Screening and Validation of Natural Product with Anti ETP-ALL Activity Using a Novel ETP-ALL Cell Line

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) was first identified in 2009 and was classified as a distinct subtype of T-cell acute lymphoblastic leukemia (T-ALL) by the WHO in 2016. Patients with ETP-ALL frequently face higher rates of relapse and treatment resistance, leading to poorer outcomes compared to individuals with other forms of T-ALL. As of now, there are no established treatment guidelines specifically for ETP-ALL.

Method: The ETP-ALL cell line was established by using continuous culture methods. Cell proliferation inhibition experiments were applied to screen the inhibitory effects of 2541 natural products from the Selleck natural product library on ETP-ALL cells. This screening occurred in three rounds with concentrations set at 1,000 nM, 100 nM, and 10 nM. Natural products that achieved an inhibitory concentration of ≥50% were advanced through subsequent rounds. Finally, natural products with an inhibition rate of ≥ 50% at a concentration of 10nM were selected for validation. In the final validation stage, we focused on products that demonstrated an inhibition rate of ≥50% at a concentration of 10 nM to confirm their efficacy against ETP-ALL.

Results: The novel ETP-ALL cell line, named ZYXY-T1, was successfully established from an ETP-ALL patient. ZYXY-T1 exhibited robust proliferation both in vitro and in vivo. The results from Flow cytometry analysis confirmed that ZYXY-T1 presents the typical characteristics of ETP-ALL, showing negative expression for CD1a, CD8, and CD5, along with positive expression for CD34, CD13, and CD33. Based on multiple diagnostic scoring systems, ZYXY-T1 met the criteria for ETP-ALL diagnosis, achieving maximum scores in four out of six scoring systems. In contrast, the Loucy cell line failed to meet ETP-ALL diagnostic criteria. RNA sequencing analyses identified six fusion genes in ZYXY-T1, including MLLT10::PICALM, AC068756.1::AC108690.1, PICALM::MLLT10, PCAT18::KCTD1, LINC02284::TMEM260, AC123912.1::ZNF493.

In the initial screening, 76 natural products demonstrated a half-maximal inhibitory concentration (IC50) of ≤1,000 nM against ETP-ALL. Following a second round of screening, 38 products exhibited IC 50 values below 100 nM. After the third round, 13 natural products were identified with IC50 values under 10 nM. Among these, natural product #1823 exhibited exceptional efficacy against ETP-ALL, with an IC50 of 0.2 nM-at least ten times lower than the other 12 compounds tested .

Conclusions: We successfully established a novel ETP-ALL cell line that reflects the typical ETP-ALL phenotype. Leveraging this cell line, we identified a potent natural product with significant anti-ETP-ALL activity through systematic screening of a natural product library.

No relevant conflicts of interest to declare.