A systematic review of sample size estimation accuracy on power in malaria cluster randomised trials measuring epidemiological outcomes.

INTRODUCTION: Cluster randomised trials (CRTs) are the gold standard for measuring the community-wide impacts of malaria control tools. CRTs rely on well-defined sample size estimations to detect statistically significant effects of trialled interventions, however these are often predicted poorly by triallists. Here, we review the accuracy of predicted parameters used in sample size calculations for malaria CRTs with epidemiological outcomes. METHODS: We searched for published malaria CRTs using four online databases in March 2022. Eligible trials included those with malaria-specific epidemiological outcomes which randomised at least six geographical clusters to study arms. Predicted and observed sample size parameters were extracted by reviewers for each trial. Pair-wise Spearman's correlation coefficients (rs) were calculated to assess the correlation between predicted and observed control-arm outcome measures and effect sizes (relative percentage reductions) between arms. Among trials which retrospectively calculated an estimate of heterogeneity in cluster outcomes, we recalculated study power according to observed trial estimates. RESULTS: Of the 1889 records identified and screened, 108 articles were eligible and comprised of 71 malaria CRTs. Among 91.5% (65/71) of trials that included sample size calculations, most estimated cluster heterogeneity using the coefficient of variation (k) (80%, 52/65) which were often predicted without using prior data (67.7%, 44/65). Predicted control-arm prevalence moderately correlated with observed control-arm prevalence (rs: 0.44, [95%CI: 0.12,0.68], p-value < 0.05], with 61.2% (19/31) of prevalence estimates overestimated. Among the minority of trials that retrospectively calculated cluster heterogeneity (20%, 13/65), empirical values contrasted with those used in sample size estimations and often compromised study power. Observed effect sizes were often smaller than had been predicted at the sample size stage (72.9%, 51/70) and were typically higher in the first, compared to the second, year of trials. Overall, effect sizes achieved by malaria interventions tested in trials decreased between 1995 and 2021. CONCLUSIONS: Study findings reveal sample size parameters in malaria CRTs were often inaccurate and resulted in underpowered studies. Future trials must strive to obtain more representative epidemiological sample size inputs to ensure interventions against malaria are adequately evaluated. REGISTRATION: This review is registered with PROSPERO (CRD42022315741).

Effectiveness and cost-effectiveness of reactive, targeted indoor residual spraying for malaria control in low-transmission settings: a cluster-randomised, non-inferiority trial in South Africa.

BACKGROUND: Increasing insecticide costs and constrained malaria budgets could make universal vector control strategies, such as indoor residual spraying (IRS), unsustainable in low-transmission settings. We investigated the effectiveness and cost-effectiveness of a reactive, targeted IRS strategy. METHODS: This cluster-randomised, open-label, non-inferiority trial compared reactive, targeted IRS with standard IRS practice in northeastern South Africa over two malaria seasons (2015-17). In standard IRS clusters, programme managers conducted annual mass spray campaigns prioritising areas using historical data, expert opinion, and other factors. In targeted IRS clusters, only houses of index cases (identified through passive surveillance) and their immediate neighbours were sprayed. The non-inferiority margin was 1 case per 1000 person-years. Health service costs of real-world implementation were modelled from primary and secondary data. Incremental costs per disability-adjusted life-year (DALY) were estimated and deterministic and probabilistic sensitivity analyses conducted. This study is registered with ClinicalTrials.gov, NCT02556242. FINDINGS: Malaria incidence was 0·95 per 1000 person-years (95% CI 0·58 to 1·32) in the standard IRS group and 1·05 per 1000 person-years (0·72 to 1·38) in the targeted IRS group, corresponding to a rate difference of 0·10 per 1000 person-years (-0·38 to 0·59), demonstrating non-inferiority for targeted IRS (p<0·0001). Per additional DALY incurred, targeted IRS saved US$7845 (2902 to 64 907), giving a 94-98% probability that switching to targeted IRS would be cost-effective relative to plausible cost-effectiveness thresholds for South Africa ($2637 to $3557 per DALY averted). Depending on the threshold used, targeted IRS would remain cost-effective at incidences of less than 2·0-2·7 per 1000 person-years. Findings were robust to plausible variation in other parameters. INTERPRETATION: Targeted IRS was non-inferior, safe, less costly, and cost-effective compared with standard IRS in this very-low-transmission setting. Saved resources could be reallocated to other malaria control and elimination activities. FUNDING: Joint Global Health Trials.

Estimating the annual dengue force of infection from the age of reporting primary infections across urban centres in endemic countries.

BACKGROUND: Stratifying dengue risk within endemic countries is crucial for allocating limited control interventions. Current methods of monitoring dengue transmission intensity rely on potentially inaccurate incidence estimates. We investigated whether incidence or alternate metrics obtained from standard, or laboratory, surveillance operations represent accurate surrogate indicators of the burden of dengue and can be used to monitor the force of infection (FOI) across urban centres. METHODS: Among those who reported and resided in 13 cities across the Philippines, we collected epidemiological data from all dengue case reports between 2014 and 2017 (N 80,043) and additional laboratory data from a cross-section of sampled case reports (N 11,906) between 2014 and 2018. At the city level, we estimated the aggregated annual FOI from age-accumulated IgG among the non-dengue reporting population using catalytic modelling. We compared city-aggregated FOI estimates to aggregated incidence and the mean age of clinically and laboratory diagnosed dengue cases using Pearson's Correlation coefficient and generated predicted FOI estimates using regression modelling. RESULTS: We observed spatial heterogeneity in the dengue average annual FOI across sampled cities, ranging from 0.054 [0.036-0.081] to 0.249 [0.223-0.279]. Compared to FOI estimates, the mean age of primary dengue infections had the strongest association (ρ -0.848, p value<0.001) followed by the mean age of those reporting with warning signs (ρ -0.642, p value 0.018). Using regression modelling, we estimated the predicted annual dengue FOI across urban centres from the age of those reporting with primary infections and revealed prominent spatio-temporal heterogeneity in transmission intensity. CONCLUSIONS: We show the mean age of those reporting with their first dengue infection or those reporting with warning signs of dengue represent superior indicators of the dengue FOI compared to crude incidence across urban centres. Our work provides a framework for national dengue surveillance to routinely monitor transmission and target control interventions to populations most in need.

A serological framework to investigate acute primary and post-primary dengue cases reporting across the Philippines.

BACKGROUND: In dengue-endemic countries, targeting limited control interventions to populations at risk of severe disease could enable increased efficiency. Individuals who have had their first (primary) dengue infection are at risk of developing more severe secondary disease, thus could be targeted for disease prevention. Currently, there is no reliable algorithm for determining primary and post-primary (infection with more than one flavivirus) status from a single serum sample. In this study, we developed and validated an immune status algorithm using single acute serum samples from reporting patients and investigated dengue immuno-epidemiological patterns across the Philippines. METHODS: During 2015/2016, a cross-sectional sample of 10,137 dengue case reports provided serum for molecular (anti-DENV PCR) and serological (anti-DENV IgM/G capture ELISA) assay. Using mixture modelling, we re-assessed IgM/G seroprevalence and estimated functional, disease day-specific, IgG:IgM ratios that categorised the reporting population as negative, historical, primary and post-primary for dengue. We validated our algorithm against WHO gold standard criteria and investigated cross-reactivity with Zika by assaying a random subset for anti-ZIKV IgM and IgG. Lastly, using our algorithm, we explored immuno-epidemiological patterns of dengue across the Philippines. RESULTS: Our modelled IgM and IgG seroprevalence thresholds were lower than kit-provided thresholds. Individuals anti-DENV PCR+ or IgM+ were classified as active dengue infections (83.1%, 6998/8425). IgG- and IgG+ active dengue infections on disease days 1 and 2 were categorised as primary and post-primary, respectively, while those on disease days 3 to 5 with IgG:IgM ratios below and above 0.45 were classified as primary and post-primary, respectively. A significant proportion of post-primary dengue infections had elevated anti-ZIKV IgG inferring previous Zika exposure. Our algorithm achieved 90.5% serological agreement with WHO standard practice. Post-primary dengue infections were more likely to be older and present with severe symptoms. Finally, we identified a spatio-temporal cluster of primary dengue case reporting in northern Luzon during 2016. CONCLUSIONS: Our dengue immune status algorithm can equip surveillance operations with the means to target dengue control efforts. The algorithm accurately identified primary dengue infections who are at risk of future severe disease.

