Journal: Oncotarget

Article Title: Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAF V600E patient-derived papillary thyroid carcinoma preclinical model

doi:

Figure Lengend Snippet: A . Experimental design of an in vitro and in vivo model of human PTC with the BRAF WT/V600E mutation. B . DNA genotyping analysis of human PTC identifies the heterozygous BRAF WT/V600E mutation. Mass spectrometry (MS) traces of human primary PTC cells. The intensity of the signal versus mass of the analyte is plotted in the background. Calls are based on an expected allelic frequency of 50%. Allele frequencies deviating from the expected values are assigned ambiguous or homozygous calls by the software. MS trace of PTC cells reveals a heterozygous BRAF WT/V600E allele (A>T). C . In a three dimensional (3D) cell culture assay using reconstituted basement membrane extracellular matrix (ECM) (Matrigel), BRAF V600E -PTC cells grew as larger cell aggregates. Normal thyroid (NT) cells transduced with BRAF V600E grew as adherent refractile cells vs. NT cells engineered with empty vector (control) which grew as spindled cells. Scale bar= 400 μ, 200 μ, 400 μ and 50 μ, respectively. D . Immunocytochemistry of representative established short-term primary human PTC cells in vitro with the heterozygous BRAF WT/V600E mutation of patient-PTC specimen (Hematoxylin-Eosin, H&E, arrows highlight nuclear clearing). Immunocytochemistry staining in the PTC cells in vitro shows cytoplasmic to membranous staining with antibodies against PAX8, TSH-receptor, and pan-keratin (marker of tumor epithelial cells and tumor purity). Desmin immunostain was negative. Scale bars= 500 μ (1000× magnification image) and 100 μ (400× magnification images). E . Inhibition of BRAF WT/V600E by vemurafenib reduces phospho(p)ERK1/2 protein expression levels. A parallel plate similar to F was set up and corresponding pERK1/2 protein levels (low exp= shorter exposure during chemiluminescence reaction; high exp= longer exposure during chemiluminescence reaction) were measured from BRAF WT/V600E -PTC cells, BRAF WT -PTC cells, or NT cells by western blotting. Densitometry analysis of the pERK1/2 protein levels in NT or PTC cells treated with 10 μM vemurafenib vs. vehicle (DMSO =Dimethyl sulfoxide, control) for 24 hours, in the corresponding western blotting (* p < 0.05, Mann-Whitney test). Primary BRAF WT -NT cells have MCL1 neutral copy number, primary BRAF WT -PTC cells have MCL1 copy number =0.9, primary non-metastatic BRAF WT/V600E -PTC1 cells have MCL1 copy number =2.14, primary BRAF WT/V600E -PTC5 cells with angio-invasion have MCL1 copy number =3, primary BRAF WT/V600E -PTC7 cells with angio-invasion have MCL1 copy number =3, primary LN metastatic/recurrent BRAF V600E -PTC cells have MCL1 copy number =3.8, KTC1 cells have MCL1 copy number =1.3 and BCPAP cells have MCL1 copy number =1.4. KTC1 cells have P16 homozygous loss. For more details regarding copy number gain/amplification (ampl.) assay see Figure and Methods. These data are representative of three independent experiments. We show these results in the 5 out of 7 short-term primary human PTC cell cultures which grew well. F . Arrows highlight change of cell shape in BRAF WT/V600E -PTC cells treated with vemurafenib vs. vehicle-treated (control) PTC cells. PTC cells with heterozygous BRAF WT/V600E or BRAF WT or NT cells were treated with 10 μM of vemurafenib or with DMSO (control) for about 24 hours. These data represent 3 independent experiments. All scale bars are=50 μ (DMSO images) and 10 μ (Vemurafenib images). Scale bars are =50 μ (BRAF WT/WT primary PTC cells and primary human normal thyroid cells images). G . Vemurafenib dose-reponse analysis: short-term primary human PTC or NT cells with BRAF V600E or with BRAF WT , as well as spontaneously immortalized human PTC and ATC cells, were treated with the indicated concentrations of vemurafenib for 48 hours, and viability was determined using the Cell Titer-Glo ATP-based luminescence assay, with DMSO-treated cells as the control. We show these results in the 5 out of 7 short-term primary human PTC cell cultures which grew well. These data represent the average ± standard deviation (error bars) of 3-5 independent replicate measurements (* p < 0.05, ** p < 0.01, *** p < 0.001, Mann-Whitney test). H . Immunocytochemistry of representative established non-immortalized primary human PTC cells with the heterozygous BRAF WT/V600E mutation or with BRAF WT , or NT cells. Immunohistochemistry staining shows cytoplasmic to membranous staining with antibodies against NG2 or PDGFRB (platelet-derived growth factor receptor-beta) in BRAF WT/V600E -PTC or BRAF WT -PTC cells. OCT3/4 immunostain was negative. Markers expression was assessed semiquantitatively using the following scoring method: 0 (negative), 1+ (1–10% positive cells), 2+ (11–50% positive cells), and 3+ (more than 50% positive cells). All scale bars are=100 μ.

