Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation

Abstract

Other than hydroxyurea, no pharmacologic agents are clinically available for fetal hemoglobin (HbF) induction in sickle cell disease (SCD). An optimal candidate would induce HbF without causing cell cycle inhibition and would act independently of hydroxyurea in order to yield additional HbF induction when combined. We explored whether inhibition of histone deacetylase (HDAC) 1 or HDAC2 could achieve these goals. In human erythroid progenitor cells, shRNA knockdown of the HDAC1 or HDAC2 genes induced gamma globin, without altering cellular proliferation in vitro, and without altering cell cycle phase. Treatment with hydroxyurea in combination with HDAC2 knockdown yielded a further increase in gamma globin expression. Additionally, when CD34+ cells were treated with both hydroxyurea and MS-275 (an inhibitor of HDAC 1, 2, and 3), an additive induction of relative gamma globin expression was achieved. Our findings support further clinical investigation of HDAC inhibitors in combination with hydroxyurea in SCD patients.

Figure 1. Inactivation of HDAC1 or HDAC2 induces gamma globin expression without blocking cellular proliferation

(A) A Western blot shows the decreased level of protein with HDAC1 knockdown (left panel) and with HDAC2 knockdown (right panel). (B) Lentiviruses expressing shRNAs targeting HDAC1 (1-A and 1-B) or HDAC2 (2-A and 2-B) effectively decreased expression of the target mRNA, and (C) increased expression of γ-globin relative to β-globin in primary human erythroid progenitor cells. (D) After infection with the shRNA-expressing lentiviruses, cells in culture were counted over the course of 14 days, and normalized to the number of cells 3 days after infection, following selection with puromycin. In panels B and C, a 2-tailed Student t test was used. *P ≤ .01.

Figure 2. Inactivation of HDAC1 or HDAC2 does not affect p21 expression or cell cycle phase

(A) Level of p21 expression was assessed by qPCR in cells harvested three days after lentiviral infection. (B) Cell cycle was analyzed by flow cytometry. Following a 30 minute pulse of BrdU, cells were collected and fixed ten days after infection. After staining with an antibody against BrdU and 7-AAD, cells were analyzed by FACS. A representative flow plot is shown in Figure S1. In panel A, a 2-tailed Student t test was used, and samples were not statistically different from controls (P > .1).

Figure 3. HDAC inactivation in combination with hydroxyurea has additive effects on γ-globin expression

(A) Primary human erythroid progenitor cells were infected with lentiviruses expressing shRNAs targeting HDAC2. Each population of infected cells was treated with either hydroxyurea (HU) or vehicle control. Expression of γ-globin relative to β-globin, measured by qPCR, was the highest in cells with both HDAC2 knockdown and HU treatment. (B) Primary human erythroid progenitor cells were treated with hydroxyurea, MS-275, or a combination of both, at multiple doses. At each dose of MS-275, the γ-globin induction is further increased by the addition of hydroxyurea, in a dose-dependent fashion.