Serology reveals heterogeneity of Plasmodium falciparum transmission in northeastern South Africa: implications for malaria elimination.

BACKGROUND: It is widely acknowledged that modifications to existing control interventions are required if South Africa is to achieve malaria elimination. Targeting indoor residual spraying (IRS) to areas where cases have been detected is one strategy currently under investigation in northeastern South Africa. This seroprevalence baseline study, nested within a targeted IRS trial, was undertaken to provide insights into malaria transmission dynamics in South Africa and evaluate whether sero-epidemiological practices have the potential to be routinely incorporated into elimination programmes. METHODS: Filter-paper blood spots, demographic and household survey data were collected from 2710 randomly selected households in 56 study wards located in the municipalities of Ba-Phalaborwa and Bushbuckridge. Blood spots were assayed for Plasmodium falciparum apical membrane antigen-1 and merozoite surface protein-119 blood-stage antigens using an enzyme linked immunosorbent assay. Seroprevalence data were analysed using a reverse catalytic model to determine malaria seroconversion rates (SCR). Geospatial cluster analysis was used to investigate transmission heterogeneity while random effects logistic regression identified risk factors associated with malaria exposure. RESULTS: The overall SCR across the entire study site was 0.012 (95% CI 0.008-0.017) per year. Contrasting SCRs, corresponding to distinct geographical regions across the study site, ranging from <0.001 (95% CI <0.001-0.005) to 0.022 (95% CI 0.008-0.062) per annum revealed prominent transmission heterogeneity. Geospatial cluster analysis of household seroprevalence and age-adjusted antibody responses detected statistically significant (p < 0.05) spatial clusters of P. falciparum exposure. Formal secondary education was associated with lower malaria exposure in the sampled population (AOR 0.72, 95% CI 0.56-0.95, p = 0.018). CONCLUSIONS: Although overall transmission intensity and exposure to malaria was low across both study sites, malaria transmission intensity was highly heterogeneous and associated with low socio-economic status in the region. Findings suggest focal targeting of interventions has the potential to be an appropriate strategy to deploy in South Africa. Furthermore, routinely incorporating sero-epidemiological practices into elimination programmes may prove useful in monitoring malaria transmission intensity in South Africa, and other countries striving for malaria elimination.

MicroRNA programs in normal and aberrant stem and progenitor cells.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

Expression of the runt homology domain of RUNX1 disrupts homeostasis of hematopoietic stem cells and induces progression to myelodysplastic syndrome.

Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain. The expression of the runt homology domain, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia. Analysis of premyelodysplastic animals found expansion of c-Kit(+)Sca-1(+)Lin(-) cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling found that RUNX1(41-214) c-Kit(+)Sca-1(+)Lin(-) cells have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of HSCs, suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.

Could prophylactic antivirals reduce dengue incidence in a high-prevalence endemic area?

Prophylactic drugs against dengue are currently under development. In this study, we explored how such prophylactic approaches might affect dengue cases in four communes of Nha Trang City, Vietnam. A community level dengue transmission survey indicated high levels of previous exposure to dengue (89.7%; 95% CI: 87.2,92.0). We fitted a spatially explicit model to an observed outbreak and simulated likely effectiveness of Case-Area Targeted Interventions (CATI) and One-Time Mass Distribution (OTMD) of drug and vector control strategies. Increasing radius and effectiveness and decreasing delay of CATI was most effective, with drugs being more effective in averting dengue cases than vector control. Using an OTMD approach early in the outbreak required the least number of treatments to avert a case, suggesting that OTMD strategies should be considered as pre-emptive rather than reactive strategies. These findings show that pre-emptive interventions can substantially reduce the burden of dengue outbreaks in endemic settings.

A phylogenetic study of dengue virus in urban Vietnam shows long-term persistence of endemic strains.

