CD9 Blockade Effectively Suppresses Disease Progression of Pediatric B-Precursor Acute Lymphoblastic Leukemia and Enhances Chemosensitivity

Despite major advances in risk-directed multi-agent chemotherapy, specific subtypes of high-risk and relapsed/refractory pediatric B-precursor acute lymphoblastic leukemia (B-ALL) remain associated with poor clinical outcomes, highlighting the need for development of novel and effective therapeutic strategies. CD9, a tetraspanin family protein, has been associated with metastasis and progression of various types of malignancies, but its prognostic relevance and therapeutic potential in pediatric B-ALL remain largely unknown. In a cohort of pediatric B-ALL cases, we identified that CD9+ patients had significantly lower 5-year overall (OS) and relapse-free survival (RFS) rates when compared with CD9- cases (OS: 81.5% vs 100%, P= 0.022; RFS: 70.7% vs 92.3%, P= 0.029). Subgroup analysis revealed remarkably poorer outcomes in CD9+ patients of the high-risk group (OS: 61.5% vs 100%, P= 0.042; RFS: 46.2% vs 88.9%, P= 0.045). A similar trend was also observed in patients of the intermediate-risk group but not in the standard-risk group. In univariate analysis, CD9 positivity, age <1 year, white cell count ≥100 × 10^9/L and poor prednisone response were associated with lower RFS rate. In multivariate analysis, CD9 positivity (HR = 6.0; P= 0.019) remained as an independent prognostic factor for lower RFS rate. The efficacy of targeting CD9 for treatment of pediatric B-ALL was evaluated in the NOD/SCID mouse xenograft model. Our results showed that administration of CD9 neutralizing antibody as a single agent substantially reduced multi-organ leukemic burden by >90% (P ≤ 0.037) and significantly prolonged survival (P ≤ 0.007) of animals xenografted with the intermediate-risk 697 (TCF3-PBX1+) and high-risk RS4;11 (MLL-AF4+), but not the standard-risk Reh (ETV6-RUNX1+) cell lines. Similarly, CD9 antibody treatment significantly decreased B-ALL progression in patient-derived xenografts with a wide spectrum of genetic and disease features, including high-risk infantile MLL-AF4+ cases as well as those with relapsed/refractory diseases (P ≤ 0.009). Importantly, CD9 antibody in combination with conventional chemotherapy consisting of vincristine, dexamethasone and L-asparaginase further prolonged animal survival, when compared to animals treated with CD9 antibody or chemotherapy alone (P ≤ 0.006). In vitro, CD9 antibody significantly reduced proliferation of B-ALL cells (P= 0.004) and markedly enhanced vincristine-, dexamethasone- and L-asparaginase-induced apoptosis (P ≤ 0.044). Mechanistic studies revealed that CD9 was involved in B-ALL migration and stromal adhesion by modulating the affinity of integrin very late antigen-4. Our results collectively suggest that expression of CD9 in pediatric B-ALL patients was associated with adverse survival outcomes and that CD9 blockade, in adjunct to chemotherapy, could be a promising strategy for treatment of high-risk and relapsed/refractory pediatric B-ALL, possibly mediated by disruption of leukemia-stroma interaction in the bone marrow microenvironment.