Highlights d Human iPSC-cardiomyocytes were used for MAP4K4 target validation and drug discovery d MAP4K4 shRNA protects hiPSC-cardiomyocytes from lethal oxidative stress d MAP4K4 inhibitors promote hiPSC-cardiomyocyte survival and function d MAP4K4 inhibition markedly reduces cardiac ischemiareperfusion injury in mice

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BackgroundNeurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved.ResultsAnalysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered.ConclusionsOur study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2β were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.

Given the poor track record to date of animal models for creating cardioprotective drugs, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been proposed as a therapeutically relevant human platform to guide target validation and cardiac drug development. Mitogen-Activated Protein Kinase Kinase Kinase Kinase-4 (MAP4K4) is an "upstream" member of the MAPK superfamily that is implicated in human cardiac muscle cell death from oxidative stress, based on gene silencing and pharmacological inhibition in hPSC-CMs. A further role for MAP4K4 was proposed in heart muscle cell death triggered by cardiotoxic anti-cancer drugs, given its reported activation in failing human hearts with doxorubicin (DOX) cardiomyopathy, and its activation acutely by DOX in cultured cardiomyocytes. Here, we report successful protection from DOX in two independent hPSC-CM lines, using two potent, highly selective MAP4K4 inhibitors. The MAP4K4 inhibitors enhanced viability and reduced apoptosis at otherwise lethal concentrations of DOX, and preserved cardiomyocyte function, as measured by spontaneous calcium transients, at sub-maximal ones. Notably, in contrast, no intereference was seen in tumor cell killing, caspase activation, or mitochondrial membrane dissipation by DOX, in human cancer cell lines. Thus, MAP4K4 is a plausible, tractable, selective therapeutic target in DOX-induced human heart muscle cell death. With earlier diagnosis and improved treatments for cancer, cardiovascular disease has become the main alternative cause of death in cancer survivors 1-4. This heightened risk is ascribable to the cardiac toxicity of several routine anti-cancer agents including, especially, anthracyclines like doxorubicin (DOX) 5. Though some relief can result from standard heart failure medications, no approved therapy apart from the iron chelator dexrazoxane addresses the responsible cytotoxic mechanisms and effector pathways operating in the damaged cardiomyocytes. For anthracyclines, these include diverse reported mediators-binding to nuclear topoisomerase 2β, thereby triggering DNA double-strand breaks, p53-dependent apoptosis, mitochondrial dysfunction, reactive oxygen species, and programmed iron-dependent cell death (ferroptosis) 1,6,7. Cardiotoxicity can be acute, early, or late; early intervention based on subclinical abnormalities may be key to averting the delayed or cumulative effects 8. Notably, acute toxicity is seen in up to 30% of patients receiving anthracyclines, soon after infusion 4. Current cardioprotection trials in cancer rely chiefly on routine heart failure medications (ACE inhibitors, β-blockers, angiotensin receptor blockers), which mitigate the symptoms and signs, with little or no demonstrated impact on cardiomyocyte death. Therapeutic progress has been hampered, in part, by the failure of animal models alone to predict clinical success in cardioprotection. For instance, nearly all strategies for protection in ischemic heart disease, a more intensively studied indication, have failed between phases I and I...

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1(Wnt1Cre) mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1(DbhiCre) animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3(+)/ Ret(+) cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells.

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growthfactor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells.