Aberrant Cytoplasmic Localization Research Articles

Could Targeting NPM1c+ Misfolding Be a Promising Strategy for Combating Acute Myeloid Leukemia?

Acute myeloid leukemia (AML) is a heterogeneous group of diseases classified into various types on the basis of distinct features concerning the morphology, cytochemistry and cytogenesis of leukemic cells. Among the different subtypes, the group "AML with gene mutations" includes the variations of the gene of the multifunctional protein nucleophosmin 1 (NPM1). These mutations are the most frequent (~30-35% of AML adult patients and less in pediatric ones) and occur predominantly in the C-terminal domain (CTD) of NPM1. The most important mutation is the insertion at W288, which determines the frame shift W288Cfs12/Ffs12/Lfs*12 and leads to the addition of 2-12 amino acids, which hamper the correct folding of NPM1. This mutation leads to the loss of the nuclear localization signal (NoLS) and to aberrant cytoplasmic localization, denoted as NPM1c+. Many investigations demonstrated that interfering with the cellular location and oligomerization status of NPM1 can influence its biological functions, including the proper buildup of the nucleolus, and therapeutic strategies have been proposed to target NPM1c+, particularly the use of drugs able to re-direct NPM1 localization. Our studies unveiled a direct link between AML mutations and the neat amyloidogenic character of the CTDs of NPM1c+. Herein, with the aim of exploiting these conformational features, novel therapeutic strategies are proposed that rely on the induction of the selective self-cytotoxicity of leukemic blasts by focusing on agents such as peptides, peptoids or small molecules able to enhance amyloid aggregation and targeting selectively AML-NPM1c+ mutations.

Nucleophosmin (NPM1) Type D Genotype Is Associated with Distinct Outcome in Acute Myeloid Leukemia - an AIEOP-BFM and COG-SWOG Intergroup Collaboration

Background: Nucleophosmin (NPM1) is a nucleo-cytoplasmic shuttling protein, predominantly located in the nucleolus that regulates a multiplicity of different biological processes. NPM1 localization in the cell is finely tuned by the nuclear export signal domain, where two tryptophan residues (Trp) are essential for the nucleolar localization. In acute myeloid leukemia (AML), several genomic subsets of NPM1 frameshift mutations of 4 base pair insertions have been identified, with the type A variant (insertion of TCTG) being the most common, followed by type B (CATG) and D (CCTG). More recently, a series of different and rarer frameshifts have been found in adult and pediatric AML. We collected and studied the NPM1 insertion genotypes in a large cohort of children with de novo AML enrolled in the AIEOP, BFM, ELAM02, NOPHO, DCOG, COG trials. Methods: NPM1 mutations were sequenced in a group of 345 pediatric patients with de novo AML. 166 patients were enrolled in recent AIEOP, BFM, ELAM02, NOPHO, DCOG trials, and 179 in the CCG/COG AML trials (CCG-2961, AAML03P1, AAML0531 and AAML1031). A cohort of 75 patients of SWOG young adult (age 18-60) AML trial (S0106) were also included as validation cohort. The Kaplan-Meier method was used to estimate the probabilities