Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

Metabolic profile of leukemia cells influences treatment efficacy of L-asparaginase.

BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us. CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.

ERG deletions in childhood acute lymphoblastic leukemia with DUX4 rearrangements are mostly polyclonal, prognostically relevant and their detection rate strongly depends on screening method sensitivity.

ERG-deletions occur recurrently in acute lymphoblastic leukemia, especially in the DUX4-rearranged subtype. The ERG-deletion was shown to positively impact prognosis of patients with IKZF1-deletion and its presence precludes assignment into IKZF1 plus group, a novel high-risk category on AIEOP-BFM ALL trials. We analyzed the impact of different methods on ERG-deletion detection rate, evaluated ERG-deletion as a potential marker for DUX4-rearranged leukemia, studied its associations with molecular and clinical characteristics within this leukemia subtype, and analyzed its clonality. Using single-nucleotide-polymorphism array, genomic polymerase chain reaction (PCR) and amplicon-sequencing we found ERG-deletion in 34% (16 of 47), 66% (33 of 50) and 78% (39 of 50) of DUX4-rearranged leukemia, respectively. False negativity of ERG-deletion by single-nucleotide-polymorphism array caused IKZF1 plus misclassification in 5 patients. No ERG-deletion was found outside the DUX4-rearranged cases. Within DUX4-rearranged leukemia, the ERG-deletion was associated with higher total number of copy-number aberrations, and, importantly, the ERG-deletion positivity by PCR was associated with better outcome [5-year event-free survival (EFS), ERG-deletion-positive 93% vs. ERG-deletion-negative 68%, P=0.022; 5-year overall survival (OS), ERG-deletion-positive 97% vs. ERG-deletion-negative 75%, P=0.029]. Ultra-deep amplicon-sequencing revealed distinct co-existing ERG-deletions in 22 of 24 patients. In conclusion, our data demonstrate inadequate sensitivity of single-nucleotide-polymorphism array for ERG-deletion detection, unacceptable for proper IKZF1 plus classification. Even using more sensitive methods (PCR/amplicon-sequencing) for its detection, ERG-deletion is absent in 22-34% of DUX4-rearranged leukemia and does not represent an adequately sensitive marker of this leukemia subtype. Importantly, the ERG-deletion potentially stratifies the DUX4-rearranged leukemia into biologically/clinically distinct subsets. Frequent polyclonal pattern of ERG-deletions shows that late origin of this lesion is more common than has been previously described.

Genomic landscape of pediatric B-other acute lymphoblastic leukemia in a consecutive European cohort.

Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia (ALL) during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations still have to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of DUX4-rearranged, BCR-ABL1-like, ZNF384-rearranged, ETV6-RUNX1-like, iAMP21 and MEF2D-rearranged subtypes was 27%, 15%, 5%, 5%, 4%, and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in DUX4-rearranged leukemia and a strong association of ZNF384-rearranged leukemia with B-myeloid immunophenotype. Of the druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in one patient. A recently described negative prognostic factor, IKZF1plus , was found in 14% of patients and was enriched in (but not exclusive for) BCR-ABL1-like subtype. PAX5 fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and only occurred in the B-rest subset, altogether accounting for 20% of the B-other group. PAX5 P80R was associated with a specific gene expression signature, potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations. This study also strengthens and widens the current knowledge of B-other ALL and provides an objective basis for optimization of current genetic diagnostics.

Two novel fusion genes, AIF1L-ETV6 and ABL1-AIF1L, result together with ETV6-ABL1 from a single chromosomal rearrangement in acute lymphoblastic leukemia with prenatal origin.

Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and 2 novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood. We cloned coding sequences of AIF1L-ETV6 and ABL1-AIF1L in-frame fusion transcripts from the patient's leukemic blasts and we show that the chimeric protein containing the DNA binding domain of ETV6 is expressed from the AIF1L-ETV6 transcript and localized in both the cytoplasm and nucleus of transfected HEK293T cells. Transcriptomic and genomic profiling of the diagnostic bone marrow sample revealed Ph-like gene expression signature and loss of the IKZF1 and CDKN2A/B genes, the typical genetic lesions accompanying ETV6-ABL1-positive ALL. The prenatal origin of the rearrangement confirms that ETV6-ABL1 is not sufficient to cause overt leukemia, even when combined with the 2 novel fusions. We did not find the AIF1L-ETV6 and ABL1-AIF1L fusions in other ETV6-ABL1-positive ALL. Nevertheless, functional studies would be needed to establish the biological role of AIF1L-ETV6 and ABL1-AIF1L and to determine whether they contribute to leukemogenesis and/or to the final leukemia phenotype.

