Periodic Reporting for period 1 - SpinSAC (hSpindly, a new regulatory protein of the SAC mechanism and its implication in tumour therapy)
Período documentado: 2021-09-01 hasta 2024-08-31
During mitosis a surveillance system, known as the spindle assembly checkpoint (SAC), is crucial to make sure that the cell does not separate its sister chromatids until all the chromosomes are correctly attached to spindle microtubules and bi-oriented. Nowadays, one of the more important strategy used for the cancer treatment is the inhibition of the microtubule dynamic to activate the SAC by the addition of antimitotic drugs. At the end, the activation of SAC for a long period of time would provoke the cell death. However, many tumor cells exhibit defects in SAC functionality, leading chromosomal instability (CIN) and resistance to the treatment. For this reason, it is becoming extremely useful to seek new proteins which are implicated directly to SAC functionality. This project has been focused on the study of hSpindly, a protein whose role in SAC regulation had not been clearly defined. Preliminary evidence suggested that hSpindly may participate in SAC activation through a mechanism independent of the stripping pathway. However, the exact mode of action remained completely unknown at the start of this fellowship. Since hSpindly is highly phosphorylated in mitosis, we hypothesized that its phosphorylation pattern could play a critical role in modulating its function.
The overall objectives of the project were:
• To identify the phosphorylation pattern of hSpindly during mitosis and production of point mutants.
• To analyze the functions of the hSpindly phosphorylations in mitosis and the role of these phosphorylations in the cell response to anti-mitotic drugs.
This project has revealed the regulatory role of hSpindly in the SAC pathway and identified threonine 552 as a critical phosphorylation site affecting its function. These findings enhance our understanding of drug resistance in cancer and pave the way for the development of personalized therapies, new biomarkers, and targeted treatments with relevance for both research and clinical applications.
The overall objectives of the project were:
• To identify the phosphorylation pattern of hSpindly during mitosis and production of point mutants.
• To analyze the functions of the hSpindly phosphorylations in mitosis and the role of these phosphorylations in the cell response to anti-mitotic drugs.
This project has revealed the regulatory role of hSpindly in the SAC pathway and identified threonine 552 as a critical phosphorylation site affecting its function. These findings enhance our understanding of drug resistance in cancer and pave the way for the development of personalized therapies, new biomarkers, and targeted treatments with relevance for both research and clinical applications.
From the beginning of the project in September 2021 until its completion in February 2025, the research has successfully achieved its primary objectives and contributed important findings to the field of mitotic regulation and cancer biology. The work has been focused on characterizing the role of hSpindly protein in the regulation of the Spindly Assembly Checkpoint (SAC) and evaluating its impact on the response of tumor cells to anti-mitotic drugs.
The project began with the identification of phosphorylation sites on hSpindly during mitosis using mass spectrometry. This was followed by the generation of inducible RPE-1 cell lines expressing wild-type and phospho-mutant forms of hSpindly fused to GFP, using the Flp-In T-REx system. These lines allowed precise control over protein expression and were central to all subsequent experiments.
Functional assays confirmed that hSpindly is a positive regulator of SAC activity, capable of recruiting Mad2 to kinetochores even in the absence of canonical scaffold proteins like Bub1 and KNL1. This revealed a novel RZZ-dependent pathway for SAC activation, previously poorly understood. Overexpression of hSpindly was shown to enhance checkpoint activity and sensitize cells to anti-mitotic drugs.
Among several candidate phosphorylation sites, threonine 552 (T552) emerged as critical for SAC function. Mutation of this residue (T552A) revealed major alterations in hSpindly's behavior: reduced kinetochore mobility, loss of oligomerization capacity (confirmed via N&B analysis), and increased resistance to anti-mitotic drugs. These findings strongly suggest that T552 phosphorylation is essential for hSpindly's function in SAC regulation and drug sensitivity.
Therefore, the key achievements and results were:
• hSpindly defined as a novel component of SAC, with the ability to regulate checkpoint signaling independently of the Bub1/KNL1 axis and the cellular response to anti-mitotic drugs.
• Threonine 552 identified as a critical phosphorylation site regulating hSpindly dynamics and SAC functionality.
• Integration of advanced microscopy techniques (RISC, N&B, confocal live-cell imaging) to study protein dynamics in real time.
• The relevance of hSpindly in the cellular response to anti-mitotic drugs.
The project´s results have been disseminated through presentations at international conferences, departmental seminars and sessions with Master’s students at the University of Seville, formal meeting with clinical research groups and several outreach activities.
The results obtained during this project provide a solid foundation for future translational research and commercial applications. The identification of hSpindly as a novel regulator of the Spindle Assembly Checkpoint (SAC) and the discovery that phosphorylation at threonine 552 is critical for its function open new opportunities for the development of diagnostic tools (such as a phospho-specific antibody) and targeted therapies for cancer.
This exploitation strategy aligns with the EU’s objectives to foster innovation in the health sector, promote personalized medicine, and reinforce the competitiveness of the European research and biotech landscape.
