The immune checkpoint controller C1q (C1QA/B/C) is a druggable oncologically relevant target: Proof-of-concept in vitro with an FDA-approved medicine, used as pioneer inhibitor of protein hydroxylation (273)

Abstract

<b>Objectives:</b> C1q, a key initiator of immune reactions (adaptive, innate), is the single most important protein mediating tolerogenic clearance of apoptotic debris (‘self-waste') and functions in tissue homeostasis, e.g., angiogenesis. Cancer cells employ locally secreted C1q to secure growth and immune escape [Cancer Immunol. Res. 7.7, 1091-1195 (2019)]. In the tissue of high-grade serous ovarian cancer (HGSOC), expression of <i>C1</i>QA, or <i>C1QB,</i> or <i>C1QC</i> is a negative prognostic indicator [OSFPreprint 10.31219/osf.io/bcv9a]. C1q consists of ABC monomers, each comprising two domains, globule, and stem, the latter aligning six of these monomers into a ‘bouquet-of-tulips.' Every ABC monomer requires multiple Pro/Lys hydroxylations by specific 2-oxoacid utilizing dioxygenases (2OUDs) to form its stem as a triple helix identical to collagen. This collagenous stem (CS) mediates all C1q functions. Several experimental agents, read off the HAG mechanism that defines the principally possible inhibitors of 2OUDs (https://pubmed.ncbi.nlm.nih.gov/6281585/), are long known to prevent CS formation in C1q, to cause intracellular accumulation of underhydroxylated collagen and thus CS-defective C1q, and to block the secretion of any C1q that is dysfunctional due to CS mis-or unfolding [Biochem. J. 248, 625-33 (1987)]. We hypothesized that HGSOC cells generate their own C1q, which is rendered CS-defective and thus intracellularly retained by the Pro/Lys hydroxylation-inhibiting medicine deferiprone (DEF), discovered per the HAG mechanism. We aimed to Identify a medicine that disrupts all C1q support for malignancy. <b>Methods:</b> Cell line cultures (KURAMOCHI [HGSOC], ARK1 [uterine serous cancer, USC], MRC5 [normal fibroblasts, NF]); RNA-seq; multi-channel flow cytometry. <b>Results:</b> KURAMOCHI, which is unique among 47 HGSOC cell lines for displaying superior genetic similarity to HGSOC tumors [Nat. Commun. 4.1, 1-10 (2013)], synthesized C1q within 48 hours of plating, as detected intracellularly by the FITC-labeled monoclonal antibody JL-1 that specifically recognizes the CS domain of C1q. At 96 hours, the flow-cytometric JL-1 signal indicated further C1q increase by at least half and mirrored C1q kinetics in MRC5, a model of NF, the major source of C1q in non-malignant tissues. DEF, ranged at its concentrations in serum during multi-year treatment of iron overload conditions, e.g., transfusion-dependent thalassemia, caused all cell lines to accumulate JL-1 reactive C1q material, even at the residual 50 µM that patients on the FDA-approved DEF regimen may maintain for most or all of 24 hours [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154842]. After 96 hours on the drug, this intracellular accumulation exceeded the JL-1 signal of C1q within untreated controls by a factor of 3 in KURAMOCHI and by a factor of 10 in ARK1 (p<0.02), whereas JL-1 reactivity in treated MRC5 had, even at 150 µM, reverted to that of untreated MRC5. In KURAMOCHI, the accumulated JL-1 reactivity coincided with a 25-fold increase in active caspase 3 at 96 hours, a level of apoptotic activity reached in ARK1 after 48 hours already. In MRC5, active caspase-3 was suppressed at 96 hours on DEF. <b>Conclusions:</b> DEF at clinically relevant concentrations disrupts C1q synthesis selectively in HGSOC and USC cells and triggers their apoptotic self-destruction; however, NF remain unaffected. Our results encourage a DEF trial in HGSOC/USC patients and the further HAG mechanism-guided development of improved 2OUD inhibitors for oncological indications.