ALBERT

Description: Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples. We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A). The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus. Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation. In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. Disclosures Schimmer: Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.

Description: The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC. Our previous work on LSC subsets from 〉80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen. Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP. Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation. Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency. In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac. In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. Disclosures Takayanagi: Kyowa Hakko Kirin Co., Ltd.: Employment.

Description: Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring. Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL. Disclosures Mullighan: Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.

Description: Background: Residing at the apex of the blood system hierarchy, hematopoietic stem cells (HSCs) are endowed with multi-potency and self-renewal potential. Hematopoietic homeostasis is tightly regulated by controlling the balance between quiescence, self-renewal and lineage-commitment of HSCs. Although many studies have profiled gene expression patterns and epigenomes of HSC and downstream progenitors, post-transcriptional regulation of determinants that control these regulatory networks is largely unknown. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs by inhibiting their translation and/or inducing their degradation. A limited number of reports suggest that miRNAs are differentially expressed across the hematopoietic hierarchy and control lineage commitment and cell fate decisions by orchestrating gene regulatory networks, however the mechanisms remain unexplored. Methods: To identify miRNA(s) that play a functional role in human hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were over-expressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. Results: As miRNAs are negative regulators of gene expression, we studied the functional impact of miR-130a on long-term hematopoietic reconstitution by enforcing its expression in CB cells using lentiviral vector containing orange fluorescent protein (OFP) reporter. At 12 and 24 weeks after transplantation, increased miR-130a expression conferred a statistically significant, competitive advantage to transduced CB cells demonstrated by increased human chimerism and the proportion of OFP+/hCD45+ cells in the injected femur (IF), bone marrow (BM) and spleen of recipient mice. Xenografts produced by miR-130 O/E showed multi-lineage engraftment with myeloid skewing at the expense of B-lymphoid development and significantly enhanced erythroid output in RF, BM and spleen. In addition, ectopic expression of miR-130a caused splenomegaly in recipient mice. Flow cytometry analysis using several markers expressed during erythroid development revealed accumulation of immature GlyA+/CD71+/CD36+ erythroid progenitors, suggesting an erythroid differentiation block. Enforced expression of miR-130a also perturbed myeloid differentiation shown by the presence of abnormal CD14+/CD66b+ myeloid cells in the BM. At the primitive and progenitor cell stages, miR-130a O/E caused significant expansion of primitive CD34+/CD38- cells and increased the proportion of immuno-phenotypic HSC. Secondary transplantation involving limited dilution analysis revealed 10-fold increase in HSC frequency, consistent with a role of miR-130a in HSC self-renewal. Analysis of chromatin accessibility surrounding the miR-130a locus across the human hematopoietic hierarchy revealed peaks of accessible chromatin in HSC and downstream progenitors that were absent in mature cells. To ascertain the molecular mechanism of miR-130a function, label-free semi-quantitative proteomics was performed to determine differentially expressed proteins between miR-130a O/E and control-transduced CD34+ CB cells. Gene set enrichment analysis (GSEA) identified top miR-130a downregulated gene sets centered on chromatin remodelling. Components of SMRT/N-CoR co-repressor complex and polycomb repressive complex (PRC2) were identified to be among the top downregulated miR-130a targets. We assessed the impact of miR-130a O/E on the global chromatin accessibility landscape by performing ATAC-seq on CD34+ CB cells transduced with miR-130a or control lentivirus. Enforced expression of miR-130a resulted in a gain of approximately 450 accessible chromatin peaks. Transcription factor DNA recognition motif analysis revealed significant enrichment of GATA3 motif in accessible sites specific to miR-130a O/E cells. Conclusion: Together, our data suggests that miR-130a regulates HSC self-renewal and lineage specification. miR-130a mediates repression of several gene networks centered on chromatin remodelling and focally reshapes the accessible chromatin landscape of HSPC. Disclosures No relevant conflicts of interest to declare.

