Glycoproteome remodeling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are urgently needed. Currently no multi-omics data set for primary MLL r patient cells exists that integrates transcriptomics, proteomics and glycomics to gain an inclusive picture of theranostic targets. Methods: We have integrated transcriptomics, proteomics and glycomics to i) obtain the first inclusive picture of primary patient BCP-ALL cells and identify molecular signatures that distinguish leukemic from normal precursor B-cells and ii) better understand the benefits and limitations of the applied technologies to deliver deep molecular sequence data across major cellular biopolymers. Results: MLL-r cells feature an extensive remodeling of their glycocalyx, with increased levels of Core 2-type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation. Notably, glycosaminoglycan remodeling from chondroitin sulfate to heparan sulfate was observed. A survival screen, to determine if glycan remodeling enzymes are redundant, identified MGAT1 and NGLY1, essential components of the N-glycosylation/degradation pathway, as highly relevant within this in vitro screening. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. While there is overall good correlation between transcriptomics and proteomics data, we demonstrate that a systematic combined multi-omics approach delivers important diagnostic information that is missed when applying a single omics technology. Conclusions: Apart from confirming well-known MLL-r BCP-ALL glycoprotein markers, our integrated multi-omics workflow discovered previously unidentified diagnostic/therapeutic protein targets.

Deubiquitinating enzyme inhibitor alleviates cyclin A1-mediated proteasome inhibitor tolerance in mixed-lineage leukemia.

Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug-tolerant cancer cells, which offer therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively, however the ways by which drug-tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed-lineage leukemia (MLL) cells. This adaptive process involved reversible downregulation of cyclin A1, which promoted PI resistance through cell-cycle arrest. PI-tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL-mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1-engaged cell-cycle regulation in PI resistance in MLL cells, and suggest that cell-cycle re-entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.

A regulatory element in the 3'-untranslated region of CEBPA is associated with myeloid/NK/T-cell leukemia.

OBJECTIVES: CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS: Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS: Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS: These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.

Heritable genetic background alters survival and phenotype of Mll-AF9-induced leukemias.

The MLL-AF9 fusion protein occurring as a result of t(9;11) translocation gives rise to pediatric and adult acute leukemias of distinct lineages, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and mixed-phenotype acute leukemia (MPAL). The mechanisms underlying how this same fusion protein results in diverse leukemia phenotypes among different individuals are not well understood. Given emerging evidence from genome-wide association studies that genetic risk factors contribute to MLL-rearranged leukemogenesis, here we tested the impact of genetic background on survival and phenotype of a well-characterized Mll-AF9 knockin mouse model. We crossed this model with five distinct inbred strains (129, A/J, C57BL/6, NOD, CAST) and tested their F1 hybrid progeny for dominant genetic effects on Mll-AF9 phenotypes. We discovered that genetic background altered peripheral blood composition, with Mll-AF9 CAST F1 having a significantly increased B-lymphocyte frequency, while the remainder of the strains exhibited myeloid-biased hematopoiesis, similar to the parental line. Genetic background also had an impact on overall survival, with Mll-AF9 A/J F1 and Mll-AF9 129 F1 having significantly shorter survival and Mll-AF9 CAST F1 having longer survival, compared with the parental line. Furthermore, we observed a range of hematologic malignancies, with Mll-AF9 A/J F1, Mll-AF9 129 F1, and Mll-AF9 B6 F1 developing exclusively myeloid cell malignancies (myeloproliferative disorder [MPD] and AML), whereas a subset of Mll-AF9 NOD F1 developed MPAL and Mll-AF9 CAST F1 developed ALL. This study provides a novel in vivo experimental model in which to evaluate the underlying mechanisms by which MLL-AF9 results in diverse leukemia phenotypes and provides definitive experimental evidence that genetic risk factors contribute to survival and phenotype of MLL-rearranged leukemogenesis.

Suppression of Mll1-Complex by Stat3/Cebpß-Induced miR-21a/21b/181b Maintains the Accumulation, Homeostasis, and Immunosuppressive Function of Polymorphonuclear Myeloid-Derived Suppressor Cells.

