Molecular Analysis of Elements of Melanoma Insensitivity to TCR-Engineered Adoptive Cell Therapy.

Metastatic melanoma accounts for the highest number of skin cancer-related deaths. Traditional treatments are ineffective due to their inability to induce tumor regression at a high rate. Newer treatments such as immune checkpoint inhibitors (ICI), targeted therapy (BRAFi and MEKi), and T cell receptor (TCR)-engineered T cells aim to increase the ability of the host immune system to recognize and eradicate tumors. ICIs inhibit negative regulatory mechanisms and boost the antitumor activity of the host's immune system, while targeted therapy directed against aberrant signaling molecules (BRAF and MEK) will block the uncontrolled proliferation and expansion of melanomas. The basis of the TCR-engineered T cell strategy is to transduce host T cells with antigen-specific TCRα/ß chains to produce high-affinity T cells for tumor-associated antigens. TCR-transgenic T cells are expanded and activated ex vivo and reinfused into patients to increase the targeting of cancer cells. While these treatments have had varyingly favorable results, their efficacy is limited due to inherent or acquired resistance. Various mechanisms explain melanoma immune-resistance, including the loss or downregulation of the MCH/peptide complex, aberrant activity of signaling pathways, and altered dynamics of apoptotic machinery. Collectively, these mechanisms confer melanoma resistance to apoptotic stimuli delivered by T cells despite a fully functional and effective antitumor immune response. Identification of biomarkers, combination treatment, and the use of CAR T cells are among the approaches that can potentially circumvent melanoma's resistance to immunotherapy.

Rescue of cell cycle progression in BRAFV600E inhibitor-resistant human melanoma by a chromatin modifier.

The BRAFV600E-specific inhibitor vemurafenib blocks mitogen-activated protein kinase pathway and induces cell cycle arrest at G0/G1 phase leading to apoptosis of melanomas. To gain an understanding of the dynamics of cell cycle regulation during vemurafenib therapy, we analyzed several vemurafenib-resistant human melanoma sublines derived from BRAFV600E harboring vemurafenib-sensitive parental lines. Vemurafenib provoked G0/G1 phase arrest in parental but not in vemurafenib-resistant sublines. We hypothesized that refractoriness of vemurafenib-resistant sublines to vemurafenib-mediated cell cycle inhibition can be partially rescued by the chromatin modifier suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid promoted G2/M arrest at expense of S phase irrespective of vemurafenib sensitivity. In parental lines, combination of suberoylanilide hydroxamic acid and vemurafenib induced both G0/G1 arrest and apoptosis, whereas in vemurafenib-resistant sublines combination induced G0/G1 as well as G2/M arrest resulting in dramatic cytostasis. Vemurafenib-resistant sublines exhibited extracellular signal-regulated protein kinases 1 and 2 but not AKT and hyperphosphorylation. Gene expression profiling revealed mitogen-activated protein kinase hyperactivation and deregulations of cyclins and cyclin-dependent kinases in vemurafenib-resistant sublines, all of which were reversed by suberoylanilide hydroxamic acid; changes that may explain the cytostatic effects of suberoylanilide hydroxamic acid. These results suggest that unresponsiveness of vemurafenib-resistant sublines to the biological effects of vemurafenib may be amenable by suberoylanilide hydroxamic acid. These in vitro results, while require further investigation, may provide rational biological basis for combination therapy in the management of vemurafenib-resistant melanoma.

Histone deacetylase inhibitor sensitizes apoptosis-resistant melanomas to cytotoxic human T lymphocytes through regulation of TRAIL/DR5 pathway.

Modern immune therapies (PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer) have remarkably improved the response rates of metastatic melanoma. These modalities rely on the killing potential of CTL as proximal mediator of antimelanoma responses. Mechanisms of tumor resistance to and the predominant cytotoxic pathway(s) used by melanoma-reactive CTL are important outcome determinants. We hypothesized that downmodulation of death receptors (DRs) in addition to aberrant apoptotic signaling might confer resistance to death signals delivered by CTL. To test these two hypotheses, we used an in vitro model of MART CTL-resistant melanoma sublines. TCR-transgenic and patient-derived CTLs used the TRAIL cytotoxic pathway through DR5. Furthermore, recombinant human TRAIL and drozitumab (anti-DR5 agonistic mAb) were used to explicitly verify the contribution of the DR5/TRAIL pathway in killing melanomas. CTL resistance was due to DR5 downregulation and an inverted ratio of pro- to antiapoptotic molecules, both of which were reversed by the histone deacetylase inhibitor suberoylanilide hydroxanic acid. Apoptosis negative (c-IAP-2 and Bcl-xL) and positive (DR5) regulators were potential incriminators partly regulating CTL sensitivity. These preclinical findings suggest that exposure to this chromatin remodeling drug of immune-resistant melanomas can skew toward an intracellular proapoptotic milieu, increase DR expression, and overcome acquired immune resistance.

