Normal Human Hematopoiesis Research Articles

Loss of FoxM1 Promotes Erythroid Differentiation through Increased Proliferation of Erythroid Progenitors

FoxM1 belongs to the fork head/winged-helix family of transcription factors and regulates a network of proliferation-associated genes including the G2/M transition, chromosome segregation, and spindle assembly. FoxM1 expression is commonly upregulated in a number of human cancers such as liver, ovarian, breast, prostate, colon, and brain tumors. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor of oncogenesis. In normal cells, FoxM1 is highly expressed in multipotent progenitor cells and inhibits differentiation of the progenitors, suggesting that FoxM1 plays in a role in the maintenance of multipotent progenitor cells. However, the exact molecular mechanism by which FoxM1 regulates stem/progenitor cells is still uncharacterized. In this study, we have examined the role of FoxM1 in normal hematopoiesis using human cord blood CD34+ cells.To investigate the role of FoxM1 in normal hematopoiesis, we infected human cord blood CD34+ cells with FoxM1 shRNA lentivirus and observed blood cell differentiation using FACS analysis with a range of cell surface markers. We found that knockdown of FoxM1 resulted in an increase of the erythroid population (CD71+/GlyA+), a decrease of the myeloid population (CD11b+), and an unchanged megakaryocyte population (CD41a+) in two phase liquid culture system. Overall, we found a 2-fold increase in the erythroid population compared to the myeloid population. Importantly, methylcellulose colony assays also demonstrated increased numbers of CFU-E colonies (2-2.5 fold increase compared to control) and decreased numbers of CFU-GM colonies in FoxM1 knockdown cells. Taken together, these findings imply a role for FoxM1 in normal erythropoiesis.To better define the function of FoxM1 in hematopoietic cells, we sorted distinct populations of cells based on their cell surface marker expression and quantitated FoxM1 expression level by RT-qPCR. FoxM1 had a 3-fold increased expression in CD71+ (erythroid) cells compared to CD11b+ (myeloid) cells. Additionally, we found FoxM1 expression was particularly elevated in the BFU-E and CFU-E stages of erythropoiesis, suggesting a functional role for FoxM1 in erythroid progenitor proliferation.Finally, to study the potential molecular mechanism of FoxM1 in normal hematopoiesis, we analyzed cell cycle progress in FoxM1 knockdown cells with DAPI staining. We found increased S and G2/M phases in FoxM1 knockdown cells, which was significant only in the CD71+ (erythroid) population and not in the unsorted cell populations. We also detected an increase of BrdU+ cells in FoxM1 knockdown CD71+ population by BrdU incorporation assay, indicating faster proliferation of erythroid progenitors with FoxM1 knockdown.These findings suggest a novel function of FoxM1 in normal human hematopoiesis, in which FoxM1 deficiency leads to increased proliferation of erythroid progenitors resulting in increased erythroid differentiation. Our data indicate that FoxM1 inhibitors, such as Thiostrepton or FDI-6, may be beneficial in treating patients with anemia. DisclosuresNo relevant conflicts of interest to declare.

Characterization of snoRNA Expression in Acute Myeloid Leukemia

Small nucleolar RNAs (snoRNAs) are small (90-300nt) non-coding guide RNAs found in all multicellular organisms. H/ACA and CD box snoRNAs localize to the nucleolar region and are part of a catalytic multicomponent protein complex selecting target RNAs based on complementarity. They are responsible for the site-specific pseudouridylation and 2' O methylation of ribosomal RNAs, respectively. ScaRNAs localize to the Cajal body and are responsible for the methylation and pseudouridylation of splicesomal RNAs U1, U2, U4, U5, and U12. There are also orphan snoRNAs without any known RNA targets. Recent studies have suggested an expanded role for snoRNAs outside of ribosomal biogenesis, including the regulation of RNA splicing and chromatin remodeling. Moreover, emerging data suggest that aberrantly expressed snoRNAs may contribute to neoplastic transformation. Here, we characterize snoRNA expression in normal human hematopoiesis and in AML.We first developed a novel strategy to identify and quantify properly processed snoRNAs. This is important, since standard RNA sequencing or array-based analyses cannot distinguish between processed snoRNAs and primary mRNA transcripts of the host genes (most snoRNAs are contained in the introns of coding genes). In brief, we take advantage of the fact that, like miRNAs, snoRNAs contain a free 3'-hydroxyl group, which allows for efficient ligation to sequencing adaptors. Following size selection (20-200 nt) to enrich for small non-coding RNAs, the libraries are sequenced on the Illumina next generation sequencing platform. We also have developed a novel analysis pipeline that maps areas of contiguous alignment in the genome, forming ab initio "clusters" representing snoRNAs. Using this sequencing assay, we first interrogated snoRNA expression in hematopoietic cells from healthy individuals. Specifically, we sequenced small RNA libraries derived from CD34+ cells, promyelocytes, neutrophils, monocytes, T-cells, and B-cells. Of the 269 known snoRNAs, 132 (49%) were expressed in one or more hematopoietic cell population. Likewise, 80 of 112 (71%) of known H/ACA box snoRNAs, and all 21 scaRNAs were expressed. In addition, we identified (and computationally validated using SnoReport, snoGPS, and an in-house snoFinder script) 8 putative novel snoRNAs (1 H/ACA box and 7 CD box snoRNAs). Most snoRNAs were stably expressed across all hematopoietic lineages. However, there were numerous examples of snoRNAs that were specifically enriched in a specific hematopoietic cell population (e.g., CD34+ cells). We also identified several snoRNAs that were up- or down-regulated during granulocytic differentiation. For example, many of the orphan C/D box snoRNAs contained in the imprinted DLK/DIO3 locus on chromosome 14q32 are significantly down-regulated during granulocytic differentiation.To determine whether snoRNAs are frequently dysregulated in AML, we next sequenced small non-coding RNAs isolated from the bone marrow of 33 patients with de novo acute myeloid leukemia (all with a normal karyotype). Using a strict 5% false discovery rate, only 9.3% of CD box snoRNAs and 0.9% of H/ACA box snoRNAs were found to have significantly increased or decreased expression compared with normal CD34+ cells (including some of the putative novel snoRNAs). Of note, no differentially expressed snoRNAs were detected comparing AML with or without DNMT3A mutations or with or without IDH1/2 mutations. We next interrogated published whole genome sequencing data to determine whether there were any cytogenetically silent genetic alterations in snoRNAs. No recurring point mutations or small insertions/deletions were detected in snoRNA genes in 50 cases of AML.In summary, we developed a new next-generation sequencing approach and analysis pipeline to quantify snoRNAs. We show that, compared with coding genes, snoRNA expression is more stable across different hematopoietic lineages, consistent with a housekeeping function. However, examples of developmentally-regulated and lineage-restricted snoRNA expression were identified. Finally, we show that a small subset of snoRNAs appear to be dysregulated in AML, although genetic alterations the specifically target snoRNAs appear to be rare in AML. DisclosuresNo relevant conflicts of interest to declare.

Introduction of the Kit Mutation into Immunodeficient Mice Enables Xenogeneic Reconstitution of Human Megakaryocyte-Erythroid Hematopoietic System

Immunodeficient mice are used to reconstitute human hematopoiesis and analyze human hematopoietic function in vivo by xenotransplantation of hematopoietic stem cells (HSCs). It is well recognized that in addition to disruption of the lymphoid system, the strain background such as NOD is critical for efficient human cell engraftment in xenotransplantation models. We have reported that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows the mouse SIRPA to bind human CD47, preventing activation of recipient macrophages to engulf human hematopoietic cells (Takenaka et al., Nature Immunol., 2006). We then developed a C57BL/6.Rag2nullIl2rgnull mouse line harboring the NOD-type Sirpa, named the BRGS mouse. The engraftment efficiency of human HSCs in BRGS mice was equal to that of NOD-Rag2nullIl2rgnull mice. This BRGS strain should be useful for further genetic modification because its background is simplified (Yamauchi et al., Blood, 2013).One of the persistent problems with the current xenograft models is that reconstituted human hematopoiesis is B-lymphoid dominant, whereas the engraftment levels of human myeloid cells, including megakaryocyte/erythrocyte lineage cells are quite low. In mouse syngeneic and allogeneic transplantation, recipients bearing mutation in the receptor tyrosine kinase Kit can accept donor HSCs without irradiation preconditioning. These data suggest that the Kit mutation impairs endogenous murine HSC self-renewal and allows donor HSCs efficiently to access to niche space. To develop a more efficient xenograft model, we further introduced a loss-of-function KitWv into the BRGS strain to deteriorate self-renewal and reconstitution capability of host HSCs. Here we show that this new mouse strain, termed the BRGSK mouse, showed very efficient human cell engraftment including myeloid, erythroid and megakaryocyte lineage cells.CD34+ cells obtained from human umbilical cord blood were transplanted into sublethally-irradiated BRGSK and BRGS mice at the age of 6-8 weeks by intrafemoral injection. At 8-12 weeks after transplantation, the average human hematopoietic chimerism defined by human CD45+ cells was more than 90% in the BM and this chimerism was maintained over 24 weeks in homozygous BRGSK recipients. Furthermore, the chimerism of human CD33+ myeloid cells in the BM was significantly improved (~5.7% in BRGS vs. ~32.5% in BRGSK). Surprisingly, CD71- CD235a+ mature erythrocytes and CD41+ platelets were detected in the BM. All human erythroid series including mature erythrocytes, CD71+ CD235a- early erythroblasts and CD71+ CD235a+ late erythroblasts were clearly seen (~0.1% in BRGS vs. ~42.2% in BRGSK). Immunohistochemical staining showed that these human erythroid cells were positive for human hemoglobin subunit alpha. Confocal immunofluorescence imaging revealed that human erythroid cells formed large erythroid islands, independently from mouse erythroid islands in the sternal marrow of recipient BRGSK mice. A number of CD41+ megakaryocytes were scattered in the BM. The BRGSK mice also well supported terminal differentiation of myeloid cells such as neutrophils, eosinophils, basophils, mast cells, monocytes, and dendritic cells. Thus, reconstitution and maintenance of human myelo-erythropoiesis is significantly improved in the new BRGSK strain. The new BRGSK xenograft model should be a strong tool to investigate normal and malignant human hematopoiesis. DisclosuresAkashi:Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy.

