Finding novel vulnerabilities of hypomorphic BRCA1 alleles

  1. Anne Schreuder
  2. Klaas de Lint
  3. Mariana M. Góis
  4. Rosalie A. Kampen
  5. Marta San Martin Alonso
  6. Ilse Nootenboom
  7. Veronica Garzero
  8. Rob M. F. Wolthuis
  9. Sylvie M. Noordermeer

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Abstract

With the recent rise in CRISPR/Cas9-mediated genome-wide synthetic lethality screens, many new synthetic lethal targets have been identified for diseases with underlying genetic causes such as tumours with BRCA1 mutations. Such screens often use full deficiency of a protein to identify novel vulnerabilities. However, patient-derived mutations not only result in loss of the protein but often also concern missense mutations with hypomorphic phenotypes. Here we study the genetic vulnerabilities of two previously described hypomorphic BRCA1 missense mutations and compare these to a BRCA1-depleted setting to study whether this affects screening for synthetic lethal interactions. Our research showed that BRCA1 I26A mutated cells have very similar vulnerabilities to BRCA1 wildtype cells, confirming its low tumorigenic effect. In contrast, the BRCA1 R1699Q mutation induced a more similar phenotype to BRCA1-deficient cells. For this mutation, we also unveiled a unique vulnerability to the loss of NDE1. Specifically in BRCA1 R1699Q mutated cells, and not BRCA1-proficient or -deficient cells, NDE1 loss leads to increased genomic instability. Altogether our findings highlight the importance to differentiate between patient-derived mutations when assessing novel treatment targets.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Anne Schreuder et al studied the genetic vulnerabilities of two previously described hypomorphic BRCA1 missense mutations- I26A and R1699Q, and compare these to a BRCA1-depleted setting to identify novel vulnerabilities of the two hypomorphic BRCA1 alleles. The authors showed that BRCA1I26A mutated cells have very similar vulnerabilities to BRCA1 wildtype cells, while the BRCA1R1699Q mutation induced a more similar phenotype to BRCA1-deficient cells. Then the authors unveiled a unique vulnerability to the loss of NDE1 with increased genomic instability specifically in BRCA1R1699Q mutated cells, but not BRCA1-proficient or -deficient cells. While the experiment design strategy is quite reasonable and the data are quite solid, some of the interpretations look not that convincing.

    Major concerns:

    1. According to your data, RAD51 IRIF were reduced to similar levels in BRCA1 depleted cells reconstituted with either BRCA1R1699Q or BRCA1I26A mutants，suggesting that both the two mutants have defects in HR (Line 110-117). And when you tested the Olaparib sensitivity, your results showed that unlike wildtype BRCA1, re-expression of BRCA1I26A only partially rescued the sensitivity, suggesting that BRCA1I26A still have the capacity to perform HR (Line 120-125). Since the function of BRCA1I26A is quite controversial in the field, the authors should explain in your experiment why RAD51 IRIF of BRCA1I26A is not correlated with its HR level, these two data should be consistent.

    Moreover, in line 194-195, you mentioned that this finding correlates with research showing that the I26A mutation does not affect tumour suppression and HR by BRCA1. It is hard to tell whether BRCA1I26A is defective or functional in HR, what's your opinion about it? If you agree that BRCA1I26A is functional in HR, then why it affects RAD51 IRIF.

