Neoadjuvant enoblituzumab in localized prostate cancer: a single-arm, phase 2 trial.

B7 homolog 3 (B7-H3; CD276), a tumor-associated antigen and possible immune checkpoint, is highly expressed in prostate cancer (PCa) and is associated with early recurrence and metastasis. Enoblituzumab is a humanized, Fc-engineered, B7-H3-targeting antibody that mediates antibody-dependent cellular cytotoxicity. In this phase 2, biomarker-rich neoadjuvant trial, 32 biological males with operable intermediate to high-risk localized PCa were enrolled to evaluate the safety, anti-tumor activity and immunogenicity of enoblituzumab when given before prostatectomy. The coprimary outcomes were safety and undetectable prostate-specific antigen (PSA) level (PSA0) 1 year postprostatectomy, and the aim was to obtain an estimate of PSA0 with reasonable precision. The primary safety endpoint was met with no notable unexpected surgical or medical complications, or surgical delay. Overall, 12% of patients experienced grade 3 adverse events and no grade 4 events occurred. The coprimary endpoint of the PSA0 rate 1 year postprostatectomy was 66% (95% confidence interval 47-81%). The use of B7-H3-targeted immunotherapy in PCa is feasible and generally safe and preliminary data suggest potential clinical activity. The present study validates B7-H3 as a rational target for therapy development in PCa with larger studies planned. The ClinicalTrials.gov identifier is NCT02923180.

Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.

Dynamic chromatin organization and regulatory interactions in human endothelial cell differentiation.

Vascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. The gene-regulatory mechanisms underpinning endothelial specialization are largely unknown, as are the roles of chromatin organization in regulating endothelial cell transcription. To investigate the relationships between chromatin organization and gene expression, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-sequencing assays at specific time points. Long-range intrachromosomal contacts increase over the course of differentiation, accompanied by widespread heteroeuchromatic compartment transitions that are tightly associated with transcription. Dynamic topologically associating domain boundaries strengthen and converge on an endothelial cell state, and function to regulate gene expression. Chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes essential to vascular biology. Chromatin dynamics guide transcription in endothelial cell development and promote the divergence of endothelial cells from cardiomyocytes.

Perturbations of the T-cell immune repertoire in kidney transplant rejection.

In this cross-sectional and longitudinal analysis of mapping the T-cell repertoire in kidney transplant recipients, we have investigated and validated T-cell clonality, immune repertoire chronology at rejection, and contemporaneous allograft biopsy quantitative tissue injury, to better understand the pathobiology of acute T-cell fraction, T-cell repertoire and antibody-mediated kidney transplant rejection. To follow the dynamic evolution of T-cell repertoire changes before and after engraftment and during biopsy-confirmed acute rejection, we sequenced 323 peripheral blood samples from 200 unique kidney transplant recipients, with (n=100) and without (n=100) biopsy-confirmed acute rejection. We report that patients who develop acute allograft rejection, have lower (p=0.01) T-cell fraction even before transplantation, followed by its rise after transplantation and at the time of acute rejection accompanied by high TCR repertoire turnover (p=0.004). Acute rejection episodes occurring after the first 6 months post-transplantation, and those with a component of antibody-mediated rejection, had the highest turnover; p=0.0016) of their T-cell repertoire. In conclusion, we validated that detecting repertoire changes in kidney transplantation correlates with post-transplant rejection episodes suggesting that T-cell receptor sequencing may provide recipient pre-transplant and post-transplant predictors of rejection risk.

Anti-spike T-cell and Antibody Responses to SARS-CoV-2 mRNA Vaccines in Patients with Hematologic Malignancies.

The anti-spike T-cell and antibody responses to SARS-CoV-2 mRNA vaccines in patients with B-cell malignancies were examined in a real-world setting. A next-generation sequencing (NGS)-based molecular assay was used to assess SARS-CoV-2-specific T-cell responses. After the second dose, 58% (166/284) of seropositive and 45% (99/221) of seronegative patients display anti-spike T cells. The percentage of patients who displayed T-cell response was higher among patients receiving mRNA-1273 vaccines compared with those receiving BNT162b2 vaccines. After the third vaccination, 40% (137/342) of patients seroconverted, although only 22% displayed sufficient antibody levels associated with the production of neutralizing antibodies. 97% (717/738) of patients who were seropositive before the third dose had markedly elevated anti-spike antibody levels. Anti-spike antibody levels, but not T-cell responses, were depressed by B cell-directed therapies. Vaccinated patients with B-cell malignancies with a poor response to SARS-CoV-2 vaccines may remain vulnerable to COVID-19 infections. SIGNIFICANCE: This study represents the first investigation of SARS-CoV-2-specific immune responses to vaccination in a patient registry using an NGS-based method for T-cell receptor repertoire-based analysis combined with anti-spike antibody assessments. Vaccinated patients with B cell-derived hematologic malignancies are likely at higher risk of infection or severe COVID-19. This article is highlighted in the In This Issue feature, p. 476.

