- Genes to Tailor Warfarin Dosage (Cover Story)
- Optimism and Caution on the Use of MSCs in Patients Undergoing HSCT for Hematologic Malignancies
- Gene Dosage and Lineage Commitment in Myeloid Leukemia
- Reworking Our Textbooks
- Epidermal Sensing of Oxygen Regulates Systemic Hypoxic Response
- Artificial Blood – Not Too Sweet
- Selecting Patients Most Likely to Respond to Therapy
- Erythrocyte Indigestion: A Surprising Role for the Mitochondrial Pathway of Apoptosis in Reticulocyte Autophagy
- Targeting NF-κB in CLL
Optimism and Caution on the Use of MSCs in Patients Undergoing HSCT for Hematologic Malignancies
By Michael Linenberger, MD, FACP
Dr. Linenberger indicated no relevant conflicts of interest.
Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371:1579-86.
Ning H, Yang F, Jiang M, et al. The correlation between cotransplantation of mesenchymal stem cells and higher recurrence rate in hematologic malignancy patients: outcome of a pilot clinical study. Leukemia. 2008;22:593-9.
Mesenchymal stem (or stromal) cells (MSCs) are culture-derived, non-hematopoietic, adherent progenitors that are defined by specific immunophenotypic features and the ability to differentiate into adipocytes, chondrocytes, or osteoblasts. In vivo, MSCs can migrate to sites of tissue injury and inflammation where they produce trophic and growth factors that facilitate repair and regeneration. MSCs also support hematopoiesis; they are relatively non-immunogenic and can down-modulate T-cell-mediated alloreactivity.1 Pilot and phase II studies in allogeneic hematopoietic stem cell transplantation (HSCT) suggest that donor or mismatched, “third party,” marrow-derived MSCs are safe and can enhance engraftment in certain patients or treat corticosteroid-refractory graft-versus-host disease (GVHD). Importantly, MSCs are also recruited to tumor microenvironments, and studies in murine or human xenograft tumor models show that systemic delivery or co-implantation of MSCs can promote malignant cell survival, proliferation, and/or metastasis.2-4 Thus, the safety of MSCs in patients undergoing HSCT for malignancies remains a major concern.
The report by LeBlanc, et al. describes a multicenter, non-randomized phase II trial of donor or third-party marrow MSCs for severe, steroid-refractory acute GVHD after myeloablative or non-myeloablative HSCT for a hematologic malignancy (78 percent), solid tumor (4 percent), or non-malignant disease (12 percent). One intravenous infusion of MSCs induced a complete response (CR) in 27 of 55 patients (49 percent), and CR occurred in 30 patients overall (55 percent). Compared with patients without CR, those with CR had significantly lower one-year transplant-related mortality (37 percent vs. 72 percent) and higher two-year survival (53 percent vs. 16 percent). Response was not related to GVHD grade, MSC source, or total MSC dose. No acute or late side effects were reported; relapse occurred in three of 43 patients (7 percent) with hematologic malignancy. In the randomized controlled trial by Ning, et al., HLA-matched donor MSCs were co-transplanted with marrow and/or peripheral blood stem cells on day zero after myeloablative conditioning for hematologic malignancies. Patients were randomized by age, disease type, stage, and prognosis. Only 10 of 15 patients allocated to the treatment arm received MSCs; their engraftment was not enhanced, but only one developed GVHD, compared with eight of 15 non-MSC control patients. The study was closed early because six of the 10 patients who received MSCs relapsed (including five recurrences by day 150), compared with three relapses in the 15 non-MSC patients. The three-year disease-free survival rates for the MSC and non-MSC groups were 30 percent and 66.7 percent, respectively (log rank p=0.035).
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| Culture-derived MSCs, which are defined by specific immunophenotypic features and their ability to differentiate into adipocytes, chondrocytes, or osteoblasts in vitro, exhibit pleiotropic functions in vivo. |
These observations add to the growing experience of using culture-expanded MSCs in HSCT. The results of LeBlanc, et al. are highly encouraging. If confirmed in current randomized clinical trials, MSCs could offer the safest and most effective salvage therapy option for steroid-resistant acute GVHD. This enthusiasm must be tempered, however, by the observations of Ning, et al. that remind us that MSCs can promote malignant cell survival and growth. Reassuringly, high relapse rates were not observed in a similar, but non-randomized, study of 46 patients with hematologic malignancies undergoing myeloablative HSCT with MSC co-transplantation on day zero,5 nor have increased relapse rates been reported after administering MSCs for GVHD. Moreover, the results of Ning, et al. may not be broadly applicable because their study groups were small and the technical limitations that prevented optimal donor MSC expansion could have introduced confounding variables. Additional clinical and pathobiological studies are needed to address whether MSCs enhance disease recurrence after HSCT, especially when co-transplanted on day zero, and if this might occur through direct cell-cell interactions, paracrine effects, and/or suppression of graft-versus-tumor alloreactivity.
