👤 Sowmya Iyer

Genetic alterations in FGFR2 drive multiple malignancies, most notably intrahepatic cholangiocarcinoma, where they occur in ∼10-15% of patients. While approved pan-FGFR inhibitors provide clinical benefit, their durability is limited by acquired, often polyclonal, on-target resistance mutations affecting key regions of the FGFR2 kinase domain, including the gatekeeper residue (V565), molecular brake residues (N550, E566, K642), and other key variants. These liabilities motivate the development of next-generation inhibitors. Given FGFR2-associated toxicities and the need for subtype selectivity, FGFR4 inhibition was prioritized as a selectivity determinant, while sparing FGFR1 was considered less critical. Guided by structure-based drug design, a reversible aminopyrimidine screening hit was optimized into a novel covalent inhibitor series active against FGFR2 wild-type and clinically relevant resistance mutations. An advanced lead Show less

We investigated naporafenib (LXH254), a pan-RAF kinase inhibitor, with or without spartalizumab, in patients with advanced solid tumors harboring MAPK pathway alterations. This first-in-human phase 1 study had two dose-escalation arms: single-agent naporafenib (starting at 100 mg once-daily [QD]) and naporafenib (starting at the recommended dose/regimen)/spartalizumab (400 mg every 4 weeks). The naporafenib/spartalizumab dose-expansion part enrolled patients with KRAS-mutated non-small cell lung cancer (NSCLC) and NRAS-mutated melanoma. The primary objectives were to establish the maximum tolerated doses (MTD)/recommended doses for expansion (RDE) and evaluate tolerability and safety. A total of 142 patients were included in the naporafenib dose-escalation (n = 87), naporafenib/spartalizumab dose-escalation (n = 12) and naporafenib/spartalizumab dose-expansion (n = 43) arms. The MTD/RDE of naporafenib was 600 mg twice-daily (BID). In naporafenib escalation, five patients experienced 7 dose-limiting toxicities: decreased platelet count (1200 mg QD); neuralgia, maculopapular rash, pruritus (600 mg BID); increased blood bilirubin, hyponatremia, peripheral sensory neuropathy (800 mg BID). No DLTs occurred in the naporafenib/spartalizumab arm: the RDE was established at 400 mg BID. The most common treatment-related adverse events were rash and dermatitis acneiform (each 24.1%; naporafenib), nausea and pruritus (each 33.3%; naporafenib/spartalizumab; escalation) and rash (39.5%; naporafenib/spartalizumab; expansion). Naporafenib reduced DUSP6 expression in tumors. Two partial responses (PRs) occurred in naporafenib escalation, and 1 complete response and 3 PRs in the naporafenib/spartalizumab NRAS-mutated melanoma and KRAS-mutated NSCLC arms, respectively. Naporafenib, with or without spartalizumab, showed an acceptable safety profile, pharmacodynamic activity and limited antitumor activity. Additional naporafenib combination therapies are currently under investigation. Show less

Activating FGFR3 alterations have been identified in up to 15-20% of muscle-invasive bladder cancer and metastatic urothelial carcinoma (mUC), and as high as 80% in nonmuscle invasive bladder cancers. FGFR3 germline mutations have also been associated with a variety of skeletal dysplasias. Achondroplasia, the most common form of dwarfism in humans, results from a G380R mutation in FGFR3. The pan-FGFR inhibitor erdafitinib was approved for the treatment of mUC with FGFR3 alterations but is limited due to FGFR isoform off-target toxicities and the development of on-target gatekeeper resistance mutations. TYRA-300 ( Show less

FGFR alterations are reported across various malignancies and might act as oncogenic drivers in multiple histologies. Erdafitinib is an oral, selective pan-FGFR tyrosine kinase inhibitor with activity in FGFR-altered advanced urothelial carcinoma. We aimed to evaluate the safety and activity of erdafitinib in previously treated patients with FGFR-altered advanced solid tumours. The single-arm, phase 2 RAGNAR study was conducted at 156 investigative centres (hospitals or oncology practices that are qualified oncology study centres) across 15 countries. The study consisted of four cohorts based on tumour histology and patient age; the results reported in this Article are for the primary cohort of the study, defined as the Broad Panel Cohort, which was histology-agnostic. We recruited patients aged 12 years or older with advanced or metastatic tumours of any histology (except urothelial cancer) with predefined FGFR1-4 alterations (mutations or fusions according to local or central testing). Eligible patients had disease progression on at least one previous line of systemic therapy and no alternative standard therapy available to them, and an Eastern Cooperative Oncology Group performance status of 0-1 (or equivalent for adolescents aged 12-17 years). Patients received once-daily oral erdafitinib (8 mg/day with provision for pharmacodynamically guided up-titration to 9 mg/day) on a continuous 21-day cycle until disease progression or intolerable toxicity. The primary endpoint was objective response rate by independent review committee according to Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, or Response Assessment In Neuro-Oncology (RANO). The primary analysis was conducted on the treated population of the Broad Panel Cohort. This ongoing study is registered with ClinicalTrials.gov, number NCT04083976. Patients were recruited between Dec 5, 2019, and Feb 15, 2022. Of 217 patients treated with erdafitinib, 97 (45%) patients were female and 120 (55%) were male. The data cutoff was Aug 15, 2022. At a median follow-up of 17·9 months (IQR 13·6-23·9), an objective response was observed in 64 (30% [95% CI 24-36]) of 217 patients across 16 distinct tumour types. The most common grade 3 or higher treatment-emergent adverse events related to erdafitinib were stomatitis (25 [12%]), palmar-plantar erythrodysaesthesia syndrome (12 [6%]), and hyperphosphataemia (11 [5%]). The most commonly occurring serious treatment-related adverse events (grade 3 or higher) were stomatitis in four (2%) patients and diarrhoea in two (1%). There were no treatment-related deaths. RAGNAR results show clinical benefit for erdafitinib in the tumour-agnostic setting in patients with advanced solid tumours with susceptible FGFR alterations who have exhausted other treatment options. These results support the continued development of FGFR inhibitors in patients with advanced solid tumours. Janssen Research & Development. Show less

