CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells

Abstract

ObjectiveCancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.DesignTo discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.ResultsThe drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.ConclusionsLY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Type-I-interferons in infection and cancer: Unanticipated dynamics with therapeutic implications

“…66 CSCs are defined as the reservoir of a chemoresistant niche within the tumor and the driving force for tumor relapse. 67,68 Mounting observations indicate a potential contribution of (2) and enhanced immunogenicity through cell surface expression of MHC-I (3) and TAAs (4), or tumor progression by inducing resistance to apoptosis (5), EMT (6), tumor-cell stemness (7), and the upregulation of immune-inhibitory signals such as PD-L1 (8). Second, acting on the vascular and lymphatic system Type-I-IFNs inhibit angiogenesis through VEGF downregulation (9).…”

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“…We were also able to demonstrate that LY2606368 preferentially depleted the CSC fraction exclusively in responsive (i.e., high or medium sensitive) CRC-SCs. CSC eradication by LY2606368 occurred (1) in vitro, as shown by the decrease in the percentage of cells positive for the colorectal CSC markers CD44v6 and ephrin B2 (EFNB2) or displaying high level of WNT activity; and (2) in vivo, as proven by the diminished tumor growth potential of LY2606368-treated primary and secondary xenografts derived from CRC-SCs [3].We then elucidated the mechanisms of CSC killing by LY2606368, showing that it involved the inhibition of CHK1 activity followed by a lethal increase in the levels of replication stress (RS). In more detail, CHK1 inhibition by LY2606368 simultaneously impaired the DNA replication process and the intra-S checkpoint resulting in DNA damage accrual, premature mitoses and cell death [3].…”

“…By combining genetic and cytogenetic profiles with sensitivity data we also found an association between LY2606368 sensitivity and either mutation(s) of the tumor protein p53 (TP53, best known as p53) or increased chromosomal content (i.e., hyperdiploidy). Accordingly, the constitutive depletion of p53 or the pharmacological induction of whole-genome redoubling sensitized previously resistant CRC-SCs to LY2606368 [3].To sum up, our results indicate that RS in CRC-SCs is promoted by (1) the abrogation of p53 functions, which leads to diminished DNA repair efficiency, unscheduled S-phase entry and/or tolerance to (and protection from cell death induced by) RS; and (2) hyperdiploidy, which increases the risks to incorrectly duplicate the DNA. …”

“…For this, we took advantage of a large panel of multicellular spheroids enriched for CSCs that were derived from colorectal cancer (CRC) patient specimens. These tumorspheres, to which we will hereafter refer to as CRC-SCs, were all characterized at genetic and cytogenetic levels [3]. A drug library highthroughput screening on three selected CRC-SCs led to the identification of LY2606368 -a novel checkpoint kinase 1 (CHEK1, best known as CHK1) and CHK2 inhibitor also known as prexasertib [4] -as one potent CRC-SC killing agent [3].…”

“…Further underscoring the relevance of CSCs in tumor pathology, (cancer) stemness gene signatures have been correlated with poor clinical outcomes [2]. The eradication of CSCs is thus required for an efficient cancer therapy.Very recently, we performed a study to identify novel anti-CSC compounds to be clinically investigated as standalone agents [3]. For this, we took advantage of a large panel of multicellular spheroids enriched for CSCs that were derived from colorectal cancer (CRC) patient specimens.…”