Search

Biallelic mutations in MAPKBP1 were recently associated with late-onset cilia-independent nephronophthisis. MAPKBP1 was found at mitotic spindle poles but could not be detected at primary cilia or centrosomes. Here, by identification and characterization of novel MAPKBP1 variants, we aimed at further investigating its role in health and disease. Genetic analysis was done by exome sequencing, homozygosity mapping, and a targeted kidney gene panel while coimmunoprecipitation was used to explore wild-type and mutant protein-protein interactions. Expression of MAPKBP1 in non-ciliated HeLa and ciliated inner medullary collecting duct cells enabled co-localization studies by fluorescence microscopy. By next generation sequencing, we identified two novel homozygous MAPKBP1 splice-site variants in patients with nephronophthisis-related chronic kidney disease. Splice-site analyses revealed truncation of C-terminal coiled-coil domains and patient-derived deletion constructs lost their ability to homodimerize and heterodimerize with paralogous WDR62. While wild-type MAPKBP1 exhibited centrosomal, basal body, and microtubule association, mutant proteins lost the latter and showed reduced recruitment to cell cycle dependent centriolar structures. Wild-type and mutant proteins had no reciprocal influence upon co-expression excluding dominant negative effects. Thus, MAPKBP1 appears to be a novel microtubule-binding protein with cell cycle dependent centriolar localization. Truncation of its coiled-coil domain is enough to abrogate its dimerization and results in severely disturbed intracellular localizations. Delineating the impact of impaired dimerization on cell cycle regulation and intracellular kidney signaling may provide new insights into common mechanisms of kidney degeneration. Thus, due to milder clinical presentation, MAPKBP1-associated nephronophthisis should be considered in adult patients with otherwise unexplained chronic kidney disease.

Acute lymphoblastic leukemia (ALL) can relapse in the extramedullary compartment, with or without medullary involvement. Response to treatment may be individual. We evaluated response to inotuzumab ozogamicin in 31 patients with relapsed/refractory B-ALL with extramedullary disease. Median age was 31 years (range, 19-81). All patients were heavily pretreated, including allogeneic hematopoietic stem cell transplantation (HSCT; n=18). Overall response rate after two cycles of inotuzumab ozogamicin was 84% (complete remission, 55%; partial remission, 29%; resistant disease, 13%; early death, 3%). The median follow-up was 29 months and median overall survival was 12.8 months. One-year and 2-year overall survival rates were 53% (95% CI: 37-76%) and 18% (95% CI: 8-43%), respectively. Age had no impact on overall survival when assessed as a continuous variable or dichotomized at 60 years. Twelve patients proceeded to allogeneic HSCT (complete remission, n=6; partial remission, n=3; resistant disease, n=3). Prior to allogeneic HSCT, eight patients received two or fewer cycles and four patients received three or four cycles of inotuzumab ozogamicin. Sinusoidal obstruction syndrome was reported in three patients, including one after transplantation. Allogeneic HSCT, evaluated as a time-dependent variable, had no impact on overall survival. Inotuzumab ozogamicin seems to be effective as a debulking strategy in relapsed/refractory ALL with extramedullary disease. However, inotuzumab ozogamicin followed by allogeneic HSCT seems not to be effective in maintaining long-term disease control.

Controlled release of active biomolecules is an attractive approach to modulate chemotactic gradients and accordingly the recruitment of cells, e.g. endothelial progenitor cells to improve wound healing or stimulate angiogenesis after myocardial infarction. Here, we developed variants of hCXCL12, also named stromal cell-derived factor 1α, a chemokine that activates the CXCR4 and consequently recruits tissue specific stem and progenitor cells. hCXCL12 variants were designed to bind to glycosaminoglycans (GAGs) with different affinities in order to modulate its release. Sixteen analogs were recombinantly produced, characterized, and tested for their GAG-binding property. The most promising variants hCXCL12 K24/K27/R41/R47A and hCXCL12 Q48K were used for release studies from starPEG-heparin-hydrogels. The reduced GAG affinity led to a fast release of hCXCL12 K24/K27/R41/R47A, whereas hCXCL12 Q48K was slowly released over 2 weeks due to its increased binding strength compared to wild type hCXCL12. Migration of Jurkat cells and early endothelial progenitor cells was proven to demonstrate the applicability of the approach to endogenously CXCR4 expressing cell types. Thus, this work offers new options for enhancing chemotactic hCXCL12 gradients by a combination of native and modified hCXCL12 variants to improve and prolong the recruitment of CXCR4-positive stem and progenitor cells to injured sites.