of overall survival (OS) and event-free survival (EFS). Results: In this cohort of 345 pediatric patients sequencing of exon 12 demonstrated multiple 4 bp insertion subtypes, with type A variant detected in 188 patients (55%), type B in 64 cases (19%) and type D mutation in 26 patients (7.5%). Other variants with the loss of the 2 Trp residues (namely A-like) were found in 42 patients (12%), whereas in 25 patients (7.5%) we identified mutations with the loss of 1 Trp residue (namely non-A-like). Comparison of disease characteristics demonstrated that NPM1 variants were prevalent in older patients (median age=13.3 years). WBC count was not significantly different among subgroups (Type A=30.3; Alike=25.2; B=16.8; D=41; non-A-like= 14.8x10 3/ul, p=0.14). FLT3-ITD prevalence was higher in Type A, B and D compared to the other Type A-like variants (42%, 44%, 46% vs. 26%); interestingly, FLT3-ITD was absent in non-A-like variants. Evaluation of clinical outcome based on NPM1 genotypes showed that although patients with type A, A-Like, non-A-like and type B had favorable outcomes, those with Type D had a significantly worse outcome with OS of 63% versus 86% for other genotypes (p=0.0053). Type D variant adverse outcome was confirmed in most of the trials and will be further examined considering different treatments. Each NPM1 variant mediated a different EFS, with most of early events occurring within the D group (EFS 51.4%), and type A (EFS 68.8%) compared to the non-A-like NPM1, A like and B mutations (79.8%, 73%, and 75.5%, respectively) (p=ns). Validation data from adult SWOG trial (SWOG0106) demonstrated similar adverse outcome with EFS of 40% for type D vs. 61.4% for other NPM1 variants, indicating D as a peculiar NPM1 genotype /Figure A). Evaluation of overlapping mutations demonstrated that except for lack of FLT3-ITD in non-A-like genotype, there was similar distribution of high-risk mutations (FLT3-ITD, WT1, etc.) in those with different genotypes, thus not explaining the underlying biology responsible for the Type D adverse outcome. Even if the aminoacidic sequence of D and A variants is the same, the different prognosis suggests a further layer of regulation. We inquired whether Type D genotype might have a different NPM1 localization pattern compared to that in Type A variant. Immunofluorescence staining of primary AML cells from patients with Type D vs. Type A variant demonstrated that, in contrast to the canonical Type A variant with aberrant cytoplasmic localization, Type D variant maintains nuclear localization (Figure B). Further studies will provide the underlying biology of Type D being a silent mutation mediating a clinical outcome like NPM1 wild type AML. Conclusion: This Eu/COG/SWOG collaborative study of over 400 pediatric and adult patients suggest a different functional biology behind NPM1 genotype where those with Type D variants have a significantly inferior outcome resembling feature of non-NPM1 mutated AML. Our data suggest that the specific genotype of NPM1 mutations can contribute to a more accurate risk-stratification, with D cases eventually shifting from standard to higher risk groups.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

