Abstract AIMS Glioma stem cells (GSCs) are believed to underpin treatment resistance in glioblastoma by virtue of their self- renewal capabilities and presumed transcriptional plasticity. Understanding how cell state diversity is established and maintained is crucial to make therapeutic gains. We have previously shown mitochondrial membrane potential (MMP) to be a functionally relevant source of extrinsic noise in embryonic and haematopoietic stem cells. This study evaluates cellular MMP as an axis of variability in GSCs. METHOD MMP was measured by flow cytometry in patient-derived GSC lines using cationic dyes that diffuse reversibly across the mitochondrial membrane in a voltage dependent manner. MMP-high and low populations (top and bottom 10-20%) were fractionated and subjected to RNA-sequencing and assays of proliferation and clonogenicity. Correlation between MMP and global transcription rate was investigated through a uridine analogue (5-EU) incorporation assay. The relationship between MMP and quiescence was assessed, with quiescence defined as inducible histone2B-GFP label retention after 10 days. Timecourse data for MMP was obtained through sequential staining. RESULTS RNA sequencing identified differential gene expression and exon utilization according to MMP status. MMP- high cells upregulated cell cycle genes, while MMP-low cells exhibited a diverse signature suggestive of a more differentiated state. A greater proportion of MMP-high cells were in cycle, but MMP-low cells formed more colonies. MMP positively correlated with global transcription rate in both cycling and quiescent GSCs. Time- course experiments revealed dynamic periodicity in cellular MMP. CONCLUSION The MMP axis reveals transcriptional and functional heterogeneity in GSCs and importantly unmasks phenotypic variability within the treatment-refractory quiescent compartment. The positive correlation between MMP and transcription rate, along with splicing differences, suggests a mechanism underlying functional diversity. Given the oscillatory dynamics of MMP, we postulate that stable-low MMP may define a deeply quiescent, therapy-resistant state. Evaluating MMP as a tractable regulator of GSC fate is warranted.

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.

The ETV6-RUNX1 onco-fusion arises in utero , initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs in vivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1(-/-) mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1(-/-) cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.

SummaryThe ETV6-RUNX1 onco-fusion arisesin utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results implicate chemical intervention in the RUNX1 program as an exciting therapeutic opportunity in ALL.

During ageing, the haematopoietic stem cell (HSC) pool expands numerically, but declines functionally. This functional decline is characterised by myeloid skewing and decreased long-term reconstitution potential and clinically manifests as anaemia, immune compromise and increased risk of clonal malignancy. We postulate that HSCs do not synchronously functionally decline with age, but instead represent a spectrum in which physiologically aged HSCs become dominant. In this study, we aimed to reveal properties that might identify physiologically young HSCs during chronological ageing and to exploit these in an attempt to rescue haematopoietic ageing. Young adult ((y), 2-3 months) and aged (old (o), >18 months) mouse HSCs were profiled and we observed a significant decrease (30-50%) in mitochondrial membrane potential (MMP) in oHSCs. Interestingly, a small fraction (15%) of oHSCs maintained a similar MMP to the bulk (70%) of yHSCs. We explored, initially at the transcriptional level, whether these MMPhigh HSCs are distinct from MMPlow HSCs. Strikingly, RNA sequencing of MMPhigh and MMPlow young and aged HSCs revealed that samples cluster by MMP over age. MMPhigh young and aged HSCs were characterised by upregulation of lymphoid and erythroid lineage markers, as well as RNA processing, MYC and E2F pathways. MMPlow young and aged HSCs were transcriptionally associated with ageing, inflammation and myeloid bias. Based on these results, we hypothesised that enhancing MMP in oHSCs might restore lineage-balanced peripheral blood (PB) output, used as a measure of functional improvement. To achieve this, we chemically enhanced MMP in oHSCs in vivo, using the mitochondrially-targeted drug mitoquinol (MQ). Interestingly, our initial experiments show that a 5-day treatment with MQ significantly shifted the B-cell/myeloid ratio in PB from 0.6 (aged) to 1.5 (MQ), in the direction of the ratio observed in young mice (2.9). This ratio change was not due to numerical depletion of myeloid cells, but due to restoration of B-cell numbers, including IgM+ B-cells commonly reduced with age. To assess whether the effect of MQ was due to HSC-intrinsic changes and could be sustained over time, HSCs were isolated from MQ-treated or untreated aged mice and transplanted into lethally irradiated recipients. Our experiments to date show that HSCs isolated from MQ-treated aged mice show superior engraftment and faster and greater B-cell reconstitution than HSCs from age-matched untreated animals, and that these improvements are stable over the 16-week assay. Based on our sequencing results indicating that RNA processing, MYC and E2F pathways are associated with MMPhigh HSCs, and previous work reporting a relationship between MMP and rate of mRNA transcription (das Neves et al., PLoS Biol. 2010; Johnston et al., PLoS Comput. Biol. 2012), we explored the possibility that MMP might orchestrate the observed changes by altering transcriptional rate of HSCs. We tested transcription rate in HSCs in vivo and found that yHSCs display a particularly high rate of mRNA transcription. Rate of transcription was significantly reduced in oHSCs compared to yHSCs, equivalent to their quantitative reduction in MMP. We could demonstrate a direct correlation between MMP and transcription rate in HSCs, by showing that MMPhigh sorted HSCs were transcribing nearly twice as fast as MMPlow sorted HSCs. Furthermore, in vivo injection with mitochondrial uncoupler CCCP caused a similar reduction in transcription rate of HSCs (>50%) as did conventional RNA Pol-II inhibitors (DRB, Flavopiridol), hereby demonstrating that transcription rate directly depends on MMP. This work indicates that mitochondrial state can separate HSCs with distinct transcriptional profiles linked to different cell fates. We speculate that changes in transcriptional profile arise from MMP-driven regulation of transcriptional rate in HSCs. This would open up the possibility that pharmacological manipulation of mitochondrial activity can alter transcriptional programs of HSCs with consequences for functionality. Disclosures No relevant conflicts of interest to declare.

