Publications by authors named "Cristina Panuzzo"

31 Publications

The Giant HECT E3 Ubiquitin Ligase HERC1 Is Aberrantly Expressed in Myeloid Related Disorders and It Is a Novel BCR-ABL1 Binding Partner.

Cancers (Basel) 2021 Jan 19;13(2). Epub 2021 Jan 19.

Department of Oncology, Medical School, University of Torino, 10043 Orbassano, Italy.

HERC E3 subfamily members are parts of the E3 ubiquitin ligases and key players for a wide range of cellular functions. Though the involvement of the Ubiquitin Proteasome System in blood disorders has been broadly studied, so far the role of large HERCs in this context remains unexplored. In the present study we examined the expression of the large HECT E3 Ubiquitin Ligase, HERC1, in blood disorders. Our findings revealed that gene expression was severely downregulated both in acute and in chronic myelogenous leukemia at diagnosis, while it is restored after complete remission achievement. Instead, in Philadelphia the negative myeloproliferative neoplasm level was peculiarly controlled, being very low in Primary Myelofibrosis and significantly upregulated in those Essential Thrombocytemia specimens harboring the mutation in the calreticulin gene. Remarkably, in CML cells mRNA level was associated with the BCR-ABL1 kinase activity and the HERC1 protein physically interacted with BCR-ABL1. Furthermore, we found that HERC1 was directly tyrosine phosphorylated by the ABL kinase. Overall and for the first time, we provide original evidence on the potential tumor-suppressing or -promoting properties, depending on the context, of in myeloid related blood disorders.
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http://dx.doi.org/10.3390/cancers13020341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832311PMC
January 2021

Genetic Screening for Potential New Targets in Chronic Myeloid Leukemia Based on Drosophila Transgenic for Human BCR-ABL1.

Cancers (Basel) 2021 Jan 14;13(2). Epub 2021 Jan 14.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9;22)(q34;q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the () sequence and the () gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the transgenic as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.
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http://dx.doi.org/10.3390/cancers13020293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830713PMC
January 2021

Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Int J Mol Sci 2020 Oct 16;21(20). Epub 2020 Oct 16.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

I is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.
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http://dx.doi.org/10.3390/ijms21207674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589297PMC
October 2020

Iron overload alters the energy metabolism in patients with myelodysplastic syndromes: results from the multicenter FISM BIOFER study.

Sci Rep 2020 06 8;10(1):9156. Epub 2020 Jun 8.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myelodysplastic syndromes (MDS) are hematological malignancies characterized by ineffective hematopoiesis and increased apoptosis in the bone marrow, which cause peripheral cytopenia. Mitochondria are key regulators of apoptosis and a site of iron accumulation that favors reactive oxygen species (ROS) production with detrimental effects on cell survival. Although the energy metabolism could represent an attractive therapeutic target, it was poorly investigated in MDS. The purpose of the study was to analyze how the presence of myelodysplastic hematopoiesis, iron overload and chelation impact on mitochondrial metabolism. We compared energy balance, OxPhos activity and efficiency, lactic dehydrogenase activity and lipid peroxidation in mononuclear cells (MNCs), isolated from 38 MDS patients and 79 healthy controls. Our data show that ATP/AMP ratio is reduced during aging and even more in MDS due to a decreased OxPhos activity associated with an increment of lipid peroxidation. Moreover, the lactate fermentation enhancement was observed in MDS and elderly subjects, probably as an attempt to restore the energy balance. The biochemical alterations of MNCs from MDS patients have been partially restored by the in vitro iron chelation, while only slight effects were observed in the age-matched control samples. By contrast, the addition of iron chelators on MNCs from young healthy subjects determined a decrement in the OxPhos efficiency and an increment of lactate fermentation and lipid peroxidation. In summary, MDS-MNCs display an altered energy metabolism associated with increased oxidative stress, due to iron accumulation. This condition could be partially restored by iron chelation.
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http://dx.doi.org/10.1038/s41598-020-66162-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280296PMC
June 2020

Mitochondria: A Galaxy in the Hematopoietic and Leukemic Stem Cell Universe.

Int J Mol Sci 2020 May 30;21(11). Epub 2020 May 30.

Department of Clinical and Biological Sciences, University of Turin, Orbassano, 10043, Italy.

Mitochondria are the main fascinating energetic source into the cells. Their number, shape, and dynamism are controlled by the cell's type and current behavior. The perturbation of the mitochondrial inward system via stress response and/or oncogenic insults could activate several trafficking molecular mechanisms with the intention to solve the problem. In this review, we aimed to clarify the crucial pathways in the mitochondrial system, dissecting the different metabolic defects, with a special emphasis on hematological malignancies. We investigated the pivotal role of mitochondria in the maintenance of hematopoietic stem cells (HSCs) and their main alterations that could induce malignant transformation, culminating in the generation of leukemic stem cells (LSCs). In addition, we presented an overview of LSCs mitochondrial dysregulated mechanisms in terms of (1) increasing in oxidative phosphorylation program (OXPHOS), as a crucial process for survival and self-renewal of LSCs,(2) low levels of reactive oxygen species (ROS), and (3) aberrant expression of B-cell lymphoma 2 (Bcl-2) with sustained mitophagy. Furthermore, these peculiarities may represent attractive new "hot spots" for mitochondrial-targeted therapy. Finally, we remark the potential of the LCS metabolic effectors to be exploited as novel therapeutic targets.
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http://dx.doi.org/10.3390/ijms21113928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312164PMC
May 2020

Landscape of Tumor Suppressor Mutations in Acute Myeloid Leukemia.

J Clin Med 2020 Mar 16;9(3). Epub 2020 Mar 16.

Department of Clinical and Biological Sciences, University of Turin, 10124 Turin, Italy.

Acute myeloid leukemia is mainly characterized by a complex and dynamic genomic instability. Next-generation sequencing has significantly improved the ability of diagnostic research to molecularly characterize and stratify patients. This detailed outcome allowed the discovery of new therapeutic targets and predictive biomarkers, which led to develop novel compounds (e.g., IDH 1 and 2 inhibitors), nowadays commonly used for the treatment of adult relapsed or refractory AML. In this review we summarize the most relevant mutations affecting tumor suppressor genes that contribute to the onset and progression of AML pathology. Epigenetic modifications (TET2, IDH1 and IDH2, DNMT3A, ASXL1, WT1, EZH2), DNA repair dysregulation (TP53, NPM1), cell cycle inhibition and deficiency in differentiation (NPM1, CEBPA, TP53 and GATA2) as a consequence of somatic mutations come out as key elements in acute myeloid leukemia and may contribute to relapse and resistance to therapies. Moreover, spliceosomal machinery mutations identified in the last years, even if in a small cohort of acute myeloid leukemia patients, suggested a new opportunity to exploit therapeutically. Targeting these cellular markers will be the main challenge in the near future in an attempt to eradicate leukemia stem cells.
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http://dx.doi.org/10.3390/jcm9030802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141302PMC
March 2020

Two conserved glycine residues in mammalian and Rictor are required for mTORC2 activity and integrity.

J Cell Sci 2019 11 14;132(22). Epub 2019 Nov 14.

Department of Oncology, University of Torino, AOU S. Luigi, 10043 Orbassano (TO), Italy

Mammalian, or mechanistic, target of rapamycin complex 2 (mTORC2) regulates a variety of vital cellular processes, and its aberrant functioning is often associated with various diseases. Rictor is a peculiar and distinguishing mTORC2 component playing a pivotal role in controlling its assembly and activity. Among extant organisms, Rictor is conserved from unicellular eukaryotes to metazoans. We replaced two distinct, but conserved, glycine residues in both the gene and its human ortholog, The two conserved residues are spaced ∼50 amino acids apart, and both are embedded within a conserved region falling in between the Ras-GEFN2 and Rictor-_V domains. The effects of point mutations on the mTORC2 activity and integrity were assessed by biochemical and functional assays. In both cases, these equivalent point mutations in the mammalian and gene impaired mTORC2 activity and integrity. Our data indicate that the two glycine residues are essential for the maintenance of mTORC2 activity and integrity in organisms that appear to be distantly related, suggesting that they have a evolutionarily conserved role in the assembly and proper mTORC2 functioning.
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http://dx.doi.org/10.1242/jcs.236505DOI Listing
November 2019

Reduced Expression of Sprouty1 Contributes to the Aberrant Proliferation and Impaired Apoptosis of Acute Myeloid Leukemia Cells.

