Abstract
Survival rates for pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL) have improved dramatically over the past 40 years approaching a current long-term survival rate of 85%. However childhood B-ALL patients continue to confront co-morbidities and their long-term consequences. For example, osteopenia and osteoporosis associated fractures are a common complication of pediatric leukemia at diagnosis, during treatment and in long-term B-ALL survivors. The STeroid-associated Osteoporosis in the Pediatric Population (STOPP) study reported that at ALL diagnosis, 16% of children and adolescents present with bone pain, vertebral compression and low vertebral Bone Mineral Density (BMD) scores, with the greatest incidence of vertebral fractures (VF) seen in the first year following diagnosis (J Clin Endocrinol Metab. 2015, 100:3408-17). Glucocorticoid treatment further elevated fracture risk in this population. These data underscore the need to identify molecular mechanism by which leukemic cells contribute to bone loss, and provide targeted therapies to limit these effects.
Our laboratory previously showed that Rag2-/- p53-/- Prkdcscid/scid triple mutant (TM) and p53-/- Prkdcscid/scid double mutant (DM) mice develop spontaneous B-ALL, but only TM animals exhibit dissemination of leukemic blasts to the leptomeninges of the CNS, a poor prognosis feature observed in pediatric and adult ALL patients. We observed that TM leukemic mice also displayed fragile vertebral bones. Using comparative transcriptome analysis, we found that RANKL (Receptor Activator of the Nuclear factor-kB Ligand), a Tumor Necrosis Factor (TNF) superfamily member ligand and a key regulator of B cell and osteoclast differentiation, was expressed at greater levels in TM compared to the DM leukemia cells. RANKL binds to its receptor RANK, which is expressed in osteoclast precursor cells. RANK-RANKL interaction induces signaling in the osteoclast precursors and drives their differentiation into mature bone resorbing osteoclasts (Proc. Natl. Acad. Sci. 1999, 96:3540-3545). Upon adoptive leukemia cell transfer into immune deficient mice, RANKL+ TM but not DM cells caused decreased vertebral trabecular bone density in the recipients. Treatment with the recombinant RANKL antagonist protein Osteoprotegerin (OPG-Fc) inhibited the growth and dissemination of RANKL+TM leukemic cells and attenuated bone destruction in the recipient mice. These data suggested that TM mouse leukemia cells cause bone loss in the absence of glucocorticoid or other chemotherapy agents.
We then examined the potential role of RANKL in osteoporosis associated with human B-ALL. RANKL mRNA was expressed by a majority of primary human adult and pediatric B-ALL. To determine whether primary patient B-ALL can cause bone loss, we transplanted RANKL+ human B-ALL samples of multiple cytogenetic high-risk subgroups (Complex, hypo-diploid and Mixed Lineage Leukemia (MLL) rearranged) into NOD.SCID.gC-/-(NSG) recipient mice. Micro-CT imaging and bone density measures in the xenotransplant recipients revealed extensive vertebral trabecular bone destruction. Immuno-histological analysis of the human B-ALL recipient mice demonstrated extensive osteoporotic damage of the long bones and marked RANKL protein expression in the long bones of mice harboring extensive human B-ALL cell burden compared to NSG control mice. To determine whether RANKL-RANK interaction was required for the B-ALL mediated bone destruction, cohorts of NSG mice engrafted with human B-ALL were treated with recombinant OPG-Fc compared to a matched Fc control protein. OPG-Fc treatment did not attenuate leukemia cell expansion and bone marrow burden, but despite bulky disease, the treatment conferred robust protection from bone destruction suggesting that RANKL was a critical mediator of this clinical complication.
Our data demonstrate a central role of the RANK-RANKL axis in B-ALL-mediated bone disease and identify an actionable therapeutic target to reduce acute and long-term morbidity. Denosumab, an anti-RANKL antibody has been approved for the treatment of bone metastasis by solid tumors and for post-menopausal osteoporosis. Our pre-clinical studies suggest that Denosumab and other agents that inhibit the RANK-RANKL pathway may be efficacious in patients with B-ALL associated bone degeneration.
Disclosures
No relevant conflicts of interest to declare.
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