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

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. Significance: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.

Noncanonical 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 (BM) and tonsil donors, performed RNA sequencing, 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 driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript used an alternative first exon to produce the poorly characterized FBXW7β isoform, previously thought to be restricted to neural tissues. The FBXW7β transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the BM. 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, FBXW7β mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7β, we used gain- and loss-of-function approaches and identified an FBXW7-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7β expression. This signature contained several members of the tumor necrosis factor superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7β expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis, and responses to anticancer therapies.

Background Gene fusion events are significant sources of somatic variation across adult and pediatric cancers and are some of the most clinically-effective therapeutic targets, yet low consensus of RNA-Seq fusion prediction algorithms makes therapeutic prioritization difficult. In addition, events such as polymerase read-throughs, mis-mapping due to gene homology, and fusions occurring in healthy normal tissue require informed filtering, making it difficult for researchers and clinicians to rapidly discern gene fusions that might be true underlying oncogenic drivers of a tumor and in some cases, appropriate targets for therapy. Results We developed annoFuse, an R package, and shinyFuse, a companion web application, to annotate, prioritize, and explore biologically-relevant expressed gene fusions, downstream of fusion calling. We validated annoFuse using a random cohort of TCGA RNA-Seq samples (N = 160) and achieved a 96% sensitivity for retention of high-confidence fusions (N = 603). annoFuse uses FusionAnnotator annotations to filter non-oncogenic and/or artifactual fusions. Then, fusions are prioritized if previously reported in TCGA and/or fusions containing gene partners that are known oncogenes, tumor suppressor genes, COSMIC genes, and/or transcription factors. We applied annoFuse to fusion calls from pediatric brain tumor RNA-Seq samples (N = 1028) provided as part of the Open Pediatric Brain Tumor Atlas (OpenPBTA) Project to determine recurrent fusions and recurrently-fused genes within different brain tumor histologies. annoFuse annotates protein domains using the PFAM database, assesses reciprocality, and annotates gene partners for kinase domain retention. As a standard function, reportFuse enables generation of a reproducible R Markdown report to summarize filtered fusions, visualize breakpoints and protein domains by transcript, and plot recurrent fusions within cohorts. Finally, we created shinyFuse for algorithm-agnostic interactive exploration and plotting of gene fusions. Conclusions annoFuse provides standardized filtering and annotation for gene fusion calls from STAR-Fusion and Arriba by merging, filtering, and prioritizing putative oncogenic fusions across large cancer datasets, as demonstrated here with data from the OpenPBTA project. We are expanding the package to be widely-applicable to other fusion algorithms and expect annoFuse to provide researchers a method for rapidly evaluating, prioritizing, and translating fusion findings in patient tumors.

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 FBXW7β isoform, previously thought to be restricted to neural tissues. The FBXW7β 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, FBXW7β mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7β, we utilized gain- and loss-of-function approaches and identified an FBXW7β-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7β 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 FBXW7β expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis and responses to anti-cancer therapies.Key pointsPreviously thought to be restricted to neural tissues, FBXW7β is the predominant FBXW7 isoform in normal and malignant human pro-B cells.FBXW7β promotes transcriptional activation of a proinflammatory gene cluster that contains TNF superfamily members.

Downregulation of surface epitopes causes post-immunotherapy 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 resistant to 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; therefore, this event 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 in vitro. Furthermore, among inotuzumab-treated pediatric B-ALL patients, we identified one non-responder in whose 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. Citation Format: Sisi Zheng, Elisabeth Gillespie, Ammar S. Naqvi, Katharina E. Hayer, Zhiwei Ang, Manuel Torres-Diz, Mathieu Quesnel-Vallières, David A. Hottman, Asen Bagashev, John A. Chukinas, Carolin Schmidt, Mukta Mukta Asnani, Rawan Shraim, Deanne M. Taylor, Susan R. Rheingold, Maureen M. O’Brien, Nathan Singh, Kristen W. Lynch, Marco Ruella, Yoseph Barash, Sarah K. Tasian, Andrei Thomas-Tikhonenko. Modulation of CD22 protein expression in childhood leukemia by pervasive splicing aberrations: Implications for CD22-directed immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6248.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.

