Remarkable Response of Hairy Cell Leukemia Variant to Single-Agent Cladribine.

Classical hairy cell leukemia (cHCL) and related mature lymphoid B-cell neoplasms including hairy cell leukemia variant (HCLv) and splenic diffuse red pulp lymphoma (SDRPL) are a rare subset of lymphoid neoplasms. cHCL accounts for around 2% of all leukemias and is characterized by a peripheral smear with large lymphoid cells with cytoplasmic projections giving the cells a hairy appearance, splenomegaly, and cytopenias. Majority of cHCL cases harbor a BRAFV600E mutation. cHCL usually responds well to single-agent purine analogs. HCLv is even rarer and constitutes around 0.4% of lymphoid malignancies. Unlike cHCL, HCLv is less responsive to standard single-agent purine analogs and typically does not harbor the BRAFV600E mutation. The "hairy cells," splenomegaly, and cytopenias are common in both. We report a case of a patient with HCLv who was treated with a single purine analog and achieved a near-complete response.

Severe Hemolysis in a COVID-19 Patient with Paroxysmal Nocturnal Hemoglobinuria.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has been demonstrated to be able to activate complement, making patients with deficiency in negative complement regulation, such as paroxysmal nocturnal hemoglobinuria (PNH), particularly vulnerable to complement-mediated cell damage. We report a case of a patient who presented with fatigue, facial swelling, and upper respiratory infection (URI) symptoms and was found to have COVID-19 with laboratory tests showing severe hemolysis and pancytopenia secondary to PNH.

Managing Massive Splenic Hematoma: Exploratory Laparotomy or Active Surveillance?

Splenic hematoma is a known complication of blunt force abdominal trauma. Traditional management of splenic hematomas has been primarily surgical. However, more recently, spleen-sparing management has been favored over surgical management for cases that meet certain criteria, with surgery now reserved for patients with complications. In this report, we present a case of a massive splenic hematoma that was managed conservatively and analyze the challenges faced in clinical decision making.

CIN or not: An approach to the evaluation and management of chronic idiopathic neutrophilia.

Neutrophilia refers to an increase in the number of circulating neutrophils in the peripheral blood. Some common etiologies include infection, inflammatory conditions, myeloproliferative disorders, malignancies, endocrinopathies, drugs, and anemia. Rare disorders such as leukocyte adhesion deficiency can also cause neutrophilia. In many cases, there is an elevation of neutrophil count that persists for months or even years with no clear underlying cause in an otherwise asymptomatic patient. This is referred to as chronic idiopathic neutrophilia (CIN). Despite being a condition encountered by many physicians, there is a paucity of literature addressing CIN. Certain conditions such as stress, exercise, smoking, obesity, and obstructive sleep apnea have been associated with CIN and may provide explanations for neutrophilia previously thought to be idiopathic. Herein, we present a review of the literature on CIN and propose a systematic approach to this commonly encountered clinical condition.

Rapid Response in a Patient with Relapsed/Refractory Multiple Myeloma Treated with BRAF/MEK Inhibitors.

Patients with relapsed and refractory multiple myeloma have a poor prognosis. The mitogen-activated protein kinase (MAPK) pathway has been implicated in the pathogenesis of multiple myeloma. Several mutations in this pathway can lead to its constitutive activation leading to oncogenesis. One such mutation is BRAFV600E which is a therapeutic target in the treatment of melanoma, lung cancer, colon cancer, thyroid cancer, and hairy cell leukemia. BRAFV600E-directed therapy currently does not have approval in multiple myeloma. It has been proposed that this mutation leads to proteasome inhibitor resistance. About 4-10% of multiple myeloma cases harbor the BRAFV600E mutation. Herein, we report a case of a patient with relapsed and refractory multiple myeloma who had a progression-free survival (PFS) of 8.5 months on BRAF-targeted therapy.

The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases.

