Leukemia-Specific Delivery of Mutant NOTCH1 Targeted Therapy
Authors: Roti Giovanni , Qi Jun , Kitara Samuel , Varca Anthony C. , Sanchez-Martin Marta , Kung Andrew L. , Ferrando Adolfo A. , Bradner James E. , Stegmaier Kimberly 
Published: October 2019

Abstract
Several studies strongly 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.
We previously demonstrated that a small-molecule inhibitor of SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), thapsigargin, had NOTCH1 dependent anti-leukemia activity in mouse models of human T-ALL. Interestingly thapsigargin preferentially inhibits the maturation of mutant NOTCH1 compared to wild type NOTCH1 and NOTCH2 receptors. This selectivity provides a therapeutic window not observed before with other Notch modulators, including γ-secretase inhibitors or antibody-based approaches.
To avoid the potential non-specific toxicityof thapsigargin secondary to intracellular calcium shifts, we leveraged the addiction of ALL to folic acid (FA), and we tagged folate to a permissive site on an active alcohol derivative of thapsigargin (8-O-debutanoylthapsigargin) via a cleavable ester linkage (JQ-FT). We explored folate conjugation of thapsigargin as an anti-cancer strategy for several reasons: the restricted expression of folate receptors (FR1-4) on cancer cells, the affinity of folic acid for FR binding and the FR endocytic mechanism that allows the internalization of large probes.
We determined that folate-conjugates, including JQ-FT, enter T-ALL cells by FR2 binding and endocytosis. Importantly, T-ALL cell lines demonstrated stronger folic acid fluorescence-tagged probe (FL-FITC) labeling compared to human CD3+ T cells, providing support for leukemia-specific targeting. To determine if these effects were FR mediated, we treated T-ALL cells with JQ-FT in the presence or absence of the competitive inhibitor FA (10 and 100 μM). Co-treatment with FA diminished the negative growth effects of JQ-FT on T-ALL cells. This result supports FR-mediated internalization of JQ-FT.
Next, we demonstrated that JQ-FT alters the growth of T-ALL cell lines, induces apoptosis and a G1/G0 arrest. Similar to thapsigargin, JQ-FT treatment decreased the levels of the activated form of NOTCH1, ICN1, and trans-membrane NOTCH1, while it increased full-length NOTCH1. Consequently, the NOTCH1 transcriptional targets, MYC, DTX1 and HES1, were repressed as measured by qRT-PCR. As predicted, the addition of free FA to the media rescued the decrement of ICN1.
To assess the translational significance of these findings, we studied patient-derived xenograft (PDX) cells. JQ-FT treatment inhibited the viability of NOTCH1-mutated PDX cells in vitro. In addition, JQ-FT treatment resulted in a loss of transmembrane NOTCH1, leading to the depletion of ICN1. In contrast, no effect was observed in PDX T-ALL cells possessing wild-type NOTCH1. Consistent with these results, no transcriptional changes were observed in NOTCH1 target genes in wild type PDX samples, supporting the notion that mutated NOTCH1 receptors are more sensitive to SERCA inhibitors, including JQ-FT treatment, than are wild type NOTCH1 receptors in primary patient T-ALL.
To explore the therapeutic efficacy of JQ-FT in vivo, we studied its effects on a syngeneic T-ALL mouse model carrying a NOTCH1 L1601P ΔPEST, a common mutation observed in the human disease. We first established the maximal tolerated dose (MTD) of JQ-FT as 60 mg/kg/day in mice administered by daily intraperitoneal injection. Notably, this MTD is 150-fold improved over our prior established MTD of unconjugated thapsigargin. Following five days of treatment, a decrease in tumor growth was observed, confirmed pathologically by a decrease in the leukemic infiltration in the bone marrow, spleen and liver, and clinically by a reduction in spleen weight. Pharmacodynamic modulation of the Notch pathway was validated by measurement of reduced ICN1 expression in T-ALL cells from JQ-FT-treated animals as compared to vehicle-treated controls.
In summary, we report the development of the first-in-class NOTCH1 inhibitor with dual selectivity for leukemia over normal cells and NOTCH1-mutant over wild-type receptors. JQ-FT is recognized by folate receptors on the leukemia cell surface and delivered into cells as a mutant NOTCH1-targeted anti-leukemic agent. In mechanistic and translational models of T-ALL, we demonstrated NOTCH1-specific inhibition in vitro and in vivo.

Disclosures
Bradner: Novartis Institutes for BioMedical Research: Employment.