Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion. Cancer Res; 70(21); 8347-56. ©2010 AACR.

have interest in AuraSense Therapeutics, which develops SNAbased technologies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors or government, and no official endorsement should be inferred. Data and materials availability: SNA conjugates and Bcl2L12-related biologicals are available upon request from C.A.M. and A.H.S. with an executed materials transfer agreement.

Significance Molecular mechanisms of therapy (apoptosis) resistance in cancer are poorly understood. Here, we have identified Bcl2-like 13 (Bcl2L13) as a ceramide synthase inhibitor that is overexpressed in glioblastoma (GBM) and other malignancies. Bcl2L13 inhibits therapy-induced apoptosis and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) and blocks CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors, thereby providing a molecular explanation for the low levels of proapoptotic ceramide species in high-grade gliomas, which are associated with poor survival. To our knowledge, this work provides the first evidence of direct regulation of CerS activity by a Bcl-2 family member and establishes the Bcl2L13–CerS axis as a target for therapeutic intervention.

Glioblastoma (GBM) is a highly aggressive and lethal brain cancer with a median survival of less than two years after diagnosis. Hallmarks of GBM tumors include soaring proliferative indices, high levels of angiogenesis, diffuse invasion into normal brain parenchyma, resistance toward therapy-induced apoptosis, and pseudopallisading necrosis. Despite the recent advances in neurosurgery, radiation therapy, and the development of targeted chemotherapeutic regimes, GBM remains one of the deadliest types of cancer. Particularly, the alkylating agent temozolomide (TMZ) in combination with radiation therapy prolonged patient survival only marginally, and clinical studies assessing efficacies of targeted therapies, foremost ATP mimetics inhibiting the activity of receptor tyrosine kinases (RTKs), revealed only few initial responders; tumor recurrence is nearly universal, and salvage therapies to combat such progression remain ineffective. Consequently, myriad preclinical and clinical studies began to define the molecular mechanisms underlying therapy resistance of GBM tumors, and pointed to the Bcl-2 protein family, in particular the atypical member Bcl2-Like 12 (Bcl2L12), as important regulators of therapy-induced cell death. This review will discuss the multi-facetedmodi operandiof Bcl-2 family proteins, describe their roles in therapy resistance of malignant glioma, and outline current and future drug development efforts to therapeutically target Bcl-2 proteins.

have interest in AuraSense Therapeutics, which develops SNAbased technologies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors or government, and no official endorsement should be inferred. Data and materials availability: SNA conjugates and Bcl2L12-related biologicals are available upon request from C.A.M. and A.H.S. with an executed materials transfer agreement.

have interest in AuraSense Therapeutics, which develops SNAbased technologies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors or government, and no official endorsement should be inferred. Data and materials availability: SNA conjugates and Bcl2L12-related biologicals are available upon request from C.A.M. and A.H.S. with an executed materials transfer agreement.

Using in silico analysis of 272 GBM samples in the TGCA data set to find important cell death mechanisms in gliomapathogenesis, we identified differential expression of the novel Bcl2-Like13 protein (Bcl2L13). Bcl2L13 shares significant structural homology with Bcl-2 and Bcl-xL, but contains a unique 250 amino acid sequence that may point to unique, non-classical functions. Several studies have shown that Bcl2L13 expression correlates with increased chemotherapeutic resistance and unfavorable treatment outcome in leukemia and ZIC1-driven liposarcomas. These clinical data suggests that Bcl2L13 likely acts as an onco-protein in multiple cancers and may act in a similar manner in GBM. Using qRT-PCR and immunohistochemical analyses, we found that Bcl2L13 was highly overexpressed in >90% of GBM samples, but absent in adjacent normal brain. Using RNAi-loss- and cDNA complementation studies in glioma cell lines and glioma stem cells (GSCs), we determined that Bcl2L13 potently inhibits therapy-induced apoptosis by inhibiting mitochondrial outer membrane permeabalization (MOMP) and post-mitochondrial caspase-3 and -7 cleavage, likely acting in a similar manner as canonical Bcl-2 family proteins. Bcl2L13 overexpression also greatly increased cellular proliferation, and immunofluorescence and cell fractionalization experiments revealed that Bcl2L13 is localized at the mitochondria. To study the impact of Bcl2L13 on the progression of gliomagenesis in vivo, we orthotopically injected glioma cell lines with enforced expression of Bcl2L13-targeting shRNAs into immunocompromised SCID mice. Strikingly, neutralization of Bcl2L13 signaling increased overall glioma-free survival, which was associated with enhanced intratumoral apoptosis and decreased proliferative indices. To molecularly elucidate the mechanism by which Bcl2L13 exerts its anti-apoptotic effects, we performed a Yeast Two Hybrid screen and identified a select number of tumor suppressors and onco-proteins including ceramide synthase 2 (CerS2), (a regulator of MOMP) and O-6-methylguanine-DNA methyltransferase (MGMT), (an important prognostic indicator of telozolomide effectiveness) as Bcl2L13 interaction partners. Co-immunoprecipitations confirmed the interactions between Bcl2L13 and these proteins. Taken together, these results reveal that Bcl2L13 represents a novel anti-apoptotic, Bcl-2-like GBM oncoprotein that inhibits apoptosis progression and promotes tumor growth by impacting mitochondrial membrane physiology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2005. doi:1538-7445.AM2012-2005