Targeting DNA repair with combined inhibition of NHEJ and MMEJ induces synthetic lethality in TP53-mutant cancers

Abstract

DNA repair pathway inhibitors are a new class of anti-cancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of non-homologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (Polymerase Theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway as key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors.

INTRODUCTION

Genome integrity is critical for cellular survival and is maintained by DNA repair pathways collectively known as the DNA damage response (DDR) (1). Loss of genomic integrity results in permanent changes to the sequence of DNA and is the source of many human diseases, notably cancer (2–7). Double-stranded breaks (DSBs) are the most deleterious form of DNA damage and can be repaired through three main DNA repair pathways: homologous recombination (HR), non-homologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ).

NHEJ occurs throughout the cell cycle and is the predominant DSB repair pathway. NHEJ is an error-prone pathway, unlike HR repair, which is homology-guided. The first step of NHEJ is recognition of the DSB by the Ku70-Ku80 heterodimer (8,9). Depending on the configuration of the DNA ends (blunt ends, 5’ overhangs, and 3’ overhangs), the Ku70-Ku80 heterodimer acts as a scaffold to which other NHEJ proteins are recruited to promote rejoining of the DNA ends (8–12). Next, DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) binds with high affinity to Ku70-Ku80 heterodimer-DNA ends (9). If the DNA break ends are compatible, either blunt or with complementary overhangs, then repair will likely be error-free with direct ligation by XRCC4-DNA ligase IV. If, however, the DNA break ends are not compatible, requiring additional processing, either resection by nucleases, or addition of nucleotides by polymerases, then repair will be error prone (13).

Since DSBs are the most deleterious form of DNA damage, targeting their repair is a potential therapeutic strategy for the treatment of cancers (1,14–16). The majority of DSBs are repaired by NHEJ, where DNA-PK is the key driver. Accordingly, inhibitors of DNA-PK activity may provide a therapeutic strategy for sensitizing cancer cells to chemotherapy or ionizing radiation (IR) (17–21). Peposertib is a novel, selective small molecule inhibitor of the serine/threonine kinase activity of DNA-PK (22). Peposertib is a potent and selective pharmacologic inhibitor of DNA-PK that competes with ATP for binding to DNA-PK with an IC50 of 0.6 nmol/L (22).

In the current report, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loss of function genetic screen to identify enhancers and suppressors of cell killing by the DNA-PK inhibitor, peposertib. Single guide RNAs (sgRNAs) directed against all reported members of the MMEJ pathway, including POLQ (encoding Polymerase Theta, POLθ), PARP1, HMCES, FEN1, XRCC1, and LIG3 were synthetically lethal with DNA-PK inhibition (23,24). We recently identified the ATPase inhibitor, novobiocin (NVB), as a first-in-class inhibitor of POLθ and the MMEJ pathway (25). As expected from the CRISPR-KO screen results, the combination of peposertib plus NVB, exhibited synergistic anti-cancer activity. This drug combination resulted in excessive DSB end-resection and ultimately apoptosis-mediated cell death, providing a molecular mechanism of the observed synthetic lethality. Interestingly, TP53-mutant tumor cells are resistant to peposertib but have elevated sensitivity to the drug combination, resulting from their elevated POLθ expression level. Accordingly, the combination of peposertib plus NVB resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, a combination of a targeted DNA-PK inhibitor plus a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors.

MATERIALS AND METHODS

Cell Lines

A549 and NCI-H460 stably expressing the Cas9 endonuclease were obtained from the Genetic Perturbation Platform (GPP) at the Broad Institute (Cambridge, USA) and were grown at 37°C in a humidified 5% CO2 incubator in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma). U2OS and RPE cells were purchased from ATCC and grown in RPMI (Gibco) supplemented with 10% FBS or in F12 (Gibco) supplemented with 10% FBS. MBA-MD-436 isogenic pairs were a gift from Dr. Shapiro and grown in RPMI (Gibco) supplemented with 10% FBS. CAPAN-1 isogenic pairs were a gift from Dr. Christopher Lord and grown in DMEM (Gibco) supplemented with 20% FBS. Mycoplasma testing was performed every 2 weeks using MycoAlert (Lonza). All experiments were performed in low passage number (less than 20) cells.

CRISPR Screens

The primary CRISPR screen was performed in pXPR_311 integrated Cas9 expressing NSCLC cell line A549 and NCI-H460 per the Genetic Perturbation Platform CRISPR screening protocol from the MIT Broad Institute (26,27). For adequate representation of each gRNA, e.g., an average of 300 cells/ sgRNA, cells were infected at MOI 0.3 in media containing 8 μg/ml of polybrene. Briefly, cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, L-glutamine and Penicillin-Streptomycin. Each cell line was infected in 12 well plates for two hours under 2000 rpm at 30C with the Brunello whole genome sgRNA pooled lentiviral library consisting of 76,441 sgRNAs targeting 19,114 different genes. Media was replaced immediately following lentiviral infection. Infected cells were then plated 15 cm dishes with 2ug/ ml of puromycin to select four days. After selection, 5e6 cells were plated for each biological replicate, and additional 5e6 cells per replicate were collected and frozen for subsequent genomic DNA extraction as time 0. Both cell lines were treated with 1 μM of peposertib (C-1358, Chemgood) or equal volume of DMSO for 17 days. Cells were re-plated with a density of 5e6 every 3 days, along with fresh DMSO or drug. At the end of screen, the cells were harvested and frozen for gDNA extraction. gDNA was isolated using an QIAGEN QIAamp DNA Blood Maxi kit, according to manufacturer’s protocol. gDNA samples were subjected to the Genetic Perturbation Platform at the Broad institution for barcoding, PCR purification and sequencing on an illumine HiSeq 2000. Similarly, the secondary CRISPR screen was also performed in these cell lines (A549 and H46) but with a subset of top hit genes from the primary screen.

Genomic DNA Preparation, Sequencing and Identification of Top Hits

Sequenced reads per million were log2-transformed by first adding one to all values, which was necessary to take the log of sgRNAs with zero reads. For analysis, these normalized data were used to determine the log2-fold change of each sgRNA for the drug treatment group relative to DMSO control group (28). The STARS gene-ranking algorithm was used to determine the top hits (https://portals.broadinstitute.org/gpp/public/software/stars); at least two sgRNAs targeting a gene had to rank within the top 10% of sgRNAs by log2-fold change in one direction or another (29). The hit genes were scored using the STARS score and the false discovery rate. Additionally, the normalized data were also analyzed with the hypergeometric distribution method (https://github.com/mhegde/volcano_plots). The volcano plots of these results were created using R (https://www.r-project.org).