Immunotherapy of Hematological Malignancies of Human B-Cell Origin with CD19 CAR T Lymphocytes.

Acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) are hematological malignancies with high incidence rates that respond relatively well to conventional therapies. However, a major issue is the clinical emergence of patients with relapsed or refractory (r/r) NHL or ALL. In such circumstances, opportunities for complete remission significantly decline and mortality rates increase. The recent FDA approval of multiple cell-based therapies, Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), Tecartus (Brexucabtagene autoleucel KTE-X19), and Breyanzi (Lisocabtagene Maraleucel), has provided hope for those with r/r NHL and ALL. These new cell-based immunotherapies use genetically engineered chimeric antigen receptor (CAR) T-cells, whose success can be attributed to CAR's high specificity in recognizing B-cell-specific CD19 surface markers present on various B-cell malignancies and the subsequent initiation of anti-tumor activity. The efficacy of these treatments has led to promising results in many clinical trials, but relapses and adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity (NT) remain pervasive, leaving areas for improvement in current and subsequent trials. In this review, we highlight the current information on traditional treatments of NHL and ALL, the design and manufacturing of various generations of CAR T-cells, the FDA approval of Kymriah, Yescarta Tecartus, and Breyanzi, and a summary of prominent clinical trials and the notable disadvantages of treatments. We further discuss approaches to potentially enhance CAR T-cell therapy for these malignancies, such as the inclusion of a suicide gene and use of FDA-approved drugs.

T cell receptor (TCR)-transgenic CD8 lymphocytes rendered insensitive to transforming growth factor beta (TGFß) signaling mediate superior tumor regression in an animal model of adoptive cell therapy.

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFß), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFß by transduction with a TGFß dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.

Regulation of apoptosis-associated genes by histone deacetylase inhibitors: implications in cancer therapy.

Anomalous gene regulation, dictated by epigenetic modifications, is a universal characteristic of cancer cells. Histone deacetylases (HDACs) are an important class of enzymes that influence gene expression by the removal of acetyl groups from histones leading to chromatin remodeling and transcriptional suppression of key apoptosis and cell cycle regulatory genes. Histone deacetylase inhibitors (HDACis) are a novel category of anticancer pharmacological agents developed to counter the actions of HDACs, thus, inducing an array of cellular consequences, such as apoptosis, cell cycle arrest, generation of reactive oxygen species, inhibition of angiogenesis, and autophagy. Suberoylanilide hydroxamic acid (SAHA, Zolinza, Vorinostat), is currently the only Food and Drug Administration-approved HDACi for the treatment of cutaneous T-cell lymphoma. SAHA and other HDACis have shown selective toxicity toward malignant cells while sparing the surrounding normal cells. In addition to this specificity, their regulation of apoptosis-associated genes and the synergistic augmentation of apoptotic events when used simultaneously with other anticancer agents such as conventional chemotherapies, radiation, inhibitors of DNA methylation, and proteasome inhibitors make HDACis potential novel arsenals in the battle against cancer. Herein I review epigenetic modifications, discuss the various mechanisms of HDACi-induced effects, in particular modulation of expression of apoptosis-associated gene products, and highlight SAHA and its antitumor functions.

Development of rituximab-resistant lymphoma clones with altered cell signaling and cross-resistance to chemotherapy.

