Precursor B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematopoietic neoplasm characterized by recurrent genetic lesions resulting in B-cell maturation arrest and malignant transformation. Even with the addition of targeted therapies to conventional treatment regimens, prognosis for adults with high risk disease remains poor, particularly for those patients with relapsed or refractory disease. Despite an arrest in B cell maturation, we previously showed that human B-ALL blasts retain the capacity for reprogramming to the myeloid lineage (McClellan et al, PNAS 2015). While the concept of forced differentiation was proposed several decades ago, no differentiation therapies have been effective in the treatment of B-ALL. Thus, we sought to investigate the therapeutic implications of myeloid lineage reprogramming of B-ALL cells.
We speculated that myeloid-reprogramming of B-ALL cells into antigen presenting cells (APCs) could induce tumor-specific T cell responses through effective presentation of aberrant tumor-associated self-peptides. To test this hypothesis, we generated murine models of B-ALL capable of reprogramming to the myeloid lineage through the inducible expression of two transcription factors, CEBPα and PU.1. Ectopic expression of these factors efficiently reprogrammed B-ALL cells into myeloid-lineage APCs, expressing myeloid markers (CD11b, CD14, CD115, and Ly6C). Reprogramming ablated the tumorigenicity of these cells as they acquired APC characteristics, including phagocytic activity and expression of antigen presentation and co-stimulation molecules: MHC-I (3.13-fold, p=0.0018), MHC-II (8.6-fold, p<0.0001), CD80 (62.1-fold, p<0.0001), CD86 (107.6-fold, p<0.0001), and CD40 (92-fold, p<0.0001). Using chicken ovalbumin as a model antigen and DO11.10 transgenic CD4+ T cells, we demonstrated that reprogrammed B-ALL cells, but not parental blasts, can process and present both endogenous and exogenous peptides for antigen-specific T cell activation.
To explore the therapeutic potential of B-ALL reprogramming, we engrafted immunodeficient (NSG) and immunocompetent syngeneic (BALB/c) mice with our B-ALL model and induced myeloid reprogramming in vivo. While B-ALL reprogramming in immunodeficient mice led to a three day extension in median survival (p=0.0016, n= 5 per group), all of the mice succumbed to their disease. Strikingly, B-ALL reprogramming in immunocompetent mice led to complete tumor regression and survival of the entire cohort 100 days post treatment (p<0.0001, n=10 per group), suggesting that reprogramming induced immune-mediated tumor eradication. Importantly, these animals were not susceptible to subsequent B-ALL re-challenge, demonstrating successful generation of durable, systemic, and protective immunity.
In order to investigate the mechanism underlying tumor eradication, we depleted BALB/c mice of CD4+ or CD8+ T cells. Depletion of either T cell population abrogated the therapeutic benefit of B-ALL reprogramming, indicating that reprogrammed B-ALL cells stimulate T cell activation in vivo. Further analysis of the CD8 T cell repertoire by TCRVb chain usage revealed significant 10.3-fold (p=0.0109, n=5 per group) expansion of a single TCRVb chain family in response to B-ALL reprogramming, consistent with an oligoclonal T cell response. Following reprogramming, a 4.01-fold increase in the frequency of infiltrating T cells is observed in the bone marrow (p=0.0028), including both activated (CD25+/CD69+) (1.62-fold, p=0.018) and effector memory (CD44+CD62L-) (1.99-fold, p=0.0097) T cells. Finally, using a dual tumor model, we demonstrated that myeloid reprogramming-dependent T cell activation eradicates malignant cells systemically, as demonstrated by regression of contralateral tumors lacking reprogramming.
Together, our data suggests that (1) B-ALL cells reprogrammed to the myeloid lineage can operate as potent APCs capable of presenting both endogenous and exogenous tumor-associated antigens, (2) in vivo B-ALL reprogramming elicits robust immune activation, dependent on both CD4+ and CD8+ T cells, and (3) B-ALL reprogramming-induced immune activation is potent, durable, tumor-eradicating, and systemic. Thus, reprogramming of B-ALL cells into APCs represents a novel immunotherapeutic strategy with potential clinical benefit for the management of B-ALL disease progression.
Disclosures
Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy.
Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes.