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RARA and RXRA contribute to myeloid maturation in both mice and humans, and deletion of Rxra and Rxrb augments leukemic growth in mice. While defining the domains of RXRA that are required for anti-leukemic effects in murine KMT2A-MLLT3 leukemia cells, we unexpectedly identified RXRA DT448/9PP as a constitutively active variant capable of inducing maturation and loss of their proliferative phenotype. RXRA DT448/9PP was associated with ligand-independent activity in reporter assays, with enhanced co-activator interactions, reduced engraftment in vivo, and activation of myeloid maturation transcriptional signatures that overlapped with those of cells treated with the potent RXRA agonist bexarotene, suggestive of constitutive activity that leads to leukemic maturation. Phenotypes of RXRA DT448/9PP appear to differ from those of two other RXRA mutations with forms of constitutive activity (F318A and S427F), in that DT448/9PP activity was resistant to mutations at critical ligand-interacting amino acids (R316A/L326A) and was resistant to pharmacological antagonists, suggesting it may be ligand-independent. These data provide further evidence that activated retinoid X receptors can regulate myeloid maturation and provide a novel constitutively active variant that may be germane for broader studies of retinoid X receptors in other settings. This work was supported by National Institutes of Health grant R01 HL128447 (JSW), by the Siteman Investment Program (JSW), the Washington University SPORE DRP (JSW and MAF), the Children’s Discovery Institute (JSW), the Alex’s Lemonade Stand Foundation Young Investigator Award (MAF), the National Institutes of Health 5K12HD07622408 (MAF), and grants from the Spanish Ministerio de Ciencia e Innovación (MCI) (SAF2017- 90604-REDT-NurCaMeIn, RTI2018-095928-BI00) (MR). Sí

The orphan nuclear receptor Nur77 is an immediate-early response gene that based on tissue and cell context is implicated in a plethora of cellular processes, including proliferation, differentiation, apoptosis, metabolism, and inflammation. Nur77 has a ligand-binding pocket that is obstructed by hydrophobic side groups. Naturally occurring, cell-endogenous ligands have not been identified, and Nur77 transcriptional activity is thought to be regulated through posttranslational modification and modulation of protein levels. To determine whether Nur77 is transcriptionally active in hematopoietic cells in vivo, we used an upstream activating sequence (UAS)-GFP transgenic reporter. We found that Nur77 is transcriptionally inactive in vivo in hematopoietic cells under basal conditions, but that activation occurs following cytokine exposure by G-CSF or IL-3. We also identified a series of serine residues required for cytokine-dependent transactivation of Nur77. Moreover, a kinase inhibitor library screen and proximity labeling-based mass spectrometry identified overlapping kinase pathways that physically interacted with Nur77 and whose inhibition abrogated cytokine-induced activation of Nur77. We determined that transcriptional activation of Nur77 by G-CSF or IL-3 requires functional JAK and mTor signaling since their inhibition leads to Nur77 transcriptional inactivation. Thus, intracellular cytokine signaling networks appear to regulate Nur77 transcriptional activity in mouse hematopoietic cells.

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The presence of natural retinoids in vivo could influence the efficacy of pharmacologic agonists and antagonists. We found that natural RXRA ligands, but not RARA ligands, were present in murine MLL-AF9-derived myelomonocytic leukemias in vivo and that the concurrent presence of receptors and ligands acted as tumor suppressors. Pharmacologic retinoid responses could be optimized by concurrent targeting RXR ligands (e.g. bexarotene) and RARA ligands (e.g. all-trans retinoic acid, ATRA), which induced either leukemic maturation or apoptosis depending on cell culture conditions. Co-repressor release from the RARA:RXRA heterodimer occurred with RARA activation, but not RXRA activation, providing an explanation for the combination synergy. Combination synergy could be replicated in additional, but not all, AML cell lines and primary samples, and was associated with improved survival in vivo, although tolerability of bexarotene administration in mice remained an issue. These data provide insight into the basal presence of natural retinoids in leukemias in vivo and a potential strategy for clinical retinoid combination regimens in leukemias beyond acute promyelocytic leukemia. This work was supported by NIH R01 HL128447 (to JSW), NIH P50 CA171963 (Project 1, to JSW and DRP) by the Siteman Investment Program (to JSW), and grants from the Spanish Ministerio de Ciencia e Innovacion (MCI) (SAF201571878-REDT-NurCaMeIn, RTI2018-095928-B100) (to MR). The CNIC is supported by the MCI and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV2015-0505). Sí

Retinoic acid (RA) signaling pathways regulate fundamental biological processes, such as cell proliferation, development, differentiation, and apoptosis. Retinoid receptors (RARs and RXRs) are ligand-dependent transcription factors. All-trans retinoic acid (ATRA) is the principal endogenous ligand for the retinoic acid receptor alpha (RARA) and is produced by the enzymatic oxidation of dietary vitamin A, whose deficiency is associated with several pathological conditions. Differentiation therapy using ATRA revolutionized the outcome of acute promyelocytic leukemia (APL), although attempts to replicate these results in other cancer types have been met with more modest results. A better knowledge of RA signaling in different leukemia contexts is required to improve initial designs. Here, we will review the RA signaling pathway in normal and malignant hematopoiesis, and will discuss the advantages and the limitations related to retinoid therapy in acute myeloid leukemia.

Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways that cooperate to restore tissue integrity and homeostasis. To facilitate healing, injuries need to be promptly treated. Recently, the interest in alternatives to antibiotics has been raised given the widespread phenomenon of antibiotic resistance. Among these alternatives, the use of silver appears to be a valid option, so a resurgence in its use has been recently observed. In particular, in contrast to ionic silver, colloidal silver, a suspension of metallic silver particles, shows antibacterial activity displaying less or no toxicity. However, the human health risks associated with exposure to silver nanoparticles (NP) appear to be conflicted, and some studies have suggested that it could be toxic in different cellular contexts. These potentially harmful effects of silver NP depend on various parameters including NP size, which commonly range from 1 to 100 nm. In this study, we analyzed the effect of a colloidal silver preparation composed of very small and homogeneous nanoparticles of 0.62 nm size, smaller than those previously tested. We found no adverse effect on the cell proliferation of HaCaT cells, even at high NP concentration. Time-lapse microscopy and indirect immunofluorescence experiments demonstrated that this preparation of colloidal silver strongly increased cell migration, re-modeled the cytoskeleton, and caused recruitment of E-cadherin at cell-cell junctions of human cultured keratinocytes.

SMC3 encodes a subunit of the cohesin complex that has canonical roles in regulating sister chromatids segregation during mitosis and meiosis. Recurrent heterozygous mutations in SMC3 have been reported in acute myeloid leukemia (AML) and other myeloid malignancies. In this study, we investigated whether the missense mutations in SMC3 might have dominant-negative effects or phenocopy loss-of-function effects by comparing the consequences of Smc3-deficient and -haploinsufficient mouse models. We found that homozygous deletion of Smc3 during embryogenesis or in adult mice led to hematopoietic failure, suggesting that SMC3 missense mutations are unlikely to be associated with simple dominant-negative phenotypes. In contrast, haploinsufficiency was tolerated during embryonic and adult hematopoiesis. Under steady-state conditions, Smc3 haploinsufficiency did not alter colony forming in methylcellulose, only modestly decreased mature myeloid cell populations, and led to limited expression changes and chromatin alteration in Lin–cKit+ bone marrow cells. However, following transplantation, engraftment, and subsequent deletion, we observed a hematopoietic competitive disadvantage across myeloid and lymphoid lineages and within the stem/progenitor compartments. This disadvantage was not affected by hematopoietic stresses, but was partially abrogated by concurrent Dnmt3a haploinsufficiency, suggesting that antecedent mutations may be required to optimize the leukemogenic potential of Smc3 mutations.

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are ligand-activated transcription factors that influence hematopoietic stem cell self-renewal and differentiation. In normal hematopoiesis, RARA and RXRA are dynamically regulated during myeloid maturation, with highest expression in mature neutrophils. In acute myeloid leukemia (AML) RARA and RXRA have parallel expression among AML subtypes, with the highest expression in M4/M5 myelomonocytic and monocytic subtypes. Using a murine reporter assay, we found that natural RXRA ligands, but not RARA ligands, were present in vivo in primary mouse myelomonocytic leukemia cells (derived with an MLL-AF9 retrovirus) and acted as tumor suppressors. RXR ligands were absent in erythroleukemia (derived with TLS-ERG) or T-cell leukemia (derived with activated Notch1), suggesting the presence of natural RXRA ligands specifically in myelomonocytic leukemia. Moreover, we found that deletion of Rxra and Rxrb was naturally selected in MLL-AF9, but not in Notch1 or TLS-ERG leukemias, and that loss of Rxra and Rxrb accelerated leukemic growth. This suggests that Rxrs act as tumor suppressors, but only when exposed to natural ligands. In MLL-AF9 derived leukemia cells, pharmacologic treatment with single-agent retinoid led to modest growth inhibition due to non-permissive activity of the RARA:RXR heterodimer, whereas concurrent activation of both RARA, with all-trans retinoic acid (a pan-RAR ligand), and RXR, by bexarotene (a pan-RXR ligand), enabled efficient co-repressor release and synergistic leukemic apoptosis. We observed that co-repressors release (SMRT/NCoR) from the RARA:RXRA heterodimer was specifically associated with RARA activation, whereas growth inhibition required RXR binding, and this occurred through apoptosis rather than maturation. Generating a series of RXRA mutant proteins we demonstrated the active contribution of RXRA to the activity of the RARA:RXRA heterodimer, specifically requiring the activation domains (AF1 and AF2) and the ability to recruit co-activator to the RXRA element. Finally, we observed a significant dose-dependent effect of combination ATRA and bexarotene treatment in in vivo in MLL-AF9 leukemic mice with a striking reduction in the tumor burden of treated mice compared to the control cohort. These data provide a strategy for clinical retinoid therapies in leukemias beyond acute promyelocytic leukemia and provide a mechanism for integrating retinoid therapy into future clinical trials. Disclosures No relevant conflicts of interest to declare.

Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene-activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y-box binding 1 (YB-1 or YBX1) oncoproteins. Here, we show that knockdown of YB-1 in spontaneously immortalized HaCaT and non-metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB-1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB-1 depletion and promotes cell survival. In summary, our results unveil a functional cross-talk between YB-1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells.

The retinoid X receptor α (RXRA) has been implicated in diverse hematological processes. To identify natural ligands of RXRA that are present in hematopoietic cells, we adapted an upstream activation sequence-green fluorescent protein (UAS-GFP) reporter mouse to detect natural RXRA ligands in vivo. We observed reporter activity in diverse types of hematopoietic cells in vivo. Reporter activity increased during granulocyte colony-stimulating factor (G-CSF)-induced granulopoiesis and after phenylhydrazine (PHZ)-induced anemia, suggesting the presence of dynamically regulated natural RXRA ligands in hematopoietic cells. Mouse plasma activated Gal4-UAS reporter cells in vitro, and plasma from mice treated with G-CSF or PHZ recapitulated the patterns of reporter activation that we observed in vivo. Plasma from mice with dietary vitamin A deficiency only mildly reduced RXRA reporter activity, whereas plasma from mice on a fatty acid restriction diet reduced reporter activity, implicating fatty acids as plasma RXRA ligands. Through differential extraction coupled with mass spectrometry, we identified the long-chain fatty acid C24:5 as a natural RXRA ligand that was greatly increased in abundance in response to hematopoietic stress. Together, these data suggest that natural RXRA ligands are present and dynamically increased in abundance in mouse hematopoietic cells in vivo. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO. for the use of the Flow Cytometry Core. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant P30 CA91842. We thank High-Throughput Screening Center at Washington University School of Medicine in St. Louis, MO. We thank Deborah Laflamme for technical assistance and Feng Gao for statistical assistance. This work was supported by NIH R01 HL128447 (JS Welch), NIH P50 CA171963 (Project 1, JS Welch), and by grants from the Spanish Ministry of Economy and Competitiveness (SAF2015-64287R, SAF2015-71878-REDT) (M Ricote). The mass spectrometry facility at Washington University is supported by NIH P30 DK020579, Daniel Ory. J.S.W., H.N. and M.R. designed experiments, performed experiments, and wrote the manuscript. H.F., O.M., G.H., M.P.M, T.E.F., G.R.B. designed and performed experiments. Sí

Y-box binding protein 1 (YBX-1 or YB-1) is an oncoprotein that promotes replicative immortality, tumor cell invasion and metastasis. The increase in the abundance of YB-1 in the cell or YB-1 translocation from the cytoplasm to the nucleus is characteristic of malignant cell growth. We have previously reported that ΔNp63α, a transcription factor that is known to play a pivotal role in keratinocyte proliferation and differentiation, promotes YB-1 nuclear accumulation. Here, we show that YB-1 is highly expressed in proliferating keratinocytes and is down-regulated during keratinocyte differentiation. ΔNp63α reduces YB-1 protein turnover and leads to accumulation of ubiquitin-conjugated YB-1 into the nucleus. Reduction of YB-1 protein level, following treatment with a DNA-damaging agent, is inhibited by ΔNp63α suggesting that YB-1 and ΔNp63α interplay can support keratinocyte proliferation and protect cells from apoptosis under genotoxic stress.

The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.

The p63 protein is a member of the p53 gene family [1].The TP63 gene encodes isoforms that contain (TA) or lack (∆N) a transactivation domain. ∆Np63α, a critical pro-proliferative factor and a marker of epidermal stemness, is the most commonly expressed p63 protein and is essential for morphogenesis of organs/tissues developing by epithelial-mesenchimal interactions such as the epidermis, teeth, hair and glands [2]. We have recently shown that ∆Np63α is a molecular partner of the Y-box binding protein 1 (YB-1). YB-1, a marker of malignant tumor, is a nucleic acid binding protein with pleiotropic functions, such as alternative splicing, regulation of transcription and translation. Although YB-1 is predominantly cytoplasmic, it is highly expressed in the nuclear compartment of proliferating keratinocytes and squamous carcinoma cells. Here we show that ∆Np63α induces the nuclear accumulation of post-translationally modified forms of YB-1, sumoylation and ubiquitination are both involved in this phenomenon. During keratinocyte differentiation, YB-1 silencing restrains cell proliferation. In proliferating HaCaT and squamous carcinoma cells, YB-1 knockdown causes dramatic cell death and detachment. Our observations suggest that ∆Np63α and YB-1 functional interaction is critically associated with the proliferation and survival of normal and transformed keratinocytes.

Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene-activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y-box binding 1 (YB-1 or YBX1) oncoproteins. Here, we show that knockdown of YB-1 in spontaneously immortalized HaCaT and non-metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB-1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB-1 depletion and promotes cell survival. In summary, our results unveil a functional cross-talk between YB-1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells.