Probing the Dissociation Pathway of a Kinetically Labile Transthyretin Mutant.

Aggregation of transthyretin (TTR) is associated with devastating amyloid diseases. Amyloidosis begins with the dissociation of the native homotetramer (a dimer of dimers) to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the process by which TTR dissociates and reassembles at neutral pH remains poorly characterized due to the low population of intermediates. Here, we use 19F-nuclear magnetic resonance (NMR) and a highly sensitive trifluoromethyl probe to determine the relative populations of the species formed by the dissociation of a destabilized variant, A25T. The A25T mutation perturbs both the strong dimer and weak dimer-dimer interfaces. A tetramer ⇌ dimer ⇌ monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. Thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by van't Hoff and 19F-NMR line shape analysis, saturation transfer, and transition state theory. Chemical shifts for the dimer and T* species are degenerate for 19F and methyl probes close to the strong dimer interface, implicating interfacial perturbation as a common structural feature of these destabilized species. All-atom molecular dynamics simulations further suggest more frequent F87 ring flipping on the nanosecond time scale in the A25T dimer than in the native A25T tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

NMR Measurements Reveal the Structural Basis of Transthyretin Destabilization by Pathogenic Mutations.

Inherited mutations of transthyretin (TTR) destabilize its structure, leading to aggregation and familial amyloid disease. Although numerous crystal structures of wild-type (WT) and mutant TTRs have been determined, they have failed to yield a comprehensive structural explanation for destabilization by pathogenic mutations. To identify structural and dynamic variations that are not readily observed in the crystal structures, we used NMR to study WT TTR and three kinetically and/or thermodynamically destabilized pathogenic variants (V30M, L55P, and V122I). Sequence-corrected chemical shifts reveal important structural differences between WT and mutant TTR. The L55P mutation linked to aggressive early onset cardiomyopathy and polyneuropathy induces substantial structural perturbations in both the DAGH and CBEF ß-sheets, whereas the V30M polyneuropathy-linked substitution perturbs primarily the CBEF sheet. In both variants, the structural perturbations propagate across the entire width of the ß-sheets from the site of mutation. Structural changes caused by the V122I cardiomyopathy-associated mutation are restricted to the immediate vicinity of the mutation site, directly perturbing the subunit interfaces. NMR relaxation dispersion measurements show that WT TTR and the three pathogenic variants undergo millisecond time scale conformational fluctuations to populate a common excited state with an altered structure in the subunit interfaces. The excited state is most highly populated in L55P. The combined application of chemical shift analysis and relaxation dispersion to these pathogenic variants reveals differences in ground state structure and in the population of a transient excited state that potentially facilitates tetramer dissociation, providing new insights into the molecular mechanism by which mutations promote TTR amyloidosis.

Conformational Dynamics of an Amyloidogenic Intermediate of Transthyretin: Implications for Structural Remodeling and Amyloid Formation.

The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer (a dimer of dimers) and partial misfolding of the resulting monomer, which assembles into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. NMR relaxation dispersion offers a unique opportunity to characterize amyloidogenic intermediates when they exchange on favorable timescales with NMR-visible ground states. Here we use NMR to characterize the structure and conformational dynamics of the monomeric F87E mutant of human TTR. Chemical shifts derived from analysis of multinuclear relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s-1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both ß-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH ß-sheet, from the inner ß-strand H, which forms the strong dimer-dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer-dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.

Cryo-Fluorescence Tomography as a Tool for Visualizing Whole-Body Inflammation Using Perfluorocarbon Nanoemulsion Tracers.

PURPOSE: We explore the use of intravenously delivered fluorescent perfluorocarbon (PFC) nanoemulsion tracers and multi-spectral cryo-fluorescence tomography (CFT) for whole-body tracer imaging in murine inflammation models. CFT is an emerging technique that provides high-resolution, three-dimensional mapping of probe localization in intact animals and tissue samples, enabling unbiased validation of probe biodistribution and minimizes reliance on laborious histological methods employing discrete tissue panels, where disseminated populations of PFC-labeled cells may be overlooked. This methodology can be used to streamline the development of new generations of non-invasive, cellular-molecular imaging probes for in vivo imaging. PROCEDURES: Mixtures of nanoemulsions with different fluorescent emission wavelengths were administered intravenously to naïve mice and models of acute inflammation, colitis, and solid tumor. Mice were euthanized 24 h post-injection, frozen en bloc, and imaged at high resolution (~ 50 µm voxels) using CFT at multiple wavelengths. RESULTS: PFC nanoemulsions were visualized using CFT within tissues of the reticuloendothelial system and inflammatory lesions, consistent with immune cell (macrophage) labeling, as previously reported in in vivo magnetic resonance and nuclear imaging studies. The CFT signals show pronounced differences among fluorescence wavelengths and tissues, presumably due to autofluorescence, differential fluorescence quenching, and scattering of incident and emitted light. CONCLUSIONS: CFT is an effective and complementary methodology to in vivo imaging for validating PFC nanoemulsion biodistribution at high spatial localization, bridging the resolution gap between in vivo imaging and histology.

Method for estimation of apoptotic cell fraction of cytotherapy using in vivo fluorine-19 magnetic resonance: pilot study in a patient with head and neck carcinoma receiving tumor-infiltrating lymphocytes labeled with perfluorocarbon nanoemulsion.

BACKGROUND: Adoptive transfer of T cells is a burgeoning cancer therapeutic approach. However, the fate of the cells, once transferred, is most often unknown. We describe the first clinical experience with a non-invasive biomarker to assay the apoptotic cell fraction (ACF) after cell therapy infusion, tested in the setting of head and neck squamous cell carcinoma (HNSCC). A patient with HNSCC received autologous tumor-infiltrating lymphocytes (TILs) labeled with a perfluorocarbon (PFC) nanoemulsion cell tracer. Nanoemulsion, released from apoptotic cells, clears through the reticuloendothelial system, particularly the Kupffer cells of the liver, and fluorine-19 (19F) magnetic resonance spectroscopy (MRS) of the liver was used to non-invasively infer the ACF. METHODS: Autologous TILs were isolated from a patient in their late 50s with relapsed, refractory human papillomavirus-mediated squamous cell carcinoma of the right tonsil, metastatic to the lung. A lung metastasis was resected for T cell harvest and expansion using a rapid expansion protocol. The expanded TILs were intracellularly labeled with PFC nanoemulsion tracer by coincubation in the final 24 hours of culture, followed by a wash step. At 22 days after intravenous infusion of TILs, quantitative single-voxel liver 19F MRS was performed in vivo using a 3T MRI system. From these data, we model the apparent ACF of the initial cell inoculant. RESULTS: We show that it is feasible to PFC-label ~70×1010 TILs (F-TILs) in a single batch in a clinical cell processing facility, while maintaining >90% cell viability and standard flow cytometry-based release criteria for phenotype and function. Based on quantitative in vivo 19F MRS measurements in the liver, we estimate that ~30% cell equivalents of adoptively transferred F-TILs have become apoptotic by 22 days post-transfer. CONCLUSIONS: Survival of the primary cell therapy product is likely to vary per patient. A non-invasive assay of ACF over time could potentially provide insight into the mechanisms of response and non-response, informing future clinical studies. This information may be useful to developers of cytotherapies and clinicians as it opens an avenue to quantify cellular product survival and engraftment.

An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain.

The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250-300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain-a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.

Assessing Oximetry Response to Chimeric Antigen Receptor T-cell Therapy against Glioma with 19F MRI in a Murine Model.

