Venetoclax and dexamethasone synergize with inotuzumab ozogamicin-induced DNA damage signaling in B-lineage ALL.

Adult patients with relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a dismal prognosis. To improve pharmacotherapy, we analyzed induction of apoptosis by venetoclax and inotuzumab ozogamicin in terms of cytotoxicity and mode of action. Flow cytometry-based analyses of mitochondrial outer membrane permeabilization (MOMP) and ataxia telangiectasia mutated activation demonstrate rapid induction of MOMP by venetoclax and DNA damage signaling by inotuzumab ozogamicin, respectively. In primary ALL samples and patient-derived xenograft (PDX) models, venetoclax and inotuzumab ozogamicin cooperated and synergized in combination with dexamethasone in vitro in all tested samples of ALL. In murine PDX models, inotuzumab ozogamicin, but not venetoclax, induced complete remission in a dose-dependent manner but constantly failed to achieve relapse-free survival. In contrast, combination therapy with venetoclax, dexamethasone, and inotuzumab ozogamicin induced long-term leukemia-free survival and treatment-free survival in all 3 ALL-PDX models tested. These data demonstrate synergistic and highly efficient pharmacotherapy in preclinical models that qualify for evaluation in clinical trials.

Increased prostaglandin-D2 in male STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation.

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)-signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.

Anthracycline-free tumor elimination in mice leads to functional and molecular cardiac recovery from cancer-induced alterations in contrast to long-lasting doxorubicin treatment effects.

Systemic effects of advanced cancer impact on the heart leading to cardiac atrophy and functional impairment. Using a murine melanoma cancer model (B16F10 melanoma cells stably transduced with a Ganciclovir (GCV)-inducible suicide gene), the present study analysed the recovery potential of cancer-induced cardiomyopathy with or without use of doxorubicin (Dox). After Dox-free tumor elimination and recovery for 70 ± 5 days, cancer-induced morphologic, functional, metabolic and molecular changes were largely reversible in mice previously bearing tumors. Moreover, grip strength and cardiac response to angiotensin II-induced high blood pressure were comparable with healthy control mice. In turn, addition of Dox (12 mg/kg BW) to melanoma-bearing mice reduced survival in the acute phase compared to GCV-alone induced recovery, while long-term effects on cardiac morphologic and functional recovery were similar. However, Dox treatment was associated with permanent changes in the cardiac gene expression pattern, especially the circadian rhythm pathway associated with the DNA damage repair system. Thus, the heart can recover from cancer-induced damage after chemotherapy-free tumor elimination. In contrast, treatment with the cardiotoxic drug Dox induces, besides well-known adverse acute effects, long-term subclinical changes in the heart, especially of circadian clock genes. Since the circadian clock is known to impact on cardiac repair mechanisms, these changes may render the heart more sensitive to additional stress during lifetime, which, at least in part, could contribute to late cardiac toxicity.

Neuraminidase-1 promotes heart failure after ischemia/reperfusion injury by affecting cardiomyocytes and invading monocytes/macrophages.

Neuraminidase (NEU)1 forms a multienzyme complex with beta-galactosidase (ß-GAL) and protective-protein/cathepsin (PPC) A, which cleaves sialic-acids from cell surface glycoconjugates. We investigated the role of NEU1 in the myocardium after ischemia/reperfusion (I/R). Three days after inducing I/R, left ventricles (LV) of male mice (3 months-old) displayed upregulated neuraminidase activity and increased NEU1, ß-GAL and PPCA expression. Mice hypomorphic for neu1 (hNEU1) had less neuraminidase activity, fewer pro-inflammatory (Lin-CD11b+F4/80+Ly-6Chigh), and more anti-inflammatory macrophages (Lin-CD11b+F4/80+Ly-6Clow) 3 days after I/R, and less LV dysfunction 14 days after I/R. WT mice transplanted with hNEU1-bone marrow (BM) and hNEU1 mice with WT-BM showed significantly better LV function 14 days after I/R compared with WT mice with WT-BM. Mice with a cardiomyocyte-specific NEU1 overexpression displayed no difference in inflammation 3 days after I/R, but showed increased cardiomyocyte hypertrophy, reduced expression and mislocalization of Connexin-43 in gap junctions, and LV dysfunction despite a similar infarct scar size to WT mice 14 days after I/R. The upregulation of NEU1 after I/R contributes to heart failure by promoting inflammation in invading monocytes/macrophages, enhancing cardiomyocyte hypertrophy, and impairing gap junction function, suggesting that systemic NEU1 inhibition may reduce heart failure after I/R.

Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in death because of respiratory or cardiac failure. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iPSC-CMs) from two DMD patients: a male and female manifesting heterozygous carrier. Dystrophin mRNA and protein expression were analysed by qRT-PCR, RNAseq, Western blot and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. X-inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation, which was maintained in female iPSC-CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC-CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration. DMD female iPSC-CMs displayed increased beat rate variability (BRV). DMD male iPSC-CMs manifested decreased If density, and DMD female and male iPSC-CMs showed increased ICa,L density. Our findings demonstrate cellular mechanisms underlying electrophysiological abnormalities and cardiac arrhythmias in DMD.

Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

miR-181a Expression in Donor T Cells Modulates Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation.

Because miR-181a has been described to alter T cell activation, we hypothesized that manipulation of miR-181a expression in donor T cells may alter acute graft-versus-host disease (aGvHD) after allogeneic bone marrow transplantation (BMT). We therefore analyzed the impact of enhanced and reduced miR-181a expression in donor T cells on aGvHD induction by lentiviral gene transfer into primary T cells and using miR-181a/b-1(-/-) T cells, respectively. BMT-recipient mice receiving donor T cells with enhanced miR-181a expression showed no signs of aGvHD and survived for the time of follow-up, whereas T cells lacking miR-181a/b-1 accelerated aGvHD. In line with these data, analysis of donor T cells in blood, secondary lymphoid organs, and target organs of aGvHD after BMT showed significantly reduced numbers of miR-181a-transduced T cells, as compared with controls. In addition, expansion of activated T cells with enhanced miR-181a expression was reduced in vitro and in vivo. We further show that anti-apoptotic BCL-2 protein expression is reduced in murine and human T cells upon overexpression of miR-181a, suggesting that regulation of BCL-2-expression by miR-181a may contribute to altered alloreactivity of T cells in aGvHD. These data indicate that proteins regulated by miR-181a may be therapeutic targets for aGvHD prevention.

Low STAT3 expression sensitizes to toxic effects of ß-adrenergic receptor stimulation in peripartum cardiomyopathy.

Aims: The benefit of the ß1-adrenergic receptor (ß1-AR) agonist dobutamine for treatment of acute heart failure in peripartum cardiomyopathy (PPCM) is controversial. Cardiac STAT3 expression is reduced in PPCM patients. Mice carrying a cardiomyocyte-restricted deletion of STAT3 (CKO) develop PPCM. We hypothesized that STAT3-dependent signalling networks may influence the response to ß-AR agonist treatment in PPCM patients and analysed this hypothesis in CKO mice. Methods and Results: Follow-up analyses in 27 patients with severe PPCM (left ventricular ejection fraction ≤25%) revealed that 19 of 20 patients not obtaining dobutamine improved cardiac function. All seven patients obtaining dobutamine received heart transplantation (n = 4) or left ventricular assist devices (n = 3). They displayed diminished myocardial triglyceride, pyruvate, and lactate content compared with non-failing controls. The ß-AR agonist isoproterenol (Iso) induced heart failure with high mortality in postpartum female, in non-pregnant female and in male CKO, but not in wild-type mice. Iso induced heart failure and high mortality in CKO mice by impairing fatty acid and glucose uptake, thereby generating a metabolic deficit. The latter was governed by disturbed STAT3-dependent signalling networks, microRNA-199a-5p, microRNA-7a-5p, insulin/glucose transporter-4, and neuregulin/ErbB signalling. The resulting cardiac energy depletion and oxidative stress promoted dysfunction and cardiomyocyte loss inducing irreversible heart failure, which could be attenuated by the ß1-AR blocker metoprolol or glucose-uptake-promoting drugs perhexiline and etomoxir. Conclusions: Iso impairs glucose uptake, induces energy depletion, oxidative stress, dysfunction, and death in STAT3-deficient cardiomyocytes mainly via ß1-AR stimulation. These cellular alterations may underlie the dobutamine-induced irreversible heart failure progression in PPCM patients who frequently display reduced cardiac STAT3 expression.

