CHK1 inhibition exacerbates replication stress induced by IGF blockade

Oncogene ARTICLE www.nature.com/onc OPEN CHK1 inhibition exacerbates replication stress induced by IGF blockade Xiaoning Wu1, Elena Seraia2, Stephanie B. Hatch 2, Xiao Wan2, Daniel V. Ebner 2, Francesca Aroldi1, Yanyan Jiang 3, ✉ Anderson J. Ryan3, Thomas Bogenrieder4,5,6, Ulrike Weyer-Czernilofsky4, Guillaume Rieunier 1,8,9 and Valentine M. Macaulay 7,9 ✉ 1234567890();,: © The Author(s) 2021 We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs. Oncogene; https://doi.org/10.1038/s41388-021-02080-1 INTRODUCTION Many cancers show aberrant signalling via the insulin-like growth factor (IGF) axis, activating type 1 IGF receptors (IGF-1Rs) and variant insulin receptors (INSRs) to signal via phosphatidylinositol 3-kinase–AKT–mammalian target of rapamycin (PI3K-AKT-mTOR) and mitogen-activated protein kinase kinase–extracellular signalregulated kinases (MEK-ERK) [1]. Through these effectors, IGFs mediate cell cycle progression, cancer cell proliferation and protection from apoptosis [1–3]. Previous studies from our group and others revealed that IGF-1R blockade sensitises human tumour cells to ionising radiation (IR) and cytotoxic drugs [4–9]. We further reported that IGF-1R depletion or inhibition delays repair of IR-induced DNA double-strand breaks (DSBs), and inhibits DSB repair via both homologous recombination (HR) and nonhomologous end-joining [5, 6]. The present study was underpinned by three observations. First, given evidence that IGFs regulate the response to IR, we also found evidence that IGF-1R depletion induced endogenous DNA lesions marked by γH2AX foci in prostate cancer cells [10]. The origin of these lesions was unclear, although γH2AX foci are known to accumulate at DSBs and stalled replication forks to recruit repair and cell signalling machineries, serving as a sensitive indicator of DNA damage and replication stress [11, 12]. Secondly, we noted that an IGF gene signature identified in MCF7 breast cancer cells included components of the replication machinery [13]. Thirdly, we recently identified an absolute requirement for IGF-1 to maintain replication integrity by regulating the function of ribonucleotide reductase [14], the rate-limiting step for dNTP production [15]. RNR contains two subunits: ribonucleotide reductase subunit M1 (RRM1) and M2 (RRM2) [16]. Acting via both PI3K-AKT and MEK-ERK-JUN pathways, we showed that IGF-1 potently upregulates RRM2 transcription [14]. Thus, IGF-1R inhibited or depleted cells downregulate RRM2 and dNTP supply, delaying replication fork progression, activating ATR/CHK1 and the replication checkpoint, and suppressing new origin firing [14], all key hallmarks of replication stress [17]. The resulting singlestranded DNA (ssDNA) lesions were found to be marked by γH2AX foci and 53BP1 nuclear bodies, which form in G1 phase to protect from erosion under-replicated DNA generated by mitotic transmission of chromosomes under replication stress [18, 19]. Finally, we showed that ssDNA lesions are converted to toxic DSBs in cells lacking functional ataxia telangiectasia mutated (ATM), likely due to failure to form 53BP1 bodies and/or a role for ATM in SSB repair or fork protection [14]. While striking, the replication stress phenotype induced by IGF blockade appeared tolerable with minor impact on viability. 1 Department of Oncology, University of Oxford, Oxford, UK. 2Target Discovery Institute, University of Oxford, Oxford, UK. 3CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK. 4Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria. 5AMAL Therapeutics, c/o Fondation pour Recherches Médicales, 1205 Geneva, Switzerland. 6Department of Urology, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich, Germany. 7Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK. 8Present address: Immunocore Ltd, Abingdon, UK. 9These authors contributed equally: Guillaume Rieunier, Valentine M. Macaulay. ✉email: guillaume. ; Received: 4 May 2021 Revised: 8 September 2021 Accepted: 12 October 2021 X. Wu et al. 2 Hypothesising that this state represents an exploitable vulnerability, we conducted a compound screen using IGF neutralising antibody xentuzumab, currently undergoing clinical evaluation with evidence of benefit in patients with oestrogen receptor positive (ER+) breast cancer and non-visceral metastases [20–22]. We tested five ER+ breast cancer cell lines with xentuzumab alone or with a compound library of inhibitors targeting cell cycle controls, replication and repair. Our recent report described screen outcomes in MCF7 cells [14]; here we describe the findings in the full cell line panel. We show that tolerable replication stress in IGFinhibited cells is exacerbated by co-targeting IGF with CHK1 or WEE1 due to profound RRM2 protein depletion, consistent with roles for these checkpoint kinases in maintaining E2F1-mediated RRM2 transcription and counteracting CDK-mediated RRM2 degradation [23–25]. This approach represents a potential treatment strategy that induces intolerable replication stress, replication catastrophe and tumour cell death. RESULTS IGF axis inhibition induces tolerable replication stress associated with therapeutic vulnerabilities Using genetic and pharmacological approaches to block IGF signalling, we recently uncovered a previously-unrecognised role for IGFs in regulating global DNA replication, with replication stress upon IGF axis blockade [14]. To confirm this effect, we first tested IGF ligand antibody xentuzumab (BI-836845), and IGF-1R tyrosine kina (...truncated)