References
- Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell. 2008;2:141-50.
- Djouad F, Plence P, Bony C, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood. 2003;102:3837-44.
- Ramasamy R, Lam EW, Soeiro I, et al. Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on in vivo tumor growth. Leukemia. 2007;21:304-10.
- Karnoub AE, Dash AB, Vo AP, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007;449:557-63.
- 5. Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant. 2005;11:389-98.
Gene Dosage and Lineage Commitment in Myeloid Leukemia
By Jerald Radich, MD
Dr. Radich indicated no relevant conflicts of interest.
Tiedt R, Hao-Shen H, Sobas MA, et al. Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice. Blood. 2008;111:3931-40.
Mutations in tyrosine kinases are a common theme in myeloid leukemia. The hallmark example is the inappropriate activation of Abl through the Bcr-Abl translocation in CML. Mutations in the FLT3 tyrosine kinase are quite common in AML. Recently, point mutations resulting in a valine to phenylalanine substitution at amino acid 617 of the JAK2 kinase (JAK2-V617F) have been found in >90 percent of cases of polycythemia vera (PV), and approximately 50 percent of primary myeloid fibrosis (PMF) and essential thrombocytosis (ET). How can one mutation be associated with three different diseases?
A recent study by Tiedt, et al. paints a fascinating picture of how the mutant gene level can actually influence the malignant phenotype. The authors used elegant genetic engineering to create three mouse models: one with a balanced expression of the wild-type JAK2 and mutant JAK2-V617F, one with relatively high JAK2-V617F, and one with very high JAK2-V617F. The mice developed a hematologic disease influenced by the relative amount of wild-type to mutant allele. Thus, mice expressing balanced expression of wild-type and mutant Jak2 developed an ET-like disease, with increases predominately in platelet counts, splenomegaly, and fibrosis in the bone marrow. Mice that expressed higher levels of mutant JAK2-V617F showed increasing levels of erythroid expansion, with a phenotype that appeared PV-like. A study of 82 patients with myeloproliferative disease and 11 healthy people showed a similar pattern as the mouse model. Quantitative RT-PCR showed the highest mutant: wild-type ratio in cases with PV, followed by PMF, then ET. Expression of the mutant and wild-type JAK2 correlated with the gene copy numbers found in the samples. Thus, cases with PV tended to have samples where the chromosomal number of mutant JAK2 was greater than wild-type.
A variation of this theme has been found in AML cases with the FLT3 mutation. Approximately 15 percent to 30 percent of AML cases with normal cytogenetics harbor FLT3 mutations characterized by a head-to-tail duplication in gene coding for the juxtamembrane region of the protein. The occurrence of this FLT3 internal tandem duplication (FLT3-ITD) alone has had a variable prognostic import across different studies and treatments. However, several studies have now shown that the allelic ratio (the ratio of mutant FLT3-ITD to wild-type allele) drives prognosis.1-3 Cases with predominately mutant FLT3-ITD have a very poor prognosis; cases with predominately wild-type allele tend not to have a poor prognosis.
These findings run counter to the conventional (and, perhaps, wrong) wisdom of leukemia being a single clonal event. If AML really is only derived from a single clone, there could only be three possible allelic ratios in respect to the FLT3 mutation in an AML sample: all wild-type; heterogyzgous wild-type and mutant; or all mutant. The fact that one can have a variety of allelic ratios in AML cases suggests that there must be multiple clones in most leukemic cases, each clone having a different state of the three conditions outlined above.
