BH-3 like motif containing inducer of cell death (BLID) is a known prognostic factor in breast cancer. The aim of the present study was to determine the significance of BLID in the outcomes of chemotherapy and mechanisms affected in BLID-deficient breast cancer cells. Reverse transcription-PCR, reverse transcription-quantitative PCR, dual-luciferase reporter and chromatin immunoprecipitation assays were used to determine the effects of drugs on BLID expression and binding of forkhead box protein O3a (FOXO3a) to the BLID promoter. RNA arrays, antibody microarrays and microRNA arrays were used to illuminate the omics features of BLID knockdown vs. isogenic control breast cancer cell lines. Kaplan-Meier plotter and receiver operating characteristic plotter tools were used to determine the prognostic and therapy response benefits of BLID expression using publicly available clinical datasets. BLID expression was induced in response to several chemotherapeutic drugs. Drug treatment resulted in increased binding of FOXO3a to the BLID promoter, and FOXO3a knockdown was associated with decreased expression of BLID. BLID depletion led to a decrease in the cytotoxicity of chemotherapeutic drugs. Through multi-omics profiling, three functionally distinct classes of effectors that were predominately influenced in BLID knockdown cells were identified: i) Genes and proteins associated with cell death and survival, including cellular inhibitor of apoptosis protein 2/baculoviral IAP repeat containing 3, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α/p110α and DFNA5/gasdermin E; ii) members of the aldo-ketoreductase family 1, specifically aldo-keto reductase family 1 member C3 implicated in drug metabolism; and iii) effectors of the interferon response, including IFNβ1, interferon-induced protein with tetratricopeptide repeats 2 and interferon-induced protein with tetratricopeptide repeats 3. Finally, higher BLID expression was associated with improved overall survival in several types of cancer and the response of breast cancer to anthracyclines. The results of the present study demonstrated that BLID is a target of FOXO3a, and BLID-deficiency in breast cancer cells was associated with modulation of cell death, survival and proliferation, chemoresistance, drug potency, and the interferon response. These findings highlighted BLID as a promising biomarker of drug response and offer a novel framework of integrative mechanisms of therapeutic resistance and disease progression.

Due to their inhibition of acetylcholinesterase, organophosphates are among the most toxic of chemicals. Pralidoxime (a.k.a 2-PAM) is the only acetylcholinesterase reactivator approved in the U.S., but 2-PAM only poorly traverses the blood–brain barrier. Previously, we have demonstrated that scL-2PAM, a nanoformulation designed to enter the brain via receptor-mediated transcytosis, is superior to unencapsulated 2-PAM for reactivating brain acetylcholinesterase, ameliorating cholinergic crisis, and improving survival rates for paraoxon-exposed mice. Here, we employ histology and transcriptome analyses to assess the ability of scL-2PAM to prevent neurological sequelae including microglial activation, expression of inflammatory cytokines, and ultimately loss of neurons in mice surviving paraoxon exposures. Levels of the mRNA encoding chemokine ligand 2 (CCL2) were significantly upregulated after paraoxon exposures, with CCL2 mRNA levels in the brain correlating well with the intensity and duration of cholinergic symptoms. Our nanoformulation of 2-PAM was found to be superior to unencapsulated 2-PAM in reducing the levels of the CCL2 transcript. Moreover, brain histology revealed that scL-2PAM was more effective than unencapsulated 2-PAM in preventing microglial activation and the subsequent loss of neurons. Thus, scL-2PAM appears to be a new and improved countermeasure for reducing neuroinflammation and mitigating brain damage in survivors of organophosphate exposures.

