Targeting Polθ Helicase with RP-3467: Preclinical Validation and Clinical Response in a gBRCA1 ovarian cancer patient with an acquired PARPi resistance mutation in TP53BP1
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eLife Assessment
Loss of POLQ function is synthetically lethal with BRCA1 or BRCA2 deficiency, as is pharmacological inhibition of its motor (ATPase) or polymerase activity. This important manuscript validates an inhibitor of the DNA polymerase theta (POLθ) motor domain in biochemical and cell-based assays and provides evidence of its efficacy against homologous recombination-deficient tumors in preclinical and clinical settings, particularly in combination with PARP inhibitors. The experiments are rigorous, the evidence is convincing, and the results are appropriately interpreted. This work will be of considerable interest to the ovarian cancer research community.
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Abstract
Homologous recombination (HR) deficiency underlies a range of cancers, and PARP inhibitors (PARPi) targeting this vulnerability have demonstrated clinical efficacy in BRCA1/2-mutant tumors. However, resistance mechanisms and tolerability issues limit their long-term clinical effectiveness. Here, we characterize RP-3467, a novel, selective small-molecule inhibitor targeting the ATPase domain of DNA polymerase theta (Polθ), the key mediator of the error-prone microhomology-mediated end joining (MMEJ) pathway. We demonstrate that RP-3467 potently suppresses Polθ enzymatic activity, abrogates MMEJ, and disrupts mitotic DNA repair in HR-deficient (HRD) models, inducing synthetic lethality. In preclinical studies, we show that RP-3467 synergizes strongly with PARPi (olaparib and rucaparib), achieving sustained tumor regression across multiple xenograft and patient-derived xenograft (PDX) models without added systemic toxicity. Mechanistically, we find that RP-3467 increases mitotic DNA damage in vivo, as evidenced by elevated micronuclei and CIP2A foci. RP-3467 was evaluated in a phase 1 clinical trial (NCT06560632) and has shown preliminary signs of clinical activity. A heavily pretreated germline BRCA1-mutant ovarian cancer patient achieved a confirmed partial response to RP-3467 plus olaparib (RECIST v1.1), with retrospective profiling revealing a TP53BP1 loss-of-function mutation previously linked to both PARPi resistance and POLQ dependency. Although broader clinical validation is needed, these early findings suggest that Polθ inhibition has the potential to address PARPi resistance and offer therapeutic benefit in HRD cancers.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study describes targeting Poltheta Helicase with a new drug RP-3467. The properties of the drug and a clinical response in a gBRCA1 Ovarian Cancer Patient with a mutation in TP53BP1 are described.
Strengths:
This is an important and interesting set of observations.
We thank the reviewer for their positive assessment and appreciate their recognition of the significance and novelty of our findings.
Weaknesses:
(1) The response of the patient with a TP53BP1 mutant tumor is of interest but requires more clarification before solid conclusions can be drawn. The presence of the TP53BP1 mutation after but not before olaparib treatment is suggestive of selection, but in the absence of demonstration of a biallelic loss, this is conjecture. It could be a …
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study describes targeting Poltheta Helicase with a new drug RP-3467. The properties of the drug and a clinical response in a gBRCA1 Ovarian Cancer Patient with a mutation in TP53BP1 are described.
Strengths:
This is an important and interesting set of observations.
We thank the reviewer for their positive assessment and appreciate their recognition of the significance and novelty of our findings.
Weaknesses:
(1) The response of the patient with a TP53BP1 mutant tumor is of interest but requires more clarification before solid conclusions can be drawn. The presence of the TP53BP1 mutation after but not before olaparib treatment is suggestive of selection, but in the absence of demonstration of a biallelic loss, this is conjecture. It could be a co-selected passenger mutation. The authors should report the TP53BP1 variant allele fraction, LOH, and orthogonal validation should be performed by targeted deep sequencing or ddPCR. This would establish the prevalence before and after selection and enhance the interpretation of the functional effects of the mutation observed.