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics. Show less

The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1β knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency. Show less

The goal of this study was to analyze the association between the FTO rs17817449 (G>T), G protein beta3 subunit (GNB3) C825T and Melanocortin 4 receptor (MC4R) A822G single nucleotide polymorphism (SNP) with obesity in Saudi subjects. The subjects were divided into 2 groups according to BMI: Obese (BMI> 29.9) and non- obese control (BMI<24.9). Genotyping of the target genes were determined by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP). We demonstrated the association of the FTO genotype TT with increased weight, BMI and leptin levels in both males and females. However, there was no association of genotype TT with fasting blood glucose, triglycerides and cholesterol levels. Regarding GNB3 rs5443 polymorphism, the likelihood of obesity was linked to the TT genotype which was also associated with increased leptin levels. On the other hand, the SNP of MC4R A822G did not exhibit any significant association with obesity among studied subjects and showed only the presence of homozygous AA genotype. The polymorphism of FTO gene rs17817449 and GNB3 gene rs5443 (C825T) may be a genetic determinant of obesity in Saudi population whereas impact of MC4R Asn274Ser change could not be detected. Show less

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. Show less

G protein-coupled receptors (GPCRs) are among the most important targets in drug discovery. In this study, we used TaqMan Low Density Arrays to profile the full GPCR repertoire of primary human macrophages differentiated from monocytes using either colony stimulating factor-1 (CSF-1/M-CSF) (CSF-1 Mϕ) or granulocyte macrophage colony stimulating factor (GM-CSF) (GM-CSF Mϕ). The overall trend was a downregulation of GPCRs during monocyte to macrophage differentiation, but a core set of 10 genes (e.g. LGR4, MRGPRF and GPR143) encoding seven transmembrane proteins were upregulated, irrespective of the differentiating agent used. Several of these upregulated GPCRs have not previously been studied in the context of macrophage biology and/or inflammation. As expected, CSF-1 Mϕ and GM-CSF Mϕ exhibited differential inflammatory cytokine profiles in response to the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS). Moreover, 15 GPCRs were differentially expressed between these cell populations in the basal state. For example, EDG1 was expressed at elevated levels in CSF-1 Mϕ versus GM-CSF Mϕ, whereas the reverse was true for EDG6. 101 GPCRs showed differential regulation over an LPS time course, with 65 of these profiles being impacted by the basal differentiation state (e.g. GPRC5A, GPRC5B). Only 14 LPS-regulated GPCRs showed asynchronous behavior (divergent LPS regulation) with respect to differentiation status. Thus, the differentiation state primarily affects the magnitude of LPS-regulated expression, rather than causing major reprogramming of GPCR gene expression profiles. Several GPCRs showing differential profiles between CSF-1 Mϕ and GM-CSF Mϕ (e.g. P2RY8, GPR92, EMR3) have not been widely investigated in macrophage biology and inflammation. Strikingly, several closely related GPCRs displayed completely opposing patterns of regulation during differentiation and/or activation (e.g. EDG1 versus EDG6, LGR4 versus LGR7, GPRC5A versus GPRC5B). We propose that selective regulation of GPCR5A and GPCR5B in CSF-1 Mϕ contributes to skewing toward the M2 macrophage phenotype. Our analysis of the GPCR repertoire expressed during primary human monocyte to macrophage differentiation and TLR4-mediated activation provides a valuable new platform for conducting future functional analyses of individual GPCRs in human macrophage inflammatory pathways. Show less

Bicuspid Aortic Valve (BAV) is a highly heritable congenital heart defect. The low frequency of BAV (1% of general population) limits our ability to perform genome-wide association studies. We present the application of four a priori SNP selection techniques, reducing the multiple-testing penalty by restricting analysis to SNPs relevant to BAV in a genome-wide SNP dataset from a cohort of 68 BAV probands and 830 control subjects. Two knowledge-based approaches, CANDID and STRING, were used to systematically identify BAV genes, and their SNPs, from the published literature, microarray expression studies and a genome scan. We additionally tested Functionally Interpolating SNPs (fitSNPs) present on the array; the fourth consisted of SNPs selected by Random Forests, a machine learning approach. These approaches reduced the multiple testing penalty by lowering the fraction of the genome probed to 0.19% of the total, while increasing the likelihood of studying SNPs within relevant BAV genes and pathways. Three loci were identified by CANDID, STRING, and fitSNPS. A haplotype within the AXIN1-PDIA2 locus (p-value of 2.926x10(-06)) and a haplotype within the Endoglin gene (p-value of 5.881x10(-04)) were found to be strongly associated with BAV. The Random Forests approach identified a SNP on chromosome 3 in association with BAV (p-value 5.061x10(-06)). The results presented here support an important role for genetic variants in BAV and provide support for additional studies in well-powered cohorts. Further, these studies demonstrate that leveraging existing expression and genomic data in the context of GWAS studies can identify biologically relevant genes and pathways associated with a congenital heart defect. Show less

We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: alpha factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu Show less