plx-4720 and Thyroid-Carcinoma--Anaplastic

Anaplastic thyroid carcinomas (ATCs) have a high prevalence of BRAF and TP53 mutations. A trial of vemurafenib in nonmelanoma BRAFV600E-mutant cancers showed significant, although short-lived, responses in ATCs, indicating that these virulent tumors remain addicted to BRAF despite their high mutation burden. To explore the mechanisms mediating acquired resistance to BRAF blockade, we generated mice with thyroid-specific deletion of p53 and dox-dependent expression of BRAFV600E, 50% of which developed ATCs after dox treatment. Upon dox withdrawal there was complete regression in all mice, although recurrences were later detected in 85% of animals. The relapsed tumors had elevated MAPK transcriptional output, and retained responses to the MEK/RAF inhibitor CH5126766 in vivo and in vitro. Whole-exome sequencing identified recurrent focal amplifications of chromosome 6, with a minimal region of overlap that included Met. Met-amplified recurrences overexpressed the receptor as well as its ligand Hgf. Growth, signaling, and viability of Met-amplified tumor cells were suppressed in vitro and in vivo by the Met kinase inhibitors PF-04217903 and crizotinib, whereas primary ATCs and Met-diploid relapses were resistant. Hence, recurrences are the rule after BRAF suppression in murine ATCs, most commonly due to activation of HGF/MET signaling, which generates exquisite dependency to MET kinase inhibitors.

Topics: Amino Acid Substitution; Animals; Antineoplastic Agents; Cell Line, Tumor; Coumarins; Crizotinib; Disease Models, Animal; Drug Resistance, Neoplasm; Genes, p53; Humans; Indoles; MAP Kinase Signaling System; Mice; Mice, Transgenic; Mutation, Missense; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Pyrazines; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Triazoles

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAF. We again utilised our mouse model of ATC to assess the combination of BRAF. Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8. Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

Topics: Animals; Antibodies, Monoclonal; B7-H1 Antigen; Cell Line, Tumor; Disease Models, Animal; Female; Flow Cytometry; Immunocompetence; Indoles; Mice; Programmed Cell Death 1 Receptor; Proto-Oncogene Proteins B-raf; Sulfonamides; Survival Rate; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tumor Microenvironment; Xenograft Model Antitumor Assays

The interaction of programmed cell death-1 and its ligand is widely studied in cancer. Monoclonal antibodies blocking these molecules have had great success but little is known about them in thyroid cancer. We investigated the role of PD-L1 in thyroid cancer with respect to BRAF mutation and MAP kinase pathway activity and the effect of anti PD-L1 antibody therapy on tumor regression and intra-tumoral immune response alone or in combination with BRAF inhibitor (BRAFi). BRAFV600E cells showed significantly higher baseline expression of PD-L1 at mRNA and protein levels compared to BRAFWT cells. MEK inhibitor treatment resulted in a decrease of PD-L1 expression across all cell lines. BRAFi treatment decreased PD-L1 expression in BRAFV600E cells, but paradoxically increased its expression in BRAFWT cells. BRAFV600E mutated patients samples had a higher level of PD-L1 mRNA compared to BRAFWT (p=0.015). Immunocompetent mice (B6129SF1/J) implanted with syngeneic 3747 BRAFV600E/WT P53-/- murine tumor cells were randomized to control, PLX4720, anti PD-L1 antibody and their combination. In this model of aggressive thyroid cancer, control tumor volume reached 782.3±174.6mm3 at two weeks. The combination dramatically reduced tumor volume to 147.3±60.8, compared to PLX4720 (439.3±188.4 mm3, P=0.023) or PD-L1 antibody (716.7±62.1, P<0.001) alone. Immunohistochemistry analysis revealed intense CD8+ CTL infiltration and cytotoxicity and favorable CD8+:Treg ratio compared to each individual treatment. Our results show anti PD-L1 treatment potentiates the effect of BRAFi on tumor regression and intensifies anti tumor immune response in an immunocompetent model of ATC. Clinical trials of this therapeutic combination may be of benefit in patients with ATC.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; B7-H1 Antigen; Cell Proliferation; Female; Humans; Indoles; Male; Mice; Middle Aged; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms

Tumor microenvironment influences targeted drug therapy. In this study we compared drug responses to RAF and MEK inhibitors on tumor cell migration in 2D and 3D culture of BRAF(V600E) mutant cell lines derived from human papillary (BCPAP) and anaplastic (SW1736) thyroid carcinomas. Scratch wounding was compared to a double-layered collagen gel model developed for analysis of directed tumor cell invasion during prolonged culture. In BCPAP both PLX4720 and U0126 inhibited growth and migration in 2D and decreased tumor cell survival in 3D. In SW1736 drugs had no effect on migration in 2D but decreased invasion in 3D, however this related to reduced growth. Dual inhibition of BRAF(V600E) and MEK reduced but did not prevent SW1736 invasion although rebound phosphorylation of ERK in response to PLX4720 was blocked by U0126. These findings indicate that anti-tumor drug effects in vitro differ depending on culture conditions (2D vs. 3D) and that the invasive features of anaplastic thyroid cancer depend on non-MEK mechanism(s).

Topics: Antineoplastic Agents; Butadienes; Carcinoma; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Survival; Humans; Indoles; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Invasiveness; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Thyroid Carcinoma, Anaplastic

Anaplastic (ATC) and refractory papillary thyroid cancer (PTC) lack effective treatments. Inhibition of either oncogenic BRAF or SRC has marked anti-tumor effects in mouse models of thyroid cancer, however, neither drug induces notable apoptosis. Here we report that the SRC-inhibitor dasatinib further sensitizes BRAFV600E-positive thyroid cancer cells to the BRAFV600E-inhibitor PLX4720. Combined treatment with PLX4720 and dasatinib synergistically inhibited proliferation and reduced migration in PTC and ATC cells. Whereas PLX4720 did not induce robust apoptosis in thyroid cancer cells, combined treatment with dasatinib induced apoptosis in 4 of 6 lines. In an immunocompetent orthotopic mouse model of ATC, combined PLX4720 and dasatinib treatment significantly reduced tumor volume relative to PLX4720 treatment alone. Immune cell infiltration was increased by PLX4720 treatment and this effect was maintained in mice treated with both PLX4720 and dasatinib. Further, combined treatment significantly increased caspase 3 cleavage in vivo relative to control or either treatment alone. In conclusion, combined PLX4720 and dasatinib treatment induces apoptosis, increases immune cell infiltration and reduces tumor volume in a preclinical model of ATC, suggesting that the combination of these FDA-approved drugs may have potential for the treatment of patients with ATC or refractory PTC.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Humans; Indoles; Mice; Phosphorylation; Proto-Oncogene Proteins B-raf; src-Family Kinases; Sulfonamides; Thyroid Carcinoma, Anaplastic

Melanoma antigen gene family (MAGE)-A4, a member of the cancer testis antigen family, has been reported in various cancers including melanoma, bladder, head and neck, oral, and lung, and is a potential target for T-cell-receptor-based immunotherapy. Baseline expression levels of the MAGE-A4 gene in thyroid cancer cell lines have not been previously studied thoroughly.. Human thyroid cancer cell lines (8505c, HTh7, BCPAP, and TPC-1) were treated with either 10 μmol/L 5'-azacytidine (Aza) or 10 μmol/L 5-AZA-2'deoxycytidine (DAC) and evaluated for various MAGEA gene expression. Later melanoma cell lines A375 and 8505c were treated with PLX4720 in combination with DAC and evaluated for MAGE-A4 expression.. Only BCPAP cells expressed moderate levels of MAGE-A3 and MAGE-A6 at baseline. Treatment with DAC/Aza induced the expression of MAGE-A4 and MAGE-A1 in 8505c cells. PLX4720 treatment did not affect MAGE-A4 expression in 8505c cells, but increased its expression in A375 cells. In contrast, addition of PLX4720 to DAC-treated 8505c cells decreased the previously induced MAGE-A4 expression by DAC in these cells. A similar decrease in MAGE-A4 expression by DAC was also seen in 8505cBRAF(-/-) cells. Although DAC treatment resulted in demethylation of the MAGE-A4 promoter in 2 CpG sites, PLX addition to DAC did not affect the demethylation status.. Demethylating agents increased the expression of MAGE genes in thyroid cancer cells. The effect of BRAFV600E inhibitors on MAGE-A4 expression suggest the role of downstream MEK/BRAF signaling in its expression apart from promoter demethylation being the sole requirement. Expression of MAGE-A4 may make immunotherapeutic intervention possible in selected patients with thyroid cancer.

