Targeting ALC1 can safely expand the therapeutic utility of PARP inhibitors across high-grade serous ovarian cancers

  1. Lindsey N. Aubuchon
  2. Deanna H. Wong
  3. Natasha M. Ramakrishnan
  4. Samantha F. Greenberg
  5. Rohan Reddy
  6. Elena Lomonosova
  7. Amanda Compadre
  8. Kathleen E. Jackson
  9. Danielle Kemper
  10. Vrutti Mehta
  11. Kellen Zoberi
  12. Dineo Khabele
  13. Elizabeth L. Christie
  14. Mary M. Mullen
  15. Priyanka Verma

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Abstract

Poly (ADP-ribose) polymerase inhibitors (PARPi) are approved for homologous recombination-deficient (HRD) high-grade serous ovarian cancers (HGSOCs), but their long-term effectiveness is limited by the emergence of resistance and hematological toxicity. Moreover, PARPi are largely ineffective in HR-proficient HGSOCs, particularly tumors with CCNE1 amplification, which exhibit marked therapeutic resistance and currently lack effective treatment options. Loss of a chromatin remodeling enzyme, Amplified in Liver Cancer 1 (ALC1), has been shown to enhance PARPi sensitivity. However, the clinical contexts in which ALC1 targeting will be clinically meaningful remain elusive. Here we demonstrate that ALC1 loss enhances PARPi sensitivity across HRD and CCNE1 -amplified serous ovarian cancer lines, xenograft and patient-derived cells. ALC1 depletion can overcome clinically relevant mechanisms of PARPi resistance while having minimal effects in BRCA -wild-type or heterozygous non-cancerous cells. Consistent with this therapeutic safety, PARPi sensitivity upon ALC1 loss can be reliably predicted by the endogenous levels of phospho-T21 RPA2, a marker for replication stress which is typically higher in ovarian cancer cells compared to their normal counterparts. Together, our studies define the clinical contexts in which the therapeutic utility of PARPi can be expanded by targeting ALC1, whose inhibitors are currently in Phase I clinical trials.

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    Reply to the reviewers

    Manuscript number: RC-2026-03474

    Corresponding author(s): Priyanka, Verma

    [Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

    If you wish to submit a preliminary revision with a revision plan, please use our "Revision Plan" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

    1. General Statements [optional]

    Point-by-point rebuttal is presented below. Reviewer’s comments are in BLACK; author’s response is in BLUE and figure numbers corresponding to the manuscript are in RED.

    2. Point-by-point description of the revisions

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

    ALC1 suppression has been shown to potentiate PARP inhibitor lethality in HR-deficient cells. Rather than revisiting the underlying mechanism, which has been characterized and remains an active area of investigation, this study aims to define the clinical contexts in which combined ALC1 and PARP inhibition may be beneficial. The clinical efficacy of PARP inhibitors, and their FDA approval, is largely restricted to HR-deficient tumors. This study dissects the combined effects of ALC1 and PARP suppression across a panel of HRD ovarian cancer cell lines, multiple classes of PARP inhibitor, and cells harboring distinct PARPi resistance mechanisms. In doing so, the authors delineate both the potential utility and the limitations of combined ALC1 and PARP inhibitor treatment in HRD ovarian cancers. The most impactful finding of the study, however, is likely the demonstration that ALC1 suppression sensitizes HR-proficient, CCNE1-amplified high-grade serous ovarian cancers to PARP inhibitors. These tumors are associated with particularly poor outcomes owing to the current absence of effective targeted therapies, making this observation of considerable clinical relevance.

    We thank the reviewer for appreciating the significance of our work in “HR-proficient, CCNE1-amplified high-grade serous ovarian cancers to PARP inhibitors” which is a critical unmet need.

    Of note, the study relies on genetic rather than pharmacological depletion of ALC1, a choice likely reflecting the current lack of a commercially available ALC1 inhibitor. While genetic suppression may not fully recapitulate the effects of combined drug treatment, it offers the advantage of not being tied to any specific compound, allowing the authors to establish more general principles. I have only a few comments.

