Differentiation agents increase the potential AraC therapy of AML by reactivating cell death pathways without enhancing ROS generation.

Acute myeloid leukemia (AML) has a poor prognosis and requires new approaches for treatment. We have reported that a combination of vitamin D-based cell differentiation agents (doxercalciferol/carnosic acid [D2/CA]) added following the cytotoxic drug arabinocytosine (AraC) increases AML cell death (CD), a model for improved therapy of this disease. Because AraC-induced CD is known to involve reactive oxygen species (ROS) generation, here we investigated if the modulation of cellular REDOX status plays a role in the enhancement of cell death (ECD) by D2/CA. Using thiol antioxidants, such as N-acetyl cysteine (NAC), we found a significant inhibition of ECD, yet this occurred in the absence of any detectable change in cellular ROS levels. In contrast, NAC reduced the vitamin D receptor (VDR) abundance and its signaling of ECD. Importantly, VDR knockdown and NAC similarly inhibited ECD without producing an additive effect. Thus, the proposed post-AraC therapy may be compromised by agents that reduce VDR levels in AML blasts.

The MAPK ERK5, but not ERK1/2, inhibits the progression of monocytic phenotype to the functioning macrophage.

Intracellular signaling pathways present targets for pharmacological agents with potential for treatment of neoplastic diseases, with some disease remissions already recorded. However, cellular compensatory mechanisms usually negate the initial success. For instance, attempts to interrupt aberrant signaling downstream of the frequently mutated ras by inhibiting ERK1/2 has shown only limited usefulness for cancer therapy. Here, we examined how ERK5, that overlaps the functions of ERK1/2 in cell proliferation and survival, functions in a manner distinct from ERK1/2 in human AML cells induced to differentiate by 1,25D-dihydroxyvitamin D3 (1,25D). Using inhibitors of ERK1/2 and of MEK5/ERK5 at concentrations specific for each kinase in HL60 and U937 cells, we observed that selective inhibition of the kinase activity of ERK5, but not of ERK1/2, in the presence of 1,25D resulted in macrophage-like cell morphology and enhancement of phagocytic activity. Importantly, this was associated with increased expression of the macrophage colony stimulating factor receptor (M-CSFR), but was not seen when M-CSFR expression was knocked down. Interestingly, inhibition of ERK1/2 led to activation of ERK5 in these cells. Our results support the hypothesis that ERK5 negatively regulates the expression of M-CSFR, and thus has a restraining function on macrophage differentiation. The addition of pharmacological inhibitors of ERK5 may influence trials of differentiation therapy of AML.

Vitamin D Control of Hematopoietic Cell Differentiation and Leukemia.

It is now well known that in the mammalian body vitamin D is converted by successive hydroxylations to 1,25-dihydroxyvitamin D (1,25D), a steroid-like hormone with pleiotropic properties. These include important contributions to the control of cell proliferation, survival and differentiation, as well as the regulation of immune responses in disease. Here, we present recent advances in current understanding of the role of 1,25D in myelopoiesis and lymphopoiesis, and the potential of 1,25D and analogs (vitamin D derivatives; VDDs) for the control of hematopoietic malignancies. The reasons for the unimpressive results of most clinical studies of the therapeutic effects of VDDs in leukemia and related diseases may include the lack of a precise rationale for the conduct of these studies. Further, clinical trials to date have generally used extremely heterogeneous patient populations and, in many cases, small numbers of patients, generally without controls. Although low calcemic VDDs have been used and combined with agents that can increase the leukemia cell killing or differentiation effects in acute leukemias, the sequencing of agents used for combination therapy should to be more clearly delineated. Most importantly, it is recommended that in future clinical trials the rationale for the basis of the enhancing action of drug combinations should be clearly articulated and the effects on anticancer immunity should also be evaluated.

ERK5 pathway regulates transcription factors important for monocytic differentiation of human myeloid leukemia cells.

