Secondary Transplantation Assays Research Articles

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

AbstractThe t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Srcap Plays an Essential Role in Normal Hematopoiesis

The incorporation of variant histones into chromatin has been revealed to be an important mechanism of epigenetic regulation. Deposition of H2A.Z, in particular, is known to mediate several essential cellular processes including chromatin organization, DNA damage response, and transcriptional regulation. Nonsense and frameshift mutations in the H2A.Z exchanger, SRCAP, have been recurrently identified in both clonal hematopoiesis and AML, suggesting the importance of this gene in both non-malignant hematopoiesis and leukemogenesis. However, the precise role of SRCAP and the impact of its dysregulation on hematopoiesis remain unknown. To investigate the role of Srcap in hematopoiesis, we generated a conditional Srcap loss of function murine model with loxP sites flanking exons 11-16. Cre-induced deletion of these exons results in a loss of the catalytic ATPase domain along with a frameshift within the remaining protein sequence. When Vav1-iCre +/-Srcap fl/+ and Srcap fl/fl mice are crossed, no Vav1-iCre +/-Srcap fl/fl pups are born, suggesting that hematopoietic-specific-loss of Srcap is embryonic lethal. At E14.5/15.5, Vav1-iCre +/-Srcap fl/fl embryos are paler than other genotypes ( Figure 1) with a reduced number of Ter119 + fetal liver cells. Interestingly, the decrease in Ter119 + cells upon Srcap loss occurs primarily in later stage erythroid progenitors, whereas the number of stage 1 erythroid progenitors is relatively well preserved, suggesting that loss of Srcap adversely impacts fetal erythroid maturation. Furthermore, Srcap loss impacts fetal liver myeloid maturation. While the number of granulocytes is unchanged in Srcap deficient embryos, they have a near complete absence of monocytes, monocyte-dendritic cell progenitors, and monocyte progenitors. In contrast to the changes observed within mature hematopoietic populations, phenotypic hematopoietic stem cells (HSCs) are maintained in Vav1-iCre +/-Srcap fl/fl fetal livers. However, their function is impaired as fetal liver HSCs fail to repopulate upon transplantation ( Figure 2). Importantly, adult HSCs isolated from Mx1-Cre +/-Srcap fl/flmice show a very similar phenotype in that they fail to maintain hematopoiesis when transplanted and treated with poly (I:C). In total, this data shows that Srcap is essential for the maintenance and proper function of both mature hematopoietic populations and hematopoietic progenitors with different lineages and developmental stages displaying varied sensitivities to Srcap loss. To investigate the cellular consequences of SRCAP loss, we knocked down SRCAP expression in hematopoietic cell lines using an inducible shRNA approach. In agreement with the results obtained from our murine model, SRCAP deficient MOLM13 cells show a baseline competitive growth disadvantage that becomes apparent within one week of culture. This is associated with increased γ-H2AX activity, suggesting enhanced DNA damage and consistent with the previously identified role of SRCAP in the cellular DNA damage response. Interestingly, murine fetal liver HSCs with loss of Srcap also exhibit increased γ-H2AX activity, and Srcap null fetal liver stage 1 erythroid progenitors display marked transcriptional upregulation of multiple p53 downstream genes. Our data demonstrate that, in hematopoietic populations, Srcap is an essential gene, with its complete loss resulting in enhanced cellular stress potentially due to increased DNA damage. In humans, nonsense and frameshift mutations in SRCAP appear to be heterozygous in nature, suggesting that the impact of SRCAP deficiency is dose dependent. Indeed, murine HSCs with heterozygous loss of Srcap maintain normal repopulating activity in both primary and secondary transplantation assays ( Figure 2). It remains to be determined how the intermediate deficiencies in SRCAP levels found in patients impact hematopoiesis and lead to clonal expansion and whether the impact of SRCAP loss on the cellular response to stress, including DNA damage, is mediated by its canonical function as a histone variant H2A.Z exchanger. These studies promise to reveal novel insights on the nature of H2A.Z-mediated epigenetic regulation in HSCs and on how its dysregulation may lead to leukemogenesis.

Polycom Group Protein Mel18 Inhibits Hematopoietic Stem Cell Self-Renewal through Repressing the Expression of Genes Important for Cellular Senescence and Proliferation

