Manuel Torres-Diz

Children's Hospital of Philadelphia

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Andrei Thomas‐Tikhonenko

University of Pennsylvania

Katharina E. Hayer

Children's Hospital of Philadelphia

Yoseph Barash

University of Pennsylvania

Sarah K. Tasian

Children's Hospital of Philadelphia

Zhiwei Ang

Children's Hospital of Philadelphia

Mathieu Quesnel-Vallières

University of Pennsylvania

Montse Sánchez‐Céspedes

Josep Carreras Leukaemia Research Institute

Priyanka Sehgal

Children's Hospital of Philadelphia

Mukta Asnani

Children's Hospital of Philadelphia

Marco Ruella

University of Pennsylvania

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University of Pennsylvania

Children's Hospital of Philadelphia

Josep Carreras Leukaemia Research Institute

Institut d'Investigació Biomédica de Bellvitge

Philadelphia University

Institut Català d'Oncologia

Harvard University

Institute of Molecular Biology

California University of Pennsylvania

Dana-Farber Cancer Institute

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Supplementary Tables from MAX Inactivation in Small Cell Lung Cancer Disrupts MYC–SWI/SNF Programs and Is Synthetic Lethal with BRG1

Octavio A. Romero Manuel Torres-Diz Eva Pros Suvi Savola Antonio Gomez

PDF file 236K, Table S1. List of the 121 lung cancer cell lines screened for MAX alterations. Information about the histopathology of each cell line, and the presence of alterations at MYC and BRG1 is also included. Grey boxes indicate that no information is available. Table S2. List of genes that are up-regulated or down-regulated upon MAX reconstitution. The values represent the n-fold change in the level of gene expression of each of the lung cancer cell lines infected with the wild type MAX relative to the controls (?). Table S3. List of genes that are up-regulated or down-regulated upon depletion of BRG1. The values represent the n-fold change in the level of gene expression of each of the lung cancer cell lines infected with the shBRG1 relative to the controls ?. Table S4. List of the cell lines included in Figure 5. The information about alterations at the indicated genes was obtained from different sources, as indicated. For data extracted from databases we applied the following criteria to define a mutation: i) mutations at tumor suppressor genes (BRG1, SMARCB1, MAX, ARID1A, PRBM1, and MGA) should be homozygous and predictive of truncated proteins, ii) for amplification at the MYC family of oncogenes, only very high levels of gene amplification have been considered to be positive. Other genes, related to MYC/MAX or to the SWI/SNF complex, have also been searched for alterations (i.e., ARID1B, ARID2, MXI, MXDs) but either no alterations were reported in the databases or the changes did not fulfill our selection criteria. CCLE, Cancer Cell Line Encyclopedia (Broad-Novartis Cancer Cell Line Encyclopedia; website, http://www.broadinstitute.org/ccle/). COSMIC, Catalogue of Somatic Mutations in Cancer (Trust Sanger Institute's Cancer Cell Line Project; website, http://cancer.sanger.ac.uk/)

ARID1A

SMARCA4

SWI/SNF

10.1158/2159-8290.22530665

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Supplementary Figures from MAX Inactivation in Small Cell Lung Cancer Disrupts MYC–SWI/SNF Programs and Is Synthetic Lethal with BRG1

