Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss... Show moreDifferentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves’ disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers. Show less
Aydemirli, M.D.; Eendenburg, J.D.H. van; Wezel, T. van; Oosting, J.; Corver, W.E.; Kapiteijn, E.; Morreau, H. 2021
Finding targetable gene fusions can expand the limited treatment options in radioactive iodine-refractory (RAI-r) thyroid cancer. To that end, we established a novel cell line `JVE404' derived from... Show moreFinding targetable gene fusions can expand the limited treatment options in radioactive iodine-refractory (RAI-r) thyroid cancer. To that end, we established a novel cell line `JVE404' derived from an advanced RAI-r papillary thyroid cancer (PTC) patient, harboring an EML4-ALK gene fusion variant 3 (v3). Different EML4-ALK gene fusions can have different clinical repercussions. JVE404 cells were evaluated for cell viability and cell signaling in response to ALK inhibitors crizotinib, ceritinib and lorlatinib, in parallel to the patient's treatment. He received, after first-line lenvatinib, crizotinib (Drug Rediscovery Protocol (DRUP) trial), and lorlatinib (compassionate use). In vitro treatment with crizotinib or ceritinib decreased viability in JVE404, but most potently and significantly only with lorlatinib. Western blot analysis showed a near total decrease of 99% and 89%, respectively, in pALK and pERK expression levels in JVE404 cells with lorlatinib, in contrast to remaining signal intensities of a half and a third of control, respectively, with crizotinib. The patient had a 6-month lasting stable disease on crizotinib, but progressive disease occurred, including the finding of cerebral metastases, at 8 months. With lorlatinib, partial response, including clinical cerebral activity, was already achieved at 11 weeks' use and ongoing partial response at 7 months. To our best knowledge, this is the first reported case describing a patient-specific targeted treatment with lorlatinib based on an EML4-ALK gene fusion v3 in a thyroid cancer patient, and own cancer cell line. Tumor-agnostic targeted therapy may provide valuable treatment options in personalized medicine. Show less
Hurthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome ... Show moreHurthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2-7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of alpha-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate-N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG. Show less
Tuin, K. van der; Garcia, M.V.; Corver, W.E.; Khalifa, M.N.; Neto, D.R.; Corssmit, E.P.M.; ... ; Morreau, H. 2019
The genomes of a wide range of cancers, including colon, breast and thyroid cancers frequently show copy number gains of chromosome 7 and rarely show loss of heterozygosity. The molecular basis for... Show moreThe genomes of a wide range of cancers, including colon, breast and thyroid cancers frequently show copy number gains of chromosome 7 and rarely show loss of heterozygosity. The molecular basis for this phenomenon is unknown. Strikingly, oncocytic follicular thyroid carcinomas can display an extreme genomic profile, with homozygosity of all chromosomes except for chromosome 7. The observation that homozygosity of chromosome 7 is never observed suggests that retention of heterozygosity is essential for cells. We hypothesized that cell survival genes are genetically imprinted on either of two copies of chromosome 7 which thwarts loss of heterozygosity at this chromosome in cancer cells. By employing a DNA methylation screen and gene expression analysis we identified six imprinted genes that force retention of heterozygosity on chromosome 7. Subsequent knockdown of gene expression showed that CALCR, COPG2, GRB10, KLF14, MEST and PEG10 were essential for cancer cell survival resulting in reduced cell proliferation, G1 -phase arrest and increased apoptosis. We propose that imprinted cell survival genes provide a genetic basis for retention of chromosome 7 heterozygosity in cancer cells. The monoallelically expressed cell survival genes identified in this study, and the cellular pathways they are involved in, offer new therapeutic targets for the treatment of tumours showing retention of heterozygosity on chromosome 7. DNA methylation data has been submitted to GEO under accession number GSE77804. Show less
