UMG1/CD3ε-bispecific T-cell engager redirects T-cell cytotoxicity against diffuse large B-cell lymphoma.

UMG1 is a unique epitope of CD43, not expressed by normal cells and tissues of haematopoietic and non-haematopoietic origin, except thymocytes and a minority (<5%) of peripheral blood T lymphocytes. By immunohistochemistry analysis of tissue microarray and pathology slides, we found high UMG1 expression in 20%-24% of diffuse large B-cell lymphomas (DLBCLs), including highly aggressive BCL2high and CD20low cases. UMG1 membrane expression was also found in DLBCL bone marrow-infiltrating cells and established cell lines. Targeting UMG1 with a novel asymmetric UMG1/CD3ε-bispecific T-cell engager (BTCE) induced redirected cytotoxicity against DLBCL cells and was synergistic with lenalidomide. We conclude that UMG1/CD3ε-BTCE is a promising therapeutic for DLBCLs.

Hit identification of novel small molecules interfering with MALAT1 triplex by a structure-based virtual screening.

Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.

A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer.

BACKGROUND: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC). METHODS: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC). RESULTS: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals. CONCLUSION: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

The First-In-Class Anti-AXL×CD3ε Pronectin™-Based Bispecific T-Cell Engager Is Active in Preclinical Models of Human Soft Tissue and Bone Sarcomas.

Sarcomas are heterogeneous malignancies with limited therapeutic options and a poor prognosis. We developed an innovative immunotherapeutic agent, a first-in-class Pronectin™-based Bispecific T-Cell Engager (pAXL×CD3ε), for the targeting of AXL, a TAM family tyrosine kinase receptor highly expressed in sarcomas. AXL expression was first analyzed by flow cytometry, qRT-PCR, and Western blot on a panel of sarcoma cell lines. The T-cell-mediated pAXL×CD3ε cytotoxicity against sarcoma cells was investigated by flow cytometry, luminescence assay, and fluorescent microscopy imaging. The activation and degranulation of T cells induced by pAXL×CD3ε were evaluated by flow cytometry. The antitumor activity induced by pAXL×CD3ε in combination with trabectedin was also investigated. In vivo activity studies of pAXL×CD3ε were performed in immunocompromised mice (NSG), engrafted with human sarcoma cells and reconstituted with human peripheral blood mononuclear cells from healthy donors. Most sarcoma cells showed high expression of AXL. pAXL×CD3ε triggered T-lymphocyte activation and induced dose-dependent T-cell-mediated cytotoxicity. The combination of pAXL×CD3ε with trabectedin increased cytotoxicity. pAXL×CD3ε inhibited the in vivo growth of human sarcoma xenografts, increasing the survival of treated mice. Our data demonstrate the antitumor efficacy of pAXL×CD3ε against sarcoma cells, providing a translational framework for the clinical development of pAXL×CD3ε in the treatment of human sarcomas, aggressive and still-incurable malignancies.

TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability, and telomere dysfunction is an important cause of acquired genomic alterations. Telomeric repeat-containing RNA (TERRA) transcripts are long non-coding RNAs involved in telomere stability through the interaction with shelterin complex. Dysregulation of TERRAs has been reported across several cancer types. We recently identified a small molecule, hit 17, which stabilizes the secondary structure of TERRA. In this study, we investigated in vitro and in vivo anti-MM activities of hit 17. METHODS: Anti-proliferative activity of hit 17 was evaluated in different MM cell lines by cell proliferation assay, and the apoptotic process was analyzed by flow cytometry. Gene and protein expressions were detected by RT-qPCR and western blotting, respectively. Microarray analysis was used to analyze the transcriptome profile. The effect of hit 17 on telomeric structure was evaluated by chromatin immunoprecipitation. Further evaluation in vivo was proceeded upon NCI-H929 and AMO-1 xenograft models. RESULTS: TERRA G4 stabilization induced in vitro dissociation of telomeric repeat-binding factor 2 (TRF2) from telomeres leading to the activation of ATM-dependent DNA damage response, cell cycle arrest, proliferation block, and apoptotic death in MM cell lines. In addition, up-regulation of TERRA transcription was observed upon DNA damage and TRF2 loss. Transcriptome analysis followed by gene set enrichment analysis (GSEA) confirmed the involvement of the above-mentioned processes and other pathways such as E2F, MYC, oxidative phosphorylation, and DNA repair genes as early events following hit 17-induced TERRA stabilization. Moreover, hit 17 exerted anti-tumor activity against MM xenograft models. CONCLUSION: Our findings provide evidence that targeting TERRA by hit 17 could represent a promising strategy for a novel therapeutic approach to MM.

