CAD204520 Targets NOTCH1 PEST Domain Mutations in Lymphoproliferative Disorders

Abstract

NOTCH1 PEST domain mutations are often seen in hematopoietic malignancies, including T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). These mutations play a key role in the development and progression of lymphoproliferative tumors by increasing the Notch signaling and, consequently, promoting cell proliferation, survival, migration, and suppressing apoptosis. There is currently no specific treatment available for cancers caused by NOTCH1 PEST domain mutations. However, several NOTCH1 inhibitors are in development. Among these, inhibition of the Sarco-endoplasmic Ca²⁺-ATPase (SERCA) showed a greater effect in NOTCH1-mutated tumors compared to the wild-type ones. One example is CAD204520, a benzimidazole derivative active in T-ALL cells harboring NOTCH1 mutations. In this study, we preclinically assessed the effect of CAD204520 in CLL and MCL models and showed that NOTCH1 PEST domain mutations sensitize cells to the anti-leukemic activity mediated by CAD204520. Additionally, we tested the potential of CAD204520 in combination with the current first-line treatment of CLL, venetoclax, and ibrutinib. CAD204520 enhanced the synergistic effect of this treatment regimen only in samples harboring the NOTCH1 PEST domain mutations, thus supporting a role for Notch inhibition in these tumors. In summary, our work provides strong support for the development of CAD204520 as a novel therapeutic approach also in chronic lymphoproliferative disorders carrying NOTCH1 PEST domain mutations, emerging as a promising molecule for combination treatment in this aggressive subset of patients.

Keywords: NOTCH1; NOTCH1 PEST domain mutations; chronic lymphocytic leukemia; lymphoproliferative disorders; mantle cell lymphoma; synergy treatment; target therapy.

Figure 1

NOTCH1 mutational status in chronic lymphocytic leukemia (CLL) primary samples and characteristics of the patient cohort: (A) The OncoPrint illustrates the distribution of gene mutations affecting individual samples. Single nucleotide polymorphisms (SNPs) are represented in red, and insertions/deletions (indels) are in blue. Each row in the OncoPrint displays the percentage distribution of relative gene mutations in the entire cohort (shown in the right histogram panel). Each column represents the total number of mutations for each patient, with a specific indication of the mutation type (upper histogram panel; red: SNP, blue: indel). The OncoPrint also provides relevant clinical, genetic, molecular, and prognostic characteristics of the patient samples collected for this study (lower panel). (B) The structure of the human NOTCH1 protein is depicted, with each colored block representing an exon. The PEST domain illustrates the distribution of PEST mutations found in CLL patient samples. (C) Patient characteristics for the collected CLL primary samples. (D) Western immunoblotting results display the expression of unprocessed full-length NOTCH1 precursor (FL), furin-processed NOTCH1 transmembrane subunit (TM), and cleaved intracellular domain (ICD) in CLL primary samples. β-Actin serves as the loading control. NOTCH1-mutated patient samples are indicated in light red.

Figure 2

CAD204520 inhibits Notch1 signaling and impairs cell growth in PEST-mutated lymphoproliferative malignancies: (A) Protein expression of NOTCH1 processed isoforms in a panel of T-ALL, MCL, and CLL cell lines. β-Actin was used as a loading control. (FL: full-length unprocessed precursor; TM: transmembrane; ICD: intracellular domain). The table shows NOTCH1 mutational status in the cell lines. (B) Effect of CAD204520 treatment for 24 h on Notch1 trafficking and activation in cell lines (CTV-1, SKW-3/KE-37, REC-1) with PEST domain mutations. β-Actin was used as a loading control. (C) Effect of CAD204520 treatment on cell viability after 72 h in NOTCH1 PEST-mutated (REC-1, SKW-3/KE-37, CTV-1) and NOTCH1 WT (JEKO-1, Granta-519, MEC1) cell lines. Error bars denote ± SD of a minimum of two replicates. (D) Comparison of the area under the curve (AUC) values after CAD204520 treatment of NOTCH1-mutated and WT cell lines. Statistical significance was determined by a non-parametric t-test (* p < 0.05). (E) Effect of CAD204520 treatment on the induction of apoptosis. Annexin V/propidium iodide staining of MCL cells after 48 h of treatment with the indicated concentrations of CAD204520. A minimum of 20000 events was collected for each condition. (F) Western immunoblot showing the expression of cleaved PARP in NOTCH1 WT (JEKO-1) and mutated (SKW-3/KE-37 and REC1) cell lines treated at the indicated concentrations of CAD204520 for 24 h. β-Actin was used as a loading control. (G) Densitometric quantification of indicated proteins in JEKO-1, SKW-3/KE-37, and REC-1 cells treated with indicated doses of CAD204520, as in Figure 2F. (H) Combined scatter and bar plot representing the AUC values of CAD204520 treatment in CLL primary samples with or without Notch1 activating pathway mutations. Statistical significance was determined using a non-parametric t-test (** p < 0.01). (I) Effect of CAD204520 treatment after 24 h on Notch1 trafficking in CLL primary samples. β-Actin was used as a loading control. (J) Histogram plots showing the percentage of live and dead cells in 6 different samples (top: 3 NOTCH1 WT samples; bottom: 3 NOTCH1-mutated samples) after 72 h of treatment with CAD204520 at indicated concentrations. Results were obtained with a luminescence-based and a flow cytometric assay, respectively. Error bars denote the SD of a minimum of two replicates. Statistical significance among groups was determined by a one-way ANOVA using Dunnett’s correction for multiple comparison testing (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 3

