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Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and transgenic mouse studies indicate that activation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease. While studying the regulation of TCL1 expression, we identified the microRNA cluster miR-4521/3676 and discovered that these two microRNAs are associated with tRNA sequences and that this region can produce two small RNAs, members of a recently identified class of small noncoding RNAs, tRNA-derived small RNAs (tsRNAs). We further proved that miR-3676 and miR-4521 are tsRNAs using Northern blot analysis. We found that, like ts-3676, ts-4521 is down-regulated and mutated in CLL. Analysis of lung cancer samples revealed that both ts-3676 and ts-4521 are down-regulated and mutated in patient tumor samples. Because tsRNAs are similar in nature to piRNAs [P-element–induced wimpy testis (Piwi)-interacting small RNAs], we investigated whether ts-3676 and ts-4521 can interact with Piwi proteins and found these two tsRNAs in complexes containing Piwi-like protein 2 (PIWIL2). To determine whether other tsRNAs are involved in cancer, we generated a custom microarray chip containing 120 tsRNAs 16 bp or more in size. Microarray hybridization experiments revealed tsRNA signatures in CLL and lung cancer, indicating that, like microRNAs, tsRNAs may have an oncogenic and/or tumor-suppressor function in hematopoietic malignancies and solid tumors. Thus, our results show that tsRNAs are dysregulated in human cancer.

The central role of the microRNA (miR) 15a/16-1 cluster in B-cell oncogenesis has been extensively demonstrated, with over two-thirds of B-cell chronic lymphocytic leukemia characterized by the deletion of the miR-15a/16-1 locus at 13q14. Despite the well-established understanding of the molecular mechanisms occurring during miR-15a/16-1 dysregulation, the oncogenic role of other miR-15/16 family members, such as the miR-15b/16-2 cluster (3q25), is still far from being elucidated. Whereas miR-15a is highly similar to miR-15b, miR-16-1 is identical to miR-16-2; thus, it could be speculated that both clusters control a similar set of target genes and may have overlapping functions. However, the biological role of miR-15b/16-2 is still controversial. We generated miR-15b/16-2 knockout mice to better understand the cluster’s role in vivo. These mice developed B-cell malignancy by age 15–18 mo with a penetrance of 60%. At this stage, mice showed significantly enlarged spleens with abnormal B cell-derived white pulp enlargement. Flow cytometric analysis demonstrated an expanded CD19+ CD5+ population in the spleen of 40% knockout mice, a characteristic of the chronic lymphocytic leukemia-associated phenotype found in humans. Of note, miR-15b/16-2 modulates theCCND2(Cyclin D2),CCND1(Cyclin D1), andIGF1R(insulin-like growth factor 1 receptor) genes involved in proliferation and antiapoptotic pathways in mouse B cells. These results are the first, to our knowledge, to suggest an important role of miR-15b/16-2 loss in the pathogenesis of B-cell chronic lymphocytic leukemia.

Resistance to platinum-based chemotherapy represents a major clinical challenge for many tumors, including epithelial ovarian cancer. Patients often experience several response-relapse events, until tumors become resistant and life expectancy drops to 12–15 months. Despite improved knowledge of the molecular determinants of platinum resistance, the lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer is not known. Here, we show that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD + ) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Our findings reveal metabolic vulnerabilities with clinical implications for a subset of platinum resistant ovarian cancers. Metabolic reprogramming is associated with cancer development and therapy resistance. Here, the authors show that downregulation of the serine biosynthesis enzyme PHGDH in a fraction of patients is associated with relapse in platinum-treated ovarian cancers and to NAD + and PARP activity upregulation.

Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 ( TCL1 ) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3′UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1 . We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19 ⁺ cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676 . Two of these mutations were loss-of-function mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 ( Tp53 ), and is codeleted with Tp53 , we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression. Significance B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia. We previously found that dysregulation of the T-cell leukemia/lymphoma 1 ( TCL1 ) oncogene is a critical contributing event in the pathogenesis of this disease. In this study we investigated molecular causes of TCL1 overexpression in CLL. We identified miR-3676 as a powerful regulator of TCL1 expression. We found that miR-3676 is down-regulated on all groups of CLLs and mutated in 1% of CLLs. Interestingly, miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 ( Tp53 ), and is codeleted with Tp53 in 17p-deleted CLL. Loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression. Thus, this study uncovers a major mechanism in the pathogenesis of CLL.

