Inflammatory signals from fatty bone marrow support DNMT3A driven clonal hematopoiesis.

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3AMut-HSCs when exposed to FBM. DNMT3AMut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3AMut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3AMut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3AMut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Comprehensive cancer predisposition testing within the prospective MASTER trial identifies hereditary cancer patients and supports treatment decisions for rare cancers.

BACKGROUND: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers. PATIENTS AND METHODS: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies. RESULTS: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation. CONCLUSIONS: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research.

Epo-induced erythroid maturation is dependent on Plcγ1 signaling.

Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Binding of erythropoietin (Epo) to its receptor (EpoR) is required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Janus kinase 2 (Jak2) tyrosine kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR-Jak2 independently of Stat5. Plcγ1-deficient pro-erythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined HSCs (Lin(-)Sca1(+)KIT(+)CD48(-)CD150(+)) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation, we assessed changes occurring at the global transcriptional and DNA methylation level after inactivation of Plcγ1. The top common downstream effector was H2afy2, which encodes for the histone variant macroH2A2 (mH2A2). Inactivation of mH2A2 expression recapitulated the effects of Plcγ1 depletion on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target mH2A2, as a 'non-canonical' Epo signaling pathway essential for erythroid differentiation.

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

Autophosphorylation-independent activation of Acanthamoeba myosin I heavy chain kinase by plasma membranes.

The three isoforms of Acanthamoeba myosin I (non-filamentous myosin with only a single heavy chain) express actin-activated Mg(2+)-ATPase activity only when phosphorylated at a single site by myosin I heavy chain kinase. The kinase is activated by autophosphorylation that is greatly stimulated by acidic phospholipids. Substantial fractions of the three myosins I and the kinase are associated in situ with membranes, and all four enzymes bind to purified membranes in vitro. We now report that when kinase and myosin I are incubated together with phosphatidylserine vesicles not only does the kinase autophosphorylate more rapidly than soluble kinase in the absence of phosphatidylserine but that, probably as a result, the kinase phosphorylates myosin I more rapidly than soluble kinase phosphorylates soluble myosin I. Similarly, plasma membrane-bound kinase phosphorylates membrane-bound myosin I and activates its actin-activated Mg(2+)-ATPase activity more rapidly than soluble kinase phosphorylates and activates soluble myosin I in the absence of membranes. However, the enhanced activity of membrane-bound kinase (which is comparable to the activity of kinase in the presence of phosphatidylserine) is not due to autophosphorylation of the membrane-bound kinase, which is very much slower than for kinase activated by phosphatidylserine vesicles.

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.

Preparation of a phospholipid-insensitive, autophosphorylation-activated catalytic fragment of Acanthamoeba myosin I heavy chain kinase.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of its single heavy chain. The activity of the myosin I heavy chain kinase is increased about 50-fold by autophosphorylation, and the rate of kinase autophosphorylation is enhanced about 20-fold by acidic phospholipids independent of the presence of Ca2+ (Brzeska, H., Lynch, T. J., and Korn, E. D. (1990) J. Biol. Chem. 265, 3591-3594). In this paper, we show that chymotryptic digestion of the kinase produces a 54-kDa fragment which contains three to four of the approximately 11 original phosphorylation sites and whose activity is greatly stimulated by autophosphorylation. However, both the rate of autophosphorylation and the kinase activity of the 54-kDa fragment are independent of phospholipid and comparable to those of intact kinase in the presence of phospholipid. These data imply that the (probably NH2-terminal) region(s) removed by proteolysis is necessary for phospholipid-sensitive inhibition of autophosphorylation of sites residing within the (probably COOH-terminal) 54-kDa fragment. The 54-kDa fragment contains the catalytic site of the kinase as well as three to four sites whose phosphorylation is necessary for full expression of kinase activity. The middle region of the kinase molecule contains proline-rich regions that are similar to the COOH-terminal tail of the kinase substrate, Acanthamoeba myosin I.

Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes.

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.

Degradation of alpha-actinin during Ca2+-sensitive proteolysis of myofibrils.

The noted loss of alpha-actinin from the Z-line of myofibrils during post-mortem autolysis, probably following the action of calcium-activated protease, has previously been attributed to its release without degradation. This report shows that in isolated myofibrils alpha-actinin is proteolysed in a Ca2+-sensitive manner presumably via the action of calcium-activated protease.

The contractile apparatus of striated muscle in the course of atrophy and regeneration. II. Myosin and actin filaments in mature rat soleus muscle regenerating after reinnervation.

Changes in the contractile apparatus of denervated rat soleus muscles were investigated during the course of reinnervation. As observed earlier, in the course of denervation atrophy the ratio of myosin to actin filaments decreases because myosin filaments disappear faster than actin filaments (Jakubiec-Puka et al. 1981 a). After reinnervation the amount of myosin filaments and myosin heavy chains (myosin HC) in the muscle increased during the first few days; the increment of actin content was negligible. The proportion of myosin HC to actin remained lower than normal for about 30 days. The excess of actin filaments frequently observed in the newly-formed myofibrils reflects this disproportion. The results show a lability of myosin and suggest some cytoskeletal role for actin filaments.

The contractile apparatus of striated muscle in the course of atrophy and regeneration. I. Myosin and actin filaments in the denervated rat soleus.

The ultrastructure of the contractile apparatus of the rat soleus muscle during the course of denervation atrophy was investigated. It was found that the ratio of thin the thick filaments increased in myofibrils of atrophying muscle fibers. Elevation of the ratio was observed as early as the second day after denervation, and became more pronounced with the progress of atrophy. Parallel measurements of the amounts of actin and myosin in the myofibrils and in the muscle protein extracts revealed a lower proportion of myosin heavy chains to actin in the fractions from denervated muscles, compared with the control values. Both the electron-microscopic observations and the biochemical evaluation of the actin content of the muscle, suggests that the elevated ratio of thin of thick filaments seen in the course of the muscle atrophy appears as the results of an earlier and more intensive disappearance of thick filaments. Thin filaments disappeared more slowly, in parallel to the decrease in muscle weight. On the basis of the results presented a mechanism of progress of "simple atrophy" of muscle in suggested.