LCK Facilitates DNA Damage Repair by Stabilizing RAD51 and BRCA1 in the Nucleus of Chemoresistant Ovarian Cancer

doi:10.21203/rs.3.rs-2223587/v1

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LCK Facilitates DNA Damage Repair by Stabilizing RAD51 and BRCA1 in the Nucleus of Chemoresistant Ovarian Cancer

https://doi.org/10.21203/rs.3.rs-2223587/v1

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Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success of this therapy in the treatment of HR repair deficient cancers has not translated to HR-proficient cancers. Our studies identify the novel role of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in the regulation of HR repair in endometrioid epithelial ovarian cancer (eEOC) model. We show that DNA damage leads to direct interaction of LCK with the HR repair proteins RAD51 and BRCA1 in a kinase dependent manner RAD51 and BRCA1 stabilization. LCK expression is induced and activated in the nucleus in response to DNA damage insult. Disruption of LCK expression attenuates RAD51, BRCA1, and BRCA2 protein expression by hampering there stability and results in inhibition of HR-mediated DNA repair including suppression of RAD51 foci formation, and augmentation of gH2AX foci formation. In contrast LCK overexpression leads to increased RAD51 and BRCA1 expression with a concomitant increase in HR DNA damage repair. Importantly, attenuation of LCK sensitizes HR-proficient eEOC cells to PARP inhibitor in cells and pre-clinical mouse studies. Collectively, our findings identify a novel therapeutic strategy to expand the utility of PARP targeted therapy in HR proficient ovarian cancer.

Health sciences/Oncology/Cancer/Cancer therapy/Chemotherapy

Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer

Epithelial ovarian cancer (EOC) is the second most common gynecologic malignancy in the United States, but the leading cause of gynecologic cancer death. It is estimated that in 2022, ~ 19,880 women in the U.S. will be diagnosed with epithelial ovarian cancer (EOC) and 12,810 will die from their disease [1]. Poly-(ADP-ribose) polymerase inhibitors (PARPi) have emerged as new therapeutic options in the treatment of ovarian cancer [2–4]. Recent studies show that this treatment has prolonged median recurrence-free survival after primary therapy by more than 24 months [5]. While the benefit of PARPi is greatest in BRCA1/2-mutant or deficient tumors, those with HR deficiencies also experience a benefit from this therapy [5]. Conversely, PARPi and chemotherapy have so far shown limited efficacy in HR-proficient ovarian cancers. Further, platinum resistance is associated with HR proficiency in EOC [6, 7]. This limited efficacy of both platinum and PARPi therapy highlights an unmet clinical need in ovarian cancer patients.

Several strategies have been assessed to expand the utility of PARPi in HR-proficient cancers [2, 8–10]. RAD51, BRCA1, and BRCA2 are critical components of the HR repair complex. Studies have focused on disrupting this complex. Cyclin-dependent kinase (CDK) proteins were shown to regulate the HR repair pathway in a lung cancer model [11]. Indeed, the CDK inhibitor dinaciclib can attenuate the expression of RAD51 and BRCA proteins resulting in the inhibition of HR repair capacity and potentiation of the pharmacological effect of PARPi [9]. However, there is no clinically approved drug for combination with PARPi for HR-proficient cancers.

Approximately 80% of endometrial cancers and 10% of ovarian cancers demonstrate endometrioid tumor histology (eEOC) [12]. A small but clinically significant proportion of eEOC display high-grade histology, advanced stage (FIGO stage III-IV), and a poor 5-year survival of 6–24%. These traits are like those of the more aggressive high-grade serous type of ovarian cancer [13]. Moreover, somatic and/or germline mutations in HR genes including BRCA1/2 occur in only a third of ovarian tumors, indicating the majority of eEOC are HR-proficient. Of note, eEOC show a considerably higher rate of resistance to platinum-based chemotherapy [14] compared to serous carcinomas and do not commonly respond to targeted therapies such as PARP inhibitors.

We previously determined that the intracellular, non-receptor tyrosine kinase, LCK regulates genes implicated in DNA repair machinery in eEOC[15]. We also demonstrated the pharmacologic inhibition of LCK attenuated expression of homologous recombination DNA damage repair genes leading to sensitization of eEOC cells to cisplatin [16]. In contrast, increased expression of LCK led to upregulation of DNA damage-repair genes and increased resistance to cisplatin. As LCK modulates RAD51, BRCA1, and BRCA2 expression, we hypothesized that blocking LCK expression or inhibiting kinase activity would sensitizes eEOC to PARPi. Here, we elucidate the mechanism of LCK in regulating HR DNA damage repair and identify a therapeutic approach to sensitize HR-proficient eEOC to PARP inhibitors.

LCK complexes with RAD51 and BRCA1 in response to DNA damage in an LCK kinase dependent manner.

We tested whether LCK directly interacts with RAD51 and BRCA1 in nuclear extracts. For this, CP70 and SKOV3 cells were transduced with a myc-tagged LCK (Fig. 1A and Supplementary Fig. S1) treated cells with/without etoposide, isolated nuclei, and performed an IP (immunoprecipitation) assay with myc antibodies. In untreated cells, neither BRCA1 nor RAD51 co-precipitated with mycLCK. In contrast, etoposide treatment resulted in co-precipitation of RAD51 and BRCA1 with mycLCK (Fig. 1B,C). In parallel, RAD51 could co-immunoprecipitate LCK from etoposide-treated OE SKOV3 cells. LCK and BRCA1 were detected in RAD51 immunoprecipitates (Fig. 1D).

