Abstract A31: Combination therapies guided by evolution of tumor heterogeneity.
Authors: Zhao Boyang , Hemann Michael T. , Lauffenburger Douglas A. 
Published: August 2015

Abstract
Successful treatment of hematologic malignancies is challenged by genetic heterogeneity of tumors and the dynamic evolution of the heterogeneity in response to targeted drugs as well as chemotherapeutics. We are applying an integrated computational/experimental approach to elucidating principles which can guide selection of effective combination therapies by mathematical analysis of how tumors evolve with respect to drug sensitivity and resistance during treatment.
In an initial pharmacological screen of targeted drugs and chemotherapeutics on murine Bcr-Abl; p19Arf-/- ALL cells, we found that crizotinib was particularly effective (IC50 = ~500 nM) with comparable efficacy to that for imatinib (IC50 = ~200 nM). Moreover, in a derivative cell line bearing spontaneous Bcr-AblT315I mutation, crizotinib exhibited efficacy comparable to that of the Bcr-AblWT parental cell line. This same Bcr-AblT315I cell line showed strong resistance to Abl inhibitors imatinib, dasatinib, nilotinib, and bosutinib. Imatinib and crizotinib also exhibited comparable in vivo efficacy, based on overall survival of syngeneic immunocompetent recipient mice transplanted with Bcr-Abl; p19Arf-/- ALL cells. Cell cycle profiles and signaling analyses suggest that crizotinib induces G2/M arrest and subsequently bim-dependent caspase-mediated apoptosis. This phenotype has been subsequently validated in the human K562 Ph+ CML and murine Baf3 Bcr-AblWT and Bcr-AblT315I cell lines.
Resistant populations were also derived through dose escalation treatments with imatinib, dasatinib, nilotinib, foretinib, and crizotinib. Bcr-Abl; p19Arf-/- ALL cells were treated at IC90, and recovered populations were subsequently split into: [a] no-drug media, with sensitivity to different sets of drugs assessed at specific time points post-recovery; and [b] new media with drug concentration at 2x the previous dose (e.g., IC90 2x). Cell populations that were resistant to dasatinib at 1x and 2x IC90 became even more sensitive to crizotinib and foretinib. The drug sensitivity does not appear to be a transient behavior as a result of intracellular rewiring, but rather a change in the tumor heterogeneity via selection of particular subpopulations, as evidenced by sustained sensitivity when these populations were analyzed 24h, 48h, 72h, 5 days, and 9 days post drug selection in no-drug media. The sensitivity was abrogated at IC90 4x and above. Cell populations resistant at these higher doses of dasatinib also became cross-resistant to imatinib and foretinib. This potentially suggests particular stages in the tumor evolution under continued drug selection for which they are extremely sensitive to other pharmacological agents.
These findings suggest a novel combination treatment strategy taking into account the evolutionary trajectories of tumor heterogeneity, with drugs dosed sequentially in a multi-course regimen. The sequential treatment is not to exploit vulnerabilities of intracellular rewiring induced by the first drug, but rather vulnerabilities of resulting expanded subpopulations upon selection by the first drug.
Citation Format: Boyang Zhao, Michael T. Hemann, Douglas A. Lauffenburger. Combination therapies guided by evolution of tumor heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A31.