Immunotherapy with rituximab (chimeric anti-CD20 monoclonal antibody, Rituxan), alone or in conjunction with chemotherapy, has significantly improved the treatment outcome of lymphoma patients. Via an elusive mechanism, a subpopulation of patients becomes unresponsive and/or relapses. To recapitulate various aspects of acquired resistance, rituximab-resistant (RR) clones were established from lymphoma lines and compared with parental cells. Surface CD20 expression was diminished in the clones. The clones neither responded to rituximab-mediated growth reduction or complement-dependent cytotoxicity nor underwent apoptosis in response to cross-linked rituximab. Rituximab failed to chemosensitize the RR clones, which exhibited constitutive hyperactivation of the nuclear factor-kappaB and extracellular signal-regulated kinase 1/2 pathways, leading to overexpression of B-cell lymphoma protein 2 (Bcl-2), Bcl-2-related gene (long alternatively spliced variant of Bcl-x gene), and myeloid cell differentiation 1 and higher drug resistance. Unlike parental cells, rituximab neither inhibited the activity of these pathways nor diminished the expression of resistant factors. Pharmacologic inhibitors of the survival pathways or Bcl-2 family members reduced the activity of these pathways, diminished antiapoptotic protein expression, and chemosensitized the RR clones. These novel in vitro results denote that continuous long-term rituximab exposure culminates in RR clones that do not respond to rituximab-mediated effects, have altered cellular signaling dynamics, and exhibit different genetic and phenotypic properties compared with parental cells. The data also reveal that although RR clones exhibit higher resistance to rituximab and cytotoxic drugs, these clones can be chemosensitized following treatment with pharmacologic inhibitors (e.g., dehydroxymethylepoxyquinomicin, bortezomib, PD098059) that target survival/antiapoptotic pathways. The findings also identify intracellular targets for potential molecular therapeutic intervention to increase treatment efficacy. The significance and potential clinical relevance of the findings are discussed.

Overcoming Resistance of Human Non-Hodgkin's Lymphoma to CD19-CAR CTL Therapy by Celecoxib and Histone Deacetylase Inhibitors.

Patients with B-cell non-Hodgkin’s lymphoma (B-NHL) who fail to respond to first-line treatment regimens or develop resistance, exhibit poor prognosis. This signifies the need to develop alternative treatment strategies. CD19-chimeric antigen receptor (CAR) T cell-redirected immunotherapy is an attractive and novel option, which has shown encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. However, the underlying mechanisms of, and approaches to overcome, acquired anti-CD19CAR CD8⁺ T cells (CTL)-resistance in NHL remain elusive. CD19CAR transduced primary human CTLs kill CD19⁺ human NHLs in a CD19- and caspase-dependent manner, mainly via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. To understand the dynamics of the development of resistance, we analyzed several anti-CD19CAR CTL-resistant NHL sublines (R-NHL) derived by serial exposure of sensitive parental lines to excessive numbers of anti-CD19CAR CTLs followed by a limiting dilution analysis. The R-NHLs retained surface CD19 expression and were efficiently recognized by CD19CAR CTLs. However, R-NHLs developed cross-resistance to CD19CAR transduced human primary CTLs and the Jurkat human T cell line, activated Jurkat, and lymphokine activated killer (LAK) cells, suggesting the acquisition of resistance is independent of CD19-loss and might be due to aberrant apoptotic machinery. We hypothesize that the R-NHL refractoriness to CD19CAR CTL killing could be partially rescued by small molecule sensitizers with apoptotic-gene regulatory effects. Chromatin modifiers and Celecoxib partially reversed the resistance of R-NHL cells to the cytotoxic effects of anti-CD19CAR CTLs and rhTRAIL. These in vitro results, though they require further examination, may provide a rational biological basis for combination treatment in the management of CD19CAR CTL-based therapy of NHL.

Reversal of Resistance in Targeted Therapy of Metastatic Melanoma: Lessons Learned from Vemurafenib (BRAFV600E-Specific Inhibitor).

Malignant melanoma is the most aggressive form of skin cancer and has a very low survival rate. Over 50% of melanomas harbor various BRAF mutations with the most common being the V600E. BRAFV600E mutation that causes constitutive activation of the MAPK pathway leading to drug-, immune-resistance, apoptosis evasion, proliferation, survival, and metastasis of melanomas. The ATP competitive BRAFV600E selective inhibitor, vemurafenib, has shown dramatic success in clinical trials; promoting tumor regression and an increase in overall survival of patients with metastatic melanoma. Regrettably, vemurafenib-resistance develops over an average of six months, which renders melanomas resistant to other therapeutic strategies. Elucidation of the underlying mechanism(s) of acquisition of vemurafenib-resistance and design of novel approaches to override resistance is the subject of intense clinical and basic research. In this review, we summarize recent developments in therapeutic approaches and clinical investigations on melanomas with BRAFV600E mutation to establish a new platform for the treatment of melanoma.