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

FOXC1 Is Derepressed to Functional Effect in Human Acute Myeloid Leukemia

The identification of genes and cellular pathways that are active in acute myeloid leukemia stem cells (AML LSC) but not normal hematopoietic stem and progenitor cells (HSPC) is essential, both for the understanding of disease biology and also for their evaluation as candidate therapeutic targets. Through in silico analysis, we identified FOXC1 as expressed in approximately 15-30% of cases of human AML in both LSCs and bulk cell populations. FOXC1 is a member of the forkhead box family of transcription factors and has essential roles in mesenchymal differentiation. Reflecting murine knockout phenotypes, in patients with Axenfeld-Rieger syndrome haploinsufficiency of FOXC1due to mutation or deletion causes developmental anterior segment abnormalities of the eye.By quantitative PCR we confirmed high level expression of FOXC1 in 17% of blast cell samples from patients with AML, and medium level expression of FOXC1 in 24% of samples (cohort size n=29). Critically, FOXC1 expression was not detected in any normal human hematopoietic cell population (including prospectively FACS-purified HSC, MPP, GMP, MEP, as well as defined mature cell populations, all from normal human bone marrow). Thus while FOXC1 is not expressed in normal human hematopoiesis, it is expressed in human leukemic hematopoiesis.To investigate whether FOXC1 derepression in AML makes a functional contribution to transformation, we initially performed knockdown (KD) experiments in human THP1 AML cells (which exhibit high level FOXC1 expression). FOXC1 KD led to loss of clonogenic potential and induction of morphological and immunophenotypic differentiation and this phenotype could be rescued by forced expression of a KD-resistant version of the gene. By contrast, FOXC1 KD in normal HSPC had no effect. Thus, FOXC1 contributes to the differentiation block in human AML cells.Forced expression of FOXC1 alone in normal murine HSPC induced a transient enhancement of clonogenic potential and myeloid differentiation block in serial replating assays, and myeloid skewing in in vivotransplantation assays. It did not however result in acute leukemia.Further quantitative PCR analyses demonstrated that high level FOXC1 expression associated strongly with high level HOXA9 expression in human AML. To determine whether co-expression of HOXA9 and FOXC1 is of functional significance, murine KIT+ HSPC were retrovirally infected with either Hoxa9 alone (with empty vector, MTV) or in pairwise combinations with FOXC1 or Meis1 (hereafter referred to as Hoxa9/MTV, Hoxa9/FOXC1 and Hoxa9/Meis1 cells, respectively) and their clonogenic potential was assessed in serial replating assays. As expected, Hoxa9 overexpression strongly augmented the clonogenic potential of BM HSPC, an effect which was enhanced by co-expression of Meis1. Importantly, the co-expression of Hoxa9 and FOXC1 also significantly enhanced the clonogenic potential and myeloid differentiation block of BM HSPC versus cells overexpressing Hoxa9 alone, as determined by immunophenotyping and colony morphology. Thus, FOXC1 and HOXA9 collaborate to enhance clonogenic potential and differentiation block in HSPC.To determine whether HOXA9 and FOXC1 collaborate to initiate leukemia, Hoxa9/MTV, Hoxa9/FOXC1 and Hoxa9/Meis1 double transduced HSPC were transplanted into irradiated congenic recipients. As expected, recipients of Hoxa9/Meis1 cells developed AML more rapidly than recipients of Hoxa9 cells (median latency 57 days versus 125 days). Strikingly, despite reduced initial engraftment levels, recipients of Hoxa9/FOXC1 cells succumbed to AML significantly earlier than mice receiving Hoxa9cells (median latency 83 days versus 125 days). In every case and in each cohort, autopsy demonstrated splenomegaly and pale BM due to infiltration of donor-derived cells of myeloid immunophenotype, confirming that these animals died from AML. These data demonstrate that FOXC1 functions to accelerate and enhance the development of AML in collaboration with HOXA9.Our functional studies are consistent with a model whereby lineage-inappropriate derepression of FOXC1 in human AML contributes to oncogenic transformation. DisclosuresNo relevant conflicts of interest to declare.

TIM-3 and Its Ligand, Galectin-9 Constitute a Pan-Myeloid Autocrine Loop to Develop and Maintain Malignant Stem Cells in Most Human Myeloid Leukemia

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on the surface of LSCs in most types of AML, and that TIM-3-targeted therapy could eradicate AML LSCs, based on in vivo xenograft experiments using patients’ AML samples (Kikushige et al, Cell Stem Cell, 2010). Since only the TIM-3+ but not the TIM-3- fraction of human AML cells can reconstitute human AML in immunodeficient mice, we hypothesized that the TIM-3 plays indispensable function to maintain AML LSCs. Galectin-9 is a ligand for TIM-3, and ligation of TIM-3 by galectin-9 has been shown to phosphorylate tyrosine residues of TIM-3 and activate Src family kinases through its SH2 domain in T cells and monocytes.We found that serum galectin-9 concentration was significantly elevated in AML patients (431.1+58.6pg/ml, n=13) but not in the healthy individuals (25.2+6.8pg/ml, n=7) or patients with B cell malignancies (36.1+15.5pg/ml, n=10). Primitive CD34+ AML cells had abundant galectin-9 protein in their cytoplasms. We then transplanted human CD34+ primitive AML cells into irradiated immunodeficient mice. Strikingly, only in mice reconstituted with human AML, but not in those with normal cord blood or human ALL cells had elevated serum levels of human galectin-9: Serum galectin-9 levels were 234.7+69.0pg/ml (n=8) in mice reconstituted with primary human AML cells, whereas 4.64+4.64pg/ml (n=12) in mice with normal human hematopoiesis. These results collectively suggested that AML cells secreted galectin-9 in vivo in an autocrine manner. We then performed transcriptome analyses of primary CD34+TIM-3+ AML cells after galectin-9 ligation. Results were further evaluated by a pathway enrichment analysis, which demonstrated that both NF-κB and β-catenin pathways were activated. In fact, galectin-9 ligation of TIM-3+ human AML cells induced activation of the NF-κB pathway via ERK and AKT phosphorylation. Activation of ERK and AKT pathways are known to inhibit the GSK3β activity, and to promote the nucleus translocation of β-catenin in several cancers. To demonstrate significant nucleus translocation of β-catenin of primary AML cells, we established a quantitation system by utilizing the Array Scan VTI system. By this system, we could formally prove that TIM-3 ligation by galectin-9 significantly promoted the nucleus translocation of β-catenin in primary AML cells.We also have extensively analyzed TIM-3 and galectin-9 interaction in cells from patients with myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and chronic myelogeneous leukemia. Strikingly, in all cases, frequencies of CD34+CD38-TIM-3+ cells dramatically increased along with disease progression from early/chronic phase to overt leukemias. Furthermore, serum levels of galectin-9 were also dramatically elevated after leukemic transformation. Significant nucleus translocation of β-catenin by galectin-9 ligation was also found in these diseases after leukemic transformation. A recent study has shown that NF-κB and β-catenin pathways could play a cooperative role in conferring the cancer-stem-cell properties to non stem cells in intestinal cancer model. These data collectively suggest that TIM-3 and galectin-9 constitutes a pan-myeloid autocrine loop to develop malignant stem cells in the vast majority of human myeloid malignancies. Thus, signaling molecules downstream of TIM-3 and galectin-9 ligation, as well as surface TIM-3 itself might be good candidates for cancer stem cell-target therapy common to most myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Enforced Expression Of Mir-125b Promotes the in vivo expansion Of Human Linneg cord Blood Multi-Lymphoid Progenitors and Leukemia Stem Cells