We thank the reviewer for this important comment. We agree that identifying a TP53BP1 mutation after olaparib treatment does not, by itself, establish whether the mutation was directly selected under therapeutic pressure or represents a co-selected event, and we do not intend to make that distinction. Instead, we report that whole-genome sequencing (WGS) identified the TP53BP1 mutation in the tumor collected at the second debulking surgery. Importantly, an additional biopsy obtained at disease progression during the POLAR trial revealed a TP53BP1 variant allele frequency of 77%, with FACETS analysis identifying copy-neutral loss of heterozygosity (CN-LOH). These findings (new data in the paper) provide additional evidence consistent with biallelic inactivation of TP53BP1 and support the biological relevance of this alteration. Nevertheless, we acknowledge that these observations do not establish whether TP53BP1 loss directly drove acquired olaparib resistance, and we clarified this distinction in the revised manuscript.
(2) To further strengthen the proposed mechanism, the authors should consider knocking in the mutation (or a similar LOF mutation) into an appropriate cell line and establish resistance to olaparib and sensitivity to the combination, as well as each drug individually. This will address the latter question of whether it is the poltheta inhibitor or the combo that is responsible for the clinical response.
We thank the reviewer for this thoughtful suggestion and agree that establishing the contribution of TP53BP1 loss to treatment sensitivity is important for interpreting the clinical response. We would like to highlight, however, that this genetic relationship has been investigated in several published studies. Genetic depletion of POLQ has been shown to be synthetic lethal with 53BP1 or Ku70 deficiency (Wyatt et al., Molecular Cell, 2016; Feng et al., Nature Communications, 2019). More directly, Zatreanu et al. (Nature Communications, 2021) demonstrated that loss of 53BP1 or Shieldin components (SHLD1/SHLD3) in BRCA1-deficient cells confers resistance to olaparib while retaining sensitivity to the POLθ inhibitor ART558 as a single agent. These findings are further supported by Krais et al. and Ronson et al. (Nature Communications, 2023), who demonstrated POLθ dependency in BRCA1/53BP1-deficient backgrounds. Collectively, these studies provide strong genetic and pharmacological support for our proposed mechanism and suggest that POLθ inhibition itself may have contributed substantially to the observed clinical response. While we acknowledge that the relative contributions of RP-3467 and olaparib cannot be definitively distinguished in this patient, we believe that the existing literature provides a strong mechanistic foundation for interpreting this clinical observation, without necessitating additional genetic modeling in the context of the present study.
(3) In the animal experiments, "synergy" is overstating the situation. Without an interaction analysis, this is an enhanced combination activity. Also, several of the animal experiments have a small N. It would be better to show the growth of tumors in individual mice.
Although we believe that minimally in BRCA2-deficient CDX models (e.g. Fig. 2a,b) the term synergy is appropriate (no/minimal single-agent activity of RP-3467 and tumor regressions in combination), we replaced the term ‘synergy’ with ‘enhanced combination activity’ due to lack of a formal interaction analysis. Data from individual animals are included as Source Data.
(4) The authors propose that Poltheta inhibition could prevent the emergence of reversion mutations. This is not addressed in the paper, and if they want to suggest this, they should provide some evidence
We thank the reviewer and agree that the manuscript presents no data bearing on reversion mutations. The discussion should not have implied otherwise. We have rewritten relevant paragraph so that the idea is presented explicitly as an untested hypothesis arising from the microhomology signatures reported at reversion breakpoints by others.
Reviewer #2 (Public review):
Summary:
The authors set out to assess the in vitro potency of a new POLQ helicase inhibitor, assess its in vitro BRCA1/2 synthetic lethal properties, and confirm how it performs in vivo in PDX and cell line xenografts. The authors also show an anecdotal patient response with a PARP inhibitor + POLQ inhibitor combination.
Strengths:
This is a novel POLQi of the helicase activity. The anecdotal clinical response in a heavily pre-treated patient is interesting.
We thank the reviewer for their positive assessment and are pleased that they recognize the novelty of targeting the POLθ helicase activity and the potential clinical significance of the response observed in a heavily pretreated patient.