The blood-brain barrier (BBB) blocks ~98% of drugs, so brain tumors tend to be refractory to chemotherapy. Transferrin receptors (TfRs) are expressed on the endothelial cell that comprise the BBB and mediate the movement of transferrin into the brain via transcytosis. Tumor cells overexpress TfRs, so encapsulating drugs within nanocomplexes that bind to TfRs has the potential to enhance both entry into the brain and delivery to tumor cells. Here, we present data on the use of TfR-targeted platform nanotechnology termed scL (for single-chain Liposome) to encapsulate five different chemotherapeutic agents. Data are provided showing that scL nanoformulations of these drugs were more effective in killing cancer cells than their unencapsulated counterparts. It is not our intention to study any one of these nanoformulations in detail but rather to highlight the general versatility of this drug delivery system. The scL formulation of TMZ (scL-TMZ) was more effective than unencapsulated TMZ against TMZ-resistant glioblastoma (GBM) and displayed improved efficacy in impeding tumor growth and extending lifespan in mice bearing intracranial GBM tumors. The scL formulation of irinotecan displayed both enhanced potency in killing lung cancer cells in culture and improved in vivo efficacy in a murine lung cancer xenograft model.

Purpose:Atypical teratoid rhabdoid tumor (ATRT) is a deadly, fast-growing form of pediatric brain cancer with poor prognosis. Most ATRTs are associated with inactivation of SMARCB1, a subunit of the chromatin remodeling complex, which is involved in developmental processes. The recent identification of SMARCB1 as a tumor suppressor gene suggests that restoration of SMARCB1 could be an effective therapeutic approach.Methods:We tested SMARCB1 gene therapy in SMARCB1-deficient rhabdoid tumor cells using a novel tumor-targeted nanomedicine (termed scL-SMARCB1) to deliver wild-type SMARCB1. Our nanomedicine is a systemically administered immuno-lipid nanoparticle that can actively cross the blood-brain barrier via transferrin receptor-mediated transcytosis and selectively target tumor cells via transferrin receptor-mediated endocytosis. We studied the antitumor activity of the scL-SMARCB1 nanocomplex either as a single agent or in combination with traditional treatment modalities in preclinical models of SMARCB1-deficient ATRT.Results:Restoration of SMARCB1 expression by the scL-SMARCB1 nanocomplex blocked proliferation, and induced senescence and apoptosis in ATRT cells. Systemic administration of the scL-SMARCB1 nanocomplex demonstrated antitumor efficacy as monotherapy in mice bearing ATRT xenografts, where the expression of exogenous SMARCB1 modulates MYC-target genes. scL-SMARCB1 demonstrated even greater antitumor efficacy when combined with either cisplatin-based chemotherapy or radiation therapy, resulting in significantly improved survival of ATRT-bearing mice.Conclusion:Collectively, our data suggest that restoring SMARCB1 function via the scL-SMARCB1 nanocomplex may lead to therapeutic benefits in ATRT patients when combined with traditional chemoradiation therapies.

Introduction:Organophosphates are among the deadliest of known chemicals based on their ability to inactivate acetylcholinesterase in neuromuscular junctions and synapses of the central and peripheral nervous systems. The consequent accumulation of acetylcholine can produce severe acute toxicities and death. Oxime antidotes act by reactivating acetylcholinesterase with the only such reactivator approved for use in the United States being 2-pyridine aldoxime methyl chloride (a.k.a., pralidoxime or 2-PAM). However, this compound does not cross the blood-brain barrier readily and so is limited in its ability to reactivate acetylcholinesterase in the brain.Methods:We have developed a novel formulation of 2-PAM by encapsulating it within a nanocomplex designed to cross the blood-brain barrier via transferrin receptor-mediated transcytosis. This nanocomplex (termed scL-2PAM) has been subjected to head-to-head comparisons with unencapsulated 2-PAM in mice exposed to paraoxon, an organophosphate with anticholinesterase activity.Results and Discussion:In mice exposed to a sublethal dose of paraoxon, scL-2PAM reduced the extent and duration of cholinergic symptoms more effectively than did unencapsulated 2-PAM. The scL-2PAM formulation was also more effective than unencapsulated 2-PAM in rescuing mice from death after exposure to otherwise-lethal levels of paraoxon. Improved survival rates in paraoxon-exposed mice were accompanied by a higher degree of reactivation of brain acetylcholinesterase.Conclusion:Our data indicate that scL-2PAM is superior to the currently used form of 2-PAM in terms of both mitigating paraoxon toxicity in mice and reactivating acetylcholinesterase in their brains.

