Spliceosome: Physiology and Disease Pathogenesis
Abstract Pre-mRNA splicing is an essential step for the expression of intron-containing genes, i.e., the majority of genes. Splicing is a high-fidelity process, as required for the correct expression of proteins. However, there is flexibility in the selection of competing splice sites, which gives rise to alternative splicing, a regulated process that greatly increases the diversity of the proteome. Splicing is catalyzed in a stepwise manner by the spliceosome, a macromolecular machine that consists of 5 small RNAs and more than 100 proteins. Key insights about the structure and dynamics of spliceosomal complexes have recently been obtained through cryo-electron microscopy studies. Dysregulated splicing can arise from mutations in the splice sites or regulatory elements of individual genes, from alterations in the levels of regulatory splicing factors (activators and repressors), or from mutations in splicing-factor genes. All of these scenarios can give rise to various cancers, depending on the affected gene and the cellular context. Interestingly, recurrent somatic heterozygous mutations in particular splicing factors involved in branchpoint-sequence and 3'-splice-site recognition have emerged as key drivers of certain myeloid neoplasias. This presentation will review relevant features of the spliceosome machinery, the functional implications for normal and pathological conditions, and the potential for novel therapies. Disclosures Krainer: Ionis Pharmaceuticals: Consultancy, Honoraria, Patents & Royalties, Research Funding; Stoke Therapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties; Cold Spring Harbor Laboratory: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; H3 Biomedicine: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients
Despite progress with immunotherapy and targeted agents, treatment of refractory disease remains challenging, in particular for patients with T-ALL. Identification of genomic lesions defining actionable targets had limited impact on patient care so far. The complexity of biological systems highlights the need to develop complementary functional approaches. We and others have established platforms with a library of 120 drugs based on current treatment and (pre-)clinical development, to detect ex-vivo drug response phenotypes on leukemia samples at single cell resolution by high content image analysis. We demonstrated that drug response profiling (DRP) identifies dependencies not predicted by genetic alterations adding a functional information layer for clinicians. Here we report first correlations with clinical outcome using DRP in a non-interventional setting. From 2016 to 2019 we performed DRP in the framework of European ALL first- and second- line protocols upon request by treating centers. Here we analyze retrospectively treatment decisions and outcome for 23 T- and 50 BCP-ALL patients. To evaluate drug responses, we compared dose response curves of individual patients to data recorded for all patients. Sensitivity and resistance were defined based on the IC50 outlier analysis using cut-offs depending on distribution (normal gaussian vs. skewed). From 73 patients tested, clinical outcome data has been available for 36 BCP- and 15 T-ALL patients. NGS data provided by the INFORM registry has been available in 8 BCP- and 2 T-ALL patients. In first line BCP-ALL patients, ex-vivo Dexamethasone response predicted clinical response to prephase prednisone (d8) and minimal residual disease (MRD) reduction measured by flow cytometry at d15 of first line AIEOP BFM 2009 induction (Fig. 1). For refractory and relapsed ALL we observed an association of DRP and response to targeted agents in 14 out of 16 patients (87.5 %; Table 1). Data for the r/r BCP-ALL cohort is limited because most patients underwent CD19 and / or CD22-directed immunotherapy. Sensitivity and resistance to Calicheamicin correlated with clinical response to Inotuzumab, suggesting functional testing to be evaluated in future studies. In contrast, lack of correlation of ex-vivo sensitivity to MEK-inhibitors with presence of RAS-pathway alterations caution the exclusive use of molecular information to predict response to these agents. Most therapeutic decisions based on DRP information were made for patients with r/r T-ALL. Bortezomib ex-vivo sensitivity correlated with clinical responses in 