    We are grateful to the reviewer for providing the unique perspective on our genetic study that “it offers the advantage of not being tied to any specific compound, allowing the authors to establish more general principles.”

    We have included this in our discussion to strengthen the study.

    The effect of ALC1 KO on PARPi sensitivity is less pronounced in OVSAHO cells (BRCA2-mutated) than in BRCA1-mutated cells. In these cells, it looks like there is an additive effect rather than synergy. 1- The authors should calculate, if possible, whether there is synergy or additive effect of ALC1-KO lethality (BLISS).

    We thank the reviewer for recognizing our limitations to perform BLISS score analysis, as our experiments were conducted at a single level of total protein depletion. Ideally, synergy assessments require a range of depletion levels to generate a full response matrix. Regardless, to address the reviewer’s concern regarding the impact of ALC1 on olaparib response in BRCA1- and BRCA2-mutant cells, we performed a BLISS score calculation under the conservative assumption that total ALC1 depletion alone has no effect on cell viability. We then employed the following formula for BLISS score calculation:

    Bliss Score =Eobs- (EA+EB-EAX EB)

    Where Eobs is viability of ALC1-depleted cells at a given drug concentration. This is observed impact upon combined loss of ALC1 and olaparib treatment.

    EA is impact on viability upon ALC1 depletion only. This was considered to be zero.

    EB is impact on viability on ALC1 WT in the presence of drug. This assesses the impact of drug alone.

    BLISS score was calculated at all non-saturating drugs concentration and then averaged to obtain a final BLISS value. We used the following cut off:

    > 10: Synergistic (the interaction is considered significant);

    -10 to 10: Additive (no significant interaction);

    __

    Olaparib

    Rucaparib

    Niraparib

    Veliparib

    Cisplatin

    UWB1.289

    22.34

    25.21

    13.24

    14.95334

    0.26

    JHOS-4

    37.27

    47.14

    26.3

    27.94

    -0.37

    OVSAHO

    19.34

    27.6

    23.2

    19.15

    7.04

    Kuramochi

    11.38

    11.98

    -3.56

    6.79

    -0.39

    We observe that ALC1 loss synergistically enhances olaparib and rucaparib response in both BRCA1- and 2-mutant cells. However, as correctly noted by the reviewer, we notice that the BLISS score is higher in BRCA1-mutant cells compared to BRCA-2 mutant, OVSAHO.

    In the revised manuscript, we have also included data for another BRCA-2-mutant cell line: KURAMOCHI (Fig.1d; Supp. Fig1b). We chose this cell line because, despite having a BRCA2-mutation, it is highly resistant to PARP inhibitors and cisplatin, owing to KRAS amplification. Notably, we observe that ALC1 loss can synergistically enhance the response of Kuramochi to olaparib and rucaparib.

    We have included a statement in the manuscript that the impact of ALC1 loss was more profound in BRCA1- versus BRCA2-settings. However, if acceptable to the reviewer, we would prefer not to include the BLISS values in the manuscript, as these calculations were not performed using the standard approach of titrating multiple levels of protein depletion.

    2- Another BRCA2-mutated cell line should be included.

    As discussed above, we have now included data from another BRCA2-mutant cell line, Kuramochi. Consistent with data in other BRCA-mutant cell lines, loss of ALC1 enhances olaparib and rucaparib sensitivity in these cells (Fig. 1d; Supp. Fig.1b).

    Minor comments: • Figure key is missing for S2C (I assume it's grey DMSO, blue olaparib)

    We apologize for this oversight. Figure key has now been included.

    Page 8: "BRCA1-mutant ovarian cancer cells eventually develop chemoresistance when exposed to PARPi for a prolonged period. Mechanistically, this is due to rewiring of ATR signaling, which enables RAD51 loading at DNA breaks and reversed forks independent of BRCA1 protein(25)." This sentence suggest this is the only existing resistance mechanism, which should be correct. Modify to "mechanistically, this CAN be due to", or "this is OFTEN due to".

    We thank for the reviewer for suggesting this important correction. This has now been fixed.

