Abstract In preclinical and mostly in vitro studies, pomalidomide (Pom) has been shown to mediate direct anti-proliferative effects on tumor cells, as well as immune-modulatory effects on T cells, NK cells and monocytes. Cereblon (CRBN), a direct cellular target for Pom has been involved in the anti-proliferative effects in tumor cells via selective degradation of Ikaros (IKZF1) and Aiolos (IKZF3). Depletion of IKZF1/IKZF3 has also been implicated in Len-mediated amplification of anti-CD3-induced IL2 production in human T cells in culture. However the impact of pomalidomide on tumor proliferation and immune activation in vivo is unknown. Here we have evaluated the clinical and pharmacodynamic effects of continuous or intermittent dosing strategies of pomalidomide/dexamethasone in lenalidomide-refractory myeloma in a randomized trial. 39 eligible patients with relapsed myeloma were randomized to therapy with Pom/Dexamethazone (following Pom alone for cycle 1), utilizing either continuous Pom dosing (2 mg-28/28 days, cohort 1, n = 19) or an intermittent dosing schedule (4 mg-21/28 days, cohort 2, n = 20). Dexamethazone was administered at 40 mg weekly at cycle 2 and beyond. Intermittent dosing strategy, despite having frequent adverse events, led to greater tumor reduction. Both cohorts experienced similar event-free and overall survival. Both regimens led to a distinct pattern but similar degree of mid-cycle immune activation as manifest by increased expression of cytokines and lytic genes in T and NK cells. Pomalidomide induced polyfunctional T cell activation, with increased proportion of co-inhibitory receptor BTLA+ T cells and Tim-3+ NK cells. Baseline levels of cereblon, ikaros and aiolos protein in tumor cells using validated IHC assay on marrow biopsies, did not correlate with response or survival. Pomalidomide treatment led to a rapid decline in Ikaros in T and NK cells in vivo as measured by intranuclear flow staining, and therapy-induced activation of CD8+ T cells correlated with clinical response. These data demonstrate that pomalidomide leads to strong and rapid immunomodulatory effects involving both innate and adaptive immunity, even in heavily pre-treated MM, which correlate with clinical anti-tumor effects. Clinicaltrials.gov-NCT01319422. Citation Format: Rituparna Das, Kartik Sehgal, Lin Zhang, Rakesh Verma, Yanhong Deng, Mehmet Kocoglu, Juan Vasquez, Srini Koduru, Yan Ren, Maria Wang, Suzana Couto, Mike Breider, Donna Hansel, Stuart Seropian, Dennis Cooper, Anjan Thakurta, Xiaopan Yao, Kavita M. Dhodapkar, Madhav V. Dhodapkar. Comparison of pomalidomide dosing strategies in lenalidomide-refractory myeloma: Impact on clinical outcome, immune activation and cereblon targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1354. doi:10.1158/1538-7445.AM2015-1354