Normal Murine Bone Marrow Research Articles

Ajania pacifica (Nakai) K. Bremer and Humphries Extract Limits MYC Expression to Induce Apoptosis in Diffuse Large B Cell Lymphoma.

The proto-oncogene MYC is frequently dysregulated in patients with diffuse large B-cell lymphoma (DLBCL) and plays a critical role in disease progression. To improve the clinical outcomes of patients with DLBCL, the development of strategies to target MYC is crucial. The use of medicinal plants for developing anticancer drugs has garnered considerable attention owing to their diverse mechanisms of action. In this study, 100 plant extracts of flora from the Republic of Korea were screened to search for novel agents with anti-DLBCL effects. Among them, Ajania pacifica (Nakai) K. Bremer and Humphries extract (APKH) efficiently suppressed the survival of DLBCL cells, while showing minimal toxicity toward normal murine bone marrow cells. APKH suppressed the expression of anti-apoptotic BCL2 family members, causing an imbalance between the pro-apoptotic and anti-apoptotic BCL2 members. This disrupted mitochondrial membrane potential, cytochrome c release, and pro-caspase-3 activation and eventually led to DLBCL cell death. Importantly, MYC expression was markedly downregulated by APKH and ectopic expression of MYC in DLBCL cells abolished the pro-apoptotic effects of APKH. These results demonstrate that APKH exerts anti-DLBCL effects by inhibiting MYC expression. Moreover, when combined with doxorubicin, an essential component of the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone), APKH synergistically enhanced the therapeutic effect of doxorubicin. This indicates that APKH may overcome drug resistance, which is common in patients with refractory/relapsed DLBCL. To identify compounds with anti-DLBCL activities in APKH, the chemical profile analysis of APKH was performed using UPLC-QTOF/MSe analysis and assessed for its anticancer activity. Based on the UPLC-QTOF/MSe chemical profiling, it is conceivable that APKH may serve as a novel agent targeting MYC and sensitizing drug-resistant DLBCL cells to CHOP chemotherapy. Further studies to elucidate how the compounds in APKH exert tumor-suppressive role in DLBCL are warranted.

Anti-Leukemic Effects of Idesia polycarpa Maxim Branch on Human B-Cell Acute Lymphoblastic Leukemia Cells

Patients with pediatric B-cell acute lymphoblastic leukemia (B-ALL) have a high survival rate, yet the prognosis of adults and patients with relapsed/refractory disease is relatively poor. Therefore, it is imperative to develop new therapeutic strategies. Here, we screened 100 plant extracts from South Korean Flora and investigated their anti-leukemic effect using CCRF-SB cells as a B-ALL model. The top cytotoxic extract identified in this screening was the Idesia polycarpa Maxim. branch (IMB), which efficiently inhibited the survival and proliferation of CCRF-SB cells, while having minimal to no impact on normal murine bone marrow cells. Mechanistically, the IMB-induced proapoptotic effect involves the increase of caspase 3/7 activity, which was shown to be associated with the disruption of the mitochondrial membrane potential (MMP) through the reduction in antiapoptotic Bcl-2 family expression. IMB also promoted the differentiation of CCRF-SB cells via the upregulation of the expression of differentiation-related genes, PAX5 and IKZF1. Given that resistance to glucocorticoid (GC) is often found in patients with relapsed/refractory ALL, we investigated whether IMB could restore GC sensitivity. IMB synergized GC to enhance apoptotic rate by increasing GC receptor expression and downmodulating mTOR and MAPK signals in CCRF-SB B-ALL cells. These results suggest that IMB has the potential to be a novel candidate for the treatment of B-ALL.

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow.

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% ± 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% ± 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% ± 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% ± 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p < 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

Germline MPO Variants Predispose to Myeloid Neoplasia: Potential Mechanisms Suggested By In Vivo and in Vitro Studies