Acute Myeloid Leukemia (AML) is a heterogeneous disease with a relapse rate of up to 80% depending on patient age and AML subtype. AML is organized as a functional cellular hierarchy and is sustained by a rare population of leukemia stem cells (LSC). Recent work suggests that LSC properties influence therapy response, overall survival, and disease relapse. In order to develop more effective novel therapies that target this rare cell population; it is imperative that we better understand LSCs at the molecular level. Although it is generally accepted that oncogenic mutations underlie cancer initiation and progression, most studies have focused on protein coding genes. However, there is increasing recognition that non-coding RNAs can also play a role in leukemogenesis. microRNAs (miRNA) are a family of small non-coding RNAs that function as important regulators of mRNA stability and translation of protein-coding genes with significant roles in maintenance of human hematopoietic stem cells (HSC) (Lechman et. al., Cell Stem Cell, 2012). To understand the functional role of miRNA in human hematopoiesis, we generated HSC- and leukemia stem cell (LSC)-specific microRNA (miRNA) profiles by microarray analysis of sorted cell fractions from umbilical cord blood (CB) and AML patient samples that have been validated in xenograft assays. We identified ten miRNA candidates over-represented in HSC and/or LSC. To determine whether these were functional and impacted on stem cell properties we transduced lineage depleted CB cells with lentivirus expressing either a candidate miRNA or control vector followed by transplantation into immune deficient mice. Three miRNAs (miR-125b, miR-130a, miR-155) conferred a competitive growth advantage while four miRNAs (miR-99a, miR133a, miR194, miR-196b) conferred a growth disadvantage. miR-125b, a top LSC array candidate, showed the most pronounced phenotype with an overt expansion of transduced cells (19% to 96.2%) and enlarged spleens (2.4 fold increase). Detailed flow cytometric analysis of the miR-125b human grafts in recipient mice revealed a greatly expanded proportion of multi-lymphoid progenitors (MLP), in comparison to HSC and multi-potent progenitors. Furthermore, upon enforced in vivo expression of miR-125b in three AML patient samples, we observed large increases in the primitive primitive CD34+CD117+ populations (CD34+: 2.4-4.6 fold increase; CD117+: 1.3-4.1 fold increase) and a decrease in the proportion of differentiated CD14+/CD15+ cells (CD14+: 6.2-7.6 fold decrease; CD15+: 1.2-6 fold decrease) in leukemic grafts. Limiting dilution assays into secondary recipients revealed up to a 34-fold increase in LSC frequency compared to control vector transduced AML cells. Overall, these data suggest that miR-125b normally functions in the limited self-renewal of lymphoid committed early progenitors and this function may be usurped during leukemogenesis to enhance LSC self-renewal. miR-125b belongs to an evolutionarily conserved family consisting of three paralogs (miR-125a; miR-125b1; miR-125b2). Recent studies present strong evidence for a role of the miR-125 family in normal and malignant murine hematopoiesis, yet comprehensive functional inconsistencies remain in regards to the precise roles for each paralog. We are currently carrying out additional enforced expression studies directly comparing these family members in vitro and in vivo in order to clarify the functional roles of miR-125a (a top HSC array candidate) and miR-125b (a top LSC array candidate) in both normal and malignant human hematopoiesis. These studies will determine whether the miR-125 family is a suitable target for therapy of hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

TIM-3 as a novel therapeutic target for eradicating acute myelogenous leukemia stem cells

Acute myelogenous leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), which represent the ultimate therapeutic target for AML. Recent studies have identified several AML LSC-specific surface antigens as candidate targets of therapeutic molecules. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, with the exception of acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In xenograft models reconstituted with human AML LSCs or HSCs, an anti-human TIM-3 mouse IgG2a antibody with cytotoxic activities eradicates AML LSCs in vivo, but does not affect normal human hematopoiesis. Thus, TIM-3 is a promising therapeutic target for the eradication of AML LSCs.

Lysophosphatidic Acid Mediates Myeloid Differentiation within the Human Bone Marrow Microenvironment

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid present in the blood and certain tissues at high concentrations; its diverse effects are mediated through differential, tissue specific expression of LPA receptors. Our goal was to determine if LPA exerts lineage-specific effects during normal human hematopoiesis. In vitro stimulation of CD34+ human hematopoietic progenitors by LPA induced myeloid differentiation but had no effect on lymphoid differentiation. LPA receptors were expressed at significantly higher levels on Common Myeloid Progenitors (CMP) than either multipotent Hematopoietic Stem/Progenitor Cells (HSPC) or Common Lymphoid Progenitors (CLP) suggesting that LPA acts on committed myeloid progenitors. Functional studies demonstrated that LPA enhanced migration, induced cell proliferation and reduced apoptosis of isolated CMP, but had no effect on either HSPC or CLP. Analysis of adult and fetal human bone marrow sections showed that PPAP2A, (the enzyme which degrades LPA) was highly expressed in the osteoblastic niche but not in the perivascular regions, whereas Autotaxin (the enzyme that synthesizes LPA) was expressed in perivascular regions of the marrow. We propose that a gradient of LPA with the highest levels in peri-sinusoidal regions and lowest near the endosteal zone, regulates the localization, proliferation and differentiation of myeloid progenitors within the bone marrow marrow.

SALL4 is a key transcription regulator in normal human hematopoiesis

Stem cell factor SALL4 is a zinc finger transcription factor. It plays vital roles in the maintenance of embryonic stem cell properties, functions as an oncogene in leukemia, and has been recently proposed to use for cord blood expansion. The mechanism(s) by which SALL4 functions in normal human hematopoiesis, including identification of its target genes, still need to be explored. Chromatin immunoprecipitation followed by microarray hybridization (ChIP-chip) was used for mapping SALL4 global gene targets in normal primary CD34+ cells. The results were then correlated with SALL4 functional studies in the CD34+ cells. More than 1000 potential SALL4 downstream target genes have been identified, and validation of binding by ChIP-quantitative polymerase chain reaction was performed for 5% of potential targets. These include genes that are involving in hematopoietic differentiation and self-renewal, such as HOXA9, RUNX1, CD34, and PTEN. Down regulation of SALL4 expression using small-hairpin RNA in these cells led to decreased in vitro myeloid colony-forming abilities and impaired in vivo engraftment. Furthermore, HOXA9 was identified to be a major SALL4 target in normal human hematopoiesis and the loss of either SALL4 or HOXA9 expression in CD34+ cells shared a similar phenotype. Taken together, SALL4 is a key regulator in normal human hematopoiesis and the mechanism of its function is at least in part through the HOXA9. Future study will determine whether modulating the SALL4/HOXA9 pathway can be used in cellular therapy such as cord blood expansion and/or myeloid engraftment.