HHEX suppresses advanced thyroid cancer by interacting with TLE3

Haematopoietically Expressed Homeobox (HHEX) gene is highly expressed in the thyroid gland and plays critical roles in the development and differentiation of the thyroid gland. While it has been indicated to be downregulated in thyroid cancer, its function and the underlying mechanism remain unclear. Herein, we observed low expression and aberrant cytoplasmic localization of HHEX in thyroid cancer cell lines. Knockdown of HHEX significantly enhanced cell proliferation, migration and invasion, while overexpression of HHEX showed the opposite effects in vitro and in vivo. These data provide evidence that HHEX is a tumor suppressor in thyroid cancer. Additionally, our results showed that HHEX overexpression upregulated the expression of sodium iodine symporter (NIS) mRNA and also enhanced NIS promoter activity, suggesting a favorable effect of HHEX in promoting thyroid cancer differentiation. Mechanistically, HHEX exerted a regulatory effect on the expression of transducin-like enhancer of split 3 (TLE3) protein, which inhibited the Wnt/β-catenin signaling pathway. Nuclear localized HHEX bound to and upregulated TLE3 expression by preventing TLE3 protein from being distributed to the cytoplasm and being ubiquitinated. In conclusion, our study suggested that restoring HHEX expression has the potential to be a new strategy in the treatment of advanced thyroid cancer.

