using-crisprcas9-gene-editing-to-investigate-oncogenic-activity

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Brigham and Women's Hospital, Harvard Medical School

Broad Institute

Harvard Medical School

Interferon-Î± (IFNÎ±) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNÎ± reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNÎ± on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNÎ± on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNÎ± treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNÎ± treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNÎ± on Jak2V617F mutant and normal hematopoiesis and suggest that IFNÎ± achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNÎ± and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy.

Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-Î± in a murine model of polycythemia vera.

L&#39;interf&eacute;ron-&alpha; (IFN&alpha;) est un traitement efficace des patients atteints de syndromes my&eacute;loprolif&eacute;ratifs n&eacute;oplasmes (NPP). En plus d&#39;induire des r&eacute;ponses h&eacute;matologiques chez la plupart des patients MPN, IFN&alpha; r&eacute;duit le fardeau all&eacute;lique JAK2V617F et peut rendre le clone mutant JAK2V617F ind&eacute;tectable chez certains patients. Le m&eacute;canisme pr&eacute;cis qui sous-tendent ces r&eacute;ponses est encore partiellement compris, et si les r&eacute;ponses mol&eacute;culaires que l&#39;on voit se produisent en raison des effets de l&#39;IFN&alpha; sur les cellules souches mutantes de JAK2V617F est l&#39;objet d&#39;un d&eacute;bat. &Agrave; l&#39;aide d&#39;un mod&egrave;le murin de MPN Jak2V617F, nous avons &eacute;tudi&eacute; les effets de l&#39;IFN&alpha; sur la multiplication de MPN Jak2V617F stem cells in vivo. Nous rapportons que le traitement de l&#39;IFN&alpha; induit des r&eacute;ponses h&eacute;matologiques dans le mod&egrave;le et entra&icirc;ne l&#39;appauvrissement de la couche des cellules Jak2V617F MPN-multiplication au fil du temps, alt&eacute;rant la transplantation de la maladie. Nous d&eacute;montrons que le traitement de l&#39;IFN&alpha; induit l&#39;activation du cycle cellulaire de Jak2V617F mutants cellules souches h&eacute;matopo&iuml;&eacute;tiques &agrave; long terme et promeut un programme de diff&eacute;renciation de lign&eacute;e &eacute;rythro&iuml;de pr&eacute;d&eacute;termin&eacute;. Ces r&eacute;sultats donnent un aper&ccedil;u sur les effets diff&eacute;rentiels de l&#39;IFN&alpha; dans Jak2V617F mutant et l&#39;h&eacute;matopo&iuml;&egrave;se normale et sugg&egrave;rent qu&#39;IFN&alpha; permet d&#39;obtenir des r&eacute;missions mol&eacute;culaires chez les patients MPN par ses effets sur les cellules souches MPN. En outre, ces r&eacute;sultats supportent la combinatoires approches th&eacute;rapeutiques en MPN en simultan&eacute;ment appauvrissant dormants JAK2V617F MPN-multiplication des cellules souches avec IFN&alpha; et en ciblant la prog&eacute;niture prolif&eacute;rant en aval avec des inhibiteurs de JAK2 ou chimioth&eacute;rapie cytotoxique.

D&eacute;pl&eacute;tion des cellules de tige sur multiplication de n&eacute;oplasmes my&eacute;loprolif&eacute;ratifs de Jak2V617F par l&#39;interf&eacute;ron-&alpha; dans un mod&egrave;le murin de la maladie de Vaquez vera.

