Tracking MAPK-Dependent CD38 Upregulation by All-Trans Retinoic Acid in Human Leukemia Using ⁸⁹Zr Immuno-PET

Anti-CD38 antibodies (Abs) are promising immunotherapeutics for hematologic malignancies, but their efficacy in leukemias often requires pharmacologic upregulation of CD38. Immuno-PET provides a noninvasive strategy to evaluate such target modulation in vivo. Cysteine site-specific ⁸⁹Zr labeling of anti-CD38 Abs was performed using deferoxamine-maleimide. CD38-specific target binding was confirmed in three human myeloma and two leukemia cell lines. Total and surface expressed CD38 levels were assessed by Western blotting and flow cytometry. Immuno-PET imaging and biodistribution studies were conducted in murine leukemia models. All-trans retinoic acid (ATRA) was used to stimulate CD38 expression. Myeloma and MOLT4 leukemia cells showed variable baseline CD38, while HL60 cells exhibited negligible levels. All tumor cells demonstrated ⁸⁹Zr-OKT10 IgG and ⁸⁹Zr-daratumumab (Fc-silenced) binding that paralleled surface CD38 expression. ATRA upregulated CD38 in all tested cells, including a marked induction in HL60 cells, all accompanied by corresponding elevations in ⁸⁹Zr-CD38 Ab binding. On ⁸⁹Zr-OKT10 IgG PET, MOLT4 tumors showed high uptake that was reduced by 67.9% with unlabeled Ab, but HL60 tumors showed low uptake and high liver accumulation, limiting ATRA assessment. ⁸⁹Zr-daratumumab produced lower liver uptake and improved MOLT4 and HL60 tumor visualization; ATRA modestly increased MOLT4 tumor uptake and substantially enhanced HL60 tumor uptake from 8.0 ± 1.7 %ID/g to 14.7 ± 3.1 %ID/g (83.8% increase; P < 0.005). Mechanistic studies demonstrated ATRA-induced ERK1/2 activation in HL60 cells that was abolished by the MAPK inhibitor U0126; U0126 also suppressed CD38 induction and ⁸⁹Zr-CD38 Ab uptakes in all tested tumor cells. Furthermore, U0126 blocked ATRA-induced HL60 tumor ⁸⁹Zr-daratumumab uptake in vivo. Western blots and immunohistochemistry confirmed ATRA-induced HL60 tumor CD38 elevation, which was partly reversed by U0126. Thus, ⁸⁹Zr immuno-PET enables noninvasive monitoring of ATRA-driven CD38 upregulation via MAPK signaling and supports its potential utility for optimizing combination strategies in leukemias.