Identification of a Musashi2 translocation as a novel oncogene in myeloid leukemia.

Myeloid leukemias, diseases marked by aggressiveness and poor outcomes, are frequently triggered by oncogenic translocations. In the case of chronic myelogenous leukemia (CML) the BCR-ABL fusion initiates chronic phase disease with second hits allowing progression to blast crisis. Although Gleevec has been transformative for CML, blast crisis CML remains relatively drug resistant. Here we show that MSI2-HOXA9, a translocation with an unknown role in cancer, can serve as a second hit in driving bcCML. Compared to BCR-ABL, BCR-ABL/MSI2-HOXA9 led to a more aggressive disease in vivo with decreased latency, increased lethality and a differentiation blockade that is a hallmark of blast crisis. Domain mapping revealed that the MSI2 RNA binding domain RRM1 had a preferential impact on growth and lethality of bcCML relative to RRM2 or the HOXA9 domain. Mechanistically, MSI2-HOXA9 triggered global downstream changes with a preferential upregulation of mitochondrial components. Consistent with this, BCR-ABL/MSI2-HOXA9 cells exhibited a significant increase in mitochondrial respiration. These data suggest that MSI2-HOXA9 acts, at least in part, by increasing expression of the mitochondrial polymerase Polrmt and augmenting mitochondrial function and basal respiration in blast crisis. Collectively, our findings demonstrate for the first time that translocations involving the stem and developmental signal MSI2 can be oncogenic, and suggest that MSI, which we found to be a frequent partner for an array of translocations, could also be a driver mutation across solid cancers.

A stem cell reporter based platform to identify and target drug resistant stem cells in myeloid leukemia.

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus, identifying the cells responsible for residual disease and leukemia re-growth is critical to better understanding how they are regulated. Here, we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of leukemia stem cells in aggressive myeloid malignancies, and provides a strategy for defining their core dependencies. Specifically, we carry out a high throughput screen using Msi2-reporter blast crisis chronic myeloid leukemia (bcCML) and identify several adhesion molecules that are preferentially expressed in therapy resistant bcCML cells and play a key role in bcCML. In particular, we focus on syndecan-1, whose deletion triggers defects in bcCML growth and propagation and markedly improves survival of transplanted mice. Further, live imaging reveals that the spatiotemporal dynamics of leukemia cells are critically dependent on syndecan signaling, as loss of this signal impairs their localization, migration and dissemination to distant sites. Finally, at a molecular level, syndecan loss directly impairs integrin ß7 function, suggesting that syndecan exerts its influence, at least in part, by coordinating integrin activity in bcCML. These data present a platform for delineating the biological underpinnings of leukemia stem cell function, and highlight the Sdc1-Itgß7 signaling axis as a key regulatory control point for bcCML growth and dissemination.

An in vivo genome-wide CRISPR screen identifies the RNA-binding protein Staufen2 as a key regulator of myeloid leukemia.

Aggressive myeloid leukemias such as blast crisis chronic myeloid leukemia and acute myeloid leukemia remain highly lethal. Here we report a genome-wide in vivo CRISPR screen to identify new dependencies in this disease. Among these, RNA-binding proteins (RBPs) in general, and the double-stranded RBP Staufen2 (Stau2) in particular, emerged as critical regulators of myeloid leukemia. In a newly developed knockout mouse, loss of Stau2 led to a profound decrease in leukemia growth and improved survival in mouse models of the disease. Further, Stau2 was required for growth of primary human blast crisis chronic myeloid leukemia and acute myeloid leukemia. Finally, integrated analysis of CRISPR, eCLIP and RNA-sequencing identified Stau2 as a regulator of chromatin-binding factors, driving global alterations in histone methylation. Collectively, these data show that in vivo CRISPR screening is an effective tool for defining new regulators of myeloid leukemia progression and identify the double-stranded RBP Stau2 as a critical dependency of myeloid malignancies.

SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors.

