Abstract B113: Selective blockade of oncogenic NOTCH1 with the new SERCA inhibitor CAD204520 in T-cell acute lymphoblastic leukemia

Abstract

Abstract Given its oncogenic role in human cancers, Notch1 signaling has garnered increased attention as a therapeutic target. Several Notch modulators, including γ-secretase inhibitors, have shown therapeutic efficacy in preclinical tumor models. However, despite this promise, few of these candidates have shown clinical benefit in patients, in part due to tissue-dependent on-target toxicities from the repression of both mutant and wild type Notch proteins. The discovery of the P-type ATPase Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) as a modulator of Notch transport and activity suggested a new approach to treat T-cell Acute Lymphoblastic Leukemia (T-ALL) an aggressive cancer dependent on aberrant Notch1 activation. In fact, thapsigargin mediated SERCA inhibitions had a stronger effect on the most common type of activating NOTCH1 mutants compared to wild type. However, the consequence of thapsigargin binding to SERCA is a rapid disruption of Ca2+ homeostasis that might cause cardiac toxicity in human, suggesting the need to identify inhibitors with better drug-like properties and reduced off-target toxicity. From a small molecule screening of 191,000 P-type ATPase modulators for inhibition of the yeast S. cerevisiae H+-ATPase Pma1, 407 hits were counter screened for their ability to target Na+/K+-ATPase and the Ca2+-ATPase proteins. CAD204520 displayed ~25 and ~79 fold greater selectivity toward human SERCA compared to Na+/K+and H+-ATPase and emerged as a candidate for the development of novel anti-NOTCH1 agents. We demonstrated that CAD204520 impairs the proliferation of a panel of lymphoid malignancies carrying activating mutations in the NOTCH1 heterodimerization domain (HD, such as T-ALL) and/or deletions in the degradation domain (PEST, such as mantle cell lymphoma (MCL)). Similar to thapsigargin, CAD204520 causes a defect in NOTCH1 trafficking that results in an accumulation of full-length NOTCH1 and in a depletion of the activated form: ICN1. Consequently, the NOTCH1 targets, MYC, DTX1, were repressed as measured by qRT-PCR. In contrast, no effect at protein level was observed in T-ALL wild type NOTCH1. To assess the in vivo efficacy of CAD204520 we established xenografts from the SKW-3/KE-37 T-ALL cell line and demonstrated that drug’s treatment reduced circulating and tissue infiltrating human leukemia cells without causing heart-related off-target effects. Consistently preclinical toxicity studies in rat cardiomyocytes showed that compared to thapsigargin, CAD204520 minimally alters cell cardiac mechanical performance (~25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo. In fact, in CD1 mice exposed to CAD204520 (30 mg/Kg, BID for 21 days) no adverse clinical symptoms were found. A tempting hypothesis to explain for the variation of Ca2+ dependent toxicities seen in our studies is that thapsigargin and CAD204520 inhibit SERCA in distinct pockets. Consistently, docking poses showed that CAD204520 binds SERCA in the trans-membrane helices M1, M3 and M4 while thapsigargin in the M3, M5 and M7 groove. Consequently, thapsigargin and CAD204520 co-treatment resulted in synergistic anti-proliferative effect in T-ALL. In conclusion, this study supports the development of tolerated SERCA inhibitors for Notch dependent cancers and extends its application to cases with mutations in the PEST degradation domain such as MCL or chronic lymphocytic leukemia. Citation Format: Marchesini Matteo, Andrea Gherli, Anne-Marie Lund Winter, Samuel Kitara, Anna Montanaro, Chiara Rompietti, Claudia Sorrentino, Donatella Stilli, Federica Rizzi, Luca Pagliaro, Kimberly Stegmaier, William Dalby-Brown, Giovanni Roti. Selective blockade of oncogenic NOTCH1 with the new SERCA inhibitor CAD204520 in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B113. doi:10.1158/1535-7163.TARG-19-B113