Contribution of Ordered Water Molecules and a Crucial Phenylalanine to Cooperative Pathway(s) in Scapharca Dimeric Hemoglobin: a Dissertation

The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis binds oxygen cooperatively and thus offers a simple model system for studying communication between two chemically identical sites. Although the individual subunits of HbI have the same myoglobin-fold as mammalian hemoglobins, the quaternary assemblage is radically different. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins. Analysis of structures of this hemoglobin in the liganded (02or CO) and unliganded states has provided a framework for understanding the role of individual amino acid side-chains in mediating cooperativity. The work presented in this dissertation has directly tested the central tenets of the proposed structural mechanism for cooperativity in HbI, illuminating the key roles played by residue Phe 97 and interface water molecules in intersubunit communication. Heterologous expression of Scapharca dimeric hemoglobin: A synthetic gene has been utilized to express recombinant RbI in Escherichia coli. The HbI apoprotein constitutes 5-10% of the total bacterial protein in this system. Addition of the heme precursor δ-aminolevulinic acid to the expression culture results in a ~3-fold increase in the production of soluble hemoglobin. Recombinant HbI has been successfully purified to homogeneity, resulting in a final yield of 80-100 mg of pure holoprotein from a 12 L expression culture. Analysis of recombinant HbI reveals its oxygen binding properties to be indistinguishable from native HbI. It was necessary to correct a protein sequence error by mutating residue Asn 56 to aspartate in order to obtain diffraction quality crystals, that are isomorphous to native HbI crystals. These recombinant HbI crystals diffract to high resolution, permitting the functional effects of mutant HbI proteins to be correlated with detailed structural analysis

Pregnancy in patients with myelofibrosis: Mayo–Florence series of 24 pregnancies in 16 women

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A globally applicable “triple A” risk model for essential thrombocythemia based on Age, Absolute neutrophil count, and Absolute lymphocyte count

: We examined the individual prognostic contribution of absolute neutrophil (ANC), lymphocyte (ALC), and monocyte (AMC) counts, on overall (OS), leukemia-free (LFS), and myelofibrosis-free (MFFS) survival in essential thrombocythemia (ET). Informative cases (N = 598; median age 59 years; females 62%) were retrospectively accrued from a Mayo Clinic database: JAK2 59%, CALR 27%, triple-negative 11%, and MPL 3%; international prognostic scoring system for ET (IPSET) risk high 21%, intermediate 42%, and low 37%; 7% (37/515) had abnormal karyotype and 10% (21/205) adverse mutations (SF3B1/SRSF2/U2AF1/TP53). At median 8.4 years, 163 (27%) deaths, 71 (12%) fibrotic, and 20 (3%) leukemic transformations were recorded. Multivariable analysis resulted in HR (95% CI) of 16.5 (9.9-27.4) for age > 70 years, 3.7 (2.3-6.0) for age 50-70 years, 2.4 (1.7-3.3) for ANC ≥8 × 109 /L, and 1.9 (1.4-2.6) for ALC <1.7 × 109 /L. The corresponding HR-based scores were 4, 2, 1, and 1, resulting in an new 4-tiered AgeAncAlc (AAA; triple A) risk model: high (5-6 points; median survival 8 years; HR 30.1, 95% CI 17.6-54), intermediate-2 (4 points; median 13.5 years; HR 12.7, 95% CI 7.1-23.0), intermediate-1 (2-3 points; median 20.7 years; HR 3.8, 95% CI 2.3-6.4) and low (0-1 points; median 47 years). The AAA model (Akaike Information Criterion [AIC] 621) performed better than IPSET (AIC 647) and was subsequently validated by an external University of Florence ET cohort (N = 485). None of the AAA variables predicted LFS while ALC <1.7 × 109 /L was associated with inferior MFFS (p = .01). Adverse mutations (p < .01) and karyotype (p < .01) displayed additional prognostic value without disqualifying the prognostic integrity of the AAA model. This study proposes a simple and globally applicable survival model for ET, which can be used as a platform for further molecular refinement. This study also suggests a potential role for immune-related biomarkers, as a prognostic tool in myeloproliferative neoplasms

The impact of thrombosis on probabilities of death and disease progression in polycythemia vera: a multistate transition analysis of 1,545 patients

: We applied a parametric Markov five-state model, on a well-characterized international cohort of 1,545 patients with polycythemia vera (PV; median age 61 years; females 51%), in order to examine the impact of incident thrombosis on the trajectory of death or disease progression. At a median follow-up of 6.9 years, 347 (23%) deaths, 50 (3%) blast phase (BP), and 138 (9%) fibrotic (post-PV MF) transformations were recorded. Incident thrombosis occurred at a rate of 2.62% pt/yr (arterial 1.59% and venous 1.05%). The probability of death, in the first 10 years, for 280 (18%) patients who developed thrombosis during follow-up was 40%, which was two-fold higher than that seen in the absence of thrombosis or any other transition state (20%; p < 0.01); the adverse impact from thrombosis was more apparent for arterial (HR 1.74; p < 0.01) vs venous thrombosis (p=NS) and was independent of other fixed (i.e., age, prior venous thrombosis, leukocytosis) or time-dependent (i.e., progression to BP or MF) risk variables. The transition probability to post-PV MF increased over time, in a linear fashion, with a rate of 5% capped at 5 and 10 years, in patients with or without incident thrombosis, respectively. The impact of thrombosis on transition probability to death or post-PV MF tapered off beyond 10 years and appeared to reverse direction of impact on MF evolution at the 12-year time point. These observations suggest thrombosis in PV to be a marker of aggressive disease biology or a disease-associated inflammatory state that is consequential to both thrombosis and disease progression

Targeting megakaryocytic induced fibrosis by AURKA inhibition in the myeloproliferative neoplasms

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF