Intracellular NAD⁺ depletion enhances bortezomib-induced anti-myeloma activity.

We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD⁺ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD⁺ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD⁺ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD⁺ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib.

Targeting NAD+ salvage pathway induces autophagy in multiple myeloma cells via mTORC1 and extracellular signal-regulated kinase (ERK1/2) inhibition.

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach.

The NAD+-dependent histone deacetylase SIRT6 promotes cytokine production and migration in pancreatic cancer cells by regulating Ca2+ responses.

Cytokine secretion by cancer cells contributes to cancer-induced symptoms and angiogenesis. Studies show that the sirtuin SIRT6 promotes inflammation by enhancing TNF expression. Here, we aimed to determine whether SIRT6 is involved in conferring an inflammatory phenotype to cancer cells and to define the mechanisms linking SIRT6 to inflammation. We show that SIRT6 enhances the expression of pro-inflammatory cyto-/chemokines, such as IL8 and TNF, and promotes cell migration in pancreatic cancer cells by enhancing Ca(2+) responses. Via its enzymatic activity, SIRT6 increases the intracellular levels of ADP-ribose, an activator of the Ca(2+) channel TRPM2. In turn, TRPM2 and Ca(2+) are shown to be involved in SIRT6-induced TNF and IL8 expression. SIRT6 increases the nuclear levels of the Ca(2+)-dependent transcription factor, nuclear factor of activated T cells (NFAT), and cyclosporin A, a calcineurin inhibitor that reduces NFAT activity, reduces TNF and IL8 expression in SIRT6-overexpressing cells. These results implicate a role for SIRT6 in the synthesis of Ca(2+)-mobilizing second messengers, in the regulation of Ca(2+)-dependent transcription factors, and in the expression of pro-inflammatory, pro-angiogenic, and chemotactic cytokines. SIRT6 inhibition may help combat cancer-induced inflammation, angiogenesis, and metastasis.

Autophagy in blood cancers: biological role and therapeutic implications.

Autophagy is a cell recycling process the molecular apparatus of which has been identified over the past decade. Autophagy allows cells to survive starvation and inhospitable conditions and plays a key role in numerous physiological functions, including hematopoiesis and immune responses. In hematologic malignancies, autophagy can either act as a chemo-resistance mechanism or have tumor suppressive functions, depending on the context. In addition, autophagy is involved in other important aspects of blood cancers as it promotes immune competence and anti-cancer immunity, and may even help enhance patient tolerance to standard treatments. Approaches exploiting autophagy, either to activate or inhibit it, could find broad application in hematologic malignancies and contribute to improved clinical outcomes. These aspects are discussed here together with a brief introduction to the molecular machinery of autophagy and to its role in blood cell physiology.

Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols.

New derivatives of 1,4-dideoxy-1,4-imino-D-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-ribitol (13, IC(50) ∼2 µM) and its C(18)-analogues (IC(50) <10 µM) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC(50) ∼8 µM) growth of JURKAT cells.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Monoclonal antibodies for non-Hodgkin's lymphoma: state of the art and perspectives.

Monoclonal antibodies have been the most successful therapeutics ever brought to cancer treatment by immune technologies. The use of monoclonal antibodies in B-cell Non-Hodgkin's lymphomas (NHL) represents the greatest example of these advances, as the introduction of the anti-CD20 antibody rituximab has had a dramatic impact on how we treat this group of diseases today. Despite this success, several questions about how to optimize the use of monoclonal antibodies in NHL remain open. The best administration schedules, as well as the optimal duration of rituximab treatment, have yet to be determined. A deeper knowledge of the mechanisms underlying resistance to rituximab is also necessary in order to improve the activity of this and of similar therapeutics. Finally, new antibodies and biological agents are entering the scene and their advantages over rituximab will have to be assessed. We will discuss these issues and present an overview of the most significant clinical studies with monoclonal antibodies for NHL treatment carried out to date.

The use of dendritic cells in cancer immunotherapy.

Cancer immunotherapy aims at eliciting an immune response directed against tumor antigens to help fight off residual tumor cells and thereby improve survival and quality of life of cancer patients. Different immunotherapeutic approaches share the use of dendritic cells (DCs) to present tumor-associated antigens to T-lymphocytes. Ex vivo generated DCs can be loaded with antigens and re-infused to the patients, or they can be used for ex vivo expansion of antitumor lymphocytes. Alternatively, methods exist to target antigens to DCs in vivo without need for ex vivo cell manipulations. The clinical studies have shown that DC administration to patients is safe and induces antigen-specific immunity. However, it seldom elicits objective clinical responses in patients with advanced-stage malignancies. Novel insights into DC and lymphocyte regulation are expected to lead to more effective vaccines in the near future. Meanwhile, efforts are directed at identifying the most appropriate clinical targets for active specific immunotherapies. Data suggests that vaccinations may indeed be beneficial when given in the adjuvant setting rather than to treat metastatic cancers. These issues are discussed here together with an overview of the DC-based antitumor immunotherapy studies.

Pegfilgrastim compared with filgrastim after autologous peripheral blood stem cell transplantation in patients with solid tumours and lymphomas.

To evaluate the safety and efficacy of pegfilgrastim administered as haematological support after autologous peripheral blood stem cell transplantation, we compared 44 patients with solid tumours and lymphomas receiving a 6-mg single dose of pegfilgrastim on day +5 after transplantation to a historical control group of 25 patients receiving filgrastim 5 microg kg(-1) day(-1) starting on day +5. There were no significant differences in haematological recovery nor in the incidence and duration of neutropenic fever. Median duration of grade 4 neutropenia in the pegfilgrastim and filgrastim group was similar. The incidence of grade III-IV mucositis was lower in pegfilgrastim than in filgrastim group due to the significant difference observed among the patients with solid tumours (p = 0.00). The only adverse event considered to be cytokine related was mild to moderate bone pain occurring during haematological recovery. According to the present study design and taking into account the current prices in our institution, the cost of the two drugs was similar in both treatment groups. In conclusion, a single injection of pegfilgrastim administered at day +5 post-transplantation shows comparable safety and efficacy profiles to daily injections of filgrastim and may be cost effective.

Histone deacetylase inhibitors affect dendritic cell differentiation and immunogenicity.

PURPOSE: Histone deacetylases (HDAC) modulate gene transcription and chromatin assembly by modifying histones at the posttranscriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies. Cumulating evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we have evaluated the effects of two HDAC inhibitors currently in clinical use, sodium valproate and MS-275, on human monocyte-derived DCs. EXPERIMENTAL DESIGN: DCs were generated from monocytes through incubation with granulocyte macrophage colony-stimulating factor and interleukin-4. DC maturation was induced by addition of polyinosinic-polycytidylic acid. DC phenotype, immunostimulatory capacity, cytokine secretion, and migratory capacity were determined by flow cytometry, mixed leukocyte reaction, ELISA, and Transwell migration assay, respectively. Nuclear translocation of RelB, IFN regulatory factor (IRF)-3, and IRF-8 were determined by immunoblotting. RESULTS: HDAC inhibition skews DC differentiation by preventing the acquisition of the DC hallmark CD1a and by affecting the expression of costimulation and adhesion molecules. In addition, macrophage inflammatory protein-3beta/chemokine, motif CC, ligand 19-induced migration, immunostimulatory capacity, and cytokine secretion by DCs are also profoundly impaired. The observed defects in DC function on exposure to HDAC inhibitors seem to reflect the obstruction of signaling through nuclear factor-kappaB, IRF-3, and IRF-8. CONCLUSIONS: HDAC inhibitors exhibit strong immunomodulatory properties in human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders.

The proteasome and its inhibitors in immune regulation and immune disorders.

The ubiquitin-proteasome pathway is a well-characterized mechanism deputed to the degradation of intracellular proteins. Proteasomal degradation intervenes in the regulation of numerous cellular functions including signal transduction, apoptosis, cell cycle, and antigen presentation. In vitro and in vivo studies have shown that both normal and malignant cells of the immune system are exquisitely affected by inhibition of proteasome activity. This property is currently exploited in the treatment of multiple myeloma and mantle cell lymphoma, two B-cell malignancies that respond to treatment with the proteasome inhibitor bortezomib. Pharmacological inhibitors of the proteasome also affect function and survival of B and T lymphocytes and of dendritic cells and were shown to reduce autoimmune and inflammatory manifestations in several models of immune-mediated disorders. The present review offers an overview of the mechanisms implicated in the immunomodulatory effects of proteasome inhibitors and discusses prospective future applications for these small molecules in immune and inflammatory diseases.

Reduced intensity conditioning for allograft after cytoreductive autograft in metastatic breast cancer.

The benefits of allografting noted in some malignant diseases might be safely extended to metastatic breast cancer by a combination of cytoreduction with high-dose chemotherapy (HDT) and autologous stem-cell transplant (ASCT) with graft-versus-tumour effect mediated by transplanted donor immune cells with nonmyeloablative allografting (reduced intensity conditioning transplantation, RICT). 17 patients with heavily pretreated disease were given tandem transplants. 13 patients sustained donor engraftment. Three had partial remission after HDT and ASCT and complete remission after RICT; they achieved full chimerism and all developed graft-versus-host disease (GVHD) before regression of cancer. Another patient did not respond to HDT and ASCT but had partial remission after RICT, giving an overall response rate of 24%. Five patients had grade II or higher acute GVHD and five had extensive chronic GVHD. No non-relapse-related deaths occurred during the first 100 days. Five patients (29%) were alive 90-2160 days (median 1320) after RICT. This two-step approach is feasible in patients with metastatic breast cancer.

Cooperative cytotoxicity of proteasome inhibitors and tumor necrosis factor-related apoptosis-inducing ligand in chemoresistant Bcl-2-overexpressing cells.

PURPOSE: Bcl-2 overexpression is frequently detected in lymphoid malignancies, being associated with poor prognosis and reduced response to therapy. Here, we evaluated whether Bcl-2 overexpression affects the cytotoxic activity of proteasome inhibitors taken alone or in association with conventional anticancer drugs or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). EXPERIMENTAL DESIGN: Jurkat cells engineered to overexpress Bcl-2 were treated with proteasome inhibitors (MG132, epoxomicin, and bortezomib), anticancer drugs (etoposide and doxorubicin), TRAIL, or combinations of these compounds. Cell death and loss of mitochondrial transmembrane potential were detected by flow cytometry. Cytosolic relocalization of cytochrome c and SMAC/Diablo, caspase cleavage, and Bcl-2 and Mcl-1 levels were determined by immunoblotting. Nuclear factor-kappaB inhibition was done by retroviral transduction with a dominant-negative mutant of IkappaBalpha. RESULTS: Bcl-2 overexpression results in significant inhibition of apoptosis in response to proteasome inhibitors, antiblastics, and TRAIL. Addition of TRAIL to proteasome inhibitors results in a synergistic cytotoxic effect in Bcl-2-overexpressing cells, whereas this result is not reproduced by the combination of proteasome inhibitors with antiblastic drugs. Importantly, proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of up-regulated Bcl-2. Bcl-2 cleavage to a fragment with putative proapoptotic activity and elimination of antiapoptotic Mcl-1 may both play a role in proteasome inhibitors-TRAIL cooperation. Conversely, nuclear factor-kappaB inhibition by proteasome inhibitors is per se insufficient to explain the observed synergy. CONCLUSIONS: Combined proteasome inhibitors and TRAIL overcome the apoptotic threshold raised by Bcl-2 and may prove useful in the treatment of chemoresistant malignancies with up-regulated Bcl-2.

Systems medicine in colorectal cancer: from a mathematical model toward a new type of clinical trial.

Current colorectal cancer (CRC) treatment guidelines are primarily based on clinical features, such as cancer stage and grade. However, outcomes may be improved using molecular treatment guidelines. Potentially useful biomarkers include driver mutations and somatically inherited alterations, signaling proteins (their expression levels and (post) translational modifications), mRNAs, micro-RNAs and long noncoding RNAs. Moving to an integrated system is potentially very relevant. To implement such an integrated system: we focus on an important region of the signaling network, immediately above the G1-S restriction point, and discuss the reconstruction of a Molecular Interaction Map and interrogating it with a dynamic mathematical model. Extensive model pretraining achieved satisfactory, validated, performance. The model helps to propose future target combination priorities, and restricts drastically the number of drugs to be finally tested at a cellular, in vivo, and clinical-trial level. Our model allows for the inclusion of the unique molecular profiles of each individual patient's tumor. While existing clinical guidelines are well established, dynamic modeling may be used for future targeted combination therapies, which may progressively become part of clinical practice within the near future. WIREs Syst Biol Med 2016, 8:314-336. doi: 10.1002/wsbm.1342 For further resources related to this article, please visit the WIREs website.

Systems Medicine in Oncology: Signaling Network Modeling and New-Generation Decision-Support Systems.

