Cytokine Release Syndrome Associated with T-Cell-Based Therapies for Hematological Malignancies: Pathophysiology, Clinical Presentation, and Treatment.

Cytokines are a broad group of small regulatory proteins with many biological functions involved in regulating the hematopoietic and immune systems. However, in pathological conditions, hyperactivation of the cytokine network constitutes the fundamental event in cytokine release syndrome (CRS). During the last few decades, the development of therapeutic monoclonal antibodies and T-cell therapies has rapidly evolved, and CRS can be a serious adverse event related to these treatments. CRS is a set of toxic adverse events that can be observed during infection or following the administration of antibodies for therapeutic purposes and, more recently, during T-cell-engaging therapies. CRS is triggered by on-target effects induced by binding of chimeric antigen receptor (CAR) T cells or bispecific antibody to its antigen and by subsequent activation of bystander immune and non-immune cells. CRS is associated with high circulating concentrations of several pro-inflammatory cytokines, including interleukins, interferons, tumor necrosis factors, colony-stimulating factors, and transforming growth factors. Recently, considerable developments have been achieved with regard to preventing and controlling CRS, but it remains an unmet clinical need. This review comprehensively summarizes the pathophysiology, clinical presentation, and treatment of CRS caused by T-cell-engaging therapies utilized in the treatment of hematological malignancies.

Citarinostat and Momelotinib co-target HDAC6 and JAK2/STAT3 in lymphoid malignant cell lines: a potential new therapeutic combination.

Histone deacetylase (HDAC) inhibitors represent an encouraging class of antitumor drugs. HDAC inhibitors induce a series of molecular and biological responses and minimal toxicity to normal cells. Citarinostat (Acy-241) is a second generation, orally administered, HDAC6-selective inhibitor. Momelotinib (CYT387) is an orally administered inhibitor of Janus kinase/signal transducer of transcription-3 (JAK/STAT3) signaling. Momelotinib showed efficacy in patients with myelofibrosis. We hypothesized that both HDAC and JAK/STAT pathways were important in lymphoproliferative disorders, and that inhibiting JAK/STAT3 and HDAC simultaneously might enhance the efficacy of momelotinib and citarinostat without increasing toxicity. Accordingly, we tested the citarinostat + momelotinib combination in lymphoid cell lines. Citarinostat + momelotinib showed strong cytotoxicity; it significantly reduced mitochondrial membrane potential, down-regulated Bcl-2 and Bcl-xL, and activated caspases 9 and 3. Caspase-8 was upregulated in only two lymphoid cell lines, which indicated activation of the extrinsic apoptotic pathway. We identified a lymphoid cell line that was only slightly sensitive to the combination treatment. We knocked down thioredoxin expression by transfecting with small interfering RNA that targeted thioredoxin. This knockdown increased cell sensitivity to the combination-induced cell death. The combination treatment reduced Bcl-2 expression, activated caspase 3, and significantly inhibited cell viability and clonogenic survival.

The Therapeutic Strategy of HDAC6 Inhibitors in Lymphoproliferative Disease.

Histone deacetylases (HDACs) are master regulators of chromatin remodeling, acting as epigenetic regulators of gene expression. In the last decade, inhibition of HDACs has become a target for specific epigenetic modifications related to cancer development. Overexpression of HDAC has been observed in several hematologic malignancies. Therefore, the observation that HDACs might play a role in various hematologic malignancies has brought to the development of HDAC inhibitors as potential antitumor agents. Recently, the class IIb, HDAC6, has emerged as one potential selective HDACi. This isoenzyme represents an important pharmacological target for selective inhibition. Its selectivity may reduce the toxicity related to the off-target effects of pan-HDAC inhibitors. HDAC6 has also been studied in cancer especially for its ability to coordinate a variety of cellular processes that are important for cancer pathogenesis. HDAC6 has been reported to be overexpressed in lymphoid cells and its inhibition has demonstrated activity in preclinical and clinical study of lymphoproliferative disease. Various studies of HDAC6 inhibitors alone and in combination with other agents provide strong scientific rationale for the evaluation of these new agents in the clinical setting of hematological malignancies. In this review, we describe the HDACs, their inhibitors, and the recent advances of HDAC6 inhibitors, their mechanisms of action and role in lymphoproliferative disorders.

Cell-Penetrating CaCO3 Nanocrystals for Improved Transport of NVP-BEZ235 across Membrane Barrier in T-Cell Lymphoma.