Weaknesses:
Most of these issues could be addressed by changes to the main text, i.e. by the authors describing limitations/caveats to interpretation, etc.
(1) The clinical anecdote is interesting but underanalyzed. It's difficult, though, to know one way or the other whether the PARPi+ POLQi treatment effect (only partial response) has anything to do with the 53bp1 mutation. There is a causative sign that the 53bp1 mutation could be the cause of PARPi resistance (the increase in 53bp1 mutation allele frequency in PARPi-naïve vs PARPi-resistant tumors), but no similar sign to show that the 53bp1 mutant tumor clone is targeted by POLQi treatment. For example, does 53bp1 mutation allele frequency fall in ascites or ctDNA during the course of treatment, or is the 53bp1 mutation lost in a tumor taken at the end of treatment?
We thank the reviewer for this suggestion. While longitudinal monitoring of the TP53BP1-mutant clone would be informative, such data are unavailable. A biopsy obtained at progression on the POLAR trial revealed a TP53BP1 variant allele frequency of 77%, with copy-neutral LOH by FACETS analysis. Importantly, extensive preclinical evidence establishes synthetic lethality between 53BP1 deficiency and POLQ inhibition (Wyatt et al., 2016; Feng et al., 2019; Zatreanu et al., 2021; Krais et al., 2023), supporting a contribution of POLθ inhibition to the clinical response. Nevertheless, determining whether the response was driven by POLθ inhibition alone or its combination with olaparib will require evaluation in a larger clinical cohort.
(2) Although the relevant work is cited, the authors should be more explicit in the introduction and state that there is already evidence from 2 papers that Combined loss of BRCA1 and 53bp1 causes enhanced sensitivity to small-molecule POLQ helicase or polymerase inhibitors.
Agreed, and adopted. We changed the introduction to state this prior evidence explicitly on first mention, naming both studies and specifying that the effect has been demonstrated for inhibitors of Polymerase theta.
(3) Figure 4. Not exacerbating existing PARPi myelosuppression is great, but the authors have to acknowledge that the myelosuppression is still present when POLQ+ PARPI is used. Single-agent POLQi doesn't give myelotoxicity, but it also does not give strong anti-tumor efficacy. i.e. it would be fair for the authors to acknowledge this.
We agree with the reviewer and have revised the text to more accurately reflect these findings. While POLθ inhibition does not exacerbate PARPi-associated myelosuppression, it does not eliminate the hematologic toxicity of PARPi treatment. We also acknowledge that POLθ inhibitor monotherapy has limited antitumor efficacy in the models tested, highlighting the importance of combination treatment and the need to balance efficacy with tolerability.
(4) The increase in reticulocytes when mice are treated with POLQi could be a sign that there is an effect on myeloid lineages, but the animals are responding by increased production of this young myeloid lineage. Alternatively, this reticulocyte increase could drive or be a sign of pathology (polycythaemia vera, etc.). It would be good to see a commentary on this in the Discussion.
We agree, and updated the language in Results, page 9, to reflect this possibility.
(5) Re Figure 4. A therapeutic advance would be to see that, compared to other PARPi combinations such as platinum + PARPi or temozolomide + PARPi, a POLQ + PARPi combination doesn't give the same myelosuppression that these combinations do (by analogy, combined platinum + PARPi gives curative responses in mice but also wipes out the adaptive immune system). The authors need to add data showing the platinum+ PARPi positive control, or at the very least add a limitations of the study section to the manuscript to explain this fairly.
We thank the reviewer for this suggestion. The substantial myelosuppression associated with combining PARP inhibitors with platinum-based chemotherapy or temozolomide has been well documented in preclinical and clinical studies, including the references cited by the reviewer (Rajan et al., 2012; PMID: 24827126). We agree that a direct comparison would be informative. However, given that these toxicities are already established in the literature, we believe that repeating these experiments is not essential to the present study. We have instead clarified that our findings demonstrate that RP-3467 does not exacerbate olaparib-associated myelosuppression under the conditions tested, without implying that the combination is free of hematologic toxicity or has been directly demonstrated to be safer than other PARPi combinations.
