Molecular consequences of acute versus chronic CDK12 loss in prostate carcinoma nominates distinct therapeutic strategies

Reviewer #1 (Public review):

Summary:

The authors were attempting to identify the molecular and cellular basis for why modulators of the HR pathway, specifically PARPi, are not effective in CDK12 deleted or mutant prostate cancers and they seek to identify new therapeutic agents to treat this subset of metastatic prostate cancer patients. Overall, this is an outstanding manuscript with a number of strengths and in my opinion represents a significant advance in the field of prostate cancer biology and experimental therapeutics.

Strengths:

The patient data cohort size and clinical annotation from Figure 1 are compelling and comprehensive in scope. The associations between tandem duplications and amplifications of oncogenes that have been well-credentialed to be drivers of cancer development and progression are fascinating and the authors identify that in those that have AR amplification for example, there is evidence for AR pathway activation. The association between CDK12 inactivation and various specific gene/pathway perturbations is fascinating and is consistent with previously published studies - it would be interesting to correlate these changes with cell line-based studies in which CDK12 is specifically deleted or inhibited with small molecules to see how many pathways/gene perturbations are shared between the clinical samples and cell and mouse models with CDK12 perturbation. The short-term inhibitor studies related to changes in HRD genes and protein expression with CDK12/13 inhibition are fascinating and suggest differential pathway effects between short inhibition of CDK12/13 and long-term loss of CDK12. The in vivo studies with the inhibitor of CDK12/13 are intriguing but not definitive

Weaknesses:

Given that there are different mutations identified at different CDK12 sites as illustrated in Figure 1B it would be nice to know which ones have been functionally classified as pathogenic and for which ones that the pathogenicity has not been determined. This would be especially interesting to perform in light of the differences in the LOH scores and WES data presented - specifically, are the pathogenic mutations vs the mutations for which true pathogenicity is unknown more likely to display LOH or TD? For the cell inhibition studies with the CDK12/13 inhibitor, more details characterizing the specificity of this molecule to these targets would be useful. Additionally, could the authors perform short-term depletion studies with a PROTAC to the target or short shRNA or non-selected pool CRISPR deletion studies of CDK12 in these same cell lines to complement their pharmacological studies with genetic depletion studies? Also perhaps performing these same inhibitor studies in CDK12/13 deleted cells to test the specificity of the molecule would be useful. Additionally, expanding these studies to additional prostate cancer cell lines or organdies models would strengthen the conclusions being made. More information should be provided about the dose and schedule chosen and the rationale for choosing those doses and schedules for the in vivo studies proposed should be presented and discussed. Was there evidence for maximal evidence of inhibition of the target CDK12/13 at the dose tested given the very modest tumor growth inhibition noted in these studies?

Reviewer #2 (Public review):

Summary:

The study explores the functional consequence of CDK12 loss in prostate cancer. While CDK12 loss has been shown to confer homologous recombination (HR) deficiency through premature intronic polyadenylation of HR genes, the response of PARPi monotherapy has failed. This study therefore performed an in-depth analysis of genomic sequencing data from mCRPC patient tumors, and showed that tumors with CDK12 loss lack pertinent HR signatures and scars. Furthermore, functional exploration in human prostate cancer cell lines showed that while the acute inhibition of CDK12 resulted in aberrant polyadenylation of HR genes like BRCA1/2, HR-specific effects were overall modest or absent in cell lines or xenografts adapted to chronic CDK12 loss. Instead, vulnerability to genetically targeting CDK13 resulted in a synthetic lethality in tumors with CDK12 loss, as shown in vivo with SR4825, a CDK12/13 inhibitor - thus serving as a potential therapeutic avenue.

