Blockade of oncogenic notch1 with the new serca inhibitor cad204520 in t-cell acute lymphoblastic leukemia

Abstract

The discovery of the P-type ATPase Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) as a bidirectional 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. SERCA inhibition employing the pan SERCA modulator thapsigargin results in a potentially cardiotoxic raise of cytosolic Ca2+, suggesting the need to identify inhibitors with better drug-like properties and reduced off-target toxicity. We developed a novel oral SERCA inhibitor, CAD204520, through medicinal chemistry optimization and crystal structure-oriented analysis describing its anti-leukemic effect in vitro and in vivo to support a SERCA-based therapeutic modality in T-ALL. From a 191000 small molecules screening targeting P-type ATPase, we identified CAD204520 which showed ~25 and ~79-fold greater selectivity toward human SERCA compared to Na+/K+ and H+-ATPase respectively and promising drug properties. Crystal structure analysis showed that CAD204520 binds to a groove at the membrane interface of SERCA, between the transmembrane helices M1, M2, M3 and M4. This protein pocket has been previously identified as a site for Ca2+ ion entry into the pump from the cytosolic side of the membrane, and compound binding at this groove locks SERCA in a Ca2+-free conformation. This mode of action, that is different from the one of thapsigargin, suggests a lower affinity for Ca2+ resulting in a diminished net increase in cytosolic Ca2+. We leveraged this therapeutic index and showed that compared to thapsigargin, CAD204520 minimally alters Ca2+ shift and fails to trigger Ca2+ dependent programs such as the unfolded protein response. We next tested how CAD204520 alters the function of cardiomyocytes and demonstrated that thapsigargin induces a greater negative effect on cardio-mechanics suggesting that the heart will probably tolerate CAD204520 modulation in vivo. CAD204520 impairs the proliferation of a panel of T-ALL cell lines carrying activating mutations both in the heterodimerization and in the PEST degradation NOTCH1 domain. Importantly, clinical samples and cell lines carrying NOTCH1 mutations including PEST deletions were more sensitive to CAD204520 compared to normal lymphocytes or wild type NOTCH1 ALL cells. CAD204520 treatment reduces the levels of the activated form of NOTCH1 as consequences of a defect in NOTCH1 trafficking. In anticipation of clinical translation and to explain general mechanisms of acquired resistance to SERCA modulators, we established a T-ALL cell line resistant to thapsigargin. We demonstrated that somatic hotspot mutations in SERCA2 ATPase pocket do not interfere with CAD204520 binding, suggesting that the activity of CAD204520 will be unlikely affected by recurrent resistance 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 off-target effects. In conclusion, this study presents CAD204520 as a novel orally bioavailable SERCA inhibitor with tolerable off-target toxicity in NOTCH1 dependent tumors. This work provides a foundation for further development of novel drugs targeting Notch-dependent hematopoietic malignancies.