Hemoglobinopathies and Malaria — Epistasis for Better or Worse
Nancy Andrews, MD, PhD
Williams TN, Mwangi TW, Wambua S, et al. Negative epistasis between the malaria-protective effects of α+-thalassemia and the sickle cell trait. Nature Genetics 2005; 37:1253-1257.
We all learned in medical school that hemoglobinopathies are prevalent, at least in part, because carriers benefit from protection against malaria. It is well established that both sickle cell trait and α+-thalassemia alter red cell physiology to put the parasite at a disadvantage. Sickle cell trait may attenuate malarial infection by reducing intracellular oxygen tension or by targeting parasite-infected cells for splenic clearance. The protective mechanism of α+-thalassemia is even less certain, but the phenomenology is undisputed. In this article, the authors asked whether the combination of sickle cell trait and α+-thalassemia (HbAS, -α/-α) would be more protective than either trait alone. Their surprising finding was that patients carrying both hemoglobinopathy traits had little more protection than individuals who had neither.
This is an elegant example of negative epistasis. Epistasis is a genetic term that describes a situation in which the effect of a change in one gene depends on what is happening in a second, unrelated gene. The Bombay blood group phenotype is a familiar example of this effect. Individuals homozygous for the Bombay phenotype lack the ability to make a precursor molecule that is needed for attachment of both A and B antigens. As a result, their genes may dictate that they should have an A or B phenotype, but the Bombay gene overrules and the blood types as O. In the situation described in this article, the α+-thalassemia genotype abrogates the strong protective effect of HbAS. The mechanism for this negative epistasis is uncertain, but the authors hypothesize that it may result from greater affinity of the α globin polypeptide for the normal β globin polypeptide. Decreased α globin in the cell might thus change the relative amounts of HbA and HbS, effectively decreasing the intracellular concentration of HbS. While this compromises protection against malaria, the co-existence of α+-thalassemia and sickle cell genes in the same gene pool may have a beneficial effect in ameliorating the anemia of sickle cell disease.
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Excited Platelet Ignores Its Status
Josef Prchal, MD
Denis MM, Tolley ND, Bunting M, et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005;122:379-91.
Denis and colleagues report on a remarkable phenomenon that destroys conventional wisdom in several respects and greatly extends our understanding of platelet function and biology in general. The platelet, which is an anucleate cell, is generated from the extrusion of megakaryocyte cytoplasm and contains pre-mRNA species (unprocessed transcripts of genes that contain as yet unspliced introns) that should only be found in the nucleus. Further, when a platelet is activated by extrinsic stimuli, it undergoes another process that should be nucleus-specific; platelets use the spliceosome complex (composed of an array of small nuclear RNPs and auxiliary proteins) to remove introns and produce mature mRNA. This mRNA can productively be translated into protein in response to exogenous stimuli, another surprising process in platelets also recently described by Andy Weyrich's laboratory. Interestingly, only a subset of platelet mRNAs contain introns, suggesting the existence of a novel selection process that megakaryocytes use for platelet formation, i.e., choosing selected mRNA as templates for proteins that can be made on demand in response to exogenous stimuli. This remarkable observation adds yet another mechanism to other nuclear independent gene expression pathways including mRNA editing1 and nonsense mediated decay2. Presumably, this novel series of processes allows platelets to rapidly respond to activating signals by processing primitive mRNAs and making them ready for translation to the peptides, thus bypassing the complexity of mRNA processing and nuclear transport to the cytoplasm. The heretofore unchallenged dogma that transcription of genomic DNA to pre-mRNA and removal of introns by the splicosome complex is nucleus-specific is demolished. The extension of this dogma, that the definite mRNA is transported out to the cytoplasm and the unprocessed intron containing pre-mRNA is degraded in nucleus, is also no longer absolute.
