ALL-043: Selective Blockade of Oncogenic NOTCH1 with the New SERCA Inhibitor CAD204520 in T-cell Acute Lymphoblastic Leukemia

Abstract

The discovery of the P-type ATPase sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggests an innovative approach for treating T-cell acute lymphoblastic leukemia (T-ALL). In fact, SERCA inhibition preferentially affects the maturation and activity of the most common class of oncogenic NOTCH1 mutants. The aim of this study was to identify inhibitors with better drug-like properties and reduced off-target toxicity for SERCA inhibition. We developed a novel oral SERCA inhibitor, CAD204520, through medicinal chemistry optimization and crystal structure-oriented analysis describing its anti-leukemic effects in vitro and in vivo to support a SERCA-based therapeutic modality in T-ALL. From a 191,000 small-molecule screening targeting P-type ATPase, we identified CAD204520 as a selective inhibitor of human SERCA compared to Na+/K+ and H+-ATPase. Crystal structure analysis showed that CAD204520 binds the transmembrane interface of SERCA between helices M1, M2, M3, and M4. CAD204520 minimally alters Ca2+ shift and fails to trigger Ca2+-dependent programs, such as the unfolded protein response. We demonstrated that CAD204520 impairs the proliferation of T-ALL cell lines carrying activating mutations of NOTCH1. Importantly, clinical samples carrying NOTCH1 mutations, including PEST deletions, were more sensitive to CAD204520 compared to normal lymphocytes or wild-type NOTCH1 ALL cells. Mechanistically, CAD204520 treatment reduces the levels of the activated form of NOTCH1 as a consequence of a defect in NOTCH1 trafficking. Next, we demonstrated that somatic hotspot mutations in the SERCA2 ATPase pocket that confer resistance to known SERCA modulators (e.g., thapsigargin) do not interfere with CAD204520 binding, suggesting that the activity of CAD204520 will be unlikely affected by recurrent resistant genetic variants. Finally, we showed that 30 mg/kg BID for 21 days is well-tolerated in vivo in CD1 mice without causing loss of weight and cardiac toxicity. In a xenograft SKW-3/KE-37 T-ALL model, CAD204520 reduces circulating and tissue-infiltrating human leukemia T-ALL cells with no heart-related or gastrointestinal toxicities. In conclusion, we present CAD204520 as a novel orally bioavailable SERCA inhibitor with tolerable off-target toxicity in NOTCH1-dependent tumors. This work provides a foundation for the further development of novel drugs targeting Notch-dependent hematopoietic malignancies.