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

Optimization of Lentiviral ß-Globin Vectors in a Forward Orientation to Enhance Therapeutic Effectiveness for Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic hemoglobin (Hgb) disorder characterized by a single pointmutation in the β-globin gene which causes Hgb polymerization under low oxygen tension and sickling of red blood cells (RBCs) which in turn drives severe systemic injury. Gene addition technologies like hematopoietic stem cell (HSC) targeted lentiviral (LV) gene therapy are among the most promising curative strategies for SCD. To expand this approach, improvements in LV titer, transduction efficiency, along with suppression of native sickle-globin, are preferred to generate successful therapeutic outcomes. One such improvement targets LV orientation: the relationship between the DNA encoding the β-globin gene in the LV and the promoter driving healthy β-globin gene expression. We created a forward-oriented β-globin-expression vector with an improved titer that results in more efficient transduction of human CD34+ cells in vitro, in human reconstituting CD34+ cells in xenografted mice, and in rhesus reconstituting CD34+ cells(Uchida N. et al., 2019). We report here on further optimization of LVs that in addition to forward orientation, also express shRNA that decreases native β-globin and sickle-globin, which could enhance the assembly of corrected Hgb tetramers. Seven unique constructs which varied in anti-β-globin shRNA sequences, packaging elements, and regulatory elements, were transduced into MEL-BB88 cells and expanded for 2 weeks. gDNA was collected for vector copy number (VCN) analysis, and treated with 10 µM hemin to induce Hgb expression. Five days after hemin-induction, cells were harvested for benzidine staining and immunoblot analysis to validate globin expression. Sca-1+ HSC/progenitor (HSPC) BM cells were isolated from Towne's humanized SCD donor mice prior to stimulation for 48 h with cytokines, then transduced at an MOI of 24 twice, 24 h apart. To assess the in vivo engraftment potential of the transduced HSPCs, 2.5x10^6 cells were transplanted into lethally irradiated CD45.1 recipient mice. Peripheral blood (PB) was sampled at 2-week intervals for PBMC chimerism and lineage engraftment, Hgb electrophoresis, Lorrca OxygenScan and OsmoScan assays, and PB VCN. Whole BM from engrafted recipients was collected at 30 weeks and used for secondary transplantation assays which were again analyzed over 24 weeks for PB engraftment, VCN, and donor Hgb expression. LVs were produced at varying titers and all successfully transduced MEL-BB88 cells with VCNs between 1 and 5 that were maintained following hemin induction. Interestingly, variations in RRE elements dramatically affected human globin expression in vitro. Furthermore, the transplanted mice engrafted fully with HSPCs transduced with multiple vectors. Notably, mice transplanted with HSPCs transduced with vector CX0066p (LCR-Enhancer-P-HBB-shmiR-3g1.223 + 3g2.30a-R1) maintained a VCN of 0.2-0.4 in PB for the 30-week duration of the primary transplant. Cellulose acetate gels of RBCs from PB showed ~40% HbA in a background of HbS. The P50 right-shifted towards normal, RBC counts increased, WBC counts decreased, and OxygenScan parameters were all corrected towards controls and away from those values obtained from mice transplanted with mock transduced HSPCs. EI max and EI min increased while pO2@95% decreased. Lastly, the RBC sickling was corrected in an in vitro sickling assay. In conclusion, based on LV titer, MEL cell expression, transduction efficiency, engraftment of modified cells, in vivo globin expression, anti-sickling activity, and oxygen transport, these constructs provide outstanding Hgb expression and phenotype correction. Consequently, these candidates serve as a solid foundation for future human gene therapy products in the treatment of SCD.

Chronic Inflammation Drives p53 Mutant Clonal Hematopoiesis Via Activating the NLRP1 Inflammasome

Individuals with somatic TP53 mutations manifest clonal hematopoiesis and are at high risk of developing hematological malignancies. However, how environmental factors influence the expansion of p53 mutant hematopoietic stem and progenitor cells (HSPCs) is unclear. We found that pI:pC or LPS treatment promotes the clonal expansion of p53 mutant HSPCs in transplantation assays. To determine the impact of LPS on HSPC function in vivo, we treated p53+/+ and p53R248W/+ mice with LPS or PBS and then performed competitive transplantation assays. LPS-treated WT BM cells show decreased engraftment in both primary and secondary transplantation assays, whereas LPS-treated p53 mutant BM cells continued to show increased engraftment. To understand how LPS-induced chronic inflammation promotes p53 mutant HSPC expansion, we examined the impact of mutant p53 on cytokine secretion from BM derived macrophages. We found that p53 mutant macrophages show increased secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-12 (p70), KC, VEGF, IP-10, CXCL-9, LIX, and MCP-1. To further investigate how chronic inflammation promotes p53 mutant HSPC expansion, we performed ATAC-seq assays in LSKs to identify differential regions of chromatin accessibility. Notably, we found that mutant p53 increases the chromatin accessibility to inflammasome genes, including Nlrp1a and Nlrp1b. Inflammasomes are multiprotein complexes that activate Caspase-1 and increase the release of pro-inflammatory cytokines such as IL-1β, leading to caspase-1-dependent death, known as pyroptosis. We found that loss of NLRP1 significantly decreased the secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-6, MCP-1, KC, M-CSF, RANTES, LIX, GM-CSF G-CSF, TNF-α and IL-12 (p40), from macrophages expressing mutant p53. Loss of NLRP1 significantly decreases the repopulating potential of HSPCs expressing mutant p53 in vivo. To further understand how chronic inflammation may confer a competitive advantage to p53 mutant HSPCs, we performed in vitro cell competition assays. We found that WT CD45.1+ cells co-cultured with p53 mutant cells show decreased replating potential compared to WT CD45.1+ cells co-cultured with WT CD45.2+ cells. We measured the levels of cytokines in the condition media utilizing cytokine arrays and found increased levels of IL-1β, IL-6, CXCL-9, and IL-4 in mutant cell cultures compared to that of the WT cells. We found that WT CD45.1+ cells co-cultured with mutant cells show increased pyroptosis. Importantly, we found that the enhanced pyroptosis of WT CD45.1+ cells seen in the mutant group is IL-1β dependent. We found that IL-1β treatment decreases the replating potential of WT BM cells, but not that of p53 mutant cells. To determine the mechanism by which p53 mutant HSPCs are resistant to IL-1β treatment, we performed RNA sequencing in p53+/+ and p53R248W/+ LSKs treated with IL-1β (10 ng/ml) or PBS in vitro for 90 minutes. GSEA analysis revealed that hematopoietic stem and progenitor cell differentiation genes are significantly upregulated in p53 mutant LSKs compared to WT LSKs following IL-1β stimulation. Notably, we found that p53 mutant LSKs treated with IL-1β are enriched with MYC target genes. Furthermore, blocking of IL-1β using IL-1β neutralizing antibody decreases the fitness of p53 mutant HSPCs. Our studies will likely establish both the NLRP1 inflammasome and the pro-inflammatory cytokine IL-1β as novel potential therapeutic targets for preventing clonal hematopoiesis development and treating hematological malignancies with TP53 mutations.

