bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication of Genetic Markers Associated with Clozapine-Induced Agranulocytosis.
Authors: Gerson Stanton L. , Dettling Michael , Dain Brad , Lachowicz Michael , Kane John , Malhotra Anil , Cascorbi Ingolf , Mosyagin Igor , Reed Carol , Athanasiou Maria
Published: April 2020

Clozapine is a highly efficacious drug for the treatment of schizophrenia, but its use is limited, in part due to the side effect of agranulocytosis. In order to reduce the incidence of clozapine-induced agranulocytosis (CIA), patients are required to submit to a blood monitoring program. A genetic component to CIA is suggested by published associations in a number of genes. A genetic test assessing the risk of CIA might allow safer and more broad use of clozapine. We previously conducted a candidate gene-based case-control study to discover genes associated with CIA. Cases were patients with an ANC<500 during clozapine treatment, and controls were patients treated for at least one year without a significant reduction in WBC or ANC. For each case, we attempted to enroll 2 age-, gender- and ethnicity-matched controls. We collected blood, medical histories and informed consent from 33 CIA cases and 54 controls. Seventy-four candidate genes were sequenced in the exons, intron-exon boundaries, 5′ untranslated region and promoter region for each of the cases and controls, and a haplotype analysis was conducted using logistic regression. A total of 20 haplotype markers in HLA-DQB1, 1 in HLA-C, 1 in NTSR1, 1 in DRD1 and 6 in CSF2RB were significant with permutation test adjusted p-values under 0.05.

A replication analysis was performed on an independently collected cohort of 49 cases and 78 controls from Germany. Cases were patients who developed an ANC<500 during clozapine treatment, and controls were patients treated for at least two years without an adverse effect on the WBC or ANC. We attempted to replicate 28 haplotype markers from 4 of the 5 genes. We did not attempt to replicate the findings from HLA-C. Permutation tests were performed for markers within genes, and the Benjamini-Hochberg FDR method was applied to the best permutation p-values from the four genes. Markers with p-values less than 0.05 after these adjustments were considered replicated. One genetic marker in HLA-DQB1 replicated further implicating this as the strongest genetic association with CIA. Markers in CSF2RB were not statistically significant, but warrant further study. The strength of the findings is sufficient to develop a diagnostic test for CIA risk. These findings may also be applicable to agranulocytosis and neutropenia induced by other drugs.
Elevated Circulating Levels of Thrombospondin-1 in Plasma Are Associated with Increased Circulating Levels of TGFb and Pro-Inflammatory Cytokines in Patients Afflicted with Rheumatoid Arthritis: Utilization of the Multiplexed Protein Profiling on Microarray by Rolling-Circle Amplification Technique.
Authors: Rico Mario C. , Manns Joanne M. , Nguyen Hien , Beharry Nicole , Reddy Meera , Uknis Audrey B. , De La Cadena Raul A.
Published: April 2020

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that affects a vast population worldwide with women being afflicted three times more than men. There is evidence for an increased risk of cardiovascular events in RA patients compared to the general population. These cardiovascular events may be associated with the chronic inflammatory state in which activation of coagulation leads to thrombin generation. Our laboratory has evidence that thrombospondin-1 (TSP1), an adhesive molecule that plays a major role in RA, promotes thrombin generation on the surface of a monocytic cell line (Isordia-Salas et al, Thromb Res.2005;116(6)). We also have documented that disrupting the TSP1 interaction on human neutrophils prevents the development of erosive arthritis in an experimental animal model (Manns et al, Arthritis and Rheumatism54(8), 2006). This observation is mediated by a novel pathway whereby TSP1 induces the up-regulation of Connective Tissue Growth Factor (CTGF). Therefore, to assess whether our in vitro and in vivo observations can be extrapolated to human disease, blood samples were collected from 20 patients afflicted with rheumatoid arthritis and 13 from healthy donors which served as the negative control. Plasma samples were separated and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to determine the levels of transforming growth factor beta (TGF-β), protrombin F1+2 fragments (F1+2) and thrombospondin-1, and by multiplexed cytokine protein profiling on microarray by rolling-circle amplification (RCA) to determine cytokine levels. The F1+2 plasma levels showed an elevated trend in the RA group (p=0.06). TSP1 plasma levels were significantly increased in the RA group compared to the normal control (p=0.0004). Pro-inflammatory cytokine levels including IL-1β (p=0.0365), IL-6 (p=0.0029), TNF-α (p=0.0339), interferon-inducible protein 10 (p=0.0003) and macrophage inflammatory protein -1α (p=0.0012) were found elevated in the RA group compared to the normal control group. Some regulatory cytokines such as transforming growth factor-β (0.06), interferon-gamma (p=0.06) and IL-13 (p=0.22) showed no statistically significance between groups, but all of them showed a trend for higher circulation levels in plasma in the RA group. In summary, comparison between normal individuals and RA patients showed an increase in the levels of cytokines in the RA afflicted patients confirming what has been reported in the literature. We were able to correlate an increase in proteolytic factors and TSP1 levels in the RA patients with an increase of pro-inflammatory cytokines. Further studies are needed to elucidate why TSP1 acts as a pro-inflammatory molecule on the neutrophil surface of the RA patients.
Anemia of Chronic Renal Failure Is Associated with Much Higher Frequency of Apoptosis in Erythroid Progenitor in a Mouse Model.
Authors: Oyake Tatsuo , Ito Shigeki , Kowata Shugo , Murai Kazunori , Sawai Takashi , Ishida Yoji
Published: April 2020

