• Hematopoietic Stem Cells: Nature’s Unpredictable Child
• Primary CNS Lymphoma: Progress and Caveats
• When Three Months May Not Be Enough: Evidence That a Subgroup of Patients With Venous Thrombosis Identified by Bio-marker Assay Benefits From Long-Term Oral Anticoagulation
• LEF-1 Expression in Congenital Neutropenia
• Susceptibility to Oxidative Stress: A Leukemic Cell’s Achilles’ Heel?
• Economy Class Syndrome - Survival of the Richest?
• Graft–Versus-Host Disease: Finding THE Cell Subset!
• Targeting Protein Homeostasis in Novel Therapeutics

Hematopoietic Stem Cells: Nature’s Unpredictable Child

Robert Lowsky, MD

Dr. Lowsky indicated no relevant conflicts of interest.

McKenzie JL, Gan OI, Doedens M, et al. Individual stem cells with highly variable proliferation and self-renewal properties comprise the human hematopoietic stem cell compartment. Nat Immunol 2006;7:1225-1233.

To maintain the enormous daily hematopoietic cell output (approximately 1 x 10¹² cells/day), terminally differentiated blood cells are continually produced from highly proliferative but short-lived progenitors, which in turn arise from a rare population of quiescent hematopoietic stem cells (HSCs). Understanding how individual HSCs contribute to blood cell formation throughout a lifetime has remained a subject of debate. In this paper, McKenzie and colleagues studied individual human HSCs and found substantial variation in proliferation kinetics and self-renewal capacity implying that HSC fate is unpredictable before they enter the more rigid downstream developmental programs.

Hematopoietic cell repopulation following sublethal irradiation of the nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse has emerged as the “gold standard” surrogate assay for studying human HSCs. The human cells that repopulate these mice are functionally defined as “SCID mouse-repopulating cells” (SRCs). SRCs express CD34 and are lineage-negative (Lin-), and distinct SRC activities can be found in the CD38- and CD38^lo subfractions. McKenzie and colleagues generated more than 600 individual human SRCs from placental and umbilical tissues that were transduced with a lentivirus to track the clonal ancestry of the human cells after injection and evaluated their repopulating activity and self-renewal over a seven-month period of analysis in serially transplanted NOD-SCID mice. Primary transplant recipients showed that only a subset of clones in the injection site (right femur) were also present in other bones, indicating that only some SRCs divided and migrated to other hematopoietic tissues. Individual SRCs collected from primary recipients were heterogeneous in terms of self-renewal, with some clones making substantial yet fluctuating contributions over time to all hematopoietic territories and cells of secondary mice, and others not engrafting after serial transplantation. Evidence that clonally related daughter cell pairs have distinct and unpredictable repopulation kinetics provides the strongest support favoring this stochastic model for hematopoiesis.

The paper by McKenzie and colleagues successfully brings together a wide range of experimental methodologies to now address the key issue of how human HSCs act in vivo. The considerable heterogeneity among stem cell fate and self-renewal implies that this unpredictability likely arises through HSC interactions with as-of-yet undefined extrinsic properties (i.e., HSC niche occupancy and cytokine exposure) or intrinsic properties (i.e., asymmetric distribution of intracellular proteins and alterations of signal transduction pathways). If HSC self-renewal and proliferation kinetics are governed by probabilistic elements, then the current strategies to identify a molecular stem cell signature by profiling global gene expression of pooled yet static HSC populations may prove unreliable. Also, as the resemblance between normal and malignant stem cells deepens, the understanding of how stem cell behavior can be modulated by extrinsic and intrinsic factors will identify novel targets for cancer therapeutics.

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Primary CNS Lymphoma: Progress and Caveats

Michael Williams, MD

Dr. Williams indicated no relevant conflicts of interest.

Gavrilovic IT, Hormigo A, Yahalom J, et al. Long-term follow-up of high-dose methotrexate-based therapy with and without whole brain irradiation for newly diagnosed primary CNS lymphoma. J Clin Oncol 2006;24:4570-4.