In recent decades, there has been a substantial increase in recognition of germline (GL) predisposition to MN. Some congenital blood disorders predispose to subsequent myeloid neoplasia (MN). Severe congenital neutropenia (SCN) and Fanconi anemia serving as prominent examples. There is emerging evidence that heterozygous variants of disease recessive conditions may constitute risk alleles for late developing MN. However, their low penetrance may preclude identification of such alterations. Our previous analysisof allelic burden among selected combined potential GL predisposition genes to MN identified that the myeloperoxidase (MPO) gene (17q22-23) carried the highest risk of pathogenic alterations (Li, S.T, Leukemia, 2020). GL MPO mutations cause MPO deficiency syndrome, one of the most common inherited disorder associated with phagocyte defects and variable clinical penetrance. Despite the high prevalence, inherited MPO deficiency patients are asymptomatic and may remain unrecognized.We investigated 3280 MN and bone marrow failure syndromes (BMF) pts including 1138 MDS, 260 MDS/MPN, 1661 AML and 221 AA for presence of MPO mutations. In total, 38 different germline MPO mutations were identified in 143 cases. With a stringent bioanalytic pipeline, 28 pathogenic/likely pathogenic variants from 100 MN patients were included in the study. Germline MPO variants were significantly enriched in MN compared to control population (294 vs. 125 per 10 4 individuals; P<.0001) with an odds ratio of 2 (95%CI=1.6-2.5, P<.0001) for AML and 1.8 (95%CI=1.4-2.4, P<.0001) for MDS. The most common pathogenic/likely pathogenic variant (46%; 46/100) was a 3' splice site of intron11 (c.2031-2A>C) followed by R569W (13/100), M519fs* (13/100) and Y173C (6/100) with none being biallelic. While no differences were found in distribution of -7/7q, del5q, or del20q, MPO variants carriers harbored less tri-8 compared to wild type counterparts (2% vs. 12%; P=.008). Only FLT3 and NRAS were significantly associated with MPO mutations.We then investigated the effects of MPO deficiency on hematopoietic function in murine model using competitive repopulation assays, whereby the difference in CD45.1/CD45.2 isotypes transplanted in to ROSA26 (tdTomato-EGFP) recipients assayed by flow cytometry allowed distinction of the 2 grafts. Mpo-/- cells gained over time proliferative advantage over normal murine bone marrow cells. Reverse combinations (graft mix vs recipient) also replicated this result. This effect was not due to increased HSC content in Mpo-/- marrow as there were no significant differences in LSK, CMP and MEPs cells. We then investigated the clonogenic consequences of Mpo-/- cells in the setting of H 2O 2 induced oxidative stress, after exposing to hydrogen peroxide. Mpo-/- cells increased clonogenic potential after second serially replating. This prompted us to investigate an MPO inhibitor, AZD5904 in human leukemia cells with different MPO expression (HL-60, high; K562, low). High MPO expressors retained higher cell viability following H 2O 2 and MPOi addition compared to those without MPOi (85±10 vs.59±8, P<.0001) while no change was observed in low MPO stage cells with or without MPOi (84±2 vs.89±10, P=0.7).In conclusion, for the first time we demonstrated that germline MPO variants constitute risk alleles for MN evolution and replicated the potential replicative advantage of MPO deficient cells in murine model and potential mechanisms of the MPO deficiency in vitro including activation of non-homologous DNA repair response and error-prone DNA repair favoring replication. Repeated cycles of stress hematopoiesis e.g., due to increased infection rate may provide conditions in which MPO variants contribute to the risk of MN. DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy.

High-Throughput Screen Identification of Albendazole As a Novel Repurposed Drug in Acute Myeloid Leukaemia

ObjectivesAcute myeloid leukaemia (AML) is a progressive malignancy that arises from impaired differentiation of hematopoietic progenitor cells. While many AML patients respond to induction therapy, relapse and refractory disease is common (Dohner et al. 2015). Recent studies have defined the driver landscape in AML (Pappaemmanuil et al. 2016)and novel targeted therapies are emerging(Erb et al. 2017). Moreover, molecular profiling has revealed the genetic complexity of leukaemia development and progression with growing evidence of mutational hierarchies that dynamically evolve during therapy(Mossner et al. 2016). Therefore, while target-driven, de novo drug design holds promise for novel AML treatments, alternative strategies for developing AML therapies should be exploited, especially those that provide both efficacy and reduced toxicities.MethodsIn order to discover novel therapies for AML, we performed an FDA drug repurposing screen using 760 drugs on primary murine cell lines developed in our laboratory; models representative of AML with a t(9;11) (MLL-AF9) translocation) and AML with a normal karyotype (HOXA9-Meis 1 -driven).ResultsWe identified 38 hits (5% of total) at 50% viability (Figure A), and these hits were validated in secondary screens. Of the candidates identified, 12 are established therapies in haematological malignancies, providing confirmation of the screen validity. These included cytarabine, danorubicin and idarubicin, standard of care therapies used in AML induction and maintenance therapy (Woelich et al. 2017). A further 12 drugs are currently used clinically in the treatment of solid tumours. The remaining 14 are novel to oncology therapy. We found the anthelmintic agent albendazole (ABZ) to have particular efficacy with limited toxicity, reducing viability and colony formation in murine leukaemia cells whilst having negligible effects in normal murine bone marrow cells. A clinically achievable low dose of ABZ (IC50 <350 nM) reduced viability in several human AML cell lines, such as THP-1 and OCI-AML3. Low dose ABZ caused G2/M arrest in AML cells, leading to caspase-dependent apoptosis as confirmed by PI and Annexin V-based flow cytometry. BH3 profiling of AML cells showed increased mitochondrial associated pro-death proteins following ABZ treatment, consistent with caspase-dependent cell death. Ex-vivo treatment of THP-1 pSLIEW cells transplanted into NSG mice showed decreased leukaemia burden and extended lifespan in the ABZ treated (315 nM) cohort (median survival = 53 days) compared to controls (30 days) (p=0.0002).DiscussionIn summary, ABZ has anti-leukaemia efficacy in both primary cells and cell lines representing a range of molecular subtypes of AML, with negligible effects shown in normal cells. Current and future studies are focused on understanding the molecular mechanism of ABZ in AML and further pre-clinical validation alone, and in combination with standard of care therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

More than neutrophils: Lin(+)Ly6G(+)IL-5Rα(+) multipotent myeloid cells (MMCs) are dominant in normal murine bone marrow and retain capacity to differentiate into eosinophils and monocytes.

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.

Siglec-F-negative resident eosinophils represent an unexpectedly dominant population in homeostatic murine bone marrow

Abstract Bone marrow, while known to be a hematopoietic site for eosinophils, also harbors resident populations of these cells. Identification of such resident eosinophils typically requires flow cytometry-assisted sorting, with Siglec-F as a key marker for identification of murine eosinophils. At baseline, we found that bone marrow resident eosinophils described as Siglec-F(+) constitute about 5% of CD45 cells. Although this identifies mature eosinophils, it may not be sufficient to represent all resident phenotypes. Using sequencing and FACS sorting approaches, we found that the Siglec-F(+) population represented only a minority of the resident eosinophil pool. Rather, we identified a lineage-positive population of IL-5Ra(+)Ly6G(+) but Siglec-F(−) cells distinct from lineage-negative EoPs (eosinophil progenitors). At 40% of all CD45(+) hematopoietic cells in adult mice, this bone marrow resident population dwarfs the Siglec-F(+) eosinophil pool. When sorted from bone marrow homogenates and treated with IL-5, these resident cells acquired all characteristics of mature eosinophils, including expression of Siglec-F and granular proteins (EPX, MPO). These tissue residents may play critical homeostatic roles in normal murine bone marrow and should be accounted for in studies of eosinophil biology.

A novel reporter construct for screening small molecule inhibitors that specifically target self-renewing cancer cells

Cancer stem cells (CSCs) are a subset of cancer cells, which possess self-renewal ability, and lead to tumor progression, metastasis, and resistance to therapy. Live detection and isolation of CSCs are important to understand the biology of CSCs as well as to screen drugs that target them. Even though CSCs are detected using surface markers, there is a lot of inconsistencies for that in a given cancer type. At the same time, self-renewal markers like ALDH1A1, OCT4A and SOX2, which are intracellular molecules, are reliable markers for CSCs in different cancers. In the present study, we generated a reporter construct for self-renewing CSCs, based on ALDH1A1 expression. Oral cancer cells harboring ALDH1A1-DsRed2 were used to screen inhibitors that target CSCs. Our results showed that Comb1, a cocktail of inhibitors for EGF and TGF-β pathways and their intermediates, effectively reduced the DsRed2 population to 34%. Our immunohistochemical analysis on primary oral cancer corroborated the importance of EGF and TGF-β pathways in sustaining CSCs. Since these two pathways are also critical for the self-renewal and differentiation of normal stem cells, Comb1 might abolish them as well. On analysis of the effect of Comb1 on normal murine bone marrow cells, there was no significant change in the stem cell self-renewal and differentiation potential in the treated group compared to untreated cells. To conclude, we claim that ALDH1A1-DsRed2 is a useful tool to detect CSCs, and Comb1 is effective in targeting CSCs without affecting normal stem cells.

RNA Binding Protein Rbmx Is Required in Acute Myeloid Leukemia By Regulating the Transcriptional Activity of the Heterochromatin Protein HP1α

