Analysis of the CDK4/6 Cell Cycle Pathway in Leiomyosarcomas as a Potential Target for Inhibition by Palbociclib.

Leiomyosarcoma (LMS) is characterized by high genomic complexity, and to date, no specific targeted therapy is available. In a genome-wide approach, we profiled genomic aberrations in a small cohort of eight primary tumours, two relapses, and eight metastases across nine different patients. We identified CDK4 amplification as a recurrent alteration in 5 out of 18 samples (27.8%). It has been previously shown that the LMS cell line SK-LMS-1 has a defect in the p16 pathway and that this cell line can be inhibited by the CDK4 and CDK6 inhibitor palbociclib. For SK-LMS-1 we confirm and for SK-UT-1 we show that both LMS cell lines express CDK4 and that, in addition, strong CDK6 expression is seen in SK-LMS-1, whereas Rb was expressed in SK-LMS-1 but not in SK-UT-1. We confirm that inhibition of SK-LMS-1 with palbociclib led to a strong decrease in protein levels of Phospho-Rb (Ser780), a decreased cell proliferation, and G0/G1-phase arrest with decreased S/G2 fractions. SK-UT-1 did not respond to palbociclib inhibition. To compare these in vitro findings with patient tissue samples, a p16, CDK4, CDK6, and p-Rb immunohistochemical staining assay of a large LMS cohort (n=99 patients with 159 samples) was performed assigning a potential responder phenotype to each patient, which we identified in 29 out of 99 (29.3%) patients. Taken together, these data show that CDK4/6 inhibitors may offer a new option for targeted therapy in a subset of LMS patients.

Boosting Antitumor Drug Efficacy with Chemically Engineered Multidomain Proteins.

A facile chemical approach integrating supramolecular chemistry, site-selective protein chemistry, and molecular biology is described to engineer synthetic multidomain protein therapeutics that sensitize cancer cells selectively to significantly enhance antitumor efficacy of existing chemotherapeutics. The desired bioactive entities are assembled via supramolecular interactions at the nanoscale into structurally ordered multiprotein complexes comprising a) multiple copies of the chemically modified cyclic peptide hormone somatostatin for selective targeting and internalization into human A549 lung cancer cells expressing SST-2 receptors and b) a new cysteine mutant of the C3bot1 (C3) enzyme from Clostridium botulinum, a Rho protein inhibitor that affects and influences intracellular Rho-mediated processes like endothelial cell migration and blood vessel formation. The multidomain protein complex, SST3-Avi-C3, retargets C3 enzyme into non-small cell lung A549 cancer cells and exhibits exceptional tumor inhibition at a concentration ≈100-fold lower than the clinically approved antibody bevacizumab (Avastin) in vivo. Notably, SST3-Avi-C3 increases tumor sensitivity to a conventional chemotherapeutic (doxorubicin) in vivo. These findings show that the integrated approach holds vast promise to expand the current repertoire of multidomain protein complexes and can pave the way to important new developments in the area of targeted and combination cancer therapy.

HOXA7, HOXA9, and HOXA10 are differentially expressed in clival and sacral chordomas.

Chordomas are rare tumours of the bone arising along the spine from clivus to sacrum. We compared three chordoma cell lines of the clivus region including the newly established clivus chordoma cell line, U-CH14, with nine chordoma cell lines originating from sacral primaries by morphology, on genomic and expression levels and with patient samples from our chordoma tissue bank. Clinically, chordomas of the clivus were generally smaller in size at presentation and patients with sacral chordomas had more metastases and more often recurrent disease. All chordoma cell lines had a typical physaliphorous morphology and expressed brachyury, S100-protein and cytokeratin. By expression analyses we detected differentially expressed genes in the clivus derived cell lines as compared to the sacral cell lines. Among these were HOXA7, HOXA9, and HOXA10 known to be important for the development of the anterior-posterior body axis. These results were confirmed by qPCR. Immunohistologically, clivus chordomas had no or very low levels of HOXA10 protein while sacral chordomas showed a strong nuclear positivity in all samples analysed. This differential expression of HOX genes in chordomas of the clivus and sacrum suggests an oncofetal mechanism in gene regulation linked to the anatomic site.