Exploiting a metabolic vulnerability in brain tumour stem cells using a brain-penetrant drug with safe profile

  1. Audrey Burban
  2. Cloe Tessier
  3. Mathis Pinglaut
  4. Joris Guyon
  5. Johanna Galvis
  6. Benjamin Dartigues
  7. Maxime Toujas
  8. Mathieu Larroquette
  9. H Artee Luchman
  10. Samuel Weiss
  11. Nathalie Nicot
  12. Barbara Klink
  13. Macha Nikolski
  14. Lucie Brisson
  15. Thomas Mathivet
  16. Andreas Bikfalvi
  17. Thomas Daubon
  18. Ahmad Sharanek

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Abstract

Glioblastoma (GB) remains one of the most treatment refractory and fatal tumour in humans. GB contains a population of self-renewing stem cells, the brain tumour stem cells (BTSC) that are highly resistant to therapy and are at the origin of tumour relapse. Here, we report, for the first time, that mubritinib potently impairs stemness and growth of patient-derived BTSCs harboring different oncogenic mutations. Mechanistically, by employing bioenergetic assays and rescue experiments, we provide compelling evidence that mubritinib acts on complex I of the electron transport chain to impair BTSC stemness pathways, self-renewal and proliferation. Global gene expression profiling revealed that mubritinib alters the proliferative, neural-progenitor-like, and the cell-cycling state signatures. We employed in vivo pharmacokinetic assays to establish that mubritinib crosses the blood-brain barrier. Using preclinical models of patient-derived and syngeneic murine orthotopic xenografts, we demonstrated that mubritinib delays GB tumourigenesis, and expands lifespan of animals. Interestingly, its combination with radiotherapy offers survival advantage to animals. Strikingly, thorough toxicological and behavioral studies in mice revealed that mubritinib does not induce any damage to normal cells and has a well-tolerated and safe profile. Our work warrants further exploration of this drug in in-human clinical trials for better management of GB tumours.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript has potential interest as a preclinical study for glioblastoma treatment. There is a substantial amount of data that is promising, but there are numerous issues that will require additional experimental effort before publication.

    Major concerns:

    1. The title is overly general and uninformative. The authors should include the drug name (mubritinib) and the specific tumour type (glioblastoma).
    2. The concept that mubritinib functions through metabolic effects is not surprising given recent publications (PMID: 37382244; PMID: 35429141; PMID: 33245718; PMID: 31287994) and that it impacts the blood-brain barrier (PMID: 36178590). However, there are limitations to the strength of this observation. Most of the experiments are associations between drug treatment and metabolic changes. The NDI1 partially rescue experiments in Figures 1h and 1i are nice but show that NDI1 expression itself increases OCR. This experiment is also performed over a very brief window of time. A better set of experiments would include measurement of cell number over a prolonged time course (Figure 2d has one time point) and to use a genetic targeting strategy against ETC complex 1.
    3. The authors observe that EGFR expressing lines are more sensitive to mubritinib. As the rescue experiments are only partially effective and ERBB family members may be targeted by mubritinib, it is critical to address the effects of mubritinib on EGFR activation and perform rescue studies, as the application of mubritinib in patients may be guided by the EGFR mutational state.
    4. The differences found with NSC responses is interesting but needs to be developed. Why are there differences in mubritinib responses? For example, do NSCs not require ETC complex 1 as much?
    5. I would suggest that the authors also compare sensitivity of the BTSCs (I would suggest a change in nomenclature as these are only from GB) and differentiated tumour cells to determine if the stem cells have greater dependence. Please use similar culture conditions.
    6. The differences in cell cycle are useful but the mechanism is lacking. The claim that self-renewal is drastically or markedly changed is overstated. The ELDAs are not striking. There is no evidence that stemness is a direct target.
    7. The in vivo effects on cell biology need greater analysis in mechanism. I am also not sure why the authors switch lines tested in different assays.
    8. The mechanism of interaction with radiation is not developed. What is happening here? Are there changes in DNA damage repair or simply growth? This is a nice observation that could be better developed.

    Minor concerns:

    1. Grammar needs attention.
    2. Please remove the overuse of "strikingly", "drastically", "importantly", etc. Most of these descriptions are overstated.
    3. The number of in vivo replicates needs to be addressed.
    4. All gene expression data should be deposited. All raw data (numeric) should be made available.
    5. Please replace all normalized data with raw data. The statistical testing was likely incorrectly performed, and this can give rise to false conclusions. I am particularly concerned about the normalization to cell numbers.

