Resistance to PSEN1-selective γ-secretase inhibitors in T-cell acute lymphoblastic leukemia

  1. Charlien Vandersmissen
  2. Sofie Demeyer
  3. Kris Jacobs
  4. Lien Boogaerts
  5. Sara Gutiérrez Fernández
  6. Heidi Segers
  7. Lucía Chávez-Gutiérrez
  8. Jan Cools

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Abstract

PSEN1-selective gamma-secretase inhibitors (GSI), such as MRK-560, are a potential option for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) with NOTCH1 activating mutations, as these show less toxicity compared to broad-spectrum GSIs. However, an important challenge with targeted therapies for cancer treatment is the rapid development of drug resistance. We therefore investigated if PSEN1 mutations could confer resistance to MRK-560 in T-ALL. We performed a CRISPR-mediated mutagenesis screen in a T-ALL cell line to identify mutations leading to MRK-560 resistance and confirmed these findings in additional cell lines. We identified 3 types of resistance mutations. Mutations at the enzyme-drug interface directly disrupt the interaction of MRK-560 with PSEN1. Mutations at the enzyme-substrate interface cause a shift in relative binding affinities towards drug and/or substrate. The third resistance mechanism involves a mutation at the enzyme-substrate interface that hinders the entrance of MRK-560 to the binding pocket. These findings contribute to the understanding of the PSEN1-selectivity of MRK-560 and can help to design other PSEN1-selective GSIs to overcome resistance in cancer therapy.

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  1. Review Commons

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    Reply to the reviewers

    Reply to reviewers

    We thank the reviewers for their constructive comments. We appreciate the insights that they have shared. The comments were very helpful and will be addressed in the following sections

    Reviewer 1:

    As this field is very specific and this study may not have a broadened readership, it would benefit to add some more layers of complexity hence potential interest for Notch signaling in general and/or T-ALL pathology: e.g. do the induced mutated cell lines are more aggressive than the parental cell lines in vivo? How well do these PSEN1 mutated cell lines respond to other drugs like CB-103 (in vivo and in vitro), especially the cell lines where more Notch1 cleavage was observed

    Response to reviewer 1:

    • Reviewer 1 suggested to test the sensitivity of resistant T-ALL cell lines with PSEN1 mutations to other NOTCH inhibitors, such as CB-103. In response, we plan to assess the sensitivity of our DND-41 Cas9, HPB-ALL Cas9 and RPMI-8402 Cas9 cell lines (WT and PSEN1 mutants: 275(A>Y), 275(A>Y)+276(Q>E), 421_422(->ILS)) to CB-103 using a proliferation assay (ATPlite luminescence assay) in the coming weeks. These data will provide insight in the resistance profile and will determine if the mutations conferring resistance to the PSEN1-selective gamma-secretase inhibitor also confer resistance to other inhibitors that target NOTCH1 directly. We will incorporate this data in the manuscript once the experiments are completed.

    Reviewer 1 was wondering whether the induced mutated T-ALL cell lines are more aggressive than the parental cell line in vivo. We did not observe a significant change in proliferation in vitro for the mutated cell lines after 10-day culture with DMSO compared to parental cell line (Fig. 3). As shown in Fig. 5, certain PSEN1 mutation can enhance affinity for the NOTCH1 substrate, thereby increasing the amount of cleaved NOTCH1. Here, we could hypothesize that these PSEN1 mutations could maybe lead to a more aggressive phenotype in patients. However, the focus of this article was to determine if PSEN1 mutations lead to MRK-560 resistance. Consequently, we believe that including these additional experiments would not significantly improve the study and animal experiments would not offer a major improvement.

    *Reviewer 2: *

    The study reports an important mechanism of resistance to THE MRK-560 inhibitor. The study might benefit from a few considerations: -Test the effect of the mutations in resistance using in vivo setting (xenograft model) -The authors should ideally introduce the mutations in patient samples and repeat some of the studies using this more relevant model. -"We identified 3 types of resistance mutations.": Could mutations in the control elements of the gene (that might affect gene expression) also lead to resistance to MRK-560? Please discuss.

    *Response to reviewer 2: *

    Reviewer 2 suggested discussing whether mutations in the control elements of the PSEN1 gene could also lead to MRK-560 resistance. In this paper, we focused on a specific resistance mechanism involving mutations in the target protein. However, the reviewer's point is valid. Therefore, we will add a new section in the discussion of the revised manuscript to explore other potential resistance mechanism to MRK-560 (PTEN deletion, PSEN2 upregulation). However, we do believe that alterations of PSEN1 expression will not be an important resistance mechanism: PSEN1 expression cannot be completely silenced because NOTCH1 cleavage is necessary for cell proliferation, and higher PSEN1 expression levels will not affect drug binding/affinity.