J Clin Med 2019 Jul 4;8(7). Epub 2019 Jul 4.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.
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http://dx.doi.org/10.3390/jcm8070972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678378PMC
July 2019

Curcumin induces apoptosis in JAK2-mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways.

J Cell Mol Med 2019 06 29;23(6):4349-4357. Epub 2019 Apr 29.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti-proliferative and pro-apoptotic effects of curcumin in JAK2 V617F-mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F-mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.
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http://dx.doi.org/10.1111/jcmm.14326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533565PMC
June 2019

Specific Monoclonal Antibody Against Bcr/Abl Out-of-Frame Alternative Proteins as Diagnostic Tool in Chronic Myelogenous Leukemia Patients.

Monoclon Antib Immunodiagn Immunother 2017 Aug 13;36(4):149-156. Epub 2017 Jul 13.

2 Department of Medical Biotechnologies and Translational Medicine, School of Medicine, University of Milan , Milan, Italy .

More recently, alternative splicing of specific genes are investigated for their therapeutic potential. In particular, we reported the existence of BCR-ABL alternative splicing isoforms, in about 80% of Philadelphia-positive patients, which lead to the expression of aberrant proteins. These fusion proteins are characterized by an orphan initial and correct Bcr portion attached to a 112 amino acid sequence, arising from the impairment in the reading frame (reading of ABL exon 4 and 5). We demonstrated that these Abl-out-of-frame (OOF) isoforms could have an immunological role with therapeutic implications. The aim of this study was to characterize a new monoclonal antibody (MAb) specific for Abl-OOF protein portion, for diagnostic use, to detect this biomarker in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) patients and to generate novel approaches in the immunotherapy. 5F11G11 MAb recognizes the OOF protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence. In addition, we demonstrate the MAb's ability to recognize the alternative hybrid Bcr/Abl fusion protein expressed in leukemic cells from CML patients, to support the possible use of 5F11G11 MAb as a diagnostic tool to select patients with Philadelphia chromosome-positive CML that could be eligible for an immunotherapeutic approach with this new antigen.
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http://dx.doi.org/10.1089/mab.2016.0054DOI Listing
August 2017

Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones.

Oncotarget 2017 May;8(22):35508-35522

Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano, Italy.

Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disorder with either indolent or aggressive clinical course. Current treatment regiments have significantly improved the overall outcomes even if higher risk subgroups - those harboring TP53 mutations or deletions of the short arm of chromosome 17 (del17p) - remain highly challenging. In the present work, we identified USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL. We demonstrated that in primary CLL samples and in CLL cell lines USP7 is: i) over-expressed through a mechanism involving miR-338-3p and miR-181b deregulation; ii) functionally activated by Casein Kinase 2 (CK2), an upstream interactor known to be deregulated in CLL; iii) effectively targeted by the USP7 inhibitor P5091. Treatment of primary CLL samples and cell lines with P5091 induces cell growth arrest and apoptosis, through the restoration of PTEN nuclear pool, both in TP53-wild type and -null environment. Importantly, PTEN acts as the main tumor suppressive mediator along the USP7-PTEN axis in a p53 dispensable manner. In conclusion, we propose USP7 as a new druggable target in CLL.
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http://dx.doi.org/10.18632/oncotarget.16348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482594PMC
May 2017

IκB-α: At the crossroad between oncogenic and tumor-suppressive signals.

Oncol Lett 2017 Feb 6;13(2):531-534. Epub 2016 Dec 6.

Department of Clinical and Biological Sciences, University of Turin, I-10043 Orbassano, Turin, Italy.

Nuclear factor κB (NF-κB) is an essential component of tumorigenesis and resistance to cancer treatments. NFKB inhibitor α (IκB-α) acts as a negative regulator of the classical NF-κB pathway through its ability to maintain the presence of NF-κB in the cytoplasm. However, IκB-α is also able to form a complex with tumor protein p53, promoting its inactivation. Recently, we demonstrated that IκB-α is able to mediate p53 nuclear exclusion and inactivation in chronic myeloid leukemia, indicating that IκB-α can modulate either oncogenic or tumor-suppressive functions, with important implications for cancer treatment. The present review describes the role of IκB-α in cancer pathogenesis, with particular attention to hematological cancers, and highlights the involvement of IκB-α in the regulation of p53 tumor-suppressive functions.
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http://dx.doi.org/10.3892/ol.2016.5465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351326PMC
February 2017

The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia.