The evidence supporting this study is based on in-depth genomic analyses of human patients, acute knockdown studies of CDK12 using a CDK12/13 inhibitors SR4835, adaptive knockout of CDK12 using LuCaP 189.4_CL and inducible re-expression of CDK12, CDK12 single clones in 22Rv1 (KO2 and KO5) and Skov3 (KO1), Tet-inducible knockdown of BRCA2 or CDK12 followed by ionizing radiation and measurement of RAD51 foci, lack of sensitivity generally to PARPi and platinum chemotherapy in cells adapted to CDK12 loss, loss of viability of CDK13 knockout in CDK12 knockout cells, and in vivo testing of SE4825 in LuCaP xenografts with intact and CDK12 loss.

Strengths:

Overall, this study is robust and of interest to the broader homologous recombination and CDK field. First, the topic is clinically relevant given the lack of PARPi response in CDK12 loss tumors. Second, the strength of the genomic analysis in CDK12 lost PCa tumors is robust with clear delineation that BRCA1/2 genes and maintenance of most genes regulating HR are intact. Specifically, the authors find that there is no mutational signature or genomic features suggestive of HR, such as those found in BRCA1/2 tumors. Lastly, novel lines are generated in this study, including de novo LuCaP 189.4_CL with CDK12 loss that can be profound for potential synthetic lethalities.

Weakness:

One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?

While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12-K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout.

Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.

The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.

Reviewer #3 (Public review):

Significance:

About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.

In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.

Strengths:

Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.

Weaknesses:

(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Given that there are different mutations identified at different CDK12 sites as illustrated in Figure 1B it would be nice to know which ones have been functionally classified as pathogenic and for which ones that the pathogenicity has not been determined. This would be especially interesting to perform in light of the differences in the LOH scores and WES data presented - specifically, are the pathogenic mutations vs the mutations for which true pathogenicity is unknown more likely to display LOH or TD?

Alterations were classified as pathogenic when resulting in frameshift, nonsense, or cause an aminoacid change likely to alter function (according to ANNOVAR). Four patients were called CDK12^BAL but were negative for TDP signatures. Three of these had CDK12 mutations downstream of the kinase domain, which may be less likely to ablate protein activity. Most functionally validated pathogenic mutations include disruption of the kinase domain (PMID: 25712099). We added a sentence to the Results section (under “Identification of genomic characteristics that associate with CDK12 loss in prostate cancer”) to highlight this caveat on pathogenic mutation calls.

For the cell inhibition studies with the CDK12/13 inhibitor, more details characterizing the specificity of this molecule to these targets would be useful. Additionally, could the authors perform short-term depletion studies with a PROTAC to the target or short shRNA or non-selected pool CRISPR deletion studies of CDK12 in these same cell lines to complement their pharmacological studies with genetic depletion studies? Also perhaps performing these same inhibitor studies in CDK12/13 deleted cells to test the specificity of the molecule would be useful.

We are not aware of a CDK12-specific PROTAC, and generate such as reagent is beyond the scope of the present study. Regarding the specificity of the CDK12/13 inhibitor molecules, additional information on the specificity and in vivo dose selection were added to the Results section (under “CDK13 is synthetic lethal in cells with biallelic CDK12 loss”). Cells with CDK12-KO did not tolerate CDK13-KO, so we were unable to generate double knockouts to test for CDK12/13 inhibitor non-specific effects.

Additionally, expanding these studies to additional prostate cancer cell lines or organdies models would strengthen the conclusions being made. More information should be provided about the dose and schedule chosen and the rationale for choosing those doses and schedules for the in vivo studies proposed should be presented and discussed. Was there evidence for maximal evidence of inhibition of the target CDK12/13 at the dose tested given the very modest tumor growth inhibition noted in these studies.

With respect to additional acute CDK12 loss models, our Tet-inducible shCDK12 models show only minor growth slowdown and do not appear to phenocopy the strong arrest or apoptosis seen with CDK12 KO or inhibition, respectively. Future work is ongoing to generate CDK12-degron regulated cell lines. We added a new immunoblot panel showing that acute CRISPR/sgRNA targeting of CDK12 does indeed lead to BRCA2 and ATM protein decrease (Fig. S4g), providing some orthogonal genomic targeting evidence of the acute HR gene effect. We are continuing efforts to collect and generate additional CDK12^BAL cell models, in both 2D and 3D culture systems, but none are presently available. We added a 3D culture drug dose curve with LuCaP189.4 exposed to THZ531 (Fig. S7m), which confirms heightened sensitivity vs two CDK12-intact lines.

Regarding assessments of CDK12 targets; as we are not aware of any unique CDK12 substrates, it is fair to ask but difficult to measure precise CDK12 inhibition by the compounds in tumors. We dosed mice using the same protocol as detailed in the original report testing SR4835 in mice (PMID: 31668947). We performed immunoblots on lysates from 3 and 28 day treated PDX tumors and did not see any consistent decreases in pRBP1(Ser2) or ATM or increases in γH2A.X (data not shown). However, we did see increases in APA usage and downregulation of DNA repair transcripts with three day treatment (Fig. 6k-l), as would be expected from on target acute effects.

Reviewer #2 (Public review)

One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?

In regard to untangling the effects of cell arrest on HR gene expression, this is a difficult question given that many HR genes, including BRCA2, are S/G2 linked. We attempted to account for those effects in the acute CDK12 inhibition experiment by including a palbociclib (CDK4/6i) control, which caused cell arrest and decreased BRCA1/2 RNA expression with no apparent 5/3’ transcript imbalance determined by qPCR (Fig. 4e,g). Though overall BRCA1 and BRCA2 mRNA levels are lower in the stable 22Rv1-CDK12-KO2 and KO5 lines, they do not show selective 3’ loss (Fig. 5c), suggesting the downregulation in these lines is mostly due to their slower growth (Fig. S4k) and not intronic polyA usage.

While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout.

As a clarification, the 22Rv1-CDK12-KO2 and 22Rv1-CDK12-KO5 are stable CRISPR knockout clonal lines that were expanded from single cells. We added a new figure to include more validation of these lines (Fig. S5). We tried multiple times to reproduce the HRd phenotype and PARPi sensitivity with siRNA and inducible shRNA lines but were unable to see clear sensitivity differences, despite seeing the expected shifts with shBRCA2 controls (data not shown). It is possible the degree of knockdown (~80%), timing (8 days), or specific cell lines used in our experiments were not sufficient to expose the acute phenotype by this method.

However, we were able to see acute HR gene decreases by inhibitor treatment (Fig. 4) or acute CRISPR (Fig. S4g).

Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.

We appreciate the complexity of the data and attempted to use multiple models to report consistency and variability. We are not able to ascertain what data would be extraneous, and elected to present data we view as relevant in the main figures while moving supporting data in the supplement.

The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 (mutant) tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.

Though the response of the CDK12^BAL models to some compounds is variable, we believe those mixed results are important and future studies may be able to better explain why some show shifts in sensitivity while others do not. We hope future studies with additional models will help determine which sensitivities are more consistently true, and perhaps provide explanations for differences between models.

Regarding SR4835, we find, and others have reported, a toxic (i.e. apoptotic) effect for in vitro treatment with dual CDK12/13 inhibitors (Fig. 4f, S4e,f); in fact, that may be why previous studies have used short timepoints in cell culture assays with these dual inhibitors. In mice, SR4835 was tolerated well but only LuCaP 189.4 showed statistically significant decreases in tumor volume and weight (Fig. 6j). We did not test PARPi responses in the PDX models, nor did we attempt engrafting the 22Rv1-CDK12-KO cell lines, but both would be worthwhile experiments in the future. Beyond CDK12^BAL tumors, we agree that CDK12/13 inhibitors could be effective in cancer therapies more generally (e.g. triggering acute HRd, loss of RNAP2 phosphorylation). We added a line to the discussion section about ongoing efforts to combine PARPi and CDK12/13i, which we expect to be synergistic in CDK12-intact tumors due to the acute loss phenotype. We certainly agree that development of a specific CDK13 inhibitor would be the ideal therapeutic option for CDK12^BAL tumors. However, CDK12 and CDK13 are 43% conserved at the protein level (PMID: 26748711), with 92% conservation in the active site (PMID: 30319007), and there are no available pharmacologic inhibitors that discriminate between CDK12 and CDK13.

