RESUMO
BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Células Clonais , Frequência do GeneRESUMO
BACKGROUND: Individuals with germline BRCA1 and BRCA2 pathogenic variants (BRCA carriers) are at high risk of developing high grade serous ovarian carcinoma (HGSC). HGSC is predominantly driven by TP53 mutations, but mutations in this gene are also commonly found in non-cancerous tissue as a feature of normal human aging. We hypothesized that HGSC predisposition in BRCA carriers may be related to increased TP53 somatic evolution, which could be detectable by ultra-deep sequencing of TP53 mutations in gynecological liquid biopsies. METHODS: Duplex sequencing was used to identify TP53 mutations with high sensitivity in peritoneal washes and cervical liquid-based cytology (LBC) collected at surgery from 60 individuals including BRCA1 and BRCA2 carriers, and non-carriers. TP53 mutation pathogenicity was compared across groups and with TP53 cancer mutations. RESULTS: TP53 mutations were more abundant in cervical LBC than in peritoneal washes but increased with age in both sample types. In peritoneal washes, but not in cervical LBC, pathogenic TP53 mutation burden was increased in BRCA1 carriers compared to non-carriers, independently of age. Five individuals shared identical pathogenic TP53 mutations in peritoneal washes and cervical LBC, but not in blood. CONCLUSIONS: Ultra-deep sequencing of TP53 mutations in peritoneal washes collected at surgery reveals increased burden of pathogenic TP53 mutations in BRCA1 carriers. This excess of pathogenic TP53 mutations might be linked to the elevated risk of HGSC in these individuals. In some patients, concordant TP53 mutations were found in peritoneal washes and cervical LBCs, but the cell of origin remains unknown and deserves further investigation.
RESUMO
Wnt signaling has a well-established role as a regulator of nervous system development, but its role in the maintenance and regulation of established synapses in the mature brain remains poorly understood. At excitatory glutamatergic synapses, NMDA receptors (NMDARs) have a fundamental role in synaptogenesis, synaptic plasticity, and learning and memory; however, it is not known what controls their number and subunit composition. Here we show that the receptor tyrosine kinase-like orphan receptor 2 (RoR2) functions as a Wnt receptor required to maintain basal NMDAR-mediated synaptic transmission. In addition, RoR2 activation by a noncanonical Wnt ligand activates PKC and JNK and acutely enhances NMDAR synaptic responses. Regulation of a key component of glutamatergic synapses through RoR2 provides a mechanism for Wnt signaling to modulate synaptic transmission, synaptic plasticity, and brain function acutely beyond embryonic development.
Assuntos
Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica , Proteínas Wnt/metabolismo , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Expressão Gênica , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Hibridização In Situ , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Microscopia Confocal , Técnicas de Patch-Clamp , Proteína Quinase C/metabolismo , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Interferência de RNA , Ratos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Somatic TP53 mutations are prevalent in normal tissue but little is known about their association with cancer risk. Cervical liquid-based cytology (LBC), commonly known as Pap test, provides an accessible gynecological sample to test the value of TP53 somatic mutations as a biomarker for high-grade serous ovarian cancer (HGSC), a cancer type mostly driven by TP53 mutations. We used ultra-deep duplex sequencing to analyze TP53 mutations in LBC and blood samples from 70 individuals (30 with and 40 without HGSC) undergoing gynecologic surgery, 30 carrying BRCA1 or BRCA2 germline pathogenic variants (BRCApv). Only 30% of the tumor mutations were found in LBC samples. However, TP53 pathogenic mutations were identified in nearly all LBC and blood samples, with only 5.4% of mutations in LBC (20/368) also found in the corresponding blood sample. TP53 mutations were more abundant in LBC than in blood and increased with age in both sample types. BRCApv carriers with HGSC had more TP53 clonal expansions in LBC than BRCApv carriers without cancer. Our results show that, while not useful for direct cancer detection, LBC samples capture TP53 mutation burden in the gynecological tract, presenting potential value for cancer risk assessment in individuals at higher hereditary risk for ovarian cancer.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Mutação em Linhagem Germinativa , Neoplasias Ovarianas , Proteína Supressora de Tumor p53 , Humanos , Feminino , Proteína Supressora de Tumor p53/genética , Mutação em Linhagem Germinativa/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/diagnóstico , Proteína BRCA2/genética , Pessoa de Meia-Idade , Proteína BRCA1/genética , Adulto , Idoso , Colo do Útero/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , CitologiaRESUMO
We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs elamipretide (SS-31) and nicotinamide mononucleotide (NMN) to reverse aging-associated changes in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are altered with age, while also inducing additional changes. Age-related increases in protein acetylation were predominantly in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling. We further assessed how these age-related changes associated with diastolic function (Ea/Aa) and systolic function (fractional shortening under higher workload) measurements from echocardiography. These results identify a subset of protein abundance and acetylation changes in muscle, mitochondrial, and structural proteins that appear to be essential in regulating diastolic function in old hearts.
Assuntos
Mononucleotídeo de Nicotinamida , Proteoma , Animais , Camundongos , Mitocôndrias/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Proteoma/metabolismo , Proteoma/farmacologiaRESUMO
Wnt signaling controls multiple biological process, particularly the embryonic development of metazoans. Sustained expression of Wnt signaling components in the mature mammalian CNS and their apparent deregulation in certain neuropathologies suggest that it also plays a part beyond embryonic development to regulate normal brain function. We describe a noncanonical Wnt/Ca2+ signaling cascade that regulates the electrophysiological intrinsic properties of rat neurons, resulting in sustained membrane depolarization and the mobilization of Ca2+ from internal stores. These effects require tyrosine kinase-like orphan receptor 2 (RoR2), activation of PLC, and voltage-gated Ca2+ channels. Activation of this signaling cascade then promotes surface expression of N-methyl-D-aspartate receptors (NMDARs) through a SNARE-dependent mechanism. This neuronal Wnt/Ca2+ signaling pathway represents a mechanism for Wnt ligands to regulate normal brain processes in the mature animal and provides a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders where NMDARs are compromised.
Assuntos
Sinalização do Cálcio , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Via de Sinalização Wnt , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Microtomia , Neurônios/citologia , Técnicas de Patch-Clamp , Transporte Proteico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/deficiência , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Técnicas de Cultura de Tecidos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMO
A murine model of cutaneous leishmaniasis with green fluorescent protein positive (GFP+) L. major enables the monitoring of parasitic load via measurements of GFP fluorescence intensity, allowing for a faster and more efficient way of monitoring the clinical outcome of photodynamic therapy (PDT). This model may provide new insights on the phototoxic aspects in PDT. Although PDT regimens may be somewhat different in humans, it is expected that the developed model will facilitate the optimization and clinical translation of PDT as a therapy for cutaneous leishmaniasis and the eventual development of topical PDT treatments for other granulomatous infections.