Serine/arginine-rich Splicing Factors Research Articles

High expression of SRSF1 facilitates osteosarcoma progression and unveils its potential mechanisms

BackgroundSRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS.MethodsSRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1.ResultsSRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS.ConclusionsOur results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.

Abstract 617: BH-30236, a novel macrocyclic CLK inhibitor modulating RNA splicing, demonstrates potent inhibition of cancer cell growth in a broad panel of cancer cell lines

Abstract Alternative splicing (AS) is a primary mechanism for mRNA transcript diversification and protein expression regulation. In cancer, altered mRNA splicing promotes oncogenic transformation, induces metastasis, and confers resistance to cancer treatment. Mutations or imbalanced expression/activity of splicing factors (SF), such as serine-arginine-rich SFs (SRSFs), often result in deregulation of RNA splicing and tumor progression. CDC2-like kinases (CLK) and dual-specificity tyrosine-regulated kinase (DYRK) are key regulators of AS via phosphorylation of SRSFs. BH-30236 is a novel macrocyclic ATP-competitive inhibitor of CLK1/2/4. It also inhibits DYRK1/2, provirus integration site for moloney leukemia virus 3 (PIM3) and FMS-like tyrosine kinase 3 (FLT3) at clinically relevant concentrations. The inhibitory activity of BH-30236 against CLK, DYRK, PIM3 and FLT3 in cancer cells was investigated via the phosphorylation inhibition of SRSFs, Tau, 4EBP1 and FLT3, respectively. A panel of 119 hematological and solid tumor cell lines were used to investigate the spectrum of BH-30236 against cancer cell growth. To understand the mechanism of anti-cancer cell growth, the effect of BH-30236 on AS and protein expression of key tumor related biomarkers were examined. In conclusion, BH-30236 effectively inhibited pSRSF (IC50 40-60 nM in IMR-32 cells), pTau (IC50 ~50 nM in SH-SY5Y cells), p4EBP1 (IC50 ~80 nM in MM1S cells) and pFLT3-ITD (IC50 0.16 nM in MV-4-11 cells). BH-30236 demonstrated broad inhibition of cancer cell growth with a median IC50 of 85.2 nM (range 0.55-393 nM) across 12 tumor types. BH-30236 showed great efficacy against cell lines derived from hematological malignancy (median IC50 23.34 nM), neuroblastoma (median IC50 25.73 nM), breast cancer (median IC50 83.80 nM), colon cancer (median IC50 85.17 nM), and lung cancer (median IC50 102.65 nM). Furthermore, BH-30236 demonstrated synergy with KRAS inhibitors in KRAS mutant cell lines, with EGFR inhibitors in RBM10 mutant H1975 cells, and with BCL2 inhibitor Venetoclax in MOLM-13 cells. Mechanistically, BH-30236 modulated AS by increasing pro-apoptotic and anti-proliferative splicing variants of key factors, such as BCL2L1, S6K, and BCLAF1. Meanwhile, BH-30236 downregulated RNA expression of SRSFs and modulated BCL2 family to increase apoptosis. These results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors as a single agent or in combination with other therapies. Citation Format: Ping Jiang, Danan Li, Nancy Ling, Dayong Zhai, Wei Deng, Eugene Rui, J. Jean Cui. BH-30236, a novel macrocyclic CLK inhibitor modulating RNA splicing, demonstrates potent inhibition of cancer cell growth in a broad panel of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 617.

SRSF10 facilitates HCC growth and metastasis by suppressing CD8+T cell infiltration and targeting SRSF10 enhances anti-PD-L1 therapy

Background and AimsRNA splicing is an essential step in regulating the gene posttranscriptional expression. Serine/arginine-rich splicing factors (SRSFs) are splicing regulators with vital roles in various tumors. Nevertheless, the expression patterns and functions of SRSFs in hepatocellular carcinoma (HCC) are not fully understood. MethodsFlow cytometry and immunofluorescent staining were used to determine the CD8+T cell infiltration. Orthotopic HCC model, lung metastasis model, DEN/CCl4 model, Srsf10△hep model, and Srsf10HepOE model were established to evaluate the role of SRSF10 in HCC and the efficacy of combination treatment. ResultsSRSF10 was one of the most survival-relevant genes among SRSF members and was an independent prognostic factor for HCC. SRSF10 facilitated HCC growth and metastasis by suppressing CD8+T cell infiltration. Mechanistically, SRSF10 down-regulated the p53 protein by preventing the exon 6 skipping (exon 7 in mouse) mediated degradation of MDM4 transcript, thus inhibiting CD8+T cell infiltration. Elimination of CD8+T cells or overexpression of MDM4 removed the inhibitory role of SRSF10 knockdown in HCC growth and metastasis. SRSF10 also inhibited the IFNα/γ signaling pathway and promoted the HIF1α-mediated up-regulation of PD-L1 in HCC. Hepatocyte-specific SRSF10 deficiency alleviated the DEN/CCl4-induced HCC progression and metastasis, whereas hepatocyte-specific SRSF10 overexpression deteriorated these effects. Finally, SRSF10 knockdown enhanced the anti-PD-L1-mediated anti-tumor activity. ConclusionsSRSF10 promoted HCC growth and metastasis by repressing CD8+T cell infiltration mediated by the MDM4-p53 axis. Furthermore, SRSF10 suppressed the IFNα/γ signaling pathway and induced the HIF1α signal mediated PD-L1 up-regulation. Targeting SRSF10 combined with anti-PD-L1 therapy showed promising efficacy.

Phase 1/2 Multicenter, Open-Label Study of CTX-712 in Patients with Relapsed/Refractory Acute Myeloid Leukemia and Higher Risk Myelodysplastic Syndromes

Background and Significance Dysregulation of alternative splicing, caused by abnormal expression or mutation of splicing factors, has been identified as an important contributor to the genesis and progression of cancer, and suggested as a novel target for anticancer treatment. CDC2-like kinases (CLK) represent a family of dual-specificity tyrosine and serine-threonine kinases that are involved in the regulation of splicing by phosphorylation of serine/arginine-rich splicing factors (SRSFs) which play a central role in exon recognition. CTX-712 is an orally available, potent, selective, and a first-in-class pan-CLK inhibitor (CLK 1-4), that inhibits phosphorylation of SRSFs resulting in significant antitumor activity in vitro and multiple xenograft models in vivo. CTX-712 formulated in capsules is currently being evaluated in an ongoing first-in-man study in Japan, evaluating tolerability and early efficacy in patients with solid tumors or hematologic malignancies (JapicCTI-184188). Preliminary data from the Japanese study were presented during ASCO 2022 and ASH 2022, and showed an acceptable safety profile (doses up to 175 mg (MTD 140 mg) twice a week in 28-day cycles, observed DLTs included dehydration, decreased platelets, hypokalemia, and pneumonia) with early signs of clinical antitumor activity (2 partial responses / 26 treated solid tumor patients, 5 complete remissions and complete remission with incomplete hematologic recovery/ 8 treated patients with AML or MDS), warranting further clinical investigations.. (J Clin Oncol (2022) 40(16_suppl):3080. Blood (2022) 140(Supplement 1):6211-2.). This abstract details a second study with CTX-712 that has just been initiated. Study Design and Methods A new film-coated tablet formulation will be evaluated in this phase 1/2 multicenter, open-label study of CTX-712 in patients with (mutated or wild type spliceosomes genes) relapsed/refractory AML, higher risk MDS (IPSS-R intermediate, high, or very high), or high marrow blast (≥5 %) MDS/MPN (including CMML). The phase 1 part of the study will be conducted at 4 US sites, and all sites are currently open for enrolment. The study allows patients with performance status 0, 1, or 2, adequate organ function, and up to four prior lines of therapy. Due to potential risk of interactions (in vitro studies suggest CYP3A4 as main metabolic pathway for CTX-712), concomitant use of strong CYP3A4 inhibitors is prohibited. Use of azole antifungals that are not strong CYP3A4 inhibitors (e.g., isavuconazole) is permitted. The phase 1 part of the study consists of dose-escalation (sequential standard 3 + 3 design), where cohorts of 3 (or 6) patients will receive ascending doses of CTX-712 once weekly in a 28-day cycle with continuous monitoring of emerging clinical PK, PD, safety, and efficacy data, to determine the recommended phase 2 dose (RP2D) based on the suggestion by the Safety Review Committee (SRC). The protocol defines five different dose levels (20-140 mg, once weekly) but also authorizes the SRC to suggest up to two interim dose levels or alternate schedules (e.g., dosing two times a week) if supported by emerging data. The suggested RP2D will be used in a confirmatory phase 1 expansion cohort where an additional 10 patients (total n = 16; 8 patients with AML and 8 patients with MDS) will be treated to gain further confidence in the selected dose level. The phase 2 part of the study will commence after the RP2D has been identified and confirmed and aims at evaluating therapeutic activity in R/R AML (Cohort 2a) or R/R HR-MDS (Cohort 2b), in addition to confirmation of the safety profile. Clinical trial registry number: NCT05732103

Molecular characteristics and clinical implications of serine/arginine-rich splicing factors in human cancer.

As critical splicing regulators, serine/arginine-rich splicing factors (SRSFs) play pivotal roles in carcinogenesis. As dysregulation of SRSFs may confer potential cancer risks, targeting SRSFs could provide important insights into cancer therapy. However, a global and comprehensive pattern to elaborate the molecular characteristics, mechanisms, and clinical links of SRSFs in a wide variety of human cancer is still lacking. In this study, a systematic analysis was conducted to reveal the molecular characteristics and clinical implications of SRSFs covering more than 10000 tumour samples of 33 human cancer types. We found that SRSFs experienced prevalent genomic alterations and expression perturbations in multiple cancer types. The DNA methylation, m6A modification, and miRNA regulation of SRSFs were all cancer context-dependent. Importantly, we found that SRSFs were strongly associated with cancer immunity, and were capable of predicting response to immunotherapy. And SRSFs had colossal potential for predicting survival in multiple cancer types, including those that have received immunotherapy. Moreover, we also found that SRSFs could indicate the drug sensitivity of targeted therapy and chemotherapy. Our research highlights the significance of SRSFs in cancer occurrence and development, and provides sufficient resources for understanding the biological characteristics of SRSFs, offering a new and unique perspective for developing cancer therapeutic strategies.

Alternative splicing induced by bacterial pore-forming toxins sharpens CIRBP-mediated cell response to Listeria infection.

Cell autonomous responses to intracellular bacteria largely depend on reorganization of gene expression. To gain isoform-level resolution of these modes of regulation, we combined long- and short-read transcriptomic analyses of the response of intestinal epithelial cells to infection by the foodborne pathogen Listeria monocytogenes. Among the most striking isoform-based types of regulation, expression of the cellular stress response regulator CIRBP (cold-inducible RNA-binding protein) and of several SRSFs (serine/arginine-rich splicing factors) switched from canonical transcripts to nonsense-mediated decay-sensitive isoforms by inclusion of 'poison exons'. We showed that damage to host cell membranes caused by bacterial pore-forming toxins (listeriolysin O, perfringolysin, streptolysin or aerolysin) led to the dephosphorylation of SRSFs via the inhibition of the kinase activity of CLK1, thereby driving CIRBP alternative splicing. CIRBP isoform usage was found to have consequences on infection, since selective repression of canonical CIRBP reduced intracellular bacterial load while that of the poison exon-containing isoform exacerbated it. Consistently, CIRBP-bound mRNAs were shifted towards stress-relevant transcripts in infected cells, with increased mRNA levels or reduced translation efficiency for some targets. Our results thus generalize the alternative splicing of CIRBP and SRSFs as a common response to biotic or abiotic stresses by extending its relevance to the context of bacterial infection.

Interplays between cis- and trans-Acting Factors for Alternative Splicing in Response to Environmental Changes during Biological Invasions of Ascidians.

Alternative splicing (AS), a pivotal biological process contributing to phenotypic plasticity, creates a bridge linking genotypes with phenotypes. Despite its importance, the AS mechanisms underlying environmental response and adaptation have not been well studied, and more importantly, the cis- and trans-acting factors influencing AS variation remain unclear. Using the model invasive congeneric ascidians, Ciona robusta, and Ciona savignyi, we compared their AS responses to environmental changes and explored the potential determinants. Our findings unveiled swift and dynamic AS changes in response to environmental challenges, and differentially alternative spliced genes (DASGs) were functionally enriched in transmembrane transport processes. Interestingly, both the prevalence and level of AS in C. robusta were lower than those observed in C. savignyi. Furthermore, these two indices were higher under temperature stresses compared to salinity stresses in C. savignyi. All the observed patterns underscore the species-specific and environmental context-dependent AS responses to environmental challenges. The dissimilarities in genomic structure and exon/intron size distributions between these two species likely contributed to the observed AS variation. Moreover, we identified a total of 11 and 9 serine/arginine-rich splicing factors (SRSFs) with conserved domains and gene structures in the genomes of C. robusta and C. savignyi, respectively. Intriguingly, our analysis revealed that all detected SRSFs did not exhibit prevalent AS regulations. Instead, we observed AS control over a set of genes related to splicing factors and spliceosome components. Altogether, our results elucidate species-specific and environmental challenge-dependent AS response patterns in closely related invasive ascidians. The identified splicing factors and spliceosome components under AS control offer promising candidates for further investigations into AS-mediated rapid responses to environmental challenges complementary to SRSFs.

Towards understandings of serine/arginine-rich splicing factors.

Serine/arginine-rich splicing factors (SRSFs) refer to twelve RNA-binding proteins which regulate splice site recognition and spliceosome assembly during precursor messenger RNA splicing. SRSFs also participate in other RNA metabolic events, such as transcription, translation and nonsense-mediated decay, during their shuttling between nucleus and cytoplasm, making them indispensable for genome diversity and cellular activity. Of note, aberrant SRSF expression and/or mutations elicit fallacies in gene splicing, leading to the generation of pathogenic gene and protein isoforms, which highlights the therapeutic potential of targeting SRSF to treat diseases. In this review, we updated current understanding of SRSF structures and functions in RNA metabolism. Next, we analyzed SRSF-induced aberrant gene expression and their pathogenic outcomes in cancers and non-tumor diseases. The development of some well-characterized SRSF inhibitors was discussed in detail. We hope this review will contribute to future studies of SRSF functions and drug development targeting SRSFs.

Alternative splicing (AS) regulation as a novel therapeutic target in soft-tissue sarcoma (STS).