Changes in T-cell subsets and clonal repertoire during chemoimmunotherapy with pembrolizumab and paclitaxel or capecitabine for metastatic triple-negative breast cancer.

BACKGROUND: Chemoimmunotherapy is a standard treatment for triple-negative breast cancer (TNBC), however, the impacts of different chemotherapies on T-cell populations, which could correlate with clinical activity, are not known. Quantifying T-cell populations with flow cytometry and T-cell receptor (TCR) immunosequencing may improve our understanding of how chemoimmunotherapy affects T-cell subsets, and to what extent clonal shifts occur during treatment. TCR immunosequencing of intratumoral T cells may facilitate the identification and monitoring of putatively tumor-reactive T-cell clones within the blood. METHODS: Blood and tumor biopsies were collected from patients with metastatic TNBC enrolled in a phase Ib clinical trial of first or second-line pembrolizumab with paclitaxel or capecitabine. Using identical biospecimen processing protocols, blood samples from a cohort of patients treated for early-stage breast cancer were obtained for comparison. Treatment-related immunological changes in peripheral blood and intratumoral T cells were characterized using flow cytometry and TCR immunosequencing. Clonal proliferation rates of T cells were compared based on intratumoral enrichment. RESULTS: When combined with pembrolizumab, paclitaxel and capecitabine resulted in similar time-dependent lymphodepletions across measured peripheral T-cell subsets. Their effects were more modest than that observed following curative-intent dose-dense anthracycline and cyclophosphamide (ddAC) (average fold-change in CD3+ cells, capecitabine: -0.42, paclitaxel: -0.56, ddAC: -1.21). No differences in T-cell clonality or richness were observed following capecitabine or paclitaxel-based treatments. Regression modeling identified differences in the emergence of novel T-cell clones that were not detected at baseline (odds compared with ddAC, capecitabine: 0.292, paclitaxel: 0.652). Pembrolizumab with paclitaxel or capecitabine expanded T-cell clones within tumors; however, these clones did not always expand within the blood. Proliferation rates within the blood were similar between clones that were enriched and those that were not enriched within tumors. CONCLUSION: Chemoimmunotherapy for metastatic TNBC with pembrolizumab and capecitabine or paclitaxel resulted in similar peripheral T-cell subset lymphodepletion without altering T-cell clonal diversity. Regression modeling methods are applicable in immune monitoring studies, such as this to identify the odds of novel T-cell clones emerging during treatment, and proliferation rates of tumor-enriched T-cell clones.

A Phase II Study to Assess the Safety and Efficacy of the Dual mTORC1/2 and PI3K Inhibitor Bimiralisib (PQR309) in Relapsed, Refractory Lymphoma.

Bimiralisib is an orally bioavailable pan-phosphatidylinositol 3-kinase and mammalian target of rapamycin inhibitor which has shown activity against lymphoma in preclinical models. This phase I/II study evaluated the response rate to bimiralisib at 2 continuous dose levels (60 mg and 80 mg) in patients with relapsed/refractory lymphoma. Fifty patients were enrolled and started treatment. The most common histologies were diffuse large B-cell lymphoma (n = 17), follicular lymphoma (n = 9), T-cell lymphoma (n = 8), and others (mostly indolent). Patients had been treated with a median of 5 prior lines of treatment and 44% were considered refractory to their last treatment. Mean duration of treatment (and standard deviations) with 60 mg once daily (o.d.) was 1.3 ± 1.2 months, and with 80 mg o.d. 3.7 ± 3.9 months. On an intention to treat analysis, the overall response rate was 14% with 10% achieving a partial response and 4% a complete response. Thirty-six percent of patients were reported as having stable disease. No dose-limiting toxicities were observed during the phase I portion of the study. Overall, 70% of patients had a grade 3 treatment emergent adverse events (TEAE) and 34% had a grade 4 TEAE; 28% of patients discontinued treatment due to toxicity. The most frequent TEAEs grade ≥3 was hyperglycemia (24%), neutropenia (20%), thrombocytopenia (22%), and diarrhea (12%). Per protocol, hyperglycemia required treatment with oral antihyperglycemic agents in 28% and with insulin in 14%. At 60 mg or 80 mg continuous dosing, bimiralisib showed modest efficacy with significant toxicity in heavily pretreated patients with various histological subtypes of lymphoma.

