Tischer, Alexander

Abstract Background : Approximately 25% of patients with primary myelofibrosis (PMF) harbor calreticulin (CALR) mutations, which have been associated with longer survival (Klampf et al. NEJM 2013). More than 80% of CALR mutated patients harbor one of two mutation variants: type 1, a 52-bp deletion (p.L367fs*46) or type 2, a 5-bp TTGTC insertion (p.K385fs*47). Recent studies have suggested phenotypic and prognostic differences between these two variants (Tefferi et al. Blood 2014, Leukemia 2014 and AJH 2014). Furthermore, data are emerging that suggest functionally-relevant structural differences between type 1 and type 2 CALR variants, including a higher alpha-helix content of the mutant C-terminus in type 2, compared to type 1 (Eder-Azanza et al. Leukemia 2014). Objectives : We used statistical models to calculate helix propensity for thirty-one unique amino acid sequences that were altered by CALR mutations and used the results to subclassify non-type 1/2 CALR mutations into “type 1-like” and “type 2-like” variants. Subsequently, we examined the prognostic relevance of these subgroups. Methods : Calculation of helix propensity, which is the percentage of residues that are predicted to be involved in the formation of an alpha-helix, was performed using AGADIR, which is a statistical approximation algorithm (Munoz et al. Biopolymers 1997). The helix tendency calculations were performed using conditions of pH 7.0, 5 and 25 °C, an ionic strength of 0.1 M and no N- or C-terminal protection. Results : 532 PMF patients were screened for JAK2, CALR and MPL mutations; the respective mutational frequencies were 58%, 24.6% and 7.3%. Among the 131 CALR-mutated cases, 98 (74.8%) harbored type 1, 15 (11.5%) type 2 and 18 (13.7%) other variants. Based on predicted helix propensity scale, the “other” CALR mutations were subclassified as type 1-like (n=12) or type 2-like (n=6) and respectively grouped with type 1 and type 2 variants, for purposes of phenotypic and prognostic comparisons. The AGADIR-derived predicted helix propensity scale was 29.69 for wild-type CALR and 8.6 or 34.17 for type 1 and type 2 mutant CALR, respectively; accordingly, CALR variants with values that are close to or above the value for wild-type CALR were classified as “type 2-like” (range 26.47-36.12) and those with values close to or below the value for type 1 as “type 1-like” (range 2.11-17.3). Comparison of “type 1/type 1-like” (n=110) and “type 2/type 2-like” (n=21) CALR mutations showed the latter to be associated with higher DIPSS-plus score (p=0.01), EZH2 mutations (p<0.01), leukocyte count >25 x 10(9)/L (p<0.01), higher circulating blast percentage (p=0.02) and palpable spleen size >10 cm (p<0.01). Comparison of “type 1/type 1-like” CALR and JAK2 mutations (n=309) showed the former to be associated with younger age, higher platelet count, lower transfusion need, higher hemoglobin level, lower leukocyte count and lower DIPSS-plus score (p<0.01 for all comparisons). None of these associations was evident during comparison of “type 2/type 2-like” CALR with JAK2 mutations. Survival was similar between patients with type 1 and “type 1-like” (p=0.8) and between type 2 and “type 2-like” (p=0.63) CALR mutations. In contrast, survival was significantly shorter in patients with type 2 (HR 2.4, 95% CI 1.2-4.8) and “type 2-like” (HR 3.2, 95% CI 1.0-10.6), when compared to those with type 1 CALR mutations. Survival was also significantly shorter with “type 2/type 2-like” vs “type 1/type 1-like” CALR mutations (p=0.003; HR 2.5, 95% CI 1.4-4.5) and the difference remained significant when analysis was adjusted for age (p=0.047), ASXL1 (p=0.003) or EZH2 (p=0.001) mutations. Similarly, compared to JAK2-mutated cases (n=309), survival was longer in patients with “type 1/type 1-like” (HR 0.4, 95% CI 0.3-0.5) but not in those with “type 2/type 2-like” (HR 0.9, 95% CI 0.5-1.6) CALR mutations; the difference in survival between JAK2 and “type 1/type 1-like” CALR mutated cases remained significant (P<0.01) when analysis was adjusted for age, ASXL1 or EZH2 mutations or DIPSS-plus score. Conclusions : CALR mutations in PMF might be subclassified into type 1-like and type 2-like variants, based on predicted helical propensity of their mutant C-terminus. The favorable impact of CALR mutations in PMF might be restricted to type 1 or “type 1-like” variants. Disclosures No relevant conflicts of interest to declare.

Introduction: Isocitrate dehydrogenase 1 and 2 (IDH1/2) are metabolic enzymes in the citric acid cycle, producing alpha-ketoglutarate (αKG). Mutations in specific regions of these genes have been characterized in gliomas, AML and chronic myeloid neoplasms such as MDS and MPN. These mutations produce an oncometabolite 2-hydroxyglutarate (2HG) that disrupts the epigenetic phenotype of myeloid cells promoting oncogenesis. While this process has been characterized for arginine hot spot (R132/R140) mutations, there exist variants of unclear significance (IDHVUS), with potential pathogenic predictions using in silico assessment models. With the availability of mutant IDH inhibitors in practice, it has become important to characterize these VUS. We carried out this study to functionally interrogate potentially pathogenic IDHVUS in MDS and MPN. Methods: After institutional approval, bone marrow (BM) DNA of patients with MDS and MPN, including overlap syndromes were subjected to next generation sequencing (29-gene, including the entire coding region of IDH1/2) by previously described methods (Patnaik et al., BCJ 2016). A sub-set of patients with available plasma [IDH wild type (IDHWT), bona fide IDH mutations (IDHMT) and IDHVUS with predicted pathogenicity (≤0.01% minor allele frequency, and predicted damaging in silico scores)] were submitted for mass spectrometry (LC-MS) based detection of IDH-related metabolites; D and L isomers of 2HG, αKG, and total 2HG. The impact of variations on protein secondary structure was assessed by the GorIV prediction algorithm. Potential alterations within the tertiary structure of the proteins was evaluated by in silico mutagenesis. Results: One hundred and seventeen patients were included in the study; median age 68 years (range, 39-94), 68% male. Of these, 40 (34%) had MDS, 42 (36%) MPN, and 35 (30%) MDS/MPN overlap syndromes. Eighty six (74%) had IDHMT, including n=28 (24%) with IDH1MT (R132C/G/H/S) (11 MDS, 8 MPN, 9 MDS/MPN) and n=59 (50%) with IDH2MT (R140G/Q/W) (22 MDS, 20 MPN, 17 MDS/MPN). IDHVUS were detected in 32 (27%) patients, including 1 IDH1MT patient, and involved IDH1 in 14 (12%) cases and IDH2 in 18 (15%) cases. All IDHVUS were missense, except for a 2 amino acid insertion in IDH2, with the most frequent VUS being IDH1Y183C (7%) and IDH2T352P (7%). While there were no phenotypic differences between IDH1/2MUT and IDH1/2VUS, in comparison to patients with IDH1/2MT, those with IDH1VUS were more likely to have higher WBC counts (median 8.1 vs 3.6 x109/L, p=0.0322), higher neutrophil counts (median 3.3 vs 1.3 x109/L, p=0.0203), and lower BM blast % (median 1 vs 3%, p=0.02061). Compared to IDH1/2MT patients, those with sole IDH1/2VUS more frequently had mutations in SF3B1 (p=0.0167), while they were associated with a lower frequency of SRSF2 mutations (p=0.0002). Interestingly, TET2 mutations were detected in only 5% of patients with IDHMT and 16% of IDHVUS patients, although without reaching statistical significance. LC-MS analysis on 75 plasma samples (n=43 IDHMT, n=9 IDHVUS, n=20 IDHWT, 3 normal) revealed a significant increase in total 2HG levels in IDHMT vs IDHVUS (p=0.0089), and IDHWT (p=0.0020) cases (Figure 2A-D). While there were no significant differences in αKG levels between the groups, the D-2HG enantiomer levels were higher in IDHMT samples vs IDHVUS (p=0.0199) and IDHWT (p=0.0055). On examination of the crystal structures of the IDH genes using PDB, while we observed a direct interaction between R132/R140/R172 and the catalytic core, the IDHVUS were located away from this active center and were hence thought to be allosteric in nature (Figure 2F). These VUS however, did demonstrate predictive steric clashes in the tertiary structure context of IDH1 using the PyMol artificial mutagenesis tool. The absence of wild-type crystal structure for IDH2precluded a similar analysis for IDH2. Median overall survival (OS) of the entire cohort (Figure 1) was 36 (range, 0-190) months, with OS being inferior in IDH1/2MT patients (34 months) vs those with IDH1/2VUS (50 months, p=0.0255). Conclusion: Our study demonstrates that in spite of predictive pathogenicity of IDH1/2VUS using in silico predictive tools and crystal studies, the lack of involvement of the arginine hot spot precludes IDH neomorphic activity, as shown by normal 2HG levels. In addition, in comparison to IDHMT patients, those with IDHVUS had fewer BM blasts and a better survival. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Abstract The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI) and 417 controls (Ludwigshafen Risk and Cardiovascular Health Study (LURIC)) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele ≤55 years revealed an elevated risk for repeated MI (Odds ratio: 2.53, 95% CI:1.07-5.98). The Odds ratio was even higher in females ≤55 years at a value of 5.93 (95% CI:1.12-31.24). Cone and plate aggregometry showed, that compared to Phe2561, Tyr2561 was associated with increased platelet aggregate size, both in probands' blood and with the recombinant variants. Inhibition of the VWF-GPIIb/IIIa interaction abrogated the effect. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared to Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF, as a result of altered association of the C-domains that disrupts the normal dimer interface. In summary, our data emphasize the functional impact of the VWF C4-domain for VWF-mediated platelet aggregation in a GPIIb/IIIa-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism. Disclosures Schneppenheim: SHIRE: Consultancy; CSL Behring: Consultancy.