Oncol Lett 2016 Nov 1;12(5):3123-3126. Epub 2016 Sep 1.

Department of Clinical and Biological Sciences, 'San Luigi Gonzaga' University Hospital, School of Medicine, University of Turin, 10043 Turin, Italy.

Philadelphia chromosome-positive (Ph) acute lymphoblastic leukemia (ALL) is driven by the p190 breakpoint cluster region (BCR)-ABL isoform. Although effectively targeted by BCR-ABL tyrosine kinase inhibitors (TKIs), ALL is associated with a less effective response to TKIs compared with chronic myeloid leukemia. Therefore, the identification of additional genes required for ALL maintenance may provide possible therapeutic targets to aid the eradication of this cancer. The present study demonstrated that p190 BCR-ABL is able to interact with the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which in turn affects p53 protein stability. Notably, the inhibition of HAUSP with small molecule inhibitors promoted the upregulation of p53 protein levels. These results suggest that HAUSP inhibitors may harbor clinically relevant implications in the treatment of Ph ALL.
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http://dx.doi.org/10.3892/ol.2016.5073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103907PMC
November 2016

Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection.

Oncotarget 2016 Nov;7(45):74082-74096

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Meningioma 1 (MN1) gene overexpression has been reported in acute myeloid leukaemia (AML) patients and identified as a negative prognostic factor. In order to characterize patients presenting gene overexpression and to verify if MN1 transcript could be a useful marker for minimal residual disease detection, MN1 was quantified in 136 AML patients with different cytogenetic risk and in 50 normal controls. In 20 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment and in 8 patients with NPM1 mutation, we performed a simultaneous analysis of MN1 and the fusion-gene transcript or NPM1 mutation during follow-up. Sequential MN1 and WT1 analysis was also performed in 13 AML patients lacking other molecular markers. The data obtained show that normal cells consistently express low levels of MN1 transcript. In contrast, high levels of MN1 expression are present in 47% of patients with normal karyotype and in all cases with inv(16). MN1 levels during follow-up were found to follow the pattern of other molecular markers (fusion gene transcripts, NPM1 and WT1). Increased MN1 expression in the BM during follow up was always found to be predictive of an impending hematological relapse.
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http://dx.doi.org/10.18632/oncotarget.12269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342037PMC
November 2016

The BCR-ABL/NF-κB signal transduction network: a long lasting relationship in Philadelphia positive Leukemias.

Oncotarget 2016 10;7(40):66287-66298

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy.

The Nuclear Factor-kappa B (NF-κB) family of transcription factors plays a key role in cancer pathogenesis due to the ability to promote cellular proliferation and survival, to induce resistance to chemotherapy and to mediate invasion and metastasis. NF-κB is recruited through different mechanisms involving either canonical (RelA/p50) or non-canonical pathways (RelB/p50 or RelB/p52), which transduce the signals originated from growth-factors, cytokines, oncogenic stress and DNA damage, bacterial and viral products or other stimuli. The pharmacological inhibition of the NF-κB pathway has clearly been associated with significant clinical activity in different cancers. Almost 20 years ago, NF-κB was described as an essential modulator of BCR-ABL signaling in Chronic Myeloid Leukemia and Philadelphia-positive Acute Lymphoblastic Leukemia. This review summarizes the role of NF-κB in BCR-ABL-mediated leukemogenesis and provides new insights on the long lasting BCR-ABL/NF-κB connection.
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http://dx.doi.org/10.18632/oncotarget.11507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323234PMC
October 2016

Unleashing the Guardian: The Targetable BCR-ABL/HAUSP/PML/PTEN Network in Chronic Myeloid Leukemia.

Curr Drug Targets 2017 ;18(4):389-395

Department of Clinical and Biological Sciences, University of Turin, regione Gonzole 10, 10043, Orbassano, Italy.