The treatment and outcomes for primary CNS lymphoma improved in the 1990s with the incorporation of high-dose methotrexate-based regimens. These gains were in part offset by neurotoxicity associated with the use of whole brain radiation therapy (WBRT), usually manifest as impaired cognitive function or overt dementia. Gavrilovic and colleagues now update the extensive Memorial Sloan-Kettering Cancer Center experience with follow-up of a consecutive non-randomized series of 57 patients treated from 1992-98 with high-dose methotrexate (MTX), procarbazine, vincristine, cytarabine, and intrathecal MTX (per Ommaya reservoir). WBRT 45 Gy was also included, although after 1995 patients > 60 years of age did not receive RT due to recognition of neurotoxicity being pronounced in this age group. Overall, 17 patients (30 percent) remain alive at the time of analysis (December 2005), 13 of whom were < 60 years at the time of initial therapy (Table).

Primary CNS lymphoma remains a significant clinical challenge. While it occurs with increased frequency among individuals with congenital or acquired immunodeficiency, it also occurs in those who are immunologically intact. Spread outside the CNS is unusual, although ocular, spinal cord, and leptomeningeal involvement may occur. As demonstrated in this long-term follow-up study, improved outcomes as compared with historical experience were realized using a combined modality regimen incorporating high-dose methotrexate and WBRT, albeit at the cost of neurotoxicity and neurocognitive decline, especially in patients > 60 years of age. The authors note that they likely underestimated the rate of neurotoxicity, as formal neuropsychiatric testing was not performed. WBRT is now deferred in many patients, especially in the older age group. Future studies will need to incorporate cognitive testing and utilize recently developed standardized staging and response criteria¹. Newer therapeutic strategies include the use of rituximab and temazolamide, which have activity in recurrent disease; these agents have been incorporated into up-front therapy ongoing trials. Recent reports have also suggested benefit for stem cell transplantation as part of initial therapy in younger patients.

1. Abrey LE, Batchelor TT, Ferreri AJM, et al. Report of an International Workshop to Standardize Baseline Evaluation and Response Criteria for Primary CNS Lymphoma. J Clin Oncol 2005;23:5034-5043.

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When Three Months May Not Be Enough: Evidence That a Subgroup of Patients With Venous Thrombosis Identified by Bio-marker Assay Benefits From Long-Term Oral Anticoagulation

Roy Silverstein, MD

Dr. Silverstein indicated no relevant conflicts of interest.

Palareti G, Cosmi B, Legnani C, et al. D-dimer testing to determine the duration of anticoagulation therapy. N Engl J Med 2006;355:1780-9.

In this multicenter prospective study conducted mainly in Italy, investigators studied ~600 patients with a first episode of well documented, unprovoked venous thromboembolism (DVT and/or PE). After completing at least three months of oral anticoagulation (OAC) therapy with vitamin K antagonists, all subjects stopped therapy and ~30d later underwent qualitative testing for d-dimer and a limited thrombophilia panel. After excluding subjects with AT3 deficiency, anti-phospholipid antibodies, liver or kidney disease, or serious life-threatening illnesses, those with positive d-dimer were randomized to either resume OAC or not, while those with negative d-dimer remained off therapy. Subjects were then followed for at least nine months. Bleeding events were rare during the relatively short period of follow-up, but interesting differences were seen with regard to recurrent VTE. Those with detectable d-dimer who remained off OAC had a 2.5-fold increased risk of recurrence (11 events per 100 patient years) compared to those with negative d-dimer. More importantly, they had a 5.4-fold increased risk compared to those with positive d-dimer who were placed back on OAC. Thus a positive d-dimer test identified a high risk group for preventable recurrence.

