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The calreticulin (CALR) exon 9 mutations are found in ∼30% of patients with essential thrombocythemia and primary myelofibrosis. Recently, we reported spontaneous immune responses against the CALR mutations. Here, we describe that CALR-mutant (CALRmut)-specific T cells are able to specifically recognize CALRmut cells. First, we established a T-cell culture specific for a CALRmut epitope. These specific T cells were able to recognize several epitopes in the CALRmut C terminus. Next, we established a CALRmut-specific CD4+ T-cell clone by limiting dilution. These CD4+ T cells recognized autologous CALRmut monocytes and hematopoietic stem cells, and T-cell recognition of target cells was dependent on the presence of CALR. Furthermore, we showed that the CALRmut response was human leukocyte antigen (HLA)-DR restricted. Finally, we demonstrated that the CALRmut-specific CD4+ T cells, despite their phenotype, were cytotoxic to autologous CALRmut cells, and that the cytotoxicity was mediated by degranulation of the T cells. In conclusion, the CALR exon 9 mutations are targets for specific T cells and thus are promising targets for cancer immune therapy such as peptide vaccination in patients harboring CALR exon 9 mutations.

CD44 is a transmembrane glycoprotein. When the CD44 gene is expressed, its pre-messenger RNA (mRNA) can be alternatively spliced into mature mRNAs that encode several CD44 isoforms. The mRNA assembles with ten standard exons, and the sixth variant exon encodes CD44v6, which engages in a variety of biological processes, including cell growth, apoptosis, migration, and angiogenesis. Mechanistically, CD44v6 interacts with hyaluronic acid (HA) or osteopontin, or it acts as a coreceptor for various cytokines, such as epidermal growth factor, vascular endothelial growth factor, hepatocyte growth factor, and C-X-C motif chemokine 12. In this context, the receptor tyrosine kinase or G protein-coupled receptor-associated signaling pathways, including mitogen-activated protein kinase/extracellular-signal-regulated kinase and phosphoinositide-3-kinase/Akt, are activated. Using these actions, homeostasis or regeneration can be facilitated among normal tissues. However, overexpression of the mature mRNA encoding CD44v6 can induce cancer progression. For example, CD44v6 assists colorectal cancer stem cells in colonization, invasion, and metastasis. Overexpression of CD44v6 predicts poor prognosis in patients with colorectal cancer, as patients with a large number of CD44v6-positive cells in their tumors are generally diagnosed at late stages. Thus, the clinical significance of CD44v6 in colorectal cancer deserves consideration. Preclinical results have indicated satisfactory efficacies of anti-CD44 therapy among several cancers, including prostate cancer, pancreatic cancer, and gastric cancer. Moreover, clinical trials aiming to evaluate the pharmacokinetics, pharmacodynamics, efficacy, and toxicity of a commercialized anti-CD44 monoclonal antibody developed by Roche (RO5429083) have been conducted among patients with CD44-expressing malignant tumors, and a clinical trial focusing on the dose escalation of this antibody is ongoing. Thus, we are hopeful that anti-CD44 therapy will be applied in the treatment of colorectal cancer in the future.

Nuclear clearance and cytoplasmic aggregation of TDP-43, initially identified in ALS-FTD, are hallmark pathological features observed across a spectrum of neurodegenerative diseases. We previously found that TDP-43 loss-of-function leads to transcriptome-wide inclusion of deleterious cryptic exons, a signature detected in presymptomatic biofluids and postmortem ALS-FTD brain tissue, but the upstream mechanisms that lead to TDP-43 dysregulation remain unclear. Here, we developed a web-based resource (SnapMine) to determine the levels of TDP-43 cryptic exon inclusion across hundreds of thousands of publicly available RNA sequencing datasets. We established cryptic exon inclusion levels across a variety of human cells and tissues to provide ground truth references for future studies on TDP-43 dysregulation. We then explored studies that were entirely unrelated to TDP-43 or neurodegeneration and found that ciclopirox olamine (CPX), an FDA-approved antifungal, can trigger the inclusion of TDP-43-associated cryptic exons in a variety of mouse and human primary cells. CPX induction of cryptic exons arises from heavy metal toxicity and oxidative stress, suggesting that similar vulnerabilities could play a role in neurodegeneration. Our work demonstrates how diverse datasets can be linked through common biological features and underscores how public archives of sequencing data remain a vastly underutilized resource with tremendous potential for uncovering novel insights into complex biological mechanisms and diseases. TDP-43 nuclear clearance and loss-of-function, a hallmark of ALS-FTD and other neurodegenerative diseases, causes widespread inclusion of harmful cryptic exons. Here, the authors developed SnapMine to analyze cryptic exon inclusion across public RNA-seq datasets and identified that the antifungal ciclopirox olamine (CPX) induces such inclusion via heavy metal toxicity and oxidative stress.

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small‐molecule SMN2 pre‐mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose‐dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients’ blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

Although targeted therapies have increased the life expectancy of patients with druggable molecular alterations directly involved in tumor development, the efficacy of these therapies is limited by acquired resistances leading to treatment failure. Most targeted therapies, including ones exploiting therapeutic antibodies and kinase inhibitors, are directed against receptor tyrosine kinases (RTKs) or major signaling hubs. Resistances to these therapies arise when inhibition of these targets is bypassed through activation of alternative signaling pathways. In recent years, activation of the receptor tyrosine kinase MET has been shown to promote resistance to various targeted therapies. This casts MET as important actor in resistance. In this review, we describe how the MET receptor triggers resistance to targeted therapies against RTKs such as EGFR, VEGFR, and HER2 and against signaling hubs such as BRAF. We also describe how MET can be its own resistance factor, as illustrated by on-target resistance of lung tumors harboring activating mutations causing MET exon 14 skipping. Interestingly, investigation of all these situations reveals functional physiological relationships between MET and the target of the therapy to which the cancer becomes resistant, suggesting that resistance stems from preexisting mechanisms. Identification of MET as a resistance factor opens the way to co-treatment strategies that are being tested in current clinical trials.

Immediate early genes are rapidly transcribed in response to neuronal activity, but the underlying mechanism is unclear. The authors show that this rapid transcription is mediated by a stalled RNA polymerase II, poised just downstream of the transcription start site. RNAi-depletion of negative elongation factor compromises the rapid transcription. Transcription of immediate early genes (IEGs) in neurons is highly sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We found that several IEGs, such as Arc (also known as Arg3.1 ), are poised for near-instantaneous transcription by the stalling of RNA polymerase II (Pol II) just downstream of the transcription start site in rat neurons. Depletion through RNA interference of negative elongation factor, a mediator of Pol II stalling, reduced the Pol II occupancy of the Arc promoter and compromised the rapid induction of Arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that were expressed later and largely lacked promoter-proximal Pol II stalling. Together, our data strongly indicate that the rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory.

In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1-3, transferrin and acid sphingomyelinase 1) and seven new genes (mgp4-10). The genes encoding mgp2-10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2-10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2-10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2-10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2-10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches.

Background:Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood.Methods:In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder.Results:We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7.Conclusions:Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice.

Treatment with tyrosine kinase inhibitors (TKI) may sequentially induce TKI‐resistant BCR‐ABL mutants in chronic myeloid leukemia (CML). Conventional PCR monitoring of BCR‐ABL is an important indicator to determine therapeutic intervention for preventing disease progression. However, PCR cannot separately quantify amounts of BCR‐ABL and its mutants, including alternatively spliced BCR‐ABL with an insertion of 35 intronic nucleotides (BCR‐ABLIns35bp) between ABL exons 8 and 9, which introduces the premature termination and loss of kinase activity. To assess the clinical impact of BCR‐ABL mutants, we performed deep sequencing analysis of BCR‐ABL transcripts of 409 samples from 37 patients with suboptimal response to frontline imatinib who were switched to nilotinib. At baseline, TKI‐resistant mutations were documented in 3 patients, whereas BCR‐ABLIns35bp was detected in all patients. After switching to nilotinib, both BCR‐ABL and BCR‐ABLIns35bp became undetectable in 3 patients who attained complete molecular response (CMR), whereas in the remaining all 34 patients, BCR‐ABLIns35bp was persistently detected, and minimal residual disease (MRD) fluctuated at low but detectable levels. PCR monitoring underestimated molecular response in 5 patients whose BCR‐ABLIns35bp was persisted, although BCR‐ABLIns35bp does not definitively mark TKI resistance. Therefore, quantification of BCR‐ABLIns35bp is useful for evaluating “functional” MRD and determining the effectiveness of TKI with accuracy. Deep sequencing analysis revealed that BCR‐ABLIns35bp persisted and fluctuated at low levels in patients who failed to achieved DMR, resulting in underestimating of molecular response status to TKI therapy. Therefore, separate quantification of BCR‐ABL, “function‐dead” BCR‐ABLIns35bp and TKI‐resistant KD mutations enables us to determine the effectiveness of TKI therapy with accuracy.