of triple-negative tumors, 18% of HER2-positive tumors and 5% of ER-positive tumors were predicted to have the first CDK4 profile wherein CDK4 phosphorylation is undetectable and to be completely unresponsive to CDK4 inhibitors. The phosphorylated CDK4 was predicted to be the major modified form in 26% of triple-negative tumors, 48% of HER2- positive tumors and 56% of ER-positive tumors. These patients should benefit the most from treatment with CDK4 inhibitors. Therefore, prediction of the CDK4 modification profile may allow extending treatment with Palbociclib to presently ineligible patients. As tumors with the third CDK4 modification profile generally present low grade and low OncotypeDX risks, the added value of including CDK4 inhibitors in their treatment compared to surgery and hormone therapy alone is questionable.
In conclusion, we identified CDK4 phosphorylation as the most direct biomarker of CDK4 inhibitor sensitivity in breast cancer and developed a promising 11-gene based surrogate marker to guide their use in the clinic.Although the specific CDK4/6 inhibitor PD0332991 (Palbociclib) was recently approved by the FDA to treat advanced ER+ breast tumors, there is yet no reliable sensitivity prediction tool. Cyclin D-CDK4/6 are the first CDK complexes to be activated in G1 phase in response to oncogenic pathways. They phosphorylate and inactivate the central cell cycle/tumor suppressor pRb. CDK4 activity requires its binding to a cyclin D (CCND1-3 genes) with which INK4 CDK4 inhibitors such as p16 (CDKN2A-D genes) compete. Although the assembly of the CDK4-cyclin D complexes was considered to be the main level of CDK4 activity control, we have shown that the activating T172-phosphorylation of CDK4 is actually the central rate-limiting event that initiates the cell cycle decision and signals the presence of active CDK4.
Here, using 2D-gel electrophoresis to separate the modified forms of CDK4, we found in breast cancer cell lines that only the CDK4 T172-phosphorylation correlates with the sensitivity to PD0332991. The only exception was in the rare case of combined CCNE1 amplification and CDKN2A loss wherein combination of PD0332991 with a CDK2 inhibitor is required to block entry in the cell cycle. Additionally, three types of CDK4 modification profile were identified by 2D-gel electrophoresis in 56 breast tumors. In the first profile, the phosphorylated CDK4 was undetectable as in normal breast samples despite a high KI67 index. In the second and third profiles, the CDK4 phosphorylation was detectable and its intensity was either above or below 90% of the intensity of a second yet unidentified form of CDK4, respectively. The proportions of these profiles differ among breast tumors according to their clinic-pathological characteristics, molecular subtypes and risk. Finally, we identified a 11-gene expression signature that faithfully predicted the CDK4 modification profiles of breast tumors and cell lines (concordance rates of 84% and 100% in the 56 analyzed breast tumor samples or cell lines respectively). All three CDK4 modification profiles were evaluated in a merged independent dataset of 4034 published gene expression profiles. In these 4034 patients, 70% of triple-negative tumors, 18% of HER2-positive tumors and 5% of ER-positive tumors were predicted to have the first CDK4 profile wherein CDK4 phosphorylation is undetectable and to be completely unresponsive to CDK4 inhibitors. The phosphorylated CDK4 was predicted to be the major modified form in 26% of triple-negative tumors, 48% of HER2- positive tumors and 56% of ER-positive tumors. These patients should benefit the most from treatment with CDK4 inhibitors. Therefore, prediction of the CDK4 modification profile may allow extending treatment with Palbociclib to presently ineligible patients. As tumors with the third CDK4 modification profile generally present low grade and low OncotypeDX risks, the added value of including CDK4 inhibitors in their treatment compared to surgery and hormone therapy alone is questionable.
In conclusion, we identified CDK4 phosphorylation as the most direct biomarker of CDK4 inhibitor sensitivity in breast cancer and developed a promising 11-gene based surrogate marker to guide their use in the clinic.
Citation Format: Raspe ES, Coulonval K, Pita J, Paternot S, Rothé F, Larsimont D, Van Laere S, Piccart M, Ignatiadis M, Sotiriou C, Roger PP. CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-02.