Antibodies and Chemicals

All antibodies and chemicals are shown in Supplemental Table 1.

Western Blotting

Cells were lysed in NETN buffer (10 mM TrisCl, pH 7.4, 1 mM EDTA, 0.05% Nonidet P-40, and protease inhibitors) containing 150 mM NaCl (30). Cell lysates were boiled in 2x Laemmli buffer and resolved on Nu-Page Bis-Tris (Invitrogen) polyacrylamide gels using MES buffer, and subsequently transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in TBST and probed with primary and appropriate enhanced chemiluminescence secondary antibodies, then detected with chemiluminescence (Western Lightning, Perkin Elmer) or appropriate LiCor secondary antibodies, and then detected on the LiCor Odyssey.

CRISPR Cas9 KO Cell Line Production

To generate knockout A549 and NCI-H460 cell lines, the indicated sgRNAs, described in Supplemental Table 1 were cloned into the pLentiCRISPR V2 viral vector.

siRNA Transfection

For siRNA transfection, cells were seeded at about 30% confluence into 6-well plates the day prior to transfection. Specific siRNA duplexes were transfected using Lipofectamine RNAiMAX Reagent Agent (Life Technologies, #13778150) according to the manufacturer’s instructions. Sequences of the siRNAs employed in this study are shown in Supplemental Table 1.

Clonogenic Assays

Cells of interest were seeded at a concentration of 1000–2500 cells per well into 6-well plates. After 24 hours, cells were continuously treated with indicated compounds for 10–14 days. Colonies were fixed (fixation solution: 5 parts methanol + 1 part acetic acid) at room temperature for 20 min and then stained with a solution of 0.5% crystal violet in methanol for 1–2 hours. Plates were imaged using the Amersham Imager 600 (GE) and colonies were quantified with Image J software (NIH). All clonogenic assays were performed in technical triplicates, with a “n” of at least three.

CellTiter-Glo Assay

Cells of interest were seeded at a concentration of 3000 cells per well into 96 well plates and treated the next day with the indicated compounds. Following 4 days of indicated treatment, the CellTiter-Glo, ATP-based, cell viability assay was performed (Promega). Survival curves were calculated by best-fit analysis of the log of the drug concentration to fold change of treated cells over vehicle-treated cells. Synergy between peposertib and novobiocin was calculated using Combenefit software as previously described (31). All survival assays were performed in technical triplicates, with a “n” of at least three.

DNA Damage Reporter Assays

Four different DNA damage reporter assays were employed in this study. NHEJ efficiency was measured using the EJ5-GFP reporter assay. HR efficiency was measured using the DR-GFP assay. MMEJ was measured using either the EJ2 or the P2A MMEJ reporter assays. SSA efficiency was measured using the SSA-GFP assay. U2OS cells with containing the appropriate DNA damage reporter assay were transfected with the indicated siRNA as described above and/or treated with peposertib, NVB, or a combination of both. 24 hours after transfection, cells were infected with an I-Sce-I adenovirus. 48 hours later, reporter assay positivity was determined by flow cytometric quantification. Technical triplicates were performed, and experiments repeated at least three times.

BrdU / ssDNA Assay, Immunofluorescence

U2OS cells were grown on coverslips in the presence of 10 uM BrdU (Sigma) for 16 hours in the presence of DMSO or the appropriate drug compound. Cells were fixed using 4% paraformaldehyde. Fixed cells were washed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 minutes at 4°C. Cells were washed with PBST and incubated with the appropriate primary antibody. Following primary incubation, coverslips were washed three times with PBST, then incubated with the appropriate secondary antibody (Alexa Fluor, Life Technologies) for 1 hour. Coverslips were then mounted on glass slides using mounting media containing DAPI (Vectashield) and foci imaged using a florescence microscope and quantified using Image J (NIH).

SMART Assay

Single molecule analysis of resection tracks was performed as previously described (32). Briefly, cells were treated with bromodeoxyuridine (BrdU, Sigma) for 24 hours. Subsequently, cells were treated with the appropriate drug compound for 24 hours and then trypsinized, counted and embedded in low melting point agarose plugs for treatment with proteinase K overnight. Then, agarose plugs were washed and digested with Agarase. Agarase-treated samples were then poured into FiberComb wells and combed onto silanized coverslips. Coverslips were probed with rat anti-BrdU antibody (Abcam) and visualized by fluorescence microscopy. Pictures were taken of at least 100 fibers per condition. DNA fibers were measured with Adobe Photoshop CC 2019. Each experiment was performed in triplicate.

DNA End-Resection Assay

U2OS cells stably expressing ER-AsiSI were used to quantify DNA end-resection. Briefly, cells were treated with the appropriate drug compound for 24 hours. During the final 4 hours of treatment, cells were treated either with or without 300 nM 4-OHT for four hours to induce DSBs at specific AsiSI sites by allowing transportation of the constitutively expressed AsiSI to the nucleus. Genomic DNA was extracted with the Qiagen DNeasy Blood & Tissue Kit. Genomic DNA was either mock digested or digested overnight with BsrGI-HF (New England Biolabs). Two microliters of digested or mock-digested samples were used as templates in 20 μl of qPCR reaction containing 10 μl of 2× SYBR green (Thermofisher), and 1 μM of each primer using a QuantStudio 7 Real-Time PCR System (Thermofisher). The percentage of ssDNA (ssDNA%) generated by resection at selected sites was then determined by calculating a △Ct for each sample by subtracting the Ct value of the mock-digested sample from the Ct value of the digested sample.

Comet Assay

Alkaline comet assays to detect ssDNA and dsDNA were performed according to the manufacturer’s instructions (Trevigen). Cells were trypsinized and suspended in cold PBS then mixed with low melting agarose (Trevigen) at a ratio of 1:10 and plated onto Cometslides (Trevigen). Electrophoresis was performed for 30 minutes at 25 volts in an alkaline buffer. Photographs of comets were captured by fluorescence microscopy and analyzed using Image J and OpenComet.

RNA Extraction, cDNA Synthesis and qPCR

Total RNA was isolated using the Qiagen RNAeasy kit. To quantify gene expression levels, equal amounts of cDNA were synthesized using the SuperScript IV Reverse Transcriptase (Life Technologies) and mixed with PrimeTime Gene Expression Master Mix (IDTDNA) and the appropriate PrimeTime qPCR Primer Assays. Actin was amplified as an internal control. The threshold crossing value was noted for each transcript and normalized to the internal control. The relative quantitation of each mRNA or miRNA was performed using the comparative Ct method.