Impaired RAS Proteolysis Drives Clonal Hematopoietic Transformation

Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells.Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D).In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients.Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L).While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. [Display omitted] DisclosuresAbdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Divergent Levels of CD112 and INKA1 Define a Subset of Human Hematopoietic Stem Cells That Resists Regenerative Stress to Preserve Stemness

The first insight into the complexity of post-transplant in vivo dynamics of hematopoietic stem cells (HSC) in humans was only recently showcased by examining longitudinal clonal contributions in gene therapy patients. Initial blood reconstitution was achieved by short-term (ST-)HSC, but in the longer-term hematopoiesis originated from long-term (LT-)HSC that only became recruited after a latency phase of 1-2 years (Scala et al 2018). Thus, deciphering the mechanisms governing how LT-HSC might resist transplant mediated activation and/or respond to the varying hematopoietic demands that occur during homeostasis would be an important goal for improving HSC cell therapies. We previously linked INKA1(=C3orf54/FAM212A) mediated PAK4 inhibition and reduced H4K16 acetylation (H4K16ac) to quiescence in human leukemia stem cells (Kaufmann, Blood 2019). Here, we interrogate the role of this signaling axis in normal human hematopoiesis. Immunostaining revealed distinct subsets defined by the dichotomy of INKA1 and H4K16ac within cord blood ST- and LT-HSC, mechanistically supported by BioID, chromatin fiber analysis and PLA data showing INKA1 interacting with the H4K16 deacetylase, SIRT1. Among quiescent LT-HSC, we found that the INKA1high fraction (~10 % of LT-HSC) had the lowest CDK6 levels and represented LT-HSC in an alternative state of quiescence. We then used protein interaction (BioID) data to show that a shared interactor of INKA1 and PAK4, CD112 could be used to sort for the subset of CD112low LT-HSC that was in this alternatively quiescent LT-HSC state (H4K16aclow, CDK6low, CellROXlow). Compared to primary cells, culture attenuates the strict dichotomy of INKA1 and H4K16ac but only in cells in which colocalization of PAK4 with both occurred, suggesting PAK4 interferes with SIRT1-INKA1 interaction thereby permitting H4K16 acetylation. In vitro time course analysis showed that H4K16ac and PAK4 levels preceded the upregulation of the G0-exit marker CDK6 implicating a role in cell cycle priming. Pseudo-time scRNAseq analysis for INKA1high versus PAK4high, CDK6high and CD112high expression in mobilized peripheral blood (modeling in vivo HSC activation) showed enrichment of early or late diffusion indicative of quiescent versus primed cell status, respectively. Strikingly, in xenograft assays CD112low LT-HSC exhibited delayed engraftment kinetics with higher secondary repopulation capacity than faster repopulating CD112high LT-HSC reflecting the subset of LT-HSC that resist early activation. Similarly, INKA1-OE or PAK4 knock-down in vivo resulted in an early restraint in engraftment levels (@4 w), whereas 20 w engraftment reached control levels. When measured in secondary transplant assays the HSC frequency was 4 to 8-fold higher in the groups that showed this early restraint. Thus, resisting early activation (latency) preserves the regenerative potential of such HSC. In vivo 5-Fluorouracil treatment at week 4 accelerated latency exit and further increased HSC frequency of INKA1-OE cells while abolishing serial transplantation potential of controls. Hence, the induction of proliferative stress via creating acute hematopoietic demand is able to lift the INKA1-OE imposed restraint resulting in increased hematopoietic output while also promoting HSC self-renewal. Collectively, our data decipher the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to preserve a stem cell reservoir. Disclosures Takayanagi: Kirin Holdings Company, Ltd: Current Employment. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

3032 – SUPPRESSION OF VASCULAR INFLAMMATION PREVENTS MYELOID BIAS OF AGED HEMATOPOIETIC STEM CELLS

Chronic inflammation within the bone marrow (BM) microenvironment has been proposed as a key driver of aging-associated hematopoietic defects including loss of hematopoietic stem cell (HSC) self-renewal, myeloid-biased HSC differentiation, and a predisposition towards development of myeloid neoplasms. In support of this idea, we recently discovered that constitutive activation of the NFKB signaling pathway within endothelial cells (ECs) of young mice leads to an inflammatory stress within the BM, resulting in loss of HSC self-renewal and a myeloid-biased output at the expense of lymphopoiesis. Since chronic vascular inflammation has emerged as a key component of the systemic aging process, we hypothesized that suppression of NFKB dependent vascular inflammation during physiological aging could temporize BM inflammation and preserve HSC health. To test this hypothesis, we utilized the Tie2.IkBss mouse model wherein the canonical NFKB signaling pathway is suppressed specifically within ECs of adult mice, and physiologically aged these mice to assess their hematopoietic function. Our findings demonstrate that aged Tie2.IkBss mice displayed a significant decrease in their peripheral blood myeloid biased output as compared to their aged littermate controls. HSC transplantation assays revealed that HSCs derived from aged Tie2.IkBss mice demonstrated a significant decrease in their myeloid-biased differentiation and displayed enhanced lymphopoiesis. Furthermore, serial transplantation revealed that HSCs from aged Tie2.IkBss mice displayed balanced multi-lineage reconstitution ability during both primary as well as secondary transplantation assays. Collectively, these results suggest that inflammatory cytokines derived from the aged vascular niche play a crucial role in promoting aging-associated myeloid bias of HSCs, and strategies directed towards suppression of chromic vascular inflammation will provide novel therapeutic interventions towards prevention of aging-associated myeloid malignancies and improving overall healthspan.

Lncrna Morrbid Regulates Myeloid Cell Survival of AML Driven By a Combination of FLT3ITD and Epigenetic Mutations

