Human Hematological Diseases Research Articles

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2305.

Label-Free High Dimensional Single Cell Morphological Profiling of Different Hematological Malignancies By Ghost Cytometry

Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern 'omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6) (Figure 1). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information (Figure 2). For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified unique cell populations across blood cell populations that were distinct from the healthy BM-MNC controls. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Keywords: flow cytometry, acute leukemia, multiple myeloma, label-free cell analysis, biomarker, liquid biopsy, ghost cytometry, artificial intelligence, machine learning Figure 1. Research design overview. Bone marrow samples from patients and non-diseased controls will be analyzed by Ghost Cytometry to assess disease by conventional markers and identify novel morphometric subpopulations. Morphological information is acquired as label-free GMI signals without any labeling. The label-free GMI information is transformed into two-dimensional space by UMAP dimensionality reduction method to identify subpopulations of interest. Both samples are stained with known cell surface markers for validation of unsupervised classification. Figure 2. Identification of disease-specific cell subpopulations by unsupervised approach. Using an unsupervised machine learning technique called UMAP, label-free morphologically distinct cellular subpopulations can be identified in B-ALL, AML, MM when compared to healthy donor controls (left panels, inside the dotted circles). For validation of the subpopulations, known cell surface markers (CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM) and cell trace marker (CellTracker) were overlaid. The intensity increases monotonically through the colormaps. The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population.

Generation of a thrombopoietin‐deficient thrombocytopenia model in zebrafish

BackgroundThe production of platelets is tightly regulated by thrombopoietin (THPO). Mutations in the THPO gene cause thrombocytopenia. Although mice lacking Thpo present with thrombocytopenia, predicting phenotypes and pathogenicity of novel THPO mutations in mice is limited. Zebrafish can be a powerful tool for fast validation and study of candidate genes of human hematological diseases and have already been used as a model of human thrombocytopenia. ObjectivesWe aim to investigate the role of Thpo in zebrafish thrombopoiesis and to establish a Thpo‐deficient zebrafish model. The model could be applied for illustrating the clinically discovered human THPO variants of which the clinical significance is not known and to evaluate the effect of THPO receptor agonists (THPO‐Ras), as well as a screening platform for new drugs. MethodsWe generated a thpo loss‐of‐function zebrafish model using CRISPR/Cas9. After disruption of zebrafish thpo, thposzy6 zebrafish presented with a significant reduction of thpo expression and developed thrombocytopenia. Furthermore, we performed in vivo studies with zebrafish with the thposzy6 mutation and found two human clinical point mutations (c.091C > T and c.112C > T) that were responsible for the thrombocytopenia phenotype. In addition, effects of THPO‐RAs used as therapeutics against thrombocytopenia were evaluated in the Tg(mpl:eGFP);thposzy6 line. Results and conclusionsZebrafish with the mutation thposzy6 presented with a significant reduction of thpo expression and developed thrombocytopenia. Thpo loss‐of‐function zebrafish model can serve as a valuable preclinical model for thrombocytopenia caused by thpo‐deficiency, as well as a tool to study human clinical THPO variants and evaluate the effect of THPO‐RAs.

3D Multicellular Spheroid for the Study of Human Hematopoietic Stem Cells: Synergistic Effect Between Oxygen Levels, Mesenchymal Stromal Cells and Endothelial Cells.

IntroductionThe human bone marrow microenvironment is composed of biological, chemical and physical factors that act in a synergistic way to modulate hematopoietic stem cell biology, such as mesenchymal stromal cells (MSCs), endothelial cells (ECs) and low oxygen levels; however, it is difficult to mimic this human microenvironment in vitro.MethodsIn this work, we developed 3D multicellular spheroid (3D-MS) for the study of human hematopoietic stem cells (HSCs) with some components of perivascular niche. HSCs were isolated from umbilical cord blood, MSCs were isolated from human bone marrow and a microvasculature EC line (CC-2811, Lonza®) was used. For the formation of a 3D structure, a magnetic levitation culture system was used. Cultures were maintained in 21%, 3% and 1% O2 for 15 days. Culture volume, sphericity index and cell viability were determined. Also, human HSC proliferation, phenotype and production of reactive oxygen species were evaluated.ResultsAfter 15 days, 3D-MS exhibited viability greater than 80%. Histology results showed structures without necrotic centers, and higher cellular proliferation with 3% O2. An increase in the expression of the CD34 antigen and other hematopoietic antigens were observed to 1% O2 with MSCs plus ECs and low ROS levels.ConclusionThese findings suggest that 3D-MS formed by MSCs, ECs and HSCs exposed to low concentrations of oxygen (1–3% O2) modulate human HSC behavior and mimics some features of the perivascular niche, which could reduce the use of animal models and deepen the relationship between the microenvironment of HSC and human hematological diseases development.