Fewer than 1% of children diagnosed with diffuse-midline glioma (DMG) survive for more than 5 years, because no effective therapies exist for these patients. Here, we sought to identify and characterize mechanisms of aberrant splicing (AS) in primary DMG tumors. We observed transcriptome-wide AS (9,805 differential splicing variations in 4,734 genes), and identified a DMG-specific splicing signature, that included known cancer genes. We hypothesize that AS of cancer genes play a role in DMG tumor formation. Assessing whether splicing factor dysregulation impacted known cancer transcripts, we discovered several splicing factors, including SRRM4, SRRM3 and RBFOX3 to be down-regulated in DMG. Additionally, we found an enrichment of binding motifs for these proteins within flanking regions of these mis-spliced exons. We also observed recurrent significant exon inclusion in tumor suppressor SMARCA4, an integral member of the SWI/SNF family of proteins involved in chromatin remodeling. Further, we identified AS in 16 of the 27 members of the SWI/SNF complex, including increased skipping of exon 7 in DPF2, representing a complete mRNA transcript switch in DMG. Since SRRM4, SRRM3 and RBFOX3 are known regulators for neural-specific microexons, we focused on microexon splicing changes, hypothesizing that these regulators may be driving microexon mis-splicing in these tumors. We identified 245 known microexons lost or gained in DMG. Moreover, a quarter of which were observed in known cancer genes, with the most frequent splice event causing gain of a clathrin-binding site in the tumor suppressor BIN1 with a concurrent loss of an out-of-frame microexon in the oncogene BAK1, presumably activating it. Altogether, our results suggest that aberrant splicing may be an alternative mechanism driving DMG tumorigenesis and we are currently molecularly validating a subset of these events with the overall goal of identifying novel therapeutic targets for DMG tumors.

SummaryMembers of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

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 these samples 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 3′UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, 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 contributes to disease pathogenesis.