Leukaemia is the most common childhood cancer and acute lymphoblastic leukaemia (ALL) is responsible for a third of all childhood cancer deaths. Despite the overall good prognosis, failure to eradicate the disease leads to relapse in ∼20% of patients and survival rates for infants are less than 50%. There is therefore a clear unmet need for less toxic and more effective targeted treatments. ETV6-RUNX1 translocation is the first-hit event in ∼25% of B-ALLs, initiating a clinically silent pre-leukaemia in utero. The translocation fuses almost the entire RUNX1 protein to the repressor domain of ETV6 and the chimeric product is proposed to mediate a block in B cell differentiation by repressing RUNX1 targets. Interestingly, the remaining RUNX1 allele is not mutated, but is instead often amplified suggesting an onco-supportive role of the endogenous RUNX1. Using genetic and pharmacological inhibition, we have discovered that RUNX1/CBF is essential for the growth and survival of B-ALL cells in vitro and in vivo. Global RNA-Seq on CBF-depleted B-ALL cells revealed significant downregulation of E2F and Myc signature genes along with a marked increase of p53 targets, consistent with these cells accumulating in G0 and subsequently becoming apoptotic. Notably, RNA-Seq and mass cytometry analyses of pro-B cells derived from an hiPSC ETV6-RUNX1 knockin model reveal a similar cell cycle trend indicating that ETV6-RUNX1-mediated inhibition of RUNX1 targets may induce a quiescence-like pre-leukaemic state while continued RUNX1 expression is essential for the survival of overt leukaemia. We propose that CBF-dependency is an Achilles heel of ALL cells that could be exploited by targeting either CBF itself or its key downstream nodes. By combining clinically tested compounds targeting multiple CBF effectors we aim to improve efficacy of potential treatments and circumvent resistance.

Hematopoietic stem cells (HSCs) emerge from aortic hemogenic endothelium through a continuum of precursors, the pro- and pre-HSCs. Studies in zebrafish have implicated blood flow in this process. In the mouse, embryos without blood flow die before transplantable HSCs appear, precluding a direct assessment of the role of flow in HSC emergence. With the recent identification of pro-HSCs that emerge early in development and can be matured ex vivo into functional HSCs, it is now possible to address this question. Here, we assessed the specification and development of the HSC lineage in Ncx1-/- embryos lacking a heartbeat and circulation. We found that hemogenic endothelium and pro-HSCs emerge at a normal frequency, although in reduced numbers. However, Ncx1-/- pro-HSCs were severely impaired in their function and did not give rise to functional HSCs ex vivo. These data indicate that although the first steps of HSC lineage development take place in the absence of circulation, HSC precursors fail to complete their maturation, possibly due to the absence of key signaling events triggered by blood flow. Single cell gene expression profiling showed an aberrant signature in pro-HSCs and hemogenic endothelium isolated from Ncx1-/- embryos, with evidence of a block in hematopoietic development. Moreover, our data showed a decrease in Jag1-mediated Notch signaling in E9.5 Ncx1-/- hemogenic endothelium and pro-HSC, with a role for Notch signaling in the maturation of pro-HSC into HSCs further supported by functional assays. Finally, we found that lack of blood flow resulted in defects in the microenvironment, with absence of peri-aortic smooth muscle and a decrease of macrophages. Exploring what other flow-related signals affect the maturation of pro-HSC along the HSC lineage will be of interest in light of the future optimizing of in vitro HSC generation for therapeutic purposes.

The RUNX1 transcription factor is a critical regulator of normal haematopoiesis and its functional disruption by point mutations, deletions or translocations is a major causative factor leading to leukaemia. In the majority of cases, genetic changes in RUNX1 are linked to loss of function classifying it broadly as a tumour suppressor. Despite this, several recent studies have reported the need for a certain level of active RUNX1 for the maintenance and propagation of acute myeloid leukaemia and acute lymphoblastic leukaemia cells, suggesting an oncosupportive role of RUNX1. Furthermore, in solid cancers, RUNX1 is overexpressed compared with normal tissue, and RUNX factors have recently been discovered to promote growth of skin, oral, breast and ovarian tumour cells, amongst others. RUNX factors have key roles in stem cell fate regulation during homeostasis and regeneration of many tissues. Cancer cells appear to have corrupted these stem cell-associated functions of RUNX factors to promote oncogenesis. Here, we discuss current knowledge on the role of RUNX genes in stem cells and as oncosupportive factors in haematological malignancies and epithelial cancers.