11546 Background: There is an urgent clinical need to identify novel treatments for advanced STS, since all the clinical trials testing chemotherapy combinations have failed to improve survival compared to doxorubicin (dox) alone. AS deregulation has been associated with cancer initiation, progression and resistance to therapy. This deregulation can occur due to mutations or imbalanced expression or activity of splicing factors (SF). The alteration SF can alter thousands of pre-mRNAs, increasing cellular complexity and facilitating tumour progression. CDC-like kinases (CLK) that phosphorylate serine-arginine-rich SF (SRSFs), are key regulators of AS. SM09419 is an oral pan CLK/DYRK inhibitor that has been shown to inhibit SRSF phosphorylation, selectively modify spliceosome activity and decrease tumour growth by multiple mechanisms including the downregulation of Wnt/β-catenin signalling, a pathway that we reported to be important in sarcoma. Methods: A panel of 9 STS cell lines, including LMS (CP0024, IEC005, AA, SK-UT-1), LPS (93T449), synovial sarcoma (MCP037) and UPS (MCP016, MCP021, MCP025) were used to analyse the effect of SM09419 (provided by Biosplice) or PRI-724 (Wnt/β-catenin inhibitor) on cell viability (MTS assays). TOP flash β-catenin/TCF-responsive reporter assay was performed to evaluate the inhibition of the Wnt/β-catenin pathway in response to SM09419. In vivo experiments tested the combination of SM09419 (25mg/kg 5 days on and 2 off, for 21 days cycle) plus IP dox (5mg/kg once a week for 21 days cycle) in LMS (IEC005) and synovial sarcoma (FJD-SS-001) PDX models. Results: Our results showed that SM09419 had superior activity in STS cells compared to specific Wnt/β-catenin inhibitors with IC50 values starting at 29.8 nM in SK-UT-1 cell line, whereas PRI-724 IC50 values were clearly higher, starting from 5.18 µM in 93T449 cells. SM09419 treatment significantly inhibited the transcriptional activity of β-catenin by 40% in the MCP021 cells, demonstrating the regulatory activity of AS inhibitors on this pathway. I n vivo studies confirmed the higher efficacy of SM09419 in monotherapy compared to dox, in inhibiting tumour growth (p < 0.05), which was potentiated when combined with dox. The combination was increasingly active in synovial sarcoma with tumour regressions in 3 out of 4 mice, including one complete response. Treatment schedule was adapted to a single IP administration of dox (5mg/kg) and 5 administrations of SM09419 (25mg/kg), to improve the tolerability of the combination. Conclusions: The effects of pan CLK/DYRK inhibition on growth and survival of STS cancer cell lines and PDX models, indicate that can STS may be therapeutically addressed with these AS-modulators. The combination of SM09419 with dox was active in STS preclinical models known for their resilience toward treatment. Based on these results, the exploration of such combinations in clinical trials is warranted.

Investigation of the effect of SRSF9 overexpression on HIV-1 production

Serine-arginine-rich splicing factors (SRSFs) are members of RNA processing proteins in the serine-arginine-rich (SR) family that could regulate the alternative splicing of the human immunodeficiency virus-1 (HIV-1). Whether SRSF9 has any effect on HIV-1 regulation requires elucidation. Here, we report for the first time the effects and mechanisms of SRSF9 on HIV-1 regulation. The overexpression of SRSF9 inhibits viral production and infectivity in both HEK293T and MT-4 cells. Deletion analysis of SRSF9 determined that the RNA regulation motif domain of SRSF9 is important for anti-HIV-1 effects. Furthermore, overexpression of SRSF9 increases multiple spliced forms of viral mRNA, such as Vpr mRNA. These data suggest that SRSF9 overexpression inhibits HIV-1 production by inducing the imbalanced HIV-1 mRNA splicing that could be exploited further for a novel HIV-1 therapeutic molecule. [BMB Reports 2022; 55(12): 639-644].

Comprehensive Molecular Analysis Identified an SRSF Family-Based Score for Prognosis and Therapy Efficiency Prediction in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma (HCC) remains one of the most common malignancies worldwide. Despite significant progress in the comprehensive management of HCC, continued efforts are still necessary to identify prognostic assessment approaches for patient-specific survival so that more appropriate treatment and management regimens can be proposed for different HCC subpopulations. Here, we comprehensively exploited the expression characteristics of 12 SRSF family members across multiple datasets. We further identified an SRSF score consisting of 18 SRSF-related genes which were associated with overall survival and drug sensitivity in HCC. Moreover, the predictive power of the SRSF score was validated in independent HCC cohorts and different HCC subgroups. We further explored the effect of SRSF11 expression on HCC cell proliferation and drug sensitivity. Overall, our study provides a novel predictive model to assess the prognosis and drug sensitivity of HCC.The serine/arginine-rich splicing factors (SRSF)-mediated alternative splicing plays an essential role in the occurrence and progression of hepatocellular carcinoma (HCC). However, the SRSF-based signature that can predict the prognosis and therapy efficiency is yet to be investigated in HCC. Here, we comprehensively assessed the landscape and prognostic significance of the SRSF family genes in HCC. Then, we screened the SRSF family-related genes for signature construction and explored their biological characteristics. We further established an SRSF score consisting of 18 SRSF-associated genes and evaluated its correlation with prognosis and drug sensitivity in HCC. The predictive power of the SRSF score was validated in independent HCC cohorts and different HCC subgroups. Moreover, we further investigated that knockdown of SRSF11, a pivotal gene in the SRSF score, inhibited CDK1-dependent proliferation and enhanced the drug sensitivity of HCC cells. Overall, our study identified a novel SRSF family-based predictive model, and we demonstrated that SRSF11 is a promising therapeutic target for HCC, which enhances our understanding of the SRSF family genes and provides valuable insights into the clinical treatment and molecular mechanisms of HCC.

An IGF-1R-mTORC1-SRPK2 signaling Axis contributes to FASN regulation in breast cancer

BackgroundFatty acid synthase (FASN) expression is associated with a more aggressive breast cancer phenotype and is regulated downstream of receptor tyrosine kinase (RTK) signaling pathways. Recently, post transcriptional regulation of lipogenic transcripts have been demonstrated as being mediated downstream of serine-arginine rich protein kinase 2 (SRPK2), which acts to phosphorylate serine-arginine rich splicing factors (SRSFs), resulting in RNA binding and various RNA regulatory processes. Though post-transcriptional regulation of FASN has been studied previously, the upstream mediators of these pathways have not been elucidated.MethodsWestern blotting and RT-qPCR were utilized to demonstrate alterations in FASN and mRNA expression upon modulation of the IGF-1-mTORC1-SRPK2 pathway by small molecule inhibitors or RNAi mediated silencing. RNA stability was accessed by using the transcriptional inhibitor actinomycin-D followed by RT-qPCR. Further, we employed RNA-immunoprecipitation to demonstrate the direct binding of SRSF-1 to FASN transcripts.ResultsIn the current study, we demonstrated an IGF-1 induced increase in FASN mRNA and protein expression that was attenuated by mTORC1 inhibition. This mTORC1 inhibition also resulted in decreases in total and nuclear p-SRPK2 in response to IGF-1 exposure. Upon SRPK2 knockdown and inhibition, we observed a decrease in FASN protein and mRNA stability, respectively, in response to IGF-1 exposure that was specific to triple negative and HER2+ breast cancer cell lines. As we explored further, IGF-1 exposure resulted in an altered localization of eGFP expressed SRSF-1, pEGFP-SRSF-1 that was rescued upon both SRPK2 knockdown and mTORC1 inhibition. Further, we observed an increase binding of SRSF-1 to FASN RNA upon IGF-1 exposure, which was abrogated by SRPK2 knockdown.ConclusionThese current findings establish a potential IGF-1-mTORC1-SRPK2-FASN axis in breast cancer, which could be a potential therapeutic target for cancers that overexpress FASN and components of the IGF-1R pathway.

Abstract P5-05-07: An IGF-1R-mTORC1-SRPK2 signaling axis contributes to FASN regulation in breast cancer

Abstract BACKGROUND: FASN expression is associated with a more aggressive breast cancer phenotype and is both transcriptionally and post-transcriptionally regulated downstream of receptor tyrosine kinase signaling pathways. Moreover, 2nd and 3rd generation FASN inhibitors are showing promise for the treatment of breast cancer in clinical trials. Lipogenic transcripts, such as FASN, can be post-transcriptionally regulated through pre-mRNA splicing mediated through serine arginine rich protein kinases (SRPKs) and their respected substrates, serine-arginine rich splicing factors (SRSFs). Recent work has highlighted the mTORC1 signaling pathway as an upstream inducer of lipogenic pre-mRNA splicing, however, the role of extracellular environmental cues, such as growth factors and their respected receptors have not been explored. Here, an IGF-1-mTORC1-SRPK2 axis is demonstrated in the FASN regulation through SRSF-1 in breast cancer. METHODS: MDA-MB-231, MCF-7 breast cancer or MCF-10A non-transformed cells were exposed to IGF-1 followed by siRNA knockdown of IGF-1R, SRPK2, or SRSF-1. FASN expression was quantified by RT-qPCR or western blot analysis and de novo palmitate was measured by U-13C glucose incorporation followed by GS-MS. For mRNA stability, cells were pretreated with actinomycin-D with either vehicle or SRPK2 inhibitor for various timepoints followed by RT-qPCR for lipogenic and glycolytic mRNA abundance. eGFP-SRSF-1 was transfected in MDA-MB-231 and MCF-7 to visualize SRSF-1 localization in response to mTORC1 inhibition and/or SRPK2 knockdown and visualized by fluorescence microscopy. For intron retention, RT-PCR was performed with FASN intron and exon specific primers and resolved on a 2.5% agarose gel. RESULTS: Both IGF-1R and SRPK2 RNAi mediated knockdown significantly reduced FASN mRNA and protein and de novo synthesized palmitate levels. Similar results were obtained with mTORC1 inhibition. IGF-1 promoted the stabilization of FASN mRNA as well as reduced intron retention. This reduction of intron retention upon IGF-1 was abolished by SRPK2 knockdown. Additionally, IGF-1 contributed to a more diffuse localization in the nucleoplasm of SRSF-1, which become more retained in nuclear speckles upon both SRPK2 knockdown and mTORC1 inhibition. CONCLUSION: These current findings establish a potential IGF-1-mTORC1-SRPK2 axis in breast cancer that contributes to metabolic programming through FASN. More specifically, SRSF-1 is the potential mediator of FASN expression through this pathway, which could be a potential therapeutic target for breast cancers that overexpress FASN and components of the IGF-1R signaling axis. Citation Format: Bryan Mcclellan, Paul Gries, Brittany Harlow, Andrew J Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. An IGF-1R-mTORC1-SRPK2 signaling axis contributes to FASN regulation in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-07.

Abstract P3-13-03: Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids

Abstract Background: Women carrying a germline BRCA1 mutation, have 60% risk of developing breast cancer during their lifetime and frequently develop triple-negative, basal-like, aggressive breast tumors. Humans produce around 150,000 different proteins from their 25,000-30,000 genes. This is accomplished by alternative splicing (AS). We hypothesized that AS in BRCA1 mutation carriers is different from those women with wild type BRCA1, therefore, we examined AS, expression of genes encoding splicing factors, and enrichment of binding motifs for RNA-binding proteins in BRCA1 carriers (BRCA1mut/+) and controls (BRCA1 normal). Methods: Prophylactic mastectomy (BRCA1mut/+) and reduction mammoplasty (BRCA1 normal) specimens were collected at Prentice Women’s Hospital of Northwestern Medicine under approval by Northwestern’s Institutional Review Board (NU15B07). We isolated organoids from these tissues, and cryopreserved them for future use. We selected organoids of premenopausal women, age-matched (28-46) without oral contraceptive usage within 3 years: BRCA1mut/+ (n=12) and BRCA1 normal (n=4). Organoids were maintained in complete MammoCult medium in ultra-low attachment plates at 37 °C, 5% CO2 for 72 hrs, and harvested for RNA extraction (NucleoSpin RNA Plus, TaKaRa). 100 ng of total RNA (RIN 7+) were used for RNA sequencing assay with the KAPA mRNA Hyper Prep Kit (Roche Corporate) and NovaSeq 6000 for 100b paired-end sequencing (Illumina, Inc). The preprocessed reads were aligned to the human genome (hg38) using STAR. Differential expression analysis (DESeq2, nominal p-value < 0.05) and pathway analysis (GSEA, size ≥30, NES >2, FDR q-value <0.05, and enriched genes ≥ 5) were performed to compare BRCA1mut/+ with BRCA1 normal. We used rMATS- turbo v4.1.0 to perform multivariate analysis of transcript splicing and chose an FDR < 0. 05, to identify significant differential alternative splicing events. To determine which RNA binding proteins and splicing factors were employed in BRCA1mut/+ tissue, we examined the cis-regulatory motifs adjacent to splice sites using rMAPs. Results: BRCA1mut/+ organoids displayed 155 upregulated genes compared to BRCA1 normal organoids (P <0.05). Among top 20 enriched pathways, we identified that expression of mRNA spliceosome (POLR2L, U2AF2, NUP188, SNRPF, CSTF3, HNRNPM, SRSF2), and, in addition, cell cycle, oxidative phosphorylation, HIV infection, and protein metabolism genes were significantly upregulated in BRCA1mut/+ organoids. We identified significant differential AS events (FDR <0.05) between BRCA1mut/+ and BRCA1 normal organoids: 2159 genes (skipped exon), 433 genes (mutually exclusive exon), 325 genes (retained intron), 304 genes (alternative 3’ splice sites), 168 genes (alternative 5’ splice sites). RNA splicing analysis indicated that highly expressed genes (SRSF2, ALG3, AP1B1 and PMF1) in BRCA1mut/+ organoids were also alternatively spliced. In addition, many other serine/arginine-rich splicing factors (SRSF) themselves (SRSF1, 2, 3, 5, 6, 7) were alternatively spliced (skipped exon events). An examination of binding motifs of RNA-binding proteins with significantly increased expression in BRCA1 carriers revealed multiple significantly enhanced and silenced regions flanking exons involved in AS. Conclusions: Our data suggest that AS of BRCA1mut/+ mammary tissue significantly differ from BRCA1 normal tissue, and may alter RNA spliceosome activity, and consequently play a role in malignant transformation and warrant further study. Citation Format: Oukseub Lee, Mi-Ran Choi, Gannon Cottone, Priyam Patel, Susan E Clare, Seema A. Khan. Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-03.

Abstract CC02-01: Regulation of FASN expression through a novel IGF-1R-mTORC1-SRPK2-SRSF1 pathway

Abstract BACKGROUND FASN expression is associated with a more aggressive breast cancer phenotype and is both transcriptionally and post-transcriptionally regulated downstream of receptor tyrosine kinase signaling pathways. Lipogenic transcripts, such as FASN, can be post-transcriptionally regulated through pre-mRNA splicing mediated through serine arginine rich protein kinases (SRPKs) and their respected substrates, serine-arginine rich splicing factors (SRSFs). Recent work has highlighted the mTORC1 signaling pathway as an upstream inducer of lipogenic pre-mRNA splicing, however, the role of extracellular environmental cues, such as growth factors and their respected receptors have not been explored. Here, an IGF-1-mTORC1-SRPK2 axis is demonstrated in the FASN regulation through SRSF-1 in breast cancer. METHODS: MDA-MB-231, MCF-7 breast cancer or MCF-10A non-transformed cells were exposed to IGF-1 followed by siRNA knockdown of IGF-1R, SRPK2, or SRSF-1. FASN expression was quantified by RT-qPCR or western blot analysis and de novo palmitate was measured by U-13C glucose incorporation followed by GS-MS. For mRNA stability, cells were pretreated with actinomycin-D with either vehicle or SRPK2 inhibitor for various timepoints followed by RT-qPCR for lipogenic and glycolytic mRNA abundance. eGFP-SRSF-1 was transfected in MDA-MB-231 and MCF-7 to visualize SRSF-1 localization in response to mTORC1 inhibition and/or SRPK2 knockdown and visualized by fluorescence microscopy. For intron retention, RT-PCR was performed with FASN intron and exon specific primers and resolved on a 2.5% agarose gel. RESULTS: Both IGF-1R and SRPK2 RNAi mediated knockdown significantly reduced FASN mRNA and protein and de novo synthesized palmitate levels. Similar results were obtained with mTORC1 inhibition. IGF-1 promoted the stabilization of FASN mRNA as well as reduced intron retention. This reduction of intron retention upon IGF-1 was abolished by SRPK2 knockdown. Additionally, IGF-1 contributed to a more diffuse localization in the nucleoplasm of SRSF-1, which become more retained in nuclear speckles upon both SRPK2 knockdown and mTORC1 inhibition. CONCLUSION These current findings establish a potential IGF-1-mTORC1-SRPK2 axis in breast cancer that contributes to metabolic programming through FASN. More specifically, SRSF-1 is the potential mediator of FASN expression through this pathway, which could be a potential therapeutic target for breast cancers that overexpress FASN and components of the IGF-1R signaling axis. Citation Format: Bryan Mcclellan, Paul Gries, Brittany Harlow, Andrew Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. Regulation of FASN expression through a novel IGF-1R-mTORC1-SRPK2-SRSF1 pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC02-01.

Abstract P231: Multigenomic characterization of context-dependent alternative splicing in normal and neoplastic cells

Abstract Alternative pre-mRNA splicing (AS) supports the dynamic and regulated diversification of cells by allowing the production of multiple distinct proteins from individual genes. Dysregulated AS is commonly associated with human malignancies, producing pathological proteomes that underpin disease initiation, progression, and emergence of therapy resistance. The CDC2-like kinases (CLKs) and dual-specificity tyrosine-regulated kinases (DYRKs) are thought to govern the efficiency and specificity of AS by directly phosphorylating serine/arginine-rich splicing factors (SRSFs) and thereby influencing pre-mRNA splice junction selection. Cirtuvivint (SM08502) is a first-in-class small molecule ATP-competitive inhibitor of CLK/DYRK kinases. To directly evaluate the contribution of these kinases to AS profiles, changes in AS following treatment with cirtuvivint followed by high-depth RNAseq analysis across >25 cell lines representing 7 tumor lineages were evaluated. Both baseline and drug-induced changes in AS events (ASEs) were measured using a multivariate analysis of transcript splicing (rMATS). Pan-CLK/DYRK inhibition was found to affect a minority of baseline ASEs, leaving the majority of spliceosome activity intact in all samples. However, the magnitude and quantity of detected drug-induced alterations were larger in a sample of tumor cells from a patient compared with adjacent non-tumorigenic cells. Moreover, most ASEs sensitive to pan-CLK/DYRK inhibition were tumor type-specific irrespective of selective presence at the gene level. Multi-omics data integration revealed sensitivity to cirtuvivint was associated with alterations in splicing genes and that drug-induced ASEs were significantly associated with disease-promoting biology across lineage and oncogenic driver contexts. Perturbed splicing of the AR-V7 variant in treatment-resistant prostate cancer and MDM2 in p53 wild-type cancers were prominent examples. These observations indicate vulnerabilities to CLK-DYRK regulated splicing span a wide range of oncogenic contexts with potential to be therapeutically addressed with pan CLK/DYRK inhibitors. Citation Format: Elizabeth A. McMillan, Krishna Sriram, Emily Creger, Carsten Merkwirth, Raffaella Pippa, Melinda Pedraza, Long Do, Vishal Deshmukh, Carine Bossard, Michael A. White. Multigenomic characterization of context-dependent alternative splicing in normal and neoplastic cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P231.

The SR Splicing Factors: Providing Perspectives on Their Evolution, Expression, Alternative Splicing, and Function in Populus trichocarpa.

Serine/arginine-rich (SR) proteins are important splicing factors in plant development and abiotic/hormone-related stresses. However, evidence that SR proteins contribute to the process in woody plants has been lacking. Using phylogenetics, gene synteny, transgenic experiments, and RNA-seq analysis, we identified 24 PtSR genes and explored their evolution, expression, and function in Popolus trichocarpa. The PtSR genes were divided into six subfamilies, generated by at least two events of genome triplication and duplication. Notably, they were constitutively expressed in roots, stems, and leaves, demonstrating their fundamental role in P. trichocarpa. Additionally, most PtSR genes (~83%) responded to at least one stress (cold, drought, salt, SA, MeJA, or ABA), and, especially, cold stress induced a dramatic perturbation in the expression and/or alternative splicing (AS) of 18 PtSR genes (~75%). Evidentially, the overexpression of PtSCL30 in Arabidopsis decreased freezing tolerance, which probably resulted from AS changes of the genes (e.g., ICE2 and COR15A) critical for cold tolerance. Moreover, the transgenic plants were salt-hypersensitive at the germination stage. These indicate that PtSCL30 may act as a negative regulator under cold and salt stress. Altogether, this study sheds light on the evolution, expression, and AS of PtSR genes, and the functional mechanisms of PtSCL30 in woody plants.