Mitogen-activated protein kinases (MAPKs) are important transducers of external signals for cell growth, survival, and other cellular responses including cell differentiation. Several MAPK cascades are known with the MEK1/2-ERK1/2, JNK, and p38MAPKs receiving most attention, but the role of MEK5-ERK5 in intracellular signaling deserves more scrutiny, as this pathway transmits signals that can complement ERK/2 signaling. We hypothesized that the ERK5 pathway plays a role in the control of monocytic differentiation, which is disturbed in myeloid leukemia. We therefore examined the cellular phenotype and key molecular events which occur when human myeloid leukemia cells, acute (AML) or chronic (CML), are forced to differentiate by vitamin D derivatives (VDDs). This study was performed using established cell lines HL60 and U937, and primary cultures of blasts from 10 patients with ML. We found that ERK5 and its direct downstream target transcription factor MEF2C are upregulated by 1,25D in parallel with monocytic differentiation. Further, inhibition of ERK5 activity by specific pharmacological agents BIX02189 and XMD8-92 alters the phenotype of these cells by reducing the abundance of the VDD-induced surface monocytic marker CD14, and concomitantly increasing surface expression of the general myeloid marker CD11b. Similar results were obtained when the expression of ERK5 was reduced by siRNA or short hairpin (sh) RNA. ERK5 inhibition resulted in an expected decrease in MEF2C activation. We also found that in AML cells the transcription factor C/EBPß is positively regulated, while C/EBPα is negatively regulated by ERK5. These findings provide new understanding of dysregulated differentiation in human myeloid leukemia.

Isoforms of p38MAPK gamma and delta contribute to differentiation of human AML cells induced by 1,25-dihydroxyvitamin D3.

Inhibition of p38MAPK alpha/beta is known to enhance 1,25-dihydroxyvitamin (1,25D)-induced monocytic differentiation, but the detailed mechanism of this effect was not clear. We now show that the enhancement of differentiation becomes apparent with slow kinetics (12-24 h). Interestingly, the inhibition of p38MAPK alpha/beta by their selective inhibitor SB202190 (SB) leads to an upregulated expression of p38MAPK isoforms gamma and delta in 1,25D-treated AML cells, in cell lines and in primary culture. Although the expression and activating phosphorylations of p38MAPK alpha are also increased by an exposure of the cells to SB, its kinase activity is blocked by SB, as shown by reduced levels of phosphorylated Hsp27, a downstream target of p38MAPK alpha. A positive role of p38MAPKs in 1,25D-induced differentiation is shown by the inhibition of differentiation by antisense oligonucleotides to all p38MAPK isoforms. Other principal branches of MAPK pathways showed early (6 h) activation of MEK/ERK by SB, followed by activation of JNK1/2 pathway and enhanced expression and/or activation of PU.1, ATF-2 differentiation-related transcription factors. Taken together with previous reports, the results indicate that 1,25D-induced differentiation is enhanced by the activation of at least three branches of MAPK pathways (ERK1/2; p38MAPK gamma/delta; JNK1/2). This activation may result from the removal of feedback inhibition of an upstream regulator of those pathways, when p38MAPK alpha and beta are inhibited by SB.

Silibinin can induce differentiation as well as enhance vitamin D3-induced differentiation of human AML cells ex vivo and regulates the levels of differentiation-related transcription factors.

Induction of terminal differentiation is a conceptually attractive approach for the therapy of neoplastic diseases. Although vitamin D derivatives (deltanoids) can induce differentiation of AML cells in vitro, so far deltanoids have not been successfully brought to the clinic, due to the likelihood of life-threatening hypercalcemia. Here, we incubated freshly obtained blood cells from patients with AML with a plant antioxidant (PAOx), silibinin (SIL), alone or together with a deltanoid. Twenty patients with AML (all subtypes except M3) were available for this study, and in 14 (70%), SIL (60 µM) either induced differentiation ex vivo, or enhanced differentiation induced by deltanoids, or both. Interestingly, SIL acting alone induced differentiation only in cases in which chromosome aberrations could not be detected. In eleven samples sufficient material was available for a limited analysis of the underlying events. Quantitative RT-PCR showed that differentiation markers were upregulated at the mRNA level by both SIL and deltanoids, suggesting that intracellular signaling pathways upstream of transcription factors (TFs) were activated by these agents. Western analysis for proteins which function as TFs in deltanoid-induced monocytic differentiation, such as members of Jun and C/EBP families, surprisingly demonstrated that SIL upregulated all these TFs in the cases tested. This suggests that although the presence of SIL may not always be sufficient to induce differentiation, it can serve as a differentiation enabling factor for blasts obtained from a large proportion of patients with AML. Thus, SIL/deltanoid combinations warrant further consideration as preventive/therapeutic regimens in human leukaemia.