Identification of a novel NPM1 mutation in acute myeloid leukemia.

e19019 Background: Nucleophosmin (NPM1) is predominant nucleolar localization. NPM1-mutant acute myeloid leukemia (AML) account for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 WHO classification. The majority of NPM1 mutations reported affect exon 12 (classical type A mutation), account for 75-80% of adult NPM1-mutated AML cases, which generate an extra nuclear export signal (NES) in the C-terminal leading to aberrant cytoplasmic localization. Similar aberrant cytoplasmic translocation has also been reported in NPM1 mutants that identified in exons 9 and 11. Identification of novel non-type A mutants is paramount for diagnosis, risk stratification, and prognosis for targeted population. Methods: Somatic mutation analysis is performed on 566 AML patients using next-generation sequencing (NGS). Immunofluorescence and immunohistochemistry were used to identify the cytological localization of mutant NPM1. HEK-293T cells transiently transfected with plasmids for ectopic expression of the GFP-mutated NPM1 fusion proteins were treated with the specific Crm1/XPO1 inhibitor leptomycin B and to evaluate the NES dependence of their subcellular localization. Results: We identified one AML patient with NPM1 mutant located on exon 5. This patient was a 59-year-old female with de novo AML, with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCCCTGGAACTGGGGAAC, named MutSong) in the middle of exon 5 [variant allele fraction (VAF), 11.9%]. It generated a new NPM1 mutant protein (p.135insALELGN), and containing a leucine-rich NES. Notably, different with all NPM1 mutant in other exons reported so far, our exon 5 mutant retained the functional c-terminal but insert one leucine-rich NES, similar finding was recently reported in NPM1 mutants that identified in exon 5 but on different position. This NPM1 mutant in exon 5 resulted in cytoplasmic localization both by confocal and immunohistochemistry in AML patient sample. HEK-293T overexpressing the new GFP-NPM1 exon 5 fusion protein indicated the aberrant localization in the cytoplasm and partially in nucleoli. Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B, indicated that the novel NES generated by exon 5 mutant is responsible for cytoplasmic localization. This patient carried WT1, IKZF1, JAK2, and NUP98 mutations, and was intravenously treated with daunorubicin plus cytarabine for induction followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, the patient died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT. Conclusions: Our finding of the novel NPM1 mutation in exon 5 supported that beside exon 12, exon 5 mutant is another NPM1 “born to be exported” mutant critical for leukemogenesis.

SHP2 participates in decidualization by activating ERK to maintain normal nuclear localization of progesterone receptor.

The establishment and maintenance of embryo implantation and pregnancy require decidualization of endometrial stromal cells. This paper reveals that SHP2 ensures the correct subcellular localization of progesterone receptor, thereby safeguarding the process of decidualization. Decidualization is the process of conversion of endometrial stromal cells into decidual stromal cells, which is caused by progesterone production that begins during the luteal phase of the menstrual cycle and then increases throughout pregnancy dedicated to support embryonic development. Decidualization deficiency is closely associated with various pregnancy complications, such as recurrent miscarriage (RM). Here, we reported that Src-homology-2-containing phospho-tyrosine phosphatase (SHP2), a key regulator in the signal transduction process downstream of various receptors, plays an indispensable role in decidualization. SHP2 expression was upregulated during decidualization. SHP2 inhibitor RMC-4550 and shRNA-mediated SHP2 reduction resulted in a decreased level of phosphorylation of ERK and aberrant cytoplasmic localization of progesterone receptor (PR), coinciding with reduced expression of IGFBP1 and various other target genes of decidualization. Solely inhibiting ERK activity recapitulated these observations. Administration of RMC-4550 led to decidualization deficiency and embryo absorption in mice. Moreover, reduced expression of SHP2 was detected in the decidua of RM patients. Our results revealed that SHP2 is key to PR's nuclear localization, thereby indispensable for decidualization and that reduced expression of SHP2 might be engaged in the pathogenesis of RM.