One of the most vexing problems managing patients with VTE is balancing the bleeding risk, expense, and inconvenience associated with prolonged OAC with the threat of recurrent thrombosis once therapy is stopped. Thrombosis risk is highest in the first year after a VTE, but it never returns to population baseline, presumably reflecting the presence of an underlying thrombophilia in many patients. Although major advances have been made in understanding genetic, biochemical, and immunologic factors related to primary thrombotic risk, screening patients for these risk factors has contributed little to our ability to predict secondary risk in an individual patient, with the possible exception of detecting antiphospholipid antibody syndrome or the rare patient with AT3 deficiency or homozygous Factor V Leiden. The PROLONG study begins to help refine therapeutic decision-making for a subset of patients. Their data suggests strongly that a simple diagnostic test (d-dimer) performed one month after stopping OAC defines a subgroup that clearly benefits from prolonged anticoagulation. Unfortunately the data do not help in deciding what to do with the 60 percent whose d-dimer is negative and who still have a substantial event rate (4.4 percent per year in this study)! It is also important to remember that this study included only subjects with first episode unprovoked VTE, representing less than half of all VTE patients seen in the ambulatory setting.

What is the pathophysiologic meaning of a persistent positive d-dimer months after a VTE? The observation that more than 50 percent of the recurrent events were contralateral suggests that circulating d-dimer might be a marker of systemic thrombophilia (i.e., an ongoing imbalance of thrombin generation) rather than of an unresolved primary thrombus. This is consistent with studies showing that residual vein thrombosis detected by sonography months after a VTE also correlates with contralateral recurrence. Recent studies have suggested that d-dimer levels tend to increase with age (and in this study those with positive d-dimer were on average 10 years older than those negative), and d-dimer is known to increase in the setting of chronic inflammatory disorders, so this test is likely to be less useful in decision making for these patient groups. Nevertheless, the PROLONG study indicates that segregating risk in VTE patients is an attainable goal, but that there is still a need to develop more sensitive and specific biomarkers to detect persistent underlying thrombophilia in subjects who have suffered VTE.

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LEF-1 Expression in Congenital Neutropenia

Lilli Petruzzelli, MD, PhD

Dr. Petruzzelli indicated no relevant conflicts of interest.

Skokowa J, Cario G, Uenalan M, et al. LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia. Nat Med 2006;12:1191-7.

Congenital neutropenia (CN) is associated with mutation of a number of genes including elastase, G-CSF receptor, GFI-1, and WASP; however, in many cases the underlying defect is not established. In this manuscript, the focus shifts to analysis of LEF-1, whose role as a transcription factor that is important in lymphocyte proliferation and differentiation is well established. The authors compare mRNA expression patterns in CD33+ cells from patients with congenital neutropenia to those from normal donors, as well as those with neutropenia from other causes, and found that there was a marked reduction in expression of LEF-1 in cells from patients with CN, but not those with other forms of neutropenia or normal neutrophils. Although half of the 13 patients with CN in this study had mutations in the ELA2 gene, all had reduced levels of LEF-1 and maturation arrest. The differences in expression level were evident beyond the blast stage and were most dramatic in the promyelocyte. Expression of LEF-1 in progenitor cells from patients with CN enables differentiation. The investigators propose that regulation occurs through the ß-catenin-independent action of LEF-1 and is likely due to a balance between proliferative and apoptotic factors.

