CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

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   Dec 18, 2023

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   Reply to the reviewers

   1. General Statements [optional]

   We are happy to receive the comments from the reviewers and grateful for their suggestions on how to improve our manuscript. We note that both reviewers find the work extensive and meaningful.

   Based on the comments from the reviewers, we have performed a comprehensive set of additional experiments, which will result in one additional figure and a substantial restructuring of two figures with new data, considerably expanding both the preclinical as well as the mechanistic findings of our manuscript.

   In short, reviewer 1 finds that we have done extensive work to understand the role of CDK12/CDK13 in glioblastoma and would …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   1. General Statements [optional]

   We are happy to receive the comments from the reviewers and grateful for their suggestions on how to improve our manuscript. We note that both reviewers find the work extensive and meaningful.

   Based on the comments from the reviewers, we have performed a comprehensive set of additional experiments, which will result in one additional figure and a substantial restructuring of two figures with new data, considerably expanding both the preclinical as well as the mechanistic findings of our manuscript.

   In short, reviewer 1 finds that we have done extensive work to understand the role of CDK12/CDK13 in glioblastoma and would like to see additional mechanistic details. Reviewer 2 recognizes the value of our work in exploring the potential usefulness of CDK12/13 inhibition in treatment of aggressive brain tumors and would like to see additional experiments, which demonstrate the efficacy of CDK12/13 inhibition in complex environments to reinforce our proof-of-concept.

   To address this feedback, our response plan includes two lines of experiments, which will strengthen both the preclinical and mechanistic parts of our work:

   1. A) We have established a migration assay using GSC G7 in organotypic mice brain slices and tested the effect of CDK12/CDK13 inhibition on glioma migration and we will include these data in the revised manuscript.
   2. B) To further understand the mechanisms involved in the transcriptional inhibition following CDK12/CDK13 inhibition on DNA replication in glioma cells, we have performed the following additional experiments:

   • Comparative mass-spectrometry to identify changes in the total and phospho-proteome. This revealed that major regulators of DNA replication and repair are impaired following CDK12/CDK13 inhibition.
   • iPond (Identification of proteins on nascent DNA) assays that demonstrate that CDK12/CDK13 inhibition changes the composition of replication forks, with a strong reduction PCNA abundance early after treatment. PCNA tethers the DNA polymerase catalytic unit to the DNA template ensuring rapid and processive DNA synthesis. This reduction of PCNA occurs before EdU incorporation/DNA replication is reduced, suggesting that loss of DNA polymerase clamping and processivity explains the subsequent arrest of DNA replication.
   • DNA fiber assays showing that the origin firing is heavily downregulated in GSCs following CDK12/CDK13 inhibition. Further analyses using immunofluorescence microscopy reveal that the markers of DNA damage response and cell cycle progression are not affected following CDK12/CDK13 inhibition at early time-points, thereby ruling out activation of cell-cycle checkpoints and/or DNA damage response as potential explanation for replication block in GSCs following CDK12/CDK13 inhibition. The results from these experiments strengthen our main findings that inhibiting CDK12/CDK13 has a potential therapeutic value in glioblastoma treatment. Our work also offers mechanistic insights into how the glioblastoma stem cells have acquired transcriptional addiction to CDK12/CDK13 involving phosphorylation of RNAPII CTD, nascent RNA synthesis and DNA replication dependent on CDK12/CDK13 activity.

   2. Description of the planned revisions

   A point-by-point plan in blue is described below.

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   *The authors in this manuscript studied the role of a transcriptional cyclin-dependent kinase CDK12/CDK13 in glioblastoma. These cyclin-dependent kinases phosphorylate at ser2 residue in the C-terminal of RNA Pol II. Pharmacological inhibition of CDK12/CDK13 kinase with inhibitor decreases cell proliferation in multiple glioma cell lines and in patient-derived organoids. The CDK12/CDK13 inhibitor also reduces tumor growth in a mouse xenograft model. Mechanistically, the authors showed that genome-wide inhibition of CDK12/CDK13 attenuates RNA Pol II phosphorylation, disrupting transcriptional elongation and decreasing cell cycle progression. So, the authors proposed that targeting CDK12/CDK13 kinases can be used as a therapeutic strategy in glioblastoma. The authors have done extensive work in this manuscript to understand the role of CDK12/CDK13 in glioblastoma, but it is still a descriptive paper lacking mechanistic details.

   RESPONSE: We appreciate the reviewer’s recognition of the extensive efforts behind this manuscript, and we are thankful for being pointed towards strengthening the mechanistic insights. In brief, we would like to corroborate our key findings that inhibition of CDK12/CDK13 abrogates RNAPII phosphorylation, nascent RNA synthesis and DNA replication. We have expanded the mechanistic characterization using the following experiments:

   • Using DNA fiber assay, we find that origin firing is heavily downregulated in GSCs following CDK12/CDK13 inhibition. Furthermore, we have done in-depth characterization of the effect of THZ531 treatment on cell cycle regulators and DNA damage response in GSCs, and found that these were not affected by CDK12/CDK13 inhibition within six hours. This indicates that activation of a cell cycle checkpoint or DDR machinery was not the reason for replication block.
   • To further characterize the rapid effect of CDK12/CDK13 inhibition, we have done comparative mass spectrometry following CDK12/CDK13 inhibition in GSCs to identify changes in total and phosphorylated proteins and identified major regulators of DNA replication and repair machinery that are strongly affected.
   • We have implemented iPOND (identification of proteins on nascent DNA) to study the effect of CDK12/CDK13 inhibition on protein composition at the replication fork. On this basis, we find that the abundance of the DNA clamp PCNA is substantially reduced after two hours of THZ531 treatment. PCNA tethers the DNA polymerases together on the fork and adds processivity to the speed of DNA replication. EdU incorporation was not affected by two hours of THZ531 treatment, and loss of PCNA from the replication fork is a likely explanation for the DNA replication block observed after six hours of THZ531 treatment.

   *Comments:

   1. Figure 1 shows that CDK12/CDK13 inhibitor decreases cell viability, colony-forming ability, cell competition assay, and cell migration. The rationale behind choosing CDK12/CDK13 inhibitor in glioma is unclear from the manuscript. What is the CDK12/CDK13 expression in multiple glioma cells vs non-glioma cells? The authors should include normal astrocytes as a control for cell viability assay. The p value is missing in numerous Figure panels. *

   RESPONSE: We have investigated the possibility of targeting transcriptional regulation in glioma cells by using inhibitors targeting transcriptional cyclin-dependent kinases which included CDK7, CDK9 and CDK12/CDK13.

   • We found that glioma cell proliferation was most sensitive to CDK12/CDK13 inhibitors compared to other cancer cells (Figure 1A), whereas there was no specificity for CDK7 and CDK9 inhibitors on glioma cell proliferation compared to other cancer cells (Supplementary figure 1D). The selective inhibition of glioma cells by CDK12/CDK13 inhibitors was the rationale for choosing CDK12/CDK13 inhibitors for further studies. This is mentioned in the introduction, and the result section has been updated to reflect this.
   • We have performed expression analyses of CDK12/CDK13 at the mRNA levels using RT-qPCR in the cell lines that are used in the study, and we did not find any correlation of CDK12/CDK13 expression in glioma versus non-glioma cells (Supplementary figure 1B). Thus, the propensity of cells to become addicted to CDK12/13 signaling for their survival seems not related to total transcript levels, but must rely on the function of CDK12/CDK13 as a selective regulator of transcriptional program required for glioblastoma proliferation.
   • We will perform the cell viability assays on normal astrocytes.
   • p-values will be added in the figure panels.

   • Figure 2A shows the expression of CDK12 by immunohistochemistry in glioblastoma tissues. Including the non-glioma tissue samples as another control and including a quantification graph with the statistics is essential. In Figure 2B-D, the authors discussed the treatment of glioma patient-derived organoids with CDK12/CDK13 inhibitors. From the Figure, the organoids are resistant to THZ531 and SR-4835 inhibitors. To rule out this possibility, the immunoblot assay with cleave PARP will be essential to execute. Again, statistics need to be included in Figure 2C-D. *

   RESPONSE: We want to point out that the immunohistochemistry for non-glioma tissue and additional controls are shown in Figure 2A, top right panel and supplementary Figure 2A.

