Complement-Mediated Selective Tumor Cell Lysis Enabled by Bi-Functional RNA Aptamers

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, the powerful effector mechanisms of the immune response can be conscripted to treat cancer. To implement this strategy, we have been developing aptamer-derived molecular adaptors to invoke synthetic immune responses against cancer cells. Here we describe multi-valent aptamers that simultaneously bind target molecules on the surface of cancer cells and an activated complement protein, which would tag the target molecules and their associated cells as “foreign” and trigger multiple effector mechanisms. Increased deposition of the complement proteins on the surface of cancer cells via aptamer binding to membrane targets could induce the formation of the membrane attack complex or cytotoxic degranulation by phagocytes and natural killer cells, thereby causing irreversible destruction of the targeted cells. Specifically, we designed and constructed a bi-functional aptamer linking EGFR and C3b/iC3b, and used it in a cell-based assay to cause lysis of MDA-MB-231 and BT-20 breast cancer cells, with either human or mouse serum as the source of complement factors.

Discovery of Novel EPO/EPOR/JAK2 Targets and Signal Transduction Factors Via Proteomic-Based Interrogations of Post-Translational Motif Modifications

Although EPO/EPOR/JAK2 actions have been intensely studied, the majority of investigations to date have relied heavily upon conventional approaches, extant reagents and murine cell models. We therefore hypothesized that important EPOR/JAK2 targets consequently remain undiscovered, and by applying (phospho)proteomic based interrogations, have uncovered intriguing sets of new candidate mediators of EPO’s effects on human erythroid progenitor cells. Specifically, following [-] vs [+] EPO challenge, total cellular tryptic or Glu-C peptides were generated, and those post-translationally modified (PTM) due to EPO at phosphotyrosine residues (p-Y), p-TPP motifs, and MAPK plus ATK signaling nodes were isolated, and identified by duplicate tandem LC-MS/MS. EPO targets furthermore were confirmed via parallel analyses using an EPOR agonist. Known EPO/EPOR/JAK2 targets first were validated (eg., JAK2, STAT5, GAB1-3, SHIP1-3, PLCgamma, p85alpha-PI3K, LNK, SHC1, SPRY1) (and further defined with regards to specific isoforms, plus known vs novel sites of EPO-induced PTM). Beyond this, newly discovered EPO/EPOR/JAK2 regulated targets included those within the following five categories: 1] Cytoskeletal targets unexpectedly as erythroid alpha and beta spectrins (pY2332, pY15) together with pY modulation of Beta-Adducin at a calmodulin binding site (plus EPO-regulated PTM of Calmodulin per se); 2] Select EPO- regulated transcription factors, including 100-fold p-Y modulation of KLF5 at a novel transcriptional activation motif; 3] EPO/EPOR/JAK2 modulation of two new protein tyrosine phosphatase targets as PTPN18 and PTPN7; 4] Rapid EPO-induction of a notable number of novel upstream molecular adaptors including C1ORF186/”RHEX”, C1ORF150, DOK1, IRS2, SH2D2A, STAM2, SPRY2,4, DLG1,3, FRS2, AMOTL1, RAI14, CTNND1, EPS15, HGS, ITSN2, and WASL – with two novel ORF factors as C1ORF186/RHEX and C1ORF150 as prime targets in human erythroid progenitors, but absent from rat, mouse, and lower vertebrate genomes. [For C1ORF186/RHEX, select cell and molecular biological properties will be summarized]. And 5] highlighted additional novel targets (eg, transporters, metabolic regulators) that, together with the above, further emphasize a previously underestimated sophistication of EPOR/JAK2 signal transduction circuits, certain of which are proposed to be central to EPO and ESA effects on anemia (and potentially, hypertensive side-effects). Disclosures No relevant conflicts of interest to declare.

The functionally diverse roles of tribbles

Tribbles are members of the pseudokinase family of proteins, with no associated kinase activity detectable to date. As tribbles appear not to function as kinases, there has been debate surrounding their functional classification. Tribbles have been proposed to function as adaptor molecules facilitating degradation of their target proteins. Tribbles have also been proposed to mediate signalling changes to MAPK (mitogen-activated protein kinase) cascades and also to function as decoy kinases interfering with the activity of known kinases. The present review discusses the functionally divergent roles of tribbles as molecular adaptors mediating degradation, changes to signalling cascades and action as decoy kinases.