Dengue virus (DENV) causes repeated outbreaks of disease in endemic areas, with patterns of local transmission strongly influenced by seasonality, importation via human movement, immunity, and vector control efforts. An understanding of how each of these interacts to enable endemic transmission (continual circulation of local virus strains) is largely unknown. There are times of the year when no cases are reported, often for extended periods of time, perhaps wrongly implying the successful eradication of a local strain from that area. Individuals who presented at a clinic or hospital in four communes in Nha Trang, Vietnam, were initially tested for DENV antigen presence. Enrolled positive individuals then had their corresponding household members invited to participate, and those who enrolled were tested for DENV. The presence of viral nucleic acid in all samples was confirmed using quantitative polymerase chain reaction, and positive samples were then whole-genome sequenced using an amplicon and target enrichment library preparation techniques and Illumina MiSeq sequencing technology. Generated consensus genome sequences were then analysed using phylogenetic tree reconstruction to categorise sequences into clades with a common ancestor, enabling investigations of both viral clade persistence and introductions. Hypothetical introduction dates were additionally assessed using a molecular clock model that calculated the time to the most recent common ancestor (TMRCA). We obtained 511 DENV whole-genome sequences covering four serotypes and more than ten distinct viral clades. For five of these clades, we had sufficient data to show that the same viral lineage persisted for at least several months. We noted that some clades persisted longer than others during the sampling time, and by comparison with other published sequences from elsewhere in Vietnam and around the world, we saw that at least two different viral lineages were introduced into the population during the study period (April 2017-2019). Next, by inferring the TMRCA from the construction of molecular clock phylogenies, we predicted that two of the viral lineages had been present in the study population for over a decade. We observed five viral lineages co-circulating in Nha Trang from three DENV serotypes, with two likely to have remained as uninterrupted transmission chains for a decade. This suggests clade cryptic persistence in the area, even during periods of low reported incidence.

Serological evaluation of the effectiveness of reactive focal mass drug administration and reactive vector control to reduce malaria transmission in Zambezi Region, Namibia: Results from a secondary analysis of a cluster randomised trial.

BACKGROUND: Due to challenges in measuring changes in malaria at low transmission, serology is increasingly being used to complement clinical and parasitological surveillance. Longitudinal studies have shown that serological markers, such as Etramp5.Ag1, can reflect spatio-temporal differences in malaria transmission. However, these markers have yet to be used as endpoints in intervention trials. METHODS: Based on data from a 2017 cluster randomised trial conducted in Zambezi Region, Namibia, evaluating the effectiveness of reactive focal mass drug administration (rfMDA) and reactive vector control (RAVC), this study conducted a secondary analysis comparing antibody responses between intervention arms as trial endpoints. Antibody responses were measured on a multiplex immunoassay, using a panel of eight serological markers of Plasmodium falciparum infection - Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175, PfMSP119, PfAMA1, and PfGLURP.R2. FINDINGS: Reductions in sero-prevalence to antigens Etramp.Ag1, PfMSP119, Rh2.2030, and PfAMA1 were observed in study arms combining rfMDA and RAVC, but only effects for Etramp5.Ag1 were statistically significant. Etramp5.Ag1 sero-prevalence was significantly lower in all intervention arms. Compared to the reference arms, adjusted prevalence ratio (aPR) for Etramp5.Ag1 was 0.78 (95%CI 0.65 - 0.91, p = 0.0007) in the rfMDA arms and 0.79 (95%CI 0.67 - 0.92, p = 0.001) in the RAVC arms. For the combined rfMDA plus RAVC intervention, aPR was 0.59 (95%CI 0.46 - 0.76, p < 0.0001). Significant reductions were also observed based on continuous antibody responses. Sero-prevalence as an endpoint was found to achieve higher study power (99.9% power to detect a 50% reduction in prevalence) compared to quantitative polymerase chain reaction (qPCR) prevalence (72.9% power to detect a 50% reduction in prevalence). INTERPRETATION: While the observed relative reduction in qPCR prevalence in the study was greater than serology, the use of serological endpoints to evaluate trial outcomes measured effect size with improved precision and study power. Serology has clear application in cluster randomised trials, particularly in settings where measuring clinical incidence or infection is less reliable due to seasonal fluctuations, limitations in health care seeking, or incomplete testing and reporting. FUNDING: This study was supported by Novartis Foundation (A122666), the Bill & Melinda Gates Foundation (OPP1160129), and the Horchow Family Fund (5,300,375,400).

Combining rapid diagnostic tests to estimate primary and post-primary dengue immune status at the point of care.

