Alessandro Canella

Microvesicles containing miRNAs promote muscle cell death in cancer cachexia via TLR7

Significance MicroRNAs (miRNAs) are small, noncoding RNAs regulating gene expression. The aberrant expression of miRNAs is commonly associated with cancer. miRNAs can be packaged in exosomes/microvesicles secreted by the cells and involved in cell-to-cell signaling and communication; tumor-secreted miRNAs promote tumor spread and growth in the surrounding microenvironment. Apoptosis is reported to take place in wasting muscle in cancer cachexia, a debilitating syndrome associated with multiple types of cancer, although the mechanism remains elusive. This study shows that tumor-secreted microvesicles contain an elevated expression of miR-21 and induce myoblast apoptosis in cancer cachexia via a Toll-like receptor 7-c-Jun N-terminal kinase-dependent pathway. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression and, in cancers, are often packaged within secreted microvesicles. The cachexia syndrome is a debilitating state of cancer that predominantly results from the loss of skeletal muscle mass, which is in part associated with apoptosis. How tumors promote apoptosis in distally located skeletal muscles has not been explored. Using both tumor cell lines and patient samples, we show that tumor-derived microvesicles induce apoptosis of skeletal muscle cells. This proapoptotic activity is mediated by a microRNA cargo, miR-21, which signals through the Toll-like 7 receptor (TLR7) on murine myoblasts to promote cell death. Furthermore, tumor microvesicles and miR-21 require c-Jun N-terminal kinase activity to regulate this apoptotic response. Together, these results describe a unique pathway by which tumor cells promote muscle loss, which might provide a great insight into elucidating the causes and treatment options of cancer cachexia.

Characterization of Multiple Myeloma Vesicles by Label-Free Relative Quantitation

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell‐to‐cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label‐free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label‐free spectral counting applied to determine relative protein abundances in EVs.

Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers.

UNLABELLED Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patients serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers. BIOLOGICAL SIGNIFICANCE Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.

HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACis) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACis have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACis were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9–5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

Histone Deacetylase Inhibitors Enhance the Therapeutic Potential of Reovirus in Multiple Myeloma.

Multiple myeloma remains incurable and the majority of patients die within 5 years of diagnosis. Reolysin, the infusible form of human reovirus (RV), is a novel viral oncolytic therapy associated with antitumor activity likely resulting from direct oncolysis and a virus-mediated antitumor immune response. Results from our phase I clinical trial investigating single agent Reolysin in patients with relapsed multiple myeloma confirmed tolerability, but no objective responses were evident, likely because the virus selectively entered the multiple myeloma cells but did not actively replicate. To date, the precise mechanisms underlying the RV infectious life cycle and its ability to induce oncolysis in patients with multiple myeloma remain unknown. Here, we report that junctional adhesion molecule 1 (JAM-1), the cellular receptor for RV, is epigenetically regulated in multiple myeloma cells. Treatment of multiple myeloma cells with clinically relevant histone deacetylase inhibitors (HDACi) results in increased JAM-1 expression as well as increased histone acetylation and RNA polymerase II recruitment to its promoter. Furthermore, our data indicate that the combination of Reolysin with HDACi, potentiates RV killing activity of multiple myeloma cells in vitro and in vivo. This study provides the molecular basis to use these agents as therapeutic tools to increase the efficacy of RV therapy in multiple myeloma. Mol Cancer Ther; 15(5); 830–41. ©2016 AACR.