Cardiomyocytes display low mitochondrial priming and are highly resistant toward cytotoxic T-cell killing.

Following heart transplantation, alloimmune responses can cause graft rejection by damaging donor vascular and parenchymal cells. However, it remains unclear whether cardiomyocytes are also directly killed by immune cells. Here, we used two-photon microscopy to investigate how graft-specific effector CD8(+) T cells interact with cardiomyocytes in a mouse heart transplantation model. Surprisingly, we observed that CD8(+) T cells are completely impaired in killing cardiomyocytes. Even after virus-mediated preactivation, antigen-specific CD8(+) T cells largely fail to lyse these cells although both cell types engage in dynamic interactions. Furthermore, we established a two-photon microscopy-based assay using intact myocardium to determine the susceptibility of cardiomyocytes to undergo apoptosis. This feature, also known as mitochondrial priming reveals an unexpected weak predisposition of cardiomyocytes to undergo apoptosis in situ. These observations together with the early exhaustion phenotype of graft-infiltrating specific T cells provide an explanation why cardiomyocytes are largely protected from direct CD8(+) T-cell-mediated killing.

Leukemogenic potency of the novel FLT3-N676K mutant.

The novel FMS-like tyrosine kinase 3 (FLT3)-N676K point mutation within the FLT3 kinase domain-1 was recently identified in 6 % of de novo acute myeloid leukemia (AML) patients with inv(16). Because FLT3-N676K was encountered almost exclusively in inv(16) AML, we investigated the transforming potential of FLT3-N676K, the cooperation between FLT3-N676K and core binding factor ß-smooth muscle myosin heavy chain (CBFß-SMMHC) (encoded by the inv(16) chimeric gene CBFB-MYH11) in inducing acute leukemia, and tested the sensitivity of FLT3-N676K-positive leukemic cells to FLT3 inhibitors. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n = 11/13, median latency 58 days), with a transforming activity similar to FLT3-internal tandem duplication (ITD) (n = 8/8), FLT3-TKD D835Y (n = 8/9), and FLT3-ITD-N676K (n = 9/9) mutations. Three out of 14 (21.4 %) C57BL/6J mice transplanted with FLT3-N676K-transduced primary hematopoietic progenitor cells developed acute leukemia (latency of 68, 77, and 273 days), while no hematological malignancy was observed in the control groups including FLT3-ITD. Moreover, co-expression of FLT3-N676K/CBFß-SMMHC did not promote acute leukemia in three independent experiments (n = 16). In comparison with FLT3-ITD, FLT3-N676K induced much higher activation of FLT3 and tended to trigger stronger phosphorylation of MAPK and AKT. Importantly, leukemic cells carrying the FLT3-N676K mutant in the absence of an ITD mutation were highly sensitive to FLT3 inhibitors AC220 and crenolanib, and crenolanib even retained activity against the AC220-resistant FLT3-ITD-N676K mutant. Taken together, the FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, further experiments investigating molecular mechanisms for leukemogenesis induced by FLT3-N676K mutation and clinical evaluation of FLT3 inhibitors in FLT3-N676K-positive AML seem warranted.

BCL6--regulated by AhR/ARNT and wild-type MEF2B--drives expression of germinal center markers MYBL1 and LMO2.

Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

Interleukin-1 assembles a proangiogenic signaling module consisting of caveolin-1, tumor necrosis factor receptor-associated factor 6, p38-mitogen-activated protein kinase (MAPK), and MAPK-activated protein kinase 2 in endothelial cells.