The complete eradication of Chronic Myeloid Leukemia is still challenging even in the era of highly selective and potent BCR-ABL tyrosine kinase inhibitors (TKIs). The 'Achilles heel' of TKI-based CML therapy is the inability of TKI to effectively target CML stem cells. Several pathways have been described to induce TKI insensitiveness in quiescent CML stem cells. In this review, we will describe the BCR-ABL/HAUSP/PML/PTEN network, whose signaling mediators converge to regulate the function of the tumor suppressor PTEN. We will also highlight the pharmacological strategies to modulate PTEN functions in order to sustain CML stem cell eradication.
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http://dx.doi.org/10.2174/1389450117666160608120527DOI Listing
September 2017

The non-genomic loss of function of tumor suppressors: an essential role in the pathogenesis of chronic myeloid leukemia chronic phase.

BMC Cancer 2016 05 16;16:314. Epub 2016 May 16.

Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, Regione Gonzole 10, 10043, Orbassano, Italy.

Background: Chronic Myeloid Leukemia was always referred as a unique cancer due to the apparent independence from tumor suppressors' deletions/mutations in the early stages of the disease. However, it is now well documented that even genetically wild-type tumor suppressors can be involved in tumorigenesis, when functionally inactivated. In particular, tumor suppressors' functions can be impaired by subtle variations of protein levels, changes in cellular compartmentalization and post-transcriptional/post-translational modifications, such as phosphorylation, acetylation, ubiquitination and sumoylation. Notably, tumor suppressors inactivation offers challenging therapeutic opportunities. The reactivation of an inactive and genetically wild-type tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells.

Main Body: Chronic Myeloid Leukemia (CML) could be considered as the paradigm for non-genomic loss of function of tumor suppressors due to the ability of BCR-ABL to directly promote functionally inactivation of several tumor suppressors.

Short Conclusion: In this review we will describe new insights on the role of FoxO, PP2A, p27, BLK, PTEN and other tumor suppressors in CML pathogenesis. Finally, we will describe strategies to promote tumor suppressors reactivation in CML.
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http://dx.doi.org/10.1186/s12885-016-2346-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869339PMC
May 2016

Protein Kinase CK2: A Targetable BCR-ABL Partner in Philadelphia Positive Leukemias.

Adv Hematol 2015 30;2015:612567. Epub 2015 Dec 30.

Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy.

BCR-ABL-mediated leukemias, either Chronic Myeloid Leukemia (CML) or Philadelphia positive Acute Lymphoblastic Leukemia (ALL), are the paradigm of targeted molecular therapy of cancer due to the impressive clinical responses obtained with BCR-ABL specific tyrosine kinase inhibitors (TKIs). However, BCR-ABL TKIs do not allow completely eradicating both CML and ALL. Furthermore, ALL therapy is associated with much worse responses to TKIs than those observed in CML. The identification of additional pathways that mediate BCR-ABL leukemogenesis is indeed mandatory to achieve synthetic lethality together with TKI. Here, we review the role of BCR-ABL/protein kinase CK2 interaction in BCR-ABL leukemias, with potentially relevant implications for therapy.
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http://dx.doi.org/10.1155/2015/612567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710905PMC
February 2016

The Role of PTEN in Myeloid Malignancies.

Hematol Rep 2015 Nov 9;7(4):5844. Epub 2015 Dec 9.

Department of Clinical and Biological Sciences, University of Turin , Orbassano, Italy.

PTEN deletion in the mouse and in the zebrafish highlights the essential role of this tumor suppressor in the development of myeloid malignancies, in particular acute myeloid leukemia and myeloproliferative disorders. In humans, extensive genetic sequences of myeloid malignancies did not reveal recurrent PTEN mutations and deletions. However, PTEN was shown to be functionally inactivated in several acute myeloid leukemia and chronic myeloid leukemia samples, through both post-trasductional modifications, changes in protein levels and cellular compartmentalization. Notably, non genomic inactivation of PTEN in myeloid malignancies could represent a challenging therapeutic opportunity for these diseases. Targeting those mechanisms that affect PTEN function could indeed promote PTEN reactivation with consequent cancer selective apoptosis induction. In this review we will describe the role of PTEN in the development of myeloid malignancies.
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http://dx.doi.org/10.4081/hr.2015.6027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691678PMC
November 2015

New alternative splicing BCR/ABL-OOF shows an oncogenic role by lack of inhibition of BCR GTPase activity and an increased of persistence of Rac activation in chronic myeloid leukemia.