High binding affinities of GAG toward extracellular regulatory proteins are governed by recognition diversity, sulfation pattern, length, and anomeric functionalization., Binding of sulfated glycosaminoglycans (GAG) to a wide spectrum of extracellular regulatory proteins is crucial for physiological processes such as cell growth, migration, tissue homeostasis and repair. Thus, GAG derivatives exhibit great relevance in the development of innovative biomaterials for tissue regeneration therapies. We present a synthetic strategy for the preparation of libraries of defined sulfated oligohyaluronans as model GAG systematically varied in length, sulfation pattern and anomeric substitution in order to elucidate the effects of these parameters on GAG recognition by regulatory proteins. Through an experimental and computational approach using fluorescence polarization, ITC, docking and molecular dynamics simulations we investigate the binding of these functionalized GAG derivatives to ten representative regulatory proteins including IL-8, IL-10, BMP-2, sclerostin, TIMP-3, CXCL-12, TGF-β, FGF-1, FGF-2, and AT-III, and we establish structure–activity relationships for GAG recognition. Binding is mainly driven by enthalpy with only minor entropic contributions. In several cases binding is determined by GAG length, and in all cases by the position and number of sulfates. Affinities strongly depend on the anomeric modification of the GAG. Highest binding affinities are effected by anomeric functionalization with large fluorophores and by GAG dimerization. Our experimental and theoretical results suggest that the diversity of GAG binding sites and modes is responsible for the observed high affinities and other binding features. The presented new insights into GAG–protein recognition will be of relevance to guide the design of GAG derivatives with customized functions for the engineering of new biomaterials.