Caspase Assay

Cells were seeded into 96-well plates at a cell density of 2000 cells/well and incubated overnight. The following day, cells were treated with 1 uM peposertib, 100 uM NVB, or the combination, 1 hour prior to ionizing radiation exposure (2 Gy). After four days of incubation, Caspase-Glo 3/7 reagent (Promega) was added. Cells were incubated in the dark for 1 hour at room temperature on a tabletop shaker. Luminescence was measured using a Molecular Devices SpectraMax M5 spectrophotometer and normalized to the DMSO-treated control.

Patient-Derived Organoids

Tumor cells were extracted from patient derived xenografts and short-term patient-derived organoid cultures were generated following the protocol described previously (Hill et al. 2019). Briefly, processed cells were diluted in specialized media for ovarian cancer organoid generation and added into each well of a 96 well plate. Drugs were dispensed in randomized manner according to a prewritten software program using a Tecan D300e digital dispenser. The effect of NVB and peposertib was studied as single agents and combinations by measuring cell death after 5 days of drug treatment using Cell Titer-Glo cell viability assay following the manufacturer’s instructions. Luminescence was measured using a Clariostar microplate reader (BMG Labtech). A very high concentration of toxin (puromycin (2mg/mL)/cycloheximide (50 μM)) was used as a positive control which killed more than 95% of PXO. For monotherapy, we tested an eight-point drug response and for combination we prepared a concentration grid and calculated synergy.

Animal Studies

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Dana-Farber Cancer Institute (protocol 08–036). For the TP53 mutant ovarian tumors (the DF59 PDX model) studies, following written informed consent, tumor ascites was collected from with ovarian cancer at the Brigham and Women’s Hospital or at DFCI under Institutional Review Board-approved protocols in accordance with the Declaration of Helsinki and Belmont Report. PDX models were established, luciferized, and propagated as previously described (Parmar et al., 2019). 10 × 10⁶ luciferized PDX cells derived from mouse ascites were injected intraperitoneally (IP) into 8-week-old female NRG mice. Tumor burden was measured by weekly BLI imaging using a Xenogen IVIS 200 system. Prior to initiation of treatment, mice were grouped according to BLI signals and subsequently randomized, so that the average tumor BLI value in each group was the same. For treatment, NVB was diluted in saline and administered via IP injection at 100 mg per kg twice daily for 16 days and then 75 mg per kg twice daily until day 28. Peposertib (EMD Serono) was formulated in a vehicle of 0.5% Methocel/0.25% Tween20 in 300 mM citrate buffer, pH 2.5, and administered orally at 100 mg per kg daily for 4 weeks.

For the A549 xenograft studies, 2 × 10⁶ A549 cells in a 1:1 mixture with Matrigel (Corning) were subcutaneously injected into the flanks of athymic nude mice. Xenografted mice were randomly divided into the appropriate treatment group. For treatment, NVB was diluted in saline and administered via IP inject at 75 mg per kg twice daily for 29 days. Tumor volume was measured every 3–4 days using digital calipers.

Immunohistochemistry

Organoids were treated (DMSO, 5 uM peposertib, 100 uM NVB, or combination) for 2 days and then resuspended into HistoGel and fixed overnight using 10% formalin solution. Histogel blocks were paraffin embedded in the specialized histopathology core at the Brigham and Women’s Hospital (BWH). Formalin fixed, paraffin embedded (FFPE) blocks were sliced to thin sections on positively charged slides, and then stained with Hematoxylin & Eosin or stained with the appropriate antibodies. FFPE sections of irradiated and unirradiated tissue blocks were used as controls. Stained slides were scanned at the BWH specialized histopathology core on an Olympus BX41 microscope equipped with a digital camera with 40X magnification. Biomarker analysis and scoring for positively stained foci was performed using image analysis software (Leica, Aperio ImageScope)

Statistical Analyses

Quantitative data were analyzed and graphed using GraphPad Prism 9 software. All data are represented as mean ± SEM calculated unless indicated otherwise. Significance was tested using the student’s T-test unless indicated otherwise. Assessment of synergy was calculated based on the Bliss model of synergy.

Data Availability

Data were generated by the authors and available on request.

RESULTS

Whole genomic CRISPR screen identifies loss of MMEJ pathway as a determinant of peposertib sensitivity

To identify synthetic lethal interactors with the highly selective DNA-PK inhibitor (Peposertib), we initially evaluated two TP53 wild-type non-small cell lung cancer cell (NSCLC) lines, A549 and H460, which were engineered to stably express the Cas9 endonuclease (26,27). Using a clonogenic assay, we analyzed the cytotoxicity resulting from their treatment with peposertib, with or without ionizing radiation (IR) (Figure S1A). Consistent with previous reports, 1 μM peposertib alone had no discernable effect on the clonogenic survival of the cells. When combined with IR (2 Gy), however, there was a significant loss of clonogenic survival (Figure S1A) (33). Using DNA repair reporter assays, peposertib inhibited NHEJ (EJ5) activity but did not inhibit HR (DR-GFP) or MMEJ (EJ2) activity (Figure S1B) (34). In both cell lines, DNA-PK was rapidly auto-phosphorylated on Ser2056 following IR exposure, and autophosphorylation was inhibited by peposertib (Figure 1A). Drug exposure did not alter the cell cycle distribution of the tumor cells (Figure S1C).

Figure 1. Whole genomic CRISPR screen identifies loss of MMEJ pathway as determinant of peposertib sensitivity.

(A) Immunoblot analysis of A549 (top) and H460 (bottom) cells treated with 1 μM peposertib at 0-, 1-, 2-, and 4-hours following irradiation to detect phosphorylation of DNA-PK. Ratio of phosphor-DNA-PK to DNA-PK calculated using Image J. (B) Schematic of the genome-wide CRISPR-Cas9 screen in lung cancer cell lines. Cells were infected with the Brunello v2 lentiviral sgRNA library at a low multiplicity of infection, in triplicate, and grown for 17 days in media treated with peposertib. Fresh media, with freshly dissolved drug, was added every three days. Created using Biorender. (C) Doubling time of A549 cells treated with 1 μM peposertib and 0 or 0.5 Gy ionizing radiation. Cells were re-plated with a density of 5e6 every 3 days, along with fresh DMSO or drug. (D) Volcano plot showing genes targeted by sgRNAs that differentially dropped out (blue) or became enriched (red) in peposertib treated versus DMSO treated control A549 cells in CRISPR screen. (E) Gene-centric visualization of the average log2 fold change (LFC) in peposertib versus DMSO-treated in both cell lines. POLQ is highlighted in a blue box. PRKDC and TP53 are highlighted in a red box. (F) Gene-centric visualization of the average log2 fold change (LFC) in peposertib versus DMSO-treated in both cell lines, highlighting the identification of the MMEJ pathway. See also Figure S1.