FLT3ITD mutations are observed in ~30% of all acute myeloid leukemia (AML) patients, primarily classified into M4/M5 subtype, and associated with poor survival. Frequently, FLT3ITD mutations are co-expressed with epigenetic mutations including TET2 and DNMT3A and portend a poor prognosis. We analyzed human RNA sequencing datasets for lncRNAs expressed in myeloid malignancies and identified novel lncRNAs including MORRBID. MORRBID is located close to BCL2L11, encoding the pro-apoptotic protein BIM. Human TCGA analysis showed MORRBID overexpression in AML patients with TET2 mutations and its association with poor overall survival. Our independent study using over 100 patients showed that AML patients with FLT3ITD mutation express significantly higher MORRBID compared to patients without such mutation. Consistently, mice lacking Tet2 or Dnmt3a but expressing Flt3ITD develop AML and express higher levels of Morrbid in Lin- Sca-1+ Kit+ (LSK) cells. We hypothesized that MORRBID may not only act as a novel prognostic and diagnostic biomarker for AML but also as a therapeutic target in AML bearing a combination of Tet2-/-;FLT3ITD/ITD (TF) and Dnmt3a-/-;FLT3ITD/ITD (DF) mutations. We generated Tet2-/-;Flt3ITD/ITD;Morrbid-/-(TFM)and Dnmt3a-/-;FLT3ITD/ITD;Morrbid-/-(DFM)mice and assessed their hematologic parameters. Loss of Morrbid in the context of TF and DF rescued majority of peripheral blood (PB) abnormalities associated with these models of AML including increased myeloid cells (neutrophils and monocytes). In addition, the infiltration of leukemia cells in lung was significantly removed in the setting of Morrbid loss. Mechanistically, a significant reduction in the expression of Bim was observed in Lin- cells derived from TF and DF mice, which was associated with increased expression of Morrbid. However, Morrbid loss increased expression of Bim, induced apoptosis and reversed the enhanced survival seen in LSK cells derived from TF and DF AML mice. Overall, the increased bone marrow (BM) cellularity observed in TF primary mice was normalized to WT levels in the absence of Morrbid, which was associated with an increase in apoptosis in both BM myeloid progenitors as well as in more mature myeloid cells. To determine if in a competitive setting, loss of Morrbid in the setting of TF leukemia stem cells would alter clonal hematopoiesis, overall engraftment and AML development, we performed a competitive transplant assay and observed complete correction in term of abnormal leukemia cell counts, including correction in PB counts, presence of Mac-1+ blasts in PB as well as overall engraftment of TFM cells 24 weeks post-transplant. The rescue in these defects associated with TF donor cells in the absence of Morrbid was observed in the PB, BM and spleen. Loss of Morrbid mitigates the clonal expansion of AML donor cells in both primary and secondary competitive transplant assays. Transplantation of whole BM cells bearing TFM cells resulted in a partial correction of the AML phenotype including correction of mature myeloid cells in PB and BM of the recipients. Importantly, the absolute number of leukemia progenitor cells, which are normally increased in the BM with TF donors, were also restored to WT levels in recipient mice with TFM donors. Finally, the reduced overall survival defects seen upon secondary transplantation of TF cells was significantly prolonged in the absence of Morrbid. Our results warrant that MORRBID is a novel prognostic/diagnostic biomarker and a therapeutic target for AML. Disclosures Carroll: Janssen Pharmaceuticals: Consultancy; Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding.

Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation

Hematopoietic stem cell (HSC) transplantation (HSCT) is required for curative therapy for patients with high-risk hematologic malignancies, and a number of non-malignant disorders including inherited bone marrow failure syndromes (iBMFS). Strategies to enhance bone marrow (BM) niche capacity to engraft donor HSC have the potential to improve HSCT outcome by decreasing graft failure rates and enabling reduction in conditioning intensity and regimen-associated complications. Several studies in animal models of iBMFS have demonstrated that BM niche dysfunction contributes to both the pathogenesis of iBMFS, as well as impaired graft function after HSCT. We hypothesize that such iBMFS mouse models are useful tools for discovering targetable niche elements critical for donor engraftment after HSCT. Here, we report the development of a novel mouse model of Shwachman-Diamond Syndrome (SDS) driven by conditional Sbds deletion, which demonstrates profound impairment of healthy donor hematopoietic engraftment after HSCT due to pathway-specific dysfunctional signaling within SBDS-deficient recipient niches. We first attempted to delete Sbds specifically in mature osteoblasts by crossing Sbdsfl/flmice with Col1a1Cre+mice. However, the Col1a1CreSbdsExc progenies are embryonic lethal at E12-E15 stage due to developmental musculoskeletal abnormalities. Alternatively, we generated an inducible SDS mouse model by crossing Sbdsfl/flmice with Mx1Cre+ mice, and inducing Sbds deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by polyinosinic-polycytidilic acid (pIpC) administration. Compared with Sbdsfl/flcontrols, Mx1CreSbdsExc mice develop significantly decreased platelet counts, an inverted peripheral blood myeloid/lymphoid cell ratio, and reduced long-term HSC within BM, consistent with stress hematopoiesis seen in BMF and myelodysplastic syndromes. To assess whether inducible SBDS deficiency impacts niche function to engraft donor HSC, we transplanted GFP+ wildtype donor BM into pIpC-treated Mx1CreSbdsExc mice and Sbdsfl/flcontrols after 1100 cGy of total body irradiation (TBI). Following transplantation, Mx1CreSbdsExc recipient mice exhibit significantly higher mortality than controls (Figure 1). The decreased survival was related to primary graft failure, as Mx1CreSbdsExc mice exhibit persistent BM aplasia after HSCT and decreased GFP+ reconstitution in competitive secondary transplantation assays. We next sought to identify the molecular and cellular defects within BM niche cells that contribute to the engraftment deficits in SBDS-deficient mice. We performed RNA-seq analysis on the BM stromal cells from irradiated Mx1CreSbdsExc mice versus controls, and the results revealed that SBDS deficiency in BM niche cells caused disrupted gene expression within osteoclast differentiation, FcγR-mediated phagocytosis, and VEGF signaling pathways. Multiplex ELISA assays showed that the BM niche of irradiated Mx1CreSbdsExc mice expresses lower levels of CXCL12, P-selectin and IGF-1, along with higher levels of G-CSF, CCL3, osteopontin and CCL9 than controls. Together, these results suggest that poor donor HSC engraftment in SBDS-deficient mice is likely caused by alterations in niche-mediated donor HSC homing/retention, bone metabolism, host monocyte survival, signaling within IGF-1 and VEGF pathways, and an increased inflammatory state within BM niches. Moreover, flow cytometry analysis showed that compared to controls, the BM niche of irradiated Mx1CreSbdsExc mice contained far fewer megakaryocytes, a hematopoietic cell component of BM niches that we previously demonstrated to be critical in promoting osteoblastic niche expansion and donor HSC engraftment. Taken together, our data demonstrated that SBDS deficiency in BM niches results in reduced capacity to engraft donor HSC. We have identified multiple molecular and cellular defects in the SBDS-deficient niche contributing to this phenotype. Such niche signaling pathway-specific deficits implicate these pathways as critical for donor engraftment during HSCT, and suggest their potential role as targets of therapeutic approaches to enhance donor engraftment and improve HSCT outcome in any condition for which HSCT is required for cure. Disclosures Olson: Merck: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