LCK interacts with BRCA1 and RAD51 in response to DNA damage. We next tested whether kinase activity and autophosphorylation of LCK is necessary for activity and DNA repair. We generated LCK mutants at lysine 273, which is necessary for the catalytic activity of the tyrosine kinase (K273R); tyrosine 394, the autophosphorylation and activation site (Y394F) [17]; and tyrosine 192, a SH2 adaptor protein binding site (Y192F) [18]. Mutants were transduced into LCK KO CP70 cells. OE and Y192F mutants retained kinase activity, while K273R and Y394F mutants lacked kinase activity. We performed IP studies in etoposide-treated cells and determined that OE and Y192F cells were able to co-immunoprecipitate BRCA1 and RAD51, whereas K273R and Y394F failed to co- immunoprecipitate BRCA1 and RAD51 (Fig. 1E). These findings indicate that in response to DNA damage, LCK with intact kinase activity interacts with a complex containing RAD51 and BRCA1 (Fig. 1F).

To test the hypothesis that LCK increases the stability of BRCA1 and RAD51 proteins, empty vector EV and OE cells were treated with cycloheximide and harvested at 0, 1, 2, 3, 5, and 6 hours followed by quantification of protein expression levels (Fig. 1G,H Supplementary Fig. S2). Half-lives of RAD51 in CP70 EV and OE cells treated with cycloheximide were 3.2 and 5.6 h, respectively. Half-lives of BRCA1 in CP70 EV and OE cells treated with cycloheximide were 54 min and 3.4 h, respectively. These studies indicate that LCK is sufficient to regulate BRCA1 and RAD51 protein expression via protein stabilization. We observed that DNA damage induction led to interaction of LCK with Rad51/BRCA1 in OC cells. Interestingly, without inducing DNA damage, LCK overexpression was sufficient to increase the stability of Rad51 and BRCA1. This findings indicated that LCK phosphorylation led to stabilization of Rad51 and BRCA1 proteins.

LCK regulation of DNA double strand break repair.

As γH2AX and RAD51 are markers of DNA damage and repair of double strand breaks, we tested for foci formation in control and etoposide-treated cells. LCK was overexpressed (OE) in CRISPR/Cas9-background (KO) cells and treated in absence or presence of etoposide then subjected to immunofluorescence analysis to detect and quantify γH2AX foci (Fig. 2A, B and Supplementary Fig. S3A). In the absence of etoposide, no γH2AX foci formation was detected. Parental CP70 (WT) cells treated with etoposide led to increased γH2AX foci compared to DMSO treatment. KO cells treated with etoposide exhibited 4–5 fold increased foci formation compared to WT. Foci formation was nearly completely blocked in etoposide treated OE cells (Fig. 2A and B).

In parallel, we assessed RAD51 foci formation in KO and OE CP70 cells treated in the absence or presence of etoposide (Fig. 2C, D and Supplementary Fig. S5B). As with γH2AX, no RAD51 foci were observed in WT, KO, or OE cells treated with DMSO. In contrast, etoposide treatment led to a significant increase in RAD51 foci in WT CP70 cells that was significantly suppressed in KO cells (Fig. 2C, D, and Supplementary Fig. S3B). RAD51 foci formation was significantly increased in OE cells treated with etoposide. This data supports the conclusion that LCK can regulate HR repair during DNA damage response (DDR).

LCK kinase activity is essential for HR repair.

We next assessed γH2AX and RAD51 foci formation in OE, K273R, Y192F, and Y394F transduced cells. OE and Y192F exhibited similar level of foci formation in response to etoposide (Fig. 2E, F, G and H), whereas K273R and Y394F showed increased γH2AX foci and reduced RAD51 foci in etoposide treated cells (Fig. 2E, F, G and H and Supplementary Fig. S4). We determined the etoposide sensitivity in CP70 cells (Fig. 2I). The IC₅₀ for etoposide was 5µM in naïve CP70 cells, 1.67µM in LCK KO, and 10.03µM in LCK OE and 8.70µM for Y192F. Interestingly, K273R and Y394F showed of IC50 values of etoposide, 2.28µM and 2.01µM (For statistical analysis see the Supplementary table14). Analyzing these data, we showed that LCK kinase facilities DNA damage repair during DDR. We performed a timed experiment with etoposide treated CP70 cells and determined that the extent of γH2AX foci was lower in OE and LCK Y192F mutant cells than in WT cells at 0 hours. Moreover, KO, Y394F, and K273R cells exhibited the highest number of γH2AX foci at all time points (Fig. 2J, and Supplementary Fig. S5). These findings lead to the hypothesis that LCK kinase activity is essential for interaction with RAD51 and BRCA1 during DNA damage response and facilitated HR repair.

DNA damage induces LCK dependent BRCA1 expression.

We next assessed the effects of DNA damage on LCK expression and activation. DNA damage in ovarian cancer cells was induced using either etoposide, ultraviolet radiation, or methyl methanesulfonate (MMS). Dose-dependent treatment of CP70 cells with etoposide or MMS led to increased LCK protein expression (Fig. 3A). Etoposide or MMS treatment in SKOV3 cells led to increased phosphorylation of LCK at pY394, while total levels of LCK protein remained unchanged (Supplementary Fig. S6A). Likewise, ultraviolet radiation of CP70 cells was sufficient to increase LCK phosphorylation (Supplementary Fig. S6B). BRCA1 and γH2AX expression was induced by etoposide or MMS, indicating increased DNA damage (Fig. 3A and Supplementary Fig. S7A, B). Because DNA damage, particularly double strand breaks (DSB), and its repair machinery are concentrated in the nucleus [19], we investigated the effects that DNA damage could induce on the accumulation of LCK in the nucleus. We found increased total LCK and pLCK in the nucleus of etoposide-treated cells (Fig. 3B and Supplementary Fig. S7C). Sub-cellular fractionation and immunofluorescence analysis both showed that pLCK was predominately localized in the nucleus of etoposide-treated cells (Fig. 3C). These findings were replicated in SKOV3 cells (Supplementary Fig. S6C). We next tested whether inhibition of LCK would be sufficient to block BRCA1 expression in etoposide-treated cells. Etoposide treatment in shCon cells showed increased BRCA1 protein expression, whereas this treatment attenuated BRCA1 expression in KD cells (Fig. 3D and Supplementary Fig. S7D). We repeated these studies in CRISPR/CAS9 KO cells and observed similar attenuation of BRCA1 in KO CP70 cells and no attenuation in parental cells (Fig. 3E and Supplementary Fig. S7E). These findings indicate that the induction of BRCA1 expression in response to DNA damage is disrupted by LCK inhibition.