Mixed-lineage leukemia 1 (MLL1), which exerts its H3K4 methyltransferase activity by interacting with WDR5, ASH2L, and RBBP5, plays a pivotal role in regulating hematopoietic stem cell homeostasis. Disrupting the integrity of MLL1-complex has been reported to be associated with acute leukemia. However, the exact role of MLL1-complex in myeloid cells is unknown. In this study, microarray analysis revealed that the core components of the Mll1-complex, Wdr5, Ash2l, and Mll1, were concurrently downregulated by tumor-secreted factors as well as GM-CSF + IL-6 during the accumulation and activation of murine myeloid-derived suppressor cells (MDSCs). These changes were further validated by quantitative RT-PCR and Western blotting both in vitro and in vivo. The expression levels of WDR5 and ASH2L were also significantly decreased in bone marrow MDSCs of lung cancer patients compared with that of healthy controls. Functionally, ectopic expression of Wdr5, Ash2l, and Mll1 (C terminus) reversed the accumulation and function of GM-CSF + IL-6-induced as well as tumor-cocultured polymorphonuclear MDSCs (PMN-MDSCs) by promoting them to differentiate into mature neutrophil-like cells. Mechanistically, GM-CSF + IL-6-activated Stat3 and Cebpß synergistically induced the expression of miR-21a, miR-21b, and miR-181b, and thus inhibited the expression of Wdr5, Ash2l, and Mll1 by targeting to their 3' untranslated regions, respectively. Furthermore, knockdown of these microRNAs also suppressed the expansion and function of GM-CSF + IL-6-induced PMN-MDSCs. Taken together, our findings indicate that the Stat3/Cebpß-miR-21a/b/181b-Mll1-complex axis may play a critical role in PMN-MDSC expansion, activation, and differentiation, and this axis may provide an effectively immunological therapeutic approach for patients with cancer or other immunological diseases.

The reciprocal world of MLL fusions: A personal view.

Over the last 15 years the Diagnostic Center of Acute Leukemia (DCAL) at the Frankfurt University has diagnosed and elucidated the Mixed Lineage Leukemia (MLL) recombinome with >100 MLL fusion partners. When analyzing all these different events, balanced chromosomal translocations were found to comprise the majority of these cases (~70%), while other types of genetic rearrangements (3-way-translocations, spliced fusions, 11q inversions, interstitial deletions or insertion of chromosomal fragments into other chromosomes) account for about 30%. In nearly all those complex cases, functional fusion proteins can be produced by transcription, splicing and translation. With a few exceptions (10 out of 102 fusion genes which were per se out-of-frame), all these genetic rearrangements produced a direct MLL fusion gene, and in 94% of cases an additional reciprocal fusion gene. So far, 114 patients (out of 2454 = ~5%) have been diagnosed only with the reciprocal fusion allele, displaying no MLL-X allele. The fact that so many MLL rearrangements bear at least two fusion alleles, but also our findings that several direct MLL fusions were either out-of-frame fusions or missing, raises the question about the function and importance of reciprocal MLL fusions. Recent findings also demonstrate the presence of reciprocal MLL fusions in sarcoma patients. Here, we want to discuss the role of reciprocal MLL fusion proteins for leukemogenesis and beyond.

The molecular functions of common and atypical MLL fusion protein complexes.

Mixed-lineage leukemia (MLL) fuses with a variety of partners to produce a functionally altered MLL complex that is not expressed in normal cells, which transforms normal hematopoietic progenitors into leukemia cells. Because more than 80 fusion partners have been identified to date, the molecular functions of MLL fusion protein complexes appear diverse. However, over the past decade, the common functions utilized for leukemic transformation have begun to be elucidated. It appears that most (if not all) MLL fusion protein complexes utilize the AF4/ENL/P-TEFb and DOT1L complexes to some extent. Based on an understanding of the underlying molecular mechanisms, several molecular targeting drugs are being developed, opening paths to novel therapies. Here, we review the recent progress made in identifying the molecular functions of various MLL fusions and categorize the numerous fusion partners into several functionally-distinct groups to help discern commonalities and differences among various MLL fusion protein complexes.

A Dysregulated DNA Methylation Landscape Linked to Gene Expression in MLL-Rearranged AML.

Translocations of the KMT2A (MLL) gene define a biologically distinct and clinically aggressive subtype of acute myeloid leukaemia (AML), marked by a characteristic gene expression profile and few cooperating mutations. Although dysregulation of the epigenetic landscape in this leukaemia is particularly interesting given the low mutation frequency, its comprehensive analysis using whole genome bisulphite sequencing (WGBS) has not been previously performed. Here we investigated epigenetic dysregulation in nine MLL-rearranged (MLL-r) AML samples by comparing them to six normal myeloid controls, using a computational method that encapsulates mean DNA methylation measurements along with analyses of methylation stochasticity. We discovered a dramatically altered epigenetic profile in MLL-r AML, associated with genome-wide hypomethylation and a markedly increased DNA methylation entropy reflecting an increasingly disordered epigenome. Methylation discordance mapped to key genes and regulatory elements that included bivalent promoters and active enhancers. Genes associated with significant changes in methylation stochasticity recapitulated known MLL-r AML expression signatures, suggesting a role for the altered epigenetic landscape in the transcriptional programme initiated by MLL translocations. Accordingly, we established statistically significant associations between discordances in methylation stochasticity and gene expression in MLL-r AML, thus providing a link between the altered epigenetic landscape and the phenotype.

