Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α.

Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.

Differentiation therapy for the treatment of t(8;21) acute myeloid leukemia using histone deacetylase inhibitors.

Epigenetic modifying enzymes such as histone deacetylases (HDACs), p300, and PRMT1 are recruited by AML1/ETO, the pathogenic protein for t(8;21) acute myeloid leukemia (AML), providing a strong molecular rationale for targeting these enzymes to treat this disease. Although early phase clinical assessment indicated that treatment with HDAC inhibitors (HDACis) may be effective in t(8;21) AML patients, rigorous preclinical studies to identify the molecular and biological events that may determine therapeutic responses have not been performed. Using an AML mouse model driven by expression of AML1/ETO9a (A/E9a), we demonstrated that treatment of mice bearing t(8;21) AML with the HDACi panobinostat caused a robust antileukemic response that did not require functional p53 nor activation of conventional apoptotic pathways. Panobinostat triggered terminal myeloid differentiation via proteasomal degradation of A/E9a. Importantly, conditional A/E9a deletion phenocopied the effects of panobinostat and other HDACis, indicating that destabilization of A/E9a is critical for the antileukemic activity of these agents.

HDAC1 and HDAC2 collectively regulate intestinal stem cell homeostasis.

Histone deacetylases (HDACs) are posttranslational modifiers that deacetylate proteins. Despite their crucial role in numerous biological processes, the use of broad-range HDAC inhibitors (HDACi), has shown clinical efficacy. However, undesired side effects highlight the necessity to better understand the biology of different HDACs and target the relevant HDACs. Using a novel mouse model, in which HDAC1 and HDAC2 can be simultaneously deleted in the intestine of adult mice, we show that the simultaneous deletion of HDAC1 and HDAC2 leads to a rapid loss of intestinal homeostasis. Importantly, this deletion cannot be sustained, and 8 days after initial ablation, stem cells that have escaped HDAC1 or HDAC2 deletion swiftly repopulate the intestinal lining. In vitro ablation of HDAC1 and HDAC2 using intestinal organoid cultures resulted in a down-regulation of multiple intestinal stem cell markers and functional loss of clonogenic capacity. Importantly, treatment of wild-type organoids with class I-specific HDACi MS-275 also induced a similar loss of stemness, providing a possible rationale for the gastrointestinal side effects often observed in HDACi-treated patients. In conclusion, these data show that HDAC1 and HDAC2 have a redundant function and are essential to maintain intestinal homeostasis.

Two novel haemoglobin variants that affect haemoglobin A1c measurement by ion-exchange chromatography.

BACKGROUND: Haemoglobin (Hb) variants are well-known factors interfering with accurate HbA1c testing. This report describes two novel Hb variants leading to inappropriate quantification of HbA1c by ion-exchange chromatography. METHODS: Glycated forms of novel Hb variants were recognised in the blood of two patients with diabetes mellitus screened by HbA1c ion-exchange chromatography. Dedicated high-resolution cation-exchange chromatography and subsequent DNA sequencing revealed the exact nature of the variants. Other common techniques for quantifying HbA1c were applied on both samples and haematological parameters were determined to judge possible pathology associated with the novel Hb variants. RESULTS: A fraction of 15% of abnormal Hb was observed in a 37-year-old female. DNA sequencing revealed a heterozygous mutation in the α1-globin gene, resulting in a leucine-to-phenylalanine amino-acid substitution (HBA1: c.301C>T, p.Leu101Phe). We named this variant Hb Weesp. The other novel variant, Hb Haelen, presented as a 40% fraction in a 63-year-old male and resulted from a heterozygous amino acid substitution in the ß-globin gene (HBB: c.335T>C, p.Val112Gly). The presence of both Hb variants resulted in aberrant separation of the Hb components, leading to an inadequate quantification of HbA1c. CONCLUSIONS: Close examination of HbA1c chromatograms revealed two novel, clinically silent Hb variants that interfere with HbA1c quantification. Healthcare providers need to be aware of the potential of such Hb variants when interpreting HbA1c results.

NKT cell adjuvant-based tumor vaccine for treatment of myc oncogene-driven mouse B-cell lymphoma.