Characterization of leukemias with ETV6-ABL1 fusion.

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.

Aspartate in tumor microenvironment and beyond: Metabolic interactions and therapeutic perspectives.

Aspartate is a proteinogenic non-essential amino acid with several essential functions in proliferating cells. It is mostly produced in a cell autonomous manner from oxalacetate via glutamate oxalacetate transaminases 1 or 2 (GOT1 or GOT2), but in some cases it can also be salvaged from the microenvironment via transporters such as SLC1A3 or by macropinocytosis. In this review we provide an overview of biosynthetic pathways that produce aspartate endogenously during proliferation. We discuss conditions that favor aspartate uptake as well as possible sources of exogenous aspartate in the microenvironment of tumors and bone marrow, where most available data have been generated. We highlight metabolic fates of aspartate, its various functions, and possible approaches to target aspartate metabolism for cancer therapy.

Targeting amino acid metabolism in cancer.

Metabolic rewiring is a characteristic hallmark of cancer cells. This phenomenon sustains uncontrolled proliferation and resistance to apoptosis by increasing nutrients and energy supply. However, reprogramming comes together with vulnerabilities that can be used against tumor and can be applied in targeted therapy. In the last years, the genetic background of tumors has been identified thoroughly and new therapies targeting those mutations tested. Nevertheless, we propose that targeting the phenotype of cancer cells could be another way of treatment aiming to avoid drug resistance and non-responsiveness of cancer patients. Amino acid metabolism is part of the altered processes in cancer cells. Amino acids are building blocks and also sensors of signaling pathways regulating main biological processes. In this comprehensive review, we described four amino acids (asparagine, arginine, methionine, and cysteine) which have been actively investigated as potential targets for anti-tumor therapy. Asparagine depletion is successfully used for decades in the treatment of acute lymphoblastic leukemia and there is a strong implication to apply it to other types of tumors. Arginine auxotrophic tumors are great candidates for arginine-starvation therapy. Higher requirement for essential amino acids such as methionine and cysteine point out promising targetable weaknesses of cancer cells.

PTEN/PI3K/Akt pathway alters sensitivity of T-cell acute lymphoblastic leukemia to L-asparaginase.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) still remains a therapeutic challenge due to relapses which are resistant to further treatment. L-asparaginase (ASNase) is a key therapy component in pediatric T-ALL and lower sensitivity of leukemia cells to this drug negatively influences overall treatment efficacy and outcome. PTEN protein deletion and/or activation of the PI3K/Akt signaling pathway leading to altered cell growth and metabolism are emerging as a common feature in T-ALL. We herein investigated the relationship amongst PTEN deletion, ASNase sensitivity and glucose metabolism in T-ALL cells. First, we found significant differences in the sensitivity to ASNase amongst T-ALL cell lines. While cell lines more sensitive to ASNase were PTEN wild type (WT) and had no detectable level of phosphorylated Akt (P-Akt), cell lines less sensitive to ASNase were PTEN-null with high P-Akt levels. Pharmacological inhibition of Akt in the PTEN-null cells rendered them more sensitive to ASNase and lowered their glycolytic function which then resembled PTEN WT cells. In primary T-ALL cells, although P-Akt level was not dependent exclusively on PTEN expression, their sensitivity to ASNase could also be increased by pharmacological inhibition of Akt. In summary, we highlight a promising therapeutic option for T-ALL patients with aberrant PTEN/PI3K/Akt signaling.

Up-regulation of homeodomain genes, DLX1 and DLX2, by FLT3 signaling.

BACKGROUND: Activating mutations in fms-like tyrosine kinase-3 (FLT3) are frequent in acute myeloid leukemia and represent both a poor prognostic feature and a therapeutic target. We have identified a previously unrecognized downstream effect of FLT3 activation, namely up-regulation of the homeodomain genes, DLX1 and DLX2. DESIGN AND METHODS: MV4;11 cells with FLT3-internal tandem duplication mutation, RS4;11 cells with wild-type FLT3 and blasts from patients with acute myeloid leukemia were used to pursue the relation between FLT3, DLX1/2 and transforming growth factor-ß (TGFß). Real-time quantitative reverse transcriptase polymerase chain reaction, western blot and reverse-phase protein array were performed to detect changes in gene and protein expression. RNA interference and MTS assays were used to study the interaction of PKC412, FLT3 inhibitor and TGFß1. RESULTS: A direct relationship between FLT3 activity and DLX1/2 expression was revealed by both inhibition and up-regulation of FLT3 signaling in MV4;11 and RS4;11 cell lines, respectively, in isolated blast cells from patients with acute myeloid leukemia, and in reverse-phase protein array assays of samples from patients with acute myeloid leukemia. Mechanistically, the link between FLT3 and DLX1 expression appears to involve MAPK signaling through the ERK and JNK pathways. To determine whether elevated DLX1 had a functional consequence, we explored the reported inhibition by DLX1 on TGFß/Smad signaling. Indeed, TGFß responses were blunted by FLT3 activation in a DLX1-dependent manner and FLT3 inhibition resulted in a time-dependent increase in nuclear phospho-Smad2. CONCLUSIONS: These findings suggest that alterations in DLX1/2 contribute to the biological consequences of FLT3 activation.