A phase 1 trial of the HDAC inhibitor AR-42 in patients with multiple myeloma and T- and B-cell lymphomas

Abstract Histone deacetylase inhibitors (HDACi) have proven activity in hematologic malignancies, and their FDA approval in multiple myeloma (MM) and T-cell lymphoma highlights the need for further development of this drug class. We investigated AR-42, an oral pan-HDACi, in a first-in-man phase 1 dose escalation clinical trial. Overall, treatment was well tolerated, no DLTs were evident, and the MTD was defined as 40 mg dosed three times weekly for three weeks of a 28-day cycle. One patient each with MM and mantle cell lymphoma demonstrated disease control for 19 and 27 months (ongoing), respectively. Treatment was associated with reduction of serum CD44, a transmembrane glycoprotein associated with steroid and immunomodulatory drug resistance in MM. Our findings indicate that AR-42 is safe and that further investigation of AR-42 in combination regimens for the treatment of patients with lymphoma and MM is warranted. Trial registration: http://clinicaltrials.gov/ct2/show/NCT01129193

Efficacy of onalespib, a long-acting second-generation HSP90 inhibitor, as a single agent and in combination with temozolomide against malignant gliomas

Purpose: HSP90, a highly conserved molecular chaperone that regulates the function of several oncogenic client proteins, is altered in glioblastoma. However, HSP90 inhibitors currently in clinical trials are short-acting, have unacceptable toxicities, or are unable to cross the blood–brain barrier (BBB). We examined the efficacy of onalespib, a potent, long-acting novel HSP90 inhibitor as a single agent and in combination with temozolomide (TMZ) against gliomas in vitro and in vivo. Experimental Design: The effect of onalespib on HSP90, its client proteins, and on the biology of glioma cell lines and patient-derived glioma-initiating cells (GSC) was determined. Brain and plasma pharmacokinetics of onalespib and its ability to inhibit HSP90 in vivo were assessed in non–tumor-bearing mice. Its efficacy as a single agent or in combination with TMZ was assessed in vitro and in vivo using zebrafish and patient-derived GSC xenograft mouse glioma models. Results: Onalespib-mediated HSP90 inhibition depleted several survival-promoting client proteins such as EGFR, EGFRvIII, and AKT, disrupted their downstream signaling, and decreased the proliferation, migration, angiogenesis, and survival of glioma cell lines and GSCs. Onalespib effectively crossed the BBB to inhibit HSP90 in vivo and extended survival as a single agent in zebrafish xenografts and in combination with TMZ in both zebrafish and GSC mouse xenografts. Conclusions: Our results demonstrate the long-acting effects of onalespib against gliomas in vitro and in vivo, which combined with its ability to cross the BBB support its development as a potential therapeutic agent in combination with TMZ against gliomas. Clin Cancer Res; 23(20); 6215–26. ©2017 AACR.

The potential diagnostic power of extracellular vesicle analysis for multiple myeloma.

ABSTRACT Multiple myeloma (MM) is a hematologic malignancy of plasma cells (PCs). In the United States, MM accounts for approximately 1% of all diagnoses and 2% of all cancer-related deaths. Although MM is a treatable disease, most patients eventually relapse, and despite the development of numerous treatment options it is still considered incurable. Mechanisms of communication between MM-PCs and bone marrow microenvironment, including cell-cell contacts and release of pro-survival factors, promote cancer cell survival and drug resistance. Recently, the importance of extracellular vesicles (EVs) as mechanisms of communication between MM cells and other cells in the microenvironment has been reported. In this review, the authors provide the update on the biology and clinical aspects of EVs in MM.

Abstract 2519: A novel role for brachyury as a key regulator of sonic hedgehog signaling (Shh) and maintenance of stemness in gliomas