We tested whether LCK inhibition is sufficient to inhibit HR DNA repair genes RAD51, BRCA1, and BRCA2 at the protein level. We inhibited LCK expression using shRNA and CRISPR in CP70 and SKOV3 cell lines. Cells were transduced with lentivirus containing shRNA control (shCon) or LCK-targeted shRNA (KD1, KD2). Additionally, we generated LCK knock-out (KO) CP70 cells via CRISPR/Cas9. LCK inhibition was confirmed by immunoblotting followed by analysis of expression of BRCA1, BRCA2, and RAD51 via western blot analysis. In CP70 cells, we observed that KD1, KD2, and KO displayed attenuated protein expression of BRCA1, BRCA2 and RAD51 when compared to shCon (Supplementary Fig. S8A). The protein expression levels of BRCA1, BRCA2, and RAD51 were similarly attenuated in LCK knock-down SKOV3 cells (Supplementary Fig. S8A).

In complementary studies, we tested whether LCK overexpression would increase RAD51, BRCA1, and BRCA2 protein expression in eEOC. LCK overexpression led to induction of RAD51, BRCA1, and BRCA2 protein expression in CP70 and SKOV3 cells (Supplementary Fig. S8A).

To test whether pharmacologic inhibition of LCK can attenuate expression of DNA damage repair proteins, we used PP2, a cell-permeable, small-molecule inhibitor of LCK kinase[20, 21]. We tested the efficacy of PP2 in CP70 and SKOV3 OE cells. PP2 attenuated pLCK at Y394, the autophosphorylation site of LCK in these cells (Supplementary Fig. S8B). γH2AX, a marker of DNA damage and replication stress, was elevated by PP2 treatment. This is indicative of either increased damage or reduced repair of DNA damage due to attenuation of BRCA1 and RAD51 expression. LCK inhibition also attenuates expression of RAD51, BRCA1, and BRCA2 in parental CP70 and SKOV3 as well as in the CRL1978 clear-cell EOC cell line (Supplementary Fig. S9A).

Lck Inhibition Attenuates Hr Repair Efficiency

The inhibition of DNA damage repair genes led us to test whether LCK inhibition impairs HR-dependent DNA repair. We utilized the DR-GFP reporter assay established in U2OS cells to measure repair efficiency[22] (Fig. 4A). U2OS cells with/without DR-GFP reporter system express endogenous LCK protein expression as shown by western blot analysis (Supplementary Fig. S9B). U2OS cells treated with PP2 leads to a dose-dependent reduction of the GFP-positive cell population when compared to DMSO-treated cell population, indicating reduced DNA repair as a consequence of LCK inhibition (Fig. 4B). Likewise, shRNA silencing of LCK led to a reduction in the GFP-positive population compared to shCon transduced cells (Fig. 4B). This indicates that LCK inhibition attenuates HR repair efficiency in cancer cells.

DNA damage leads to activation of several repair pathways including PARP, HR, and NHEJ [23]. As our studies indicated LCK inhibition attenuates HR repair proteins, we assessed LCK’s impact on the expression of alternative DNA repair pathways, including PARP and NHEJ, in CP70 and SKOV3 cells. The LCK inhibitor PP2 did not inhibit PARylation in CP70 and SKOV3 cells (Fig. 4C). The Ku80 and Ku70 proteins are a critical component of the NHEJ pathway[24]. After PP2 treatment, Ku80 protein expression was elevated in CP70, but not in SKOV3 (Fig. 4D). Furthermore, Ku70 protein expression was not changed in either CP70 or SKOV3 after PP2 exposure (Fig. 4D). In parallel, Ku70 and Ku80 expression levels in CRL1978 cells did not change following PP2 treatment (Supplementary Fig. S9C). These findings indicate that LCK inhibition targets HR repair proteins independent of induction of NHEJ repair mechanisms (Fig. 4E).

LCK inhibition augments PARPi induced DNA damage and genomic instability.

We performed single cell gel electrophoresis (alkaline COMET) assay to quantify the extent of double and single strand DNA breaks by visualizing tail area [25]. CP70 and SKOV3 cells were incubated with either PP2, olaparib, or both. Treated cells were processed and stained with SYBR Gold to detect and measure the tail moment (Fig. 7A). PP2 and olaparib alone displayed a comparable increase in comet tails compared to DMSO (Fig. 5B). The combination of PP2 and olaparib induced a fourfold increase in comet tail area compared to monotherapy (Fig. 5B).

PARP inhibitors have been reported to induce genomic instability, leading to chromosomal aberration and DNA damage in cancer cells[26, 27]. Chromosomal damage can be detected by chromosomal breaks, gaps, and radial formations. We identified multiple breaks, gaps, and radial formation in PP2 and olaparib-treated cells (Fig. 5C). PP2 and olaparib displayed a comparable increase in chromosomal damage when compared to DMSO (Fig. 5D). The combination of PP2 and olaparib displayed increased chromosomal damage in both CP70 and SKOV3 cells (Fig. 5D).

