Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting ...histone deacetylases to target genes. We present data that low expression of GFI1 is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1 expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. Leukemic cells from animals that express low levels of GFI1 show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1 expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KD mice and from AML patients with low GFI1 levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1 expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.
The growth of malignant cells is not only driven by cell-intrinsic factors, but also by the surrounding stroma. Monocytes/Macrophages play an important role in the onset and progression of solid ...cancers. However, little is known about their role in the development of acute myeloid leukemia, a malignant disease characterized by an aberrant development of the myeloid compartment of the hematopoietic system. It is also unclear which factors are responsible for changing the status of macrophage polarization, thus supporting the growth of malignant cells instead of inhibiting it. We report herein that acute myeloid leukemia leads to the invasion of acute myeloid leukemia-associated macrophages into the bone marrow and spleen of leukemic patients and mice. In different leukemic mouse models, these macrophages support the in vitro expansion of acute myeloid leukemia cell lines better than macrophages from non-leukemic mice. The grade of macrophage infiltration correlates in vivo with the survival of the mice. We found that the transcriptional repressor Growth factor independence 1 is crucial in the process of macrophage polarization, since its absence impedes macrophage polarization towards a leukemia supporting state and favors an anti-tumor state both in vitro and in vivo These results not only suggest that acute myeloid leukemia-associated macrophages play an important role in the progression of acute myeloid leukemia, but also implicate Growth factor independence 1 as a pivotal factor in macrophage polarization. These data may provide new insights and opportunities for novel therapies for acute myeloid leukemia.
Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as ...myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of
in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of
, and latency was further decreased when
was conditionally deleted. Loss of
significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of
led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.
The differentiation of haematopoietic cells is regulated by a plethora of so-called transcription factors (TFs). Mutations in genes encoding TFs or graded reduction in their expression levels can ...induce the development of various malignant diseases such as acute myeloid leukaemia (AML). Growth Factor Independence 1 (GFI1) is a transcriptional repressor with key roles in haematopoiesis, including regulating self-renewal of haematopoietic stem cells (HSCs) as well as myeloid and lymphoid differentiation. Analysis of AML patients and different AML mouse models with reduced GFI1 gene expression levels revealed a direct link between low GFI1 protein level and accelerated AML development and inferior prognosis. Here, we report that upregulated expression of GFI1 in several widely used leukemic cell lines inhibits their growth and decreases the ability to generate colonies in vitro. Similarly, elevated expression of GFI1 impedes the in vitro expansion of murine pre-leukemic cells. Using a humanized AML model, we demonstrate that upregulation of GFI1 expression leads to myeloid differentiation morphologically and immunophenotypically, increased level of apoptosis and reduction in number of cKit
cells. These results suggest that increasing GFI1 level in leukemic cells with low GFI1 expression level could be a therapeutic approach.
The differentiation of hematopoietic stem cells to mature cells is essential for the function of the hematopoietic system. Disturbance of this process can lead to the emergence of Acute Myeloid ...Leukemia (AML). AML is characterized by an accumulation of immature, malignant blasts, which disturb the function of the “normal” hematopoietic cells. The differentiation to myeloid cells is regulated among others by transcription factors. Growth factor independence 1 (GFI1) is such a hematopoietic transcription factor regulating the differentiation of myeloid cells.
We sought to investigate whether different Gfi1 levels are causative for emergence of AML and how different levels of Gfi1 might influence the prognosis of patients. Using published expression array data, we observed that Gfi1 is expressed at a lower expression level in blast cells and in leukemic stem cells compared to the control non-malignant cells and stem cells.
We then correlated Gfi1 expression level in blast cells of patients from different centers in with the event free survival. In Essen and Dresden (Germany), low expression levels in blast cells were (n=39) associated with an inferior prognosis (EFS 9 months for low expression compared to 42 months; p=0.0095).
We confirmed our observation with an independent cohort from Rotterdam and Nijmegen. Patients with low Gfi1 expression (n=32) had an inferior event free survival (9 months) compared to patients with higher Gfi1 levels (n=144; 17 months; p=0.02). To further investigate how different levels of Gfi1 might influence initiation and progression of leukemia, we used mice expressing Gfi1 at different levels, i.e. Gfi1 deficient mice (Gfi1KO), mice heterozygous for Gfi1 (Gfi1 het) or mice expressing Gfi1 only at 20% of normal Gfi1 expression levels (Gfi1KD). We used different murine AML models to examine the role of Gfi1 in AML development. First we crossed these mice with Nup98HoxD13 mice that recapitulate MDS disease course. We observed that knockdown of Gfi1 (Gfi1KD n=15, P=0.05) and heterozygosity of Gfi1 (Gfi1 het) (n=12) accelerated AML development and were associated with higher blast cell number compared to Gfi1 wt mice (n=16). Interestingly, complete loss of Gfi1 (Gfi1KO, n=16) inhibited leukemia development.
To confirm our findings, we used an independent approach. It has been shown previously that enforced retroviral expression of certain onco-fusion proteins such as MLL-AF9 or AML1-ETO9a or proteins such as MN1 can cause AML in mice. To this end we transduced lineage negative (Lin-) cells from the different mouse strains with retroviruses overexpressing these different proteins and plated the Lin-cells in methycellulose media. Similar to above, transduced Gfi1 KD cells generated more colonies and proliferated at higher levels than wt or Gfi1 KO cells (ranging between 1,5 to 4 fold KD compared to wt, depending on oncofusionprotein or oncogene, p=0.05). We also transplanted these cells into lethally irradiated mice. Again, mice transplanted with MLL-AF9 transduced Gfi1 KD cells (n=6) developed leukemia faster than mice transplanted with transduced wt (n=8; p= 0.05). We hypothesized that if lower expression of Gfi1 promotes leukemia formation, then overexpression of Gfi1 should inhibit leukemia formation.
To this end we transduced Lin neg cells simultaneously with retroviruses overexpressing either MLL-AF9 or AMLETO9a and either Gfi1 or an empty vector. The cells were then plated in methycellulose and cells overexpressing Gfi1 generated fewer colonies (between 3-5 fold less, depending on oncogene, p=0.01) than cells transduced with an empty control vector.
Thus, up to now our data suggests that decreased levels of Gfi1 influence prognosis of AML development and are involved in the pathogenesis of AML. On a molecular level, we found that knock-down and complete loss of Gfi1 leads to deregulation of genes in AML development such as HoxA9. However, whereas Gfi1KD cells show a normal response to apoptotic events, complete loss of Gfi1 is associated with a very high level of spontaneous apoptosis, possibly explaining why knock-down but not complete loss of Gfi1 accelerates AML development. In summary we demonstrate that Gfi1 plays a crucial role in AML development depending on the expression level, which in turn might explain the role of Gfi1 in human AML.
No relevant conflicts of interest to declare.
Biomechanical research demonstrates increased subscapularis abduction range of motion (ROM) when the tendon’s upper two-thirds is repaired over-the-top of the center of rotation during reverse ...shoulder arthroplasty (RSA). This study compares the clinical outcomes of patients undergoing RSA with over-the-top subscapularis repair (OTTR) to patients without repair.
We retrospectively reviewed 97 consecutive RSAs with either OTTR of the subscapularis (N = 75) or no repair (N = 22). Repair was attempted in all patients but not performed if the subscapularis could not be brought to the over-the-top position in 20° of external rotation (ER) and 30° of abduction. Improvements in ROM were compared to the minimal clinically important difference for RSA.
The mean follow-up was 3.8 ± 1.6 years. Demographics were similar between groups. Preoperatively, patients undergoing repair had greater ER when compared to those without repair (15 ± 16° vs. 5 ± 12°, P = .003). Postoperatively, patients undergoing repair had greater forward elevation (132 ± 21° vs. 126 ± 22°, P = .268) and abduction (114 ± 26° vs. 106 ± 23°, P = .193) with both exceeding the minimal clinically important difference (−2.9° and −1.9°, respectively); however, not statistically significant. Patients with repair were more frequently able to reach the small of their back postoperatively (65% vs. 21%, P = .006) but had less improvement in ER (13 ± 20° vs. 24 ± 20°, P = .028). Postoperative outcome scores, complications, and reoperations were similar between groups.
OTTR of the subscapularis in RSA had similar ROM and outcome scores compared to no repair, but a significantly larger proportion of patients with repair achieved functional internal rotation to the small of the back. ER limitations seen after conventional repair may also apply to this novel technique, but without a corresponding detrimental effect on forward elevation or abduction.
Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type ...1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRβ, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding–defective TRβ leads to disruption of the hypothalamic–pituitary–thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.
Objective
Progressive hepatic fibrosis can be considered the final stage of chronic liver disease. Hepatic stellate cells (HSC) play a central role in liver fibrogenesis. Thyroid hormones (TH, e.g. ...thyroxine; T4 and triiodothyronine; T3) significantly affect development, growth, cell differentiation and metabolism through activation of TH receptor α and/or β (TRα/β). Here, we evaluated the influence of TH in hepatic fibrogenesis.
Design
Human liver tissue was obtained from explanted livers following transplantation. TRα‐deficient (TRα‐KO) and wild‐type (WT) mice were fed a control or a profibrogenic methionine‐choline deficient (MCD) diet. Liver tissue was assessed by qRT‐PCR for fibrogenic gene expression. In vitro, HSC were treated with TGFβ in the presence or absence of T3. HSC with stable TRα knockdown and TRα deficient mouse embryonic fibroblasts (MEF) were used to determine receptor‐specific function. Activation of HSC and MEF was assessed using the wound healing assay, Western blotting, and qRT‐PCR.