Hypoxia up-regulates VEGF ligand and downregulates VEGF soluble receptor mRNA expression in bovine granulosa cells in vitro

Oxygen concentration (02) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 02 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis. The aim of this study was to examine the effect of 02 concentration on granulosa cell expression of the VEGF-system components. Bovine granulosa cells were isolated from medium-sized follicles (4–7 mm in diameter), placed in McCoy 5a medium supplemented with 10 ng/mL of insulin, 1 ng/mL of IGF-I, and 1 ng/mL of FSH, and cultured in four well plates (500 thousand cells per well), on three separate occasions. Culture plates were placed in gas-impermeable jars with a gas mixture containing either 2%, or 5% of O2, or under atmospheric air condition inside an incubator (20% of 02). Media was replaced at 48 h of culture and cells from the plate in each oxygen concentration were pooled for RNA extraction after 96 h. The number of mRNA copies for the VEGF-system components - including ligands (VEGF120, VEGF120b, VEGF165 and VEGF165b), enzymes (cyclin-dependent like kinases-1, CLK1 and serine–arginine protein kinase 1, SRPK1), splicing factors (serine–arginine-rich splicing factors, SRSF1 and SRSF6), and the membrane-bound (VEGFR1, VEGFR2) and soluble forms of the receptors (sVEGFR1 and sVEGFR2) were quantified by qPCR. Granulosa cells cultured with low 02 (2%) had a higher expression of VEGF ligands (P < 0.05) when compared to cells cultured at 20% 02. VEGF164b mRNA was absent in granulosa cells from all culture conditions. The 2 and 5% 02 levels, which coincide with physiological concentrations, in the ovarian follicle, induced higher SRSF6 expression than atmospheric 02 concentrations (20%, P < 0.05). In contrast, mRNA copies for SRPK1, CLK1, SRSF1, VEGFR1 or VEGFR2 did not differ between 02 culture conditions. (P > 0.05). Nonetheless, mRNA copies for the soluble receptors, sVEGFR1 and sVEGFR2, linearly increased (P < 0.05) with 02 concentration. These results suggest that when cultured under hypoxic conditions, granulosa cells may develop an autocrine milieu that favors VEGF’s biological effects on their survival and function.

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241.

Abstract 3521: Transcriptome analysis of TCGA prostate cancer samples identifies an association of poorer survival and aggressive disease biology with CDC-like kinase (CLK) expression and spliceosome regulation

Abstract In prostate cancer, alternative splicing of mRNA and spliceosome activity are implicated in several areas of disease pathogenesis. This is exemplified by the strong association of androgen receptor splice variants with treatment resistance and poor clinical outcome in castration-resistant disease. Therefore, pharmacologic targeting of spliceosome-regulating proteins such as CLKs and serine/arginine-rich splicing factors (SRSFs) represents a novel treatment approach for prostate cancer. To evaluate the therapeutic potential of inhibiting CLK activity in prostate cancer, the association between splicing-related gene expression and survival was investigated in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data collection (N=495). Survival analysis of RNA-seq data assessed 17,879 genes to measure their association with progression-free interval (PFI). Using transcript per million as the metric for normalized gene expression, age-adjusted Cox proportional hazards regression models were performed for each gene (R v3.6.0, coxph v2.43-3). A total of 3,145 genes significantly correlated with worse prognosis (P-adj&amp;lt;0.10, Cox coefficient &amp;gt;0). CLK1 (P-adj=0.0218, HR=1.5939), CLK2 (P-adj=0.001298, HR=2.1393), and SRSF2 (P-adj=0.00167, HR=3.2917) were found to be positively associated with poorer PFI, ranking 1202, 400, and 437, respectively. Reactome pathway analysis of the significant gene set showed that mRNA splicing and processing accounted for 5 of the 19 pathways that were strongly associated with poorer PFI. An additional pathway analysis (GSEA v.3.0, MSigDB v6.2) of tumors categorized by PTEN status to assess relationship with disease severity showed that mRNA splicing (P-adj=0.0243, NES=1.7714) was enriched in PTEN-null vs. PTEN-wt tumors. Other pathways of interest, including Wnt signaling (P-adj=0.0187, NES=1.846), cell cycle (P-adj=0.0124, NES=1.974), chromatin remodeling (P-adj=0.0135, NES=1.901), DNA damage repair (P-adj=0.013974, NES=1.8934), and PTEN regulation (P-adj=0.0230, NES=1.7861), were also enriched in PTEN-null tumors. Lastly, a survival analysis within all TCGA-PRAD patients showed that low CLK1 (P=0.03) and CLK2 (P=0.0004) expression, individually, were associated with a better prognosis vs. their high-expressing counterparts. Analysis of CLK3 and CLK4 expression did not reach statistical significance. Collectively, these findings revealed an association of spliceosome activity and CLK1/2 expression with aggressive disease biology in prostate cancer. A Phase 1 study of SM08502, a novel, small-molecule pan-CLK inhibitor, in subjects with advanced solid tumors is ongoing (NCT03355066). This analysis nominates prostate cancer as a tumor type worth further exploring for the clinical activity of SM08502. Citation Format: Shawn Cho, Atish D. Choudhury, Catherine Fleener, Long Do, Carine Bossard, Heekyung Chung, Timothy J. Phalen, Steven Cha. Transcriptome analysis of TCGA prostate cancer samples identifies an association of poorer survival and aggressive disease biology with CDC-like kinase (CLK) expression and spliceosome regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3521.