Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies

Abstract

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

Figure 1. Identification of an activating NOTCH3 NRR mutation in TALL-1 cells

(A) T-ALL cell lines HPB-ALL (NOTCH1 NRR mutant) SUPT11 (NOTCH1 wild-type) and TALL-1 (NOTCH 1 wild-type) were treated with a GSI for 6-7 days and proliferation monitored by Cell Titer-Glo assay. (B) Protein lysates were generated from untreated T-ALL cell lines and lysates probed with antibodies for NOTCH 3, ICD1 and α-tubulin. # TM-ICD3 is the transmembrane-domain-ICD3 subunit produced by furin cleavage of NOTCH3, while * ICD3 is the intracellular domain of Notch3 produced by successive metalloprotease and gamma secretase cleavage. (C) Alignment of a portion of the NOTCH 1, NOTCH 2 and NOTCH 3 NRR domains. The S1580 residue of NOTCH3 and corresponding serine residues in NOTCH1 and NOTCH2 are highlighted in red. (D) HeLa cell lines with stable expression of GAL4-Luciferase reporter were generated expressing either wild-type or mutant (S1580L) NOTCH 3-GAL4-VP16. Cells were treated with either DMSO or 10μM DAPT for 24hr prior to measuring GAL4-Luciferase activity. The level of NOTCH 3 receptors on the cell surface was determined by binding of anti-NOTCH3 APC labeled antibody to cells expressing mutant and endogenous NOTCH3 and assessed by flow cytometry.

Figure 2. ICD3 antibody detects activated NOTCH 3 in TALL-1 cells and other lines with NOTCH3 NRR and PEST mutations

(A) Protein lysates were generated from untreated T-ALL cell lines and lysates probed with antibodies for ICD3, ICD1 and α-tubulin. (B) TALL-1 cells were treated with the DMSO or 10μM DAPT for 72hr prior to generation of protein lysates for Western blotting with ICD3 antibody and α-tubulin. (C) Protein lysates were generated from untreated cell lines with NRR mutations and lysates probed with antibodies for ICD3 and α-tubulin. (D) Protein lysates were generated from untreated cell lines with PEST mutations and lysates probed with antibodies for ICD3 and α-tubulin.

Figure 3. Identification and characterization of NOTCH3 inhibitory antibodies

(A) TALL-1 cells were treated with 10μg/ml IgG control antibody or NOTCH3 NRR antibodies (A4, MOR20337, MOR20350, and MOR20358) or NOTCH3 LBD antibody (MOR20364) for 72hrs prior to quantitation of DTX1 mRNA levels by qRT-PCR. Positive and negative control TALL-1 cells were treated with DMSO or 10μM DAPT for 72hrs. (B) Effect of treatment of TALL-1 cells and MDA-MB468 cells with NOTCH3 NRR antibodies on levels of ICD3. (C) Effect of treatment of TALL-1 cells with NOTCH3 NRR antibodies on proliferation.

Figure 4. Crystallographic determination of epitopes by MOR20350 and MOR20358

(A) Overall structures of MOR20350 and MOR20358 binding to NOTCH3 NRR. (B) Structural superposition of MOR20350 and MOR20358 complexes on NOTCH3 NRR, showing non-overlapping epitopes of the two antibodies. (C) Epitopes of MOR20350, MOR20358 and A4 (red and cyan) are shown on the surface of NOTCH3 NRR. The buried surface areas of MOR20350 and MOR20358 binding to NOTCH3 NRR are also listed.

Figure 5. NOTCH3 NRR antibodies inhibit NOTCH3 signaling in vivo and are efficacious in a TALL-1 xenograft model

Mice bearing luciferized TALL-1 xenografts were dosed intravenously with a single 20mg/kg dose of the indicated antibody. Tumors were harvested 72hr later and analyzed for (A) expression of DTX1 mRNA by qRT-PCR (NRR antibodies are shown in red while LBD antibodies are shown in grey) or (B) ICD3 and total NOTCH3 by Western blot. In addition, a portion of tumor was fixed in formalin and analyzed by immunohistochemical staining with the ICD3 antibody or an isotype control IgG (inset). (C) Mice bearing luciferized TALL-1 xenografts were dosed intravenously with the indicated antibodies at 20mg/kg twice per week starting on day 11 after implantation of tumor cells. Tumor size was monitored with the Xenogen in vivo imaging system. Images of mice treated with the control IgG (3207), MOR20350 and MOR20358 on day 29 as well as mice treated with MOR20350 on day 43 post-implantation are shown.

Figure 6. NOTCH3 signaling is activated in T-ALL patient samples

(A) ICD3 immunohistochemistry from 2 T-ALL patient samples (i and ii) and the TALL-1 cell line (iii). (B) 24 T-ALL PDX (patient derived xenograft) samples were analyzed for presence of total NOTCH3, ICD3, total NOTCH1and ICD1 protein by Western blotting. For total NOTCH3, the bands at ∼80 kDa represent the TM-ICD or ICD3 while the bands at ∼270 kDa represent the full-length NOTCH3. For total NOTCH1 the bands at ∼100-120 kDa represent the TM-ICD or ICD1 while the bands at ∼300 kDa represent the full-length NOTCH1 or furin processed NOTCH1. TALL-1 and CUTLL1 cell lysates were used as positive controls for ICD3 and ICD1 respectively.