CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

A longitudinal single-cell atlas of treatment response in pediatric AML.

Pediatric acute myeloid leukemia (pAML) is characterized by heterogeneous cellular composition, driver alterations and prognosis. Characterization of this heterogeneity and how it affects treatment response remains understudied in pediatric patients. We used single-cell RNA sequencing and single-cell ATAC sequencing to profile 28 patients representing different pAML subtypes at diagnosis, remission and relapse. At diagnosis, cellular composition differed between genetic subgroups. Upon relapse, cellular hierarchies transitioned toward a more primitive state regardless of subtype. Primitive cells in the relapsed tumor were distinct compared to cells at diagnosis, with under-representation of myeloid transcriptional programs and over-representation of other lineage programs. In some patients, this was accompanied by the appearance of a B-lymphoid-like hierarchy. Our data thus reveal the emergence of apparent subtype-specific plasticity upon treatment and inform on potentially targetable processes.

Deciphering the Significance of CD56 Expression in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group.

BACKGROUND: In patients with acute myeloid leukemia (AML), CD56 expression has been associated with adverse clinical outcome. We reported on a phenotype associated with very poor prognosis (RAM) in children enrolled in the Children's Oncology Group trial AAML0531 (Brodersen et al. Leukemia 30 (2016) 2077-2080). RAM is also characterized in part by high-intensity expression of the CD56 antigen. Herein, we investigate underlying biological and clinical differences among CD56-positive AMLs for patients in AAML0531. METHODS: For 769 newly diagnosed pediatric patients with de novo AML enrolled in AAML0531, bone marrow specimens were submitted for flow cytometric analysis. For each patient, an immunophenotypic expression profile (IEP) was defined by mean fluorescent intensities of assayed surface antigens. Unsupervised hierarchical clustering analysis (HCA) was completed to group patients with similar immunophenotypes. Clusters were then evaluated for CD56 expression. Principal component analysis (PCA) was subsequently applied to determine whether CD56-positive patient groups were nonoverlapping. RESULTS: HCA of IEPs revealed three unique phenotypic clusters of patients with CD56-positive AML, and PCA showed that these three cohorts are distinct. Cohort 1 (N = 77) showed a prevalence of t(8;21) patients (72%), Cohort 2 (N = 52) a prevalence of 11q23 patients (69%), and Cohort 3 (RAM) (N = 16) a prevalence of patients with co-occurrence of the CBFA2T3-GLIS2 fusion transcript (63%). The 5-year event-free survival (EFS) for Cohorts 1, 2, and 3 were 69, 39, and 19%, respectively. CONCLUSIONS: When leukemia is considered by its multidimensional immunophenotype and not by the expression of a single antigen, correlations are seen between genotype and there are significant differences in patient outcomes. © 2019 International Clinical Cytometry Society.

BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response.

The endoplasmic reticulum (ER) is classically linked to metabolic homeostasis via the activation of unfolded protein response (UPR), which is instructed by multiple transcriptional regulatory cascades. BRCA1 associated protein 1 (BAP1) is a tumor suppressor with de-ubiquitinating enzyme activity and has been implicated in chromatin regulation of gene expression. Here we show that BAP1 inhibits cell death induced by unresolved metabolic stress. This prosurvival role of BAP1 depends on its de-ubiquitinating activity and correlates with its ability to dampen the metabolic stress-induced UPR transcriptional network. BAP1 inhibits glucose deprivation-induced reactive oxygen species and ATP depletion, two cellular events contributing to the ER stress-induced cell death. In line with this, Bap1 KO mice are more sensitive to tunicamycin-induced renal damage. Mechanically, we show that BAP1 represses metabolic stress-induced UPR and cell death through activating transcription factor 3 (ATF3) and C/EBP homologous protein (CHOP), and reveal that BAP1 binds to ATF3 and CHOP promoters and inhibits their transcription. Taken together, our results establish a previously unappreciated role of BAP1 in modulating the cellular adaptability to metabolic stress and uncover a pivotal function of BAP1 in the regulation of the ER stress gene-regulatory network. Our study may also provide new conceptual framework for further understanding BAP1 function in cancer.

BAF180 regulates cellular senescence and hematopoietic stem cell homeostasis through p21.