Background: Mutations in splicing factor (SF) genes cause myelodysplastic syndromes and chronic lymphocytic leukemia (CLL). While such mutations had not been found in B-cell acute lymphoblastic leukemia (B-ALL), we previously reported that alternative splicing of the CD19 transcript is a robust mechanism of resistance to CD19-directed immunotherapy in children with B-ALL. We thus hypothesized that additional mRNAs may be alternatively spliced in these leukemias. Results: Using flow cytometry-based cell purification protocols, deep RNA sequencing (RNA-seq), and the MAJIQ algorithm, we compared transcriptomes of CD19+/CD34+ pro-B cells from normal bone marrow donors to 18 primary pediatric B-ALL samples. We found 4,000-5,000 differential local splice variations (LSV) per leukemia sample, with 279 LSVs in 241 genes differentially spliced in every B-ALL sample. The consistently mis-spliced genes were significantly enriched in the RNA splicing pathway components and encoded ~100 different SFs, many from the SRSF and hnRNP families. Since aberrant LSVs in hnRNPA1 mRNA were present in 100% of B-ALL samples, we knocked down this transcript in a B-lymphoblastoid cell line using siRNA and defined 213 robust hnRNPA1-dependent events. Nearly 30% of the hnRNPA1-dependent LSVs were detectable in B-ALL samples, with one of the affected genes being DICER1, which is commonly mutated or down-regulated in many hematologic malignancies and included in the COSMIC dataset. We next asked how many other COSMIC genes are affected by aberrant splicing. We discovered 81 LSVs (mainly hnRNPA1-independent) in 41 COSMIC genes, including FBXW7, which was alternatively spliced in all 18 primary B-ALL samples. We were able to confirm 77 out of 81 of these LSVs in at least one of the two large independent RNA-seq B-ALL datasets generated by the TARGET Consortium and St Jude Children's Research Hospital. In fact, the twenty most common B-ALL drivers showed much higher prevalence of aberrant splicing than of somatic mutations. Conclusions: B-ALL has widespread changes in splicing, likely due to the aberrant exon usage by SF-encoding transcripts. Aberrant splicing also affects most known B-ALL drivers, suggesting that this type of post-transcriptional regulation contributes to disease pathogenesis.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy with historically poor outcomes and no established standard of care. Nearly 100% of patients with BPDCN overexpress CD123, and targeting CD123 has therefore emerged as an attractive therapeutic target. UCART123v1 is an allogeneic "off the shelf" product composed of genetically modified T-cells expressing an anti-CD123 CAR and a RQR8 depletion ligand, which confers susceptibility to rituximab. The expression of the T-cell receptor αβ (TCRαβ) is abrogated through the inactivation of the TRAC gene, using Cellectis' TALEN® gene-editing technology. We have previously reported the selective in vitro anti-tumor activity of UCART123v1 cells against primary BPDCN samples using cytotoxicity and T-cell degranulation assays, as well as the secretion of IFNγ and other cytokines (IL2, IL5, IL6, IL-13 and TNF-α) by UCART123v1 cells when cultured in the presence of BPDCN cells (Cai et al, 2017 ASH). To evaluate anti-tumor activity of UCART123v1 cells in vivo, we established two relapsed BPDCN patient-derived xenografts (PDX1 and 2) in NSG-SGM3 mice. In PDX-1 model, mice were randomized upon tumour engraftment (D21 after primary BPDCN injection) into 4 groups and received an IV injection of either vehicle, 10×106 TCRαβ KO control T-cells, or UCART123v1 cells (3×106 or 10×106 cells). Mice from vehicle group died by D53 after BPDCN injection with high tumor burden in PB, spleen and BM. 3 out of 9 (33%) mice treated with 3×106 and 6 out of 9 (67%) mice treated with 10×106 UCART123v1 were alive and disease-free at the end of the study (D299 after primary BPDCN injection). In PDX-2 model, which received the same treatment as PDX-1 (at D19 after primary BPDCN cell injection), all vehicle-treated mice died by D49. UCART123v1 therapy extended survival of treated mice to 104-241 days, but tumors relapsed at 90-155 days (Fig. 1A). The relapses in UCART123v1 treated mice were associated with the emergence of CD123-, CD56+CD45+ BPDCN cells infiltrating spleens and BMs (Fig. 1B). To understand the molecular basis for CD123 loss, we isolated RNA from CD123+ cells from two of the vehicle-treated mice and CD123- cells from four of the UCART123v1-treated mice and performed RT-PCR and RNA-sequencing. The cells from all samples were hCD45+ and hCD56+, indicating leukemic origin. These analyses detected the presence of full-length transcripts (exons 2-12) in both CD123+ control samples (Sample 1 and 2in Fig. 1C). In 2 of the 4 CD123- samples, CD123 transcripts were absent, as were transcripts of neighbouring genes (samples 3 and 9 in Fig. 1C). RNA-sequencing reads aligned to Genome Browser tracks for CD123 and housekeeping gene GPI showed no reads present for CD123 but reads present for GPI in the two samples with CD123 loss. The aCGH (Array‐Based Comparative Genomic Hybridization) results showed that samples 3 and 9 (CD123-) had large regional deletions on chromosome X, which includes the CD123 gene. In another sample (sample 5), the splicing analysis algorithm MAJIQ detected CD123 transcripts containing only exons 2-9, indicating premature transcription termination. If translated, this truncated transcript would produce a protein isoform lacking the transmembrane domain in exon 10. Finally, MAJIQ also revealed canonical splicing of exon 2 to exon 3 in all CD123+ samples but a sharp increase in skipping from exon 2 to exon 5 in sample 16 (Fig. 1D). This exon-skipping event preserves the open-reading frame and yields the previously reported transcript variant 2. Per UniProt, the resultant protein will retain the ligand-binding domain but lack several glycosylation sites and two beta sheets in the extracellular domain, potentially compromising recognition by UCART123v1 cells. The aCGH and FISH results further showed that this patient sample harbored TP53 deletion, which could have contributed to DNA instability observed in different mice engrafted with these tumor cells. In summary, allogeneic anti-CD123 CAR T therapy resulted in eradication of BPDCN in vitro and in increased disease-free survival in primary BPDCN PDX models. However, CD123 loss was observed in one PDX model harboring a TP53 deletion. These results provide preclinical proof-of-principle that UCART123v1 cells have potent anti-BPDCN activity, and indicate potential mechanisms leading to antigen loss and disease relapse. Disclosures Galetto: Cellectis Inc: Employment. Gouble:Cellectis: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Neelapu:Cellectis: Research Funding; Celgene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; BMS: Research Funding; Poseida: Research Funding; Novartis: Consultancy; Karus: Research Funding; Acerta: Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Merck: Consultancy, Research Funding; Unum Therapeutics: Consultancy, Research Funding; Precision Biosciences: Consultancy; Cell Medica: Consultancy; Allogene: Consultancy. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. Kantarjian:BMS: Research Funding; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Agios: Honoraria, Research Funding; Novartis: Research Funding; Immunogen: Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Takeda: Honoraria; Astex: Research Funding; Daiichi-Sankyo: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding. Guzman:Cellectis: Research Funding; Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; SeqRx: Consultancy. Pemmaraju:Stemline Therapeutics: Consultancy, Honoraria, Research Funding; samus: Research Funding; plexxikon: Research Funding; incyte: Consultancy, Research Funding; affymetrix: Research Funding; sagerstrong: Research Funding; Daiichi-Sankyo: Research Funding; cellectis: Research Funding; celgene: Consultancy, Honoraria; abbvie: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Research Funding; mustangbio: Consultancy, Research Funding. Konopleva:Genentech: Honoraria, Research Funding; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties.