A novel class of ZNF384 aberrations in acute leukemia.

Fusion of the ZNF384 gene as the 3' partner to several different 5' partner genes occurs recurrently in B-cell precursor acute lymphoblastic and mixed phenotype B/myeloid leukemia. These canonical fusions (ZNF384r) contain the complete ZNF384 coding sequence and are associated with a specific gene expression signature. Cases with this signature, but without canonical ZNF384 fusions (ZNF384r-like cases), have been described previously. Although some have been shown to harbor ZNF362 fusions, the primary aberrations remain unknown in a major proportion. We studied 3 patients with the ZNF384r signature and unknown primary genetic background and identified a previously unknown class of genetic aberration affecting the last exon of ZNF384 and resulting in disruption of the C-terminal portion of the ZNF384 protein. Importantly, in 2 cases, the ZNF384 aberration, indel, was missed during the bioinformatic analysis but revealed by the manual, targeted reanalysis. Two cases with the novel aberrations had a mixed (B/myeloid) immunophenotype commonly associated with canonical ZNF384 fusions. In conclusion, we present leukemia cases with a novel class of ZNF384 aberrations that phenocopy leukemia with ZNF384r. Therefore, we show that part of the so-called ZNF384r-like cases represent the same genetic subtype as leukemia with canonical ZNF384 fusions.

HOX gene expression in phenotypic and genotypic subgroups and low HOXA gene expression as an adverse prognostic factor in pediatric ALL.

BACKGROUND: HOX genes play an important role in both normal lymphopoiesis and leukemogenesis. However, HOX expression patterns in leukemia cells compared to normal lymphoid progenitors have not been systematically studied in acute lymphoblastic leukemia (ALL) subtypes. PROCEDURE: The RNA expression levels of HOXA, HOXB, and CDX1/2 genes were analyzed by qRT-PCR in a cohort of 61 diagnostic pediatric ALL samples and FACS-sorted subpopulations of normal lymphoid progenitors. RESULTS: The RNA expression of HOXA7-10, HOXA13, and HOXB2-4 genes was exclusively detected in leukemic cells and immature progenitors. The RNA expression of HOXB6 and CDX2 genes was exclusively detected in leukemic cells but not in B-lineage cells at any of the studied developmental stages. HOXA3-4, HOXA7, and HOXB3-4 genes were differentially expressed between BCP-ALL and T-ALL subgroups, and among genotypically defined MLL/AF4, TEL/AML1, BCR/ABL, hyperdiploid and normal karyotype subgroups. However, this differential expression did not define specific clusters in hierarchical cluster analysis. HOXA7 gene was low expressed at the RNA level in patients with hyperdiploid leukemia, whereas HOXB7 and CDX2 genes were low expressed in TEL/AML1-positive and BCR/ABL-positive cases, respectively. In contrast to previous findings in acute myeloid leukemia, high HOXA RNA expression was associated with an excellent prognosis in Cox's regression model (P = 0.03). In MLL/AF4-positive ALL, lower HOXA RNA expression correlated with the methylation status of their promoters. CONCLUSIONS: HOX gene RNA expression cannot discriminate leukemia subgroups or relative maturity of leukemic cells. However, HOXA RNA expression correlates with prognosis, and particular HOX genes are expressed in specific genotypically characterized subgroups.

The identification of (ETV6)/RUNX1-regulated genes in lymphopoiesis using histone deacetylase inhibitors in ETV6/RUNX1-positive lymphoid leukemic cells.

