Lyn Kinase Is Constitutively Activated, Controls the mTOR/p70S6K/4E-BP1 Pathway and Regulates Cell Proliferation in Acute Myeloid Leukemia.

We have recently demonstrated that the mTOR/4E-BP1/p70S6K pathway is constitutively activated in AML cells but not in their normal counterpart. Rapamycin inhibits AML cell proliferation in 60% of cases and induces transient blood and marrow blast clearance in refractory AML patients (

PP2 (Src Familly Kinases, SFK inhibitor), herbimycin A (pan TK) but not SU6656 (pp60 c-src), AG1296 (c-Kit, PDGFR, FLT3) and AG490 (JAK2) inhibited the phosphorylation of p70S6K and 4E-BP1 in KG1 cell line. PP2 consistently inhibited the phosphorylation of mTOR targets in 30 fresh AML samples. Using immunostaining with anti-phosphotyrosine antibody, we detected a high level of tyrosine phosphorylations in all AML samples (in contrast to normal CD34⁺cells) and consistently found a signal at 55–60 kD which may correspond to SFK. Moreover, PP2 dramatically reduced the global level of tyrosine phosphorylations suggesting that SFK play a role on TKs activation cascade and could act upstream mTOR.

Lyn, Hck and Fgr were expressed in 100%,78% and 67% of cases while Lck and Fyn were weakly or not expressed in AML cells. All samples showed phosphorylation on Y416 residue demonstrating the constitutive activation of SFK. We also used flow cytometry to detect SFK phosphorylation in the more immature compartment of the leukemic clone. In 4/5 cases, the CD34⁺CD38⁻CD123⁺ cell sub population displayed a high level of phosphorylation on Y416 that was inhibited by PP2. In KG1, PP2 inhibited cell growth in a time and dose dependent manner. This effect was mainly due to a cell cycle arrest in G1 and to a lesser extent, apoptosis induction. In clonogenic assays, PP2 inhibited the capacity of fresh AML cells to generate AML-CFU and induced apoptosis in short term culture (n=6).

Because the two Lyn kinase isoforms were expressed in all cases and corresponded to the two bands detected by p-SrcY416 antibody, we thought to determine if this this kinase was the major component of SFK in AML. Lyn was highly phosphorylated on Y416 after immunoprecipitation. Specific down regulation by si-RNA to Lyn induced a significant decrease of p70S6K and 4E-BP1 phosphorylation and inhibited cell proliferation in KG1 and U937. Using confocal microscopy, Lyn was mainly localized all over the plasma membrane and colocalized with anti-p-SrcY416 whereas its distribution in normal CD34⁺ cells was restricted to several distinct points across the membrane.

These data demonstrate that SFK and particularly Lyn kinase, are aberrantly deregulated in AML cells, control cell proliferation at least in part through mTOR pathway and represent a new target for the treatment of this disease.