The work here not only characterizes factors that may be important in cyclic neutropenia, but also establishes a role for the transcription factor LEF-1 in myelopoiesis. An earlier report by Li et al.¹ demonstrated that LEF-1 was expressed in bone marrow cells and myeloid cell lines and identified a LEF-1 binding domain that was mutated in two patients with severe chronic neutropenia. Of interest is that LEF-1 binding to the promoter is enhanced by the mutation that was uncovered in both of the patients described and results in increased elastase production. In contrast, in the patients studied in this manuscript, diminished LEF-1 expression — and thus activity — are thought to be critical for the maturation arrest, diminished proliferation, and decreased cell survival that is observed in these patients. Here, the investigators used two techniques to confirm that LEF-1 expression was necessary for maturation of myeloid precursors — expression in progenitors from patients with CN enabled differentiation; and disruption of its expression by small hair-pin RNAs resulting in maturation arrest, diminished proliferation, and decreased cell survival. In probing for mechanisms, LEF-1 overexpression enhances CEBP expression independently of G-CSF that normally regulates its expression. Of note is that G-CSF had little effect on LEF-1 levels under “physiologic” concentrations but is hypothesized to upregulate its expression at pharmacologic doses. This manuscript characterizes a group of patients with congenital neutropenia that have diminished LEF-1 expression. It remains to be seen whether this finding is consistent among a larger cohort of patients. Nonetheless, the data presented here demonstrate that its expression level plays a role in myeloid cell differentiation and implicate it in neutropenia. Since analysis of the promoter region LEF-1 has not identified a potential mutation site, the next step will be to determine the post-transcriptional regulatory step that may be altered in these patients.

1. Li F-Q, Person RE, Takemaru K-I et al. Lymphoid enhancer factor-1 links two hereditary leukemia syndromes through core-binding factor regulation of ELA2. J Biol Chem 2004;279:2873–2884.

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Susceptibility to Oxidative Stress: A Leukemic Cell’s Achilles’ Heel?

Steven Grant, MD

Dr. Grant indicated no relevant conflicts of interest.

Trachootham D, Zhou Y, Zhang H, et al. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer Cell 2006;10:241-52.

In a recent study published in Cancer Cell, Trachootham et al. reported that expression of mutant oncogenic proteins, including Bcr/Abl in the case of hematopoietic cells, or H-Ras in the case of epithelial tumors (i.e., ovarian cancer), not only induced transformation but also triggered an increase in levels of reactive oxygen intermediaries (reactive oxygen species - or ROS). As a consequence, cells expressing these mutant oncoproteins were significantly more sensitive to the lethal effects of agents that disrupted cellular oxidative injury defense mechanisms than their normal counterparts. Specifically, exposure to ß-phenylethyl isothiocyanate (PEITC), a compound which disables the GSH anti-oxidant system, caused significantly more apoptosis in transformed versus wild-type cells. The authors conclude that oncogenic transformation may be accompanied by perturbations in redox homeostasis, and that this phenomenon could represent the tumor cell’s “Achilles’ heel,” rendering it selectively vulnerable to therapeutic intervention.

If validated, these findings could have particularly important implications for the treatment of hematologic malignancies. It has long been known that tumor cells may display higher levels of ROS than their normal counterparts. Interpretation of the significance of this phenomenon has been complicated by evidence that ROS play diverse and, on occasion, opposing roles in cellular survival and behavior. For example, at high concentrations, ROS damage DNA and lipid membranes and induce mitochondrial dysfunction culminating in apoptosis. However, at lower concentrations, ROS can act as signaling molecules and may contribute to cell proliferation among other functions. In the case of Bcr/Abl+ hematopoietic malignancies (e.g., CML), ROS induced by the Bcr/Abl oncoprotein have been implicated in the induction of mutations responsible for disease progression or drug resistance. Thus, the net effect of ROS generation may depend upon multiple factors, including cell context, the degree of oxidative injury, and perhaps the nature of the inciting stimulus.

The possibility that transformed cells display greater susceptibility to oxidative damage takes on added significance in view of emerging insights into the mechanism of action of several novel “targeted agents” and provides a possible theoretical basis for their therapeutic selectivity. In this context, histone deacetylase inhibitors (HDACIs), which are currently undergoing extensive evaluation in the treatment of hematologic malignancies, are known to kill leukemic cells through the selective induction of oxidative injury. In addition, proteasome inhibitors like Bortezomib, which in preclinical studies preferentially kill transformed cells, can also exert their lethal effects through induction of ROS. Other studies involving agents like 2-medroxyestradiol, arsenic trioxide, or the tyrphostin adaphostin, administered alone or in combination with other targeted agents, point to oxidative injury as a basis for therapeutic selectivity.