TIM‐3 as a therapeutic target for malignant stem cells in acute myelogenous leukemia

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Recent studies have shown that many AML LSC-specific surface antigens could be such candidates. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In mouse models reconstituted with human AML LSCs or human hematopoietic stem cells, a human TIM-3 mouse IgG2a antibody with complement-dependent and antibody-dependent cellular cytotoxic activities eradicates AML LSCs in vivo but does not affect normal human hematopoiesis. Thus, TIM-3 is one of the promising targets to eradicate AML LSCs.

TIM-3 Is a Promising Target to Eradicate Acute Myeloid Leukemia Stem Cells Sparing Normal Hematopoietic Stem Cells

Abstract 559Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for permanent cure. To selectively kill AML LSCs sparing normal hematopoietic stem cells (HSCs), one of the most practical approaches is to target the AML LSCs-specific surface or functionally indispensable molecules. Based on differential transcriptome analysis of prospectively-purified CD34+CD38− LSCs from AML patient samples and normal HSCs, we found that T-cell immunoglobulin mucin-3 (TIM-3) was highly expressed in AML LSCs but not in normal HSCs (Kikushige et al., Cell Stem Cell, 2010). In normal hematopoiesis, TIM-3 is mainly expressed in mature monocytes and a fraction of NK cells, but not in granulocytes, T cells or B cells. In the bone marrow, TIM-3 is expressed only in a fraction of granulocyte/macrophage progenitors (GMPs) at a low level, but not in HSCs, common myeloid progenitors, or megakaryocyte/erythrocyte progenitors. In contrast, in human AML, TIM-3 was expressed on cell surface of the vast majority of CD34+CD38− LSCs and CD34+CD38+ leukemic progenitors in AML of most FAB types, except for acute promyelocytic leukemia (M3). FACS-sorted TIM-3+ but not TIM-3− AML cells reconstituted human AML in the immunodeficient mice, indicating that the TIM-3+ population contains most of functional LSCs. To selectively eradicate TIM-3-expressing AML LSCs, we established an anti-human TIM-3 mouse IgG2a antibody, ATIK2a, possessing antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities in leukemia cell lines transfected with TIM-3. We first tested the effect of ATIK2a treatment on reconstitution of normal HSCs in a xenograft model. ATIK2a was intraperitoneally injected to the mice once a week after 12 hours of transplantation of human CD34+ cells. Injection of ATIK2a did not affect reconstitution of normal human hematopoiesis except removing TIM-3-expressing mature monocytes. In contrast, injection of TIM-3 to the mice transplanted with human AML samples markedly reduced leukemic repopulation. In some mice transplanted with AML bone marrow, only normal hematopoiesis was reconstituted after anti-TIM-3 antibody treatment, suggesting that the antibody selectively killed AML cells, sparing residual normal HSCs. To further test the inhibitory effect of ATIK2a on established human AML, eight weeks after transplantation of human AML cells, engraftment of human AML cells was confirmed by blood sampling and thereafter ATIK2a was injected to these mice. In all cases tested, ATIK2a treatment significantly reduced human TIM-3+ AML fraction as well as the CD34+CD38− LSCs fraction. In addition, to verify the anti-AML LSCs effect of ATIK2a treatment, human CD45+AML cells from the primary recipients were re-transplanted into secondary recipients. All mice transplanted from primary recipients treated with control IgG developed AML, whereas none of mice transplanted with cells from ATIK2a-treated primary recipients developed AML, suggesting that functional LSCs were effectively eliminated by ATIK2a treatment in primary recipients.Thus, TIM-3 is a promising surface molecule to target AML LSCs. Our experiments strongly suggest that targeting this molecule by monoclonal antibody treatment is a practical approach to eradicate human AML. Disclosures:No relevant conflicts of interest to declare.

Distinct Regulation of β- and γ-Catenin throughout Hematopoietic Development Contrasts with Their Cooperative Roles In Acute Myeloid Leukemia.

AbstractAbstract 1573Wnt proteins are important developmental regulators and are known to play a role in maintenance of hematopoietic stem cells (HSC). Wnt signaling has also been identified as one of the most frequently dysregulated processes associated with acute myeloid leukemia (AML), though the significance of this observation is as yet poorly understood. Here we investigate the role of two Wnt signaling proteins; β-catenin and γ-catenin and their respective roles in both normal human hematopoiesis and in AML. These proteins have dual and overlapping roles as transcriptional activators of Wnt target genes in the nucleus, and as structural components of the cytoskeleton.To determine the potential scope of influence of these proteins, we first examined their expression levels and subcellular location throughout normal human hematopoiesis using multi-parameter flow cytometric analysis and confocal microscopy. As expected β-catenin was strongly expressed in human cord blood derived HSC (212 MFI ±124, n=6) and at lower levels in differentiated subsets; surprisingly however β-catenin expression was maintained in granulocytic (1182 MFI±568) and monocytic cells (284 MFI±107). Nuclear localization was independent of cytoplasmic expression level, being strongly nuclear-localized in early progenitors and predominantly cytoplasmic in differentiated cells (58%±5 nuclear-localized in CD34+ cells vs 27%±1 in granulocytes, P=0.008). The expression pattern of γ-catenin was similar to β-catenin but showed a reciprocal pattern of subcellular localization, with levels of nuclear γ-catenin strongest in differentiated cells (10%±2 in CD34+ cells vs 44%±3 in monocytes P=0.0005). These data imply complementary roles for β and γ-catenin in normal hematopoiesis and show that nuclear localization of these proteins is regulated independently and irrespective of their expression level.In AML patients, β-catenin dysregulation has been previously reported; however, we also observed frequent overexpression of γ-catenin (over 5 fold in 25% of patients). This overexpression was associated with lower remission rates (OR 1.23 per log increase, P=0.03, CI 1.02–1.49) arising from resistant disease (OR 1.57 per log increase, P=0.003, CI 1.16–2.14) in a cohort of 243 AML patients adjusted for baseline diagnostic variables. In contrast to normal hematopoiesis, we found that nuclear localization of γ-catenin correlated with nuclear localization of β-catenin in AML (R=0.5, n=59) suggesting that the capacity to independently regulate the nuclear entry/retention of these catenins is disrupted in AML. To investigate this, we examined the effect of ectopic overexpression of γ-catenin in normal cord blood derived CD34+ cells and AML cell lines. Three-fold overexpression of γ-catenin failed to induce nuclear translocation of γ- or β-catenin in normal progenitors, which exhibited no major developmental defects. In contrast, in 3 of 4 AML cell lines, overexpression of γ-catenin strongly promoted its nuclear localization (9-16 fold) and was associated with a block in agonist-induced differentiation - a phenotype previously associated with β-catenin. In accord with this, we found that as in primary AML, nuclear translocation of γ-catenin in AML cell lines was associated with translocation of β-catenin (2-22 fold).In conclusion, we propose that in normal hematopoiesis, nuclear translocation of β- and γ-catenin is tightly and independently regulated for each catenin. In contrast, most AML cells lack this regulation resulting in correlated nuclear levels of β- and γ-catenin. In addition, we found while overexpression of γ-catenin has little consequence for normal cells; in malignant cells γ-catenin facilitates nuclear translocation of β-catenin. Disclosures:No relevant conflicts of interest to declare.