BAF180 (also called PBRM1), a subunit of the SWI/SNF complex, plays critical roles in the regulation of chromatin remodeling and gene transcription, and is frequently mutated in several human cancers. However, the role of mammalian BAF180 in tumor suppression and tissue maintenance in vivo remains largely unknown. Here, using a conditional somatic knockout approach, we explored the cellular and organismal functions of BAF180 in mouse. BAF180 deletion in primary mouse embryonic fibroblasts (MEFs) triggers profound cell cycle arrest, premature cellular senescence, without affecting DNA damage response or chromosomal integrity. While somatic deletion of BAF180 in adult mice does not provoke tumor development, BAF180 deficient mice exhibit defects in hematopoietic system characterized by progressive reduction of hematopoietic stem cells (HSCs), defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. BAF180 deletion results in elevated p21 expression in both MEFs and HSCs. Mechanistically, we showed that BAF180 binds to p21 promoter, and BAF180 deletion enhances the binding of modified histones associated with transcriptional activation on p21 promoter. Deletion of p21 rescues cell cycle arrest and premature senescence in BAF180 deficient MEFs, and partially rescues hematopoietic defects in BAF180 deficient mice. Together, our study identifies BAF180 as a critical regulator of cellular senescence and HSC homeostasis, which is at least partially regulated through BAF180-mediated suppression of p21 expression. Our results also suggest that senescence triggered by BAF180 inactivation may serve as a failsafe mechanism to restrain BAF180 deficiency-associated tumor development, providing a conceptual framework to further understand BAF180 function in tumor biology.

COUP-TFII inhibits TGF-ß-induced growth barrier to promote prostate tumorigenesis.

Mutations in phosphatase and tensin homologue (PTEN) or genomic alterations in the phosphatidylinositol-3-OH kinase-signalling pathway are the most common genetic alterations reported in human prostate cancer. However, the precise mechanism underlying how indolent tumours with PTEN alterations acquire metastatic potential remains poorly understood. Recent studies suggest that upregulation of transforming growth factor (TGF)-ß signalling triggered by PTEN loss will form a growth barrier as a defence mechanism to constrain prostate cancer progression, underscoring that TGF-ß signalling might represent a pre-invasive checkpoint to prevent PTEN-mediated prostate tumorigenesis. Here we show that COUP transcription factor II (COUP-TFII, also known as NR2F2), a member of the nuclear receptor superfamily, serves as a key regulator to inhibit SMAD4-dependent transcription, and consequently overrides the TGF-ß-dependent checkpoint for PTEN-null indolent tumours. Overexpression of COUP-TFII in the mouse prostate epithelium cooperates with PTEN deletion to augment malignant progression and produce an aggressive metastasis-prone tumour. The functional counteraction between COUP-TFII and SMAD4 is reinforced by genetically engineered mouse models in which conditional loss of SMAD4 diminishes the inhibitory effects elicited by COUP-TFII ablation. The biological significance of COUP-TFII in prostate carcinogenesis is substantiated by patient sample analysis, in which COUP-TFII expression or activity is tightly correlated with tumour recurrence and disease progression, whereas it is inversely associated with TGF-ß signalling. These findings reveal that the destruction of the TGF-ß-dependent barrier by COUP-TFII is crucial for the progression of PTEN-mutant prostate cancer into a life-threatening disease, and supports COUP-TFII as a potential drug target for the intervention of metastatic human prostate cancer.

Protein phosphatase 4 cooperates with Smads to promote BMP signaling in dorsoventral patterning of zebrafish embryos.

BMP signals play pivotal roles in dorsoventral patterning of vertebrate embryos. The role of Ppp4c, the catalytic subunit of ubiquitous protein phosphatase 4, in vertebrate embryonic development and underlying mechanisms is poorly understood. Here, we demonstrate that knockdown of zebrafish ppp4cb and/or ppp4ca inhibits ventral development in embryos and also blocks ventralizing activity of ectopic Smad5. Biochemical analyses reveal that Ppp4c is a direct binding partner and transcriptional coactivator of Smad1/Smad5. In response to BMP, Ppp4c is recruited to the Smad1-occupied promoter, and its phosphatase activity is essential in inhibiting HDAC3 activity and, consequently, potentiating transcriptional activation. Consistently, genetic or chemical interference of Hdac3 expression or activity compromises the dorsalizing phenotype induced by ppp4cb knockdown. We conclude that Ppp4c is a critical positive regulator of BMP/Smad signaling during embryonic dorsoventral pattern formation in zebrafish.

PPM1A dephosphorylates RanBP3 to enable efficient nuclear export of Smad2 and Smad3.

Smad2 and Smad3 (Smad2/3) are essential signal transducers and transcription factors in the canonical transforming growth factor-ß (TGF-ß) signalling pathway. Active Smad2/3 signalling in the nucleus is terminated by dephosphorylation and subsequent nuclear export of Smad2/3. Here we report that protein phosphatase PPM1A regulates the nuclear export of Smad2/3 through targeting nuclear exporter RanBP3. PPM1A directly interacted with and dephosphorylated RanBP3 at Ser 58 in vitro and in vivo. Consistently, RanBP3 phosphorylation was elevated in PPM1A-null mouse embryonic fibroblasts. Dephosphorylation of RanBP3 at Ser 58 promoted its ability to export Smad2/3 and terminate TGF-ß responses. Our findings indicate the critical role of PPM1A in maximizing exporter activity of RanBP3 for efficient termination of canonical TGF-ß signalling.

