Fan Liu

Heterodimeric JAK–STAT activation as a mechanism of persistence to JAK2 inhibitor therapy

The identification of somatic activating mutations in JAK2 (refsu20091–4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK–STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation.

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferative neoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase originally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 more strongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylate PRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphorylation in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cells using shRNA, we observed increased colony formation and erythroid differentiation. These results indicate that phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.

The Leukemogenicity of AML1-ETO Is Dependent on Site-Specific Lysine Acetylation

A protein that drives the growth of leukemia does so only when it carries a specific posttranslational modification. The chromosomal translocations found in acute myelogenous leukemia (AML) generate oncogenic fusion transcription factors with aberrant transcriptional regulatory properties. Although therapeutic targeting of most leukemia fusion proteins remains elusive, the posttranslational modifications that control their function could be targetable. We found that AML1-ETO, the fusion protein generated by the t(8;21) translocation, is acetylated by the transcriptional coactivator p300 in leukemia cells isolated from t(8;21) AML patients, and that this acetylation is essential for its self-renewal–promoting effects in human cord blood CD34+ cells and its leukemogenicity in mouse models. Inhibition of p300 abrogates the acetylation of AML1-ETO and impairs its ability to promote leukemic transformation. Thus, lysine acetyltransferases represent a potential therapeutic target in AML.

Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: Possible role in 20q - Polycythemia vera

L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34(+) cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34(+)) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34(+) cells as well as in 20q- cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q-) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

Beyond transcription factors: how oncogenic signalling reshapes the epigenetic landscape

Cancer, once thought to be caused largely by genetic alterations, is now considered to be a mixed genetic and epigenetic disease. The epigenetic landscape, which is dictated by covalent DNA and histone modifications, is profoundly altered in transformed cells. These abnormalities may arise from mutations in, or altered expression of, chromatin modifiers. Recent reports on the interplay between cellular signalling pathways and chromatin modifications add another layer of complexity to the already complex regulation of the epigenome. In this Review, we discuss these new studies and how the insights they provide can contribute to a better understanding of the molecular pathogenesis of neoplasia.

Akt Phosphorylates the Transcriptional Repressor Bmi1 to Block Its Effects on the Tumor-Suppressing Ink4a-Arf Locus

Akt counteracts growth-promoting signals by stimulating the transcription of tumor suppressor genes. Silencing the Silencer The Polycomb group protein Bmi1 transcriptionally silences the Ink4a-Arf locus and thus decreases the abundance of the tumor suppressor proteins p16 and p19. Liu et al. found that phosphorylation of Bmi1 by the kinase Akt causes it to dissociate from the Ink4a-Arf locus, which results in increased abundance of p16 and p19 that decreases cellular proliferation, tumor growth, and self-renewal of stem and progenitor cells. Thus, Akt, which is typically activated downstream of growth-promoting signals, can mediate a feedback loop that ultimately attenuates these growth signals. The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16Ink4a and the tumor suppressor p19Arf. Bmi1 plays a key role in oncogenesis and stem cell self-renewal. We report that phosphorylation of human Bmi1 at Ser316 by Akt impaired its function by triggering its dissociation from the Ink4a-Arf locus, which resulted in decreased ubiquitylation of histone H2A and the inability of Bmi1 to promote cellular proliferation and tumor growth. Moreover, Akt-mediated phosphorylation of Bmi1 also inhibited its ability to promote self-renewal of hematopoietic stem and progenitor cells. Our study provides a mechanism for the increased abundance of p16Ink4a and p19Arf seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.

Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis

Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells.

Post-translational modifications of Runx1 regulate its activity in the cell

In this report we review the current knowledge of the interaction of RUNX1(AML1) with serine/threonine kinases, lysine and arginine methyltransferases, lysine acetyltransferases, and histone deacetylases. We also discuss the effect of RUNX1-ETO fusion gene on DNA methylation. RUNX1 post-transcriptional modification can affect its role in influencing differentiation and self-renewal of hematopoietic cells. The goal of these studies is to develop targets for improved leukemia therapy.

Regulation of AKT signaling by Id1 controls t(8;21) leukemia initiation and progression

Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia.

Arginine methyltransferases in normal and malignant hematopoiesis

Arginine methylation is an abundant covalent modification that regulates diverse cellular processes, including transcription, translation, DNA repair, and RNA processing. The enzymes that catalyze these marks are known as the protein arginine methyltransferases (PRMTs), and they can generate asymmetric dimethyl arginine (type I arginine methyltransferases), symmetric dimethylarginine (type II arginine methyltransferases), or monomethyarginine (type III arginine methyltransferases). The PRMTs are capable of modifying diverse substrates, from histone components to specific nuclear and cytoplasmic proteins. Additionally, the PRMTs can orchestrate chromatin remodeling by blocking the docking of other epigenetic modifying enzymes or by recruiting them to specific gene loci. In the hematopoietic system, PRMTs can regulate cell behavior, including the critical balance between stem cell self-renewal and differentiation, in at least two critical ways, via (i) the covalent modification of transcription factors and (ii) the regulation of histone modifications at promoters critical to cell fate determination. Given these important functions, it is not surprising that these processes are altered in hematopoietic malignancies, such as acute myeloid leukemia, where they promote increased self-renewal and impair hematopoietic stem and progenitor cell differentiation.