5 T-ALL patients (Fig. 2). Both patients predicted to respond to Bortezomib and treated on Bortezomib + Venetoclax experienced good MRD response providing a bridge to stem cell transplantation (SCT). However, these patients relapsed after SCT emphasizing the need for additional consolidative therapeutic elements for heavily pretreated patients. In line with previous reports, we confirmed a T-ALL with high sensitivity to Dasatinib (IC50 1.9 nM; Fig. 3). Dasatinib monotherapy induced a molecular remission. A 2nd T-ALL showing an ex- vivo Dasatinib IC50 at 80 nM was refractory to treatment with Dasatinib + Daunoxome-FLAG. A 3rd ABL1-fusion positive T-ALL, ex-vivo resistant to Imatinib and Dasatinib, had only short- term response to Imatinib + chemotherapy. Finally, treating a T-ALL patient based on high sensitivity to the XPO1 inhibitor Selinexor as 4th line monotherapy led to significant decrease of PB blasts from d1 25 G/L to 0.7 G/L at d13 of treatment (Fig. 4). The patient experienced improved quality of life, minimizing need of hospitalization with stable disease for 3 months on maintenance with Selinexor. Given the promising preclinical data with this class of agents and current lack of established biomarkers, we propose that DRP should be evaluated for this class of agents. In conclusion, we established first associations between DRP and clinical response for various agents providing a rationale for the evaluation of DRP in prospective clinical trials. Integration of molecular and functional information may improve the selection of more specific treatment options for patients with resistant disease. The international BFM Study Group and ITCC Consortium are planning an international multiarm early clinical trial for treatment of r/r ALL patients that will include DRP for evaluation in order to improve selection of targeted therapy. Disclosures Cario: Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. Hrusak:Amgen: Other: MRD investigations funded by Amgen, Research Funding. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. von Stackelberg:Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau. Jacoby:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lonza: Membership on an entity's Board of Directors or advisory committees. Bourquin:Servier: Other: Travel Support.
Abstract Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.
Abstract CML patients should be screened for Abl kinase domain (KD) mutations before changing tyrosine kinase inhibitor (TKI) therapy to ensure that the second-line TKI will be effective against mutations that arose on the first drug. Sanger sequencing (SS) can confidently detect mutations present in >20% of BCR-ABL molecules but will fail to detect minor resistant sub-clones. If missed, these minor sub-clones may be further selected by the second-line TKI and cause treatment failure. The T315I mutation can be particularly problematic as it is resistant to most TKIs except ponatinib. It seems reasonable to detect all mutations if possible, and avoid second line drugs that are known to be ineffective in their presence. We have developed a next generation sequencing strategy (Illumina MiSeq, 2 x 300 bp) that enables confident detection of all Abl KD mutations present at a level of at least 1% in the BCR-ABL cDNA, this level being 3-5x above the background calling-error rate. Two 500 bp PCR products are sufficient to cover the entire kinase domain (aa M237 to E505). With 300 bp paired-end sequencing of the products, a 65 bp region containing the 315 codon (codons 306-326) is sequenced on both strands. By excluding base changes that are not corroborated on both strands, mutations in this region can be detected with a 10-fold higher accuracy (in 0.1% of BCR-ABL molecules). Samples with low disease burden (1% BCR-ABL/ABL positivity) can also be amplified sufficiently with 50 cycles of PCR, minimising artefactual DNA polymerase-induced mutations. Indexing allows the simultaneous analysis of 80 PCR's in a single MiSeq run (Abl KD of 40 patients). Important aspects of the method are: 50% PhiX DNA is added to the library to increase complexity, and the flow cell is seeded at low density (300,000 clusters per mm2) to reduce sequencing errors.Overlapping paired reads are combined to produce a single FASTQ sequence (modified FLASH source code). Any bases in the overlapping region that do not agree with their counterpart