Purpose: To assess the cell-specific, intracellular partial pressure of oxygen (Po2) dynamics of both tumor and chimeric antigen receptor (CAR) T cells in a murine immunotherapy model. Materials and Methods: Human glioblastoma cells or human T cells were intracellularly labeled with perfluorocarbon nanoemulsion droplet sensors prior to in vivo injection in severe combined immunodeficient mice to measure Po2 in the two cell types in response to treatment. Two main sets of experiments were performed: (a) mice were injected in the flank with perfluorocarbon-labeled human glioblastoma cells and were then inoculated with either CAR T cells or untransduced T cells or were untreated 5 days after tumor inoculation; and (b) mice with unlabeled glioblastoma tumors were inoculated with perfluorocarbon-labeled CAR T cells or untransduced T cells 5 days after tumor inoculation. Longitudinal fluorine 19 (19F) spin-lattice relaxation time measurements of the tumor mass were used to ascertain absolute Po2 in vivo. Results were analyzed for significance using an analysis of variance, a linear mixed-effect model, and a Pearson correlation coefficient test, as appropriate. Results: The intracellular tumor cell Po2 temporal dynamics exhibited delayed, transient hyperoxia at 3 days after infusion of CAR T cells, commensurate with significant tumor cell killing and CAR T-cell infiltration, as observed by bioluminescence imaging and histologic findings. Conversely, no significant changes were detected in CAR or untransduced T-cell intracellular Po2 over time in tumor using these same methods. Moreover, it was observed that the total 19F tumor cell signal quenches with treatment, consistent with rapid tissue clearance of probe from apoptotic tumor cells. Conclusion: Cell-specific Po2 measurements using perfluorocarbon probes can provide insights into effector cell function and tumor response in cellular immunotherapeutic cancer models.Keywords: Animal Studies, MR-Imaging, MR-Spectroscopy, Molecular Imaging-Cancer, Molecular Imaging-Immunotherapy Supplemental material is available for this article. © RSNA, 2021See also commentary by Bulte in this issue.

Metallofluorocarbon Nanoemulsion for Inflammatory Macrophage Detection via PET and MRI.

Inflammation is associated with a range of serious human conditions, including autoimmune and cardiovascular diseases and cancer. The ability to image active inflammatory processes greatly enhances our ability to diagnose and treat these diseases at an early stage. We describe molecular compositions enabling sensitive and precise imaging of inflammatory hotspots in vivo. Methods: A functionalized nanoemulsion with a fluorocarbon-encapsulated radiometal chelate (FERM) was developed to serve as a platform for multimodal imaging probe development. The 19F-containing FERM nanoemulsion encapsulates 89Zr in the fluorous oil via a fluorinated hydroxamic acid chelate. Simple mixing of the radiometal with the preformed aqueous nanoemulsion before use yields FERM, a stable in vivo cell tracer, enabling whole-body 89Zr PET and 19F MRI after a single intravenous injection. Results: The FERM nanoemulsion was intrinsically taken up by phagocytic immune cells, particularly macrophages, with high specificity. FERM stability was demonstrated by a high correlation between the 19F and 89Zr content in the blood (correlation coefficient > 0.99). Image sensitivity at a low dose (37 kBq) was observed in a rodent model of acute infection. The versatility of FERM was further demonstrated in models of inflammatory bowel disease and 4T1 tumor. Conclusion: Multimodal detection using FERM yields robust whole-body lesion detection and leverages the strengths of combined PET and 19F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases.

BCOR Binding to MLL-AF9 Is Essential for Leukemia via Altered EYA1, SIX, and MYC Activity.

MLL is a target of chromosomal translocations in acute leukemias with poor prognosis. The common MLL fusion partner AF9 (MLLT3) can directly bind to AF4, DOT1L, BCOR, and CBX8. To delineate the relevance of BCOR and CBX8 binding to MLL-AF9 for leukemogenesis, here we determine protein structures of AF9 complexes with CBX8 and BCOR, and show that binding of all four partners to AF9 is mutually exclusive. Using the structural analyses, we identify point mutations that selectively disrupt AF9 interactions with BCOR and CBX8. In bone marrow stem/progenitor cells expressing point mutant CBX8 or point mutant MLL-AF9, we show that disruption of direct CBX8/MLL-AF9 binding does not impact in vitro cell proliferation, whereas loss of direct BCOR/MLL-AF9 binding causes partial differentiation and increased proliferation. Strikingly, loss of MLL-AF9/BCOR binding abrogated its leukemogenic potential in a mouse model. The MLL-AF9 mutant deficient for BCOR binding reduces the expression of the EYA1 phosphatase and the protein level of c-Myc. Reduction in BCOR binding to MLL-AF9 alters a MYC-driven gene expression program, as well as altering expression of SIX-regulated genes, likely contributing to the observed reduction in the leukemia-initiating cell population.

Tankyrase inhibition sensitizes cells to CDK4 blockade.

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.

Distinct Colorectal Cancer-Associated APC Mutations Dictate Response to Tankyrase Inhibition.

