Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

m6A RNA methylation regulates mitochondrial function.

RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Interferon and myeloproliferative neoplasms: Evolving therapeutic approaches.

Interferons (IFNs) are a diverse group of cytokines whose potent antitumor effects have piqued the interest of scientists for decades. Some of the most sustained clinical accomplishments have been in the field of myeloproliferative neoplasms (MPNs). Here, we discuss how both historical and novel breakthroughs in our understanding of IFN function may lead to more effective therapies for MPNs. The particular relevance and importance of modulating the novel IFN-regulated ULK1 pathway to optimize IFN responses is highlighted.

Interferon signaling in cancer. Non-canonical pathways and control of intracellular immune checkpoints.

The interferons (IFNs) are cytokines with important antineoplastic and immune modulatory effects. These cytokines have been conserved through evolution as important elements of the immune surveillance against cancer. Despite this, defining their precise and specific roles in the generation of antitumor responses remains challenging. Emerging evidence suggests the existence of previously unknown roles for IFNs in the control of the immune response against cancer that may redefine our understanding on how these cytokines function. Beyond the engagement of classical JAK-STAT signaling pathways that promote transcription and expression of gene products, the IFNs engage multiple other signaling cascades to generate products that mediate biological responses and outcomes. There is recent emerging evidence indicating that IFNs control the expression of both traditional immune checkpoints like the PD-L1/PD1 axis, but also less well understood "intracellular" immune checkpoints whose targeting may define new approaches for the treatment of malignancies.

Role of Biomarkers in the Management of Acute Myeloid Leukemia.

Despite many recent advances in treatment options, acute myeloid leukemia (AML) still has a high mortality rate. One important issue in optimizing outcomes for AML patients lies in the limited ability to predict response to specific therapies, duration of response, and likelihood of relapse. With evolving genetic characterization and improving molecular definitions, the ability to predict outcomes and long-term prognosis is slowly improving. The majority of the currently used prognostic assessments relate to molecular and chromosomal abnormalities, as well as response to initial therapy. These risk categories, however, do not account for a large amount of the variability in AML. Laboratory techniques now utilized in the clinic extend beyond bone marrow morphology and single gene sequencing, to next-generation sequencing of large gene panels and multiparameter flow cytometry, among others. Other technologic advances, such as gene expression analysis, have yet to demonstrate enough predictive and prognostic power to be employed in clinical medicine outside of clinical trials, but may be incorporated into the clinic in the future. In this review, we discuss the utility of current biomarkers, and present novel biomarker techniques and strategies that are in development for AML patients. Measurable residual disease (MRD) is a powerful prognostic tool that is increasingly being incorporated into clinical practice, and there are some exciting emerging biomarker technologies that have the potential to improve prognostic power in AML. As AML continues to be a difficult-to-treat disease with poor outcomes in many subtypes, advances in biomarkers that lead to better treatment decisions are greatly needed.

An aberrantly sustained emergency granulopoiesis response accelerates postchemotherapy relapse in MLL1-rearranged acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) with mixed lineage leukemia 1 (MLL1) gene rearrangement is characterized by increased expression of a set of homeodomain transcription factors, including homeobox A9 (HOXA9) and HOXA10. The target genes for these regulators include fibroblast growth factor 2 (FGF2) and Ariadne RBR E3 ubiquitin ligase 2 (ARIH2). FGF2 induces leukemia stem cell expansion in MLL1-rearranged AML. ARIH2 encodes TRIAD1, an E3 ubiquitin ligase required for termination of emergency granulopoiesis and leukemia suppressor function in MLL1-rearranged AML. Receptor tyrosine kinases (RTKs), including the FGF receptor, are TRIAD1 substrates that are possibly relevant to these activities. Using transcriptome analysis, we found increased activity of innate immune response pathways and RTK signaling in bone marrow progenitors from mice with MLL1-rearranged AML. We hypothesized that sustained RTK signaling, because of decreased TRIAD1 activity, impairs termination of emergency granulopoiesis during the innate immune response and contributes to leukemogenesis in this AML subtype. Consistent with this, we found aberrantly sustained emergency granulopoiesis in a murine model of MLL1-rearranged AML, associated with accelerated leukemogenesis. Treating these mice with an inhibitor of TRIAD1-substrate RTKs terminated emergency granulopoiesis, delayed leukemogenesis during emergency granulopoiesis, and normalized innate immune responses when combined with chemotherapy. Emergency granulopoiesis also hastened postchemotherapy relapse in mice with MLL1-rearranged AML, but remission was sustained by ongoing RTK inhibition. Our findings suggest that the physiological stress of infectious challenges may drive AML progression in molecularly defined subsets and identify RTK inhibition as a potential therapeutic approach to counteract this process.

