Exploiting Synthetic Lethality between Germline BRCA1 Haploinsufficiency and PARP Inhibition in JAK2V617F-Positive Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are rare hematologic disorders characterized by clonal hematopoiesis. Familial clustering is observed in a subset of cases, with a notable proportion exhibiting heterozygous germline mutations in DNA double-strand break repair genes (e.g., BRCA1). We investigated the therapeutic potential of targeting BRCA1 haploinsufficiency alongside the JAK2V617F driver mutation. We assessed the efficacy of combining the PARP inhibitor olaparib with interferon-alpha (IFNα) in CRISPR/Cas9-engineered Brca1+/− Jak2V617F-positive 32D cells. Olaparib treatment induced a higher number of DNA double-strand breaks, as demonstrated by γH2AX analysis through Western blot (p = 0.024), flow cytometry (p = 0.013), and confocal microscopy (p = 0.071). RAD51 foci formation was impaired in Brca1+/− cells compared to Brca1+/+ cells, indicating impaired homologous recombination repair due to Brca1 haploinsufficiency. Importantly, olaparib enhanced apoptosis while diminishing cell proliferation and viability in Brca1+/− cells compared to Brca1+/+ cells. These effects were further potentiated by IFNα. Olaparib induced interferon-stimulated genes and increased endogenous production of IFNα in Brca1+/− cells. These responses were abrogated by STING inhibition. In conclusion, our findings suggest that the combination of olaparib and IFNα presents a promising therapeutic strategy for MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.


Introduction
Myeloproliferative neoplasms (MPN) are a rare type of blood cancer and a disorder of excess progenitor cell production induced by clonal hematopoietic stem cells (HSCs).Among the group of the BCR-ABL1-negative MPN, the three most frequent subtypes are essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF).With the exception of a minority of triple-negative patients, these disease entities are typically induced by one of three classical driver mutations in the genes encoding the tyrosine kinase Janus kinase 2 (JAK2), the chaperone calreticulin (CALR), or the thrombopoietin receptor MPL, the most frequent being the JAK2V617F mutation [1].Although MPN patients generally exhibit a more favorable prognosis compared to other hematologic malignancies, they face an increased risk of severe thrombotic and hemorrhagic events, leading to increased mortality [2].
Additionally, secondary myelofibrosis or transformation into acute myeloid leukemia (AML) can occur, significantly worsening the prognosis [3].Hence, there is an urgent need for effective treatment strategies to enhance patients' quality of life and improve complication-free survival.Currently, allogeneic HSC transplantation remains the only curative therapeutic option.However, due to its high morbidity and mortality, only few patients are eligible for this intervention [4].
MPN mostly occur sporadically, but in 7.6% of the cases, MPN are associated with a familial incidence [5].In these familial MPN, affected family members may present with different MPN subtypes or driver mutations, implying a shared genetic predisposition for acquiring these driver mutations [6].Utilizing whole-exome sequencing, we could demonstrate that four out of five families with familial MPN presented with germline mutations in genes involved in DNA double-strand break (DSB) repair-associated genes (i.e., BRCA1, BRCA2, ATM, and CHEK2), suggesting that these germline mutations might increase the risk of acquiring a somatic MPN driver mutation [7].
BRCA1 or BRCA2 (BRCA1/2)-mutated cancer cells are effectively targeted by poly ADP ribose polymerase (PARP) inhibitors, leveraging the principle of synthetic lethality [8,9].This concept was first described in 1922 by Calvin Bridges, who observed that the simultaneous loss of two distinct genes was lethal for flies, while the loss of one alone was not [10].In the context of BRCA1/2-mutated cancer cells, PARP plays a pivotal role in DNA singlestrand break (SSB) repair.Inhibiting PARP impedes the repair of DNA SSBs, causing them to progress into more severe DSBs.In BRCA1/2-mutated cells, characterized by defective homologous recombination repair (HRR), PARP inhibition induces a substantial load of DSBs that surpass their repair capacity.This results in the accumulation of DNA damage, ultimately triggering apoptosis through the phenomenon of synthetic lethality existing between BRCA1/2 mutations and PARP inhibition.