CAD204520 preferentially inhibits cells with NOTCH1 PEST mutations: (A) Outline of the cell-based competition assay: REC-1 cells were transduced with green fluorescent protein (GFP). REC-1-GFP+ cells were sorted and co-cultured in a 1:1 ratio with JEKO-1 cells, then treated with CAD204520 at various concentrations for 72 h. (B) Normalized effects of CAD204520 on cell viability in co-cultured REC-1-GFP+ and JEKO-1 cells treated for 72 h. Statistical significance was determined by a two-way ANOVA. Error bars represent ± SD of a minimum of three replicates (* p < 0.05; *** p < 0.001; **** p < 0.0001). (C) Design of the in vivo CAD204520 study: ten NSG mice were subcutaneously injected with REC-1 cells in the left flank and JEKO-1 cells in the right flank. On day 0, mice were randomized into two groups, with the former receiving CAD204520 at 45 mg/kg (day 1–5 “on”; day 6–7 “off”; day 8–12 “on”) via oral gavage, and the latter receiving the vehicle. (D) Effect of CAD204520 administration on JEKO-1 and REC-1 tumor size fold change at different time points (mean ± SD of the five different mice treated with the vehicle or CAD204520). Statistical significance was determined using a non-parametric t-test (* p < 0.05; ** p < 0.01). (E) Effect of CAD204520 administration on JEKO-1 and REC-1 tumor weight at sacrifice (mean ± SD of the five different mice treated with the vehicle or CAD204520). Statistical significance was determined using a non-parametric t-test (* p < 0.05). (F) Effect of daily administration of 45 mg/kg of CAD204520 or the vehicle on body weight. (G) Immunohistochemical analysis of REC-1 and JEKO-1 tumor masses in the murine model treated with the vehicle or CAD204520 at 45 mg/kg for 10 administrations. The tumor masses from all mice were examined. Formalin-fixed, paraffin-embedded tissue sections were stained with NOTCH1 and Ki-67 antibodies. Scale bars: 50 µm. Representative results for one control animal and one CAD204520-treated animal are shown. (H) Quantification of NOTCH1 and Ki-67 protein expression of the immunohistochemical analysis shown in Figure 3G (**** p < 0.0001).

Figure 4

CAD204520 increases the effect of venetoclax–ibrutinib treatment in NOTCH1 PEST-mutated samples: (A) Volcano surface plots of primary CLL samples with NOTCH1 WT (CLL#2) and NOTCH1 PEST mutation (CLL#20 and CLL#25) treated with venetoclax and ibrutinib. Each point represents an independent measurement. The plots illustrate the HSA analysis generated using the Combenefit script in MATLAB R201. The colorimetric scale represents the level of drug antagonism or synergism. (B) Volcano surface plots of primary CLL samples with NOTCH1 WT (CLL#2) NOTCH1 PEST mutation (CLL#20 and CLL#25) treated with venetoclax and ibrutinib plus 2 µM of CAD204520. Each point represents an independent measurement. The plots illustrate the HSA analysis generated using the Combenefit script in MATLAB R201. The colorimetric scale represents the level of drug antagonism or synergism. (C) Circular plots of 3-drug combinations in a NOTCH1 WT primary sample (CLL#2) and two NOTCH1-mutated primary samples (CLL#20 and CLL#25). The innermost rings represent the 5 drug concentrations in 3 color gradients (CAD204520 = yellow, venetoclax = cyan, ibrutinib = pink). The fourth ring represents the effect in terms of inhibition percentage for any given combination. The outermost ring represents a harmonized synergy score performed with the Bliss model. Red indicates a positive synergistic score (pointing toward synergy), while blue represents a negative score (pointing toward antagonism). Zero indicates the absence of interaction. (D) Circular plots of 3-drug combinations following the same format as described in (C). The outermost ring represents a harmonized synergy score performed with the HSA model. (E) Circular plots of 3-drug combinations following the same format as described in (C). The outermost ring represents a harmonized synergy score performed with the ZIP model. (F) Heatmaps of samples CLL#2, CLL#20, and CLL#25 (from left to right) displaying the linear fold change between combinations of venetoclax-ibrutinib-CAD204520 (columns) compared to venetoclax-ibrutinib combinations (rows). Each cell contains the fold change between the 3-drug combination effect and the 2-drug combination effect at the same doses of the first 2 drugs. A positive fold change indicates a gain in inhibition of the 3-drug combination compared to the 2-drug combination, while a negative fold change indicates a loss of inhibition of the 3 drugs compared to the combination without CAD204520.