Many questions should soon be answered by follow-up studies. Is this described phenomenon a platelet-specific mechanism, or does a similar mechanism also exist in neutrophils3 and macrophages that have also been shown to synthesize proteins after cytokine or chemokine stimuli? Does it also exist in neuronal outgrowth that share some other structural and functional similarities with megakaryocytes? How does pre-mRNA get out of the nucleus? Do other nuclear-dependent gene-expression mechanisms also take place in platelets and other cells, such as nonsense mediated decay that is also thought to require nuclear component for its initiation2? In summary, this elegant, carefully documented work is a beautiful example of the escape from the rigid dogma and adds another previously unforeseen flexibility to generate life complexity. Is this mechanism exaggerated in clonal thrombopoiesis such as polycythemia vera and essential thrombocythemia where thrombosis and bleeding are yet unexplained major causes of morbidity and mortality? There is no doubt that this important work will have practical implications for better understanding thrombosis, hemostasis, and inflammatory conditions wherein platelets play a central role. The remaining questions will have to be unraveled by future studies and other investigators. Sadly, Melvin Denis was an MD/PhD student who did not live to enjoy the publication of this seminal work after losing his life in an avalanche accident.
- Chen L, Chan L. Control of apolipoprotein B mRNA editing: implication of mRNA at various maturation stages. J Theor Biol 1996;183:391-407.
- Holbrook JA, Neu-Yilik G, Hentze MW, Kulozik AE. Nonsense-mediated decay approaches the clinic. Nat Genet 2004;36:801-8.
- Lindemann SW, Yost CC, Denis MM, et al. Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs. Proc Natl Acad Sci S A 2004;101:7076-81.
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MicroRNAs and CLL Prognosis
Michael Williams, MD
Calin GA, Ferracin M, Cimmino A, et al. A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 2005;353:1793-801.
MicroRNAs are specifically encoded small RNAs(~19-25 nucleotides) which regulate gene expression, including oncogenes and tumor suppressor genes, via interaction with messenger RNA. Calin et al. assessed microRNA profiles in 94 patients with CLL and correlated the findings with known CLL prognostic markers including ZAP70 expression and immunoglobulin heavy chain variable gene (IgVH) mutation status. MicroRNA profiles were determined by a microchip containing the precursor and active forms of 76 microRNAs. An expression signature of 13 microRNAs discriminated between patients with a favorable (ZAP70-, IgVH mutated; n=47) versus unfavorable (ZAP70+, IgVH unmutated; n=36) clinical course. High versus low level expression of nine of the 13 microRNAs was also found to correlate with time from diagnosis to initial therapy. Eleven of 75 patients were found to have germ-line or somatic mutations in a subset of the microRNAs, which may prove to be relevant to altered regulatory function and CLL pathogenesis.
MicroRNAs function by blocking messenger RNA (mRNA) translation via complementary base-pairing or by promoting mRNA destruction by nucleases, thus regulating the expression of specific proteins. This novel regulatory mechanism was completely unrecognized until its discovery ten years ago. As noted by Chen in an accompanying editorial (N Engl J Med 2005; 353:1768-71), an estimated 1,000 microRNA genes appear in the human genome. These often occur in clusters, including a cluster at the chromosome 13q14 locus which is frequently deleted in CLL and other lymphoproliferative neoplasms. Altered microRNA may dysregulate oncogene and tumor suppressor gene function, and thus play an important role in cancer pathogenesis. Since microRNAs also are involved in the regulation of cellular development and differentiation, including hematopoiesis, their altered expression may contribute to the neoplastic process on many levels. To date, cancer pathogenesis research has largely focused on oncogene dysregulation via point mutation, gene amplification, or chromosomal translocation, and on loss-of-function mutations in tumor suppressor genes via gene deletion or point mutations. The report by Calin and colleagues is an important advance in linking this novel microRNA regulatory mechanism to clinical and phenotypic findings in CLL, warranting further functional and correlative analyses of RNA interference in lymphoid malignancies and in other cancers.