Coupling of dephosphorylation and nuclear export of Smads in TGF-beta signaling.

In eukaryotes, regulation of signaling mediators/effectors in the nucleus is one of the principal mechanisms that govern duration and strength of signaling. Smads are a family of structurally related intracellular proteins that serve as signaling effectors for transforming growth factor beta (TGF-beta) and TGF-beta-related proteins. Accumulating evidence demonstrates that Smads possess intrinsic nucleocytoplasmic shuttling capacity, which enables them to transmit TGF-beta signals from cell membrane to nucleus. We recently identified two important steps in the termination of nuclear Smad signaling. The first step is initiated by a serine/threonine phosphatase PPM1A that dephosphorylates Smad2/3 in the nucleus, thereby shutting down signaling capacity of phosphorylated Smad2/3. The second step involves nuclear export of dephosphorylated Smad2/3 with the aid of nuclear protein RanBP3 to terminate Smad signaling. This chapter introduces methods for examining nuclear export of Smad2/3 in TGF-beta signaling.

Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signaling.

Smad2 and Smad3 (Smad2/3) are key intracellular signal transducers for TGF-beta signaling, and their transcriptional activities are controlled through reversible phosphorylation and nucleocytoplasmic shuttling. However, the precise mechanism underlying nuclear export of Smad2/3 remains elusive. Here we report the essential function of RanBP3 in selective nuclear export of Smad2/3 in the TGF-beta pathway. RanBP3 directly recognizes dephosphorylated Smad2/3, which results from the activity of nuclear Smad phosphatases, and mediates nuclear export of Smad2/3 in a Ran-dependent manner. As a result, increased expression of RanBP3 inhibits TGF-beta signaling in mammalian cells and Xenopus embryos. Conversely, depletion of RanBP3 expression or dominant-negative inhibition of RanBP3 enhances TGFbeta-induced antiproliferative and transcriptional responses. In conclusion, our study supports a definitive role for RanBP3 in mediating Smad2/3 nuclear export and terminating TGF-beta signaling.

A new kid on the TGFbeta block: TAZ controls Smad nucleocytoplasmic shuttling.

A recent study from Varelas et al. in Nature Cell Biology reveals a role for the transcriptional regulator TAZ in TGFbeta signaling. Not only does TAZ couple phospho-Smads to the transcriptional machinery, it is also essential for their nuclear accumulation.

BCL6 represses Smad signaling in transforming growth factor-beta resistance.

Transforming growth factor-beta (TGF-beta) controls a wide spectrum of cellular processes. Deregulation of TGF-beta signaling contributes to the pathogenesis of many diseases including cancer and autoimmune diseases. TGF-beta signaling is generally mediated through intracellular signal transducers and transcription factors called Smads. Herein, we have identified the oncoprotein BCL6 as a transcriptional corepressor of tumor suppressor Smad4. BCL6 physically interacts with Smad3 and Smad4, disrupts the Smad-p300 interaction, and represses the transcriptional activity of Smad4. In accordance, B-cell lymphoma cells with a high expression level of BCL6 were found to be refractory to TGF-beta antiproliferative response, whereas knockdown of BCL6 expression in B-cell lymphoma cells partially restores the TGF-beta responses. This study provides strong evidence that overexpression of BCL6 contributes to TGF-beta resistance in B-cell lymphoma.

Erbin inhibits transforming growth factor beta signaling through a novel Smad-interacting domain.

Smad proteins are critical intracellular signaling mediators for the transforming growth factor beta (TGFbeta) superfamily. Here, we report that Erbin (for "ErbB2/Her2-interacting protein"), which contains leucine-rich repeats and a PDZ (PSD-95/DLG/ZO-1) domain, interacts specifically with Smad3 and, to a lesser extent, with Smad2 through a novel Smad-interacting domain (SID) adjacent to its PDZ domain. Increased expression of Erbin does not affect the level of TGFbeta-induced phosphorylation of Smad2/Smad3, but it physically sequesters Smad2/Smad3 from their association with Smad4 and hence negatively modulates TGFbeta-dependent transcriptional responses and cell growth inhibition. An isoform of Erbin encoded by an alternatively spliced transcript in human tissues lacks this SID and fails to inhibit TGFbeta responses. Consistently, knockdown of the endogenous Erbin gene with short hairpin RNA enhances TGFbeta-induced antiproliferative and transcriptional responses. In addition, Erbin suppresses activin/Smad2-dependent, but not BMP/Smad1-mediated, induction of endogenous gene expression in Xenopus embryos. Therefore, these results define Erbin as a novel negative modulator of Smad2/Smad3 functions and expand the physiological role of Erbin to the regulation of TGFbeta signaling.

