ALBERT

Description: Chromosome instability (CIN) is deleterious to normal cells because of the burden of aneuploidy. However, most human solid tumors have an abnormal karyotype implying that gain and loss of chromosomes by cancer cells confers a selective advantage. CIN can be induced in the mouse by inactivating the spindle assembly checkpoint. This is lethal in the germline but we show here that adult T cells and hepatocytes can survive conditional inactivation of the Mad2l1 SAC gene and resulting CIN. This causes rapid onset of acute lymphoblastic leukemia (T-ALL) and progressive development of hepatocellular carcinoma (HCC), both lethal diseases. The resulting DNA copy number variation and patterns of chromosome loss and gain are tumor-type specific, suggesting differential selective pressures on the two tumor cell types.

Description: Abstract 2581 Oxidative metabolism generates intracellular energy and metabolic intermediates necessary to promote the growth of AML cells. Recently, we demonstrated that AML cells are uniquely sensitive to inhibition of mitochondrial translation. Therefore, we characterized the structure and metabolic function of the mitochondria in AML and normal hematopoietic cells. Compared to normal cells (n = 10), 1° AML cells (n = 12) had increased mitochondrial mass and increased levels of the NRF-1, TFAM, EF-Tu and Myc, genes that positively regulate mitochondrial biogenesis. By transmission electron microscopy, we demonstrated that the mitochondria in 1°AML cells were larger in area, but fewer in number compared to normal CD34+ cells. Given the dysregulated mitochondrial biogenesis in 1° AML cells, we examined the activity and reserve capacity of the respiratory complexes in 1° AML and normal cells. When normalized for mitochondrial mass, 1°AML cells (n = 12) had reduced activity of respiratory complexes III, IV and V compared to normal cells (n = 10). Thus, despite the increased mitochondrial mass in AML, respiratory chain complex activity did not increase proportionately. Next, we evaluated the spare reserve capacity in AML cell lines, 1° AML samples, and normal cells. Spare reserve capacity reflects the difference between basal and maximal respiratory rate and was determined by measuring oxygen consumption after treatment with oligomycin to block ATP synthesis and FCCP to uncouple ATP synthesis from the electron transport chain. The spare reserve capacity in AML cells and 1o samples was lower than normal hematopoietic cells. In order to determine the reserve capacity in individual respiratory complexes, we evaluated the rate of oxygen consumption in 1°AML and normal cells by treating the cells with increasing concentrations of the complex I, III IV, and V inhibitors, rotenone, antimycin, sodium azide, and oligomycin, respectively, and measuring changes in oxygen consumption. AML cells displayed less reserve capacity in the individual complexes compared to normal hematopoietic cells, and the differences were most striking for complexes III and IV. Consistent with the reduced reserve capacity, AML cells were more sensitive to respiratory chain inhibitors. We then employed a genetic approach to investigate the relationship between mitochondrial mass and spare reserve capacity using P493 Burkitt's cells with inducible myc as we and others have previously shown that myc regulates mitochondrial mass. Compared to myc knockdown cells, myc +P493 cells had increased mitochondrial mass, larger mitochondria, increased basal oxygen consumption, but reduced activity of respiratory complexes III, IV and V when normalized for mitochondrial mass, compared to myc - cells. In addition, myc expressing cells had less spare reserve capacity in their respiratory chain. Thus, in this isogenic cell line, increased mitochondrial mass was not accompanied by a proportionate increase in respiratory chain activity resulting in decreased spare reserve capacity. Given the reduced reserve capacity in AML cells, we evaluated the effects of increasing electron flux through respiratory chain. We speculated that the low spare reserve capacity would render AML cells more vulnerable to oxidative stress. To test this strategy AML cells and 1° samples as well as normal cells were treated increasing concentrations of the fatty acid substrate palmitate or the TCA cycle substrate dimethyl succinate. Consistent with our hypothesis, treatment with palmitate or dimethyl succinate transiently increased oxygen consumption and decreased spare reserve capacity in AML but not normal cells. Subsequently these treatments, increased reactive oxygen and induced cell death preferentially in AML cells and 1° samples compare to normal hematopoietic cells. Moreover, this treatment preferentially reduced the clonogenic growth of 1° AML cells over normal cells and reduced the engraftment of 1°AML but not normal cells into immune deficient mice. In summary, compared to normal hematopoietic cells, AML cells have greater mitochondrial mass but respiratory chain activity does not increase proportionately. The lack of proportionate rise in respiratory complex activity results in reduced spare reserve capacity in the respiratory complexes and greater sensitivity to oxidative stress. These data highlight a unique metabolic vulnerability in AML. Disclosures: No relevant conflicts of interest to declare.

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: 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: Key Points AML cells have increased mitochondrial mass, low respiratory chain complex activities, and low spare reserve capacity compared with normal cells. AML cells have heightened sensitivity to inhibitors of the respiratory chain complexes and oxidative stressors.

