Target receptor identification and subsequent treatment of resected brain tumors with encapsulated and engineered allogeneic stem cells.

Cellular therapies offer a promising therapeutic strategy for the highly malignant brain tumor, glioblastoma (GBM). However, their clinical translation is limited by the lack of effective target identification and stringent testing in pre-clinical models that replicate standard treatment in GBM patients. In this study, we show the detection of cell surface death receptor (DR) target on CD146-enriched circulating tumor cells (CTC) captured from the blood of mice bearing GBM and patients diagnosed with GBM. Next, we developed allogeneic "off-the-shelf" clinical-grade bifunctional mesenchymal stem cells (MSCBif) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSCBif (EnMSCBif) has a profound therapeutic efficacy in mice bearing patient-derived invasive, primary and recurrent GBM tumors following surgical resection. Activation of the kill switch enhances the efficacy of MSCBif and results in their elimination post-tumor treatment which can be tracked by positron emission tomography (PET) imaging. This study establishes a foundation towards a clinical trial of EnMSCBif in primary and recurrent GBM patients.

CRISPR-enhanced engineering of therapy-sensitive cancer cells for self-targeting of primary and metastatic tumors.

Tumor cells engineered to express therapeutic agents have shown promise to treat cancer. However, their potential to target cell surface receptors specific to the tumor site and their posttreatment fate have not been explored. We created therapeutic tumor cells expressing ligands specific to primary and recurrent tumor sites (receptor self-targeted tumor cells) and extensively characterized two different approaches using (i) therapy-resistant cancer cells, engineered with secretable death receptor-targeting ligands for "off-the-shelf" therapy in primary tumor settings, and (ii) therapy-sensitive cancer cells, which were CRISPR-engineered to knock out therapy-specific cell surface receptors before engineering with receptor self-targeted ligands and reapplied in autologous models of recurrent or metastatic disease. We show that both approaches allow high expression of targeted ligands that induce tumor cell killing and translate into marked survival benefits in mouse models of multiple cancer types. Safe elimination of therapeutic cancer cells after treatment was achieved by co-engineering with a prodrug-converting suicide system, which also allowed for real-time in vivo positron emission tomography imaging of therapeutic tumor cell fate. This study demonstrates self-tumor tropism of engineered cancer cells and their therapeutic potential when engineered with receptor self-targeted molecules, and it establishes a roadmap toward a safe clinical translation for different cancer types in primary, recurrent, and metastatic settings.

microRNA-7 upregulates death receptor 5 and primes resistant brain tumors to caspase-mediated apoptosis.

Background: MicroRNAs (miRs) are known to play a pivotal role in tumorigenesis, controlling cell proliferation and apoptosis. In this study, we investigated the potential of miR-7 to prime resistant tumor cells to apoptosis in glioblastoma (GBM). Methods: We created constitutive and regulatable miR-7 expression vectors and utilized pharmacological inhibition of caspases and genetic loss of function to study the effect of forced expression of miR-7 on death receptor (DR) pathways in a cohort of GBM with established resistance to tumor necrosis factor apoptosis inducing ligand (TRAIL) and in patient-derived primary GBM stem cell (GSC) lines. We engineered adeno-associated virus (AAV)-miR-7 and stem cell (SC) releasing secretable (S)-TRAIL and utilized real time in vivo imaging and neuropathology to understand the effect of the combined treatment of AAV-miR-7 and SC-S-TRAIL in vitro and in mouse models of GBM from TRAIL-resistant GSC. Results: We show that expression of miR-7 in GBM cells results in downregulation of epidermal growth factor receptor and phosphorylated Akt and activation of nuclear factor-kappaB signaling. This leads to an upregulation of DR5, ultimately priming resistant GBM cells to DR-ligand, TRAIL-induced apoptotic cell death. In vivo, a single administration of AAV-miR-7 significantly decreases tumor volumes, upregulates DR5, and enables SC-delivered S-TRAIL to eradicate GBM xenografts generated from patient-derived TRAIL-resistant GSC, significantly improving survival of mice. Conclusions: This study identifies the unique role of miR-7 in linking cell proliferation to death pathways that can be targeted simultaneously to effectively eliminate GBM, thus presenting a promising strategy for treating GBM.

Tumor Resection Recruits Effector T Cells and Boosts Therapeutic Efficacy of Encapsulated Stem Cells Expressing IFNß in Glioblastomas.

