Biology subjects

Thomas-Tikhonenko, A.

Publications and source records attributed to Thomas-Tikhonenko, A..

16 recordsLinked to original sources

bioRxiv2026

An ancestral haplotype of P2RX5 yields a B-cell surface marker and a promising multi-lineage immunotherapy target

While CD19- and BCMA-directed immunotherapies have improved outcomes for B-lymphoid and plasma cell malignancies, frequent relapses with antigen loss/downregulation highlight the need for new targets. Here, using transcriptomic datasets and newly-developed monoclonal antibodies, we show that P2RX5, long considered a pseudogene in humans, encodes a stable protein in 80% of individuals of African descent carrying the ancestral haplotype. Like CD19, P2RX5 displays B-cell lineage-restricted expression in normal tissues. Unlike CD19, P2RX5 is expressed not only in B-cell neoplasms, but also in T-cell leukemia (T-ALL) and multiple myeloma (MM). We developed P2RX5-directed bispecific T-cell engagers and CAR T cells, which killed T-ALL cells with no evidence of T-cell fratricide. These agents were non-inferior to FDA-approved CD19- and BCMA-directed immunotherapeutics in cell culture and xenograft models of Burkitt lymphoma and MM, while maintaining potency against CD19- and BCMA-negative variants. Hence, P2RX5 is a unique multi-lineage target for frontline or salvage immunotherapy.

cancer biology↗

bioRxiv2025

The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies.

CD19-CAR-T-cells emerge as a major therapeutic option for relapsed/refractory B-cell-derived malignancies, however approximately half of patients eventually relapse. To identify resistance-driving factors, we repeatedly exposed B-cell lymphoma/B-cell acute lymphoblastic leukemia to 4-1BB/CD28-based CD19-CAR-T-cells in vitro. Generated models revealed costimulatory domain-dependent differences in CD19 loss. While CD19-4-1BB-CAR-T-cells induced combination epitope/total CD19 protein loss, CD19-CD28-CAR-T-cells did not drive antigen-escape. Consistent with observations in patients relapsing after CD19-4-1BB-CAR-T-cells, we identified CD19 frameshift/missense mutations affecting residues critical for FMC63 epitope recognition. Mathematical simulations revealed that differences between CD19-4-1BB- and CD19-CD28-CAR-T-cells activity against low-antigen-expressing tumor contribute to heterogeneous therapeutic responses. By integrating in vitro and in silico data, we propose a biological scenario where CD19-4-1BB-CAR-T-cells fail to eliminate low-antigen tumor cells, fostering CAR-resistance. These findings offer mechanistic insight into the observed clinical differences between axi-cel (CD28-based) and tisa-cel (4-1BB-based)-treated B-cell lymphoma patients and advance our understanding on CAR-T resistance. Furthermore, we underscore the need for specific FMC63 epitope detection to deliver information on antigen levels accessible for CD19-CAR-T-cells. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/640707v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1728c4org.highwire.dtl.DTLVardef@759660org.highwire.dtl.DTLVardef@1e3a0deorg.highwire.dtl.DTLVardef@15653e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Neuronal cell adhesion molecule (NRCAM) variant defined by microexon skipping is an essential, antigenically distinct, and targetable surface proteoform in high-grade glioma

To overcome the paucity of known tumor-specific surface antigens in pediatric high-grade glioma (pHGG), we contrasted splicing patterns in pHGGs and normal brain samples. Among alternative splicing events affecting extracellular protein domains, the most pervasive alteration was the skipping of [≤]30 nucleotide-long exons. Several of these skipped microexons mapped to L1-IgCAM family members, such as NRCAM. Bulk and single-nuclei short- and long-read RNA-seq revealed uniform skipping of NRCAM microexons 5 and 19 in virtually every pHGG sample. Importantly, the {Delta}ex5{Delta}ex19 (but not the full-length) NRCAM proteoform was essential for pHGG cell migration and invasion in vitro and tumor growth in vivo. We developed a monoclonal antibody selective for {Delta}ex5{Delta}ex19 NRCAM and demonstrated that "painting" of pHGG cells with this antibody enables killing by T cells armed with an FcRI-based universal immune receptor. Thus, pHGG-specific NRCAM and possibly other L1-IgCAM proteoforms are promising and highly selective targets for adoptive immunotherapies.