(6) Figure 2 might give the impression that the new POLQi gives a similar, if not greater, BRCA2 synthetic lethal effect than a PARPi (e.g. 2d-f). It would help readers to see the full saruparib and olaparib dose-response curves, not (as per 2 A, B, C) just the single point estimates.
The ZIP synergy matrices in Extended Data Figure 2 were acquired across a full concentration grid for both agents, and therefore already contain the complete single-agent dose–response for olaparib and for saruparib in each line.
(7) Why was DOTC42510 not profiled for PARPi?
We apologize for the poor signposting that made this data hard to find. DOTC24510 cells were profiled with olaparib: the combination growth data appear in Extended Data Figure 2c and the corresponding ZIP synergy plot in Extended Data Figure 2d. The main-text sentence reporting the olaparib combination does name DOTC24510 alongside the two colorectal lines, but cites only the main-figure panels, which do not include this line.
(8) Figure 2f shows error bars on an n=2 dataset - one cannot estimate variance from only an n=2 dataset range.
We thank the reviewer for pointing this out. We agree that an n=2 dataset is insufficient for a reliable estimate of variability. We revised Figure 2f to show the data points without error bars and clarify the sample size in the figure legend.
(9) Is there a statistical analysis on Figure 2G?
The data originate from two independent experiments, so we did not consider it appropriate to perform a formal statistical analysis. We note, though, that the data were consistent between the two replicates and that a robust difference was observed between the combination and single-agent arms.
(10) Figure 3. Olaparib 25 mg/kg QD is well below the MTD in mice. This makes the olaparib response in HCT116 BRCA2 k/o xenografts look modest when compared to the combinations.
We used 25 mg/kg to highlight the strong combination potential between RP-3467 and olaparib. That said, Fig. 3b shows that even when a PARP inhibitor (saruparib in this case) is used at its maximally efficacious dose (PMID: 35929986), the RP-3467 combination has the potential to outperform PARPi monotherapy.
Reviewer #3 (Public review):
Summary:
The authors seek to validate the effectiveness of an inhibitor of the Pol theta helicase-like domain ATPase activity in its ability to ablate MMEJ activity in cell line models. They then confirm the inhibitor is synthetic lethal in a variety of BRCA1/2 deficient cancer models (and one PALB2 model). Finally, they report apparent effectiveness in treatment of a single patient that failed therapy with PARPi due to 53BP1 deficiency, a hypothesized special use-case for targeting pol theta.
Strengths:
It is a clear bench-to-bedside translation of mechanistic-oriented basic science to a clinical therapy. The experiments are largely rigorously performed. The approaches employed are impressively diverse, from in vitro to cell-line model, to preclinical model, to clinical application. The interpretations made are well supported by the available data.
We sincerely thank the reviewer for their thoughtful and encouraging assessment of our work. We are particularly pleased that the reviewer recognizes the translational significance of the study, the breadth and rigor of the experimental approaches, and the strength of the conclusions. We appreciate these positive comments and the constructive suggestions, which have helped us further strengthen the manuscript.
Weaknesses:
The addition of a few missing details would improve the usefulness to the reader. Additionally, the rationale behind some approaches could be clarified.
-
eLife Assessment
Loss of POLQ function is synthetically lethal with BRCA1 or BRCA2 deficiency, as is pharmacological inhibition of its motor (ATPase) or polymerase activity. This important manuscript validates an inhibitor of the DNA polymerase theta (POLθ) motor domain in biochemical and cell-based assays and provides evidence of its efficacy against homologous recombination-deficient tumors in preclinical and clinical settings, particularly in combination with PARP inhibitors. The experiments are rigorous, the evidence is convincing, and the results are appropriately interpreted. This work will be of considerable interest to the ovarian cancer research community.
-
Reviewer #1 (Public review):
Summary:
This study describes targeting Poltheta Helicase with a new drug RP-3467. The properties of the drug and a clinical response in a gBRCA1 Ovarian Cancer Patient with a mutation in TP53BP1 are described.
Strengths:
This is an important and interesting set of observations.