The Trithorax-Group Protein ASH1L Regulates Hematopoietic Stem Cell Homeostasis Independently of Its Histone Methyltransferase Activity

Absent, small or homeotic discs 1-like (Ash1l) encodes the mammalian homolog of a Trithorax-group protein with a conserved SET domain that harbors histone H3 lysine 36 dimethyltransferase activity. Using mice with constitutive knockdown of Ash1l due to the insertion of a “gene trap” cassette in the first intron (Ash1lGT/GT mice), we previously reported that Ash1l is a critical regulator of quiescence and self-renewal in adult hematopoietic stem cells (HSCs). While wild-type HSCs could readily establish long-term bone marrow reconstitution after transplantation into irradiated recipients, Ash1l-deficient HSCs had markedly decreased quiescence and failed to establish long-term bone marrow reconstitution (Jones, Chase et al., JCI 2015). Here, we addressed three important questions to better understand the role of Ash1l in hematopoiesis: 1) What is the impact of complete, as opposed to partial, Ash1l loss on adult hematopoiesis? 2) Does Ash1l regulate HSC homeostasis in a cell-autonomous manner? 3) Is the catalytic activity of ASH1L required for its function?To move beyond the limitations of the constitutive knockdown Ash1lGT allele and address the first two questions, we studied newly generated mice carrying conditional knockout (cKO) Ash1l alleles (exon 13 flanked by loxP sites) along with the Mx1-Cre transgene. Induction of Mx1-Cre expression with poly(I:C) in Mx1-Cre+Ash1lf/f mice resulted in nonsense-mediated decay of Ash1l mRNA in hematopoietic cells, thereby completely knocking out Ash1l in adult hematopoietic stem and progenitor cells. Four weeks after inducing Ash1l inactivation in the hematopoietic compartment, we observed a profound depletion of HSCs and multipotent progenitors in Ash1l cKO mice similar to the phenotype of Ash1lGT/GTmice, indicating that conditional Ash1l knockout has a comparable impact on adult hematopoiesis to that of constitutive Ash1l knockdown. Of note, overt hematopoietic failure in steady-state conditions was not observed in either model. To address the second question, we transplanted a mixture of wild-type and Mx1-Cre+Ash1lf/f bone marrow into irradiated wild-type hosts, allowed donor bone marrow to occupy the wild-type niche and establish hematopoiesis, then induced Cre-mediated excision to inactivate Ash1l in Mx1-Cre+Ash1lf/f hematopoietic cells. Upon Cre induction, donor-derived Ash1lΔ/Δ HSCs and myeloid progeny were depleted or outcompeted by wild-type cells, consistent with the model that Ash1l regulates HSC homeostasis in a cell-autonomous manner. Given that Ash1l encodes a SET domain, we next sought to directly examine whether its catalytic activity is required for its role in regulating HSCs. We studied an Ash1l allele with an in-frame deletion of exon 11 and 12, resulting in preserved expression of ASH1L with internally deleted SET domain (ΔSET) (Miyazaki et al., PLOS Genetics 2013). Homozygous ΔSET mice were viable, and phenotypic analysis of adult ΔSET mice revealed normal frequencies of HSCs and multipotent progenitors, in contrast to our observations in Ash1lGT/GTand Ash1l cKO mice. Furthermore, transplanting ΔSET donor bone marrow into irradiated wild-type hosts resulted in sustained long-term reconstitution throughout primary, secondary and tertiary competitive transplantation assays, consistent with preserved HSC function and no alterations in cell cycle regulation. These findings establish that Ash1l regulates HSC homeostasis independently of its SET domain and histone methyltransferase activity. As ASH1L is a very large protein encoding multiple chromatin binding domains, we speculate that ASH1L may serve as a platform to recruit other partners to form novel protein complex(es) that regulate genes critical for HSC homeostasis. DisclosuresNo relevant conflicts of interest to declare.

Reduction in Bone Marrow Insulin-like Growth Factor-1 Levels Enhances Long-Term Hematopoietic Stem Cell Engraftment Following Bone Marrow Transplantation