The contribution of fish models in understanding of blood disorders

Human and fish peripheral blood contains erythrocytes or red blood cells, leukocytes or white blood cells and platelets or thrombocytes. Although the fish present the same types of blood cells as humans, studies have highlighted some morphological, ultrastructural and cytochemical differences in blood cells of the two groups of vertebrates. In both human and fish hematological tests are indispensable tools which provide a considerable amount of useful information for evaluation of the general state of health. Although there are limited amount of studies on blood cells in fish, over the years especially Danio rerio, has become an powerful model organism for studying human hematological diseases, as anemia, leukemia, myeloproliferative diseases and others human hematological diseases. Thereby, fish are ideal model organisms for studying hematological diseases due to external fertilization, brief development time, large number of embryos and the results can be extrapolated to mammals, including human. Furthermore, ikaros, rag-1, rag-2 and lck genes that are found in human were observed in Danio rerio revealing the spatio-temporal proximity between these groups of vertebrates. Therefore, studies realized on fish can offer essential information about molecular mechanism which causes the appearance of numerous human hematological diseases and discovery of possible therapeutic methods. This review provides a comparative overview of blood cells in fish and humans and describes zebrafish mutants as models of human hematologic disorders.

Zebra “Fishing” the Role of Granulin in Hematopoiesis

Granulin (GRN) is a multifunctional protein with anti-inflammatory properties and involved in neurological diseases and tumorigenesis. It contains several cysteine-rich motifs that are unique to this protein, which are conserved from sponges to humans indicating their ancient evolutionary origin. Despite being highly expressed by certain hematopoietic cell lineages, the role that GRN plays in hematopoiesis has reminded elusive. The multifunctional nature of this protein, together with its wide expression in all mammalian cell types has challenged the characterization of its functional role in hematopoiesis due to its effects on other tissues. Therefore, we took advantage of the whole genomic duplication of the zebrafish (Danio rerio) and the high conservation of the cysteine-rich motifs among the zebrafish and human granulins to address this knowledge gap and explore their role in hematopoiesis in vivo. The whole genome duplication that separated teleost fish from mammals resulted in two copies of the granulin gene in the zebrafish (Granulin a and Granulin b, Grna and Grnb respectively). This has allowed us an unprecedented view into the function of this protein in hematopoiesis. We show that like mammals, grnb transcripts are found in all cell types, including hematopoietic cells. In contrast, grna is restricted to hematopoietic cells, including myeloid populations. The distinct cell expression of grna and grnb suggests that, in the zebrafish, grna evolved to specifically function in hematopoiesis, while grnb may have taken on the rest of the biological roles assigned to the mammalian granulin. The zebrafish is an animal model with unique advantages for in vivo studies. Its external development allows us to circumvent the challenges of in utero experimentation required using mammals, permitting the use of non-invasive imaging techniques to study developmental hematopoiesis. In addition, more than 70% of genes identified in the zebrafish are conserved in humans. These, together with its high conservation with the human hematopoietic system has led to a greater understanding and prevention of human hematologic diseases by using this elegant animal model. These unique advantages of the zebrafish, in addition to its genetic amenability allowed us to generate Grna and Grnb single mutants and identify their impact in the hematopoietic system in vivo. While the absence of Grnb did not affect the development of the hematopoietic system, lack of Grna led to decreased differentiation of myeloid precursors into neutrophils and macrophages. Therefore, Grna knockout allowed us to disrupt the hematopoietic function of granulin while keeping unaltered its function in the brain and other non-hematopoietic tissues. Although viable, adult Grna mutants developed kidney marrow (the fish analogous to the mammalian bone marrow) failure, with increased progenitors and decreased mature myeloid cells. Mechanistically, we found that pu.1, the main transcription factor that leads to myeloid differentiation, directly bound grna enhancers, upregulating its expression. We have demonstrated that Grna enhanced myeloid gene expression, and decreased gata1 expression thereby facilitating myeloid differentiation and inhibiting the erythroid genetic program. Finally, we show that these findings in the zebrafish are also conserved in humans. Altogether, we have identified the hematopoietic role of granulin without disturbing its biological functions in other tissues. We have unveiled a powerful and novel master regulator for myeloid differentiation that could potentially be utilized for the treatment of hematological disorders such as neutropenia and leukemia. Disclosures No relevant conflicts of interest to declare.