The possibility that the Bcr/Abl kinase or dysregulated RAS, which is frequently mutated in hematopoietic malignancies, might predispose cells to oxidative injury-induced cell death has very obvious clinical implications, particularly in diseases like leukemia. It raises the possibility that a) certain agents might preferentially induce ROS in leukemic cells¹, and b) leukemic cells may be intrinsically less capable of surviving these insults. It also suggests that strategies combining novel agents, each of which may preferentially induce ROS in transformed cells², might be a particularly appropriate strategy in this setting. In view of ongoing efforts to develop such clinical strategies, answers to these questions should begin to emerge in the near future.

1. Huang P, Feng L, Oldham EA, et al. Superoxide dismutase as a target for the selective killing of cancer cells. Nature 2000;407:390-395.
2. Gao N, Rahmani M, Shi X, et al. Synergistic antileukemic interactions between 2-medroxyestradiol (2-ME) and histone deacetylase inhibitors involves Akt down-regulation and oxidative stress. Blood 2006;107:241-249.

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Economy Class Syndrome - Survival of the Richest?

Charles Abrams, MD

Dr. Abrams indicated no relevant conflicts of interest.

Schreijer AJM, Cannegieter SC, Meijers JCM, et al. Activation of coagulation system during air travel: a crossover study. Lancet 2006;367:832-38.

Toff WD, Jones CI, Ford I, et al. Effect of hypobaric hypoxia, simulating conditions during long-haul air travel, on coagulation, fibrinolysis, platelet function, and endothelial activation. JAMA 2006;295:2251-61.

The association between long-distance travel and pulmonary embolism has been recognized for over half a century. The mechanism for the linkage between these two events is unclear. In addition to stasis of blood caused by immobility while in the economy class compartment of the plane, investigators have speculated that the unique air travel environment makes long-distance air travel particularly risky. In a study published in Lancet earlier this year, F.R. Rosendaal and colleagues compared changes in parameters of coagulation and fibrinolysis in subjects during an eight-hour plane flight, eight hours of sitting while watching a cinema marathon, or eight hours of normal activities. In that study, thrombin-antithrombin complex levels (a measure of activated coagulation) increased significantly only in the subjects while they traveled by air. The changes in thrombin-antithrombin complexes were almost exclusively identified in the subgroup of subjects who were predisposed to thrombosis (e.g., Factor V Leiden carriers who also took oral contraceptives). This study suggested that airplane travel per se, rather than mere economy-class confinement, activated the coagulation system contributing to the thrombosis.

Since reduced oxygen tension and cabin pressures are potential environmental risk factors that could affect the coagulation system, W.D. Toff et al. analyzed subjects before and after eight seated hours in a hypobaric and slightly hypoxic chamber. These subjects did not have any known prothrombotic risk factors. Again, changes in parameters of coagulation and fibrinolysis were assayed including thrombin-antithrombin complex levels. In seeming contrast to the Lancet study, these authors did not find any effect of hypobaric hypoxia on the coagulation system. The potential discrepancy between these two studies may be explained by an environmental factor encountered during air travel that is not due to low oxygen tension or cabin pressure. Alternatively, the effect of these conditions on coagulation may be so subtle that it is only detectable in subjects who are already mildly pro-coagulable.

In 1940, Keith Simpson, an Assistant Lecturer in Forensic Medicine in London, noted a recent six-fold increase in lethal pulmonary embolisms (see Case Record from Lancet December 14, 1940, page 744). Dr. Simpson speculated that prolonged sitting on deck chairs in public air-raid shelters contributed to this epidemic. He was further convinced that providing bunks, instead of chairs, would help alleviate this early example of “economy class syndrome.” Since that time, the association between confinement and thromboembolic disease has been recognized during car trips, air flights, and train travel. The death of David Bloom, a NBC reporter who suffered a pulmonary embolism while riding in an armored vehicle within Iraq, brought this problem to national attention. It has been estimated that the risk of a symptomatic thrombosis developing as a result of travel longer than four hours is between 1:2000 and 1:6000. However, it remains controversial whether mechanical confinement alone accounts for all of the risk.