AZD1222/ChAdOx1 nCoV-19 vaccination induces a polyfunctional spike protein-specific TH1 response with a diverse TCR repertoire.

AZD1222 (ChAdOx1 nCoV-19), a replication-deficient simian adenovirus­vectored vaccine, has demonstrated safety, efficacy, and immunogenicity against coronavirus disease 2019 in clinical trials and real-world studies. We characterized CD4+ and CD8+ T cell responses induced by AZD1222 vaccination in peripheral blood mononuclear cells from 296 unique vaccine recipients aged 18 to 85 years who enrolled in the phase 2/3 COV002 trial. Total spike protein­specific CD4+ T cell helper type 1 (TH1) and CD8+ T cell responses were increased in AZD1222-vaccinated adults of all ages after two doses of AZD1222. CD4+ TH2 responses after AZD1222 vaccination were not detected. Furthermore, AZD1222-specific TH1 and CD8+ T cells both displayed a high degree of polyfunctionality in all adult age groups. T cell receptor ß (TCRß) sequences from vaccinated participants mapped against TCR sequences known to react to SARS-CoV-2 revealed substantial breadth and depth across the SARS-CoV-2 spike protein for both AZD1222-induced CD4+ and CD8+ T cell responses. Overall, AZD1222 vaccination induced a polyfunctional TH1-dominated T cell response, with broad CD4+ and CD8+ T cell coverage across the SARS-CoV-2 spike protein.

Impact of HLA type, age and chronic viral infection on peripheral T-cell receptor sharing between unrelated individuals.

The human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, factors such as thymic selection and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCRß variable region of 426 healthy individuals, we find that, on average, fewer than 1% of TCRß clones are shared between individuals, consistent with largely private TCRß repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCRß clones, with each additional shared HLA allele contributing to an increase in ~0.01% of the total shared TCRß clones, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCRß clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCRß clone sharing, indicating that the pattern of private TCRß clonal expansion is a general feature of the T-cell response to other infectious antigens as well. However, increased age also correlates with increased sharing among the lowest frequency clones, consistent with decreased repertoire diversity in older individuals. Together, all of these factors contribute to shaping the TCRß repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.

T-cell mediated immunity after AZD1222 vaccination: A polyfunctional spike-specific Th1 response with a diverse TCR repertoire.

AZD1222 (ChAdOx1 nCoV-19), a replication-deficient simian adenovirus-vectored vaccine, has demonstrated safety, efficacy, and immunogenicity against coronavirus disease 2019 (COVID-19) in clinical trials and real-world studies. We characterized CD4+ and CD8+ T-cell responses induced by AZD1222 vaccination in peripheral blood mononuclear cells (PBMCs) from 280 unique vaccine recipients aged 18-85 years who enrolled in the phase 2/3 COV002 trial. Total spike-specific CD4+ T cell helper type 1 (Th1) and CD8+ T-cell responses were significantly increased in AZD1222-vaccinated adults of all ages following two doses of AZD1222. CD4+ Th2 responses following AZD1222 vaccination were not detected. Furthermore, AZD1222-specific Th1 and CD8+ T cells both displayed a high degree of polyfunctionality in all adult age groups. T-cell receptor (TCR) ß sequences from vaccinated participants mapped against TCR sequences known to react to SARS-CoV-2 revealed substantial breadth and depth across the SARS-CoV-2 spike protein for the AZD1222-induced CD4+ and CD8+ T-cell responses. Overall, AZD1222 vaccination induced a robust, polyfunctional Th1-dominated T-cell response, with broad CD4+ and CD8+ T-cell coverage across the SARS-CoV-2 spike protein. ONE SENTENCE SUMMARY: Polyfunctional CD4+ and CD8+ T-cell responses are elicited against the SARS-CoV-2 spike protein following vaccination with AZD1222.

Pre-transplant T-cell Clonality: An Observational Study of a Biomarker for Prediction of Sepsis in Liver Transplant Recipients.

