Vulnerability to low-dose combination of irinotecan and niraparib in ATM-mutated colorectal cancer

Colorectal cancer (CRC) is the third most frequent malignancy and the second leading cause of cancer death worldwide, accounting for more than 1.8 million new cases and 880,000 deaths in 2018 [1].

Despite the increase in therapeutic options for the treatment of metastatic CRC (mCRC), cytotoxic chemotherapy using fluoropyrimidines, oxaliplatin, and irinotecan still constitutes the mainstay of the treatment [2]. These genotoxic drugs function by inducing direct or indirect DNA damage, which lead to cell cycle block and cell death. Nevertheless, DNA damage is recognized by different DNA repair pathways such as mismatch repair (MMR), nucleotide excision repair (NER), and homologous recombination repair (HRR) [3]. All these mechanisms of DNA damage response (DDR) are crucial for the survival of normal cells and are often deregulated in cancer, allowing for the accumulation of mutations that are ultimately associated to cancer progression and therapeutic resistance [4]. For these reasons, blocking DDR has been widely investigated in the context of cancer treatments as a strategy to potentiate radiotherapy/chemotherapy-induced DNA damage and overcome drug resistance. Indeed, several compounds that interfere with the different mechanisms of DNA repair are already available or in advanced clinical testing [5]. Among these compounds, Poly-ADP-Ribose Polymerase inhibitors (PARPi) were the first to be approved in clinical practice and exhibit a strong activity in ovarian and in other cancers characterized by a functional impairment of HRR known as homologous recombination deficiency (HRD) [6‐8]. In the last years, several biomarkers were used to predict HRD and sensitivity to PARP inhibitors, most of them focused on finding genetic alteration in HRR-associated genes (mainly BRCA1/2, but also ATM, PALB2 and others) and/or on analyzing the genomic scars associated to HRD (loss of heterozygosity, large scale transitions, subchromosomal allelic imbalance) [9]. Moreover, mutational signature single base substitution 3 (SBS3) has been associated to HRD and correlates with PARPi sensitivity [10].

Initial preclinical data reported PARPi efficacy also in CRC cell lines, apparently increased in case of microsatellite instability (MSI). Notably, in the largest analyzed preclinical cohort, including more than 100 human CRC cell lines, no clear association was evidenced between olaparib sensitivity (13% of MSS cell lines screened) and either mutations in HR genes or mutational signatures [11]. However, a phase II clinical trial with olaparib did not show any benefit in chemorefractory mCRC patients [12, 13]. In addition, several preclinical reports evidenced promising synergism of PARP inhibitors in combination with oxaliplatin or irinotecan in CRC, independently from microsatellite status [14‐16]. However, similarly to previous cases, a phase Ib clinical trial failed to identify any benefits for the combination of the PARP inhibitor olaparib and irinotecan in chemorefractory mCRC [17]. Very disappointedly, this study evidenced a high-grade hematological toxicity that led to a significant dose reduction, which probably hampered the efficacy in this unselected population [17]. These results might be explained by the low prevalence of biallelic loss in genes involved in homologous recombination in CRC, which is less than 3% versus more than 50% in ovarian cancer [18]. Nevertheless, 26% of mCRC patients from the large MSK IMPACT database exhibit at least one mutation in HRR genes, the most frequent being ATM (8%) and BRCA2 (8%) [19]. Taking this into account, in this study we hypothesized that HRR genes mutations might represent a vulnerability to the combination of PARPi/chemotherapy in CRC. Thus, we have systematically evaluated the synergism between the PARPi niraparib and three genotoxic agents approved for mCRC in a panel of 12 human CRC cell lines representative of the main molecular subtypes, showing that irinotecan is the best candidate for combination therapy. Interestingly, in our work we have identified ATM mutations as a common genetic background associated with niraparib/ irinotecan combination efficacy. We further confirmed that a low dose combination is very effective in an in vivo model and in primary 3D cultures obtained from fresh CRC surgical specimens, which are mutated in ATM or in its downstream pathway. Moreover, we showed that a malfunction of HRR due to heterozygous mutations in ATM is responsible for such effects.

