Cell-cycle–dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling

Abstract

IntroductionThe deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells including myeloid leukemias. 1,2 RAS proto-oncogenes (H-RAS, N-RAS, and K-RAS) encode 21-kd G-proteins that play key roles in signal transduction, proliferation, differentiation, and malignant transformation. 3-5 RAS proteins are produced as cytoplasmatic precursors, which require several posttranslational modifications (eg, prenylation, proteolysis, carboxymethylation, and palmitoylation) for membrane binding and full biologic activity. 3-8 RAS functions as a biologic switch that relays signals from ligand-stimulated tyrosine kinase, cytokine, and heterotrimeric G-protein-coupled receptors to cytoplasmatic mitgen-activated protein kinase (MAPK) cascades. In its activated, GTP-bound state, RAS binds to and activates effector molecules such as Rafs, MEKK, PI-3K, and Ral-GEF. [3][4][5][8][9][10][11][12][13][14] Raf kinases (A-Raf, B-Raf, c-Raf-1) selectively phosphorylate and activate MAPK kinases (MAPKK) MEK-1/2 in the MAPK/ERK pathway. [13][14][15][16][17] MEK-1/2 are dual specificity kinases that activate MAPKs (ERK-1/2). 18,19 The best-characterized ERK substrates are cytoplasmic phospholipase A 2 (cPLA 2 ), ribosomal protein S6 kinases (RSKs), and transcription factors Elk-1 and CREB-1. [18][19][20] The importance of deregulation of ERK signaling in the molecular pathogenesis of myeloid leukemias is underscored by the positioning of several oncogene and tumor suppressor gene products on this pathway. 5,[21][22][23] RAS mutations are frequent genetic aberrations found in 20% to 30% of all human tumors, although the incidences in tumor type vary greatly. 1,2 The most commonly observed RAS mutations arise at sites critical for RAS regulation, namely codons 12, 13, and 61. 1,2,5,9 Additionally, mutations occur at codons 15, 16, 18, and 31. 24,25 These mutations result in abrogation of normal intrinsic or GAP-stimulated GTPase activity of RAS, leading to increased half-lives of mutant RAS-GTP. 5,9,26 Transformation results, at least in part, from deregulated stimulation of mitogenic signal transduction pathways. 1,2,5 The highest incidences of RAS mutations were detected in carcinomas of pancreas (90%), thyroid (50%), colon (50%), and lung (30%). RAS mutations are also frequently observed in myelodysplastic syndromes, acute myeloid leukemias (AML), juvenile myelomonocytic myeloid leukemia (JMML), and multiple myelomas (20%-40%). 1,2,5,[21][22][23] N-RAS is mutated in the majority of cases and presence of the mutation is not associated with any particular FAB type, cytogenetic abnormality, or clinical feature including prognosis. 22 In addition to activation by mutation, RAS is also deregulated in myeloid leukemias by constitutive activation of proto-oncogenes such as receptor or nonreceptor tyrosine kinases (RTKs and NRTKs) or inactivation of tumor suppressor genes. 5,[21][22][23] RTKs are constitutively activated by single point mutations (eg, colonystimulating factor-1 [CSF-1] receptor and c...

Downmodulation of ERK activity inhibits the proliferation and induces the apoptosis of primary acute myelogenous leukemia blasts

“…Our paper, together with those of Baines et al, 37 Morgan et al, 38 and Milella et al, 21 strongly suggests that anti-MEK1 inhibitors are promising agents in the treatment of AML. Our paper has made further comment on some of the unusual aspects of MEK1 inhibition in AML.…”

“…21,22,37,38 Milella et al and Bonati et al have demonstrated that selective downmodulation of MEK1 kinase significantly reduces proliferation and induces apoptosis of leukemic cell lines and primary leukemic blasts.…”

Antitumor activity of sorafenib in FLT3-driven leukemic cells

“…20 On that last point, other studies on pharmacological inhibition of the Ras/Raf/Mek pathway in AML cells in vitro have reported conflicting data, with some studies showing mostly cell cycle arrest without immediate significant induction of apoptosis in treated cells 21,22 whereas other studies have demonstrated induction of apoptosis. 23,24 Sorafenib also has potent activity against kinases reported to potentially be contributing to AML physiopathology such as PDGFR, KIT and VEGFR. [25][26][27] Although the selective antiproliferative and proapoptotic effects of sorafenib in FLT3-dependent cells versus FLT3-independent cells as well as the potent inhibition of Stat5 phosphorylation, a major downstream target of FLT3-ITD, 28 argue for FLT3 as an important driver of the activities reported, it is likely that the inhibition of other kinases by sorafenib may also contribute to the observed antiproliferative effects.…”

Identification of Ki23819, a highly potent inhibitor of kinase activity of mutant FLT3 receptor tyrosine kinase

“…Of note, THP-1 and HL60 cells possess N-RAS mutation, and MV4-11 cells possess K-RAS mutation, while K562 cells express BCR/ABL fusion protein, all of which activate the downstream ERK pathway. 34 These results suggest that Ki23819 specifically suppresses the growth of FLT3-ITD-positive cells among various types of cells with the activated ERK pathway. We next compared the growthinhibitory effect of Ki23819 to that of SU11248, another FLT3 inhibitor.…”