   Regarding the next statement, we do not think that there is any indication that the organoids (GBOs) are resistant to THZ531 and SR-4835. We would like to stress that data presented on Fig 2B-D shows the efficacy of THZ531, abemaciclib and SR-4835 inhibitors in GBOs. GBOs showed high resistance only to lomustine. We apologize for any part of the figure which may lack clarity and lead to potential misconceptions. We would very much like to improve on this, if we are able to identify which figure component that may give the impression that the organoids are resistant to THZ531 and SR-4835. One option would be to remove the 0 hr time point in Figure 2B, if that is the cause for misinterpretation. To emphasize the drug efficacy better, we plan to perform the following amendments to the revised manuscript:

   • We will provide statistical analysis of the IC50 and AUC analysis in the supplementary table xxx. These analyses will further highlight the robustness of the evaluation of drug responses in comparison to lomustine.
   • We will provide one-way Annova comparison of the efficacy of the four assessed drugs in Fig 3D.
   • The cell viability assay applied in GBOs is based on the CellTiterGlow technology, which is applicable to small organoid cultures of

   • The mouse subcutaneous xenograft experiment was carried out in U87 cells with CDK12/CDK13 inhibitors. However, the glioma stem cells are a more appropriate model for glioma biology, and it is not clear why authors suddenly chose U87 cells. Again, statistics are absent in multiple sub-panels. *

   *RESPONSE: We note reviewer’s acknowledgement of using GSCs as a more appropriate model for glioma biology and we want to emphasize that in this work, we have used 15 different glioma patient derived glioma cells (11 GSCs in Figure-1 and 4 GBOs in Figure-2) from two different research environments to show that CDK12/CDK13 inhibition compromises glioma proliferation in vitro. GSCs/GBOs used in our study are xenografted orthotopically in the brain to model glioma in vivo and since our drugs do not sufficiently cross the BBB, the GSCs/GBOs were not considered for the in vivo validation and instead, a subcutaneous xenograft model was best to assess the efficacy of the drug(s). Considering that these models require a high number of cells (eight million cells per xenograft were used in our experiment), we had to base our decision on feasibility and chose a type of cells that could be propagated to the required extent. Considering the reviewer’s criticism, we are open to moving the xenograft data are presented to the supplementary section. Appropriate statistics will be done and shown.

   • The authors have performed CUT & RUN experiments in G7 cells with CDK12/CDK13 inhibitors and decided to use 1hr and 6hr time points for the assay. Although the inhibitor THZ531 is supposed to inhibit RNA Pol II phosphorylation at the Ser2 residue, it decreases the Pol II phosphorylation at the ser5 residue quite a bit. Therefore, it is crucial to determine the effect coming from ser2 vs ser5 phosphorylation and gene expression regulation. **

   RESPONSE: This is a good point. To address the relationship further, we will perform quantitation of Ser2 and Ser5 signals as well as the changes in these over time. We will then correlate this to the transcriptional changes to assess which of the relationships that are most strongly correlated. In addition, we will perform non-parametric statistical testing of significance of ranked data.

   • There are a lot of supplementary Figures where axes are not labeled correctly or missing. **

   RESPONSE: This will be addressed.

   • The statistical section needs to be included in the manuscript. **

   RESPONSE: This will be included.

   *Reviewer #1 (Significance (Required)): **

   In this manuscript, the authors studied the role of CDK12/CDK13 in glioblastoma and performed extensive studies to uncover the importance of these kinases in glioblastoma. Understanding more mechanistic details of how these kinases are involved in glioma progression will uncover more therapeutic opportunities in glioblastoma.

   *Reviewer #2 (Evidence, reproducibility and clarity (Required)): **

   *Summary: ** Lier et al. present a set of results showing that pharmacological inhibition of CDK12/13, cyclin-dependent kinases that phosphorylate RNA polymerase II (RNAPII), alters the proliferative behavior and transcriptional program of glioblastoma cells. A set of 2D and 3D cultures of patient-derived cell lines with stem-like properties (GSC), as well as subcutaneous xenografts of the U87 cell line, were used as in vitro and in vivo models, respectively. Among the CDKs tested, only CDK13 expression was found to be associated with worse patient survival, while CDK12-immunoreactive cells were detected in patient glioblastoma tissues. The response of GSCs to the CDK12 and CDK13 inhibitor TZH541 included cell cycle blockade and decreased migration. Reduction in RNAPII phosphorylation in TZH541-treated cells was verified using one of the GSC lines. Genome-wide exploration of the transcriptional consequences of TZH541 treatment of 2 GSCs using CUT&RUN and SLAM-seq technologies revealed major transcriptional repression, particularly of genes associated with cell proliferation. *

   *Main comments: ** Although I found this study very interesting, I noted points requiring clarification, particularly in order to fully support the authors conclusions. My recommendations focus on the glioblastoma cell biology experiments, my area of expertise.

   RESPONSE: We are grateful for the reviewer's keen interest in our manuscript and appreciate various insightful observations on the challenges within glioblastoma biology. Recognizing the necessity of validating CDK12/CDK13 requirements in complex environments, we have undertaken a migration assay using GSC, G7 cells in organotypic mice brain slices. The ongoing assessment of CDK12/CDK13 inhibition on glioma migration will be included in the revised manuscript. We have also more carefully explained how the organoid models used in this study address the requested need to recapitulate the complexity seen in the patient tissue and tumor environment. Moreover, we have related immunohistochemistry assessments of CDK12 levels to the proliferation marker Ki-67. Finally, we have strengthened the mechanistic insights provided in the manuscript by the inclusion of new proteomics data, iPond data on nascent chromatin, and chromatin fiber assays, altogether showing that replication origins firing as well as PCNA function is heavily reduced and identifying key proteins in DNA replication that are affected. These points are thoroughly discussed and explained in the comments below.

   1. • The rationale for studying only CDK12 expression in patient glioblastoma tissues needs clarification. In contrast with CDK13, the authors found no association between CDK12 expression levels and patient survival (Sup Fig. 1A). Do the authors obtain similar results using independent datasets of glioblastoma tissue transcriptomes (e.g. CGGA)? With regard to the major effect of CDK12/13 inhibition on glioblastoma cell proliferation, determining whether CDK12/13 expression is observed in proliferating areas of the patients' tumor tissues (Ki67 IHC) would help support the authors' conclusion that their "results provide proof-of-concept for the potential of CDK12 and CDK13 as therapeutic targets for glioblastoma". The main data regarding CDK expression the status in patients' tumors and their possible association with patient survival should be rearranged in the same figure and described in the same paragraph of the results. * RESPONSE: We have performed our analyses on CGGA dataset, which matches with the TCGA data. We will show analyses from both TCGA and CGGA in Sup Fig. 1.

   CDK12 and CDK13 are functionally redundant, which is one of the reasons that they do not score in genome-wide CRISPR/Cas9 dropout screens. As a result, GSC proliferation is only partially dependent on the individual expression of CDK12 and CDK13, as we observe in Figure 1E. However, GSCs are dependent on the combined CDK12/CDK13 activity and therefore are sensitive to inhibitors targeting both. Possibly, this functional redundancy makes the interpretation of the relationship between the individual expression of CDK12/CDK13 and glioma patient survival less straightforward.

   With regards to the immunohistochemistry (IHC) staining evaluating the expression of CDK12 and CDK13 in glioma patient samples, we tested several antibodies for both CDK12 and CDK13. However, we were only able to identify an antibody for CDK12 which worked reliably in IHC.

   We will perform Ki-67 IHC to test whether CDK12 expression matches with proliferative areas of the tumor tissues.

   • Fig.1 caption "Inhibition of CDK12/13 specifically affects proliferation of glioma cells" is not entirely consistent with the results. This inhibition also appears to induce cell death, at least in some of the GSC tested, as indicated with cell counts (Fig. 1C., sup Fig.1 G) and an 8-fold increase in the % of apoptotic cells after a 24h-TZH treatment shown in Fig. 5E. All data concerning the effects of TZH on proliferation and survival (including detailed effects on the cell cycle) should be brought together rather than split between the 1st and last figure. *

   RESPONSE: We appreciate these comments and will be addressed it in the manuscript.

   *3. The reason for which serum-treated GSC were used should be explicated (sup Fig. 1C). Serum being usually used to trigger GSC "differentiation", did the authors want to verify whether CDK12/13 inhibitors affected GSC in a specific manner? If yes, it is necessary to demonstrate that serum-treated GSC have lost their stem-like properties. *

   RESPONSE: This is a good point that we appreciate being able to expound on. GSCs are grown in serum-free media with N2 and B27 supplements together with EGF/FGFb whereas the control cells, including breast cancer and Hela/U2OS cells are grown in media containing serum. Serum-containing media was used to assess whether the diverse set of macromolecules present in serum would affect the bioavailability and/or response to the drug, and our data clearly demonstrated that this was not the case and that glioma stem cells are susceptible to the drug regardless of serum presence. In order to minimize the effect of serum on GSC differentiation, serum was added in the media immediately before the drug treatment.

