Abstract C71: Overcoming mechanism(s) of resistance to active-site TOR inhibitors in leukemia and lymphoma.

Abstract

Abstract Introduction: The PI3K/AKT/TOR signaling network has been the focus of intense research in the cancer field as its activity is commonly elevated across cancers including leukemia and lymphoma. As an integral effector of the network, the target of rapamycin (TOR) functions as the catalytic subunit of two distinct protein complexes, TORC1 and TORC2. Both complexes promote survival of hematological malignancies through protein translation (TORC1) and AKT activation (TORC2). Unlike the classical allosteric TOR inhibitors (e.g. rapamycin), a new class of active-site TOR inhibitors (asTORi) more completely inhibits TOR enzymatic function in both TORC −1 and −2 complexes. TORC2 inhibition correlates with accumulation of FOXO transcription factors in the nucleus. Our lab showed that asTORi kill mouse leukemia and inhibit proliferation of human leukemia cells in vitro and in vivo. Here we investigated mechanisms of resistance to asTORi-mediated cell death in a human leukemia as well as a panel of human lymphoma cell lines. We show that asTORi induce nuclear localization of FOXO and transcription of cytostatic (i.e. p27) but not pro-apoptotic (ex. TRAIL) FOXO target genes. Combining asTORi with pharmacologically achievable concentrations of the histone deacetylase inhibitor (HDACi) upregulates transcription of TRAIL and dramatically enhances death of human leukemia cells in vitro. Similarly, asTORi potentiate a small molecule antagonist of the anti-apoptotic Bcl-2 family proteins whose expression is commonly elevated in lymphoma. Material and Methods: Cell Lines and reagents: The mouse p190 and human SUP-B15 cell lines are Ph+ B precursor acute lymphoblastic leukemia cells. The human lymphoma cell lines OCI-LY1, SUDHL-4, SUDHL-6 and RCK8 are germinal-center diffuse large-B-cell non-Hodgkins lymphoma (DLBCL). The HDACi (Vorinostat) and BCL-2 antagonist (ABT-263) were purchased from Merck & Co and Selleck. The active-site TOR inhibitors were generously provided by Intellikine Inc. Cell cycle and death analysis: Flow cytometry was used in conjunction with 7-amino actinomycin D or annexin V staining along with forward scatter profile to assay cell viability. Flow cytometry and propidium iodide staining was used to measure DNA content for cell cycle analysis. qPCR: RNA was isolated using trizol method and analyzed by real time PCR. Subcellular localization: SUP-B15 and OCI-LY1 cell lines were fractionated into cytosolic and nuclear fraction then probed with anti-FOXO1 antibody. Anti -actin and -histone antibodies were used to confirm the purity of the fractions. Conclusion:The human leukemia cell line SUP-B15 and DLBCL cell lines survive inhibition of TOR by asTORi.asTORi promotes nuclear localization of FOXO and transcription of the cytostatic effector and FOXO target gene, p27.Combining HDACi with asTORi promotes transcription of TRAIL, a pro-apoptotic FOXO target gene, and markedly enhances apoptosis in SUP-B15 cells.asTORi potentiates cytotoxic effects of the BCL-2 inhibitor in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C71.