The majority of colorectal cancers show hyperactivated WNT signaling due to inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor. Genetically restoring APC suppresses WNT and induces rapid and sustained tumor regression, implying that reengaging this endogenous tumor-suppressive mechanism may be an effective therapeutic strategy. Here, using new animal models, human cell lines, and ex vivo organoid cultures, we show that tankyrase (TNKS) inhibition can control WNT hyperactivation and provide long-term tumor control in vivo, but that effective responses are critically dependent on how APC is disrupted. Mutant APC proteins truncated within the mutation cluster region physically engage the destruction complex and suppress the WNT transcriptional program, while APC variants with early truncations (e.g., Apc Min) show limited interaction with AXIN1 and ß-catenin, and do not respond to TNKS blockade. Together, this work shows that TNKS inhibition, like APC restoration, can reestablish endogenous control of WNT/ß-catenin signaling, but that APC genotype is a crucial determinant of this response. SIGNIFICANCE: This study reveals how subtle changes to the mutations in a critical colorectal tumor suppressor, APC, influence the cellular response to a targeted therapy. It underscores how investigating the specific genetic alterations that occur in human cancer can identify important biological mechanisms of drug response and resistance.This article is highlighted in the In This Issue feature, p. 1325.

Wnt Signaling and Colorectal Cancer.

The WNT signaling pathway is a critical mediator of tissue homeostasis and repair, and frequently co-opted during tumor development. Almost all colorectal cancers (CRC) demonstrate hyperactivation of the WNT pathway, which in many cases is believed to be the initiating and driving event. In this short review, we provide a focused overview of recent developments in our understanding of the WNT pathway in CRC, describe new research tools that are enabling a deeper understanding of WNT biology, and outline ongoing efforts to target this pathway therapeutically.

Mechanisms of resistance to systemic therapy in metastatic castration-resistant prostate cancer.

Patients with metastatic castration-resistant prostate cancer (mCPRC) now have an unprecedented number of approved treatment options, including chemotherapies (docetaxel, cabazitaxel), androgen receptor (AR)-targeted therapies (enzalutamide, abiraterone), a radioisotope (radium-223) and a cancer vaccine (sipuleucel-T). However, the optimal treatment sequencing pathway is unknown, and this problem is exacerbated by the issues of primary and acquired resistance. This review focuses on mechanisms of resistance to AR-targeted therapies and taxane-based chemotherapy. Patients treated with abiraterone, enzalutamide, docetaxel or cabazitaxel may present with primary resistance, or eventually acquire resistance when on treatment. Multiple resistance mechanisms to AR-targeted agents have been proposed, including: intratumoral androgen production, amplification, mutation, or expression of AR splice variants, increased steroidogenesis, upregulation of signals downstream of the AR, and development of androgen-independent tumor cells. Known mechanisms of resistance to chemotherapy are distinct, and include: tubulin alterations, increased expression of multidrug resistance genes, TMPRSS2-ERG fusion genes, kinesins, cytokines, and components of other signaling pathways, and epithelial-mesenchymal transition. Utilizing this information, biomarkers of resistance/response have the potential to direct treatment decisions. Expression of the AR splice variant AR-V7 may predict resistance to AR-targeted agents, but available biomarker assays are yet to be prospectively validated in the clinic. Ongoing prospective trials are evaluating the sequential use of different drugs, or combination regimens, and the results of these studies, combined with a deeper understanding of mechanisms of primary and acquired resistance to treatment, have the potential to drive future treatment decisions in mCRPC.

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis.

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ~3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering.

Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4.

Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.

Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding.

Mixed lineage leukemia (MLL) fusion proteins cause oncogenic transformation of hematopoietic cells by constitutive recruitment of elongation factors to HOX promoters, resulting in overexpression of target genes. The structural basis of transactivation by MLL fusion partners remains undetermined. We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding. We determined the structure of the AF9 AHD in complex with the elongation factor AF4 and show that aliphatic residues, which are conserved in each of the AF9 binding partners, form an integral part of the hydrophobic core of the complex. Nuclear magnetic resonance relaxation measurements show that AF9 retains significant dynamic behavior which may facilitate exchange between disordered partners. We propose that AF9 functions as a signaling hub that regulates transcription through dynamic recruitment of cofactors in normal hematopoiesis and in acute leukemia.

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production.

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea.

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102-153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141-236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53-76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.