Matthew Ndonwi

Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo

Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼ 11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

The Kunitz-3 domain of TFPI-α is required for protein S–dependent enhancement of factor Xa inhibition

Protein S (PS) enhances the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-alpha (TFPI-alpha) in the presence of Ca(2+) and phospholipids. Altered forms of recombinant TFPI-alpha were used to determine the structures within TFPI-alpha that may be involved in this PS-dependent effect. Wild-type TFPI-alpha (TFPI(WT)), TFPI-alpha lacking the K3 domain (TFPI-(DeltaK3)), and TFPI-alpha containing a single amino acid change at the putative P1 residue of K3 (R199L, TFPI(K3P1)) produced equivalent FXa inhibition in the absence of PS, whereas the response in FXa inhibition produced by PS was reduced with TFPI(K3P1) (EC(50) 61.8 +/- 13.4nM vs 8.0 +/- 0.4nM for TFPI(WT)) and not detectable with TFPI-(DeltaK3). Ligand blotting and surface plasmon resonance experiments demonstrated that FXa bound TFPI(WT) and TFPI-(DeltaK3) but not the isolated K3 domain, whereas PS bound TFPI(WT) and the K3 domain but not TFPI-(DeltaK3). Addition of TFPI(WT), TFPI(K3P1), or TFPI-(DeltaK3) produced comparable prolongation of FXa-induced coagulation in PS-deficient plasma, but the anticoagulant effect of TFPI(WT) was substantially greater than that of TFPI(K3P1) > TFPI-(DeltaK3) in normal plasma and PS-deficient plasma reconstituted with PS. We conclude that the PS-mediated enhancement of FXa inhibition by TFPI-alpha involves an interaction between PS and TFPI-alpha, which requires the K3 domain of TFPI-alpha.

Protein S enhances the tissue factor pathway inhibitor inhibition of factor Xa but not its inhibition of factor VIIa–tissue factor

The tissue factor (TF) pathway of blood coagulation is initiated when factor VIIa binds to TF and proteolytically activates FIX and FX. The key regulator of this pathway is TF pathway inhibitor (TFPI), a multivalent Kunitz-type proteinase inhibitor that directly inhibits FXa and produces FXa-dependent feedback inhibition of the FVIIa–TF complex. In the final quaternary inhibitory complex containing FXa, TFPI, FVIIa and TF, the Kunitz-2 domain of TFPI interacts with FXa and the Kunitz-1 domain interacts with FVIIa. The formation of this complex is frequently described as a two-step process in which TFPI binds to FXa, and the FXa–TFPI complex subsequently binds to FVIIa–TF. Kinetic studies, however, strongly suggest that TFPI interacts with the tertiary FVIIa– TF–FXa complex before FXa is released from the FVIIa–TF complex after the cleavage of FX [1]. Protein S is a vitamin K-dependent protein that not only serves as a cofactor for the inactivation of FVa and FVIIIa by activated protein C (APC), but also appears to possess APCindependent anticoagulant activity [2,3]. Recently, Hackeng et al.made the important observation that protein S enhances the interaction of TFPI with FXa in the presence of Ca and phospholipids, and suggested that protein S-mediated enhancement of the FXa–TFPI interaction may also accelerate the inhibition of FVIIa–TF by TFPI [4]. Studies in which the inhibition of FVIIa–TF by TFPI is assessed by the generation of FXa, however, must be interpreted with caution, due to the inhibition of FXa by TFPI. Therefore, we have re-examined the effect of protein S on FVIIa–TF inhibition by TFPI using methods designed to minimize the confounding inhibitory effect of TFPI on FXa activity. First, the effect of protein S on TFPI inhibition of FXa was confirmed by repeating the experiments ofHackeng et al. [4], in which FXawas incubated with substrate S-2222 in the presence of TFPI, Ca and phospholipids with or without protein S, and S-2222 hydrolysis was followed by measuring the pnitroaniline released (DA405) at various concentrations of protein S. Protein S significantly enhanced the TFPI inhibition of FXa, with an apparent IC50 (concentration achieving 50% potentiation of the TFPI inhibition of FXa) of 10 nM (Fig. 1A). Maximum stimulation was observed at 160 nM protein S (Fig. 1A, inset). Protein S did not inhibit FXa in the absence of TFPI (data not shown). Similar results were obtained with protein S purchased from two independent vendors, Haematologic Technologies Inc. (Essex Junction, VT, USA) and Enzyme Research Laboratories (Southbend, IN, USA). These findings are consistent with those reported by Hackeng et al. [4], and confirm that protein S stimulates the TFPI inhibition of FXa in the presence of Ca and phospholipids. The effect of protein S on TFPI inhibition of FVIIa–TF was similarly reassessed by following the TFPI inhibition of the FVIIa–TF-catalyzed FX activation in the presence and absence of protein S. For these experiments, FX and TFPI were incubated with FVIIa–TF with or without protein S in reaction buffer containing Ca and phospholipids. At various time intervals, aliquots of the reaction were withdrawn and quenched with EDTA. Hackeng et al. [4] subsequently measured the FXa generated by amidolytic assay. Our replication of this assay is presented in Fig. 1B. As is evident from these data, protein S alone does not inhibit the FVIIa–TF activation of FX. Nevertheless, when compared to TFPI alone, the combination of TFPI with protein S appears to dramatically reduce FVIIa–TF activity. However, given the relatively higher molar concentration of TFPI than the quantity of FXa generated, the result could represent the potentiation of the TFPI inhibition of FXa by protein S. We therefore reassessed FXa generated in this system using a different approach. In this method, quenched reaction aliquots are diluted in buffer containing excess TFPI to completely complex the FXa generated to TFPI. TFPI–FXa complexes are then measured with the Imubind TFPI/Xa enzyme-linked immunosorbent assay kit from American Diagnostica Inc. (Stamford, CT, USA). In contrast to data obtained using the amidolytic assay Correspondence: George Broze Jr, Division of Hematology, Washington University, Campus Box 8125, 4940 Parkview Place, Saint Louis, MO 63110, USA. Tel.: +1 314 362 8811; fax: +1 314 362 8813. E-mail: gbroze@im.wustl.edu

