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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.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL). Clinical translation of SERCA inhibitors has been hampered by the risk of adverse cardiac events. In this work, Marchesini and Gherli et al. identified a tolerable oral available SERCA inhibitor, CAD204520, and showed that modulation of clinically relevant NOTCH1 mutations in T cell acute lymphoblastic leukemia and mantle cell lymphoma.

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

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 oncogenic NOTCH1 suggested a new approach to treat T-cell Acute Lymphoblastic Leukemia (T-ALL). In fact, thapsigargin mediated SERCA inhibition 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 increase in cytosolic Ca2+ and a depletion of Ca2+ stored in the endoplasmic reticulum (ER). This shift of Ca2+ efflux 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 CAD204520 displayed ~25 and ~79-fold greater selectivity toward human SERCA compared to Na+/K+ and H+-ATPase respectively and emerged as a candidate for the development of novel anti-NOTCH1 agents. In a series of in vitro studies, we showed 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)). Importantly, T-ALL lymphoblasts from patients carrying NOTCH1 mutations were more sensitive to CAD204520 suppression compared with normal lymphocytes or ALL wild type NOTCH1. 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 and DTX1, were repressed as measured by qRT-PCR. In contrast, we observed no effect at protein level in T-ALL wild type NOTCH1 suggesting that clinically relevant NOTCH1 mutations including PEST deletions are more sensitive to CAD204250 treatment than wild type proteins. The next question is whether CAD204520 activity has limitations in vivo due to Ca2+ shifts. Thus we analysed cardiac mechanics in isolated rat cardiomyocytes and showed that compared to thapsigargin, CAD204520 minimally alters myocardial performance (~25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo. This effect may be caused to differences between Ca2+ mediated ER stress upon thapsigargin or CAD204520 treatment. To test this hypothesis, we treated T-ALL cell lines at equimolar doses of CAD204520 and thapsigargin. We showed that, despite a similar effect on NOTCH1 protein levels, CAD204520 does not induce the unfolded protein response (UPR) pathway as measured by the phosphorylation of eIF2α or the induction of the ER chaperone BIP. This result suggests that CAD204520 retains ATPase activity, responsible for Notch inhibition, decoupled from the Ca2+ transport activity responsible for the off-target effects. A tempting hypothesis to explain these differences 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. To test CAD204520 in vivo we studied its effect in CD1 mice and established a maximum tolerated dose. Mice exposed to CAD204520 for 21 days at 30 mg/Kg BID showed no decrease in body weight and, importantly no effect on cardiac hemodynamic. Finally, to assess the in vivo efficacy of CAD204520 we xenografted SKW-3/KE-37 T-ALL cell line in L-2Rγ null mice and demonstrated that drug's treatment reduced circulating and tissue infiltrating human leukemia T-ALL cells without causing heart-related off target effects. 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. Disclosures Lund Winter: CaDo Biotechnology IvS: Employment. Stegmaier:Novartis: Research Funding; Rigel Pharmaceuticals: Consultancy. Dalby-Brown:CaDo Biotechnology IvS: Employment.