Dasatinib Synergizes With ATRA To Trigger Granulocytic Differentiation In ATRA Resistant Acute Promyelocytic Leukemia Cell Lines

All-trans retinoic acid(ATRA)resistance has been a critical problem in acute promyelocytic leukemia(APL)relapsed patients.In this study,dasatinib was shown to synergize with ATRA to trigger terminal granulocytic differentiation in ATRA resistant APL cell lines NB4-R1 and NB4-R2.The combined treatment activated RAF-1,MEK and ERK as well as enhanced ATRA-promoted up-regulation of the protein level of PU.1,C/EBP? and C/EBP?.U0126(MEK specific inhibitor)and sorafenib tosylate(RAF-1 specific inhibitor)suppressed the combined treatment-induced differentiation,ERK phosphorylation and the up-regulation of C/EBPs and PU.1.Sorafenib tosylate also attenuated the MEK activity.However,the combined treatment did not enhance the activity of Ras,the classical upstream molecule of RAF-1.Ras inhibitor salirasib neither blocked MEK activation nor inhibited differentiation.Therefore,the combined treatment induced differentiation was via Ras independent RAF-1/MEK/ERK mediated the modulation of C/EBPs and PU.1.Due to the negative role of Src family kinase(SFK)in myeloid differentiation and the inhibitory effect of dasanitib on SFK,we further investigated the relationship between the inhibition of SFK and the activation of RAF-1 to find out the upstream regulator of RAF-1.Three hours earlier than RAF-1 activation,dasatinib suppressed the activity of Lyn,the predominant activated SFK in NB4-R1 and NB4-R2 cells,accompanied by the dephosphorylation of RAF-1 inhibitory site S259.Furthermore,another SFK inhibitor,PP2 did suppress Lyn activity and mimicked the effect of dasatinib on ATRA-induced differentiation as well as decreased phosphorylation of RAF-1 at S259.Thus,it was suggested that Lyn inhibition might activate RAF-1 by the dephosphorylation of RAF at S259 and lead to differentiation.In conclusion,the combination of dasatinib and ATRA could release RAF-1 inhibition by suppressing Lyn,activate RAF-1/MEK/ERK,enhance the protein level of C/EBPs and PU.1 and lead to differentiation.