This concept is already exploited clinically in familial breast and ovarian cancer, where patients often harbor a heterozygous BRCA1/2 germline mutation and develop cancer after a somatic loss of the second allele.These cancer cells can be targeted specifically with PARP inhibitors, while non-malignant cells, which have retained one functional allele, remain unaffected [11].While it is widely accepted that the loss of the second allele is a prerequisite for carcinogenicity in breast cancer and ovarian cancer, BRCA1 haploinsufficient cells show functional deficits under challenging conditions, such as replicative stress [12,13].In the context of MPN, driver mutations, such as JAK2V617F, induce replicative stress and genetic instability [14][15][16][17].Building upon this knowledge, we postulated that PARP inhibition induces synthetic lethality, specifically in BRCA1 haploinsufficient JAK2V617F-positive cells.
Recent research utilizing an ataxia telangiectasia mutated (ATM) model, with ATM serving as the apex kinase regulating BRCA1, has unveiled that DNA damage induced by ATM loss-of-function mutations primes the type I interferon system via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway [18].Interferon-alpha (IFNα) is a widely used therapeutic option in the treatment of MPN and is recognized for its ability to selectively target malignant stem cells, evoke long-lasting deep molecular remissions, and induce DNA stress [19][20][21].With this understanding, we hypothesized that MPN cells harboring heterozygous BRCA1 mutations exhibit heightened responsiveness to IFNα treatment and that IFNα could potentially enhance the sensitivity of MPN cells with DSB repair gene haploinsufficiency to PARP inhibitors.

DNA DSBs Are Elevated in Brca1 +/− Jak2V617F Cells and Further Amplified by Olaparib
The first aim of this study was to assess whether 32D Jak2V617F Brca1 +/− cells exhibited elevated numbers of DNA DSBs compared to their Jak2V617F Brca1 +/+ counterparts.
We also investigated whether olaparib, IFNα, or their combination induced more DSBs selectively in Brca1 +/− cells.For this purpose, cells were treated with olaparib and/or IFNα for 24 h and analyzed for the frequency of γH2AX-positive cells by flow cytometry (Figure 1A).While olaparib alone exhibited a stronger impact on inducing DSBs than IFNα, the combined treatment showed the most pronounced impact.When compared to Brca1 +/+ control cells, percentages of Jak2V617F Brca1 +/− cells positive for γH2AX were significantly higher when treated with olaparib and the combination of olaparib and IFNα.

DNA DSBs Are Elevated in Brca1 +/− Jak2V617F Cells and Further Amplified by Olaparib
The first aim of this study was to assess whether 32D Jak2V617F Brca1 +/− cells exhibited elevated numbers of DNA DSBs compared to their Jak2V617F Brca1 +/+ counterparts.We also investigated whether olaparib, IFNα, or their combination induced more DSBs selectively in Brca1 +/− cells.For this purpose, cells were treated with olaparib and/or IFNα for 24 h and analyzed for the frequency of γH2AX-positive cells by flow cytometry (Figure 1A).While olaparib alone exhibited a stronger impact on inducing DSBs than IFNα, the combined treatment showed the most pronounced impact.When compared to Brca1 +/+ control cells, percentages of Jak2V617F Brca1 +/− cells positive for γH2AX were significantly higher when treated with olaparib and the combination of olaparib and IFNα.Jak2V617F Brca1 +/+ and Brca1 +/− cells were treated with olaparib (ola, 10 µM), interferon-alpha (IFNα, 10,000 U/mL), or DMSO for 24 h.As a negative control, cells were treated with the ATM inhibitor Ku55933 (Ku, 10 µM) and as a positive control, cells were treated with the topoisomerase II inhibitor (A) Jak2V617F Brca1 +/+ and Brca1 +/− cells were treated with olaparib (ola, 10 µM), interferon-alpha (IFNα, 10,000 U/mL), or DMSO for 24 h.As a negative control, cells were treated with the ATM inhibitor Ku55933 (Ku, 10 µM) and as a positive control, cells were treated with the topoisomerase II inhibitor etoposide (eto, 10 µM).After treatment, the cells were stained with a phospho-histone H2A.X (Ser139) monoclonal antibody and FxCycle™ Violet stain and then analyzed by flow cytometry (n = 4 of two clones respectively, exemplary flow cytometry plots see Figure S1).(B) Protein levels of γH2AX and protein PARylation were analyzed in Jak2V617F Brca1 +/+ and Brca1 +/− cells after incubation for 4 h with olaparib (10 µM), IFNα (10,000 U/mL), and DMSO by Western blot (WB) using GAPDH as a loading control.(C) γH2AX and (D) PAR densitometric analyses from four independent WB experiments (relative to GAPDH as loading control and relative to Brca1 +/+ DMSO, n = 4 with two clones each).Data are presented as mean ± SD and significances defined as: * p < 0.05 and ** p < 0.01, ns means no significance.