Inhibition of antithrombin by Plasmodium falciparum histidine-rich protein II.

Histidine-rich protein II (HRPII) is an abundant protein released into the bloodstream by Plasmodium falciparum, the parasite that causes the most severe form of human malaria. Here, we report that HRPII binds tightly and selectively to coagulation-active glycosaminoglycans (dermatan sulfate, heparan sulfate, and heparin) and inhibits antithrombin (AT). In purified systems, recombinant HRPII neutralized the heparin-catalyzed inhibition of factor Xa and thrombin by AT in a Zn(2+)-dependent manner. The observed 50% inhibitory concentration (IC(50)) for the HRPII neutralization of AT activity is approximately 30nM for factor Xa inhibition and 90nM for thrombin inhibition. Zn(2+) was required for these reactions with a distribution coefficient (K(d)) of approximately 7μM. Substituting Zn(2+) with Cu(2+), but not with Ca(2+), Mg(2+), or Fe(2+), maintained the HRPII effect. HRPII attenuated the prolongation in plasma clotting time induced by heparin, suggesting that HRPII inhibits AT activity by preventing its stimulation by heparin. In the microvasculature, where erythrocytes infected with P falciparum are sequestered, high levels of released HRPII may bind cellular glycosaminoglycans, prevent their interaction with AT, and thereby contribute to the procoagulant state associated with P falciparum infection.

Mutant U2AF1-expressing cells are sensitive to pharmacological modulation of the spliceosome

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations—drug and mutation—compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.