   • The viability of patient-derived 3D organoids (GBO) was assessed by measuring ATP production. It is therefore not possible to distinguish between decreased cell proliferation and increased cell death as responsible for the signal decrease. This limitation in the interpretation of the results needs to be made explicit. I was also misled by the use of GBO. This abbreviation is currently used to designate fragments of patient tumor tissue amplified in culture, which retain the cellular heterogeneity and the extracellular matrix of the original tumor and therefore provide an actual ex vivo model of the tumor. To avoid any misunderstanding, I recommend referring to experimental models obtained from dissociated patient-derived cell lines as "3D organoids" or "cellular spheroids", and avoiding to designate them as ex vivo models since they do not recapitulate the complexity of the tumor. *

   RESPONSE: We apologize for providing insufficient details concerning our GBO modelling, and we have now updated the description in the methods to avoid misconceptions and unclarity. Our GBOs are not derived from cell lines. We derive GBOs from patient tumors by short-term culture of tissue fragments in 3D conditions. Such organoids are of a very primary nature and contain extracellular matrix and tumor microenvironment components. To avoid propagation in vitro, we perform implantation of GBOs to immunodeficient animals to create patient-derived orthotopic xenografts (PDOXs). We have established that serial propagation of patient material via series of short-term GBO cultures and PDOXs allow for multiplication of GBM patient tumors without major clonal selection and genetic/phenotypic adaptation (Golebiewska, 2020, DOI: 10.1007/s00401-020-02226-7). To perform robust drug screening ex vivo in GBOs, we further developed a specific protocol based on the material isolated directly from well-established and characterized PDOXs (Oudin, 2021, DOI: 10.1016/j.xpro.2021.100534). The protocol includes reconstitution of 3D GBOs of uniform size, which allows for reliable ex vivo readouts. Importantly, GBM primary cells are able to reassemble into 3D structures of heterogeneous nature, including reconstitution of extracellular matrix. In the revised manuscript, we will provide a clear description of the GBO modelling in the material and methods as well as in the associated results.

   • Although the abstract contains a statement indicating that CDK12/13 genetic ablation inhibits cell migration, I did not find the corresponding results in the article. The demonstration that CDK12/13 inhibition decreases cell migration is weaker than the demonstration of its effect on proliferation. Contrary to the experiments evaluating cell proliferation, cell migration was assessed using a single technical approach. Moreover, the method used to assay TZH effects on cell migration rather measures cell motility than cell migration over long distances in a 3D and complex environment as observed in diffuse glioma. Since these data add nothing significant to the article, I would delete them. *

   RESPONSE: We thank the reviewer for pointing out the comment in first sentence, which is addressed in the abstract now.

   It is correct that strictly speaking our assay measured the effect of CDK12/CDK13 inhibition on glioma motility rather than migration, we have corrected this sentence in the abstract. We have however also now strengthened the methodology in the manuscript by establishing and using migration assays of GSC G7 cells on organotypic mouse brain slices. Organotypic mouse brain slices have a preserved cytoarchitecture that allows analysis of migration over longer distances in a physiological environment. We are currently analyzing the data. These results will be included in the revised manuscript.

   • In my opinion, the information from the in vivo experiments is limited and should be presented in a supplementary rather than a main figure. The data were obtained with a single cell model, U87 cells of uncertain origin, and using subcutaneous xenografts that provide an environment totally different from the patient's actual tumor. In this context, the data obtained provide little information on the response of cancer cells in a complex and specific environment well known to promote tumor growth and resistance to therapies. I understand that the use of intracerebral xenografts is not feasible, since the inhibitor does not appear to reach the brain. With this technical limitation, an alternative would be to deliver the compound directly inside the brain tumor. A cannula can be implanted into the tumor after it has formed, and connected to an Alzet minipump filled with the drug. These experiments are technically difficult, however, and success is not guaranteed. Another alternative would be to use GBO, as described by Jacob et al (2019) as a surrogate for tumor tissue, provided the authors can obtain tissue fragments from patient surgical resections or intracerebral xenografts of patient-derived cell lines. These alternatives are optional. *

   RESPONSE: We thank the reviewer for pointing out the difficulties in testing currently available compounds in vivo. Following the reviewers’ comments, we are open to placing the in vivo experiments in U87 xenografts in the supplementary material. We would like to reemphasize the clinical significance of our data in GBOs (please see the response above), which relies on models of equal complexity compared to the Jacob’s protocol and represent 3D compact and complex structures ex vivo derived from the GBM patient tumors propagated as orthotopic patient-derived xenografts.

   *Minor comments: **

   • Fig. 4A and Fig. 5E-F: Results from a single experiment? If yes, they must be repeated at least once.*

   RESPONSE: They are representative of a minimum of three independent biological experiments, which will be mentioned in the manuscript.

   *- For the sake of clarity, all y-axes in graphs presenting MTT or CellTiter-Glo assay results should be labeled "cell viability index", as they only provide a measure of overall cell or organoid metabolic activity, and thus an indirect assessment of cell viability. *

   RESPONSE: We thank the reviewer for this suggestion and will incorporate it in the revision.

   *- Statistical analyses are missing for 3 of the 4 cell lines presented in Figure 1F. *

   RESPONSE: This will be addressed.

   *- Some GO terms are truncated in sup Fig. 3. *

   *RESPONSE: This will be fixed in the revised ms.

   - The legend to Fig. 5B-D shows the mean and SD of 2 replicates. Please show individual points.

   RESPONSE: This suggestion will be addressed in the revision.

   *- Sup Fig1 D-F: unit of concentration is missing (M?) ** *RESPONSE: This is addressed.

   *Reviewer #2 (Significance (Required)): **

   Significance: Despite growing interest in the roles of CDK12/13 roles in cancers and their targeting for cancer therapy, their involvement in glioblastoma growth remains unexplored. The results presented in this study outline the potential of CDK12/13 inhibition in controlling the growth of glioblastoma, at least in vitro, and thus provide meaningful information on its potential usefulness for this aggressive brain tumor with a high proliferation rate. Obtaining the full proof-of-concept that CDK12/13 constitute relevant targets for glioblastoma therapies will however require additional experiments demonstrating efficacy of CDK12/13 inhibition in complex environments, as encountered in the patients' tumor. *

   3. Description of the revisions that have already been incorporated in the transferred manuscript

   We have addressed following of the reviewers’ comments.

   Reviewer-1:

   • Major comment-1 is partially incorporated in the text.

   • Major comments-5 and 6 are incorporated. Reviewer-2:

   • Major comment 1 is partially addressed.

   • Major comment 2, 3 and 4 are addressed in writing.

   • Major comment 5 is partially addressed in writing.

   • Major comment 6 is addressed.

   • All minor comments are incorporated in writing.

   4. Description of analyses that authors prefer not to carry out

   Read the original source
2. 

   Review Commons

   Dec 18, 2023

   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

   Summary:

   Lier et al. present a set of results showing that pharmacological inhibition of CDK12/13, cyclin-dependent kinases that phosphorylate RNA polymerase II (RNAPII), alters the proliferative behavior and transcriptional program of glioblastoma cells. A set of 2D and 3D cultures of patient-derived cell lines with stem-like properties (GSC), as well as subcutaneous xenografts of the U87 cell line, were used as in vitro and in vivo models, respectively. Among the CDKs tested, only CDK13 expression was found to be associated with worse patient survival, while CDK12-immunoreactive cells were detected in patient glioblastoma tissues. …

   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

   Summary:

   Lier et al. present a set of results showing that pharmacological inhibition of CDK12/13, cyclin-dependent kinases that phosphorylate RNA polymerase II (RNAPII), alters the proliferative behavior and transcriptional program of glioblastoma cells. A set of 2D and 3D cultures of patient-derived cell lines with stem-like properties (GSC), as well as subcutaneous xenografts of the U87 cell line, were used as in vitro and in vivo models, respectively. Among the CDKs tested, only CDK13 expression was found to be associated with worse patient survival, while CDK12-immunoreactive cells were detected in patient glioblastoma tissues. The response of GSCs to the CDK12 and CDK13 inhibitor TZH541 included cell cycle blockade and decreased migration. Reduction in RNAPII phosphorylation in TZH541-treated cells was verified using one of the GSC lines. Genome-wide exploration of the transcriptional consequences of TZH541 treatment of 2 GSCs using CUT&RUN and SLAM-seq technologies revealed major transcriptional repression, particularly of genes associated with cell proliferation.

   Main comments:

   Although I found this study very interesting, I noted points requiring clarification, particularly in order to fully support the authors conclusions. My recommendations focus on the glioblastoma cell biology experiments, my area of expertise.

   • The rationale for studying only CDK12 expression in patient glioblastoma tissues needs clarification. In contrast with CDK13, the authors found no association between CDK12 expression levels and patient survival (Sup Fig. 1A). Do the authors obtain similar results using independent datasets of glioblastoma tissue transcriptomes (e.g. CGGA)? With regard to the major effect of CDK12/13 inhibition on glioblastoma cell proliferation, determining whether CDK12/13 expression is observed in proliferating areas of the patients' tumor tissues (Ki67 IHC) would help support the authors' conclusion that their "results provide proof-of-concept for the potential of CDK12 and CDK13 as therapeutic targets for glioblastoma". The main data regarding CDK expression the status in patients' tumors and their possible association with patient survival should be rearranged in the same figure and described in the same paragraph of the results.
   • Fig.1 caption "Inhibition of CDK12/13 specifically affects proliferation of glioma cells" is not entirely consistent with the results. This inhibition also appears to induce cell death, at least in some of the GSC tested, as indicated with cell counts (Fig. 1C., sup Fig.1 G) and an 8-fold increase in the % of apoptotic cells after a 24h-TZH treatment shown in Fig. 5E. All data concerning the effects of TZH on proliferation and survival (including detailed effects on the cell cycle) should be brought together rather than split between the 1st and last figure.
   • The reason for which serum-treated GSC were used should be explicated (sup Fig. 1C). Serum being usually used to trigger GSC "differentiation", did the authors want to verify whether CDK12/13 inhibitors affected GSC in a specific manner? If yes, it is necessary to demonstrate that serum-treated GSC have lost their stem-like properties.
   • The viability of patient-derived 3D organoids (GBO) was assessed by measuring ATP production. It is therefore not possible to distinguish between decreased cell proliferation and increased cell death as responsible for the signal decrease. This limitation in the interpretation of the results needs to be made explicit. I was also misled by the use of GBO. This abbreviation is currently used to designate fragments of patient tumor tissue amplified in culture, which retain the cellular heterogeneity and the extracellular matrix of the original tumor and therefore provide an actual ex vivo model of the tumor. To avoid any misunderstanding, I recommend referring to experimental models obtained from dissociated patient-derived cell lines as "3D organoids" or "cellular spheroids", and avoiding to designate them as ex vivo models since they do not recapitulate the complexity of the tumor.
   • Although the abstract contains a statement indicating that CDK12/13 genetic ablation inhibits cell migration, I did not find the corresponding results in the article. The demonstration that CDK12/13 inhibition decreases cell migration is weaker than the demonstration of its effect on proliferation. Contrary to the experiments evaluating cell proliferation, cell migration was assessed using a single technical approach. Moreover, the method used to assay TZH effects on cell migration rather measures cell motility than cell migration over long distances in a 3D and complex environment as observed in diffuse glioma. Since these data add nothing significant to the article, I would delete them.
   • In my opinion, the information from the in vivo experiments is limited and should be presented in a supplementary rather than a main figure. The data were obtained with a single cell model, U87 cells of uncertain origin, and using subcutaneous xenografts that provide an environment totally different from the patient's actual tumor. In this context, the data obtained provide little information on the response of cancer cells in a complex and specific environment well known to promote tumor growth and resistance to therapies. I understand that the use of intracerebral xenografts is not feasible, since the inhibitor does not appear to reach the brain. With this technical limitation, an alternative would be to deliver the compound directly inside the brain tumor. A cannula can be implanted into the tumor after it has formed, and connected to an Alzet minipump filled with the drug. These experiments are technically difficult, however, and success is not guaranteed. Another alternative would be to use GBO, as described by Jacob et al (2019) as a surrogate for tumor tissue, provided the authors can obtain tissue fragments from patient surgical resections or intracerebral xenografts of patient-derived cell lines. These alternatives are optional.

   Minor comments:

   • Fig. 4A and Fig. 5E-F: Results from a single experiment? If yes, they must be repeated at least once.
   • For the sake of clarity, all y-axes in graphs presenting MTT or CellTiter-Glo assay results should be labeled "cell viability index", as they only provide a measure of overall cell or organoid metabolic activity, and thus an indirect assessment of cell viability.
   • Statistical analyses are missing for 3 of the 4 cell lines presented in Figure 1F.
   • Some GO terms are truncated in sup Fig. 3.
   • The legend to Fig. 5B-D shows the mean and SD of 2 replicates. Please show individual points.
   • Sup Fig1 D-F: unit of concentration is missing (M?)

   Significance

   Despite growing interest in the roles of CDK12/13 roles in cancers and their targeting for cancer therapy, their involvement in glioblastoma growth remains unexplored. The results presented in this study outline the potential of CDK12/13 inhibition in controlling the growth of glioblastoma, at least in vitro, and thus provide meaningful information on its potential usefulness for this aggressive brain tumor with a high proliferation rate. Obtaining the full proof-of-concept that CDK12/13 constitute relevant targets for glioblastoma therapies will however require additional experiments demonstrating efficacy of CDK12/13 inhibition in complex environments, as encountered in the patients' tumor.

   Read the original source
3. 

   Review Commons

   Dec 18, 2023

   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

   The authors in this manuscript studied the role of a transcriptional cyclin-dependent kinase CDK12/CDK13 in glioblastoma. These cyclin-dependent kinases phosphorylate at ser2 residue in the C-terminal of RNA Pol II. Pharmacological inhibition of CDK12/CDK13 kinase with inhibitor decreases cell proliferation in multiple glioma cell lines and in patient-derived organoids. The CDK12/CDK13 inhibitor also reduces tumor growth in a mouse xenograft model. Mechanistically, the authors showed that genome-wide inhibition of CDK12/CDK13 attenuates RNA Pol II phosphorylation, disrupting transcriptional elongation and decreasing cell cycle …

   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

   The authors in this manuscript studied the role of a transcriptional cyclin-dependent kinase CDK12/CDK13 in glioblastoma. These cyclin-dependent kinases phosphorylate at ser2 residue in the C-terminal of RNA Pol II. Pharmacological inhibition of CDK12/CDK13 kinase with inhibitor decreases cell proliferation in multiple glioma cell lines and in patient-derived organoids. The CDK12/CDK13 inhibitor also reduces tumor growth in a mouse xenograft model. Mechanistically, the authors showed that genome-wide inhibition of CDK12/CDK13 attenuates RNA Pol II phosphorylation, disrupting transcriptional elongation and decreasing cell cycle progression. So, the authors proposed that targeting CDK12/CDK13 kinases can be used as a therapeutic strategy in glioblastoma. The authors have done extensive work in this manuscript to understand the role of CDK12/CDK13 in glioblastoma, but it is still a descriptive paper lacking mechanistic details.

   Comments:

   1. Figure 1 shows that CDK12/CDK13 inhibitor decreases cell viability, colony-forming ability, cell competition assay, and cell migration. The rationale behind choosing CDK12/CDK13 inhibitor in glioma is unclear from the manuscript. What is the CDK12/CDK13 expression in multiple glioma cells vs non-glioma cells? The authors should include normal astrocytes as a control for cell viability assay. The p value is missing in numerous Figure panels.
   2. Figure 2A shows the expression of CDK12 by immunohistochemistry in glioblastoma tissues. Including the non-glioma tissue samples as another control and including a quantification graph with the statistics is essential. In Figure 2B-D, the authors discussed the treatment of glioma patient-derived organoids with CDK12/CDK13 inhibitors. From the Figure, the organoids are resistant to THZ531 and SR-4835 inhibitors. To rule out this possibility, the immunoblot assay with cleave PARP will be essential to execute. Again, statistics need to be included in Figure 2C-D.
   3. The mouse subcutaneous xenograft experiment was carried out in U87 cells with CDK12/CDK13 inhibitors. However, the glioma stem cells are a more appropriate model for glioma biology, and it is not clear why authors suddenly chose U87 cells. Again, statistics are absent in multiple sub-panels.
   4. The authors have performed CUT & RUN experiments in G7 cells with CDK12/CDK13 inhibitors and decided to use 1hr and 6hr time points for the assay. Although the inhibitor THZ531 is supposed to inhibit RNA Pol II phosphorylation at the Ser2 residue, it decreases the Pol II phosphorylation at the ser5 residue quite a bit. Therefore, it is crucial to determine the effect coming from ser2 vs ser5 phosphorylation and gene expression regulation.
   5. There are a lot of supplementary Figures where axes are not labeled correctly or missing.
   6. The statistical section needs to be included in the manuscript.

   Significance

   In this manuscript, the authors studied the role of CDK12/CDK13 in glioblastoma and performed extensive studies to uncover the importance of these kinases in glioblastoma. Understanding more mechanistic details of how these kinases are involved in glioma progression will uncover more therapeutic opportunities in glioblastoma.

   Read the original source
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   Version published to 10.1101/2023.07.14.548985v1 on bioRxiv

   Jul 17, 2023