First-line sunitinib treatment modification in patients with mRCC: nationwide analysis of the Swedish population.

Aim: Assess first-line sunitinib dosing for treatment of metastatic renal cell carcinoma in Swedish clinical practice (2006-2019).Materials & methods: Retrospective analysis of three sunitinib dosing regimens: 2-weeks on, 1-week off (2:1 Start); standard 4-weeks on, 2-weeks off (4:2) and 4:2 start with switch to 2:1 (2:1 Switch).Results: Time-to-treatment discontinuation (95% CI) differed significantly (p < 0.001): 6.2 (5.6-7.2), 13.9 (8.1-20.6) and 4.6 (4.3-5.6) months for 2:1 Start (n = 320), 2:1 Switch (n = 71) and 4:2 (n = 704), respectively. Overall survival (95% CI) differed significantly (p < 0.001): 21.8 (18.1-26.1), 32.2 (25.1-48.3) and 13.5 (12.3-15.8) months for 2:1 Start (n = 320), 2:1 Switch (n = 71) and 4:2 (n = 704), respectively.Conclusion: Alternative dosing does not compromise clinical efficacy and may provide advantages in terms of improved treatment outcomes. However, due to the changing treatment patterns during this long-term study, and the absence of patient risk category data, caution is required when interpreting the main outcomes.

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Real-world experience of second-line axitinib in metastatic renal cell carcinoma: analysis of the Swedish population.

Aim: Assess the time-to-treatment discontinuation (TTD) and overall survival (OS) in a Swedish metastatic renal cell carcinoma (mRCC) nationwide cohort who received second-line axitinib.Methods: Retrospective analysis of 110 patients with mRCC treated with second-line axitinib in Sweden (2012-2019). Patients included in the study received axitinib after mainly first-line sunitinib or pazopanib.Results: The median (95% CI) TTD of patients who received second-line axitinib was 5.2 (3.7-6.1) months with 6 (5.5%) patients still receiving treatment at the time of analysis. Median (95% CI) OS was 12.2 (7.7-14.2) months.Conclusion: The results are consistent with previous findings in mRCC and add to the evidence demonstrating efficacy of second-line axitinib, after failure of a prior anti-angiogenic therapy in a real-world setting.Clinical Trial Registration: NCT04669366 (ClinicalTrials.gov).

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Prevalence of diabetes and hospitalization due to poor glycemic control in people with bladder cancer or renal cell carcinoma in Sweden.

BACKGROUND: Bladder cancer (BC) and Renal cell carcinoma (RCC) are the most common urogenital cancers among both sexes, with a yearly global incidence of around 500 000 each. Both BC and RCC have been linked to diabetes. Poor glycemic control (malglycemia) is a serious consequence of diabetes and a possible consequence of systemic treatments used in BC and RCC. The objective of this study was to investigate the prevalence of diabetes and use of hospital-based care for malglycemia in people with BC or RCC. METHODS: This Swedish retrospective population-based register study used national health-data registers for longitudinal data on cancer incidence covering 15 years, use of hospital-based health care, and filled prescriptions of outpatient medications. Study endpoints included co-prevalence of diabetes in individuals with BC/RCC, healthcare resource utilization due to malglycemia, use of systemic corticosteroids, and changes in diabetes management for people with concomitant type 2 diabetes. RESULTS: We identified 36,620 and 15,581 individuals diagnosed with BC and RCC, respectively, between 2006 and 2019. The proportion of individuals registered with diabetes was 24% in BC and 23% in RCC. An association between BC/RCC and poor glycemic control was found, although the number of malglycemic events in hospital-based care were few (65/59 per 1000 individuals with diabetes and BC/RCC respectively with at least one event). An earlier switch to insulin-based diabetes management was observed in BC/RCC compared to matched individuals with type 2 diabetes but no cancer. The results also indicated an association between steroid treatment and poor glycemic control, and that systemic corticosteroids were more common among people with BC/RCC compared to diabetes controls. CONCLUSION: The high prevalence of diabetes and increased use of systemic corticosteroid treatment observed in this large national study highlights the need for specific clinical management, risk-assessment, and monitoring of individuals with BC/RCC and diabetes.

Anabaena sensory rhodopsin is a light-driven unidirectional rotor.