Octavio A. Romero Manuel Torres-Diz Eva Pros Suvi Savola Antonio Gomez

PDF file 422K, Fig. S1. Radio charts of the multiplex ligation-dependent probe amplification (MLPA), depicting intragenic deletions at the indicated lung cancer cell lines and lung primary tumor. The five exons of the MAX gene and the names and the relative location of the different probes used in the MLPA are also shown. The ratio charts depicting a the analysis of a normal DNA, an intragenic deletion of exons from 3 to 5 for the Lu134 cell line and deletion of the entire coding region for the Lu165 cell line are included. The radio chart for the tumorgraft from the same individual as the Lu134 cells is also depicted, ruling out the possibility of it being a cellculture artifact. Fig. S2. A, Cell proliferation, measured using MTT assays, was significantly greater in the cells carrying the empty vector (? control cells) than in those expressing ectopic MAX, from the parental H1417 cells. Lines represent the number of viable cells relative to the total number of cells at 0 h. Error bars, standard deviation *p<0.001. B, Western blot of total lysates of MAX in H1299tr-BRG1wt and H1299tr-BRG1mut cells, treated with (1 ng/μl) to allow BRG1 expression, depicts the ectopic expression of MAX in a hormone-free (HF) environment, or in the presence of glucocorticoids (GC) at the indicated concentrations. In the left panel, cells transfected with the 5'UTR-MAX constructs exhibit decreased levels of ectopic MAX in the BRG1mut cells compared with BRG1wt cells. On the right, cells transfected with the MAX construct lacking 5'UTR show similar levels of ectopic MAX regardless of the status of BRG1. C, Western blots illustrate the reduction of MAX upon depletion of BRG1 in the indicated lung cancer cells carrying amplification of MYC in an HF environment and at the indicated hours after treatment with GC (5 μM). TUBULIN is included as a loading control. Fig. S3. Enriched gene ontology (GO) classifications (p<0.05 for all categories shown) among genes up-regulated in the MAX gene-expression profile (from Table S2). Enrichment (represented by several GO categories) is significant for genes annotated as being involved in neural development and in glucose/carbohydrate metabolism

10.1158/2159-8290.22530671.v1

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MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex

Proceedings of the National Academy of Sciences (2021)

Paula Llabata Manuel Torres-Diz Antonio Gómez Laureano Tomás-Daza Octavio A. Romero

The MYC axis is disrupted in cancer, predominantly through activation of the MYC family oncogenes but also through inactivation of the MYC partner MAX or of the MAX partner MGA. MGA and MAX are also members of the polycomb repressive complex, ncPRC1.6. Here, we use genetically modified MAX-deficient small-cell lung cancer (SCLC) cells and carry out genome-wide and proteomics analyses to study the tumor suppressor function of MAX. We find that MAX mutant SCLCs have ASCL1 or NEUROD1 or combined ASCL1/NEUROD1 characteristics and lack MYC transcriptional activity. MAX restitution triggers prodifferentiation expression profiles that shift when MAX and oncogenic MYC are coexpressed. Although ncPRC1.6 can be formed, the lack of MAX restricts global MGA occupancy, selectively driving its recruitment toward E2F6-binding motifs. Conversely, MAX restitution enhances MGA occupancy to repress genes involved in different functions, including stem cell and DNA repair/replication. Collectively, these findings reveal that MAX mutant SCLCs have either ASCL1 or NEUROD1 or combined characteristics and are MYC independent and exhibit deficient ncPRC1.6-mediated gene repression.

10.1073/pnas.2024824118

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Alternative splicing of its 5’-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Zhiwei Ang Luca Paruzzo Katharina E. Hayer Carolin Schmidt Manuel Torres-Diz

Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the

10.1101/2023.02.19.529123

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Additional file 3 of Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts

Figshare (2021)

Laura Schulz Manuel Torres-Diz Mariela Cortés-López Katharina E. Hayer Mukta Asnani

Additional file 3: Table S1. Putative falsitrons detected from Oxford Nanopore Technologies (ONT) sequencing data for the five commonly used cell lines A549, HCT116, HepG2, K562 and MCF-7, as well as the B-lymphoblastoid cell line GM12878.

Transcription

RNA-Seq

10.6084/m9.figshare.14870359

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Supplementary Figure 1 and 2 from Colorectal Cancer-Associated Smad4 R361 Hotspot Mutations Boost Wnt/β-Catenin Signaling through Enhanced Smad4–LEF1 Binding

Claudia B. Lanauze Priyanka Sehgal Katharina E. Hayer Manuel Torres-Diz James A. Pippin

S1. Wnt signaling in CRC cell lines with Smad4 R361 mutations. S2. Wnt signaling in CRC cell lines with Smad4 R361 mutations.

