Analysis of cancer mutations introduced into the Drosophila Notch Negative Regulatory Region uncovers a diversity of regulatory outcomes

Reviewer #1 (Public review):

Summary:

In their paper, Shimizu and Baron describe the signaling potential of cancer gain-of-function Notch alleles using the Drosophila Notch transfected in S2 cells. These cells do not express Notch or the ligand Dl or Dx, which are all transfected. With this simple cellular system, the authors have previously shown that it is possible to measure Notch signaling levels by using a reporter for the 3 main types of signaling outputs, basal signaling, ligand-induced signaling and ligand-independent signaling regulated by deltex. The authors proceed to test 22 cancer mutations for the above-mentioned 3 outputs. The mutation is considered a cluster in the negative regulatory region (NRR) that is composed of 3 LNR repeats wrapping around the HD domain. This arrangement shields the S2 cleavage site that starts the activation reaction.

The main findings are:

(1) Figure 1: the cell system can recapture ectopic activation of 3 existing Drosophila alleles validated in vivo.

(2) Figure 2: Some of the HD mutants do show ectopic activation that is not induced by Dl or Dx, arguing that these mutations fully expose the S2 site. Some of the HD mutants do not show ectopic activation in this system, a fact that is suggested to be related to retention in the secretory pathway.

(3) Figure 3: Some of the LNR mutants do show ectopic activation that is induced by Dl or Dx, arguing that these might partially expose the S2 site.

(4) Figure 4-6: 3 sites of the LNR3 on the surface that are involved in receptor heterodimerization, if mutated to A, are found to cause ectopic activation that is induced by Dl or Dx. This is not due to changes in their dimerization ability, and these mutants are found to be expressed at a higher level than WT, possibly due to decreased levels of protein degradation.

Strengths and Weaknesses:

The paper is very clearly written, and the experiments are robust, complete, and controlled. It is somewhat limited in scope, considering that Figure 1 and 5 could be supplementary data (setup of the system and negative data). However, the comparative approach and the controlled and well-known system allow the extraction of meaningful information in a field that has struggled to find specific anticancer approaches. In this sense, the authors contribute limited but highly valuable information.

Reviewer #2 (Public review):

Summary:

This ambitious study introduced 22 mutations corresponding to amino acid substitution mutations known to induce cancer in human Notch1, located within the Negative Regulatory Region, into the Drosophila Notch gene. It comprehensively examined their effects on activity, intracellular transport, protein levels, and stability. The results revealed that the impact of amino acid substitutions within the Negative Regulatory Region can be grouped based on their location, differing between the Heterodimerization Domain and the Lin12/Notch Repeat. These findings provide important insights into elucidating the mechanisms by which amino acid substitution mutations in human Notch1 cause leukemia and cancer.

Strengths:

In this study, the authors successfully measured the activity of amino acid-substituted Notch with high precision by effectively leveraging the advantages of their previously established experimental system. Furthermore, they clearly demonstrated ligand-dependent and Deltex-dependent properties.

Weaknesses:

Amino acid substitution mutations exhibit interesting effects depending on their position, so interest naturally turns to the mechanisms generating these differences. Unfortunately, however, elucidating these mechanisms will require considerable time in the future. Therefore, it is reasonable to conclude that questions regarding the mechanism fall outside the scope of this paper.

Reviewer #3 (Public review):

Summary:

Overall, the work is fine; however, I find it very preliminary. To the best of my understanding, to make any claims for altered Notch signaling from this study that is physiologically relevant remains to be discerned.

Strengths:

This manuscript systematically analyzes cancer-associated mutations in the Negative Regulatory Region (NRR) of Drosophila Notch to reveal diverse regulatory mechanisms with implications for cancer modelling and therapy development. The study introduces cancer-associated mutations equivalent to human NOTCH1 mutations, covering a broad spectrum across the LNR and HD domains. The authors use rigorous phenotypic assays to classify their functional outcomes. By leveraging the S2 cell-based assay platform, the work identifies mechanistic differences between mutations that disrupt the LNR-HD interface, core HD, and LNR surface domains, enhancing understanding of Notch regulation. The discovery that certain HD and LNR-HD interface mutations (e.g., R1626Q and E1705P) in Drosophila mirror the constitutive activation and synergy with PEST deletion seen in mammalian T-ALL is nice and provides a platform for future cancer modelling. Surface-exposed LNR-C mutations were shown to increase Notch protein stability and decrease turnover, suggesting a previously unappreciated regulatory layer distinct from canonical cleavage-exposure mechanisms. By linking mutant-specific mechanistic diversity to differential signaling properties, the work directly informs targeted approaches for modulating Notch activity in cancer cells.

Weaknesses:

While this is indeed an exciting set of observations, the work is entirely cell-line-based, and is the primary reason why this approach dampens the enthusiasm for the study. The analysis is confined to Drosophila S2 cells, which may not fully recapitulate tissue or organism-level regulatory complexity observed in vivo. Some Drosophila HD domain mutants accumulate in the secretory pathway and do not phenocopy human T-ALL mutations. Possibly due to limitations on physiological inputs that S2 cells cannot account for, or species-specific differences such as the absence of S1 cleavage.

Thus, the findings may not translate directly to understanding Notch 1 function in mammalian cancer models. While the manuscript highlights mechanistic variety, the functional significance of these mutations for hematopoietic malignancies or developmental contexts in live animals remains untested. Overall, the work does not yet provide evidence for altered Notch signaling that is physiologically relevant.