    Reviewer #1 (Significance (Required)):

    ALC1 inhibitors have been developed and clinical trials are starting. The significance of this manuscript lies in establishing the clinical potential for combined ALC1-PARP inhibition in high grade serous ovarian cancer. Especially, the authors demonstrate that combined ALC1 suppression with PARP inhibition efficiently kills HR-proficient CCNE1-amplified ovarian cancers, which represent 20% of ovarian cancers and are resistant to current therapies.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ The manuscript by Lindsey et al. explores the role of ALCN1 (Amplified in Liver Cancer 1) loss in enhancing the sensitivity of PARPi in ovariar carcinomas, including BRCA1/2 mutated tumors (both sensitive and resistant to platinum) as well as cyclin E amplified settings. The data are interesting but the in some cases there is an overinterpretation of the results. I have listed below my major concerns.

    We appreciate that the reviewer finds our data interesting. We also appreciate the reviewer insightful comments and have addressed them below.

    Figure 1. Could the authors demonstrate that OVASAHO cells are BRC2 muted? Indeed, I have always though they were BRCA wt type (10.1016/j.ygyno.2015.08.017).

    OVSAHO cells have a homozygous deletion in the BRCA2 gene (PMID:23839242), which could be the reason why a mutation was not detected in the study referred to by the reviewer (PMID: 26321251). We have now included the Domcke et al; 2013 reference in manuscript. The loss of BRCA2 expression in OVSAHO is also evident in our blots (Fig. 1a), as well as in data from protein atlas analysis.

    While the data on cisplatin suggest that indeed ALC1 loss do not impact its sensitivity, I disagree with the statant that "the correlation between dispensability of ALC1 in platinum response suggests that this chromatin remodeler likely does not contribute to MMEJ (page 6)" or " is dispensable for HR (page 7). Indeed, it is has to be stressed that cisplatin induced DNA damage (interstrand crosslinks) are substrates also for nucleotide excision repair, that has a key role in repairing these lesions.

    We agree with the reviewer that transcription-coupled NER is the key pathway for the resolution of cisplatin-induced damage. We therefore have revised this statement in the manuscript as “Our data showing the dispensability of ALC1 in cisplatin response, both in BRCA1 and 2-mutant settings, is consistent with previous reports demonstrating the dispensability of this remodeler for MMEJ or transcription-coupled nucleotide excision repair.” We have cited previous work where ALC1 has been shown to be dispensable for MMEJ or TC-NER. Similarly, we have modified the text on page 7 as “Furthermore, ALC1 loss did not impact sensitivity to cisplatin in HRP cyclin E1-high cells. This observation is consistent with previous studies showing its dispensability for HR repair.”

    Figure 2. Please explain better why niraparib is not active in cyclinE1-high cells.

    Our comprehensive studies examining the impact of ALC1 depletion on PARPi response uncover the generalized theme that targeting is most effective in enhancing sensitivity of olaparib and rucaparib, which have moderate PARP1/2 trapping ability, as compared to niraparib and talazoparib, which are strong trappers. One possible explanation could be that moderate PARP1/2 trappers are more amenable for combination strategies because their effects do not reach full saturation, preserving a dynamic range that allows for additive or synergistic enhancement. This was included in the discussion section of the manuscript.

    It is not clear to me if the authors consider a cyclin E "gain" an overexpressing tumor (i.e. OVCAR8). The authors need to show the response to PARPi in one (possibly two) cell lines with very low expression of cyclin E and knock-down of ALC1.

    We have present data in multiple BRCA1-WT cell lines with very low expression of cyclin E compared to OVCAR8. These include: FT282 cell line (Fig. 4), two FT282 clones of BRCA1-/+ FT cells (Fig. 5), and full length BRCA1 addback UWB1.289 (Fig. 3c). Additionally, we have added immunoblotting data showing that in OVCAR8, the level of cyclin E1 protein and activity as assessed by pCdk2 is comparable to OVCAR3 and OVCAR4, two CCNE1-amplified lines (Fig. S2d). In contrast, FT 282 and UWB1.289 BRCA1 add back cells have low levels of cyclin E and thus low pCdk2.

    The deletion of ALC1 do interfere with tumor take and tumor growth? No clear is the in vivo experiments.

    Tumor uptake: We injected OVCAR8 cells in mice three days post-transduction of sgALC1. Depletion of ALC1 is only achieved at 14 days post transduction. This explains why tumor uptake is not impacted. We do not observe a significant impact of ALC1 loss on tumors derived from OVCAR8 cells. This is consistent with the dispensability of ALC1 in the proliferation of HR-proficient cells (PMID: 33333017; PMID: 33462394). We have added text in the manuscript to clarify this point.

    Injecting OVCAR8 cells in the peritoneum is not associated with the formation of ascites?

    We thank the reviewer to bring up this important point. The objective of this study is to examine how ALC1 loss can enhance PARPi responses and therefore we chose an earlier time point (~50 days) to assess the impact on tumor growth. Ascites formation upon intraperitoneal injection of OVCAR8 cells has primarily been reported at late stages of disease development. For example, Anirban Mitra et al. (2015) (PMID: 26050922) reported consistent ascites formation, but only at extended timepoints (up to ~90 days post-injection). Similarly, Yong-Tae Shen et al. (2019) (PMID: 31117198) injected 5-10 x106 cells and observed ascites emergence beginning around day 49, with progressive accumulation toward the endpoint, indicating that fluid buildup coincides with advanced peritoneal dissemination. In contrast, studies using comparable inoculation doses (e.g., 1×10⁶ cells) and shorter observation periods (~6 weeks) such as Luis Hernandez et al. (2016) (PMID: 27235858) did not report detectable ascites. Taken together, these findings suggest that, while OVCAR8 cells can generate ascites, this phenotype typically manifests at later stages of disease progression and is not expected within shorter experimental windows. Therefore, the absence of ascites in our model is consistent with the study design and timeframe, rather than indicative of a failure of tumor establishment.

    We have added relevant discussion in the results section to clarify this point.

    How was tumor weight calculated?

    Tumor burden was quantified by direct collection and measurement of peritoneal tumor nodules. For the sacrificed mice, all visible tumor nodules within the peritoneal cavity were carefully excised, counted, and pooled per animal. The total tumor weight was then determined by weighing the combined mass of all collected nodules using an analytical balance. Thus, “tumor weight” represents the cumulative mass of macroscopic peritoneal implants per mouse. No estimations or indirect calculations were used. This has now been elaborated on in the methods section.

    It seems that tumors grow as solid mass, but how were nodulesAll mice at endpoint exhibited disseminated peritoneal disease, characterized by multiple tumor nodules and invasion into the peritoneal wall. Tumor nodules were quantified by direct visual inspection during necropsy. Small nodules ( Why survival curves were not shown?

    Survival analysis was not included because the study was designed with a predefined experimental endpoint to enable controlled comparison of tumor burden across groups. Animals were therefore euthanized at the same timepoint rather than followed longitudinally to survival. As a result, Kaplan–Meier analysis was not applicable to this experimental design. We agree that survival is an important outcome and would be valuable in future studies specifically powered and designed for that purpose.

    The dose of 50mgr/kg every third day is a very low olaparib dose. Generally the in vivo dosing is 100mgr/kg , 5 days a week for 4 weeks (doi: 10.1158/1535-7163.MCT-21-0420; 10.1158/2767-9764.CRC-22-0423).

    We agree that higher doses of olaparib (e.g., 100 mg/kg, 5 days/week) are commonly used and have demonstrated single-agent efficacy in vivo. In this study, however, our objective was to specifically evaluate the combinatorial effect of olaparib with genetic knock-out of ALC1. To enable this, we intentionally employed a reduced dosing regimen (50 mg/kg every third day) to minimize single-agent activity. This approach allowed us to establish a condition in which olaparib in sgAAVS1 control tumors had limited impact on tumor burden, thereby providing a dynamic range in which to detect potential sensitization effects mediated by sgALC1. Using a fully efficacious dose would likely mask such interactions by producing a near-maximal response in the control group. Thus, the selected dosing strategy reflects a deliberate experimental design to assess potentiation effects rather than to model maximal therapeutic efficacy of olaparib as a monotherapy.