Insufficient production of erythropoietin (EPO) is one of the important causes of anemia of patients with chronic renal failure (CRF). Recombinant human erythropoietin (rh-EPO) administration results in a dramatic improvement of anemia in these patients. In ASH meeting 2001, we reported that excessive apoptosis in erythroid progenitor was observed in anemia of CRF patients before rh-EPO administration. In this study, we established a mouse model of anemia of CRF to confirm the hypothesis that excessive apoptosis in erythroid progenitor induced anemia. C57B6 male mice was treated with adenine-rich diets (ADRD) for 8 weeks. Serum blood urea nitrogen (BUN), creatinine (Crt) and complete blood count (CBC) were measured every week. The frequency of apoptosis in erythroid progenitor and mature erythroblasts were evaluated by three color flow cytometric analysis. Apoptotic cells were analyzed by AnnexinV. The cell populations of erythroid progenitor were identified as CD34(+)erythropoietin-receptor (EPO-R)(+) cells and mature erythroblasts identified as CD34(-)EPO-R(+) cells. Serum BUN and Crt levels began to elevate from 2 weeks after ADRD administration (BUN; 40.1±4.6, 73.2±14.4, 150.7±11.6, 164.0±8.4, 193.0±23.3mg/dl, Crt; 0.06±0.04, 0.30±0.03, 0.51±0.07, 0.68±0.09, 0.80±0.17mg/dl, n=5: mean±SD at 0, 2, 4, 6 and 8 weeks after ADRD administration, respectively). Hb and Ht began to decrease from 6 and 4 weeks after ADRD administration (Hb; 13.6±0.4, 13.3±0.3, 13.2±0.2, 10.2±0.5, 8.3±0.5 g/dl, Ht; 52.4±1.4, 49.4±1.7, 43.6±0.2, 35.4±1.8, 30.8±1.6%, n=5: mean±SD at 0, 2, 4, 6 and 8 weeks after ADRD administration, respectively). Serum EPO levels were not elevated after ADRD administration, regardless of anemia. Much higher frequency of apoptosis was observed in erythroid progenitor from 4 to 8 weeks after ADRD administration, compared to that at 0 week (CD34(+)EPO-R(+); 18.2±7.4, 41.7±7.3, 56.0±4.8, 76.4±5.3, 70.1±6.9, 73.8±8.7%, n=5, mean±SD at 0, 4, 5, 6, 7 and 8 weeks after ADRD administration, p<0.05, respectively). While, the frequency of apoptosis was not high in mature erythroblasts after ADRD administration (CD34(-)EPO-R(+); 1.58±1.4, 0.21±0.1, 0.58±0.1, 0.77±0.7, 0.49±0.2, 0.25±0.2%, n=5, mean±SD at 0, 4, 5, 6, 7 and 8 weeks after ADRD administration, N.S, respectively). Pathological examination of kidney showed that tubular damage was diffusely observed from 2 to 8 weeks after ADRD administration, and tubular necrosis was suspected as the major cause of renal failure.

Our findings suggested that excessive apoptosis occurred mainly in CD34(+) erythroid progenitor by insufficient production of EPO, which induced anemia in mice with CRF.
The V617F JAK2 Mutation Is Very Uncommon in Idiopathic Erythrocytosis.
Authors: Cassinat Bruno , Parquet Nathalie , Kiladjian Jean-Jacques , Massonnet Gerald , Menot Marie-Laurence , Chomienne Christine
Published: April 2020

The V617F JAK2 mutation is highly frequent in Polycythemia Vera (PV) patients. Indeed, we and others found the mutation in 70 to 90% of PV patients. However one of the most difficult differential diagnoses of PV is the idiopathic erythrocytosis (IE), as this diagnosis is based on negative criteria and exclusion of PV. However the differential diagnosis is of the utmost importance because IE is not considered as a clonal disease and should not transform to PV. Our centre is specialized in the diagnosis of myeloproliferative diseases, thus we have reviewed 22 cases with pure idiopathic erythrocytosis in the aim of defining the impact of V617F JAK2 mutation in this pathology. Median age of the patients was 46 (range: 29 to 67). Patients were diagnosed on the basis of an elevation of the hematocrit and a raised red cell mass without any identifiable cause of secondary erythrocytosis. PV diagnosis has been carefully excluded according to revised Pearson’s criteria. Median excess of red cell mass was +35% (range: +25% to +104%). Median hematocrit was 54% (range: 49% to 56%), median WBC (x109/L) was 6180 (range: 3500 – 8300) and median platelet count (x109/L) was 240 (range: 174 – 358). Serum Epo level was under or within the normal range in all patients except one case in whom an unexplained elevated level (x2 the upper normal limit) was found. Finally no splenomegaly was observed in these 22 patients. Because of the importance of a correct diagnosis distinguishing between PV and IE it was very important to test whether the JAK2 mutation could allow a correct classification. We have analysed DNA isolated from peripheral blood granulocytes. V617F JAK2 mutation was detected using a quantitative PCR and Taqman probes with a sensitivity of 2–4%. All of the 22 patients with IE were tested negative. This result confirms

that IE is a distinct entity from PV, and also confirms the potential for the V617F JAK2 mutation detection to help in the differential diagnosis of erythrocytosis. Indeed, the presence of a JAK2 mutation in the context of erythrocytosis with increased red cell mass is highly specific of PV.
Cardiac Biomarkers Predict for Ability To Tolerate and Complete Therapy with Lenalidomide ± Dexamathosone in AL Amyloidosis.
Authors: Dispenzieri Angela , Lacy Martha , Zeldenrust Steven , Hayman Suzanne R. , Kumar Shaji , Geyer Susan M. , Lust John A. , Allred Jacob B. , Witzig Thomas E. , Rajkumar S. Vincent , Greipp Philip R. , Russell Stephen J. , Kabat Brian , Gertz Morie Abraham
Published: April 2020

Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. Lenalidomide, especially in conjunction with dexamethasone, has been shown to be highly active in patients with multiple myeloma.

