KC-4715

TMD8-BTK-T474I-KI-1A1 Cell Line

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Background of TMD8-BTK-T474I-KI-1A1 Cell Line

Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor (BCR) signaling pathway, playing a pivotal role in B-cell development, differentiation, and survival. Due to its central role in B-cell malignancies, BTK has become a key therapeutic target, with inhibitors like ibrutinib and acalabrutinib showing significant clinical efficacy in treating chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and other B-cell disorders. However, the emergence of resistance mutations, such as BTK-T474I, poses a significant challenge to the long-term effectiveness of these therapies. The BTK-T474I mutation is a point mutation in the BTK gene, where threonine at position 474 is replaced by isoleucine. This mutation is located in the kinase domain of BTK, a region critical for the binding of covalent BTK inhibitors. The substitution of threonine with isoleucine alters the steric and electronic properties of the binding pocket, reducing the affinity of covalent inhibitors like ibrutinib. As a result, patients harboring this mutation often exhibit resistance to first-generation BTK inhibitors, leading to disease progression. Understanding the structural and functional implications of the BTK-T474I mutation is essential for developing next-generation BTK inhibitors that can overcome resistance. Structural studies have shown that the T474I mutation disrupts the formation of a hydrogen bond between BTK and ibrutinib, which is crucial for the inhibitor's covalent binding. This insight has guided the design of non-covalent BTK inhibitors, such as pirtobrutinib (LOXO-305), which can effectively target both wild-type and mutant forms of BTK, including T474I. The clinical significance of BTK-T474I underscores the need for ongoing research into resistance mechanisms and the development of novel therapeutic strategies. By elucidating the molecular basis of resistance and designing inhibitors that can circumvent these challenges, researchers aim to improve outcomes for patients with B-cell malignancies.

Specifications

Catalog Number	KC-4715
Cell Line Name	TMD8-BTK-T474I-KI-1A1 Cell Line
Host Cell Line	TMD8
Description	Stable TMD8 clone expressing endogenous BTK gene bearing T474I mutations, No.1A1
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	Lymphoblast
Subculture	Split saturated culture 1:3-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

TMD8-BTK-T474I-KI-1A1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-BTK-T474I-KI-1A1 cell line stable clone using PCR sequencing..

Figure 2: Characterization of TMD8-BTK-T474I-KI-1A1 cell line stable clone using RT-PCR sequencing..

Figure 3: Characterization of dose-response curves for BTK inhibitors on TMD8 and TMD8-BTK-T474I-KI-1A1 cells.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:3-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Woyach, J. A., et al. (2014). Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib. New England Journal of Medicine, 370(24), 2286-2294.
Wang, E., et al. (2019). Mechanisms of resistance to noncovalent Bruton's tyrosine kinase inhibitors. Blood Advances, 3(15), 2316-2325.
Wu, J., et al. (2021). Structural basis of BTK inhibition by pirtobrutinib (LOXO-305) and its potential to overcome resistance mutations. Journal of Medicinal Chemistry, 64(12), 8110-8123.