BH3 mimetics and notch inhibitors as a dual strategy in T-cell acute lymphoblastic leukemia (T-ALL)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoproliferative disorder caused by the malignant transformation of T-cell progenitors. Rare NOTCH3 mutations, but frequent NOTCH1-activating mutations and the overexpression of NOTCH3 in over 60% of cases, constitutively activate the Notch signaling pathway, promoting leukemic cell proliferation and survival. Another mechanism involved in disease progression is the upregulation of anti-apoptotic BCL2 family proteins, which helps cells evade programmed cell death. As a result, Notch and BCL2 family proteins have emerged as strategic targets for the development of new therapeutic strategies. Notch inhibition with γ-secretase inhibitors (GSIs) has a limited application due to severe gastrointestinal toxicity. Navitoclax (ABT-263), a pan-BCL-2 inhibitor, has been shown to cause dose-dependent thrombocytopenia. This has led to the development of Venetoclax (ABT-199), a more effective, yet selective, BCL-2 inhibitor. This study aims to evaluate the effects of two new experimental drugs: IS21, a pan-inhibitor of anti-apoptotic BCL2 family proteins, and CAD204520, a SERCA pump inhibitor that has been described so far as selectively targeting mutant Notch1. We tested IS21 on human Notch1- (Jurkat, Molt3), Notch3-dependent (TALL1) and Notch-independent (Loucy) T-ALL cell lines and a murine cell line, N3-232T, derived from our T-ALL mice model overexpressing the intracellular active form of Notch3 (N3-ICtg). Our results showed a dose dependent reduction in cell viability (5–20 μM) in Loucy, TALL1, Molt3, and 232 cells at 48 hours. When combined with standard chemotherapeutic agents (doxorubicin, vincristine), IS21 at a lower concentration (10μM) enhanced their efficacy, overcoming Jurkat cell drug resistance. In parallel, the effect of CAD204520 was evaluated on human cell lines (2.5–10 μM) and murine 232 cells (2–6 μM), showing decreased cell viability, increased apoptosis, and G1 cell cycle arrest at 72 hours. Dual targeting with SERCA inhibitors and the FDA-approved ABT-199 has already been investigated in chronic lymphocytic leukemia (CLL) patient samples harboring Notch1 mutations; In our future studies, we plan to evaluate the individual and then the combined effects of CAD204520 with ABT 199 or IS21 in vitro in human and murine T-ALL cell lines and then in vivo on our N3-ICtg mice. Outcomes from these experiments may extend this study to Early T-cell Precursor-ALL (ETP-ALL), a particularly aggressive T-ALL subtype marked by elevated BCL2 expression, as well as other Notch3-dependent tumor contexts. We hypothesize that this strategy could represent an innovative approach to improve therapeutic outcomes in refractory T-ALL