Methods: We studied the toxicity and efficacy of lenalidomide in patients with symptomatic AL. Patients received single agent lenalidomide. If progression by 3 months or no evidence of hematologic response after 3 cycles, dexamethasone was added. Originally, twenty-three patients (Cohort 1) were enrolled according to study design. Because of a significant early drop out rate and notable activity of the regimen, the trial was modified to include an additional 15 patients (Cohort 2). Baseline characteristics and adverse events are available for all enrolled patients, but at the time of this writing, response data are available for Cohort 1 patients due to short follow-up of Cohort 2, but will be updated by the time of the meeting.

Results: Median age was 64 years, with 69% male. Twenty-three were previously treated. Organ involvement was cardiac (67%), renal (64%), hepatic (17%), nerve (17%). Thirty-three, twenty-two, and forty-four percent of patients were cardiac biomarker stage 1, 2, and, 3 respectively. Of the 37 patients, one was a cancel, and 6 have not yet made it through 3 months of protocol treatment and event monitoring. The respective median follow-ups for Cohorts 1 and 2 are 17 and 3.4 months. Of the remaining, 30 patients, within the first 3 cycles of therapy fifteen patients discontinued treatment: 7 early deaths and 8 adverse events or other causes. Three additional patients died 0.5 to 2 months after stopping treatment. The best predictor for early withdrawal and/or death was baseline NT-proBNP and cardiac biomarker staging system (cut-offs for serum troponin T <0.035 ng/ml and NT-proBNP <332 pg/ml--Stage I neither above cut-off; Stage III, both above cut-off; and Stage II, one above cut-off).

Figure1 Figure1. Figure 2 Figure 2.

Of the twenty 22 patients assessed for response ten patients responded to treatment for an overall response rate of 45%, including 23% organ responders. Among the patients with organ responses, there were four renal responses, two cardiac responses and two liver responses. All but one of the responders had dexamethasone added to their treatment program. The most common grade 3−4 adverse events at least possibly attributable to lenalidomide were neutropenia (38%), thrombocytopenia (21%), fatigue (15%), and rash (12%).

Conclusions: Lenalidomide and dexamethasone has significant activity in patients with AL with an overall response rate of 45%. Baseline NT−proBNP may be an effective eligibility screening strategy for subsequent trials.
Anemia in Centenarians Is Associated with Elevated Serum 2−Methylcitric Acid but Not Other Measures of Cobalamin Deficiency or Renal Status.
Authors: Stabler Sally P. , Allen Robert H. , Johnson Mary Ann , Hausman Dorothy B. , Davey Adam , Poon Leonard W.
Published: April 2020

Anemia in seniors is associated with increased mortality and frailty. It is important to determine the cause of anemia in order to initiate specific therapies. We studied cobalamin (Cbl) and folate status with other clinical and laboratory parameters associated with anemia in 234 centenarians from north Georgia aged 98–108 years, 197 female, 46 African-American. The Cbl and folate dependant metabolites included methylmalonic acid (MMA), total homocysteine (Hcys) and 2-methylcitric acid (MC). MMA, MC and Hcys are elevated in clinical Cbl deficiency. MC is also elevated with even mild renal insufficiency. Metabolites were assayed by capillary gas chromatography/mass spectrometry. The median hemoglobin (Hgb) and hematocrit (Hct) were 12.0 g/dL and 35.2 %. Severe anemia (Hgb < 8 g/dL) occurred in only 1 white female with serum ferritin of 10 ng/mL. The 10th percentile for hemoglobin was 10.0 g/dL, and in this group, serum MC was increased (p = 0.021), albumin trended lower (p = 0.056) but serum folate, Cbl, Hcys, MMA, and creatinine were not significantly different. Univariate analysis between variables for the whole cohort showed that Hgb was inversely correlated with Hcys (p = 0.028), MC (p = 0.003), creatinine (p = 0.030) and directly with albumin (p = 0.001). In multivariate analysis, albumin was the strongest positive predictor of Hgb and MC was significantly inversely related, whereas creatinine and Hcys were no longer independent determinants. Elevated MMA with low Cbl was common in this cohort but not associated with anemia. Even those above the 90th percentile for MMA (844–8080 nmol/L) actually had higher Hgb as compared to the rest of the cohort, 12.3 vs. 12.0 mg/dL (p = 0.096). Folate status was high in this cohort (50th percentile (12.9 ng/mL) and those taking a multivitamin (33%) had 1.5 fold higher serum folate (p < 0.001), 17 % lower Hcy (p = 0.001) and 24 % lower MMA (p = 0.045) but no difference in Hgb. The only racial difference was that MMA was lower in African-Americans (p <0.001) and the only sex difference was that creatinine was higher in males (p = 0.025). Those with MC above the 90th percentile (> 415 nmol/L) had lower Hgb (p = 0.038), higher creatinine (p = 0.003), Hcy (p = 0.003) and MMA (p = 0.060). Ferritin was less than 50 ng/mL in 10/22 subjects with the lowest 10th percentile Hgb value (< 10.0 g/dL). One subject had apparent folate deficiency and 7/22 had serum creatinine > 1.3 mg/dL. Two subjects with MCV > 100 fl had renal insufficiency. In conclusion, 1. High serum 2-methylcitric acid and low serum albumin predict anemia in centenarians. 2. Serum 2-methylcitric acid may be a sensitive indicator of renal failure- associated anemia. 3. Cbl deficiency was not associated with anemia in this cohort with high folate status.
Hypoxia Inhibits HIV-1 Transcription.
Authors: Nekhai Sergei , Sharroya Charles , Ammosova Tatyana , Nui Xiaomei , Jerebtsova Marina , Gordeuk Victor R.
Published: April 2020