    Figure 4. I could not find the data of the minimal impact of ALC1 in UWB1.289 cells. What the author refer to? They refer to the fact that ALC1 deletion di not cause any cell growth alteration or to something else? But were there the data?

    The minimal impact being referred to was PARPi responses in BRCA1-proficient UWB1.289. We have now fixed the statement to read: “The minimal impact of ALC1 in BRCA1-proficient UWB1.289 cells on PARPi responses suggested that targeting this remodeler may have minimal impact on normal healthy cells.” and included the relevant figure number (Fig.3c) for clarity.

    The modest increment in pRPA in hTER-FT282 is statistically significant and not very different from what observed in UWB.289, suggesting that ACL1 deletion could indeed impact normal cells. These data should be interpreted more conservatively.

    The increase in pRPA levels upon ALC1 loss in hTERT FT282 BRCA1 het cells and UWB1.289 cells is 1.2 and 1.4 respectively. This is consistent with the literature that BRCA1-/+ het cells have compromised replication stress response. Unresolved replication stress gets processed into double-strand breaks (DSBs). Consistent with the proficiency of hTERT FT282 BRCA1-/+ het cells in DSBs repair, ALC1 deficiency does not increase yh2ax in these cells. Hence, despite an increase in pRPAS33 signal in hTERT FT282 BRCA1 het cells, these cells can resolve downstream breaks. In contrast, a profound, 1.7-fold increase in yh2ax signal was observed upon ALC1 loss in BRCA-mutant UWB1.289 cells, reinforcing that ALC1 loss has a more profound response in BRCA-mutant cancer cells.

    To align with the reviewer’s suggestion, we have removed the word “modest’ and have retained the fold differences in the median values.

    Figure 6. Questionable is the OS as endpoint in this heterogeneous patient population (treated in front line and recurrent) and in my opionion OS, much more than PFS, is influences by the many different treatment these patients underwent and that could influence the OS. Why not considering PFS after/or on PARPi treatment? The authors should clarify the patient population, Indeed, 48 patients were treated with PARPI and were platinum sensitive and possibly HRD. What patients are the HPR patients? How many were they? It is not clear the HRP and high replication stress cohort were treated with PARPi? How many of these were Cyclin E amplified or with high levels? Figure 6F should also include, beside UVB+BRCA1, other tumor cells with no Cyclin E overexpression and non BRCA mutation or HRD. The discussion of limitations should be addressed to strengthen the manuscript.

    We thank the reviewer and agree that PFS is often preferred for evaluating treatment-specific effects. However, in this cohort, PFS was not a reliable endpoint for several reasons. Tumor samples were obtained at diagnosis, whereas PARPi was administered later, in either the frontline maintenance or recurrent setting, introducing temporal and prognostic heterogeneity that limits the interpretability of PFS. These factors confound attribution of PFS specifically to PARPi response. We therefore selected OS from the time of PARPi exposure as a more consistently defined endpoint across this heterogeneous cohort, while acknowledging its limitations.

    Reviewer #2 (Significance (Required)):

    The manuscript by Lindsey et al. explores the role of ALCN1 (Amplified in Liver Cancer 1) loss in enhancing the sensitivity of PARPi in ovarian carcinomas, including BRCA1/2 mutated tumors (both sensitive and resistant to platinum) as well as cyclin E amplified settings. The data are interesting but the in some cases there is an overinterpretation of the results.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ The manuscript by Aubuchon, Wong et al. presents strong insights into the value of ALC1 as novel target for sensitization strategies against PARPi. The authors show that a PARPi resistance is reversible when ALC1 is knocked down and convincingly highlight the genetic circumstances for these approaches. Also, the authors point out that especially the weak PARP-trappers olaparib and rucaparib could benefit from concomitant ALC1 inhibiton and high levels of replication stress by elevated p-T21 RPA2 could serve as biomarker in clinical settings. Furthermore, the authors show that benign fallopian tube cells are not affected by ALC1-kd, which is an important finding for in vivo approaches.

    We thank the reviewer for acknowledging that our work provides “strong insights” and makes “important finding for in vivo approaches”.