Reviewer #3 (Public review):

It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

Thank you for the feedback. It is a keen observation that homozygous deletions of CDK12 are not typical, though many mutations are upstream frameshifts that are expected to lead to loss of functional protein and mRNA via nonsense mediated decay. LuCaP189.4, our only natural mutant model, has two upstream frameshifts leading to complete protein loss (Fig 5b, S4h-i). We also added a caveat previously mentioned (in response to Reviewer 1) that mutations downstream of the kinase domain may be less likely to be fully pathogenic. For upstream missense mutations, the possibility of neuromorphic function remains an intriguing possibility that cannot be ruled out and would not be captured in our current models. Hopefully additional models can be developed, both natural and engineered, to help dissect that question in future studies.

It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

We concur. Another caveat with the CRISPR models, beyond reliance on upstream frameshift mutations, is the simultaneous loss of alleles. In human tumors, there may be a period of single copy loss before the second hit that may provide a window for adaptation. It is possible that sequential loss is far easier for a cell to tolerate than acute bi-allelic inactivation. We agree that the question of what (if any) cooperating genetic alterations are required to tolerate CDK12 loss is an important one that we plant to further explore in future work.

Recommendations for Authors:

Reviewer #1 (Recommendations for Authors):

The authors have thoroughly addressed all issues of data availability, reagents, in vivo protocols, and animal approvals associated with the studies presented in this manuscript. Specific comments and experimental suggestions that in my opinion would strengthen the conclusions of this interesting and compelling manuscript are included above

Reviewer #2 (Recommendations for the authors):

The authors were thorough in their studies. As a general note, switching between the cell lines is often overwhelming in interpreting the data given cell-to-cell variability in response. If possible, consolidating the text/conclusions in results would improve the readability of the manuscript.

The variety of cell lines and models is perhaps expansive at times, but we hope the inclusion of these different models helps support the conclusions.

Is it possible to knockout CDK12 acutely using a degron-based approach, instead of utilizing an inhibitor that targets both CDK12/13?

There is a HeLa cell line made with analog-sensitive CDK12 (Bartkowiak, Yan, and Greenleaf 2016) but we were unaware of any such prostate lines at the time of this work. We are attempting to develop engineered prostate lines with specific CDK12 degradation but do not yet have them available.

How do the authors address a lower BRCA1/2 level in for example 22RV1-K05, does this cell line have increased sensitivity to PARPi over its parental 22RV1 line? Could this be added to Figure 5h/i?

The lower BRCA2 levels in 22Rv1CDK12-KO5 is likely due to the slower growth rate (Fig. S4k), as BRCA2 expression is S/G2 linked. While the mRNA level of BRCA2 overall is lower in the KO5 line, we do not observe the 5’/3’ transcript imbalance (Fig. 5c). The 22Rv1-CDK12-KO lines did not show increased sensitivity to carboplatin, while inducible shBRCA2 did (Fig. S7a), so we do not believe this lower BRCA2 confers functional HRd. We did test the KO lines with olaparib (Fig. S7d) and saw a modest increased sensitivity compared to parental 22Rv1, but not to the extent measured in the BRCA1 mutant line UWB1.289.

What is the clonality of the LuCAP 189.4 lines upon derivation? Is biallelic CDK12 loss seen in all cells?

We do not know the exact clonality of the LuCAP 189.4 PDX or CL model, but we do see highly uniform CDK12 protein loss in these cells (quantified by IHC staining, data not shown).

The authors state that 22RV1-K02/05 has an increased growth arrest to CDK13 inhibition. However, in Figure 6h, it appears the viability is not significantly different compared to the parental 22RV1 line. Similar aspects noted in 189.4-vec/CDK12?

