PS1174 MTORC2 ACTIVATION AND AKT NUCLEAR LOCALIZATION UNDER HYPOXIA COULD SUSTAIN THE LEUKEMIA CANCER STEM CELLS
Authors: Panuzzo C. , pironi L. , pergolizzi B. , petiti J. , rosso V. , Ali M.S. , calabrese C. , signorino E. , cartellà A. , bracco E. , cilloni D. 
Published: June 2019

Background:

Since more than a decade tyrosine kinase inhibitors (TKIs), selectively targeting BCR‐ABL, revolutionized the treatment of Chronic Myeloid Leukemia (CML). Nevertheless, relapse can occur because either the immature myeloid progenitor or the leukemic stem cell responsible from the disease are insensitive to TKI. mTor pathway is directly activated by BCR‐ABL and is inhibition synergize with imatinib to induce apoptosis and overcome TKI mutation resistance. There are two distinct multi‐subunit complexes with non‐overlapping functions: mTORC1 and mTORC2. Both complexes act coupling extracellular cues with intracellular pathways controlling a variety of cellular functions.

Aims:

The identification of new targets “stemness specific” to inhibit will offer the opportunity to attach cancer stem cells, that remain largely resistant to the majority of current anticancer therapeutics, with potential impact on oncology field. Conscious of the difficulties in handling CML HSC due to the paucity of material we have explored the role of the mTORC2‐Akt signalling in Ph+ cell line under hypoxic condition because low oxygen concentration represents a major hallmark of the bone marrow stem cell niche.

Methods:

Hypoxia exposure was performed by incubation of K562 cell line in 5% CO2, 1% Oxygen, 94% N2 condition for different time point (20 and 40 hours). Cell viability and proliferation rate was investigate by MTT assay and Tripan blue count. Total cell lyses, nuclear and cytoplasm fractions were used to carry out western blot to analyse mTORC1 and mTORC2 pathway and to investigate cellular delocalization of TOR component in hypoxia condition. Immunofluorescence were performed on K562 slices. RNA were extract to analyse genes known to be involved in hypoxia pathway. Knock down Rictor K562 were used to investigate the TORC2 relevance under hypoxic condition.

Results:

We found that in low oxygen condition all the mTORC1 substrates are strongly inactivate while mTORC2 substrates shown high phosphorylation and consequently activation. The interesting data is the Akt status which present a unique 473 phsphorylation, not accompanied by T308. By analyzing the canonical Akt substrate as Foxo3a we observed a pattern that confirm the Akt status: Foxo3a in not phosphorylated confirming the need of and Akt full active form to be phosphorilated. The Foxo3a status under hypoxia enforce our “stem like” model, since is well known its relevance in stem cell fate. The quantitative mRNA analysis showed a significant increase in SOX genes (SOX2 and SOX17) which would appear to be negatively regulated by mTORC1, and directly responsible of ”stemness“ phenotype. By investigating protein compartmentalization we observed that Rictor, mTor and the best mTORC2 substrate AktSer473, rilocalize inside nucleous during hypoxia. Finally the inactivation of Rictor by sort hairpin (sh) drastically reduced the AktSer473 status and induce a more “proliferative and metabolic” cells pattern.

Summary/Conclusion:

In conclusion, the present study shown that mTORC2 plays a major dominant role, when compared to mTORC1, under the environmental conditions that mimic the stem cell hypoxic niche stress. Indeed, we noticed 1) a sharp differential phosphorylation status of the mTORC1 vs mTORC2 substrates under hypoxic when compared to the normoxic conditions; 2) these data have been validated by silencing Rictor, and thus specifically and selectively inactivating the mTORC2; 3) hypoxia triggers a sub‐cellular re‐localization of the mTORC2 components within the nuclear compartment, strenghthen a new TORC‐AKT‐stemness axis.