Several studies support the development of NOTCH1 inhibitors for targeted cancer therapy. This idea is most compelling for T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) where activating mutations of NOTCH1 are present in 55–60% of the cases, and cancer dependency has been well established. With the identification of Sarco/Endoplasmic Reticulum Calcium ATPase as a modulator of gain-of-function NOTCH1 mutations, the development of SERCA inhibitors represent a unique opportunity for Notch dependent tumors.
Because SERCA plays a critical role in Ca2+ in cardiac physiology, inhibition of this pump may increase the risk of heart failure limiting the development of this class of compounds for cancer therapeutics. In this work, we describe CAD204520, a new selective SERCA inhibitor with a tolerable toxicity profile and NOTCH1 on-target anti leukemia effect both in vitro and in vivo.
From a small molecule screening of 191,000 compounds for inhibition of the fungal 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 greater selectivity toward human SERCA and emerged as a lead candidate for the development of novel anti-NOTCH1 agents. Because altered Ca2+ levels may cause heart failure we investigated the effect of CAD204520 on rat cardiomyocyte and performed pharmacokinetics studies in CD1 mice. Finally, we assessed the effects of CAD204520 in a xenografted T-ALL leukemia model.
We previously described that thapsigargin mediated SERCA inhibition affects the proliferation of NOTCH1 mutated T-ALL compared to wild types. CAD204520 retains this therapeutic index in 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)). In addition, CAD204520 induces apoptosis and a G1/G0 arrest both in cell line and primary T-ALL samples. Similar to thapsigargin, CAD204520 treatment decreased the levels of the activated form of NOTCH1, ICN1, while it increased full-length NOTCH1. The NOTCH1 targets, MYC, DTX1, were consequently repressed as measured by qRT-PCR. In contrast, no effect at protein level was observed in T-ALL wild type NOTCH1.
To test for the effect of CAD204520 on cardiac tissue, we performed toxicity studies in isolated rat cardiomyocytes and demonstrated that compared to thapsigargin, CAD204520 minimally alters cell mechanical performance (∼25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo. Consistently in CD1 mice exposed (30 mg/Kg, BID for 21 days) no adverse clinical symptoms were found. Based on the chemical structure, we hypothesized that CAD204520 locks SERCA in a binding site different to that of thapsigargin. Interestingly, thapsigargin and CAD204520 co-treatment resulted in synergistic anti-proliferative effect in T-ALL. This result suggests that the two molecules inhibit SERCA simultaneously but in different pockets explaining for the variation of Ca2+ dependent toxicities seen in our studies.
Finally, to assess the in vivo efficacy of CAD204520 we established a T-ALL derived xenografts from SKW-3/KE-37 human T-ALL cell and demonstrated that drug's treatment reduced circulating human T-ALL cells.
In conclusion, this study supports the development of tolerated SERCA inhibitors for Notch-dependent cancers and extends its application to cases with PEST mutations such as MCL or chronic lymphocytic leukemia.