Pediatric B-cell acute lymphoblastic leukemias (B-ALL) are striking for their low mutational burdens. Ostensibly, this paucity of mutation-associated neoantigens could limit the development of novel immunotherapies. However, our lab has previously shown that globally aberrant splicing is a hallmark of B-ALL, as compared to normal pro-B cells. Here we hypothesize that alternative splicing represents a new source of targetable neo-epitopes, distinct from missense mutations. To test this hypothesis, we constructed a pipeline for neo-epitope discovery, validation, and therapeutic development. First, we obtained a very large RNA-seq dataset covering over 400 pediatric B-ALL samples from St. Jude Children's Research Hospital. For normal controls, RNA-seq was performed on 4 pro-B cell fractions from bone marrow donors. We then applied a bioinformatic splicing analysis, MAJIQ 2.0, to deeply mine the dataset for local splicing variants (LSVs) unique to B-ALL. To bypass MHC presentation barriers, we focused on LSVs mapping to exons encoding extracellular protein domains (ectoLSVs). This filtering yielded a list of 914 ectoLSVs in 430 genes. These ectoLSVs were further filtered for those that preserved opening reading frames, occurred with high prevalence, and demonstrated a large differential expression between B-ALL and normal controls. One such prominent event was the increased skipping in B-ALL of CD22 of exon 5-6 (Δex5-6). We validated this event at the RNA level with RT-PCR in 18 out of 18 primary B-ALL samples from CHOP Biobank. Using Nanopore-based long read RNA sequencing, we confirmed the Δex5-6 event exists within a larger translatable transcript. BALB/c mice were then immunized against a peptide containing the junction site of exon 4 and 7 to generate hybridomas; several monoclonal antibody (mAb) against CD22 Δex5-6 were successfully obtained. To validate the specificity of our mAbs, we used CRISPR to delete endogenous CD22 in OCI-Ly10 cells and reconstituted the cells with either CD22 Δex5-6 or full length CD22. The mAb demonstrated remarkable specificity for CD22 Δex5-6 and did not bind to the full-length isoform. We also demonstrated that at least one of these mAbs (clone 3A3) specifically binds to endogenously expressed CD22 Δex5-6 in multiple B-ALL cell lines by immunoprecipitation and Western blot analysis. Our next steps are to develop 3A3-based antibody-drug conjugates and chimeric antigen receptors for further therapeutic testing. In summary, our results validate that 1) our current discovery pipeline is able to identify targetable splicing-derived neo-epitopes, and that 2) antibodies with impressive specificity can be generated against such neo-epitopes. Furthermore, this new paradigm has the promise of increasing the repertoire of highly specific immunotherapy targets in B-ALL, despite its low mutation burden. Of note, this strategy could also be carried forward into therapeutic development for many other cancers beyond B-ALL. Disclosures No relevant conflicts of interest to declare.