In our work, we have analyzed the activity of the combination between standard-of-care chemotherapeutics used in CRC and niraparib in a panel of 12 cell lines that encompasses the 4 consensus molecular subtypes, the different microsatellite status, and the different KRAS and BRAF mutational profiles. Niraparib was selected for this study due to its optimal pharmacodynamic and pharmacokinetic properties in patients, showing a strong PARP trapping activity (stronger than olaparib), but also a long half-life (> 24 h) and a wide therapeutic interval [29, 33]. Niraparib in vitro sensitivity was in line with previous reports in CRC cell lines, with mean IC₅₀ values above the trough plasma concentration of this drug at the commonly used dose of 300 mg/die in monotherapy trials (Table 1, Fig. 1a), showing no association with microsatellite status (Supplementary figure 1) [6, 16, 29]. Moreover, transcriptional classification using the CMS classifiers did not evidence any correlation with sensitivity to the PARPi (Supplementary figure 1). SN38, the active metabolite of irinotecan, was shown to be the best partner for combination with niraparib, and the sensitivity to this combination was independent from microsatellite status or CMS (Fig. 1b, Supplementary Table 1, Fig. 2). Mutational profiles in 29 relevant genes in the HRR pathway were obtained for the cell lines included in the panel from public databases and were correlated with sensitivity to niraparib or the niraparib/SN38 combination. Notably, ATM and BRCA2, that constitute the most frequently mutated HR genes in large mCRC patients datasets such as MSKCC or COSMIC [19, 34], are also recurrently mutated in our panel (Table 1 and Supplementary Table 2). The functional prediction of these genetic alterations showed that in our cell panel all ATM mutations (5/5) were functionally relevant, compared with only 2/5 BRCA2-mutated cell lines carrying mutations with a pathogenic significance (Supplementary Table 3). However, in our panel, non-synonymous mutations in ATM or BRCA2 genes were not associated to a significant higher sensitivity to niraparib used as single agent (Supplementary figure 1). This may seem counterintuitive, as both ATM and BRCA2 inactivation are associated to homologous recombination deficiency and synthetic lethality with PARP inhibition [7], and a previous work demonstrated that ATM depletion using shRNAs induces sensitivity to PARP inhibitors [35]. However, it must be noted that a mutation in ATM does not correspond to ATM loss, though a decreased protein level was found in case of heterozygous ATM mutations in CRC cell lines compared to the wild type counterpart [35]. Nevertheless, we evidenced a significant association between the presence of ATM mutations and synergism with the niraparib-SN38 combination, while no correlation is present with microsatellite status, CMS or BRCA2 mutations (Fig. 2).

One possible explanation for the increased sensitivity to the combination in ATM-mutated cell lines lies in the relative insufficiency of timely DNA repair in these cells in case of double strand break damage overload. This dysfunction is unveiled by the combination treatment but not by single agent SN38, since most SN38-induced single strand breaks can be repaired before precipitating into DSBs [3]. Notably, both DSB recognition and HRR are intact in ATM-mutant cell lines, as evidenced by γH2Ax and Rad51 immunostaining (Fig. 4a-b), though persistent HR engagement upon combination treatment reflects a delay in the resolution of DSBs 24 h after treatment washout in case of heterozygous ATM mutations (Fig. 4c-d), possibly unveiling a haploinsufficient phenotype. The delay in repairing DSBs and the following cell cycle arrest are also evident at the cell cycle analysis in HCT116 compared to LS1034 cell lines (Supplementary Figure 3).

In this framework, the inhibition of both PARP and topoisomerase 1 is fundamental to achieve the effect in ATM-mutated CRCs, even if myelotoxicity characterizes a major concern that has hampered the development of other PARP inhibitors/chemotherapy combinations [17]. For this reason, we investigated whether the molecular subgroup characterized by the presence of pathogenic mutations in ATM exhibits an increased sensitivity to the niraparib/irinotecan combination at lower doses. Indeed, low drug concentrations of niraparib and SN38 induced a clonogenic arrest in vitro in synergistic cell lines (HCT15, LOVO, LIM1215, SW948, HCT116, SW480) (Fig. 3a-b). In mice models, low doses of niraparib and irinotecan were effective and synergistic in ATM-mutated HCT116 xenograft, in which they induced complete tumor regressions in 60% of treated mice (Fig. 3e). The low doses used for the mice experiments are predicted to achieve plasma concentrations of 100 nM for niraparib and less than 25 nM for SN38, concentrations that are way below the desirable ones in monotherapy, achieved with oral doses of 40 mg/die for niraparib and less than 100 mg/iv for irinotecan [29‐32, 36].

Finally, we have also confirmed our findings in a relevant translational model using patient-derived 3D spheroids, in which the presence of a pathogenic mutation in ATM or in its downstream effector CHEK2 confer increased sensitivity to the combination of niraparib and SN38 (Fig. 5).

Recently, several papers have investigated the role of ATM mutations in cancer susceptibility and prognosis, suggesting that such mutations induce an attenuated cancer-predisposing phenotype in heterozygote carriers, that represent up to 1–2% of the general population [37], while presenting a favorable prognostic effect in CRCs bearing such heterozygous alterations [38]. Taken together, these evidences underpin a possible role for ATM heterozygous mutations in cancer therapy [39, 40]. Recently, a phase I study investigating different schedules for the combination of irinotecan and the PARP inhibitor rucaparib in refractory cancers bearing mutations in HRR genes was presented, showing how pulse schedules are clinically feasible and that the patient population with ATM-mutated cancers exhibits the greatest benefit from the combination [41].

Our work shows that there is a molecularly defined subpopulation of CRCs bearing heterozygous mutations in ATM, accounting for up to 12% of patients [42], that may benefit from a combination treatment with niraparib and irinotecan used at low doses, suggesting a new potential approach in the treatment of colorectal cancer.