Rituximab (chimeric anti-CD20 monoclonal antibody) inhibits the constitutive nuclear factor-{kappa}B signaling pathway in non-Hodgkin's lymphoma B-cell lines: role in sensitization to chemotherapeutic drug-induced apoptosis.

The chimeric anti-CD20 antibody rituximab (Rituxan, IDEC-C2B8) is widely used in the clinical treatment of patients with non-Hodgkin's lymphoma (NHL). Rituximab sensitizes NHL B-cell lines to drug-induced apoptosis via down-regulation of Bcl-x(L) expression. We hypothesized that the mechanism by which rituximab down-regulates Bcl-x(L) may be, in part, due to inhibition of constitutive nuclear factor-kappaB (NF-kappaB) activity that regulates Bcl-x(L) expression. This hypothesis was tested in CD20(+) drug-resistant Ramos (Bcl-2(-)/Bcl-x(L)(+)) and Daudi (Bcl-2(+)/Bcl-x(L)(+)) cell lines. Rituximab decreased the phosphorylation of NF-kappaB-inducing kinase, IkappaB kinase, and IkappaB-alpha, diminished IKK kinase activity, and decreased NF-kappaB DNA binding activity. These events occurred with similar kinetics and were observed 3 to 6 hours post-rituximab treatment. Rituximab significantly up-regulated Raf-1 kinase inhibitor protein expression, thus interrupting the NF-kappaB signaling pathway concomitant with Bcl-x(L) and Bfl-1/A1 down-regulation. The role of NF-kappaB in the regulation of Bcl-x(L) transcription was shown using promoter reporter assays in which deletion of the two-tandem NF-kappaB binding sites in the upstream promoter region significantly reduced the luciferase activity. This was further corroborated by using IkappaB superrepressor cells and by NF-kappaB-specific inhibitors. The direct role of Bcl-x(L) in drug resistance was assessed by using Bcl-x(L)-overexpressing cells, which exhibited higher drug resistance that was partially reversed by rituximab. Rituximab-mediated inhibition of the NF-kappaB signaling pathway and chemosensitization was corroborated by the use of specific inhibitors. These findings reveal a novel pathway mediated by rituximab through Raf-1 kinase inhibitor protein induction that negatively regulates the constitutive NF-kappaB pathway and chemosensitization of the NHL B-cells.

Combination of celecoxib (Celebrex®) and CD19 CAR-redirected CTL immunotherapy for the treatment of B-cell non-Hodgkin's lymphomas.

The nonsteroidal anti-inflammatory drug (NSAID) Celecoxib (Celebrex®) received Food and Drug Administration (FDA) approval in 1998 for treatment of osteoarthritis and rheumatoid arthritis, and in recent years, its use has been extended to various types of malignancies, such as breast, colon, and urinary cancers. To maintain the survival of malignant B cells, non-Hodgkin's Lymphoma (NHL) is highly dependent on inflammatory microenvironment, and is inhibited by celecoxib. Celecoxib hinders tumor growth interacting with various apoptotic genes, such as cyclooxygenase-2 (Cox-2), B-cell lymphoma 2 (Bcl-2) family, phosphor-inositide-3 kinase/serine-threonine-specific protein kinase (PI3K/Akt), and inhibitors of apoptosis proteins (IAP) family. CD19-redirected chimeric antigen-receptor (CD19 CAR) T cell therapy has shown promise in the treatment of B cell malignancies. Considering its regulatory effect on apoptotic gene products in various tumor types, Celecoxib is a promising drug to be used in combination with CD19 CAR T cell therapy to optimize immunotherapy of NHL.

Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention.