cancer biology↗

bioRxiv2024

The splicing modulator CLK1 is a candidate oncogenic dependency in pediatric high-grade gliomas

BackgroundPediatric brain tumors are the leading cause of disease-related mortality in children, yet many aggressive tumors lack effective therapies. RNA splicing is a hallmark of cancer, but it has not yet been systematically studied in pediatric brain tumors. MethodsWe analyzed 729 pediatric brain tumors spanning histologies and molecular subtypes to quantify differential tumor splicing. We developed the Splicing Burden Index (SBI) to enable cross-sample comparisons and performed hierarchical clustering of highly variable splice events to define splicing-informed tumor groups. These were integrated with clinical outcomes, pathway activity, and proteogenomic data. Recurrent splice events were prioritized for predicted functional impact, and in vitro perturbation studies were performed targeting the splicing kinase CDC-like kinase 1 (CLK1). ResultsSBI revealed substantial inter- and intra-histology heterogeneity. Clusters were enriched for histologies and molecular subtypes, several of which were independently associated with survival beyond histology and clinical covariates. Spliceosome pathway activity varied across clusters and was associated with worse survival, yet was not correlated with SBI, indicating distinct dimensions of splicing dysregulation. Functional prioritization identified a recurrent in CLK1 exon 4, required for canonical kinase activity. CLK1 exon 4 inclusion followed an oncofetal pattern and showed context-dependent associations with outcome distinct from total CLK1 expression. Pharmacologic inhibition and exon 4-specific perturbation of CLK1 reduced tumor cell viability and disrupted cancer-relevant splicing and transcriptional programs. ConclusionsThis study systematically characterizes splicing in pediatric brain tumors, identifies splicing-informed subgroups, and prioritizes CLK1 exon 4 as an oncofetal tumor-specific event, motivating further preclinical exploration. Key Points[bullet] Splicing analysis of 729 pediatric CNS tumors identifies splicing-defined clusters. [bullet]CLK1 exon 4 inclusion is widespread and developmentally regulated. [bullet]Exon-level CLK1 regulation shows context-dependent links to prognosis in aggressive CNS tumors.

genomics↗

bioRxiv2023

Contrasting and Combining Transcriptome Complexity Captured by Short and Long RNA Sequencing Reads

Mapping transcriptomic variations using either short or long reads RNA sequencing is a staple of genomic research. Long reads are able to capture entire isoforms and overcome repetitive regions, while short reads still provides improved coverage and error rates. Yet how to quantitatively compare the technologies, can we combine those, and what may be the benefit of such a combined view remain open questions. We tackle these questions by first creating a pipeline to assess matched long and short reads data using a variety of transcriptome statistics. We find that across datasets, algorithms and technologies, matched short reads data detects roughly 50% more splice junctions, with 10-30% of the splice junctions included at 20% or more are missed by long reads. In contrast, long reads detect many more intron retention events, pointing to the benefit of combining the technologies. We introduce MAJIQ-L, an extension of the MAJIQ software to enable a unified view of transcriptome variations from both technologies and demonstrate its benefits. Our software can be used to assess any future long reads technology or algorithm, and combine it with short reads data for improved transcriptome analysis.

bioinformatics↗

bioRxiv2023

A Novel Aberrantly Spliced Gain-of-Function NT5C2 Isoform Contributes to Thiopurine Resistance in Acute Lymphoblastic Leukemia

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B- ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.

cancer biology↗

bioRxiv2023

Alternative splicing of its 5' untranslated region controls CD20 mRNA translation and enables resistance to CD20-directed immunotherapies

Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5 untranslated regions (5-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies. Key PointsO_LIIn normal & malignant human B cells, CD20 mRNA is alternatively spliced into four 5-UTR isoforms, some of which are translation-deficient. C_LIO_LIThe balance between translation-deficient and -competent isoforms modulates CD20 protein levels & responses to CD20-directed immunotherapies C_LI Explanation of NoveltyWe discovered that in normal and malignant B-cells, CD20 mRNA is alternatively spliced to generate four distinct 5-UTRs, including the longer translation-deficient V1 variant. Cells predominantly expressing V1 were still sensitive to CD20-targeting chimeric antigen receptor T-cells. However, they were resistant to the bispecific anti-CD3/CD20 antibody mosunetuzumab, and the shift to V1 were observed in CD20-negative post-mosunetuzumab relapses of follicular lymphoma.