Glioblastomas are the most common and aggressive form of adult primary brain tumors and are associated with a dismal prognosis. Accumulating evidences suggest that a subset of therapy-resistant cancer stem cells are responsible for recurrence in tumors targeting which may help in gliomas. We identified a significant overexpression of Brachyury (T-box transcription factor) in correlation with glioma histological grade when compared to non-tumor brain controls, with the highest expression being in grade IV gliomas. We also observed increased expression of Brachyury in several glioma cell lines and stem cells (GSCs) when compared to normal human astrocytes. Serum-induced differentiation of GSCs resulted in loss of Brachyury expression along with decreased stem-cell marker levels in these cells, which prompted us to study the potential role of Brachyury in glioma stemness and to delineate the underlying signaling mechanisms. Specific shRNA-mediated knockdown of Brachyury significantly decreased proliferation, migration and invasion in glioma cell lines. Brachyury suppression led to inhibition of sphere formation of GSCs and decreased stem cells marker expression including CD133, nestin and Nanog. Brachyury knockdown also inhibited the activation of sonic hedgehog (Shh) signaling and decreased Gli1 levels in these cells. On the other hand, stable overexpression of Brachyury resulted in the activation of Shh pathway and elevated stem cell marker expression in GSCs. Based on these in vitro findings, our future studies will be focused on determining the role of Brachyury in orthotopic tumor growth in vivo. In summary, our data reveals a novel role for Brachyury in the regulation of tumor invasiveness and stemness, and indicates a potential for therapeutic targeting of Brachyury in patients with gliomas. Citation Format: Divya Kesanakurti, Alessandro Canella, Jihong Xu, Vinay K. Puduvalli. A novel role for brachyury as a key regulator of sonic hedgehog signaling (Shh) and maintenance of stemness in gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2519.

Abstract 4158: Novel PAK4-mediated regulation of endothelial CXCL1/CXCR2 signaling and angiogenesis in glioblastoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Increased endothelial cell proliferation and aberrant neovascularization are pathological hallmarks of glioblastoma that correlate with clinical outcome. Recent studies have also implicated ionizing radiation (IR)-induced angiogenesis as a potential cause for tumor recurrence and metastases in several malignancies. Its impact in gliomas has not been characterized. Factors that drive endothelial proliferation, angiogenesis and interaction of endothelial cells with tumor cells in the context of radiotherapy are potentially novel targets for glioma therapy. Our previous studies demonstrated high levels of expression of p21-activated kinase 4 (PAK4) in gliomas; analyses of tumor cell medium (CM) prepared from shRNA-mediated PAK4-knockdown cells using an angiogenesis antibody array showed a significant decrease in key angiogenic factors including CXCL1. Treatment of human brain microvascular endothelial cells (HBMECs) with CM from PAK4-knockdown cells (PAK4-kd CM) significantly inhibited proliferation, migration and microtubule formation compared with CM from control and scrambled vector-expressing glioma cells. PAK4-kd CM treatment also suppressed CXCR2 activation and decreased the expression levels of p-STAT3, VEGF, MMP-2 and cyclinD1 in HBMECs. Additionally, treatment with CM from ionizing radiation (IR, 8 Gy)-treated cells resulted in elevated CXCL1, MMP-2 and VEGF levels in HBMECs, and induced high levels of endothelial capillary network formation on matrigel, indicating an IR-induced angiogenesis in these cells. Conversely, PAK4-kd inhibited IR-induced angiogenesis and decreased p-STAT3, VEGF and MMP-2 levels in IR-treated HBMECS. Further, in vivo experiments with orthotopic intracranial tumors in nude mice showed growth suppression in PAK4-knockdown tumors compared to control tumors. Immunohistochemical analyses of brain tumor sections revealed a significant decrease in CXCL1, VEGF and MMP-2 in PAK4.sh tumors. In summary, our studies emphasize a novel regulation of angiogenic switch and endothelial CXCL1/CXCR2 signaling by PAK4 in tumor cells, thereby, suggests the role of PAK4 as a critical mediator of tumor-endothelial cell interactions in glioblastoma. Abrogation of key angiogenic signaling after IR treatment and inhibition of in vivo tumor growth by PAK4 suppression indicates its therapeutic potential in the treatment of glioma. Citation Format: Divya Kesanakurti, Jihong Xu, Alessandro Canella, Prabhakaran Nagarajan, Balveen Kaur, Vinay K. Puduvalli. Novel PAK4-mediated regulation of endothelial CXCL1/CXCR2 signaling and angiogenesis in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2015-4158