Radiolabeled Cdk4/6 inhibitors for molecular imaging of tumors

Overexpression of cell-cycle regulating cyclin-dependent kinases 4 and 6 (Cdk4/6) and deregulation of Cdk4/6-pRb-E2F pathway are common aspects in human tumors. The aim of our study was the evaluation of pyrido[2,3 d]pyrimidin-7-one derivatives (CKIA and CKIE) concerning their efficacy and suitability as small molecule Cdk4/6 inhibitors and, after iodine-124 ([124I]CKIA) or fluorine-18 ([18F]CKIE) radiolabeling, as radiotracers for Cdk4/6 imaging in tumors by positron emission tomography (PET).
CKIA and CKIE were analyzed concerning their biological properties (effects on cell growth, cell cycle distribution, Cdk4/6 mediated pRb-Ser780 phosphorylation, mRNA expression of pRb affected genes E2F-1 and PCNA) and radiopharmacological properties (cellular radiotracer uptake and PET studies) using human tumor cell lines HT-29, a colorectal adenocarcinoma cell line, FaDu, a head and neck squamous cell carcinoma cell line, and THP-1, an acute monocytic leukemia cell line, as well as phorbol ester TPA-activated THP-1 cells, as model of tumor-associated macrophages.
CKIA and CKIE were identified as potent inhibitors of Cdk4/6-pRb-E2F pathway due to decreased Cdk4/6 specific phosphorylation at pRb Ser780 and downregulation of E2F-1 and PCNA mRNA expression in HT-29, FaDu and THP-1 tumor cells. This resulted in arrest of these tumor cell lines in G1 phase of the cell cycle and growth inhibition. Otherwise, in non-proliferating TPA-activated THP-1 macrophages no change of cell-cycle distribution after treatment with CKIA and CKIE was observed. Furthermore, TPA-activated THP-1 macrophages showed lower Cdk4 mRNA and protein levels, than other tumor cell lines. In vitro radiotracer uptake studies using [124I]CKIA and [18F]CKIE demonstrated tumor cell uptake, which could be blocked with both nonradioactive CKIA and CKIE. However, THP-1 macrophages showed similar radiotracer uptake like other tumor cells. Preliminary small animal PET studies in mouse tumor xenograft models further analyzed the hypothesis that radiolabeled Cdk4/6 inhibitors are suitable tracers for molecular imaging of tumors