Oncoscience 2015 11;2(10):880-91. Epub 2015 Nov 11.

Department of Clinical and Biological Sciences, San Luigi Hospital, University of Turin, Orbassano, Turin, Italy.

In Chronic Myeloid Leukemia 80% of patients present alternative splice variants involving BCR exons 1, 13 or 14 and ABL exon 4, with a consequent impairment in the reading frame of the ABL gene. Therefore BCR/ABL fusion proteins (BCR/ABL-OOF) are characterized by an in-frame BCR portion followed by an amino acids sequence arising from the out of frame (OOF) reading of the ABL gene. The product of this new transcript contains the characteristic BCR domains while lacking the COOH-terminal Rho GTPase GAP domain. The present work aims to characterize the protein functionality in terms of cytoskeleton (re-)modelling, adhesion and activation of canonical oncogenic signalling pathways. Here, we show that BCR/ABL-OOF has a peculiar endosomal localization which affects EGF receptor activation and turnover. Moreover, we demonstrate that BCR/ABL-OOF expression leads to aberrant cellular adhesion due to the activation of Rac GTPase, increase in cellular proliferation, migration and survival. When overexpressed in a BCR/ABL positive cell line, BCR/ABL-OOF induces hyperactivation of Rac signaling axis offering a therapeutic window for Rac-targeted therapy. Our data support a critical role of BCR/ABL-OOF in leukemogenesis and identify a subset of patients that may benefit from Rac-targeted therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671955PMC
http://dx.doi.org/10.18632/oncoscience.260DOI Listing
December 2015

Non genomic loss of function of tumor suppressors in CML: BCR-ABL promotes IκBα mediated p53 nuclear exclusion.

Oncotarget 2015 Sep;6(28):25217-25

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy.

Tumor suppressor function can be modulated by subtle variation of expression levels, proper cellular compartmentalization and post-translational modifications, such as phosphorylation, acetylation and sumoylation. The non-genomic loss of function of tumor suppressors offers a challenging therapeutic opportunity. The reactivation of a tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells. The identification of mechanisms that affect tumor suppressor functions is therefore essential. In this work, we show that BCR-ABL promotes the accumulation of the NFKBIA gene product, IκBα, in the cytosol through physical interaction and stabilization of the protein. Furthermore, BCR-ABL/IκBα complex acts as a scaffold protein favoring p53 nuclear exclusion. We therefore identify a novel BCR-ABL/IκBα/p53 network, whereby BCR-ABL functionally inactivates a key tumor suppressor.
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http://dx.doi.org/10.18632/oncotarget.4611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694826PMC
September 2015

Morgana acts as an oncosuppressor in chronic myeloid leukemia.

Blood 2015 Apr 12;125(14):2245-53. Epub 2015 Feb 12.

Department of Molecular Biotechnology and Health Sciences, and.

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.
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http://dx.doi.org/10.1182/blood-2014-05-575001DOI Listing
April 2015

BCR-ABL inactivates cytosolic PTEN through Casein Kinase II mediated tail phosphorylation.

Cell Cycle 2015 ;14(7):973-9

a Department of Clinical and Biological Sciences; San Luigi Hospital ; Orbassano - Turin University ; Turin , Italy.

The tumor suppressive function of PTEN is exerted within 2 different cellular compartments. In the cytosol-membrane, it negatively regulates PI3K-AKT pathway through the de-phosphorylation of phosphatidylinositol (3,4,5)-triphosphate (PIP3), therefore blocking one of the major signaling transduction pathways in tumorigenesis. In the nucleus, PTEN controls genomic stability and cellular proliferation through phosphatase independent mechanisms. Importantly, impairments in PTEN cellular compartmentalization, changes in protein levels and post-transductional modifications affect PTEN tumor suppressive functions. Targeting mechanisms that inactivate PTEN promotes apoptosis induction of cancer cells, without affecting normal cells, with appealing therapeutic implications. Recently, we have shown that BCR-ABL promotes PTEN nuclear exclusion by favoring HAUSP mediated PTEN de-ubiquitination in Chronic Myeloid Leukemia. Here, we show that nuclear exclusion of PTEN is associated with PTEN inactivation in the cytoplasm of CML cells. In particular, BCR-ABL promotes Casein Kinase II-mediated PTEN tail phosphorylation with consequent inhibition of the phosphatase activity toward PIP3. Targeting Casein Kinase II promotes PTEN reactivation with apoptosis induction. We therefore propose a novel BCR-ABL/CKII/PTEN pathway as a potential target to achieve synthetic lethality with tyrosine kinase inhibitors.
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http://dx.doi.org/10.1080/15384101.2015.1006970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615112PMC
December 2015