To confirm and complement the findings obtained from the flow cytometry analysis, we conducted Western blot experiments using Jak2V617F Brca1 +/+ and Brca1 +/− cells after in vitro treatment with olaparib and/or IFNα for 4 h (Figure 1B-D).In Brca1 +/− cells, we observed a significant increase in γH2AX levels following treatment with DMSO, olaparib and the olaparib/IFNα combination, in comparison to Brca1 +/+ cells (Figure 1B,C).Additionally, basal levels of protein poly ADP-ribosylation (PARylation), a post-translational modification at DNA lesions catalyzed by PARP, were significantly higher in the Brca1 +/− cells compared to the Brca1 +/+ cells (Figure 1B,D).As expected, PARylation was effectively inhibited by olaparib treatment.

Impaired HRR Mechanism and Suppressed Proliferation and Viability in Brca1 +/− Jak2V617F Cells upon Treatment with Olaparib and IFNα
To investigate the impact of Brca1 haploinsufficiency on HRR within Jak2V617F cells, we analyzed the formation of γH2AX and RAD51 foci using immunofluorescence and confocal microscopy, following 24 h treatment with olaparib or DMSO.RAD51 foci are crucial indicators of HRR functionality and are commonly examined to assess impairment of this repair pathway [22].As expected, olaparib treatment prominently triggered the formation of γH2AX foci (Figure 2A).Although not reaching statistical significance, there was a numerical enhancement in the induction of γH2AX foci in Brca1 +/− cells in comparison to Brca1 +/+ cells (Figure 2B).When analyzing RAD51 foci numbers, we observed an induction by olaparib, which was significantly higher in Brca1 +/+ cells (Figure 2C).Olaparib-treated Brca1 +/− cells displayed an increased number of γH2AX foci without a corresponding RAD51 focus compared with Brca1 +/− cells (p = 0.1378; Figure S1K).
Having demonstrated the induction of DSBs by both olaparib and IFNα in Jak2V617F cells, with a notably higher effect in Brca1 +/− cells compared to Brca1 +/+ cells, we examined the effects of olaparib and IFNα on cell proliferation and cell viability.Our findings revealed that olaparib, IFNα, and their combination significantly reduced cell viability and cell proliferation in Brca1 +/− Jak2V617F cells compared to Brca1 +/+ Jak2V617F cells (Figures 3A and S2A).Most importantly, and in line with the findings of our preceding experiments, proliferation, and viability were considerably more impaired in Brca1 +/− cells than in Brca1 +/+ cells when subjected to olaparib, IFNα, or the combination.
