A combination of PARP and CHK1 inhibitors efficiently antagonizes MYCN-driven tumors

MYCN drives aggressive behavior and refractoriness to chemotherapy, in several tumors. Since MYCN inactivation in clinical settings is not achievable, alternative vulnerabilities of MYCN-driven tumors need to be explored to identify more effective and less toxic therapies. We previously demonstrated that PARP inhibitors enhance MYCN-induced replication stress and promote mitotic catastrophe, counteracted by CHK1. Here, we showed that PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of SHH-dependent medulloblastoma, their combination being more effective in MYCN amplified and MYCN overexpressing cells compared to MYCN non-amplified cells. Although the MYCN amplified IMR-32 cell line carrying the p.Val2716Ala ATM mutation showed the highest sensitivity to the drug combination, this was not related to ATM status, as indicated by CRISPR/Cas9-based correction of the mutation. Suboptimal doses of the CHK1 inhibitor MK-8776 plus the PARP inhibitor olaparib led to a MYCN-dependent accumulation of DNA damage and cell death in vitro and significantly reduced the growth of four in vivo models of MYCN-driven tumors, without major toxicities. Our data highlight the combination of PARP and CHK1 inhibitors as a new potential chemo-free strategy to treat MYCN-driven tumors, which might be promptly translated into clinical trials.


CRISPR/Cas9
sgRNA specific to the c.8147 T>C mutation (mutATM-sgRNA) and the single-stranded donor oligonucleotides (ssODN) were designed using the informatics platform Benchling (Benchling, 2019, https://benchling.com). PAM sequence (AGG) was located three nucleotides downstream of the c.8147 T>C mutation. ssODN contained the reverted form of the c.8147 T>C mutation and two additional silent "blocking mutations" on the seed sequence to avoid Cas9 re-cutting (Paquet D, Nature 2016). The mutATM-sgRNA and scRNA (Origene, Rockville, MD, USA) were cloned into the LentiCRISPR v2 plasmid (Addgene plasmid #52961). IMR-32 cells were co-transfected with mutATM-sgRNA or scRNA-LentiCRISPR v2 plasmid and ssODN using Lipofectamine2000 Transfection Reagent (#11668027 Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. To select mutATM-sgRNA/scRNA-LentiCRISPR v2 positive cells, puromycin (1 µg/ml) was added to the medium 24 hours after transfection, for 72 hours. After selection, cells were harvested and seeded in 96-well plates (0.5 cells/well) to obtain monoclonal cell populations. To perform allele-specific PCR screening, DNA was extracted from monoclonal populations using NucleoSpin Tissue (#740952 Macherey-Nagel, Dueren, Germany) according to the manufacturer's instructions. Allele-specific PCR primers were designed on the WT sequence, on the c.8147 T>C mutated sequence and on the sequence of the ssODN (provided in supplemental information). The ssODN positive clones and the scramble clones were sequenced through Sanger method (ABI 3100 Genetic Analyzer, Applied Biosystems, Warrington, UK) to confirm the results. Predicted off-target gene loci were also analyzed by Sanger sequencing to exclude off-target mutations.

In vivo studies
For in vivo experiments, olaparib and MK-8776 were dissolved in DMSO and diluted into 2hydroxy-propyl-β-cyclodextrin (2-HPβC)/PBS solution just before injection and administered by intraperitoneal (ip) injection at 50 mg/Kg and 25 mg/kg, respectively.
For the subcutaneous xenograft, IMR-32 cells (4x10 6 cells) and LAN-5 (2.5x10 6 cells) were suspended in an equal volume of medium and matrigel (BD Biosciences, Heidelberg, Germany) and injected in the posterior flanks of 4-weeks-old female BALB/c nude mice (Nu/Nu, Charles River Laboratories, Lecco, Italy). When the tumors reached a size of approximately 150 mm 3 , animals were randomly divided into 4 groups and injected with vehicle, olaparib, MK-8776 or their combination and different sets of animals were used to test the early biochemical responses or the long-term efficacy of the drugs. For short term analyses, animals were injected twice with a 24 hours interval (30 hours time point) or every day for 4 days (5 days time point) and then sacrificed to collect tumor tissues for WB or immunohistochemical analysis. Animals for long term experiments were treated every day, 6 days/week. Animals were sacrificed after 14 days of treatment and tumor tissues were collected for weight measurement and macroscopic examination. In survival studies, mice were sacrificed when the tumor size reached 2 cm 3 or at latest on day 40. The time span between the first treatment and the sacrifice was used as a surrogate for survival. MS (50% survival) was determined by GraphPad Prism 7 software (La Jolla, CA). %ILS was calculated by the formula: (median survival of treated mice -median survival of control mice) / median survival of control mice. In all cases, tumor growth was monitored by caliper size measurement and calculated by the formula length x width x 0.5 x (length + width).
For orthotopic xenografts, IMR-32 were transfected with luciferase to obtain IMR-32-luc cells. Five-weeks-old female athymic Foxn1nu mice (Envigo, Bresso, Italy) were subjected to laparotomy and inoculated with 1x10 6 IMR-32-luc cells into the left adrenal gland capsule, as previously described (Pastorino F, Cancer research 2003). Luciferase activity was visualized by in vivo bioluminescent imaging (BLI, IVIS Caliper Life Sciences, Hopkinton, MA) after a 10-min incubation with 150 µg/mL of D-luciferin (Caliper Life Sciences), as described (Cossu I, Biomaterials 2015). Tumor-bearing mice were randomized into 4 groups for efficacy studies (n=10 animals/group) and systemic toxicity evaluation (n=3 animals/group). Starting one week after tumor cells implantation, MK-8776 and olaparib, as single agents or in combination, were given every day, 6 days/week, for 4 weeks. Mice were monitored 2-3 times weekly and euthanized before showing signs of illness/suffering, such as paraplegia, dehydration, severe weight loss (>15%) or abdominal dilatation, according to the ethical committee of the Italian Ministry of Health. The time span between the first treatment and the sacrifice was used as a surrogate for survival.
For the systemic toxicity experiment, mice were anaesthetized with xylezine 48h following the last treatment and blood was collected into either anticoagulant-free tubes or K3EDTA coated tubes through the retro-orbital sinus from each mouse, for clinical chemistry and hematological evaluations. These analyses were performed at the Mouse Clinic, IRCCS Ospedale San Raffaele, Milan. Finally, mice were sacrificed, tumors and healthy organs (liver, spleen, heart, kidney) were recovered, fixed in formalin and embedded in paraffin for subsequent histopathological examination.
For the medulloblastoma mouse model, post-natal day 5 (P5) human GFAP promoter-driven CREmediated Ptch-KO (Ptch -/-) mice (Yang ZJ, Cancer cell 2008) were ip injected with vehicle (n=5), olaparib (n=4), MK-8776 (n=5) or their combination (n=5), every day, for 4 days. Dealing with P5 mice, MK-8776 concentration was further reduced to 12.5 mg/Kg. At the end of the experiment (P9), animals were sacrificed and cerebella were measured by caliper and collected for histopathological examination. Animals were checked regularly, and no signs of illness/suffering were observed until the end of the treatment.
For in vivo experiments Tukey outlier box plot has been used to identify and exclude outliers. Animal experiments were approved by the ethical committee of the Italian Ministry of Health (protocol n.: n°379/2016-PR and n.: 661/2016-PR) in compliance with the "ARRIVE" guidelines (Animals Research: Reporting in Vivo Experiments).
Briefly, antigen-retrieval treatment was applied, when required, using PT link instrument (DakoCytomation, Glostrup, Denmark). Endogenous peroxidase activity was quenched with hydrogen peroxide (3%) for 10 min at room temperature. After blocking with unrelated antiserum, tissue slides were incubated with the primary monoclonal antibodies and/or antiserum in a moist chamber at 4°C. After washing with phosphate buffered saline (PBS), slides analyzed for phosphorylated-H2A.XSer139 and cleaved caspase-3 primary antibodies were incubated with secondary antibodies and EnVision™ FLEX/HRP reagent (Agilent, California, USA). Instead, samples analyzed for anti-CD31 were incubated with a goat anti rat-HRP conjugated antibody (#A24555, TermoFisher Scientific, UK), revealed with 3,3'-diaminobenzidine (Dako EnVision FLEX System; Agilent) as previously reported (Peplau E, Thyroid 2020).
Immunohistochemical staining was assessed and scored by two independent pathologists who were blinded to the clinicopathological data. Discrepancies were resolved by consensus.