PURPOSE: Chimeric transcription factor ETV6/RUNX1 (TEL/AML1) is believed to cause pathologic block in lymphoid cell development via interaction with corepressor complex and histone deacetylase. We wanted to show the regulatory effect of ETV6/RUNX1 and its reversibility by histone deacetylase inhibitors (HDACi), as well as to identify potential ETV6/RUNX1-regulated genes. EXPERIMENTAL DESIGN: We used luciferase assay to show the interaction of ETV6/RUNX1 protein, ETV6/RUNX1-regulated gene, and HDACi. To identify ETV6/RUNX1-regulated genes, we used expression profiling and HDACi in lymphoid cells. Next, using the flow cytometry and quantitative reverse transcription-PCR, we measured differentiation changes in gene and protein expression after HDACi treatment. RESULTS: Luciferase assay showed repression of granzyme B expression by ETV6/RUNX1 protein and the reversibility of this effect by HDACi. Proving this regulatory role of ETV6/RUNX1, we identified, using complex statistical analysis, 25 genes that are potentially regulated by ETV6/RUNX1 protein. In four selected genes with known role in the cell cycle regulation (JunD, ACK1, PDGFRB, and TCF4), we confirmed expression changes after HDACi by quantitative analysis. After HDACi treatment, ETV6/RUNX1-positive cells showed immunophenotype changes resembling differentiation process compared with other leukemic cells (BCR/ABL, ETV6/PDGFRB positive). Moreover, ETV6/RUNX1-positive leukemic cells accumulated in G(1)-G(0) phase after HDACi whereas other B-lineage leukemic cell lines showed rather unspecific changes including induction of apoptosis and decreased proliferation. CONCLUSIONS: Presented data support the hypothesis that HDACi affect ETV6/RUNX1-positive cells via direct interaction with ETV6/RUNX1 protein and that treatment with HDACi may release aberrant transcription activity caused by ETV6/RUNX1 chimeric transcription factor.

Assessment of the Metabolic Profile of Primary Leukemia Cells.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Altered Metabolism of Leukemic Cells: New Therapeutic Opportunity.

The cancer metabolic program alters bioenergetic processes to meet the higher demands of tumor cells for biomass production, nucleotide synthesis, and NADPH-balancing redox homeostasis. It is widely accepted that cancer cells mostly utilize glycolysis, as opposed to normal cells, in which oxidative phosphorylation is the most employed bioenergetic process. Still, studies examining cancer metabolism had been overlooked for many decades, and it was only recently discovered that metabolic alterations affect both the oncogenic potential and therapeutic response. Since most of the published works concern solid tumors, in this comprehensive review, we aim to summarize knowledge about the metabolism of leukemia cells. Leukemia is a malignant disease that ranks first and fifth in cancer-related deaths in children and adults, respectively. Current treatment has reached its limits due to toxicity, and there has been a need for new therapeutic approaches. One of the possible scenarios is improved use of established drugs and another is to introduce new druggable targets. Herein, we aim to describe the complexity of leukemia metabolism and highlight cellular processes that could be targeted therapeutically and enhance the effectiveness of current treatments.

Low HOX gene expression in PML-RARα-positive leukemia results from suppressed histone demethylation.

Homeobox (HOX) genes are frequently dysregulated in leukemia. Previous studies have shown that aberrant HOX gene expression accompanies leukemogenesis and affects disease progression and leukemia patient survival. Patients with acute myeloid leukemia (AML) bearing PML-RARα fusion gene have distinct HOX gene signature in comparison to other subtypes of AML patients, although the mechanism of transcription regulation is not completely understood. We previously found an association between the mRNA levels of HOX genes and those of the histone demethylases JMJD3 and UTX in PML-RARα- positive leukemia patients. Here, we demonstrate that the release of the PML-RARα-mediated block in PML-RARα-positive myeloid leukemia cells increased both JMJD3 and HOX gene expression, while inhibition of JMJD3 using the specific inhibitor GSK-J4 reversed the effect. This effect was driven specifically through PML-RARα fusion protein since expression changes did not occur in cells with mutated RARα and was independent of differentiation. We confirmed that gene expression levels were inversely correlated with alterations in H3K27me3 histone marks localized at HOX gene promoters. Furthermore, data from chromatin immunoprecipitation followed by sequencing broaden a list of clustered HOX genes regulated by JMJD3 in PML-RARα-positive leukemic cells. Interestingly, the combination of GSK-J4 and all-trans retinoic acid (ATRA) significantly increased PML-RARα-positive cell apoptosis compared with ATRA treatment alone. This effect was also observed in ATRA-resistant NB4 clones, which may provide a new therapeutic opportunity for patients with acute promyelocytic leukemia (APL) resistant to current treatment. The results of our study reveal the mechanism of HOX gene expression regulation and contribute to our understanding of APL pathogenesis.