Efficient donor hematopoietic stem cell (HSC) engraftment is critical for successful bone marrow transplantation (BMT) and requires interactions between donor HSC and host-derived HSC niche elements. Due to disruption of perivascular stem cell niches induced by myeloablative BMT conditioning, initial engraftment of HSC following BMT occurs in niches located near the endosteum. We have previously shown that myeloablative BMT conditioning with total body irradiation (TBI) induces expansion of endosteal niche osteolineage cells in a time course correlating with increases in bone marrow (BM) levels of insulin-like growth factor (IGF)-1. We also have shown that inhibition of IGF-1 Receptor (IGF1R) signaling blocks osteolineage cell expansion after TBI, resulting in poor engraftment of long-term (LT)-HSC after BMT and suggesting a critical role for IGF-1/IGF1R interactions in regulating engraftment after BMT. We now present data in which we have defined the cellular sources of BM IGF-1 following TBI and have surprisingly discovered that reduction in BM IGF-1 levels promotes LT-HSC retention at baseline and donor LT-HSC engraftment following BMT.Using mice with floxed IGF-1 alleles (IGF1L/L) and with tissue-specific expression of Cre-recombinase, we generated models with deletion of IGF-1 derived from mature osteolineage cells (Col1A1CreIGF1Exc), hematopoietic cells (CD45CreIGF1Exc), or liver (AlbCreIGF1Exc). We additionally generated a conditional IGF-1 deletion model (MX1CreIGF1Exc) in which polyI:polyC (pI:pC) treatment disrupts IGF-1 production from all 3 of the above sources.By qPCR analysis, CD45CreIGF1Exc BM showed marked reduction (>95%) in BM IGF-1 mRNA at baseline and following TBI, suggesting that hematopoietic cells produce most of the locally derived IGF-1 in BM. Unexpectedly however, neither deletion of osteoblast-derived (Col1A1CreIGF1Exc) nor hematopoietic cell-derived (CD45CreIGF1Exc) IGF-1 significantly reduced BM extracellular or intracellular IGF-1 protein levels at baseline, or extracellular BM IGF-1 levels following TBI. In contrast, elimination of liver-derived IGF-1 in AlbCreIGF1Exc mice not only reduced plasma IGF-1 levels by 80%, but surprisingly reduced BM extracellular IGF-1 protein levels by ≥80% both at baseline and 48 hours post-TBI, and reduced BM intracellular IGF-1 protein levels by >80% and >60% at baseline and post-TBI, respectively. These data suggest that most IGF-1 protein in BM during BMT is derived from liver. Conditional IGF-1 deletion in MX1CreIGF1Exc mice resulted in severe reduction of both BM IGF-1 mRNA and protein levels, combining effects seen in CD45CreIGF1Exc and AlbCreIGF1Exc mice. None of these models, however, showed deficits in TBI-induced endosteal cell expansion, suggesting that other ligands or perhaps only low levels of IGF-1 are required to induce IGFR1 signaling-dependent endosteal cell expansion.Unexpectedly, reduction of BM IGF-1 in ALBCreIGF1Exc and MX1CreIGF1Exc mice caused a 3-4 fold increase (% of total BM) in BM Lin-cKit+Sca1+CD48-CD150+ or Lin-cKit+Sca1+CD34-CD135- HSC. ALBCreIGF1Exc BM displayed enhanced multi-lineage long-term repopulating capacity in competitive transplant assays, proving that these cells are functional LT-HSC. Given this baseline LT-HSC enhancement, we asked if reduction in BM IGF-1 levels impacted capacity of ALBCreIGF1Exc BM niches to engraft donor HSC after BMT. Using competitive secondary transplantation assays to assess numbers of engrafted WT GFP+ LT-HSC 3 weeks after primary BMT into ALBCreIGF1Exc or control mice, we found that reduced IGF-1 in the ALBCreIGF1Exc BM microenvironment resulted in >2.5 fold enhancement of donor LT-HSC engraftment. In an initial search of downstream mediators of this effect, we discovered that reduction of IGF-1 levels in MX1CreIGF1Exc BM results in a 2.5-fold increase in post-TBI CXCL12 mRNA expression, suggesting that reducing BM IGF-1 may enhance donor LT-HSC engraftment by promoting LT-HSC homing and maintenance within HSC niches.Taken together our data define a critical role for IGF-1/IGF1R interactions in regulating the efficiency of donor HSC engraftment following BMT. Therapeutic strategies to reduce BM IGF-1 may prove to be a valuable approach to improving engraftment following clinical BMT. DisclosuresNo relevant conflicts of interest to declare.

Ap2a2 Is Crucial for LT-HSC Quiescence

Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150+48-LSK GFPhigh as opposed to the more cycling GFPlow HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150+48-LSK GFPhigh HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150+48-LSK long-term (LT-) versus CD150-48-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150+48-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function. DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Analysis of Clonal Dynamics in Childhood ALL Reveals a Key Role for Transcriptional Intratumor Heterogeneity in Driving Resistance to Chemotherapy

Tumors exhibit intratumor heterogeneity in both genetic make-up and phenotypic traits such as morphology, cell cycle status and gene expression. Although heterogeneity has been long recognized, its biological and clinical significance is not fully understood. To determine the relative contribution of genetic versus epigenetic heterogeneity to therapeutic resistance and relapse in childhood acute lymphoblastic leukemia (cALL), we have developed a mouse model that affords "real-time" longitudinal analysis of subclonal dynamics.The mouse model allows the engraftment of primary leukemic cells from an individual patient into multiple xenograft recipients, half of which are then treated with a combination of vincristine and dexamethasone. By assessing the reproducibility of independent outcomes, one can distinguish between deterministic and stochastic mechanisms of selection during therapy. We have used automated multicolor fluorescence in situ hybridization (mFISH) for the most frequent drivers of the leukemia (ETV6, RUNX1, PAX5, P16) to track the fate of individual genetic subclones. By comparing clonal composition in control and treated mice pre- and post-chemotherapy we demonstrate that, while treatment of sensitive ALLs results in a striking reduction in leukemic burden, the overall extent of genetic diversity (measured by mean of the Shannon index of diversity) is unaffected. This suggests that resistance in ALL may be largely independent of genetic variegation. In light of recent data demonstrating aberrant RAG activation in ETV6-RUNX1 leukemia, we reasoned that our results could reflect convergent evolution. Because RAG mediated deletions recurrently target the same key B-cell pathways, it is possible that genetically distinct clones could display convergent phenotypes. To test this hypothesis, and extend our mutational analysis to whole genome resolution, we performed single-cell whole genome sequencing. We sequenced matching diagnostic and relapse samples, and further validated our mFISH data. This reveals unprecedented levels of intratumor heterogeneity, with individual cells carrying on average 37 copy number variations (CNVs), a large number of which are private. By resolving the genomic breakpoints of lesions to known drivers of disease we formally proved that convergent evolution can happen. Thus, we identified clones carrying the same lesion but distinct breakpoints, highlighting their independent origins.Functional analysis based on limiting dilution secondary transplantation assays showed that chemotherapy enriches for cells with tumor propagating potential. Transcriptome analysis (RNAseq) of treated and untreated cells revealed that the former have distinct signatures shared amongst metastatic sites. Chemo-resistant cells displayed differences in cell cycle status and transcription rate, as well as differential activation of key signaling pathways (MAPK, Rap1 and Jak-Stat), and expression of cell adhesion molecules. Through gene set enrichment analysis we observed that resistant cells have features characteristic of more primitive hematopoietic cells, including concomitant up-regulation of hematopoietic stem cells and lympho-myeloid progenitors markers. Interestingly, following secondary transplantation global gene expression of treated cells largely reverted to a treatment-naïve signature.Overall this analysis provides novel insight into the contribution of genetic and epigenetic heterogeneity to resistance to cytotoxic chemotherapy. In the case of cALL phenotypic heterogeneity appears to play a larger role than genetic diversity, particularly with regards to cell cycle state and developmental stage. Our data suggest that resistance is driven by a pre-existing population of immature cells with a distinct global gene expression signature and higher tumor propagating potential, and that genetic heterogeneity in cALL arises through independent acquisition of CNVs affecting the same gene or genes within the same pathway, thereby driving convergent phenotypic evolution. Such a view is consistent with the observation that distinct subclones appear to respond to chemotherapy-derived environmental pressure in a similar manner. DisclosuresNo relevant conflicts of interest to declare.