Marrow-derived macrophages are highly phagocytic, but whether they can also traffic into solid tumors and engulf cancer cells is questionable, given the well-known limitations of tumor-associated macrophages (TAMs). Here, SIRPα on macrophages from mouse and human marrow was inhibited to block recognition of its ligand, the "marker of self" CD47 on all other cells. These macrophages were then systemically injected into mice with fluorescent human tumors that had been antibody targeted. Within days, the tumors regressed, and single-cell fluorescence analyses showed that the more the macrophages engulfed, the more they accumulated within regressing tumors. Human-marrow-derived macrophages engorged on the human tumors, while TAMs were minimally phagocytic, even toward CD47-knockdown tumors. Past studies had opsonized tumors in situ with antibody and/or relied on mouse TAMs but had not injected SIRPα-inhibited cells; also, unlike past injections of anti-CD47, blood parameters remained normal and safe. Consistent with tumor-selective engorge-and-accumulate processes in vivo, phagocytosis in vitro inhibited macrophage migration through micropores that mimic features of dense 3D tissue. Accumulation of SIRPα-inhibited macrophages in tumors favored tumor regression for 1-2 weeks, but donor macrophages quickly differentiated toward non-phagocytic, high-SIRPα TAMs. Analyses of macrophages on soft (like marrow) or stiff (like solid tumors) collagenous gels demonstrated a stiffness-driven, retinoic-acid-modulated upregulation of SIRPα and the mechanosensitive nuclear marker lamin-A. Mechanosensitive differentiation was similarly evident in vivo and likely limited the anti-tumor effects, as confirmed by re-initiation of tumor regression by fresh injections of SIRPα-inhibited macrophages. Macrophage motility, phagocytosis, and differentiation in vivo are thus coupled.

Cross-linked matrix rigidity and soluble retinoids synergize in nuclear lamina regulation of stem cell differentiation.

Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here the rigidity of cross-linked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nanofilms serve as a model matrix that MSCs can easily deform unless the film is enzymatically cross-linked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by retinoic acid receptor (RAR) transcription factors, but soft matrix prevents any response to any retinoids. Rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and for RARG-specific antagonist to increase or maintain expression of lamin-A as well as for RARG-agonist to repress expression. A progerin allele of lamin-A is regulated in the same manner in iPSC-derived MSCs. Rigid matrices are further required for eventual expression of osteogenic markers, and RARG-antagonist strongly drives lamin-A-dependent osteogenesis on rigid substrates, with pretreated xenografts calcifying in vivo to a similar extent as native bone. Proteomics-detected targets of mechanosensitive lamin-A and retinoids underscore the convergent synergy of insoluble and soluble cues in differentiation.

Filomicelles from aromatic diblock copolymers increase paclitaxel-induced tumor cell death and aneuploidy compared with aliphatic copolymers.

AIM: In order to improve the delivery of aromatic drugs by micellar assemblies, and particularly by long and flexible filomicelles, aromatic groups were integrated into the hydrophobic block of a degradable diblock copolymer. MATERIALS & METHODS: Aromatic filomicelles were formed by self-directed assembly of amphiphilic diblock copolymer PEG-PBCL with suitable block ratios. Worm-like filomicelles with an aromatic core were loaded with a common chemotherapeutic, Paclitaxel, for tests of release as well as effects on cancer cell lines in vitro and in vivo. RESULTS: Aromatic filomicelles loaded more Paclitaxel than analogous aliphatic systems. Cell death and aneuploidy of surviving cells (which indicates toxicity) were highest for carcinoma lines treated in vitro with the new filomicelles. Initial tests in vivo also suggest more potent tumor shrinkage. CONCLUSION: Flexible filomicelles with an aromatic core form an efficient drug delivery system that leads to higher cell death than previously reported systems, while inducing aneuploidy in surviving cells.

Macrophage engulfment of a cell or nanoparticle is regulated by unavoidable opsonization, a species-specific 'Marker of Self' CD47, and target physical properties.

Professional phagocytes of the mononuclear phagocyte system (MPS), especially ubiquitous macrophages, are commonly thought to engulf or not a target based strictly on 'eat me' molecules such as Antibodies. The target might be a viable 'self' cell or a drug-delivering nanoparticle, or it might be a cancer cell or a microbe. 'Marker of Self' CD47 signals into a macrophage to inhibit the acto-myosin cytoskeleton that makes engulfment efficient. In adhesion of any cell, the same machinery is generally activated by rigidity of target surfaces, and recent results confirm phagocytosis is likewise driven by the rigidity typical of microbes and many synthetics. Basic insights are already being applied in order to make macrophages eat cancer or to delay nanoparticle clearance for better drug delivery and imaging.

Myosin-II repression favors pre/proplatelets but shear activation generates platelets and fails in macrothrombocytopenia.