on the other strand are labelled "N" and given a quality score (Q score) of 20.Combined sequences are quality parsed (FASTX Tool Kit) to exclude sequences that do not have a Q score of at least 20 at all basesParsed high quality sequences are compared to the reference sequence. We have sequenced the BCR-ABL KD of patients who were sub-optimal responders (BCR-ABL/ABL ratio of >1% at >= 11 months on therapy) in the NCRI SPIRIT 2 trial of first-line imatinib vs dasatinib. Of 60 sub-optimally responding imatinib patients, 6 (10%) had high level mutations (in >20% of BCR-ABL molecules): T315I, L387F, G250E, N331D, M244V x 2. The patients with L387F and G250E were switched to dasatinib and proceeded to respond well. The patient with T315I was also initially switched to dasatinib but failed to respond (BCR-ABL/ABL 29% after 1 year). This patient eventually received ponatinib with good response (BCR-ABL/ABL <1%). One patient with M244V was switched to nilotinib. Initially this caused relative selection of a pre-existing M387F mutated clone, this mutation increasing from <1% to 55% of BCR-ABL molecules sequenced. However the patient eventually responded well to the drug, indicating that M387F causes only partial resistance to nilotinib. 11/60 (18.3%) imatinib-treated patients had low level mutations present in <20% of BCR-ABL molecules and multiple low-level mutations were seen in 2 patients. One patient had a BCR-ABL/ABL ratio of 40% and 4 non-compound mutations (Y253H - 18%, M244V- 6.5%, K285E - 5.6%, Y312C - 2.6%). All were undetectable by SS. This patient was discontinued from the study and received nilotinib. Nilotinib, which is known to be ineffective against Y253H, caused selection of the Y253H clone to 90% of BCR-ABL molecules and an increase in the BCR-ABL/ABL ratio to 61%. This patient was subsequently switched to ponatinib and responded well (BCR-ABL/ABL ratio < 1%) before undergoing allogeneic transplantation. Of 28 dasatinib-treated patients, 14 had low level mutations including one patient with a T315I of 3.4%. No high-level or compound mutations have so far been discovered in this group at this time point (>11 months). Our study demonstrates the value of using 2 x 300 bp paired-end sequencing to detect high and low level mutations, even in patients with low-level disease burden, to guide the choice of an appropriate second-line TKI. Disclosures Dickson: Ariad: Research Funding. Kennedy:Ariad: Research Funding. Cork:Roche: Research Funding; Ariad: Research Funding; BMS: Research Funding; Novartis: Research Funding. Hedgley:Roche: Research Funding; BMS: Research Funding; Ariad: Research Funding; Novartis: Research Funding. Copland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Holyoake:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. O'Brien:BMS: Consultancy, Honoraria, Research Funding; Pzifer: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ramashoye:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Mutations in splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) are identified in over 50% of patients diagnosed with myelodysplastic syndrome (MDS). U2AF1 is a U2 auxiliary factor that forms a heterodimer with U2AF2 for the recognition of the 3' splice site (SS) and results in the subsequent recruitment of U2snRNPs during pre mRNA splicing. U2AF1 mutations are present in 11% of MDS and its presence is correlated with an increased risk of progression to AML. Non-canonical mutations are rarely seen in U2AF1 but two highly conserved hotspots (S34, Q157) are frequently seen and result in distinct downstream effects. We performed NGS analysis of a large cohort of patients with myeloid neoplasms (MN; n=3,328) and identified 238 patients with U2AF1 mutations (7%). We analyzed the somatic mutational profile, clonal hierarchy, and splicing profile of patients with U2AF1S34 (n=99), U2AF1Q157 (n=119), and others (n=20; M1/*, A26T/V, R35Q, R118C, E124K, F150L, E152G, C154S, R156H, M172L). The mutational spectrum of U2AF1S34 and U2AF1Q157 was equally dismal but pretty distinct. U2AF1 S34 cases were mostly co-mutated for DNMT3A (5%), TET2 (4%), RUNX1 (2%), ASXL1, CBL, ETV6, KRAS, NRAS (1.3%), STAG2 (1%), CUX1, and TP53 (<1%) while U2AF1Q157 had higher numbers of mutations in ASXL1 (10%), SETBP1, TET2 (3%), NRAS (2%), DNMT3A, PHF6 (2%), JAK2 (2%), CBL, EZH2, TP53 (2%), IDH2, RUNX1, STAG2 (2%), KRAS (1%), and IDH1 (<1%). Mutational