Immunomodulators are effective in controlling hematologic malignancy by initiating or reactivating host antitumor immunity to otherwise poorly immunogenic and immune suppressive cancers. We aimed to boost antitumor immunity in B-cell lymphoma by developing a tumor cell vaccine incorporating α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells. In the Eµ-myc transgenic mouse model, single therapeutic vaccination of irradiated, α-GalCer-loaded autologous tumor cells was sufficient to significantly inhibit growth of established tumors and prolong survival. Vaccine-induced antilymphoma immunity required NKT cells, NK cells, and CD8 T cells, and early IL-12-dependent production of IFN-γ. CD4 T cells, gamma/delta T cells, and IL-18 were not critical. Vaccine treatment induced a large systemic spike of IFN-γ and transient peripheral expansion of both NKT cells and NK cells, the major sources of IFN-γ. Furthermore, this vaccine approach was assessed in several other hematopoietic tumor models and was also therapeutically effective against AML-ETO9a acute myeloid leukemia. Replacing α-GalCer with ß-mannosylceramide resulted in prolonged protection against Eµ-myc lymphoma. Overall, our results demonstrate a potent immune adjuvant effect of NKT cell ligands in therapeutic anticancer vaccination against oncogene-driven lymphomas, and this work supports clinical investigation of NKT cell-based immunotherapy in patients with hematologic malignancies.

Regulation of stem cell self-renewal and differentiation by Wnt and Notch are conserved throughout the adenoma-carcinoma sequence in the colon.

BACKGROUND: Colon cancer stem cells are shown to be the self-renewing cells within a tumor that give rise to all lineages of more differentiated tumor cells. In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining. This suggests that, despite the numerous genetic aberrations and morphological changes that have occurred during cancer initiation and progression, a remnant homeostatic regulation persists. FINDINGS: Using a number of human and mouse intestinal-derived organoid cultures from normal, adenoma and cancerous tissues, we show here that Notch signals coordinate self-renewal and lineage determination not only in normal, but also at the adenoma and carcinoma stage in both mice and humans. Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease. CONCLUSION: These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.

Asymmetric cell division of T cells upon antigen presentation uses multiple conserved mechanisms.

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Antitumor activities and on-target toxicities mediated by a TRAIL receptor agonist following cotreatment with panobinostat.

The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.

The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy.

LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Emu-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X(L) protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Emu-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.

Correction to: Combining the differentiating effect of panobinostat with the apoptotic effect of arsenic trioxide leads to significant survival benefit in a model of t(8;21) acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via the original article.

Serpins in T cell immunity.

Serine protease inhibitors (serpins) are a family of proteins that are important in the regulation of several biological processes. This mainly involves the inhibition of serine proteases, although some serpins inhibit a different class of proteases or even function without inhibitory activity. In contrast to other protease inhibitor families, serpins inhibit their target proteases by a specific mechanism, which depends on a change in conformation. This review primarily focuses on one subgroup of serpins--ovalbumin (ov)-serpins. Different than most members of the family, this group of serpins lacks secretion signal sequences and therefore, mainly functions intracellularly. In addition to expression in most normal tissues, ov-serpins can be found in multiple different cells of the immune system. Interestingly, expression of ov-serpins in these cells is tightly regulated, indicating a role for these serpins in the regulation of immune responses. The role of serpins in the immune response will be the topic of this review.

Discovery of biomarkers for the presence and progression of left ventricular diastolic dysfunction and HEart faiLure with Preserved ejection Fraction in patients at risk for cardiovascular disease: rationale and design of the HELPFul case-cohort study in a Dutch cardiology outpatient clinic.