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: The gene regulatory networks (GRN) governing maintenance and expansion of normal and leukemic human hematopoietic stem-cells (HSC and LSC) are not well understood. Typically, GRNs are inferred from gene expression (GE) data of a limited subset of pre-selected genes implicated to be relevant to the cell types being studied. Such data are commonly derived from relatively homogeneous cell populations or cell lines, which do not reflect the heterogeneity of primary human samples. Importantly, there are currently no GRNs that directly interrogate the transcriptional circuitry controlling human HSC/LSC. To gain insight into the determinants of stem cell function in human HSC/LSC, we developed a unique method for building GRNs that employs GE and chromatin accessibility (ATAC-Seq) data derived from n=17 highly purified human umbilical cord blood hematopoietic stem and progenitor cell populations (hUCB-HSPC) and n=64 functionally-validated LSC-enriched and LSC-depleted cell fractions sorted from AML patient samples. Estimates of HSC/LSC frequencies based on limiting dilution xenotransplantation assays were also incorporated with statistical learning approaches to infer GRN models. Specifically, we determined transcription factor (TF) motif occurrence in HSC/LSC-enriched open chromatin regions near genes that are more highly expressed in stem versus non-stem profiles (P

Description: Understanding the mechanisms underlying the abnormal differentiation of human acute myeloid leukemia (AML) may reveal novel therapies to eradicate leukemic stem cells (LSC), which are often resistant to standard treatments and contribute to relapse. Cellular metabolism is recognized as a hallmark of cancer and is known to be distinct between hematopoietic stem cells (HSC) and downstream progenitors. In particular, sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that regulates proliferation and survival and is implicated in HSC egress, lymphocyte trafficking and mouse lymphoid lineage determination primarily through binding to S1PR1, one of five S1P G-protein coupled receptors. However, sphingolipids are understudied in human LSC and HSC. We recently found that sphingolipid perturbation governs HSC self-renewal and influences lineage outcome. Here, we show that S1PR3 governs myeloid commitment of LSC in a subset of human AML and is thus an attractive therapeutic target. Lipidomic analysis of LSC+ and LSC‒ fractions derived from AML patient samples and validated by xenotransplantation assays showed distinct sphingolipid alterations when compared to each other and to normal human cord blood (CB) CD34+CD38‒ stem cells and CD34+CD38+ progenitor populations. Interestingly, LSC+ fractions have increased S1P levels, prompting us to wonder if S1P signaling contributes to LSC maintenance. To gain a better understanding of the role of S1P in stemness, we examined S1P signaling in normal CB. Analysis of gene expression of S1PR1-5 and S1P transporters (SPNS2 and MFSD2B) within a comprehensive transcriptional roadmap of human hematopoiesis comprising thirteen normal populations from HSC to mature blood lineages demonstrated distinct expression patterns in specific blood lineages. S1PR1 and MFSD2B were most highly expressed in lymphoid and erythroid lineages, respectively, consistent with murine data. Notably, S1PR3 expression is specific to mature monocytes and granulocytes in human CB. S1PR3 protein was absent from the surface of long-term (LT) and short-term (ST)-HSC as determined by flow cytometry. Remarkably, lentiviral overexpression (OE) of S1PR3 was sufficient to promote myelopoiesis at the expense of erythropoiesis from LT- and ST-HSC in vitro in liquid culture, single cell assays and colony forming assays. To ascertain the mechanisms promoting myeloid commitment, we performed gene expression profiling by RNA-seq of LT- and ST-HSC following S1PR3 OE. This yielded a subset of shared genes similarly upregulated following S1PR3 OE relative to controls, including the known myeloid differentiation and AML-associated factors Early Growth Response 1 and 2 (EGR1/2) and Tribbles 1 (TRIB1). Thus, promiscuous expression of S1PR3 promotes a myeloid fate program in human HSC. S1PR3 protein expression was higher in AML patient samples relative to human CB and bone marrow cells by flow cytometry. Bioinformatic analysis of three independent AML cohorts revealed that AML patient samples with high S1PR3 gene expression also had high EGR2 and TRIB1 expression. Limiting dilution xenotransplantation assays of LSC-containing fractions sorted based on surface expression of S1PR3 demonstrated lower LSC frequency in S1PR3high versus S1PR3low/- LSC fractions. Moreover, S1PR3 OE in LSC+ fractions virtually abolished leukemic engraftment in xenotranplantation assays. These results suggest that higher S1PR3 levels in LSC are associated with a more mature myeloid state and that further increasing S1PR3 levels disrupts LSC maintenance. Treatment of mice bearing primary AML xenografts with FTY720, a S1P mimetic that targets S1P receptors including S1PR3, decreased leukemia burden in a subset of patient samples tested, including those obtained from relapsed and treatment-refractory patients. 70% of AML samples tested showed decreased LSC frequency in serial repopulation assays following FTY720 treatment. Importantly, treatment with FTY720 did not alter normal hematopoietic xenografts, demonstrating the existence of a therapeutic window. Collectively, our results provide the first direct evidence that sphingolipids govern myeloid commitment in human HSC and LSC, and demonstrate the potential of S1PR3 as a novel therapeutic target in AML for eradication of LSC while sparing HSC. Disclosures No relevant conflicts of interest to declare.