Acute myeloid leukemia (AML) is characterized by a block in the development of myeloid cells, often due to dysregulation of genes involved in key processes including self-renewal, proliferation, and differentiation. Somatic mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to be important in hematological malignancies. For example, our group and others have recently determined that increased expression of MUSASHI-2 and SYNCRIP drives aggressive leukemia. To discover novel RBP regulators of leukemia, we performed an in vivo pooled shRNA screen of 127 MSI2 direct protein interactors and associated genes (Vu et al. Nat Gen. 2017). In this screen, shRNAs specific to the RBP RBMX (RNA binding motif protein, X-linked) were selectively depleted in murine MLL-AF9 driven leukemia. RBMX has been implicated in regulating alternative splicing, chromatin cohesion, and DNA-damage response, but its function in hematopoiesis and leukemia is not known. We confirmed that depletion of RBMX with shRNAs in murine MLL-AF9 leukemia cells resulted in reduced myeloid colony formation, increased apoptosis, and increased differentiation as determined by flow analysis of myeloid cell surface markers Gr-1 and Mac-1 (mean of 61-65% shRNA versus mean of 12.95% control). Furthermore, RBMX is highly expressed among human myeloid leukemia cell lines (n=10/11) and primary AML patient samples (n=2/4). Depletion of RBMX with shRNAs led to a dramatic decrease in cell proliferation and 3-fold induction of apoptosis in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX depletion in AML cells induced myeloid differentiation and significantly delayed leukemogenesis cells in vivo (median survival of 51.5 days in control versus median ‘not reached’ in shRNA1 and shRNA2). To determine if there is a differential requirement of RBMX in survival of leukemia cells compared to normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBMX with shRNAs in normal murine bone marrow c-Kit+ cells and found no significant changes in colony formation. Depleting RBMX with shRNAs in human cord blood derived CD34+ HSPCs resulted in reduced colony formation but no increase in apoptosis. Thus, these data suggest that there is a differential requirement for RBMX in myeloid leukemia cells compared to normal cells. To uncover the mechanism of RBMX function, we performed RNA-sequencing of human AML cells (MOLM-13) depleted for RBMX. Gene set enrichment analysis demonstrated a loss of cell cycle and DNA repair associated programs in RBMX depleted cells. Complex chromosomal karyotyping analysis of these cells revealed increased metaphases with breaks and gaps (mean of 30.67% shRNA versus mean of 13.33% control) and irregular chromatin compaction (mean of 47.67%shRNA versus mean of 20% control), while cell cycle analysis showed significantly increased S-phase arrest as determined by flow analysis of Hoechst stained cells (mean of 37-40% shRNA versus of 24.18% control). Reanalysis of RBMX transcriptome-wide binding sites in 293T cells revealed that RBMX directly binds to heterochromatin protein HP1α transcripts (Liu et al. Nucleic Acids Res. 2017). HP1α, also called CBX5, is a key heterochromatin protein that binds to histone H3-K9 tri-methylation marks to promote heterochromatin formation, which is critical in chromatin condensation and chromosome segregation. HP1α has also been determined to be required for MLL leukemia stem cell maintenance. We demonstrated that RBMX depletion resulted in a significant decrease of HP1α mRNA expression without affecting its mRNA stability in AML cells. We confirmed that RBMX depletion reduced the protein abundance of HP1α. Moreover, overexpression of HP1α rescued the effect of RBMX depletion on cell growth and apoptosis. Our study finds that RBMX binds to HP1α mRNA and regulates the transcriptional activity of the HP1α locus, which then maintains proper chromatin compaction in leukemia cells. Overall, we determine that RBMX function is critical for myeloid leukemia survival and has potential as a novel therapeutic target in AML. DisclosuresNo relevant conflicts of interest to declare.

Mathematical modeling of erythrocyte chimerism informs genetic intervention strategies for sickle cell disease.

Recent advances in gene therapy and genome-engineering technologies offer the opportunity to correct sickle cell disease (SCD), a heritable disorder caused by a point mutation in the β-globin gene. The developmental switch from fetal γ-globin to adult β-globin is governed in part by the transcription factor (TF) BCL11A. This TF has been proposed as a therapeutic target for reactivation of γ-globin and concomitant reduction of β-sickle globin. In this and other approaches, genetic alteration of a portion of the hematopoietic stem cell (HSC) compartment leads to a mixture of sickling and corrected red blood cells (RBCs) in periphery. To reverse the sickling phenotype, a certain proportion of corrected RBCs is necessary; the degree of HSC alteration required to achieve a desired fraction of corrected RBCs remains unknown. To address this issue, we developed a mathematical model describing aging and survival of sickle-susceptible and normal RBCs; the former can have a selective survival advantage leading to their overrepresentation. We identified the level of bone marrow chimerism required for successful stem cell-based gene therapies in SCD. Our findings were further informed using an experimental mouse model, where we transplanted mixtures of Berkeley SCD and normal murine bone marrow cells to establish chimeric grafts in murine hosts. Our integrative theoretical and experimental approach identifies the target frequency of HSC alterations required for effective treatment of sickling syndromes in humans. Our work replaces episodic observations of such target frequencies with a mathematical modeling framework that covers a large and continuous spectrum of chimerism conditions. Am. J. Hematol. 91:931-937, 2016. © 2016 Wiley Periodicals, Inc.