Owing to their nano-sized porous structure, CaCO3 nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3 NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

Ruxolitinib combined with vorinostat suppresses tumor growth and alters metabolic phenotype in hematological diseases.

JAK-2 dysregulation plays an important role as an oncogenic driver, and is thus a promising therapeutic target in hematological malignancies. Ruxolitinib is a pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3. Vorinostat is an HDAC inhibitor that reduces JAK-2 expression, thus affecting JAK-2 mRNA expression and increasing JAK-2 proteasomal deterioration. Here we hypothesized that the combination of ruxolitinib and vorinostat could have synergistic effects against hematological disease. We tested combinations of low doses of ruxolitinib and vorinostat in 12 cell lines, and observed highly synergistic cytotoxic action in six cell lines, which was maintained for up to 120 h in the presence of stromal cells. The sensitivity of the six cell lines may be explained by the broad effects of the drug combination, which can affect various targets. Treatment with the combination of ruxolitinib and vorinostat appeared to induce a possible reversal of the Warburg effect, with associated ROS production, apoptotic events, and growth inhibition. Decreased glucose metabolism may have markedly sensitized the six more susceptible cell lines to combined treatment. Therapeutic inhibition of the JAK/STAT pathway seems to offer substantial anti-tumor benefit, and combined therapy with ruxolitinib and vorinostat may represent a promising novel therapeutic modality for hematological neoplasms.

Ricolinostat, a selective HDAC6 inhibitor, shows anti-lymphoma cell activity alone and in combination with bendamustine.

Histone deacetylase inhibitors (HDACis) have emerged as a new class of anticancer agents, targeting the biological process including cell cycle and apoptosis. We investigated and explained the anticancer effects of an HDAC6 inhibitor, ricolinostat alone and in combination with bendamustine in lymphoma cell lines. Cell viability was measured by MTT assay. Apoptosis, reactive oxygen species (ROS) generation, Bcl-2 protein expression, cell cycle progression and tubuline expression were determined by flow cytometry. The effects of ricolinostat alone and in combination on the caspases, PI3K/Akt, Bcl-2 pathways, ER stress and UPR were assessed by immunoblotting. Ricolinostat shows anti lymphoma activity when used as single agent and its capability to induce apoptosis is synergistically potentiated by the bendamustine in lymphoma cell lines. Drug combination reduced the proportion of cells in the G0/G1 and S phases and caused an increase of "sub-G0/G1" peak. The synergistic effect accompanied with the increased ROS, activation of caspase-8, -9, and -3, the cleavage of PARP and modulated by Bcl-2 proteins family. In addition, the exposure of ricolinostat induced the acetylation level of α-tubulin, the extend of which was not further modified by bendamustine. Finally, the apoptosis effect of ricolinostat/bendamustine may be mediated by a corresponding effect on microtubule stabilization. Our data suggest that ricolinostat in combination with bendamustine may be a novel combination with potential for use as an antitumor agent in lymphoma.

The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines.

We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas-299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou-Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, -9, -3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies.

Activity of BKM120 and BEZ235 against Lymphoma Cells.

Non-Hodgkin lymphomas encompass a heterogeneous group of cancers, with 85-90% arising from B lymphocytes and the remainder deriving from T lymphocytes or NK lymphocytes. These tumors are molecularly and clinically heterogeneous, showing dramatically different responses and outcomes with standard therapies. Deregulated PI3K signaling is linked to oncogenesis and disease progression in hematologic malignancies and in a variety of solid tumors and apparently enhances resistance to antineoplastic therapy, resulting in a poor prognosis. Here, we have evaluated and compared the effects of the pan-PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitor BEZ235 on mantle, follicular, and T-cell lymphomas. Our results suggest that BKM120 and BEZ235 can effectively inhibit lymphoma cell proliferation by causing cell cycle arrest and can lead to cell death by inducing apoptosis and autophagy mediated by ROS accumulation. Despite great advances in lymphoma therapy after the introduction of monoclonal antibodies, many patients still die from disease progression. Therefore, novel treatment approaches are needed. BKM120 and BEZ235 alone and in combination are very effective against lymphoma cells in vitro. If further studies confirm their effectiveness in animal models, they may be promising candidates for development as new drugs.

The combination of bortezomib with enzastaurin or lenalidomide enhances cytotoxicity in follicular and mantle cell lymphoma cell lines.

