Abstract B62: Multiple genomic aberrations in BCR-ABL inhibitor-resistant cells lead to sensitivity towards MEK inhibition
Authors: Wagle Marie-Claire , Wongchenko Matthew J. , Ramakrishnan Vanitha , Hampton Garret , Yan Yibing 
Published: March 2013

Purpose: Chronic myeloid leukemia (CML) can be effectively treated with BCR-ABL inhibitors such as imatinib, dasatinib, and nilotinib; however, resistance to these inhibitors develops over time causing patients to relapse. Recently, MEK inhibition was shown to synergize with BCR-ABL inhibitors to inhibit resistant CML cells harboring the T315I mutation (1). In this study we characterized mechanisms of resistance to BCR-ABL inhibitor in acquired resistant cells without BCR-ABL mutation by genomic and phosphoprotein profiling. We further evaluated the sensitivity of these resistant clones to MEK inhibitor, GDC0973, PI3K inhibitor, GDC0941, alone or in combination.

Methods: The CML cell K562 was grown in an increasing concentration of imatinib or/and dasatinib to induce acquired resistance. Clones were isolated from the resistant pool and the GI50 of each clone was measured using the Cell-Titer Glo® viability assay. Genomic DNA from selected clones was tested using Oncoscan® (Affymetrix), which surveys whole genome for copy number variation (CNV) and 400 oncogenic mutations. Cell lysates were subjected to reverse phase protein array (RPPA) analysis of phosphorylation levels of ∼100 proteins. Genomic mutation and CNV profiles, as well as protein phosphorylation patterns of the clones were compared to the parental cells to identify potential pathways involved in resistance.

Results: Four imatinib-resistant clones with varying GI50s (4–30 times higher than parental cells) were fully characterized with both Oncoscan and RPPA. None contained the T315I mutation. A genomic mutation scan showed that all the clones had multiple mutations affecting various signaling pathways including TGFβ (Smad-2), Wnt (APC), PI3K (PIK3CA) and EGFR. Likewise, whole genome copy number scan showed that CNV occurred in multiple genes including copy number gains in ADAM29, BMP2, Rspondin4 (Wnt) and PKA in multiple clones, and copy number losses in ADAMTS14, LIM-kinase and FzD9 (Wnt) in multiple clones. The protein phosphorylation patterns also showed multiple changes in the resistant clones. Three of the resistant clones (K14, 15 and 25) had 6-10-fold increased pMEK and pERK, relative to the parental cells. MEk inhibition resulted in a cytostatic response in the parental cells (GI50: 1900 nM), whereas resistant clones 14, 15, and 25 were highly sensitive to GDC0973-mediated apoptosis, with GI50s ranging from 60–75 nM. Clone 6 had lower pMEK and pERK levels than the others, but was still sensitive to GDC0973 (GI50: 600 nM). Phosphorylation of proteins in the PI3K pathway such as pS6, p70S6K and p-mTOR were elevated in all of the clones as compared to the parental cells (7–11 fold); however, none of these clones were sensitive to GDC0941 alone. Our data suggest that either GDC0973 or GDC0973 alone or in combination with GDC0941 could be effective at inducing cell death in resistant clones despite their various genetic backgrounds.

Conclusion: The MEK inhibitor GDC0973 caused robust cellular apoptosis in all of the resistant clones in contrast to the cytostatic effect in parental cells. All of the clones had elevated PI3K signaling as shown by enhanced PI3K substrate phosphorylation. Despite the many genetic aberrations that occurred as cells became resistant; most noticeably changes in Wnt, PKA, ADAMs and BMP2 in all of the clones, resistant cells appear to channel escape/survival signals through the MAPK and/or PI3K pathway rendering them sensitive to GDC0973 alone or in combination with GDC0941.