314. Mathematical Modeling of Erythrocyte Chimerism Informs Clinical Strategies for Sickle Cell Disease

Recent advances in gene therapy and genome-engineering technologies offer the opportunity to correct sickle cell disease (SCD), which originates from a point mutation in the gamma-globin gene. The developmental switch from fetal gamma-globin to adult beta-globin is governed in part by the transcription factor (TF) BCL11A. This TF can be a therapeutic target for reactivation of corrected gamma-globin and concomitant reduction of sickling beta-globin. Genetic alteration of a portion of the hematopoietic stem cell (HSC) compartment would lead to a mixture of sickling and corrected peripheral red blood cell (RBC) populations. The degree of HSC alteration required to achieve a desired stable fraction of corrected RBCs with high gamma-gobin remained unclear. We developed a mathematical model that describes aging and survival of normal and sickling RBCs: a survival advantage of non-sickled RBCs over sickled RBCs leads to their overrepresentation in periphery. The math model considers HSC and age-structured RBC populations, and we aimed to validate it in an experimental mouse model by transplanting mixtures of Berkeley SCD mouse model and normal murine bone marrow cells to establish chimeric grafts in murine hosts. The math model identified the level of bone marrow chimerism required for successful stem cell-based gene therapies in SCD. We found an equation for the stable fraction of normal RBCs in periphery as a function of the fraction of genetically altered HSCs, predicting that gene therapy leading to 40% altered HSCs results in 55% non-sickling RBCs in periphery (Figure 1A, lineFigure 1A, line). These findings were confirmed in the experimental mouse setting, where we achieved altered HSC mixtures between 0.5 and 38%, leading to RBC fractions between 7 and 62% (Figure 1A, data pointsFigure 1A, data points). We found that a mathematical model incorporating a constant RBC selection approach was sufficient to predict pheripheral chimerism. This approach was then used to make SCD patient-predictions: 10% HSC alteration leads to 40% normal RBCs in periphery, 30% leads to over 70% normal RBCs (medians, assuming variable S cell life span, Figure 1BFigure 1B), which depend on the mean life spans of sickling (median 20 days) and normal (110 days) RBCs. Our integrative approach informs the target frequency of HSC genetic alterations likely required for effective treatment of sickling syndromes in humans.View Large Image | Download PowerPoint Slide

The Pharmacological Inhibition of KDM1A Displays Preclinical Efficacy in AML and MDS By Inducing Myelomonocytic Differentiation

Background: Histone methylation is one of the major systems of epigenetics and reversibly regulated by lysine (K) specific methyltransferases (KMTs) and demethylases (KDMs). Dysregulation of KMTs such as EZH2 and MLL play a key role in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) while the precise function of KDMs remains unclear.KDM1A is the first reported histone demethylase, which mainly catalyzes demethylation of mono- and di-methylated lysine 4 of histone 3 (H3K4me1 and me2 respectively). According to recent reports, the inhibition of KDM1A either alone or in combination of all-trans retinoic acid is effective for AML expressing MLL-AF9 or for several types of AML respectively, suggesting KDM1A could be a therapeutic target of AML. However, other reports argue that KDM1A is essential in hematopoiesis, raising concern that KDM1A-targeted therapy could lead to severe hematological toxicity. Here, we try to clarify what types of leukemia can be ameliorated by the pharmacological inhibition of KDM1A with a possible therapeutic window and what are functional and molecular mechanisms utilizing highly selective KDM1A inhibitors we have newly designed.Results: First we validated the effect of our novel inhibitors on murine leukemia cells harboring MLL-AF9. In accordance with a previous report, our novel KDM1A inhibitors suppressed cell proliferation, diminished clonogenic capacity and induced G1-S cell cycle arrest and myelomonocytic differentiation but not apoptosis in quite low concentration that clonogenicity of normal murine bone marrow cells was spared. Next we examined the effect of these drugs on diverse types of human myeloid leukemia cells and found that our drugs were particularly effective in erythroid leukemia cells (HEL), megakaryocytic leukemia cells (CMK11-5), and a blastic subline from a MDS patient with complex karyotype (MDS-L). MDS-L cells were changed phenotypically and morphologically towards myelomonocytic differentiation such as the increase of CD11b expression level and the induction of neutrophil-like cells. HEL and CMK cells which are negative for myelomonocytic markers also gained CD11b expression and decreased erythroid markers such as CD235a and CD71. These data suggest that the inhibition of KDM1A induces myeloid differentiation across various types of leukemia.To investigate an underlying molecular basis for the cell fate conversion in HEL and myeloid differentiation in MDS-L by the inhibition of KDM1A, we performed gene expression profiling and analyzed a change of gene signatures. The expression pattern of transcriptional factors was changed from the erythroid signature (e.g. GATA1 and TAL1) to the myeloid signature (e.g. SPI1 and CEBPA) in HEL. Gene Set Enrichment Analysis (GSEA) showed that the myeloid differentiation-associated gene signature was positively enriched and the leukemia stem cell-associated gene signature was negatively enriched in both HEL and MDS-L.Finally, we investigated the effect of our KDM1A inhibitors on primary human samples such as AML with MLL-AF9 and MDS in the phase of overt leukemia (MDS/AML) with complex karyotype. The colony formation capacity was clearly impaired in relatively low concentration that normal colonies were spared. We transplanted primary MDS/AML cells with complex karyotype to immunodeficient mice and treated with a KDM1A inhibitor or vehicles after confirming the engraftment. In one MDS/AML case, all mice treated with vehicles (n=4) died of anemia and the increase of human leukemia cells within four months while 3 of 4 mice treated with a KDM1A inhibitor have survived for more than six months. Also in another case, both of two vehicle-treated mice died while all drug-treated mice had survived for more than six months. At day 200 after transplantation, we sacrificed all survived mice treated with a KDM1A inhibitor and found that human blasts were displaced from the bone marrow of treated mice. Those data suggest that our KDM1A inhibitor is effective in vivo and have a possibility for the clinical application.Conclusion: Our study suggests that KDM1A involves with myeloid differentiation and the leukemia stem cell signature and that the pharmacological inhibition of KDM1A by highly selective inhibitors is a promising way to ameliorate AML with poor prognosis such as erythroleukemia and MDS/AML with complex karyotype, without impairing normal hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Hydrogen peroxide (H2O2) induces leukemic but not normal hematopoietic cell death in a dose-dependent manner

