Globin modification in HUDEP-2 and SS-HUDEP-2 cells by MetAP2 knockdown or inhibition. The N termini of α-globin and β-globin are modified with iMet and acetyl-iMet in wild-type HUDEP-2 cells treated with lentivirus expressing MetAP2-specific shRNA or in HUDEP-2 and SS-HUDEP-2²⁴  cells treated with MetAP2 inhibitors. (A) Analysis by LC-MS confirms that wild-type HUDEP-2 cells do not have baseline globin modification. (B) Globin modification in subclone H3 from a pool of lentivirus-treated HUDEP-2 cells. MetAP2 expression in clone H3 is decreased ∼99% based on western blot analysis (supplemental Table 3). Because of the smaller sample size, nonglobin background peaks are more evident in this sample. (C) Treatment of HUDEP-2 cells during differentiation with the selective covalent MetAP2 inhibitor CKD-732 (10 nM) leads to high levels of HbA modification (∼80%) on both globins (85% α-globin and 74% β-globin modification). Similar results were obtained in 2 independent studies. (D) SS-HUDEP-2 cells were differentiated for 7 days in the indicated concentrations of the covalent, irreversible MetAP2 inhibitor ZGN-1061,²⁰  to increase modification of HbS by iMet retention at concentrations that decrease cell viability (88% α-globin and 59.5% β^S-globin maximum modification). (E) In contrast, a reversible inhibitor of MetAP2, Compound I,²¹  increases HbS modification at concentrations that do not affect cell viability (93.5% α-globin and 63% β^S-globin maximum modification). Similar results were obtained in 2 independent studies.