    *Reviewer 2 suggested validating the resistance mutations in vivo using cell line xenograft or patient-derived xenograft mouse models. Our study aimed to investigate if PSEN1 mutations could confer resistance to MRK-560. We demonstrated in 2 different cell models (mouse embryonic fibroblasts and T-ALL cell lines) that the identified PSEN1 mutations resulted in higher levels of cleaved NOTCH1 compared to WT cells following MRK-560 treatment, confirming resistance. Additionally, we validated the resistance mechanisms, including the direct disruption of the drug binding pocket, a well-established resistance mechanism observed in patients treated with other targeted therapies. While in vivo validation would likely confirm the in vitro findings, we believe it requires extensive resources and would add only a marginal value to the study. *

    *Reviewer 3: *

    • Major comments
    1. The authors have limited their CRISPR mutant analysis to PS1 in this paper. The molecular studies are fine, but it is unclear whether such mutants can be generated by MRK560 treatment. To clarify the significance of these mutants in vivo, they should discuss whether the mutations identified in this study are observed in cancer patients or cultured cells treated with MRK560.
    2. Many of the mutants examined are similar to familial Alzheimer's disease, such as those that increase Aβ42 production (Fig. 5F). Concerning this mechanism, we would appreciate the discussion on how to establish cancer treatment strategies for patients with familial Alzheimer's disease.
    3. The analysis in this paper shows that mutations in a single PSEN1 allele are sufficient to acquire resistance to MRK560. On the other hand, since duplication of genes can occur in cancer cells, there is a possibility that cancer cells with multiple PSEN1 alleles or mutants with elevated PSEN1 expression (mutations in the promoter region) may arise. In such cases, we would like to see experimental evidence as to whether resistance to MRK560 is canceled or whether mutant PS1 is selectively incorporated into functional γ-secretase.*

    *Response to reviewer 3: *

    Reviewer 3 raised a valid concern regarding the use of MRK-560 in patients with pre-existing PSEN1 mutations, particularly those with familial Alzheimer's disease, who can also develop leukemia. This is a good comment, as these patients may exhibit primary resistance to PSEN1-selective inhibitors. Consequently, we will expand our discussion to address the possibility of primary resistance to MRK-560 due to inherent PSEN1 mutations.

    Reviewer 3 suggested to discuss whether the identified mutations could be acquired during MRK-560 treatment in cell lines and/or patients. Currently, no T-ALL patients have been treated with PSEN1-selective g-secretase inhibitors and hence, no data on PSEN1 mutations in patients is available (PSEN1 is also not screened at diagnosis of T-ALL patients). However, it is known that patients treated with other targeted drugs, such as imatinib (B-ALL patients) or IDH inhibitors (AML), acquired point mutations in the target gene/protein in response to treatment, leading to resistance.1,2,3,4 Here, we also demonstrated that mutations in PSEN1 can result in MRK-560 resistance, indicating a similar resistance mechanism to those previously described and further increasing the likelihood that PSEN1 mutations will arise in patients treated with PSEN1-selective g-secretase inhibitors. Predicting these resistance mutations offers the possibility to test already other inhibitors that can overcome or prevent such resistance.

    Reviewer 3 inquired about the impact of PSEN1 gene duplications on MRK-560 resistance. First, it is important to note that PSEN1 gene duplications are not observed in T-ALL patients. Additionally, PSEN1 mutations are predominantly heterozygous and dominant. Consequently, duplication of either WT or mutated PSEN1 allele is unlikely to influence resistance to MRK-560. We believe that investigating the effect of gene duplication on MRK-560 resistance is out of scope for this paper

    1*Lyczek, A., Berger, B. T., Rangwala, A. M., et al. *Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance. Proc. Natl. Acad. Sci. U. S. A. 2021; 118 (46);.

    2Melo, J. V. & Chuah, C. Resistance to imatinib mesylate in chronic myeloid leukaemia. Cancer Lett. 2007; 249 (2); 121-132

    3Issa, G. C. & DiNardo, C. D. Acute myeloid leukemia with IDH1 and IDH2 mutations: 2021 treatment algorithm. Blood Cancer J. 2021; 11 (107); 1-7.