Targeting Transcriptional Co-Activator PGC-1a for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is a FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU). Transcriptional co-activator PGC-1α has recently been showed to play a crucial role in globin gene regulation and erythropoiesis by our group. Loss of function in the PGC-1α knock out mice significantly reduced the expression of murine embryonic εy- and βh1-globin genes; both of them are homologues of the human ε- and γ-globin genes. Forced overexpression of PGC-1α in vitroby adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. These results directly validate a role for PGC-1α in human γ-globin induction and provide significant support for the hypothesis that modulating PGC-1α activity, or the signaling pathways that it regulates, in SCD patients may be an effective approach that could therapeutically benefit individuals with SCD. Here we report the effects of PGC-1α in inducing HbF in human primary erythoid progenitor CD34+cells. We infected CD34+cells with lentivirus carrying PGC-1α and found that over-expression of PGC-1α resulted in significantly increased F-cells in the culture, from 1.6% up to 41.1%. We further discovered several PGC-1α agonists that are highly selective for PGC-1α. We found that one of the PGC-1α agonists, Compound SR-18292, induces PGC-1α mRNA expression in a dose dependent manner (2.6-fold at 1 μM, 5.7-fold at 10 μM) in cultured CD34+cells without effecting cell viability. Consistent with the increase of PGC-1α levels, the percentage of F-cells increased from 2.76% (vehicle control) to 32.1% when cells exposed to 1 µM SR-18292, which is comparable to that of 25 µM HU (32.2%). The mean fluorescence intensity of F-cells in SR-18292 treated cells was higher than in controls, suggesting that the treatment not only increased the number of F-cells but also increased the concentration of HbF in F-cells. The increased levels of PGC-1α reactivates the expression of fetal γ-globin genes resulting in significant HbF accumulation in adult erythrocytes, suggesting that the transcriptional co-activator PGC-1α comprises a new molecular target for possible therapeutic intervention in treating SCD. Disclosures No relevant conflicts of interest to declare.

Regulating Transcriptional Co-Activator PGC-1α for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is the FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU).We recently identified PPARγ co-activator (PGC-1α) as a new protein involved in the regulation of the fetal globin genes. Forced overexpression of PGC-1α in vitro by adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. Furthermore, up-regulation of PGC-1α by a small molecular agonist (Compound Z) in human umbilical cord blood-derived erythroid progenitor (HUDEP-1) cells markedly increases γ-globin gene expression and HbF synthesis. The highest response was achieved when HUDEP-1 cells were treated with 5µM Compound Z for 2 days, which results in 66.6% HbF+ cells compared to vehicle control (29.5% HbF+ cells). The effect of Compound Z in inducing HbF was further validated in erythroblasts derived from cultures of normal adults' CD34+ cells as well as in iPSC-derived sickle erythroblasts (SS24 cells). These data suggest that modulating PGC-1α activity may be effectively applied to the treatment of SCD since enhanced HbF synthesis would alleviate pathophysiological effects of SCD. DisclosuresNo relevant conflicts of interest to declare.

A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells.