OBJECTIVE: This study investigated the ability of pre-transplant T-cell clonality to predict sepsis after liver transplant (LT). SUMMARY BACKGROUND DATA: Sepsis is a leading cause of death in LT recipients. Currently, no biomarkers predict sepsis before clinical symptom manifestation. METHODS: Between December 2013 and March 2018, our institution performed 478 LTs. After exclusions (eg, patients with marginal donor livers, autoimmune disorders, nonabdominal multi-organ, and liver retransplantations), 180 consecutive LT were enrolled. T-cell characterization was assessed within 48 hours before LT (immunoSEQ Assay, Adaptive Biotechnologies, Seattle, WA). Sepsis-2 and Sepsis-3 cases, defined by presence of acute infection plus ≥2 SIRS criteria, or clinical documentation of sepsis, were identified by chart review. Receiver-operating characteristic analyses determined optimal T-cell repertoire clonality for predicting post-LT sepsis. Kaplan-Meier and Cox proportional hazard modeling assessed outcome-associated prognostic variables. RESULTS: Patients with baseline T-cell repertoire clonality ≥0.072 were 3.82 (1.25, 11.40; P = 0.02), and 2.40 (1.00, 5.75; P = 0.049) times more likely to develop sepsis 3 and 12 months post-LT, respectively, when compared to recipients with lower (<0.072) clonality. T-cell repertoire clonality was the only predictor of sepsis 3 months post-LT in multivariate analysis (C-Statistic, 0.75). Adequate treatment resulted in equivalent survival rates between both groups: (93.4% vs 96.2%, respectively, P = 0.41) at 12 months post-LT. CONCLUSIONS: T-cell repertoire clonality is a novel biomarker predictor of sepsis before development of clinical symptoms. Early sepsis monitoring and management may reduce post-LT mortality. These findings have implications for developing sepsis-prevention protocols in transplantation and potentially other populations.

Safety and Efficacy of Consolidation Therapy with Ipilimumab Plus Nivolumab after Autologous Stem Cell Transplantation.

Autologous hematopoietic stem cell transplantation (ASCT) is a standard-of-care treatment for many hematologic malignancies. Progression of disease after ASCT is the primary cause of treatment failure. In this Phase Ib trial, we studied the safety and clinical effect of combined checkpoint inhibition therapy (CPIT) with ipilimumab and nivolumab as a consolidation strategy after ASCT for patients with high-risk diffuse large B cell lymphoma (DLBCL), mature T cell lymphoma (TCL), and multiple myeloma (MM). Starting at 14 to 28 days after ASCT, patients received ipilimumab (1 mg/kg i.v. on day 1 of weeks 1, 4, 7, 10, 16, and 22) and nivolumab (3 mg/kg i.v. on day 1 of weeks 1, 4, 7, 10, 12, 14, 16, 18, 20, 22, 24, and 26). Patients received a median of 5 doses of ipilimumab and 8 doses of nivolumab. Thirty-five patients were included in the intent-to-treat population. Ninety-four percent of the patients experienced immune-related adverse events (irAEs) of any grade. Ninety-seven percent of irAEs resolved spontaneously or after holding study drugs and instituting high-dose corticosteroid therapy. Progression-free and overall survival at 18 months post-ASCT for each disease cohort were 85.7% and 100% for primary refractory DLBCL, 28.6% and 57.1% for relapsed DLBCL, not evaluable and 80% for frontline TCL, 25% and 75% for relapsed TCL, 57.1% and 87% for high-risk transplant-naïve MM, and 40% and 100% for MM relapsed within 3 years of first ASCT. We conclude that combined CPIT appears to be tolerable as a consolidation strategy after ASCT and in addition to the potential clinical efficacy observed in some subsets of disease, T cell receptor repertoire, T regulatory cell phenotype, and gut microbiota profiles provide a biologic rationale warranting further study of this approach.

Results of a UK National Cancer Research Institute Phase II study of brentuximab vedotin using a response-adapted design in the first-line treatment of patients with classical Hodgkin lymphoma unsuitable for chemotherapy due to age, frailty or comorbidity (BREVITY).