OBJECTIVE: Interleukin-1ß (IL-1ß) is a major cytokine linking inflammation and angiogenesis in pathological vascular processes, such as atherosclerosis and tumor neoangiogenesis. However, signaling pathways mediating IL-1ß-induced proangiogenic processes in endothelial cells (ECs) have barely been elucidated yet. Therefore, the present study investigated IL-1ß-induced proangiogenic signaling in ECs. METHODS AND RESULTS: IL-1ß potently induced tube formation and migration of ECs. This was associated with and dependent on activation of p38-mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) as determined by pharmacological inhibition and gene silencing. Furthermore, silencing of the adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) (lentiviral short hairpin RNA) inhibited these IL-1ß-induced processes. Moreover, IL-1ß promoted translocation of TRAF6 to insoluble cellular fractions (containing membrane rafts/caveolae) and interaction of TRAF6 with caveolin-1. Accordingly, cellular cholesterol depletion (cyclodextrin) and silencing of caveolin-1 (small interfering RNA) inhibited IL-1ß-induced activation of p38-MAPK and MK2, as well as IL-1ß-induced tube formation and migration. Finally, silencing of TRAF6 and MK2 deficiency inhibited IL-1ß-induced microvessel outgrowth in murine aortic rings ex vivo, and deficiency of MK2 or caveolin-1 significantly reduced IL-1ß-induced angiogenesis in mice in vivo (Matrigel plug assay). CONCLUSIONS: IL-1ß assembles a proangiogenic signaling module consisting of caveolin-1, TRAF6, p38-MAPK, and MK2 in ECs, representing a potential target to intervene into angiogenesis-dependent processes and diseases.

LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia.

We demonstrate here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells. We initially documented the importance of this transcription factor in the bone marrow of individuals with severe congenital neutropenia (CN) with a 'differentiation block' at the promyelocytic stage of myelopoiesis. LEF-1 expression was greatly reduced or even absent in CN arrested promyelocytes, resulting in defective expression of the LEF-1 target genes CCND1, MYC and BIRC5, encoding cyclin D1 (ref. 2), c-Myc and survivin, respectively. In contrast, healthy individuals showed highest LEF-1 expression in promyelocytes. Reconstitution of LEF-1 in early hematopoietic progenitors of two individuals with CN corrected the defective myelopoiesis and resulted in the differentiation of these progenitors into mature granulocytes. Repression of endogenous LEF-1 by specific short hairpin RNA inhibited proliferation and induced apoptosis of CD34(+) progenitors from healthy individuals and of cells from two myeloid lines (HL-60 and K562). C/EBPalpha, a key transcription factor in granulopoiesis, was directly regulated by LEF-1. These observations indicate that LEF-1 is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN.

Cabergoline treatment promotes myocardial recovery in peripartum cardiomyopathy.