Based on this analysis, we assessed whether the LCK inhibitor PP2 could synergize with a PARPi, olaparib, to augment the DNA damage response (Fig. 5E). Olaparib treatment led to an increase in BRCA1 expression and a detectable increase in γH2AX expression in SKOV3 cells (Fig. 5E). Co-treatment with PP2 was sufficient to suppress BRCA1 expression and significantly augment γH2AX expression in a dose-dependent manner (Fig. 5E). Our findings indicate LCK inhibition leads to HR deficiency. As proof of concept, we tested the impact of LCK silencing on the efficacy of olaparib in SKOV3 and CP70 cells via colony formation assay. Olaparib sensitivity was analyzed in parental (WT), KO, OE (Fig. 5F). We quantified colony formation and observed that IC50 value of Olaparib were as following: CP70 parental: 0.97µM, CP70 LCK KO: 0.13 µM and CP70 LCK OE (In KO background): 1.34 µM. (Fig. 5F, Supplementary Fig. S10). We replicated these findings by silencing with shRNA in CP70 and SKOV3 cells (Supplementary Fig. S11A and B). shCon, KD1, and KD2 cells were treated with various concentrations of olaparib and plated for colony formation. In CP70 cells, silencing LCK inhibited colony formation with greater efficiency in olaparib-treated compared to shCon treated cells (Supplementary Fig. S11A, B). Similarly, in SKOV3 cells, the number of colonies were significantly decreased after olaparib treatment in KD1 and KD2 cells as compared to shCon cells (Supplementary Fig. S11C, D). These findings support the hypothesis that olaparib has a higher efficacy in LCK-deficient cancer cells and indicate that LCK inhibition is sufficient to sensitize eEOC to PARPi.

LCK inhibition potentiates therapeutic efficacy of PARPi in in vivo

To test whether LCK impacts olaparib efficacy in pre-clinical models of eEOC, we injected KO and OE CP70 cells into mice and once tumors were detected, we treated with 3 courses of 5-day Olaparib 50 mg/kg (Fig. 6A). KO and OE CP70 exhibited nearly identical tumor growth in vehicle-treated mice (Fig. 6B and C). Olaparib treatment led to significant tumor suppression of in OE mice and to complete suppression of tumor growth in KO mice (Fig. 6B and C). These findings indicate that LCK inhibition potentiates olaparib synthetic lethality.

We next performed a molecular analysis on tumor sections from OE and KO cells treated without and with olaparib. TUNEL assay was used to detect apoptotic DNA fragmentation and indicated no positive cells (green fluorescence) in OE and KO tumors, indicating no apoptotic cells (Fig. 6D, Supplementary Fig. S12). Tumors from olaparib-treated mice exhibited a few TUNEL-positive cells present in OE tumors, whereas most cells were TUNEL-positive in KO tumors. Next, tumors were assessed for presence of γH2AX in tissue sections by immunohistochemistry (Fig. 6E). Vehicle treated mice exhibited low levels of γH2AX in both KO and OE cohort (Fig. 6E). Tumors from olaparib-treated mice exhibited low levels of γH2AX expression in OE, whereas most cells were positive in KO group (Fig. 6E). These findings indicate multiple DNA double-strand breaks were generated due to suppression of LCK and inhibition of PARP. We next assayed for expression of CD31, an indicator of microvessel (angiogenesis) density and of tumor mass and growth. Vehicle treated mice exhibited high CD31 positive staining in both KO and OE cohort (Fig. 6F). Tumors from olaparib-treated mice exhibited high levels of CD31 expression in OE, whereas there was no detectable CD31 in the KO group (Fig. 6F). These findings suggested that olaparib was also sufficient to inhibit tumor angiogenesis, corroborating previous findings which showed that PARP facilitates tumor vascularization by augmenting CD31 and VEGF [28].

Endometrioid ovarian cancer, while rare, lacks effective therapeutic strategies and are HR proficient. Our studies identified a strategy to induce HR deficiency in endometrioid ovarian cancer. We uncover the complex formation of LCK with BRCA1 and RAD51 in response to DNA damage supported by co-immunoprecipitation. We additionally find that interaction of LCK with BRCA1 and RAD51 requires kinase activity or phosphorylation on Y394, indicating active LCK is necessary for complex formation [17]. In contrast, phosphorylation on Y192 is not required for complex formation indicating the SH2 domain is not involved in DNA damage response. Previous study described that kinase dead LCK remains in closed and inactive conformation [29]. Phosphorylation at Y394 of LCK results stabilized and open structure leading to enhanced kinase activity and substrate binding [30, 31]. So it is speculated that the open and active form of LCK having intact kinase activity may facilitates the interactions with RAD51 and BRCA1 proteins. Along with this LCK was found to stabilize RAD51 and BRCA1 proteins. Moreover, kinase activity and autophosphorylation are necessary for functional DNA repair, as shown by γH2AX and RAD51 foci formation assays. These findings indicate that LCK kinase activity and autophosphorylation is essential to allow for interaction with HR repair proteins BRCA1 and RAD51 during DNA damage response. We discovered that inhibition or disruption of the non-receptor tyrosine kinase LCK attenuates the expression of HR proteins RAD51, BRCA1, and BRCA2 in eEOC. This complements our previous study showing that LCK overexpression or LCK inhibition modulated the mRNA levels of HR DNA repair genes including RAD51, BRCA1, and BRCA2 [15]. Here, we showed that LCK modulates HR genes at the protein level. This leads to functional consequences as the inhibition of LCK, via shRNA or pharmacologic inhibitor, leads to inhibition of DNA damage repair as assessed using the established DR-GFP assay in U2OS osteosarcoma cells. LCK does not impact PARP and NHEJ repairs, the alternate mechanisms for repair of double strand breaks by direct ligation independent of an homologous template [24]. The NHEJ repair proteins, Ku70 and Ku80, did not have their expression levels impacted by PP2 treatment. LCK kinase activity is necessary for maintaining HR proficiency. Finally, we demonstrated that LCK disruption is sufficient to sensitize endometrioid ovarian cancer cells to olaparib. These findings are consistent with our data indicating that LCK inhibition leads to chemosensitization to cisplatin treatment in endometrioid ovarian cancer.

We determined for the first time that the LCK protein is upregulated in response to DNA damage in eEOC. DNA damage by etoposide, methyl methanesulfonate (MMS), and by UV radiation induces LCK protein expression. This is also corroborated by previous findings that indicate fractionated radiation induces stem cell populations in human gliomas to display LCK activation [32]. Phosphorylation of LCK (pY394) was elevated in etoposide, MMS, and UV-treated SKOV3 cells and in UV-treated CP70 cells as compared to untreated cells. We found that DNA damage led to nuclear localization of both total and pY394 LCK protein, a finding supported by immunofluorescence analysis of pY394 LCK following DNA damage. This finding is unprecedented as LCK is localized to the inner leaflet of the cell membrane on microdomains [33]. Our findings are supported by previous studies have found constitutively active LCK in the nucleus where it binds at the promoter region of LIM domain only 2 (LMO2) leading to gene expression[34]. These findings are significant as we show that LCK is activated by DNA damage, leading to nuclear translocation and subsequent activation of HR repair pathways.

LCK regulates HR repair in response to DNA damage and its inhibition potentiates the activity of PARPi to induce synthetic lethality. The simultaneous inhibition of LCK and PARP with pharmacological agents PP2 and olaparib showed significantly more DNA damage and chromosomal aberration compared to only either PP2 or olaparib treatment in eEOC cells. PP2 treatment was sufficient to attenuate DNA repair, augmenting the effect of olaparib in ovarian cancer cells. Finally, in vivo studies showed that olaparib efficacy was enhanced in CP70 LCK KO compared to OE tumor bearing mice. This provides evidence for proof of concept for utility of LCK inhibitors to disrupt HR DNA damage repair. Indeed, several strategies are currently being explored in the clinic to increase use of PARP targeted therapy in HR proficient cancers. CDK1 and CDK12 inhibition led to HR deficiency by decreasing HR repair proteins RAD51, BRCA1, and BRCA2 in lung cancer[8]. Further, inhibition of BET proteins also led to attenuation of RAD51 and BRCA1 proteins in breast, ovarian, and prostate cancer models [2]. PI3K inhibition is also sufficient to reduce BRCA1 and BRCA2 expression, hampering HR repair in triple-negative breast cancer [35]. Other reported targets are HSP90 [36] and VEGFR3 [37] shown to attenuate RAD51, BRCA1, and BRCA2 expressions in ovarian cancer. Concurrent inhibition of LCK enhanced the efficacy of PARPi in HR proficient cancer models in preclinical settings. Clinical trials are ongoing to assess the efficacy of PARPi in combination with CDK1/12 inhibitors, PI3K inhibitor, and VEGFR3 inhibitors [3]. Our findings complement these studies and identify an alternate signaling pathway for enhancing PARP targeted therapy in eEOC.

These findings provide an innovative new strategy for inducing an HR-deficient status in an otherwise HR-proficient tumor. We identify targeted therapies that compromise HR repair genes and augment sensitivity to PARPi. Our study defines the mechanistic impact of LCK and potentially other non-receptor tyrosine kinases in regulation of HR repair that is apparently crucial to ovarian cancer’s response to chemotherapy and PARP inhibitors. This study highlights new clinical applications that target LCK, expanding PARPi utility. The studies provide support for future clinical studies using combination of LCK inhibitors with PARP inhibitors to improve patient outcomes.

Cell lines and culture conditions

Cisplatin resistant eEOC cancer cells CP70 were a gift from Analisa Difeo (University of Michigan) and SKOV3 were purchased from American Type Culture Collection (ATCC). Others cell lines used in this study are mentioned in resource table. Cells were grown in DMEM and McCoy's 5A media respectively, supplemented with 10% fetal bovine serum at 37°C in humidified incubator in 5% CO2. Cells were tested and confirmed as mycoplasma contamination negative on a quarterly basis. Cells were passaged by treatment with trypsin/EDTA solution when they reached 80–90% confluence and further passaged or seeded for experiments.

Chemicals And Reagents

We used several pharmacological agents in our study. The PARP inhibitor (Olaparib) [38], the LCK inhibitor (PP2) [20, 21] and the radiomimetic drug, etoposide [39] were purchased as shown in the resource table. Inhibitors were dissolved in 100% DMSO to make stock concentrations and kept at -20°C until use. The details of chemical, reagents, primary antibodies, and secondary antibody details are outlined in the resource table (Supplementary table S12).

Plasmid Construct Mutants

Myc-tagged LCK containing plasmid was generated using pENTR/D-TOPO cloning kit (Thermo Scientific) according to manufacturer instructions. Briefly, Myc-LCK gene block was purchased from Integrated DNA Technologies (IDT, USA). Myc-LCK was cloned into pENTR/D-TOPO vector. The entry clone was further transferred into a destination vector, pLenti CMV Puro DEST (Addgene). The plasmid was validated by DNA sequencing (Eurofins). LCK mutants 192F, Y394F, 273R were generated by site directed mutagenesis and sequenced. Each mutant was cloned into a lenti viral vector, pLenti CMV Puro DEST (Addgene) for subsequent use.

Lentivirus Production

Lentiviral particles for LCK silencing were generated using established lab protocols[15]. Briefly HEK293T cells were seeded into 6 well plates. The next day cells were transfected with pRSV-Rev, pMDLg/pRRE, pMD2.G and lentivral vector expressing shRNA for targeting LCK (KD1, TRCN0000426292, KD2, TRCN0000001599). Following 24h incubation, transfection media was replaced with fresh DMEM medium. 48h post transfection, lentiviral particle containing media was filtered to remove cell debris and added to CP70 and SKOV3 cells. Fresh media was subsequently added to the HEK-293T transfection plates and incubated for an additional 24 hours followed by filtration and addition to further CP70 and SKOV3 cells. Transduced CP70 and SKOV3 cells were identified using 1.5ug/ml and 2ug/ml puromycin (Thermo Scientific) selection respectively.

