Methotrexate Polyglutamate Concentrations as a Possible Predictive Marker for Effectiveness of Methotrexate Therapy in Patients with Sarcoidosis: A Pilot Study.

INTRODUCTION: Methotrexate (MTX), a folate antagonist, is often used as second-line treatment in patients with sarcoidosis. Effectiveness of MTX has large inter-patient variability and at present therapeutic drug monitoring (TDM) of MTX is not possible. Upon administration, MTX is actively transported into cells and metabolized to its active forms by adding glutamate residues forming MTXPG(n=1-5) resulting in enhanced cellular retention. In this study we address the question whether different MTXPG(n) concentrations in red blood cells (RBC) of patients with sarcoidosis after 3 months of MTX therapy correlate with response to treatment. METHODS: We retrospectively included patients with sarcoidosis that had started on MTX therapy and from whom blood samples and FDG-PET/CT were available 3 and 6-12 months after MTX initiation, respectively. FDG-uptake was measured by SUVmax in the heart, lungs and thoracic lymph nodes. Changes in SUVmax was used to determine anti-inflammatory response after 6-12 months of MTX therapy. MTXPG(n) concentrations were measured from whole blood RBC using an LC-MS/MS method. Pearson correlation coefficients were calculated to evaluate the relationship between changes in the SUVmax and MTXPG(n) concentrations. RESULTS: We included 42 sarcoidosis patients treated with MTX (15 mg/week); 31 with cardiac sarcoidosis and 11 with pulmonary sarcoidosis. In MTXPG3 and MTXPG4 a significant negative relation between the absolute changes in SUVmax and MTXPG(n) was found r = - 0.312 (n = 42, p = 0.047) for MTXPG3 and r = - 0.336 (n = 42, p = 0.031 for MTXPG4). The other MTXPG(n) did not correlate to changes in SUVmax. CONCLUSION: These results suggest a relation between MTXPG(n) concentrations and the anti-inflammatory effect in patients with sarcoidosis. Further prospective validation is warranted, but if measuring MTXPG concentrations could predict treatment effect of MTX this would be a step in the direction of personalized medicine.

ASXL2 regulates hematopoiesis in mice and its deficiency promotes myeloid expansion.

Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.

Methotrexate accumulation in target intestinal mucosa and white blood cells differs from non-target red blood cells of patients with Crohn's disease.

BACKGROUND: Intracellular methotrexate polyglutamates (MTX-PGs) concentrations are measurable in red blood cells (RBCs) during MTX treatment. MTX-PG3 concentrations correlate with efficacy in patients with Crohn's disease (CD). Since RBCs are not involved in pathogenesis of CD and lack extended MTX metabolism, we determined MTX-PGs accumulation in peripheral blood mononuclear cells (PBMCs: effector cells) and intestinal mucosa (target cells) and compared those with RBCs as a potential more precise biomarker. METHODS: In a multicentre prospective cohort study, blood samples of patients with CD were collected during the first year of MTX therapy. Mucosal biopsies were obtained from non-inflamed rectum and/or inflamed intestine. MTX-PGs concentrations in mucosa, PBMCs and RBCs were measured by liquid chromatography-tandem mass spectrometry. RESULTS: From 80 patients with CD, a total of 27 mucosal biopsies, 9 PBMC and 212 RBC samples were collected. From 12 weeks of MTX therapy onwards, MTX-PG3 was the most predominant species (33%) in RBCs. In PBMCs, the distribution was skewed towards MTX-PG1 (48%), which accounted for an 18 times higher concentration than in RBCs. Long-chain MTX-PGs were highly present in mucosa: 21% of MTX-PGtotal was MTX-PG5. MTX-PG6 was measurable in all biopsies. CONCLUSIONS: MTX-PG patterns differ between mucosa, PBMCs and RBCs of patients with CD.

Therapeutic drug monitoring of methotrexate in patients with Crohn's disease.

