Abstract 1029: MAPK-mediated immunomodulation in disseminated murine Emu-TCL1 chronic lymphocytic leukemia
Authors: Lakshmanan Aparna , Tsai Yo-Ting , Lehman Amy , Sass Ellen J. , Tran Minh , Lucas Fabienne , Long Meixiao , Harrington Bonnie K. , Perle Krista La , Coppola Vincenzo , Lozanski Gerard , Muthusamy Natarajan , Byrd John C. , Grever Michael R. , Lucas David M. 
Published: July 2017

Abstract
Work in melanoma and other solid tumors shows mutations activating MAPK pathway not only promote malignancy by promoting survival and proliferation but also are immunosuppressive than wild-type (WT) counterparts. However, relevance of MAPK-activating mutations to immune modulation in disseminated cancers such as leukemia is uncertain, and the mechanisms by which this occurs are likely to differ. BRAF activating mutation is found in nearly all hairy cell leukemia cases, 4% of chronic lymphocytic leukemia (CLL) and 9% CLL cases when including other MAPK-activating mutations such as KRAS and NRAS. To assess immunomodulatory effects of BRAF-mutant CLL cells, we co-cultured dox-inducible OSU-CLL cells transfected with WT or BRAFV600E constructs with healthy donor T cells incubated with anti-CD3/anti-CD28 antibodies, and T cell proliferation, cytokine production and expression of surface proteins were assessed by flow cytometry. We also employed a transgenic mouse model of BRAF B cell leukemia. CD19-Cre x BRAFV600E or CD19-Cre only mice were crossed with the well-characterized Emu-TCL1 model of CLL to obtain mice with spontaneous B cell leukemia expressing either WT BRAF (CD19-Cre xTCL1) or mutant BRAF (BRAFV600E xCD19-Cre xTCL1) under the native BRAF promoter. For adoptive transfer (AT) experiments, leukemia cells (2e7) from transgenic mice were engrafted intravenously into syngeneic healthy adult animals. Blood and spleen cells were examined by flow cytometry. OSU-CLL cells expressing BRAFV600E more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. TNF levels were higher in BRAFV600E-expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E-expressing cells on T cell proliferation. Proliferation was rescued by the BRAFV600E inhibitor dabrafenib, further supporting the contribution of mutant BRAF to this effect. Impact of BRAFV600E mutation on myeloid compartment was also evaluated using AT model. Mice were engrafted with BRAFWT or BRAFV600E leukemia cells, and upon achieving similar disease loads, BRAFV600E leukemic B cells caused 2.2-fold increase in PD-L1 expressing-peripheral myeloid cells (p&lt;0.001; n=25 WT; 11 mutant). Additionally, there was an increase in F4/80+ macrophages (11%; p=0.002), CD11b+Ly6CintLy6Ghi MDSCs (8.5%, p=0.014) and decrease in CD11b+Ly6CloLy6Glo patrolling monocytes (11%; p=0.031; n=33 WT; 40 mutant) in spleens of mice with BRAFV600E leukemia, indicating that this mutation influences the myeloid cell compartment as well. Ongoing studies are investigating the mechanism of these effects and the potential for pharmacologic reversal. Together, these results demonstrate immunosuppressive impact of BRAF in B-cell leukemia.
Citation Format: Aparna Lakshmanan, Yo-Ting Tsai, Amy Lehman, Ellen J. Sass, Minh Tran, Fabienne Lucas, Meixiao Long, Bonnie K. Harrington, Krista La Perle, Vincenzo Coppola, Gerard Lozanski, Natarajan Muthusamy, John C. Byrd, Michael R. Grever, David M. Lucas. MAPK-mediated immunomodulation in disseminated murine Emu-TCL1 chronic lymphocytic leukemia [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 1029. doi:10.1158/1538-7445.AM2017-1029