Generation Of Crispr/cas9 Ko Cells

CP70 cells were used to generate LCK CRISPR/Cas9 knockout cells according to the manufacturer protocol (Santa Cruz Biotechnology). Briefly, cells were transfected with GFP labelled LCK CRISPR/Cas9 plasmid using lipofectamine 3000 (Thermo Scientific) in the presence of antibiotic-free, FBS-enriched, Optimem media. Following transfection, cells were kept in transfection medium for 24h, then replaced with fresh culture media. After an additional 24h, transfected cells were screened for GFP expression using a flow cytometer, and the GFP^+/high population was isolated and plated as single cells into a 96 well plate. Cells were grown and expanded in accordance with standard culture techniques as stated above, followed by western blotting for LCK protein expression with anti-LCK antibody (0.5 µg/mL, R & D Systems). Clones with the lowest LCK expression compared to parental cells were considered LCK KO cells.

Western Blot Analysis

Western blot analysis was performed as reported with modifications as follows[40, 41]. Briefly, cancer cells were washed with chilled Dulbecco’s phosphate buffered saline (PBS) two times at the end of treatment. NP-40 lysis buffer (Invitrogen) was added dropwise to the plates and placed on ice for 10 minutes. The NP-40 lysis buffer contains 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% Nonidet™ P40 (NP40), 0.02% NaN3 and was supplemented with 1mM PMSF and 2 µg/ml protease cocktail inhibitor (PCI) (Sigma Aldrich). Cells were then collected in a 1.5 mL centrifuge tube by scraping, and incubated on ice for one hour with occasional vortexing. Lysates were centrifuged at 10,000rpm for 10 min at 4°C. Protein concentration was measured of each lysate supernatant by BCA kit analysis (Thermo Scientific). Protein samples were then prepared using 6x Laemmli dye containing BME (β-mercapto ethanol) and boiled for 5-10min. Protein samples were subjected to SDS-PAGE gel electrophoresis using pre-made gradient gels (4–20%, Biorad). Proteins were transferred by wet transfer to a PVDF membrane (Millipore) at 4°C overnight. Membranes were then blocked in 5% BSA in TBST for one hour at room temperature, and subsequently, incubated overnight at 4°C in the following primary antibodies: T-LCK (1:1000 R&D Systems), T-LCK (1:1000 Proteintech), P-LCK 394 (1:1000 R&D Systems), RAD51 (1:1000, Proteintech), BRCA1 (1:500, EMD Millipore), BRCA2 (1:500, EMD Millipore), γH2AX (1:1000 Cell Signaling Technology), GAPDH (1:5000 Proteintech), and β-actin (1:4000 Proteintech). After primary antibody incubation membranes were washed three times with TBST (Tris-buffered saline containing 0.1% tween 20) washing buffer on a platform shaker. Membranes were incubated with HRP-conjugated rabbit (1:25000) or mouse (1:25000) secondary antibodies for one hour at room temperature, followed by three washes with TBST buffer. Chemiluminescence reagent (PerkinElmer) was added to detect immobilized proteins in PVDF membranes utilizing the ChemiDoc imaging system. Densitometry was performed using Image J software.

Nuclear Protein Isolation And Co-immunoprecipitation Analysis

CP70 and SKOV3 cells transduced with Myc-tagged LCK were treated with etoposide (10µM) or DMSO for 24h followed by replacement with fresh serum-enriched media for an additional 24h. Cells were collected and washed with cold PBS two times, scraped and centrifuged. Cell pellets were then lysed with cytoplasmic and nuclear extraction buffers according to manufacturer protocols (NE-PER Nuclear and Cytoplasmic Extraction Kit, Thermo Scientific). Protein concentrations of nuclear lysates were estimated using the BCA method outlined above. For co-immunoprecipitation, nuclear protein lysates were incubated with 3µg anti-Myc antibody (Proteintech) or 3µg control antibody (Cell Signaling Technology) overnight at 4°C with gentle rocking. Pre-cleaned protein A/G agarose beads (Thermo Scientific) were added to the lysates and incubated for 4h at 4°C on a rotating mixer. Beads were then collected by centrifugation and washed three times with chilled NP-40 lysis buffer. 6x Laemmli buffer (Alfa Aesar) containing BME was added and beads were boiled for 5 minutes. Samples were separated on SDS-PAGE and processed for western blot analysis as outlined above. Further, LCK overexpression (OE) SKOV3 (without Myc tagged) cells were treated with etoposide (10µM) for 24h. Then, serum-enriched media was added to replace drug-containing media and kept for another 24h. Then, cells were collected, and nuclear lysates were prepared. Further immunoprecipitation/co-immunoprecipitation was performed after RAD51 pulled down as described above.

Gene Conversion Assay

Gene conversion assay or DR-GFP assay was performed according to reported methods[42]. Human osteosarcoma U2OS cells stably transfected with DR-GFP plasmid and I-SceI endonuclease expression vector pCBASce were kindly provided by Maria Jasin at Memorial Sloan-Kettering Cancer Center. Cells were treated with, PP2 (5, 7, and 10µM) or DMSO vehicle for 48h. Cells were then transfected with I-SceI endonuclease expression vector pCBASce using Lipofectamine 3000. In a separate set of experiments, U2OS cells with DR-GFP integration were transfected with shCon, LCK KD1 or KD2 for 24h and incubated in serum enriched medium for another 24h. Cells were further transfected with I-SceI plasmid for 24h followed by incubation with serum enriched medium for 24h. Live cells (Live/Dead dye kit, Thermo Scientific) were analyzed with a flow cytometer to estimate the percentage of GFP-positive cells.