Of interest, the allelic data suggest not only the case of a loss of the normal FLT3 (resulting in one mutant gene, no wild-type), but in some cases, a duplication of the mutant gene. How does a patient develop two copies of a mutant gene? Bad luck twice? In some cases of malignancy, wild-type alleles are dropped through chromosome loss (for example, deletion of an arm of chromosome 17 eliminates a copy of the p53 tumor suppressor gene). This loss of heterozygosity, however, does not appear to be the case in the JAK2 and FLT3 story. Here, rather, the process of chromosomal repair causes a duplication of the mutant allele (see Figure). In this situation, a double-stranded DNA break takes place, and, in order to facilitate repair, a second copy of one of the genes is made. In the process of repair, one of the copies of the genes is lost. If the recombination process selects the wild-type gene to duplicate, then the cell has two copies of the wild-type gene, and order is restored. If, in this process, the wild-type gene is selected, then the cell has two copies of the mutated gene.
Thus, the mere presence of a mutated tyrosine kinase does not tell the whole story in myeloid malignancies. The dosage level of the mutated gene can affect the disease phenotype and the biology of response. Once again in science and medicine, the more we know, the more we need to know.
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An example of DNA repair causing a duplication of a mutated gene.
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References
- Whitman SP, Archer KJ, Feng L, et al. Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study. Cancer Res. 2001;61:7233-9.
- Meshinchi S, Alonzo TA, Stirewalt DL, et al. Clinical implications of FLT3 mutations in pediatric AML. Blood. 2006;108:3654-61.
- Gale RE, Green C, Allen C, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood. 2008;111:2776-84.
Reworking Our Textbooks
By Nelson Chao, MD
Dr. Chao indicated no relevant conflicts of interest.
Wada H, Masuda K, Satoh R, et al. Adult T-cell progenitors retain myeloid potential. Nature. 2008;452:768-72.
Bell JJ, Bhandoola A. The earliest thymic progenitors for T cells possess myeloid lineage potential. Nature. 2008;452:764-7.
Who’s your daddy? This seems like a simple question, but in hematopoiesis the answer may not be so simple. We all know that blood is made up of red cells, white cells, and platelets. They all are derived from a hematopoietic stem cell. The red cells and platelets are derived from a common precursor. White blood cells, on the other hand, come in many different flavors with a very specialized function. They also seem to segregate broadly into differentiated myeloid and lymphoid cells through a series of less well-understood intermediates. The bifurcation or restricted commitment of these two lineages is thought to occur early on in differentiation. The myeloid lineages are derived from a common myeloid progenitor (CMP), and the lymphocytes are derived from a common lymphoid progenitor (CLP). This picture came from the description of the CLP, which was able to give rise to T, B, and NK cells but not to myeloid cells. The cartoon is well entrenched in our thinking of hematopoietic development and in every slide set of hematopoiesis.
But, is it correct? Two recent publications have challenged this dogma and provide definitive data that the picture is not so cut-and-dried. Before delving into the data, a very brief review of thymopoiesis is needed. While all the other blood elements mature in the marrow spaces, T-cell development is thought to proceed from a CLP that migrates from the bone marrow to the thymus where a tightly orchestrated set of events results in the release of a naïve T cell that has been positively selected (to recognize the appropriate MHC molecules) and negatively selected (to remove auto-reactive T cells). The T-cell precursor begins as double-negative (DN) 1 (DN1 [CD44+CD25-CD117+], then DN2 (CD44+CD25+CD117+), and DN3 (CD44-CD25+), CD4/CD8 double-positive, and finally single-positive (CD4 or CD8) naïve T cell. Based on this elegant knowledge of T-cell development, there should not be any question as to whether these T-cell precursors could give rise to myeloid cells.
However, using clonal analysis with single-cell assays, these two group of investigators demonstrated that a substantial number of early T-cell precursors in the thymus at the DN1 and DN2 stage (prior to T-cell receptor rearrangement) have myeloid potential, which is lost at the DN3 stage. These myeloid cells were predominantly macrophages, but granulocytes and dendritic cells were also observed. Transfer of DN1 cells into T-cell-deficient mice demonstrated that up to one-third of the macrophages were derived from the T-cell precursors. Even more surprising was that these myeloid cells demonstrated rearrangement of the T-cell receptor and even expressed RAG recombinase, the enzyme necessary to create T-cell receptor diversity. Taken together, these data demonstrate that the early T-cell precursor is not yet fully committed to becoming only T cells.