Equally controversial is whether prophylactic anticoagulation is worthwhile to prevent thrombotic complications of long-distance travel. Some physicians advocate prophylactic therapy for their patients as well as for themselves. The use of elastic stockings certainly reduces the risk of leg edema and may provide some prophylactic benefit to prevent significant deep vein thrombosis. Aspirin has been advocated by some authorities, but failed to demonstrate efficacy in the LONFLIT III study (Angiology 2002;53:1-6). Low-molecular-weight heparin appears to reduce the incidence of thromboembolic disease, but it is also associated with a small risk of hemorrhage that is close to the risk of thrombosis during air travel. For most patients, the small benefit of prophylactic therapy does not significantly outweigh the risk. For patients with high prothrombotic risk factors (such as history of venous thrombosis, metastatic cancer, certain inherited thrombophilias, and perhaps hormonal therapy), prophylactic low-molecular-weight heparin therapy is a reasonable option for patients planning long-distance travel.

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Graft–Versus-Host Disease: Finding THE Cell Subset!

Gérard Socié, MD, PhD

Dr. Socié indicated no relevant conflicts of interest.

Zhang Y, Joe G, Hexner E, Zhu J, Emerson SG. Host-reactive CD8+ memory stem cells in graft-versus-host disease. Nat Med 2005;11:1299-305.

Graft-versus-host disease (GVHD) remains a devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT), and little progress has emerged since the introduction many years ago of calcineurin inhibitors (cyclosporine and tacrolimus) as prophylaxis. When it develops, treatment with steroids is efficacious in only 40 to 50 percent of the cases, and the prognosis of steroid-resistant GVHD remains very poor with less than 30 percent long-term survivors. There is thus a desperate need to improve understanding of this complex disease.

One major paper was published by Zhang and colleagues in the fall of 2005. In this study, the authors identify in a mouse model a new subset of CD8+ T lymphocytes that generate and sustain all allogeneic T-cell subsets in GVHD reactions, while self-renewing themselves (inducing GVHD upon transfer into secondary recipients). These CD8+T cells persist throughout the course of GVHD, are generated in the initial phase in response to alloantigen and dendritic cells, and require IL-15. Their long life, ability to self-renew, and multi-potentiality define these cells as being memory stem cells.

Why is this study not just another rodent model with poor clinical implications? Two main reasons, at least from my point of view:

1. Identification of selective drug targets within the GVHD memory stem cell could be used for eradicating ongoing GVHD,and thus break the deadly circle of donor T-cell activation against the host target cells, while sparing a T-cell subset engaged against infectious agents (viral and fungal, in particular).
2. Discovery of such a cell subset has much broader applications and implications beyond just GVHD. Memory stem cells will be important targets for understanding and influencing diverse chronic immune reactions, including autoimmune diseases and solid organ transplant rejection.

So now what are the obstacles to overcome from a clinical perspective before moving from the bench to the bedside?

1. The first objective must surely be to identify and characterize whether these memory stem cells actually do exist in humans.
2. The relationship of this subset of cells to other cells responsible for the graft-versus-leukemia (GVL) observed after allogeneic HSCT is of major importance. Does the memory stem cell identified in this model setting differ from the cell that is responsible for the GVL effect both in rodent models and, most importantly, in human beings?

These questions aside, this paper nevertheless opens the door of a new and highly exciting era in the field of GVHD in particular, and in the world of immunology in general.

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Targeting Protein Homeostasis in Novel Therapeutics

Kenneth Anderson, MD

Dr. Anderson indicated no relevant conflicts of interest.

Catley L, Weisberg L, Kiziltepe T, et al. Aggresome induction by proteasome inhibitor bortezomib and a tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH 589 are synergistic in myeloma cells. Blood 2006;108:3441-9.