rank showed: U2AF1S34 was ancestral in 38% of the cases followed by secondary DNMT3A and NRAS (5%, both). Ancestral U2AF1Q157 was found in 35%, with ASXL1 (19%) being the most common secondary hit. Subclonal U2AF1S34 (44%) was most commonly preceded by DNMT3A (21%), while secondary U2AF1Q157 (40%) had ASXL1 (28%) as the most common first hit. U2AF1S34 and U2AF1Q157 were co-dominant to a miscellanea of mutations. U2AF1S34 cases had a shorter OS than U2AF1Q157 cases (n=82 vs. n=101; 20 vs. 25 mo.; P=.002). Ancestral U2AF1S34 or U2AF1Q157 defined a dismal prognosis compared to secondary U2AF1S34 or U2AF1Q157 (OS: n=63 vs. n=86; 29 vs. 37 mo.; P=.03). To investigate the effects of both mutations on splicing patterns, we analyzed RNA-Seq profiles, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events that were classified as skipping exon (SE), retained intron (RI), and 3' or 5' alternative SS (A3SS, A5SS) (Hershberger, ASH 2019). Overall, 675 AS events in 430 genes were scored significant (pFDR<.05) when the splicing inclusion/exclusion difference was ±10%. U2AF1S34 and U2AF1Q157 caused an equal fraction of SE (79% U2AF1S34; 72% U2AF1Q157). Only 4% of the genes were commonly misspliced by both mutations, while the rest of the genes were uniquely spliced according to each mutation. Some exemplary genes misspliced by both mutations were DDX3X (an RNA helicase) showing a consistent SE at exon 5 and an RI 3-5a and CCNG1 (cell growth regulation) which was enriched for RI 7-7. Among others, U2AF1S34 uniquely affected the splicing of TET2 (SE e3); cell cycle regulators, CDC37L1 (SE), CCNC (SE e8), and HDAC3 (SE e6). We then investigated whether U2AF1 mutations might affect the splicing of other RNA splicing components. This mechanism would lead to the loss of regulation of the spliceosome complex. U2AF1S34 produces selective RIs in SRSF2 (4-4a) and A5SS in SRSF6 (7a and 7). Tumor suppressors and proto-oncogenes were also found to be misspliced by U2AF1S34 including PTEN (RI 3-Ua), CTNNB1 (RI 15-19; 3'SS (19 and 19a), and CCNL1 (RI 4-U). Major regulators of splicing factor activity are phosphatases like PP1R12A and PPP1R12B, which showed an RI 8-7 and an A3SS, respectively. Among genes exclusively misspliced by U2AF1Q157, we identified DEAD-Box helicases [DDX17 (RI), DDX59 (SE e8), DHX29 (RI)], ALAS family members (ALAS1, SE e6; ALAS2, SE e5) and UTX (KDM6A; SE e16). U2AF1Q157 affected the missplicing of DYRK1A (SE e7), a kinase known to phosphorylate SF3B1 at T434 site. In sum, our study suggests that while concurrent mutations in splicing factors lead to lethality, the presence of mutations (as the case of U2AF1S34 and U2AF1Q157) and consequent missplicing of other splicing factors are permissive events in MN and might represent novel mechanisms of disease pathogenesis. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
Abstract Background Nonsense-mediated decay (NMD) is a cellular quality control system that degrades mRNAs containing premature termination codons (PTCs) as well as ~10% of normal mRNAs (Kurosaki and Maquat, 2016). NMD thus prevents translation of misfolded proteins, and potential activation of the unfolded protein response (UPR). Mutations in splicing factors such as SF3B1, SRSF2, U2AF1 and ZRSR2 found in hematological as well as solid tumors, can lead to generation of aberrant mRNAs that contain PTCs. Aberrant splicing patterns in cancer cells can possibly result in increased pressure on the NMD machinery. CC-115, a potent inhibitor of mTOR kinase (TORK) and of DNA-dependent protein kinase, (DNA-PK; Mortensen et al., 2015; Tsuji et al., 2017), is in clinical development for the treatment of solid and hematologic malignancies (Thijssen et al., 2016). Preclinical data revealed an additional target of CC-115 and its differential effect on NMD. Our hypothesis was that a subset of tumor cells, especially hematologic tumors with high protein production and/or splicing factor mutations, would be susceptible to NMD inhibition by CC-115. Methods In total, 141 cell lines were screened for sensitivity to CC-115-mediated inhibition of proliferation and induction of cell death, in comparison to specific inhibition of TORK (CC-223). Isogenic DNA-PK knockout cell lines HCT116/HCT116 DNA-PK-/- and M059K/M059J DNA-PK-/- were treated with CC-115 and CC-223. Activity on NMD in vivo was tested using HCT-116 xenograft tumors treated with Vehicle or