The implementation of multiconfigurational quantum chemistry methods into a quantum-mechanics/molecular-mechanics protocol has allowed the construction of a realistic computer model for the sensory rhodopsin of the cyanobacterium Anabaena PCC 7120. The model, which reproduces the absorption spectra of both the all-trans and 13-cis forms of the protein and their associated K and L intermediates, is employed to investigate the light-driven steps of the photochromic cycle exhibited by the protein. It is found that the photoisomerizations of the all-trans and 13-cis retinal chromophores occur through unidirectional, counterclockwise 180° rotations of the =C14-C15= moiety with respect to the Lys210-linked end of the chromophore axis. Thus, the sequential interconversions of the all-trans and 13-cis forms during a single photochromic cycle yield a complete (360°) unidirectional rotation of the =C14-C15= moiety. This finding implies that Anabaena sensory rhodopsin is a biological realization of a light-driven molecular rotor.

Extracellular signal-regulated kinase 8 (ERK8) controls estrogen-related receptor α (ERRα) cellular localization and inhibits its transcriptional activity.

ERK8 (MAPK15) is a large MAP kinase already implicated in the regulation of the functions of different nuclear receptors and in cellular proliferation and transformation. Here, we identify ERRα as a novel ERK8-interacting protein. As a consequence of such interaction, ERK8 induces CRM1-dependent translocation of ERRα to the cytoplasm and inhibits its transcriptional activity. Also, we identify in ERK8 two LXXLL motifs, typical of agonist-bound nuclear receptor corepressors, as necessary features for this MAP kinase to interact with ERRα and to regulate its cellular localization and transcriptional activity. Ultimately, we demonstrate that ERK8 is able to counteract, in immortalized human mammary cells, ERRα activation induced by the EGF receptor pathway, often deregulated in breast cancer. Altogether, these results reveal a novel function for ERK8 as a bona fide ERRα corepressor, involved in control of its cellular localization by nuclear exclusion, and suggest a key role for this MAP kinase in the regulation of the biological activities of this nuclear receptor.

Initial excited-state relaxation of the bilin chromophores of phytochromes: a computational study.

The geometric relaxation following light absorption of the biliverdin, phycocyanobilin and phytochromobilin tetrapyrrole chromophores of bacterial, cyanobacterial and plant phytochromes has been investigated using density functional theory methods. Considering stereoisomers relevant for both red-absorbing Pr and far-red-absorbing Pfr forms of the photoreceptor, it is found that the initial excited-state evolution is dominated by torsional motion at the C10-C11 bond. This holds true for all three chromophores and irrespective of which configuration the chromophores adopt. This finding suggests that the photochromic cycling of phytochromes between their Pr and Pfr forms, which is known to be governed by Z/E photoisomerizations at the C15-C16 bond, relies on interactions between the chromophore and the protein to prevent photoisomerizations at C10-C11. Further, it is found that the uneven distribution of positive charge between the pyrrole rings is a major factor for the photochemical reactivity of the C10-C11 bond.

Excited-state modeling of the astaxanthin dimer predicts a minor contribution from exciton coupling to the bathochromic shift in crustacyanin.

The >0.50 eV bathochromic shift of the absorption by the carotenoid astaxanthin in crustacyanin, the carotenoprotein responsible for the coloration of lobster shell, is believed to result from ground-state polarization of the chromophore and/or exciton coupling between the two chromophores of each protein subunit. Here, the contribution of exciton coupling to the shift is quantified using quantum chemical calculations that do not rely on the dipole-dipole approximation but rather consider the full astaxanthin dimer in the geometry relevant for the protein-bound state. Employing a variety of methods, it is found that the exciton coupling amounts to 0.04 eV only. Furthermore, even when more closely aggregated dimers than the one in the protein are considered, the predicted couplings remain small (0.05-0.09 eV). These findings demonstrate that the bathochromic shift cannot be explained in terms of exciton coupling. It is therefore argued that polarization is likely to be the dominant mechanism, a notion supported by the fact that calculations carried out at the same levels of theory identify a scenario (hydrogen bonding with a histidine residue) whose contribution to the shift vastly exceeds that of exciton coupling.

Relationship between the excited state relaxation paths of rhodopsin and isorhodopsin.

The pigment Isorhodopsin, an analogue of the visual pigment Rhodopsin, is investigated via quantum-mechanics/molecular-mechanics computations based on an ab initio multiconfigurational quantum chemical treatment. The limited <5 kcal mol(-1) error found for the spectral parameters allows for a nearly quantitative analysis of the excited-state structure and reactivity of its 9-cis-retinal chromophore. We demonstrate that, similar to Rhodopsin, Isorhodopsin features a shallow photoisomerization path. However, the structure of the reaction coordinate appears to be reversed. In fact, while the coordinate still corresponds to an asynchronous crankshaft motion, the dominant isomerization component involves a counterclockwise, rather than clockwise, twisting of the 9-cis bond. Similarly, the minor component involves a clockwise, rather than counterclockwise, twisting of the 11-trans bond. Ultimately, these results indicate that Rhodopsin and Isorhodopsin relax along a common excited-state potential energy valley starting from opposite ends. The fact that the central and lowest energy region of such valley runs along a segment of the intersection space between the ground and excited states of the protein explains why the pigments decay at distinctive conical intersection structures.