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Rebuilding Immunity After Chemotherapy
Robert Lowsky, MD, FRCPC
Rapoport AP, Stadtmauer EA, Aqui N, et al. Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer. Nat Med 2005;11:1230-7.
The immune deficiency that follows high dose chemotherapy and autologous hematopoietic progenitor cell transplantation (HCT) is well described. Antibody levels against infectious organisms steadily decline during the first year after transplantation, and early post-transplantation vaccination against preventable diseases is not recommended because the successful immunization rate is very low. As a consequence, opportunistic infections remain a major cause of morbidity and mortality after autologous HCT. The immune deficiency is even more pronounced in recipients of allogeneic HCT owing to the immune-suppression medication required to prevent graft-versus-host disease. Rapoport and colleagues now report a strategy that helps restore immunity in lymphopenic cancer patients receiving high-dose chemotherapy and autologous HCT.
In this clinical study, patients with advanced-stage multiple myeloma were treated with high-dose chemotherapy and autologous HCT. To overcome the immune deficiency associated with HCT, autologous T cells were harvested by apheresis, stimulated and expanded ex vivo before transplantation, and transferred back to the patients at designated time points after HCT. The reconstitution of CD4+ and CD8+ T-cell counts was significantly accelerated by an early post HCT T-cell infusion. A cohort of the study patients also received the 7-valent pneumococcal conjugate vaccine (PCV) 10 days prior to the collection of the autologous T cells. The infusion of the PCV-primed T cells shortly after transplantation combined with early post-transplant booster immunizations led to the induction of clinically relevant immunity within a month after transplantation. Immune assays showed that this group of patients had accelerated restoration of CD4+ T-cell function as T-cell proliferation in response to antigens including those not contained in the vaccine (staphylococcal enterotoxin B and cytomegalovirus antigens) was increased. Thus, combined pre-transplantation vaccine and early post-transplantation adoptive T-cell transfer with booster immunizations enables the development of enhanced memory T-cell responses.
This paper is important because it offers a platform to help restore impaired adaptive immunity in cancer patients. After high-dose chemotherapy and autologous HCT, innate immunity, composed of granulocytes, natural killer cells, and monocytes, is generally restored within a few weeks. Adaptive immunity, however, acquired over the individual's lifetime exposure to infections and vaccines, is lost as a result of the high-dose preparative regimen and is not overcome by the infusion of the memory B and T cells contained in the autologous graft. Rapoport and colleagues show that mature T cells in cancer patients can be ex vivo expanded, and that their infusions in the early post-transplant period accelerate the numerical recovery of T cells with a broad repertoire. The combination of antigen-primed T cells and early post-transplantation booster vaccinations led to enhanced immunity to the specific pathogen. The mechanism for the enhanced immunity remains to be fully determined; the relative importance of ex vivo expansion of PVC-specific memory T cells compared to the homeostatic in vivo expansion following their infusion in lymphopenic patients is unclear. The cytokine milieu of the patient may be relevant to the in vivo expansion and function of the infused T cells. Ultimately, the strategy developed by Rapoport et al. is exciting as it brings the goal of rebuilding immunity after cytotoxic therapy to the forefront and offers clues for designing studies using T cells and tumor vaccines to eradicate minimal residual disease after autologous HCT.
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"Slug"ging It Out Until the End
Lilli Petruzzelli, MD, PhD
Wu WS, Heinrichs S, Xu D, et al. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005;123:641-53.