BACKGROUND: Characterising dengue virus (DENV) infection history at the point of care is challenging as it relies on intensive laboratory techniques. We investigated how combining different rapid diagnostic tests (RDTs) can be used to accurately determine the primary and post-primary DENV immune status of reporting patients during diagnosis. METHODS AND FINDINGS: Serum from cross-sectional surveys of acute suspected dengue patients in Indonesia (N:200) and Vietnam (N: 1,217) were assayed using dengue laboratory assays and RDTs. Using logistic regression modelling, we determined the probability of being DENV NS1, IgM and IgG RDT positive according to corresponding laboratory viremia, IgM and IgG ELISA metrics. Laboratory test thresholds for RDT positivity/negativity were calculated using Youden's J index and were utilized to estimate the RDT outcomes in patients from the Philippines, where only data for viremia, IgM and IgG were available (N:28,326). Lastly, the probabilities of being primary or post-primary according to every outcome using all RDTs, by day of fever, were calculated. Combining NS1, IgM and IgG RDTs captured 94.6% (52/55) and 95.4% (104/109) of laboratory-confirmed primary and post-primary DENV cases, respectively, during the first 5 days of fever. Laboratory test predicted, and actual, RDT outcomes had high agreement (79.5% (159/200)). Among patients from the Philippines, different combinations of estimated RDT outcomes were indicative of post-primary and primary immune status. Overall, IgG RDT positive results were confirmatory of post-primary infections. In contrast, IgG RDT negative results were suggestive of both primary and post-primary infections on days 1-2 of fever, yet were confirmatory of primary infections on days 3-5 of fever. CONCLUSION: We demonstrate how the primary and post-primary DENV immune status of reporting patients can be estimated at the point of care by combining NS1, IgM and IgG RDTs and considering the days since symptoms onset. This framework has the potential to strengthen surveillance operations and dengue prognosis, particularly in low resource settings.

AML1/ETO oncoprotein is directed to AML1 binding regions and co-localizes with AML1 and HEB on its targets.

A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.

Serological Evidence of Widespread Zika Transmission across the Philippines.

Zika virus (ZIKV) exposure across flavivirus-endemic countries, including the Philippines, remains largely unknown despite sporadic case reporting and environmental suitability for transmission. Using laboratory surveillance data from 2016, 997 serum samples were randomly selected from suspected dengue (DENV) case reports across the Philippines and assayed for serological markers of short-term (IgM) and long-term (IgG) ZIKV exposure. Using mixture models, we re-evaluated ZIKV IgM/G seroprevalence thresholds and used catalytic models to quantify the force of infection (attack rate, AR) from age-accumulated ZIKV exposure. While we observed extensive ZIKV/DENV IgG cross-reactivity, not all individuals with active DENV presented with elevated ZIKV IgG, and a proportion of dengue-negative cases (DENV IgG-) were ZIKV IgG-positive (14.3%, 9/63). We identified evidence of long-term, yet not short-term, ZIKV exposure across Philippine regions (ZIKV IgG+: 31.5%, 314/997) which was geographically uncorrelated with DENV exposure. In contrast to the DENV AR (12.7% (95%CI: 9.1-17.4%)), the ZIKV AR was lower (5.7% (95%CI: 3-11%)) across the country. Our results provide evidence of widespread ZIKV exposure across the Philippines and suggest the need for studies to identify ZIKV infection risk factors over time to better prepare for potential future outbreaks.

AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex.

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree.

Phosphorylation of AML1/RUNX1 regulates its degradation and nuclear matrix association.

The acute myeloid leukemia 1 (AML1) transcription factors are key regulators of hematopoietic differentiation. Cellular AML1c protein is found in the nucleus and can be separated into two fractions, one soluble in buffers containing salt and nonionic detergent and the other insoluble and tightly bound to the nuclear matrix. We find that the AML1c protein is modified by both phosphorylation and ubiquitination. Our studies show that the majority of the ubiquitinated AML1c is associated with the insoluble nuclear matrix. Treatment of cells with the proteasome inhibitor PS341 (Velcade, Bortezomib) increases the levels of ubiquitinated AML1c. Mutation of the four phosphorylation sites necessary for transcriptional regulation (serine 276, serine 293, serine 303, and threonine 300) mimics the effects of the proteasome inhibitor, increasing the levels of ubiquitinated, matrix-bound AML1c. We find that the soluble and insoluble forms of AML1c are degraded at a similar rate. However, mutation of these four serine/threonine residues statistically increases the half-life of the matrix-associated AML1c. Thus, phosphorylation of AML1c on specific serine/threonine residues controls both transcriptional activity and rate of degradation.