Beta-catenin

10.1158/1541-7786.22526730

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Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

Blood Cancer Discovery (2021)

Sisi Zheng Elisabeth Gillespie Ammar S. Naqvi Katharina E. Hayer Zhiwei Ang

Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy.

CD22

Minigene

10.1158/2643-3230.bcd-21-0087

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MYC Hyperactivates Wnt Signaling in APC/CTNNB1-Mutated Colorectal Cancer Cells through miR-92a–Dependent Repression of DKK3

Molecular Cancer Research (2021)

Priyanka Sehgal Claudia Lanauze Xin Wang Katharina E. Hayer Manuel Torres-Diz

Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding β-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major β-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 β-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 miRNAs, 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/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. IMPLICATIONS: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

LRP6

LRP5

10.1158/1541-7786.mcr-21-0666

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Data from Colorectal Cancer-Associated Smad4 R361 Hotspot Mutations Boost Wnt/β-Catenin Signaling through Enhanced Smad4–LEF1 Binding

Claudia B. Lanauze Priyanka Sehgal Katharina E. Hayer Manuel Torres-Diz James A. Pippin

<div>AbstractAbout 10% to 30% of patients with colorectal cancer harbor either loss of or missense mutations in SMAD4, a critical component of the TGFβ signaling pathway. The pathophysiologic function of missense mutations in Smad4 is not fully understood. They usually map to the MH2 domain, specifically to residues that are involved in heterodimeric complex formation with regulatory Smads (such as Smad2/3) and ensuing transcriptional activation. These detrimental effects suggest that SMAD4 missense mutations can be categorized as loss-of-function. However, they tend to cluster in a few hotspots, which is more consistent with them acting by a gain-of-function mechanism. In this study, we investigated the functional role of Smad4 R361 mutants by re-expressing two R361 Smad4 variants in several Smad4-null colorectal cancer cell lines. As predicted, R361 mutations disrupted Smad2/3-Smad4 heteromeric complex formation and abolished canonical TGFβ signaling. In that, they were similar to SMAD4 loss. However, RNA sequencing and subsequent RT-PCR assays revealed that Smad4mut cells acquired a gene signature associated with enhanced Lef1 protein function and increased Wnt signaling. Mechanistically, Smad4 mutant proteins retained binding to Lef1 protein and drove a commensurate increase in downstream Wnt signaling as measured by TOP/FOP luciferase assay and Wnt-dependent cell motility. Consistent with these findings, human colorectal cancers with SMAD4 missense mutations were less likely to acquire activating mutations in the key Wnt pathway gene CTNNB1 (encoding β-catenin) than colorectal cancers with truncating SMAD4 nonsense mutations.Implications:Our studies suggest that in colorectal cancer hotspot mutations in Smad4 confer enhanced Wnt signaling and possibly heightened sensitivity to Wnt pathway inhibitors.</div>

Beta-catenin

10.1158/1541-7786.c.6545193.v1

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An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia

Cancer Research (2024)

Manuel Torres-Diz Clara Reglero Catherine D. Falkenstein Annette Castro Katharina E. Hayer

Abstract 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 sequencing 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, antifolates, 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 resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-mercaptopurine in B-ALL cells both in vitro and in vivo. Furthermore, both NT5C2ex6a and the R238W variant induced collateral sensitivity to the inosine monophosphate dehydrogenase inhibitor mizoribine. These results ascribe to splicing perturbations an important role in chemotherapy resistance in relapsed B-ALL and suggest that inosine monophosphate dehydrogenase inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias. Significance: Alternative splicing is a potent mechanism of acquired drug resistance in relapsed/refractory acute lymphoblastic leukemias that has diagnostic and therapeutic implications for patients who lack mutations in known chemoresistance genes.

Thiopurine methyltransferase

Exon skipping

10.1158/0008-5472.can-23-3804

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