The mechanism by which cells that are subjected to agents that induce DNA damage and subsequently undergo either cell cycle arrest and repair or cell death has been elusive. In particular, the involvement of p53 in either pathway, whether in a cancer cell or normal tissue, has been under intense scrutiny, and the studies presented in this manuscript bring our understanding of its role a step forward. In hematopoietic cells, γ-irradiation results in upregulation of p53, and this event induces cell cycle arrest and subsequent DNA repair or can affect cellular events that lead to apoptotic cell death. Using a mouse model, the authors extend their initial work to demonstrate that loss of Slug, a member of the snail family of zinc-finger transcription factors, renders both mature and precursor hematopoietic cells more sensitive to radiation. They show that expression of the transcriptional repressor, Slug, is induced by p53 and protects hematopoietic precursors from p53-mediated apoptosis. PUMA, a cytoplasmic factor that enhances p53-dependent apoptosis, is also induced by p53. Here, they show that Slug down-modulates PUMA expression but not p53 activation. Their results place Slug downstream of p53 activation and demonstrate that it directly affects PUMA expression. Finally, mice that lack Slug but have PUMA expression are more sensitive to radiation; however, in mice where Slug and PUMA are both missing, survival is preserved and increased survival of hematopoietic precursors was noted.
Integrating the effects of p53 on cell survival and apoptotic cell death, and understanding how it modulates these choices in normal and malignant cells, is critically important. The work here sheds light on why late myeloid cells that express low levels of Slug are more sensitive to radiation-induced cell death, whereas early hematopoietic precursors where levels are high are less sensitive and are likely to follow a pathway of cell cycle arrest and repair rather than apoptosis. These findings, combined with work from others, are important not only in shedding light on the molecular details of p53-dependent apoptosis but also for highlighting areas where treatment may be targeted. Looking for agents that up-regulate Slug may help protect cells, and, in particular, hematopoietic cells, from chemotherapy and radiation damage and enable higher doses of therapy or combined modalities that are at the present time too toxic. The question will be what effect increasing Slug will have on the tumor cell. It is not yet known whether down-modulating PUMA alone will be sufficient to block apoptosis and whether other signaling cascades will be affected by Slug that not only modulate p53-associated events and apoptosis but also cellular repair. Nonetheless, the work has shed light on both the transcriptional and cytoplasmic effects of p53.
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Location, Location, Location: c-kit‾ Stem Cells in the Adult Liver
Stephen Emerson, MD, PhD
Kotton DN, Fabian AJ, and Mulligan RC. A novel stem-cell population in adult liver with potent hematopoietic-reconstitution activity. Blood 2005; 106: 1574 - 1580.
Kotton and his colleagues use dye-exclusion to isolate a population of very rare stem cells within the adult mouse liver, which have nearly equal potency to true bone marrow stem cells to restore hematopoiesis following intravenous infusion into irradiated hosts (i.e. stem cell transplantation). These adult liver stem cells themselves derive from bone marrow stem cells, but do not express the stem cell factor receptor, c-kit. The presence, physiology, and capacity of stem cells in non-hematopoietic tissues are key issues of interest among scientists, physicians, and the lay public. One central mystery in this growing field is the relation between hematopoietic stem cells (HSC) in the bone marrow and those in other organs. Are the rare HSCs found in other tissues simply contaminants from blood, or are they a distinct local population? If a distinct HSC population can be identified, does it originate in that non-hematopoietic tissue, or does it arise by migration and differentiation from the bone marrow? Can the same stem cell generate organ-specific cells in its organ of residence and hematopoietic cells when it migrates to bone marrow?
In this report, Kotton et al. capitalized on the capacity of stem cell populations to actively export a class of small molecules that include the fluorescent dye Hoescht as a first step to purifying adult fetal liver stem cells. They further segregated these cells, which they term liver side population (LISP) tip cells, into CD45+ and CD45- subsets, and found that the CD45+LISP cells were essentially as effective as purified bone marrow SP cells in bone marrow transplantation rescue assays, whereas all other liver and blood cell populations were ineffective. Although they function similarly to bone marrow stem cells after transplantation, freshly isolated LISP tip cells did not express c-kit, the receptor for the stem cell factor. This strongly suggests that LISP tip cells are not simply bone marrow stem cells that have been mobilized, circulate, and are passively trapped in the liver. On the other hand, isolating murine LISP tip cells after allogeneic bone marrow transplantation shows that at least some of them derive from the bone marrow donor, suggesting the fascinating conclusion that bone marrow stem cells can give rise to LISP tip cells, and vice versa.
One direct implication of these results is that liver transplants always have the potential to create bone marrow chimeras in their recipients. If such stable mixed chimeras can be routinely created in organ recipients, the patient's immune system might learn to become tolerant of the transplanted organ, without need for immunosuppression. Understanding the fate of LISP tip cells after organ transplantation and learning how to regulate LISP tip cell migration to encourage graft tolerance will therefore be an important and exciting next avenue of investigation. For, in immune tolerance as in stem cell biology, the secret to the behavior of these cells will likely be location, location, location.
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Two Waves of GVL After Transplantation?
Peter Lee, MD
Reddy P, Maeda Y, Liu C, et al. A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses. Nature Medicine 2005; 11:1244-1249.
Strategies to elicit an effective graft-versus-leukemia (GVL) response without graft-versus-host disease (GVHD) post-allogeneic bone marrow transplantation or stem cell transplantation (BMT) have been an elusive goal. Host antigen presenting cells (APCs) have been shown to be crucial in inducing GVHD. Using a series of mouse BMT models, Reddy et al. shed insight on the role of host versus donor APCs in GVL and raised intriguing questions about the kinetics of GVL over time. The authors first established that in their model, inoculation of syngeneic tumor cells into syngeneic BMT recipients led to no GVHD but death from tumor, while the majority of allogeneic (minor histocompatibility, mHC) recipients developed GVHD but not disseminated tumor. To generate differential alloantigen expression between hematopoietic cells and epithelial target tissues, they generated bone marrow chimeric mice with beta-2-microglobulin-deficient (unable to express MHC I and thereby present antigens) syngeneic cells. When given allogeneic BMT, these chimeric recipients did not develop GVHD, but died of tumor progression. This suggests that MHC I expression (and thereby antigen presentation) by host hematopoietic cells was necessary for both GVHD and GVL in this model. To further dissect the relative contributions of T cells versus APCs, T cells and APCs of the same or different genetic backgrounds were given in BMT. When donor T cells were allogeneic to both APCs and tumor but syngeneic to host tissues, moderate GVHD and robust GVL response developed. In contrast, when donor T cells were allogeneic to both APC and target tissues but syngeneic to tumor, no GVL developed and mice died from tumor. When donor T cells were allogeneic to APCs but syngeneic to target tissues and tumor, recipients developed GVHD without GVL. Together, these data show the important role of APCs in both GVHD and GVL, and the need for shared alloantigens on tumor cells and APCs in driving optimal GVL responses. Lastly, to address the relative roles of residual host APCs early versus engrafted donor APCs late post-BMT, tumor cell numbers were titrated down such that recipients survived longer to observe possible late GVL effects. When tumor burden was reduced, small but significant GVL responses from donor APCs could be detected.
This study confirms and extends previous findings of the critical and central role of APCs in driving GVL and GVHD. It also demonstrates the prominent role of alloantigens in driving not only GVHD, but also GVL. Furthermore, it raises an intriguing possibility that GVL may arise in two waves. In the first (acute) wave, GVL is mediated by residual host APC presenting host mHC and leukemia antigens. In the second (chronic) wave, GVL is mediated by donor APC presenting host mHC and leukemia antigens from residual leukemic cells. This is somewhat akin to acute and chronic GVHD and raises issues regarding the optimal timing of donor lymphocyte infusion (DLI) to maintain the late GVL response. Going back to the elusive goal of separating GVL from GVHD, this study suggests that these processes share key common mechanisms BE APCs and alloantigen expression. Hence, while strategies which block APC function may reduce GVHD, they may also reduce GVL and should be used with caution in the clinical setting.
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