BACKGROUND: Therapeutic drug monitoring (TDM) has the potential to improve efficacy and diminish side effects. Measuring methotrexate-polyglutamate (MTX-PG) in erythrocytes might enable TDM for methotrexate in patients with Crohn's disease (CD). AIM: To investigate the relationship between MTX-PGs and methotrexate drug survival, efficacy and toxicity METHODS: In a multicentre prospective cohort study, patients with CD starting subcutaneous methotrexate without biologics were included and followed for 12 months. Primary outcome was subcutaneous methotrexate discontinuation or requirement for step-up therapy. Secondary outcomes included faecal calprotectin (FCP), Harvey Bradshaw Index (HBI), hepatotoxicity and gastrointestinal intolerance. Erythrocyte MTX-PGs were analysed at weeks 8, 12, 24 and 52 or upon treatment discontinuation. RESULTS: We included 80 patients with CD (mean age 55 ± 13y, 35% male) with a median FCP of 268 µg/g (IQR 73-480). After the 12-month visit, 21 patients (26%) were still on subcutaneous methotrexate monotherapy. Twenty-one patients stopped because of disease activity, 29 because of toxicity, and four for both reasons. Five patients ended study participation or stopped methotrexate for another reason. A higher MTX-PG3 concentration was associated with a higher rate of methotrexate drug survival (HR 0.86, 95% CI 0.75-0.99), lower FCP (ß -3.7, SE 1.3, p < 0.01) and with biochemical response (FCP ≤250 if baseline >250 µg/g; OR 1.1, 95% CI 1.0-1.3). Higher MTX-PGs were associated with less gastrointestinal intolerance. There was no robust association between MTX-PGs and HBI or hepatotoxicity. CONCLUSIONS: Higher MTX-PG3 concentrations are related to better methotrexate drug survival and decreased FCP levels. Therefore, MTX-PG3 could be used for TDM if a target concentration can be established.

A Model for Work Intensity in a Pediatric Training Program.

Background: The Accreditation Council for Graduate Medical Education (ACGME) requires residency programs to monitor scheduling, work intensity, and work compression. Objective: We aimed to create a model for assessing intern work intensity by examining patient and clinical factors in our electronic health systems using multiple linear regression. Methods: We identified measurable factors that may contribute to resident work intensity within our electronic health systems. In the spring of 2021, we surveyed interns on pediatric hospital medicine rotations each weekday over 5 blocks to rank their daily work intensity on a scale from -100 (bored) to +100 (exasperated). We queried our electronic systems to identify patient care activities completed by study participants on days they were surveyed. We used multiple linear regression to identify factors that correlate with subjective scores of work intensity. Results: Nineteen unique interns provided 102 survey responses (28.3% response rate) during the study period. The mean work intensity score was 9.82 (SD=44.27). We identified 19 candidate variables for the regression model. The most significantly associated variables from our univariate regression model were text messages (ß=0.432, P<.0009, R2=0.105), orders entered (ß=0.207, P<.0002, R2=0.128), and consults ordered (ß=0.268, P=.022, R2=0.053). Stepwise regression produced a reduced model (R2=0.247) including text messages (ß=0.379, P=.002), patient transfers (ß=-1.405, P=.15), orders entered (ß=0.186, P<.001), and national patients (ß=-0.873, P=.035). Conclusions: Our study demonstrates that data extracted from electronic systems can be used to estimate resident work intensity.

Hype or hope - Can combination therapies with third-generation EGFR-TKIs help overcome acquired resistance and improve outcomes in EGFR-mutant advanced/metastatic NSCLC?

Three generations of epidermal growth factor receptor - tyrosine kinase inhibitors (EGFR-TKIs) have been developed for treating advanced/metastatic non-small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations, while a fourth generation is undergoing preclinical assessment. Although initially effective, acquired resistance to EGFR-TKIs usually arises within a year due to the emergence of clones harboring multiple resistance mechanisms. Therefore, the combination of EGFR-TKIs with other therapeutic agents has emerged as a potential strategy to overcome resistance and improve clinical outcomes. However, results obtained so far are ambiguous and ideal therapies for patients who experience disease progression during treatment with EGFR-TKIs remain elusive. This review provides an updated landscape of EGFR-TKIs, along with a description of the mechanisms causing resistance to these drugs. Moreover, it discusses the current knowledge, limitations, and future perspective regarding the use of EGFR-TKIs in combination with other anticancer agents, supporting the need for bench-to-bedside approaches in selected populations.