What are the ramifications? These early T-cell precursors could explain the origin of some leukemias, which are biphenotypic. But more importantly, it is clear that a progenitor’s potential can be different from what actually occurs in vivo. Simple characterization of such precursors may not fully describe their potential. We do not know what the molecular signals are in this case or whether it is similar to the need for PAX5 expression for B-cell lineage commitment, where a single switch may determine the fate of the cell. However, these data go a long way in getting our lineages right.
Epidermal Sensing of Oxygen Regulates Systemic Hypoxic Response
By Heather Gilbert, MD, and Josef T. Prchal, MD
Drs. Gilbert and Prchal indicated no relevant conflicts of interest.
Boutin AT, Weidemann A, Fu Z, et al. Epidermal sensing of oxygen is essential for systemic hypoxic response. Cell. 2008;133:223-34.
Amphibians respond to changes in environmental oxygen at least in part through their skin, and frogs can use their permeable skin to derive oxygen directly from the atmosphere. Mammalian skin, however, has generally been thought of as an impermeable barrier, with no direct communication between outside environment and inner respiratory physiology. Mammals are known to sense changes in oxygen pressure by carotid bodies that regulate cardiovascular and respiratory response and by the kidneys and liver that regulate erythropoiesis by erythropoietin production. Boutin and colleagues, however, have created a series of experiments that demonstrate unanticipated regulation of erythropoiesis by novel regulation of renal erythropoietin production via epidermal O2 sensing.
The erythropoietin (EPO) gene is one of many “hypoxia-regulated” genes whose expression is controlled by the master transcription factors, hypoxia-inducible factors-1 and -2 (HIF-1 and HIF-2), each composed of dimers of α and β subunits. Only the HIF α subunits are regulated by hypoxia, and their expression is controlled post-transcriptionally. Under normoxic conditions, the prolyl residues of HIFs are hydroxylated by the enzyme prolyl hydroxylase, which allows the von Hippel-Lindau protein (pVHL) to bind to HIF α, leading to rapid degradation by the ubiquitin-proteasome pathway. During hypoxic conditions, HIF α is stabilized (by not being targeted for proteasome degradation) and forms a transcriptional complex with HIF β that leads to increased expression of multiple target genes involved in diverse processes, including cell proliferation and survival, metabolism, angiogenesis, and erythropoiesis. HIF-1α and HIF-2α exhibit high sequence homology but have different mRNA expression patterns. HIF-1α is expressed ubiquitously, whereas HIF-2α expression is restricted to certain tissues. Both HIF-1α and HIF-2α are regulated by identical mechanisms during hypoxia and form a heterodimer with the same HIF-β subunit. HIF-1 is the principal regulator of EPO gene transcription in the kidney. In other tissues, such as brain and liver (that generates ~ 20 percent of circulating erythropoietin), EPO gene transcription is HIF-2-dependent.
Boutin and colleagues created a mouse with conditional deletion of Vhl in epidermal keratinocytes, which caused cutaneous vasodilation and increased expression of Hif-1α and Hif-2α. Although keratinocytes do not make erythropoietin, the erythropoietin level was nonetheless found to be increased, and the mouse became polycythemic. Further studies of this epidermal Vhl knockout mouse revealed that the elevated levels of hif-1 caused upregulation of inducible nitric oxide synthase, which in turn led to increased cutaneous nitric oxide (NO), a potent vasodilator. This NO-induced skin vasodilation resulted in decreased perfusion of other organs, most notably the liver, with subsequent hypoxia-induced, increased expression of hepatic Hif-2α, which in turn caused increased expression of the Epo gene. In follow-up experiments using mice with wild-type Vhl, the authors deleted the cutaneous genes for either Hif-1α or Hif-2α. Unexpectedly, under conditions of normoxia, the loss of Hif-1α and Hif-2α had no effect on erythropoietin levels. Under hypoxic conditions, however, the Hif-1α epidermal knockout mice did not display an appropriate increase in renal Epo gene transcription and were unable to mount an appropriate renal erythropoietin response. These experiments show the importance of epidermal hif in sensing environmental oxygen levels and regulating systemic hypoxic responses in mice, with physiologic regulation mediated primarily by hif-1α while the pathologic loss of Vhl is mediated primarily by hif-2α.