Perturbation of Methionine/S-adenosylmethionine Metabolism as a Novel Vulnerability in MLL Rearranged Leukemia.

Leukemias bearing mixed lineage leukemia (MLL) rearrangement (MLL-R) resulting in expression of oncogenic MLL fusion proteins (MLL-FPs) represent an especially aggressive disease subtype with the worst overall prognoses and chemotherapeutic response. MLL-R leukemias are uniquely dependent on the epigenetic function of the H3K79 methyltransferase DOT1L, which is misdirected by MLL-FPs activating gene expression, driving transformation and leukemogenesis. Given the functional necessity of these leukemias to maintain adequate methylation potential allowing aberrant activating histone methylation to proceed, driving leukemic gene expression, we investigated perturbation of methionine (Met)/S-adenosylmethionine (SAM) metabolism as a novel therapeutic paradigm for MLL-R leukemia. Disruption of Met/SAM metabolism, by either methionine deprivation or pharmacologic inhibition of downstream metabolism, reduced overall cellular methylation potential, reduced relative cell numbers, and induced apoptosis selectively in established MLL-AF4 cell lines or MLL-AF6-expressing patient blasts but not in BCR-ABL-driven K562 cells. Global histone methylation dynamics were altered, with a profound loss of requisite H3K79 methylation, indicating inhibition of DOT1L function. Relative occupancy of the repressive H3K27me3 modification was increased at the DOT1L promoter in MLL-R cells, and DOT1L mRNA and protein expression was reduced. Finally, pharmacologic inhibition of Met/SAM metabolism significantly prolonged survival in an advanced, clinically relevant patient-derived MLL-R leukemia xenograft model, in combination with cytotoxic induction chemotherapy. Our findings provide support for further investigation into the development of highly specific allosteric inhibitors of enzymatic mediators of Met/SAM metabolism or dietary manipulation of methionine levels. Such inhibitors may lead to enhanced treatment outcomes for MLL-R leukemia, along with cytotoxic chemotherapy or DOT1L inhibitors.

A Transcription Factor Addiction in Leukemia Imposed by the MLL Promoter Sequence.

The Mixed Lineage Leukemia gene (MLL) is altered in leukemia by chromosomal translocations to produce oncoproteins composed of the MLL N-terminus fused to the C-terminus of a partner protein. Here, we used domain-focused CRISPR screening to identify ZFP64 as an essential transcription factor in MLL-rearranged leukemia. We show that the critical function of ZFP64 in leukemia is to maintain MLL expression via binding to the MLL promoter, which is the most enriched location of ZFP64 occupancy in the human genome. The specificity of ZFP64 for MLL is accounted for by an exceptional density of ZFP64 motifs embedded within the MLL promoter. These findings demonstrate how a sequence anomaly of an oncogene promoter can impose a transcriptional addiction in cancer.

LSD1 inhibition exerts its antileukemic effect by recommissioning PU.1- and C/EBPα-dependent enhancers in AML.

Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine-specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. A common feature of many of these targeted agents is their ability to induce myeloid differentiation, suggesting that multiple paths toward a myeloid gene expression program can be engaged to relieve the differentiation blockade that is uniformly seen in AML. We performed a comparative assessment of chromatin dynamics during the treatment of mixed lineage leukemia (MLL)-AF9-driven murine leukemias and MLL-rearranged patient-derived xenografts using 2 distinct but effective differentiation-inducing targeted epigenetic therapies, the LSD1 inhibitor GSK-LSD1 and the DOT1L inhibitor EPZ4777. Intriguingly, GSK-LSD1 treatment caused global gains in chromatin accessibility, whereas treatment with EPZ4777 caused global losses in accessibility. We captured PU.1 and C/EBPα motif signatures at LSD1 inhibitor-induced dynamic sites and chromatin immunoprecipitation coupled with high-throughput sequencing revealed co-occupancy of these myeloid transcription factors at these sites. Functionally, we confirmed that diminished expression of PU.1 or genetic deletion of C/EBPα in MLL-AF9 cells generates resistance of these leukemias to LSD1 inhibition. These findings reveal that pharmacologic inhibition of LSD1 represents a unique path to overcome the differentiation block in AML for therapeutic benefit.