Primary central nervous system marginal zone B-cell lymphoma arising from the dural meninges: A case report and review of literature.

Primary central nervous system (CNS) marginal zone B-cell lymphoma (MZBCL) arising from the dural meninges is a rare but indolent disease. This malignancy can present in various ways, hence making it difficult to diagnose. Biopsy results dictate an appropriate treatment plan, which commonly consists of a combination of surgical resection, whole brain radiotherapy and systemic therapy.

Autoimmune Diseases and Rosai-Dorfman Disease Coexist More Commonly than Expected: Two Case Reports.

BACKGROUND The educational objective of this study was to describe 2 case reports in which patients were found to have an autoimmune disease concomitantly with a rare, benign histiocytic disorder known as Rosai-Dorfman disease (RDD). It is unclear if there is an underlying association between autoimmune disease and RDD. Lymphadenopathy, although most frequently seen bilaterally in the cervical region in RDD, may be present anywhere. A biopsy with histologic confirmation is required to not only evaluate for malignancy in these cases, but also necessary to diagnose RDD. CASE REPORT We describe 2 cases in which RDD was found incidentally in 2 patients who concomitantly had known autoimmune diseases. The first patient's history included Factor II deficiency, antiphospholipid syndrome, and autoimmune hemolytic anemia; whereas the second patient had a positive antinuclear antibody test, elevated rheumatoid factor, positive lupus anticoagulant, and positive beta-2 glycoprotein 1 antibodies, as well as positive anticardiolipin antibody panel, immune mediated thrombocytopenia, and pernicious anemia. Lymphadenopathy and an enlarged mass were seen in these cases respectively, which were histologically proven to be RDD. Steroid therapy was the mainstay of treatment. CONCLUSIONS Autoimmune diseases are relatively common in the general population and it appears that RDD coexists more often than suspected. When lymphadenopathy or a mass is seen, especially in those with other autoimmune diseases, RDD should remain within the differential diagnosis. Further research is required to determine characteristics and optimal management of RDD. We have observed in the cases presented, that if the autoimmune disease is well controlled, RDD can be an indolent disease.

Large granular lymphocytic leukemia complicating autoimmune polyglandular syndrome type 1 in siblings.

Autoimmune polyglandular syndrome type 1 (APS1) is a rare autosomal recessive disorder, and large granular lymphocytic leukemia (LGLL) may, even more rarely, complicate APS1. LGLL may be subtle in presentation, but it is imperative to recognize LGLL in APS1 promptly, as outcome may otherwise be fatal, as described herein.

JNK1 as a signaling node in VDR-BRAF induction of cell death in AML.

Numerous clinical studies of vitamin D, its derivatives or analogs, have failed to clearly demonstrate sustained benefits when used for the treatment of human malignant diseases. However, given the strong preclinical evidence of anti-neoplastic activity and the epidemiological associations suggesting that vitamin D compounds may have a place in cancer therapy, attempts are continuing to devise new approaches to their therapeutic use. This laboratory has developed a strategy to enhance the effectiveness of the currently standard therapy of Acute Myeloid Leukemia (AML) by the immediate addition of the vitamin D2 analog Doxercalciferol combined with the plant polyphenol-derived Carnosic acid to AML cells previously treated with Cytarabine (AraC). Enhancement of AML cell death was noted to be dependent on VDR and BRAF kinase. Here we document that the stress-related kinase JNK is an important additional component of cell death enhancement in this protocol. Either the Knock-down or the inhibition of JNK activity reduced the enhancement of AraC-induced cell death, and we show that JNK signaling to the apoptosis regulator BIM and Caspase executioners of cell death are downstream of VDR and BRAF. A clear understanding of the molecular basis for the increased efficacy of AraC in the therapy of AML is expected to bring this regimen to a clinical trial.