immunology↗

bioRxiv2022

Mapping and modeling human colorectal carcinoma interactions with the tumor microenvironment

The initiation and progression of cancer are inextricably linked to the tumor microenvironment (TME). Understanding the function of specific cancer-TME interactions poses a major challenge due in part to the complexity of the in vivo microenvironment. Here we predict cancer-TME interactions from single cell transcriptomic maps of both human colorectal cancers (CRCs) and mouse CRC models, ask how these interactions are altered in established, long-term human tumor organoid (tumoroid) cultures, and functionally recapitulate human myeloid-carcinoma interactions in vitro. Tumoroid cultures suppress gene expression programs involved in promoting inflammation and immune cell migration through receptor-ligand interactions, providing a reductive platform for re-establishing carcinoma-immune cell interactions in vitro. Introduction of human monocyte-derived macrophages into tumoroid cultures instructs macrophages to acquire pro-tumorigenic gene expression programs similar to those observed in vivo. This includes hallmark induction of SPP1, encoding Osteopontin, an extracellular CD44 ligand with established oncogenic effects. Taken together, these findings offer a framework for understanding CRC-TME interactions and provide a reductionist tool for modeling specific aspects of these interactions.

cancer biology↗

bioRxiv2022

FBXW7β isoform drives transcriptional activation of a proinflammatory TNF cluster in normal and malignant pro-B cells

Non-canonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow and tonsil donors, performed RNA-seq, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a cancer driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript utilized an alternative first exon to produce the poorly characterized FBXW7{beta} isoform, previously thought to be restricted to neural tissues. The FBXW7{beta} transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the bone marrow. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7{beta} mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7{beta}, we utilized gain- and loss-of-function approaches and identified an FBXW7{beta}-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7{beta} expression. This signature contained several members of the TNF superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7{beta} expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis and responses to anti-cancer therapies. Key pointsO_LIPreviously thought to be restricted to neural tissues, FBXW7{beta} is the predominant FBXW7 isoform in normal and malignant human pro-B cells. C_LIO_LIFBXW7{beta} promotes transcriptional activation of a proinflammatory gene cluster that contains TNF superfamily members. C_LI

cell biology↗

bioRxiv2021

A convergent malignant phenotype in B-cell acute lymphoblastic leukemia involving the splicing factor SRRM1

A significant proportion of infant B-cell acute lymphoblastic leukemia (B-ALL) patients remains with a dismal prognosis due to yet undetermined mechanisms. We performed a comprehensive multicohort analysis of gene expression, gene fusions, and RNA splicing alterations to uncover molecular signatures potentially linked to the observed poor outcome. We identified 87 fusions with significant allele frequency across patients and shared functional impacts, suggesting common mechanisms across fusions. We further identified a gene expression signature that predicts high risk independently of the gene fusion background and includes the upregulation of the splicing factor SRRM1. Experiments in B-ALL cell lines provided further evidence for the role of SRRM1 on cell survival, proliferation, and invasion. Supplementary analysis revealed that SRRM1 potentially modulates splicing events associated with poor outcomes through protein-protein interactions with other splicing factors. Our findings reveal a potential convergent mechanism of aberrant RNA processing that sustains a malignant phenotype independently of the underlying gene fusion, and that could potentially complement current clinical strategies in infant B-ALL.

cancer biology↗

bioRxiv2021

Identifying common transcriptome signatures of cancer by interpreting deep learning models

Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified, signifying that cancer cases display common hallmark molecular features. It is not clear however whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types. Here, in order to agnostically identify transcriptomic features that are commonly shared between cancer types, we used RNA-Seq datasets encompassing thousands of samples from 19 healthy tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression or splice junction use, to distinguish between healthy and tumor samples. All three models achieve high precision, recall and accuracy on test sets derived from 13 datasets used during training and on an independent test dataset, indicating that our models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints, suggesting that they have important cellular functions. Importantly, we found that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer. Finally, our results also highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features across a large array of solid tumor types. The transcriptomic features that we highlight here define cancer signatures that may reflect causal variations or consequences of disease state, or a combination of both.

