Brachyury Evidence Summary

Brachyury in Chordoma

Nuclear Brachyury (T) expression has emerged as a “sensitive and fairly specific” diagnostic marker for chordoma.¹ A growing body of literature suggests that it contributes significantly to chordoma pathogenesis. With its known role in other cancers and high expression in chordomas, the T gene has risen to the forefront of chordoma therapeutic targets. This page contains a summary of published research investigating the role of Brachyury in chordoma and evaluating its inhibition as a method for treating the disease.

• Molecular Evidence
• Preclinical Evidence
• Clinical Evidence
• Open Clinical Trials

Molecular Evidence

Copy Number Variation

• Chromosome 6 gains and partial polysomies are common chromosomal aberrations observed in chordoma tumors and cell lines.^{2 3 4 5 6}
• The brachyury gene shows minor allelic gains and is amplified in some sporadic chordomas.^{5 6 7 8 9}
• Somatic copy number gain of brachyury was found in 12/27 (44%) patients and positively correlated with Brachyury expression. This suggests copy number alteration is a mechanism driving over expression of brachyury in some patients.¹⁰
• Copy number gain of brachyury was preserved in a patient-derived xenograft (PDX) model of clival chordoma and was found to increase through 3 generations, highlighting the importance of brachyury expression in chordoma tumor maintenance and biology.¹¹
• Amplification of ETS2, an upstream regulator of brachyury, has also been detected in a number of chordomas.⁸

Germline and Somatic Mutations

• Germline tandem duplication of the Brachyury gene is associated with familial chordoma^12 13 and was identified by Tarpey et al. in 3/11 sporadic chordoma cases by whole genome sequencing and 6/28 sporadic chordomas by targeted sequencing.¹⁴
• The common nonsynonymous SNP rs2305089 is strongly associated with chordoma risk and has been found in all familial chordomas analyzed.^13 15 The SNP is not associated with the T-isoform ratio in chordoma cells, indicating that it is not involved in alternative splicing.¹⁶ rs2305089 has also been found in a number of non-familial chordomas and has prognostic significance for overall survival.^17 18 19
• Also associated with chordoma risk in familiar cases is the common SNP rs1056048. In sporadic cases, rs3816300 is significantly associated with risk when evaluated in combination with rs2305089 (discussed above).^13 15
• No mutations have been found to account for differential expression of brachyury in chordomas.⁸

Gene Expression

• Brachyury is highly expressed in chordomas but not in other mesenchymal neoplasms or in a wide variety of normal tissues (with the exception of minor expression in the testes and thyroid).^{20 21 22 23 24}
• Brachyury mRNA levels are high in all cells in nearly every chordoma tumor regardless of the status of chromosome 6 or tumor location.^2 25
• Increased expression of brachyury mRNA is associated with shorter progression-free survival.¹⁰
• A 2015 study found that the long isoform of the T-gene was the dominant form expressed by chordoma tumor samples and cell lines, and that the long to short isoform ratio was significantly different in chordoma samples versus notochord controls.¹⁶
• The microRNA miR-219-5p directly targets the 3′ UTR of brachyury mRNA and functions as a tumor suppressor in chordoma. miR-219-5p is downregulated in chordoma patients, resulting in increased expression of brachyury, and its expression is correlated with tumor extent and recurrence.²⁶
• Transcriptome microarray analysis of 4 tumors with high brachyury expression versus 4 tumors with low brachyury expression found that the PI3K/AKT signaling pathway is significantly upregulated when brachyury is overexpressed.¹⁰

Protein Expression

• Brachyury protein expression is detected in virtually all chordoma tumors and cell lines evaluated.^{10 27 28} Expression is detected in not just conventional but also in poorly differentiated chordomas and miR-219-5p has been shown to regulate protein expression of brachyury. Multiple studies have found that protein expression level is not associated with tumor recurrence, suggesting that it is a sensitive marker for chordoma rather than a prognostic factor.²⁹
• Brachyury is also expressed in benign notochord cell tumor (BNCT), which is regarded as a benign counterpart of chordoma. It has been shown that at least some BNCTs are precursors of chordoma.³⁰
• Fetal notochordal cell rests (NCRs), also known as benign notochordal cell tumors (BNCT), from which chordoma cells are hypothesized to arise, have not been found to expression brachyury. Expression is absent even in NCRs that coexist with chordoma tumor components (which themselves show high nuclear brachyury expression).³¹
• Brachyury regulates the expression and activity of the YAP protein to control stemness and proliferation in chordoma. YAP is a key regulator of tissue growth and homeostasis and its transcription is directly activated by brachyury. Chordoma tissues and cell lines have a positive correlation between brachyury and YAP protein levels and these levels correlate with downstream targets of YAP such as MCL-1, cyclin D1, and CTGF.³²