RVBs Are Required for Assembling a Functional TIP60 Complex

RVB1/RVB2 (RuvBL1/RuvBL2 or pontin/reptin) are enigmatic AAA+ATPase proteins that are present in multiple cellular complexes. Although they have been implicated in many cellular functions, the exact molecular function of RVB proteins in the various complexes is not clear. TIP60 complex (TIP60.com) is a tumor suppressor chromatin-remodeling complex containing RVB proteins. RVBs are required for the lysine acetyltransferase activity of TIP60.com but not for that of the pure recombinant TIP60 polypeptide. Here we describe two molecular functions of RVBs in TIP60.com. First, RVBs negate the repression of catalytic activity of TIP60 by another protein in TIP60.com, p400. RVBs competitively displace the SNF2 domain of p400 from the TIP60 polypeptide. In addition RVBs are also required for heat stability of TIP60.com by a p400-independent pathway. RVB1 and RVB2 are redundant with each other for these functions and do not require their ATPase activities. Thus, RVB proteins act as molecular adaptors that can substitute for one another to facilitate the optimal assembly, heat stability, and function of the TIP60 complex.

Skin Basement Membrane: The Foundation of Epidermal Integrity—BM Functions and Diverse Roles of Bridging Molecules Nidogen and Perlecan

The epidermis functions in skin as first defense line or barrier against environmental impacts, resting on extracellular matrix (ECM) of the dermis underneath. Both compartments are connected by the basement membrane (BM), composed of a set of distinct glycoproteins and proteoglycans. Herein we are reviewing molecular aspects of BM structure, composition, and function regarding not only (i) the dermoepidermal interface but also (ii) the resident microvasculature, primarily focusing on theper senonscaffold forming components perlecan and nidogen-1 and nidogen-2. Depletion or functional deficiencies of any BM component are lethal at some stage of development or around birth, though BM defects vary between organs and tissues. Lethality problems were overcome by developmental stage- and skin-specific gene targeting or by cell grafting and organotypic (3D) cocultures of normal or defective cells, which allows recapitulating BM formationde novo. Thus, evidence is accumulating that BM assembly and turnover rely on mechanical properties and composition of the adjacent ECM and the dynamics of molecular assembly, including further “minor” local components, nidogens largely functioning as catalysts or molecular adaptors and perlecan as bridging stabilizer. Collectively, orchestration of BM assembly, remodeling, and the role of individual players herein are determined by the developmental, tissue-specific, or functional context.

Spry1 as a novel regulator of erythropoiesis, EPO/EPOR target, and suppressor of JAK2

Sprouty proteins are established modifiers of receptor tyrosine kinase (RTK) signaling and play important roles in vasculogenesis, bone morphogenesis, and renal uteric branching. Little is understood, however, concerning possible roles for these molecular adaptors during hematopoiesis. Within erythroid lineage, Spry1 was observed to be selectively and highly expressed at CFU-e to erythroblast stages. In analyses of possible functional roles, an Mx1-Cre approach was applied to conditionally delete Spry1. At steady state, Spry1 deletion selectively perturbed erythroid development and led to reticulocytosis plus heightened splenic erythropoiesis. When challenged by hemolysis, Spry1-null mice exhibited worsened anemia and delayed recovery. During short-term marrow transplantation, Spry1-null donor marrow also failed to efficiently rescue the erythron. In each anemia model, however, hyperexpansion of erythroid progenitors was observed. Spry function depends on phosphorylation of a conserved N-terminal PY motif. Through an LC-MS/MS approach, Spry1 was discovered to be regulated via the erythropoietin receptor (EPOR), with marked EPO-induced Spry1-PY53 phosphorylation observed. When EPOR signaling pathways were analyzed within Spry1-deficient erythroid progenitors, hyperactivation of not only Erk1,2 but also Jak2 was observed. Studies implicate Spry1 as a novel regulator of erythropoiesis during anemia, transducer of EPOR signals, and candidate suppressor of Jak2 activity.

Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.

Abstract 777 The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p<0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71high, Ter119pos precursors in Mx1-Cre; Spry1flox/flox animals compared to Mx1-Cre;Spry1flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form KitnegCD71highTer119neg and KitnegCD71highTer119pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation. Disclosures: No relevant conflicts of interest to declare.