The first epidermal growth factor‐like domains of factor Xa and factor IXa are important for the activation of the factor VII–tissue factor complex

Summary.  During tissue factor (TF)‐induced coagulation, the factor (F)VIIa–TF complex activates factor (F)X and factor (F)IX. Through positive feedback, the generated FXa and FIXa activate FVII–TF. The first epidermal growth factor‐like (EGF1) domains of FX and FIX serve as important TF‐recognition motifs when FVIIa–TF activates FX or FIX. Here, we investigated the role of EGF1 domains of FXa and FIXa during the activation of FVII–TF and inhibition by tissue factor pathway inhibitor (TFPI). FXaPCEGF1 (EGF1 domain of FXa replaced with that of protein C), and FXaQ49P (EGF1 domain mutant with impaired calcium‐binding), and the corresponding FIXa mutants were generated, and their abilities to activate FVII–TF were compared with the wild‐type (WT) enzymes. In the absence of TF, the rates of FVII activation were similar between WT enzymes and mutant FXa and FIXa proteases. In the presence of either soluble TF (sTF) or relipidated TF, each mutant of FXa or FIXa activated FVII–TF at a slower rate than the corresponding WT enzyme. Kinetics of inhibition of the amidolytic activity of WT and the mutant FXa proteases by either two‐domain or full‐length TFPI were similar. However, compared with the complex of TFPI–FXaWT, the abilities of the complexes of TFPI–FXa mutants to inhibit FVIIa–TF were impaired. We conclude that the EGF1 domains of FXa and FIXa are important for the activation of FVII–TF and for the formation of FVIIa–TF–FXa–TFPI complex.

Substitution of the Gla Domain in Factor X with That of Protein C Impairs Its Interaction with Factor VIIa/Tissue Factor LACK OF COMPARABLE EFFECT BY SIMILAR SUBSTITUTION IN FACTOR IX

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of γ-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FXPCGla and FIXPCGla (Gla domain replaced with that of protein C), FXPCEGF1 and FIXPCEGF1 (EGF1 domain replaced with that of protein C), as well as FXPCGla/EGF1 and FIXPCGla/EGF1 (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIXPCGla was minimally affected, and the reciprocal activation of FVII/TF by FIXaPCGla was normal; however, both reactions were impaired for the FIXPCEGF1 and FIXPCGla/EGF1 mutants. Predictably, FXIa activation of FIXPCEGF1 was normal, whereas it was impaired for the FIXPCGla and FIXPCGla/EGF1 mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Functional analysis of protein Z (Arg255His) and protein Z-dependent protease inhibitor (Lys25Arg and Ser40Gly) polymorphisms

1117–1121. Ravandi, F., Jorgensen, J.L., O’Brien, S.M, Verstovsek, S., Koller, C.A., Faderl, S., Giles, F.J., Ferrajoli, A., Wierda, W.G., Odinga, S., Huang, X., Thomas, D.A., Freireich, E.J., Jones, D., Keating, M.J. & Kantarjian, A.M. (2006) Eradication of minimal residual disease in hairy cell leukemia. Blood, 107, 4658–4662. Tallman, M.S., Hakimian, D., Rademaker, A.W., Zanzig, C., Wollins, E., Rose, E. & Peterson, L.C. (1996) Relapse of hairy cell leukemia after 2-chlorodeoxyadenosine: long-term follow up of the Northwestern University experience. Blood, 88, 1954–1959. Thomas., D.A., O’Brien, S., Bueso-Ramos, C., Faderl, S., Keating, M.J., Giles, F.G., Cortes, J & Kantarjian, H.M. (2003) Rituximab in relapsed or refractory Hairy Cell Leukemia. Blood, 102, 3906– 3911. Wheaton, S., Tallman, M.S., Hakimian, D. & Peterson, L. (1996) Minimal residual disease may predict bone marrow relapse in patients with hairy cell leukemia treated with 2-chlorodeoxyadenosine. Blood, 87, 1556–1560.

Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.

Reduced levels of Hspa9 attenuate Stat5 activation in mouse B cells

HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndrome (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. Although homozygous knockout of Hspa9 is embryonically lethal, mice with heterozygous deletion of Hspa9 (Hspa9(+/-)) are viable and have a 50% reduction in Hspa9 expression. Hspa9(+/-) mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9(+/-) mice have a cell-intrinsic reduction in bone marrow colony-forming unit-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9(+/-) B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNA interference and observed reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B lymphopoiesis in vivo. Knockdown of Hspa9 in an interleukin 7 (IL-7)-dependent mouse B-cell line reduced signal transducer and activator of transcription 5 (Stat5) phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B cells. Collectively, these data imply a role for Hspa9 in B lymphopoiesis and Stat5 activation downstream of IL-7 signaling.