L&#39;interferone-&alpha; (IFN&alpha;) &egrave; un trattamento efficace dei pazienti con neoplasie mieloproliferative (MPNs). Oltre a indurre risposte ematologiche nella maggior parte dei pazienti MPN, IFN&alpha; riduce l&#39;onere di JAK2V617F allelica e pu&ograve; rendere il clone mutante di JAK2V617F non rilevabili in alcuni pazienti. Il preciso meccanismo alla base di queste risposte &egrave; inteso in modo incompleto e se le risposte molecolari che sono visto verificano a causa di effetti di IFN&alpha; sulle cellule staminali mutante JAK2V617F &egrave; dibattuto. Utilizzando un modello murino di Jak2V617F MPN, abbiamo studiato gli effetti di IFN&alpha; Jak2V617F MPN-moltiplicazione delle cellule staminali in vivo. Segnaliamo che IFN&alpha; trattamento induce risposte ematologiche nel modello e provoca deplezione delle cellule Jak2V617F MPN-moltiplicazione nel corso del tempo, compromettendo trapianto di malattia. Dimostriamo che il trattamento IFN&alpha; induce attivazione ciclo cellulare di Jak2V617F mutante a lungo termine cellule staminali ematopoietiche e promuove un programma di differenziazione eritroide predeterminato-lineage. Questi risultati forniscono approfondimenti gli effetti differenziali di IFN&alpha; Jak2V617F mutante e ematopoiesi normale e suggeriscono che l&#39;IFN&alpha; raggiunge remissioni molecolari in pazienti MPN attraverso i suoi effetti sulle cellule staminali MPN. Inoltre, questi risultati supportano combinatoriali approcci terapeutici in MPN contemporaneamente che riducono le dormienti JAK2V617F MPN-moltiplicazione cellule staminali con IFN&alpha; e targeting proliferante progenie a valle con JAK2 inibitori o chemioterapia citotossica.

Deplezione di Jak2V617F mieloproliferative neoplasia-moltiplicazione cellule staminali da interferone-&alpha; in un modello murino di policitemia vera.

JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs). Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU), a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability.

Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN), but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of patients with CALR-mutant MPN. We further show that the thrombopoietin receptor MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN.

Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation.

Chronic myeloproliferative neoplasms (MPN) comprise a spectrum of clonal neoplastic disorders characterized by overproduction of terminally differentiated cells of the myeloid lineage. A common genetic basis for the BCR-ABL-negative MPN disorders was elucidated in 2005 with the identification of the JAK2V617F mutation in the majority of MPN patients. The discovery of JAK2V617F had a dramatic impact on the diagnosis and treatment of MPN. Testing for JAK2 mutations is now included in the World Health Organization (WHO) criteria for the diagnosis of MPN, and in 2011 the oral JAK2 kinase inhibitor ruxolitinib became the first Food and Drug Administration (FDA)-approved drug for the treatment of myelofibrosis. The drug is now also approved in Europe and Canada.

Underlying mechanisms of the JAK2V617F mutation in the pathogenesis of myeloproliferative neoplasms.

Following the discovery of the JAK2V617F mutation, Janus kinase (JAK) 2 inhibitors were developed as rationally designed therapy in myeloproliferative neoplasms (MPNs). Although JAK2 inhibitors have clinical efficacy in MPN, they are not clonally selective for the JAK2V617F-mutant cells. Because activated JAK-signal transducer and activator of transcription (STAT) signaling is a common feature of MPN, JAK2 inhibitors are efficacious regardless of the specific MPN phenotypic driver mutation. The Food and Drug Administration approved the JAK1/JAK2 inhibitor, ruxolitinib, for the treatment of myelofibrosis and polycythemia vera. Additional JAK2 inhibitors are currently in advanced phased clinical trials.

The Development and Use of Janus Kinase 2 Inhibitors for the Treatment of Myeloproliferative Neoplasms.

Kinase Inhibitors in the Treatment of Myeloid Malignancies.

Mutations in calreticulin (CALR) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms (MPN). Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C-terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells, binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C-terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extra-cellular domain of MPL and that three tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth while its chaperone and polypeptide binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in MPN.

Ann Mullally

Division of Hematology,
Department of Medicine,
Division of Hematology, Department of Medicine

Brigham and Women's Hospital, Harvard Medical School

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Ann Mullally

Division of Hematology,Department of Medicine,Division of Hematology, Department of Medicine

Brigham and Women's Hospital, Harvard Medical School

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Division of Hematology,
Department of Medicine,
Division of Hematology, Department of Medicine