We next devised a genome-wide CRISPR screen to identify genes which, when disrupted, would increase sensitivity to the DNA-PK inhibitor, peposertib (Figure 1B). The screen was conducted in A549 and H460, in the presence or absence of peposertib. In order to identify sgRNAs that target genes involved in sensitivity to peposertib, we chose a moderate concentration (1 μM Peposertib) of drug that does not kill the cells as a monotherapy but slightly increases the doubling time (Figures 1C and S1D) (29). Cells were collected pre- and post-drug selection, and genomic DNA was extracted. The sgRNA sequences were PCR amplified and Illumina sequenced. Top hits among the gene knockouts that conferred drug sensitivity were identified.

The screen identified several genes from both cell lines that modulated cell killing by peposertib (Figures 1D and S1E). As expected, sgRNAs against the peposertib target itself (PRKDC) conferred peposertib resistance. Genes that were hits in both cell lines were of special interest (Figure 1E). Many hits corresponded to specific signaling pathways (Figure S1F), and the top hits were in DNA repair pathways. Analysis of the two screens identified POLQ as a significant synthetic lethal interactor with peposertib (Figure 1E). POLQ encodes polymerase theta (POLθ), a unique DNA polymerase that promotes MMEJ and is a known synthetic lethal interactor with HR repair deficiency (35,36). Rescreening the top hits in a secondary screen validated POLQ as a peposertib synthetic lethal interactor (Figures S1G and S1H)

The MMEJ pathway is a stepwise DSB repair pathway (24). The pathway is initiated by 5′ to 3′ end-resection, involving near the DSB, exposing short regions of complementary sequences termed microhomologies. Additional genes, including POLQ, PARP1, HMCES, FEN1, XRCC1, and LIG3, are involved in the downstream events in MMEJ (24). Interestingly, our screens identified all of these known members of the MMEJ pathway as potential synthetic lethal interactors with peposertib-mediated DNA-PK inhibition (Figure 1E and 1F).

CRISPR-mediated knockout of individual MMEJ genes in A549 and H460 cells next validated the screening results (Figures 2A–E and S2A and B). Immunoblotting with the corresponding antibody confirmed protein knockdown by CRISPR knockout of POLQ, HMCES, FEN1, XRCC1, LIG3, and PARP1 (Figures S2C–H). As predicted, knockout of these individual MMEJ genes increased the sensitivity to peposertib-mediated DNA-PK inhibition (Figures 2A–E and Figure S2B). The MMEJ pathway, also termed alternative end-joining, is a backup mechanism for repairing DSBs when both HR and NHEJ were unavailable (37,38). Accordingly, loss of DNA-PK mediated by peposertib, results in greater cellular dependence on MMEJ for repair of DSBs, similar to the observed hyper-dependence of HR-deficient cells on MMEJ (35,36).

Figure 2. Loss of MMEJ pathway confers sensitivity to peposertib-mediated DNA-PK inhibition in lung cancer cell lines.

(A-E) Clonogenic assays of control and knockout (KO) clones after treatment with DNA-PK inhibitor (peposertib). Survival assays in A549 cells (left panel), and H460 cells (right panel) for HMCES (A), POLQ (B), XRCC1 (C), FEN1 (D), and LIG3 (E). Data are shown as mean +/− SEM from three independent experiments. Significance determined by two-tailed paired t-tests.

Pharmacologic inhibition of DNA-PK increases MMEJ activity and sensitizes cancer cells to POLθ inhibition

We recently identified novobiocin (NVB) as a first-in-class small molecule inhibitor of POLθ’s ATPase activity, that selectively kills HR-deficient cancer cells and overcomes PARP inhibitor resistance (25). NVB specifically inhibits POLθ with little effect on another DNA-repair related ATPase, SMARCA1L. NVB inhibits POLθ with an IC50 of 24 μM (25). Accordingly, NVB-mediated POLθ inhibition might be synthetically lethal with peposertib-mediated DNA-PK inhibition as well, based on our CRISPR screen results. To test this hypothesis, A549 and H460 cell lines were exposed to the combination of peposertib and NVB. As expected, addition of NVB to increasing concentrations of peposertib enhanced the cytotoxicity in both A549 and H460 cell lines (Figures 3A and 3B). To eliminate potential off-target effects of peposertib, we performed clonogenic survival assays in A549 and H460 which had been engineered with a CRISPR-knockout of PRKDC (encoding DNA-PK) (Figure S3A). Knockout of DNA-PK rendered both cell lines more sensitive to low concentrations of NVB (Figures 3C and 3D). Furthermore, to reduce the possibility of off-target effects of NVB, we generated A549 with a CRISPR-knockout of POLQ. As predicted, these A549 POLQ knockout cells were more resistant to NVB treatment than wild-type cells (Figure S3B), further confirming POLθ as the specific target of NVB. We also assessed the synergy of a combination of peposertib and NVB treatment in A549 and H460 cells lines, using the Bliss synergy model (39). The combination of peposertib plus NVB resulted in synergistic cytotoxicity in both cell lines (Figures 3E–F and S3C and D). To extend these findings, we also employed ART558, a recently identified allosteric inhibitor of the POLθ polymerase domain, in combination with peposertib or in A549 and H460 cells (40). Consistently, the combination of ART558 with peposertib or treatment of A549 or H460 with a CRISPR-knockout of POLQ resulted increased cytotoxicity (Figures S3E–G).

Figure 3. Pharmacologic inhibition of DNA-PK increases MMEJ activity and sensitizes cancer cells to POLθ inhibition.

(A-D) Clonogenic assays of A549 (A) and H460 (B) cells treated with peposertib alone or with peposertib and 100 μM NVB. Clonogenic assays of A549 DNA-PK KO (C) and H460 DNA-PK KO (D) treated with NVB. (E-F) Bliss synergy/antagonism levels on the experimental combination dose response surface (right) for A549 (E) and H460 (F) cells treated with graded concentrations of peposertib and NVB.Viability was assessed at 5 days using CellTiter-Glo. Bliss scores greater than zero (green/blue shading) indicate synergy. Synergy/antagonism between the drugs was determined using Combenefit software. (G) U2OS cells containing a MMEJ reporter assay were treated with 5 μM peposertib and transduced with an adenovirus containing I-SceI. The percentage of E2-Crimson+GFP+ cells were quantified by FACS after 5 days of growth. (H) Examination of POLθprotein expression in A549 cells treated with increasing concentrations of peposertib as indicated and immunoblotted with the indicated antibodies. Quantification of signal emitted from the POLθ protein band normalized to Tubulin is shown below each lane. (I) RNA was isolated from A549 cells treated with increasing concentrations of peposertib as indicated, and the transcript level of POLQ was quantified relative to ACTIN by qPCR. (J) Examination of POLθ protein expression in A549 DNA-PK KO cells immunoblotted with the indicated antibodies. Quantification of signal emitted from the POLθ protein band normalized to Tubulin is shown below each lane. Data are shown as mean +/− SEM from three independent experiments. Significance determined by two-tailed paired t-tests.