Article Snippet: For the duplex real-time PCR reaction, we used a TaqMan based copy number assay specific for: (i) MCL1 (myeloid cell leukemia sequence 1, gene aliases: BCL2L3 , EAT , MCL1-ES , MCL1L , MCL1S , Mcl-1 , TM , bcl2-L-3 , mcl1/EAT ) (NCBI location: Chr.1:150547027-150552214; assay gene location: exon 1; cytoband: 1q21.3; assay reference genome Location: Chr.1:150552073 on NCBI build 37) (Life Technologies, USA, assay ID: Hs01326481_cn, cat#4400291) in 58 samples and (ii) P16 (cyclin-dependent kinase inhibitor 2A or CDKN2A , gene aliases: ARF, CDK4I, CDKN2, CMM2, INK4, INK4A, MLM, MTS-1, MTS1, P14, P14ARF, P16, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16 ) (NCBI location: Chr.9:21967751-21994490; assay gene location: exon 2; cytoband: 9p21.3; assay reference genome Location: Chr.9:21974968 on NCBI build 37) (Life Technologies, USA, assay ID: Hs00237642_cn, cat#4400291) in 51 samples for which there was sufficient DNA available.

Techniques: In Vitro, In Vivo, Mutagenesis, Mass Spectrometry, Software, Cell Culture, Membrane, Transduction, Plasmid Preparation, Control, Immunocytochemistry, Staining, Marker, Inhibition, Expressing, Western Blot, MANN-WHITNEY, Amplification, Luminescence Assay, Standard Deviation, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAF V600E patient-derived papillary thyroid carcinoma preclinical model

doi:

