Acute myeloid leukemias with UBTF tandem duplications are sensitive to menin inhibitors.

ABSTRACT: UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

Large-scale integration of artificial atoms in hybrid photonic circuits.

A central challenge in developing quantum computers and long-range quantum networks is the distribution of entanglement across many individually controllable qubits1. Colour centres in diamond have emerged as leading solid-state 'artificial atom' qubits2,3 because they enable on-demand remote entanglement4, coherent control of over ten ancillae qubits with minute-long coherence times5 and memory-enhanced quantum communication6. A critical next step is to integrate large numbers of artificial atoms with photonic architectures to enable large-scale quantum information processing systems. So far, these efforts have been stymied by qubit inhomogeneities, low device yield and complex device requirements. Here we introduce a process for the high-yield heterogeneous integration of 'quantum microchiplets'-diamond waveguide arrays containing highly coherent colour centres-on a photonic integrated circuit (PIC). We use this process to realize a 128-channel, defect-free array of germanium-vacancy and silicon-vacancy colour centres in an aluminium nitride PIC. Photoluminescence spectroscopy reveals long-term, stable and narrow average optical linewidths of 54 megahertz (146 megahertz) for germanium-vacancy (silicon-vacancy) emitters, close to the lifetime-limited linewidth of 32 megahertz (93 megahertz). We show that inhomogeneities of individual colour centre optical transitions can be compensated in situ by integrated tuning over 50 gigahertz without linewidth degradation. The ability to assemble large numbers of nearly indistinguishable and tunable artificial atoms into phase-stable PICs marks a key step towards multiplexed quantum repeaters7,8 and general-purpose quantum processors9-12.

Large-scale optical characterization of solid-state quantum emitters.

Solid-state quantum emitters have emerged as a leading quantum memory for quantum networking applications. However, standard optical characterization techniques are neither efficient nor repeatable at scale. Here we introduce and demonstrate spectroscopic techniques that enable large-scale, automated characterization of colour centres. We first demonstrate the ability to track colour centres by registering them to a fabricated machine-readable global coordinate system, enabling a systematic comparison of the same colour centre sites over many experiments. We then implement resonant photoluminescence excitation in a widefield cryogenic microscope to parallelize resonant spectroscopy, achieving two orders of magnitude speed-up over confocal microscopy. Finally, we demonstrate automated chip-scale characterization of colour centres and devices at room temperature, imaging thousands of microscope fields of view. These tools will enable the accelerated identification of useful quantum emitters at chip scale, enabling advances in scaling up colour centre platforms for quantum information applications, materials science and device design and characterization.

UBTF tandem duplications in pediatric myelodysplastic syndrome and acute myeloid leukemia: implications for clinical screening and diagnosis.

Recent genomic studies in adult and pediatric acute myeloid leukemia (AML) demonstrated recurrent in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF). These alterations, which account for approximately 4.3% of AML in childhood and about 3% in adult AML aged <60 years of age, are subtype-defining and associated with poor outcomes. Here, we provide a comprehensive investigation into the clinicopathological features of UBTF-TD myeloid neoplasms in childhood, including 89 unique pediatric AML and 6 myelodysplastic syndrome (MDS) cases harboring a tandem duplication in exon 13 of UBTF. We demonstrate that UBTF-TD myeloid tumors are associated with dysplastic features, low bone marrow blast infiltration, and low white blood cell count. Furthermore, using bulk and single-cell analyses, we confirm that UBTF-TD is an early and clonal event associated with a distinct transcriptional profile, whereas the acquisition of FLT3 or WT1 mutations is associated with more stem cell-like programs. Lastly, we report rare duplications within exon 9 of UBTF that phenocopy exon 13 duplications, expanding the spectrum of UBTF alterations in pediatric myeloid tumors. Collectively, we comprehensively characterize pediatric AML and MDS with UBTF-TD, and highlight key clinical and pathologic features that distinguish this new entity from other molecular subtypes of AML.