Purpose: Despite tumor resection being the first-line clinical care for glioblastoma (GBM) patients, nearly all preclinical immune therapy models intend to treat established GBM. Characterizing cytoreductive surgery-induced immune response combined with the administration of immune cytokines has the potential of offering a new treatment paradigm of immune therapy for GBMs.Experimental Design: We developed syngeneic orthotopic mouse GBM models of tumor resection and characterized the immune response of intact and resected tumors. We also created a highly secretable variant of immune cytokine IFNß to enhance its release from engineered mouse mesenchymal stem cells (MSC-IFNß) and assessed whether surgical resection of intracranial GBM tumor significantly enhanced the antitumor efficacy of targeted on-site delivery of encapsulated MSC-IFNß.Results: We show that tumor debulking results in substantial reduction of myeloid-derived suppressor cells (MDSC) and simultaneous recruitment of CD4/CD8 T cells. This immune response significantly enhanced the antitumor efficacy of locally delivered encapsulated MSC-IFNß via enhanced selective postsurgical infiltration of CD8 T cells and directly induced cell-cycle arrest in tumor cells, resulting in increased survival of mice. Utilizing encapsulated human MSC-IFNß in resected orthotopic tumor xenografts of patient-derived GBM, we further show that IFNß induces cell-cycle arrest followed by apoptosis, resulting in increased survival in immunocompromised mice despite their absence of an intact immune system.Conclusions: This study demonstrates the importance of syngeneic tumor resection models in developing cancer immunotherapies and emphasizes the translational potential of local delivery of immunotherapeutic agents in treating cancer. Clin Cancer Res; 23(22); 7047-58. ©2017 AACR.

Engineered bifunctional proteins and stem cells: next generation of targeted cancer therapeutics.

Redundant survival signaling pathways and their crosstalk within tumor and/or between tumor and their microenvironment are key impediments to developing effective targeted therapies for cancer. Therefore developing therapeutics that target multiple receptor signaling pathways in tumors and utilizing efficient platforms to deliver such therapeutics are critical to the success of future targeted therapies. During the past two decades, a number of bifunctional multi-targeting antibodies, fusion proteins, and oncolytic viruses have been developed and various stem cell types have been engineered to efficiently deliver them to tumors. In this review, we discuss the design and efficacy of therapeutics targeting multiple pathways in tumors and the therapeutic potential of therapeutic stem cells engineered with bifunctional agents.

Multifunctional receptor-targeting antibodies for cancer therapy.

During the past decade, monospecific antibodies targeting cell-surface receptors in different tumour types have achieved substantial success and have been at the forefront of cancer treatment. However, redundant signalling and crosstalk between different pathways within tumour cells and between tumour cells and their microenvironment can limit the efficacy of receptor-targeted monospecific-based therapies. Advances in antibody engineering technologies have enabled strategies that simultaneously target multiple receptors to circumvent the limitations of conventional monospecific therapies and achieve enhanced therapeutic efficacy. In the past 5 years, a range of multifunctional, receptor-targeting, antibody-based molecules have emerged, which allow targeting of multiple surface receptors on tumour cells and endothelial or immune cells in the tumour microenvironment. In this Review, we discuss the rationales and strategies for the use of multifunctional receptor-targeting antibodies, their mechanisms of action, and the promises and challenges they hold as cancer therapeutics. This knowledge provides opportunities to improve current targeted therapy outcomes for patients with cancer.

Antiangiogenic variant of TSP-1 targets tumor cells in glioblastomas.

Three type-1 repeat (3TSR) domain of thrombospondin-1 is known to have anti-angiogenic effects by targeting tumor-associated endothelial cells, but its effect on tumor cells is unknown. This study explored the potential of 3TSR to target glioblastoma (GBM) cells in vitro and in vivo. We show that 3TSR upregulates death receptor (DR) 4/5 expression in a CD36-dependent manner and primes resistant GBMs to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8/3/7 mediated apoptosis. We engineered human mesenchymal stem cells (MSC) for on-site delivery of 3TSR and a potent and secretable variant of TRAIL (S-TRAIL) in an effort to simultaneously target tumor cells and associated endothelial cells and circumvent issues of systemic delivery of drugs across the blood-brain barrier. We show that MSC-3TSR/S-TRAIL inhibits tumor growth in an expanded spectrum of GBMs. In vivo, a single administration of MSC-3TSR/S-TRAIL significantly targets both tumor cells and vascular component of GBMs, inhibits tumor progression, and extends survival of mice bearing highly vascularized GBM. The ability of 3TSR/S-TRAIL to simultaneously act on tumor cells and tumor-associated endothelial cells offers a great potential to target a broad spectrum of cancers and translate 3TSR/TRAIL therapies into clinics.

