Anna Baruzzi

Expression of spliced oncogenic Ikaros isoforms in Philadelphia-positive acute lymphoblastic leukemia patients treated with tyrosine kinase inhibitors: implications for a new mechanism of resistance

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph+ acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph+ ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph+ ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph+ ALL patients.

The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck−/−fgr−/− neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck−/−fgr−/− mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-μm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-μm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck−/−fgr−/− neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca2+ concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck−/−fgr−/− neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck−/−fgr−/− neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.

NLRP3 Inflammasome Activation in Dialyzed Chronic Kidney Disease Patients

To assess whether NLR pyrin domain-containing protein 3 (NLRP3) inflammasome, a multiprotein complex that mediates the activation of caspase-1 (CASP-1) and pro-inflammatory cytokines IL-18 and IL-1β, could be involved in the chronic inflammatory state observed in chronic kidney disease patients undergoing hemodialysis treatment (CKD-HD), we employed several biomolecular techniques including RT-PCR, western blot, FACS analysis, confocal microscopy and microarray. Interestingly, peripheral blood mononuclear cells from 15 CKD-HD patients showed higher mRNA levels of NLRP3, CASP-1, ASC, IL-1β, IL-18 and P2X7receptor compared to 15 healthy subjects. Western blotting analysis confirmed the above results. In particular, active forms of CASP-1, IL1-β and IL-18 resulted significantly up-regulated in CKD-HD versus controls. Additionally, elevated mitochondrial ROS level, colocalization of NLRP3/ASC/mitochondria in peripheral blood mononuclear cells from CKD-HD patients and down-regulation of CASP-1, IL1-β and IL-18 protein levels in immune-cells of CKD-HD patients stimulated with LPS/ATP in presence of mitoTEMPO, inhibitor of mitochondrial ROS production, suggested a possible role of this organelle in the aforementioned CKD-associated inflammasome activation. Then, microarray analysis confirmed, in an independent microarray study cohort, that NLRP3 and CASP-1, along with other inflammasome-related genes, were up-regulated in 17 CKD-HD patients and they were able to clearly discriminate these patients from 5 healthy subjects. All together these data showed, for the first time, that NLRP3 inflammasome was activated in uremic patients undergoing dialysis treatment and they suggested that this unphysiological condition could be possibly induced by mitochondrial dysfunction.

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method

Ikaros is a member of the Kruppel family of zinc finger DNA-binding proteins. The findings of this study demonstrated that both aberrant splicing and genomic deletion leading to different non-DNA-binding transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia. Background Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. Design and Methods In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. Results We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. Conclusions Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Mesenchymal/stromal gene expression signature relates to basal-like breast cancers, identifies bone metastasis and predicts resistance to therapies.

Background Mounting clinical and experimental evidence suggests that the shift of carcinomas towards a mesenchymal phenotype is a common paradigm for both resistance to therapy and tumor recurrence. However, the mesenchymalization of carcinomas has not yet entered clinical practice as a crucial diagnostic paradigm. Methodology/Principal Findings By integrating in silico and in vitro studies with our epithelial and mesenchymal tumor models, we compare herein crucial molecular pathways of previously described carcinoma-derived mesenchymal tumor cells (A17) with that of both carcinomas and other mesenchymal phenotypes, such as mesenchymal stem cells (MSCs), breast stroma, and various types of sarcomas. We identified three mesenchymal/stromal-signatures which A17 cells shares with MSCs and breast stroma. By using a recently developed computational approach with publicly available microarray data, we show that these signatures: 1) significantly relates to basal-like breast cancer subtypes; 2) significantly relates to bone metastasis; 3) are up-regulated after hormonal treatment; 4) predict resistance to neoadjuvant therapies. Conclusions/Significance Our results demonstrate that mesenchymalization is an intrinsic property of the most aggressive tumors and it relates to therapy resistance as well as bone metastasis.

Different isoforms of the B-cell mutator activation-induced cytidine deaminase are aberrantly expressed in BCR-ABL1-positive acute lymphoblastic leukemia patients

The main reason for the unfavorable clinical outcome of BCR–ABL1-positive acute lymphoblastic leukemia (ALL) is genetic instability. However, how normal B-cell precursors acquire the genetic changes that lead to transformation has not yet been completely defined. We investigated the expression of the activation-induced cytidine deaminase (AID) and its role in clinical outcome in 61 adult BCR–ABL1-positive ALL patients. AID expression was detected in 36 patients (59%); it correlated with the BCR–ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different AID splice variants were identified: full-length isoform; AIDΔE4a, with a 30-bp deletion of exon 4; AIDΔE4, with the exon 4 deletion; AIDins3, with the retention of intron 3; AIDΔE3-E4 isoform without deaminase activity. AID-FL predominantly showed cytoplasmic localization, as did the AID-ΔE4a and AID-ΔE3E4 variants, whereas the C-terminal-truncated AID-ΔE4 showed a slightly increased nuclear localization pattern. AID expression correlated with a higher number of copy number alterations identified in genome-wide analysis using a single-nucleotide polymorphism array. However, the expression of AID at diagnosis was not associated with a worse prognosis. In conclusion, BCR–ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutators outside the immunoglobulin (Ig) gene loci in promoting genetic instability.

c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration.

MINT‐7296608: Integrin beta‐1 (uniprotkb:P09055) physically interacts (MI:0914) with Hck (uniprotkb:P08103), Abl (uniprotkb:P00520) and Fgr (uniprotkb:P14234) by anti bait coimmunoprecipitation (MI:0006) MINT‐7296596: Integrin beta‐1 (uniprotkb:P09055) physically interacts (MI:0914) with Fgr (uniprotkb:P14234) and Abl (uniprotkb:P00520) by anti bait coimmunoprecipitation (MI:0006)

Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype

Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p<0.01). Protein assay confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.

The tyrosine kinase Abl is a component of macrophage podosomes and is required for podosome formation and function

Myeloid leukocytes form actin‐based plasma membrane protrusions, called podosomes, that are implicated in myeloid cell recruitment into tissues and cell migration within the interstitium. In this study, we show that tyrosine kinases of the Abl family are present in podosomes formed by murine and human macrophages. Silencing of Abl expression in bone marrow‐derived macrophages and monocyte‐derived macrophages by siRNA or Abl enzymatic inhibition with imatinib resulted in the disassembly of macrophage podosomes and the reduction of their capacity to degrade an extracellular matrix and migrate through matrigel matrices and endothelial cell monolayers. Additionally, macrophages deficient in Src‐family kinases, which cross‐talk with Abl in regulating macrophage migration, also demonstrated podosome disassembly. These findings suggest that podosome disassembly induced by Abl targeting may inhibit podosome‐dependent functions such as leukocyte recruitment into inflammatory sites and osteoclast‐dependent bone resorption.

Sos1 Regulates Macrophage Podosome Assembly and Macrophage Invasive Capacity

Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.