Weaknesses:
(1) The response of the patient with a TP53BP1 mutant tumor is of interest but requires more clarification before solid conclusions can be drawn. The presence of the TP53BP1 mutation after but not before olaparib treatment is suggestive of selection, but in the absence of demonstration of a biallelic loss, this is conjecture. It could be a co-selected passenger mutation. The authors should report the TP53BP1 variant allele fraction, LOH, and orthogonal validation should be performed by targeted deep sequencing or ddPCR. …
Reviewer #1 (Public review):
Summary:
This study describes targeting Poltheta Helicase with a new drug RP-3467. The properties of the drug and a clinical response in a gBRCA1 Ovarian Cancer Patient with a mutation in TP53BP1 are described.
Strengths:
This is an important and interesting set of observations.
Weaknesses:
(1) The response of the patient with a TP53BP1 mutant tumor is of interest but requires more clarification before solid conclusions can be drawn. The presence of the TP53BP1 mutation after but not before olaparib treatment is suggestive of selection, but in the absence of demonstration of a biallelic loss, this is conjecture. It could be a co-selected passenger mutation. The authors should report the TP53BP1 variant allele fraction, LOH, and orthogonal validation should be performed by targeted deep sequencing or ddPCR. This would establish the prevalence before and after selection and enhance the interpretation of the functional effects of the mutation observed.
(2) To further strengthen the proposed mechanism, the authors should consider knocking in the mutation (or a similar LOF mutation) into an appropriate cell line and establish resistance to olaparib and sensitivity to the combination, as well as each drug individually. This will address the latter question of whether it is the poltheta inhibitor or the combo that is responsible for the clinical response.
(3) In the animal experiments, "synergy" is overstating the situation. Without an interaction analysis, this is an enhanced combination activity. Also, several of the animal experiments have a small N. It would be better to show the growth of tumors in individual mice.
(4) The authors propose that Poltheta inhibition could prevent the emergence of reversion mutations. This is not addressed in the paper, and if they want to suggest this, they should provide some evidence.
-
Reviewer #2 (Public review):
Summary:
The authors set out to assess the in vitro potency of a new POLQ helicase inhibitor, assess its in vitro BRCA1/2 synthetic lethal properties, and confirm how it performs in vivo in PDX and cell line xenografts. The authors also show an anecdotal patient response with a PARP inhibitor + POLQ inhibitor combination.
Strengths:
This is a novel POLQi of the helicase activity. The anecdotal clinical response in a heavily pre-treated patient is interesting.
Weaknesses:
Most of these issues could be addressed by changes to the main text, i.e. by the authors describing limitations/caveats to interpretation, etc.
(1) The clinical anecdote is interesting but underanalyzed. It's difficult, though, to know one way or the other whether the PARPi+ POLQi treatment effect (only partial response) has anything to do …
Reviewer #2 (Public review):
Summary:
The authors set out to assess the in vitro potency of a new POLQ helicase inhibitor, assess its in vitro BRCA1/2 synthetic lethal properties, and confirm how it performs in vivo in PDX and cell line xenografts. The authors also show an anecdotal patient response with a PARP inhibitor + POLQ inhibitor combination.
Strengths:
This is a novel POLQi of the helicase activity. The anecdotal clinical response in a heavily pre-treated patient is interesting.
Weaknesses:
Most of these issues could be addressed by changes to the main text, i.e. by the authors describing limitations/caveats to interpretation, etc.
(1) The clinical anecdote is interesting but underanalyzed. It's difficult, though, to know one way or the other whether the PARPi+ POLQi treatment effect (only partial response) has anything to do with the 53bp1 mutation. There is a causative sign that the 53bp1 mutation could be the cause of PARPi resistance (the increase in 53bp1 mutation allele frequency in PARPi-naïve vs PARPi-resistant tumors), but no similar sign to show that the 53bp1 mutant tumor clone is targeted by POLQi treatment. For example, does 53bp1 mutation allele frequency fall in ascites or ctDNA during the course of treatment, or is the 53bp1 mutation lost in a tumor taken at the end of treatment?