Supplementary Table S1, S3-8

Supplementary Methods, References, Figure legends, Table S2

Because lung cancer remains the most common and lethal of cancers, novel therapeutic approaches are urgently needed. RB94 is a truncated form of retinoblastoma tumor suppressor protein with elevated anti-tumor efficacy. Our investigational nanomedicine (termed scL-RB94) is a tumor-targeted liposomal formulation of a plasmid containing the gene encoding RB94. In this research, we studied anti-tumor and immune modulation activities of scL-RB94 nanocomplex in preclinical models of human non-small cell lung cancer (NSCLC). Systemic treatment with scL-RB94 of mice bearing human NSCLC tumors significantly inhibited tumor growth by lowering proliferation and increasing apoptosis of tumor cells in vivo. scL-RB94 treatment also boosted anti-tumor immune responses by upregulating immune recognition molecules and recruiting innate immune cells such as natural killer (NK) cells. Antibody-mediated depletion of NK cells blunted the anti-tumor activity of scL-RB94, suggesting that NK cells were crucial for the observed anti-tumor activity in these xenograft models. Treatment with scL-RB94 also altered the polarization of tumor-associated macrophages by reducing immune-suppressive M2 macrophages to lower immune suppression in the tumor microenvironment. Collectively, our data suggest that the efficacy of scL-RB94 against NSCLC is due to an induction of tumor cell death as well as enhancement of innate anti-tumor immunity.

AbstractPurpose: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). Experimental Design: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. Results: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. Conclusions: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.

Background Over 96% of high-grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63, which shares many of the downstream targets and functions of p53, is rarely mutated in cancer. Methods A novel strategy is presented for circumventing alterations in p53 by inducing the tumor-suppressor isoform TAp63 (transactivation domain of tumor protein p63) through its direct downstream target, microRNA-130b (miR-130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer. Results Treatment with miR-130b resulted in: 1) decreased migration/invasion in HEYA8 cells (p53 wild-type) and disruption of multicellular spheroids in OVCAR8 cells (p53-mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B-cell lymphoma (Bcl)-inhibitor B-cell lymphoma 2-like protein 11 (BIM). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR-130b. In vivo, combined miR-130b plus CDDP exhibited greater therapeutic efficacy than miR-130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR-130b in 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP-treated mice, and 20% of DOPC-miR-130b plus CDDP-treated mice were living tumor free. Systemic injections of scL-miR-130b plus CDDP in a clinically tested, tumor-targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts. Conclusions The miR-130b/TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad-spectrum therapeutic options for the majority of p53-altered cancers.

The tumor suppressor p53 responds to genotoxic and oncogenic stresses by inducing cell cycle arrest and apoptosis. Recent studies suggest that p53 also participates in the regulation of cellular immune responses. Here, we have investigated the potential of p53 gene therapy to augment immune checkpoint inhibition by combining an anti-programmed cell death protein 1 (PD1) antibody with SGT-53, our investigational nanomedicine carrying a plasmid encoding human wild-type p53. In three syngeneic mouse tumor models examined including a breast cancer, a non-small cell lung carcinoma, and a glioblastoma, SGT-53 sensitized otherwise refractory tumors to anti-PD1 antibody. The involvement of p53 in enhancing anti-PD1 immunotherapy appears to be multifaceted, since SGT-53 treatment increased tumor immunogenicity, enhanced both innate and adaptive immune responses, and reduced tumor-induced immunosuppression in a 4T1 breast tumor model. In addition, SGT-53 alleviates a fatal xenogeneic hypersensitivity associated with the anti-PD1 antibody in this model. Our data suggest that restoring p53 function by SGT-53 is able to boost anti-tumor immunity to augment anti-PD1 therapy by sensitizing tumors otherwise insensitive to anti-PD1 immunotherapy while reducing immune-related adverse events.

