Nanoparticle targeting of neutrophil glycolysis prevents lung ischemia-reperfusion injury.

Neutrophils exacerbate pulmonary ischemia-reperfusion injury (IRI) resulting in poor short and long-term outcomes for lung transplant recipients. Glycolysis powers neutrophil activation, but it remains unclear if neutrophil-specific targeting of this pathway will inhibit IRI. Lipid nanoparticles containing the glycolysis flux inhibitor 2-deoxyglucose (2-DG) were conjugated to neutrophil-specific Ly6G antibodies (NP-Ly6G[2-DG]). Intravenously administered NP-Ly6G(2-DG) to mice exhibited high specificity for circulating neutrophils. NP-Ly6G(2-DG)-treated neutrophils were unable to adapt to hypoglycemic conditions of the lung airspace environment as evident by the loss of demand-induced glycolysis, reductions in glycogen and ATP content, and an increased vulnerability to apoptosis. NP-Ly6G(2-DG) treatment inhibited pulmonary IRI following hilar occlusion and orthotopic lung transplantation. IRI protection was associated with less airspace neutrophil extracellular trap generation, reduced intragraft neutrophilia, and enhanced alveolar macrophage efferocytotic clearance of neutrophils. Collectively, our data show that pharmacologically targeting glycolysis in neutrophils inhibits their activation and survival leading to reduced pulmonary IRI.

Liposomal phytohemagglutinin: In vivo T-cell activator as a novel pan-cancer immunotherapy.

Immunotherapy is an attractive approach for treating cancer. T-cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T-cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T-cell exhaustion.

Synthesis, equilibrium, and biological study of a C-7 glucose boronic acid derivative as a potential candidate for boron neutron capture therapy.

The synthesis of d-glucoheptose derivative containing a boronic moiety is described herein. Starting from benzyl 6,7-dideoxy-2,3,4-tri-O-benzyl-ß-d-gluco-ept-6-enopyranoside, the introduction of the boronic acid was performed through a metathesis reaction by using MIDA vinyl boronic acid and the 2nd generation Grubbs catalyst. Hydrogenation led to the final product in only two reaction steps. This new sugar-containing boronic acid in the skeleton could mimic carbohydrate behavior and follow the glucose uptake in living cells. The in vitro toxicity tests performed in fibroblasts and glioma tumor cell lines showed minimal toxicity. Boron uptake measured using ICP-MS was minimal in fibroblasts, while in glioma cells showed a value of 6 ng of total boron accumulation per mg of cells, implying that compound 1a is able to accumulate selectively in the tumor tissues compared to normal.

Thermal Sensitive Liposomes Improve Delivery of Boronated Agents for Boron Neutron Capture Therapy.

PURPOSE: Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation requires sufficient tumor boron delivery while minimizing nonspecific accumulation. METHODS: Thermal sensitive liposomes (TSLs) were designed to have a stable drug payload at physiological temperatures but engineered to have high permeability under mild hyperthermia. RESULTS: We found that TSLs improved the tumor-specific delivery of boronophenylalanine (BPA) and boronated 2-nitroimidazole derivative B-381 in D54 glioma cells. Uniquely, the 2-nitroimidazole moiety extended the tumor retention of boron content compared to BPA. CONCLUSION: This is the first study to show the delivery of boronated compounds using TSLs for BNCT, and these results will provide the basis of future clinical trials using TSLs for BNCT.

Nanoparticle delivery systems, general approaches, and their implementation in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that remains incurable, with relapse rates >90%. The main limiting factor for the effective use of chemotherapies in MM is the serious side effects caused by these drugs. The emphasis in cancer treatment has shifted from cytotoxic, non-specific chemotherapies to molecularly targeted and rationally designed therapies showing greater efficacy and fewer side effects. Traditional chemotherapy has shown several disadvantages such as lack of targeting capabilities, systemic toxicity, and side effects; low therapeutic index, as well as most anticancer drugs, has poor water solubility. Nanoparticle delivery systems (NPs) are capable of targeting large doses of chemotherapies into the target area while sparing healthy tissues, overcoming the limitations of traditional chemotherapy. Here, we review the current state of the art in nanoparticle-based strategies designed to treat MM. Many nanoparticle delivery systems have been studied for myeloma using non-targeted NPs (liposomes, polymeric NPs, and inorganic NPs), triggered NPs, as well as targeted NPs (VLA-4, ABC drug transporters, bone microenvironment targeting). The results in preclinical and clinical studies are promising; however, there remains much to be learned in the emerging field of nanomedicine in myeloma.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

CXCR7-dependent angiogenic mononuclear cell trafficking regulates tumor progression in multiple myeloma.

