Are changes in patient-reported outcomes prognostic for diffuse large B-Cell lymphoma survival? Results from the GOYA trial.

In this hypothesis-generating analysis, we examined whether longitudinal changes in patient-reported outcomes (PROs), such as symptoms, over time would be prognostic for progression-free survival (PFS) and overall survival (OS) in patients with diffuse large B-cell lymphoma (DLBCL) who were newly treated with obinutuzumab (G) in combination with CHOP (G-CHOP) or rituximab (R) with CHOP (R-CHOP), in the GOYA Phase 3 trial (NCT01287741). Our results show that from the study baseline to cycle 3 day 1, every 1-point increase (worsening) in fever symptoms was associated with a 41% higher risk of death (hazard ratio [HR], 1.41; P = 0.01). Every 1-point increase (worsening) in lumps or swelling symptoms was associated with a 27% higher risk of disease progression or death (PFS events) (HR, 1.27; P = 0.01) and a 29% higher risk of death (OS events) (HR, 1.29; P = 0.02). No significant associations were observed between survival and changes in other symptoms, such as itching. Our study suggests that changes in some PROs are related to survival in DLBCL patients.

Real-world impact of patient-reported outcome measurement on overall survival, healthcare use and treatment discontinuation in cancer patients.

Aim: The purpose of this retrospective, population-based, observational cohort analysis was to assess whether routine patient-reported outcomes (PRO) monitoring alone has an impact on real-world overall survival (OS) and hospitalizations among individuals diagnosed with lung, breast or colorectal cancer. The importance of follow-up care in post-PRO data collection was also discussed. Patients & methods: Administrative databases covering 17 cancer centers from Alberta, Canada were queried and individuals ≥18 years old and diagnosed with lung, breast or colorectal cancer from 1 January 2016 to 31 December 2019 were included and followed until 31 December 2020. Patients were stratified by whether they received routine PRO monitoring initiated within 120 days of diagnosis and matched 1:1 with use of propensity scores based on baseline characteristics. OS was assessed from the index date to death, and the respective Kaplan-Meier curves were estimated along with hazard ratios from Cox Proportional Hazard Models. Linear and logistic regression models were used to estimate mean differences and odds ratios (OR) respectively for healthcare resource utilization events including cancer physician visits, emergency department visits and outpatient ambulatory care encounters. Results: 4800 patients were included in each matched cohort. There was no statistically significant difference between PRO monitoring and non-monitoring cohorts in OS (HR = 1.01; 95% CI: 0.93-1.09; p = 0.836) and treatment discontinuation (OR = 0.98; 95% CI: 0.85-1.12; p = 0.75). Median OS was 51.5 months for unmonitored cohort (95% CI: 47.5-NA) versus 50.6 months for monitored cohort (95% CI: 47.6-55.7). Compared with PRO-monitored patients, unmonitored patients were associated with lower hospitalization risks (OR = 1.12; 95% CI: 1.03-1.22; p = 0.01). However, PRO-monitored patients experienced significantly fewer physician visits in comparison to unmonitored patients (MD = -1.036; 95% CI: -1.288 to -0.784, p < 0.001). Conclusion: Our results show that capturing patient-reported symptoms alone reduced the number of physician visits but neither reduced hospitalizations nor improved OS in this real-world cancer population. To drive more meaningful clinical impact, PRO monitoring programs must be met with rigorous follow-up response to the identified symptoms.

Current Diagnostic Pathways for Alzheimer's Disease: A Cross-Sectional Real-World Study Across Six Countries.

