In search for novel treatment strategies, we investigated AMPK signaling as potential target for ALL therapy. Our results, together with our previously published report [3
] reveal that activation of AMPK by AICAR induces a compensatory survival response through activation of Akt at both of its functional residues Ser473 and Thr308. Although phosphorylation of Akt at both residues is critical for maximum catalytic activity [9
], it has been established that phosphorylation of Thr308 is sufficient to activate its kinase activity and support cell survival [34
]. We show that the mechanism of Akt activation in ALL cells is mediated in part by AMPK-induced phosphorylation of IRS-1 at Ser794, the immediate downstream effectors of the IGF-1R signaling cascade, and also in part by AMPK-induced inhibition of mTOR and its downstream feedback loop inhibition of IRS-1 (Ser312). Direct interaction between P-AMPK (Thr172) and phosphorylation of IRS-1 at Ser794 has been shown to occur in several systems such as cell lines [31
], and insulin-resistant animal models [46
], but the biological relevance of this phosphorylation event is still not clear. Different functions have been reported for AMPK-induced IGF-1R phosphorylation with some reporting a positive effect on PI3K/Akt signaling [31
] whereas others reported a negative effect [45
]. Additive activation of AMPK and Akt has been shown to regulate important biological functions such as angiogenesis and glucose metabolism [48
], suggesting that positive interactions exist between AMPK and Akt as we report here. Other reports demonstrated that Akt could negatively regulate AMPK activity by direct binding and phosphorylation of AMPK at Ser485 [50
]. These opposite effects reflect the complexity of the signaling cross-talk that exists between AMPK, IRS-1, and downstream activation of Akt.
It is clear from our studies that phosphorylation of Akt at Thr308 in AICAR-treated ALL cells occurs via direct AMPK down-regulation of mTOR and activation of the IGF-1R/IRS-1 signaling cascade. This compensatory mechanism promotes cell survival because inhibition of IGF-1R activity in either presence or absence of AICAR decreases P-IRS-1 (Ser794) and P-Akt (Thr308) levels and significantly increases apoptotic cell death. The signaling cascade triggered by activation of tyrosine kinase receptor leading to phosphorylation of IRS-1 and subsequent activation Akt at Thr308 have been extensively studied and is mediated by the downstream PI3K and PDK1 kinases [16
]. Additionally, we demonstrate that phosphorylation of Akt is also dependent on AMPK since inhibition of AMPK activity with compound-C clearly decreased P-Akt at both residues. AMPK has been shown to inhibit mTORC1 activity by two different mechanisms: one through activation of the TSC2, which promotes downstream inhibition of the mTOR activator Rheb [9
], and the other through direct phosphorylation of Raptor at Ser792 blocking mTORC1 activation [55
]. Additional studies demonstrated that phosphorylation of Akt at Ser473 was mediated by mTORC2, a complex formed by the association of rictor, mSin1, mLST8, with mTOR [7
]. Among mTORC1 and mTORC2, mTOR is the only critical factor that is shared by both complexes [9
]. Thus, it is tempting to speculate that by down-regulating mTORC1, AMPK could increase the availability of mTOR and favor the formation of mTORC2, which would promote phosphorylation of Akt at Ser473. A similar mechanism was proposed for activation of Akt by AMPK in macrophages expressing a constitutively active form of AMPK [58
]. Nevertheless, we can not rule out the possibility that a distinct mechanism independent of mTORC2 might be involved in this process.
The data presented herein shows that activity of IGF-1R/IRS-1 was higher in NALM6 vs
. CCRF-CEM cells, and that their expression also differs within Bp-ALL REH and SupB15 subtypes characterized by the non-random translocations t[12;21], and t[9;22]. More important, these differences correlated with reduction in P-IRS-1 (Ser794) and P-Akt (Ser473 and Thr308), and degree of induction of apoptotic death resulting from the pharmacological inhibition of IGF-1R. Our results raise the intriguing possibility that cell lineage of origin and/or presence of selected non-random translocations may influence response to therapy in ALL cells treated with inhibitors of IGF-1R. This possibility needs to be investigated using primary samples from patients with ALL. It is also possible that the level of Akt activation in these cells may also dictate their degree of sensitivity to IGF-1R inhibition. For instance, it is well known that the CCRF-CEM cell line carries a mutation inactivating PTEN [35
] and that REH cells born a PTEN deletion [36
], both leading to increased reliance on Akt signaling for cell survival. In addition, SupB15 cells express high levels of P-Akt because the expression of the BCR-ABL gene fusion inhibits PP1α, a serine phosphatase that negatively regulates the PI3K/Akt pathway [37
]. Interestingly, the expression level of P-Akt was the lowest in NALM6 cells which was also the most sensitive to the IGF-1R inhibitor HNMPA(AM)3
as compared to all of the other cell lines examined, therefore suggesting that Akt provides a mechanism to escape cell death following IGF-1R inhibition.
To further assess whether IGF-1R signaling may be influenced by biological pathways closely linked to cell lineage and non-random chromosomal translocations, we have mined existing gene expression databases from childhood ALL patients http://www.stjuderesearch.org/data/ALL1
, and found that the expression of relevant IGF-1 regulatory carriers such as IGFBP2 and IGFBP4 appear to be significantly differentially expressed in ALL in a phenotype specific manner. The known correlation between these carriers and IGF-1 suggested to us that differences in IGF-1 signaling may exist in ALL, and impact critical oncogenic and survival signaling pathways. Interestingly, IGF-1R signaling has been linked to cell lineage of origin in ALL. For instance, significant differences in the expression of the IGF-1 system components IGF-II, IGFBP-2, IGFBP-4 and IGFBP-5 have been described between B-lineage and T-lineage ALL [28
]. IGFBP-2 was identified as the major regulatory carrier in childhood leukemia and exhibited an inverse correlation with IGF-1 levels [59
], suggesting that activation of IGF-1R signaling may confer ALL cells a survival advantage and influence induction of apoptosis. Emerging literature suggests that IGF-1R signaling may also be influenced by non-random translocations in ALL [60
]. For instance, leukemia cells expressing the translocation t(9;22) encoding for the BCR-ABL fusion not only exhibit a higher degree of resistance to chemotherapeutic drugs but also were shown to induce autocrine IGF-1 signaling. Thus, it is clear that IGF-1R pathway may provide ALL cells a survival advantage through its crosstalk with other critical metabolic networks.
The identification of potential cross-talk within compensatory survival pathways in ALL cells prompted us to developed simultaneous co-targeting strategies to induce cell death in ALL cells. We demonstrated that co-targeting IGF-1R and downstream pathways (AMPK & IGF-1R, mTOR & IGF-1R, and AMPK & Akt) led to synergistic growth inhibition in ALL cell models. This is consistent with the study of Bertrand et al
] that demonstrated that blocking IGF-1R activity using an antibody synergized with inhibitors of PI3K/Akt/mTOR pathway by suppressing the IGF-1R-induced Akt phosphorylation, and consequently promoted apoptosis in hematopoietic cells. Among the three drug combinations tested, only the one co-targeting AMPK and Akt resulted in synergistic induction of cell death. This can be explained in part by differences in the mechanism of action between AIX vs
, with AIX being more effective in inactivating Akt. Taken together, rationally designed simultaneous targeting of key factors within the AMPK, IGF-1R, and mTOR pathways leads to synergistic induction of cell growth inhibition by blocking compensatory survival responses triggered by treatment with single agents. Nevertheless, of the combinations strategies tested only co-targeting AMPK plus Akt lead to synergistic induction of apoptosis.