We reasoned that the peposertib-mediated inhibition of DNA-PK would render the cells more dependent on MMEJ-mediated DNA repair. To test this hypothesis, we first assessed the effect of peposertib treatment on a cell-based MMEJ reporter (41). As predicted, we observed an increase in MMEJ reporter activity upon treatment with peposertib (Figure 3G) (41). We next assessed the effect of peposertib treatment on POLQ expression, a known predictive and quantitative biomarker of NVB responsiveness (25). Interestingly, peposertib treatment caused a dose-dependent increase in both POLθ protein expression and POLQ mRNA expression in A549 cells (Figures 3H–I). Similar results were observed in A549 DNA-PK knockout cells (Figures 3J and S3H). Thus, upon peposertib-mediated inhibition of DNA-PK, cells compensate by increasing POLθ expression and POLθ-mediated MMEJ, rendering them hypersensitive to NVB.

Mechanism of synthetic lethal interaction: Combination of NHEJ and MMEJ inhibitors generates toxic levels of DNA end-resection

Among the three main DSB repair pathways, NHEJ preferentially repairs unresected DSBs, whereas MMEJ and HR require nucleolytic DNA end-resection of 5’ ends generating a 3’ ssDNA overhangs (24). Since DNA-PK is critical for the NHEJ pathway (Figure S1C), we reasoned that inhibition of DNA-PK with peposertib might increase the cellular dependence on DNA end-resection and activate the other two DSB repair pathways (34). To test this hypothesis, we evaluated the role of DNA-PK inhibition on DNA end-resection using multiple complementary assays. Phosphorylation of the ssDNA binding protein RPA on S33 (p-RPA) is a biomarker of single strand DNA generation at sites of resected DSBs (42). As predicted, treatment of U2OS cells with peposertib induced more fluorescent p-RPA than DMSO vehicle treatment (Figures 4A–B). To monitor ssDNA formation more directly, we labelled genomic DNA with 5-bromo-2′-deoxyuridine (BrdU). BrdU can be detected by immunofluorescence with an anti-BrdU antibody under native conditions but only if the DNA is single-stranded, thereby allowing quantification of end-resection in cells (43). Again, treatment of U2OS cells with peposertib resulted in more DSB end resection, as visualized by enhanced BrdU fluorescence, consistent with the enhancement of p-RPA foci (Figures S4A–B). To further quantify the extent of DNA end-resection after peposertib treatment, we employed a quantitative PCR (qPCR) based method in which the AsiSI restriction enzyme is fused with a hormone-binding domain in U2OS cells. Upon treatment with tamoxifen, AsiSI translocates to the nucleus and generates DSBs at a sequence-specific site (Figure S4C) (44). Treatment with peposertib increased the level of ssDNA measured at two sites, 355 bp and 1618 bp from the induced DSB (Figures 4C and S4D). Taken together, peposertib-mediated inhibition of DNA-PK prevents NHEJ, resulting in an increase in DSB DNA end-resection.

Figure 4. Mechanism of synthetic lethal interaction: Combination of NHEJ and MMEJ inhibitors generates toxic levels of DNA end-resection.

(A) Immunofluorescence was performed on U2OS cells treated with 0, 5, and 10 μM peposertib as indicated using an antibody to phosphorylated RPA (p-RPAS33) to evaluate foci formation one hour after exposure to 5 Gy ionizing radiation. (B) Quantification of the p-RPA fluorescence intensity in (A) relative to no treatment (0 uM peposertib). (C) ssDNA quantified at 355 bp downstream of the AsiSI-induced DSB site after indicated drug treatment (5 μM peposertib) and DSB induction (4-hydroxy-tamoxifen, + OHT) or no DSB induction (- OHT). (D) Immunofluorescence was performed on U2OS cells treated with 100 μM Novobiocin (NVB), 5 μM peposertib, or a combination of 100 μM NVB and 5 μM peposertib, using an antibody to γH2AX to evaluate foci formation. (E) Quantification of the γH2AX fluorescence intensity in (D) relative to no treatment. (F) Immunofluorescence was performed on U2OS cells treated with 100 μM Novobiocin (NVB), 5 μM peposertib, or a combination of 100 μM NVB and 5 μM peposertib, using an antibody to p-RPA to evaluate foci formation. (G) Quantification of the p-RPA fluorescence intensity in (F) relative to no treatment. (H) Quantification of ssDNA at 355 bp downstream of the AsiSI-induced DSB site after indicated drug treatment (5 μM peposertib, 100 μM NVB, or combination) and DSB induction (4-hydroxy-tamoxifen, + OHT) or no DSB induction (- OHT). (I) Quantification of the change in ssDNA length of A549 cells after indicated drug overnight. (J) Comet assay images and quantification of the tail moment after indicated drug treatment. (K) Examination of cleaved Caspase 9 protein expression in A549 cells treated with peposertib, NVB, or the combination and immunoblotted with the indicated antibodies. (L) Quantification of ssDNA at 355 bp downstream of the AsiSI-induced DSB site after indicated siRNA transfection, drug treatment and DSB induction (4-hydroxy-tamoxifen, + OHT) or no DSB induction (- OHT (M) Clonogenic assays of A549 cells after indicated siRNA transfection and drug treatment. Data are shown as mean +/− SEM from three independent experiments. Significance determined by two-tailed paired t-tests.

