The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

Quantitative Age-specific Variability of Plasma Proteins in Healthy Neonates, Children and Adults.

Human blood plasma is a complex biological fluid containing soluble proteins, sugars, hormones, electrolytes, and dissolved gasses. As plasma interacts with a wide array of bodily systems, changes in protein expression, or the presence or absence of specific proteins are regularly used in the clinic as a molecular biomarker tool. A large body of literature exists detailing proteomic changes in pathologic contexts, however little research has been conducted on the quantitation of the plasma proteome in age-specific, healthy subjects, especially in pediatrics. In this study, we utilized SWATH-MS to identify and quantify proteins in the blood plasma of healthy neonates, infants under 1 year of age, children between 1-5 years, and adults. We identified more than 100 proteins that showed significant differential expression levels across these age groups, and we analyzed variation in protein expression across the age spectrum. The plasma proteomic profiles of neonates were strikingly dissimilar to the older children and adults. By extracting the SWATH data against a large human spectral library we increased protein identification more than 6-fold (940 proteins) and confirmed the concentrations of several of these using ELISA. The results of this study map the variation in expression of proteins and pathways often implicated in disease, and so have significant clinical implication.

RNA kinetics influence the response to transcriptional perturbation in leukaemia cell lines.

Therapeutic targeting of dysregulated transcription has emerged as a promising strategy for the treatment of cancers, such as leukaemias. The therapeutic response to small molecule inhibitors of Bromodomain-Containing Proteins (BRD), such as BRD2 and BRD4, P300/cAMP-response element binding protein (CBP) and Cyclin Dependent Kinases (CDKs), is generally attributed to the selective disruption of oncogenic gene expression driven by enhancers, super-enhancers (SEs) and lineage-specific transcription factors (TFs), including the c-MYC oncogene. The selectivity of compounds targeting the transcriptional machinery may be further shaped by post-transcriptional processes. To quantitatively assess the contribution of post-transcriptional regulation in responses to transcription inhibition, we performed multi-omics analyses to accurately measure mRNA production and decay kinetics. We demonstrate that it is not only the selective disruption of mRNA production, but rather mRNA decay rates that largely influence the selectivity associated with transcriptional inhibition. Accordingly, genes down-regulated with transcriptional inhibitors are largely characterized by extremely rapid mRNA production and turnover. In line with this notion, stabilization of the c-MYC transcript through swapping of its 3' untranslated region (UTR) rendered c-MYC insensitive to transcriptional targeting. This failed to negate the impact on c-MYC downstream targets and did not abrogate therapeutic responses. Finally, we provide evidence that modulating post-transcriptional pathways, such as through ELAVL1 targeting, can sensitize long-lived mRNAs to transcriptional inhibition and be considered as a combination therapy approach in leukaemia. Taken together, these data demonstrate that mRNA kinetics influence the therapeutic response to transcriptional perturbation and can be modulated for novel therapeutic outcomes using transcriptional agents in leukaemia.

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress.

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

SFPQ-ABL1 and BCR-ABL1 use different signaling networks to drive B-cell acute lymphoblastic leukemia.

Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype of B-cell ALL characterized by a gene expression profile resembling Philadelphia chromosome-positive ALL (Ph+ ALL) in the absence of BCR-ABL1. Tyrosine kinase-activating fusions, some involving ABL1, are recurrent drivers of Ph-like ALL and are targetable with tyrosine kinase inhibitors (TKIs). We identified a rare instance of SFPQ-ABL1 in a child with Ph-like ALL. SFPQ-ABL1 expressed in cytokine-dependent cell lines was sufficient to transform cells and these cells were sensitive to ABL1-targeting TKIs. In contrast to BCR-ABL1, SFPQ-ABL1 localized to the nuclear compartment and was a weaker driver of cellular proliferation. Phosphoproteomics analysis showed upregulation of cell cycle, DNA replication, and spliceosome pathways, and downregulation of signal transduction pathways, including ErbB, NF-κB, vascular endothelial growth factor (VEGF), and MAPK signaling in SFPQ-ABL1-expressing cells compared with BCR-ABL1-expressing cells. SFPQ-ABL1 expression did not activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling and was associated with phosphorylation of G2/M cell cycle proteins. SFPQ-ABL1 was sensitive to navitoclax and S-63845 and promotes cell survival by maintaining expression of Mcl-1 and Bcl-xL. SFPQ-ABL1 has functionally distinct mechanisms by which it drives ALL, including subcellular localization, proliferative capacity, and activation of cellular pathways. These findings highlight the role that fusion partners have in mediating the function of ABL1 fusions.

The Combination of Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Delays KMT2A-Rearranged Leukemia Progression.

Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Serine Biosynthesis Is a Metabolic Vulnerability in FLT3-ITD-Driven Acute Myeloid Leukemia.

Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD-driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo. Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD-induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. SIGNIFICANCE: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD-driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD-driven AMLs to chemotherapy.This article is highlighted in the In This Issue feature, p. 1307.

Mathematical modelling indicates that lower activity of the haemostatic system in neonates is primarily due to lower prothrombin concentration.

Haemostasis is governed by a highly complex system of interacting proteins. Due to the central role of thrombin, thrombin generation and specifically the thrombin generation curve (TGC) is commonly used as an indicator of haemostatic activity. Functional characteristics of the haemostatic system in neonates and children are significantly different compared with adults; at the same time plasma levels of haemostatic proteins vary considerably with age. However, relating one to the other has been difficult, both due to significant inter-individual differences for individuals of similar age and the complexity of the biochemical reactions underlying haemostasis. Mathematical modelling has been very successful at representing the biochemistry of blood clotting. In this study we address the challenge of large inter-individual variability by parameterising the Hockin-Mann model with data from individual patients, across different age groups from neonates to adults. Calculating TGCs for each patient of a specific age group provides us with insight into the variability of haemostatic activity across that age group. From our model we observe that two commonly used metrics for haemostatic activity are significantly lower in neonates than in older patients. Because both metrics are strongly determined by prothrombin and prothrombin levels are considerably lower in neonates we conclude that decreased haemostatic activity in neonates is due to lower prothrombin availability.

Blood biomarkers in paediatric mild traumatic brain injury: a systematic review.

OBJECTIVE: To summarize all current studies focusing on blood biomarkers in paediatric mild traumatic brain injury (mTBI) and to outline the possible use of blood biomarkers for diagnostic, prognostic and monitoring purposes within this setting. METHODS: A systematic review following the PRISMA guidelines was conducted using the MEDLINE, PubMed and EMBASE databases. RESULTS: A total of 21 studies were included in the review, encompassing a total of 14 different biomarkers. Seventeen (81%) of these studies found a significant association between biomarker concentration and mTBI characteristics, however results from studies to date are diverse and at times conflicting. CONCLUSION: GFAP appears to be a promising blood biomarker for the prognosis and monitoring of mTBI, whereas UCH-L1 appears more promising at mTBI diagnosis. Despite this, the overall heterogeneity in assessed biomarkers, study design and measurement tools has made drawing specific conclusions challenging. Future research will require more uniform study design and methodological approaches to allow for the comparison, corroboration and validation of blood biomarkers within the context of paediatric mTBI.