ALBERT

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: Therapy resistance and relapse in acute myeloid leukemia (AML) are driven by leukemia stem cells (LSCs). Recent evidence highlighting functional and genetic heterogeneity among LSC subclones underscores the importance of capturing the entire LSC compartment in studies of LSC biology. Although LSCs are often enriched in the CD34+CD38- cell fraction, they are frequently detected in other phenotypic fractions, and in some cases are restricted to the CD34- population. In order to discover novel LSC markers, we examined genes differentially expressed between functionally-validated LSC+ and LSC- cell fractions obtained from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs. CD200 expression in 57 primary AML samples was analyzed by flow cytometry using anti-human CD200 clone 1B9(kindly provided by Trillium Therapeutics Inc.). CD200 was present on a greater proportion of CD45dim blasts compared to more differentiated CD45high non-blast populations (54.4% versus 21.7%, p

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: Introduction Multiple myeloma (MM) is characterised by the malignant expansion of clonal plasma cells in the bone marrow (BM). We and others have used massive parallel sequencing to describe the somatic aberrations acquired in different subclones in newly diagnosed MM (NDMM). These studies have showed that chemotherapy has an impact on intra-clonal heterogeneity, but more analyses are required in paired presentation/relapse samples and samples from multiple sites at the same and different time points. Materials and methods We have studied 49 paired presentation/relapse patients from a series of 463 NDMM patients entered into the Myeloma XI trial (NCT01554852). To understand the impact of spatial separation within the MM clone and the consideration that MM is a metastatic disease, we examined BM aspirates and compared them to targeted biopsies from extramedullary disease sites in 9 MM patients. These cases were 1 patient with samples bilaterally collected from the hip during the course of the disease, 4 MM cases with plasma cell leukemia (PCL), 3 MM cases with plasmacytomas, 1 MM patient with ascites, and 1 MM case with pleural effusion. DNA from both BM and peripheral blood samples were used for whole exome sequencing plus a pull down of the MYC, IGH, IGL and IGK loci following the SureSelect Target Enrichment System for Illumina Paired-End Sequencing Library v1.5. Exome reads were used to call single nucleotide variants, indels, translocations, and copy number aberrations. Mean sequencing depth was 59.3x. The proportion of mutant tumor cells carrying a mutation was inferred. The presence and proportion of subclones will be defined using bioinformatics tools. Results For the 463 NDMM samples, the following 15 significantly mutated genes are seen KRAS (n=103 mutations), NRAS (n=88), LTB (n=53), DIS3 (n=49), BRAF (n=37), EGR1 (n=22), FAM46C (n=20), IRF4 (n=19), TRAF3 (n=17), HIST1H1E (n=16), TP53 and FGFR3 (n=14), CYLD (n=13), MAX (n=12), and RB1 (n=5). These mutations were seen within all clonal cells and at subclonal levels, consistent with the mutations being acquired at different time points and being associated with different subclonal fitness. We show that NDMM have a mean number of exonic mutations of 61.1±13.0, in contrast to samples taken at the time of relapse, which show an average of 80.6±25.4, Figure 1A. We report diverse patterns of subclonal evolution: no change, subclonal tiding, and subclonal tiding with new subclones arising. We are currently examining samples taken during clinical remission to track subclones at the time of response. For patient with multiple samples taken at different timepoints, 77 mutations were shared across all samples but, of note, specific mutations were seen at the same timepoint in different sites (13/1662 R2R vs 13/1662 R2L), which illustrates the impact of sampling differences in reporting mutation calls and differential response to therapy, Figure 1B. This is also observed in a plasmacytoma case with both a BM aspirate sample containing 11 mutations (including NRAS c.183A〉T and BRAF c.1783T〉C), and a femur plasmacytoma with 18 mutations, of which only 2 are shared with the BM sample, Figure 3. One of these shared lesions is BRAF c.1783T〉C, the cancer clonal fraction of which increases ten-fold, suggesting that the sub-clone with this mutation disseminated from the BM and founded the plasmacytoma. Conclusion Our preliminary data demonstrate that MM subclones not only respond differently to clinical treatment, but also have different biological properties leading to cause extramedullary disease. To our knowledge, this is the first comprehensive genetic analysis of the spatio-temporal heterogeneity in myeloma and reveals genetic differences due to sampling bias. Figure 1. (A) Number of mutations in MM patients at clinical presentation and relapse. Each patient sample is represented by a dot. Lines and error bars correspond to the average and the standard error of the mean values, respectively. Difference was not statistically significant (p 〉0.05, t-test). (B) MM patient analysed at presentation and following two relapses (top). The number of mutations increases through disease (bottom, left panel). Venn plot shows the number of shared and specific mutations for each time point (bottom, right panel). (C) Case with a MM sample (green) and a femur plasmacytoma (blue). Venn plot shows shared and specific mutations to the bone marrow or the plasmacytoma site. Figure 1. (A) Number of mutations in MM patients at clinical presentation and relapse. Each patient sample is represented by a dot. Lines and error bars correspond to the average and the standard error of the mean values, respectively. Difference was not statistically significant (p 〉0.05, t-test). (B) MM patient analysed at presentation and following two relapses (top). The number of mutations increases through disease (bottom, left panel). Venn plot shows the number of shared and specific mutations for each time point (bottom, right panel). (C) Case with a MM sample (green) and a femur plasmacytoma (blue). Venn plot shows shared and specific mutations to the bone marrow or the plasmacytoma site. Disclosures Jones: Celgene: Other: Travel support, Research Funding. Peterson:University of Arkansas for Medical Sciences: Employment. Brioli:Celgene: Honoraria; Janssen: Honoraria. Pawlyn:Celgene: Honoraria, Other: Travel support; The Institute of Cancer Research: Employment. Gregory:Janssen: Honoraria; Celgene: Honoraria. Davies:Onyx-Amgen: Membership on an entity's Board of Directors or advisory committees; Array-Biopharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda-Millennium: Membership on an entity's Board of Directors or advisory committees; University of Arkansas for Medical Sciences: Employment. Morgan:CancerNet: Honoraria; University of Arkansas for Medical Sciences: Employment; MMRF: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Weisman Institute: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda-Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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: Key Points Coexistent hyperdiploidy or t(11;14) does not abrogate the poor prognosis associated with adverse cytogenetics in myeloma patients. Single-cell analysis reveals that hyperdiploidy may precede IGH translocation in the clonal history of a proportion of patients with both.

Description: Hematopoietic stem cells (HSC), at the apex of the blood hierarchy, generate a series of increasingly committed progenitors and ultimately produce terminally differentiated cells at the bottom of the hierarchy. Although HSC and their immediate downstream, multipotent progenitors (MPP) have full multilineage differentiation capacity, only HSC have the capacity for long term self-renewal. Thus comparison of HSC with downstream progenitors that exhibit gradual fate restriction by assuming the identity of a mature blood cell is central to defining how the stemness state is established and how lineage commitment is executed. Interestingly, few genes are differentially expressed between human HSC and MPP, suggesting that some important stem cell mechanisms are in a permissive state and therefore difficult to identify simply by differential gene expression analysis. We hypothesized that these permissive genetic regions could be identified by chromatin analysis. To identify permissive genomic regions, we delineated regions of accessible chromatin through ATAC-seq assays in HSC, MPP, committed progenitors and seven mature lineages from human Cord Blood. DNA recognition motif analysis across accessible chromatin in each developmental stage identified both known and unknown candidate master regulatory transcription factors for each step of lineage development. The DNA recognition motif for CTCF, a crucial effector of chromatin interactions, is enriched in accessible chromatin associated with progenitors but not in HSC, even though CTCF expression is not significantly different among stem and progenitor populations. Knockdown of CTCF in HSC by lentiviral vector system increased the proportion of HSC in G0 cell cycle and consequently reduced the number of mature cells generated in both conventional and high sensitivity single cell assays. The development of differentiated lymphoid and megakaryocytic lineages was affected. Few effects were seen when CTCF was silenced in MPP or more downstream progenitors. Collectively, our results suggest that CTCF is a "gate keeper factor" that regulates transition from dormant to active stages in HSC, and might control stem cell pool throughout life. In parallel to normal blood hierarchies, AML is also composed of a hierarchy with leukemia stem cells and non-stem-like leukemia cells; LSC and non-LSC fractions sorted from 24 AML patients have also been subjected to ATAC-seq. Currently we are comparing HSC and non-HSC with LSC and non-LSC fractions to identify the similarity and differences between normal HSC and LSC. Overall, our study is revealing insights into the stemness state of normal and leukemic stem cells in primary human tissues. Disclosures Takayanagi: Kyowa Hakko Kirin: Employment.