The clinical application of rituximab (chimeric mouse anti-human CD20 mAb, Rituxan, IDEC-C2B8), alone and/or combined with chemotherapy, has significantly ameliorated the treatment outcome of patients with relapsed and refractory low-grade or follicular non-Hodgkin's lymphoma (NHL). The exact in vivo mechanisms of action of rituximab are not fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis have been suggested. We have proposed that modifications of the cellular signaling pathways by rituximab may be crucial for its clinical response. The B-cell restricted cell surface phosphoprotein CD20 is involved in many cellular signaling events including proliferation, activation, differentiation, and apoptosis upon crosslinking. Monomeric rituximab chemosensitizes drug-resistant NHL cells via selective downregulation of antiapoptotic factors through the type II mitochondrial apoptotic pathway. Several signaling pathways are affected by rituximab which are implicated in the underlying molecular mechanisms of chemosensitization. ARL (acquired immunodeficiency syndrome (AIDS)-related lymphoma) and non-ARL cell lines have been examined as in vitro model systems. In ARL, rituximab diminishes the activity of the p38MAPK signaling pathway resulting in inhibition of the interleukin (IL)-10/IL-10R autocrine/paracrine cytokine autoregulatory loop leading to the inhibition of constitutive STAT-3 activity and subsequent downregulation of Bcl-2 expression leading to chemosensitization. Rituximab upregulates Raf-1 kinase inhibitor protein (RKIP) expression in non-ARL cells. Through physical association with Raf-1 and nuclear factor kappaB (NF-kappa B)-inducing kinase (NIK), RKIP negatively regulates two major survival pathways, namely, the extracellular signal-regulated kinase1/2 (ERK1/2) and the NF-kappa B pathways, respectively. Downmodulation of the ERK1/2 and NF-kappa B pathways inhibits the transcriptional activity of AP-1 and NF-kappa B transcription factors, respectively, both of which lead to the downregulation of Bcl-(xL) (Bcl-2 related gene (long alternatively spliced variant of Bcl-x gene)) transcription and expression and sensitization to drug-induced apoptosis. Bcl-(xL)-overexpressing cells corroborated the pivotal role of Bcl-(xL) in chemosensitization. The specificity of rituximab-mediated signaling and functional effects were corroborated by the use of specific pharmacological inhibitors. Many patients do not respond and/or relapse and the mechanisms of unresponsiveness are unknown. Rituximab-resistant B-NHL clones were generated to investigate the acquired resistance to rituximab-mediated signaling, and chemosensitization. Resistant clones display different phenotypic, genetic and functional properties compared to wild-type cells. This review summarizes the data highlighting a novel role of rituximab as a signal-inducing antibody and as a chemosensitizing agent through negative regulation of major survival pathways. Studies presented herein also reveal several intracellular targets modified by rituximab, which can be exploited for therapeutic and prognostic purposes in the treatment of patients with rituximab- and drug-refractory NHL.

Rituximab (chimeric anti-CD20) sensitizes B-NHL cell lines to Fas-induced apoptosis.

Rituximab (chimeric anti-CD20 monoclonal antibodies) is currently being used in the treatment of B non-Hodgkin's lymphoma (NHL). We have recently reported that rituximab triggers and modifies various intracellular signaling pathways in NHL B-cell lines, resulting in reverting the chemoresistant phenotype to a sensitive phenotype. This study investigated whether rituximab also modifies intracellular signaling pathways resulting in the sensitization of NHL cells to Fas-induced apoptosis. Treatment of the Fas-resistant NHL cell lines (2F7, Ramos and Raji) with rituximab sensitized the cells to CH-11 (FasL agonist mAb)-induced apoptosis and synergy was achieved. Fas expression was upregulated by rituximab as early as 6 h post-treatment as determined by flow cytometry, reverse transcriptase-polymerase chain reaction and Western blot. Rituximab inhibited both the expression and activity of the transcription repressor Yin-Yang 1 (YY1) that negatively regulates Fas transcription. Inhibition of YY1 resulted in the upregulation of Fas expression and sensitization of the tumor cells to CH-11-induced apoptosis. The downregulation of YY1 expression was the result of rituximab-induced inhibition of both the p38 mitogen-activated protein kinase (MAPK) signaling pathway and constitutive nuclear factor kappa of B cells (NF-kappaB) activity. The involvement of NF-kappaB and YY1 in the regulation of Fas expression was corroborated by the use of Ramos cells with a dominant-active inhibitor of NF-kappaB (Ramos IkappaB-estrogen receptor (ER) mutant) and by silencing YY1 with YY1 siRNA, respectively. Further, the role of rituximab-mediated inhibition of the p38 MAPK/NF-kappaB/YY1 pathway in the regulation of Fas and sensitization to CH-11-induced apoptosis was validated by the use of specific chemical inhibitors of this pathway and which mimicked rituximab-mediated effects. These findings provide a novel mechanism of rituximab-mediated activity by sensitizing NHL cells to Fas-induced apoptosis.

Inhibition of the Raf-MEK1/2-ERK1/2 signaling pathway, Bcl-xL down-regulation, and chemosensitization of non-Hodgkin's lymphoma B cells by Rituximab.