AIMS: Peripartum cardiomyopathy (PPCM) is a rare heart disease, occurring in previously heart-healthy women during the last month of pregnancy or the first months after delivery due to left ventricular (LV) systolic dysfunction. A common pathomechanistic pathway of PPCM includes increased oxidative stress and the subsequent generation of a cleaved prolactin fragment (16 kDa PRL), which promotes the onset of heart failure (HF) in a microRNA (miR)-146a-dependent manner. Inhibition of prolactin secretion with the dopamine D2 receptor (D2R) agonist bromocriptine combined with standard HF therapy supports cardiac recovery. This study examined whether treatment with the more selective D2R agonist cabergoline prevents HF development in an experimental PPCM mouse model and might be used as an alternative treatment regime for PPCM. METHODS AND RESULTS: Postpartum (PP) female PPCM-prone mice with a cardiomyocyte restricted STAT3-deficiency (αMHC-Cretg/+ ; Stat3fl/fl ; CKO) were treated over two consecutive nursing periods with cabergoline (CKO Cab, 0.5 mg/kg/day) and were compared with bromocriptine treated CKO (CKO Br) and postpartum-matched WT and CKO mice. Cabergoline treatment in CKO PP mice preserved cardiac function [fractional shortening (FS): CKO Cab: 34.5 ± 9.4% vs. CKO: 22.1 ± 9%, P < 0.05] and prevented the development of cardiac hypertrophy, fibrosis, and inflammation as effective as bromocriptine therapy (FS: CKO Br: 33.4 ± 5.6%). The myocardial up-regulation of the PPCM biomarkers plasminogen inhibitor activator 1 (PAI-1) and miR-146a were prevented by both cabergoline and bromocriptine therapy. A small cohort of three PPCM patients from the German PPCM Registry was treated with cabergoline (1 mg per week for 2 weeks, followed by 0.5 mg per week for another 6 weeks) due to a temporary unavailability of bromocriptine. All PPCM patients initially presented with a severely reduced LV ejection fraction (LVEF: 26 ± 2%). However, at 6 months of follow-up, LV function (LVEF: 56 ± 2%) fully recovered in all three PPCM patients, and no adverse events were detected. CONCLUSIONS: In the experimental PPCM mouse model, the selective D2R agonist cabergoline prevents the onset of postpartum HF similar to bromocriptine. In PPCM patients, cabergoline treatment was safe and effective as all patients fully recovered. Cabergoline might serve as a promising alternative to bromocriptine. However, these findings are based on experimental data and a small case series and thus have to be interpreted with caution and should be validated in a larger clinical trial.

Signal transducer and activator of transcription 3-mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for ubiquitin-conjugating enzymes.

AIMS: Mice with a cardiomyocyte (CM)-restricted knockout of signal transducer and activator of transcription 3 (STAT3-KO) develop spontaneous heart failure. We investigated the impact of STAT3-mediated regulation of microRNAs for pathophysiological alterations in the heart. METHODS AND RESULTS: MicroRNAchip and qRT-PCR analysis revealed elevated cardiac expression of miR-199a in STAT3-KO mice. Lentiviral shRNA-mediated STAT3-knock-down in neonatal rat CMs markedly increased miR-199a promoter activity and miR-199a levels indicative of a suppressive effect of STAT3 on miR-199a transcription. Up-regulated miR-199a in CM by pre-miR-199a transfection (pre-miR-199a-CM) reduced expression of components of the ubiquitin-proteasome system (UPS), i.e. the ubiquitin-conjugating enzymes Ube2g1 (mRNA and protein) and Ube2i (protein). Pre-miR-199a-CM or CM with siRNA-mediated down-regulation of Ube2i and Ube2g1 (siRNA-Ube2i/2g1-CM) displayed massive down-regulation of α- and ß-myosin heavy chain expression associated with disrupted sarcomere structures. In addition, protein arginine methyltransferase I (PRMT-I) expression and asymmetric dimethylarginine (ADMA) synthesis were increased in pre-miR-199a-CM or in siRNA-Ube2i/2g1-CM. Increased ADMA in cell culture supernatant (SN) from pre-miR-199a-CM or siRNA-Ube2i/2g1-CM lowered nitric oxide (NO) bioavailability of rat cardiac endothelial cells while lowering ADMA concentration in CM SNs by the PRMT inhibitor arginine methyltransferase inhibitor 1 (AMI-1) (100 µM) improved NO bioavailability. In STAT3-KO hearts Ube2i and Ube2g1 expression were markedly reduced. Human terminal failing hearts harbouring low STAT3 protein levels displayed increased miR-199a levels and decreased Ube2g1 expression. CONCLUSION: This study identifies a novel pathophysiological circuit in the heart between reduced STAT3 protein levels, increased miR-199a expression, and subsequent impairment of the UPS that disrupts CM sarcomere structure and impairs via the release of ADMA endothelial cell function.

Chemotherapy-Free Targeted Anti-BCR-ABL+ Acute Lymphoblastic Leukemia Therapy May Benefit the Heart.