Xenotransplantation models provide powerful tools for the investigation of human normal and malignant hematopoiesis. However, current models do not extensively recapitulate human bone marrow (BM) microenvironment components and exhibit limited engraftment of many human leukemias and other disorders. Here, we describe a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment formed by in situ differentiation of human BM-derived mesenchymal stromal cells. In these humanized ossicles, we observed robust engraftment of normal human hematopoietic stem and progenitor cells, and detected extensive engraftment of diverse primary acute myeloid leukemia samples at levels much greater than in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in substantially higher leukemia-initiating cell frequencies. We also observed robust engraftment of acute promyelocytic leukemia and myelofibrosis leading to the identification of leukemia-initiating cells in hematopoietic stem cells in myelofibrosis. This humanized ossicle xenotransplantation approach provides a novel system to model human hematologic disease.

Genome-Wide Crispr-Cas9 Screen Identifies Functionally Relevant Micro-RNAs in FLT3-ITD+ AML

Mammalian microRNA expression is dysregulated in many human cancers, including hematologic malignancies. MicroRNAs are small non-coding RNAs that repress their target genes by binding to the 3' UTR sites in their respective mRNA targets, leading to transcript degradation and/or preventing translation. MicroRNAs play a variety of physiologic roles within the hematopoietic compartment, and a few microRNAs have even been shown to drive leukemogenesis when deleted or expressed at abnormally high levels. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a poor prognosis due to increased cellular proliferation and survival of the hematopoietic stem and progenitor cells (HSPCs). MicroRNA expression has been shown to be highly dysregulated in FLT3-ITD+ AML, however the functional relevance of many of these microRNAs on leukemic phenotypes remains unclear. In this study, we took an unbiased approach to determine which microRNAs, and which of their putative targets, are involved in FLT3-ITD+ AML cellular growth.Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of FLT3-ITD+ leukemia cells (MV4-11 cells) over a 23-day time course. By comparing library representation from genomic DNA isolated from the final time point versus the initial time point, we were able to identify a number of protein-coding gene and microRNA candidates that either promoted or suppressed FLT3-ITD+ AML cellular growth.Our screen identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote FLT3-ITD+ AML cellular growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. We also performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in FLT3-ITD+ cells. We validated one of our targets, miR-155, as a critical regulator of FLT3-ITD+ AML cell growth in vitro. miR-155 knockout cells displayed a competitive growth disadvantage compared to miR-155 wild type cells. Further analysis revealed that deletion of miR-155 in MV4-11 cells led to decreased STAT5 activation, a key signaling intermediate known to promote cell survival and proliferation in FLT3-ITD+ AML. Finally, we found that miR-155 promotes FLT3-ITD-mediated myeloproliferation in vivo. FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis compared to their FLT3-ITD miR-155+/+ counterparts. This phenotype was attributed to miR-155's role in promoting proliferation of the HSPC and myeloid progenitor cell compartments in the bone marrow.Our CRISPR-Cas9 screen identified a subset of microRNAs that regulate FLT3-ITD+ cell growth, and extensively validated miR-155 as a promoter of FLT3-ITD+ cell proliferation. These findings were validated both in vitro and in vivo, and suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use could rapidly advance our understanding of how microRNAs contribute to human hematological diseases. DisclosuresNo relevant conflicts of interest to declare.

In Vitro and In Vivo downregulation of Leukemogenesis by FMNL1 Inhibition In Namalwa Cell Line