INTRODUCTION: Left ventricular diastolic dysfunction (LVDD) is a common condition in both sexes that may deteriorate into heart failure (HF) with preserved ejection fraction (pEF), although this seems to happen more often in women than in men. Both LVDD and HFpEF often go unrecognised, necessitating the discovery of biomarkers that aid both the identification of individuals with LVDD at risk of developing HF and identification of individuals most likely to benefit from treatment. METHODS AND ANALYSIS: HELPFul is an ongoing case-cohort study at a Dutch cardiology outpatient clinic enrolling patients aged 45 years and older without history of cardiovascular disease, who were referred by the general practitioner for cardiac evaluation. We included a random sample of patients and enriched the cohort with cases (defined as an E/e' ≥8 measured with echocardiography). Information about medical history, cardiovascular risk factors, electrocardiography, echocardiography, exercise test performance, common carotid intima-media thickness measurement and standard cardiovascular biomarkers was obtained from the routine care data collected by the cardiology outpatient clinic. Study procedure consists of extensive venous blood collection for biobanking and additional standardised questionnaires. Follow-up will consist of standardised questionnaires by mail and linkage to regional and national registries. We will perform cardiac magnetic resonance imaging and coronary CT angiography in a subgroup of patients to investigate the extent of macrovascular and microvascular coronary disease. ETHICS AND DISSEMINATION: The study protocol was approved by the Institutional Review Board of the University Medical Center Utrecht. Results will be disseminated through national and international conferences and in peer-reviewed journals in cardiovascular disease. TRIAL REGISTRATION: NTR6016;Pre-results.

Long-term clinical parameters after switching to nocturnal haemodialysis: a Dutch propensity-score-matched cohort study comparing patients on nocturnal haemodialysis with patients on three-times-a-week haemodialysis/haemodiafiltration.

OBJECTIVES: Nocturnal haemodialysis (NHD), characterised by 8-hour sessions ≥3 times a week, is known to improve clinical parameters in the short term compared with conventional-schedule haemodialysis (HD), generally 3×3.5-4 hours a week. We studied long-term effects of NHD and used patients on conventional HD/haemodiafiltration (HDF) as controls. DESIGN: Four-year prospective follow-up of patients who switched to NHD; we compared patients with patients on HD/HDF using propensity score matching. SETTING: 28 Dutch dialysis centres. PARTICIPANTS: We included 159 patients starting with NHD any time since 2004, aged 56.7±12.9 years, with median dialysis vintage 2.3 (0.9-5.1) years. We propensity-score matched 100 patients on NHD to 100 on HD/HDF. PRIMARY AND SECONDARY OUTCOME MEASURES: Control of hypertension (predialysis blood pressure, number of antihypertensives), phosphate (phosphate, number of phosphate binders), nutritional status and inflammation (albumin, C reactive protein and postdialysis weight) and anaemia (erythropoiesis-stimulating agent (ESA) resistance). RESULTS: Switching to NHD was associated with a non-significant reduction of antihypertensives compared with HD/HDF (OR <2 types 2.17, 95% CI 0.86 to 5.50, P=0.11); and a prolonged lower need for phosphate binders (OR <2 types 1.83, 95% CI 1.10 to 3.03, P=0.02). NHD was not associated with significant changes in blood pressure or phosphate. NHD was associated with significantly higher albumin over time compared with HD/HDF (0.70 g/L/year, 95% CI 0.10 to 1.30, P=0.02). ESA resistance decreased significantly in NHD compared with HD/HDF, resulting in a 33% lower ESA dose in the long term. CONCLUSIONS: After switching to NHD, the lower need for antihypertensives, phosphate binders and ESA persists for at least 4 years. These sustained improvements in NHD contrast significantly with the course of these parameters during continued treatment with conventional-schedule HD and HDF. NHD provides an optimal form of dialysis, also suitable for patients expected to have a long waiting time for transplantation or those convicted to indefinite dialysis.

Clustering of risk factors and the risk of incident cardiovascular disease in Asian and Caucasian populations: results from the Asia Pacific Cohort Studies Collaboration.

