Expansion of tumor-reactive CD8+ T cell clonotypes occurs in the spleen in response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) enhances T cell responses against cancer, leading to long-term survival in a fraction of patients. CD8+ T cell differentiation in response to chronic antigen stimulation is highly complex, and it remains unclear precisely which T cell differentiation states at which anatomic sites are critical for the response to ICB. We identified an intermediate-exhausted population in the white pulp of the spleen that underwent substantial expansion in response to ICB and gave rise to tumor-infiltrating clonotypes. Increased systemic antigen redirected differentiation of this population toward a more circulatory exhausted KLR state, whereas a lack of cross-presented tumor antigen reduced its differentiation in the spleen. An analogous population of exhausted KLR CD8+ T cells in human blood samples exhibited diminished tumor-trafficking ability. Collectively, our data demonstrate the critical role of antigen density within the spleen for the differentiation and expansion of T cell clonotypes in response to ICB.

Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer.

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

14-3-3 and Smad2/3 are crucial mediators of atypical-PKCs: Implications for neuroblastoma progression.

Neuroblastoma (NB) is a cancer that develops in the neuroblasts. It is the most common cancer in children under the age of 1 year, accounting for approximately 6% of all cancers. The prognosis of NB is linked to both age and degree of cell differentiation. This results in a range of survival rates for patients, with outcomes ranging from recurrence and mortality to high survival rates and tumor regression. Our previous work indicated that PKC-ι promotes cell proliferation in NB cells through the PKC-ι/Cdk7/Cdk2 cascade. We report on two atypical protein kinase inhibitors as potential therapeutic candidates against BE(2)-C and BE(2)-M17 cells: a PKC-ι-specific 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide and a PKC-ζ specific 8-hydroxy-1,3,6-naphthalenetrisulfonic acid. Both compounds induced apoptosis and retarded the epithelial-mesenchymal transition (EMT) of NB cells. Proteins 14-3-3 and Smad2/3 acted as central regulators of aPKC-driven progression in BE(2)-C and BE(2)-M17 cells in relation to the Akt1/NF-κB and TGF-ß pathways. Data indicates that aPKCs upregulate Akt1/NF-κB and TGF-ß pathways in NB cells through an association with 14-3-3 and Smad2/3 that can be diminished by aPKC inhibitors. In summary, both inhibitors appear to be promising potential neuroblastoma therapeutics and merit further research.

Telomerase inhibition is an effective therapeutic strategy in TERT promoter-mutant glioblastoma models with low tumor volume.

BACKGROUND: Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma. Telomerase is therefore considered to be a promising therapeutic target against this tumor. However, an important limitation of this strategy is that cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We, therefore, hypothesize that telomerase inhibition would only be effective in glioblastomas with low tumor burden. METHODS: We used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient-derived models. We then measured viability using serial proliferation assays. We also assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Finally, we used a doxycycline-inducible CRISPR interference system to knock down TERT expression in vivo early and late in tumor development. RESULTS: Upon TERT inactivation, glioblastoma cells lose their proliferative ability over time and exhibit telomere shortening and chromatin bridge formation. In vivo, survival is only prolonged when TERT knockdown is induced shortly after tumor implantation, but not when the tumor burden is high. CONCLUSIONS: Our results support the idea that telomerase inhibition would be most effective at treating glioblastomas with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation.

ATRX modulates the escape from a telomere crisis.

Telomerase activity is the principal telomere maintenance mechanism in human cancers, however 15% of cancers utilise a recombination-based mechanism referred to as alternative lengthening of telomeres (ALT) that leads to long and heterogenous telomere length distributions. Loss-of-function mutations in the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) gene are frequently found in ALT cancers. Here, we demonstrate that the loss of ATRX, coupled with telomere dysfunction during crisis, is sufficient to initiate activation of the ALT pathway and that it confers replicative immortality in human fibroblasts. Additionally, loss of ATRX combined with a telomere-driven crisis in HCT116 epithelial cancer cells led to the initiation of an ALT-like pathway. In these cells, a rapid and precise telomeric elongation and the induction of C-circles was observed; however, this process was transient and the telomeres ultimately continued to erode such that the cells either died or the escape from crisis was associated with telomerase activation. In both of these instances, telomere sequencing revealed that all alleles, irrespective of whether they were elongated, were enriched in variant repeat types, that appeared to be cell-line specific. Thus, our data show that the loss of ATRX combined with telomere dysfunction during crisis induces the ALT pathway in fibroblasts and enables a transient activation of ALT in epithelial cells.

E-cigarette vapor exposure in utero causes long-term pulmonary effects in offspring.

The in utero environment is sensitive to toxicant exposure, altering the health and growth of the fetus, and thus sensitive to contaminant exposure. Though recent clinical data suggest that e-cigarette use does no further harm to birth outcomes than a nicotine patch, this does not account for the effects of vaping during pregnancy on the long-term health of offspring. Pregnant mice were exposed to: 1) e-cigarette vapor with nicotine (PV + Nic; 2% Nic in 50:50 propylene glycol: vegetable glycerin), 2) e-cigarette vapor without nicotine [PV; (50:50 propylene glycol:vegetable glycerin)], or 3) HEPA filtered air (FA). Dams were removed from exposure upon giving birth. At 5 mo of age, pulmonary function tests on the offspring revealed female and male mice from the PV group had greater lung stiffness (Ers) and alveolar stiffness (H) compared with the FA group. Furthermore, baseline compliance (Crs) was reduced in female mice from the PV group and in male mice from the PV and PV + Nic groups. Lastly, female mice had decreased forced expiratory volume (FEV0.1) in the PV group, but not in the male groups, compared with the FA group. Lung histology revealed increased collagen deposition around the vessels/airways and in alveolar tissue in PV and PV + Nic groups. Furthermore, goblet hyperplasia was observed in PV male and PV/PV + Nic female mice. Our work shows that in utero exposure to e-cigarette vapor, regardless of nicotine presence, causes lung dysfunction and structural impairments that persist in the offspring to adulthood.

Identification of Highly Cross-Reactive Mimotopes for a Public T Cell Response in Murine Melanoma.

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

POLQ suppresses genome instability and alterations in DNA repeat tract lengths.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ - /- cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ - / - than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ - /- cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ - / - cells. In conjunction with the expanded telomere lengths of POLQ - /- cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination.

The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.

Radiation-induced neuroinflammation: a potential protective role for poly(ADP-ribose) polymerase inhibitors?

Radiotherapy (RT) plays a fundamental role in the treatment of glioblastoma (GBM). GBM are notoriously invasive and harbor a subpopulation of cells with stem-like features which exhibit upregulation of the DNA damage response (DDR) and are radioresistant. High radiation doses are therefore delivered to large brain volumes and are known to extend survival but also cause delayed toxicity with 50%-90% of patients developing neurocognitive dysfunction. Emerging evidence identifies neuroinflammation as a critical mediator of the adverse effects of RT on cognitive function. In addition to its well-established role in promoting repair of radiation-induced DNA damage, activation of poly(ADP-ribose) polymerase (PARP) can exacerbate neuroinflammation by promoting secretion of inflammatory mediators. Therefore, PARP represents an intriguing mechanistic link between radiation-induced activation of the DDR and subsequent neuroinflammation. PARP inhibitors (PARPi) have emerged as promising new agents for GBM when given in combination with RT, with multiple preclinical studies demonstrating radiosensitizing effects and at least 3 compounds being evaluated in clinical trials. We propose that concomitant use of PARPi could reduce radiation-induced neuroinflammation and reduce the severity of radiation-induced cognitive dysfunction while at the same time improving tumor control by enhancing radiosensitivity.

Dysgu: efficient structural variant calling using short or long reads.

Structural variation (SV) plays a fundamental role in genome evolution and can underlie inherited or acquired diseases such as cancer. Long-read sequencing technologies have led to improvements in the characterization of structural variants (SVs), although paired-end sequencing offers better scalability. Here, we present dysgu, which calls SVs or indels using paired-end or long reads. Dysgu detects signals from alignment gaps, discordant and supplementary mappings, and generates consensus contigs, before classifying events using machine learning. Additional SVs are identified by remapping of anomalous sequences. Dysgu outperforms existing state-of-the-art tools using paired-end or long-reads, offering high sensitivity and precision whilst being among the fastest tools to run. We find that combining low coverage paired-end and long-reads is competitive in terms of performance with long-reads at higher coverage values.

Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer.

In non­small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ­producing, tumor-infiltrating CD8+ T cells. However, not all tumors with a CD8+ T cell infiltrate respond to ICB, and little is known about the mechanisms governing ICB resistance in T cell­infiltrated NSCLC. We used an orthotopic NSCLC mouse model to study ICB-refractory CD8+ T cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors revealed that lung cancer­specific tumor-infiltrating CD8+ T cells exhibited clonal expansion but lacked expression of genes associated with effector and exhausted T cell responses, indicating that they underwent a differentiation program distinct from conventional T cell exhaustion. This lung cancer­specific T cell dysfunction program was established early during priming in the mediastinal lymph node and was characterized by robust proliferation but a failed up-regulation of effector and exhausted T cell characteristics. Intriguingly, CD8+ T cells from patients with NSCLC expressed an analogous gene expression program, which appeared distinct from conventional T cell exhaustion. Administration of recombinant interleukin-2 (IL-2) and IL-12 was sufficient to restore effector T cell differentiation and induce control of KP lung tumors. These findings imply that a CD8+ T cell differentiation trajectory, activated during T cell priming in the mediastinal lymph node, limits the response of CD8+ T cells to ICB and thereby may contribute to failure of ICB in a subset T cell­infiltrated NSCLC.

Accuracy of medial-to-lateral transcondylar screw placement using an aiming device and preoperative computer tomography planning for the treatment of humeral intracondylar fissure.