Background: Diagnostic pathways for patients presenting with cognitive complaints may vary across geographies. Objective: To describe diagnostic pathways of patients presenting with cognitive complaints across 6 countries. Methods: This real-world, cross-sectional study analyzed chart-extracted data from healthcare providers (HCPs) for 6,744 patients across China, France, Germany, Spain, UK, and the US. Results: Most common symptoms at presentation were cognitive (memory/amnestic; 89.86%), followed by physical/behavioral (87.13%). Clinical/cognitive tests were used in > 95%, with Mini-Mental State Examination being the most common cognitive test (79.0%). Blood tests for APOE ɛ4/other mutations, or to rule out treatable causes, were used in half of the patients. Clinical and cognitive tests were used at higher frequency at earlier visits, and amyloid PET/CSF biomarker testing at higher frequency at later visits. The latter were ordered at low rates even by specialists (across countries, 5.7% to 28.7% for amyloid PET and 5.0% to 27.3% for CSF testing). Approximately half the patients received a diagnosis (52.1% of which were Alzheimer's disease [AD]). Factors that influenced risk of not receiving a diagnosis were HCP type (higher for primary care physicians versus specialists) and region (highest in China and Germany). Conclusion: These data highlight variability in AD diagnostic pathways across countries and provider types. About 45% of patients are referred/told to 'watch and wait'. Improvements can be made in the use of amyloid PET and CSF testing. Efforts should focus on further defining biomarkers for those at risk for AD, and on dismantling barriers such low testing capacity and reimbursement challenges.

Real-World Biomarker Test Utilization and Subsequent Treatment in Patients with Early-Stage Non-small Cell Lung Cancer in the United States, 2011-2021.

INTRODUCTION: Biomarker testing is increasingly crucial for patients with early-stage non-small cell lung cancer (eNSCLC). We explored biomarker test utilization and subsequent treatment in eNSCLC patients in the real-world setting. METHODS: Using COTA's oncology database, this retrospective observational study included adult patients ≥ 18 years old diagnosed with eNSCLC (disease stage 0-IIIA) between January 1, 2011 and December 31, 2021. Date of first eNSCLC diagnosis was the study index date. We reported testing rates by index year for patients who received any biomarker test within 6 months of eNSCLC diagnosis and by each molecular marker. We also evaluated treatments received among patients receiving the five most common biomarker tests. RESULTS: Among the 1031 eNSCLC patients included in the analysis, 764 (74.1%) received ≥ 1 biomarker test within 6 months of eNSCLC diagnosis. Overall, epidermal growth factor receptor (EGFR; 64%), anaplastic lymphoma kinase (ALK; 60%), programmed death receptor ligand 1 (PD-L1; 48%), ROS proto-oncogene 1 (ROS1; 46%), B-Raf proto-oncogene (40%), mesenchymal epithelial transition factor receptor (35%), Kirsten rat sarcoma viral oncogene (29%), RET proto-oncogene (22%), human epidermal growth factor receptor 2 (21%), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (20%) were the 10 most frequently tested biomarkers. The proportion of patients undergoing biomarker testing rose from 55.3% in 2011 to 88.1% in 2021. The most common testing methods were Sanger sequencing for EGFR (244, 37%), FISH (fluorescence in situ hybridization) for ALK (464, 75%) and ROS1 (357, 76%), immunohistochemical assay for PD-L1 (450, 90%), and next-generation sequencing testing for other biomarkers. Almost all the 763 patients who received the five most common biomarker tests had a test before the initiation of a systemic treatment. CONCLUSION: This study suggests a high biomarker testing rate among patients with eNSCLC in the US, with testing rates for various biomarkers increasing over the past decade, indicating a continuous trend towards the personalization of treatment decisions.

A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway.

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO2-) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

Adherence to guidelines-recommended diagnostic testing was associated with overall survival in patients with diffuse large B-cell lymphoma after rituximab-based treatment: an observational cohort study.