Association of Kaiso and partner proteins in oral squamous cell carcinoma

Objectives:1. Identification of protein expression and subcellular localization of E-cadherin (E-cad), p120 catenin (P120ctn), and Kaiso in oral cancer (OC). 2. To study the protein expression of cyclin D1 and c-Myc (Kaiso targets) and determine their relationship with the expression and localization of Kaiso. MethodsHistological grading was performed in accordance with Broder's criteria. Expression and localization data for E-cad, p120ctn, Kaiso, cyclin D1, and c-Myc were acquired using immunohistochemistry. Data were analyzed using SPSS version 21. The chi-square test was used to measure the statistical significance of associations, with p < 0.05 as statistically significant. ResultsOf 47 OC cases, 36% showed low E-cad expression and 34% showed low p120ctn. Low Kaiso expression was recognized in 78% of tumor specimens. Aberrant cytoplasmic localization of p120ctn was seen in 80.8% cases. Cytoplasmic Kaiso localization was appreciated in 87% of tumor tissues, whereas 29.7% lacked any nuclear Kaiso. Kaiso expression was significantly associated with the expression of cyclin D1 but not with c-Myc. ConclusionThe present study identified a change in the localization of Kaiso in OC. The significance of this in relation to OC and tumor prognosis needs to be investigated with further studies using larger sample sizes and more sensitive molecular tools.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

EAPB0503, an Imidazoquinoxaline Derivative Modulates SENP3/ARF Mediated SUMOylation, and Induces NPM1c Degradation in NPM1 Mutant AML.

Nucleophosmin-1 (NPM1) is a pleiotropic protein involved in numerous cellular processes. NPM1 shuttles between the nucleus and the cytoplasm, but exhibits a predominant nucleolar localization, where its fate and functions are exquisitely controlled by dynamic post-translational modifications (PTM). Sentrin/SUMO Specific Peptidase 3 (SENP3) and ARF are two nucleolar proteins involved in NPM1 PTMs. SENP3 antagonizes ARF-mediated NPM1 SUMOylation, to promote ribosomal biogenesis. In Acute Myeloid Leukemia (AML), NPM1 is frequently mutated, and exhibits an aberrant cytoplasmic localization (NPM1c). NPM1c mutations define a separate AML entity with good prognosis in some AML patients, rendering NPM1c as a potential therapeutic target. SENP3-mediated NPM1 de-SUMOylation induces resistance to therapy in NPM1c AML. Here, we demonstrate that the imidazoquinoxaline EAPB0503 prolongs the survival and results in selective reduction in the leukemia burden of NPM1c AML xenograft mice. Indeed, EAPB0503 selectively downregulates HDM2 expression and activates the p53 pathway in NPM1c expressing cells, resulting in apoptosis. Importantly, we unraveled that NPM1c expressing cells exhibit low basal levels of SUMOylation paralleled with high SENP3 and low ARF basal levels. EAPB0503 reverted these molecular players by inducing NPM1c SUMOylation and ubiquitylation, leading to its proteasomal degradation. EAPB0503-induced NPM1c SUMOylation is concurrent with SENP3 downregulation and ARF upregulation in NPM1c expressing cells. Collectively, these results provide a strong rationale for testing therapies modulating NPM1c post-translational modifications in the management of NPM1c AML.

Variable Anterior Segment Dysgenesis and Cardiac Anomalies Caused by a Novel Truncating Variant of FOXC1.

Anterior segment dysgenesis (ASD) encompasses a wide spectrum of developmental abnormalities of the anterior ocular segment, including congenital cataract, iris hypoplasia, aniridia, iridocorneal synechiae, as well as Peters, Axenfeld, and Rieger anomalies. Here, we report a large five-generation Caucasian family exhibiting atypical syndromic ASD segregating with a novel truncating variant of FOXC1. The family history is consistent with highly variable autosomal dominant symptoms including isolated glaucoma, iris hypoplasia, aniridia, cataract, hypothyroidism, and congenital heart anomalies. Whole-exome sequencing revealed a novel variant [c.313_314insA; p.(Tyr105*)] in FOXC1 that disrupts the α-helical region of the DNA-binding forkhead box domain. In vitro studies using a heterologous cell system revealed aberrant cytoplasmic localization of FOXC1 harboring the Tyr105* variant, likely precluding downstream transcription function. Meta-analysis of the literature highlighted the intrafamilial variability related to FOXC1 truncating alleles. This study highlights the clinical variability in ASD and signifies the importance of combining both clinical and molecular analysis approaches to establish a complete diagnosis.

