Eukaryotic initiation factor 4B is a multi-functional RNA binding protein that regulates histone mRNAs.

RNA binding proteins drive proliferation and tumorigenesis by regulating the translation and stability of specific subsets of messenger RNAs (mRNAs). We have investigated the role of eukaryotic initiation factor 4B (eIF4B) in this process and identify 10-fold more RNA binding sites for eIF4B in tumour cells from patients with diffuse large B-cell lymphoma compared to control B cells and, using individual-nucleotide resolution UV cross-linking and immunoprecipitation, find that eIF4B binds the entire length of mRNA transcripts. eIF4B stimulates the helicase activity of eIF4A, thereby promoting the unwinding of RNA structure within the 5' untranslated regions of mRNAs. We have found that, in addition to its well-documented role in mRNA translation, eIF4B additionally interacts with proteins associated with RNA turnover, including UPF1 (up-frameshift protein 1), which plays a key role in histone mRNA degradation at the end of S phase. Consistent with these data, we locate an eIF4B binding site upstream of the stem-loop structure in histone mRNAs and show that decreased eIF4B expression alters histone mRNA turnover and delays cell cycle progression through S phase. Collectively, these data provide insight into how eIF4B promotes tumorigenesis.

T cell-redirecting therapies in hematological malignancies: Current developments and novel strategies for improved targeting.

T cell-redirecting therapies (TCRTs), such as chimeric antigen receptor (CAR) or T cell receptor (TCR) T cells and T cell engagers, have emerged as a highly effective treatment modality, particularly in the B and plasma cell-malignancy setting. However, many patients fail to achieve deep and durable responses; while the lack of truly unique tumor antigens, and concurrent on-target/off-tumor toxicities, have hindered the development of TCRTs for many other cancers. In this review, we discuss the recent developments in TCRT targets for hematological malignancies, as well as novel targeting strategies that aim to address these, and other, challenges.

Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade.

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination.

Unbiased cell surface proteomics identifies SEMA4A as an effective immunotherapy target for myeloma.

The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.

RNA-seq of newly diagnosed patients in the PADIMAC study leads to a bortezomib/lenalidomide decision signature.

Improving outcomes in multiple myeloma will involve not only development of new therapies but also better use of existing treatments. We performed RNA sequencing on samples from newly diagnosed patients enrolled in the phase 2 PADIMAC (Bortezomib, Adriamycin, and Dexamethasone Therapy for Previously Untreated Patients with Multiple Myeloma: Impact of Minimal Residual Disease in Patients with Deferred ASCT) study. Using synthetic annealing and the large margin nearest neighbor algorithm, we developed and trained a 7-gene signature to predict treatment outcome. We tested the signature in independent cohorts treated with bortezomib- and lenalidomide-based therapies. The signature was capable of distinguishing which patients would respond better to which regimen. In the CoMMpass data set, patients who were treated correctly according to the signature had a better progression-free survival (median, 20.1 months vs not reached; hazard ratio [HR], 0.40; confidence interval [CI], 0.23-0.72; P = .0012) and overall survival (median, 30.7 months vs not reached; HR, 0.41; CI, 0.21-0.80; P = .0049) than those who were not. Indeed, the outcome for these correctly treated patients was noninferior to that for those treated with combined bortezomib, lenalidomide, and dexamethasone, arguably the standard of care in the United States but not widely available elsewhere. The small size of the signature will facilitate clinical translation, thus enabling more targeted drug regimens to be delivered in myeloma.

Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy.

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions.

Initial genome sequencing and analysis of multiple myeloma.

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.

Analysis of multiple genomic sequence alignments: a web resource, online tools, and lessons learned from analysis of mammalian SCL loci.

Comparative analysis of genomic sequences is becoming a standard technique for studying gene regulation. However, only a limited number of tools are currently available for the analysis of multiple genomic sequences. An extensive data set for the testing and training of such tools is provided by the SCL gene locus. Here we have expanded the data set to eight vertebrate species by sequencing the dog SCL locus and by annotating the dog and rat SCL loci. To provide a resource for the bioinformatics community, all SCL sequences and functional annotations, comprising a collation of the extensive experimental evidence pertaining to SCL regulation, have been made available via a Web server. A Web interface to new tools specifically designed for the display and analysis of multiple sequence alignments was also implemented. The unique SCL data set and new sequence comparison tools allowed us to perform a rigorous examination of the true benefits of multiple sequence comparisons. We demonstrate that multiple sequence alignments are, overall, superior to pairwise alignments for identification of mammalian regulatory regions. In the search for individual transcription factor binding sites, multiple alignments markedly increase the signal-to-noise ratio compared to pairwise alignments.

Comparative and functional analyses of LYL1 loci establish marsupial sequences as a model for phylogenetic footprinting.

Comparative genomic sequence analysis is a powerful technique for identifying regulatory regions in genomic DNA. However, its utility largely depends on the evolutionary distances between the species involved. Here we describe the screening of a genomic BAC library from the stripe-faced dunnart, Sminthopsis macroura, formerly known as the narrow-footed marsupial mouse. We isolated a clone containing the LYL1 locus, completely sequenced the 60.6-kb insert, and compared it with orthologous human and mouse sequences. Noncoding homology was substantially reduced in the human/dunnart analysis compared with human/mouse, yet we could readily identify all promoters and exons. Human/mouse/dunnart alignments of the LYL1 candidate promoter allowed us to identify putative transcription factor binding sites, revealing a pattern highly reminiscent of critical regulatory regions of the LYL1 paralogue, SCL. This newly identified LYL1 promoter showed strong activity in myeloid progenitor cells and was bound in vivo by Fli1, Elf1, and Gata2-transcription factors all previously shown to bind to the SCL stem cell enhancer. This study represents the first large-scale comparative analysis involving marsupial genomic sequence and demonstrates that such comparisons provide a powerful approach to characterizing mammalian regulatory elements.

Transcriptional regulation of the stem cell leukemia gene (SCL)--comparative analysis of five vertebrate SCL loci.

The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hematopoiesis and vasculogenesis and a pattern of expression that is highly conserved between mammals and zebrafish. Here we report the isolation and characterization of the zebrafish SCL locus together with the identification of three neighboring genes, IER5, MAP17, and MUPP1. This region spans 68 kb and comprises the longest zebrafish genomic sequence currently available for comparison with mammalian, chicken, and pufferfish sequences. Our data show conserved synteny between zebrafish and mammalian SCL and MAP17 loci, thus suggesting the likely genomic domain necessary for the conserved pattern of SCL expression. Long-range comparative sequence analysis/phylogenetic footprinting was used to identify noncoding conserved sequences representing candidate transcriptional regulatory elements. The SCL promoter/enhancer, exon 1, and the poly(A) region were highly conserved, but no homology to other known mouse SCL enhancers was detected in the zebrafish sequence. A combined homology/structure analysis of the poly(A) region predicted consistent structural features, suggesting a conserved functional role in mRNA regulation. Analysis of the SCL promoter/enhancer revealed five motifs, which were conserved from zebrafish to mammals, and each of which is essential for the appropriate pattern or level of SCL transcription.