Although these experiments were carried out using mice, the question of whether these results suggest a broader role for mammalian skin in general is compelling. While mice are not people, the partial inhibition of VHL in humans causes Chuvash polycythemia,1 a condition with a complex phenotype, the pathophysiological and molecular basis of which is not yet fully defined, but includes elevated erythropoietin levels and an increased risk of thrombosis that remains unexplained.2 This human phenotype underlines the essential importance of HIF sensing in controlling multiple physiologic pathways, and future studies looking at whether human skin, in particular, responds directly to decreases in atmospheric oxygen via HIF mediation may provide a basis for the development of new strategies for the treatment of anemia, hypoxia, and oxygen delivery in humans.
References
- Ang SO, Chen H, Hirota K, et al. Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia. Nature Genet. 2002;32:614-21.
- Gordeuk VR, Sergueeva AI, Miasnikova GY, et al. Congenital disorder of oxygen-sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood. 2004;103:3924-32.
Artificial Blood – Not Too Sweet
By Charles Abrams, MD
Dr. Abrams indicated no relevant conflicts of interest.
Natanson C, Kern SJ, Lurie P, et al. Cell-free hemoglobin-based blood substitutes and risk of myocardial infarction and death: a meta-analysis. JAMA. 2008. [Epub ahead of print]
Soon after William Harvey described the circulation of blood in the early 17th century, Christopher Wren unsuccessfully experimented with replacing wine for blood. During a cholera outbreak in the late 19th century, Gaillard Thomas also failed in his attempts to substitute milk for blood. It was not until 1933 that the first successful blood substitution experiments were performed. William Ruthrauff Amberson of the University of Tennessee Medical School used hemolyzed red blood cells for an exchange transfusion in a cat and demonstrated that he could keep the animal alive for 36 hours. Unfortunately, infusion of a similar product into humans produced oliguria and bradycardia. These toxicities were initially thought to be due to the lipids within erythrocyte membranes. Yet, when Dr. Amberson infused hemoglobin free of red-cell membranes into a hemorrhaging post-partum woman who had depleted her hospital’s inventory of cross-matched blood, she developed bradycardia and hypertension, and ultimately died from renal failure. These pioneering studies that spanned three centuries have demonstrated that the ideal blood substitute is an elusive goal.
Today, the blood supply in the United States is safe and usually sufficient to meet the needs of our patients. However, the current system does have two large fundamental problems. First, our reliance on altruistic blood donation creates seasonal shortages during the summer and winter holidays. Second, evolving and emerging infections will always endanger the safety of transfused human products. In theory, a red blood cell substitute would obviate both of these issues. A high-quality substitute would also be helpful for use in patients who are difficult to cross match, for individuals who will not accept transfusions of human products, and for emergency infusions at the scenes of trauma (civilian and military).
Most modern blood substitutes are either perfluorocarbons or hemoglobin-based oxygen carriers. Perfluorocarbons are non-water soluble, biologically inert, artificial, organic fluids with a high solubility for oxygen. Gas molecules are not chemically bound to perfluorocarbons, but instead are absorbed and released by simple diffusion. A large phase III trial using the perfluorocarbon, Oxygent, was halted early because of an increase in stroke rates in patients who were undergoing cardiopulmonary bypass. Hemoglobin-based oxygen carriers have held more promise. Four different methods have been used to avoid the toxicities induced by free hemoglobin: 1) cross-linking of the alpha chains, 2) polymerization of the hemoglobin chain tetramers, 3) conjugation of the hemoglobin to a larger molecule such as polyethylene glycol, and 4) encapsulating hemoglobin within liposomes. Since 1996, at least a dozen trials have been performed that analyzed the utility of these agents in a variety of clinical settings.
In a systematic review of the available literature on purified hemoglobin-based blood substitutes published since 1980, Natanson, et al. identified 70 trials, focusing only on the 16 randomized controlled trials involving 3,711 patients who received one of five cell-free hemoglobin products. One product, HemAssist, was cross-linked hemoglobin; three products, Hemopure, Hemolink, and PolyHeme, were polymerized hemoglobin; and one product, Hemospan, contained hemoglobin conjugated to polyethylene glycol. Disappointingly, but not surprisingly, the use of any of these products was associated with an almost three-fold increased risk of myocardial infarctions. Overall mortality was only mildly worse (relative risk 1.30) in subjects exposed to the blood substitutes. Further analysis did not indicate that any one hemoglobin product or any one indication for therapy was particularly worse than any of the others. These results demonstrate that the use of the available cell-free hemoglobin products is associated with too much morbidity, and an unacceptable rate of morbidity, to be of any clinical benefit.
