Acute T-cell lymphoblastic leukemia (T-ALL) is a haematological disease caused by malignant transformation of T-cell precursors, common in children but also affecting adults. T-cell development relies on Notch signaling, guiding lymphoid progenitors into thymocytes and mature T-cells. Activating Notch1 mutations and/or Notch3 overexpression are frequent in T-ALL, making Notch a key therapeutic target. Previous γ-secretase inhibitors (GSIs) blocked N-ICD release but caused gastrointestinal toxicity due to off-target effects on wild-type Notch. Since mutations often affect the PEST domain, reducing N-ICD degradation, new strategies focus on mutated receptors. SERCA inhibitors, like Thapsigargin, disrupt Ca2+ homeostasis to suppress Notch signaling, but with high toxicity. However, CAD204520, a new SERCA inhibitor, selectively targets mutant Notch1, inhibiting T-ALL growth with lower off-target effects, representing a promising therapeutic advancement. SERCA inhibitors disrupt Notch folding and cause ER/Golgi accumulation, reducing full-length Notch1 at the cell surface and its access to γ-secretase complex.

Human T-ALL cell lines with different Notch genetic background (NOTCH3-mutated and NOTCH wild type) and the murine cell line derived from the thymus of NOTCH3 transgenic mice (N3-232 T-cells) will be treated with CAD204520 (2.5-10 μM for human cell lines and 2-6 μM for murine one) to assess dose-dependent effects on viability (measured with Trypan Blue), apoptosis (through Annexin-V/Propidium-Iodide staining), and cell cycle progression (with PI staining) after 72 hours of treatment. Notch signaling inhibition will be confirmed through Western Blot analysis and qRT-PCR. Finally, we will analyse the effect of CAD204520 on Notch3 membrane expression.

Studies on T-ALL cell lines show dose-dependent sensitivity to CAD204520, with 50% viability reduction at concentrations between 5-7.5 μM in human cells and 4-5 μM in NOTCH3 transgenic murine N3-232 T-cells. The drug induces G1 arrest and increases the apoptotic rate in a concentration-dependent manner. Initially, designed to target Notch1 PEST mutations, CAD204520 also targets Notch3, reducing the receptor protein levels and its presence on the plasma membrane as drug concentration increases.

Compared to previous SERCA inhibitors, CAD204520 demonstrates lower off-target effects, offering a safer and more targeted strategy for treating Notch-driven T-ALL. These findings highlight CAD204520 as a valuable candidate for future preclinical and clinical investigations. Future perspectives include testing the drug in Notch3-overexpressing mice, a T-cell leukaemia model, to assess disease progression and confirm the reduction of side effects.