Over the last few years, studies have suggested that oxidative stress plays a role in the regulation of hematopoietic cell homeostasis. In particular, the effects of hydrogen peroxide (H2O2) range from hematopoietic cell proliferation to cell death, depending on its concentration in the intracellular milieu. In this work, we evaluated the effects of an oxidative environment on normal and leukemic hematopoietic cells by stimulating normal human (umbilical cord blood) and murine (bone marrow) hematopoietic cells, as well as human myeloid leukemic cells (HL-60 lineage), upon H2O2 stimulus. Total cell populations and primitive subsets were evaluated for each cell type. H2O2 stimulus induces HL-60 cell death, whereas the viability of human and murine normal cells was not affected. The effects of H2O2 stimulus on hematopoietic stem/progenitor cell subsets were examined and the normal primitive cells were found to be unaffected; however, the percentage of leukemic stem cells (LSC) increased in response to H2O2, while clonogenic ability of these cells to generate myeloid clones was inhibited. In addition, H2O2 stimulus caused a decrease in the levels of p-AKT in HL-60 cells, which most likely mediates the observed decrease of viability. In summary, we found that at low concentrations, H2O2 preferentially affects both the LSC subset and total HL-60 cells without damage normal cells.

Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells

Abstract 553In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures:No relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia-Associated PAX5 Mutations Induce Aberrant B Cell Development

