Targeting histone demethylation to knock down <i>HOXA-AS2</i> and <i>HOXA-AS3</i> , outcome-determining lncRNAs in multiple malignancies: Effect of deferiprone, a mechanism-based disruptor of KDM6B catalysis.

e15153 Background: HOXAantisense RNAs 2 and 3 ( HOXA-AS2 , -AS3 ) are lncRNAs that determine outcome in major malignancies ( HOXA-AS2: 10.3892/ol.2024.14359; HOXA-AS3: 10.1007/s12094-022-02920-w). They promote immune tolerance and chemoresistance, blocking apoptosis ( HOXA-AS2: 10.1159/000495387; HOXA-AS3: 10.1038/s41389-019-0170-y). No small molecule in clinical use affects their expression. lncRNAs ‘sponge’ miRNAs (10.3389/fcell.2020.00085) and encode cancer-relevant peptides, like those mediating cisplatin resistance (10.1186/s13045-024-01591-0); HOXA-AS2 and -AS3 offer 89 ORFs. Histone lysyl demethylases (KDMs) are master regulators of HOX gene expression (10.1038/nature06145) and - except for KDM1 - stoichiometrically consume molecular oxygen and 2-oxoglutarate as non-heme mono-iron dioxygenases (10.1039/c8cc04057e). Their catalytic pathway was resolved in 1982 at the orbital level of each reactant atom (HAG mechanism, classifying all possible inhibitors [10.1016/0022-5193(82)90320-4]); its two authors reduced the HAG coordinates to immediate practice by identifying the HAG-inhibitory pharmacophores PDCA and NOG, each in global use today. The predictive power of HAG coordinates also identified deferiprone (DEF), orally administered to alleviate transfusional iron overload in thalassemia, as a pioneer HAG inhibitor within the library of FDA-EMA approved medicines (10.2174/0929867033457601). We tested the hypothesis that KDM inhibition by DEF also decreases KDM expression, resulting in knockdown of HOXA-AS2 and HOXA-AS3 . Methods: Repeated RNA-Seq and flow cytometry of ARK1-USC at 12/24/48 hours (control; DEF and deferoxamine [DFOX] at serum concentrations). Results: Among the 17 KDM genes expressed, KDM6B was most reactive: 100 µM DEF (clinically maintained by the drug’s TID po regimen), cut KDM6B expression to less than half of controls within 48 hrs (FDR p &lt; 0.0000); the medicinal iron chelator DFOX had no effect. DEF-mediated KDM6B knockdown coincided with marked reductions vs . control of HOXA-AS2 expression ( - 2.2 fold, FDR p &lt; 0.0000) and HOXA-AS3 expression ( - 4.1 fold, FDR p &lt; 0.0000); DFOX had no effect. Their DEF-mediated KDM6B knockdown associated with the expression of proapoptotic genes at 48 hrs, e.g. those encoding BH3-only apoptosis promotors ( HRK [ + 5.6 fold, FDR p &lt; 0.0000], PMAIP1 [ + 2.1 fold, FDR p &lt; 0.0000], BIK [ + 2.1 fold, FDR p &lt; 0.000000]) and of apoptosis executioners caspase 3 and caspase 7. Flow cytometry at 96 hrs detected, via active caspase 3 and cleaved PARP, rapid progression of apoptotic death-by-DEF in malignant ARK1, yet arrest of apoptosis and reversal of apoptosis markers in DEF-treated, non-malignant fibroblasts (MRC5). Conclusions: FDA/EMA-approved DEF is first-in-class for control of HOXA-AS2 and HOXA-AS3 expression in malignant cells.