OBJECTIVE: To assess the relationship between risk factor clusters and cardiovascular disease (CVD) incidence in Asian and Caucasian populations and to estimate the burden of CVD attributable to each cluster. SETTING: Asia Pacific Cohort Studies Collaboration. PARTICIPANTS: Individual participant data from 34 population-based cohorts, involving 314 024 participants without a history of CVD at baseline. OUTCOME MEASURES: Clusters were 11 possible combinations of four individual risk factors (current smoking, overweight, blood pressure (BP) and total cholesterol). Cox regression models were used to obtain adjusted HRs and 95% CIs for CVD associated with individual risk factors and risk factor clusters. Population-attributable fractions (PAFs) were calculated. RESULTS: During a mean follow-up of 7 years, 6203 CVD events were recorded. The ranking of HRs and PAFs was similar for Australia and New Zealand (ANZ) and Asia; clusters including BP consistently showed the highest HRs and PAFs. The BP-smoking cluster had the highest HR for people with two risk factors: 4.13 (3.56 to 4.80) for Asia and 3.07 (2.23 to 4.23) for ANZ. Corresponding PAFs were 24% and 11%, respectively. For individuals with three risk factors, the BP-smoking-cholesterol cluster had the highest HR (4.67 (3.92 to 5.57) for Asia and 3.49 (2.69 to 4.53) for ANZ). Corresponding PAFs were 13% and 10%. CONCLUSIONS: Risk factor clusters act similarly on CVD risk in Asian and Caucasian populations. Clusters including elevated BP were associated with the highest excess risk of CVD.

Population distribution of traditional and the emerging cardiovascular risk factors carotid plaque and IMT: the REFINE-Reykjavik study with comparison with the Tromsø study.

OBJECTIVES: Population statistics for carotid plaque and cardiovascular risk factors reported in scientific journals are usually presented as averages for the population or age and sex adjusted, rather than sex and age groups. Important population differences about atherosclerosis and cardiovascular risk factors may thus be missed. We compare the distribution of cardiovascular risk factors, carotids plaque and carotid intima-media thickness (CIMT) in two population-based studies. METHODS: Carotid artery atherosclerotic plaque prevalence and risk factors levels for cardiovascular disease by sex in 5-year age groups from the Risk Evaluation For Infarct Estimates Reykjavik study (REFINE-Reykjavik study) were compared with data from the Tromsø 6 study. RESULTS: The threshold of carotid plaque presence in the Tromsø 6 study fell between minimal and moderate plaque defined in the REFINE-Reykjavik study reflecting carotid plaque prevalence. The prevalence of minimal carotid plaque in the REFINE-Reykjavik study was 47% in men (40-69 years old) and 38% in women and 11% in men and 7% in women of moderate plaque. The prevalence of any plaque in the Tromsø 6 study was 35% in men and 27% in women. The mean (CIMT) was similar in the studies. In the Tromsø 6 study mean systolic blood pressure was 8 mm Hg higher in men and 10 mm Hg higher in women, mean low-density lipoprotein was 0.5 mmol/L higher in men and 0.3 mmol/L higher in women and the prevalence of smoking was 4% higher in men and 9% higher in women. However, body mass index was 0.8 kg/m2 higher in men and 0.9 kg/m2 in women in the REFINE-Reykjavik study. CONCLUSION: Comparison between Iceland and Norway revealed differences in the prevalence of carotid plaque, which was assumed to be due to different definition of plaque. However, clinically significant differences in conventional cardiovascular risk factors were seen. This underscores the importance of detailed comparison of population data across different populations.

Proteinase inhibitor-9 expression is induced by maturation in dendritic cells via p38 MAP kinase.

Dendritic cells (DC) are crucial for the induction of CD8(+) T cell responses. However, as DC present antigen, they may also be at risk for being killed by recent activated CD8+ T cells. Previously we have shown that lipopolysaccharide (LPS)- or CD40L-stimulated monocyte-derived DC express high levels of the granzyme B-inhibitory serpin proteinase inhibitor-9 (PI-9). Furthermore we found that its murine homolog serine protease inhibitor-6 (SPI-6) protected murine DC against cytotoxic T lymphocyte-induced apoptosis. These data suggest that PI-9/SPI-6 may regulate survival of DC when they are under attack by effector cells. We therefore analyzed how PI-9 is regulated upon stimulation with several Toll-like receptor ligands. We found that the expression of PI-9 correlated with maturation, as measured with CD86 levels, but not with the production of interleukin-12-p70. Using LPS as stimulus, we also showed that the induction of PI-9 is dependent on the p38 mitogen-activated protein kinase signaling pathway. The data suggest that PI-9 is tightly linked to maturation and may allow DC to exert their function in a potentially hostile environment.

Prognosis of patients with dementia: results from a prospective nationwide registry linkage study in the Netherlands.