Preclinical Evidence

In-vitro Efficacy

• shRNA-mediated brachyury knockdown in chordoma cell lines induces growth arrest and apoptosis, decreases cell proliferation capacity, and leads cells to exhibit a more differentiated morphology.^{2 5 10 32 33 34}
• shRNA-mediated brachyury knockdown coupled with administration of FGF2 led to reduced FGF secretion and inhibited phosphorylation of FRS2 (FGFR2 substrate), reduced MEK/ERK phosphorylation, and blocked the effects of FGF2 on caspase 3 activity, DNA fragmentation, and cell growth, suggesting that brachyury is necessary for FGFR/MEK/Erk-mediated chordoma cell growth and survival. It also blocked the effects of FGF on EMT by upregulating e-cadherin expression and downregulating snail and slug expression, suggesting that brachyury mediated the FGF-driven EMT in chordoma cells.³⁵
• shRNA-mediated brachyury knockdown in chordoma cell lines resulted in a dramatic decrease in the expression of YAP protein and mRNA along with the YAP targets MCL-1, cyclin D1, CTGF, CYR61, ANKRD1 and c-MYC.³²
• shRNA-mediated brachyury knockdown in the chordoma cell line JHC7 resulted in a significant decrease in the mRNA levels of stemness-related genes including ABCG2, OCT4, SOX2, and ABCB1.³²
• shRNA-mediated brachyury knockdown in two primary chordoma cell lines resulted in the cell lines becoming sensitive to Paclitaxel-induced apoptosis. Brachyury is believed to confer chemoresistance in part by upregulating CA9 expression, which is known to reduce the chemosensitivity of various malignancies by disturbing the tumor microenvironment.³⁶
• Extreme limiting dilution assays in JHC7 cells showed a significant reduction in self-renewing capacity and stem cell frequency in cells with shRNA-mediated brachyury knockdown compared to control cells.³²
• Afatinib: Afatinib was found to promote degradation of brachyury and EGFR protein, both of which are crucial to chordoma cell growth.³⁴
• GSK-J4 and KDOBA67: GSK-J4 and KDOBA67 mainly target KDM6A (also known as UTX) and KMD6B (also known as JMJD3). These compounds induced downregulation of T at the transcriptional and protein level in 5/5 cell lines tested.³⁷
• Brachyury was the top hit in CRISPR-Cas9 loss-of-function screens in two chordoma cell lines (UM-Chor1, MUG-Chor1) and sgRNA-mediated brachyury repression reduced cell proliferation in UM-Chor1, MUG-Chor1, and U-CH2 cell lines.³⁸
• Brachyury expression was highest among all super-enhancer-associated transcription factors in chordoma cell lines and brachyury-associated super-enhancers were also observed in patient-derived chordoma tumors.³⁸
• The CDK7/12/13 inhibitor THZ1 reduced cellular brachyury protein levels in MUG-Chor1 and UM-Chor1 cells in a concentration- and time-dependent manner. THZ1 treatment also substantially reduced mRNA expression of brachyury.³⁸

In-vivo Efficacy

• A recent study by Zhu et al. using shRNA-mediated brachyury knockdown in transgenic mouse models found that, although brachyury knockdown in notochord precursor cells resulted in vertebral malformation, loss did not prevent notochordal progenitors from proliferating or from taking on invasive characteristics. Cells still proliferated at a high rate and had features of aggressive tumor cells. The authors suggest that their findings call for further studies to understand whether targeting brachyury is effective in reducing aggressive tumor behaviors, despite chordomas’ notochordal origins.³⁹
• 5/6 mice injected with JHC7 chordoma cells developed tumors within 1 year compared to 0/6 mice injected with JHC7 cells in which brachyury expression had been decreased via shRNA.³²
• A xenograft mouse model of the human chordoma cell line CH22 was sensitive to transcriptional CDK inhibitors and compound treatment induced downregulation of brachyury expression. THZ1 treatment led to a significant tumor-suppressive effect.³⁸

Clinical Evidence

Phase I Trial

• GI-6031 (heat-killed yeast vaccine): Among the 10 evaluable chordoma patients enrolled in this dose-escalation trial, median progression-free survival was 253 days. Six receiving highest dose levels exhibited immune responses. Two showed evidence of disease control, and five of the remaining eight showed evidence of clinical benefit after 5 months.⁴⁰

Open Clinical Trials

The following clinical trials have been launched to explore whether targeting brachyury is effective in treating chordoma patients. Visit our Clinical Trials page to view a list of other clinical trials available to chordoma patients and to find out who to contact if you wish to participate.