Inhibition of POLθ is also known to increase DNA end-resection (25,40). As predicted, exposure of U2OS cells to NVB caused a dose-dependent increase in p-RPA foci and BrdU foci, markers of ssDNA (Figures S4E–H) (42,43). A quantitative increase in ssDNA was also observed after NVB treatment, as assessed by the ER-AsiSI DSB assay (Figure S4I). Since both DNA-PK inhibition and POLθ inhibition increase DSB end-resection, we hypothesized that the mechanism of synthetic lethality in the cells with dual inhibition of NHEJ and MMEJ might be the generation of toxic levels of DNA end-resected intermediates. Indeed, significantly more γH2AX foci were generated from the combination of peposertib and NVB than from either treatment alone (Figures 4D–E). This finding is consistent with our hypothesis that combined treatment with peposertib and NVB generates more DNA damage. Next, we assessed the level of resected DNA by measuring the level of p-RPA after treatment with DMSO, peposertib, NVB, or the combination of peposertib and NVB. A significant increase in p-RPA foci was observed following the combination of peposertib and NVB, compared with either treatment alone (Figures 4F–G). To determine the extent of DNA end-resection, we again employed the AsiSI-induced DSB assay and quantified the production of ssDNA. The combination of peposertib and NVB significantly increased the production of ssDNA, compared to either treatment alone (Figures 4H and S4J). Finally, we assayed DNA end-resection using the single molecule analysis of resection tracts (SMART) method (32). Treatment of A549 cells with the combination of peposertib and NVB significantly increased the amount of ssDNA generated by DNA end-resection (Figure 4I). A similar result was also obtained in A549 DNA-PK KO cells treated with NVB (Figure S4K).

In a comet assay, the combination of peposertib plus NVB caused increased tail moments, reflecting an increase in DNA damage (Figure 4J). The induction of apoptosis was also measured by the production of cleaved caspases. The observed synergy between peposertib and NVB correlated with this induction of apoptosis (Figures 4K and S4L). Consistent with the role of drug-enhanced extensive end-resection as the mechanism of synthetic lethality, depletion of the DNA end-resection nucleases, EXO1 or BLM-DNA2, reduced DNA end-resection and significantly limited the toxicity of the drug combination (Figures 4L–M and S4M–R). Furthermore, combination of peposertib and NVB, but not monotherapy, resulted in extensive accumulation of RAD51 foci, another measure of enhanced DNA end-resection and HR repair (Figures S4S). Taken together, these results indicate that enhanced DNA end-resection, resulting from the combination of peposertib and NVB, is the key initiating event leading to cell death.

Novobiocin-mediated POLθ inhibition sensitizes TP53-deficient tumor cells and TP53-deficient PDX-derived tumor organoids to peposertib

We next identified sgRNAs in our genome-wide CRISPR knockout screen which confer peposertib resistance (Figure 1). Interestingly, sgRNAs directed against TP53 significantly increased the growth and survival of both A549 and H460 cells following peposertib exposure (Figures 1D and S1E). This finding was consistent with previous reports demonstrating that TP53 deficiency can suppress the cytotoxic activity of a DNA-PK inhibitor (17,33,45,46). We reasoned that TP53-deficient cells might acquire resistance to DNA-PKi through a compensatory upregulation of POLQ expression and an overall upregulation of MMEJ-mediated DSB repair. Accordingly, inhibition of POLθ with NVB might re-sensitize TP53-deficient cancer cells to peposertib.

To test this hypothesis, we examined a pair of isogenic retinal pigment epithelium (RPE) cell lines which differ in their TP53 expression. These RPE versus RPE-TP53-KO cells were treated with increasing concentrations of peposertib. As predicted from the CRISPR screen and consistent with a previous report, the RPE-TP53-KO cells, unlike the parental RPE cells expressing wild-type TP53, exhibited increased resistance to DNA-PK inhibition (Figure 5A) and increased sensitivity to POLθ inhibition (Figure 5B) (45). Importantly, the RPE-P53-KO cells were re-sensitized to peposertib by exposure to NVB (Figure 5C). Indeed, the combination of peposertib and NVB significantly reduced the IC50 of the peposertib from 2.053 μM to 0.2948 μM. (Figure S5A). Furthermore, the area under the curve (AUC) was the least for RPE-P53-KO cells treated with the combination of peposertib plus NVB, compared to monotherapy or RPE cells expressing wildtype TP53, indicative of increased cell killing (Figure S5B). Similarly, in both A549 and H460-cell lines, depletion of TP53 correlated with increased expression of POLQ, increased sensitivity to NVB, and increased sensitivity to the drug combination, as shown by Bliss synergy curves (Figures S5C–K) (39). The mechanism of this re-sensitization is due, at least in part, to increased POLθ expression, which is a consequence of cellular TP53 deficiency as previously reported (45). Pharmacological inhibition of NHEJ by peposertib led to increased POLθ expression and dependence on MMEJ (Figure 5D). Taken together, our results demonstrate that the combination of peposertib plus NVB is especially effective in killing TP53-deficient tumor cells. To extend these results, two additional TP53 mutant and HR-deficient cell lines (MDA-MB-436 and CAPAN-1), remained hypersensitive to the combination of peposertib plus NVB, even after their complementation with the wildtype BRCA1 or BRCA2 cDNAs, respectively (Figures S5L–M).

Figure 5. Novobiocin-mediated POLθ inhibition sensitizes TP53-deficient tumor cells and TP53-deficient PDX-derived tumor organoids to peposertib.

(A) Clonogenic assays of RPE and RPE P53 KO cell lines after treatment with increasing concentrations of peposertib. (B) Clonogenic assays of RPE and RPE P53 KO cell lines after treatment with increasing concentrations of NVB (C) Clonogenic assays of RPE and RPE P53 KO cell lines after treatment with increasing concentrations of peposertib and 100 μM NVB. (D) RNA was isolated from RPE RPE P53 KO cells treated with peposertib and the transcript level of POLQ was quantified relative to ACTIN by qPCR. (E-F) Bliss synergy/antagonism levels on the experimental combination dose response surface for DF59 (E) and DF68 (F) PXOs treated with graded concentrations of peposertib and NVB. Viability was assessed at 5 days using CellTiter-Glo. Bliss scores greater than zero (green/blue shading) indicate synergy. Synergy/antagonism between the drugs was determined using Combenefit software. IC50s for DF59 (E) and DF68 (F) PXOs in bottom panel. Representative γH2AX (top panel) p-RPA (bottom panel) immunohistochemical staining in DF59 (G) and DF68 (J) PXOs after vehicle, peposertib, NVB, or combination peposertib and NVB treatment. Quantification of γH2AX and p-RPA H-score for DF59 PXOs (H, I) and for DF68 (K, L). Data are shown as mean +/− SEM from three independent experiments. Significance determined by two-tailed paired t-tests.