We found that 22Rv1 KO2/KO5 have growth arrest with sgCDK13 and cell death with CDK12/13 inhibitor. We did notice that SR4835 did not show the differential effects we anticipated (Fig. 6h), as was seen with THZ531 (Fig. 6i). SR4835 is a non-covalent inhibitor, while THZ531 is a covalent binder, so there are some functional differences between these compounds that might explain the lack of differential effects in the isogenic lines in a 4 day in vitro assay. We included the SR4835 in vitro data because it was used for the in vivo experiment. THZ531 is not suited for animal use.

Could the authors comment on SR4835 response in vivo as a function of tumor growth rate?

The in vivo SR4835 treated LuCaP189.4 did show signs of reduced proliferation with decreased Cell Cycle and DNA Replication in the RNA-seq signatures, but a more detailed investigation into cell cycle arrest vs apoptotic response has yet to be fully explored. We plan to conduct additional PDX experiments if we can obtain a selective CDK13 inhibitor.

Do the authors explore TDPs in their isogenic cell lines?

We performed low coverage WGS on the 22Rv1 KO clones and added that to the paper (Fig. S5c). We did not see any obvious signs of TDP. We suspect the phenotype takes longer to accumulate and is not apparent within the ~20 passages our clones underwent in culture. This would be consistent with the tumor analysis (Fig. 2b) showing increase in TDs from primary to metastatic tumors, suggesting TDs accumulate over time.

A future study may allow for screening synthetic lethals in the context of CDK12 loss in the presence or absence of SR4835 inhibition.

We are actively pursuing experiments to identify new synthetic lethal targets by CRISPR and drug screens in CDK12 loss models and hope to report those in a future study.

Reviewer #3 (Recommendations for the authors):

As discussed above, the authors may wish to adjust their terminology to "CDK12-altered" rather than "CDK12 lost" or "CDK12-inactivated" to leave open the possibility that some tumors may retain residual CDK12 function or adopt neomorphic functions.

Thank you for the additional comments and feedback. The possibility of neomorphic CDK12 allele function is important. As our models were all complete protein loss mutations, we decided to retain “biallelic loss” as our preferred nomenclature, but the note is well taken.

The plots in Figures 1f-h are interesting and suggest that certain cancer genes (especially oncogenes) are recurrently gained in CDK12-altered tumors. It may be interesting to look at this on the individual level rather than the cohort level to see whether any groups of oncogenes tend to be gained together in an individual patient - this could inform whether certain combinations of cancer drivers cooperate with CDK12 alteration to drive oncogenesis.

Thank you for the idea of looking at the patient-level for TDP-enriched oncogenes. A preliminary assessment did not identify recurrent co-gained oncogenes. We will continue these analyses as additional patient tumors with CDK12 alterations are identified.

The finding that AR gene or enhancer are recurrently gained with TDP is interesting and I am curious whether the authors have thoughts on whether these alterations can also be seen in the 1-2% of CDK12altered primary prostate cancers that are treatment naïve, and where AR pathway alterations are not as frequently seen.

We did not focus on CDK12 altered primary prostate cancers, but we did check if there is AR amplification enrichment in the 6 CDK12^BAL cases of the TCGA-PRAD dataset and did not identify enrichment. However, with such small numbers we would hesitate to draw any hard conclusions.

It could be interesting to more comprehensively characterize some of the CDK12 KO-adapted lines in Figure 5 (e.g. by WES or WGS) to determine whether they exhibit the TDP and/or whether they have acquired any secondary mutations that allow them to adapt to CDK12 loss.

We are planning to do further genomics characterization of the CDK12-KO lines and will hopefully include that in a future study. Genomic analyses of the 22Rv1 clones (see copy number plots in Fig. S5c) did not identify a TDP. We plan to repeat the genomic assessments over additional cell passages and we have planned additional experiments designed to understand why some cells tolerate CDK12 loss and others do not.