Abstract 1485Formins represent a family of proteins indispensable for fundamental actin-dependent processes; it has been suggested that they represent a group of attractive drug targets for the treatment of inflammation and malignant diseases. Formins are known for their ability to act as effectors of Rho family GTPases, proteins involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematological diseases such as leukemia. FMNL1 is a formin-related protein that is restrictedly expressed in hematopoietic-lineage-derived cells and overexpressed in malignant lymphoid cells such as chronic and acute lymphoid leukemia. This restricted expression supports the idea that FMNL1 may be an attractive target for novel therapies in such diseases. FMNL1 has recently been shown to interact with srGAP2, a RhoGAP family member, and this complex is temporally regulated by Rac1. Herein, we evaluated the role of FMNL1, by gene silencing, in proliferation, cell cycle, clonal growth, migration and transendothelial migration (TEM), and apoptosis in Namalwa cells, an acute lymphoblastic leukemia cell line. We also accessed the role of FMNL1 in tumor growth using a NOD/SCID xenograft model. Moreover, we confirmed the endogenous association of FMNL1 with Rac1, and studied Rac1 activity in leukemic cells lacking FMNL1 expression for the first time. To inhibit FMNL1 in the Namalwa cell line, specific shRNA-expressing lentiviral vectors targeting the FMNL1 gene (shFMNL1) or no specific sequence (shControl) were used. Cell growth was measured using the MTT colorimetric reduction in five replicates. Colony formation was carried out in semisolid methylcellulose medium and was detected after 8 days of culture by adding 1mg/mL of MTT reagent and scored by Image J quantification software. Both assays were carried out in lentiviral transduced cells treated, or not, with different concentrations of rapamycin (10 or 100nM). Migration assays were performed in triplicate using 8-mm transwells, and for TEM, HUVECs were seeded onto fibronectin-coated 5-mm transwell filters. The lower compartment for both assays was filled with SDF-1. Annexin-PI was used to access apoptosis. NOD/SCID mice were implanted with lentiviral transduced Namalwa cells in the dorsal subcutaneous site of the animal. FMNL1 association with Rac1 protein was analyzed by immunoprecipitation, and Rac1 activity was measured by a PAK-pull down assay, both followed by Western blotting. P value <0.05 was considered statistically significant. Inhibition of FMNL1 in Namalwa cells resulted in a significant decrease in proliferation of 40%, when compared with control cells (P <0.01). Interestingly, the combination of FMNL1 inhibition/rapamycin treatment showed a significantly higher reduction in cell proliferation (50%) when compared with FMNL1 inhibited cells alone (P =0.008) or rapamycin treated cells (P =0.007). Inhibition of FMNL1 induced cell cycle delay in the S phase in Namalwa cells and a consequent decrease in the number of cells in G2/M. A significant decrease of 30% in the number of colonies was observed in shFMNL1, as compared to shControl cells (P <0.0001). Again, rapamycin treatment resulted in the synergic inhibition of colony formation by 50% (P =0.007). FMNL1 silencing also resulted in a significant decrease (62%, P <0.01) in transwell and a decrease (20%) in the TEM assay. Apoptosis was unaffected by FMNL1 knockdown. In vivo experiments, the ShFMNL1 cell injected group showed a significant decrease in tumor growth during the experimental period (15 days after injection), when compared to the shControl cell injected group (P =0.002). FMNL1 was found to be associated with Rac1 in Namalwa cells, by immunoprecipitation, performed also in a reverse assay. Interestingly, Rac1 presented a four-fold increase in its activity in shFMNL1 cells in three independent PAK-pull down experiments. Aberrant Rac1 activity is known in tumorigenesis; however, in this leukemia cell model, inhibition of FMNL1 bypassed Rac1 activity. Our findings indicate that FMNL1 participates in the regulation of leukemogenesis, through different cell activities, including proliferation, colony formation, migration and tranwell migration of Namalwa cells, suggesting that FMNL1 represents a possible therapeutic target together with rapamycin, based on their synergic effects. Disclosures:No relevant conflicts of interest to declare.

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

AbstractEmerging metabolomic tools can now be used to establish metabolic signatures of specialized circulating hematopoietic cells in physiologic or pathologic conditions and in human hematologic diseases. To determine metabolomes of normal and sickle cell erythrocytes, we used an extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry–based metabolite profiling method. Comparison of these 2 metabolomes identified major changes in metabolites produced by (1) endogenous glycolysis characterized by accumulation of many glycolytic intermediates; (2) endogenous glutathione and ascorbate metabolisms characterized by accumulation of ascorbate metabolism intermediates, such as diketogulonic acid and decreased levels of both glutathione and glutathione disulfide; (3) membrane turnover, such as carnitine, or membrane transport characteristics, such as amino acids; and (4) exogenous arginine and NO metabolisms, such as spermine, spermidine, or citrulline. Finally, metabolomic analysis of young and old normal red blood cells indicates metabolites whose levels are directly related to sickle cell disease. These results show the relevance of metabolic profiling for the follow-up of sickle cell patients or other red blood cell diseases and pinpoint the importance of metabolomics to further depict the pathophysiology of human hematologic diseases.

