An in vivo "turning model" reveals new RanBP9 interactions in lung macrophages.

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.

Serine metabolism remodeling after platinum-based chemotherapy identifies vulnerabilities in a subgroup of resistant ovarian cancers.

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.

Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma.

The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance. In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation. We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics. Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models. Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy. In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines. Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion. Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.

Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m6A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.

The TLR7/8/9 Antagonist IMO-8503 Inhibits Cancer-Induced Cachexia.

: Muscle wasting is a feature of the cachexia syndrome, which contributes significantly to the mortality of patients with cancer. We have previously demonstrated that miR-21 is secreted through extracellular vesicles (EV) by lung and pancreatic cancer cells and promotes JNK-dependent cell death through its binding to the TLR7 receptor in murine myoblasts. Here, we evaluate the ability of IMO-8503, a TLR7, 8, and 9 antagonist, to inhibit cancer-induced cachexia. Using EVs isolated from lung and pancreatic cancer cells and from patient plasma samples, we demonstrate that IMO-8503 inhibits cell death induced by circulating miRNAs with no significant toxicity. Intraperitoneal administration of the antagonist in a murine model for Lewis lung carcinoma (LLC-induced cachexia) strongly impaired several cachexia-related features, such as the expression of Pax7 as well as caspase-3 and PARP cleavage in skeletal muscles, and significantly prevented the loss of lean mass in tumor-bearing mice. IMO-8503 also impaired circulating miRNA-induced cell death in human primary myoblasts. Taken together, our findings strongly indicate that IMO-8503 serves as a potential therapy for the treatment of cancer cachexia. SIGNIFICANCE: Cancer-associated cachexia is a significant problem for patients with cancer that remain poorly understood, understudied, and inadequately treated; these findings report a potential new therapeutic for the treatment of TLR7-mediated cancer cachexia.

Increased SHISA3 expression characterizes chronic lymphocytic leukemia patients sensitive to lenalidomide.

Lenalidomide is a therapeutically effective drug in chronic lymphocytic leukemia (CLL). Twenty-seven CLL patients were treated with lenalidomide in a phase II clinical trial. Ten patients were grouped as responders (R) and 6 as nonresponders (NR). We evaluated T lymphocytes, NK, monocytes and dendritic cells at baseline and after treatment. A gene expression analysis was performed on 16 CLL samples collected before treatment. The levels of immune cells or immune-related cytokines are not different between R and NR patients. However, CLL patients sensitive to lenalidomide clearly show a peculiar gene expression profile in leukemic cells. The most up-regulated gene (fold change = +23 in R vs. NR) is Wnt inhibitor SHISA homolog 3 (SHISA3). SHISA3highCLL are characterized by a restrained activation of Wnt signaling and sensibility to lenalidomide-induced apoptosis. In conclusion, SHISA3 is a candidate gene for the identification of CLL patients who will benefit of lenalidomide treatment as single agent.

Selective targeting of point-mutated KRAS through artificial microRNAs.

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.

Targeting BTK through microRNA in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer.

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.

HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

miR-15b/16-2 deletion promotes B-cell malignancies.

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 the CCND2 (Cyclin D2), CCND1 (Cyclin D1), and IGF1R (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.

TCL1 targeting miR-3676 is codeleted with tumor protein p53 in chronic lymphocytic leukemia.

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.

Genetic subclonal complexity and miR125a-5p down-regulation identify a subset of patients with inferior outcome in low-risk CLL patients.

The majority of patients with chronic lymphocytic leukemia (CLL) and favorable prognostic features live for long periods without treatment. However, unexpected disease progression is observed in some cases. In a cohort of untreated CD38- CLL patients with normal FISH or isolated 13q- we found that, by fluorescence in situ hybridization (FISH), 16/28 cases presented, within immunomagnetic sorted CD38+ cells, genetic lesions undetectable in the CD38- fraction. These patients showed a shorter time to first treatment (TTFT, p=0.0162) in comparison to cases without FISH lesions in CD38+ cells. Patients with FISH abnormalities in CD38+ cells showed a distinctive microRNA profile, characterized by the down-regulation of miR-125a-5p both in the CD38- and CD38+ populations. In an independent cohort of 71 consecutive untreated CD38- CLL with normal FISH or isolated 13q-, a lower miR125a-5p expression was associated with a shorter TTFT both in univariate and multivariate analysis (p=0.003 and 0.016, respectively) and with a higher prevalence of mutations (7/12 vs 0/8, p=0.015) as assessed by next-generation sequencing. In conclusion, our data showed previously unrecognized subclonal heterogeneity within the CD38+ fraction of CD38- CLL patients with low-risk FISH findings and suggested an association between down-regulated miR-125a-5p expression, genetic complexity and worse outcome.

Endothelium-mediated survival of leukemic cells and angiogenesis-related factors are affected by lenalidomide treatment in chronic lymphocytic leukemia.

Lenalidomide is an IMID immunomodulatory agent clinically active in patients with chronic lymphocytic leukemia (CLL). We evaluated the activity of lenalidomide inside an in vitro coculture system of endothelial and CLL cells. Lenalidomide was able to inhibit CLL survival advantage mediated by endothelial contact. Moreover, the marked increase of in vitro angiogenesis determined by CLL-derived conditioned media was reduced by lenalidomide. We also analyzed peripheral blood collected from 27 patients with relapsed or refractory CLL being treated with lenalidomide within a phase II trial. Plasma levels of VEGF and THBS-1 decreased, whereas Ang2 and Ang increased during treatment. Patients who respond to lenalidomide showed a more pronounced decrease of VEGF and bFGF than did patients with stable or progressive disease (p = 0.007 and p = 0.005). Furthermore, lenalidomide reduced circulating endothelial cells and endothelial progenitors by increasing the percentage of apoptotic cells. Conversely, for six matched bone marrow biopsies available before and after treatment, we did not detect any modification in vessel density, suggesting a possible mechanism of vessel normalization rather than regression. In conclusion, our study provides further evidence that the anti-CLL effect of lenalidomide is mediated through the alteration of microenvironmental elements, implying the modulation of several angiogenesis-related factors and disruption of CLL crosstalk with endothelial cells.

The HIV-1 Tat protein induces the activation of CD8+ T cells and affects in vivo the magnitude and kinetics of antiviral responses.

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.

miR deregulation in CLL.

B-cell chronic lymphocytic leukemia (CLL) is the most frequent human leukemia and it occurs in two forms, indolent and aggressive. Although clinical features and genetic abnormalities in CLL are well documented, molecular details underlying the disease are still under investigation.MicroRNAs are small noncoding RNAs involved in a variety of cellular processes and expressed in a tissue-specific manner. MicroRNAs have the ability to regulate gene expression. In physiological conditions, microRNAs act as gene expression controllers by targeting the mRNA or inhibiting its translation. Their deregulation can lead to an alteration of the expression level of many genes which can induce the development or promote the progression of tumors.In CLL, microRNAs can function as oncogenes, tumor suppressor genes, and/or can be used as markers for disease onset/progression. For example, in indolent CLL, 13q14 deletions targeting miR-15/16 initiate the disease, while in aggressive CLL miR-181 targets the critical TCL1 oncogene and can also be used as a progression marker.Here we discuss the foremost findings about the role of microRNAs in CLL pathogenesis, and how this knowledge can be used to identify new approaches to treat CLL.

A Sleeping Beauty screen reveals NF-kB activation in CLL mouse model.

TCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eµ-TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL.