Preclinical evaluation of <scp>CDK4</scp> phosphorylation predicts high sensitivity of pleural mesotheliomas to <scp>CDK4</scp>/6 inhibition

Paternot, Sabine; Raspé, Eric; Meiller, Clément; Tarabichi, Maxime; Assié, Jean-Baptiste; Libert, Frédérick; Remmelink, Myriam; Bisteau, Xavier; Pauwels, Patrick; Blum, Yuna; Stang, Nolwenn Le; Tabone‐Eglinger, Séverine; Galateau-Sallé, Françoise; Blanquart, Christophe; Meerbeeck, Jan P. van; Berghmans, Thierry; Jean, Didier; Roger, Pierre P.

doi:10.1002/1878-0261.13351

Cited by 6 publications

(14 citation statements)

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“…By contrast, the homologous T177 phosphorylation on CDK6 is not regulated and is generally absent or weak ( 13 , 25 ). In our previous studies in breast tumors ( 26 ) and pleural mesotheliomas ( 27 ), we observed a variable presence of CDK4 T172-phosphorylation in most tumors. Its absence however, was also observed in some highly proliferative tumors, in association with the main mechanisms of resistance to CDK4/6i, including RB defects or inactivation, and high expressions of CDKN2A (p16), CCNE1 (cyclin E1) and E2F1 ( 26 – 29 ).…”

Section: Introductionmentioning

confidence: 66%

“…This latter form does not incorporate [32P] phosphate and binds to p16 but only weakly to cyclins D ( 13 ). The abundance ratio of the T172-phosphorylated form (spot 3) over the form separated in spot 2 allowed us to define three types of profiles (L for low, H for high and A for absent phosphorylated CDK4), as previously reported ( 26 , 27 ). All WDTC exhibited the T172-phosphorylated form of CDK4 (n=29).…”

Section: Resultsmentioning

confidence: 89%

“…CDK4 was detected by immunoblotting from whole protein extracts separated by 2D-gel electrophoresis. CDK4 was thus resolved by its charge into three main forms as first characterized in thyroid primary cultures ( 13 , 21 , 51 ) and in other tumor types ( 26 , 27 ). The most basic form (spot 1) is the native CDK4.…”

Section: Resultsmentioning

confidence: 99%

“…RNA yield and purity were assessed using a Fragment Analyzer 5200 (Agilent Technologies, Massy, France). 10ng to 100 ng of RNA was used for cDNA libraries and sequences production, as previously described ( 27 ). Homo_sapiens.GRCh38.90.gtf annotations and Homo_sapiens.GRCh38.90.dna.primary-assembly.fa sequence files downloaded from ftp.Ensembl.org were used for reads alignments.…”

Section: Methodsmentioning

confidence: 99%

“…Then it bins the rest of the genome in 30 kb bins and count the number of reads with MAPQ > 30 falling in each bin to obtain a read-count track genome-wide. We used a normal diploid sample (L2), previously profiled with the same panel ( 27 ), to normalize the read counts. The number of reads in each bin is divided by the average number to get a ratio r, which can be expressed as a function of the purity ρ, the average number of copies (or average ploidy ψ) and the local number of DNA copies nT: r = (n_T ρ + 2(1−ρ))/ψ.…”

Section: Methodsmentioning

confidence: 99%

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CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas

Pita,

Raspé,

Coulonval

et al. 2023

Front. Endocrinol.

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BackgroundCDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers.MethodsWe analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays.ResultsCDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes.ConclusionOur study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.

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