Increased Proliferation Induced by Constitutive Activation of the Src-Kinase Lyn and Aberrant mTOR Signaling in AML Is Abrogated by the EGFR-Inhibitor Erlotinib.
Authors: Boehrer Simone , Galluzzi Lorenzo , Ades Lionel , Fouassier Celine , Gardin Claude , Fenaux Pierre , Kroemer Guido 
Published: October 2019

Abstract 3813

Poster Board III-749

Background

Aberrant activation of intracellular tyrosine kinases (such as the Src family kinases, SFK) as well as constitutive activation of mTOR signaling contribute to leukemogenesis by inducing pro-proliferative signals in the majority of AML cases. Both pathways are considered – due to their restricted deregulation in neoplastic cells – attractive therapeutic targets. We and others provided evidence that the epidermal-growth-factor-receptor (EGFR)-inhibitor erlotinib has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008), but its mode of action in these EGFR-negative blasts remained elusive. We thus tested the hypothesis that erlotinib exhibits anti-leukemic activity by targeting SFK.

Methods

In erlotinib-sensitive KG-1 cells and ex vivo patient AML blast cells, expression and phosphorylation status of individual SFK (Lyn, Syk) was tested under incubation with erlotinib by immunobloting and FACS-analysis, while concomitantly assessing proliferation/cell cycle distribution and apoptosis (FACS-analysis). In addition, experiments were recapitulated in the presence of SRC-family inhibitors (PP2, Piceatannol, BAY 61-3606) and the mTOR-inhibitor rapamycine. Induction of autophagy was determined by immunoblot analysis and by ultastructural assessment using electron microscopy (EM).

Results

erlotinib abrogated the constitutive phosphorylation of the SFK Lyn (immunoblot, FACS) in KG-1 and in patient-derived AML cells, while having no impact on p-Syk (reported to be decreased by its “sister” compound gefitinib) or its downstream target p-Cbl. Moreover, inhibition of p-Lyn by erlotinib was accompanied by a cell cycle arrest in G1, an effect recapitulated to the same extent by the SFK inhibitor PP2 (10mM), but not by the Syk-inhibitors Piceatannol (10mM) or BAY 61-3606 (50nM). Co-incubation of KG-1 cells with PP2 and erlotinib had no additive/synergistic effects on the extent of G1 arrest, in accordance with the notion, that both agents function by inhibiting the same target/pathway. Since there is evidence that aberrant Lyn activation in AML confers its pro-proliferative effects via mTOR signaling, we next assessed the phosphorylation status of the mTOR targets p70S6 kinase (Thr389) and 4E-BP1 (Ser65). As demonstrated after 4h of incubation by immunoblot analysis, 10mM erlotinib significantly diminished phosphorylation of the G1-regulating kinase p70S6 and of the translation repressor 4E-PB1. Noteworthy, whereas 10nM of the mTOR inhibitor rapamycin induced the same extent of G1-arrest as 10mM erlotinib (ie an increase of about 10% compared to controls), co-incubation of KG-1 cells with both rapamycin and erlotinib further increased G1-arrest (by 15-20% compared to controls). Since inhibition of aberrant mTOR activation can lead to autophagy (“self-eating”), we next assessed parameters of autophagy under erlotinib. We showed that in KG-1 cells, erlotinib up-regulates expression of LCII (immunoblot) and induces the appearance of autophagic vacuolation (EM), both correlates of autophagy.

Conclusions

These results delineate the pathway by which the EGFR-inhibitor erlotinib exhibits antileukemic activity in EGFR-negative AML, and demonstrate erlotinib's capacity to antagonize the pro-survival effects of aberrant Lyn- and mTOR-mediated signaling. We therefore provide a mechanistic explanation to the therapeutic benefit of erlotinib in AML/MDS patients, and also give the first evidence of a potential benefit of its combination with inhibitors of mTOR signaling.

Disclosures:

Off Label Use: Erlotinib in not approved in the treatment of AML or MDS. Fenaux:cELGENE: Research Funding; AMGEN: Research Funding.