Rituximab (Rituxan, IDEC-C2B8) has been shown to sensitize non-Hodgkin's lymphoma (NHL) cell lines to chemotherapeutic drug-induced apoptosis. Rituximab treatment of Bcl-2-deficient Ramos cells and Bcl-2-expressing Daudi cells selectively decreases Bcl-(xL) expression and sensitizes the cells to paclitaxel-induced apoptosis. This study delineates the signaling pathway involved in rituximab-mediated Bcl-(xL) down-regulation in Ramos and Daudi NHL B cells. We hypothesized that rituximab may interfere with the extracellular signal-regulated kinase (ERK) 1/2 pathway, leading to decreased Bcl-(xL) expression. Rituximab (20 microg/mL) inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK) 1/2 and reduced the phosphorylation of the components of the ERK1/2 pathway (Raf-1, MEK1/2, and ERK1/2) and decreased activator protein-1 DNA binding activity and Bcl-(xL) gene expression. These events occurred with similar kinetics and were observed 3 to 6 hours after rituximab treatment. Rituximab-mediated effects were corroborated by using specific inhibitors of the ERK1/2 pathway, which also reduced Bcl-(xL) levels and sensitized the NHL B cells to paclitaxel-induced apoptosis. Previous findings implicated a negative regulatory role of the Raf-1 kinase inhibitor protein (RKIP) on the ERK1/2 pathway. Rituximab treatment of NHL B cells significantly up-regulated RKIP expression, thus interrupting the ERK1/2 signaling pathway through the physical association between Raf-1 and RKIP, which was concomitant with Bcl-(xL) down-regulation. These novel findings reveal a signaling pathway triggered by rituximab, whereby rituximab-mediated up-regulation of RKIP adversely regulates the activity of the ERK1/2 pathway, Bcl-(xL) expression, and subsequent chemosensitization of drug-refractory NHL B cells. The significance of these findings is discussed.

CD19 chimeric antigen receptor (CD19 CAR)-redirected adoptive T-cell immunotherapy for the treatment of relapsed or refractory B-cell Non-Hodgkin's Lymphomas.

Recovery rates for B-cell Non-Hodgkin's Lymphoma (NHL) are up to 70% with current standard-of-care treatments including rituximab (chimeric anti-CD20 monoclonal antibody) in combination with chemotherapy (R-CHOP). However, patients who do not respond to first-line treatment or develop resistance have a very poor prognosis. This signifies the need for the development of an optimal treatment approach for relapsed/refractory B-NHL. Novel CD19- chimeric antigen receptor (CAR) T-cell redirected immunotherapy is an attractive option for this subset of patients. Anti-CD19 CAR T-cell therapy has already had remarkable efficacy in various leukemias as well as encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. In going forward with additional clinical trials, complementary treatments that may circumvent potential resistance mechanisms should be used alongside anti-CD19 T-cells in order to prevent relapse with resistant strains of disease. Some such supplementary tactics include conditioning with lymphodepletion agents, sensitizing with kinase inhibitors and Bcl-2 inhibitors, enhancing function with multispecific CAR T-cells and CD40 ligand-expressing CAR T-cells, and safeguarding with lymphoma stem cell-targeted treatments. A therapy regimen involving anti-CD19 CAR T-cells and one or more auxiliary treatments could dramatically improve prognoses for patients with relapsed/refractory B-cell NHL. This approach has the potential to revolutionize B-NHL salvage therapy in much the same way rituximab did for first-line treatments.

PD-1 inhibition and treatment of advanced melanoma-role of pembrolizumab.

Remarkable clinical responses have been seen in patients with metastatic melanoma with targeted therapy (BRAFi vemurafenib, MEKi) and with modern immune cell-based approaches such as TCR engineered adoptive cell transfer (ACT) and earlier experiences with high-dose IL-2. The proximal mediators of these immune therapies are tumor-reactive CTL. Various mechanisms of resistance to immune-mediated apoptotic signals have been described, including phenotypic changes, effector cell exhaustion, functional tolerance, deficiencies in Ag processing and presentation, and mutation or down-regulation of antigenic epitopes. The immune system and drugs eradicate tumors via apoptosis. Therefore, tumors' resistance to apoptosis may be a determining factor that limits the efficacy of immunotherapies. It is predicted that these therapies have limited efficacy in patients whose melanomas have developed resistance to targeted therapy such as vemurafenib. Upregulation of the immune checkpoint molecule CTLA-4 on activated T cells and its interaction with CD80/86 blocks T cell activation. The fully humanized mAb ipilimumab blocks this interaction, resulting in sustained T cell stimulation. Likewise, the programmed death receptor 1 (PD-1) is another member of the B7:CD28 family of costimulatory molecules that regulates T cell activation, whose ligand (PD-L1) is expressed on melanomas. The human anti-PD-1 mAb, Pembrolizumab, overcomes tolerance, has a favorable pharmacokinetics profile with minimal undesired toxic side effects and has shown remarkable improvement in melanoma therapy. This review focuses on recent advances in the development of various anti-PD-1 checkpoint blockade antibodies and will summarize recent clinical data using immune checkpoint blocking antibodies.

Raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis.

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains the major obstacle in the successful treatment of cancer patients. Tumor cells acquire resistance to apoptotic stimuli and it has been demonstrated that conventional therapies exert their cytotoxic activities primarily by inducing apoptosis in the cells. Resistance to radiation and chemotherapeutic drugs has led to the development of immunotherapy and gene therapy approaches with the intent of overcoming resistance to drugs and radiation as well as enhancing the specificity to eliminate tumor cells. However, cytotoxic lymphocytes primarily kill by apoptosis and, therefore, drug-resistant tumor cells may also be cross-resistant to immunotherapy. To evade apoptosis, tumor cells have adopted various mechanisms that interfere with the apoptotic signaling pathways and promote constitutive activation of cellular proliferation and survival pathways. Thus, modifications of the antiapoptotic genes in cancer cells are warranted for the effectiveness of conventional therapies as well as novel immunotherapeutic approaches. Such modifications will avert the resistant phenotype of the tumor cells and will render them susceptible to apoptosis. Current studies, both in vitro and preclinically in vivo, have been aimed at the modification and regulation of expression of apoptosis-related gene products and their activities. A novel protein designated Raf-1 kinase inhibitor protein (RKIP) has been partially characterized. RKIP is a member of the phosphatidylethanolamine-binding protein family. RKIP has been shown to disrupt the Raf-1-MEK1/2 [mitogen-activated protein kinase-ERK (extracellular signal-regulated kinase) kinase-1/2]-ERK1/2 and NF-kappaB signaling pathways, via physical interaction with Raf-1-MEK1/2 and NF-kappaB-inducing kinase or transforming growth factor beta-activated kinase-1, respectively, thereby abrogating the survival and antiapoptotic properties of these signaling pathways. In addition, RKIP has been shown to act as a signal modifier that enhances receptor signaling by inhibiting G protein-coupled receptor kinase-2. By regulating cell signaling, growth, and survival through its expression and activity, RKIP is considered to play a pivotal role in cancer, regulating apoptosis induced by drugs or immune-mediated stimuli. Overexpression of RKIP sensitizes tumor cells to chemotherapeutic drug-induced apoptosis. Also, induction of RKIP by drugs or anti-receptor antibodies sensitizes cancer cells to drug-induced apoptosis. In this review, we discuss the discovery, structure, function, and significance of RKIP in cancer.

Resveratrol modifies the expression of apoptotic regulatory proteins and sensitizes non-Hodgkin's lymphoma and multiple myeloma cell lines to paclitaxel-induced apoptosis.