Formin-Like 1 (FMNL1) Associates with Rac1 and Negatively Regulates Neoplastic Growth and Migration in Leukemia Cell Lines.

AbstractAbstract 1030FMNL1 belongs to a conserved family of formin-related proteins, has high restricted expression in malignant lymphoid derived cells and has been described as a tumor associated antigen in chronic lymphoid leukemia (CLL). Overexpression of FMNL1 in CLL has already been show to correlate with adverse clinical and biological parameters, particularly in young patients (E Calpe et al, ASH 2006, abstract submitted). Formins are known for their ability to act as effectors of Rho family GTPases, proteins that regulate multiple pathways involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as leukemia. FMNL1 function and regulation have not yet been well characterized; its restricted expression suggests that FMNL1 represents an attractive target for novel immunotherapies in hematopoietic malignant disorders. Herein, we evaluated the association of FMNL1 with the three most studied Rho GTPase proteins (Rac1, RhoA and Cdc42) and the role of FMNL1, by gene silencing, in proliferation, migration and cell cycle in acute lymphoblastic leukemia cell lines. We also tested the cumulative effect of FMNL1 silencing and rapamycin treatment in proliferation assays. FMNL1 association with Rho GTPases proteins was analyzed by immunoprecipitation, followed by western blot. To inhibit FMNL1, specific shRNA-expressing lentiviral vector targeting the FMNL1 gene or no specific sequence (control) were used. Cell growth was measured using the MTT colorimetric reduction method in cells treated or not with different concentrations of rapamycin (10 or 100nM) in five replicates. Migration assays were performed in triplicate using 5-mm Transwells and the lower compartment was filled with 600 mL 0.5% BSA/RPMI containing 100ng/mL SDF-1. The experiments were performed in two cell lines: Jurkat and Namalwa. P value <0.05 was considered statistically significant. The levels of FMNL1 mRNA and protein in FMNL1 knockdown cells were reduced by approximately 70% for both cell lines tested. Inhibition of FMNL1 in Jurkat and Namalwa cells resulted in a significant decrease of proliferation by 40% for both cell lines when compared with control cells (P<0.01). Interesting, in both cell lines, the combination of FMNL1 inhibition/rapamycin treatment showed higher reduction in cell proliferation when compared with FMNL1 inhibited cells alone (P=0.008) or rapamycin treated cells (P=0.007). Inhibition of FMNL1 resulted in an accumulation of Namalwa cells in the S phase and a consequent decrease of cells in G2/M. FMNL1 silencing also resulted in a significant decrease by 62% of cell migration for both cell lines when compared to control cells (P=<0.01). Among the Rho GTPase proteins tested, FMNL1 was found to be associated with Rac1, but not with RhoA and Cdc42. The immunoprecipitation results suggest FMNL1 as an effector of Rac1. Indeed, similar effect obtained with FMNL1 silencing has already been shown with Rac1 knockdown, which also suppresses leukemia cell migration and growth. Our findings indicate that FMNL1 participates in the regulation of proliferation and migration of Jurkat and Namalwa cell lines, which suggests that FMNL1 represents an attractive therapeutic target. Based on the result of FMNL1 inhibition/rapamycin combination, we hypothesized that combinatorial inhibition of these pathways would be effective for the treatment of lymphoid malignancies. Supported by Fapesp and CNPq. Disclosures:No relevant conflicts of interest to declare.