To further determine the effect of TP53 expression on POLQ expression, we next generated and analyzed primary human tumor organoid cultures from TP53-deficient high-grade serous ovarian carcinoma (HGSOCs). Therapeutic drug responses of human tumor organoids have been shown to correlate, at least in part, with their corresponding patient responses in the clinic (47–49). HGSOCs were chosen since 96% of these tumors are TP53 mutated leading to deficiency in TP53 function and expression, though with the caveat that there may remain some low levels of TP53 function not seen with CRISPR-knockout of TP53. (50). Patient derived xenograft derived organoids (PXOs) were generated from tumor cells from two different TP53 mutant ovarian cancer patient-derived xenograft (PDX) models, DF59 and DF68 (51,52). DF59 has no discernible TP53 protein expression by Western blot analysis, whereas DF68 has reduced TP53 protein expression (53).

To determine whether combination of peposertib and NVB would act synergistically in an organoid culture, we treated these two TP53-deficient PXO models with peposertib, NVB, or the drug combination. Both organoid models were sensitive to monotherapy with either peposertib or NVB but the combination treatment, at the monotherapy doses, resulted in a significant increase in lethality as assessed by cell viability using the Bliss synergy model (39). The combination of peposertib and NVB resulted in synergistic cytotoxicity in both PXOs (Figures 5E–F, top panels). Indeed, the combination of peposertib and NVB significantly reduced the IC₅₀, thereby improving the therapeutic index of the two drugs (Figures 5E–F, bottom panels). We next sought to correlate the tumor suppressive effects with the molecular mechanisms described in Figure 4. Indeed, both DF59 and DF68 PXOs exhibited a statistically significant increase in γH2AX and p-RPA staining by immunohistochemistry upon treatment with the combination of peposertib and NVB, consistent with an increase in DSB end resection (Figures 5G–L). Next, based on the synthetic lethal mechanism of increased DSB end-resection, we performed SMART assays on DF59 PXOs. We observed a marked increase in SMART fiber length in the combination treatment group compared to peposertib-alone treatment or NVB-alone treatment, consistent with enhanced DSB end-resection (Figure S5N–O). Thus, we conclude that peposertib and NVB act synergistically in TP53-deficient ovarian cancer organoid models by generating toxic levels of DSB DNA end-resection and cell death.

Combined peposertib and NVB kills TP53-deficient patient-derived xenograft (PDXs) in vivo

We next determined whether the combination of DNA-PK and POLθ inhibition would be an effective treatment for a TP53-deficient tumors in vivo (Figure 6). Mice bearing TP53 mutant ovarian tumors (the DF59 PDX model) were treated with peposertib, NVB, or the drug combination, and tumor growth was monitored by bioluminescence imaging (BLI) (Figure 6A). Peposertib alone did not significantly affect the growth of these TP53-deficient HGSOC tumors, consistent with the inability of a DNA-PK inhibitor to kill TP53-mutant tumor cells in vitro (Figure 6B). NVB demonstrated significant monotherapy tumor growth inhibition in TP53-deficient HGSOC tumors in vivo, consistent with our previous report (25). Critically, the combination of these two targeted DNA repair inhibitors further enhanced tumor growth inhibition despite the lack of peposertib monotherapy efficacy. Interestingly, the targeted drug combination alone killed the tumor cells in vivo, and no additional cytotoxic therapy, such as IR or an additional chemotherapeutic agent, was required to achieve this synergy. Again, the enhanced anti-tumor activity of the combination is caused in part by the increased expression of POLQ generated by peposertib exposure (Figure 6C). This was evident in the TP53-mutant cells, which likely have intrinsic peposertib resistance and high baseline levels of POLQ expression (Figures 5 and S5). Therefore, the addition of NVB reverses the resistance of TP53-mutant cancer cells to peposertib. The enhancement of tumor killing by the drug combination was also demonstrated by the pharmacodynamic increase in DSB end-resection (p-RPA level) and increased DNA damage (γH2AX) observed in extracted tumor cells from the treated mice (Figure 6D). Importantly, no significant toxicity to normal mouse tissue was observed from this drug combination as evidenced by comparable increase in bodyweights of mice during treatment and hematological examination of mice following 28 days of treatment (Figures S6A–B). Given the lack of DF59 tumor growth inhibition with peposertib monotherapy despite signs of increased DNA end-resection, we treated A549, A549 DNA-PK KO, and A549 DNA-PK P53 DKO xenograft-bearing nude mice with vehicle or NVB. Consistently, tumor growth inhibition after 28 days of treatment was greatest for NVB treated A549 DNA-PK P53 DKO xenografted mice (Figures S6C–D).

Figure 6. Combined peposertib and NVB kills TP53-deficient patient-derived xenografts (PDXs) in vivo.

(A) DF59 patient-derived luciferized tumor cells were injected into NRG mice (n = 8–10 mice per group). Randomized mice were treated with DMSO (vehicle), peposertib (100 mg/kg BID, orally), NVB (100 mg/kg BID, transitioned to 75 mg/kg BID on day 16, intraperitoneally), or a combination for 4 weeks. Tumor growth was monitored weekly by bioluminescence (BLI). Representative images of tumor burden on day 0 and day 28 are shown. (B) Quantification of tumor volume shown are mean +/− SEM. Log2 (fold change in tumor size) values were calculated using the formula log2 (T ÷ T0), where T is the tumor volume at a day 28 (T28), and T0 is the initial tumor volume. (C) RNA was isolated from PDX ascitic tumor cells treated as indicated, and the transcript level of POLQ was quantified relative to ACTIN by qPCR. Three mice per treatment group. (D) Examination of indicated protein expression in PDX ascitic tumor cells treated as indicated and immunoblotted with the indicated antibodies. Three mice per treatment group.

DISCUSSION

The DNA Damage Response maintains genomic integrity by responding to endogenous and exogenous DNA damage and activating specific DNA repair pathways. DNA damage is repaired by one of at least six pathways acting on specific types of DNA damage. Cells become dependent on an alternative pathway if the primary DNA repair pathway is lost due to mutation, deletion, epigenetic silencing, or inhibition with a targeted drug. Indeed, HR-deficient tumor cells are dependent on PARP1-mediated base excision repair or POLθ-mediated MMEJ (25,35,36,54). Here we sought to identify a DNA repair pathway that is upregulated following cellular exposure to peposertib, a targeted and highly selective inhibitor of the kinase DNA-PK in the NHEJ pathway (22,33). Through a whole-genome CRISPR screen, we demonstrated that loss of POLQ, or loss of other components of the MMEJ pathway, are synergistic with peposertib or with genetic knockdown of NHEJ both in vitro and in vivo. Consistent with this result, POLQ mRNA and POLθ protein expression are upregulated after peposertib exposure. Also, as expected, the combination of peposertib plus POLθ inhibitors, novobiocin (NVB) or ART558, killed cancer cells. The synergy of peposertib plus NVB was observed across multiple cancer cell lines, including those with either homologous recombination repair proficiency or deficiency.