PURPOSE: This study assessed the impact of adherence to guidelines-recommended diagnostic testing on treatment selection and overall survival (OS) in patients with diffuse large B-cell lymphoma (DLBCL) initiated on rituximab-based first line of treatment (1-LOT). METHODS: This retrospective cohort study used a nationwide electronic health record-derived de-identified database, including diagnostic testing information on immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and karyotype analysis that were abstracted from pathology reports or clinical visit notes, where available. The study included patients above 18 years old who were diagnosed with DLBCL between January 2011 and December 2019 and initiated on rituximab-based 1-LOT. Patients were classified into 'non-adherence,' 'partial-adherence' and 'complete-adherence' groups according to the evidence/documentation of a confirmed known result for IHC and molecular profiling tests (FISH and karyotyping) on a selection of the markers prior to the initiation of 1-LOT. Logistic regression was used to evaluate associations of adherence to diagnostic testing with 1-LOT between R-CHOP and other rituximab-based regimens. Median OS after the start of rituximab-based 1-LOT was calculated using the Kaplan-Meier method. Multivariable-adjusted Cox proportional hazards regression was used to assess the risk of all-cause death after initiation of 1-LOT by the degrees of adherence to guidelines-recommended diagnostic testing. RESULTS: In total, 3730 patients with DLBCL who initiated on rituximab-based 1-LOT were included. No association was found between adherence to guidelines-recommended diagnostic testing and treatment selection of 1-LOT for R-CHOP versus other rituximab-based regimens. Patients with a higher degree of adherence to guidelines-recommended diagnostic testing survived longer (median OS at 5.1, 6.9 and 7.1 years for 'non-adherence,' 'partial-adherence' and 'complete-adherence' groups, respectively [log-rank p < 0.001]) and had a decreased mortality risk (multivariable-adjusted hazard ratio with 95% confidence intervals at 0.83 [0.70-0.99] for 'partial-adherence' and 0.77 [0.64-0.91] for 'complete-adherence' groups, respectively). CONCLUSION: Patients' adherence to guidelines-recommended diagnostic testing were associated with better survival benefit, reinforcing the need for adoption of diagnostic testing guidelines in routine clinical care.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

National burden of achondroplasia: an analysis of the National Inpatient and Nationwide Ambulatory Surgery Samples.

Background: To estimate the cost of US hospital admissions and outpatient surgeries associated with achondroplasia. Materials & methods: Using 2017 data from nationally representative databases, this study identifies hospital admissions and outpatient encounters with an achondroplasia diagnosis. Descriptive measures are reported. Results: There were 1985 achondroplasia admissions nationwide. The most frequent admissions were neonatal care (33.7%) in children and musculoskeletal (22.7%) in adults. Average hospital length of stay was 6.8 days, 2.2 days longer than the US mean. Total mean inpatient costs were US$19,959, $7789 greater than the US mean. In the outpatient setting, children 5-14 years accounted for 56.9% of procedures. Conclusion: Achondroplasia is a serious condition with a wide range of lifelong complications frequently requiring hospitalization and surgical intervention.

Biologic initiation rates in systemic-naive psoriasis patients after first-line apremilast versus methotrexate use.

Aim: To compare rates of biologic initiation after commencing treatment with apremilast (APR) versus methotrexate (MTX) in systemic-naive patients with psoriasis (PsO). Methods: This was a retrospective cohort study of systemic-naive patients with PsO who initiated treatment with APR or MTX between 1 January 2015 and 31 March 2018. Outcomes: Adjusted rates of biologic initiation during follow-up were compared by logistic and Cox regressions. Results: APR initiators had 58% lower likelihood of biologic initiation (odds ratio: 0.42; 95% CI: 0.37-0.48; p < 0.001), lower adjusted biologic initiation rate (14.4% [95% CI: 13.2-15.7%] vs 28.6% [95% CI: 26.8-30.5%]), lower risk of biologic initiation (hazard ratio: 0.45; 95% CI: 0.40-0.51; p < 0.001) compared with MTX initiators. Conclusion: Systemic-naive patients with PsO have a lower rate of biologic initiation over 1 year following APR initiation.

Kinetic, Inhibition, and Structural Characterization of a Malonate Semialdehyde Decarboxylase-like Protein from Calothrix sp. PCC 6303: A Gateway to the non-Pro1 Tautomerase Superfamily Members.

The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (∼85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Šresolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues can play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences.

SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

Induced phase separation of mutant NF2 imprisons the cGAS-STING machinery to abrogate antitumor immunity.

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.

Kinetic and Structural Analysis of Two Linkers in the Tautomerase Superfamily: Analysis and Implications.