The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

AbstractAbstract 739Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures:No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Stem Cells Escape Innate Immune Surveillance by Macrophages through Interaction with SIRPα

AbstractAbstract 499 Introduction:Using a genome-wide positional cloning approach, we previously identified NOD-derived Sirpα as the gene required for support of normal human hematopoiesis in NOD.SCID xenotransplantation assays. NOD.SCID mice bearing other polymorphic Sirpα alleles (e.g. NOD.NOR-Idd13.SCID) do not support human engraftment due to lack of interaction between CD47 expressed on human hematopoietic cells and SIRPα expressed on host macrophages. CD47-SIRPα interaction is also critical for engraftment of human acute myeloid leukemia stem cells (AML-SC): homing, engraftment and migration are all significantly impaired in NOD.NOR-Idd13.SCID compared to NOD.SCID mice. However, AML-SC from different patients are heterogeneous in their ability to engraft NOD.NOR-Idd13.SCID mice, suggesting variable ability to evade innate immune surveillance. We undertook investigations of the mechanisms underlying the decreased engraftment ability of AML-SC in NOD.NOR-Idd13.SCID mice, a better understanding of which will aid in optimizing potential therapeutic approaches aimed at disrupting CD47-SIRPα signaling in AML. Result:To investigate the cellular components of innate immunity that impair AML-SC function, we depleted macrophages in NOD.NOR-Idd13.SCID mice by treatment with clodronate liposomes. Following intravenous injection, homing of AML cells (n=2 patient samples) to bone marrow (BM) and spleen of NOD.NOR-Idd13.SCID mice was not detectable in control mice but was increased in clodronate-treated mice to levels similar to those seen in clodronate-treated NOD.SCID mice. Thus, impaired homing resulting from lack of interaction between CD47 on AML cells and SIRPα on macrophages is rescued by macrophage depletion.To examine the role of macrophages in engraftment and migration of AML-SC, we transplanted NOD.NOR-Idd13.SCID mice intrafemorally with primary AML cells (n=3 patient samples) and treated them weekly with clodronate for 8 weeks. In previous experiments, these AML samples were unable to engraft NOD.NOR-Idd13.SCID mice, or could only engraft the injected femur without migration to other hematopoietic sites. In contrast, clodronate-mediated macrophage depletion in these mice allowed substantial leukemic engraftment by all samples not only in the injected femur, but also in non-injected bones, the spleen and peripheral blood. Thus, evasion of innate immune surveillance by macrophages through interactions between CD47 on AML cells and SIRPα on macrophages is critical for engraftment of AML-SC.To investigate the mechanism of macrophage-mediated impairment of AML-SC function in NOD.NOR-Idd13.SCID mice, we established an in vitro phagocytosis assay using BM-derived macrophages from NOD.SCID and NOD.NOR-Idd13.SCID mice. NOD.NOR-Idd13.SCID macrophages prestimulated with interferon-γ and lipopolysaccharide consistently showed higher levels of phagocytosis of human AML cells in vitro compared to NOD.SCID macrophages. This finding suggests that macrophages eliminate AML cells in vivo through phagocytosis, a process that is inhibited by CD47-SIRPα signaling.The critical importance of CD47-SIRPα signaling for AML-SC function led us to develop a novel human SIRPα (hSIRPα)-Fc fusion protein antagonist for therapy of AML. In pilot studies, in vitro pretreatment of AML cells efficiently blocked homing, engraftment and dissemination following xenotransplantation into NOD.SCID mice. More importantly, in vivo hSIRPα-Fc treatment of NOD.SCID mice repopulated with human AML resulted in dramatic decrease of the leukemic burden in all hematopoietic tissues. Conclusion:Our data show that the interaction between CD47 on AML-SC and SIRPα expressed on host macrophages is critical for the leukemic clone to evade macrophage-mediated innate immune attack. Disruption of this signaling axis, as with hSIRPα-Fc fusion proteins, is an effective therapeutic approach for eradication of AML-SC. Disclosures:Theocharides: Trillium Therapeutics Inc. (TTI): Patents & Royalties, Research Funding. Jin: Trillium Therapeutics Inc. (TTI): Patents & Royalties, Research Funding. Dick: Trillium Therapeutics Inc. (TTI): Patents & Royalties, Research Funding. Danska: Trillium Therapeutics Inc. (TTI): Patents & Royalties, Research Funding. Wang: Trillium Therapeutics Inc. (TTI): Patents & Royalties, Research Funding.