    Significance

    This manuscript has potential interest as a preclinical study for glioblastoma treatment. There is a substantial amount of data that is promising, but there are numerous issues that will require additional experimental effort before publication.

    Major concerns:

    1. The title is overly general and uninformative. The authors should include the drug name (mubritinib) and the specific tumour type (glioblastoma).
    2. The concept that mubritinib functions through metabolic effects is not surprising given recent publications (PMID: 37382244; PMID: 35429141; PMID: 33245718; PMID: 31287994) and that it impacts the blood-brain barrier (PMID: 36178590). However, there are limitations to the strength of this observation. Most of the experiments are associations between drug treatment and metabolic changes. The NDI1 partially rescue experiments in Figures 1h and 1i are nice but show that NDI1 expression itself increases OCR. This experiment is also performed over a very brief window of time. A better set of experiments would include measurement of cell number over a prolonged time course (Figure 2d has one time point) and to use a genetic targeting strategy against ETC complex 1.
    3. The authors observe that EGFR expressing lines are more sensitive to mubritinib. As the rescue experiments are only partially effective and ERBB family members may be targeted by mubritinib, it is critical to address the effects of mubritinib on EGFR activation and perform rescue studies, as the application of mubritinib in patients may be guided by the EGFR mutational state.
    4. The differences found with NSC responses is interesting but needs to be developed. Why are there differences in mubritinib responses? For example, do NSCs not require ETC complex 1 as much?
    5. I would suggest that the authors also compare sensitivity of the BTSCs (I would suggest a change in nomenclature as these are only from GB) and differentiated tumour cells to determine if the stem cells have greater dependence. Please use similar culture conditions.
    6. The differences in cell cycle are useful but the mechanism is lacking. The claim that self-renewal is drastically or markedly changed is overstated. The ELDAs are not striking. There is no evidence that stemness is a direct target.
    7. The in vivo effects on cell biology need greater analysis in mechanism. I am also not sure why the authors switch lines tested in different assays.
    8. The mechanism of interaction with radiation is not developed. What is happening here? Are there changes in DNA damage repair or simply growth? This is a nice observation that could be better developed.

    Minor concerns:

    1. Grammar needs attention.
    2. Please remove the overuse of "strikingly", "drastically", "importantly", etc. Most of these descriptions are overstated.
    3. The number of in vivo replicates needs to be addressed.
    4. All gene expression data should be deposited. All raw data (numeric) should be made available.
    5. Please replace all normalized data with raw data. The statistical testing was likely incorrectly performed, and this can give rise to false conclusions. I am particularly concerned about the normalization to cell numbers.
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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript Burban et al explore the effect of the mitochondrial oxidative phosphorylation (OXPHOS) inhibitor Mubritinib on patient derived glioblastoma stem cells and murine xenografts. The authors first show that i) Mubritinib is an inhibitor of OXPHOS in brain tumor stem cells (BTSC), ii) that it impairs cell growth and self-renewal of patient-derived BTSC with different genetic background and iii) has an effect on the expression of genes related to stemness. In addition, the authors convincingly show that Mubritinib is a brain penetrant drug, and by transplanting luciferase expressing BTSC into the brain of immunodeficient mice they show it delays GB tumorigenesis and the animal lifespan (either alone, or more efficiently if combined with IR treatment). Finally, by performing toxicological and behavioral studies in mice models, Burban et al demonstrate that Mubritinib has a well-tolerated and safe profile and does not induce damage to healthy cells.

    The manuscript is well written and organized and the data is clearly presented. The results are convincing, but a few additional experiments and controls would be beneficial to support the claims of the paper, most of which are easily addressable.

    Main comments:

    • Finding suitable control cells for BTSC experiments is a widely acknowledged challenge in the field. However, in line with other studies, it is recommended that the authors consider using a non-oncogenic NSC as control line to demonstrate that the effects reported in Figure 1 and Figure 2 are more pronounced in BTSC compared to NSC (as it was done in Suppl Fig3).
    • Figure 5 presents a significant finding indicating that Mubritinib enhances the sensitivity of GB tumors to IR. Considering that Temozolomide (TMZ) is the primary chemotherapy drug for GB patients, it would be crucial to investigate the potential outcomes of a combined treatment involving Mubritinib and TMZ. This will help determine if the combination exhibits promising results, comparable to what is demonstrated in Figures 5d, 5g, and 5i for Mubritinib and IR. Such experiment would reinforce the drug's potential for clinical trials in GB treatment.