Several studies indicate the importance of colonic microbiota in metabolic and inflammatory disorders and importance of diet on microbiota composition. The effects of alcohol, one of the prominent components of diet, on colonic bacterial composition is largely unknown. Mounting evidence suggests that gut-derived bacterial endotoxins are cofactors for alcohol-induced tissue injury and organ failure like alcoholic liver disease (ALD) that only occur in a subset of alcoholics. We hypothesized that chronic alcohol consumption results in alterations of the gut microbiome in a subgroup of alcoholics, and this may be responsible for the observed inflammatory state and endotoxemia in alcoholics. Thus we interrogated the mucosa-associated colonic microbiome in 48 alcoholics with and without ALD as well as 18 healthy subjects. Colonic biopsy samples from subjects were analyzed for microbiota composition using length heterogeneity PCR fingerprinting and multitag pyrosequencing. A subgroup of alcoholics have an altered colonic microbiome (dysbiosis). The alcoholics with dysbiosis had lower median abundances of Bacteroidetes and higher ones of Proteobacteria. The observed alterations appear to correlate with high levels of serum endotoxin in a subset of the samples. Network topology analysis indicated that alcohol use is correlated with decreased connectivity of the microbial network, and this alteration is seen even after an extended period of sobriety. We show that the colonic mucosa-associated bacterial microbiome is altered in a subset of alcoholics. The altered microbiota composition is persistent and correlates with endotoxemia in a subgroup of alcoholics.

Summary MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation [1–3]. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription [4–8], processing [9–11], and stability [12, 13], with miRNA deregulation linked with diseases [14] and neurodegenerative disorders [15]. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage [16–18] to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC) [19, 20]. Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3′ end, suggesting a novel biogenesis mechanism involving 3′ end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3′→5′ exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34 . Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler . These findings suggest that Nbr-mediated 3′ end processing represents a critical step in miRNA maturation that impacts miRNA diversity.

Since the initial annotation of miRNAs from cloned short RNAs by the Ambros, Tuschl, and Bartel groups in 2001, more than a hundred studies have sought to identify additional miRNAs in various species. We report here a meta-analysis of short RNA data from Drosophila melanogaster, aggregating published libraries with 76 data sets that we generated for the modENCODE project. In total, we began with more than 1 billion raw reads from 187 libraries comprising diverse developmental stages, specific tissue- and cell-types, mutant conditions, and/or Argonaute immunoprecipitations. We elucidated several features of known miRNA loci, including multiple phased byproducts of cropping and dicing, abundant alternative 5′ termini of certain miRNAs, frequent 3′ untemplated additions, and potential editing events. We also identified 49 novel genomic locations of miRNA production, and 61 additional candidate loci with limited evidence for miRNA biogenesis. Although these loci broaden the Drosophila miRNA catalog, this work supports the notion that a restricted set of cellular transcripts is competent to be specifically processed by the Drosha/Dicer-1 pathway. Unexpectedly, we detected miRNA production from coding and untranslated regions of mRNAs and found the phenomenon of miRNA production from the antisense strand of known loci to be common. Altogether, this study lays a comprehensive foundation for the study of miRNA diversity and evolution in a complex animal model.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy with historically poor outcomes and no established standard of care. Nearly 100% of patients with BPDCN overexpress CD123, and targeting CD123 emerged as an attractive therapeutic target given its differential expression on BPDCN cell surface. UCART123 product (Cellectis) uses genetically modified allogeneic T-cells (derived from healthy donors, so-called “off the shelf”) containing an anti-CD123 CAR and a RQR8 depletion ligand that confers susceptibility to rituximab. The expression of the T-cell receptor (TCR) is abrogated through the inactivation of the TCRα constant gene, using Cellectis' TALEN® gene-editing technology. We have previously reported the selective in vitro anti-tumor activity of UCART123 cells against CD123+ primary BPDCN samples using cytotoxicity assays, T-cell degranulation assay and the secretion of IFNγ and other cytokines (IL2, IL5, IL6, IL-13 and TNF-α) by UCART123 cells when cultured in the presence of BPDCN cells (Tianyu Cai, 2017 ASH). However, UCART123 had minimum toxicity against normal bone marrow cells. To evaluate in vivo anti-tumor activity of UCART123 cells, we established two patient-derived xenografts (PDX1-2) from patients with relapsed BPDCN in NSG-SGM3 mice. In PDX-1 model, all mice in vehicle-treated group died by D53, with high tumor burden in peripheral blood, spleen and bone marrow. Three out of 9 (33%) mice treated with 3×106 UCART123 and Six out of 9 (67%) mice treated with 10×106 UCART123 were alive and disease-free at the end of the study (D299). In PDX-2 model, while UCART123 similarly extended survival of the mice (D104-241), relapses occurred in all treatment cohorts at D90-155. Flow cytometric analysis showed that all of the relapses were associated with emergence of CD123- BPDCN clones (95-96% CD123-). To understand the molecular basis for loss of CD123 surface expression, we isolated RNA from two CD123 positive samples from vehicle group and two CD123 negative samples from 1×106 UCART123 group. RT-PCR and RNA-seq detected the presence of full-length transcripts containing exons 1-12 in both CD123 positive samples. In one of the two CD123 (-) samples, CD123 transcripts were completely absent, along with loss of transcripts of neighboring genes. In another CD123 (-) sample, CD123 transcripts containing only exons 1-9 were detected, indicating the presence of a truncation. Interestingly, if translated, this transcript would produce a protein isoform lacking the transmembrane domain (Ex 10). In summary, UCART123 therapy results in BPDCN eradication and long-term disease-free survival in a subset of primary BPDCN PDX models. However, loss of CD123 through diverse genetic mechanisms could lead to escape from UCART123 therapy and cause relapses. A phase I trial of UCART123 in BPDCN is opened for enrollment (NCT03203369). Citation Format: Tianyu Cai, Kathryn L. Black, Ammar Naqvi, Roman Galetto, Agnès Gouble, Julianne Smith, Antonio Cavazos, Lina Han, Qi Zhang, Vinitha Kuruvilla, Sergej Naumovich Sergej Konoplev, Sattva S. Neelapu, Andrew A. Lane, Monica L. Guzman, Hagop Kantarjian, Andrei Thomas-Tikhonenko, Naveen Pemmaraju, Marina Konopleva. Preclinical efficacy of allogeneic anti-CD123 CAR T-cells for the therapy of blastic plasmacytoid dendritic cell neoplasm (BPDCN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2560.