The emerging scenario of immunotherapy for T-cell Acute Lymphoblastic Leukemia: advances, challenges and future perspectives.

T-cell acute lymphoblastic leukemia (T-ALL) is a challenging pediatric and adult haematologic disease still associated with an unsatisfactory cure rate. Unlike B-ALL, the availability of novel therapeutic options to definitively improve the life expectancy for relapsed/resistant patients is poor. Indeed, the shared expression of surface targets among normal and neoplastic T-cells still limits the efficacy and may induce fratricide effects, hampering the use of innovative immunotherapeutic strategies. However, novel monoclonal antibodies, bispecific T-cell engagers (BTCEs), and chimeric antigen receptors (CAR) T-cells recently showed encouraging results and some of them are in an advanced stage of pre-clinical development or are currently under investigation in clinical trials. Here, we review this exciting scenario focusing on most relevant advances, challenges, and perspectives of the emerging landscape of immunotherapy of T-cell malignancies.

Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin.

BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.

The New Microtubule-Targeting Agent SIX2G Induces Immunogenic Cell Death in Multiple Myeloma.

Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.

A Novel Bispecific T-Cell Engager (CD1a x CD3ε) BTCE Is Effective against Cortical-Derived T Cell Acute Lymphoblastic Leukemia (T-ALL) Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy burdened by poor prognosis. While huge progress of immunotherapy has recently improved the outcome of B-cell malignancies, the lack of tumor-restricted T-cell antigens still hampers its progress in T-ALL. Therefore, innovative immunotherapeutic agents are eagerly awaited. To this end, we generated a novel asymmetric (2 + 1) bispecific T-cell engager (BTCE) targeting CD1a and CD3ε (CD1a x CD3ε) starting from the development of a novel mAb named UMG2. UMG2 mAb reacts against CD1a, a glycoprotein highly expressed by cortical T-ALL cells. Importantly, no UMG2 binding was found on normal T-cells. CD1a x CD3ε induced high T-cell mediated cytotoxicity against CD1a+ T-ALL cells in vitro, as demonstrated by the concentration-dependent increase of T-cell proliferation, degranulation, induction of cell surface activation markers, and secretion of pro-inflammatory cytokines. Most importantly, in a PBMC-reconstituted NGS mouse model bearing human T-ALL, CD1a x CD3ε significantly inhibited the growth of human T-ALL xenografts, translating into a significant survival advantage of treated animals. In conclusion, CD1a x CD3ε is a novel BTCE highly active against CD1a-expressing cortical-derived T-ALL cells suitable for clinical development as an effective therapeutic option for this rare and aggressive disease.

miR-22 Modulates Lenalidomide Activity by Counteracting MYC Addiction in Multiple Myeloma.

Background: MYC is a master regulator of multiple myeloma (MM) by orchestrating several pro-tumoral pathways, including reprograming of the miRNA transcriptome. MYC is also involved in the acquirement of resistance to anti-MM drugs, including immunomodulatory imide drugs (IMiDs). Methods: In silico analysis was performed on MM proprietary and on public MMRF-CoMMpass datasets. Western blot and chromatin immunoprecipitation (ChIP) experiments were performed to validate miR-22 repression induced by MYC. Cell viability and apoptosis assays were used to evaluate lenalidomide sensitization after miR-22 overexpression. Results: We found an inverse correlation between MYC and miR-22 expression, which is associated with poor outcome in IMiD-treated MM patients. Mechanistically, we showed that MYC represses transcription of miR-22, which, in turn, targets MYC, thus establishing a feed-forward loop. Interestingly, we found that IMiD lenalidomide increases miR-22 expression by reducing MYC repression and, most importantly, that the combination of lenalidomide with miR-22 mimics results in a synergistic direct and NK-mediated cytotoxic activity. Conclusions: Taken together, our findings indicate that: (1) low miR-22 expression could represent a potential predictive biomarker of poor lenalidomide response in MM patients; and (2) miR-22 reduces MYC oncogenic activity, thus triggering a novel synthetic lethality loop, which sensitizes MM cells to lenalidomide.

miRNAs and lncRNAs as Novel Therapeutic Targets to Improve Cancer Immunotherapy.

Immunotherapy is presently one of the most promising areas of investigation and development for the treatment of cancer. While immune checkpoint-blocking monoclonal antibodies and chimeric antigen receptor (CAR) T-cell-based therapy have recently provided in some cases valuable therapeutic options, the goal of cure has not yet been achieved for most malignancies and more efforts are urgently needed. Noncoding RNAs (ncRNA), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), regulate several biological processes via selective targeting of crucial molecular signaling pathways. Recently, the key roles of miRNA and lncRNAs as regulators of the immune-response in cancer have progressively emerged, since they may act (i) by shaping the intrinsic tumor cell and microenvironment (TME) properties; (ii) by regulating angiogenesis, immune-escape, epithelial-to-mesenchymal transition, invasion, and drug resistance; and (iii) by acting as potential biomarkers for prognostic assessment and prediction of response to immunotherapy. In this review, we provide an overview on the role of ncRNAs in modulating the immune response and the TME. We discuss the potential use of ncRNAs as potential biomarkers or as targets for development or clinical translation of new therapeutics. Finally, we discuss the potential combinatory approaches based on ncRNA targeting agents and tumor immune-checkpoint inhibitor antibodies or CAR-T for the experimental treatment of human cancer.

Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor cure rate for relapsed/resistant patients. Due to the lack of T-cell restricted targetable antigens, effective immune-therapeutics are not presently available and the treatment of chemo-refractory T-ALL is still an unmet clinical need. To develop novel immune-therapy for T-ALL, we generated an afucosylated monoclonal antibody (mAb) (ahuUMG1) and two different bispecific T-cell engagers (BTCEs) against UMG1, a unique CD43-epitope highly and selectively expressed by T-ALL cells from pediatric and adult patients. METHODS: UMG1 expression was assessed by immunohistochemistry (IHC) on a wide panel of normal tissue microarrays (TMAs), and by flow cytometry on healthy peripheral blood/bone marrow-derived cells, on 10 different T-ALL cell lines, and on 110 T-ALL primary patient-derived cells. CD43-UMG1 binding site was defined through a peptide microarray scanning. ahuUMG1 was generated by Genetic Glyco-Engineering technology from a novel humanized mAb directed against UMG1 (huUMG1). BTCEs were generated as IgG1-(scFv)2 constructs with bivalent (2+2) or monovalent (2+1) CD3ε arms. Antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and redirected T-cell cytotoxicity assays were analysed by flow cytometry. In vivo antitumor activity of ahUMG1 and UMG1-BTCEs was investigated in NSG mice against subcutaneous and orthotopic xenografts of human T-ALL. RESULTS: Among 110 T-ALL patient-derived samples, 53 (48.1%) stained positive (24% of TI/TII, 82% of TIII and 42.8% of TIV). Importantly, no expression of UMG1-epitope was found in normal tissues/cells, excluding cortical thymocytes and a minority (<5%) of peripheral blood T lymphocytes. ahUMG1 induced strong ADCC and ADCP on T-ALL cells in vitro, which translated in antitumor activity in vivo and significantly extended survival of treated mice. Both UMG1-BTCEs demonstrated highly effective killing activity against T-ALL cells in vitro. We demonstrated that this effect was specifically exerted by engaged activated T cells. Moreover, UMG1-BTCEs effectively antagonized tumor growth at concentrations >2 log lower as compared with ahuUMG1, with significant mice survival advantage in different T-ALL models in vivo. CONCLUSION: Altogether our findings, including the safe UMG1-epitope expression profile, provide a framework for the clinical development of these innovative immune-therapeutics for this still orphan disease.

