Crosstalk between CEP72 germline polymorphism and NOTCH1 somatic mutations alters the sensitivity of leukemia cells and normal T cells to vincristine, influencing the prognosis of pediatric T-ALL

Abstract

Abstract Background: Vincristine is one of the most important agents for pediatric acute lymphoblastic leukemia (ALL), but its extended use in maintenance therapy didn't offer additional benefit. Findings of the CCCG-ALL-2015 trial, comparing shortened vincristine and dexamethasone duration in later maintenance with standard maintenance, confirmed this view. As CEP72 SNP rs924607 C>T potentially increasing the cytotoxicity of Vincristine against leukemia cells. We analyzed its influence on prognosis of CCCG-ALL-2015 cohort. Methods: This study included 945 pediatric ALL cases with enough bio-samples from Shanghai Children's Medical Center and the Second Affiliated Hospital of Anhui Medical University treated according to CCCG-ALL-2015 protocol as the discovery cohort. While, additional 100 T-ALL cases from the SCCCG-ALL-2016 cohort of the South China Children's Cancer Group were included as the validation cohort. PCR-Sanger sequencing was performed to genotype the CEP72 rs924607 locus and NOTCH1 among cases. Survival and relapse were estimated using the Kaplan-Meier method and compared with the log-rank test. CEP72 knocking down, γ secretase DAPT and NICD was overexpression in T-ALL cell line Jurkat and B-ALL cell lines Nalm-6 and Reh were applied to study CEP72, NOTCH1, Vincristine and T cell cytotoxicity and their functional interaction. Results: Consistent with previous whole cohort reports, vincristine duration reduction showed no impact on outcomes in our 945-case sub-cohort (B-ALL: N=812; T-ALL: N=133), stratified by risk category (LR: N=459; IR: N=465). Furthermore, CEP72 genotype similarly lacked prognostic significance across risk groups or randomized arms, while immunotype-specific analyses revealed striking divergence: B-ALL patients with mutant genotypes (TT/CT,N=444) exhibited superior EFS/CIR versus wild-type (CC, N=368), particularly in the IR subgroup (mutant: N=180 vs wild-type: N=161; HR for EFS=0.63, 95% CI: 0.43-0.94; p=0.02). While in T-ALL patients, those with the CEP72 mutant genotype (N=86) showed inferior outcomes compared to wild-type cases (N=47) including EFS (HR=2.84, 95% CI: 1.47-5.48, P=0.0092), CIR, and OS. Stratified by randomized arms, patients with standard maintenance who had mutant CEP72 showed the poorest outcomes, while patients treated by shortened vincristine, the CEP72 genotype did not affect patient outcomes. Validation in the independent SCCCG-ALL-2016 cohort treated by standard maintenance confirmed the adverse prognostic impact of CEP72 mutations (EFS HR=2.4, 95% CI: 1.06-5.43; p=0.034). Unable to explain CEP72 deficiency on the outcome of T-ALL by sensitivity of leukemia cells to vincristine, we hypothesized that CEP72-deficient immune cells might be more susceptible to vincristine. To test this, we used shRNA to interfere CEP72 expression in normal T cells and found that the blinatumomab dependent or independent cytotoxicity against Reh and Nalm-6 were significantly reduced. However, after knocking down CEP72 expression in the T-ALL cell line Jurkat and the B-ALL cell lines Nalm-6 and Reh, unlike the increased sensitivity of Nalm-6 and Reh to vincristine, Jurkat did not show increased sensitivity to vincristine. Since NOTCH1 activate mutations are the most common drivers in T-ALL, we supposed that NOTCH1 alterations might modulate vincristine response in leukemia cells, while normal T cells without NOTCH1 mutation remained affected by the CEP72 genotype. As we expected, standard maintenance treated patients, both in CCCG-ALL-2015 and SCCCG-ALL-2016 cohort, simultaneous harboring CEP72 loss-of-function and NOTCH1 mutations had the worst prognosis. In vitro experiments confirmed this interaction: γ-secretase inhibitor (DAPT) treatment increased vincristine sensitivity in NOTCH1-mutated Jurkat cells, whereas NICD overexpression conferred resistance in NOTCH1-wild-type B-ALL lines (Nalm-6/Reh). Conclusions: T-ALL with NOTCH1 mutations exhibit increased resistance to vincristine, while normal T cells remain influenced by the CEP72 genotype. As a result, T-ALL patients with somatic NOTCH1 mutations who carry the germline loss-of-function polymorphism rs924607 experience worse prognosis when treated with more vincristine. This phenomenon reveals the crosstalk between somatic mutations and germline variants, as well as between chemotherapy and the immune microenvironment, collectively impacts T-ALL prognosis.