Background RNA-related applications of the next-generation sequencing (NGS) technologies require context-specific interpretations: e.g., sequence mismatches may indicate sites of RNA editing, or uneven read coverage often points to mature form of microRNA. Existing visualization tools traditionally show RNA molecules in two dimensions, with their base pairing and the resulting secondary structure. However, it is not straightforward to combine a linear NGS data display with the 2-D RNA depictions. Results We present a novel approach for interactive representation of nucleotide substitutions and modifications in the transcribed genome. With the focus on RNA secondary structure in the context of NGS data, it provides intuitive visualization of genomic environment, sequence reads, nucleotide polymorphisms and editing events integrated with the structural and functional elements of both coding and non-coding RNA molecules. Using our approach we present and discuss examples and general trends of polymorphisms and editing in the context of the secondary structure of microRNAs. As expected, most of the substitutions comprised A to G and C to T events, consistent with typical RNA editing patterns. However, we did not observe prevalence of editing in double-stranded regions of the microRNA stem-loop. We describe novel prominent editing event candidates, observed across several small RNA libraries of Drosophila melanogaster. Conclusions In contrast to the existing general tools for NGS data visualization, the power of our approach is not only in the display of read alignments and their counts, but the integration of RNA secondary structure, sequencing depth, and rates/patterns of editing or other modifications. It provides a comprehensive picture, important for large-scale studies and detailed analyses, helping to gain insight into the intricate relationships between different events in RNA biogenesis.

MicroRNAs (miRNAs) are 20- to ∼24-nucleotide (nt) small RNAs that impact a variety of biological processes, from development to age-associated events. To study the role of miRNAs in aging, studies have profiled the levels of miRNAs with time. However, evidence suggests that miRNAs show heterogeneity in length and sequence in different biological contexts. Here, by examining the expression pattern of miRNAs by Northern blot analysis, we found that Drosophila miRNAs show distinct isoform pattern changes with age. Surprisingly, an increase of some miRNAs reflects increased 2′-O-methylation of select isoforms. Small RNA deep sequencing revealed a global increase of miRNAs loaded into Ago2, but not into Ago1, with age. Our data suggest increased loading of miRNAs into Ago2, but not Ago1, with age, indicating a mechanism for differential loading of miRNAs with age between Ago1 and Ago2. Mutations in Hen1 and Ago2, which lack 2′-O-methylation of miRNAs, result in accelerated neurodegeneration and shorter life span, suggesting a potential impact of the age-associated increase of 2′-O-methylation of small RNAs on age-associated processes. Our study highlights that miRNA 2′-O-methylation at the 3′ end is modulated by differential partitioning of miRNAs between Ago1 and Ago2 with age and that this process, along with other functions of Ago2, might impact age-associated events in Drosophila.