Rad51 And γh2ax Nuclei Staining And Analysis

Laser scanning confocal microscopy was performed to detect RAD51 and γH2AX foci in cancer cells. Briefly, ovarian cancer cell, CP70 were grown on coverslips and treated with 10µM etoposide or vehicle for 24h followed by an additional 24h in drug-free media. Coverslips were washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS. Cells were permeabilized with 0.01% triton-X 100 (Fisher Scientific) for 5 minutes followed by a wash with chilled PBS and blocked with 3% goat serum (Thermo Scientific) for 1h at room temperature. Cells were incubated with anti-RAD51 (1:250, Abcam) or anti-γH2AX (1:300, Cell Signaling Technology) antibodies overnight at 4°C in a humidified chamber. Next, cover slips were washed 3X with PBS. Alexa fluorescent conjugated secondary antibodies were added to the coverslips and incubated for 1h. Coverslips were washed 3x and mounted with DAPI containing Vectashield (Vector Lab). Images were captured by confocal microscope at 63x magnification in oil emersion (Leica SP8 confocal microscope). RAD51 and γH2AX foci were counted on 20 representative cells by image J software.

Metaphase Spread Analysis

Metaphase spread analysis was performed on LCKi and PARPi treated cells using established methods [27]. Briefly, cells were treated with LCK inhibitor, PP2 (5µM) (Selleck Chemicals) and PARP inhibitor, olaparib (3µM) (Selleck Chemicals) for 48h. After treatment, cells were harvested. Cells were treated with colcemid (50ng/ml) (Sigma) for 1.5h then washed with PBS and placed in 0.075 mol/L KCl (Sigma) solution for 20min. Subsequently, cells were washed with PBS and fixed in carnoy fixative solution (Methanol: acetic acid 3:1) added dropwise followed by one hour incubation. Cell pellets were collected, and fixative solution was added and incubated at 4°C for 24h. Cell pellets were collected and small amount fixative solution was added and cell suspension was slowly dropped on glass slides and allowed to dry at 37°C. Slides were then stained with Giemsa solution (Sigma Aldrich). Images were captured at 100X magnification with a bright field microscope. Abnormalities in chromosomes were quantified (Chromosomal break, gap, radial formation) by visually counting five nuclei per treatment group.

Single Cell Electrophoresis Assay

Single cell electrophoresis assay or comet assay was performed according to a previously reported method[43]. This experiment was performed following the manufacturer’s instructions (Trevigen). Briefly, CP70 and SKOV3 eEOC cells were treated with 5µM PP2 and/or 3µM Olaparib for 48h. Comet LMAgarose was melted at 90°C for 10min in a water bath then cooled for 20min to 37°C. Treated cells were detached from plates using trypsin. Serum enriched media was added to neutralize the trypsin. Cell suspension was washed twice with chilled 1X Ca⁺⁺ and Mg⁺⁺ free PBS, and subsequently suspended at 1 X 10⁵ cells/ml in chilled 1X PBS buffer (Free of Ca + + and Mg++). For the alkaline comet assay, the cell suspension was mixed with molten LMAgarose at 37°C. Immediately, 50µL LMAgarose mix was spread on glass microscope slides and incubated at 4°C for 30 min in the dark. Slides were incubated in lysis solution (provided in kit) overnight at 4°C. The next day comet slides were incubated in alkaline unwinding solution at room temperature for 20 min. Agarose gel electrophoresis (21volts for 30min) was performed using alkaline electrophoresis protocol. After electrophoresis, slides were briefly immersed in distilled water twice and then immersed in 70% ethyl alcohol for 5 min. Slides were dried and 100µl of SYBR gold (excitation/emission is 496 nm/522 nm) was added on agarose and kept for 30 min in the dark. Slides were washed with water, dried, and visualized by fluorescence microscopy.

Colony Formation Assay

The pharmacological effect of olaparib in cancer cells was investigated by colony formation assay according to the earlier reported method[27]. Briefly, ovarian cancer cells CP70 and SKOV3 cells (WT, sh, LCK OE, LCK KO, LCK KD and mutants) were placed on 12 well plates. The next day, cells were treated with olaparib in a dose dependent manner for 12 days. During this time the media was changed every day using fresh drug. At the end of experiment, PBS was added to the well to wash the colony. Then, cells were fixed with 4% paraformaldehyde solution for 10min. Cells were then washed two times with PBS and incubated in 0.2% crystal violet solution for one hour at room temperature. After incubation cells were washed three times with PBS. Then, the images of six well plates were captured and number of colony forming area was analyzed by Image J software.

Cell Titer Glo Viability Assay

CP70 (WT, OE, KD, KO and LCK mutants, Y394F, K273R and Y192F) cells were collected after trypsinization. Cells were counted and 4000 cells were plated in each well of a 96 well plate. Cells were then treated with etoposide for 48h in a dose dependent manner. Control cells were treated with vehicle (DMSO). After the drug treatment, Cell TiterGlo® Luminescent Cell viability assay reagent (Promega) mixture was prepared and added to the cells to lyse them for 10 minutes shaking and luminescence was measured via luminometer. Cell viability percentage was calculated as luminescence of treated group/luminescence of vehicle treated group×100.

Nhej Gene Expression

To check for NHEJ expression, CP70 and SKOV3 cells were grown on 60mm petri dishes until 70% confluent. Cells were then treated with PP2 in a dose dependent manner (5, 7 and 10µM) for 48h and collected by scraping and Western blot was performed to assess protein expression of NHEJ markers Ku70 and Ku80.