The hypoxic response is an important component of the body’s reaction to impaired tissue oxygenation associated with the anemia and vasoocclusive episodes of sickle cell disease (SCD). It has been reported that HIV infection progresses relatively slowly in patients with SCD (Am J Hematol1998;59:199–207). HIV-1 Tat protein promotes transcription of HIV-1 genes by inducing CDK9/cyclin T1 to phosphorylate the C-terminal domain of RNA polymerase-II. Our previous studies indicate that protein phosphatase-1 (PP1) promotes Tat-induced HIV-1 transcription (J Biol Chem2003;278:32189–941), apparently by interacting with HIV-1 Tat, for disruption of this interaction prevents induction of transcription (J Biol Chem2005;280:36364–71). Recently PP1 activity was shown to be decreased in hypoxia in part through increased association of PP1 with NIPP1 (J Cell Physiol 2006 Jul 6; Epub ahead of print). We hypothesized that decreased PP1 activity during hypoxia would reduce HIV-1 transcription and viral replication, and we have obtained experimental results consistent with this hypothesis.Increased expression of nuclear inhibitor of PP1 (NIPP1) was associated with inhibition of HIV-1 transcription and viral replication.Low oxygen tension (3%–6% O2) was associated with transient inhibition of HIV-1 transcription in transfected 293T and HeLa cells.Low oxygen tension was associated with inhibition of HIV-1 transcription in CEM T cells infected with pseudotyped HIV-1 virus.

We are now planning to examine the possible role of altered PP1 activity in the observed inhibition of HIV-1 transcription during hypoxia. Thus, a clinical insight from patients with SCD has pointed the way to investigating the influence of hypoxia on HIV transcription. An improved understanding of how oxygen status influences viral activation versus inactivation might open new possibilities for treatment of hidden HIV-1 reservoirs that harbor non-replicating HIV-1 virus.
The Role of EHZF in Erythroid Differentiation of K562 Cells.
Authors: Matsubara Etsuko , Sakai Ikuya , Yamanouchi Jun , Azuma Taichi , Narumi Hiroshi , Hato Takaaki , Hasegawa Hitoshi , Yasukawa Masaki
Published: April 2020

Early hematopoietic zinc finger protein (EHZF), the human homolog to mouse Evi3, is a transcription factor with 30 zinc fingers and a highly conserved N-terminal FOG repression motif. The EHZF mRNA expression is abundant in early hematopoietic progenitors and declines during differentiation. In most of the leukemic cells from acute myelogenous leukemia patients, significant levels of EHZF mRNA are detected. These findings suggest that EHZF play an important role in hematopoietic differentiation.

In the present study we investigated whether siRNA-mediated depletion of EHZF affected the erythroid differentiation of K562 cells. Three kind of EHZF siRNA were designed and their expression vectors were constructed. After transient expression of these siRNAs by electroporation, hemoglobinization of K562 cells were analyzed by Benzidine staining. In all of these siRNAs introduced cells, the levels of hemoglobinizations were abundant compared to control cells, which suggested that EHZF might influence the erythroid differentiation of K562 cells. To further analyze the effects of EHZF siRNA in erythroid differentiation, we established K562 clones in which EHZF was depleted by siRNA. EHZF siRNA expression vectors were introduced into K562 cells by electroporation and the expression of EHZF mRNA were examined by RT-PCR in G418 resistant clones. The depressions of EHZF mRNA in the clones were confirmed by Northern blotting. We isolated several EHZF depleted clones in each kind of EHZF siRNA introduced cells. Most of the EHZF depleted clones showed marked hemoglobinization by Benzidine staining compared to control clones, and surface expressions of glycophorin A were also increased in these clones. These results confirmed the relevant role of EHZF in erythroid differentiation of K562 cells. Comparison of mRNA expressions between an EHZF depleted clone and a control clone using DNA array showed increase expressions of hemoglobin alpha, beta, delta mRNA in EHZF depleted clone compared to control clone. Several transcription factors which are involved in erythroid differentiation also showed the difference mRNA levels between two clones.

Studies are underway to elucidate the mechanisms underlying the role of EHZF in erythroid differentiation, the relationship to other transcription factors (GATA-1, GFi-1B, FOG-1 etc) which involved in erythroid differentiation.
Adaptor Protein Lnk Negatively Controls the Likelihood of Self-Renewal in Hematopoietic Stem Cells.
Authors: Ema Hideo , Seita Jun , Ooehara Jun , Iseki Akiko , Takano Hina , Nakauchi Hiromitsu
Published: April 2020