Topics: Antigens, Neoplasm; Azacitidine; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Gene Expression; Humans; Immunotherapy; Indoles; MAP Kinase Signaling System; Melanoma; Neoplasm Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins B-raf; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms

Human anaplastic thyroid cancer (ATC) is a lethal disease with an advanced clinical presentation and median survival of 3 months. The BRAF(V600E) oncoprotein is a potent transforming factor that causes human thyroid cancer cell progression in vitro and in vivo; therefore, we sought to target this oncoprotein in a late intervention model of ATC in vivo. We used the human ATC cell line 8505c, which harbors the BRAF(V600E) and TP53(R248G) mutations. Immunocompromised mice were randomized to receive the selective anti-BRAF(V600E) inhibitor, PLX4720, or vehicle by oral gavage 28 d after tumor implantation, 1 wk before all animals typically die due to widespread metastatic lung disease and neck compressive symptoms in this model. Mice were euthanized weekly to evaluate tumor volume and metastases. Control mice showed progressive tumor growth and lung metastases by 35 d after tumor implantation. At that time, all control mice had large tumors, were cachectic, and were euthanized due to their tumor-related weight loss. PLX4720-treated mice, however, showed a significant decrease in tumor volume and lung metastases in addition to a reversal of tumor-related weight loss. Mouse survival was extended to 49 d in PLX4720-treated animals. PLX4720 treatment inhibited cell cycle progression from 28 d to 49 d in vivo. PLX4720 induces striking tumor regression and reversal of cachexia in an in vivo model of advanced thyroid cancer that harbors the BRAF(V600E) mutation.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Line; Disease Models, Animal; Female; Humans; Indoles; Lung Neoplasms; Mice; Mice, SCID; Neoplasm Invasiveness; Proto-Oncogene Proteins B-raf; Random Allocation; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Weight Loss

B-Raf(V600E) is a frequent mutation in anaplastic thyroid cancers and is a novel therapeutic target. We hypothesized that PLX4720 (an inhibitor of B-Raf(V600E)) and thyroidectomy would extend survival and would decrease tumor burden in a mouse model.. Orthotopic anaplastic thyroid tumors were induced in severe combined immunodeficient mice. Mice were treated with PLX4720 or vehicle after 7 days of tumor growth, and thyroidectomy or sham surgery was performed at day 14. The neck space was re-explored, and tumor volume was measured at day 35. Mice were sacrificed when they lost >25% of their initial weight.. All 5 mice that received the vehicle developed cachexia, had invasive tumors (average 61 mm(3))and were sacrificed by day 35. All 6 mice receiving PLX4720 + sham had small tumors (average 1.3 mm(3)) and maintained their weight. Three out of 6 mice receiving PLX4720+thyroidectomy had no evidence of tumor at 35 days; the other 3 mice had small tumors (average 1.4 mm(3)) and showed no signs of metastatic disease. All mice treated with PLX4720 were alive and well-appearing at 50 days.. Thyroidectomy with neoadjuvant PLX4720 could be an effective therapeutic strategy for early anaplastic thyroid cancers that harbor the B-Raf(V600E) mutation and are refractory to conventional therapeutic modalities.

Topics: Animals; Body Weight; Cell Division; Combined Modality Therapy; Cost of Illness; Disease Models, Animal; Indoles; Mice; Neoadjuvant Therapy; Neoplasm Staging; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins B-raf; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Thyroidectomy