Figure Lengend Snippet: A . Graphical representation of the log2 ratio of sequence coverage in tumor versus reference for fragments sequenced using a targeted exome sequencing strategy. BRAF V600E -PTC primary cells or patient specimens harboring the BRAF V600E mutation were compared with PTC or NT samples with BRAF WT . This analysis revealed 1q somatic copy number alterations (SCNAs) (i.e. copy number gain/amplifications) in metastatic/recurrent neck or mediastinal lymph nodes (LN). B . Detailed view of sequenced exons in 1q, including the MCL1 gene highlighted by asterisks, in metastatic/recurrent mediastinal LN (lymph node) BRAF V600E -PTC primary cells and patient specimens harboring the BRAF V600E mutation compared with PTC or NT samples with BRAF WT . Probes that were colored or shaded blue or marked by blue triangles were called as gained by the analysis software. C . MCL1 somatic copy number analysis normalized in 31 samples which included PTC and normal thyroid samples, and some established primary PTC or NT cell cultures derived from same patients' cohort, from independent patients. Histogram shows MCL1 copy number assay results in: 1 DNA control sample from healthy man; 1 primary NT (normal thyroid) cell culture; 3 primary PTC cell cultures (2 with BRAF WT/V600E and 1 with BRAF WT ), 1 mediastinal LN metastatic BRAF V600E -PTC cell culture; 5 spontaneously immortalized cell lines established from patients with BRAF V600E (i.e., KTC1 and BCPAP) or BRAF WT (TPC1) PTC, or BRAF V600E -positive anaplastic thyroid cancer (ATC) (i.e. 8505c and SW1736); 5 NT (normal thyroid) tissue samples, 31 PTC (23 BRAF V600E -PTC and 8 BRAF WT -PTC), 17 LN metastatic PTC (9 BRAF V600E -LN and 8 BRAF WT -LN) and 5 distant metastatic samples from patients with PTC (1 from lungs, 1 from bone and 3 from adrenal glands). Results were normalized against two separate reference, housekeeping (reference) genes: GAPDH and RNAase-P . These data represent the average ± standard error mean (error bars) of 2–3 independent replicate measurements. D . Box plot analysis using: 5 NT (normal thyroid) tissue samples, 31 PTC (23 BRAF V600E -PTC and 8 BRAF WT -PTC), 17 LN metastatic PTC (9 BRAF V600E -LN and 8 BRAF WT -LN) and 5 distant metastatic samples from patients with PTC (1 from lungs, 1 from bone and 3 from adrenal glands). These data represent the average ± standard deviation of 2–3 independent replicate measurements (* p < 0.05, ** p < 0.01, *** p < 0.01, one-way ANOVA test). E . Immunohistochemistry shows strong and diffuse MCL1 protein expression in the cytosol and stippled nuclear staining in all neck LN metastatic PTC samples harboring BRAF V600E (n=3) (scoring, 3+) and primary PTC with BRAF V600E (n=4) (scoring, 3+), whereas NT samples (n=3) (scoring, 1+) showed focal and weak nuclear MCL1 localization. Primary BRAF V600E -PTC (scoring, 0) or LN metastatic BRAF V600E -PTC (scoring, 0) samples did not show significant change in cytoplasmic cleaved caspase 3 (cCasp3) protein levels compared with NT samples (scoring, 0). Protein expression was assessed semi-quantitatively using the following scoring method: 0 (negative), 1+ (1–10% positive cells), 2+ (11–50% positive cells), and 3+ (more than 50% positive cells). All scale bars are=400 μ. F . Proposed mechanisms of LN metastatic PTC spreading from primary PTC harboring the BRAF V600E mutation along with MCL1 copy number gain/amplification. G . Graphical representation of next generation sequencing, targeted-exome sequencing results showing loss of P16 (marked by red bars) in the chromosome 9p in PTC (i.e. heterozygous BRAF WT/V600E -positive human KTC1 PTC-derived cells with P16 homozygous loss) or in ATC samples with distant metastasis (i.e. homozygous BRAF V600E -positive 8505c ATC cells) compared with metastatic/recurrent LN BRAF V600E -PTC sample (LN1-T) or derived primary cells (e.g. LN0-PTC), BRAF V600E -PTC sample (e.g. PTC-7), BRAF WT -PTC sample (e.g. PTC-S2), or NT sample with BRAF WT (e.g. N1-T) which show neutral copy number without loss of P16 . The top red bar shows the aggregate SCNAs for all samples. H . P16 somatic copy number alteration analysis normalized in: 1 DNA control sample from healthy man; 3 primary PTC cell cultures (2 with BRAF WT/V600E and 1 with BRAF WT ), 1 mediastinal LN metastatic BRAF V600E -PTC cell culture; 4 spontaneously immortalized cell lines established from patients with BRAF V600E (i.e., KTC1 and BCPAP) or BRAF WT (TPC1) PTC, or BRAF V600E -positive anaplastic thyroid cancer (ATC) (i.e. 8505c); 4 NT (normal thyroid) tissue samples, 27 PTC (19 BRAF V600E -PTC and 8 BRAF WT -PTC), 15 LN metastatic PTC (7 BRAF V600E -LN and 8 BRAF WT -LN), 6 distant metastatic samples from patients with PTC (2 from lungs, 1 from bone and 3 from adrenal glands). Results were normalized against two separate reference, housekeeping (reference) genes: GAPDH and RNAase-P . Histogram shows P16 copy number assay. These data represent the average ± standard error mean (error bars) of 2–3 independent replicate measurements. I . Box plot analysis using: 4 NT (normal thyroid) tissue samples, 27 PTC (19 BRAF V600E -PTC and 8 BRAF WT -PTC), 15 LN metastatic PTC (7 BRAF V600E -LN and 8 BRAF WT -LN), 6 distant metastatic samples from patients with PTC (2 from lungs, 1 from bone and 3 from adrenal glands). These data represent the average ± standard deviation of 2–3 independent replicate measurements (* p < 0.05, ** p < 0.01, *** p < 0.01, one-way ANOVA test). J . Immunocytochemistry shows loss of P16 protein expression in human KTC1 PTC-derived cells ( BRAF WT/V600E spontaneously immortalized metastatic PTC cells) compared with primary NT cells which show P16 protein focal staining in the nuclei. All scale bars are=100 μ. K . Proposed mechanisms of metastatic spreading from primary PTC harboring the BRAF V600E mutation along with loss of P16 .