Abstract Von Willebrand disease (VWD) is the most common inherited human bleeding disorder. It is caused by deficiencies or defects in the plasma protein von Willebrand factor (vWF). The current classification of VWD consists of six distinct types. Type 1 and 3 result in a quantitative vWF deficiency in patients while the four type 2 variants (type 2A, 2B, 2M & 2N) are caused by qualitative defects in vWF. The von Willebrand factor is a multimeric multidomain glycoprotein that is secreted into blood from vascular endothelial cells. The protein initiates platelet adhesion at sites of cardiovascular injury. In vWF three essential domains could be identified which are responsible for the interaction with platelets and subendothelial tissue. While the A1 domain is responsible for the interaction with platelets, the A3-domain interacts with collagen from the subendothelial tissue. The A2-domain contains a cleavage site for the zinc protease AdamTS13 which regulates the size and the function of the vWF multimers by cleaving the A2-domain. We have recently studied the effect of several type 2B (= gain of function, stronger interaction with platelets) and type 2M (= loss of function, weak or no interaction with platelets) mutations in the vWF A1-domain and found clear evidence that these mutations cause a misfolding of this domain resulting in either gain- or loss of function (Tischer et al. (2014) Biophys. J. Accepted article). Hence type 2M and 2B VWD are clearly protein folding disorder diseases such like Alzheimer or various Amyloidoses. However since our group has obtained the A1-domain recombinantly from E.coli, it was unclear whether indeed misfolding of A1 is occurring or whether it is the result of the expression of the proteins in bacteria. To investigate this issue we have expressed triple domains in mammalian HEK293 cells consisting of A1, A2 and A3 and harboring the type 2B mutation V1314D and the 2B mutation F1369I in the A1 domain. The impact of the mutations on the biological function was determined in shear flow-dependent assays by observing the translocation of platelets on surface-immobilized A1- or triple domain. Using high speed video microscopy we were able to obtain statistical valid parameters for platelet translocation such as mean pause times and velocities. While F1369I did not support platelet translocation at all, V1314D was found to almost immobilize platelets in the flow chamber. The triple domain constructs harboring the mutations were found to have very similar functional features as the single domain mutants. F1369I triple domain did not support platelet translocation whereas V1314D triple domain immobilized platelets in the flow chamber at all applied shear rates. Structural characterization of the single A1 domains and of the triple domains resulted in evidence for massive misfolding in the A1 domain induced by the mutations. Therefore, all attempts to understand VWD and a potential drug development are required to account for non-native conformations of the A1 domain. Disclosures No relevant conflicts of interest to declare.

Abstract The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands’ blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.

AbstractProtein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non‐2‐state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two‐state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs‐Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147‐60). We resolve this thermodynamic conundrum using a Clausius‐Clapeyron analysis of the urea‐temperature phase diagram that defines how and the urea m‐value interconvert through the slope of cm versus T, . This relationship permits the calculation of at low temperature from m‐values obtained through iso‐thermal urea denaturation and high temperature m‐values from obtained through iso‐urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of and the m‐value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea‐expanded and thermally‐compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide‐intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼–13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this “cooperative collapse” of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.