    As the manuscript covers a broad experimental field, I would only suggest a few additional experiments to further strengthen the overall story:

    1. How does an ALC1 knock-down affect the expression of PARP1 and if so, how does this contribute to the effects seen by ALC1-kd? The authors could add Western Blot experiments for cell lines belonging to the respective groups that are distinguished in the manuscript: BRCA wt, BRCA mutated and Cyclin E1-high cancer cells and also a benign fallopian tube cell line.

    This was an interesting point brought up by the reviewer. To address this, we examined and compared total PARP1 protein levels in BRCA1 add-back UWB1.289, BRCA1-mutant UWB1.289, cyclin E1-high OVCAR8, and FT282, between ALC1 WT and depleted cells. However, we do not observe any consistent alteration in PARP1 level upon ALC1 depletion (Fig. Supp. Fig. 6a, b).

    In some of the Western Blot data, it also looks like BRCA1 expression is affected by ALC1 kd. The authors could provide some quantified protein expression or qPCR data if there is a correlation between both expressions.

    To address the reviewer’s question, we quantified changes in BRCA1 levels upon ALC1 loss across all cell lines used in this study. As expected, BRCA1 levels were higher in UWB del 11q and Cyclin E1-overexpressing cell lines. In contrast, cell lines harboring heterozygous BRCA1 mutations or BRCA1 promoter methylation were among those with the lowest BRCA1 expression. This trend provides us confidence in reliably quantifying our immunoblotting data. Although minor fluctuations in BRCA1 protein levels were observed following ALC1 depletion, no consistent trend towards either an increase or decrease was evident (Fig. Supp. Fig. 6c). Likewise, when cell lines were grouped according to their sensitivity to PARP inhibition upon ALC1 loss, no clear pattern emerged (Fig. Supp. Fig. 6d). Together, these data suggest that ALC1 depletion does not substantially affect BRCA1 protein levels, consistent with our previous RNA-seq and functional studies indicating that this chromatin remodeler is dispensable for transcriptional regulation or homologous recombination (PMID: 33462394).

    To further strengthen the hypothesis that the effects of strong PARP-trappers are not improved by ALC1 kd, the authors should add data regarding the viability of the cells presented in Figure 3b upon treatment with niraparib and talazoparib in sgALC1 cells (versus vector control). Also, the authors should add cell viability data using talazoparib for the sgALC1 OVCAR cell lines (versus vector control) in Figure 2 and Supplement Figure 3.

    Sensitivity to niraparib and talazoparib upon ALC1 depletion have now been added in Figure 3b, and for OVCAR lines in Supplement Figure 3. As correctly pointed by the reviewer, we consistently observe that impact of ALC1 loss is more profound on olaparib and rucaparib compared to niraparib and talazoparib.

    Some minor points I noticed while reading the manuscript:

    We apologize for the oversight and thank the review for pointing this out.

    • in Figure 3b, both graphs have the same title. I think the right one should be "SYr14" instead of "SYr12" again

    Fixed.

    • In the heading of Figure 2 an "in" is missing

    Fixed.

    • There are some citations, that seem to be made with another citation style (superscript numbers) than numbers in brackets across the manuscript.

    Fixed.

    Reviewer #3 (Significance (Required)):

    The most important aspect resulting from this manuscript is that ALC1 inhbitors could improve the response to some PARPi without damaging healthy cells. Thereby, the authors also mention the limitation of the use of ALC1 as a target and offer a potential biomarker for combinatory approaches. This study offers a very detailed insight into the potential role of ALC1 as a target for sensitization approaches under the different genetic conditions that can occur in HGSOC. These novel insights contribute to further broaden the therapeutic options by PARPi in clinical settings if the results can be approved by in vivo trials.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Aubuchon, Wong et al. presents strong insights into the value of ALC1 as novel target for sensitization strategies against PARPi. The authors show that a PARPi resistance is reversible when ALC1 is knocked down and convincingly highlight the genetic circumstances for these approaches. Also, the authors point out that especially the weak PARP-trappers olaparib and rucaparib could benefit from concomitant ALC1 inhibiton and high levels of replication stress by elevated p-T21 RPA2 could serve as biomarker in clinical settings. Furthermore, the authors show that benign fallopian tube cells are not affected by ALC1-kd, which is an important finding for in vivo approaches.