Combinatorial Genetic Uncovers DOCK1/RAC2 As Specific Targets for the Treatment of NPM1;Cohesin Mutated AML

Acute myeloid leukemia (AML) is an aggressive malignancy of the blood and bone marrow resulting from the accumulation of multiple serially acquired mutations. The mutational complexity of the disease makes AML difficult to treat, contributing to a low 5-year survival rate. A better understanding of how different mutations interact with one another to influence disease characteristics is critical and may result in the development of targeted therapies to improve patient outcome. The most common recurrent somatic mutation in AML affects the gene NPM1 and occurs in 25-30% of patients. This mutation results in the aberrant cytoplasmic localization of the protein and is termed NPM1cA (Falini et al. 2005, Cancer Genome Atlas Research Network 2013). NPM1cA is considered to be a driver of AML, however Npm1 cA/+ mice only develop disease after a long latency (median 18 months), suggesting that other cooperating mutations are required for AML development (Vassiliou et al. 2011).Approximately 50% of patients with an NPM1cA mutation also harbor a mutation in one of four members of the cohesin complex (STAG2, SMC3, SMC1A, and RAD21). Mutations in cohesin genes are mutually exclusive and result in haploinsufficiency of the complex. As cohesin is known to regulate gene expression by facilitating promoter-enhancer interactions and three-dimensional genome organization, we wished to determine how cohesin mutation influences AML biology and gene expression in the presence of NPM1cA. We utilized the inducible Npm1 cAflox/+ and Smc3 flox/+ mouse models to examine this genetic interaction. We and others have shown that cohesin mutations result in enhanced hematopoietic stem and progenitor cell (HSPC) self-renewal (Mazumdar et al. 2015, Viny et al. 2015, Mullenders et al. 2015, Galeev et al. 2016, Fisher et al. 2017). Consistent with this, Npm1 cA/+;Smc3 Δ/+ HSPCs show enhanced self-renewal in vitro over HSPCs harboring either single mutation. Despite a shared role in self-renewal, Npm1 cA/+;Smc3 Δ/+mice developed AML with similar latency as Npm1 cA/+ mice. Interestingly, however, Npm1 cA/+;Smc3 Δ/+ HSPCs exhibited dysregulation of a unique set of genes compared to cells from either single mutant, suggesting that the Npm1 cA;cohesin mutational combination uniquely alters the transcriptional environment. Further, Npm1 cA/+;Smc3 Δ/+ leukemias had a completely unique spectrum of acquired mutations compared to Npm1 cA/+ leukemias, suggesting that the addition of Smc3 haploinsufficiency to Npm1 cA/+alters AML evolution and that different driver mutations result in the accumulation of very different somatic mutations.Among the uniquely upregulated genes in Npm1 cA/+;Smc3 Δ/+ HSPCs is Dock1, a guanine nucleotide exchange factor (GEF) for Rac1/2. As high Dock1 expression has been associated with low overall and disease-free survival in multiple cohorts of AML patients (Lee et al. 2017, Zhang et al. 2019), we hypothesized that Dock1 would be a novel target for the treatment of Npm1cA; cohesin mut leukemias. Consistent with this hypothesis, We found that knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis in an Npm1 cA/+;Smc3 Δ/+leukemic line, but not in an Npm1 cAsingle mutant line. Higher Rac activity was also observed in Npm1 cA/+;Smc3 Δ/+ vs. Npm1 cA/+ leukemic lines. We found that DN Rac2 specifically impacted both the growth and apoptosis of an Npm1 cA/+;Smc3 Δ/+line, suggesting that Dock1 functions primarily through Rac2 to regulate survival. Importantly, the Dock1/Rac pathway is specifically targetable in Npm1 cA/+;Smc3 Δ/+ AMLs in vitro and in vivo, as knockdown of Dock1 resulted in prolonged latency in a Npm1 cA/+;Smc3 Δ/+ transplant model and slowed the growth and enhanced apoptosis of an Npm1 cA/+;Smc3 Δ/+, but not an Npm1 cA/+, leukemic line. Further, small molecule inhibitors of Dock and Rac had similar effects. Our results suggest that Dock1/Rac2 represent unique targets for the treatment of patients harboring the NPM1cA;cohesin mutational combination and provide validity to the concept that combinatorial genetics can uncover novel precision oncology targets. DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Expression of N-Terminal-Deficient E-Cadherin Protein in Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) of the breast is characterized by the discohesive growth of tumor cells, which is mainly associated with the complete loss of E-cadherin (E-cad) expression. However, some aberrant expression patterns of E-cad protein that are inconsistent with their morphologies have been reported in ILC. We report herein ILC cases expressing a new type of abnormal E-cad protein that lacks the N-terminal domain, but conserves the C-terminal domain on the cell membrane. Immunohistochemical staining of 299 ILC cases using specific antibodies against the N-terminal or C-terminal region of E-cad revealed that 227 (76%) cases showed loss of the membranous expression of both terminuses (N-/C-) and 72 (24%) cases showed expression of only the C-terminus (N-/C+). In all cases, the expression of p120-catenin and β-catenin coincided with the expression of the C-terminus of E-cad. Clinicopathologic analysis revealed that N-/C+ expression in ILC cells was significantly associated with the histologic subtype (especially mixed-type ILC with another histologic type) and immunohistochemical molecular subtype (especially the triple-negative subtype), but not with prognostic factors (pT or pN). In addition, 12 of 15 cases (80%) with aberrant cytoplasmic localization of the N-terminal of E-cad showed diffuse membranous expression of the C-terminal domain. Additional immunohistochemistry using an antibody recognizing the extracellular juxtamembrane region showed that 28 (39%) of the N-/C+ cases had lost membranous expression, suggesting diversity in the deletion pattern of the N-terminal region. Our findings provide a novel mechanism for the loss of E-cad function because of N-terminal-deficient E-cad protein in ILC.