In vivo animal study in NSG mice

In vivo antitumor efficacy of Olaparib was tested in NSG (NOD severe combined immunodeficient (SCID) IL2R gamma) mice. This study was approved (IACUC Protocol# 2707) by Institutional Animal Care and Use Committee (IACUC), Cleveland Clinic Lerner Research Institute. Animals were procured from BRU (Biological Response Unit) facility of the Cleveland Clinic. All cells used in this study were transfected (Lentiviral transfection) with pCDH-EF1a-eFFly-mCherry plasmid. Mice were injected with CP70 LCK knockout cells or CP70 LCK overexpression cells intraperitoneally (Cells: 0.5x10⁶). After that mice were divided into four groups-1. Mice: CP70 LCK KO: Vehicle (n = 8), 2. Mice: CP70 LCK KO: Olaparib (50mg/kg) (n = 8), 3. Mice: CP70 LCK OE: Vehicle (n = 8), 4. Mice: CP70 LCK KO: Olaparib (50mg/kg) (n = 8). Olaparib was dissolved in ddH₂O containing 4% DMSO and 30% polyethelene glycol (PEG300) and injected intraperitoneally. Animals were treated with olaparib for 5days/week. Bioluminescence of the tumor were measured by in vivo imaging system (IVIS Spectrum CT, PerkinElmer). At the end of the experiment, mice were sacrificed according to the protocol of Institutional Animal Care and Use Committee (IACUC), Cleveland Clinic Lerner Research Institute. Tumor tissues were collected and preserved in 10% formalin solution.

Immunohistochemistry And Tunel Assay

Immunohistochemical (IHC) analysis and TUNEL assay of tumor sections were performed according to the earlier reported method[44]. Formalin fixed tissues were sent to histology core to make thin slice (around 5micron) of tissue embedded on glass slides. Next, slides were dipped in Histo-Clear to deparaffinize. Then sections were rehydrated in gradient ethanol (100%, 95%, 80% and 60% ethanol 5 min for each bath). For antigen retrieval, sections were then put in Tris-EDTA buffer (pH9) and boiled in a pressure cooker for 10min. Then slides were put for an hour to cool. Slides were then put into 0.1% triton X-100 solution for 10 min to permeabilize. After washing with PBS three times, tissue sections were blocked with blocking buffer (5% goat serum and 0.1% triton X-100 in 1X TBST) at room temperature for one hour. Then antibodies were added to the tissues and incubated for overnight at 4°C. Next day, sections were washed three times with PBS. Then diluted 100 µl Peroxidase Labeled Polymer was added to the tissues and incubated for 30 min at room temperature. Now sections were washed three times with PBS. Then diluted DAB chromogen was added to the tissue and incubated for 2-5min until desired color was generated. Sections were washed three times and then hematoxylin staining was performed. After washing with PBS, tissue sections were dried and mounted with Cytoseal. Images were captured using upright microscope at 20x magnification.

For TUNEL assay, tissue sections were processed for antigen retrieval and permeabilization as discussed earlier. Sections were washed three times with PBS. Then enzyme solution was prepared according to the manufacturer instructions. Slides were then incubated in the enzyme for 60min at 37°C. Sections were washed three times with PBS and mounted with Vectashield containing DAPI.

Software And Statistics

Graph pad prism software was utilized for graph preparation and to determine statistical significance (detailed in each figure legend). Image J was used for quantification of data. Each experiment was performed at least three times. p-value less than 0.05 was considered significant.

Data availability

Data generated by the authors are included within the text and supplementary files or will be made available upon request to the corresponding author. Other reagents can be available upon request to the corresponding author.

Acknowledgments

Authors thank the members of Reizes and Lathia Laboratories for helping to improve the scientific quality of the manuscript. We would like to thank Alex Myers for extensive editing the manuscript. We would like to thanks Dr. Debjit Khan from Fox Laboratory for insights on complex formation assays. We thank Dr. Gauravi Deshpande for helping in image acquisition by confocal microscopy, Amy Graham and Eric Schultz for flow cytometry, and we would also like to thank Dr. Suparna Mazumdar (Cleveland Clinic) for giving constructive comments of this study. Research in the Reizes laboratory is funded by Center of Research Excellence in Gynecologic Cancer from the Cleveland Clinic Foundation, VeloSano Bike to Cure, and the Laura J. Fogarty Endowed Chair for Uterine Cancer Research. Dr. Gong is supported by and NIH/NCI grant (R01 CA222195).

Author Contribution

OR: Conceptualization, funding acquisition, reviewing the data, writing and editing the manuscript, and approval of final manuscript draft, GD: Data generation, design of experiment and writing the manuscript, RB: Data generation, design of experiment and writing the manuscript, CB: Generation of LCK mutants, RA: Generation of LCK construct, EE: Manuscript editing and formal analysis, KCT: Manuscript editing and formal analysis, KMC: LCK construct generation and formal review, AJP: Immunohistochemistry analysis, PGR: Manuscript editing and formal analysis, JL: Manuscript editing and formal analysis, ZG: Providing insight in various experiments and DR-GFP construct, reagents. GD and RB have contributed equally to this work.

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LCK Facilitates DNA Damage Repair by Stabilizing RAD51 and BRCA1 in the Nucleus of Chemoresistant Ovarian Cancer

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Abstract

Figures

Introduction

Results

LCK complexes with RAD51 and BRCA1 in response to DNA damage in an LCK kinase dependent manner.

LCK regulation of DNA double strand break repair.

LCK kinase activity is essential for HR repair.

DNA damage induces LCK dependent BRCA1 expression.

Lck Inhibition Attenuates Hr Repair Efficiency

LCK inhibition augments PARPi induced DNA damage and genomic instability.

LCK inhibition potentiates therapeutic efficacy of PARPi in in vivo

Discussion

Methods

Cell lines and culture conditions

Chemicals And Reagents

Plasmid Construct Mutants

Lentivirus Production

Generation Of Crispr/cas9 Ko Cells

Western Blot Analysis

Nuclear Protein Isolation And Co-immunoprecipitation Analysis

Gene Conversion Assay

Rad51 And γh2ax Nuclei Staining And Analysis

Metaphase Spread Analysis

Single Cell Electrophoresis Assay

Colony Formation Assay

Cell Titer Glo Viability Assay

Nhej Gene Expression

In vivo animal study in NSG mice

Immunohistochemistry And Tunel Assay

Software And Statistics

Declarations

References

Additional Declarations

Supplementary Files

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