Asen Bagashev

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy

UNLABELLED The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Immune dysregulation in severe influenza

Among previously healthy children with severe influenza, the mechanisms leading to increased pathology are not understood. We hypothesized that children with severe influenza would have high levels of circulating cytokines. To examine this, we recruited patients with severe influenza and examined plasma cytokine levels as well as the ability of peripheral blood cells to respond to stimuli. Ten patients with severe influenza were enrolled during the 2005–2007 influenza seasons. We evaluated plasma cytokine levels, circulating NK cells, and responses to TLR ligands during the illness. We compared these patients with five patients with moderate influenza, six patients with respiratory syncytial virus (RSV), and 24 noninfected controls. Patients with influenza showed depressed responses to TLR ligands when compared with RSV patients and healthy controls (P<0.05). These normalized when retested during a convalescent phase. Plasma levels of IL‐6, IL‐12, and IFN‐γ were elevated in influenza patients compared with controls (P<0.05). A compromised ability to produce TNF‐α was reproduced by in vitro infection, and the magnitude of the effect correlated with the multiplicity of infection and induction of IFN regulatory factor 4 expression. Aberrant, systemic, innate responses to TLR ligands during influenza infection may be a consequence of specific viral attributes such as a high inoculum or rapid replication and may underlie the known susceptibility of influenza‐infected patients to secondary bacterial infections.

Leucine-Rich Repeat (in Flightless I) Interacting Protein-1 Regulates a Rapid Type I Interferon Response

The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.

Monocyte polarization: the relationship of genome-wide changes in H4 acetylation with polarization

The character of monocytes is both molded by and contributes to ongoing immune responses. We hypothesized that monocyte polarization could have durable qualities and these would be mediated partly by changes in the chromatin. We defined genome-wide expression and histone H4 acetylation (H4ac) changes after γ-interferon (IFN), α-IFN and interleukin-4 treatment. To identify genes with altered potential for expression, we stimulated polarized monocytes and identified genes up- or downregulated after polarization and stimulation but not either treatment alone. We also defined durability after an 18-h or 3-day washout. Genes uniquely regulated after the combination of polarization and stimulus were durably altered, with 51% of the effects being durable. This gene set was highly enriched for cytokine-induced alterations in H4ac, with P-values ranging from 10−24 to 10−37. Certain regulons defined by patterns of expression were also associated with altered H4ac, with P-values ranging from 10−4 to 10−29. Networking software revealed a high density of mitogen-activated protein (MAP) kinase nodes in these clusters. Therefore, some changes in monocyte gene expression were sustained over a 3-day period. These durably altered gene sets were enriched for changes in H4ac and were associated with potential MAP kinase effects.

Aberrant splicing in B-cell acute lymphoblastic leukemia

Abstract 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.