The failure of therapeutic agents to cross the blood-brain barrier (BBB) has been a major impediment in the treatment of neurological disorders and brain tumors. We have addressed this issue using an immunoliposome nanocomplex (designated scL) that delivers therapeutic nucleic acids across the BBB into the deep brain via transcytosis mediated by transferrin receptors. We validated brain delivery of payloads after systemic administration by monitoring uptake of fluorescently labeled payloads and by confirming up- or down-modulation of specific target gene expression in the brain, mainly in neuronal cells. As proof of concept for the therapeutic potential of our delivery system, we employed scL delivering an siRNA targeting tumor necrosis factor alpha to suppress neuroinflammation and neuronal apoptosis and to protect mice in lethal endotoxemia triggered by bacterial lipopolysaccharide. Brain delivery of therapeutic payloads via scL has major implications for the development of treatments for neurological disorders and brain tumors.

Abstract The triple-negative breast cancer (TNBC) is an aggressive form of breast cancer frequently seen in African American women and BRCA1 mutation carriers. The TNBC tumors often relapse with distant metastases following standard chemotherapy. It is now evident that discoveries of new mechanisms and approaches that explain and target breast cancer biology are urgently needed for durable intervention of metastatic disease. Earlier, we have demonstrated that BLID, BH-3 Like motif containing Inducer of cell Death, is a strong prognostic factor in invasive breast cancer. Frequent lack of BLID has been associated with TNBC, African American ethnicity and younger women. Significant correlations exist between BLID negative breast cancer and declines in overall survival, local relapse-free survival and distant metastasis-free survival. Recently, BLID has been shown to inhibit breast cancer cell growth and metastasis. The purpose of this study was to investigate the role of BLID in response of breast cancer cells to chemotherapeutic drugs. In the dose response and time course studies, BLID mRNA expression was found to be induced by chemotherapeutic drugs. Expression of BLID cDNA nanocomplex (scLBLID) resulted in significant increase in chemosensitivity in SKBr3 and MDA-MB-231 cells, and a comparison of BLID with p53 showed that the chemosensitization effect of BLID was significantly greater than that of p53. Consistently, BLID knockdown led to reversal of drug-induced cytotoxicity. In the ChIP-PCR and ChIP-qPCR assays, drug treatment of breast cancer cells resulted in an increased binding of pro-apoptotic transcription factor FOXO3a to the BLID promoter, and the reversal of drug-induced BLID reporter activity was seen in presence of FOXO3a siRNA. Furthermore, siRNA silencing of FOXO3a was found to be associated with decrease in endogenous BLID mRNA expression. Remarkably, we found that expression of central tumor suppressor microRNA miR34a also resulted in increased BLID mRNA expression and drug toxicity in breast cancer cells. Because lack of BLID expression has been associated with poor prognosis in breast cancer patients, we reasoned that the silencing of BLID may reveal as yet unknown changes in gene expression that may drive breast cancer cell proliferation and therapy resistance. In this context, the mRNA array profiling studies showed that BLID knockdown in MDA-MB-231 cells was associated with increased expression of the oncogenic/anti-apoptotic molecules CYP1B1, BIRC3 and CSF1, and decreased expression of the anti-oncogenic/apoptotic molecules AKAP12, DFNA5 and CHRDL1. Our data suggest that chemotherapeutic drugs induce BLID expression via activation of FOXO3a, and the BLID signaling axis downstream of FOXO3a and miR34a is a novel integrative mechanism of breast cancer response to chemotherapy. SY and RH are equal contributors in this study. Citation Format: Sivaramakrishna Yadavalli, Rong Hu, Antonina Rait, Xin Li, Esther Chang, Robert Clarke, Usha Kasid. BLID is a novel drug-inducible apoptotic molecule: Identification of an integrative mechanism of chemosensitivity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2164. doi:10.1158/1538-7445.AM2017-2164