    As the manuscript covers a broad experimental field, I would only suggest a few additional experiments to further strengthen the overall story:

    1. How does an ALC1 knock-down affect the expression of PARP1 and if so, how does this contribute to the effects seen by ALC1-kd? The authors could add Western Blot experiments for cell lines belonging to the respective groups that are distinguihed in the manuscript: BRCA wt, BRCA mutated and Cyclin E1-high cancer cells and also a benign fallopian tube cell line.
    2. In some of the Western Blot data, it also looks like BRCA1 expression is affected by ALC1 kd. The authors could provide some quantified protein expression or qPCR data if there is a correlation between both expressions.
    3. To further strengthen the hypothesis that the effects of strong PARP-trappers are not improved by ALC1 kd, the authors should add data regarding the viability of the cells presented in Figure 3b upon treatment with niraparib and talazoparib in sgALC1 cells (versus vector control). Also, the authors should add cell viability data using talazoparib for the sgALC1 OVCAR cell lines (versus vector control) in Figure 2 and Supplement Figure 3.

    Some minor points I noticed while reading the manuscript:

    • in Figure 3b, both graphs have the same title. I think the right one should be "SYr14" instead of "SYr12" again
    • In the heading of Figure 2 an "in" is missing
    • There are some citations, that seem to be made with another citation style (superscript numbers) than numbers in brackets across the manuscript.

    Significance

    The most important aspect resulting from this manuscript is that ALC1 inhbitors could improve the response to some PARPi without damaging healthy cells. Thereby, the authors also mention the limitation of the use of ALC1 as a target and offer a potential biomarker for combinatory approaches. This study offers a very detailed insight into the potential role of ALC1 as a target for sensitization approaches under the different genetic conditions that can occur in HGSOC.

    These novel insights contribute to further broaden the therapeutic options by PARPi in clinical settings if the results can be approved by in vivo trials.

  3. Review Commons

    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

    The manuscript by Lindsey et al. explores the role of ALCN1 (Amplified in Liver Cancer 1) loss in enhancing the sensitivity of PARPi in ovariar carcinomas, including BRCA1/2 mutated tumors (both sensitive and resistant to platinum) as well as cyclin E amplified settings.

    The data are interesting but the in some cases there is an overinterpretation of the results. I have listed below my major concerns

    Figure 1. Could the authors demonstrate that OVASAHO cells are BRC2 muted? Indeed, I have always though they were BRCA wt type (10.1016/j.ygyno.2015.08.017). While the data on cisplatin suggest that indeed ALC1 loss do not impact its sensitivity, I disagree with the statant that "the correlation between dispensability of ALC1 in platinum response suggests that this chromatin remodeler likely does not contribute to MMEJ (page 6)" or " is dispensable for HR (page 7). Indeed, it is has to be stressed that cisplatin induced DNA damage (interstrand crosslinks) are substrates also for nucleotide excision repair, that has a key role in repairing these lesions. Figure 2. Please explain better why niraparib is not active in cyclinE1-high cells. It is not clear to me if the authors consider a cyclin E "gain" an overexpressing tumor (i.e. OVCAR8). The authors need to show the response to PARPi in one (possibly two) cell lines with very low expression of cyclin E and knock-down of ALC1.
    The deletion of ALC1 do interfere with tumor take and tumor growth? No clear is the in vivo experiments. Injecting OVCAR8 cells in the peritoneum is not associated with the formation of ascites? How was tumor weight calculated? It seems that tumors grow as solid mass, but how were nodules<1mm quantified? Please clarify. Why survival curves were not shown? The dose of 50mgr/kg every third day is a very low olaparib dose. Generally the in vivo dosing is 100mgr/kg , 5 days a week for 4 weeks (doi: 10.1158/1535-7163.MCT-21-0420; 10.1158/2767-9764.CRC-22-0423).

    Figure 4. I could not find the data of the minimal impact of ALC1 in UWB1.289 cells. What the author refer to? They refer to the fact that ALC1 deletion di not cause any cell growth alteration or to something else? But were there the data? The modest increment in pRPA in hTER-FT282 is statistically significant and not very different from what observed in UWB.289, suggesting that ACL1 deletion could indeed impact normal cells. These data should be interpreted more conservatively.