"Creation of mouse models for human diseases"

To translate research findings into medicine and drug development, we have been performing in vivo research using primary human samples. Development of mouse models with reconstituted human immunity would be critical to overcome technical and ethical constraints associated with in vivo human research. To this end, we have created humanized mice by intravenously injecting purified human hematopoietic stem cells (HSCs) into immune-compromised NOD/SCID/IL2rgKO newborns. This xenogeneic transplantation system allows long-term engraftment and multi-lineage differentiation of human HSCs. The humanized mouse fully reconstituted with human myeloid and lymphoid subsets is expected to serve as an in vivo platform for the investigation of human immune function. In addition to understanding normal human hematopoiesis and immunity, the use of humanized mice is expected to allow investigators to translate their research findings into therapeutic and pharmaceutical development. As a possibility for such translation, we developed an in vivo model of human acute myeloid leukemia (AML), a disease in which the majority of patients succumb to relapse. Using the model, we examined the pathogenesis of AML and tried to clarify the role of AML stem cells in chemotherapy resistance and relapse. Humanized mouse model for both normal and diseased immuno-hematopoietic system is opening a new era in translational medicine.

Blockage of SDF-1-CXCR4 Axis by AMD 3100 Can Be a Novel Therapy for Acute Lymphoblastic Leukemia by Targeting the Extramedullary Sites of Leukemic Cells.

Abstract 981Poster Board I-3 Background and Purpose:Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although the accumulated progresses in treatment regimen have raised the 5-year survival rate as high as 80% for whole pediatric patients, only poor prognosis, an overall survival rate of 30%, can be still now expected for the patients with relapsed diseases. Widespread extramedullary involvement such as liver, spleen, lymph nodes and central nervous system invasion is a well-known characteristic of ALL related to poor prognosis. Recently, bone marrow (BM) microenvironments supporting leukemic cells have been widely noticed as an important element which influences on treatment response and relapse of disease. Although the mechanism of extramedullary dissemination has been the most crucial issues in the study of leukemia, it still remains incompletely understood. In this study, we established a novel murine model of human ALL with NOD/SCID/γc null (NOG) mouse. Using this model, we examined the involvement of SDF-1-CXCR4 signaling axis in hepatomegary development in ALL. ResultPrimary bone marrow samples were collected from 13 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOD/SCID/γc null (NOG) mice, a transgenic mouse with severe combined immunodeficiency and IL-2 receptor chain allelic mutation showing high potential to reconstitute the normal human hematopoietic system. Primary samples from 10 out of 13 patients were successfully engrafted into mice without any conditioning such as prior irradiation and DNA-damaging agents medication, and those engrafted leukemic cells were able to be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as >80% blood chimerism and conserved blast phenotypes through serial transplantations. Moreover, extramedullary organs including liver, spleen and kidneys showed the leukemic invasion consistent with donor ALL disease. In contrast, no normal human hematopoiesis was observed in our xenotransplantation system without conditioning.CXCR4 is a known regulator of lymphocyte migration and has been suggested to be important for proliferation of normal leucocytes and leukemic cells. CXCR4 expression and function of leukemic cells in NOG mice were confirmed by flow cytometry and in vitro chemotaxis assays towards its known chemokine ligand SDF-1. Immunohistorical analysis of liver reveals that SDF-1 was detectable only in biliary duct endotherial cells. Finally, we demonstrated directly the effect of SDF-1-CXCR4 axis in our model by using the CXCR4 inhibitor AMD3100 in vivo and in vitro. Discussion:NOG mice model for engraftment of human leukemic cells provides significant insights into the biology of ALL and allows us to answer various questions concerning the molecular mechanism of extramedullaly invasion. This non-conditioning approach may prevent possible damage to the host microenvironment, thereby providing a more natural model for growth of human leukemic cells in mice. Our present study on the involvement of SDF-1-CXCR4 axis in ALL dissemination could rink to the novel therapies in future which target the extramedullary sites in order to perfectly overcome ALL. Disclosures:No relevant conflicts of interest to declare.

NFI-A directs the fate of hematopoietic progenitors to the erythroid or granulocytic lineage and controls β-globin and G-CSF receptor expression

AbstractIt is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the β-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.

