PLCG2 exon-skipped variants: insights into their potential role in chronic lymphocytic leukemia

Area of Science:

• Hematology
• Oncology
• Molecular Biology

Background:

• Bruton tyrosine kinase inhibitors (BTKi) are effective treatments for chronic lymphocytic leukemia (CLL).
• Acquired resistance to BTKi is a significant clinical challenge in CLL management.
• Understanding resistance mechanisms is crucial for improving patient outcomes.

Purpose of the Study:

• To investigate novel genetic alterations in Phospholipase C gamma 2 (PLCG2) associated with BTKi resistance in CLL.
• To characterize the functional impact of identified PLCG2 variants on cellular activity and drug sensitivity.
• To explore the prevalence and clinical relevance of these variants in CLL patient samples.

Main Methods:

• Long-term treatment of REC-1 CLL cells with tirabrutinib to induce resistance.
• Sanger sequencing and deletion analysis to identify genetic alterations in PLCG2.
• Analysis of alternative splicing variants using RT-PCR and sequencing.
• Assessment of phospholipase activity and anti-IgM-mediated calcium flux.
• Expression analysis of PLCG2 variants in primary CLL samples from patients before and after targeted therapy.

Main Results:

• A splice-site mutation (c.2236-1G>T) and a splice-site deletion (c.2236-23_2238) in PLCG2 were identified in BTKi-resistant CLL cells and patient samples, leading to the exon-skipped variant ∆21.
• The ∆21 variant exhibited enhanced phospholipase activity, increased calcium flux, and resistance to BTK inhibition, while remaining sensitive to PLCγ inhibitors.
• Widespread expression of other exon-skipped variants (∆22, ∆20-22) was observed in CLL, with ∆20-22 being more prevalent than in healthy donors.
• Expression of ∆20-22 decreased after venetoclax treatment but remained unchanged after ibrutinib treatment, suggesting differential drug selection pressure.

Conclusions:

• Novel PLCG2 splice-site alterations and exon-skipped variants contribute to BTKi resistance in CLL, independent of classical BTK or PLCG2 mutations.
• These findings highlight the potential of targeting PLCγ or related pathways for managing BTKi-resistant CLL.
• Understanding these resistance mechanisms can inform future treatment strategies and patient selection for targeted therapies.