Thereafter, we studied the effect of olaparib and IFNα on Jak2V617F Brca1 +/− and Brca1 +/+ cells using MTT assays as an indicator of cell viability, proliferation, and cytotoxicity Figures 3B-D and S2B-D).These assays revealed that the reduction in metabolic activity was significantly more pronounced in Jak2V617F Brca1 +/− cells than in Jak2V617F Brca1  3C and S2B,C), or the combination of both drugs (Figure 3D).We also analyzed the effect of olaparib and IFNα on the metabolic activity of 32D Jak2 wildtype (WT) cells.Interestingly, olaparib and IFNα exhibited a weaker impact on the metabolic activity of Jak2WT cells, with olaparib still demonstrating a stronger effect on Brca1 +/− cells than on Brca1 +/+ cells (Figure S3).However, for IFNα, no difference between Brca1 +/− and Brca1 +/+ cells was observed.Moreover, by comparing the mean relative absorbances from the MTT assays with Jak2WT cells to those with Jak2V617F cells (Table S1), we found that both olaparib and IFNα led to a more pronounced impairment of metabolic activity in Jak2V617F than in Jak2WT cells.Moreover, olaparib treatment had a stronger impact on Jak2V617F cells compared to Jak2WT cells, which was not observed for IFNα treatment.the effects of olaparib and IFNα on cell proliferation and cell viability.Our findings revealed that olaparib, IFNα, and their combination significantly reduced cell viability and cell proliferation in Brca1 +/− Jak2V617F cells compared to Brca1 +/+ Jak2V617F cells (Figures 3A and S2A).Most importantly, and in line with the findings of our preceding experiments, proliferation, and viability were considerably more impaired in Brca1 +/− cells than in Brca1 +/+ cells when subjected to olaparib, IFNα, or the combination.To investigate the potential of olaparib and IFNα to induce apoptosis in Jak2V617F Brca1 +/− and Brca1 +/+ cells, we utilized Annexin V/7-AAD apoptosis assays (Figure 3E).IFNα induced apoptosis to a greater extent than olaparib, whereas the combination provoked the strongest response.By calculating the coefficient of drug interaction (CDI), we observed higher synergistic effects of olaparib and IFNα in Brca1 +/− cells than in Brca1 +/+ cells in MTT (Figure S4A) and apoptosis (Figure S4B) assays.
To assess the impact of olaparib and IFNα on the cell cycle of Jak2V617F Brca1 +/+ and Brca1 +/− cells, we analyzed the fraction of cells in G 0 /G 1 or G 2 /M phase with FxCycle Violet by flow cytometry after 24 h of treatment with olaparib, IFNα, or their combination.A pronounced cell cycle arrest in G 0 /G 1 was induced in Brca1 +/− cells following single treatments of olaparib and IFNα, and this effect was notably enhanced by the combination treatment (Figure S5A).In contrast, the reduction in the fraction of Brca1 +/+ cells in G 2 /M phase was less pronounced.Additionally, we analyzed the expression of negative cell cycle regulators, specifically p16 (i.e., CDKN2A gene) and p21 (i.e., CDKN1A gene).p16, also known as cyclin-dependent kinase inhibitor 2A, inhibits the progression from the G 1 phase to the S phase, and p21, also called cyclin-dependent kinase inhibitor 1, is involved in inhibiting the progression through the G 1 , the S, and the G 2 phases.The upregulation of p16 and p21 was significantly more pronounced in Brca1 +/− cells when treated with both olaparib and IFNα, corroborating the flow cytometric cell cycle analysis (Figure S5B).
In summary, the results affirm our hypothesis that Jak2V617F Brca1 +/− cells exhibit a higher number of DSBs compared with Brca1 +/+ cells.Notably, we found that this susceptibility to DSBs is specifically heightened in Jak2V617F Brca1 +/− cells through PARP inhibition.Furthermore, olaparib and IFNα preferentially compromised the metabolic activity, proliferation, viability, and cell cycle progression of Jak2V617F Brca1 +/− cells.These findings underscore the elevated sensitivity of Brca1 +/− cells to these therapeutic interventions.

