Pomalidomide augments fetal hemoglobin production without the myelosuppressive effects of hydroxyurea in transgenic sickle cell mice

Abstract

Pharmacologic induction of fetal hemoglobin (HbF) expression is an effective treatment strategy for sickle cell disease (SCD) and β-thalassemia. Pomalidomide is a potent structural analog of thalidomide and member of a new class of immunomodulatory drugs. Recent reports demonstrated that pomalidomide reduced or eliminated transfusion requirements in certain hematologic malignancies and induced HbF ex vivo in CD34(+) progenitor cells from healthy and SCD donors. We investigated the effects of pomalidomide on erythropoiesis and hemoglobin synthesis in a transgenic mouse model of SCD. We found that 8 weeks of treatment with pomalidomide induced modest increases of HbF with similar efficacy as hydroxyurea. However, in stark contrast to hydroxyurea's myelosuppressive effects, pomalidomide augmented erythropoiesis and preserved bone marrow function. Surprisingly, combinatory therapy with both drugs failed to mitigate hydroxyurea's myelotoxic effects and caused loss of HbF induction. These findings support further evaluation of pomalidomide as a novel therapy for SCD.

Figure 1

Pomalidomide regulates HbF synthesis and erythropoiesis in transgenic sickle cell mice. Homozygous sickle cell mice were treated with vehicle, pomalidomide, or hydroxyurea for 8 weeks by intraperitoneal injection and killed for analysis. (A) HbF protein levels as a percentage of total hemoglobin were determined by high performance liquid chromatography at the end of the study to avoid artifactual increases of HbF from repeated blood draws. F cells were analyzed by flow cytometry after immunolabeling formalin-fixed RBCs with anti–human fluorescein isothiocyanate-conjugated HbF antibody. (B) Representative images depicting the M:E ratio in hematoxylin and eosin-stained bone marrow sections of the proximal femur. Erythroid cells are recognized by their round, dense, and deeply basophilic nuclei. All images were collected using a Zeiss Axioplan 2 microscope and a Plan-Apochromat 63×/1.4 oil objective. (C) Analysis of bone marrow cellularity, M:E ratio, and megakaryocyte counts. Cellularity in the active treatment groups was determined in reference to the 100% cellular marrow in vehicle-treated sickle cell mice. Megakaryocytes were counted in 5 low magnification optical fields per animal and converted to megakaryocytes/mm². All sections were analyzed by 2 blinded investigators. Veh indicates vehicle; PL, pomalidomide; HU, hydroxyurea; C/HD, combination high-dose treatment (10 mg/kg pomalidomide, 100 mg/kg hydroxyurea); and C/LD, combination low-dose treatment (10 mg/kg pomalidomide, 10 mg/kg hydroxyurea). *Significantly different from Veh and HU (P < .05). **Significantly different from Veh and PL (P < .01).