Selecting Patients Most Likely to Respond to Therapy
By Kenneth Anderson, MD
Dr. Anderson indicated no relevant conflicts of interest.
Mateo G, Montalbán MA, Vidriales MB, et al. Prognostic value of immunophenotyping multiple myeloma: a study by the GEM and the PETHEMA cooperative study groups on patients uniformly treated with high-dose therapy. J Clin Oncol. 2008.[Epub ahead of print]
Multiple myeloma (MM) is a heterogeneous disease with a broad range of biological and clinical features.1,2 Multiple studies have therefore attempted to identify genetic and biologic markers, as well as clinical characteristics to define subgroups of patients. For example, prognostic factors such as beta 2 microglobulin and albumin form the basis of the International Staging System,3 which is predictive of disease course. Conventional cytogenetics can identify patients with adverse outcome to conventional low- and high-dose therapies (i.e., 4;14 translocation or deletion of chromosome 13).4 More recent studies have used microarray profiling5 and array comparative genomic hybridization6 to form the basis for defining mRNA-based and DNA-based prognostic subgroups of myeloma. Importantly, prognostic factors must be defined in a particular clinical context. For example, in patients treated with novel therapies such as bortezomib, chromosome 13 deletion and 4;14 translocation are no longer of adverse prognostic import.7 Moreover, recent studies have utilized microarray profiling to define patient populations with gene signatures predictive of response to specific therapies (i.e., bortezomib).8
Mateo and colleagues have recently carried out a prospective study of 685 newly diagnosed patients with myeloma treated uniformly with six alternating cycles of vincristine, BCNU, melphalan, cytoxan, and prednisone (VBMCP) alternating with vincristine, BCNU, adriamycin, and prednisone (VBAP) therapy, followed by melphalan 200mg/m2 and autologous stem cell transplantation. The median progression-free survival (PFS) and overall survival (OS) were 37 and 67 months, respectively. In order to delineate patient subgroups with differential outcome, CD138 positive bone marrow plasma cells were purified and immunophenotyped prospectively as well. Importantly, CD19+CD28+CD117-phenotype on CD138+BM plasma cells predicted poor outcome, with significantly shorter PFS and OS. An immunophenotype-based staging system, defined on the basis of CD28 and CD117 expression on tumor cells, identified poor-, intermediate-, and good-risk patient subgroups with significantly different PFS and OS. In addition to its clinical utility, understanding the biologic significance of the observed antigen-expression profiles conferring this differential outcome may both delineate mechanisms of sensitivity versus resistance to therapy and also yield insights into MM pathogenesis. Moreover, correlation of these phenotype profiles with microarray profiling and aCGH analysis may identify, and yield, new insights into MM pathogenesis to be exploited in novel, single agent or combination targeted therapeutics.
Several novel therapies are now available for myeloma, and the challenge is to use them most effectively. These investigators are to be congratulated on their major effort of prospectively purifying patient tumor cells for phenotypic analysis and correlation with clinical outcome. Although VBMCP/VBAP is no longer utilized and incorporation of novel therapies such as bortezomib into the initial therapy pre-transplant improves response post high-dose melphalan and autologous stem cell transplantation,9 this study by Mateo and coworkers is an example for future efforts of how correlative science can inform the selection of patients most likely to benefit from novel therapeutics.
References
- Kuehl WM, Bergsagel PL. Multiple myeloma: evolving genetic events and host interactions. Nat Rev Cancer. 2002;2:175-87.
- Hideshima T, Mitsiades C, Tonon G, et al. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007;7:585-98.
- Greipp PR, San Miguel J, Durie BG, et al. International staging system for multiple myeloma. J Clin Oncol. 2005;23:3412-20.
- Greipp PR. Prognosis in myeloma. Mayo Clin Proc. 1994;69:895-902.
- Bergsagel PL, Kuehl WM, Zhan F, et al. Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood. 2005 Jul 1;106:296-303.
- Carrasco DR, Tonon G, Huang Y, et al. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients. Cancer Cell. 2006;9:313-25.