Nuclear Export Inhibitor KPT-8602 Is Highly Active Against Leukemic Blasts and Leukemia-Initiating Cells in Patient-Derived Xenograft Models of AML

Currently available combination chemotherapy for AML induces complete remission in about 75% of younger and 45% of older patients, but often fails to induce long-term disease-free survival and is toxic to normal hematopoietic cells. The frequent disease relapse observed in chemotherapy treated AML patients is thought to occur through the inability of the existing drugs to specifically target the self-renewing leukemia-initiating cells (LICs). These cells have the functional capacity to replenish AML blasts. An attractive target for AML therapy is the nuclear export protein exportin 1 (XPO1), which is functionally required for continuous nuclear export of a subset of proteins and mRNAs. Small molecule Selective Inhibitor of Nuclear Export (SINE) compounds covalently bind to Cysteine528 in the cargo-binding groove of XPO1 and inhibit nuclear export. The orally bioavailable clinical SINE compound, selinexor (KPT-330), is currently in Phase 1 and 2 clinical trials in adult patients with AML (NCT01607892, NCT02088541, NCT02249091, NCT02403310, NCT02093403, NCT02485535, NCT02299518, NCT02416908, NCT02212561, and NCT01607892) and in a Phase 1 trial for relapsed childhood ALL and AML (NCT02091245). The results of these trials are encouraging, as they have demonstrated that selinexor alone or in combination is active in inducing remission in patients with relapsed or refractory AML. Here, we explore the anti-leukemia activity of a next generation SINE compound, KPT-8602.In preclinical toxicology studies as well as clinical trials, noted adverse events for selinexor are anorexia with weight loss, fatigue and thrombocytopenia, which limit the frequency of dosing to no more than every other day, three times a week. KPT-8602 is orally bioavailable and has similar pharmacokinetic properties to selinexor, with reduced brain penetration. Preliminary toxicology studies in rats and monkeys suggest that KPT-8602 has a substantially better tolerability profile, with reduced fatigue, thrombocytopenia, and anorexia compared to selinexor. Thus, KPT-8602 can be dosed orally on a daily basis for 5 days each week. The increased drug exposure with daily dosing and better tolerability of KPT-8602 leads directly to superior efficacy in pre-clinical models compared with selinexor.To define the anti-leukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant pre-clinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts were treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 was highly active against blast cells from three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML)), as evidenced by a reduction in leukemic engraftment in primary mice after treatment (Fig. 1A-C). At these doses, KPT-8602 demonstrated higher anti-leukemic activity than selinexor, with no leukemic cells detected in the bone marrow of two of the eight AML-CN PDX treated mice (Fig. 1A-C). In secondary transplantation assays, KPT-8602 greatly reduced the frequency of LICs in the PDX model derived from AML-CN cells, indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs (Fig. 1D). In mice engrafted with normal cord blood human CD34+ cells, KPT-8602 reduced bone marrow human CD45+ cell numbers (Fig. 1E), but the impact on normal hematopoietic stem and progenitor cells, as determined by secondary transplantation assays (Fig. 1E), was modest when compared to the LIC reduction after KPT-8602 treatment. These findings demonstrate that KPT-8602 has better tolerability, perhaps through decreased CNS penetration, and is better at eliminating both AML LIC and blast cells when compared to selinexor. Therefore, KPT-8602 should be tested for efficacy and tolerability in patients with relapsed and refractory AML, with the goal to overcome a difficult obstacle in curing AML; destroying the LIC compartment while sparing normal hematopoietic cells. [Display omitted] DisclosuresEtchin:Karyopharm: Research Funding. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Ellis:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics, Inc: Employment. Stone:Agios: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Karyopharm: Consultancy; Celator: Consultancy; Novartis: Research Funding; Merck: Consultancy; Juno: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Roche/Genetech: Consultancy; Celgene: Consultancy. DeAngelo:Bristol Myers Squibb: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Ariad: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Look:Karyopharm Therapeutics, Inc: Research Funding.

Polycomb Repressive Complex 1 Enhances HSC Self-Renewal through Repressing the Canonical Wnt Signaling Pathway