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response.

Simple SummaryNucleophosmin (NPM) is one of the most abundant nucleolar proteins and its mutations frequently occur in acute myeloid leukemia (AML). The mutations cause aberrant cytoplasmic localization of mutated protein (NPMmut) and often mediate dislocation of NPM interaction partners. Tumor suppressor p53 is known to interact with NPM in response to genotoxic stress and its cytoplasmic localization is an unfavorable prognostic factor in cancers. This study aims to characterize the NPM-p53 interaction and to elucidate the effect of the NPM mutations on p53 localization and expression in live cells. In addition, the cellular dynamics of NPMmut and p53 after treatment with nuclear export inhibitor Selinexor is described and the mechanism of the Selinexor action proposed. Our results contribute to a better understanding of the oncogenic potential of NPM mutations.Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.

Nmp1 and Cohesin Mutations Cooperate to Generate Acute Myeloid Leukemia

Acute myelogenous leukemia (AML) is an aggressive malignancy characterized by the accumulation of myeloid blasts in the bone marrow and blood as a result of multiple serially acquired genetic mutations. The most common recurrent somatic mutation in AML affects the gene NPM1 and occurs in 25-30% of patients. This mutation results in the aberrant cytoplasmic localization of the protein and is termed NPM1c (Falini et al. 2005, Cancer Genome Atlas Research Network 2013). NPM1c is considered to be a driver of AML, however mice carrying the most common variant, NPM1cA, only develop disease after a long latency (median 18 months), suggesting that other cooperating mutations are required for AML development (Vassiliou et al. 2011).Heterozygous mutations in one of four members of the cohesin complex (RAD21, SMC3, STAG1, and SMC1A) are commonly found in patients with AML. Different cohesin gene mutations are mutually exclusive and are frequently found in combination with NPM1 mutations (Cancer Genome Atlas Research Network 2013). We and others have shown that cohesin mutations result in enhanced hematopoietic stem and progenitor cell (HSPC) self-renewal and that aberrant expression of HOXA7 and HOXA9 contribute to this phenotype (Mazumdar et al. 2015, Viny et al. 2015, Mullenders et al. 2015, Galeev et al. 2016, Fisher et al. 2017). To explore the relationship between cohesin haploinsufficiency and Npm1cA in the development of AML, we crossed the inducible Npm1cA-flox/+ and Smc3flox/+ mouse models (Vassiliou et al. 2011 and Viny et al. 2015). Our pilot studies indicate that approximately 75% of Npm1cA/+; Smc3Δ/- mice develop AML, with a shorter latency observed compared to Npm1cA/+ only mice (8 months vs. 18 months). Surprisingly, while single-mutant Npm1cA/+ and Smc3 Δ/- mice both show increased HSPC self-renewal and HoxA gene overexpression, minimal synergy was observed in Npm1cA/+; Smc3 Δ/- mice with respect HoxA7 and HoxA9 expression. In an effort to uncover changes in gene transcription that may contribute to the accelerated development of AML observed in double-mutant mice, we performed RNA sequencing on bone marrow from WT, Npm1cA/+, Smc3 Δ/-, and Npm1cA/+; Smc3 Δ/- mice (three from each genotype). Interestingly, additive changes in gene expression were not seen in the Npm1cA/+; Smc3 Δ/- mice. Instead, upregulation of a set of unique genes was observed. Such genes include factors known to play a role in hematopoiesis or myeloid cell differentiation, including Fli1 and Csf3r. We hypothesize that Smc3 haploinsufficiency alters gene expression in Npm1cA/+ mice, resulting in the upregulation of a unique set of genes that contribute to enhanced AML development. Our work provides important insights into how combined mutations can promote AML development by altering the transcriptome. In addition, it provides a tractable model system to explore the mechanism by which NPM1 mutations cause leukemia. DisclosuresLevine:Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Roche: Research Funding.

Aberrant cytoplasmic localization of ARID1B activates ERK signaling and promotes oncogenesis.

The ARID1B (BAF250b) subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. We employed in silico prediction, intracellular fluorescence and cellular fractionation-based subcellular localization analyses to identify the ARID1B nuclear localization signal (NLS). A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised in its canonical transcription activation and tumor suppressive functions, as expected. Surprisingly however, cytoplasmic localization appeared to induce a gain of oncogenic function for ARID1B, as evidenced from several cell line- and mouse xenograft-based assays. Mechanistically, cytoplasm-localized ARID1B could bind c-RAF (RAF1) and PPP1CA causing stimulation of RAF-ERK signaling and β-catenin (CTNNB1) transcription activity. ARID1B harboring NLS mutations derived from tumor samples also exhibited aberrant cytoplasmic localization and acquired a neo-morphic oncogenic function via activation of RAF-ERK signaling. Furthermore, immunohistochemistry on a tissue microarray revealed significant correlation of ARID1B cytoplasmic localization with increased levels of active forms of ERK1 and ERK2 (also known as MAPK3 and MAPK1) and of β-catenin, as well as with advanced tumor stage and lymph node positivity in human primary pancreatic tumor tissues. ARID1B therefore promotes oncogenesis through cytoplasm-based gain-of-function mechanisms in addition to dysregulation in the nucleus.This article has an associated First Person interview with the first author of the paper.

First person – Srinivas Animireddy

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Srinivas Animireddy is first author on ‘Aberrant cytoplasmic localization of ARID1B activates ERK signaling and promotes oncogenesis’, published in JCS. Srinivas conducted the research described in this article while a PhD student in Dr Murali Dharan Bashyam's lab at the Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India. He is now a postdoc in the lab of Dr Blaine Bartholomew at the MD Anderson Cancer Center, Smithville, TX, investigating the role of chromatin remodelers in development and diseases.

Cleavage of the APE1 N-Terminal Domain in Acute Myeloid Leukemia Cells Is Associated with Proteasomal Activity.

Apurinic/apyrimidinic endonuclease 1 (APE1), the main mammalian AP-endonuclease for the resolution of DNA damages through the base excision repair (BER) pathway, acts as a multifunctional protein in different key cellular processes. The signals to ensure temporo-spatial regulation of APE1 towards a specific function are still a matter of debate. Several studies have suggested that post-translational modifications (PTMs) act as dynamic molecular mechanisms for controlling APE1 functionality. Interestingly, the N-terminal region of APE1 is a disordered portion functioning as an interface for protein binding, as an acceptor site for PTMs and as a target of proteolytic cleavage. We previously demonstrated a cytoplasmic accumulation of truncated APE1 in acute myeloid leukemia (AML) cells in association with a mutated form of nucleophosmin having aberrant cytoplasmic localization (NPM1c+). Here, we mapped the proteolytic sites of APE1 in AML cells at Lys31 and Lys32 and showed that substitution of Lys27, 31, 32 and 35 with alanine impairs proteolysis. We found that the loss of the APE1 N-terminal domain in AML cells is dependent on the proteasome, but not on granzyme A/K as described previously. The present work identified the proteasome as a contributing machinery involved in APE1 cleavage in AML cells, suggesting that acetylation can modulate this process.

Nucleophosmin in leukemia: Consequences of anchor loss.

Nucleophosmin (NPM), one of the most abundant nucleolar proteins, has crucial functions in ribosome biogenesis, cell cycle control, and DNA-damage repair. In human cells, NPM occurs mainly in oligomers. It functions as a chaperone, undergoes numerous interactions and forms part of many protein complexes. Although NPM role in carcinogenesis is not fully elucidated, a variety of tumor suppressor as well as oncogenic activities were described. NPM is overexpressed, fused with other proteins, or mutated in various tumor types. In the acute myeloid leukemia (AML), characteristic mutations in NPM1 gene, leading to modification of NPM C-terminus, are the most frequent genetic aberration. Although multiple mutation types of NPM are found in AML, they are all characterized by aberrant cytoplasmic localization of the mutated protein. In this review, current knowledge of the structure and function of NPM is presented in relation to its interaction network, in particular to the interaction with other nucleolar proteins and with proteins active in apoptosis. Possible molecular mechanisms of NPM mutation-driven leukemogenesis and NPM therapeutic targeting are discussed. Finally, recent findings concerning the immunogenicity of the mutated NPM and specific immunological features of AML patients with NPM mutation are summarized.

Mutations in ILK, encoding integrin-linked kinase, are associated with arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy is a genetic heart muscle disorder characterized by fibro-fatty replacement of cardiomyocytes leading to life-threatening ventricular arrhythmias, heart failure, and sudden cardiac death. Mutations in genes encoding cardiac junctional proteins are known to cause about half of cases, while remaining genetic causes are unknown. Using exome sequencing, we identified 2 missense variants (p.H33N and p.H77Y) that were predicted to be damaging in the integrin-linked kinase (ILK) gene in 2 unrelated families. The p.H33N variant was found to be de novo. ILK links integrins and the actin cytoskeleton, and is essential for the maintenance of normal cardiac function. Both of the new variants are located in the ILK ankyrin repeat domain, which binds to the first LIM domain of the adaptor proteins PINCH1 and PINCH2. In silico binding studies proposed that the human variants disrupt the ILK-PINCH complex. Recombinant mutant ILK expressed in H9c2 rat myoblast cells shows aberrant prominent cytoplasmic localization compared to the wild-type. Expression of human wild-type and mutant ILK under the control of the cardiac-specific cmlc2 promotor in zebrafish shows that p.H77Y and p.P70L, a variant previously reported in a dilated cardiomyopathy family, cause cardiac dysfunction and death by about 2-3 weeks of age. Our findings provide genetic and functional evidence that ILK is a cardiomyopathy disease gene and highlight its relevance for diagnosis and genetic counseling of inherited cardiomyopathies.

AML-associated mutation of nucleophosmin compromises its interaction with nucleolin.

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.