AbstractAbstract 10Over 60% of pediatric B progenitor acute lymphoblastic leukemia (ALL) cases contain somatic mutations in genes regulating B cell development, with PAX5 being the most common target of mutation (>32% of cases). The transcription factor PAX5 is required for commitment and maintenance of the B lymphoid lineage. A variety of PAX5 mutations has been identified including, mono-allelic deletions, sequence mutations, internal deletions, frame-shift mutations and translocations. We have previously shown that these PAX5 mutations result in reduced transcriptional activity either as a result of haploinsufficiency or the generation of altered PAX5 isoforms with reduced DNA-binding and/or transcriptional activity. However, the direct effect of the mutations on normal B cell development remains unknown. To address this question, we assessed the ability of a series of PAX5 mutations to rescue normal B cell development in Pax5-/- bone marrow (BM) cells using a murine in vitro culture system.Whole BM or transduced cells were grown in IL-7 producing stromal-supported cultures for two weeks and then assessed for their extent of B cell differentiation using flow cytometry. Under these in vitro conditions, both Pax5+/+ and Pax5+/− BM cells differentiated to a Hardy fraction D pre-B cell stage of differentiation (CD43−/B220+/CD19+/BP1+), with only a slight decrease in the level of expression of BP1 detected in the Pax5+/− cells. By contrast, Pax5-/- cells failed to undergo significant differentiation under these in vitro growth conditions and were arrested at an early pro-B stage of development (CD43+/−/B220+/CD19−/BP1−). To assess the biological activity of the identified PAX5 mutants, we then transduced lineage-depleted BM cells from Pax5+/+, Pax5+/− and Pax5-/- mice with MSCV-based retroviral vectors expressing either wild type (WT) or mutant PAX5 followed by in vitro culture. Three classes of PAX5 mutations were assessed: DNA binding domain mutations (P80R, P34Q, and V26G), an internal deletion mutation (Δe6-8), and translocation-induced PAX5 chimeric genes (PAX5-ETV6, PAX5-FOXP1 and PAX5-ZNF521). As expected, expression of WTPAX5 resulted in full rescue of Pax5-/- cells and induced no significant effects on the ability of Pax5+/+ and +/− cells to differentiate. By contrast, PAX5 DNA-binding domain mutants resulted in only partial rescue of Pax5-/- cells, with P80R inducing B220+/CD19−/BP1−, P34Q producing B220+/CD19+ cells with weak BP1 expression, and V26G yielding CD19+/BP1+ cells with minimally reduced levels of BP1. Similarly, expression of Δe6-8 resulted in partial rescue with the expansion of B220+/CD19+/−/BP1− cells. In stark contrast, expression of the translocation encoded PAX5 fusion proteins failed to induce any evidence of rescue. Moreover, these fusion proteins induced only minimal perturbations in the ability of Pax5+/+ and +/− cells to differentiate, suggesting that these fusion proteins were weak competitive inhibitors of normal Pax5 transcriptional activity under intra-cellular conditions. To further characterize the effects of these PAX5 mutations on B cell differentiation, we next analyzed the gene expression patterns of the resultant cell populations using the Mouse Genome 430 2.0 Arrays (Affymetrix) and compared the profiles to those obtained from purified Hardy fractions from normal murine BM. The expression signatures of the Pax5-/- cells were identical to those for normal Hardy fraction A and shifted to the signature of Hardy fraction C following rescue with WTPAX5. Transduction of Pax5-/- cells with either V26G or P34Q resulted in a near complete rescue with expression signatures similar to those obtained for Hardy fractions B/C. By contrast, transduction with P80R or Δe6-8 yielded a more incomplete rescue with expression profiles that were between Hardy fractions A and B. Interestingly, a number of genes within the B cell receptor signaling pathway were altered in cells rescued by P80R and Δe6-8, including the down regulation of CD19, Btk and Blnk. In summary, our data demonstrate that leukemia-associated PAX5 mutations have a graded effect on the transcriptional network that controls normal B cell development and differentiation. Defining the differential target gene specificity of the various PAX5 mutants should provide valuable insights into the molecular mechanisms through which these genetic lesions contribute to leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Long Term Humoral Immunity Is Dependent on CD28 Expression In Plasma Cells

AbstractAbstract 1737Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, is has been shown normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment including cell-to-cell interactions. Leading us to rationalize, modulating this interaction could either enhance antibody production for cancer vaccine development or conversely compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC plays an essential role. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases the survival and IgG production of LLPC. These observations led us to directly investigate the role of CD28 in LLPC survival as well as cell-cell interactions with CD80/CD86+ bone marrow derived dendritic cells (BMDC). Utilizing normal murine bone marrow and splenic PC as our model system we further investigated the role of CD28 in LLPC function and survival. We have previously shown, in vitro serum starvation experiments, direct activation of CD28 increased survival of LLPC by 12-fold (p<0.05), whereas CD28 activation of SLPC did not induce survival. Addition of BMDC improved the survival of LLPC 2-fold over that seen with media alone, and resulted in a significant increase in IgG production (p<0.001). In contrast, CD28-/- PC had no increase in survival when cocultured with BMDC, suggesting a direct role for CD28 in PC-DC interaction. Consistent with these findings we now show that in vivo, vaccinated bone marrow CD28-/-:μMT chimeras had significantly reduces long-term antibody titers and LLPC (but not SLPC) survival from t1/2 of 426 to 63 days. Additionally, LLPC CD28 modulates the microenvironment by inducing CD80/CD86+ stromal cell production of the supportive cytokine IL-6 (p<0.001 vs. BMDC/PC alone), which was abrogated by blocking CD80 and CD86 (p<0.05). From the above experiments we hypothesized IL-6 was playing a significant role in the survival of LLPC, however to our surprise LLPC cocultured with WT or IL-6-/- BMDC maintained equivalent LLPC numbers, interestingly however LLPC cocultures with BMDC showed a 3-fold increase of IgG compared to LLPC cocultured with IL-6-/- BMDC (p<0.001). These data suggest CD28 is a key molecular component in LLPC survival, whereas IL-6 contributes to Ig production. Our data demonstrates that signaling through CD28 directly supports the survival of LLPC, sustaining long term protective antibody titers. These findings suggest CD28 plays an important role in maintaining the quality of protective durable humoral immunity. Strategies to augment CD28 signaling may lead to greater LLPC survival and persistent antibody titers in cancer vaccine development. Conversely, blocking CD28 signaling could compromise the survival of transformed myeloma cells which are critically dependent on the bone marrow microenvironment. Disclosures:Boise:University of Chicago: Patents & Royalties.