Expression of troponin subunits in the rat renal afferent arteriole.

Vascular smooth muscle cells of the renal afferent arteriole are unusual in that they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure in order to protect the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for, in part, by exclusive expression, at the protein level, of the "fast" (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole which express exclusively the "slow" (A) isoforms (Shiraishi et al. (2003) FASEB. J. 17, 2284-2286). As contraction of the more rapidly contracting striated (skeletal and cardiac) muscles is regulated by the thin filament-associated troponin (Tn) system, we hypothesized that Tn or a Tn-like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+ -binding subunit), TnI (inhibitory subunit), and TnT (tropomyosin-binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast-twitch skeletal muscle and slow-twitch skeletal muscle/cardiac TnC isoforms and slow-twitch skeletal muscle and cardiac TnI isoforms were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by cDNA sequencing. Furthermore, cardiac and slow-twitch skeletal muscle TnI isoforms, but not fast-twitch skeletal muscle TnI, were detected in isolated afferent arterioles at the protein level by proximity ligation assay. Finally, striated muscle myosin II heavy chain expression was identified in isolated rat afferent arterioles by RT-PCR. We conclude that, in addition to Ca2+ -mediated phosphorylation of myosin II regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.

Validation of chemical genetics for the study of zipper-interacting protein kinase signaling.

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr kinase that mediates a variety of cellular functions. Analogue-sensitive kinase technology was applied to the study of ZIPK signaling in coronary artery smooth muscle cells. ZIPK was engineered in the ATP-binding pocket by substitution of a bulky gatekeeper amino acid (Leu93) with glycine. Cell-permeable derivatives of pyrazolo[3,4-d]pyrimidine provided effective inhibition of L93G-ZIPK (1NM-PP1, IC50 , 1.0 µM; 3MB-PP1, IC50 , 2.0 µM; and 1NA-PP1, IC50 , 8.6 µM) but only 3MB-PP1 had inhibitory potential (IC50 > 10 µM) toward wild-type ZIPK. Each of the compounds also attenuated Rho-associated coiled-coil containing protein kinase (ROCK) activity under experimental conditions found to be optimal for inhibition of L93G-ZIPK. In silico molecular simulations showed effective docking of 1NM-PP1 into ZIPK following mutational enlargement of the ATP-binding pocket. Molecular simulation of 1NM-PP1 docking in the ATP-binding pocket of ROCK was also completed. The 1NM-PP1 inhibitor was selected as the optimal compound for selective chemical genetics in smooth muscle cells since it displayed the highest potency for L93G-ZIPK relative to WT-ZIPK and the weakest off-target effects against other relevant kinases. Finally, the 1NM-PP1 and L93G-ZIPK pairing was effectively applied in vascular smooth muscle cells to manipulate the phosphorylation level of LC20, a previously defined target of ZIPK.

Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

Phosphorylation of the myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.

A role for the tyrosine kinase Pyk2 in depolarization-induced contraction of vascular smooth muscle.

Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca(2+)-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K(+)-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K(+), Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca(2+), elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca(2+) channel blocker nifedipine inhibited peak and sustained K(+)-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K(+)-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca(2+) activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.

Potential Role of Glycogen Synthase Kinase-3ß in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3ß, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3ß results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3ß regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3ß inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3ß inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3ß inhibitors were mimicked by transfection with GSK-3ß siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3ß mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3ß in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3ß reduces myocardin transcriptional activity, suggesting a role for GSK-3ß in myocardin transcriptional activity and smooth muscle differentiation.

Cytoskeletal reorganization evoked by Rho-associated kinase- and protein kinase C-catalyzed phosphorylation of cofilin and heat shock protein 27, respectively, contributes to myogenic constriction of rat cerebral arteries.

Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC20) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC(20), calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.

The GPER agonist G-1 induces mitotic arrest and apoptosis in human vascular smooth muscle cells independent of GPER.

