Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing.

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

PDP1 is a key metabolic gatekeeper and modulator of drug resistance in FLT3-ITD-positive acute myeloid leukemia.

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20-30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML.

Dataset of single nucleotide polymorphisms of immune-associated genes in patients with SARS-CoV-2 infection.

The SARS-CoV-2 pandemic has affected nations globally leading to illness, death, and economic downturn. Why disease severity, ranging from no symptoms to the requirement for extracorporeal membrane oxygenation, varies between patients is still incompletely understood. Consequently, we aimed at understanding the impact of genetic factors on disease severity in infection with SARS-CoV-2. Here, we provide data on demographics, ABO blood group, human leukocyte antigen (HLA) type, as well as next-generation sequencing data of genes in the natural killer cell receptor family, the renin-angiotensin-aldosterone and kallikrein-kinin systems and others in 159 patients with SARS-CoV-2 infection, stratified into seven categories of disease severity. We provide single-nucleotide polymorphism (SNP) data on the patients and a protein structural analysis as a case study on a SNP in the SIGLEC7 gene, which was significantly associated with the clinical score. Our data represent a resource for correlation analyses involving genetic factors and disease severity and may help predict outcomes in infections with future SARS-CoV-2 variants and aid vaccine adaptation.

Distinct and targetable role of calcium-sensing receptor in leukaemia.

Haematopoietic stem cells (HSC) reside in the bone marrow microenvironment (BMM), where they respond to extracellular calcium [eCa2+] via the G-protein coupled calcium-sensing receptor (CaSR). Here we show that a calcium gradient exists in this BMM, and that [eCa2+] and response to [eCa2+] differ between leukaemias. CaSR influences the location of MLL-AF9+ acute myeloid leukaemia (AML) cells within this niche and differentially impacts MLL-AF9+ AML versus BCR-ABL1+ leukaemias. Deficiency of CaSR reduces AML leukaemic stem cells (LSC) 6.5-fold. CaSR interacts with filamin A, a crosslinker of actin filaments, affects stemness-associated factors and modulates pERK, ß-catenin and c-MYC signaling and intracellular levels of [Ca2+] in MLL-AF9+ AML cells. Combination treatment of cytarabine plus CaSR-inhibition in various models may be superior to cytarabine alone. Our studies suggest CaSR to be a differential and targetable factor in leukaemia progression influencing self-renewal of AML LSC via [eCa2+] cues from the BMM.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1ß condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

Mycobacterium tuberculosis hijacks ubiquitin to inhibit pyroptosis.

Chai et al.1 reveal that the eukaryotic-like effector protein PtpB from Mycobacterium tuberculosis (MTB) dephosphorylates phospholipid membrane proteins, which prevents membrane localization of cleaved gasdermin D, inhibiting pyroptosis and cytokine release by infected macrophages to enable MTB immune evasion.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia.

Lipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL-AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.

The age of the bone marrow microenvironment influences B-cell acute lymphoblastic leukemia progression via CXCR5-CXCL13.

B-cell acute lymphoblastic leukemia (B-ALL) occurs most commonly in children, whereas chronic myeloid leukemia is more frequent in adults. The myeloid bias of hematopoiesis in elderly individuals has been considered causative, but the age of the bone marrow microenvironment (BMM) may be contributory. Using various murine models of B-ALL in young vs old mice, we recapitulated B-ALL preponderance in children vs adults. We showed differential effects of young vs old BM macrophages on B-ALL cell function. Molecular profiling using RNA- and ATAC-sequencing revealed pronounced differences in young vs old BMM-derived macrophages and enrichment for gene sets associated with inflammation. In concordance with the role of C-X-C motif chemokine (CXCL) 13 for disease-associated B-cell chemoattraction, we found CXCL13 to be highly expressed in young macrophages on a translational compared with a transcriptional level. Inhibition of CXCL13 in BM macrophages impaired leukemia cell migration and decreased the proliferation of cocultured B-ALL cells, whereas recombinant CXCL13 increased pAKT and B-ALL cell expansion. Pretreatment of B-ALL-initiating cells with CXCL13 accelerated B-ALL progression. Deficiency of Cxcr5, the receptor for CXCL13, on B-ALL-initiating cells prolonged murine survival, whereas high expression of CXCR5 in pediatric B-ALL may predict central nervous system relapse. CXCL13 staining was increased in bone sections from pediatric compared with adult patients with B-ALL. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCR5-CXCL13 axis may act as prognostic marker and an attractive novel target for the treatment of B-ALL.

Recent advances in understanding chronic myeloid leukemia: where do we stand?

While the need for complete eradication of leukemic stem cells (LSCs) in chronic myeloid leukemia may be controversial, it is agreed that remaining LSCs are the cause of relapse and disease progression. Current efforts are focused on the understanding of the persistence of immunophenotypically defined LSCs, which feature abnormalities in signaling pathways relating to autophagy, metabolism, epigenetics, and others and are influenced by leukemia cell-extrinsic factors such as the immune and bone marrow microenvironments. In sum, these elements modulate response and resistance to therapies and the clinical condition of treatment-free remission (TFR), the newly established goal in CML treatment, once the patient has achieved a durable molecular remission after treatment with tyrosine kinase inhibitors. Novel combination therapies based on these identified vulnerabilities of LSCs, aimed at the induction or maintenance of TFR, are being developed, while other research is directed at the elucidation of factors mediating progression to blast crisis.