Great progresses are promised for the development of stem cell-based regenerative medicine if we can manipulate stem cell self-renewal. Thus, one of the central tasks in stem cell biology is to understand how stem cell fate is determined. Hematopoietic stem cells (HSCs) are the best studied stem cells. Their in vivo self-renewal has been extensively studied, but its in vitro recapitulation remains so difficult. We previously reported that HSCs undergo asymmetrical self-renewal division in culture with stem cell factor (SCF) and thrombopoietin (TPO). Since then, we have sought any condition in which HSCs can symmetrically self-renew. We now report in vitro symmetrical self-renewal division of HSCs in the absence of Lnk. Lnk is an adaptor protein containing praline-rich domain, pleckstrin homology domain, and Src homology domain. Lnk-deficient mice have over 10-fold HSCs due to increased self-renewal capacity. CD34−Kit+Sca-1+Lin− cells were purified from bone marrow of wild-type or Lnk-deficient B6 mice, and were subjected to serum-free single cell cultures in the presence of a variety of cyokines. We found that Lnk-deficient CD34−Kit+Sca-1+Lin− cells are hypersensitive to TPO. Repopulating activity in 40 CD34−Kit+Sca-1+Lin− cells from Lnk-deficient mice increased after 3 day-culture with TPO or with SCF and TPO, but not after 3 day-culture with SCF. In contrast, repopulating activity in 40 CD34−Kit+Sca-1+Lin− cells from wild type mice did not significantly change after 3 day-culture with SCF, TPO, or SCF and TPO. Moreover, paired daughter cell-experiments clearly showed that Lnk-deficient, but not wild-type long-term repopulating cells are able to undergo symmetrical self-renewal division at least once in the presence of SCF and TPO. These data suggest that Lnk acts just like a tuner in the regulation of HSC self-renewal downstream of TPO/Mpl signaling. We further investigated TPO-mediated signal transduction pathways in CD34−Kit+Sca-1+Lin− cells. To this end, we developed a novel assay which allowed us to analyze signal transduction in a very limited number of cells. We detected enhanced up-regulation of STAT5 and Akt pathways, and inversely enhanced down-regulation of p38 MAPK pathway in Lnk-deficient CD34−Kit+Sca-1+Lin− cells, as compared with normal ones. These data suggest that these combinational changes in signal transduction lead to initiation of self-renewal in HSCs. We propose that stem cell self-renewal is determined by a balance of positive and negative signals in multiple signal transduction pathways rather than by any particular self-renewal signals.
Spp1 (Osteopntin) Is Essential for the Early Niche Formation in the Bone Marrow.
Authors: Mizukami Takuo , Hamaguchi Isao , Kuramitsu Madoka , Momose Haruka , Takizawa Kazuya , Okada Seiji , Noce Toshiaki , Yamaguchi Kazunari
Published: April 2020

Hematopoietic stem cells (HSCs) have a potential to differentiate into variety types of blood cells, and have an ability of self-renewing in the bone marrow (BM). All these fates are regulated by intrinsic genetic pathways in the micro-enviroment (niche) where HSCs are located. Osteoblasts are thought to be the hematopoietic niche which regulates HSC function in the BM. To clarify the molecular basis of hematopoietic niche system, we compared the two somatic stem cells (Germ line stem cell (GS) and HSC) using cDNA suppression subtractive hybridization, and identified 12 stem cell-specific genes which are expressed both in the HSCs and GS. Among these genes, we analyzed Spp1, since it is a major glycoprotein produced by osteoblast and it has been reported to be a negative regulator of hematopoietic niche size in the BM.

In situ Hybridization (ISH) with Spp1-specific probes demonstrated that Spp1-expressing osteobalsts were located at the endosteal region of trabecular bone (TB). In the TB of adult mouce, Spp1-expressing hematopoietic cells attached with Spp1-expressing osteobalsts. Double ISH analysis revealed that Spp1 and Tie2 were partially co-expressed in the TB. These data suggests that Spp1 is an important molecule of hematopoietic niche system which were produced and interacted both HSCs and niche cell.

When we analyzed spp1 knock out mouse (Spp1−/−), adult Spp1−/− mice (8 weeks) appeared to be normal and fertile. However, the abnormal TB formation was observed in the younger Spp1−/− mice (2 weeks). A number of osteobalsts were increased and formed a multi-layer of osteoblasts at the endosteal region of TB. Along with the morphological change of the TB, distribution pattern of CD34, Tie2 and Bmi-1 expressing HSC was unusual. Tie2 expressing cells of Spp1−/− mouse were mainly located at vascular-enriched zone in the bone marrow, but few Tie2 expressing cells at osteoblastic zone. These data suggest that HSC and osteobalst do not form the correct niche in the Spp1−/− TB, and Spp1 is thought to be essential for the initial niche formation. To confirm the function of Spp1 at the initiation phase of niche formation, we performed the bone marrow transplantation. Mononuclear cells (1×105 cells) from Ly5.1 mice were transplanted into lethally irradiated Ly5.2 mice. At 12 days after transplantation, donor hematopoietic cells were observed in the recipient TB. At this day 12, the expression of Spp1 was up-regulated in the endosteal region of TB, however, Spp1 expression was decreased in the course of reconstitution. Thus, the findings regarding the hematopoietic niche formation in Spp1−/− mice and in the irradiated mice allow to understand the Spp1 function in the initial phase of niche formation in the bone marrow.
Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.
Authors: Rizo Aleksandra , Vellenga Edo , de Haan Gerald , Schuringa Jan Jacob
Published: April 2020

Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC).

In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors.

After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.
The Hematopoietic Activity of the Homeobox Gene HOXB4 Depends on a N-Terminal Proline-Rich Region In Vitro and In Vivo.
Authors: Cusan Monica , Deshpande Aniruddha , Hiddemann Wolfgang , Humphries R. Keith , Buske Christian , Feuring-Buske Michaela
Published: April 2020

HOXB4 belongs to the family of homeobox transcription factors, which play a key role in hematopoietic development. The expression of HOXB4 induces a significant increase of long-term repopulating stem cells (SC) in human and mouse models, without perturbing hematopoietic differentiation. So far the underlying mechanisms of the SC amplificatory impact of HOXB4 are poorly understood. In an attempt to understand the unique characteristics of HOXB4, we performed a mutational study by deleting its proline - rich N-terminus, which has been described to act as a transcriptional activation domain in many other proteins, like non-homeobox genes (e.g. p53, AP2) and other homeobox genes (e.g. HOXD4 and HOXA13). We performed in vitro and in vivo experiments transducing murine 5-FU enriched HSCs with the pMSCV-IRES-GFP based retroviral vector harbouring the HOXB4 wild-type (wt) and several mutants, including a Δproline HOXB4 mutant (ΔP), where the proline-rich sequence between the aminoacidic positions 71–123 is deleted.