Identification and targeting of novel CDK9 complexes in acute myeloid leukemia.

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

Sirtuin 2-mediated deacetylation of cyclin-dependent kinase 9 promotes STAT1 signaling in type I interferon responses.

Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities. Transcription of these interferon-stimulated genes (ISGs) occurs upon activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. Phosphorylation and acetylation are both events crucial to tightly regulate expression of ISGs. Here, using mouse embryonic fibroblasts and an array of biochemical methods including immunoblotting and kinase assays, we show that sirtuin 2 (SIRT2), a member of the NAD-dependent protein deacetylase family, is involved in type I IFN signaling. We found that SIRT2 deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN-dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727. We also found that SIRT2 is subsequently required for the transcription of ISGs and for IFN-driven antiproliferative responses in both normal and malignant cells. These findings establish the existence of a previously unreported signaling pathway whose function is essential for the control of JAK-STAT signaling and the regulation of IFN responses. Our findings suggest that targeting sirtuin activities may offer an avenue in the development of therapies for managing immune-related diseases and cancer.

Association of a novel circulating tumor DNA next-generating sequencing platform with circulating tumor cells (CTCs) and CTC clusters in metastatic breast cancer.

PURPOSE: Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), can be used to understand disease prognosis, tumor heterogeneity, and dynamic response to treatment in metastatic breast cancer (MBC). We explored a novel, 180-gene ctDNA panel and the association of this platform with CTCs and CTC clusters. METHODS: A total of 40 samples from 22 patients with MBC were included in the study. For the primary analysis, all patients had ctDNA sequencing using the PredicinePLUS™ platform. CTCs and CTC clusters were examined using the CellSearch™ System. Clinical and pathological variables were reported using descriptive analyses. Associations between CTC count and specific genomic alterations were tested using the Mann-Whitney U test. RESULTS: Of 43 sequenced patients, 40 (93%) had at least one detectable genomic alteration with a median of 6 (range 1-22). Fifty-seven different genes were altered, and the landscape of genomic alterations was representative of MBC, including the commonly encountered alterations TP53, PTEN, PIK3CA, ATM, BRCA1, CCND1, ESR1, and MYC. In patients with predominantly hormone-receptor-positive MBC, the number of CTCs was significantly associated with alterations in ESR1 (P < 0.005), GATA3 (P < 0.05), CDH1 (P < 0.0005), and CCND1 (P < 0.05) (Mann-Whitney U test). Thirty-six percent of patients had CTC clusters, which were associated with alterations in CDH1, CCND1, and BRCA1 (all P < 0.05, Mann-Whitney U test). In an independent validation cohort, CTC enumeration confirmed significant associations with ESR1 and GATA3, while CTC clusters were significantly associated with CDH1. CONCLUSIONS: We report on a novel ctDNA platform that detected genomic alterations in the vast majority of tested patients, further indicating potential clinical utility for capturing disease heterogeneity and for disease monitoring. Detection of CTCs and CTC clusters was associated with particular genomic profiles.

Policy Challenges and Opportunities to Address Changing Paradigms in Cancer Care Delivery.

Multiple factors are forcing the healthcare delivery system to change. A movement toward value-based payment models is shifting these systems to team-based integration and coordination of care for better efficiencies and outcomes. Workforce shortages are stressing access and quality of care for patients with cancer and survivors, and their families and caregivers. Innovative therapies are expensive, forcing payers and employers to prioritize resources. Patients are advocating for care models centered on their needs rather than those of providers. In response, payment policies have recently focused on the promotion of alternative payment models that incentivize coordinated, high-quality care with consideration for value and controlling the increasing overall costs associated with cancer and its treatment. Given the multitude of factors confounding cancer care, NCCN convened a multistakeholder working group to examine the challenges and opportunities presented by changing paradigms in cancer care delivery. The group identified key challenges and developed policy recommendations to address 4 high-visibility topics in cancer care delivery. The findings and recommendations were then presented at the NCCN Policy Summit: Policy Challenges and Opportunities to Address Changing Paradigms in Cancer Care Delivery in September 2018, and multistakeholder roundtable panel discussions explored these findings and recommendations along with additional items. This article encapsulates the discussion from the NCCN Working Group meetings and the NCCN Policy Summit, including multistakeholder policy recommendations on delivery issues in cancer care designed to help inform national policies moving forward.

Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses.

The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

Merestinib blocks Mnk kinase activity in acute myeloid leukemia progenitors and exhibits antileukemic effects in vitro and in vivo.

Mitogen-activated protein kinase interacting protein kinases (Mnks) play important roles in the development and progression of acute myeloid leukemia (AML) by regulating eukaryotic translation initiation factor 4E (eIF4E) activation. Inhibiting Mnk1/2-induced phosphorylation of eIF4E may represent a unique approach for the treatment of AML. We provide evidence for antileukemic effects of merestinib, an orally bioavailable multikinase inhibitor with suppressive effects on Mnk activity. Our studies show that merestinib effectively blocks eIF4E phosphorylation in AML cells and suppresses primitive leukemic progenitors from AML patients in vitro and in an AML xenograft model in vivo. Our findings provide evidence for potent preclinical antileukemic properties of merestinib and support its clinical development for the treatment of patients with AML.

Intersection of mTOR and STAT signaling in immunity.

Optimal regulation of immune networks is essential for the generation of effective immune responses, and defects in such networks can lead to immunodeficiency while uncontrolled responses can result in autoimmune disorders. mTOR and STAT signaling cascades are key regulators of the differentiation and function of cells of the immune system. Both pathways act as sensors and transducers of environmental stimuli, and recent evidence has revealed points of crosstalk between these pathways, highlighting synergistic regulation of immune cell differentiation and function. We review here the current understanding of mTOR and STAT interactions in T cells and innate immune cells, and discuss potential mechanisms underlying these events. We further outline models for the intersection of these pathways in the regulation of immunity and highlight important areas for future research.

The Interferon Consensus Sequence Binding Protein (Icsbp/Irf8) Is Required for Termination of Emergency Granulopoiesis.

Emergency granulopoiesis occurs in response to infectious or inflammatory challenge and is a component of the innate immune response. Some molecular events involved in initiating emergency granulopoiesis are known, but termination of this process is less well defined. In this study, we found that the interferon consensus sequence binding protein (Icsbp/Irf8) was required to terminate emergency granulopoiesis. Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. Expression of Icsbp is decreased in chronic myeloid leukemia, and Icsbp(-/-) mice exhibit progressive granulocytosis with evolution to blast crisis, similar to the course of human chronic myeloid leukemia. In this study, we found aberrantly sustained granulocyte production in Icsbp(-/-) mice after stimulation of an emergency granulopoiesis response. Icsbp represses transcription of the genes encoding Fas-associated phosphatase 1 (Fap1) and growth arrest-specific 2 (Gas2) and activates genes encoding Fanconi C and F. After stimulation of emergency granulopoiesis, we found increased and sustained expression of Fap1 and Gas2 in bone marrow myeloid progenitor cells from Icsbp(-/-) mice in comparison with the wild type. This was associated with resistance to Fas-induced apoptosis and increased ß-catenin activity in these cells. We also found that repeated episodes of emergency granulopoiesis accelerated progression to acute myeloid leukemia in Icsbp(-/-) mice. This was associated with impaired Fanconi C and F expression and increased sensitivity to DNA damage in bone marrow myeloid progenitors. Our results suggest that impaired Icsbp expression enhances leukemogenesis by deregulating processes that normally limit granulocyte expansion during the innate immune response.

Interferon γ (IFNγ) Signaling via Mechanistic Target of Rapamycin Complex 2 (mTORC2) and Regulatory Effects in the Generation of Type II Interferon Biological Responses.

We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFNγ signaling and type II IFN responses.

Dual targeting of eIF4E by blocking MNK and mTOR pathways in leukemia.

Dysregulation of mRNA translation leads to aberrant activation of cellular pathways that promote expansion and survival of leukemic clones. A key element of the initiation translation complex is eIF4E (eukaryotic translation initiation factor 4E). The mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways play important roles in the regulation of eIF4E expression and downstream functional outcomes. Mitogen-activated protein kinase interacting protein kinases (Mnks) control translation by phosphorylation of eIF4E, whereas the mTOR kinase phosphorylates/de-activates the eIF4E inhibitor, 4E-BP1, to release translational repression. Both pathways are often abnormally activated in leukemia cells and promote cell survival events by controlling expression of oncogenic proteins. Targeting these pathways may provide approaches to avoid aberrant proliferation and neoplastic transformation.

Regulatory effects of SKAR in interferon α signaling and its role in the generation of type I IFN responses.

We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.