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple ß-α-ß structural scaffold. Most members are constructed from a single-core ß-α-ß motif or two consecutively fused ß-α-ß motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

The protein phosphatase PPM1A dephosphorylates and activates YAP to govern mammalian intestinal and liver regeneration.

The Hippo-YAP pathway responds to diverse environmental cues to manage tissue homeostasis, organ regeneration, tumorigenesis, and immunity. However, how phosphatase(s) directly target Yes-associated protein (YAP) and determine its physiological activity are still inconclusive. Here, we utilized an unbiased phosphatome screening and identified protein phosphatase magnesium-dependent 1A (PPM1A/PP2Cα) as the bona fide and physiological YAP phosphatase. We found that PPM1A was associated with YAP/TAZ in both the cytoplasm and the nucleus to directly eliminate phospho-S127 on YAP, which conferring YAP the nuclear distribution and transcription potency. Accordingly, genetic ablation or depletion of PPM1A in cells, organoids, and mice elicited an enhanced YAP/TAZ cytoplasmic retention and resulted in the diminished cell proliferation, severe gut regeneration defects in colitis, and impeded liver regeneration upon injury. These regeneration defects in murine model were largely rescued via a genetic large tumor suppressor kinase 1 (LATS1) deficiency or the pharmacological inhibition of Hippo-YAP signaling. Therefore, we identify a physiological phosphatase of YAP/TAZ, describe its critical effects in YAP/TAZ cellular distribution, and demonstrate its physiological roles in mammalian organ regeneration.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis.

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.

Visible Light Mediated Bidirectional Control over Carbonic Anhydrase Activity in Cells and in Vivo Using Azobenzenesulfonamides.

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation. Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.

Structural Motifs for CTD Kinase Specificity on RNA Polymerase II during Eukaryotic Transcription.

The phosphorylation states of RNA polymerase II coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The individual residues of the repetitive heptad of the C-terminal domain (CTD) of the biggest subunit of RNA polymerase II are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles. The mechanism of how the kinases place phosphorylation on the correct serine is not well understood. In this paper, we use biochemical assays, mass spectrometry, molecular modeling, and structural analysis to understand the structural elements determining which serine of the CTD heptad is subject to phosphorylation. We identified three motifs in the activation/P+1 loops differentiating the intrinsic specificity of CTD in various CTD kinases. We characterized the enzyme specificity of the CTD kinases-CDK7 as Ser5-specific, Erk2 with dual specificity for Ser2 and Ser5, and Dyrk1a as a Ser2-specific kinase. We also show that the specificities of kinases are malleable and can be modified by incorporating mutations in their activation/P+1 loops that alter the interactions of the three motifs. Our results provide an important clue to the understanding of post-translational modification of RNA polymerase II temporally during active transcription.

Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

Structural Basis for the Asymmetry of a 4-Oxalocrotonate Tautomerase Trimer.

Tautomerase superfamily (TSF) members are constructed from a single ß-α-ß unit or two consecutively joined ß-α-ß units. This pattern prevails throughout the superfamily consisting of more than 11000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4-OT) subgroup and trimers are found in the other four subgroups. One exception is a subset of sequences that are double the length of the short 4-OTs in the 4-OT subgroup, where the coded proteins form trimers. Characterization of two members revealed an interesting dichotomy. One is a symmetric trimer, whereas the other is an asymmetric trimer. One monomer is flipped 180° relative to the other two monomers so that three unique protein-protein interfaces are created that are composed of different residues. A bioinformatics analysis of the fused 4-OT subset shows a further division into two clusters with a total of 133 sequences. The analysis showed that members of one cluster (86 sequences) have more salt bridges if the asymmetric trimer forms, whereas the members of the other cluster (47 sequences) have more salt bridges if the symmetric trimer forms. This hypothesis was examined by the kinetic and structural characterization of two proteins within each cluster. As predicted, all four proteins function as 4-OTs, where two assemble into asymmetric trimers (designated R7 and F6) and two form symmetric trimers (designated W0 and Q0). These findings can be extended to the other sequences in the two clusters in the fused 4-OT subset, thereby annotating their oligomer properties and activities.