In vitro the ΔP-HOXB4 mutant led to a 3.2fold decrease in the number of hematopoietic cells harvested after 1 week of liquid expansion compared to the HOXB4 wt (n=3; p<0.05). Furthermore, deletion of the proline - rich region resulted in a 7.5fold decrease in the total number of CFCs per 500 cells plated (n=3; p<0.03), compared to the HOXB4 wt. The ΔP-HOXB4 mutant also generated less secondary colonies (18.5 versus 103 per 500 cells initially plated; n=2; p<0.0001) compared to the HOXB4-wt. Of note and in contrast to the ΔP-HOXB4 mutant, the deletion of the PBX interacting domain (Pbx-HOXB4, aa W→A) or deletion of the C-terminal stretch (aa 221 −251; cDel-HOXB4) did not reduce the formation of primary colonies compared to HOXB4 wt (n=3; 178 and 124 versus 226 colonies per 500 cells initially plated, respectively). On the level of the short-term repopulating stem cells constitutive expression of HOXB4 wt (n=3) induced on average a 1,565fold increase in the frequency of ΔCFU-S in comparison to the GFP control (n=11) (10,645 versus 6.8 colonies/45,000 input cells; p<0.0000001). The deletion on the N-terminal proline-rich region of HOXB4 (n=14) led to a significant decrease in the Δ-CFU-S frequency in comparison to the HOXB4-wt (mean 42fold; p<0.000001), while it still generated significantly more Δ-CFU-S compared to the GFP control (252 versus 6.8/45,000 input cells; p<0.001).

Taken together, these results characterize the N-terminal proline - rich stretch between aminoacidic positions 71–123 as a domain, which is highly relevant for the hematopoietic activity of HOXB4.
Differential Signaling of G Protein-Coupled Chemotactic Receptors (CXCR4, cysLT1 and S1P1) Expressed in Hematopoietic Progenitor Cells.
Authors: Xue Xingkui , Boehmler Andreas , Kanz Lothar , Seitz Gabriele , Mohle Robert
Published: April 2020

We have previously demonstrated consistent and high expression of the G protein-coupled receptors (GPCR) CXCR4, cysLT1 and S1P1 in human hematopoetic progenitor and stem cells (HPC). All of these receptors induced chemotactic responses in mobilized CD34+ HPC. As their ligands are present in the hematopoietic microenvironment, these GPCR may be involved in HPC homing and/or proliferation. Ligands for CXCR4 (SDF-1) and cysLT1 (cys-leukotrienes) are both produced by stromal cells, while the S1P1 ligand (sphingosine 1-phosphate, S1P) is released by megakaryocytes and platelets. As GPCR may activate multiple signaling pathways resulting in different functional effects, we compared the signaling capacities of CXCR4, cysLT1 and S1P1 in CD34+ HPC and CD34+ progenitor cell lines. At optimal ligand concentrations, cysLT1 mediated the strongest intracellular calcium fluxes, in contrast to CXCR4 (intermediate response) and S1P1 (almost absent calcium fluxes). Pertussis toxin (PTX) only partially inhibited cysLT1-mediated calcium signaling, indicating coupling of the receptor to both Gi and Gq proteins, while CXCR4-mediated responses were exclusively PTX-sensitive and therefore only Gi-dependent. Both cysLT1 and CXCR4 induced phosphorylation of the proliferation-related p44/42 (Erk) MAP kinase. Again, much stronger responses were mediated by cysLT1, which again were only partially PTX-sensitive. No Erk/MAP kinase activation was mediated by S1P1. We could demonstrate that pyk2, a kinase that links calcium signaling with Erk/MAP kinase activation, was involved in Gq-mediated signaling of cysLT1, as its phosphorylation was not inhibited by PTX. The fact that cysLT1 employs both Gi and Gq-mediated signaling pathways, while CXCR4 signaling was exclusively Gi-dependent, may explain why enhanced proliferation of CD34+ hematopoietic progenitor cells in cytokine-supplemented serum-free liquid cultures was only seen in response to ligands of cysLT1, but not in response to the CXCR4 ligand SDF-1. Activation of CXCR4 resulted in phosphorylation of Akt, which was not observed in response to ligands of cysLT1, while both GPCR induced translocation of NFkappaB to the nucleus. We conclude that in CD34+ HPC, Gi protein-mediated signaling of CXCR4 is mainly involved in cell migration, while Gi and Gq-mediated signaling of cysLT1 also affects cell proliferation predominantly by combined and profound activation of the Erk/MAP kinase pathway. The signaling capacity of S1P1 however was limited compared to the other GPCRs.
The GEF Lsc Regulates Hematopoietic Stem Cell Motility, Mobilization and Recruitment.
Authors: Petit Isabelle , Kaul Prashant , Lerner Daniel J. , Rafii Shahin
Published: April 2020