The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21(Cip1), p27(Kip1) and p57(Kip2). Their kinase inhibitory activities are mediated by a homologous N-terminal kinase inhibitory domain. The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the kinase inhibitory domain and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, as well as its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation.

Probing the role of nascent helicity in p27 function as a cell cycle regulator.

p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27.

Electrostatically accelerated coupled binding and folding of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27(Kip1) (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering) but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition.

Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein.

p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.

The role of the LH subdomain in the function of the Cip/Kip cyclin-dependent kinase regulators.

The Cip/Kip protein family, which includes p27, p21, and p57, modulates the activity of cyclin-dependent kinases (Cdks). A domain within these proteins, termed the kinase inhibitory domain (KID), is necessary and sufficient for Cdk inhibition. The KID consists of a cyclin-binding subdomain (termed D1) and a Cdk-binding subdomain (termed D2) joined by a 22-residue linker subdomain (termed LH). Before binding the Cdks, D1 and D2 are largely unstructured and the LH subdomain exhibits nascent helical characteristics. Curiously, although the sequence of the linker subdomain is not highly conserved within the family, its nascent helical structure is conserved. In this study, we explored the role of this structural conservation in interactions with cyclin-dependent kinase 2 (Cdk2) and cyclin A. We constructed chimeric p27-KID molecules in which the p27 LH subdomain was replaced with the corresponding segments of either p21 or p57. The chimeric molecules bind and inhibit Cdk2 in a manner similar to wild-type p27-KID. However, the extent of enthalpy/entropy compensation associated with these interactions was dramatically different, indicating different extents of LH subdomain folding upon binding. Our results indicate that the different LH subdomains, despite their sequence and thermodynamic differences, play similar roles in binding and inhibiting Cdk2/cyclin A.

Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21.

Traditionally, well-defined three-dimensional structure has been thought to be essential for protein function. However, myriad biological functions are performed by highly dynamic, intrinsically disordered proteins (IDPs). IDPs often fold upon binding their biological targets and frequently show 'binding diversity' by targeting multiple ligands. We sought to understand the physical basis of IDP binding diversity and report here that the cyclin-dependent kinase (Cdk) inhibitor p21(Cip1) adaptively binds to and inhibits the various Cdk-cyclin complexes that regulate eukaryotic cell division. Using results from NMR spectroscopy and biochemical and cellular assays, we show that structural adaptability of a helical subdomain within p21, termed LH, enables two other subdomains, D1 and D2, to specifically bind conserved surface features of the cyclin and Cdk subunits, respectively, within otherwise structurally distinct Cdk-cyclin complexes. Adaptive folding upon binding is likely to mediate the diverse biological functions of the thousands of IDPs present in eukaryotes.

Quantification of the vitamin D receptor-coregulator interaction.

The vitamin D receptor (VDR) regulates a diverse set of genes that control processes including bone mineral homeostasis, immune function, and hair follicle cycling. Upon binding to its natural ligand, 1alpha,25(OH)(2)D(3), the VDR undergoes a conformational change that allows the release of corepressor proteins and the binding of coactivator proteins necessary for gene transcription. We report the first comprehensive evaluation of the interaction of the VDR with a library of coregulator binding motifs in the presence of two ligands, the natural ligand 1alpha,25(OH)(2)D(3) and a synthetic, nonsecosteroidal agonist LG190178. We show that the VDR has relatively high affinity for the second and third LxxLL motifs of SRC1, SRC2, and SRC3 and second LxxLL motif of DRIP205. This pattern is distinct in comparison to other nuclear receptors. The pattern of VDR-coregulator binding affinities was very similar for the two agonists investigated, suggesting that the biologic functions of LG190178 and 1alpha,25(OH)(2)D(3) are similar. Hairless binds the VDR in the presence of ligand through a LxxLL motif (Hr-1), repressing transcription in the presence and absence of ligand. The VDR binding patterns identified in this study may be used to predict functional differences among different tissues expressing different sets of coregulators, thus facilitating the goal of developing tissue- and gene-specific vitamin D response modulators.

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding.

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.