Megakaryocyte ploidy and the generation of pre/proplatelets are both increased in culture by pharmacologic inhibition of myosin-II, but nonmuscle myosin-IIA (MIIA) mutations paradoxically cause MYH9-related diseases (MYH9-RD) that adversely affect platelets. In marrow, megakaryocytes extend projections into the microcirculation, where shear facilitates fragmentation to large pre/proplatelets, suggesting that fluid stresses and myosin-II activity somehow couple in platelet biogenesis. Here, in bulk shear, plateletlike particles generated from megakaryocytes are maximized at a shear stress typical of that in the microcirculation and after treatment with a myosin-II inhibitor. MIIA activity in static cells is naturally repressed through phosphorylation at Serine-1943, but shear decreases phosphorylation, consistent with MIIA activation and localization to platelet cortex. Micropipette aspiration of cells shows myosin-II accumulates at stressed sites, but its inhibition prevents such mechanoactivation and facilitates generation of CD41(+) fragments similar in size to pre/proplatelets. MYH9-RD mutants phenocopy inhibition, revealing a dominant negative effect. MIIA is diffuse in the large platelets of a MYH9-RD patient with macrothrombocytopenia and is also diffuse in normal pre/proplatelets treated with inhibitor that blocks in vitro division to small platelets. The findings explain the large platelets in MYH9-RD and the near-normal thrombocrit of patients. Myosin-II regulation thus controls platelet size and number.

Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin.

Tissue microenvironments are characterized not only in terms of chemical composition but also by collective properties such as stiffness, which influences the contractility of a cell, its adherent morphology, and even differentiation. The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant. Here we show in single-cell analyses that matrix stiffness couples to myosin-II activity to promote lamin-A,C dephosphorylation at Ser22, which regulates turnover, lamina physical properties, and actomyosin expression. Lamin-A,C phosphorylation is low in interphase versus dividing cells, and its levels rise with states of nuclear rounding in which myosin-II generates little to no tension. Phosphorylated lamin-A,C localizes to nucleoplasm, and phosphorylation is enriched on lamin-A,C fragments and is suppressed by a cyclin-dependent kinase (CDK) inhibitor. Lamin-A,C knockdown in primary MSCs suppresses transcripts predominantly among actomyosin genes, especially in the serum response factor (SRF) pathway. Levels of myosin-IIA thus parallel levels of lamin-A,C, with phosphosite mutants revealing a key role for phosphoregulation. In modeling the system as a parsimonious gene circuit, we show that tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.

Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival.

Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A:B producing extruded nuclear shapes after migration. Because the juxtaposed A and B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, subpopulations with different A:B levels sorted in 3D migration. However, net migration was also biphasic in lamin-A, as wild-type lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress resistance. In vivo xenografts proved consistent with A:B-based cell sorting, and intermediate A:B-enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.

Contractile forces sustain and polarize hematopoiesis from stem and progenitor cells.

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Lamins regulate cell trafficking and lineage maturation of adult human hematopoietic cells.

Hematopoietic stem and progenitor cells, as well as nucleated erythroblasts and megakaryocytes, reside preferentially in adult marrow microenvironments whereas other blood cells readily cross the endothelial barrier into the circulation. Because the nucleus is the largest organelle in blood cells, we hypothesized that (i) cell sorting across microporous barriers is regulated by nuclear deformability as controlled by lamin-A and -B, and (ii) lamin levels directly modulate hematopoietic programs. Mass spectrometry-calibrated intracellular flow cytometry indeed reveals a lamin expression map that partitions human blood lineages between marrow and circulating compartments (P = 0.00006). B-type lamins are highly variable and predominate only in CD34(+) cells, but migration through micropores and nuclear flexibility in micropipette aspiration both appear limited by lamin-A:B stoichiometry across hematopoietic lineages. Differentiation is also modulated by overexpression or knockdown of lamins as well as retinoic acid addition, which regulates lamin-A transcription. In particular, erythroid differentiation is promoted by high lamin-A and low lamin-B1 expression whereas megakaryocytes of high ploidy are inhibited by lamin suppression. Lamins thus contribute to both trafficking and differentiation.

Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation.

Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.

Mechanobiology of bone marrow stem cells: from myosin-II forces to compliance of matrix and nucleus in cell forms and fates.

Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly affect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells.

Dynamic domains in polymersomes: mixtures of polyanionic and neutral diblocks respond more rapidly to changes in calcium than to pH.

Chemical triggering of membrane domain dynamics is of broad relevance to cell signaling through lipid bilayers and might also be exploited in application of phase-separated vesicles. Here we describe the morphodynamics and remixing kinetics of spotted polymersomes made with mixtures of polyanionic and neutral amphiphiles plus calcium. Addition of the calcium chelator EDTA to vesicle dispersions produced a decrease in domain size within minutes, whereas increasing the pH with NaOH led to the viscous fingering of domains and decreased domain size over hours. Although the latter suggests that the charge of the polyanion contributes to domain formation, the remixing of more negative chains at high pH is surprising. Domain roughening at high pH is also accelerated by EDTA, which highlights the dominance of cross-bridging. Importantly, even though vesicles were perturbed only externally, the inner and outer leaflets remain coupled throughout, consistent with molecular dynamics simulations and suggestive of an order-disorder transition that underlies the remixing kinetics.

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes.

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)--seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbß3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.