Estrogen-receptor-alpha exchange and chromatin dynamics are ligand- and domain-dependent.

We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor alpha (ERalpha) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERalpha- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in k(off). ERalpha-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERalpha DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.

Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B.

The molecular machinery required for autophagy is highly conserved in all eukaryotes as seen by the high degree of conservation of proteins involved in the formation of the autophagosome membranes. Recently, both yeast Apg8p and its rat homologue Map1lc3 were identified as essential constituents of autophagosome membrane as a processed form. In addition, both the yeast and human proteins exist in two modified forms produced by a series of post-translational modifications including a critical C-terminal cleavage after a conserved Gly residue, and the smaller processed form is associated with the autophagosome membranes. Herein, we report the identification and characterization of three human orthologs of the rat Map1LC3, named MAP1LC3A, MAP1LC3B, and MAP1LC3C. We show that the three isoforms of human MAP1LC3 exhibit distinct expression patterns in different human tissues. Importantly, we found that the three isoforms of MAP1LC3 differ in their post-translation modifications. Although MAP1LC3A and MAP1LC3C are produced by the proteolytic cleavage after the conserved C-terminal Gly residue, like their rat counterpart, MAP1LC3B does not undergo C-terminal cleavage and exists in a single modified form. The essential site for the distinct post-translation modification of MAP1LC3B is Lys-122 rather than the conserved Gly-120. Subcellular localization by cell fractionation and immunofluorescence revealed that three human isoforms are associated with membranes involved in the autophagic pathway. These results revealed different regulation of the three human isoforms of MAP1LC3 and implicate that the three isoforms may have different physiological functions.

Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues.

Digestion and detoxification are the two most important functions of the liver, and liver cells always keep a high metabolism level and active vesicular traffic. The malfunction of the vesicular traffic system might be a cause of the abnormal biological behavior of cancerous liver cells. The Ras superfamily is known to regulate various steps of vesicular traffic in eukaryotic cells. It would be significant to determine the change of vesicular transport molecules such as the members of Ras superfamily in carcinogenesis of liver cells. In the present study, we have cloned nine novel genes encoding human small GTPases: RAB1B, RAB4B, RAB10, RAB22A, RAB24, RAB25 ARL5, SARA1, and SARA2, among which the former six belong to the RAB family and the latter three belong to the ARF/SAR1 family. The identification of these new genes has greatly enlarged the pool of the Ras superfamily. It is interesting to find that they are upregulated in most of the 11 hepatocellular carcinoma and 1 cholangiohepatoma cases. Furthermore, the expression in 16 normal human adult tissues, the chromosome loci, and the gene structures of the nine genes are also described. The above findings could be valuable for understanding the vesicular transport system and elucidating the molecular basis of liver cancer carcinogenesis.

Molecular cloning, genomic organization, and mapping of beta 4GalT-VIb, a brain abundant member of beta 4-galactosyltransferase gene family, to human chromosome 18q12.1.

In the present study, a brain abundant member of beta 4-galactosyltransferase gene family with an open reading frame encoding 343 amino acids was cloned and identified from a human leukemia cell cDNA library. The putative protein sequence is about 94.8 and 94.2% identical to the 382-amino-acid mouse and rat beta 4-galactosyltransferase respectively and also contains cysteine residues previously shown to be important for the function of the gene family members. This cDNA (tentatively termed beta 4GalT-VIb) is identical to a recently reported cDNA (tentatively termed beta 4GalT-VIa) of human beta 4-galactosyltransferase except lacking one exon, suggesting that these two cDNAs are two different alternative transcripts of the same gene. Northern hybridization showed that the new alternative transcript, beta 4GalT-VIb, is expressed in all 16 human tissues tested with highest level in brain and rich level in testis, thymus and pancreas, whereas weak expression was detected in lung. The beta 4GalT-VIb gene was located to human chromosome 18q12.1 between markers WI-9180 and SGC35630 by radiation hybrid mapping. The genomic organization and adjacent gene content of beta 4GalT-VIb were identified by comparing its cDNA sequence with three genomic sequences AC017100, AP002474 and AP001336, which showed that beta 4GalT-VIb spans an approximately 58 kb region and is composed of 8 exons. In addition, the most conserved motif composed of 41 residues, LXYX3FGGVSXL(T/S)X2 QFX2INGFPNX(Y/F)WGWGGEDDDX2NR, was defined according to 17 sequences of beta 4GalTs from seven different organisms for the first time.

Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction.

Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.