Mixed-Phenotype Acute Leukemia: Diagnostic Criteria and Pitfalls.

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous category in the World Health Organization classification that comprises acute leukemias with discrete admixed populations of myeloid and lymphoid blasts ("bilineal") or with extensive coexpression of lymphoid and myeloid markers in a single blast population ("biphenotypic"). Flow cytometric findings suggestive of MPAL are often met with consternation by pathologists and oncologists alike, owing to unfamiliarity with the disease and uncertainty about how MPAL fits into established paradigms for treatment of acute leukemia. The purpose of this review is to explain the diagnostic criteria for MPAL, summarize its biological and clinical features, and address common diagnostic pitfalls of these unusual leukemias.

A Review of the Scaffold Protein Menin and its Role in Hepatobiliary Pathology.

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome with neuroendocrine tumorigenesis of the parathyroid glands, pituitary gland, and pancreatic islet cells. The MEN1 gene codes for the canonical tumor suppressor protein, menin. Its protein structure has recently been crystallized, and it has been investigated in a multitude of other tissues. In this review, we summarize recent advancements in understanding the structure of the menin protein and its function as a scaffold protein in histone modification and epigenetic gene regulation. Furthermore, we explore its role in hepatobiliary autoimmune diseases, cancers, and metabolic diseases. In particular, we discuss how menin expression and function are regulated by extracellular signaling factors and nuclear receptor activation in various hepatic cell types. How the many signaling pathways and tissue types affect menin's diverse functions is not fully understood. We show that small-molecule inhibitors affecting menin function can shed light on menin's broad role in pathophysiology and elucidate distinct menin-dependent processes. This review reveals menin's often dichotomous function through analysis of its role in multiple disease processes and could potentially lead to novel small-molecule therapies in the treatment of cholangiocarcinoma or biliary autoimmune diseases.

MLL-ENL-mediated leukemia initiation at the interface of lymphoid commitment.

Translocations involving the mixed lineage leukemia-1 are recurrent events in acute leukemia and associate with lymphoid (ALL), myeloid (AML) or mixed lineage (MLL) subtypes. Despite an association with ALL in humans, murine MLL fusion models are persistently restricted to AML. We here explored this issue using an inducible mixed lineage leukemia-eleven nineteen leukemia (MLL-ENL) mouse model. Although multiple progenitor cell types with myeloid potential are potent AML leukemia-initiating cells, also the earliest lymphoid progenitors were capable of initiating AML. This ability to evoke a latent myeloid potential in the earliest lymphoid progenitors was lost upon further lymphoid commitment. At the same time, more downstream/committed lymphoid precursors also failed to initiate lymphoid leukemia. Co-expression of MLL-ENL with a constitutively active RAS allele, the most common co-mutation in MLL fusion leukemias, could influence on both disease latency and lineage assignment of developing leukemia in what appears to be a mutation-order-dependent manner. Finally, CEBPB-mediated transdifferentation of committed and otherwise leukemia-incompetent B-cell progenitors imbued these cells with leukemic competence for AML. Therefore, apart from providing detailed insight into the differential responsiveness of candidate target cells to a first-hit MLL fusion event, our data warrants caution to therapeutic approaches based on the concept of transdifferentiation.

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Mixed-phenotype acute leukemia: current challenges in diagnosis and therapy.

PURPOSE OF REVIEW: Mixed-phenotype acute leukemia (MPAL) is a rare disease that poses many diagnostic and therapeutic challenges. Patients with MPAL are considered to have poor outcomes. The difficulties in classifying this leukemia, the lack of prospectively collected data concerning therapeutic outcomes, and rare incidence result in much uncertainty as to the best approach for patients with MPAL. RECENT FINDINGS: Recent studies demonstrated that most MPALs are associated with cytogenetic abnormalities; genetic sequencing studies disclose a high frequency of somatic mutations in genes encoding epigenetic regulators, tumor suppressors, and transcription factors. The limited available data suggest that higher remission rates are achieved with acute lymphoblastic leukemia-like induction regimens compared with acute myeloid leukemia-type approaches. Allogeneic transplantation in first remission may be associated with improved survival compared with consolidation chemotherapy. SUMMARY: Advances in understanding the genetic landscape of MPAL may allow a more biologically driven classification of this heterogeneous group of leukemias in the future that will lead to optimized therapies for individual patients. Most data that inform therapy are based on retrospective, uncontrolled studies; prospective trials that incorporate targeted approaches based on genetics and immunophenotype are needed.

Low-dose salinomycin induces anti-leukemic responses in AML and MLL.

Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response.Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.