Targeting carbonic anhydrase IX acidifies intracellular pH, disrupts redox homeostasis, and creates vulnerability to ferroptosis.

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.

The ability of tumor cells to alter their metabolism to support survival and growth presents a challenge to effectively treat cancers. Carbonic anhydrase IX (CAIX) is a hypoxia-induced, metabolic enzyme that plays a crucial role in pH regulation in tumor cells. Recently, through a synthetic lethal screen, we identified CAIX to play an important role in redox homeostasis. In this study, we show that CAIX interacts with the glutamine (Gln) transporter, solute carrier family 1 member 5 (SLC1A5), and coordinately functions to maintain redox homeostasis through the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis. Inhibition of CAIX increases Gln uptake by SLC1A5 and concomitantly increases GSH levels. The combined inhibition of CAIX activity and Gln metabolism or the GSH/GPX4 axis results in an increase in lipid peroxidation and induces ferroptosis, both in vitro and in vivo. Thus, this study demonstrates cotargeting of CAIX and Gln metabolism as a potential strategy to induce ferroptosis in tumor cells.

Tumour associated neutrophils (TANs) promote metastasis through interactions of Neutrophil Extracellular Traps (NETs) with tumour cells. However, molecular details surrounding the interactions between NETs and Pancreatic Ductal Adenocarcinoma (PDAC) cells are poorly understood. Here, we examine the contribution of NETs in the progression of PDAC, which is characterized by high metastatic propensity. We carry out consensus clustering and pathway enrichment analysis of NET-related genes in an integrated cohort of 369 resectable and metastatic PDAC patient tumour samples, and compile two gene expression signatures comprising of either, integrin-actin cytoskeleton and Epithelial to Mesenchymal Transition (EMT) signaling, or cell death signaling, which identifies patients with very poor to better overall survival, respectively. Tumour Infiltrating neutrophils and NETs associate with ITGB1, CCDC25 and ILK, within clinical and experimental PDAC tumours. Functionally, exposure of PDAC cells to NETs identifies a cytoskeletal dynamic-associated CCDC25-ITGB1-ILK signaling complex which stimulates EMT and migration/invasion. NETosis-driven experimental metastasis to the lungs of PDAC cells delivered through the tail vein of female non-obese diabetic (NOD) scid gamma (NSG) mice is significantly inhibited by ILK knock down. Our data identify novel NET-related gene expression signatures for PDAC patient stratification, and reveal targetable signaling axes to prevent and treat disease progression.

Combating resistance to targeted therapy remains a major challenge to improving lung cancer care. Epithelial-mesenchymal transition (EMT) in tumour cells is an established non-genetic resistance mechanism to EGFR tyrosine kinase inhibitors (TKI) that is also associated with worse outcome in patients. Here we demonstrate that integrin-linked kinase (ILK) is an important driver of EMT-mediated TKI resistance in lung adenocarcinoma (LUAD) by promoting a drug-tolerant persister (DTP) cell phenotype. Our results indicate that high ILK expression is associated with EMT in LUAD patients and that genetic suppression of ILK can limit EMT progression and reduce the viability of DTP cells by impairing YAP activation, ultimately improving osimertinib (Osi) sensitivity in LUAD cells. Importantly, LUAD cells with high ILK expression are able to persist during EGFR-TKI treatment, acquiring additional genetic and phenotypic alterations to develop EGFR-TKI resistance. To improve clinical translatability of our findings, we showed that pharmacological inhibition of ILK can suppress EMT and improve Osi response in LUAD cells. Lastly, we found that strong immunohistochemistry staining of ILK in patient biopsies was significantly associated with and may be used to predict receptor tyrosine kinase-independent mechanisms of EGFR-TKI resistance. Overall, our results suggest that ILK is an important regulator of EGFR-TKI response and may be exploited as a predictor for acquired resistance, providing evidence for co-targeting ILK with EGFR to better control minimal residual disease and EGFR-TKI resistance in lung cancer.

The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.