The project began with the identification of phosphorylation sites on hSpindly during mitosis using mass spectrometry. This was followed by the generation of inducible RPE-1 cell lines expressing wild-type and phospho-mutant forms of hSpindly fused to GFP, using the Flp-In T-REx system. These lines allowed precise control over protein expression and were central to all subsequent experiments.
Functional assays confirmed that hSpindly is a positive regulator of SAC activity, capable of recruiting Mad2 to kinetochores even in the absence of canonical scaffold proteins like Bub1 and KNL1. This revealed a novel RZZ-dependent pathway for SAC activation, previously poorly understood. Overexpression of hSpindly was shown to enhance checkpoint activity and sensitize cells to anti-mitotic drugs.
Among several candidate phosphorylation sites, threonine 552 (T552) emerged as critical for SAC function. Mutation of this residue (T552A) revealed major alterations in hSpindly's behavior: reduced kinetochore mobility, loss of oligomerization capacity (confirmed via N&B analysis), and increased resistance to anti-mitotic drugs. These findings strongly suggest that T552 phosphorylation is essential for hSpindly's function in SAC regulation and drug sensitivity.
Therefore, the key achievements and results were:
• hSpindly defined as a novel component of SAC, with the ability to regulate checkpoint signaling independently of the Bub1/KNL1 axis and the cellular response to anti-mitotic drugs.
• Threonine 552 identified as a critical phosphorylation site regulating hSpindly dynamics and SAC functionality.
• Integration of advanced microscopy techniques (RISC, N&B, confocal live-cell imaging) to study protein dynamics in real time.
• The relevance of hSpindly in the cellular response to anti-mitotic drugs.
The project´s results have been disseminated through presentations at international conferences, departmental seminars and sessions with Master’s students at the University of Seville, formal meeting with clinical research groups and several outreach activities.
The results obtained during this project provide a solid foundation for future translational research and commercial applications. The identification of hSpindly as a novel regulator of the Spindle Assembly Checkpoint (SAC) and the discovery that phosphorylation at threonine 552 is critical for its function open new opportunities for the development of diagnostic tools (such as a phospho-specific antibody) and targeted therapies for cancer.
This exploitation strategy aligns with the EU’s objectives to foster innovation in the health sector, promote personalized medicine, and reinforce the competitiveness of the European research and biotech landscape.
This project has significantly advanced the current understanding of Spindle Assembly Checkpoint (SAC) regulation, particularly by uncovering the previously unknown role of hSpindly as a key modulator of this mechanism. Prior to this work, the involvement of hSpindly in SAC activation was unclear and largely unexplored. The identification of hSpindly as a positive regulator of SAC, capable of recruiting SAC components like Mad2 to kinetochores independently of the canonical Bub1/KNL1 pathway, represents a notable advance beyond the state of the art. Even more, the alteration of a single phosphorylation site on hSpindly is sufficient to affect the cellular response to anti-mitotic treatments. These findings not only reveal a new layer of SAC regulation but also identify a novel biomarker for cancers that display resistance to mitotic inhibitors.
The project is expected to have a strong future impact by enabling the publication of high-impact scientific findings, validating a phospho-specific antibody as a potential diagnostic tool, and advancing cell models for preclinical testing. Additionally, the groundwork laid during the fellowship will support the creation of a spin-off company focused on developing personalized cancer therapies based on SAC regulation.
From a socio-economic perspective, this project addresses a critical challenge in oncology: the resistance of tumors to standard therapies. By providing tools to better predict therapeutic response and identify new molecular targets, the research contributes to improving cancer treatment efficiency, reducing the use of ineffective drugs, and minimizing side effects for patients.
This aligns closely with European healthcare goals under initiatives such as Europe’s Beating Cancer Plan, and supports the move towards personalized medicine, which is expected to reshape healthcare systems and reduce long-term treatment costs.
In conclusion, the project not only pushes the boundaries of scientific knowledge in cell cycle and cancer research but also lays the foundation for future translational and clinical applications with tangible societal and economic benefits.
The project is expected to have a strong future impact by enabling the publication of high-impact scientific findings, validating a phospho-specific antibody as a potential diagnostic tool, and advancing cell models for preclinical testing. Additionally, the groundwork laid during the fellowship will support the creation of a spin-off company focused on developing personalized cancer therapies based on SAC regulation.
From a socio-economic perspective, this project addresses a critical challenge in oncology: the resistance of tumors to standard therapies. By providing tools to better predict therapeutic response and identify new molecular targets, the research contributes to improving cancer treatment efficiency, reducing the use of ineffective drugs, and minimizing side effects for patients.
This aligns closely with European healthcare goals under initiatives such as Europe’s Beating Cancer Plan, and supports the move towards personalized medicine, which is expected to reshape healthcare systems and reduce long-term treatment costs.
In conclusion, the project not only pushes the boundaries of scientific knowledge in cell cycle and cancer research but also lays the foundation for future translational and clinical applications with tangible societal and economic benefits.