The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Targeting hedgehog signaling reduces self-renewal in embryonal rhabdomyosarcoma.

Current treatment regimens for rhabdomyosarcoma (RMS), the most common pediatric soft tissue cancer, rely on conventional chemotherapy, and although they show clinical benefit, there is a significant risk of adverse side effects and secondary tumors later in life. Therefore, identifying and targeting sub-populations with higher tumorigenic potential and self-renewing capacity would offer improved patient management strategies. Hedgehog signaling has been linked to the development of embryonal RMS (ERMS) through mouse genetics and rare human syndromes. However, activating mutations in this pathway in sporadic RMS are rare and therefore the contribution of hedgehog signaling to oncogenesis remains unclear. Here, we show by genetic loss- and gain-of-function experiments and the use of clinically relevant small molecule modulators that hedgehog signaling is important for controlling self-renewal of a subpopulation of RMS cells in vitro and tumor initiation in vivo. In addition, hedgehog activity altered chemoresistance, motility and differentiation status. The core stem cell gene NANOG was determined to be important for ERMS self-renewal, possibly acting downstream of hedgehog signaling. Crucially, evaluating the presence of a subpopulation of tumor-propagating cells in patient biopsies identified by GLI1 and NANOG expression had prognostic significance. Hence, this work identifies novel functional aspects of hedgehog signaling in ERMS, redefines the rationale for its targeting as means to control ERMS self-renewal and underscores the importance of studying functional tumor heterogeneity in pediatric cancers.

Analysis of the oligomeric state and transactivation potential of TAp73α.

The proteins p73 and p63 are members of the p53 protein family and are involved in important developmental processes. Their high sequence identity with the tumor suppressor p53 has suggested that they act as tumor suppressors as well. While p63 has a crucial role in the maintenance of epithelial stem cells and in the quality control of oocytes without a clear role as a tumor suppressor, p73's tumor suppressor activity is well documented. In a recent study we have shown that the transcriptional activity of TAp63α, the isoform responsible for the quality control in oocytes, is regulated by its oligomeric state. The protein forms an inactive, dimeric and compact conformation in resting oocytes, while the detection of DNA damage leads to the formation of an active, tetrameric and open conformation. p73 shows a high sequence identity to p63, including those domains that are crucial in stabilizing its inactive state, thus suggesting that p73's activity might be regulated by its oligomeric state as well. Here, we have investigated the oligomeric state of TAp73α by size exclusion chromatography and detailed domain interaction mapping, and show that in contrast to p63, TAp73α is a constitutive open tetramer. However, its transactivation potential depends on the cellular background and the promoter context. These results imply that the regulation of p73's transcriptional activity might be more closely related to p53 than to p63.

p21 Downregulation is an important component of PAX3/FKHR oncogenicity and its reactivation by HDAC inhibitors enhances combination treatment.

A number of drugs developed against cancer-specific molecular targets have been shown to offer survival benefits alone or in combination with standard treatments, especially for those cases in which tumor pathogenesis is dominated by a single molecular abnormality. One example for such a tumor type is alveolar rhabdomyosarcoma (aRMS), which is characterized by a specific translocation creating the oncogenic PAX3/FKHR transcription factor, believed to be the molecular basis of the disease. Recently, we were able to show that the small molecule inhibitor PKC412 (midostaurin) shows strong antitumor activity against aRMS by reducing the transcriptional activity of PAX3/FKHR. In this study, we screened for combination strategies that are superior to PKC412-only treatment and found that the combination of PKC412 with histone deacetylase inhibitors like valproic acid (VPA) synergistically induced apoptosis resulting in suppressed aRMS tumor growth in vivo. We provide evidence that the antitumor effect on combination treatment is achieved by VPA-induced reactivation of p21, which is downregulated in aRMS cells by destabilization of the transcriptional regulator EGR1 by PAX3/FKHR. Our study highlights a possible mechanism behind the increased efficacy and indicates that different arms of PAX3/FKHR oncogenicity can be exploited therapeutically by the specific combination of drugs to increase their therapeutic potential.

The C-terminus of p63 contains multiple regulatory elements with different functions.

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ∼13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63.

Conformational stability and activity of p73 require a second helix in the tetramerization domain.

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.

New insights to the MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

KIF5C: a new binding partner for protein kinase CK2 with a preference for the CK2alpha' subunit.

Protein kinase CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in eukaryotic cells. CK2 is a constitutively active tetrameric enzyme composed of two catalytic alpha and/or alpha'-subunits and two regulatory beta-subunits. There is increasing evidence that the individual subunits may have independent functions and that they are asymmetrically distributed inside the cell. To gain a better understanding of the functions of the individual subunits, we employed a yeast-two-hybrid screen with CK2alpha and CK2alpha'. We identified the motor neuron protein KIF5C as a new binding partner for CK2. The interaction found in the yeast-two-hybrid screen was confirmed by co-sedimentation analysis on a sucrose density gradient and by co-immunoprecipitation analysis. Pull-down experiments and surface plasmon resonance spectrometry revealed a direct binding of KIF5C to CK2alpha'. Co-localization studies with neuroblastoma cells, bone marrow and with primary neurons confirmed the biochemical analysis that KIF5C preferentially bound to CK2alpha'.

Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia.

Detection of minimal residual disease (MRD) is the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood acute lymphoblastic leukemia (ALL). The 10-year update on the I-BFM-SG MRD study 91 demonstrates stable results (event-free survival), that is, standard risk group (MRD-SR) 93%, intermediate risk group (MRD-IR) 74%, and high risk group (MRD-HR) 16%. In multicenter trial AIEOP-BFM ALL 2000, patients were stratified by MRD detection using quantitative PCR after induction (TP1) and consolidation treatment (TP2). From 1 July 2000 to 31 October 2004, PCR target identification was performed in 3341 patients: 2365 (71%) patients had two or more sensitive targets (< or =10(-4)), 671 (20%) patients revealed only one sensitive target, 217 (6%) patients had targets with lower sensitivity, and 88 (3%) patients had no targets. MRD-based risk group assignment was feasible in 2594 (78%) patients: 40% were classified as MRD-SR (two sensitive targets, MRD negativity at both time points), 8% as MRD-HR (MRD > or =10(-3) at TP2), and 52% as MRD-IR. The remaining 823 patients were stratified according to clinical risk features: HR (n=108) and IR (n=715). In conclusion, MRD-PCR-based stratification using stringent criteria is feasible in almost 80% of patients in an international multicenter trial.

A novel yeast-based tool to detect mutagenic and recombinogenic effects simultaneously.

In this work, we describe a new yeast-based assay to allow efficient detection of a comprehensive spectrum of genotoxicity events. The constructed diploid Saccharomyces cerevisiae strain allows the simultaneous monitoring of forward mutations, mitotic recombination events and chromosome loss or non-disjunction by direct selection in an easy and highly reproducible approach. The strain contains a DNA module consisting of a single functional copy of the URA3 gene and the kanMX4 gene inserted at the ADE2 locus on the right arm of chromosome XV. The changes of the genotype within the marker region were primarily selected on 5-fluoroorotic acid (5-FOA) agar plates. Further simple phenotypic tests of the 5-FOA-resistant ura3 clones make it possible to analyze the genetic configuration in detail (e.g. point mutations in URA3, gene conversion, crossing-over and chromosome loss). We demonstrate the successful application of our test system by studying the effects of well-known genotoxic agents (UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, aniline and benomyl). We found that the various agents induced mutations and recombination events with different relative frequencies. The integration of the module has generated a hot spot region of mutation and recombination at the borders of the artificially integrated URA3 kanMX4 cassette, which makes the system more sensitive towards DNA-damaging agents. Unlike other test systems, our S. cerevisiae strain is capable to detect a mutagenic effect caused by aniline.

Protein kinase CK2 interacts with the splicing factor hPrp3p.

Numerous signalling pathways in cells are influenced by the ubiquitous Ser/Thr protein kinase CK2. Protein kinase CK2 is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. Several of the known CK2 substrates are proteins known to regulate transcriptional events. Here, we describe that protein kinase CK2 interacts with the splicing factor hPrp3p, which is important for the assembly of the spliceosome. In a two-hybrid screen hPrp3p is exclusively bound to the catalytic alpha- or alpha'-subunits of CK2 but not to the regulatory beta-subunit. The interaction was confirmed by coimmunoprecipitation experiments in vitro and in vivo. Moreover, both proteins colocalized in nuclear speckles which is typical for splicing factor compartments within the nucleus. Phosphorylation experiments revealed that hPrp3p is also a substrate of protein kinase CK2. The main phosphorylation site was mapped to C-terminal residues. In vitro and in vivo splicing assays showed that the splicing activity is significantly influenced by the CK2-hPrp3p interaction. Thus, these data showed that CK2 is involved in the regulation of RNA processing.

Comparative expression profiling identifies an in vivo target gene signature with TFAP2B as a mediator of the survival function of PAX3/FKHR.

The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric transcription factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.

The LIM-only protein FHL2 is a negative regulator of E4F1.

The E1A-targeted transcription factor E4F1 is a key player in the control of mammalian embryonic and somatic cell proliferation and survival. Mouse embryos lacking E4F die at an early developmental stage, whereas enforced expression of E4F1 in various cell lines inhibits cell cycle progression. E4F1-antiproliferative effects have been shown to depend on its capacity to repress transcription and to interact with pRb and p53. Here we show that full-length E4F1 protein (p120(E4F1)) but not its E1A-activated and truncated form (p50(E4F1)), interacts directly in vitro and in vivo with the LIM-only protein FHL2, the product of the p53-responsive gene FHL2/DRAL (downregulated in rhabdomyosarcoma Lim protein). This E4F1-FHL2 association occurs in the nuclear compartment and inhibits the capacity of E4F1 to block cell proliferation. Consistent with this effect, ectopic expression of FHL2 inhibits E4F1 repressive effects on transcription and correlates with a reduction of nuclear E4F1-p53 complexes. Overall, these results suggest that FHL2/DRAL is an inhibitor of E4F1 activity. Finally, we show that endogenous E4F1-FHL2 complexes form in U2OS cells upon UV-light-induced nuclear accumulation of FHL2.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Prenatal origin of separate evolution of leukemia in identical twins.

Several studies involving identical twins with concordant leukemia and retrospective scrutiny of archived neonatal blood spots have shown that the TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia (ALL) frequently arises before birth. A prenatal origin of childhood leukemia was further supported by the detection of clonotypic immunoglobulin gene rearrangements on neonatal blood spots of children with various other subtypes of ALL. However, no comprehensive study is available linking these clonotypic events. We describe a pair of 5-year-old monozygotic twins with concordant TEL-AML1-positive ALL. Separate leukemic clones were identified in the diagnostic samples since distinct IGH and IGK-Kde gene rearrangements could be detected. Additional differences characterizing the leukemic clones included an aberration of the second, nonrearranged TEL allele observed in one twin only. Interestingly, both the identical TEL-AML1 fusion sequence and distinct immunoglobulin gene rearrangements were identified on the neonatal blood spots indicating that separate preleukemic clones evolved already before birth. Finally, we compared the reported twins with an additional 31 children with ALL by using the microarray technology. Gene expression profiling provided evidence that leukemia in twins harbours the same subtype-typical feature as TEL-AML1-positive leukemia in singletons suggesting that the leukemogenesis model might also be applicable generally.

The in vivo form of the murine class VI POU protein Emb is larger than that encoded by previously described transcripts.

The class VI POU domain family member known as Emb in the mouse (rat Brn5 or human mPOU/TCFbeta1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5' RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the -COOH terminal, and an -NH(2) terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene.

[Systematic development of a scale for determination of health-related quality of life in multiple trauma patients. The Polytrauma Outcome (POLO) Chart]. / Systematische Entwicklung eines Messinstruments zur Erfassung der gesundheitsbezogenen Lebensqualität beim polytraumatisierten Patienten. Die Polytrauma-Outcome-(POLO-)Chart.

Even years after having sustained multiple injuries patients often suffer from its sequelae. These comprise restrictions in physical function, but also pain, social and psychological impairments. Although the Meran Consensus Conference in 1990 defined the contents of "quality of life" (QoL) measures in surgery, still no instrument is available for the valid assessment of all relevant QoL domains in multiple injured patients. This paper describes the systematic development of a modular instrument for the assessment of health related QoL. Within three phases (phase I: generation of items, phase II: item reduction, phase III: pre-testing in 70 multiple injured and control patients) a questionnaire of 57 items was developed, which measures all relevant trauma-related aspects of QoL after acute hospital care. In combination with the Glascow Outcome Scale (GOS), the EUROQOL and the SF-36, the newly developed instrument builds the Polytrauma Outcome Chart (POLO-Chart) which will also be used as "Part E" for outcome assessment within the "Trauma registry" of the German Society for Trauma Surgery. In phase IV, the POLO-Chart will finally be validated in five trauma centres (Celle, Essen, Hanover, Cologne und Munich).