The G protein-coupled estrogen receptor (GPER) has been implicated in the regulation of smooth muscle cell (SMC) proliferation. The GPER selective agonist G-1 has been a useful tool for exploring the biological roles of GPER in a variety of experimental settings, including SMC proliferation. The present study, originally designed to investigate cellular and signaling mechanisms underlying the regulatory role of GPER in vascular SMC proliferation using G-1, unexpectedly revealed off-target effects of G-1. G-1(1-10 µM) inhibited bromodeoxyuridine (BrdU) incorporation of human SMCs and caused G2/M cell accumulation. G-1 treatment also increased mitotic index concurrent with a decrease in phosphorylation of Cdk1 (Tyr 15) and an increase in phosphorylation of the mitotic checkpoint protein BuBR1. Furthermore, G-1 caused microtubule disruption, mitotic spindle damage, and tubulin depolymerization. G-1 induced cell apoptosis as indicated by the appearance of TUNEL-positive and annexin V-positive cells with enhanced cleavage of caspases 3 and 9. However, neither the GPER antagonist G-15 nor the MAPK kinase inhibitor PD98059 prevented these G-1 effects. Down-regulation of GPER or p44/42 MAPK with siRNA transfection also did not affect the G-1-induced apoptosis. We conclude that G-1 inhibits proliferation of SMCs through mechanisms involving mitotic arrest and apoptosis, independent of GPER and the MAPK pathway.

Endothelin-1, but not angiotensin II, induces afferent arteriolar myosin diphosphorylation as a potential contributor to prolonged vasoconstriction.

Bolus administration of endothelin-1 elicits long-lasting renal afferent arteriolar vasoconstriction, in contrast to transient constriction induced by angiotensin II. Vasoconstriction is generally evoked by myosin regulatory light chain (LC20) phosphorylation at Ser19 by myosin light chain kinase (MLCK), which is enhanced by Rho-associated kinase (ROCK)-mediated inhibition of myosin light chain phosphatase (MLCP). LC20 can be diphosphorylated at Ser19 and Thr18, resulting in reduced rates of dephosphorylation and relaxation. Here we tested whether LC20 diphosphorylation contributes to sustained endothelin-1 but not transient angiotensin II-induced vasoconstriction. Endothelin-1 treatment of isolated arterioles elicited a concentration- and time-dependent increase in LC20 diphosphorylation at Thr18 and Ser19. Inhibition of MLCK or ROCK reduced endothelin-1-evoked LC20 mono- and diphosphorylation. Pretreatment with an ETB but not an ETA receptor antagonist abolished LC20 diphosphorylation, and an ETB receptor agonist induced LC20 diphosphorylation. In contrast, angiotensin II caused phosphorylation exclusively at Ser19. Thus, endothelin-1 and angiotensin II induce afferent arteriolar constriction via LC20 phosphorylation at Ser19 due to calcium activation of MLCK and ROCK-mediated inhibition of MLCP. Endothelin-1, but not angiotensin II, induces phosphorylation of LC20 at Thr18. This could contribute to the prolonged vasoconstrictor response to endothelin-1.

A role for the Ca(2+)-dependent tyrosine kinase Pyk2 in tonic depolarization-induced vascular smooth muscle contraction.

Depolarization of the plasma membrane is a key mechanism of activation of contraction of vascular smooth muscle. This is commonly achieved in isolated, de-endothelialized vascular smooth muscle strips by increasing extracellular [K(+)] (replacing Na(+) by K(+)) and leads to a rapid phasic contraction followed by a sustained tonic contraction. The initial phasic contractile response is due to opening of voltage-gated Ca(2+) channels and entry of extracellular Ca(2+), which binds to calmodulin, leading to activation of myosin light chain kinase, phosphorylation of the regulatory light chains of myosin II at Ser19 and cross-bridge cycling. The subsequent tonic contractile response involves, in addition to myosin light chain kinase activation, Ca(2+)-induced Ca(2+) sensitization whereby Ca(2+) entry activates the RhoA/Rho-associated kinase pathway leading to phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase) and inhibition of the phosphatase. Investigations into the mechanism of activation of RhoA by Ca(2+) have implicated a genistein-sensitive tyrosine kinase, and recent evidence indicates this to be the Ca(2+)-dependent tyrosine kinase, Pyk2.