    Figure 6. Questionable is the OS as endpoint in this heterogeneous patient population (treated in front line and recurrent) and in my opionion OS, much more than PFS, is influences by the many different treatment these patients underwent and that could influence the OS. Why not considering PFS after/or on PARPi treatment? The authors should clarify the patient population, Indeed, 48 patients were treated with PARPI and were platinum sensitive and possibly HRD. What patients are the HPR patients? How many were they? It is not clear the HRP and high replication stress cohort were treated with PARPi? How many of these were Cyclin E amplified or with high levels? Figure 6F should also include, beside UVB+BRCA1, other tumor cells with no Cyclin E overexpression and non BRCA mutation or HRD.

    The discussion of limitations should be addressed to strengthen the manuscript.

    Significance

    The manuscript by Lindsey et al. explores the role of ALCN1 (Amplified in Liver Cancer 1) loss in enhancing the sensitivity of PARPi in ovarian carcinomas, including BRCA1/2 mutated tumors (both sensitive and resistant to platinum) as well as cyclin E amplified settings. The data are interesting but the in some cases there is an overinterpretation of the results.

  4. Review Commons

    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

    ALC1 suppression has been shown to potentiate PARP inhibitor lethality in HR-deficient cells. Rather than revisiting the underlying mechanism, which has been characterized and remains an active area of investigation, this study aims to define the clinical contexts in which combined ALC1 and PARP inhibition may be beneficial. The clinical efficacy of PARP inhibitors, and their FDA approval, is largely restricted to HR-deficient tumors. This study dissects the combined effects of ALC1 and PARP suppression across a panel of HRD ovarian cancer cell lines, multiple classes of PARP inhibitor, and cells harboring distinct PARPi resistance mechanisms. In doing so, the authors delineate both the potential utility and the limitations of combined ALC1 and PARP inhibitor treatment in HRD ovarian cancers. The most impactful finding of the study, however, is likely the demonstration that ALC1 suppression sensitizes HR-proficient, CCNE1-amplified high-grade serous ovarian cancers to PARP inhibitors. These tumors are associated with particularly poor outcomes owing to the current absence of effective targeted therapies, making this observation of considerable clinical relevance. Of note, the study relies on genetic rather than pharmacological depletion of ALC1, a choice likely reflecting the current lack of a commercially available ALC1 inhibitor. While genetic suppression may not fully recapitulate the effects of combined drug treatment, it t offers the advantage of not being tied to any specific compound, allowing the authors to establish more general principles. I have only a few comments.

    The effect of ALC1 KO on PARPi sensitivity is less pronounced in OVSAHO cells (BRCA2-mutated) than in BRCA1-mutated cells. In these cells, it looks like there is an additive effect rather than synergy.

    1. The authors should calculate, if possible, whether there is synergy or additive effect of ALC1-KO lethality (BLISS).
    2. Another BRCA2-mutated cell line should be included.

    Minor comments:

    • Figure key is missing for S2C (I assume it's grey DMSO, blue olaparib)
    • Page 8: "BRCA1-mutant ovarian cancer cells eventually develop chemoresistance when exposed to PARPi for a prolonged period. Mechanistically, this is due to rewiring of ATR signaling, which enables RAD51 loading at DNA breaks and reversed forks independent of BRCA1 protein(25)." This sentence suggest this is the only existing resistance mechanism, which should be correct. Modify to "mechanistically, this CAN be due to", or "this is OFTEN due to".

    Significance

    ALC1 inhibitors have been developed and clinical trials are starting. The significance of this manuscript lies in establishing the clinical potential for combined ALC1-PARP inhibition in high grade serous ovarian cancer. Especially, the authors demonstrate that combined ALC1 suppression with PARP inhibition efficiently kills HR-proficient CCNE1-amplified ovarian cancers, which represent 20% of ovarian cancers and are resistant to current therapies.

  5. Version published to 10.64898/2025.12.04.692458 on bioRxiv