Olaparib Induces IFNα
Signaling via Activation of the cGAS-STING Pathway Specifically in Brca1 +/− Jak2V617F Cells Intriguingly, recent evidence has suggested a link between DNA damage and the activation of the cGAS-STING pathway, which is one major pathway responsible for driving the production of IFNα in response to cytosolic microbial and self-DNA [23].This pathway is part of the innate immune system and detects cytosolic microbial DNA but can also be activated by an accumulation of DNA damage, leading to the release of DNA into the cytoplasm and subsequent activation of the cGAS-STING pathway resulting in increased production of IFNα [18].Therefore, we postulated that (a) the cGAS-STING pathway is constitutively active in Jak2V617F Brca1 +/− cells and further augmented by olaparib treatment and (b) that the activated STING-pathway results in an elevated production of IFNα (Figure 4).
To test the hypothesis that the STING pathway is activated by cytoplasmic DNA as a result of increased DSBs in Jak2V617F Brca1 +/− cells, we analyzed transcriptional levels of Sting1 and several interferon-responsive genes (Stat1, Irf7, Mx1, Oas1a, and Isg15) in Jak2V617F Brca1 +/− and Jak2V617F Brca1 +/+ cells after treatment with olaparib, IFNα, or the combination (Figure 5).As expected, we observed an upregulation of all interferonresponsive genes after treatment with IFNα, and mRNA levels of Sting1 were not elevated in Brca1 +/− cells upon treatments but even downregulated in the basal condition.However, interestingly, olaparib treatment induced the expression of Irf7, Mx1, Oas1a, and Isg15 in Jak2V617F Brca1 +/− cells compared to Jak2V617F Brca1 +/+ cells.Moreover, the basal expression of Isg15 and Mx1 was elevated in Brca1 +/− compared to Brca1 +/+ cells, and when treated with both olaparib and IFNα, Stat1, Oas1a, and Isg15 exhibited significant upregulation in Brca1 +/− cells.
into the cytoplasm and subsequent activation of the cGAS-STING pathway resulting in increased production of IFNα [18].Therefore, we postulated that (a) the cGAS-STING pathway is constitutively active in Jak2V617F Brca1 +/− cells and further augmented by olaparib treatment and (b) that the activated STING-pathway results in an elevated production of IFNα (Figure 4).To test the hypothesis that the STING pathway is activated by cytoplasmic DNA as a result of increased DSBs in Jak2V617F Brca1 +/− cells, we analyzed transcriptional levels of Sting1 and several interferon-responsive genes (Stat1, Irf7, Mx1, Oas1a, and Isg15) in Jak2V617F Brca1 +/− and Jak2V617F Brca1 +/+ cells after treatment with olaparib, IFNα, or the combination (Figure 5).As expected, we observed an upregulation of all interferon-responsive genes after treatment with IFNα, and mRNA levels of Sting1 were not elevated To confirm that the induction of the interferon-responsive genes was indeed a result of cGAS-STING pathway activation, we conducted a secretion assay, as this pathway is also known to induce heightened expression and secretion of IFNα.Supernatant harvested from olaparib-or DMSO-treated Jak2V617F Brca1 +/− and Brca1 +/+ cells, was added to freshly seeded Jak2V617F Brca1 +/+ cells.The expression of interferon-responsive genes was then assessed by RT-qPCR to determine whether olaparib-treated cells had secreted IFNα (Figure 6A).We found that, indeed, basal levels of Oas1a and Isg15 were upregulated in those cells that have received supernatant from Jak2V617F Brca1 +/− cells compared to Jak2V617F Brca1 +/+ cells.This effect was further enhanced by olaparib treatment.Importantly, this effect was not observed when the cells were preincubated with an IFNαR1 blocking antibody before adding the supernatant, confirming that Brca1 haploinsufficiency in Jak2V617F Brca1 +/− cells stimulates an increased production of IFNα.