Article Snippet: For the duplex real-time PCR reaction, we used a TaqMan based copy number assay specific for: (i) MCL1 (myeloid cell leukemia sequence 1, gene aliases: BCL2L3 , EAT , MCL1-ES , MCL1L , MCL1S , Mcl-1 , TM , bcl2-L-3 , mcl1/EAT ) (NCBI location: Chr.1:150547027-150552214; assay gene location: exon 1; cytoband: 1q21.3; assay reference genome Location: Chr.1:150552073 on NCBI build 37) (Life Technologies, USA, assay ID: Hs01326481_cn, cat#4400291) in 58 samples and (ii) P16 (cyclin-dependent kinase inhibitor 2A or CDKN2A , gene aliases: ARF, CDK4I, CDKN2, CMM2, INK4, INK4A, MLM, MTS-1, MTS1, P14, P14ARF, P16, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16 ) (NCBI location: Chr.9:21967751-21994490; assay gene location: exon 2; cytoband: 9p21.3; assay reference genome Location: Chr.9:21974968 on NCBI build 37) (Life Technologies, USA, assay ID: Hs00237642_cn, cat#4400291) in 51 samples for which there was sufficient DNA available.

Techniques: Sequencing, Mutagenesis, Software, Derivative Assay, Control, Cell Culture, Standard Deviation, Immunohistochemistry, Expressing, Staining, Amplification, Next-Generation Sequencing, Immunocytochemistry

Journal: Oncotarget

Article Title: Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAF V600E patient-derived papillary thyroid carcinoma preclinical model

doi:

Figure Lengend Snippet: A . Flow cytometry analysis of non-synchronized cells normalized to the number of events in each condition shows that 10 μM vemurafenib or 100 nM obatoclax at 48 hours post-treatment partially increased sub-G1 (apoptosis) in representative patient-derived angioinvasive PTC cells (i.e. PTC7) harboring the BRAF WT/V600E mutation and with MCL1 copy number gain/amplifications compared to vehicle-treated (DMSO, control) cells. Five μM vemurafenib and 100 nM obatoclax combined treatment significantly increased the percent of PTC cells in sub-G1 compared to vehicle-treated (DMSO, control) cells. These data represent the average ± standard deviation (error bars) of 2 independent experiments replicate measurements (* p < 0.05, ** p < 0.01). B . Arrows highlight change of cell shape (rounded up and detached) in angioinvasive BRAF WT/V600E -PTC cells (i.e. PTC7) with MCL1 copy number gain/amplifications treated for about 48 hours with vemurafenib (10 μM), obatoclax (100 nM), or vemurafenib (5 μM) + obatoclax (100 nM) compared to vehicle-treated PTC cells (DMSO, control). All scale bars are=400 μ. C . Flow cytometry analysis of non-synchronized cells normalized to the number of events in each condition at 48 hours post-treatment shows that 10 μM vemurafenib or 100 nM obatoclax significantly increased sub-G1 in metastatic PTC patient-derived KTC1 cells harboring the BRAF WT/V600E mutation and with a lower copy number of MCL1 and with P16 homozygous loss compared to vehicle-treated (DMSO, control) cells. Five μM vemurafenib and 100 nM obatoclax combined treatment strongly increased the percent of KTC1 cells in sub-G1 compared to vehicle-treated (DMSO, control) cells. These data represent the average ± standard deviation (error bars) of 2 independent experiments replicate measurements (* p < 0.05, ** p < 0.01, *** p < 0.01). D . Arrows highlight change of cell shape (rounded up and detached) in metastatic BRAF WT/V600E -KTC1 cells in the presence of vemurafenib (10 μM), obatoclax (100 nM), or vemurafenib (5 μM) + obatoclax (100 nM) compared to vehicle-treated PTC cells (DMSO, control). All scale bars are=400 μ.

Article Snippet: For the duplex real-time PCR reaction, we used a TaqMan based copy number assay specific for: (i) MCL1 (myeloid cell leukemia sequence 1, gene aliases: BCL2L3 , EAT , MCL1-ES , MCL1L , MCL1S , Mcl-1 , TM , bcl2-L-3 , mcl1/EAT ) (NCBI location: Chr.1:150547027-150552214; assay gene location: exon 1; cytoband: 1q21.3; assay reference genome Location: Chr.1:150552073 on NCBI build 37) (Life Technologies, USA, assay ID: Hs01326481_cn, cat#4400291) in 58 samples and (ii) P16 (cyclin-dependent kinase inhibitor 2A or CDKN2A , gene aliases: ARF, CDK4I, CDKN2, CMM2, INK4, INK4A, MLM, MTS-1, MTS1, P14, P14ARF, P16, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16 ) (NCBI location: Chr.9:21967751-21994490; assay gene location: exon 2; cytoband: 9p21.3; assay reference genome Location: Chr.9:21974968 on NCBI build 37) (Life Technologies, USA, assay ID: Hs00237642_cn, cat#4400291) in 51 samples for which there was sufficient DNA available.

Techniques: Flow Cytometry, Derivative Assay, Mutagenesis, Control, Standard Deviation