We have been using the Roche GS-FLX sequencing platform to produce tens of thousands of sequencing reads from samples of both bacterial communities (microbiome) and fungal communities (mycobiome) of stool, gut mucosa, vaginal washes, and oral washes from a large number of subjects. This vast volume of data from diverse sources has necessitated the development of an analysis pipeline in order to systematically and rapidly identify the taxa within the samples and to correlate the sample data with clinical and environmental features. Specifically, we have developed automated analytical tools for data tracking, taxonomical analysis, and feature clustering of bacteria in the human microbiome and demonstrate the pipeline using Cervical Vaginal Lavage (CVL) samples. This analysis pipeline will not only provide insight to our specific CVL dataset, but is applicable to other microbiome samples and will ultimately broaden our understanding of how the microbiome influences human health.

In this article, we used a network-based approach to characterize the microflora abundance in colonic mucosal samples and correlate potential interactions between the identified species with respect to the healthy and diseased states. We analyzed the modelled network by computing several local and global network statistics, identified recurring patterns or motifs, fit the network models to a family of well-studied graph models. This study has demonstrated, for the first time, an approach that differentiated the gut microbiota in alcoholic subjects and healthy subjects using topological network analysis of the gut microbiome.

The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants. Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease., Author Summary We characterized the fungal microbiome (mycobiome) of the oral cavity in healthy individuals. Our results demonstrate that the fungal component of the oral microbiome is diverse as revealed by the presence of 74 culturable and 11 non-culturable fungal genera in the oral cavity. A total of 101 species were identified, with between 9 and 23 culturable species present in each person. Fifteen genera (which included four known pathogenic fungi and non-culturable organisms) were present in ≥20% of the tested samples; Candida species were the most frequently obtained genera, isolated from 75% of all study participants, followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). The remaining fungi detected in the oral wash samples represent organisms likely originating from the environment. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease.

Background Development of sequencing technologies and supporting computation enable discovery of small RNA molecules that previously escaped detection or were ignored due to low count numbers. While the focus in the analysis of small RNA libraries has been primarily on microRNAs (miRNAs), recent studies have reported findings of fragments of transfer RNAs (tRFs) across a range of organisms. Results Here we describe Drosophila melanogaster tRFs, which appear to have a number of structural and functional features similar to those of miRNAs but are less abundant. As is the case with miRNAs, (i) tRFs seem to have distinct isoforms preferentially originating from 5’ or 3’ end of a precursor molecule (in this case, tRNA), (ii) ends of tRFs appear to contain short “seed” sequences matching conserved regions across 12 Drosophila genomes, preferentially in 3’ UTRs but also in introns and exons; (iii) tRFs display specific isoform loading into Ago1 and Ago2 and thus likely function in RISC complexes; (iii) levels of loading in Ago1 and Ago2 differ considerably; and (iv) both tRF expression and loading appear to be age-dependent, indicating potential regulatory changes from young to adult organisms. Conclusions We found that Drosophila tRF reads mapped to both nuclear and mitochondrial tRNA genes for all 20 amino acids, while previous studies have usually reported fragments from only a few tRNAs. These tRFs show a number of similarities with miRNAs, including seed sequences. Based on complementarity with conserved Drosophila regions we identified such seed sequences and their possible targets with matches in the 3’UTR regions. Strikingly, the potential target genes of the most abundant tRFs show significant Gene Ontology enrichment in development and neuronal function. The latter suggests that involvement of tRFs in the RNA interfering pathway may play a role in brain activity or brain changes with age. Reviewers This article was reviewed by Eugene Koonin, Neil Smalheiser and Alexander Kel. Electronic supplementary material The online version of this article (doi:10.1186/s13062-015-0081-6) contains supplementary material, which is available to authorized users.