Intrinsic therapeutic resistance especially in cancer stem cells (CSCs) together with extensive tumor cell infiltration and restricted permeation of the blood-brain barrier (BBB) by drugs may all contribute to the treatment failure in patients with glioblastoma multiforme (GBM). Accumulating evidence suggests that long non-coding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a role in tumor cell infiltration and therapeutic resistance of GBM. Using our tumor-targeted nanocomplex, we have modulated the expression of MALAT1 and investigated its impact on GBM cells. Importantly, our nanocomplex is able to target CSCs that are considered to be the prime culprits in therapeutic resistance and recurrence of GBM. Attenuation of MALAT1 by RNA interference significantly lowered the growth, motility and stemness of GBM cells. In addition, silencing of MALAT1 clearly improved the sensitivity of GBM cells to chemotherapeutic agents including the current first-line therapy of GBM [temozolomide (TMZ)]. In animal models of GBM, tumor involution with a modest but statistically significant survival benefit was achieved with concurrent treatment of TMZ and nanocomplex-mediated silencing of MALAT1. These results suggest that combining standard TMZ treatment with lncRNA-targeting therapies using our nanocomplex could substantially enhance the very poor prognosis for GBM patients.

Development of temozolomide (TMZ) resistance contributes to the poor prognosis for glioblastoma multiforme (GBM) patients. It was previously demonstrated that delivery of exogenous wild-type tumor suppressor gene p53 via a tumor-targeted nanocomplex (SGT-53) which crosses the blood-brain barrier could sensitize highly TMZ-resistant GBM tumors to TMZ. Here we assessed whether SGT-53 could inhibit development of TMZ resistance. SGT-53 significantly chemosensitized TMZ-sensitive human GBM cell lines (U87 and U251), in vitro and in vivo. Furthermore, in an intracranial GBM tumor model, two cycles of concurrent treatment with systemically administered SGT-53 and TMZ inhibited tumor growth, increased apoptosis and most importantly, significantly prolonged median survival. In contrast TMZ alone had no significant effect on median survival compared to a single cycle of TMZ. These results suggest that combining SGT-53 with TMZ appears to limit development of TMZ resistance, prolonging its anti-tumor effect and could be a more effective therapy for GBM.From the Clinical Editor: Using human glioblastoma multiforma cell lines, this research team demonstrated that the delivery of exogenous wild-type tumor suppressor gene p53 via a tumor-targeted nanocomplex limited the development of temozolomide resistance and prolonged its anti-tumor effect, which may enable future human application of this or similar techniques. (C) 2015 Elsevier Inc. All rights reserved.

Although temozolomide (TMZ) is the current first-line chemotherapy for glioblastoma multiforme (GBM), most patients either do not respond or ultimately fail TMZ treatment. Both intrinsic tumor resistance and limited access of TMZ to brain tumors as a result of the blood-brain barrier (BBB) contribute to poor response and ultimately to poor prognosis for GBM patients. We have developed a "dual-targeting" nanomedicine that both actively crosses the BBB and actively targets cancer cells once in the brain parenchyma. This nanomedicine (termed scL-TMZ) is sized similar to 40 nm and comprised of a cationic liposome (DOTAP:DOPE) encapsulating TMZ. The surface of liposome is decorated with anti-transferrin receptor single-chain antibody fragments to facilitate the crossing of the BBB by the scL-TMZ in addition to targeting GBM in the brain. This novel formulation was found to be markedly more effective than standard TMZ in both TMZ-resistant and TMZ-sensitive GBM. Encapsulation of TMZ also markedly enhanced its efficacy in killing a variety of non-GBM tumor cells. The scL-TMZ nanocomplex was shown to target cancer stem cells, which have been linked to both drug resistance and recurrence in GBM. Most significantly, systemically administered scL-TMZ significantly prolonged survival in mice bearing intracranial GBM tumors. The improved efficacy of scL-TMZ compared to standard TMZ was accompanied by reduced toxicity, so we conclude that the scL-TMZ nanomedicine holds great promise as a more effective therapy for GBM and other tumor types. (C) 2015 Elsevier Ireland Ltd. All rights reserved.