Gain-of-function mutations in NOTCH1, encoding a trans-membrane receptor involved in a transcriptional activation complex, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Thus, targeting NOTCH1 represents one promising therapeutic opportunity in T-ALL. The implementation of NOTCH1 pathway antagonists, however, poses several challenges secondary to concerns about inhibiting normal Notch receptors, leading to gut toxicity and secondary malignancies.We recently reported the results of a gene-expression based, NOTCH1 chemical genomic loss-of-function screen and a cell-based screen for cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA (Sarco-endoplasmic reticulum Ca2+ ATPases) emerged at the intersection of these complementary approaches. We discovered that SERCA inhibition impairs the maturation of mutated NOTCH1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human T-ALL cells. A small-molecule SERCA inhibitor, thapsigargin, a sesquiterpene-g-lactone, had on-target activity in mouse models of human T-ALL and also interfered with Notch signaling in Drosophila. Remarkably, thapsigargin preferentially inhibited mutated NOTCH1 receptors. This selectivity provides a therapeutic window not observed with other Notch pathway inhibitors, such as gamma-secretase inhibitors, that are equipotent inhibitors of mutated and wild-type receptors.While thapsigargin is a highly potent tool compound, in its native form, it is unlikely to have direct clinical application because of its global effects on intracellular Ca2+. Aberrant expression of folate receptors (FR) on the surface of cancer cells, including leukemia, has previously been reported, and efforts have been undertaken to leverage folate receptor-mediated endocytosis to deliver cancer therapeutics in vivo. We therefore developed a thapsigargin derivative conjugated with folic acid (JQ-FT) for preclinical testing in T-ALL.The FR gene family includes four members: FOLR1-4. The high affinity of FOLR1 and FOLR2 for folate binding, their endocytic capacity and their restricted expression pattern have prompted evaluation of folate-drug conjugates as a therapeutic approach in cancer. Analysis of gene-expression profiling suggested that FOLR2 is more highly and consistently expressed compared to FOLR1 in human T-ALL cell lines. We confirmed this finding by qRT-PCR across a panel of T-ALL cell lines and three primary patient samples. Surface expression of FOLR1 and FOLR2 measured by flow cytometry paralleled the transcriptional findings. We next validated the dependency of the uptake of a folate-FITC probe in T-ALL cells on active endocytosis via FR. First, we demonstrated that treatment of T-ALL cells with increasing concentrations of folate-FITC corresponds to an increase in measurable fluorescent signal by flow cytometry. The uptake is specific for FR as overexpression of FOLR1 or FOLR2 leads to increased, and folic acid competition decreased, fluorescent signal. Moreover, active endocytosis is supported by data demonstrating that folate-FITC fluorescence is not eliminated with an acidic wash step and that cold temperature does diminish internalization of the chemical probe.Next we demonstrated that increasing concentrations of JQ-FT impede the growth of T-ALL cells and induce apoptosis as measured by the activation of caspase 2. Moreover, the overexpression of ICN1, the intracellular active form of NOTCH1, partially rescues the small-molecule anti-proliferative effect indicating that the JQ-FT retains the on target activity similar to the underivatized thapsigargin. As observed with thapsigargin treatment of T-ALL cells, JQ-FT treatment decreased the levels of the intracellular active form of NOTCH1, ICN1, and the furin-processed transmembrane form of NOTCH1, while the full length precursor accumulated.In summary, these pre-clinical studies confirm the selective uptake of a folate-conjugated thapsigargin by an FR-mediated mechanism with the suppression of mutated NOTCH1 maturation and support the ongoing testing of this approach in mouse models of NOTCH1-mutated T-ALL. If successful, this approach would enhance the therapeutic window of thapsigargin as a NOTCH1 inhibitor providing dual selectivity: leukemia over normal cell and NOTCH1 mutated over wild-type receptors. DisclosuresNo relevant conflicts of interest to declare.