Alterations in the Cell Cycle of Acute Lymphoblastic Leukemia Cells Resistant to Everolimus

Disease relapse following treatment still occurs in a significant minority of children and the majority of adult patients with acute lymphoblastic leukemia. The inability to further intensify current treatments due to dose limiting toxicities of chemotherapeutic agents demands the development of new agents. Preclinical studies using the mTOR inhibitor everolimus, while promising, revealed that resistance can emerge following prolonged treatment in vivo. Our study aims to identify the mechanisms of resistance and explore potential avenues to overcome it.

Mice bearing xenografts sensitive or resistant to everolimus were treated with everolimus or placebo 24hrs prior to cull. Proteins were isolated from recovered spleen cells, fluorescently labelled and separated using 2D-DIGE. Protein gels were scanned using a Typhoon scanner and analysed using DeCyder software. Differences in protein regulation were considered significant if the relative fluorescence was altered 1.5 fold with a p-value of <0.05 across 3 biological replicates. Significantly regulated proteins were excised for identification by MALDI/TOF-TOF mass spectrometry. To assess cell cycle mice treated as above were administered BrdU by intra-peritoneal injection 1 hour prior to cull.Protein expression and cell cycle was analysed by flow cytometry using a BD LSRFortessa.

Fourteen proteins were differentially regulated in the resistant as compared to the sensitive xenograft, with 5 being down and 9 up regulated. Proteomic analysis revealed increased expression of stathmin-1 in resistant cells (p=0.002). Increased expression of stathmin-1 has been associated with increased proliferation and poor prognosis in a number of malignancies. Stathmin-1 destabilizes microtubules and is inactivated by phosphorylation mediated by CDK1 and 2. eEF2, a downstream target of mTOR through the S6 kinase which drives protein production by promoting elongation of peptides, was also increased in the resistant cells (p=0.043).

Cell cycle analysis demonstrated that resistant xenograft cells were more proliferative in vivo, consistent with increased stathmin-1 expression, in all tissues but most notably in the spleen. Furthermore, analysis of ALL cells recovered from spleens, showed that resistant xenograft cells had decreased phosphorylation of stathmin-1 at serine 38 and 63 indicating increased activity. Although everolimus inhibited proliferation in both sensitive and resistant cells, the resistant cells remained more proliferative reflecting the reduced survival of these mice. Expression and the kinase activity, as indicted by the activating phosphorylation, of CDK1 and CDK2 were decreased (p<0.001 and p<0.02 respectively) in resistant cells treated with everolimus.

Everolimus resistant ALL cells exhibited increased proliferation with elevated levels of the active form of stathmin-1. The reduction in active CDK1 and CDK2 in mTOR inhibitor resistant cells despite increased proliferation is paradoxical and additional work is required to determine how this contributes to drug resistance. Prospective targets to overcome the resistance in ALL cells may be translatable to other diseases where mTOR inhibition is currently being used in order to further intensify their effectiveness in treating the disease in question.

Figure 1.

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Loss of CDK1 in ALL xenograft cells resistant to everolimus following treatment. Western blotting of protein lysates for total CDK1 and phospho-CDK1 (Thr161). Blots run with 3 biological replicates and p-values calculated by students t-test. p<0.05.

Figure 1.

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Loss of CDK1 in ALL xenograft cells resistant to everolimus following treatment. Western blotting of protein lysates for total CDK1 and phospho-CDK1 (Thr161). Blots run with 3 biological replicates and p-values calculated by students t-test. p<0.05.

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