(2) Although the relevant work is cited, the authors should be more explicit in the introduction and state that there is already evidence from 2 papers that Combined loss of BRCA1 and 53bp1 causes enhanced sensitivity to small-molecule POLQ helicase or polymerase inhibitors.
(3) Figure 4. Not exacerbating existing PARPi myelosuppression is great, but the authors have to acknowledge that the myelosuppression is still present when POLQ+ PARPI is used. Single-agent POLQi doesn't give myelotoxicity, but it also does not give strong anti-tumor efficacy. i.e. it would be fair for the authors to acknowledge this.
(4) The increase in reticulocytes when mice are treated with POLQi could be a sign that there is an effect on myeloid lineages, but the animals are responding by increased production of this young myeloid lineage. Alternatively, this reticulocyte increase could drive or be a sign of pathology (polycythaemia vera, etc.). It would be good to see a commentary on this in the Discussion.
(5) Re Figure 4. A therapeutic advance would be to see that, compared to other PARPi combinations such as platinum + PARPi or temozolomide + PARPi, a POLQ + PARPi combination doesn't give the same myelosuppression that these combinations do (by analogy, combined platinum + PARPi gives curative responses in mice but also wipes out the adaptive immune system). The authors need to add data showing the platinum+ PARPi positive control, or at the very least add a limitations of the study section to the manuscript to explain this fairly.
(6) Figure 2 might give the impression that the new POLQi gives a similar, if not greater, BRCA2 synthetic lethal effect than a PARPi (e.g. 2d-f). It would help readers to see the full saruparib and olaparib dose-response curves, not (as per 2 A, B, C) just the single point estimates.
(7) Why was DOTC42510 not profiled for PARPi?
(8) Figure 2f shows error bars on an n=2 dataset - one cannot estimate variance from only an n=2 dataset range.
(9) Is there a statistical analysis on Figure 2G?
(10) Figure 3. Olaparib 25 mg/kg QD is well below the MTD in mice. This makes the olaparib response in HCT116 BRCA2 k/o xenografts look modest when compared to the combinations.
-
Reviewer #3 (Public review):
Summary:
The authors seek to validate the effectiveness of an inhibitor of the Pol theta helicase-like domain ATPase activity in its ability to ablate MMEJ activity in cell line models. They then confirm the inhibitor is synthetic lethal in a variety of BRCA1/2 deficient cancer models (and one PALB2 model). Finally, they report apparent effectiveness in treatment of a single patient that failed therapy with PARPi due to 53BP1 deficiency, a hypothesized special use-case for targeting pol theta.
Strengths:
It is a clear bench-to-bedside translation of mechanistic-oriented basic science to a clinical therapy. The experiments are largely rigorously performed. The approaches employed are impressively diverse, from in vitro to cell-line model, to preclinical model, to clinical application. The interpretations made …
Reviewer #3 (Public review):
Summary:
The authors seek to validate the effectiveness of an inhibitor of the Pol theta helicase-like domain ATPase activity in its ability to ablate MMEJ activity in cell line models. They then confirm the inhibitor is synthetic lethal in a variety of BRCA1/2 deficient cancer models (and one PALB2 model). Finally, they report apparent effectiveness in treatment of a single patient that failed therapy with PARPi due to 53BP1 deficiency, a hypothesized special use-case for targeting pol theta.
Strengths:
It is a clear bench-to-bedside translation of mechanistic-oriented basic science to a clinical therapy. The experiments are largely rigorously performed. The approaches employed are impressively diverse, from in vitro to cell-line model, to preclinical model, to clinical application. The interpretations made are well supported by the available data.
Weaknesses:
The addition of a few missing details would improve the usefulness to the reader. Additionally, the rationale behind some approaches could be clarified.
-
Targeting Polθ Helicase with RP-3467: Preclinical Validation and Clinical Response in a gBRCA1 ovarian cancer patient with an acquired PARPi resistance mutation in TP53BP1 https://www.medrxiv.org/content/10.64898/2026.09.16.26363059v1
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