RUNX1-ETO induces a type I interferon response which negatively effects t(8;21)-induced increased self-renewal and leukemia development.

The 8;21 translocation is the most common chromosomal aberration occurring in acute myeloid leukemia (AML). This translocation causes expression of the RUNX1-ETO (AML1-ETO) fusion protein, which cooperates with additional mutations in leukemia development. We report here that interferons (IFNs) and IFN-stimulated genes are a group of genes consistently up-regulated by RUNX1-ETO in both human and murine models. RUNX1-ETO-induced up-regulation of IFN-stimulated genes occurs primarily via type I IFN signaling with a requirement for the IFNAR complex. Addition of exogenous IFN in vitro significantly reduces the increase in self-renewal potential induced by both RUNX1-ETO and its leukemogenic splicing isoform RUNX1-ETO9a. Finally, loss of type I IFN signaling via knockout of Ifnar1 significantly accelerates leukemogenesis in a t(8;21) murine model. This demonstrates the role of increased IFN signaling as an important factor inhibiting t(8;21) fusion protein function and leukemia development and supports the use of type I IFNs in the treatment of AML.

A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy.

The 8;21 chromosomal translocation occurs in 15% to 40% of patients with the FAB M2 subtype of acute myeloid leukemia (AML). This chromosomal abnormality fuses part of the AML1/RUNX1 gene to the ETO/MTG8 gene and generates the AML1-ETO protein. We previously identified a C-terminal truncated AML1-ETO protein (AEtr) in a mouse leukemia model. AEtr is almost identical to the AML1-ETO exon 9a isoform expressed in leukemia patients. Here, we describe a novel function of AEtr in the development of aneuploidy through spindle checkpoint attenuation. AEtr cells had a reduced mitotic index following nocodazole treatment, suggesting a failure in a subset of cells to arrest in mitosis with a functional spindle checkpoint. Additionally, primary leukemia cells and cell lines expressing AEtr were aneuploid. Moreover, AEtr cells had reduced levels of several spindle checkpoint proteins including BubR1 and securin following treatment with the spindle poison nocodazole. These results suggest that inactivation of the spindle checkpoint may contribute to the development of aneuploidy described in t(8;21) leukemia patients.

Acute myeloid leukemia with the 8q22;21q22 translocation: secondary mutational events and alternative t(8;21) transcripts.

Nonrandom and somatically acquired chromosomal translocations can be identified in nearly 50% of human acute myeloid leukemias. One common chromosomal translocation in this disease is the 8q22;21q22 translocation. It involves the AML1 (RUNX1) gene on chromosome 21 and the ETO (MTG8, RUNX1T1) gene on chromosome 8 generating the AML1-ETO fusion proteins. In this review, we survey recent advances made involving secondary mutational events and alternative t(8;21) transcripts in relation to understanding AML1-ETO leukemogenesis.

Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation.

We find that phorbol ester (PE) treatment of K562 cells greatly stimulates promoters (T cell receptor beta, myeloperoxidase, macrophage colony-stimulating factor receptor, and granulocyte macrophage colony-stimulating factor receptor) containing AML1 transcription factor binding sites. This stimulation of AML1c transcriptional activity is mediated by direct phosphorylation of the AML1c molecule on multiple phosphorylation sites. Eleven AML1c (S/T)P sites in the transcriptional activating domain are phosphorylated at a basal level in untreated K562 cells; treatment of the K562 cells with PE results in increased phosphorylation at five of these sites (serines 276, 293, 303, 462, and threonine 300). Mutation of these five sites to alanine inhibits PE-induced transcriptional activity; mutation of the sites to an acidic amino acid, aspartic acid, stimulates constitutive activity. Single mutations in four amino acids or double mutations (serines 276 and 293 or threonine 300 and serine 303) have little effect on AML1c transcriptional activity. Inhibitor assays suggest that the ERK family of protein kinases is activated by PEs to phosphorylate the (S/T)P sites within the AML1c molecule and markedly enhance the transcriptional activity of AML1c.