In vitro anti-proliferative and anti-bacterial properties of new C7 benzoate derivatives of pinocembrin.

In the present work, the in vitro anti-proliferative and anti-bacterial activities of three semi-synthetic benzoate pinocembrin derivatives, isolated from the aerial parts of Glycyrrhiza glabra L., were investigated. As occurs in most natural compounds, the bioavailability of pinocembrin is very poor, therefore it should be improved by chemical strategies aimed to prolong its shelf life and, consequently, its activity. On this basis, three benzoate derivatives of pinocembrin (a1-a3) were synthesised and assayed in order to ascertain their biological value. Among them, compound a1 showed the highest anti-proliferative activity on a wide panel of cancer cell lines, as well as low toxic effects on non-malignant breast cells. The calculated IC50 values in HeLa and SKBR3 cells were 8.5 and 12.7 µM, respectively. Briefly, a1 treatment increased ROS levels, induced mitochondrial membrane damage leading to necrotic death of HeLa cells. Moreover, a1 displayed a promising anti-bacterial activity.

miR-21 antagonism abrogates Th17 tumor promoting functions in multiple myeloma.

Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.

Quercetin and its Natural Sources in Wound Healing Management.

Giving a glance to the report of Wound Care Market by Product updated in 2017, we can see that wound care market is expected to reach USD 22.01 billion by 2022 from USD 18.35 billion at a CAGR of 3.7%. Numerous factors are driving the growth of this market, including the increasing prevalence of chronic wounds and acute wounds, increasing aged population, rising R&D activities and advancement in the field of wound care research. Advanced wound management products are accounted for the largest market share in 2017. These evidences mean that the wound care research represents a Clinical Emergency other than an interesting Marketing tool. Drug therapies so far fight efficaciously with the opportunistic pathologies derived from chronic wounds, although an unsolved challenge is still finding a useful remedy to correct the impaired wound healing process and overcome the chronic wound state, to avoid bacterial rising and severe pain. Traditional medicinal plants have been widely used in the management of wounds and different plant extracts have been evaluated for their wound healing properties through both in vitro and in vivo studies. Their phytochemical components in particular quercetin, contribute to their remedial properties in wound repair. Quercetin has important biological activities related to the improvement of the wound healing process. The present review discusses and focuses on the latest findings of the wound healing properties of quercetin, alone or as a part of plant extract, and its role as a new frontier in wound repair.

From Vegetable Waste to New Agents for Potential Health Applications: Antioxidant Properties and Effects of Extracts, Fractions and Pinocembrin from Glycyrrhiza glabra L. Aerial Parts on Viability of Five Human Cancer Cell Lines.

Glycyrrhiza glabra cultivation and harvesting produces substantial quantities of aerial parts as waste. With the aim to prospect an innovative valorization of these byproducts, the aerial parts were harvested in May and October and analyzed for their chemical profile, antioxidant properties, and effects on viability of five cancer cell lines. Pinocembrin was the main constituent. A significant protection of lipid peroxidation was observed with the May total extract (IC50 of 4.2 ± 0.4 µg/mL at 30 min of incubation). The effects on viability of HeLa, MCF-7, MDA-MB-231, Caco-2, and PC3 human cancer cells were investigated. All samples shown a remarkable activity with IC50 values below 25 µg/mL. Samples from plants harvested in May exhibited greater activity than those harvested in October. MCF-7 and HeLa were the most sensitive cells with IC50 in the range 2.73-3.01 and 3.28-5.53 µg/mL, respectively. G. glabra aerial parts represent a good source of valuable products.