    Minor comments:

    • The authors should specify early in the paper what is the number of samples of patient-derived BTSC they use and the fact that their genetic mutations are known (this information is summarized in the supplemental table 1, but only reported later in the manuscript). This information is important and should be clearly stated at the beginning of the manuscript.
    • In Figure 2a the inhibition at 20nM is significant but not very pronounced. Based on Figure 1 I would have expected to see a stronger effect at this concentration range. Can the authors comment/provide an explanation for this discrepancy?
    • The EdU incorporation experiment presented in Figure2h-I should be repeat with lower concentrations of the drug (in most of the assays the effect of Mubritinib is detectable at much lower concentrations).
    • Since the authors have done RNA-seq on the samples why don't they report the specific subtypes of their samples in the text and in Suppl Table 1 (Proneural, Neural, Classical or Mesenchymal) ? It is known that different molecular subtypes respond differently to treatments; therefore this information would be essential to understand if Mubritinib is effective on a wide range of GB subtypes.
    • In Figure2b and Supplemental Figure1b-c : instead of correlating the effect with genetic mutations, it would be more relevant if the authors could correlate the data with the molecular subtypes inferred by RNA-seq (see my comment above)
    • Regarding the RNA-seq experiment the authors should report what is the percentage (and numbers) of genes that change expression. Is there for example a preference for up- or down- regulation? It would be interesting to see a Gene Ontology (GO) analysis for the up-regulated genes versus a GO analysis of the down-regulated genes to confirm that the relevant categories show dysregulation as expected (e.g. enrichment for cell cycle and stemness genes in the down-regulated list, etc ).
    • In Figure2 m-o the difference between CTL and Mubritinib treated cells do not seem substantial, although it is shown as statistically relevant. Can the authors specify the percentage to be able to better assess the differences?
    • Add p-value for Figure3a-c
    • In the western blot in Supplemental Figure 3b Vinculin shows twice
    • Change" Given that Mubritinib is already completed a phase I clinical trial" into "...has completed..."

    Referees cross-commenting

    I agree with other reviewers that more data is needed to determine if mubritinib could be an effective treatment for various GB subtypes. The models used in this study do not encompass the full spectrum of GBM genetics. The authors should repreat the experiments using models that represent the major genetic/transcriptional subtypes of GBMs and clearly label and identify them in the study. Specifically, the authors should include models like 'classical/EGFR-amplified', 'mesenchymal', 'proneural/PDGFR amplified'. Alternatively, it is advisable to refrain from asserting that mubritinib is effective across genetic alterations in the manuscript.

    Significance

    Considering the limited effectiveness of existing treatments, it is crucial to explore alternative approaches to improve patient outcomes. This study demonstrates promising potential for the clinical translation of Mubritinib in GB treatment.

    A major limitation of this study is the narrow numbers of patient-derived samples used and absence of a proper control cell line. Unfortunately, as evidenced by the existing literature in the field, selecting a control cell line for glioma stem cells research is challenging due to the unknown cell-of-origin for this type of tumor. In addition, all the toxicity/safety tests were performed in mice models and it is difficult to predict how this would translate into human patients. However, the fact that a phase I clinical trial has already been completed for Mubritinib (in the context of a different type of tumor) is encouraging.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors report the use of mubritinib, a drug targeting complex I of the mitochondrial electron transport chain, to halt the proliferation of brain tumor stem cells (BTSCs) isolated as neurospheres from glioblastomas. They demonstrate that mubritinib crosses the blood-brain barrier, and show that this drug delays GBM tumorigenesis and extends lifespan in mouse models that were generated by transplantation of human BTSCs or mouse cell lines. They also provide evidence that this potentially harmful drug is well-tolerated by mice.