in Brca1 +/− cells upon treatments but even downregulated in the basal condition.However, interestingly, olaparib treatment induced the expression of Irf7, Mx1, Oas1a, and Isg15 in Jak2V617F Brca1 +/− cells compared to Jak2V617F Brca1 +/+ cells.Moreover, the basal expression of Isg15 and Mx1 was elevated in Brca1 +/− compared to Brca1 +/+ cells, and when treated with both olaparib and IFNα, Stat1, Oas1a, and Isg15 exhibited significant upregulation in Brca1 +/− cells.those cells that have received supernatant from Jak2V617F Brca1 +/− cells compared to Jak2V617F Brca1 +/+ cells.This effect was further enhanced by olaparib treatment.Importantly, this effect was not observed when the cells were preincubated with an IFNαR1 blocking antibody before adding the supernatant, confirming that Brca1 haploinsufficiency in Jak2V617F Brca1 +/− cells stimulates an increased production of IFNα.Finally, to verify that the upregulation of interferon-responsive genes and the increased production of IFNα are indeed mediated through the cGAS-STING pathway, we treated Jak2V617F Brca1 +/− and Brca1 +/+ cells with olaparib and the STING inhibitor H-151.Subsequently, we analyzed the expression of three interferon-stimulated genes (ISGs): Mx1, Oas1a, and Isg15 (Figure 6B).Intriguingly, we observed that the increased ISG expression levels in Brca1 +/− cells after treatment with olaparib were antagonized by STING inhibition, except for Oas1a.These findings indicate that DNA damage induced by PARP inhibition leads to an activation of the cGAS-STING pathway with subsequent increased production of IFNα in Jak2V617F-positive Brca1 haploinsufficient cells.This unique interplay potentially renders these cells more susceptible to IFNα and other MPN-directed treatments.

Discussion
Loss-of-function mutations of DNA repair-associated genes play a role in many types of cancer, but their potential significance in MPN has only recently been suggested by a whole-exome sequencing study of our group, in which heterozygous germline mutations in DSB repair genes have been identified in four out of five families with familial MPN [7].Moreover, even in patients with sporadic MPN, the natural incidence of germline mutations in DSB repair genes would be expected to be at least 0.1-0.5% since this is the combined incidence of BRCA1 and BRCA2 mutations in the general population [24].As it is already known that cancers harboring germline mutations in DSB repair-associated genes are susceptible to PARP inhibition, it is tempting to consider whether this principle could also be translated to patients with familial or sporadic MPN.Currently, no clear recommendations on how to manage these MPN patients are available.
In this study, we demonstrated that heterozygous Brca1 mutations in 32D Jak2V617F cells result in impaired HRR, suggesting a potential therapeutic vulnerability that could be exploited by PARP inhibitors.As previously reported, JAK2V617F induces DNA damage [14,15], and our findings suggest that Jak2V617F-positive cells harboring an additional heterozygous Brca1 mutation experience even greater DNA damage, surpassing the capacity of the defective DSB repair machinery.This effect is notably amplified by olaparib, with significantly elevated γH2AX levels as demonstrated by Western blot and flow cytometry, further supported by close-to-significant immunofluorescence analyses (p = 0.07).Based on these findings, we propose that PARP inhibition could offer a promising therapeutic strategy for familial MPN patients carrying DSB repair-associated germline mutations by exploiting synthetic lethality.Furthermore, we observed an activation of the STING pathway in olaparib-treated Brca1 +/− Jak2V617F cells, leading to an increased secretion of IFNα.This cytokine, which has emerged as a standard treatment in MPN, exhibited enhanced efficacy in Brca1 +/− Jak2V617F cells compared to Brca1 +/+ cells.While IFNα is capable of inducing long-lasting deep molecular remission and promoting the cycling of dormant stem cells [25,26], the combination of IFNα with olaparib presents a potential synergistic drug combination worthy of further investigation.