We analyzed the combination of a proteasome inhibitor (bortezomib) with enzastaurin (PKC/AKT-inhibitor) or lenalidomide (immunomodulatory agent) for the inhibition of proliferation and induction of apoptosis in B-cell lymphoma cell lines and primary malignant cells. The effects of bortezomib, enzastaurin or lenalidomide, alone or in combinations, on cell viability and apoptosis were evaluated using the Cell Proliferation Kit and flow cytometry analysis. The interaction between drugs was examined by the Chou-Talalay method. Cell cycle analysis was performed by flow cytometry. The PI3K/AKT, PKC and MAPK/ERK signaling pathways were analyzed using western blot. Bortezomib with either enzastaurin or lenalidomide synergistically induced anti-proliferative and pro-apoptotic effects in B-cell lymphoma cells, even in the presence of the bone marrow microenvironment. The direct cytotoxicity is mediated by signaling events involving the PI3K/AKT, PKC and MAPK/ERK pathways leading to cell death. The significant increase of apoptosis was mediated by an increased ratio of pro-apoptotic proteins (Bax, Bad and Bim) to anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), triggering the cleavage of caspases -3, -9, -8 and PARP. Further evaluation of the combination of bortezomib with enzastaurin or lenalidomide for the treatment of B-cell lymphoma is warranted, with the goal to improve the quality of life and survival of patients.

NVP-BEZ235 alone and in combination in mantle cell lymphoma: an effective therapeutic strategy.

OBJECTIVES: Mantle cell lymphoma (MCL) is a distinct subtype of B-cell lymphoma; the complete response rate for standard therapies in use today is 85 - 90%. NVP-BEZ235 inhibits the PI3K/Akt/mTOR signaling axis at the level of both PI3K and mTOR. In this study, we analyzed the inhibitory effects of NVP-BEZ235 on mantle cell lines and its effects in combination with enzastaurin, everolimus and perifosine. METHODS: The effects of NVP-BEZ235 on cell proliferation and apoptosis were evaluated using MTT assay and flow cytometry analysis. The cell cycle analysis was performed applying BrdU incorporation. Western blot analysis was utilized for phosphorylation status evaluation of protein kinases. The interaction between NVP-BEZ235 and enzastaurin, everolimus and perifosine was examined by Chou-Talalay method. RESULTS: NVP-BEZ235 induced significant increase of apoptosis, both via intrinsic and extrinsic pathways. We found that NVP-BEZ235 inhibited mantle cells growth by induction of G1 arrest. NVP-BEZ235 exerts its antitumor activity even when mantle cells were in contact with bone marrow microenvironment. Enzastaurin, everolimus and perifosine enhanced the cytotoxicity triggered by NVP-BEZ235. CONCLUSIONS: The above results encourage clinical development of NVP-BEZ235 in combination and the possible inclusion of patients with mantle lymphoma in Phase I/II clinical trials.

Combination of low doses of enzastaurin and lenalidomide has synergistic activity in B-non-Hodgkin lymphoma cell lines.

Less toxic and more active treatments are needed for indolent lymphomas as there is no curative treatment, and patients eventually die due to complications related to their disease. The purpose of the present study was to assess the antitumour activity of the combination of low doses of Enzastaurin and Lenalidomide (Revlimid) on B-lymphoma cell lines. The combination of Enzastaurin and Lenalidomide, at doses as low as 1 µM, showed strong synergism against indolent lymphomas by reducing cell growth, producing an increase in G0-G1 phase followed by significant decrease in S phase, increasing apoptosis, and inhibiting PI3K/AKT, PKC and MAPK/ERK pathways. These preclinical findings, together with promising results obtained with Lenalidomide for the treatment of non-Hodgkin lymphoma, suggest that further evaluation of the combination of Enzastaurin and Lenalidomide for the treatment of indolent lymphomas is warranted. These compounds, with a favourable toxicity profile, are not classic chemotherapeutic agents, causing severe side effects, and could be considered an example of a new innovative attempt of an anti-cancer 'soft treatment'.

Effects of enzastaurin, alone or in combination, on signaling pathway controlling growth and survival of B-cell lymphoma cell lines.

