Abstract 1356: Variable drug responses characterize the functional heterogeneity of Nf1 null tumors
Authors: Pucciarelli Daniela , Krishnamurthi Ganesh , Braunstein Steve , Nakamura Jean L. 
Published: July 2017

Introduction: Neurofibromatosis type 1 (NF1) is a autosomal dominant disease with a predisposition to cancer. Biallelic inactivation of the NF1 gene increases risk of developing brain tumors, leukemia, neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Somatic mutations in the NF1 gene are also associated with sporadic malignancies including glioblastoma, neuroblastoma, and melanoma. The NF1 tumor suppressor gene encodes the RAS GTPase-activating protein (GAP) neurofibromin. Loss of NF1 results hyperactivation of Ras, MAPK and PI3K signaling pathways components, representing potential therapeutic targets. However, the variable response of NF1 mutant tumors to MAPK and mTOR inhibitors suggests the intrinsic heterogeneity of NF1 mutant tumors. We hypothesized that defining alternative mechanisms and functional sub-classes of Nf1-mutant tumors on the basis of their variable drug sensitivity will produce pre-clinical therapeutics data that will inform clinical trials.

Methods: We previously generated mouse models in which we mutagenized Nf1 heterozygous mice with radiation, recapitulating the susceptibility of patients with NF1 to radiation-induced cancers. These models produced solid tumors such as mammary carcinomas, squamous cell carcinomas, and soft tissue sarcomas, which we determined to be Nf1 null. Cell lines established from these tumors were characterized by a drug sensitivity screen using a custom 94 compound drug library. Drug responses indicate that Nf1 mutant tumor cell lines organize into functional groups sharing similar drug sensitivities. Six candidate drugs each targeting distinct components of MAPK and PI3K signaling pathways were selected from the drug library. After 24 hours of exposure to each inhibitor or control, cells were analyzed for cell proliferation, cell cycle changes, and cell death. Western blotting were performed to determine whether drug exposures produce alterations in their predicted biochemical pathways such as PI3K/Akt, and MAPK pathways.

Results: Nf1 loss did not predict uniform sensitivity of cell lines to treatment with MAPK and mTOR inhibitors. Each compound induced differential effects on viability of Nf1 null tumors cell lines. High variability and cell line-dependent cytotoxic and cytostatic effects were also observed. Although phosphorylated Akt(S473), S6, and p44/42 MAPK varied widely among all untreated Nf1 null cell lines, this did not predict their drug sensitivity. In vitro drug sensitivity data indicates heterogeneous differential sensitivities of Nf1 mutant tumor cells to different drug classes, independent of tumor histology, permitting segregation into functional groups.

Conclusion: Tumor cell lines driven by Nf1 loss demonstrate heterogeneous responses to Ras pathway inhibition, which may be explained by mechanisms of tumor formation after Nf1 loss involve multiple alternative pathways.

Citation Format: Daniela Pucciarelli, Ganesh Krishnamurthi, Steve Braunstein, Jean L. Nakamura. Variable drug responses characterize the functional heterogeneity of Nf1 null tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1356. doi:10.1158/1538-7445.AM2017-1356