Targeting connective tissue growth factor (CTGF) in acute lymphoblastic leukemia preclinical models: anti-CTGF monoclonal antibody attenuates leukemia growth

Abstract

Connective tissue growth factor (CTGF/CCN2) is involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in precursor B-acute lymphoblastic leukemia (ALL) and that increased expression of CTGF is associated with inferior outcome in B-ALL. In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knockdown CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA. Silencing of CTGF resulted in significant suppression of leukemia cell growth compared to control vector, which was associated with AKT/mTOR inactivation and increased levels of cyclin-dependent kinase inhibitor p27. CTGF knockdown sensitized ALL cells to vincristine and methotrexate. Treatment with an anti-CTGF monoclonal antibody, FG-3019, significantly prolonged survival of mice injected with primary xenograft B-ALL cells when co-treated with conventional chemotherapy (vincristine, L-asparaginase and dexamethasone). Data suggest that CTGF represents a targetable molecular aberration in B-ALL, and blocking CTGF signaling in conjunction with administration of chemotherapy may represent a novel therapeutic approach for ALL patients.

Figure 1. CTGF is highly expressed in precursor-B ALL cells

(A, B) Real-time PCR measurement of steady state expression of CTGF in ALL cell lines (A) and patient precursor-B ALL (B precursor) and T-ALL (T) samples (B). The abundance of mRNA was normalized to that of ABL1. Results are expressed as mean ± SD of triplicate measurements in cell lines and mean of duplicate measurements in patient samples. Precursor-B ALL cells expressed high levels of CTGF mRNA.

Figure 2. CTGF knockdown inhibits ALL cell proliferation in vitro

(A, B) CTGF expression levels (A) and growth curves (B) of RS4;11 cells expressing either empty vector (EV) or CTGF shRNA (shCTGF). The abundance of mRNA was normalized to that of ABL1. Cell proliferation was analyzed by counting absolute cell numbers with a Vi-Cell XR cell counter. Results are expressed as mean ± SD of triplicate measurements. Statistical significances are denoted as follows: **P < 0.01, ***P < 0.001. (C) CTGF knockdown led to inactivated AKT/mTOR components and increased levels of p27. Protein expression was determined by Western blotting.

Figure 3. CTGF knockdown enhances chemotherapy-induced apoptosis

RS4;11 and REH cells expressing either empty vector (EV) or CTGF shRNA (shCTGF) cells were treated for 48 hours with the indicated concentrations of vincristine (VCR) and methotrexate (MTX), and Annexin V-positive fractions were measured. Results are expressed as mean ± SD of triplicate measurements. Statistically significant differences are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. FG-3019 significantly prolongs overall survival in patient-derived human ALL xenograft models when combined with conventional chemotherapy

(A) Schematic representation of the in vivo xenograft experiment and chemotherapy/FG-3019 administration. (B) Overall survival of control (hIgG-treated) and FG-3019-treated mice with or without conventional chemotherapy (vincristine, L-asparaginase and dexamethasone, VXL) in the ALL-2 and ALL-10 xenograft model. X axis, days post initiation of treatment (day 84 for ALL-2, day 3 for ALL-10).