The CXCR4/stromal cell-derived factor-1 (SDF-1) axis is essential for cell trafficking and has been shown to regulate tumor progression and metastasis in many tumors including multiple myeloma (MM). A second chemokine receptor for SDF-1, CXCR7 was discovered recently and found on activated endothelial cells. We examined the role of CXCR7 in angiogenic mononuclear cells (AMCs) trafficking in MM. Our data demonstrate that AMCs are circulating in patients with MM and in vivo studies show that they specifically home to areas of MM tumor growth. CXCR7 expression is important for regulating trafficking and homing of AMCs into areas of MM tumor growth and neoangiogenesis. We demonstrate that the CXCR7 inhibitor, POL6926, abrogated trafficking of AMCs to areas of MM tumor progression leading to a significant inhibition of tumor progression. These effects were through regulation of endothelial cells and not through a direct tumor effect, indicating that targeting a bone marrow microenvironmental cell can lead to a delay in MM tumor progression. In conclusion, our studies demonstrate that CXCR7 may play an important role in the regulation of tumor progression in MM through an indirect effect on the recruitment of AMCs to areas of MM tumor growth in the bone marrow niche.

A Hypoxia-Targeted Boron Neutron Capture Therapy Agent for the Treatment of Glioma.

PURPOSE: Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells. METHODS: B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo. RESULTS: B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively. CONCLUSIONS: B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on hypoxic tumor metabolism. Further studies are warranted to evaluate B-381 and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma.

Advancements in Tumor Targeting Strategies for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a promising cancer therapy modality that utilizes the nuclear capture reaction of epithermal neutrons by boron-10 resulting in a localized nuclear fission reaction and subsequent cell death. Since cellular destruction is limited to approximately the diameter of a single cell, primarily only cells in the neutron field with significant boron accumulation will be damaged. However, the emergence of BNCT as a prominent therapy has in large part been hindered by a paucity of tumor selective boron containing agents. While L-boronophenylalanine and sodium borocaptate are the most commonly investigated clinical agents, new agents are desperately needed due to their suboptimal tumor selectivity. This review will highlight the various strategies to improve tumor boron delivery including: nucleoside and carbohydrate analogs, unnatural amino acids, porphyrins, antibody-dendrimer conjugates, cationic polymers, cell-membrane penetrating peptides, liposomes and nanoparticles.

PI3KCA plays a major role in multiple myeloma and its inhibition with BYL719 decreases proliferation, synergizes with other therapies and overcomes stroma-induced resistance.

The phosphatidylinositide 3-kinase (PI3K) pathway is activated and correlated with drug resistance in multiple myeloma (MM). In the present study we investigated the role of PI3KCA (PI3K-α) in the progression and drug resistance in MM. We showed that the gene expression of PI3KCA isoform was higher in MM compared to normal subjects. BYL719, a novel and specific PI3KCA inhibitor inhibited the survival of primary MM cells and cell lines but not normal peripheral blood mononuclear cells. BYL719 induced the apoptosis of MM cells and inhibited their cell cycle by causing G1 arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells cycle signalling, and induced apoptosis signalling in MM cells. Finally, BYL719 synergized with bortezomib and carfilzomib, and overcame drug resistance induced by bone marrow stroma. These results were confirmed using in silico simulation of MM cell lines, BYL719 and bortezomib, and showed similar trends in survival, proliferation, apoptosis, cell signalling and synergy with drugs. In conclusion, PI3KCA plays a major role in proliferation and drug resistance of MM cells, the effects of which were inhibited with BYL719. These results provide a preclinical basis for a future clinical trial of BYL719 in MM as a single agent or in combination with other drugs.

Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma.