Two different perspectives are the main focus of this book chapter: (1) A perspective that looks to the future, with the goal of devising rational associations of targeted inhibitors against distinct altered signaling-network pathways. This goal implies a sufficiently in-depth molecular diagnosis of the personal cancer of a given patient. A sufficiently robust and extended dynamic modeling will suggest rational combinations of the abovementioned oncoprotein inhibitors. The work toward new selective drugs, in the field of medicinal chemistry, is very intensive. Rational associations of selective drug inhibitors will become progressively a more realistic goal within the next 3-5 years. Toward the possibility of an implementation in standard oncologic structures of technologically sufficiently advanced countries, new (legal) rules probably will have to be established through a consensus process, at the level of both diagnostic and therapeutic behaviors.(2) The cancer patient of today is not the patient of 5-10 years from now. How to support the choice of the most convenient (and already clinically allowed) treatment for an individual cancer patient, as of today? We will consider the present level of artificial intelligence (AI) sophistication and the continuous feeding, updating, and integration of cancer-related new data, in AI systems. We will also report briefly about one of the most important projects in this field: IBM Watson US Cancer Centers. Allowing for a temporal shift, in the long term the two perspectives should move in the same direction, with a necessary time lag between them.

Tumor necrosis factor-related apoptosis-inducing ligand cooperates with anticancer drugs to overcome chemoresistance in antiapoptotic Bcl-2 family members expressing jurkat cells.

PURPOSE: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-x(L) overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model EXPERIMENTAL DESIGN: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-x(L) overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC(6) and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. RESULTS: Bcl-2 and Bcl-x(L) overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-x(L) Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation CONCLUSIONS: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.

APO866 Increases Antitumor Activity of Cyclosporin-A by Inducing Mitochondrial and Endoplasmic Reticulum Stress in Leukemia Cells.

PURPOSE: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have antileukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current antileukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here, we evaluated the antileukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. EXPERIMENTAL DESIGN: The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n = 6; B-CLL, n = 19), and healthy leukocytes. Intracellular nicotinamide adenine dinucleotide (NAD(+)) and ATP levels, mitochondrial transmembrane potential (ΔΨ(m)), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. RESULTS: The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34(+) progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD(+) and ATP shortage, and induced ΔΨ(m) dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. CONCLUSIONS: APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including acute myelogenous leukemia (AML) and B-cell chronic lymphocytic leukemia (B-CLL) samples. Further evaluations of the combination of these agents in clinical setting should be considered.

Advances in dynamic modeling of colorectal cancer signaling-network regions, a path toward targeted therapies.

The interconnected network of pathways downstream of the TGFß, WNT and EGF-families of receptor ligands play an important role in colorectal cancer pathogenesis.We studied and implemented dynamic simulations of multiple downstream pathways and described the section of the signaling network considered as a Molecular Interaction Map (MIM). Our simulations used Ordinary Differential Equations (ODEs), which involved 447 reactants and their interactions.Starting from an initial "physiologic condition", the model can be adapted to simulate individual pathologic cancer conditions implementing alterations/mutations in relevant onco-proteins. We verified some salient model predictions using the mutated colorectal cancer lines HCT116 and HT29. We measured the amount of MYC and CCND1 mRNAs and AKT and ERK phosphorylated proteins, in response to individual or combination onco-protein inhibitor treatments. Experimental and simulation results were well correlated. Recent independently published results were also predicted by our model.Even in the presence of an approximate and incomplete signaling network information, a predictive dynamic modeling seems already possible. An important long term road seems to be open and can be pursued further, by incremental steps, toward even larger and better parameterized MIMs. Personalized treatment strategies with rational associations of signaling-proteins inhibitors, could become a realistic goal.

Dendritic cells transfected with tumor RNA for the induction of antitumor CTL in colorectal cancer.

Dendritic cells (DC) are the most potent antigen-presenting cells known, currently tested for vaccination studies in cancer patients. The use of tumor-derived RNA to load DC overcomes the requirement of defined HLA types and the identification of tumor antigens expressed by the tumors. Here, we show that human monocyte-derived DC generated under serum-free conditions by GM-CSF, IL-4 and TNF-alpha acquire a mature phenotype and expression of the chemokine receptor CCR-7, which plays a pivotal role in DC migration to the afferent lymph nodes. We demonstrate the feasibility of total RNA transfection into such DC using the renal cell carcinoma (RCC) cell line N43-EGFP, which was stably transfected with an EGFP-encoding vector. Moreover, we show that DC transfected with RNA from colorectal cancer cells present HLA class I-restricted antigenic epitopes to induce a primary antitumor CTL response in vitro. Interestingly, the CTL induced by SW480 RNA also recognized another colon cancer line, HCT116, and the RCC line A498. Our results confirm the feasibility of total RNA transfection of serum-free generated DC for the induction of CTL against colon cancer and RCC cells, and support the relevance of shared tumor rejection epitopes between colorectal cancer and RCC.