Epithelial ovarian cancer (EOC) metastasizes predominantly through multicellular aggregates known as spheroids, which disseminate within the peritoneal cavity and initiate secondary disease upon reattachment at distant sites. EOC spheroids resist detachment-induced cell death by upregulating stress responses including AMP-activated protein kinase (AMPK) signaling and AMPK-dependent macroautophagy (autophagy), highlighting these pathways as potential therapeutic targets. Previously, we used a pharmacological approach to putatively identify Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CAMKKβ, encoded by CAMKK2) as the primary activator of AMPK in EOC spheroids. Herein we have generated CAMKK2 knockout EOC cell lines via CRISPR-Cas9 genome editing to confirm this function of CAMKKβ and explore the impacts of its loss using in vitro and in vivo models of metastatic EOC. CAMKK2 knockout spheroids exhibited decreased AMPK activation, autophagic flux, cell viability, and metastatic potential relative to parental spheroids, and intraperitoneal xenograft tumours lacking CAMKKβ grew slower than their CAMKKβ-intact counterparts. Effect magnitudes varied between cell line models, suggesting context-dependent roles for CAMKKβ in EOC and rationalizing further studies to characterize the underlying mechanisms. Altogether, our findings highlight CAMKKβ as an important contributor to metabolic reprogramming in EOC spheroids and as a potential therapeutic target in the setting of advanced disease.

Background/Objectives: Epithelial ovarian cancer (EOC) is often diagnosed at advanced stages, with metastasis driven by spheroid dissemination within the peritoneal cavity. We previously demonstrated that autophagy supports spheroid cell survival and suggest that it contributes to chemoresistance. Unc-51-like autophagy activating kinase 1 (ULK1), a key regulator of autophagy, has emerged as a promising therapeutic target. Here, we evaluated the effects of ULK1 inhibition via MRT68921, alone and in combination with afatinib—a tyrosine kinase inhibitor (TKI) known to induce pro-survival autophagy—in EOC. Methods: High-grade serous (HGSOC) and ovarian clear cell carcinoma (OCCC) cell lines were cultured under adherent and spheroid conditions. Immunoblotting confirmed on-target effects and modulation of autophagy. Autophagic flux was assessed using mCherry-eGFP-LC3 reporter assays. We assessed 96 dose combinations of MRT68921 and afatinib using drug combination matrices, with synergy evaluated via Synergy Finder. Promising combinations were evaluated across multiple EOC spheroid models and patient ascites-derived organoids. Results: MRT68921 inhibited ULK1 activity and reduced autophagic flux in a context-dependent manner while afatinib alone induced autophagy. Their combination produced synergistic effects at select concentrations, impairing spheroid reattachment and viability. However, MRT68921 alone significantly reduced viability across multiple EOC models, including patient ascites-derived organoids. Conclusions: This study is the first to evaluate the combined effects of MRT68921 and afatinib in epithelial ovarian cancer. Our findings demonstrate that ULK1 inhibition via MRT68921 consistently reduces cell viability across multiple ovarian cancer models, supporting ULK1 as a promising therapeutic target. In contrast, combination with afatinib produced limited and context-dependent effects, indicating that further investigation is needed to identify optimal combination strategies for ULK1-targeted therapies.

Background/Objectives: Epithelial ovarian cancer (EOC) is often diagnosed at advanced stages, with metastasis driven by spheroid dissemination within the peritoneal cavity. We previously demonstrated that autophagy supports spheroid cell survival and suggest that it contributes to chemoresistance. Unc-51-like autophagy activating kinase 1 (ULK1), a key regulator of autophagy, has emerged as a promising therapeutic target. Here, we eval-uated the effects of ULK1 inhibition via MRT68921, alone and in combination with afatinib—a tyrosine kinase inhibitor (TKI) known to induce pro-survival autophagy—in EOC. Methods: High-grade serous (HGSOC) and ovarian clear cell carcinoma (OCCC) cell lines were cultured under ad-herent and spheroid conditions. Immunoblotting confirmed on-target effects and modulation of autophagy. Autophagic flux was assessed using mCherry-eGFP-LC3 reporter assays. We assessed 96 dose combinations of MRT68921 and afatinib using drug combination matrices, with synergy evaluated via Synergy Finder. Promising combinations were evaluated across multiple EOC spheroid models and patient ascites-derived organoids. Results: MRT68921 inhibited ULK1 activity and reduced autophagic flux in a con-text-dependent manner while afatinib alone induced autophagy. Their combination produced synergistic effects at select concentrations, impairing spheroid reattachment and viability. However, MRT68921 alone significantly reduced viability across multiple EOC models, including patient ascites-derived organoids. Conclusions: This study is the first to evaluate the combined effects of MRT68921 and afatinib in EOC. Our findings support ULK1 inhibition as a therapeutic strategy with potential enhanced efficacy when combined with afatinib. These results warrant further preclinical investigation to explore the translational potential of ULK1-targeted therapies alone and in combination with TKIs.

Metastasis, chemoresistance, and tumour recurrence are facilitated by cancer stem cells (CSCs), a small subpopulation of cells capable of regenerating a primary tumour while maintaining the tumour’s genetic and phenotypic features. CSCs can be identified by the expression of specific markers; however, the CSC population in ovarian clear cell carcinoma (OCCC), a rare histotype of ovarian cancer, remains poorly defined. Given the well-established role that CSCs play in cancer progression and metastasis, it is critical to identify reliable markers of CSCs in OCCC. Here, we endeavoured to determine whether ALDH1 expression could be used to define OCCC stem cells in OCCC cell lines using a variety of methods including assessing ALDH1A1 expression in spheroids generated under distinct conditions. We also generated and used chemo-resistant cell lines to assess the enrichment of cancer stem cells. Human OCCC cell lines were enriched for CSCs using selective culture conditions and drug resistance methods. CSC-enriched spheroids demonstrated increased expression of stemness markers NANOG and SOX2, while ALDH1A1 expression was enriched only in drug-resistant cell lines, relative to parental cell lines. RNA-seq analyses of CSC-media-derived spheroids versus standard media spheroids provided novel data supporting CSC enrichment and identified transcription factors induced by CSC media. These findings highlight the ambiguous role of ALDH1A1 as a CSC marker in OCCC and demonstrates the utility of CSC enrichment methods for identifying CSC populations in OCCC cell lines.