Based on published data and on the results presented here, the mechanism of synthetic lethality between combination NHEJ and MMEJ inhibition is an increase in DNA DSB end resection leading to loss of genetic material, for example by utilization of mutagenic repair processes such as single-strand annealing, and subsequent cellular death through apoptosis (Figure S6E) (25,40). Cells have several regulatory processes that normally limit the amount of DSB end resection, and these activities function in a stepwise manner (Figure S6F). Under normal circumstances, DSBs can be repaired rapidly by NHEJ and blunt end ligation (1). Because, NHEJ occurs throughout the cell cycle, it is the predominant DSB repair pathway and can quickly join unresected ends, thereby limiting DNA end-resection. Thus, NHEJ is the first barrier to DNA end-resection, and inhibition of DNA-PK with peposertib eliminates this barrier. A second barrier to end resection is the 53BP1-Shieldin complex which further limits resection and fortifies blunt end ligation by NHEJ (55–60). This complex promotes NHEJ by protecting DNA ends from additional resection. Indeed, depletion of 53BP1 or other SHLD proteins increases DSB end-resection, thereby providing a mechanism of HR restoration in BRCA1-deficient cells as well as a predictive biomarker of POLθ inhibitor response (61–63) (Figure S6F). Finally, if a low level of DNA end-resection occurs, producing short 3’ overhangs and the preferred substrate of POLθ-mediated MMEJ, the POLθ-mediated MMEJ pathway further limits DNA end-resection by performing MMEJ repair. Thus, MMEJ-mediated repair provides a third barrier to DSB end-resection (64). Based on this model, disruption of NHEJ with peposertib and disruption of MMEJ with NVB results in a massive level of DSB end-resection and ultimately to apoptosis (Figure S6F).

Prior studies have demonstrated that MMEJ is a backup mechanism for repairing DSBs when NHEJ is not available. When NHEJ is genetically perturbed, there is a compensatory increase in POLθ-mediated MMEJ activity (37,38). Our findings are consistent with these prior reports and demonstrate that pharmacological inhibition of NHEJ by peposertib directly increases POLQ mRNA and POLθ protein expression, resulting in increased MMEJ activity and increased dependence on POLθ. We have previously reported that increased POLQ expression is an important predictive biomarker of responsiveness to POLθ inhibition by NVB (25). Indeed, low-dose treatment with peposertib potentiated NVB tumor killing. Thus, our study indicates that upregulation of POLθ-mediated MMEJ activity is a major intrinsic resistance mechanism to the use of peposertib.

DNA-PKi resistance can be overcome when peposertib is combined with a MMEJ inhibitor (NVB). Furthermore, peposertib, amongst other DNA-PK inhibitors, is currently undergoing early phase clinical trial investigation as a potentiating agent in combination with radiation or chemotherapy (65). In general, the use of chemotherapy and/or radiation therapy in conjunction with targeted therapy has been limited by toxicity (66). Importantly, our study did not require supplemental radiation or cytotoxic chemotherapy to induce DNA damage. The combination of the targeted NHEJ inhibitor (peposertib) and the MMEJ inhibitor (NVB) alone induced DNA damage and led to tumor killing via apoptosis. These results suggest that a combination of a NHEJ inhibitor (peposertib) and a MMEJ inhibitor (NVB, or ART558) will be both safely tolerated and effective in treating cancer in patients.

Prior work by a number of groups has demonstrated that TP53 deficiency can suppress the toxicity of DNA-PK inhibition (17,33,45,46). Consistently, our whole-genome CRISPR knockout screen identified TP53 loss as a resistance mechanism resulting from peposertib-mediated DNA-PK inhibition. TP53 is the most commonly mutated tumor suppressor, occurring in 50% of newly diagnosed solid tumors (67). TP53 deficiency is associated with resistance to DNA damaging agents and DNA repair pathway inhibitors (68,69). Furthermore, TP53 deficiency in cancers portends a worse prognosis (68,70). Kumar et al. has demonstrated that POLQ mRNA expression is increased upon TP53 deletion and that there is a correlation between POLQ mRNA overexpression and TP53 mutation status in a wide variety of cancers in The Cancer Genome Atlas dataset (45). Our findings are consistent with this report and extend the results by using the specific POLθ inhibitor, NVB. We hypothesized that TP53-deficient cancer cells acquire resistance to DNA-PK inhibition through a compensatory upregulation of POLQ expression and an overall upregulation of MMEJ-mediated DSB repair. Accordingly, the combination of peposertib plus NVB alone resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models, without the need for additional cytotoxic therapy. Accordingly, we show that inhibition of POLθ with NVB re-sensitizes TP53-deficient cancer cells to peposertib. Thus, our findings establish a strong rationale for the combination of two targeted DNA damage repair inhibitors (NHEJ-peposertib) and (MMEJ-novobiocin) in TP53-deficient cancers.

Predictive biomarkers are critical to the successful development of targeted DNA repair in inhibitors (71). These biomarkers aid in the identification of cancer patients whose tumors are most likely to be sensitive or resistant to the targeted therapy. In our current study, we propose a model in which multiple cellular barriers block DNA end-resection. When these barriers are disrupted, there is the accumulation of toxic DSB end-resected DNA, leading to cell death. This model also predicts responsiveness to NVB-mediated POLθ inhibition. The first barrier to DNA end-resection is NHEJ mediated by DNA-PK. We show that DNA-PK inhibition by peposertib increases DNA end-resection, POLQ expression, POLθ activity, and ultimately sensitivity to NVB. Thus, loss of NHEJ is a predictive biomarker for responsiveness to NVB. Similarly, the 53BP1-Shieldin complex is a second barrier to DNA end-resection and its loss predicts response to POLθ inhibition (25,40,72). We previously reported that POLQ expression, and hence MMEJ activity, is the third barrier to DNA end-resection, correlating with response to NVB treatment. Thus, high POLQ expression is a strong predictive biomarker of POLθ inhibitor response. Because loss of these barriers to DNA end-resection results in increased DSB end-resection, assessment of p-RPA accumulation could serve as a pharmacodynamic biomarker for POLθ inhibition. Finally, as noted above, TP53 deficiency or biallelic mutation results in increased POLθ expression and sensitivity to POLθ inhibition and is an important predictive biomarker of response to the DNA-PK and POLθ inhibitor combination. How TP53 normally suppresses POLQ expression remains an important unanswered question. Thus, our study supports the investigation of the combination of targeted NHEJ and MMEJ inhibitors in TP53-mutant cancers.

Significance:

Combined inhibition of NHEJ and MMEJ using two non-toxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.