    1. In line 175, the authors validated the synthetic lethal interaction between BRCA1 and CSA in BRCA1-depleted RPE1 cells and BRCA1-mutated HCC1937 cells (Figure 2D, Supplemental figure 2A, B, C). Actually, HCC1937 is a both BRCA1 and FAM35A-mutated cell line (DOI: 10.15252/embj.201899543), it is a HR functional cell line that does not response to PARPi. According to your CRSIPR data in Table1 and others publications, loss of 53BP1 or its downstream factors such as C20ORF196, FAM35A are synthetic survival with BRCA1 loss. If CSA is synthetic lethal with BRCA1 loss in HCC1937, suggesting that CSA is not simply synthetic lethal with BRCA1 loss of function, at least not only synthetic lethal with HR deficiency. CSA maybe a promising drug target for treating with the PARPi resistant or the PARPi non-response patients. The authors should mention it in the manuscript.
    2. RPE1 hTERT P53-/- BRCA1-/- cells have very severe cell growth defects compared with RPE1 hTERT P53-/- BRCA1+/+ cells. Did you see a growth defect or a certain cell death when you induce acute BRCA1 depletion? In Figure 1C, you only showed the survival rate compared with PARPi non-treatment group. Can you also show the growth curve of all these cell lines?
    3. Based on your CRISPR screen results from Table 1,2,3, you made the conclusion that BRCA1I26A exhibits vulnerabilities similar to BRCA1-proficient cells and BRCA1R1699Q exhibits vulnerabilities similar to BRCA1-deficient cells. However, when looked at the data carefully, XRCC1 and several FA genes were all found as synthetic lethality hits with BRCA1-deficient, BRCA1I26A, BRCA1R1699Q. And the known genes such as TP53BP1 and ATMIN were found beneficial for survival in the all three screens. If BRCA1I26A exhibits vulnerabilities similar to BRCA1-proficient cells, then why the known hits in the screen are same with BRCA1-deficient cells. Loss of NDE1 is specifically toxic to cells expressing BRCA1R1699Q. Did you find any target that specifically toxic to cells expressing BRCA1I26A?

    It is hard to tell whether your conclusion is correct. Of course, the three cells have some same and also different genetic background, you may consider how to separate the difference. Separate the difference will benefit the treatment for patients with different BRCA1 mutation background.

    Minor concerns:

    1. RPE1 hTERT P53-/- BRCA1-/- cell line has very clear BRCA1 depletion background. Why at the beginning, you did not choose to use RPE1 hTERT P53-/- BRCA1-/- cells reconstituted with BRCA1 wildtype, BRCA1R1699Q or BRCA1I26A mutants to perform the CRISPR screens? What are the advantages of your strategy by first depletion of BRCA1 with auxin and then inducible express BRCA1 constructs? It looks much more complicated.
    2. In Supplemental Figure 2C, a blot to detect BRCA1 should be included.

    Significance

    If the conclusions are correct, the new findings will tell the importance to differentiate between patient-derived mutations when assessing novel treatment targets.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Exploiting synthetic lethality based on functional correlations has the potential to significantly improve the survival of cancer patients by reducing resistance to targeted therapies and increasing anti-tumour efficacy when combined with other treatment modalities. Schreuder et al., aim to identifying novel vulnerabilities of patient-derived mutations that could improve patient stratification based on a specific genetic background. Precisely, they established a model system to perform a genome-wide CRISPR-Cas9 KO screen to identify genomic vulnerabilities of BRCA1 variants with reported hypomorphic phenotypes, namely BRCA1 R1699Q and BRCA1 I26A in engineered RPE1 hTERT cells with AID tag. Using this system the authors were able to confirm known synthetic lethal genes reported in literature (e.g. APEX2, PARP1, POLQ) comparing cells with acute BRCA loss and BRCA1 deficiency. Moreover, the screen identified two genes, CSA and GPX4 that were not previously described as synthetic lethal with BRCA1 loss. What is potentially interesting, but marginally explored, is the identification of a unique synthetic vulnerability of cells expressing BRCA1 R1699Q mutant and NDE1 gene encoding for a dynamic scaffold protein essential in neocortical neurogenesis and heterochromatin patterning by H4K20me3, whose loss of function results in nuclear architecture aberrations and DNA double-strand breaks (Chomiak et al., iScience 2022). Accordingly, cells ablated of NDE1 and expressing BRCA1 R1699Q mutant show less proliferation of cells expressing either BRCA1 WT or BRCA1-depleted. Furthermore, cells lacking NDE1 show increased genomic instability by means of increased micronuclei and anaphase bridges compared to BRCA1 proficient and BRCA1 R1699Q mutant.