CC-115. Dependence on CC-115 sensitivity was determined using CRISPR/Cas9 technology of apoptosis or UPR genes in various MM cell lines. RNA sequencing was used for identification of potential targets in sensitive and resistant cell lines. Results A subset of cancer cell lines underwent cell death at sub-micromolar concentrations of CC-115 due to inhibition of NMD, but this was independent of mutations in splicing factors such as SF3B1. We next focused on MM cells as these generally produce high levels of (immunoglobulin) proteins and are prone to ER stress, and therefore potentially susceptible to NMD inhibition. Indeed, treatment with CC-115 resulted in activation of the UPR independent of TORK and DNA-PK inhibition, and cell death in 11/12 MM cell lines. Activity of CC-115 correlated strongly with cell death by the known ER-stress inducer, thapsigargin. Cell death by CC-115 occurred by the mitochondrial pathway of apoptosis, as it depended on caspase activity and the presence of Bax-Bak. Analysis of RNA sequencing data is ongoing and has indicated potential targets dictating sensitivity to CC-115-mediated cell death. Conclusions We describe that hematologic tumors with high protein production are specifically sensitive to CC-115, a novel and clinically exploitable inhibitor of NMD. This might lead to application in malignancies that depend on NMD to avoid excessive protein stress, such as multiple myeloma. Disclosures Garrick: Celgene: Employment. Trowe:Celgene: Employment. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Eldering:Celgene: Research Funding. Filvaroff:Celgene: Employment.
Abstract Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer and despite improved survival rates, relapsed ALL is still among the most common causes of cancer death in children. Although changes in the expression of specific genes have been linked to chemotherapeutic resistance, relatively little is understood of the pharmacogenomic impact of the noncoding, cis-regulatory landscape governing gene regulation. Glucocorticoids (GCs; i.e. steroids) are a mainstay of contemporary, multi-drug chemotherapy in ALL, and GC resistance is predictive of both relapse and poor clinical outcome in ALL. Because GCs function through activation of glucocorticoid receptor (GR), a nuclear receptor transcription factor that interacts directly with cis-regulatory elements, unveiling the glucocorticoid gene regulatory network (GC-GRN) in leukemia cells is crucial to understanding not only the biological mechanism of apoptosis, but also illuminating gene regulatory mechanisms contributing to GC resistance. To test the hypothesis that alterations to the GC-GRN are important contributors to steroid resistance in ALL, we comprehensively mapped cellular responses to GCs in human ALL cell lines using >100 independent functional genomic datasets. This comprehensive approach uncovered thousands of genes and cis-regulatory elements that were responsive to GCs, and further identified >38,000 high-confidence glucocorticoid response elements (GREs) in the ALL genome. A closer examination of these data revealed GR binding profiles that were consistent with the long-range flexible billboard model of gene regulation. By further integrating our results with genetic and epigenetic data in primary ALL cells from patients enrolled on St. Jude clinical trials, we identified 45 DNA sequence variants associated with ex vivo GC resistance that map to GREs and functionally validated an associated variant within the TLE1 gene locus. We also uncovered 1929 accessible chromatin sites (FDR<0.1) in primary ALL cells that were associated with ex vivo GC resistance, and these GC-resistance accessible chromatin sites were highly enriched at GREs determined from ALL cell lines (p<2.2x10 -16). High-throughput pharmacogenomic CRISPRi screening in human ALL cell lines with a library of >10,000 sgRNAs targeting >1000 GR binding events at putative GC-resistance accessible chromatin sites identified a subset of GR binding sites implicated in GC resistance. Overall, these data indicate that GCs initiate pervasive, genome-wide effects on the leukemia epigenome and transcriptome, and that genetic and epigenetic alterations to GREs are mechanisms contributing to GC resistance in childhood ALL. Disclosures Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee. Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months.
Abstract Myelodysplastic syndromes (MDS) are unique among cancers because of the frequent occurrence of somatic mutations impacting spliceosome machinery. At least 65% of MDS patients harbor a mutation in one of several splicing factors including U2AF1, SF3B1 and SRSF2. Whole exome sequencing of MDS bone marrow uncovered somatic frameshift mutations in LUC7L2, the mammalian ortholog of a yeast splicing factor. LUC7L2 is located in the most commonly deleted region of chromosome 7. Deletions and frameshifts lead to haploinsufficient expression and therefore it can be approximated that a combined 14% of MDS patients have low expression of LUC7L2. Restoring expression of LUC7L2 in del(7q)-iPSCs partially rescues the differentiation of iPSCs into CD45+ myeloid progenitors. Although perhaps partly due to associated losses of other genes on chromosome 7, low expression of LUC7L2 correlates with a poorer patient prognosis, so its haploinsufficiency may play an important role in bone marrow failure. While U2AF1, SF3B1, and SRSF2 are well-characterized splicing factors, the function of LUC7L2 in pre-mRNA splicing is unexamined and its role in the MDS pathogenesis is undefined. We hypothesize that low expression of LUC7L2 results in the aberrant splicing of oncogenes and tumor suppressor gene transcripts thus reducing expression or altering function and contributing to the pathogenesis of MDS. We have characterized LUC7L2 as an alternative splicing regulatory protein that plays a repressive role in the regulation of alternative RNA splicing. We generated HEK-293 cells overexpressing V5-tagged LUC7L2 for immunoprecipitation-mass spectrometry, to ascertain protein interactions with LUC7L2. LUC7L2 co-immunoprecipitated with splicing regulators which are involved in splice site recognition. We performed cross-linking-IP-high-throughput-sequencing (CLIP-seq) to identify LUC7L2 binding sites on RNA. We identified 301 LUC7L2 RNA-binding sites as well as binding sites on U1 and U2 which is common for splicing regulatory proteins. Metagene analysis of these binding sites showed that LUC7L2 bound near splice sites in exonic sequences. We knocked down LUC7L2 expression in HEK293 and K562 cells to phenocopy the frameshifts and deletions observed in MDS patients. We used a PCR-based assay to measure the splicing efficiency of introns near LUC7L2-binding sites. Knockdown of LUC7L2 increased the splicing efficiency of 8/13 selected introns; this suggests that LUC7L2 represses selective splice site usage. We also performed RNA-seq to characterize global mis-splicing events. Analysis of RNA transcripts revealed a multitude of splicing changes, including enhanced exclusion of alternative introns. Knockdown LUC7L2 cells exhibited-altered expression of other splicing factors; this could have further contributed to the vast number of splicing changes observed. To identify specific splicing changes that could contribute to the pathogenesis of MDS, we compared the splicing profiles of LUC7L2-knockdown in K562 cells with RNA-seq data from K562 cells expressing U2AF1S34F, SRSF2P95H or SF3B1K700E. This analysis yielded several exon-skipping splicing patterns in cancer-relevant transcripts, such as oncogene PRC1, splicing factor PTBP1 and MRPL33. Additionally, we noticed commonly mis-spliced transcripts among the four datasets in which the missplicing events occurred in the functional domain, potentially conferring a functional change. Surprisingly, we observed missplicing of U2AF1 in LUC7L2-knockdown, SRSF2P95H, and SF3B1K700E K562 cells, which altered the length of the RNA-recognition UHM domain by inclusion of a mutually exclusive exon or retention of an intron. In this way, low expression of LUC7L2, or point mutants U2AF1S34F, SRSF2P95H, and SF3B1K700E,could alter U2AF1 function as a distal convergence point. In summary, we identified a novel splicing factor implicated in the pathogenesis of MDS. We characterized LUC7L2 as a splicing repressor and discovered many splicing changes caused by low expression of LUC7L2. Several genes were also mis-spliced in U2AF1S34F, SRSF2P95H and SF3B1K700E K562 cells targeting these for further study. Commonly mis-spliced targets such as U2AF1 may indicate that some of the novel therapeutics may have spliceosome mutation agnostic effects. If this applies to the LUC7L2 mutations, then they may also be effective in del7/del7q cases. Disclosures Carraway: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.
