Targeting of Glutamine Pathways Restores Sensitivity in Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

Introduction:

Venetoclax is a BH3-mimetic shown to specifically inhibit BCL-2 and has activity in hematologic malignancies addicted to BCL-2, including Ph+ ALL. However, when used as a single agent resistance may rapidly emerge. We previously found that venetoclax resistant Ph+ ALL cell lines increase expression of the anti-apoptotic proteins MCL-1 and BCL-xL yet have enhanced sensitivity to mTOR inhibitors, by an unknown mechanism. In these experiments we sought to define the specific pathways that contribute to enhanced sensitivity to mTOR inhibitors, as well as to identify targets to prevent the emergence of drug resistance.

Methods:

A venetoclax resistant cell line (SUPVR2) was generated as previously described using the parent Ph+ALL cell line SUPB15. Briefly, SUPVR2 have an IC50 to venetoclax of >1000 nM as compared to <10 nM in SUPB15. Colorimetric MTS assay was used as a marker of cell viability to determine the sensitivity of SUPB15 vs the SUPVR2 towards a panel of small molecule inhibitors affecting various targets of the PI3K / AKT / mTOR pathway. Protein expression for mTOR pathway members including phosphor mTOR, total mTOR, pAKT, pPI3K, RAG, LAMTOR, as well as downstream targets of the mTORC1 pathway s6kinase, 4EBP1 and HIF1a were assessed via immunoblot. Quantification of LAMTOR expression was also assessed via qPCR. Glucose and amino acid deprivation studies were performed by culturing cells in nutrient free media for 48 hours, and then assessing for protein expression of RAG, LAMTOR, BCL-xL and mTOR. To assess the effects of glutamine deprivation on venetoclax sensitivity, cells were cultured in nutrient free media for 72 hours and then assessed for viability via MTS assay. Cells were additionally cultured with 100nM venetoclax in nutrient free media and then assessed for apoptosis via annexin V staining at 24, 48 and 72 hours. Expression of both pro and anti-apoptotic members of the BCL-2 family of proteins were assessed with immunoblots.

Results:

SUPVR2 cells demonstrated enhanced sensitivity to the mTOR inhibitors rapamycin, INK-128, PP-242 and BEZ-235, however had no sensitivity to selective inhibitors of the PI3K pathway or AKT pathway. Co-treatment of SUPVR2 with mTOR inhibitors or HIF1a inhibitors restored sensitivity to venetoclax. Immunoblot analysis showed no difference in the levels of p-AKT or p-PI3K between SUPB15 cells and SUPVR2 cells, however SUPVR2 cells had elevated levels of total mTOR as well as p-mTOR (s2448), and increased expression of the downstream targets of the mTORC1 pathway S6 kinase, 4EBP1 and HIF1a. Given the lack of change in the phosphorylation of PI3K and AKT as well as insensitivity to their respective inhibitors, we suspected mTORC1 activation via a glutamine-addiction pathway circumventing PI3K/AKT. We found increased expression of the RAG and LAMTOR family proteins in SUPVR2 cells as compared to SUPB15, at both the protein and mRNA level. Glutamine deprivation decreased expression of both RAG and LAMTOR, and partially restored sensitivity to venetoclax, decreasing the IC50 from >1000nM in resistant cells to <50nM. Venetoclax treatment of SUPVR2 cells in glutamine deficient media also enhanced apoptosis to near the degree of the parental cell line by 24 hours, while levels of BCL-xL but not MCL-1 were reduced after glutamine deprivation.

Conclusions:

The venetoclax resistant SUPVR2 cells have enhanced sensitivity to inhibitors of the mTOR pathway, due to specific upregulation of the mTORC1 pathway circumventing the PI3K/AKT pathway through enhanced glutamine addiction. Glutamine deprivation thereby reduced upregulation of the mTORC1 pathway proteins, and restored sensitivity to venetoclax in part by reducing levels of BCL-xL. Treatment of venetoclax resistant cells with either inhibitors of the mTORC1 pathway, HIF1a, or glutamine deprivation restored sensitivity, and may prevent the emergence of venetoclax resistance in Ph+ ALL.