RANBP9 and RANBP10, also called Scorpins, are essential components of the C-terminal to LisH (CTLH) complex, an evolutionarily conserved poorly investigated multisubunit E3 ligase. Their role in non-small cell lung cancer (NSCLC) is unknown. In this study, first we used stable loss-of function and overexpression inducible cell lines to investigate the ability of either RANBP9 or RANBP10 to form their own functional CTLH complex. Then, we probed lysates from patient tumors and analyzed data from publicly available repositories to investigate the expression of RANBP9 and RANBP10. Finally, we used inducible cell lines in vitro to recapitulate the expression observed in patients and investigate the changes of the proteome and the ubiquitylome associated with either RANBP9 or RANBP10 in NSCLC. Here, we show that the two Scorpins are both expressed in NSCLC cells and that either of them can independently support the formation of the CTLH complex. Short-term experiments revealed that the RANBP9 and RANBP10 proteins balance each other in terms of expression, and the acute overexpression of one or the other results in significant reshaping of the NSCLC cell proteome and ubiquitylome. A higher RANBP9/RANBP10 ratio is associated with greater proliferation in both NSCLC cell lines and patients. Acute increased expression of RANBP10 slows NSCLC cell proliferation and decreases the level of proliferation-associated proteins, including key players in DNA replication. We present evidence that the Scorpins act as partial antagonists and work together as one sophisticated rheostat to modulate the CTLH complex ubiquitylation output, which regulates cell proliferation and other key biological processes in NSCLC. These results suggest that the two Scorpins can be considered as targets for the treatment of NSCLC.

Introduction Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Most nucleolar proteins are essential; thus, their abrogation cannot be achieved through conventional approaches. This technical obstacle has limited our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Methods We applied the Auxin Inducible Degron (AID) proteolytic system, paired with CRISPR/Cas9 knock-in gene-editing, to obtain an unprecedented characterization of the biological activities of Nucleolin (NCL), one of the most abundant nucleolar proteins, in Triple Negative Breast Cancer (TNBC) cells. Then, we combined live-cell imaging, RNA-sequencing, and quantitative proteomics, to characterize the impact of NCL acute abrogation on the behavior of TNBC cells. Finally, we used in silico analyses to validate NCL molecular role in TNBC patients. Results Acute abrogation of endogenous NCL impacted both the transcriptome and the proteome of TNBC cells, particularly affecting critical players involved in ribosome biogenesis and in cell cycle progression. Unexpectedly, NCL depletion limited cancer cell ability to effectively complete cytokinesis, ultimately leading to the accumulation of bi-nucleated cells. In silico analyses confirmed that the levels of regulators of cell cycle progression and chromosome segregation correlated with NCL abundance in TNBC patients. Finally, NCL degradation enhanced the activity of pharmaceutical inhibitors of cellular mitosis, such as the Anaphase Promoting Complex inhibitor APCin. Conclusions Our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

Background Fludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria. Results By comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance. Conclusions This is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies.

T cells are functionally compromised during HIV infection despite their increased activation and proliferation. Although T cell hyperactivation is one of the best predictive markers for disease progression, its causes are poorly understood. Anti-tat natural immunity as well as anti-tat antibodies induced by Tat immunization protect from progression to AIDS and reverse signs of immune activation in HIV-infected patients suggesting a role of Tat in T cell dysfunctionality. The Tat protein of HIV-1 is known to induce, in vitro, the activation of CD4(+) T lymphocytes, but its role on CD8(+) T cells and how these effects modulate, in vivo, the immune response to pathogens are not known. To characterize the role of Tat in T cell hyperactivation and dysfunction, we examined the effect of Tat on CD8(+) T cell responses and antiviral immunity in different ex vivo and in vivo models of antigenic stimulation, including HSV infection. We demonstrate for the first time that the presence of Tat during priming of CD8(+) T cells favors the activation of antigen-specific CTLs. Effector CD8(+) T cells generated in the presence of Tat undergo an enhanced and prolonged expansion that turns to a partial dysfunctionality at the peak of the response, and worsens HSV acute infection. Moreover, Tat favors the development of effector memory CD8(+) T cells and a transient loss of B cells, two hallmarks of the chronic immune activation observed in HIV-infected patients. Our data provide evidence that Tat affects CD8(+) T cell responses to co-pathogens and suggest that Tat may contribute to the CD8(+) T cell hyperactivation observed in HIV-infected individuals.

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.