Recurrent ETNK1 mutations in atypical chronic myeloid leukemia.

Blood 2015 Jan 24;125(3):499-503. Epub 2014 Oct 24.

Department of Health Sciences, University of Milano-Bicocca, Monza, Italy;

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.
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http://dx.doi.org/10.1182/blood-2014-06-579466DOI Listing
January 2015

BCR-ABL promotes PTEN downregulation in chronic myeloid leukemia.

PLoS One 2014 24;9(10):e110682. Epub 2014 Oct 24.

Department of Clinical and Biological Sciences, San Luigi Hospital, University of Turin, Orbassano, Turin, Italy.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) translocation coding for the chimeric protein p210 BCR-ABL. The tumor suppressor PTEN plays a critical role in the pathogenesis of CML chronic phase, through non genomic loss of function mechanisms, such as protein down-regulation and impaired nuclear/cytoplasmic shuttling. Here we demonstrate that BCR-ABL promotes PTEN downregulation through a MEK dependent pathway. Furthermore, we describe a novel not recurrent N212D-PTEN point mutation found in the EM2 blast crisis cell line.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208795PMC
June 2015

HAUSP compartmentalization in chronic myeloid leukemia.

Eur J Haematol 2015 Apr 3;94(4):318-21. Epub 2014 Sep 3.

Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, Orbassano, Italy.

Introduction: PTEN plays an essential role in the pathogenesis of chronic myeloid leukemia. Recently, we have shown that BCR-ABL promotes PTEN nuclear exclusion, through the modulation of HAUSP activity.

Objectives: Here, we investigate HAUSP cellular compartmentalization in primary CML samples.

Results: While in normal CD34 positive cells HAUSP is expressed mostly in the nucleus, in CML CD34 cells HAUSP is expressed both in the nuclear bodies and in the cytoplasm.

Conclusions: This observation suggests that HAUSP behaves as a shuttling protein in CML. It can bind to BCR-ABL in the cytosol, where it is phosphorylated on tyrosine residues, and it maintains the proper compartmentalization in the nuclear bodies, where it acts as part of a PML network to regulate PTEN de-ubiquitination.
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http://dx.doi.org/10.1111/ejh.12422DOI Listing
April 2015

Kinase-inhibitor-insensitive cancer stem cells in chronic myeloid leukemia.

Expert Opin Biol Ther 2014 Mar 3;14(3):287-99. Epub 2014 Jan 3.

University Turin, San Luigi Hospital, Division of Hematology and Internal Medicine, Department of Oncology , Orbassano - Turin , Italy

Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the translocation t(9;22), coding for the chimeric protein BCR-ABL. The development of BCR-ABL tyrosine kinase inhibitors (TKIs) has dramatically revolutionized and improved CML therapy. However, TKI-based therapy faces a major challenge: the insensitivity of CML leukemic stem cells (LSCs) to TKIs. In particular, while CML progenitor cells and differentiated cells are oncogene addicted, BCR-ABL tyrosine kinase is dispensable for CML LSC survival and maintenance. Notably, in CML, additional cellular mechanisms promote LSC survival and maintenance, rendering these cells able to survive even in the presence of TKI and to eventually promote relapse.

Areas Covered: This review will focus on the mechanisms of LSC insensitivity to TKI and on the strategies to obtain synthetic lethality with combination therapies.

Expert Opinion: Several pathways have been proposed to promote LSC maintenance and described as ideal targets to induce CML LSC exhaustion in combination with TKI. Ongoing clinical trials designed to target some of these pathways will assess which molecular target is relevant for in vivo human LSC survival in a new 'stem-cell targeting' perspective.
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http://dx.doi.org/10.1517/14712598.2014.867323DOI Listing
March 2014

Characterization of a monoclonal antibody specific for novel Bcr/Abl out-of-frame fusion proteins.

Hybridoma (Larchmt) 2011 Jun;30(3):261-9

Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy.

The new tumor-specific antigens Bcr/Abl-OOF, identified in Philadelphia chromosome (Ph)-positive leukemia cells, are derived from an alternative splicing event involving BCR exons 1, 13, or 14 and ABL exons 4 and 5. The COOH-terminus of these transcription products contain an amino acid portion derived from an out-of-frame (OOF) reading of the ABL gene; these variants are expressed in Ph-positive chronic myelogenous leukemia (CML) and acute lymphocytic leukemia patients. Previously, we confirmed the presence of out-of-frame peptide-specific T cells in the peripheral blood of CML patients with the ability to lyse primary autologous CML cells. We also demonstrated that the out-of-frame Abl portion was immunogenic in HLA-A2.1 transgenic mice. Here we describe the production and characterization of monoclonal antibody 1D8G8, a new tool for localization and functional studies of the tumor antigen Bcr/Abl-OOF. This antibody recognizes the out-of-frame protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence, and binds to a specific epitope of this antigen presented in association with HLA-A2.1 molecules at the surface of these cells, as demonstrated by flow cytometry. Thus this MAb could be useful to better understand how this new protein presents in Ph-positive cells beside the canonical Bcr/Abl fusion proteins.
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http://dx.doi.org/10.1089/hyb.2010.0122DOI Listing
June 2011

FOXO transcription factor activity is partially retained in quiescent CML stem cells and induced by tyrosine kinase inhibitors in CML progenitor cells.

Blood 2009 Dec 1. Epub 2009 Dec 1.

Paul O'Gorman Leukaemia Research Centre, Medical Faculty, University of Glasgow, Glasgow, UK.

Chronic Myeloid Leukaemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL. ABL-specific tyrosine kinase inhibitors (TKIs), whilst effective against mature CML cells, induce little apoptosis in stem/progenitor cells. However, in stem/progenitor cells TKIs exert potent anti-proliferative effects through a poorly understood mechanism. We showed that in CD34(+) CML cells FOXO1, 3a and 4 (FOXOs) were phosphorylated, predominantly cytoplasmic and inactive, consequent to BCR-ABL expression. TKIs decreased phosphorylation of FOXOs, leading to their re-localisation from cytoplasm (inactive) to nucleus (active), thus inducing G1 arrest. Of key importance, despite BCR-ABL activity, primitive quiescent CML stem cells showed low levels of FOXO phosphorylation and predominant nuclear localisation, resembling the pattern in normal stem cells. These results demonstrate for the first time that TKI-induced G1 arrest in CML progenitor cells is mediated by re-activation of FOXOs, whilst quiescence of CML stem cells is regulated by sustained FOXO activity. These data contribute to our understanding of CML stem cell quiescence and TKI activity, suggesting new strategies to target CML stem/progenitor cells by preventing or reversing this effect.
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http://dx.doi.org/10.1182/blood-2009-06-226621DOI Listing
December 2009

Imatinib resistance in CML.

Cancer Lett 2009 Feb 23;274(1):1-9. Epub 2008 Jul 23.

Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, San Luigi Hospital, 10043 Orbassano, Turin, Italy.

Imatinib is, at present, the first-choice treatment for patients with chronic myeloid leukaemia in chronic phase. Despite the impressive rate of complete haematological response and complete cytogenetical remissions, some cases show primary resistance or relapse after an initial response (secondary or acquired resistance). The most common mechanisms responsible for this resistance are BCR/ABL kinase domain mutations, BCR/ABL amplification and over-expression and clonal evolution with activation of additional oncogenic pathways. Here, we describe the molecular basis of imatinib resistance, the significance of molecular monitoring and the current efforts to overcome imatinib resistance, ranging from the development of new drugs to the stimulation of an immune response against the disease.
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http://dx.doi.org/10.1016/j.canlet.2008.06.003DOI Listing
February 2009