Abstract Introduction. Despite the recent approval of several drugs for the treatment of AML, the 3 + 7 regimens remain as the standard of care for many patients. Its lack of efficacy represents the main cause of death, since only 10% of patients who show refractoriness/relapse overcome the disease. Therefore, there is still an urgent need for seeking more effective treatments. Aberrant RNA splicing has been described in AML, but its relevance as mechanism of resistance is unclear. In this study, we deepen the mechanism of resistance to cytarabine and the role of splicing factors SR proteins, involved in the spliceosome functionality, to seek more effective therapies for AML. Methods. First, the expression levels of genes encoding SR proteins were analyzed with the GEPIA2 platform, comparing the data from the TCGA-LAML (AML patients) and GTEx (healthy) projects. Then, the gene expression of one of the most overexpressed genes, SRRM2, was validated by qPCR in samples of AML patients compared to controls and other myeloid disorders, as MDS and MPN (n=54). The resistance-associated phospho-proteomic profile was analyzed by LC-MS / MS after IMAC enrichment in paired samples from 3 AML patients. The expression of SR proteins and their phosphorylated forms was studied by immunohistochemistry (IHC) before and after resistance in paired bone marrow samples from 3 AML patients. We also analyzed by IHC the prognostic value of phospho-SR proteins at the moment of diagnosis in 64 patients with different responses to cytarabine (non-responders and responders). In order to validate an altered function of SR proteins, the analysis of the differential use of exons of paired samples from 25 AML patients was performed using RNAseq. Then, we evaluated in vitro the efficacy of some splicing modulators, and its combination with other approved drugs, in cytarabine-sensitive and resistant cells. The combination of H3B-8800, a spliceosome inhibitor, with venetoclax was tested in ex vivo samples from AML patients and healthy donors. Results. We found that the gene expression levels of SRSF9, SRSF12 and SRRM2 were altered in AML (Fig 1A-B). Immunohistochemical studies revealed that, although at the protein level no differences were found in SR proteins expression between the diagnosis and relapse moment, an increase in the levels of phosphorylated SR proteins was associated at the time of relapse (Fig 1C). Indeed, the phosphorylation levels of SRRM2, among other SR proteins, were found to be increased during cytarabine resistance by phospho-proteomics (Fig 1D). Moreover, the phosphorylation levels of SR proteins predicted the response to cytarabine treatment, as AML patients that were non-responders presented significantly higher levels compared to responders ones (Fig 1E). The observed alterations in the phosphorylation of these proteins were correlated with a differential use of exons in some of their known targets, when comparing the diagnostic condition and drug resistance moment. Based on this evidence, the efficacy of combining different therapeutic options was evaluated in vitro using sensitive or cytarabine-resistant cell models (Fig 1F). The combination of H3B-8800 together with venetoclax was the most effective in vitro and also presented synergic effects ex vivo in AML patients samples (Fig 1G). Furthermore, this combination did not show toxicity over healthy hematopoietic progenitors, since the same doses that were effective in AML did not show toxicity in a healthy context (Fig 1H). Conclusions. The results of this work shed light on the role of the RNA splicing process in cytarabine resistance in AML. Interestingly, the high levels of phosphorylated splicing factors SR proteins at diagnosis in refractory patients, would allow us to use them as a predictive biomarker of response to cytarabine treatment. Otherwise, due to the need to search effective and safe treatments in this disease, we have found that the combination of splicing inhibitors with venetoclax should be a good strategy for the treatment of AML. Acknowledgment. This work has been possible thanks to the granting of the project PI19/01518 from the Carlos III Health Institute and the CRIS Against Cancer Foundation. ML.M. enjoys a research grant from the Spanish Society of Hematology and Hemotherapy and R.GV. a FPU grant from the Ministry of Science, Innovation and Universities. Figure 1 Figure 1. Disclosures Sanchez: Altum sequencing: Current Employment. Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria.