Description: The hematopoietic stem cells (HSC) field has long been perplexed by how the blood system d (~10e12 cells produced daily) - yet hematologic malignancies remain relatively rare. The risk of malignancy is mitigated in part by a complex hierarchy in which the quiescent long-term hematopoietic stem cells (LT-HSC) with high self-renewal capacity undergo a restricted number of cell divisions. Nonetheless, such a high production demand over a lifetime raises an inherent risk of malignancy due to DNA replication errors, misfolded proteins and metabolic stress that cause cellular damage in HSC. Previously, HSC dormancy, largely thought to be controlled by transcription factor networks, was held responsible for preventing mutation acquisition. However, recent studies suggest that LT-HSC contain critical cellular networks centered around the coordination of distinct HSC metabolic programs with proteostasis, which serve as crucial decision nodes to balance persistence or culling of HSC for lifelong blood production. While HSC culling mechanisms are known, the linkage between cellular stress programs and the self-renewal properties that underlie human HSC persistence remains unknown. Here, we ask how this HSC fate choice is influenced by lipid biosynthesis - an underexplored area of HSC metabolism. We observed a distinct sphingolipid transcriptional signature in human HSC and examined the consequences of sphingolipid perturbation in human cord blood (CB) stem cells during ex vivo activation. DEGS1 (Delta 4-Desaturase, Sphingolipid 1) is the final enzyme in de novo sphingolipid synthesis, converting dihydroceramide (dhCer) to ceramide (Cer); ablation of DEGS1 either genetically or by treatment with the synthetic retinoid fenretinide/N-(4-hydroxyphenyl) retinamide (4HPR) is sufficient to activate autophagy in mouse cells and human cell lines. DEGS1 gene expression was higher in HSC than in progenitors and was significantly increased in LT-HSC following 6 hours of cytokine stimulation, suggesting that it plays a role in cellular activation. Sphingolipid composition was altered in CB cultured with 4HPR for 8 days with an increase in dhCer levels and decrease in Cer levels shown by lipidomics. Remarkably, 4HPR treatment significantly increased in vitro colony forming efficiency from LT-HSC (50% over control), but not from short-term HSC or granulocyte-macrophage progenitors. Ex vivo 4HPR treatment of CB followed by serial xenotransplantation resulted in a 2.5-fold increase in long-term repopulation cell (LTRC) frequency over control-treated cells, suggesting that 4HPR treatment affects HSC self-renewal. RNA-seq analysis showed that 4HPR activates a set of proteostatic quality control (QC) programs that coalesce around the unfolded protein response (UPR) and autophagy, the latter confirmed by immunofluorescence and flow cytometry in CB stem cells. Ex vivo culture perturbs these programs and results in loss of chromatin accessibility at sites associated with uncultured LT-HSC as determined by ATAC-Seq. Addition of 4HPR to the culture activates these proteostatic programs to sustain immunophenotypic and functional HSC. These results suggest that ceramide, the central component to all sphingolipids, may act as a "lipid biostat" for measuring cellular stress and activating stress responses. We further asked if 4HPR could synergize with known compounds to enhance HSC self-renewal. Treatment of CB with a combination of 4HPR plus CD34+ agonists UM171 and StemRegenin-1 during ex vivo culture maintains a chromatin state more similar to uncultured LT-HSC as demonstrated by ATAC-seq, and led to a 4-fold increase in serial repopulating ability in xenotransplant assays over treatment with UM171 and SR1 alone. These results suggest that sphingolipid perturbation not only activates proteostatic mechanisms that protect HSC organelles from damage incurred during cellular activation, but also regulates the landscape of chromatin accessibility in cultured HSC when combined with CD34+ agonists. This work identifies a new linkage between sphingolipid metabolism, proteostatic QC systems and HSC self-renewal, and identifies novel strategies by which to expand HSC numbers and improve HSC quality for clinical applications. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding.

Description: There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

Description: Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.