An Investigation of Biological Processes Underlying Neratinib-induced Gut Injury

Abstract

Neratinib, a small-molecule tyrosine kinase inhibitor (TKI) that irreversibly binds to human epidermal growth factor receptors 1,2, and 4 (HER1, 2 and 4), was recently approved as an adjuvant therapy for patients with HER2-amplified or -overexpressed (HER2-positive) breast cancer. However, in clinical practice, more than 90% of patients receiving neratinib experience mild-to-severe symptoms of gut toxicity including abdominal pain and diarrhoea. Despite a highly prevalent complication in gut health, the underlying biological processes of how neratinib causes gut injury, especially in the colon, leading to symptoms of gut toxicity remains unclear. Here, using archived colon tissues collected from healthy female Albino Wistar rats dosed with neratinib (50 mg/kg) daily for 28 consecutive days, we found that the severity of colonic injury, especially degeneration of surface lining colonocytes and infiltration of immune cells, was more pronounced in the distal than in the proximal colon. To better understand biological processes underlying neratinib-induced cell death, we leveraged previously published bulk RNA-sequencing and CRISPR-screening datasets of neratinib-treated mouse TBCP-1 breast cancer and human glioblastoma SF298 cell line and human glioblastoma T895 xenograft. Gene ontology (GO) term and KEGG pathway analyses suggested that a type of cell death induced by neratinib was likely context specific. Specifically, neratinib stimulates ferritinophagy-mediated ferroptosis in TBCP1 and T895 cells, whereas apoptosis in SF298 cells. To identify whether ferroptosis or apoptosis was potentially induced by neratinib in the rat colon, we integrated the analyses from immunohistochemical staining (Caspase-3 to detect apoptosis, FTH1 and 4HNE to detect ferroptosis) on paraffin-embedded rat colons, and RT-qPCR (markers for iron homeostasis: Fth1 and Tfrc; markers for lipid peroxidation: Acsl4 and Alox15; and marker for general ROS: Nox1) on cryopreserved rat colons. Our findings suggested that ferritinophagy-mediated ferroptosis, but less likely apoptosis, was a potential underlying histopathological feature of colonic injury in rat treated with neratinib. We further wanted to ascertain that colonic epithelial cells could undergo ferroptosis as a direct consequence of neratinib treatment by utilising SW48 colorectal cancer cell line. Although SW48 cells were resistant to neratinib treatment, the observation of vacuoles formation at supra-clinical concentration (10 μM) suggests that this cell line could undergo autophagy-mediated cell death. Thus, we switched our efforts to utilise the 3-dimensional (3D) mouse colonic organoids to circumvent the mutation complexities associated with neratinib resistance in SW48 cells. We showed that organoids can be terminally differentiated which might potentially be exploited as a model system for future mechanistic investigations. In a context of neratinib-induced ferroptosis, such as in mouse TBCP1 and human SKBR3 HER2-positive breast cancer cell lines, by using published LINC-KINOME scan datasets and the alignment of published X-ray crystal structures, we proposed that inhibiting kinase activity of mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) by neratinib might be essential for potentiating ferroptosis, but not other TKI, such as lapatinib, which is linked to apoptosis. Overall, the findings from this research suggest that a type of cell death, i.e. apoptosis or ferroptosis, induced by neratinib may be cell-type specific. Ferroptosis is a potential underlying feature of colon injury. Targeting the molecular machinery underlying neratinibinduced ferroptosis, especially the initiating event of cell death such as perturbed redox regulation at specific subcellular compartments, may serve as an exciting platform for future supportive therapies and drug discovery to mitigate toxicity while enhancing the efficacy of similar or emerging anti-cancer therapeutics.