Results
TRα and TRβ expression is downregulated in the liver during hepatic fibrogenesis in humans and mice. TRα represents the dominant isoform in HSC. In vitro, T3 blunted TGFβ‐induced expression of fibrogenic genes in HSC and abrogated wound healing by modulating TGFβ signalling, which depended on TRα presence. In vivo, TRα‐KO enhanced MCD diet‐induced liver fibrogenesis.
Conclusion
These observations indicate that TH action in non‐parenchymal cells is highly relevant. The interaction of TRα with TH regulates the phenotype of HSC via the TGFβ signalling pathway. Thus, the TH–TR axis may be a valuable target for future therapy of liver fibrosis.
Little information exists regarding the benefit of computer navigation in shoulder arthroplasty in the clinical setting. This study aimed to quantify how computer navigation affects the number and ...length of screws used during in vivo reverse total shoulder arthroplasty (RSA) placement.
We performed a retrospective review of a research database to identify patients who underwent primary RSA before and after the use of computer navigation between January 1, 2015, and December 31, 2019. One hundred consecutive RSAs were selected from the computer navigation implantation date; then, 100 consecutive sex-matched RSAs were chosen prior to navigation implantation in reverse chronologic order. Baseplate augmentations were chosen based on surgeon discretion, with the goal of restoring version to within 10° of neutral and inclination to neutral or slightly inferior with removal of the smallest amount of subchondral bone possible. Screws were placed with the goal of ≥3 screws with good purchase and were added as needed, with up to 5 screws used. We compared demographic factors, comorbidities, preoperative diagnosis, number of screws, screw length, number of wasted screws, and number of cases with bone graft used behind the baseplate between the 2 groups. We used the χ2 test for bivariate analysis and the Student t test for continuous variables.
A total of 200 RSAs were included, with 100 primary RSAs (mean age, 69.3 years) performed prior to computer navigation compared with 100 primary RSAs (mean age, 69.7 years) performed using computer navigation. The total number of screws used in RSAs without computer navigation was 414; the total used in the computer navigation cases was 344. RSAs placed with computer navigation used significantly fewer screws per case (3.4 screws vs. 4.1 screws, P < .001) and had a significantly greater average screw length (35.0 mm vs. 32.6 mm, P < .001). Three screws were implanted in 61% of computer navigation cases vs. 1% of cases without computer navigation (P < .001). Screws ≥ 30 mm in length were more commonly used in patients undergoing RSA using computer navigation (84.6% vs. 73.7%, P < .001).
This study shows that computer navigation in RSA leads to longer and fewer glenoid baseplate screws being implanted. Computer navigation appears to assist with better screw placement, which may have similar clinical benefits of better glenoid fixation. Additionally, using fewer screws can save glenoid bone stock, avoid added glenoid stress risers, and decrease operative time.
The purpose of this study was to evaluate the relationship between humeral lengthening and clinical outcomes after reverse shoulder arthroplasty (RSA) with stratification based on measurement method ...and implant design.
This systematic review was performed using PRISMA-P guidelines. PubMed/Medline, Cochrane Trials, and Embase were queried for articles evaluating the relationship between humeral lengthening and clinical outcomes inclusive of range of motion (ROM), strength, outcome scores, and pertinent complications (acromial and scapular spine fractures, nerve injury) after RSA. The relationship between humeral lengthening and clinical outcomes was reported descriptively overall and stratified by measurement method and implant design (globally medialized vs. lateralized). A positive association was defined as increased humeral lengthening being associated with greater ROM, outcome scores, or a greater incidence of complications, whereas a negative association denoted that increased humeral lengthening was associated with poorer ROM, outcome scores, or a lower incidence of complications. Meta-analysis was performed to compare humeral lengthening between patients with and without fractures of the acromion or scapular spine.
Twenty-two studies were included. Humeral lengthening was assessed as the acromiohumeral distance (AHD), the distance from the acromion to the greater tuberosity (AGT), the acromion to the deltoid tuberosity (ADT), and the acromion to the distal humerus (ADH). Of 11 studies that assessed forward elevation, a positive association with humeral lengthening was found in 6, a negative association was found in 1, and 4 studies reported no association. Of studies assessing internal rotation (n = 9), external rotation (n = 7), and abduction (n = 4), all either identified a positive or lack of association with humeral lengthening. Studies assessing outcome scores (n = 11) found either a positive (n = 5) or no (n = 6) association with humeral lengthening. Of the studies that assessed fractures of the acromion and/or scapular spine (n = 6), 2 identified a positive association with humeral lengthening, 1 identified a negative association, and 3 identified no association. The single study that assessed the incidence of nerve injury identified a positive association with humeral lengthening. Meta-analysis was possible for AGT (n = 2) and AHD (n = 2); greater humeral lengthening was found in patients with fractures for studies using the AGT (mean difference 4.5 mm, 95% CI 0.7-8.3) but not the AHD. Limited study inclusion and heterogeneity prohibited identification of trends based on method of measuring humeral lengthening and implant design.
The relationship between humeral lengthening and clinical outcomes after RSA remains unclear and requires future investigation using a standardized assessment method.
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