Sabree, Shakoora

TNF receptor-associated factor 3 (TRAF3) is an adapter protein that inhibits many signals that promote B cell survival and activation. Mice with a B cell-specific TRAF3 deficiency and humans with a rare haploinsufficiency in TRAF3 have enhanced development of BCLs as they age. Loss-of-function mutations in TRAF3 are common in B cell malignancies. Recent studies show that pharmacological inhibition of the enzyme glycogen synthase kinase 3 (GSK3), which regulates cellular growth, survival, and metabolism, inhibits growth and survival of BCL-derived B cells. In this study, we found that TRAF3 and GSK3 associated in B cells. The relative levels of TRAF3 in BCL cell lines correlated positively with the ratio of inactive to total GSK3β, and negatively correlated with susceptibility to GSK3 inhibition by the GSK3 inhibitory drug 9-ING-41, currently in clinical trials. Uniquely in BCLs with low TRAF3, GSK3 inhibition caused increased loss of the TRAF3-regulated, anti-apoptotic protein Mcl-1. GSK3 inhibition also blocked hyperresponsiveness to IL-6 receptor signaling in TRAF3-deficient BCL cells. Together, these results support the utility of 9-ING-41 as a treatment for BCL, and suggest that a decrease or loss of TRAF3 in BCLs could act as a biomarker for increased susceptibility to GSK3 inhibitor treatment.

Abstract CMP-001 is a virus-like particle containing a TLR9 agonist (class A CpG ODN) encapsulated by the Qβ capsid. Preclinical studies and early phase clinical trials of intralesional injection with CMP-001, combined with anti-PD1 antibody, demonstrate promising results. Prior work by our group has demonstrated that the immunostimulatory effects of CMP-001 are dependent on generation of an anti-Qβ antibody response which results in opsonization of CMP-001 by plasmacytoid dendritic cells (pDCs) and production of IFNα. While a variety of cell types including monocytes and B cells can take up CMP-001, the immune response to CMP-001 is based in large part on rapid induction of IFNα production by pDCs. The current studies were designed to assess the downstream effects of CMP-001 and anti-Qβ antibody on various arms of the immune response using normal donor human peripheral blood mononuclear cells (PBMCs) as a model. Stimulation of PBMCs with CMP-001 and anti-Qβ antibody induced production of IFNα from pDCs as previously observed. This IFNα production occurred very rapidly (within 2 hours) and was short lived, but had long term secondary effects, including induction of CXCL10 production by monocytes. Single cell RNA sequencing done 24 hours after stimulation demonstrated additional downstream changes including the following: Upregulation of CXCL10, MIP1-α, IL1-RA and IDO by monocytes. Downregulation of CD14 by monocytes. Upregulation of a broad variety of interferon response genes in various cell types including NK cells and T cells. We conclude the primary effect of in situ immunization with CMP-001 is to induce rapid production of IFNα production by pDCs. This results in significant downstream effects on various cell populations that contribute to the anti-tumor immune response. Understanding these pathways could help identify the most promising combination immunotherapies involving CMP-001. Ongoing studies are exploring the kinetics of these responses and whether similar changes are observed in the tumor microenvironment. Citation Format: Shakoora Sabree, Andrew Voigt, Laura Rogers, Sue Blackwell, Caitlin Lemke-Miltner, George J. Weiner. Evaluation of the complexity of the immune response to CMP-001, a TLR9 agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6666.