IFNα selectively induces cycling of the JAK2V617F HSC, leading to cell cycle stressassociated genomic instability and increased susceptibility towards PARP inhibition, particularly in cells with defective DNA repair mechanisms.To our knowledge, this therapeutic approach of combining PARP inhibitors with IFNα has not been investigated yet and holds potential for application in non-familial MPN patients as well, as BRCA1 is epigenetically inactivated in 40% of all MPN samples analyzed [22], and alterations in DNA repair genes are a frequent feature in MPN patients [27].This suggests that effective treatment options targeting defective DNA repair mechanisms might extend beyond familial MPN cases to encompass a larger cohort of MPN patients who could benefit from this targeted therapeutic approach.Supporting this notion, the effectiveness of PARP inhibitors in MPN with no detected DSB repair-associated gene mutation has already been demonstrated [28].
The cGAS-STING pathway has been recognized as an activator of the antitumor immune response [29], and in triple-negative breast cancer, the efficacy of olaparib depends on the activation of the cGAS-STING pathway, which recruits CD8 + T cells into the tumor microenvironment, thereby triggering an antitumor immune response [30].Our findings indicate that olaparib induces an upregulation of the cGAS-STING pathway in Brca1 +/− cells, leading to an increase in intrinsic IFNα production.
Our study on murine Jak2V617F-positive 32D cells indicates the potential relevance of Brca1 haploinsufficiency to human MPN disease.Given the rarity of MPN and the low prevalence of BRCA1 mutations in the general population, identifying MPN patients harboring BRCA1 mutations is challenging.To address this, we are actively conducting an extensive screening on patients with familial MPN.Subsequently, we plan to validate our mechanistic findings in further studies, utilizing primary MPN patient samples harboring BRCA1 mutations and haploinsufficient Brca1 animal models to assess how the concept of synthetic lethality between BRCA1 haploinsufficiency and PARP inhibition translates clinically into JAK2V617F-driven MPN.Considering that BRCA1 mutations in patients with familial MPN extend beyond the bone marrow, data from triple-negative breast and ovarian cancer patients harboring heterozygous germline BRCA1 mutations who have undergone olaparib treatment might aid in estimating treatment-associated side effects on BRCA1-haploinsufficient non-cancerous cells.Nevertheless, our MTT assays have already demonstrated that both olaparib and the combination with IFNα exert a more pronounced effect on Brca1 +/− cells with an additional Jak2V617F driver mutation when compared to Brca1 +/− Jak2WT cells.Although PARP inhibitors are already approved for the treatment of other cancers and their efficacy and safety have been assessed in clinical trials [31,32], in vivo studies are required to assess the efficacy and safety of PARP inhibitors, both alone and in combination with IFNα, for the treatment in MPN.Additionally, considering that PARP inhibitors may impair DNA damage repair and pose a carcinogenic risk for homologous recombination proficient cells [33], further studies are required to estimate the risk of developing secondary cancers.This is underlined by reports suggesting an increased risk of developing AML or myelodysplastic syndrome in breast or ovarian cancer patients treated with PARP inhibitors over extended periods [34].In conclusion, our findings suggest the potential of combining olaparib and IFNα as a promising therapeutic strategy in MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.

Cell Lines
32D Jak2V617F Brca1 +/− cells were generated by CRISPR/Cas9 using two guideRNAs targeting Brca1 exon 10 (CD.Cas9.LFMV2350.AA (AGTCCAAAGGTGACAGCTAA) and CD.Cas9.LFMV2350.AB (GGTTAAGCGCGTGTCTCAAG) and Cas9 nuclease according to the manufacturer's protocol (IDT technologies, Coralville, IA, USA).Parental 32D cells (RRID:CVCL_0118, DSMZ, Braunschweig, Germany) were retrovirally transduced with pMSCV-Jak2V617F-IRES-GFP.Clones were screened by Sanger sequencing (genomic DNA and mRNA) for frameshift mutations inducing premature stop codons in the same region as the human BRCA1 c.2722G>T Glu908* missense mutation that was identified in our whole-exome sequencing analysis in familial MPN [7].Off-target analysis was performed as described in the Supplementary Materials and relevant off-target effects of the CRISPR/Cas9 process were ruled out.In all experiments, two different clones of each genotype were examined.