- Jagannath S, Richardson PG, Sonneveld P, et al. Bortezomib appears to overcome the poor prognosis conferred by chromosome 13 deletion in phase 2 and 3 trials. Leukemia. 2007;21:151-7.
- Mulligan G, Mitsiades C, Bryant B, et al. Gene expression profiling and correlation with outcome in clinical trials of the proteasome inhibitor bortezomib. Blood. 2007;109:3177-88.
- Harousseau JL, Mathiot C, Attal M, et al. VELCADE/dexamethasone versus VAD as induction treatment prior to autologous stem cell transplantation in newly diagnosed multiple myeloma: updated results of the IFM 2005/01 trial. Blood (Annual Meeting Abstract). 2007;110:139a.
Erythrocyte Indigestion: A Surprising Role for the Mitochondrial Pathway of Apoptosis in Reticulocyte Autophagy
By Mark Koury, MD, and Charles Parker, MD
Sandoval H, Thiagarajan P, Dasgupta SK, et al. Essential role for Nix in autophagic maturation of erythroid cells. Nature. 2008. Epub ahead of print.
Schweers RL, Zhang J, Randall MS, et al. NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Proc Natl Acad Sci USA. 2007; 104:19500-5.
Kundu M, Lindsten T, Yang CY, et al. Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood. 2008. [Epub ahead of print]
Many dramatic changes of terminal mammalian erythroid cell differentiation occur during reticulocyte maturation, including completion of hemoglobin synthesis, degradation of internal organelles, conversion from aerobic to anaerobic metabolism, and acquisition of a uniform biconcave discoid shape. Recent publications have shed light on the role of autophagy, an intracellular process by which organelles are degraded, in mitochondrial loss during reticulocyte maturation. Two of the studies have demonstrated that knockout mice deficient in Nix, a BH3 domain-only member of the Bcl2 family of proteins, have retarded degradation and clearance of mitochondria in reticulocytes. This inability to degrade mitochondria leads to a shortened erythrocyte survival and anemia (i.e., partially compensated hemolytic anemia). Because Bcl2 and its family of proteins are key regulators of the mitochondrial pathway of apoptosis, the role of Nix in reticulocyte mitochondrial degradation suggests that it can mediate either apoptosis or survival, depending upon circumstances of the individual cell.
In nutrient-deprived cells, autophagy may be an alternative to apoptosis in that essential metabolites required for survival of the nutrient deprivation are salvaged by degrading organelles such as the mitochondria and recycling the crucial metabolic products.1 Similar to nutrient deprivation, maturating reticulocytes reach a crucial stage when death can result if the autophagic process is disrupted. Mitochondria appear to be both degraded and extruded from the maturing reticulocyte by autophagy.2 (See figure.) The failure of mitochondria to undergo autophagy in reticulocytes appears to be detrimental because a similar hemolytic anemia with mitochondria-retaining erythrocytes as found in Nix knockout mice was found in knockout mice with deficiency of Ulk1, the mammalian homologue of atg 1p, a mitochondrial autophagy regulatory protein in yeast.
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| Model of Nix-mediated autophagic clearance of mitochondria from reticulocytes. Nix interacts with mitochondria in reticulocytes that have completed heme synthesis. Nix induces depolarization of the inner mitochondrial membrane, and the depolarized mitochondrion is sequestrated in a double membrane structure termed the autophagosome. The autophagosome subsequently fuses with a lysosome forming the autolysosome. Proteolytic enzymes from the lysosome degrade the inner membrane of the autolysosome and partially degrade the sequestered depolarized mitochondrion. The non-degraded contents of the autolysosome are extruded from the reticulocyte. |
In the sequence of events in mitochondrial autophagy in reticulocytes, Nix acts at the stage of mitochondrial depolarization and targeting for inclusion in autophagosomes. Nix interacts with the outer mitochondrial membrane, leading to loss of inner membrane polarization.3 Reticulocytes from Nix-deficient mice retain polarized mitochondria, but they are localized to areas adjacent to autophagosomes in reticulocytes. This result suggests that targeting of the mitochondria to the autophagosomes may be intact, but Nix’s induction of mitochondrial depolarization is required for normal mitochondrial incorporation into autophagosomes. Further evidence for this role of Nix in mitochondrial depolarization is that chemical depolarization of mitochondria in reticulocytes from Nix-deficient mice leads to their autophagic clearance. Unlike reticulocytes and erythrocytes from Nix-deficient mice, those from Ulk1-deficient mice have retention of ribosomes and mitochondria. Furthermore, the retained mitochondria in the Ulk1-deficient mice are not localized to areas adjacent to autophagosomes, suggesting that Ulk1-deficient mice have a defect at a different point than do Nix-deficient mice in the reticulocyte autophagy pathway.