    4Zhuang, X., Pei, H. Z., Li, T., et al. The Molecular Mechanisms of Resistance to IDH Inhibitors in Acute Myeloid Leukemia. Front. Oncol. 2022; 12 (931462);.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this paper, the authors comprehensively investigated the mechanism of resistance acquisition to MRK560, a PS1-specific γ-secretase inhibitor, in T-ALL cells by CRISPR screening. They found multiple mutants and explored their molecular mechanisms based on the 3D structure of γ-secretase. They found that the mutants can be classified into three groups: those that inhibit the binding of PSEN1 to the compound, those that inhibit the binding of PSEN1 to the substrate, and those that inhibit the binding of MRK560.

    Major comments

    1. The authors have limited their CRISPR mutant analysis to PS1 in this paper. The molecular studies are fine, but it is unclear whether such mutants can be generated by MRK560 treatment. To clarify the significance of these mutants in vivo, they should discuss whether the mutations identified in this study are observed in cancer patients or cultured cells treated with MRK560.
    2. Many of the mutants examined are similar to familial Alzheimer's disease, such as those that increase Aβ42 production (Fig. 5F). Concerning this mechanism, we would appreciate the discussion on how to establish cancer treatment strategies for patients with familial Alzheimer's disease.
    3. The analysis in this paper shows that mutations in a single PSEN1 allele are sufficient to acquire resistance to MRK560. On the other hand, since duplication of genes can occur in cancer cells, there is a possibility that cancer cells with multiple PSEN1 alleles or mutants with elevated PSEN1 expression (mutations in the promoter region) may arise. In such cases, we would like to see experimental evidence as to whether resistance to MRK560 is canceled or whether mutant PS1 is selectively incorporated into functional γ-secretase.

    Significance

    The results of this paper advance our understanding of the molecular mechanisms by which mutations in the PSEN1 gene may lead to the acquisition of γ-secretase inhibitor resistance in T-ALL treatment strategies. On the other hand, this study alone cannot be generalized to the development of T-ALL treatment strategies in terms of gene mutation acquisition in cancer cells, because mutations in the non-coding region of the PSEN1 gene and mutants of other γ-secretase components as well as PSEN1 can occur.

    Some of the mutations found have not been previously identified and provide new insights into our understanding of the mechanisms by which PSEN1 exerts its activity. However, the mutants obtained are based on structural analysis of the MRK560 complex PSEN1, which has already been analyzed and does not provide major advances in mechanistic insights of γ-secretase.

    Given that this paper is primarily a pharmacological analysis and is limited to γ-secretase and T-ALL, the intended audience for this paper is likely to be researchers involved in cancer-related research and pharmacological research.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The study reports an important mechanism of resistance to THE MRK-560 inhibitor. The study might benefit from a few considerations:

    • Test the effect of the mutations in resistance using in vivo setting (xenograft model)
    • The authors should ideally introduce the mutations in patient samples and repeat some of the studies using this more relevant model.
    • "We identified 3 types of resistance mutations.": Could mutations in the control elements of the gene (that might affect gene expression) also lead to resistance to MRK-560? Please discuss.

    Significance

    It is a well-designed study

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Vandersmissen et al., performed a CRISPR-mediated mutagenesis screen to identify presenilin (PSEN1) mutations that could lead to the resistance of PSEN1-selective gamma-secretase inhibitors such as MRK-560, in T-ALL. In general, the manuscript is well-written, and the experiments performed do support their claims and are well done. The authors even went on to interrogate that the mutations that confer the drug resistance were located at the enzyme-substrate interface that caused a shift in relative binding affinities towards MRK-560 and/or substrate. Another resistance mechanism involved a mutation at the enzyme-substrate interface that hindered the entrance of MRK-560 to the binding pocket. This study is quite unusual in the sense that PSEN1 mutations that confer resistance to MRK-560 in T-ALL have yet to be reported, as far as this reviewer is aware. Hence, the authors created a potential problem that has yet to exist. Although cancers do develop resistance to drugs, whether naturally occurring MRK-560-resistant T-ALL samples would be the same as described in this study is unknown. Nevertheless, it is an interesting study and can set the foundation for future studies.

    Significance

    As this field is very specific and this study may not have a broadened readership, it would benefit to add some more layers of complexity hence potential interest for Notch signaling in general and/or T-ALL pathology: e.g. do the induced mutated cell lines are more aggressive than the parental cell lines in vivo? How well do these PSEN1 mutated cell lines respond to other drugs like CB-103 (in vivo and in vitro), especially the cell lines where more Notch1 cleavage was observed?

  5. Version published to 10.1101/2024.03.01.582944v1 on bioRxiv