Cell Proliferation Assay
The different cell clones were seeded in a concentration of 2 × 10 5 cells/mL in RPMI-1640 medium with 10% FCS, 5 ng/mL mIL-3, and 1% penicillin/streptomycin and treated with the following drugs: IFNα (10,000 U/mL), olaparib (10 µM), or the combination of IFNα and olaparib.Then, the cells were analyzed with a CASY-TTC cell analyzer (OMNI Life Science, Bremen, Germany) after 24 h, 48 h, and 72 h.

RT-qPCR
Reverse transcriptase quantitative PCR (RT-qPCR) for the analysis of cDNA transcripts was performed as previously published (20).Primer sequences are given in the Supplementary Materials (Table S3).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.4.0 (GraphPad Software, Boston, MA, USA) to calculate independent t-tests or, in case of multiple comparisons and, unless otherwise stated, two-way ANOVAs (Tukey post hoc test).The results are displayed as individual values with mean and standard deviation (SD) if not indicated otherwise.

Conclusions
In summary, our study has demonstrated that Brca1 haploinsufficiency induces DNA damage in Jak2V617F-positive cells while priming the type I interferon system via STING, rendering them more susceptible to olaparib treatment, whether applied as a standalone therapy or in conjunction with IFNα.This combined therapeutic approach, which targets DSB repair mechanisms, presents the potential for the treatment of MPN patients with germline DSB repair gene mutations, including those with familial and sporadic MPN.Further investigations are needed to fully elucidate the clinical implications of our findings and to assess the feasibility of implementing genetic counseling for DSB repair germline mutational testing in all MPN patients [7].

Figure 4 .
Figure 4. Hypothesis: PARP inhibition activates the cGAS-STING pathway in Jak2V617F Brca1 +/− cells.(1) The Jak2V617F mutation leads to the formation of reactive oxygen species and thereby causes DNA stress, resulting in an increased number of single-and double-strand breaks (SSBs/DSBs).(2) PARP inhibition suppresses the repair of SSBs, resulting in the formation of doublestrand breaks (DSBs) that also cannot be repaired sufficiently due to the Brca1 haploinsufficiency.(3) The accumulation of DSBs results in the translocation of DNA fragments into the cytoplasm.(4) cGAS recognizes cytosolic DNA and, upon detection of cytosolic DNA, synthesizes cGAMP.(5) cGAMP is detected by STING and, together with TBK1, phosphorylates and activates the transcription factors NF-κB and IRF3.(6) NF-κB and IRF3 translocate into the nucleus and induce the production of proinflammatory cytokines (e.g., TNF, IL-6, type I interferons).(7) Those cytokines are secreted into the interstitium where they bind to the membrane receptors of the original and the surrounding cells and hence induce a proinflammatory state.Created with BioRender.com.

Figure 4 .
Figure 4. Hypothesis: PARP inhibition activates the cGAS-STING pathway in Jak2V617F Brca1 +/− cells.(1) The Jak2V617F mutation leads to the formation of reactive oxygen species and thereby causes DNA stress, resulting in an increased number of single-and double-strand breaks (SSBs/DSBs).(2) PARP inhibition suppresses the repair of SSBs, resulting in the formation of double-strand breaks (DSBs) that also cannot be repaired sufficiently due to the Brca1 haploinsufficiency.(3) The accumulation of DSBs results in the translocation of DNA fragments into the cytoplasm.(4) cGAS recognizes cytosolic DNA and, upon detection of cytosolic DNA, synthesizes cGAMP.(5) cGAMP is detected by STING and, together with TBK1, phosphorylates and activates the transcription factors NF-κB and IRF3.(6) NF-κB and IRF3 translocate into the nucleus and induce the production of proinflammatory cytokines (e.g., TNF, IL-6, type I interferons).(7) Those cytokines are secreted into the interstitium where they bind to the membrane receptors of the original and the surrounding cells and hence induce a proinflammatory state.Created with BioRender.com(accessed on 17 October 2023.