Abnormal Rho-associated kinase activity contributes to the dysfunctional myogenic response of cerebral arteries in type 2 diabetes.

The structural and functional integrity of the brain, and therefore, cognition, are critically dependent on the appropriate control of blood flow within the cerebral circulation. Inadequate flow leads to ischemia, whereas excessive flow causes small vessel rupture and (or) blood-brain-barrier disruption. Cerebral blood flow is controlled through the interplay of several physiological mechanisms that regulate the contractile state of vascular smooth muscle cells (VSMCs) within the walls of cerebral resistance arteries and arterioles. The myogenic response of cerebral VSMCs is a key mechanism that is responsible for maintaining constant blood flow during variations in systemic pressure, i.e., flow autoregulation. Inappropriate myogenic control of cerebral blood flow is associated with, and prognostic of, neurological deterioration and poor outcome in patients with several conditions, including type 2 diabetes. Here, we review recent advances in our understanding of the role of inappropriate Rho-associated kinase activity as a cause of impaired myogenic regulation of cerebral arterial diameter in type 2 diabetes.

Structural analysis of a calmodulin variant from rice: the C-terminal extension of OsCaM61 regulates its calcium binding and enzyme activation properties.

OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca(2+)] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca(2+). NMR dynamics data for OsCaM61 indicate that the two lobes of the CaM domain act together unlike the independent behavior of the lobes seen in mammalian CaM and soybean CaM4. Also, data demonstrate that the positively charged nuclear localization signal region in the tail in apo-OsCaM61 is helical, whereas it becomes flexible in the Ca(2+)-saturated protein. The extra helix in apo-OsCaM61 provides additional interactions in the C-lobe and increases the structural stability of the closed apo conformation. This leads to a decrease in the Ca(2+) binding affinity of EF-hands III and IV in OsCaM61. In Ca(2+)-OsCaM61, the basic nuclear localization signal cluster adopts an extended conformation, exposing the C-terminal extension for prenylation or enabling OsCaM61 to be transferred to the nucleus. Moreover, Ser(172) and Ala(173), residues in the tail, interact with different regions of the protein. These interactions affect the ability of OsCaM61 to activate different target proteins. Altogether, our data show that the tail is not simply a linker between the prenyl group and the protein but that it also provides a new regulatory mechanism that some plants have developed to fine-tune Ca(2+) signaling events.

Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia.

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

A new genomic framework to categorize pediatric acute myeloid leukemia.

Recent studies on pediatric acute myeloid leukemia (pAML) have revealed pediatric-specific driver alterations, many of which are underrepresented in the current classification schemas. To comprehensively define the genomic landscape of pAML, we systematically categorized 887 pAML into 23 mutually distinct molecular categories, including new major entities such as UBTF or BCL11B, covering 91.4% of the cohort. These molecular categories were associated with unique expression profiles and mutational patterns. For instance, molecular categories characterized by specific HOXA or HOXB expression signatures showed distinct mutation patterns of RAS pathway genes, FLT3 or WT1, suggesting shared biological mechanisms. We show that molecular categories were strongly associated with clinical outcomes using two independent cohorts, leading to the establishment of a new prognostic framework for pAML based on these updated molecular categories and minimal residual disease. Together, this comprehensive diagnostic and prognostic framework forms the basis for future classification of pAML and treatment strategies.

Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²âºCaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries.

Abstract The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca(2+) entry via voltage-gated Ca(2+) channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca(2+) sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca(2+) sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca(2+) sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC20 phosphorylation, and a ROK- and PKC-evoked increase in actin polymerization) contribute to force generation in the myogenic response of skeletal muscle arterioles.