The ubiquitin proteasome pathway is a major system for degradation of proteins, and the proteasome inhibitor, Bortezomib, has now been FDA-approved for treatment of relapsed as well as relapsed and refractory multiple myeloma. In spite of its remarkable activity, some patients do not respond, and those who do respond eventually acquire resistance. Moreover, clinical activity outside of myeloma has been limited. We are now beginning to uncover clues to address this problem. Specifically, an alternative mechanism for degradation of ubiquinated proteins is via the aggresome/lysosome/autophagy pathway¹. In this cascade, histone deacetylase (HDAC) 6 binds to ubiquinated protein on the one hand, and to dynein microtubule complexes on the other, thereby shuttling the ubiquinated protein to its degradation via the aggresome/lysosome/-autophagy mechanism. The relative roles of these two pathways of protein degradation, both in normal and cancer cells, as well as their substrate specificities, are not yet defined. Previous studies in myeloma have shown that blockade of the aggresome cascade with tubulin deacetylase inhibitor tubacin triggers a compensatory increase in proteasomal degradation of ubiquinated proteins, whereas inhibition of proteasomal degradation of ubiquinated proteins with Bortezomib induces increased aggresomal activity². Importantly, inhibiting both aggresomal and proteasomal activity with tubacin and Bortezomib, respectively, triggers synergistic myeloma cell cytotoxicity, associated with significant accumulation of polyubiquinated proteins.

In this study, Catley and colleagues move this concept from bench to bedside towards a derived clinical trial. They show that the novel hydroxamic acid derivative histone deacetylase inhibitor LBH 589 induces apoptosis of myeloma cells resistant to conventional and novel therapies, as well as tubulin hyperacetylation, at clinically achievable concentrations. Conversely, Bortezomib triggers increased aggresome formation. When used in combination, both Bortezomib-induced aggresome formation and LBH 589-induced a tubulin hyperacetylation are augmented. Importantly, combined LBH 589 and Bortezomib treatment induces synergistic cytotoxicity against myeloma cell lines and patient cells, including those sensitive and resistant to conventional and novel therapies.

This study provides insight into the mechanisms of synergistic cytotoxicity of combined Bortezomib and LBH 589 in myeloma. Excitingly, it suggests that either intrinsic or acquired resistance to Bortezomib may be overcome by addition of LBH 589, thereby broadly expanding the spectrum of myeloma patients who respond. It is not at present clear to what extent ubiquinated protein degradation via the aggresome/lysosome/autophagy mechanism mediates resistance to proteasome inhibitors in cancers outside of myeloma. However, Nawrocki and colleagues have recently shown that Bortezomib induces aggresomes in pancreatic cancer cells, and that Bortezomib-induced aggresome formation was inhibited by either HDAC 6 small interfering RNA or HDAC inhibitors, resulting in synergistic cytotoxicity³. Importantly, the therapeutic index of LBH 589, Bortezomib, and combined use is favorable, since normal cells are not dependent on multiple mechanisms of ubiquinated protein degradation. Clinical trials of LBH 589 in myeloma are beginning, with trials of combined LBH589 and Bortezomib to quickly follow, based upon this study. Targeting protein homeostasis in this fashion therefore has great potential to improve outcome, not only of patients with myeloma, but those with solid tumors as well.

1. Hideshima T, Bradner JE, Chauhan D, Anderson KC. Intracellular protein degradation and its therapeutic implications. Clin Cancer Res 2005;11:8530-8533.
2. Hideshima T, Bradner J, Wong J, et al. Small molecule inhibition of proteasome and aggresome function induces synergistic anti-tumor activity in multiple myeloma: therapeutic implications. Proc Natl Acad Sci USA 2005;102:8567-8572.
3. Nawrocki ST, Carew JS, Pino MS, et al. Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells. Cancer Res 2006;66:3773-3381.

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