Pan-Histone Deacetylase (HDAC) Inhibitor LBH589 Depletes CXCR4 Levels and Signaling, Exerting Synergistic Anti-Leukemia Activity in Combination with CXCR4 Antagonists.

The trafficking and mobilization of normal hematopoietic progenitors and their leukemic counterparts is programmed in part by chemotactic gradients of CXCL12, which are transduced by CXCR4. This mechanism also results in decreased sensitivity to pro-apoptotic and anti-leukemic agents, mediated through CXCR4 activation of Akt and Raf phosphorylation and/or harboring in a microenvironmental niche. Both CXCL12 and CXCR4 are overexpressed in AML and expression of CXCR4 has been associated with a poor prognosis. Moreover, inhibition of CXCR4 with AMD3100 enhanced the sensitivity of leukemic myeloblasts to anti-leukemic agents. We therefore explored therapeutic mechanisms to decrease CXCR4 expression and tested them in combination with AMD3100 as well as a new generation CXCR4 inverse agonist, FC131. Exposure to 10 to 50 nM of the pan-HDAC inhibitor LBH589 (Novartis) depleted mRNA and protein levels of CXCR4 in the cultured human acute leukemia OCI-AML3, HL-60 and Jurkat cells, as well as in primary AML cells in a dose and time-dependent manner. LBH589 depleted CXCR4 levels in the presence or absence of 10 nM of CXCL12 in the culture medium. LBH589 mediated depletion of CXCR4 levels was partly due to decreased CXCR4 mRNA levels (by RT-PCR analysis). LBH589 induced acetylation of heat shock protein (hsp) 90 and attenuated the binding of CXCR4 to hsp90 with subsequent degradation of CXCR4 by the proteasome. LBH589 treatment also increased hsp70 levels and acetylation, as well as its binding to CXCR4, which also resulted in increased extra-cellular, cell surface co-localization of CXCR4 and hsp70. This was markedly inhibited by siRNA-mediated knockdown of hsp70 in HL-60 cells. While exposure of cultured and primary AML cells to CXCL12 markedly increased cytosolic levels of p-AKT and p-ERK1/2, co-treatment with LBH589 markedly attenuated the phosphorylation of AKT and ERK1/2 induced by CXCL12, resulting in apoptosis of up to 50% of cultured and primary AML cells. Treatment with AMD3100 (10 uM for 24 hours) alone decreased levels of CXCR4, p-AKT and p-ERK1/2, without significantly increasing apoptosis of AML cells. Notably, co-treatment with LBH589 and AMD3100 caused greater depletion of CXCR4, p-AKT and p-ERK1/2 levels, and exerted synergistic apoptotic effects against AML cells with combination indices of < 1.0 utilizing isobologram and median effect analyses. Co-treatment with LBH589 and FC131 (10 nM for 24 hours), which is a more potent CXCR4 antagonist than AMD3100, also induced synergistic apoptosis of cultured and primary AML cells. Taken together, these findings provide direct evidence that CXCR4 is a novel target depleted by LBH589 in AML cells. Furthermore, our in vitro findings highlight the novel combination of LBH589 and a CXCR4 antagonist, AMD3100 or FC131, exerts a synergistic effect on acute leukemia cells. These findings strongly support the in vivo testing of this synergistic combination in the therapy of human acute leukemias that express CXCR4.