Julien Ablain

A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.

CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation

Visualizing the beginnings of melanoma In cancer biology, a tumor begins from a single cell within a group of precancerous cells that share genetic mutations. Kaufman et al. used a zebrafish melanoma model to visualize cancer initiation (see the Perspective by Boumahdi and Blanpain). They used a fluorescent reporter that specifically lit up neural crest progenitors that are only present during embryogenesis or during adult melanoma tumor formation. The appearance of this tumor correlated with a set of gene regulatory elements, called super-enhancers, whose identification and manipulation may prove beneficial in detecting and preventing melanoma initiation. Science, this issue p. 10.1126/science.aad2197; see also p. 453 Melanocytes with oncogenic or tumor suppressor mutations revert to expressing the crestin gene early in melanoma formation. [Also see Perspective by Boumahdi and Blanpain] INTRODUCTION The “cancerized field” concept posits that cells in a given tissue sharing an oncogenic mutation are cancer-prone, yet only discreet clones within the field initiate tumors. Studying the process of cancer initiation has remained challenging because of (i) the rarity of these events, (ii) the difficulty of visiualizing initiating clones in living organisms, and (iii) the transient nature of a newly transformed clone emerging before it expands to form an early tumor. A more complete understanding of the molecular processes that regulate cancer initiation could provide important prognostic information about which precancerous lesions are most prone to becoming cancer and also implicate druggable molecular pathways that, when inhibited, may prevent the cancer from ever starting. RATIONALE The majority of benign nevi carry oncogenic BRAFV600E mutations and can be considered a cancerized field of melanocytes, but they only rarely convert to melanoma. In an effort to define events that initiate cancer, we used a melanoma model in the zebrafish in which the human BRAFV600E oncogene is driven by the melanocyte-specific mitfa promoter. When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward. RESULTS We developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors. We show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. Early crestin+ patches of cells expand and are transplantable in a manner consistent with their possessing tumorigenic activity, and they exhibit a gene expression pattern consistent with the NCP identity readout by the crestin reporter. The crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, whereas CRISPR/Cas9 targeting of sox10 delayed melanoma onset. We show activation of super-enhancers at NCP genes in both zebrafish and human melanomas, identifying an epigenetic mechanism for control of this NCP signature leading to melanoma. CONCLUSION This work using our zebrafish melanoma model and in vivo reporter of NCP identity allows us to see cancer from its birth as a single cell and shows the importance of NCP-state reemergence as a key event in melanoma initiation from a field of cancer-prone melanocytes. Thus, in addition to the typical fixed genetic alterations in oncogenes and tumor supressors that are required for cancer development, the reemergence of progenitor identity may be an additional rate-limiting step in the formation of melanoma. Preventing NCP reemergence in a field of cancer-prone melanocytes may thus prove therapeutically useful, and the association of NCP genes with super-enhancer regulatory elements implicates the associated druggable epigenetic machinery in this process. Neural crest reporter expression in melanoma. The crestin:EGFP transgene is specifically expressed in melanoma in BRAFV600E/p53 mutant melanoma-prone zebrafish. (Top) A single cell expressing crestin:EGFP expands into a small patch of cells over the course of 2 weeks, capturing the initiation of melanoma formation (bracket). (Bottom) A fully formed melanoma specifically expresses crestin:EGFP, whereas the rest of the fish remains EGFP-negative. The “cancerized field” concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAFV600E mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

Revisiting the differentiation paradigm in acute promyelocytic leukemia

As the result of intense clinical and basic research, acute promyelocytic leukemia (APL) has progressively evolved from a deadly to a curable disease. Historically, efforts aimed at understanding the molecular bases for therapy response have repeatedly illuminated APL pathogenesis. The classic model attributes this therapeutic success to the transcriptional reactivation elicited by retinoic acid and the resulting overcoming of the differentiation block characteristic of APL blasts. However, in clinical practice, retinoic acid by itself only rarely yields prolonged remissions, even though it induces massive differentiation. In contrast, as a single agent, arsenic trioxide neither directly activates transcription nor triggers terminal differentiation ex vivo, but cures many patients. Here we review the evidence from recent ex vivo and in vivo studies that allow a reassessment of the role of differentiation in APL cure. We discuss alternative models in which PML-RARA degradation and the subsequent loss of APL cell self-renewal play central roles. Rather than therapy aimed at inducing differentiation, targeting cancer cell self-renewal may represent a more effective goal, achievable by a broader range of therapeutic agents.

Activation of a promyelocytic leukemia–tumor protein 53 axis underlies acute promyelocytic leukemia cure

Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)–retinoic acid receptor-α (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml–transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA–driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies.

Therapy-induced selective loss of leukemia-initiating activity in murine adult T cell leukemia

Treatment with a combination of interferon-α and arsenic trioxide ablates leukemia-initiating activity before reducing primary tumor bulk in a murine model of adult T cell leukemia.

Of fish and men: using zebrafish to fight human diseases

Long restricted to the field of developmental biology, the use of the zebrafish (Danio rerio) has extended to the study of human pathogenesis. Fostered by the rapid adaptation of new technologies, the design and analysis of fish models of human diseases have contributed important findings that are now making their way from aquariums to clinics. Here we outline the clinical relevance of the zebrafish as a model organism.

Comprehensive genomic screens identify a role for PLZF-RARα as a positive regulator of cell proliferation via direct regulation of c-MYC

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.

Uncoupling RARA transcriptional activation and degradation clarifies the bases for APL response to therapies

Synthetic retinoids activate RARA- or PML/RARA-dependent transcription, but fail to degrade RARA or PML/RARA protein, which is insufficient for eradication of acute promyelocytic leukemia.

The Drug-Induced Degradation of Oncoproteins: An Unexpected Achilles' Heel of Cancer Cells?

Many targeted therapies against cancer are aimed at inhibiting the enzymatic activity of kinases. Thus far, this approach has undoubtedly yielded significant clinical improvements, but has only rarely achieved cures. Other drugs, which selectively elicit proteasome-dependent degradation of oncoproteins, induce the loss of cancer cell self-renewal and promote cell differentiation and/or apoptosis. In acute promyelocytic leukemia, the cooperative degradation of PML/RARA by arsenic and retinoic acid cures most patients. In this condition and others, drug-induced proteolysis of oncoproteins is feasible and underlies improved clinical outcome. Several transcription factors, nuclear receptors, or fusion proteins driving cancer growth could be candidates for proteolysis-based drug-discovery programs.

Retinoic acid signaling in cancer: The parable of acute promyelocytic leukemia

Inevitably fatal some 40 years, acute promyelocytic leukemia (APL) can now be cured in more than 95% of cases. This clinical success story is tightly linked to tremendous progress in our understanding of retinoic acid (RA) signaling. The discovery of retinoic acid receptor alpha (RARA) was followed by the cloning of the chromosomal translocations driving APL, all of which involve RARA. Since then, new findings on the biology of nuclear receptors have progressively enlightened the basis for the clinical efficacy of RA in APL. Reciprocally, the disease offered a range of angles to approach the cellular and molecular mechanisms of RA action. This virtuous circle contributed to make APL one of the best‐understood cancers from both clinical and biological standpoints. Yet, some important questions remain unanswered including how lessons learnt from RA‐triggered APL cure can help design new therapies for other malignancies.