309 MYC Inhibition Overcomes IMiD Resistance in Heterogeneous Multiple Myeloma Populations

Abstract

OBJECTIVES/GOALS: Immunomodulatory drugs (IMiDs) are critical to multiple myeloma (MM) disease control. IMiDs act by inducing Cereblon-dependent degradation of IKZF1 and IKZF3, which leads to IRF4 and MYC downregulation (collectively termed the “Ikaros axis”). We therefore hypothesized that IMiD treatment fails to downregulate the Ikaros axis in IMiD resistant MM. METHODS/STUDY POPULATION: To measure IMiD-induced Ikaros axis downregulation, we designed an intracellular flow cytometry assay that measured relative protein levels of IKZF1, IKZF3, IRF4 and MYC in MM cells following ex vivo treatment with the IMiD Pomalidomide (Pom). We established this assay using Pom-sensitive parental and dose-escalated Pom-resistant MM cell lines before assessing Ikaros axis downregulation in CD38+CD138+ MM cells in patient samples (bone marrow aspirates). To assess the Ikaros axis in the context of MM intratumoral heterogeneity, we used a 35-marker mass cytometry panel to simultaneously characterize MM subpopulations in patient samples. Lastly, we determined ex vivo drug sensitivity in patient samples via flow cytometry. RESULTS/ANTICIPATED RESULTS: Our hypothesis was supported in MM cell lines, as resistant lines showed no IMiD-induced decrease in any Ikaros axis proteins. However, when assessed in patient samples, Pom treatment caused a significant decrease in IKZF1, IKZF3 and IRF4 regardless of IMiD sensitivity. Mass cytometry in patient samples revealed that individual Ikaros axis proteins were differentially expressed between subpopulations. When correlating this with ex vivo Pom sensitivity of MM subpopulations, we observed that low IKZF1 and IKZF3 corresponded to Pom resistance. Interestingly, most of these resistant populations still expressed MYC. We therefore assessed whether IMiD resistant MM was MYC dependent by treating with MYCi975. In 88% (7/8) of patient samples tested, IMiD resistant MM cells were sensitive to MYC inhibition. DISCUSSION/SIGNIFICANCE: While our findings did not support our initial hypothesis, our data suggest a mechanism where MYC expression becomes Ikaros axis independent to drive IMiD resistance, and resistant MM is still dependent on MYC. This suggests targeting MYC directly or indirectly via a mechanism to be determined may be an effective strategy to eradicate IMiD resistant MM.