EGFR-Targeted Magnetic Nanovectors Recognize, in Vivo, Head and Neck Squamous Cells Carcinoma-Derived Tumors.

Head and neck squamous cell carcinomas (HNSCC) are a diverse group of tumors with high morbidity and mortality that have remained mostly unchanged over the past decades. The epidermal growth factor receptor (EGFR) is often overexpressed and activated in these tumors and strongly contributes to their pathogenesis. Still, EGFR-targeted therapies such as monoclonal antibodies and kinase inhibitors have demonstrated only limited improvements in the clinical outcome of this disease. Here, we take advantage of the extraordinary affinity of EGF for its cognate receptor to specifically target magnetite-containing nanoparticles to HNSCC cells and mediate, in vitro, their cellular upload. On the basis of this, we show efficient accumulation, in vivo, of such nanoparticles in subcutaneous xenograft tumor tissues in sufficient amounts to be able to mediate visualization by magnetic resonance imaging. Overall, our EGF-coated nanosystem may warrant, in the near future, novel and very efficient theranostic approaches to HNSCC.

MAPK15 upregulation promotes cell proliferation and prevents DNA damage in male germ cell tumors.

Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest.To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase.In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a "stress support" autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors.

MAPK15 mediates BCR-ABL1-induced autophagy and regulates oncogene-dependent cell proliferation and tumor formation.

A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel "druggable" link between BCR-ABL1 and human CML.

Acidic extracellular pH neutralizes the autophagy-inhibiting activity of chloroquine: implications for cancer therapies.

Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared with cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.

Structure prediction and validation of the ERK8 kinase domain.

Extracellular signal-regulated kinase 8 (ERK8) has been already implicated in cell transformation and in the protection of genomic integrity and, therefore, proposed as a novel potential therapeutic target for cancer. In the absence of a crystal structure, we developed a three-dimensional model for its kinase domain. To validate our model we applied a structure-based virtual screening protocol consisting of pharmacophore screening and molecular docking. Experimental characterization of the hit compounds confirmed that a high percentage of the identified scaffolds was able to inhibit ERK8. We also confirmed an ATP competitive mechanism of action for the two best-performing molecules. Ultimately, we identified an ERK8 drug-resistant "gatekeeper" mutant that corroborated the predicted molecular binding mode, confirming the reliability of the generated structure. We expect that our model will be a valuable tool for the development of specific ERK8 kinase inhibitors.

MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.

Protein Influence on Electronic Spectra Modeled by Multipoles and Polarizabilities.

We have developed automatic methods to calculate multipoles and anisotropic polarizabilities for all atoms and bond centers in a protein and to include such a model in the calculation of electronic properties at any level of quantum mechanical theory. This approach is applied for the calculation of the electronic spectra of retinal in rhodopsin at the CASPT2//CASSCF level (second-order multiconfigurational perturbation theory) for the wild-type protein, as well as two mutants and isorhodopsin in QM/MM structures based on two crystal structures. We also perform a detailed investigation of the importance and distance dependence of the multipoles and the polarizabilities for both the absolute and the relative absorption energies. It is shown that the model of the surrounding protein strongly influences the spectrum and that different models give widely different results. For example, the Amber 1994 and 2003 force fields give excitation energies that differ by up to 16 kJ/mol. For accurate excitation energies, multipoles up to quadrupoles and anisotropic polarizabilities are needed. However, interactions with residues more than 10 Å from the chromophore can be treated with a standard polarizable force field without any dipoles or quadrupoles.

The color of rhodopsins at the ab initio multiconfigurational perturbation theory resolution.

We demonstrate that "brute force" quantum-mechanics/molecular-mechanics computations based on ab initio (i.e., first principles) multiconfigurational perturbation theory can reproduce the absorption maxima of a set of modified bovine rhodopsins with an accuracy allowing for the analysis of the factors determining their colors. In particular, we show that the theory accounts for the changes in excitation energy even when the proteins display the same charge distribution. Three color-tuning mechanisms, leading to changes of close magnitude, are demonstrated to operate in these conditions. The first is based on the change of the conformation of the conjugated backbone of the retinal chromophore. The second operates through the control of the distance between the positive charge residing on the chromophore and the carboxylate counterion. Finally, the third mechanism operates through the changes in orientation of the chromophore relative to the protein. These results offer perspectives for the unbiased computational design of mutants or chemically modified proteins with wanted optical properties.