Metabolomic analysis of normal and sickle cell erythrocytes

Metabolic signatures of specialized circulating hematopoietic cells in physiological or human hematological diseases start to be described. We use a simple and highly reproductive extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry based metabolites profiling method to determine metabolomes of normal and sickle cell erythrocytes. Sickle cell erythrocytes and normal erythrocytes metabolomes display major differences in glycolysis, in glutathione, in ascorbate metabolisms and in metabolites associated to membranes turnover. In addition, the amounts of metabolites derived from urea cycle and NO metabolism that partly take place within erythrocyte were different between normal and sickle cell erythrocytes. These results show that metabolic profiling of red blood cell diseases can now be determined and might indicate new biomarkers that can be used for the follow-up of sickle cell patients.

Generation and Characterization of Transgenic Mice Expressing MplW515L.

Abstract 3901Poster Board III-837Myeloproliferative neoplasias (MPNs) are a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). JAK2V617F, a gain-of-function mutation of tyrosine kinase JAK2, is found in over 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Attempt to identify other signaling components involving the JAK2 signaling pathways has led to discovery of acquired mutations of Mpl, the receptor of thrombopoietin, in 5-10% patients with PMF and ET. To prove the pathogenesis of Mpl mutants, we have generated transgenic mice expressing the most frequently occurred Mpl mutant designated MplW515L by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. We obtained three lines of MplW515L transgenic mice which all displayed similar hematological abnormalities. As expected, the mice developed ET- and PMF-like phenotypes with much elevated platelet counts, severe splenomegaly/hepatomegaly, and bone marrow/spleen myelofibrosis. Interestingly, these mice also had markedly increased white blood cells in the peripheral blood, majority of which are IgD-positive mature B-cells. Histochemical staining and flow cytometric analyses revealed infiltrations of megkaryocytes and B cells into the spleen, the presence of megkaryocytes and erythroid blast cells in the liver, and infiltrations of the bone marrow with B-cells. Reticulin staining revealed that MplW515L transgenic mice developed profound myelofibrosis in the bone marrow and spleen. In vitro hematopoietic colony assays demonstrated increased numbers of hematopoietic progenitor cells including BFU-E, CFU-GM, CFU-Mk, and CFU-Pre-B in the bone marrow, mobilization of these stem/progenitor cells to peripheral blood and spleen, and their autonomous growth in the absence of growth factors and cytokines. Finally, transplantation of bone marrow cells from MplW515L mice into irradiated normal mice installed the aforementioned phenotypes into the recipient mice, indicating that expression of MplW515L altered the activity of hematopoietic stem cells. Together, our data demonstrated that transgenic expression of MplW515L not only causes PMF- and ET-like phenotypes but also lymphoproliferative disorders. Considering that Mpl is expressed in hematopoietic stem cells and that oncogenic gene mutations are often associated with alteration of gene expression, we believe that MplW515L may be involved in a wider spectrum of human hematological diseases than MPNs. Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanisms of Spindle Checkpoint-Apoptosis Linkage and Karyotypic Stability in Stem Cells.

Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.

Progress toward vector design for hematopoietic stem cell gene therapy.

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy because of their capacity for self renewal and the wide systemic distribution of their progeny. Sustained expression of transgenes at clinically relevant levels in the progeny of HSCs would provide novel and potentially curative treatments for a wide range of inherited and acquired blood diseases. Recent improvements in retroviral transduction protocols have resulted in the first successful amelioration of a human hematologic disease--a form of severe combined immunodeficiency--by HSC gene transfer. However, continued advances in gene transfer technology are necessary if the inherent promise of HSC gene therapy is to be fully realized. Ongoing efforts are focused on modifying oncoretroviral vector designs and pseudotyping with alternative envelope proteins. In addition, because of their ability to transduce non-divided cells, safety-modified human immunodeficiency virus-1-based lentiviral vectors have emerged as promising tools for gene modification of HSCs, which reside primarily in the G0/G1 phase of the cell cycle. Irrespective of these advances, accumulated data indicate that stably integrated transgenes are frequently subject to position-effect variegation and extinction of expression. Therefore, the extent to which genetic control elements such as chromatin domain insulators and scaffold/matrix attachment regions in conjunction with posttranscriptional regulatory elements will result in enhanced probability and level of transgene expression is under active investigation. Collectively, these developments increase the likelihood that HSC gene transfer will ultimately become an effective therapeutic strategy.