A Phase 1 and Correlative Biological Study of CSL360 (anti-CD123 mAb) in AML

CD123 (IL-3Rα) is a phenotypic marker of putative leukemic stem cells (LSC) in AML (Jordan, Leukemia 2000; 14:1777). We and others have found that CD34+38− cells from AML patients (pts) express high levels of CD123, in contrast to absence of expression on CD34+38− cells in normal individuals. Binding of CD123 by monoclonal antibody (mAb) 7G3 inhibits IL-3-dependent signalling and proliferation in vitro. In a NOD-SCID xenograft model 7G3 inhibits human AML engraftment, but not normal human hematopoiesis (Lock ASH 2007; Abs161). CSL360, a recombinant chimeric IgG1 mAb derived from 7G3, binds the same epitope. CSL360 concentrations ≥ 0.1μg/mL in vitro inhibited 90% AML cell growth in the presence of supraphysiological IL-3 levels. Preclinical toxicology studies with doses up to 100 mg/kg weekly × 4 in cynomolgus monkeys showed no CSL360-related effects in clinical signs, hematology, chemistry, urinalysis, gross pathology or histopathology. A Phase 1 study of safety, pharmacokinetics (PK) and bioactivity of CSL360 in relapsed, refractory or high risk AML began in March 2007. Pts receive 12 weekly iv infusions if not withdrawn early due to treatment-related toxicity or disease progression. Additional treatments may be given to pts who achieve a response. Bone marrow aspirates/trephine samples are obtained at screening, after dose 3 and before doses 5 and 11. More than 180 infusions have been administered to 26 AML pts (21 M, 5 F; 17 de novo, 8 MDS-related, 1 treatment-related AML) in 5 dose level cohorts: 0.1, 0.3, 1.0, 3.0 and 10 mg/kg. There is no intra-patient dose escalation. PK parameters over the dose range, estimated in 19 pts over 7 days after doses 1 and 4, were linear with dose-proportional increases in the AUC and Cmax; dose 1 Cmax ranged from 0.62 – 287.33 μg/mL and dose 4 Cmax from 1.02 – 178.22 μg/mL. CSL360 mean plasma half-life (dose 1, 83 ± 33 h; dose 4, 117 ± 59 h) appears to be independent of dose and treatment number. Dose 1 systemic clearance (0.21 ± 0.16 L/h) and volume of distribution (0.39 ± 0.22 L/kg) were relatively low, consistent with this size IgG. In all pre-treatment samples anti-CSL360 antibody titers were negative, determined by enzyme immunoassay. Anti-CSL360 antibodies were detected post-treatment in 8/12 pts; these antibodies have not been fully quantified or characterised. CSL360 has been well tolerated; a MTD has not been defined. Seven pts received all 12 doses, 13 pts were withdrawn due to progressive disease or investigator's decision, 3 pts were withdrawn in association with infections, 2 pts withdrew consent, and 1 pt is ongoing. Three serious adverse events have been considered possibly related or related to CSL360: 1 invasive fungal infection (Gr 5), and 2 infusion reactions (Gr 2; hospitalised). Other adverse events are consistent with expectations for the disease population. Of 8 pts in the 3 mg/kg and 10 mg/kg cohorts who are evaluable for response after ≥ 4 doses, 1 complete response (CR) has been observed. A 22 yr old male, de novo FAB M1 cytogenetically normal AML, who had relapsed post-2 allogeneic SCT, achieved a morphologic leukemia free state after 3 doses (3.0 mg/kg) and CR after 12 doses, sustained for > 9 weeks. The pt received 17 doses before withdrawal to treat co-morbidities. Flow cytometry studies with anti-CD123 antibodies demonstrated dose-dependent CSL360 coating of both AML blasts and LSC. Saturation of target antigen on marrow and blood cells was observed 1 day after dosing at 0.3mg/kg, associated with decreased expression of CD123 detected by an antibody to a different epitope. At higher dose levels saturation of CD123 was maintained 7 days post dosing, associated with ongoing reduction in surface CD123 expression. In a representative sample, plasma from a pt treated at 10 mg/kg specifically inhibited IL-3-induced proliferation of AML blasts ex vivo, indicating sufficient circulating concentration of CSL360 to inhibit IL-3 mediated effects in vivo. Effects of CSL360 on proliferation and apoptosis of AML cells in treated patients are being investigated. These preliminary results show anti-CD123 mAb therapy with CSL360 is safe and tolerable; biological effects have been demonstrated; a sustained CR was achieved in 1 advanced, refractory AML pt. The study is continuing, with 20 evaluable patients to be accrued and treated at 10 mg/kg weekly; at this dose level the PK and correlative assays predict that complete blockade of IL-3 signalling through CD123 can be achieved in vivo.

SHP-2 Knockdown Results in Profound Inhibition of Human CD34+ Hematopoietic Stem/Progenitor Cell Survival, Proliferation and Differentiation in Response to Growth Factor Stimulation

SHP-2 (ptpn11), a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase, is expressed at high levels in hematopoietic cells and regulates downstream signaling from growth factor (GF) receptors. SHP-2 has been shown to play an important role in murine hematopoiesis. Moreover, several SHP-2 activating mutations have been identified in myeloid malignancies and there is interest in the development of SHP-2 inhibitors for cancer treatment. On the other hand previous report suggested that SHP-2 inhibition was associated with enhanced GF responsiveness in human hematopoietic cell lines. However the role of SHP-2 signaling in normal human hematopoietic stem and progenitor cell growth has not been studied. Here we investigated the function of SHP-2 in normal human hematopoiesis by inhibiting SHP-2 expression in cord blood (CB) CD34+ cells with stable SHP-2 shRNA expression. We transduced CB CD34+ cells with lentivirus vectors coexpressing SHP-2 specific shRNAs (Si-1 or Si-2) or a control shRNA (Ctrl) and RFP and selected RFP expressing CD34+ cells by flow cytometry sorting. We observed >80% inhibition of SHP-2 expression by Western blotting in Si-1 or Si-2 shRNA transduced cells compared with Ctrl shRNA transduced cells. We observed that culture with increasing concentrations of GF was associated with markedly reduced GF-induced stimulation of proliferation of SHP-2-knockdown CD34+ cells compared to controls. In addition we observed significantly increased apoptosis of SHP-2-knockdown CD34+ cells cultured under low and high GF conditions compared to controls, but little increase in apoptosis in GF-deprived cells, indicating markedly reduced response of SHP-2-knockdown cells to GF-mediated promotion of cell survival. SHP-2-knockdown CD34+ cells also demonstrated significantly reduced expansion in cell numbers following culture in high GF conditions compared with controls (115.3, 25.5 and 10.4 fold expansion for Ctrl, Si-1 and Si-2 at day 7). Analysis of the nature of cells generated in GF culture showed significantly reduced generation of both myeloid (CD33+, CD11b+ and CD14+) and erythroid cells from SHP-2-knockdown CD34+ cells compared with controls, with relatively greater inhibition of myeloid compared with erythroid differentiation. On the other hand CD34+ cell numbers were retained at levels similar to controls after culture. We also observed significantly reduced cell expansion and differentiation and higher apoptotic rates of SHP-2-knockdown cells cultured under either myeloid promoting (IL-3+SCF+G-CSF+GM-CSF) or erythroid promoting (SCF+EPO) GF conditions. SHP-2-knockdown cells demonstrated reduced activation of MAPK and STAT5 but not Akt on Western blotting that was associated with reduced MCL-1 expression, consistent with their reduced GF mediated proliferation and survival. Expression of the transcription factors SCL1, GATA-1, NF-E2 and FOG-1 was increased in SHP-2 knockdown CD34+ cells compared to controls, consistent with the relatively higher retention of CD34+ and erythroid cells compared with myeloid cells after culture. In conclusion, we show that SHP-2 knockdown in human CD34+ cells results in markedly decreased responsiveness to GF stimulation with significantly increased apoptosis, markedly diminished proliferation and reduced generation of differentiated cells during GF culture. A relative retention of the CD34+ cell population was seen despite increased apoptosis, which may be the result of reduced cell turnover and altered transcription factor expression in SHP-2-knockdown cells, and is in contrast to reduced stem cell self-renewal observed following SHP-2 knockdown in murine models. These results indicate a critical role for SHP-2 in GF mediated signaling responses in human hematopoietic stem/progenitor cells. These studies also caution that therapeutic SHP-2 inhibition could be associated with significant hematopoietic toxicity.