In Chronic Myeloid Leukemia Cytotoxic T Cell Responses to BMI-1 Protein Correlate with Higher Expression of BMI-1 in Leukemia Progenitors, and May Contribute to Improved Outcome After Allogeneic Stem Cell Transplantation.

Abstract 192

The polycomb group (PcG) proteins BMI-1 and EZH2 are key regulators of self-renewal processes in normal and leukemic stem cells. Of these two PcG proteins, BMI-1 is more highly expressed in chronic myeloid leukemia (CML) than in normal stem cells, and is associated with a more rapid disease progression in patients who are treated with drug therapy alone, implying that an increased level of “stem-ness” conferred by BMI-1 contributes to leukemogenicity. Conversely, CML patients with high BMI-1 expression prior to allogeneic stem cell transplantation (SCT) have better overall survival post-transplant (Mohty, et al, Blood 2008). To investigate the potential of PcG proteins as leukemia-associated antigens, and targets for graft-versus-leukemia (GVL) effects, we studied a cohort of 86 CML patients (54 chronic phase, 32 advanced phase) who received T-cell depleted SCT with T-cell add-back on day 45-100 post-SCT from HLA-identical sibling donors. Using quantitative real-time PCR, we measured the expression of EZH2, BMI-1 and its target for repression, CDKN2A (encoding p16INK4A) in CD34+ progenitors and their CD34-negative counterparts. Using flowcytometric detection of intracellular cytokines IFN-γ or TNF-α, and degranulation marker CD107a, in CD8+ cytotoxic T lymphocytes (CTL), we assessed immune responses to BMI-1 (TLQDIVYKL and CLPSPSTPV) and EZH2 (YMCSFLFNL and SQADALKYV) peptides in 25 HLA-A*0201+ patient-donor pairs. Seven of 17 (41%) HLA-A*0201+ CML patients had native immune responses to BMI-1 peptide, which was associated with higher BMI-1 expression in CD34+ progenitors (p=0.04, Mann-Whitney U test). Five of 25 (20%) healthy HLA-A*0201+ sibling donors had detectable immune responses to BMI-1 peptide. EZH2 was less immunogenic compared to BMI-1 in both patients and donors. The majority of peptide-specific CTLs analyzed by peptide-specific dextramers had central memory phenotype. BMI-1- or EZH2-specific T cells were readily detected after 7-day cultures using an ELISPOT assay in 75% of donors or patients where peptide-specific CTLs were detected ex-vivo. A higher expression of BMI-1 in CML patients pre-SCT and correspondingly lower expression of its target for repression, CDKN2A, was associated with improved leukemia-free survival (p=0.01), and reduced disease-related death (p=0.0001). In four HLA-A*0201+ patients whose donors had immune responses to PcG peptides, BMI-1 or EZH2-specific T cell responses were detected in the first 120 days post-SCT. CML patients who had donors with immune responses to BMI-1 peptide had improved leukemia-free survival compared to patients whose donors were non-responders (80% vs. 60% respectively). Immune responses to PcG proteins, in particular to BMI-1, may be relevant for disease control by GVL effects. Unlike CTLs specific for primary granular proteins such as proteinase 3 and elastase, which are also highly expressed in CML cells, CTLs against BMI-1 and EZH2 may be less susceptible to selective deletion processes resulting in tolerance, as these proteins are less ubiquitously expressed by mature progenitors which are expanded in CML, and are therefore good GVL and immunotherapy target candidates in CML. Furthermore, these CTLs have the potential to target leukemia stem cells.