    The ability of mubritinib (initially conceived as a ERBB2 inhibitor) to block complex I of the electron transport chain was previously identified in acute myeloid leukemia, where this drug proved to selectively inhibit a subset of cases relying on oxidative phosphorylation (OXPHOS). However, the use of mubritinib is novel in GBM, a lethal malignancy for which a few therapeutic options are available, and no substantial progress has been reported since 2005. In addition, the authors show that accumulation of mubritinib in the brain tissue allows for the use of a reduced dose of mubritinib, thereby reducing the risk of the deleterious effects that blunt enthusiasms about targeting of mitochondrial respiration.

    However, this manuscript presents significant weaknesses that are detailed below. In particular, the models used appear insufficient to support the conclusion, stated in the abstract, that the drug can be effective in GBMs with different oncogenic mutations. Based on the provided evidence, the claim that 'mubritinib potently impairs stemness and growth of patient-derived BTSCs harboring different oncogenic mutations' should be removed, or supported by using BTSCs that adequately represent the major GBM subtypes identified by genetic and transcriptional analysis. Specifically, BTSCs harboring EGFR amplification (displayed by 40% of GBMs) would need to be added. Equally, it is inappropriate to conclude (Discussion, page 14) that the models used, displaying EGFR mutations, are representative of widespread EGFR alterations: indeed the EGFR alteration observed in 40-50% of patients is EGFR amplification, whose pathogenic effects can not be recapitulated by the EGFR mutation harbored in the models used. If models harboring EGFR amplification are unavailable to these authors, making unrealistic to repeat the experiments in at least two different EGFR-amplified BTSCs in the timeframe allowed for revision, not only the claim that mubritinib inhibits BTSCs harboring different oncogenic mutations should be removed, but lack of experiments in EGFR-amplified BTSCs should be discussed as a limitation of this study. In addition, as detailed below, in vitro experiments should be expanded to corroborate mechanistic aspects of the drug, safety should be better demonstrated and some aspects of in vivo experiments should be clarified. Overall, the methodology seems sufficiently detailed to allow reproduction. The experiments are adequately repeated and the statistical analysis is appropriate, but in one case detailed below.

    Major Point N.1

    Figure 2a and 2c. Although statistically significant, the effect of mubritinib at 20 nM is biologically of limited significance in a subset of BTSCs, where the drug reduces viability by less than 25% (Fig. 2a). Therefore, the correlation between oxygen consumption rate (OCR) and mubritinib sensitivity (% of live cells), shown in Fig. 2c, should be presented for all mubritinib doses, and particularly for the dose of 500 nM, which is utilized in subsequent experiments. Additionally, it would be useful to display the correlation not only between viability and basal OCR but also with maximal OCR. This analysis could identify varying levels of sensitivity to ECT inhibition, aligning with the expectation that different BTSCs may exhibit varying degrees of dependency on mitochondrial OXPHOS. This would suggest that different GBMs may require different dosages of the drug. In all the experiments presented in the manuscript, the authors use the lines exhibiting the highest mubritinib sensitivity (BTSC 53, BTSC73), which might not be representative of all GBMs. This selection bias need explicit clarification in the text.

    Major Point N.2.

    In Fig. 2b, the statistics is significantly biased as it is calculated based on technical replicates, rather than on a significant number of independent models featuring either wild-type or mutated EGFR. Presented in this manner, this analysis is unacceptable. Additionally, as noted previously, the models used in this manuscript do not represent the overall GBM genetics, particularly due to lack of EGFR amplified models, which correspond to 40% of cases, and cannot be recapitulated by EGFR mutations. In general, the number of models is too small to draw any conclusion regarding the relationship between genetics and mubritinib sensitivity (including conclusions concerning TP53, or the MGMT status, shown in supplementary figures 1b-c). If the authors intend to claim that GBMs are sensitive to mubritinib independently of the genetic status, they should repeat their experiments by using models representative of the major genetic/transcriptional subtypes of GBMs, by clearly characterizing and identifying them in the experiments: e.g. 'classical/EGFR-amplified'; 'mesenchymal'; 'proneural/PDGFR amplified'. Otherwise (more realistically), it is suggested to remove claims that mubritinib is effective independently of genetic alterations throughout the manuscript (including the abstract and the discussion).