The anti-proliferative effect of neurokinin-A on hematopoietic progenitor cells is partly mediated by p53 activating the 5' flanking region of neurokinin-2 receptor.

The bone marrow (BM) is home to at least two stem cells, hematopoietic (HSC) and mesenchymal. Hematopoiesis is partly regulated through neurokinin-1 (NK-1) and NK-2 belonging to the family of G-protein/7-transmembrane receptors. NK-1 and NK-2 show preference for the neurotransmitters, substance P (SP) and neurokinin-A (NK-A), respectively. Hematopoietic suppression mediated by NK-A could be partly explained through the production of TGF-beta1 and MIP-1alpha. This study further characterizes mechanisms by which NK-A inhibits progenitor cell proliferation. The study addresses the hypothesis that p53 is a mediator of NK-A activation and this occurs partly through p53-mediated expression of NK-2. The studies first analyzed two consensus sequences for p53 in supershift assays. Reporter gene assays with NK-2 gene constructs and p53 expressing wild-type and mutant vectors, combined with cell proliferation assays, show NK-A activating p53 to inhibit the proliferation of K562 progenitors. These effects were reversed by hematopoietic stimulators, GM-CSF and SP. Verification studies with human CD34+/CD38- and CD34+/CD38+ BM progenitors show similar mechanisms with the expression of p21. This study reports on p53 as central to NK-A-NK-2 interaction in cell cycle quiescence of hematopoietic progenitors. These effects are reversed by at least two hematopoietic stimulators, SP and GM-CSF, with concomitant downregulation of p53.

BRAF signals to pro-apoptotic BIM to enhance AraC cytotoxicity induced in AML cells by Vitamin D-based differentiation agents.

Vitamin D has so far not fulfilled its early promise as an antineoplastic agent, in spite of compelling in vitro data. With the aim of bringing vitamin D or its derivatives (VDDs) effectively to the clinic, we developed a two-pronged approach. First, by adding the plant-derived Carnosic Acid (CA) to a vitamin D2 derivative Doxercalciferol we increased its differentiation potency without increasing it hypercalcemic properties. Second, we added these two agents together to AML cells already treated with Cytarabine (AraC), the standard drug for the treatment of patients with AML. We now report that BRAF, a part of the MAPK signaling pathway, is required for the optimally increased cell death in this system and acts upstream of BIM, the regulator of the caspase cascade that leads to cell death by apoptosis. It is proposed that this therapeutic regimen should be tested in a clinical trial.

Evidence of both von Willebrand factor deposition and factor V deposition onto AL amyloid as the cause of a severe bleeding diathesis.

: Acquired coagulopathies are common; uncommonly, adsorption of coagulation factors from the circulation into the tissues by pathologic amyloid exceeds the body's ability to produce factor and results in acquired factor deficiency. When amyloidosis does cause a coagulopathy, it is most often acquired factor X deficiency, but there are rare reports of amyloidosis being associated with other acquired factor deficiencies. We investigated a case of a severe bleeding diathesis, the cause of which was combined acquired factor V deficiency and concomitant acquired von Willebrand syndrome. Studies revealed prolonged prothrombin time and activated partial thromboplastin time. Mixing patient plasma with normal plasma corrected both the prothrombin time and activated partial thromboplastin time. Assays showed decreased factor V activity of 27%; Ristocetin cofactor activity was decreased at 49%, but von Willebrand antigen was elevated at 213%. Multimer analysis was consistent with type 2 von Willebrand syndrome. Lymph node biopsy documented amyloid light chain type (AL) amyloidosis; extraction of protein from the lymph node documented AL lambda light chain amyloid. Marrow biopsy documented IgG lambda myeloma. Immunohistochemical staining of the lymph node, using investigational polyvalent antibodies, demonstrated that both von Willebrand factor and factor V were identifiable in areas of amyloid deposition, providing evidence that these coagulation factors were adsorbed to the amyloid protein, resulting in accelerated clearance from the circulation, previously reported to be the mechanism of cases of acquired factor X deficiency in the setting of amyloidosis. Although there are case reports of acquired von Willebrand syndrome because of amyloidosis and case reports of acquired factor V deficiency because of amyloidosis, this appears to be the first reported case of concomitant acquired von Willebrand syndrome and acquired factor V deficiency because of amyloidosis, and the first report of localization of both von Willebrand protein and factor V protein to AL amyloid as a cause of a severe bleeding diathesis.

