Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults, affecting 20,000 people in the United States and resulting in nearly 12,000 deaths [1]. Although AML accounts for about 1% of all cancers, its incidence is increasing [2], and AML patients have a remission rate of ∼67% [1] with the 5-year relative survival rate of just 30.5% [3]. These poor patient outcomes can be attributed to tumor relapse. Notably, AML is characterized by an increase in the number of myeloid cells in the bone marrow and an arrest in their maturation, resulting in hematopoietic insufficiency [4]. In other words, AML cells proliferate rapidly in the bone marrow, depleting nutrients necessary for hematopoiesis, resulting in the inability to produce enough healthy blood cells.

One of the major characteristics of AML is that it is a disseminated cancer with immunosuppressive pathways. Recent studies showed that systemic inoculation of AML cells induces T cell tolerance in an antigen-specific manner [5,6]. Mice inoculated intravenously (i.v.) with C1498.SIY, a murine AML model expressing model antigen SIY, generated lower SIY-specific T cell responses than mice inoculated with C1498.SIY subcutaneously (s.c.) [5,6]. This observation indicates the stark difference in immune surveillance between disseminated cancer and solid cancer. Taking this one step further, i.v. inoculation of C1498.SIY cells, followed by s.c. inoculation of C1498.SIY cells resulted in significantly decreased SIY-specific T cell responses, compared with only s.c. inoculation of C1498.SIY cells [5], showing that AML cells actively promoted T cell dysfunction. However, agonistic anti-CD40 antibody, which activates antigen-presenting cells (APCs), reversed T cell tolerance and extended animal survival [5]. These results demonstrate that T cell tolerance observed in mice inoculated i.v. with C1498.SIY cells was likely regulated by tolerogenic host APCs in the innate immune system. Thus, activation of innate and effector immune cells may offer a potential strategy to elicit strong immune responses against AML.

Current AML therapies do not effectively activate innate immune cells. The standard of care treatments for AML is a chemotherapeutic approach – the “3 + 7” regimen (3 days of daunorubicin +7 days of cytarabine) and allogeneic hematopoietic cell transplantation [7,8]. In addition to these, targeted therapies have gained attention as the pathophysiological molecular subsets of AML have been identified [8]. Some of these experimental therapeutics include hypomethylating agents, fms-like tyrosine kinase 3 (FLT3) inhibitors, IDH inhibitors, TP53 modulator, menin inhibitors, T cell engagers, chimeric antigen receptor (CAR)-T cells, and immune checkpoints blockers (ICBs) [8].

One approach to effectively activate innate immune cells is through activation of the Stimulator of Interferon Genes (STING) pathway. Cyclic GMP-AMP synthase (cGAS) senses damaged double stranded DNA in the cytosol which catalyzes the production of cyclic [G(2′,5′)pA(3′,5′)p] (cGAMP). cGAMP serves as a secondary messenger and binds to the STING adaptor protein, resulting in the production of Type I Interferons (Type IFNs). While Curran, et al. showed that i.v. administration of a proprietary STING agonist, dithio-(RP, RP)-[cyclic[A(2′,5′) pA(3′,5′)p]] (ML RR-S2), induced a significant level of Type I IFN-β in vivo, this required a high dose of ML RR-S2 at 100 μg, which resulted in only 25% survival rate in a mouse AML model based on C1498.SIY cells expressing an exogenous antigen, SIY [9]. Therefore, there is a strong need for a delivery system that can exert robust anti-tumor efficacy in a murine model of AML.

We previously reported the development of a STING-activating nanoparticle, termed CMP, and shown their robust efficacy in various models of solid cancer. CMP is a lipid-based nanoparticle loaded with a bacterial STING agonist, cyclic di-AMP (CDA), and manganese ions (Mn2+), which increased Type I IFN activities of STING agonists [10]. Monotherapy with CMP significantly increased STING activation, induced Type I IFNs, reversed immunosuppression in the tumor microenvironment (TME), and exerted strong anti-tumor efficacy in murine models of solid cancer [10]. Here, we have employed the CMP platform as a monotherapy or in combination with ICB to activate innate immune cells and effector T cells in an aggressive model of disseminated C1498 AML [11,12]. Given the ability of systemic AML to induce T cell tolerance [5], we sought to overcome this challenge through STING activation and ICB employment. We report that CMP i.v. therapy led to the accumulation of CMP in the lymphoid tissues and bone marrow, increased Type I IFN production, decreased immunosuppressive cytokines, and induced robust innate and effector T cell responses, while inhibiting the growth of systemic C1498 AML. Furthermore, CMP plus anti-CTLA-4 ICB combination therapy promoted a stronger cytotoxic response and increased immunological memory against C1498 AML.