Genome-wide histone acetylation analysis reveals altered transcriptional regulation in the Parkinson's disease brain.

BACKGROUND: Parkinson's disease (PD) is a complex, age-related neurodegenerative disorder of largely unknown etiology. PD is strongly associated with mitochondrial respiratory dysfunction, which can lead to epigenetic dysregulation and specifically altered histone acetylation. Nevertheless, and despite the emerging role of epigenetics in age-related brain disorders, the question of whether aberrant histone acetylation is involved in PD remains unresolved. METHODS: We studied fresh-frozen brain tissue from two independent cohorts of individuals with idiopathic PD (n = 28) and neurologically healthy controls (n = 21). We performed comprehensive immunoblotting to identify histone sites with altered acetylation levels in PD, followed by chromatin immunoprecipitation sequencing (ChIP-seq). RNA sequencing data from the same individuals was used to assess the impact of altered histone acetylation on gene expression. RESULTS: Immunoblotting analyses revealed increased acetylation at several histone sites in PD, with the most prominent change observed for H3K27, a marker of active promoters and enhancers. ChIP-seq analysis further indicated that H3K27 hyperacetylation in the PD brain is a genome-wide phenomenon with a strong predilection for genes implicated in the disease, including SNCA, PARK7, PRKN and MAPT. Integration of the ChIP-seq with transcriptomic data from the same individuals revealed that the correlation between promoter H3K27 acetylation and gene expression is attenuated in PD patients, suggesting that H3K27 acetylation may be decoupled from transcription in the PD brain. Strikingly, this decoupling was most pronounced among nuclear-encoded mitochondrial genes, corroborating the notion that impaired crosstalk between the nucleus and mitochondria is involved in the pathogenesis of PD. Our findings independently replicated in the two cohorts. CONCLUSIONS: Our findings strongly suggest that aberrant histone acetylation and altered transcriptional regulation are involved in the pathophysiology of PD. We demonstrate that PD-associated genes are particularly prone to epigenetic dysregulation and identify novel epigenetic signatures associated with the disease.

Identification of Small Molecule Enhancers of Immunotherapy for Melanoma.

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.

Chromatin remodeling mediated by ARID1A is indispensable for normal hematopoiesis in mice.

Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.

A Unique Heterozygous CARD11 Mutation Combines Pathogenic Features of Both Gain- and Loss-of-Function Patients in a Four-Generation Family.

CARD11 is a lymphocyte-specific scaffold molecule required for proper activation of B- and T-cells in response to antigen. Germline gain-of-function (GOF) mutations in the CARD11 gene cause a unique B cell lymphoproliferative disorder known as B cell Expansion with NF-κB and T cell Anergy (BENTA). In contrast, patients carrying loss-of-function (LOF), dominant negative (DN) CARD11 mutations present with severe atopic disease. Interestingly, both GOF and DN CARD11 variants cause primary immunodeficiency, with recurrent bacterial and viral infections, likely resulting from impaired adaptive immune responses. This report describes a unique four-generation family harboring a novel heterozygous germline indel mutation in CARD11 (c.701-713delinsT), leading to one altered amino acid and a deletion of 4 others (p.His234_Lys238delinsLeu). Strikingly, affected members exhibit both moderate B cell lymphocytosis and atopic dermatitis/allergies. Ectopic expression of this CARD11 variant stimulated constitutive NF-κB activity in T cell lines, similar to other BENTA patient mutations. However, unlike other GOF mutants, this variant significantly impeded the ability of wild-type CARD11 to induce NF-κB activation following antigen receptor ligation. Patient lymphocytes display marked intrinsic defects in B cell differentiation and reduced T cell responsiveness in vitro. Collectively, these data imply that a single heterozygous CARD11 mutation can convey both GOF and DN signaling effects, manifesting in a blended BENTA phenotype with atopic features. Our findings further emphasize the importance of balanced CARD11 signaling for normal immune responses.

Sorting Tubules Regulate Blood-Brain Barrier Transcytosis.

Transcytosis across the blood-brain barrier (BBB) regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs) remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab) sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab) sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome.

Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.