Enhancement of arabinocytosine (AraC) toxicity to AML cells by a differentiation agent combination.

Arabinocytosine (AraC, also known as cytarabine) is one of the mainstays of AML therapy, but like other DNA damaging therapeutic agents it is rarely curative by itself. There is an emerging realization that the therapeutic outcomes may be improved by combining AraC with other compounds. Here we report that the addition of a differentiating agent combination immediately following AraC damage to AML blasts, selectively increases the cell kill. The experiments were performed using cultured cells from established cell lines of AML (HL60 and U937). The cells were exposed to 100nM AraC, a concentration which produced approximately 25-50% cell kill, followed by a combination of 100nM 1alpha-hydroxyvitamin D2 (1-D2) and 10µM carnosic acid (CA), which together can serve as a powerful differentiating agent combination for AML cells, but are not toxic alone. AraC-induced cell death, measured by annexin V/propidium iodide, was significantly (p<0.01) increased by the 1-D2/CA combination in both cell lines, but not by 1-D2 or CA alone. The enhancement of cell death occurred by both apoptosis and necrosis, was associated with increased DNA damage and with higher levels of DNA damage response (DDR) activated marker Chk1, but the expression of p27, a cell cycle inhibitor protein, was not enhanced by 1-D2/CA. The principal finding is that a vitamin D analog 1-D2 combined with a plant-derived antioxidant CA can markedly augment the cytotoxic action of AraC, an anti-leukemia therapeutic agent.

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts.

Acute Myeloid Leukemia (AML) has grave prognosis due to aggressive nature of the disease, the toxicity of standard treatment, and overall low cure rates. We recently showed that AML cells in established culture treated with Cytarabine (AraC) and a differentiation agent combination show enhancement of AraC cytotoxicity. Here we elucidate molecular changes which underlie this observation with focus on AML blasts in primary culture. The cells were treated with AraC at concentrations achievable in clinical settings, and followed by the addition of Doxercalciferol, a vitamin D2 derivative (D2), together with Carnosic acid (CA), a plant-derived antioxidant. Importantly, although AraC is also toxic to normal bone marrow cell population, the enhanced cell kill by D2/CA was limited to malignant blasts. This enhancement of cell death was associated with activation of the monocytic differentiation program as shown by molecular markers, and the increased expression of vitamin D receptor (VDR). Apoptosis elicited by this treatment is caspase-dependent, and the optimal blast killing required the increased expression of the apoptosis regulator Bim. These data suggest that testing of this regimen in the clinic is warranted.

The rationale for deltanoids in therapy for myeloid leukemia: role of KSR-MAPK-C/EBP pathway.

The evidence for the promising potential for derivatives of Vitamin D (deltanoids) in the treatment of myeloid leukemias is increasing, but currently is not matched by the understanding of the precise mechanisms by which these anti-neoplastic effects are achieved. Unlike solid tumors in which growth retardation by deltanoids appears to result from inhibition of cell proliferation and the promotion of cell death by apoptosis, control of myeloid leukemia proliferation by deltanoids results from the induction of differentiation of the immature myelo-monocytic cells towards functional monocytic cells. We present here the accumulating evidence that a pathway that is initiated by deltanoid activation of Vitamin D receptor (VDR) and leads to monocytic differentiation of human myeloblastic HL60 cells, includes the MEK-ERK and JNK mitogen-activated protein kinases (MAPKs), their positive and negative regulators and a downstream effector C/EBPbeta. As in other cells, the abundance of VDR protein increases shortly after an exposure of HL60 cells to 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)). Other early events include a parallel upregulation of kinase suppressor of Ras (KSR-1) and the activation of the ERK MAPK pathway and data suggest that KSR-1 acts to amplify the signal provided by low concentrations of 1alpha,25(OH)(2) D(3). Maintenance of monocytic differentiation may be enhanced by JNK, but diminished by p38, MAPK signaling. Downstream, one of the targets of these pathways is C/EBPbeta, which can directly interact with the promoter for CD14, a gene characteristically expressed in monocytes. Importantly, in freshly obtained acute myeloid leukemia (AML)-M2 cells exposed to PRI-2191, a novel deltanoid with a modified side chain, upregulation of C/EBPbeta paralleled the induction of monocytic differentiation. These data provide a basis for the hypothesis that deltanoid-induced upregulation of C/EBPbeta bypasses the block to granulocytic differentiation in myeloid leukemia cells by redirecting the cells to monocytic differentiation.