Further studies of maturing reticulocytes have the potential to determine the specific range, targeting, and fate of the organelles that are removed by autophagy. This information will not only interest those studying erythropoiesis, but also interest those researchers and physicians interested in other cellular processes that involve autophagy, such as differentiation, aging, and survival following chemical or physical stress.
References
1. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol. 2004;36:2405-19.
2. Koury MJ, Koury ST, Kopsombut P, et al. In vitro maturation of nascent reticulocytes to erythrocytes. Blood. 2005;105:2168-74.
3. Diwan A, Koesters AG, Odley AM, et al. Unrestrained erythroblast development in Nix-/- mice reveals a mechanism for apoptotic modulation of erythropoiesis. Proc Natl Acad Sci USA. 2007;104:6794-9.
Targeting NF-κB in CLL
By Steven Grant, MD
Dr. Grant indicated no relevant conflicts of interest.
Hewamana S, Alghazal S, Lin TT, et al. The NF-κB subunit Rel A is associated with in vitro survival and clinical disease progression in chronic lymphocytic leukemia and represents a promising therapeutic target. Blood. 2008;111:4681-9.
The NF-κB family of transcription factors has been implicated in diverse cellular processes, including cell proliferation, differentiation, survival, and inflammatory responses, among numerous others. At least three NF-κB cascades have been characterized: the classical or canonical pathway, which is induced by cytokines such as TNFα; the non-classical, or alternative pathway, which is triggered by BAFF and CD40 ligation; and the atypical pathway, which is engaged by DNA damage. Activation of NF-κB leads to transcription of numerous genes, many of which (for example, XIAP and Bcl-xL) serve survival functions. Not unexpectedly, NF-κB activation occurs in many tumor types, particularly hematologic malignancies. For example, multiple myeloma cells have long been thought to depend heavily upon NF-κB activation for their survival, and several recent studies have documented a high incidence of abnormalities involving genes associated with NF-κB activation in patient-derived myeloma cells. In addition, NF-κB activation is characteristic of AML cells in general, as well as in AML stem cells. A corollary of these observations is that NF-κB represents a logical candidate for therapeutic intervention.
CLL is an accumulative disease of mature, differentiated lymphocytes. Although activation of the classical and alternative pathways has been implicated in CLL cell survival, the clinical relevance of these observations has not been clearly defined. However, a recent study by Hewamana, et al. may shed significant light on this issue. In this study, the authors examined NF-κB DNA binding, reflected by EMSA assays, in cells from a series of patients with CLL and sought correlations with more established prognostic indicators. They also tested whether the extent of NF-κB activation predicted resistance of cells to conventional and novel agents. While the authors observed considerable inter-sample variability in basal NF-κB activation status, clear correlations were observed between activation and certain known negative prognostic indicators (e.g., high white count, short doubling time), although not between others (e.g., ZAP-70 expression). Interestingly, cells exhibiting high basal NF-κB activity were less sensitive to the established agent fludarabine, but more sensitive to the novel agent, LC-1, a parthenolide analog that inhibits IKK and has recently been shown to be active against AML stem cells.
One implication of this study is that, as recently suggested in the case of other hematologic malignancies such as multiple myeloma and AML, the NF-κB pathway may not only be an important prognostic determinant in CLL, but could also represent a logical target for pharmacologic intervention in this disorder. In this context, the results of a recent preclinical study suggested that synergistic interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors in primary CLL cells involved, at least in part, NF-κB inactivation.1 The broader implication of the present study is that, as more sophisticated gene and protein profiling classification systems are developed in CLL and other hematologic malignancies, their ultimate benefit may lie in guiding the development of more rational, mechanism-based, targeted forms of therapy.
References
- Dai Y, Chen S, Kramer LB, et al. Interactions between bortezomib and romidepsin and belinostat in chronic lymphocytic leukemia cells. Clin Cancer Res. 2008;14:549-58.