Resveratrol (trans-3,4,5-trihydroxystilbene) has received attention for its potential chemopreventive and antitumor effects in experimental systems. Recent evidence suggests that paclitaxel, alone or in combination with other drugs, can be effectively used in the treatment of non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). This study investigated whether resveratrol can sensitize NHL and MM cell lines to paclitaxel-mediated apoptosis and to delineate the underlying molecular mechanism of sensitization. Both resveratrol and paclitaxel negatively modulated tumor cell growth by arresting the cells at the G(2)-M phase of the cell cycle. Low concentrations of resveratrol exerted a sensitizing effect on drug-refractory NHL and MM cells to apoptosis induced by paclitaxel. Resveratrol selectively down-regulated the expression of antiapoptotic proteins Bcl-x(L) and myeloid cell differentiation factor-1 (Mcl-1) and up-regulated the expression of proapoptotic proteins Bax and apoptosis protease activating factor-1 (Apaf-1). Paclitaxel down-regulated the expression of Bcl-x(L), Mcl-1, and cellular inhibitor of apoptosis protein-1 antiapoptotic proteins and up-regulated Bid and Apaf-1. Combination treatment resulted in apoptosis through the formation of tBid, mitochondrial membrane depolarization, cytosolic release of cytochrome c and Smac/DIABLO, activation of the caspase cascade, and cleavage of poly(adenosine diphosphate-ribose) polymerase. Combination of resveratrol with paclitaxel had minimal cytotoxicity against quiescent and mitogenically stimulated human peripheral blood mononuclear cells. Inhibition of Bcl-x(L) expression by resveratrol was critical for chemosensitization and its functional impairment mimics resveratrol-mediated sensitization to paclitaxel-induced apoptosis. Inhibition of Bcl-x(L) expression by resveratrol was due to the inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and diminished activator protein-1-dependent Bcl-x(L) expression. The findings by resveratrol were corroborated with inhibitors of the ERK1/2 pathway. This study demonstrates that in resistant NHL and MM cell lines resveratrol and paclitaxel selectively modify the expression of regulatory proteins in the apoptotic signaling pathway and the combination, via functional complementation, results in synergistic apoptotic activity.

Rituximab (anti-CD20) selectively modifies Bcl-xL and apoptosis protease activating factor-1 (Apaf-1) expression and sensitizes human non-Hodgkin's lymphoma B cell lines to paclitaxel-induced apoptosis.

The anti-CD20 monoclonal antibody rituximab (Rituxan, IDEC-C2B8) has shown promising results in the clinical treatment of a subset of patients with low grade or follicular non-Hodgkin's lymphoma (NHL). However, chemotherapy- and rituximab-refractory NHL patients may benefit from a regimen in which rituximab acts as a sensitizing agent. This study examined the apoptotic signaling mediated by rituximab on rituximab- and paclitaxel-resistant CD20(+) NHL B cell lines (Ramos, Raji, Daudi, and 2F7). Treatment with either rituximab (20 micro g/ml) or paclitaxel (0.1-1000 nM) inhibited viable cell recovery of NHL lines. Neither rituximab nor paclitaxel induced significant apoptosis, although the combination treatment resulted in synergy in apoptosis. Rituximab selectively down-regulated Bcl-xL and induced apoptosis protease activating factor 1 (Apaf-1) expressions in Ramos cells. Paclitaxel down-regulated the expression of Bcl-xL and inhibitor of apoptosis proteins (c-IAP-1) and up-regulated the expression of Bad and Apaf-1. The combination treatment resulted in the formation of truncated Bid, cytosolic accumulation of cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI, activation of caspase-9, caspase-7, caspase-3, and cleavage of poly(ADP-ribose) polymerase. The findings identify two potential novel intracellular targets of rituximab-mediated signaling in Ramos NHL cells (i.e., Bcl-xL and Apaf-1). Further, the findings show that both rituximab and paclitaxel selectively modify the expression pattern of proteins involved in the apoptosis signal transduction pathway and, through functional complementation, the combination results in synergy in apoptosis. The potential therapeutic significance of these findings is discussed.

Rituximab-mediated sensitization of B-non-Hodgkin's lymphoma (NHL) to cytotoxicity induced by paclitaxel, gemcitabine, and vinorelbine.

Non-Hodgkin's lymphoma (NHL) responds initially to chemotherapy, however, the disease often relapses and acquires a chemoresistant phenotype. Third line treatment options such as paclitaxel, gemcitabine, and vinorelbine have been tried in patients with refractory or relapsed NHL with antilymphoma activity. Currently, rituximab (anti-CD20) has been approved for treatment of indolent NHL, with significant activity in a wide spectrum of B-cell malignancies, though a percentage of patients do not respond to rituximab treatment. Previous findings from our laboratory have demonstrated that rituximab can sensitize drug-resistant NHL B-lymphoma in vitro to some chemotherapeutic drugs. Hence, in this study, we examined the effect of combination treatment of rituximab and the drugs paclitaxel, gemcitabine, and vinorelbine on various NHL cell lines. Our findings indicate that pretreatment of NHL tumor cells with rituximab sensitizes drug-resistant NHL tumor cells to drug-mediated cytotoxicity. These findings suggest the potential clinical application of combination treatments of rituximab and paclitaxel, gemcitabine or vinorelbine in the treatment of drug/rituximab refractory non-Hodgkin's lymphoma.