OBJECTIVE: To report mortality risks of dementia based on national hospital registry data, and to put these risks into perspective by comparing them with those in the general population and following cardiovascular diseases. DESIGN: Prospective cohort study from 1 January 2000 through 31 December 2010. SETTING: Hospital-based cohort. PARTICIPANTS: A nationwide hospital-based cohort of 59,201 patients with clinical diagnosis of dementia (admitted to a hospital or visiting a day clinic) was constructed (38.7% men, 81.4 years (SD 7.0)). MAIN OUTCOMES AND MEASURES: 1-year and 5-year age-specific and sex-specific mortality risks were reported for patients with dementia visiting a day clinic compared with the general population; for patients hospitalised with dementia compared with patients hospitalised for acute myocardial infarction (AMI), heart failure or stroke, these were presented as absolute and relative risks (RRs). RESULTS: 1-year mortality was 38.3% in men and 30.5% in women. 5-year risk was 65.4% and 58.5%, respectively. Mortality risks were significantly higher in patients with dementia admitted to the hospital than in those visiting a day clinic (1-year RR 3.29, 95% CI 3.16 to 3.42; and 5-year RR 1.79, 95% CI 1.76 to 1.83). Compared with the general population, mortality risks were significantly higher among patients visiting a day clinic (1-year RR for women 2.99, 95% CI 2.84 to 3.14; and for men 3.94, 95% CI 3.74 to 4.16). 5-year RRs were somewhat lower, but still significant. Results were more pronounced at younger ages. Mortality risks among admitted patients were comparable or even exceeded those of cardiovascular diseases (1-year RR for women with dementia vs AMI 1.24, 95% CI 1.19 to 1.29; vs heart failure 1.05, 95% CI 1.02 to 1.08; vs stroke 1.07, 95% CI 1.04 to 1.10). 5-year RRs were comparable. For men, RRs were slightly higher. CONCLUSIONS: Dementia has a poor prognosis as compared with other diseases and the general population. The risks among admitted patients even exceeded those following cardiovascular diseases.

Combining the differentiating effect of panobinostat with the apoptotic effect of arsenic trioxide leads to significant survival benefit in a model of t(8;21) acute myeloid leukemia.

BACKGROUND: One of the most frequently found abnormalities in acute myeloid leukemia (AML) is the t(8;21)(q22;q22) translocation, which is seen in around 15% of patients. This translocation results in the production of the AML1/ETO (A/E) fusion protein and commonly involves cooperating activating mutations of RAS. AE9a encodes a C-terminally truncated A/E protein of 575 amino acids that retains the ability to recruit histone deacetylases (HDACs). Expression of AE9a leads to rapid development of leukemia in experimental mouse systems. We have recently shown that treatment of mice bearing A/E9a;Nras (G12D) tumors with the histone deacetylase inhibitor (HDACi) panobinostat leads to degradation of the A/E9a fusion protein, cell cycle arrest, differentiation of AML blasts into mature granulocytes and prolonged survival. Herein, we sought to enhance this therapeutic effect. FINDINGS: Combined treatment of mice bearing A/E9a;Nras (G12D) leukemias with panobinostat and arsenic trioxide (ATO) resulted in a significant survival advantage compared to mice treated with either agent alone. Moreover, some of the mice treated with the panobinostat/ATO combination showed complete tumor responses and remained in remission for over 220 days. Panobinostat caused differentiation of A/E9a;Nras (G12D) cells while ATO induced apoptosis of the leukemic cells, an effect that was enhanced following co-treatment with panobinostat. CONCLUSIONS: Our results indicate that leukemic blast differentiation mediated by panobinostat combined with induction of apoptosis by ATO could be therapeutically beneficial and should be considered for patients with t(8;21) AML.

Detection of apoptotic cells using immunohistochemistry.

Immunohistochemistry is commonly used to show the presence of apoptotic cells in situ. In this protocol, B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples are fixed and sectioned, and fragmented DNA (a feature of apoptotic cells) is end-labeled by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Immunohistochemical methods are then used to detect the labeled DNA and identify B-cell lymphoma cells in the last stage of apoptosis. Because the assay can lead to false-positive results, it is advisable to carry out an additional assay (e.g., immunohistochemistry for active caspase-3) to confirm the presence of apoptotic cells.