Summary We show that imatinib nilotinib and dasatinib possess weak off-target

Summary We show that imatinib nilotinib and dasatinib possess weak off-target activity against RAF and therefore drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. tyrosine kinase. The normal function(s) of BCR are unclear but ABL is a cytosolic/nuclear tyrosine kinase that regulates stress responses cell growth and differentiation. Critically fusion of ABL to BCR generates a constitutively active kinase that drives transformation and leukemogenesis by phosphorylating substrates such as CRKL and STAT5 and activating pathways such as NFkB and RAS/RAF/MEK/ERK (Deininger et al. 2000 The clinical management of CML was revolutionized by imatinib a small molecule ABL inhibitor (Druker et al. 2001 Imatinib mediates remission in the majority of CML patients but patients can develop resistance through acquired point mutations that block imatinib binding to BCR-ABL. Fortunately most imatinib-resistant BCR-ABL mutants are sensitive to nilotinib and dasatinib next-generation drugs that provide vital second-line treatments (Kantarjian et al. 2010 However substitution of threonine 315 in ABL for isoleucine (BCR-ABLT315I) generates a protein that is resistant to all three drugs and this mutant remains a persistent clinical problem for the long-term CML management. Pan-ABL inhibitors effective against BCR-ABLT315I are undergoing clinical trials (reviewed in O’Hare et al. 2011 but compound mutants (two or more mutations in the same protein) are resistant to all current ABL inhibitors and may represent a future obstacle for CML management (O’Hare et al. 2009 Eide et al. 2011 Furthermore patients can develop resistance that is mediated by BCR-ABL-independent mechanisms and for these patients treatment options are limited (reviewed in Bixby and Talpaz 2011 The RAS/RAF/MEK/ERK pathway promotes CML cell survival (Goga et al. 1995 RAS is a small membrane bound G-protein and RAF MEK and ERK are sequentially activated protein kinases. There are three genes (and genes (and is mutated in about half of melanomas and at a lower frequency in several other cancers (Wellbrock et al. 2004 BRAF Elesclomol inhibitors such as vemurafenib (PLX4032 RG7204) mediate dramatic responses in BRAF mutant melanoma patients but not in BRAF wild-type patients (Flaherty et al. 2010 validating mutant BRAF as a therapeutic target in melanoma. However these drugs also reveal an unexpected paradox because while they inhibit MEK and ERK in cells expressing oncogenic BRAF they activate MEK and ERK in cells expressing oncogenic RAS (Halaban et al. 2010 Hatzivassiliou et al. 2010 Heidorn et al. 2010 Poulikakos et al. 2010 This is Elesclomol because in the presence of oncogenic RAS BRAF inhibition drives BRAF binding to CRAF resulting in Elesclomol BRAF acting as a scaffold to facilitate CRAF Rabbit polyclonal to PITPNM2. hyper-activation by stimulating critical events such as serine 338 (S338) phosphorylation (Hatzivassiliou et al. 2010 Heidorn et al. 2010 Paradoxical activation of the pathway can also be achieved by CRAF inhibition which drives CRAF homodimerization consisting of drug-bound monomers that facilitate the activation of drug-free monomer through scaffold functions or conformational changes (Poulikakos et al. 2010 Thus under some circumstances RAF inhibitors drive paradoxical activation of BRAF and CRAF to accelerate tumorigenesis by hyper-activating MEK and ERK (Hatzivassiliou Elesclomol et al. 2010 Heidorn et al. 2010 Here we investigated if other kinase inhibitors can also drive paradoxical activation of RAF MEK and ERK. Surprisingly we found that imatinib nilotinib and dasatinib hyper-activated BRAF CRAF MEK and ERK in cells expressing oncogenic RAS or BCR-ABLT315I. We therefore investigated the underlying mechanisms and examined how this affected the growth of leukemia cells. Results Imatinib nilotinib and dasatinib activate RAF MEK and ERK in RAS mutant cells To initiate our study we treated D04 cells a melanoma line that expresses NRASQ61L with a variety of protein kinase inhibitors and investigated their effects on the MEK/ERK pathway by measuring MEK and ERK phosphorylation by western blot. The majority of compounds tested did not affect MEK or ERK phosphorylation (Fig S1A) but surprisingly imatinib nilotinib and dasatinib stimulated robust MEK and ERK phosphorylation at concentrations as low as 100nM (Fig 1A). Since the peak plasma/serum concentrations of imatinib nilotinib and dasatinib are ～5μM 4 and 90nM respectively.