UCSF

Ras pathway mutations are prevalent in T lineage leukemias and the Ras/PI3 kinase/PTEN/AKT pathway plays an important role in the maintenance and evolution of this disease. Second-site mutants of Ras are a unique biological system that allows us to study the effects of distinct downstream Ras effectors for cancer initiation and maintenance. By adding chemical inhibitors to this genetically modified system, further information regarding the cells requirements for pathway activation can be assessed.

To accomplish this goal, second site cell lines were exposed to the MEK inhibitor PD0325901, PI3 kinase (PI3K) inhibitor GDC-0941, and the AKT inhibitor MK2206 and proliferation assays were performed. No significant differences were seen between mutational groups when lines were exposed to inhibitors of PI3K or AKT. However, exposing T-ALL cells to PD0325901 revealed significant differences between lines with PTEN expression and those lacking PTEN, despite equal inhibition of the RAF/MEK/ERK pathway. Cells expressing PTEN are uniquely sensitive to MEK inhibition and the cells respond to PD0325901 by undergoing apoptosis.

Dual inhibition of AKT and MEK causes cell lines that are resistant to MEK inhibition to become sensitive, despite significantly higher basal pathway activation. These data implicate AKT as an important mediator of resistance to PD0325901.

Pro-survival proteins BCL-xl and MCL-1 are expressed at low levels in cell lines with PTEN expression, and in the presence of PD0325901, the levels decrease. However, in PTEN negative lines that all show resistance to PD0325901, expression of these proteins is high and remains high or increases in the presence of drug. Our results argue that AKT is activated by loss of PTEN expression, and that AKT mediates drug sensitivity through BCL-xl and MCL-1.

We implicate loss of PTEN expression as integral to PD0325901 resistance in T-ALL cells. DNA sequencing of most lines lacking PTEN expression did not uncover mutations, so we sought to determine whether methylation and epigenetic silencing were the mechanism of Pten silencing. Bisulfite sequencing, 5 azacytidine treatment, Trichostatin A treatment, and EpiQ analysis all came back negative, leading us to believe that methylation is not the cause of PTEN silencing in our cells.