    Major Point N.3

    Fig. 2k-l present a transcriptional analysis with questionable representativeness as it is performed on the single line BTSC147 from a recurrent GBM, which is unlikely to represent primary GBMs. The analysis appears overly descriptive and fails to add significant information beyond the observation that mubritinib induces a proliferative arrest, as assessed in biological experiments. Additionally, the claim that the Neftel 'Neural Progenitor Cell' signature is altered in a biologically significant manner after only 24 hours of mubritinib treatment seems questionable. As such, this analysis should be moved to supplementary information. A more intriguing alternative would be to compare groups of BTSCs that exhibit high or low sensitivity to mubritinib and attempt to identify gene sets that can correlate with and possibly contribute to explain differences in drug sensitivity.

    Major point N. 4

    Figure 3. LDA need to be measured at longer timepoints (14-21 days vs. 7 days shown).

    Major point N. 5

    Supplementary Figure 3c aims to demonstrate that neural stem cells are unaffected by mubritinib merely by showing stem markers in western blots, which is insufficient. To provide convincing evidence, an LDA should be performed using human Neural Progenitor Cells.

    Major Point N. 6

    Page 9. The mechanistic nexus between OXPHOS inhibition and radiosensitization described by the authors remains unclear. In particular, the link between enrichment in OXPHOS proteins observed in recurrent vs.primary GBMs on the one hand, and downregulation of homologous recombination related-pathways on the other hand is difficult to grasp. The authors should endeavor to more clearly explain how mubritinib can interfere with the adaptive response to ionizing radiation, thereby providing a rationale for experiments combining the two treatments. As noted in the discussion (page 14), 'targeting mitochondrial respiration is an emerging strategy to overcome radioresistance in the tumour hypoxic areas'. Thus, a plausible mechanism of mubritinib-induced radiosensitization may involve reducing oxygen consumption, thereby leaving more oxygen available for diffusion and improving radiation response (by increasing generation of reactive oxygen species). To provide convincing mechanistic evidence, the authors should include in vitro experiments assessing the ability of mubritinib to radiosensitize BTSC in both normoxic and hypoxic conditions. LDA or radiobiological clonogenic assays showing the effect of combination treatment on stem cell frequency are recommended.

    Major Point N. 7

    Fig. 5d. Concerning the scheme of in vivo treatment, it is unclear why irradiation is administered 5 days after the beginning of mubritinib treatment, considering that mubritinib reaches its peak brain concentration much earlier, as shown in Fig. 4d. Furthermore, if mubritinib alone is effective against the tumor, comparing tumors that have been treated with IR alone at the same time-point as those treated with IR + mubritinib seems inappropriate. This is because, in the latter scenario, IR is applied to tumors that are likely reduced in volume compared to those treated with vehicle prior to IR. This discrepancy could introduce bias in the evaluation of the combined treatment's efficacy.

    Major Point N. 8.

    Figure 6b. The methodology employed to measure the effect of mubritinib on human neural progenitor cells should be the same as that used for BTSC. Moreover, a positive control (a treatment inducing death, such as bosentan for hepatocytes) needs to be added.

    Minor points

    Minor point N.1

    Please use consistent units (nM or uM) for mubritinib.

    Minor point N.2

    ND1 expression in transduced cells should be shown by western blot (in Supplementary Figures).

    Significance

    This manuscript reports preclinical results on the use of mubritinib, a drug targeting the mithochondrial electron chain transport complex 1, to halt proliferation of glioblastoma (GBM) stem cells in vitro and treat experimental GBMs generated by stem cell transplantation. Given that the standard therapy for GBM still relies on a limited number of conventional options, evidence demonstrating the preclinical effectiveness of mubritinib could exert a significant translational impact. Mubritinib has not yet been proposed for GBM treatment, but there is convincing evidence that the drug may be effective in subsets of acute myeloid leukemia that rely on oxidative phosphorylation (Baccelli et al., Cancer Cell 36:84, 2019. PMID: 31287994). Data provided in this manuscript on potential effectiveness of mubritinib are overall convincing. However, in its current form, the manuscript present major limitations regarding the representativeness of models used, which do not support the claim that mubritinib could be universally useful in GBM, and regarding mechanistic aspects of mubritinib combination with radiotherapy. These and other aspects could be addressed through additional experiments.

    The audience interested in the reported findings includes preclinical and clinical neuro-oncologists. My field of expertise is biology and genetics of glioblastoma stem cells and generation of in vitro and in vivo GBM preclinical models.

  5. Version published to 10.1101/2024.01.15.574967v1 on bioRxiv