Targeted therapies are currently considered the best cost-benefit anti-cancer treatment. In hematological malignancies, however, relapse rates and non-hematopoietic side effects including cardiotoxicity remain high. Here, we describe significant heart damage due to advanced acute lymphoblastic leukemia (ALL) with t(9;22) encoding the bcr-abl oncogene (BCR-ABL+ ALL) in murine xenotransplantation models. Echocardiography reveals severe cardiac dysfunction with impaired left ventricular function and reduced heart and cardiomyocyte dimensions associated with increased apoptosis. This cardiac damage is fully reversible, but cardiac recovery depends on the therapy used to induce ALL remission. Chemotherapy-free combination therapy with dasatinib (DAS), venetoclax (VEN) (targeting the BCR-ABL oncoprotein and mitochondrial B-cell CLL/Lymphoma 2 (BCL2), respectively), and dexamethasone (DEX) can fully revert cardiac defects, whereas the depletion of otherwise identical ALL in a genetic model using herpes simplex virus type 1 thymidine kinase (HSV-TK) cannot. Mechanistically, dexamethasone induces a pro-apoptotic BCL2-interacting mediator of cell death (BIM) expression and apoptosis in ALL cells but enhances pro-survival B-cell lymphoma extra-large (BCLXL) expression in cardiomyocytes and clinical recovery with the reversion of cardiac atrophy. These data demonstrate that therapies designed to optimize apoptosis induction in ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery. In the future, combining the careful clinical monitoring of cardiotoxicity in leukemic patients with the further characterization of organ-specific side effects and signaling pathways activated by malignancy and/or anti-tumor therapies seems reasonable.

A unique role of p53 haploinsufficiency or loss in the development of acute myeloid leukemia with FLT3-ITD mutation.

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.

Continuous glycoprotein-130-mediated signal transducer and activator of transcription-3 activation promotes inflammation, left ventricular rupture, and adverse outcome in subacute myocardial infarction.

BACKGROUND: In patients with myocardial infarction, high serum levels of interleukin-6 cytokines predict a poor outcome. The common receptor of interleukin-6 cytokines, glycoprotein-130 (gp130), signals via janus kinase/signal transducer and activator of transcription (STAT), cytoplasmic protein tyrosine phosphatase/extracellular signal-regulated kinase, and phosphoinositide-3-kinase/Akt pathways, and the regulation of these pathways depends at least in part on the gp130 tyrosine-757 residue. By analyzing cardiomyocyte-specific gp130(Y757F) mutant mice, we investigated the effect of disturbed gp130 signaling after myocardial infarction. METHODS AND RESULTS: The cardiomyocyte-restricted alpha-myosin heavy chain-Cre-recombinase-loxP system was used to generate mice with gp130(Y757F) mutant cardiomyocytes (alphaMHC-Cre(tg/-);gp130(fl/Y757F) [Y(757)F]); all other cells carried at least 1 functional gp130 gene, ensuring normal gp130 signaling. Y(757)F mice displayed normal cardiac function and morphology at 3 months of age comparable to their nonmutant littermates. In response to myocardial infarction, Y(757)F mice displayed higher mortality associated with increased left ventricular rupture rate, sustained cardiac inflammation, and heart failure. These adverse effects were associated with prolonged and enhanced STAT3 activation and increased expression of interleukin-6 and of the complement-activating mannose-binding lectin C. Pharmacological inhibition of the complement system by cobra venom factor attenuated inflammation, prevented left ventricular rupture, and improved cardiac function in Y(757)F mice. Stronger effects were observed with a genetic reduction of STAT3 (STAT3(flox/+)) restricted to cardiomyocytes in Y(757)F mice, which prevented extensive upregulation of interleukin-6, complement activation, and sustained inflammation and lowered left ventricular rupture rate, heart failure, and mortality in subacute myocardial infarction. CONCLUSIONS: Impaired downregulation of gp130-mediated STAT3 activation in subacute infarction promotes cardiac inflammation, adverse remodeling, and heart failure, suggesting a potential causative role of high interleukin-6 serum levels after myocardial infarction.