C-Myb Associates with MLL1 through Menin, Augments MLL1's H3K4 Methylation Activity, and Regulates Hoxa9 and Meis1 Gene Expression in Primary Human Leukemia Cells.

Abstract 448The c-myb proto-oncogene was first identified as the cellular homologue of the v-myb oncogene carried by the avian leukemia viruses AMV, and E26. c-myb encodes a transcription factor, c-Myb, that is highly expressed in immature hematopoietic cells. In such primitive cells, c-Myb has been found to exert an important role in lineage fate selection, cell cycle progression, and differentiation of both myeloid, B, and T lymphoid progenitor cells. c-Myb is also highly expressed in many leukemia cells and on this basis has been implicated in leukemic transformation. Despite intensive study, a mechanisms based understanding for c-Myb's myriad effects on blood cell development has yet to be fully achieved though c-Myb's ability to interact with a variety of transcriptionally active co-factors, such as p300, CBP, and FLASH, as well as to modulate its own expression, have all been reported to contribute to its activities. Therefore, we undertook a series of biochemical, molecular, and clinical studies to further address c-Myb's role in leukemic hematopoiesis. Using in vitro translated proteins and nuclear extracts from leukemic cells in immunoprecipitation (IP) assays, we found that c-Myb is associated with MLL1, the SET1 proteins WDR5, RbBp5, and Ash2L, and menin, all of which form a complex with histone methyltransferase (HMT) activity. c-Myb associated with the MLL1 and SET1 proteins through menin, which served as an adapter protein by interacting (as previously shown) with the extreme amino terminus of the MLL1 protein, and, as we show, with a region around the c-Myb transactivation domain (aa 194-325). We demonstrated in vitro with purified proteins and an H3 peptide, that c-Myb contributed to the HMT activity of the MLL1 complex. In leukemia patients being treated with a c-myb targeted antisense oligodeoxynucleotide (ASODN), and in leukemic cell lines, silencing c-myb evoked a significant decrease in H3K4 methylation demonstrating biological relevance of this observation. The decrease in H3K4 methylation is the direct result of silencing c-myb and is not due to changes in cell proliferation, and could not be reproduced by silencing B-myb. Also, we confirmed that c-Myb is a downstream target of HoxA9, and Meis 1, but showed unexpectedly that leukemic blasts derived from the c-myb ASODN treated patients, and c-myb siRNA treated cell lines, decrease c-myb expression also led to a decrease in Hoxa9 and Meis1 expression. This suggested the presence of an autoregulatory feedback loop between c-Myb and HoxA9. This finding too was specific for c-myb and not associated with a block in proliferation or silencing B-myb. Finally, disrupting the c-Myb-MLL1 interaction impairs localization of MLL1 and menin on the Hoxa9 gene promoter, as well as the MLL-ENL induced transformation of normal murine bone marrow cells. In summary, our results bring new insights regarding c-Myb function in human hematopoietic cells, suggest new mechanisms whereby c-Myb may contribute to cell transformation, and suggest new therapeutic targets for the treatment of acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Functional Characterization of a Continuous Natural Killer Cell Line, KIL, In Vitro and in Adoptive Immunotherapy.

We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (Dehart et al. Blood 2005; 105:3521). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3−NK1.1+CD122+ phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg− pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivo tumoricidal potential of KIL C.2, we used a C57BL/6 → BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferase tumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107 TCD-WBM along with A20-luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106 donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107 per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy.

Point Mutations in Two EVI1 Zn Fingers Abolish EVI1-GATA1 Interaction and Allow Erythroid Differentiation of Murine Bone Marrow Cells

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Murine Embryonic Stem Cells Secrete Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis and Stimulate Colony Formation of Murine Hematopoietic Progenitor Cells

Stromal cell-derived factor (SDF)-1/CXCL12, released by murine embryonic stem (ES) cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells. Conditioned medium (CM) from murine ES cells growing in the presence of leukemia inhibitory factor (LIF) was generated while the ES cells were in an undifferentiated Oct-4 expressing state. ES cell-CM enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased apoptosis of murine bone marrow c-kit(+)lin- cells. ES CM contained interleukin (IL)-1alpha, IL-10, IL-11, macrophage-colony stimulating factor (CSF), oncostatin M, stem cell factor, vascular endothelial growth factor, as well as a number of chemokines and other proteins, some of which are known to enhance survival/anti-apoptosis of progenitors. Irradiation of ES cells enhanced release of some proteins and decreased release of others. IL-6, FGF-9, and TNF-alpha, not detected prior to irradiation was found after ES cells were irradiated. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins, results which may be of physiological and/or practical significance.