SUMMARY Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA calcium channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies “credential” SERCA as a therapeutic target in cancers associated with NOTCH1 mutations.

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on the experiences and limitations of a novel MSCV-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBz). This phase I dose-escalation trial enrolled children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose-finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives, including CAR T-cell expansion and cytokine profiling, and laboratory investigations, were also analyzed. Twenty patients, ages 5.4-34.6 years, with B-ALL received CD19.22.BBz. The complete response (CR) rate was 60% (12/20) in the full cohort and 71.4% (10/14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having grade 3 CRS and only 1 experiencing any neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% CI: 42.4-94.9%) and 57.7% (95% CI: 22.1-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBz compared to EF1a-CD22.BBz prompted laboratory investigations comparing EF1a versus MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBz, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28z/CD22.BBz construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBz in a heavily pre-treated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations. (Clinicaltrials.gov NCT03448393)

Isothermal titration calorimetry (ITC) was used to quantify the thermodynamics of Pb(2+) and Zn(2+) binding to metallothionein-3 (MT-3). Pb(2+) binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (ΔG) and enthalpy (ΔH). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extracted in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodynamics. Metal binding is entropically favoured, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb(2+) binding to both apo MT-3 and Zn7MT-3 is thermodynamically favourable, and implicate MT-3 in neuronal lead biochemistry.

NOTCH1 is an attractive cancer target, particularly in T cell acute lymphoblastic leukemia (T-ALL), with activating mutations in this gene identified in more than 50% of cases. In this study, Roti et al. describe the synthesis, characterization, and validation of JQ-FT, a first-in-class NOTCH1 inhibitor that has dual selectivity for leukemia over normal cells and NOTCH1 mutants over wild-type receptors.