    Major comments:

    1. The authors claim that cells expressing BRCA1-I26A are largely HR-proficient, based on a milder effect on Olaparib sensitivity compared to cells expressing BRCA1-R1699A (Fig. 1C). However, I26A mutant cells are defective in RAD51 IRIF (Fig. 1B), indicative of an HR defect. Recently it has been shown that BRCA1 RING mutations that do not impact BARD1 binding, including I26A, render BRCA1 unable to accumulate to DNA damage sites and unable to form RAD51 foci when such mutation is combined with mutations that disable RAP80-BRCA1 interaction (Sherker et al., 2021). How do the authors explain this discrepancy with the literature?
    2. The reduction in survival following CSA depletion in BRCA1-proficient vs. -deficient cells is only 20% (Figure 2 and S2B). In my opinion, such a minor difference is not supporting the notion of a SL interaction between BRCA1 and CSA. To substantiate CSA as synthetic lethal hit, I would recommend the authors comparing the effect of CSA loss to that of EXO1 or BLM loss, both genes recently identified by the same group as SL partners of BRCA1 using the same experimental screening set up (van de Kooij et al, 2024). Moreover, validation data for GPX4 is missing.
    3. Similar to the minor effect observed for CSA, DOT1L and OTUD5 depletions caused rather mild and/or divergent phenotypes between the two sgRNAs used (Figure 4B), rather arguing against robust SL interactions between those genes and BRCA1 deficiency that could be therapeutically exploited.
    4. To strengthen their conclusion in Figure 4C the authors should perform complementation experiments with NDE1 WT and, ideally, with NDE1 mutant(s). On a related note, are NDE1 knock-out cells expressing BRCA1-R1699A more sensitive to PARPi?
    5. Graphs shown in Fig. 1A-C, Fig. 4B, S2D, S3B, S3E and S3F are lacking proper statistical analysis of the differences. Some experiments have only been repeated twice (e.g. Figure 1C), precluding running statistical tests.

    Minor comments:

    1. The authors should include representative images for results shown in Fig.1 A-C
    2. The authors should add immunoblots for BRCA1 in Fig. S2C to indicate successful BRCA1 cDNA complementation in HCC1937 cells.
    3. Most numbers in the Venn diagram shown in Figure 3A cannot be read when printed.
    4. In the western blots shown in Supplemental Figure 1A, the electrophoretic mobility of BRCA1 variants expressed in RPE1 is quite variable. Could the authors indicate in the Figure (e.g. with arrowheads) which bands represent endogenous and which transgenic BRCA1. Moreover, in BRCA-wt complemented cells there are two bands following auxin/DOX addition, whereas there is one band observed in cells expressing BRCA1 hypomorphic variants
    5. Line 229 please correct "BRCA1-proficient" to "BRCA1-depleted".

    Significance

    General assessment:

    This manuscript starts with an attractive hypothesis, which is the generation of a cellular system to study patient-derived hypomorphic BRCA1 missense mutations rather than using BRCA1 knockout cells. Performing CRISPR/Cas9-mediated genome-wide synthetic lethal screens in this system allowed uncovering genetic vulnerabilities of cells expressing BRCA1-R1699A, a pathogenic mutant identified in several cancer patients. The data are of good quality and the manuscript is coherent and generally well written (few typos). However, some data describe mainly negative results (e.g. BRCA1-I26A mutant) or weak phenotypes while other more interesting aspects are not rigorously exploited (e.g. NDE1 SL) and therefore need to be interpreted with more caution and extended by additional experiments.

    Advance:

    BRCA1-R1699Q is classified as a pathogenic variant despite its low penetrance and intermediate cancer risk in breast and ovarian cancer compared to other variants. A recent case report highlighted the unique clinical outcome of a patient with the BRCA1 R1699Q variant, suggesting that this variant may differ from others in terms of cancer risk and drug response (Saito et al., Cancer Treatment and Research Communications 2022). These findings underscore the need for further studies to confirm these observations and to elucidate the specific mechanisms underlying the response to platinum agents and PARP inhibitors in patients with the BRCA1 R1699Q variant. The manuscript proposed by the authors has the potential to help understanding how BRCA1 missense mutations can contribute to determine treatment sensitivity and pave the way to patient stratification.

    Audience:

    This manuscript is suitable for a specialized, basic research audience.

  4. Version published to 10.1101/2024.05.24.595688v1 on bioRxiv