Crosstalk between neurokinin receptors is relevant to hematopoietic regulation: cloning and characterization of neurokinin-2 promoter.

Neurokinin (NK)-1 and NK-2 receptors regulate hematopoiesis by interacting with neurotransmitters that belong to the tachykinin. This report studies the relationship between NK-1 and NK-2 in primary human bone marrow (BM) stroma, which supports hematopoiesis. Use of NK receptor antagonists and deficient stromal cells indicate that the neurotransmitter, substance P (SP), could exert dual hematopoietic effects (inhibitory or stimulatory), depending on the interacting receptor and crosstalk between NK-1 and NK-2. Cloning and identification of the minimal promoter for NK-2 and comparison with NK-1 promoter showed that the hematopoietic functions of NK receptors involve receptor crosstalk and the particular cytokine (IL-3, GM-CSF, TGF-beta or IL-1alpha). Crosstalk between NK-1 and NK-2 adds to communication within neural-hematopoietic axis.

Hematopoietic growth factor inducible neurokinin-1 type: a transmembrane protein that is similar to neurokinin 1 interacts with substance P.

Neurokinin 1 (NK-1) is a member of seven transmembrane G protein-coupled receptors. NK-1 interacts with peptides belonging to the tachykinin family and showed preference for substance P (SP). NK-1 is induced in bone marrow (BM) stroma. NK-1-SP interactions could lead to changes in the functions of lymphohematopoietic stem cell (LHSC). This report describes the cloning and characterization of a cDNA clone isolated after screening of three cDNA libraries with an NK-1-specific probe. Based on its expression, the cDNA clone was designated hematopoietic growth factor inducible neurokinin-1 type (HGFIN). Computational analyses predicted that HGFIN is transmembrane with the carboxyl terminal extracellular. Proteomic studies with purified HGFIN and SP showed noncovalent interactions. HGFIN-SP interactions were supported by transient expression of HGFIN in CHO cells. Transient expression of HGFIN in unstimulated BM fibroblasts led to the induction of endogenous NK-1. Since NK-1 expression in BM fibroblasts requires cell stimulation, these studies suggest that there might be intracellular crosstalk between NK-1 and HGFIN. Northern analyses with total RNA from different BM cell subsets showed that HGFIN was preferentially expressed in differentiated cells. This suggests that HGFIN might be involved in the maturation of LHSC. HGFIN was detected in several other tissues, but not in brain where NK-1 is constitutively expressed.

The neuronal cyclin-dependent kinase 5 activator p35Nck5a and Cdk5 activity in monocytic cells.

Cyclin-dependent kinase 5 (Cdk5) and its activator p35Nck5a are an odd but interesting couple. Recent work has established that in spite of their names, Cdk5 is clearly not cyclin-dependent, while p35Nck5a is not exclusively neuronal. Indeed, p35Nck5a and/or Cdk5 activity has been demonstrated to be present in human and rat promyelocytic cell lines, and in normal human monocytes. Further, monocytes from mice in which p35Nck5a gene was deleted do not express non-specific esterase, an enzyme present in normal monocytes, while transfection of Cdk5 together with p35Nck5a into myeloblastic cells leads to the expression of this marker of the monocytic phenotype. Thus, the Cdk5/p35Nck5a complex appears to play an important role in monocytic differentiation.