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. While studies on individual PRC1 component suggest that PRC1 plays an important role in hematopoiesis, how PRC1 regulates transcriptional repression in hematopoietic stem cells (HSCs) is largely unknown. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the PRC1 complex. Bmi1 is a positive regulator of HSC self-renewal; however, the role of Mel18 in hematopoiesis has been controversial. To determine whether Bmi1 and Mel18 play redundant or distinct role in HSC self-renewal, we have generated Bmi1 and Mel18 conditional knockout mice. While acute deletion of Mel18 affects neither HSC frequency nor lineage commitment, we found that Mel18-deficent hematopoietic progenitor cells showed enhanced replating potential compared to wild type cells. To determine the role of Mel18 in HSC self-renewal, we performed serial HSC transplantation assays and found that the repopulating ability of Mel18-/- HSCs was significantly higher than WT HSCs in both primary and secondary transplantation assays, demonstrating that the loss of Mel18 enhances HSC self-renewal in vivo. We hypothesize that loss of Bmi1 and Mel18 in hematopoietic stem cells will disrupt PRC1 complex and impairs HSC self-renewal. To determine the role of PRC1 complex in HSCs, we analyzed the HSC behavior in Bmi1 and Mel18 double-deficient mice. While we found that Bmi1-deficient HSCs showed decreased repopulating potential compared to WT HSCs 16 weeks following transplantation, loss of both Bmi1 and Mel18 in HSCs resulted in even more severe self-renewal defects. In addition, loss of both Bmi1 and Mel18 resulted in decreased myeloid differentiation and increased B cell differentiation compared to WT, Mel18 KO, and Bmi1 KO mice. These data demonstrate that Bmi1 and Mel18 have non-overlapping role in HSC maintenance and lineage commitment.Given that Bmi1 plays a dominant role in the PRC1 complex, we decided to identify Bmi1 target genes in hematopoietic stem cells to understand how PRC1 complex regulates HSC self-renewal. We performed transcript profiling assays to compare gene expression in HSCs isolated from wild type and Bmi1-/- mice. The Ingenuity Pathways indicates that the canonical Wnt signaling is significantly elevated in Bmi1 null HSCs compared to WT HSCs. We confirmed the upregulation of several genes of the Wnt pathway in Bmi1 null HSCs by quantitative real-time PCR analysis. To determine whether Bmi1 can repress the activation of Wnt signaling in cells, we utilized the Top-Flash Wnt reporter system. Stimulation of 293T cells with Wnt3a activates the Wnt reporter and this activation can be efficiently repressed by Bmi1. Furthermore, we detected the association of Bmi1 with the Lef1, Tcf4, and Axin2 promoters in Baf3 cells by ChIP experiment. Thus, Bmi1 directly represses the expression of several Wnt genes in hematopoietic cells. To determine the functional significance of activation of Wnt signaling in Bmi1 null HSCs, we have generated R26StopFL Bmi1-Apcf/f-Mx1-Cre+ and Bmi1f/f-Ctnnb1f/f-Mx1-Cre+ mice. Loss of Apc in hematopoietic cells activates the Wnt signaling pathway and impairs HSC self-renewal. We found that expressing three-copies of Bmi1 from the Rosa26 locus enhanced the self-renewal capabilities of Apc deficient HSCs in transplantation assays. Ctnnb1 encodes b-catenin and loss of Ctnnb1 in HSCs diminishes Wnt signaling. Acute deletion of Bmi1 in hematopoietic compartments resulted in decreased bone marrow cellularity and enhanced apoptosis of hematopoietic stem and progenitor cells. Deletion of Ctnnb1 in Bmi1 null hematopoietic cells rescued these defects. Thus, impaired HSC self-renewal seen in Bmi1 null mice is, at least in part, due to activation of the canonical Wnt signaling pathway.Taken together, we demonstrate that PRC1 complex enhances HSC self-renewal through inhibiting the canonical Wnt signaling. DisclosuresNo relevant conflicts of interest to declare.

MLL leukemia induction by genome editing of human CD34+ hematopoietic cells

AbstractChromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease–mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.

Abstract 4445: Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells

Abstract Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are toxic to normal tissues, prompting the need to develop new targeted therapies. The frequent disease relapse that is observed in patients with AML is thought to occur because of the inability of the existing drugs to target the self-renewing leukemia-initiating cells (LICs). An attractive new strategy for AML therapy is inhibition of the nuclear export protein exporter 1 (XPO1), or CRM1. XPO1 regulates export of proteins that contain leucine-rich nuclear export signals (NES), including protein adaptors that mediate transport of RNA. XPO1 cargo encompass tumor suppressor proteins, cell cycle regulators, and apoptotic proteins. Recently, small molecule inhibitors of nuclear export (SINE) that inhibit the export function of XPO1 by targeting Cys528 in its NES-binding groove, were developed using an in silico molecular modeling. Selinexor (KPT-330), the orally bioavailable SINE compound, is in Phase 1 and 2 studies in adult patients with AML (NCT01607892 and NCT02088541) and in a Phase 1 study for relapsed childhood ALL and AML initiated in March 2014 (NCT02091245). To define the anti-leukemic activity of selinexor against primary AML blasts and LICs in a clinically relevant setting, we established mouse models of primary human leukemia, or patient-derived xenografts (PDX), in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcγnull (NSG) mice. Mice engrafted with leukemic blasts were treated with either vehicle or selinexor. Selinexor was highly active against blast cells from two of the three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN) and complex karyotype AML (AML-CK1 and AML-CK2)), as evidenced by a reduction in leukemic engraftment in primary mice after 4 weeks of treatment. Secondary transplantation assays indicated that selinexor greatly reduced the frequency of LICs in PDX models derived from all three patients (6- to 430- fold reduction compared to controls), indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs. These findings show that selinexor has potent activity against LICs, even when it has only moderate activity against the bulk AML cell population. Furthermore, preliminary results of combination studies of selinexor with Ara-C, a standard chemotherapeutic agent, demonstrate synergistic effect of the two drugs against LICs in a PDX model of AML-CN. Importantly, 4 weeks of selinexor treatment demonstrated minimal toxicity in mice engrafted with normal human CD34+ hematopoietic cells. These findings demonstrate that inhibition of nuclear export with selinexor overcomes an important obstacle to cure of AML, which is to destroy the very critical LIC compartment while sparing normal hematopoietic cells. Citation Format: Julia Etchin, Bonnie Thi Le, Alla Berezovskaya, Amy S. Conway, Weihsu C. Chen, Alex Kentsis, Marc R. Mansour, Richard M. Stone, Ilene A. Galinsky, Daniel J. DeAngelo, Dilara McCauley, Michael Kauffman, Sharon Shacham, Jean CY Wang, Andrew L. Kung, Thomas Look. Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2015-4445

Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning

AbstractSuccessful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL–dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.