A role for metaphase spindle elongation forces in correction of merotelic kinetochore attachments.

During mitosis, equal segregation of chromosomes depends on proper kinetochore-microtubule attachments. Merotelic kinetochore orientation, in which a single kinetochore binds microtubules from both spindle poles [1], is a major cause of chromosome instability [2], which is commonly observed in solid tumors [3, 4]. Using the fission yeast Schizosaccharomyces pombe, we show that a proper force balance between kinesin motors on interpolar spindle microtubules is critical for correcting merotelic attachments. Inhibition of the plus-end-directed spindle elongation motors kinesin-5 (Cut7) and kinesin-6 (Klp9) reduces spindle length, tension at kinetochores, and the frequency of merotelic attachments. In contrast, merotely is increased by deletion of the minus-end-directed kinesin-14 (Klp2) or overexpression of Klp9. Also, Cdk1 regulates spindle elongation forces to promote merotelic correction by phosphorylating and inhibiting Klp9. The role of spindle elongation motors in merotelic correction is conserved, because partial inhibition of the human kinesin-5 homolog Eg5 using the drug monastrol reduces spindle length and lagging chromosome frequency in both normal (RPE-1) and tumor (CaCo-2) cells. These findings reveal unexpected links between spindle forces and correction of merotelic attachments and show that pharmacological manipulation of spindle elongation forces might be used to reduce chromosome instability in cancer cells.

Phosphorylation state defines discrete roles for monopolin in chromosome attachment and spindle elongation.

BACKGROUND: It is unknown how oscillations in Cdk1 activity drive the dramatic changes in chromosome and spindle dynamics that occur at the metaphase/anaphase transition. RESULTS: We show that the Schizosaccharomyces pombe monopolin complex has distinct functions in metaphase and anaphase that are determined by the phosphorylation state of its Mde4 subunit. When Cdk1 activity is high in metaphase, Mde4 is hyperphosphorylated on Cdk1 phosphorylation sites and localizes to kinetochores. A nonphosphorylatable mutant of Mde4 does not localize to kinetochores, appears prematurely on the metaphase spindle, and interferes with spindle dynamics and chromosome segregation, illustrating the importance of Cdk1 phosphorylation in regulating metaphase monopolin activity. When Cdk1 activity drops in anaphase, dephosphorylation of Mde4 triggers monopolin localization to the mitotic spindle, where it promotes spindle elongation and integrity, coupling the late mitotic loss of Cdk1 activity to anaphase spindle dynamics. CONCLUSIONS: Together, these findings illustrate how the sequential phosphorylation and dephosphorylation of monopolin helps ensure the orderly execution of discrete steps in mitosis.

The nucleolar Net1/Cfi1-related protein Dnt1 antagonizes the septation initiation network in fission yeast.

The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe, and Saccharomyces cerevisiae, respectively. One function of these pathways is to keep the Cdc14-family phosphatase, called Clp1 in S. pombe, from being sequestered and inhibited in the nucleolus. In S. pombe, the SIN and Clp1 act as part of a cytokinesis checkpoint that allows cells to cope with cytokinesis defects. The SIN promotes checkpoint function by 1) keeping Clp1 out of the nucleolus, 2) maintaining the cytokinetic apparatus, and 3) halting the cell cycle until cytokinesis is completed. In a screen for suppressors of the SIN mutant cytokinesis checkpoint defect, we identified a novel nucleolar protein called Dnt1 and other nucleolar proteins, including Rrn5 and Nuc1, which are known to be required for rDNA transcription. Dnt1 shows sequence homology to Net1/Cfi1, which encodes the nucleolar inhibitor of Cdc14 in budding yeast. Like Net1/Cfi1, Dnt1 is required for rDNA silencing and minichromosome maintenance, and both Dnt1 and Net1/Cfi1 negatively regulate the homologous SIN and MEN pathways. Unlike Net1/Cfi1, which regulates the MEN through the Cdc14 phosphatase, Dnt1 can inhibit SIN signaling independently of Clp1, suggesting a novel connection between the nucleolus and the SIN pathway.