Background: Extramedullary B-cell acute lymphoblastic leukemia (EM-ALL) is a rare occurrence characterized by dismal outcome and no defined therapeutic approach. The monoclonal antibody inotuzumab ozogamicin (InO) is approved for treatment of CD22+ B-ALL. Aims: To characterize a series of relapsed/refractory (r/r) adult EM-ALL patients (pts) and evaluate outcome after treatment with InO. Methods: We studied 31 r/r pts (median age, 31 years; range, 19-81 years), who were treated with InO between 2015 and 2021 within a compassionate use program (n=7) or on-label after FDA or EMA approval (n=24) All pts were CD22 positive at relapse/progressive disease. Up to 6 InO cycles (≤2 cycles, n=19; 3-4 cycles, n=7; 5-6 cycles, n=5) were administered according to the previously approved regimen. EM response assessment was performed by CT or PET-CT. Prior therapy consisted of intensive chemotherapy +/- tyrosine kinase inhibitors. Allogeneic hematopoietic stem cell transplantation (allo-SCT) was performed in 18 pts (first line or at relapse, n=9, each). Prior to InO, blinatumomab was administered in n=14 and local irradiation in n=5 pts. Results: Overall, pts had in median 2 EM manifestations (range, 1-9). Localization of EM disease is shown in Table 1. In addition to EM disease, n=16 (52%) pts had a relapse in bone marrow. At the time of r/r EM-ALL median white blood cell and platelet counts were 5.1/nl (range, 0.04-24.7/nl) and 110.5/nl (range, 6-337/nl), respectively. Fifteen pts (48%) were female; ECOG was ≤ 2 in 29 pts and 3 in 2 pts. Cytogenetic analysis at the time of r/r EM-ALL was available in 13 (42%) pts. Of those, 6 pts had a normal karyotype, 4 were complex, 2 pts displayed a t(9;22) and 1 had an additional X-chromosome. Seven (23%) of the 31 pts had no response assessment after the first induction cycle including 1 patient who died at day 11 of the first InO cycle due to cerebral hemorrhage. Complete remission assessed by PET-CT (CR; including EM and hematological/bone marrow CR) after the first InO cycle was achieved in 10 of 24 assessed pts (42%), 9 pts (37.5%) had a partial remission (PR), 2 (8%) had stable disease (SD) and 3 (12.5%) showed resistant/progressive disease (RD/PD). After 2 InO cycles, CR was achieved in 17 of 31 pts (55%), PR in 9 (29%); 1 patient (3%) experienced early death and 4 pts with SD+RD/PD did not receive further InO treatment (13%). Median follow-up was 29 months (95%-CI, 21 months - not reached) and median overall survival (OS) 12.8 months (95%-CI, 9.9-16.2 months; Figure 1). One-year and 2-years OS rates were 53% (95%-CI, 37-76%) and 18% (95%-CI, 8-43%), respectively. In Cox regression analysis age as a continuous variable had no impact on OS (P=0.83). This was also true when using 60 years as cut-off (P=0.2). Twelve pts went on to allo-SCT (CR, n=6; PR, n=3; PD, n=3). Prior to allo-SCT 8 pts received ≤2 InO cycles and 4 pts ≤4 cycles. Sinusoidal obstruction syndrome (SOS) was reported in 1 patient after transplant; conditioning in this patient consisted of treosulfan/fludarabine/thiotepa. In pts achieving a CR after InO treatment (n=16), median OS was 10 months with no difference (P=0.80) in relapse-free survival (RFS) if an allo-SCT was performed (n=6) or not (n=10). There was no difference on OS (P=0.08) or RFS (P=0.2) if pts had EM manifestations only as compared to EM disease and bone marrow involvement. In patients with CR/PR after InO treatment, relapse occurred in 10 of 26 pts (38%; after allo-SCT, n=3); of those, all except than one succumbed of their disease. Two pts died in remission (sepsis, SOS/multi-organ failure, n=1; each). One patient experienced a molecular relapse, which could be successfully treated with InO again. Ten pts are in ongoing CR (n=9) or PR (n=1), including the patient with prior molecular relapse and InO re-exposure. Conclusions: This outcome analysis demonstrates that treatment with InO is an effective and promising approach in r/r-ALL patients with EM disease. However, allo-SCT alone seems not to be effective in maintaining disease control. Thus, autologous chimeric antigen receptor T-cells or advanced bi-specific antibodies as consolidation therapy should be evaluated in the future. Figure 1 Figure 1. Disclosures Webster: Pfizer: Consultancy; AmGen: Consultancy. Brunner: Keros Therapeutics: Consultancy; GSK: Research Funding; AstraZeneca: Research Funding; Aprea: Research Funding; Agios: Consultancy; Acceleron: Consultancy; Janssen: Research Funding; Takeda: Consultancy, Research Funding; BMS/Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Levis: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Astellas Pharma, Daiichi-Sankyo, FujiFilm, and Menarini: Honoraria; Pfizer: Consultancy, Honoraria; Takeda: Honoraria; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Honoraria; Astellas and FujiFilm: Research Funding. Schlenk: Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding; Abbvie: Honoraria. Papayannidis: Pfizer, Amgen, Novartis: Honoraria.

The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule.

Protein ligation allows the introduction of a wide range of modifications into proteins that are not accessible by mutagenesis. This includes non-proteinogenic amino acids and even backbone modification. This review summarizes recent reports on modified chemokine variants by ligation technologies and includes the development of the first protein with a full secondary structure motif exchanged by a helix that exclusively consists of β-amino acids. Furthermore the first protein activatable by light by rearrangement of a depsi-peptide bond is described. Combining different ligation methods, immobilization and specific release of chemokines were achieved, which is of major importance for the gradient forming activity of chemokines. Examples are shown for CXCL8 (interleukin 8, IL-8) and CXCL12 (stromal derived factor 1, SDF 1) including their chemical and structural characterization as well as the most frequently used assays.