Lsc is a Rho GTPase guanine nucleotide exchange factor (RhoGEF) that physically and functionally links G-protein coupled receptors (GPCR) to the monomeric GTPase RhoA in mature hematopoietic and other cells. Lsc−/− (LscKO) mice have a peripheral leukocytosis, abnormal neutrophil and B cell motility, and immune response deficiencies. Although Lsc is required for neutrophil homeostasis, its role in hematopoietic stem and progenitor cells is unknown. In this study, we have used LscKO mice to determine if Lsc is required for normal stem cell motility and mobilization. Initially, we used immunofluorescence labeling to demonstrate that hematopoietic stem and progenitor cells express Lsc. This suggested that Lsc may be required for normal hematopoietic stem and progenitor cell migration. Stromal-cell derived factor-1 (SDF-1) is a potent chemokine for hematopoietic stem cells and activates the CXCR4 GPCR. It has been reported that Lsc is not required for SDF-1-stimulated migration of mature murine T and B cells. However, using a bare-filter transwell assay, we found that while LscKO Sca-1+ cells and Sca-1+Lin- cells have normal spontaneous migration, they have significantly increased SDF-1-stimulated migration compared to their wild-type (WT) counterparts, 1.4 and 2.3 fold, respectively. We then demonstrated that adhesion of LscKO Sca-1+ cells to bone marrow (BM) stromal MS-5 cells was normal, indicating that impaired adhesion was not responsible for the abnormal SDF-1-stimulated migration. Using colony assay, we demonstrated that LscKO mice have a normal number of circulating peripheral stem and progenitor cells. Strikingly, after 5 days of G-CSF administration, LscKO mice have 1.6 fold and 2.3 fold the number of peripheral mature WBC and stem and progenitor cells (colony forming units), respectively, compared to WT mice. Recruitment of BM CXCR4+ pro-angiogenic stem and progenitor cells has been linked to enhanced tumor angiogenesis. Because LscKO BM cells had abnormal SDF-1-stimulated migration and mobilization, we hypothesized that Lsc might regulate tumor angiogenesis as well. To this end, we assessed tumor growth in LscKO mice by injecting congenic Lewis lung carcinoma cells subcutaneously into LscKO mice and WT controls. Preliminary experiments revealed that tumors were 3.3 times larger in the LscKO mice as compared to WT mice. Quantification of the tumor vessels with anti-CD31 staining demonstrated that the tumors in LscKO mice were 1.4 fold more vascularized than controls. In summary, our results demonstrate that the Rho GEF Lsc is essential for normal hematopoietic stem cell migration and mobilization. In addition, we propose that absence of Lsc facilitates tumor growth by promoting BM stem and progenitor cell recruitment to the neo-angiogenic vessels, possibly augmenting tumor vascularization.
A NUP98-HOX Fusion Gene Containing the Homeodomain of HOXA10 Promotes Significant Expansion of Primitive Human Hematopoietic Cells in Extended Cultures.
Authors: Imren Suzan , Sauvageau Guy , Eaves Connie J. , Humphries R. Keith
Published: April 2020

Expanding human hematopoietic stem cells (HSCs) in vitro is a major goal in clinical hematology but remains a major challenge due to the potent differentiating activity of known cytokines. We recently demonstrated that a NUP98-HOX fusion gene containing only the homeodomain (hd) of HOXA10 (NUP98-HOXA10hd) is a powerful stimulator of murine HSC expansion in vitro - causing >1000-fold net HSC increases in 10 days (Sekulovic et al, ISEH 2005). To investigate the proliferative effect of NUP98-HOXA10hd on primitive human hematopoietic cells, highly enriched CD34+ cord blood cells were prestimulated overnight and exposed to self-inactivating MNDUSNUP98-HOXA10hd pgkGFP or control pgkGFP lentiviruses for 6h. The gene transfer efficiency into CD34+ cells determined 4 days after infection was 56 ± 5% for NUP98-HOXA10hd and 66 ± 5% for the GFP control. GFP+ cells were sorted on day 5 and then maintained for another 5 days in serum-free cultures containing Flt3-ligand, Steel factor, IL-3, IL-6 and G-CSF. An aliquot of each was then plated into “primary” colony-forming cell (CFC) assay cultures. No difference was detected in either the numbers or the types of colonies generated in these primary CFC assays of the 10-day cultured cells from the NUP98-HOXA10hd and control arms. However, when these primary CFC assays were replated into secondary CFC assays, the number of colonies obtained from the NUP98-HOXA10hd-transduced cells was 5-fold higher as compared to the GFP-control transduced cells and, upon replating into tertiary CFC assays, this difference increased to over a 100-fold. To determine the effect of NUP98-HOXA10hd on more primitive hematopoietic cells, 104 day-10 GFP+ cells were co-cultured on mouse fibroblast feeders engineered to produce human SF, IL-3 and G-SCF. At the end of 6 weeks, 13-fold more cells were recovered from the cultures initiated with NUP98-HOXA10hd-transduced cells than from the control cultures (474,000 ± 190,000 vs 37,000 ± 16,000, 3 experiments). CFC outputs were also greatly enhanced (21-fold more CFC than in the controls cultures, range=20–80, 3 experiments). Moreover, the proportion of progenitors in the assays of the cultures initiated with NUP98-HOXA10hd cells that were multi-lineage (CFU-GEMM) was >10-fold higher as compared to the CFCs obtained from the control cultures (8 ± 3% vs 0.7 ± 0.7%). When this experiment was repeated using limiting dilutions of initial day-10 cells, the frequency of NUP98-HOXA10hd-transduced cells able to generate CFCs another 6 weeks later was 10-fold higher as compared to the day-10 GFP control-transduced cells. These findings document an unprecedented potency of NUP98-HOXA10hd for stimulating the ex-vivo expansion of very primitive pluripotent human hematopoietic cells.
Bcr-Abl Inhibitors Induce Expression of HoxA10, Playing a Role as an Inhibitor of the Proliferation through PI3K/PKB Pathway in Chronic Myelogenous Leukemia Cells.
Authors: Sugimoto Yuya , Nakamura Satoki , Ono Takaaki , Kobayashi Miki , Sahara Naohi , Shinjo Kaori , Shigeno Kazuyuki , Ohnishi Kazunori
Published: April 2020