Abstract RNA splicing dysregulation is a hallmark of chronic lymphocytic leukemia (CLL). Although somatic mutations in SF3B1 or U1 snRNA present in >20% of CLL patients, general splicing defects cannot be fully explained by genetic alterations of spliceosome alone. We reported that splicing factors are upregulated at protein, but not RNA, level in CLL compared to normal B cells by an integrated transcriptomic and proteomic analysis. This highlights a post-transcriptional layer of regulation that controls the abundance of splicing factors and contributes to RNA splicing dysregulation in CLL, with mechanism that has yet to be elucidated. To discover these regulators, we performed weighted correlation network analysis and found expression of many splicing factors strongly correlated with the abundance of METTL3. METTL3 is an RNA methyltransferase that modifies N 6-methyladenosine (m 6A) on mRNA and regulates the translation of m 6A-installed transcripts. Primary CLL cells have consistent upregulated protein expression of METTL3 and exhibit an increased m 6A level on mRNA. To identify m 6A modification sites in normal and CLL B cells, we performed m 6A sensitive RNase sequencing and found transcripts with differential m 6A modification are highly enriched in the RNA splicing pathway (q=1.8E-4). Consistent with this, these transcripts appear to have a higher m 6A density. These results raised up a possibility that METTL3 translationally controls the expression of splicing factors through m 6A modification. To examine whether METTL3 impacts splicing factor expression, we performed an integrated ribosome profiling (Ribo-seq) and RNA sequencing using CLL cell line HG3 with or without METTL3. Knockout (KO) of METTL3 decreased overall translation efficiency with RNA splicing as the most significantly affected pathway (q=2.1E-52). We further confirmed that KO of METTL3 or treatment with METTL3 inhibitor STM2457 decreases the expression of many splicing factors in HG3 cells. However, we detected no protein changes in known METTL3 targets (BCL2, MYC), highlighting splicing factors as the preferred targets of METTL3 in CLL. Moreover, overexpression of wildtype but not catalytic mutant METTL3 restored splicing factor expression defects in METTL3 KO cells, demonstrating that the regulation of splicing factor expression is methyltransferase activity dependent. To dissect the mechanism of how METTL3 preferentially influences the expression of splicing factors via m 6A modification, we mapped out METTL3 responding m 6A sites using methylated RNA immunoprecipitation sequencing (MeRIP-seq). Consistent with its role as an methyltransferase, we detected 5875 hypomethylated and 1409 hypermethylated sites upon METTL3 KO. Most downregulated splicing factors harbored hypomethylation around the stop codon region, including SF3B1, SF3A2 and SR proteins, indicating they are direct targets of METTL3. To validate this association, we utilized m 6A editing platform dCasRx-METTL3 to install m 6A at the stop codon region of endogenous SF3B1 transcripts, leading to increased SF3B1 protein expression. These results provided evidence that METTL3 directly regulates splicing factor protein expression via m 6A mediated translational control. Unexpectedly, we also discovered that hypermethylated transcripts are highly enriched in RNA splicing with most affected sites localized at the CDS regions. The hypermethylation of these transcripts can be caused by a combined upregulation of m 6A writer (METTL16) and eraser (ALKBH5) protein upon KO of METTL3. To determine how hypermethylation affects expression of splicing factors, we conducted an integrated MeRIP-seq and Ribo-seq analysis and observed a convergence of increased ribosomal density and hypermethylated adenosines. Harnessing dCasRx-METTL3 platform, we installed m 6A at the CDS region of SF3A3 and confirmed the downregulation of protein by immunoblot. This implicates that, as an alternative mechanism, METTL3 regulates the translation of splicing factors via m 6A mediated decoding process. Altogether, our results uncovered a novel regulatory axis of METTL3 as a regulator for splicing dysregulation in CLL. We propose that METTL3 regulates splicing factor expression through m 6A-mediated translational control. Our study highlights a post-transcriptional layer of m 6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Disclosures Brown: Gilead, Loxo/Lilly, SecuraBio, Sun, TG Therapeutics: Research Funding; Invectys: Other: Data Safety Monitoring Committee Service; Abbvie, Acerta/Astra-Zeneca, Beigene, Bristol-Myers Squibb/Juno/Celgene, Catapult, Eli Lilly, Genentech/Roche, Janssen, MEI Pharma, Morphosys AG, Nextcea, Novartis, Pfizer, Rigel: Consultancy. Danilov: Abbvie: Consultancy, Honoraria; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Rigel Pharm: Honoraria; Genentech: Consultancy, Honoraria, Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding. Siddiqi: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Speakers Bureau; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Oncternal: Research Funding; TG Therapeutics: Research Funding.
