A minimally invasive diagnostic assay for early detection of Alzheimer's disease (AD) is required to select optimal patient groups in clinical trials, monitor disease progression and response to ...treatment, and to better plan patient clinical care. Blood is an attractive source for biomarkers due to minimal discomfort to the patient, encouraging greater compliance in clinical trials and frequent testing. MiRNAs belong to the class of non-coding regulatory RNA molecules of ∼22 nt length and are now recognized to regulate ∼60% of all known genes through post-transcriptional gene silencing (RNAi). They have potential as useful biomarkers for clinical use because of their stability and ease of detection in many tissues, especially blood. Circulating profiles of miRNAs have been shown to discriminate different tumor types, indicate staging and progression of the disease and to be useful as prognostic markers. Recently their role in neurodegenerative diseases, both as diagnostic biomarkers as well as explaining basic disease etiology has come into focus. Here we report the discovery and validation of a unique circulating 7-miRNA signature (hsa-let-7d-5p, hsa-let-7g-5p, hsa-miR-15b-5p, hsa-miR-142-3p, hsa-miR-191-5p, hsa-miR-301a-3p and hsa-miR-545-3p) in plasma, which could distinguish AD patients from normal controls (NC) with >95% accuracy (AUC of 0.953). There was a >2 fold difference for all signature miRNAs between the AD and NC samples, with p-values<0.05. Pathway analysis, taking into account enriched target mRNAs for these signature miRNAs was also carried out, suggesting that the disturbance of multiple enzymatic pathways including lipid metabolism could play a role in AD etiology.
Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing ...these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.
Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR ...inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19.
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Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the ...spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3′ splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3′ ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3′ ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.
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•SF3B1 hotspot mutations are neomorphic and induce aberrant 3′ splice site selection•Mutant SF3B1 utilizes a different branch point than that used by wild-type SF3B1•SF3B1 mutants require the canonical 3′ splice site to induce aberrant splicing•∼50% of aberrant mRNAs undergo NMD leading to downregulation of canonical isoforms
Darman et al. report that SF3B1 mutations found in cancer induce aberrant 3′ splice site selection. To induce aberrant splicing, mutant SF3B1 requires canonical 3′ splice site but utilizes a different branch point than wild-type SF3B1. Approximately 50% of the aberrant mRNAs undergo NMD resulting in downregulation of canonical transcripts.
We present a protocol for in vitro production of recombinant lentiviruses using COS-1 African green monkey kidney epithelial cells and HEK293T human embryonic kidney epithelial cells as packaging ...cells. COS-1 and HEK293T express SV40 large T antigen, amplifying transfected circular plasmids harboring SV40 replication origin. Support protocols for evaluation of transfection efficiency by in situ β-galactosidase enzyme activity assay and titer of infection-capable virions are also provided. Advantages of using COS-1 packaging cells over the standard HEK293T cells for contamination-sensitive applications or automated processing are discussed.
Alberta is the driest province in Canada; however, residents do not often recognize the relationship between their own water uses and the health of the watershed they reside in. Therefore, there is a ...recognised need for water literacy education in the province. The researcher asked five members of water education organizations in central and southern Alberta how they work to increase water literacy within their regions. The programs primarily use experiential methods, combined with discussion and information to engage participants in connecting their own water usages to the watershed. This research looked at ways language, stories, and metaphors, as well as the hidden curriculum of systems thinking, are used in the programs to foster water literacy. However, due to the generally low water literacy of participants, the programs are often limited to basic water education which restricts educator’s ability to create deeper awareness of the holistic systems connected to water.
Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 ...hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700E or SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E (the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700E vs. SF3B1WT or SF3B1G742D identified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700E cells only. This is consistent with the role of SF3B1K700E in RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700E cells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS.
Buonamici:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.
Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. ...Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA).
comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERα
and ERα
cell lines.
, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERα
and ERα
breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERα
and ERα
cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.
Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERα
and ERα
, promotes a unique antagonist conformation, and demonstrates improved
and
activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors.
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Recently, heterozygous mutations in several spliceosome genes have been observed in hematological and solid cancers, but their functional role in these diseases is not well understood. Among these, ...SF3B1 is the most commonly mutated spliceosome gene in myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). SF3B1 is part of the U2 complex involved in the recognition of the 3’ splice sites (3’ss) during early spliceosome assembly.
To determine the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1MUT) or wild-type SF3B1 (SF3B1WT) in breast cancer, melanoma, CLL and MDS. This analysis revealed significantly increased usage of aberrant 3’ss in SF3B1MUT samples. In addition, the aberrantly spliced exons carry a proximal splice acceptor (SA) 15 to 21 nucleotides upstream of the canonical SA with a weak and short polypyrimidine tract. Using ectopic expression and allele-specific RNAi, we confirmed that mutations in SF3B1 are sufficient and required for these aberrant splicing events which suggests a neomorphic splicing activity of SF3B1MUT. Furthermore, a common aberrant splicing profile was shared across different hotspot mutations and diseases; however, unique aberrant splicing profiles were also observed in each disease suggesting lineage and disease specific effects. In particular, gene-set enrichment analysis of aberrantly spliced and differentially expressed genes in mutant vs. wild type samples identified genes that regulate cell differentiation and epigenetics in MDS, pathways/processes known to be dysregulated in myeloid malignancies. To study the impact of SF3B1MUT on differentiation processes, we used the well-established TF-1 model of erythroid differentiation. SF3B1K700E (the most common mutation in MDS and CLL), SF3B1G742D (a mutation found in CLL but not MDS patients), SF3B1K700R (a mutation unable to induce aberrant splicing) and SF3B1WT were overexpressed in TF-1 to study erythoid differentiation post erythropoietin (EPO) exposure. EPO treatment, as expected, induced differentiation in TF-1 cells transduced with SF3B1WT and SF3B1K700R. Consistent with a possible mechanism in MDS, SF3B1K700E transduction blocked differentiation of TF-1 cells. Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Ongoing studies comparing splicing aberrations induced by SF3B1K700E and SF3B1G742D in TF-1 cell differentiation will be described.
Finally, we evaluated a potent and selective modulator of SF3B1 that inhibits both canonical and neomorphic splicing activities in vitro and in vivo. The SF3B1 modulator induced tumor regression in SF3B1MUT xenografts and increased the overall survival of animals bearing SF3B1MUT xenografts at well tolerated doses. Taken together, our data suggest that SF3B1 mutations impair cell differentiation and that splicing modulators hold promise for the treatment of cancers with SF3B1 mutations, including CLL and MDS.
Buonamici:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Lim:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Aicher:H3 Biomedicine: Employment. Aird:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Berkenblit:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Erik:H3 Biomedicine: Employment. Corson:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Keaney:H3 Biomedicine: Employment. Kumar:Eisai: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Park:H3 Biomedicine: Employment. Puyang:H3 Biomedicine: Employment. Selvaraj:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Mizui:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.
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