[Background] Homeobox (Hox) genes are grouped together in 4 clusters, A to D. Recent studies has shown that the Hox proteins are important in the control of differentiation and proliferation in hematopoietic cells. It is reported that HoxA10 has been expressed in all types acute myeloid leukemias (except for APL) but not in acute lymphoid leukemias, implicating an important role as a regulator of myeloid progenitors. Moreover, overexpression of HoxA10 induses the proliferation of early progenitor cells and leads to the development of myeloid leukemias. However, we found that Bcr-Abl inhibitors increased the expression of HoxA10 gene in CML cells. In this study, we investigated the mechanism controlling HoxA10 in CML cells treated with Bcr-Abl inhibitors.

[Methods] The cells used in this study were Ph1 positive CML cells, K562 and Meg01, and U937 cells for a Ph1 negative cell. We used AMN107 and BMS354825 for Bcr-Abl inhibitors, LY294002 for a PI3K inhibitor, PP2 for a Src kinase inhibitor, and SB203580 for a p38 MAP kinase inhibitor. For analysis of HoxA10 mRNA and protein, RT-PCR and western blot were performed in K562, Meg-01 and U937 cells, which untreated or treated with AMN107, BMS354825, LY294002, PP2, or SB203580 respectively. We then attempted to localize the intracellular locations of HoxA10 in K562 and Meg01, which untreated or treated with AMN107, BMS354825, or LY294002 by using conforcal fluorescence microscopy. Finally, for analysis of proliferation in K562, Meg-01 and U937 transfected with siRNA HoxA10, MTT assays were performed in untransfected or transfected K562, Meg-01 and U937 treated with or without AMN107, BMS354825, orLY294002.

[Results] Both K562 and Meg01 cells expressed HoxA10 mRNA and protein at lower level compared to U937 cells. Interestingly, treatment with AMN107, BMS354825, or LY294002 increased the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. In contrast, treatment with PP2 or SB203580 did not affect the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. The fluorescence of HoxA10 was more strongly observed in the area corresponding to the cell’s cytoplasm than nucleus, and the treatment with AMN107, BMS354825, or LY294002 increased the fluorescence in nucleus of K562 and Meg01 cells in a time-dependent manner. In K562 and Meg01 cells transfected with the siRNA HoxA10, treatment with AMN107 or BMS354825 slightly inhibited the proliferation compared to K562 and Meg01 transfected with control siRNA.

[Conclusions] In this study, we showed that Bcr-Abl inhibitors induced the expression of HoxA10, and HoxA10 was regulated by PI3K/P[Kappa[B pathway in CML cells. This finding indicates a new insight in the regulation of cell proliferation via the PI3K/PKB signal pathway in CML cells.
Generation of Specific Resistance Profiles in FLT3-ITD and FIP1L1-PDGFRalpha towards Specifically Acting Tyrosine Kinase Inhibitors.
Authors: von Bubnoff Nikolas , Thoene Silvia , Gorantla Sivahari P. , Saenger Jana , Peschel Christian , Duyster Justus
Published: April 2020

BCR-ABL kinase domain mutations constitute the major mechanism of resistance in patients with chronic myelogenous leukemia treated with the ABL kinase inhibitor imatinib. Mutations causing resistance to therapeutic kinase inhibition were also identified in other target kinases in various malignant diseases, such as FLT3-ITD in acute myelogenous leukemia, cKit in gastrointestinal stromal tumors, EGFR in patients with lung cancer, and FIP1L1-PDGFRalpha in hypereosinophilic syndrome. Thus, mutations in kinase domains seem to be a general mechanism of resistance to therapeutically applicated tyrosine kinase inhibitors. We recently developed a cell-based screening strategy that allows one to predict the pattern and relative abundance of BCR-ABL resistance mutations emerging in the presence of imatinib, and the novel ABL kinase inhibitor AMN107 (nilotinib). We therefore intended to determine, if this method would also allow the generation of resistant cell clones with other oncogeneic tyrosine kinases as targets in the presence of specifically acting kinase inhibitors. When FLT3-ITD and su5614 were used as drug/target combination in our cell-based method, the frequency of resistant clones in the presence of su5614 at 10 times the IC50 was 0.17 per million cells. In 40 per cent of resistant clones, point mutations were detected leading to amino acid exchanges within the FLT3-ITD split kinase domain. The yield of resistant clones was increased by the factor of 14 to 2.37 per million cells by adding ethyl-nitrosourea (ENU), a potent inducer of point mutations. Also, the proportion of mutant clones increased from 40 to 74 per cent. In 83 mutant clones that were examined so far, we detected eight exchanges affecting kinase domain two (TK2) of the split kinase domain within or shortly behind the FLT3-ITD activation loop (A-loop). We did not detect exchanges affecting TK1. We next examined whether resistant clones would also come up with FIP1L1-PDGFRalpha-transformed cells in the presence of imatinib. Again, the yield of resistant clones increased when cells were pretreated with ENU, and a proportion of resistant clones contained mutations in the FIP1L1-PDGFRalpha kinase domain, affecting the nucleotide-binding loop (P-loop) and A-loop. We conclude that cell-based resistance screening is a simple and powerful tool that allows prediction of resistance mutations towards kinase inhibitors in various relevant oncogeneic kinases.