cancer biology↗

bioRxiv2021

High-throughput mutagenesis identifies mutations and RNA binding proteins controlling CD19 splicing and CART-19 therapy resistance

During CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy. HighlightsO_LIMutations in relapsed CART-19 patients lead to CD19 mis-splicing C_LIO_LIHigh-throughput mutagenesis uncovers ~200 single point mutations with a potential role in CART-19 therapy resistance C_LIO_LIMany mutations generate non-functional CD19 proteins by activating cryptic splice sites C_LIO_LIRNA-binding proteins such as PTBP1 are key to the expression of properly spliced, CART-19 immunotherapy-sensitive isoforms C_LI

molecular biology↗

bioRxiv2021

MYC hyperactivates WNT signaling in APC/CTNNB1-mutated colorectal cancer cells through miR-92a-dependent repression of DKK3

Activation of Wnt signaling is among the earliest events of colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding {beta}-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations in APC, a major {beta}-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct {beta}-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that Myc suppresses Dkk3 transcription through one of mir-17-92 cluster microRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in APC-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration and invasion and metastasis in syngeneic orthotopic mouse colon cancer model. Our results suggest that the MYC->miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feedforward oncogenic loop. SIGNIFICANCECommon APC and CTNNB1 mutations activate Wnt signaling in colorectal cancers. Here we demonstrate that further potentiation of this pathway involves microRNA-dependent repression of the DKK3 gene by the Myc oncoprotein.

cancer biology↗

bioRxiv2020

MOCCASIN: A method for correcting for known and unknown confounders in RNA splicing analysis

While the effects of confounders on gene expression analysis have been extensively studied there is a lack of equivalent analysis and tools for RNA splicing analysis. Here we assess the effect of confounders in two large public RNA-Seq datasets (TARGET, ENCODE), develop a new method, MOCCASIN, to correct the effect of both known and unknown confounders on RNA splicing quantification, and demonstrate MOCCASINs effectiveness on both synthetic and real data.

genomics↗

bioRxiv2019

The role of SRSF3 splicing factor in generating circular RNAs

Circular RNAs (circRNAs) represent a novel class of non-coding RNAs that are emerging as potentially important regulators of gene expression. circRNAs are typically generated from host gene transcripts through a non-canonical back-splicing mechanism, whose regulation is still not well understood. To explore regulation of circRNAs in cancer, we generated sequence data from RNase R-resistant transcripts in human p493-6 B-lymphoid cells and identified thousands of novel as well as previously identified circRNAs. Approximately 40% of expressed genes generated a circRNA, with half of them generating multiple isoforms, suggesting the involvement of alternative back-splicing and regulatory RNA-binding proteins (RBPs). We observed that genes generating circRNAs with back-spliced exonic junctions were enriched for RBP recognition motifs, including multiple splicing factors, most notably SRSF3, a splicing factor known to promote exon inclusion. To test the role of SRSF3 role in circRNA production, we performed traditional RNA-seq in p493-6 B-lymphoid cells with and without SRSF3 knockdown, and identified 926 mRNA transcripts, whose canonical splicing was affected by SRSF3. We found that a subset (205) of these SRSF3 targets served as host transcripts for circRNA, suggesting that SRSF3 may regulate exon circularization. Since this splicing factor is deregulated in hematologic malignancies, we hypothesize that SRSF3-dependent circRNAs, similar to their mRNA counterparts, might contribute to the pathogenesis of lymphomas and leukemias.

molecular biology↗

bioRxiv2017

Aberrant splicing in B-cell acute lymphoblastic leukemia

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing them to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ~100 SFs, e.g. hnRNPA1. hnRNPA1 3UTR was pervasively misspliced, yielding the transcript subject to nonsense-mediated decay. Thus, we knocked it down in B-lymphoblastoid cells, identified 213 hnRNPA1-dependent splicing events, and defined the hnRNPA1 splicing signature in pediatric leukemias. One of its elements was DICER1, a known tumor suppressor gene; its LSVs were consistent with reduced translation of DICER1 mRNA. Additionally, we searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 genes. 77 LSVs were confirmed using two large independent B-ALL RNA-seq datasets. In fact, the twenty most common B-ALL drivers showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contribute to disease pathogenesis.

cancer biology↗