Immunochemotherapies have improved outcomes in indolent lymphoma. However, response durations progressively shorten following each treatment, and the majority of patients eventually die from the disease. Thus, new, less toxic, and more active treatments are needed. Protein kinase C (PKC), which has been repeatedly implicated in B-cell lymphoma progression, may be a new target for lymphoma cell growth inhibition. Enzastaurin, a PKC-beta inhibitor, has toxic effects on a variety of cancer cells. The purpose of the present study was to assess the antitumor activity of enzastaurin on B-cell lymphoma cell lines and to investigate the underlying antitumor mechanisms. Enzastaurin induced apoptosis and inhibited phosphorylation of PKC, RSK, AKT, and downstream proteins. Moreover, our results reveal a new mechanism for enzastaurin-induced apoptosis via BAD activation. Finally, enzastaurin synergizes in its effects with chlorambucil and fludarabine, respectively. Taken together, our results strongly support clinical evaluation of enzastaurin in patients with B-cell lymphoma.

Molecular targeting of the PKC-beta inhibitor enzastaurin (LY317615) in multiple myeloma involves a coordinated downregulation of MYC and IRF4 expression.

The protein kinase C (PKC) pathway has been shown to play a role in the regulation of cell proliferation in several haematological malignancies, including multiple myeloma (MM). Recent data have shown that a PKC inhibitor, enzastaurin, has antiproliferative and proapoptotic activity in a large panel of human myeloma cell lines (HMCLs). In order to further characterise the effect of enzastaurin in MM, we performed gene expression profiling of enzastaurin-treated KMS-26 cell line. We identified 62 upregulated and 32 downregulated genes that are mainly involved in cellular adhesion (CXCL12, CXCR4), apoptosis (CTSB, TRAF5, BCL2L1), cell proliferation (IGF1, GADD45A, BCMA (B-cell maturation antigen), CDC20), transcription regulation (MYC, MX11, IRF4), immune and defence responses. Subsequent validation by Western blotting of selected genes in four enzastaurin-treated HMCLs was consistent with our microarray analysis. Our data indicate that enzastaurin may affect important processes involved in the proliferation and survival of malignant plasma cells as well as in their interactions with the bone marrow microenvironment and provide a preclinical rationale for the potential role of this drug in the treatment of MM.

The oral protein-kinase C beta inhibitor enzastaurin (LY317615) suppresses signalling through the AKT pathway, inhibits proliferation and induces apoptosis in multiple myeloma cell lines.

Deregulation of the protein kinase C (PKC) signalling pathway has been implicated in tumor progression. Here we investigated the PKC inhibitor enzastaurin for its activity against multiple myeloma (MM) cells. Enzastaurin suppresses cell proliferation in a large panel of human myeloma cell lines (HMCLs), with IC50 values ranging from 1.3 to 12.5 microM and induces apoptosis, which is prevented by the ZVAD-fmk broad caspase inhibitor. These results are consistent with decreased phosphorylation of AKT and GSK3-beta, a downstream target of the AKT pathway and a pharmacodynamic marker for enzastaurin. Furthermore, enzastaurin cytotoxicity is retained when HMCLs were cocultured with multipotent mesenchymal stromal cells. Enzastaurin has additive or synergistic cytotoxic effects with bortezomib or thalidomide. Considering the strong anti-myeloma activity of enzastaurin in vitro and in animal models and its safe toxicity profile, phase II studies in MM patients of enzastaurin alone or in combination with other drugs are warranted.

Biological effects of Atra and Arsenic Trioxide on short term cultures of non-M3 leukemic blasts.

The efficacy of All-Trans Retinoic Acid (Atra) and Arsenic Trioxide (As(2)O(3)) in the treatment of Acute Promyelocytic Leukemia (APL) is well known. Further, these drugs inhibit cell growth and induce apoptosis in several cell lines, but few data are reported on leukemic blasts. The aim of this study was to evaluate the biological effects on non-M3 Acute Myeloid Leukemia (AML) cells. Blasts of six patients, after exposition to Atra and As(2)O(3) were tested for growth inhibition, induction of apoptosis and change in cell cycle distribution, evaluating cell viability, percentage of apoptotic cells and of blasts positive for Ki-67 and BrdU. In the present study we demonstrated that either Atra or As(2)O(3) inhibit leukemic cells proliferation by induction of apoptosis. The effects are time and dose dependent. We did not observe additive or synergistic effects with the combination of the drugs. Further, our results showed that Atra and As(2)O(3) have effects on cell cycle distribution reducing S-phase and proliferating cells. These results should be taken in to account preparing future laboratory and clinical experimental protocols that associate these drugs with antineoplastic agents with different cell cycle specificity.