OBJECTIVE: To determine the accuracy of transcondylar screws placed from medial-to-lateral using preoperative planning on computed tomography (CT) and an aiming device in elbows with humeral intracondylar fissures (HIFs). STUDY DESIGN: Retrospective case series. ANIMALS: Twenty-five client-owned dogs with HIF. METHODS: A 4.5-mm transcondylar screw was placed in 34 elbows with HIF. humeral condylar diameter (HCD) was measured at the humeral condylar isthmus on CT. Entry and exit points were planned at 0.3 × HCD cranial and 0.2 × HCD distal to the medial epicondyle and 0.3 × HCD cranial and 0.3 × HCD distal to the lateral epicondyle. An aiming device was used to guide drilling from the medial entry point to the lateral exit point. The difference between planned and actual screw entry and exit points, and the angular deviation of the actual screw axis from the planned screw axis, was assessed on the postoperative CT scans. RESULTS: Thirty-three out of 34 screws were completely within the humeral condyle. Thirteen out of 34 screws were placed less than 2 mm from planned entry and exit points in both the transverse and the frontal plane. The axis of the screw deviated by a mean of 3.2° (transverse plane) and 3.5° (frontal plane) from the planned axis. CONCLUSION: Humeral transcondylar screws placed with the technique tested here were entirely within the humeral condyle in 33 out of 34 elbows. CLINICAL SIGNIFICANCE: Use of CT planning and an aiming device allows accurate placement of transcondylar screws from medial-to-lateral in dogs with HIF.

UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.

Two Consecutive Invasive Surgeries Utilizing Zymogen Protein C (ZPC) That Enhanced Patient Safety and Reduced Costs.

This case report describes a major surgical procedure for a protein C-deficient, hypercoagulable patient who underwent two back-to-back invasive surgeries, hip replacement, and spinal stenosis correction. The patient, an 84-year-old male with a history of deep vein thromboses (DVT) and pulmonary emboli (PE), was treated pre-, peri-, and postoperatively with zymogen protein C (ZPC-Baxter, International) and recovered without clotting or increased bleeding. During the procedure, the patient was not administered any other anticoagulants. There have now been several case reports on different patients with unrelated teams in various locations worldwide using zymogen protein C during surgical procedures. Thus, this procedure is becoming a viable choice for patients with a high probability of clotting during and after invasive surgery. This case focuses on accomplishing safer surgery and reducing costs, by using less ZPC while accomplishing two surgeries in one procedure. As a result, this procedure might be useful for many medical situations where acquired protein C deficiency could be a problem (e.g., sepsis, pregnancy, etc.). This approach may have greater application to medical conditions other than protein C deficiency, where clotting and inflammation can become issues.

High-throughput STELA provides a rapid test for the diagnosis of telomere biology disorders.

Telomere biology disorders are complex clinical conditions that arise due to mutations in genes required for telomere maintenance. Telomere length has been utilised as part of the diagnostic work-up of patients with these diseases; here, we have tested the utility of high-throughput STELA (HT-STELA) for this purpose. HT-STELA was applied to a cohort of unaffected individuals (n = 171) and a retrospective cohort of mutation carriers (n = 172). HT-STELA displayed a low measurement error with inter- and intra-assay coefficient of variance of 2.3% and 1.8%, respectively. Whilst telomere length in unaffected individuals declined as a function of age, telomere length in mutation carriers appeared to increase due to a preponderance of shorter telomeres detected in younger individuals (< 20 years of age). These individuals were more severely affected, and age-adjusted telomere length differentials could be used to stratify the cohort for overall survival (Hazard Ratio = 5.6 (1.5-20.5); p < 0.0001). Telomere lengths of asymptomatic mutation carriers were shorter than controls (p < 0.0001), but longer than symptomatic mutation carriers (p < 0.0001) and telomere length heterogeneity was dependent on the diagnosis and mutational status. Our data show that the ability of HT-STELA to detect short telomere lengths, that are not readily detected with other methods, means it can provide powerful diagnostic discrimination and prognostic information. The rapid format, with a low measurement error, demonstrates that HT-STELA is a new high-quality laboratory test for the clinical diagnosis of an underlying telomeropathy.

Tracking telomere fusions through crisis reveals conflict between DNA transcription and the DNA damage response.

Identifying attributes that distinguish pre-malignant from senescent cells provides opportunities for targeted disease eradication and revival of anti-tumour immunity. We modelled a telomere-driven crisis in four human fibroblast lines, sampling at multiple time points to delineate genomic rearrangements and transcriptome developments that characterize the transition from dynamic proliferation into replicative crisis. Progression through crisis was associated with abundant intra-chromosomal telomere fusions with increasing asymmetry and reduced microhomology usage, suggesting shifts in DNA repair capacity. Eroded telomeres also fused with genomic loci actively engaged in transcription, with particular enrichment in long genes. Both gross copy number alterations and transcriptional responses to crisis likely underpin the elevated frequencies of telomere fusion with chromosomes 9, 16, 17, 19 and most exceptionally, chromosome 12. Juxtaposition of crisis-regulated genes with loci undergoing de novo recombination exposes the collusive contributions of cellular stress responses to the evolving cancer genome.