The Hedgehog (Hh) pathway is required for cell-fate determination during the embryonic life, as well as cell growth and differentiation in the adult organism, where the inappropriate activation has been implicated in several cancers. Here we demonstrate that Hh signaling plays a significant role in growth and survival of multiple myeloma (MM) cells. We observed that CD138(+) MM cells express Hh genes and confirmed Smoothened (Smo)-dependent Hh signaling in MM using a novel synthetic Smo inhibitor, NVP-LDE225 (Novartis), which decreased MM cell viability by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of Hh activity. In addition, we detected a nuclear localization of Gli1 in MM cells, which is completely abrogated by Forskolin, a Gli1-modulating compound, confirming Smo-independent mechanisms leading to Hh activation in MM. Finally, we identified that bone marrow stromal cells are a source of the Shh ligand, although they are resistant to the Hh inhibitor because of defective Smo expression and Ptch1 up-regulation. Further in vitro as well as in vivo studies showed antitumor efficacy of NVP-LDE225 in combination with bortezomib. Altogether, our data demonstrate activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for testing Hh inhibitors in clinical trials to improve MM patient outcome.

P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment.

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in the pathogenesis of MM and in the development of drug resistance by MM cells. Selectins are involved in extravasation and homing of leukocytes to target organs. In the present study, we focused on adhesion dynamics that involve P-selectin glycoprotein ligand-1 (PSGL-1) on MM cells and its interaction with selectins in the BM microenvironment. We show that PSGL-1 is highly expressed on MM cells and regulates the adhesion and homing of MM cells to cells in the BM microenvironment in vitro and in vivo. This interaction involves both endothelial cells and BM stromal cells. Using loss-of-function studies and the small-molecule pan-selectin inhibitor GMI-1070, we show that PSGL-1 regulates the activation of integrins and downstream signaling. We also document that this interaction regulates MM-cell proliferation in coculture with BM microenvironmental cells and the development of drug resistance. Furthermore, inhibiting this interaction with GMI-1070 enhances the sensitization of MM cells to bortezomib in vitro and in vivo. These data highlight the critical contribution of PSGL-1 to the regulation of growth, dissemination, and drug resistance in MM in the context of the BM microenvironment.

Hypoxia promotes dissemination of multiple myeloma through acquisition of epithelial to mesenchymal transition-like features.

The spread of multiple myeloma (MM) involves (re)circulation into the peripheral blood and (re)entrance or homing of MM cells into new sites of the BM. Hypoxia in solid tumors was shown to promote metastasis through activation of proteins involved in the epithelial-mesenchymal transition (EMT) process. We hypothesized that MM-associated hypoxic conditions activate EMT-related proteins and promote metastasis of MM cells. In the present study, we have shown that hypoxia activates EMT-related machinery in MM cells, decreases the expression of E-cadherin, and, consequently, decreases the adhesion of MM cells to the BM and enhances egress of MM cells to the circulation. In parallel, hypoxia increased the expression of CXCR4, consequently increasing the migration and homing of circulating MM cells to new BM niches. Further studies to manipulate hypoxia to regulate tumor dissemination as a therapeutic strategy are warranted.

LNA-mediated anti-miR-155 silencing in low-grade B-cell lymphomas.

miR-155 acts as an oncogenic miR in B-cell lymphoproliferative disorders, including Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. However, efficient targeting of miRs in tumor cells in vivo remains a significant challenge for the development of miR-155-based therapeutics for the treatment of B-cell malignancies. In the present study, we show that an 8-mer locked nucleic acid anti-miR-155 oligonucleotide targeting the seed region of miR-155 inhibits WM and chronic lymphocytic leukemia cell proliferation in vitro. Moreover, anti-miR-155 delivered systemically showed uptake in the BM CD19(+) cells of WM-engrafted mice, resulting in the up-regulation of several miR-155 target mRNAs in these cells, and decreased tumor growth significantly in vivo. We also found miR-155 levels to be elevated in stromal cells from WM patients compared with control samples. Interestingly, stromal cells from miR-155-knockout mice led to significant inhibition of WM tumor growth, indicating that miR-155 may also contribute to WM proliferation through BM microenvironmental cells. The results of the present study highlight the therapeutic potential of anti-miR-155-mediated inhibition of miR-155 in the treatment of WM.

Targeting survival and cell trafficking in multiple myeloma and Waldenstrom macroglobulinemia using pan-class I PI3K inhibitor, buparlisib.

The phosphatidylinositol-3 kinase (PI3K) pathway is activated in multiple myeloma (MM) and Waldenstrom Macroglobulenima (WM), and plays a crucial role in tumor progression and drug resistance. In this study, we characterized the role of pan-class I PI3K inhibition on cell trafficking and survival of MM and WM cells. We tested the effect of pan-class I PI3K inhibition by siRNA silencing or pharmacologic inhibition with buparlisib on MM cell survival, apoptosis and cell cycle in vitro and tumor growth and mobilization of MM cells in vivo. We then evaluated buparlisib-dependent mechanisms of induced MM cell mobilization. Moreover, the effect of buparlisib on cell survival, apoptosis, and adhesion of WM cells to bone marrow stromal cells (BMSCs) has been evaluated. We showed that buparlisib induced toxicity in MM cells, supported by induction of apoptosis and cell cycle arrest. Buparlisib was also found to reduce tumor progression in vivo. Importantly, buparlisib enhanced MM cell mobilization in vivo which was driven by decreased adhesion of MM cells to BMSCs and increased chemotaxis via up-regulation of CXCR4 expression. Similar to its effects on MM cells, buparlisib also induced cell survival and apoptosis, and decreased adhesion in WM cells. These data highlight the critical contribution of class I PI3K signaling to the regulation of survival and cell dissemination in B-cell malignancies.

Integrin ß7-mediated regulation of multiple myeloma cell adhesion, migration, and invasion.

Integrin-ß7 (ITGB7) mRNA is detected in multiple myeloma (MM) cells and its presence is correlated with MAF gene activation. Although the involvement of several integrin family members in MM-stoma cell interaction is well documented, the specific biologic functions regulated by integrin-ß7 in MM are largely unknown. Clinically, we have correlated integrin-ß7 expression in MM with poor survival outcomes post autologous stem cell transplantation and postsalvage therapy with bortezomib. Functionally, we have found that shRNA-mediated silencing of ITGB7 reduces MM-cell adhesion to extra-cellular matrix elements (fibronectin, E-cadherin) and reverses cell-adhesion-mediated drug resistance (CAM-DR) sensitizing them to bortezomib and melphalan. In addition, ITGB7 silencing abrogated MM-cell transwell migration in response to SDF1α gradients, reduced vessel density in xenografted tumors, and altered MM cells in vivo homing into the BM. Mechanistically, ITGB7 knockdown inhibited focal adhesion kinase (FAK) and Src phosphorylation, Rac1 activation, and SUMOylation, reduced VEGF production in MM-BM stem cell cocultures and attenuated p65-NF-κB activity. Our findings support a role for integrin-ß7 in MM-cell adhesion, migration, and BM homing, and pave the way for a novel therapeutic approach targeting this molecule.

Defining the role of TORC1/2 in multiple myeloma.

Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment to regulate multiple cellular processes. Rapamycin and its analogs have not shown significant activity in multiple myeloma (MM), likely because of the lack of inhibition of TORC2. In the present study, we investigated the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. TORC1/2 knock-down led to significant inhibition of the proliferation of MM cells, even in the presence of BM stromal cells. We also tested INK128, a dual TORC1/2 inhibitor, as a new therapeutic agent against these MM cell lines. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin), even in the presence of cytokines or stromal cells. In vitro and in vivo studies showed that p-4EBP1 and p-Akt inhibition could be predictive markers of TORC2 inhibition in MM cell lines. Dual TORC1/2 inhibition showed better inhibition of adhesion to BM microenvironmental cells and inhibition of homing in vivo. These studies form the basis for further clinical testing of TORC1/2 inhibitors in MM.

Fatty acid binding proteins in multiple myeloma.

Fatty acid binding proteins (FABPs) transport fatty acids (FA) into cells as an energy source, and their inhibition suppressed tumor proliferation in solid tumors. Multiple myeloma (MM) is a hematologic malignancy, known for disrupted protein metabolism including high proteasome activity, where proteasome inhibitors made a dramatic improvement in its treatment. Recent discovery found FABPs as a novel metabolic pathway in MM, which will have an impact on understanding the biology and on therapeutic application in MM.

Selective inhibition of chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in Waldenstrom macroglobulinemia.

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.