Chronic Myeloid Leukemia: A Model Disease of the Past, Present and Future.

Chronic myeloid leukemia (CML) has been a "model disease" with a long history. Beginning with the first discovery of leukemia and the description of the Philadelphia Chromosome and ending with the current goal of achieving treatment-free remission after targeted therapies, we describe here the journey of CML, focusing on molecular pathways relating to signaling, metabolism and the bone marrow microenvironment. We highlight current strategies for combination therapies aimed at eradicating the CML stem cell; hopefully the final destination of this long voyage.

The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.

The mRNA m6A reader YTHDF2 suppresses proinflammatory pathways and sustains hematopoietic stem cell function.

The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.

"Caught in the net": the extracellular matrix of the bone marrow in normal hematopoiesis and leukemia.

The influence of the bone marrow microenvironment on normal hematopoiesis, but also leukemia, has largely been accepted. However, the focus has been predominantly on the role of various cell types or cytokines maintaining hematopoietic stem cells or protecting leukemia stem cells from different therapies. A frequently overlooked component of the bone marrow microenvironment is the extracellular matrix, which not only provides a mechanical scaffold, but also serves as a source of growth factors. We discuss here how extracellular matrix proteins directly or indirectly modulate hematopoietic stem cell physiology and influence leukemia progression. It is hoped that existing and future studies on this topic may propel forward the possibility of augmenting normal hematopoiesis and improving therapies for leukemia, for instance, by targeting of the extracellular matrix in the bone marrow.

Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia.

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin ß3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin ß3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

Bone marrow niches in haematological malignancies.

Haematological malignancies were previously thought to be driven solely by genetic or epigenetic lesions within haematopoietic cells. However, the niches that maintain and regulate daily production of blood and immune cells are now increasingly being recognized as having an important role in the pathogenesis and chemoresistance of haematological malignancies. Within haematopoietic cells, the accumulation of a small number of recurrent mutations initiates malignancy. Concomitantly, specific alterations of the niches, which support haematopoietic stem cells and their progeny, can act as predisposition events, facilitating mutant haematopoietic cell survival and expansion as well as contributing to malignancy progression and providing protection of malignant cells from chemotherapy, ultimately leading to relapse. In this Perspective, we summarize our current understanding of the composition and function of the specialized haematopoietic niches of the bone marrow during health and disease. We discuss disease mechanisms (rather than malignancy subtypes) to provide a comprehensive description of key niche-associated pathways that are shared across multiple haematological malignancies. These mechanisms include primary driver mutations in bone marrow niche cells, changes associated with increased hypoxia, angiogenesis and inflammation as well as metabolic reprogramming by stromal niche cells. Consequently, remodelling of bone marrow niches can facilitate immune evasion and activation of survival pathways favouring malignant haematopoietic cell maintenance, defence against excessive reactive oxygen species and protection from chemotherapy. Lastly, we suggest guidelines for the handling and biobanking of patient samples and analysis of the niche to ensure that basic research identifying therapeutic targets can be more efficiently translated to the clinic. The hope is that integrating knowledge of how bone marrow niches contribute to haematological disease predisposition, initiation, progression and response to therapy into future clinical practice will likely improve the treatment of these disorders.

Bone marrow niche-derived extracellular matrix-degrading enzymes influence the progression of B-cell acute lymphoblastic leukemia.

Specific and reciprocal interactions with the bone marrow microenvironment (BMM) govern the course of hematological malignancies. Matrix metalloproteinase-9 (MMP-9), secreted by leukemia cells, facilitates tumor progression via remodeling of the extracellular matrix (ECM) of the BMM. Hypothesizing that leukemias may instruct the BMM to degrade the ECM, we show, that MMP-9-deficiency in the BMM prolongs survival of mice with BCR-ABL1-induced B-cell acute lymphoblastic leukemia (B-ALL) compared with controls and reduces leukemia-initiating cells. MMP-9-deficiency in the BMM leads to reduced degradation of proteins of the ECM and reduced invasion of B-ALL. Using various in vivo and in vitro assays, as well as recipient mice deficient for the receptor for tumor necrosis factor (TNF) α (TNFR1) we demonstrate that B-ALL cells induce MMP-9-expression in mesenchymal stem cells (MSC) and possibly other cells of the BMM via a release of TNFα. MMP-9-expression in MSC is mediated by activation of nuclear factor kappa B (NF-κB) downstream of TNFR1. Consistently, knockdown of TNF-α in B-ALL-initiating cells or pharmacological inhibition of MMP-9 led to significant prolongation of survival in mice with B-ALL. In summary, leukemia cell-derived Tnfα induced MMP-9-expression by the BMM promoting B-ALL progression. Inhibition of MMP-9 may act as an adjunct to existing therapies.

The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin - SCL/TAL1 - CD44 axis.

The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries for leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells. We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of leukemic stem cells in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1+ cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and proto-oncogene Scl/Tal1 in leukemia-initiating cells. We implicate SCL/TAL1 as an indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - which could be exploited therapeutically in the future.