Silencing B7-H3 mitigates tumor aggressiveness in group 3 medulloblastoma - PROJECT SUMMARY/ABSTRACT Medulloblastoma (MB) is the leading cause of cancer-related mortality in childhood. Divided into four subgroups, patients with group 3 tumors (G3MB) have the lowest survival rate (<50% at 5 years). This has been attributed to a lack of targeted therapies and high-risk features, like deletions along the short arm of chromosome 17. These deletions target critical tumor suppressor genes like miR-1253, a brain-enriched microRNA active during cerebellar development. Restoring miR-1253 expression in G3MB cell lines reduced cancer cell growth and aggressiveness, inhibiting several oncoproteins, including B7-H3, which has been strongly linked with metastasis and immune evasion in many aggressive cancers. Silencing B7-H3 expression in G3MB cells dramatically reduced their growth and invasiveness, as well as the expression of SLUG, a JAK2/STAT3 signaling product associated with metastasis. This kindled our interest in finding drug compounds to silence B7-H3 signaling as a novel way to treat G3MB tumors. By screening an extensive collection of molecules known to interact with B7-H3, one highly potent compound, B7-H3-Ni1 (Ni1), was identified, which inhibited G3MB cancer cell viability at low micromolar concentrations. The overall objective of this proposal is to demonstrate a mechanistic proof-of-concept for B7- H3 inhibition as a therapeutic approach against G3MB. We hypothesize that B7-H3-Ni1 can suppress G3MB tumor growth and aggressiveness by activating immune cell function and inhibiting JAK/STAT signaling pathways associated with metastasis. In Aim 1, we will map the molecular binding of Ni1 to B7-H3. For immune activation, a syngeneic G3MB cell line (MP-1) co-cultured with mouse Tregs, CTLs, or NK cells will be treated with vehicle, Ni1, or si-B7-H3 to study the effect of B7-H3 blockade on immune cell degranulation, target cell lysis, cytokine production, and cancer cell viability. For JAK2/STAT3 signaling, we will measure the impact of vehicle vs. Ni1 vs. si-B7-H3 on metastasis- associated signaling partners of B7-H3, metastasis-associated effectors of JAK2/STAT3 signaling, and apoptotic markers. Impact on cancer cell growth, viability, and invasiveness will also be assessed in vitro. Then, we will examine if Ni1 signaling is enhanced by a STAT3 inhibitor (WP1066). A B7-H3 knockout (B7-H3KO) G3MB cell line will be incorporated into all in vitro studies. In Aim 2, an immunocompetent G3MB mouse model that most closely recapitulates human disease will be used to illustrate the immunostimulatory and anti-neoplastic properties of Ni1. We expect to demonstrate that treatment with Ni mitigates tumor burden and longevity by downregulating JAK2/STAT3-associated metastasis signaling. At completion, our studies will address a critical gap for G3MB patients who suffer disproportionately high mortality rates amongst MB patients by characterizing a novel anti-tumor agent that may be efficacious and well-tolerated. By identifying a new druggable target (B7- H3), we may unlock the potential to transform the treatment of advanced MB tumors.