Standard treatment for classical Hodgkin lymphoma (cHL) is poorly tolerated in older patients and results disappointing. We assessed safety and efficacy of brentuximab vedotin (BV), in previously untreated patients with cHL unfit for standard treatment due to age, frailty or comorbidity. The primary outcome was complete metabolic response (CMR) by positron emission tomography/computed tomography after four BV cycles (PET4). The secondary outcomes included progression-free survival (PFS), overall survival (OS), and toxicity. In all, 35 patients with a median age of 77 years and median total Cumulative Illness Rating Scale for Geriatrics (CIRS-G) score of 6 were evaluable for toxicity and 31 for response. A median of four cycles were given (range one-16). In all, 14 patients required dose reduction due to toxicity and 11 patients stopped treatment due to adverse events (AEs). A total of 716 AEs were reported, of which 626 (88%) were Grade 1/2 and 27 (77%) patients had at least one AE Grade ≥3. At PET4, CMR was 25·8% [95% confidence interval (CI) 13·7-42.2%] and objective response rate 83·9% (95% CI 63·7-90·8%). Median PFS was 7·3 months (95% CI 5·2-9·0), and OS 19·5 months. Our results suggest that BV monotherapy is tolerable but suboptimal in the front-line therapy of elderly or comorbid patients with cHL. Combining BV with other agents may be more effective. Trial Registration: Clinicaltrials.gov identifier: NCT02567851.

T cell fraction impacts oncologic outcomes in human papillomavirus associated oropharyngeal squamous cell carcinoma.

BACKGROUND: We investigated T cell clonality (TCC) and T cell fraction (TCF) in human papilloma virus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) progressors [cases] vs. non-progressors [controls]. METHODS: This nested case-control study included patients undergoing intent-to-cure surgery ± adjuvant therapy from 6/1/2007-10/3/2016. Patients experiencing local/regional/distant disease (progressors), and a consecutive sample of non-progressors were matched (2 controls: 1 case) on tumor subsite, T-stage and number of metastatic lymph nodes. We performed imunosequencing of the CDR3 regions of human TCRß chains. RESULTS: 34 progressors and 65 non-progressors were included. There was no statistically significant difference in baseline TCF (range: 0.039-1.084) and TCC (range: 0.007-0.240) (p > 0.05). Female sex was associated with higher TCF (p = 0.03), while extranodal extension (ENE) was associated with lower TCF (p = 0.01). There was a positive correlation between tumor size and clonality (R = 0.34, p < 0.01). The strongest predictor of progression-free survival (PFS) was TCF (HR 0.80, 95%CI 0.66-0.96, p = 0.02). The strongest predictors of cancer specific survival (CSS) were TCF (HR0.69, 95%CI 0.47-1.00, p < 0.05) and Adult Comorbidity Evaluation-27 (ACE-27) score (p < 0.05). Similarly, the strongest predictors of overall survival (OS) were TCF (HR 0.62, 95%CI 0.43-0.91, p = 0.01) and ACE-27 score (p = 0.03). On multivariable modeling, TCF ≥ 0.4 was independently associated with PFS (HR 0.34, 95%CI 0.14-0.85, p = 0.02) while an ACE-27 score of ≥ 2 independently predicted CSS (HR 3.85, 95%CI 1.07-13.85, p = 0.04) and OS (HR 3.51, 95%CI 1.10-11.20, p = 0.03). CONCLUSIONS: In patients with HPV(+)OPSCC, TCF was higher in female patients and those without ENE, suggesting differential immune responses. Lower TCF was significantly and independently associated with disease progression. Better ACE-27 scores appear to predict improved oncologic control.

Multiple mutations at exon 2 of RHOA detected in plasma from patients with peripheral T-cell lymphoma.

The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencing of lymph node samples, but there has been little work on the mutational load that is present in cell-free DNA (cfDNA) from plasma. We report targeted sequencing of cfDNA from PTCL patients to demonstrate c.50G>T (p.Gly17Val) in RHOA as previously described in angioimmunoblastic T-cell lymphoma (AITL) and a group of PTCL not otherwise specified (NOS) but also detect novel mutations at c.73A>G (p.Phe25Leu) and c.48A>T (p.Cys16*) of exon 2, which were confirmed by Sanger sequencing. In a group of AITL and PTCL-NOS analyzed by droplet digital polymerase chain reaction, 63% (12/19) showed c.50G>T (p.Gly17Val), 53% (10/19) c.73A>G (p.Phe25Leu), and 37% (7/19) c.48A>T (pCys16*). Sequencing of lymph node tissue in 3 out of 9 cases confirmed the presence of c.73A>G (p.Phe25Leu). Inspection of individual sequencing reads from individual patients showed that a single RHOA allele could contain >1 mutation, suggesting haplotypes of mutations at RHOA. Serial sampling showed changes to RHOA mutational frequency with treatment and the apparent occurrence of clones bearing specific haplotypes associated with relapse. Therefore, sequencing of RHOA from cfDNA has revealed new mutations and haplotypes. The clinical significance of these findings will need to be explored in clinical trials, but liquid biopsy might have potential for guiding treatment decisions in PTCL.

Evolution of retrovirus-infected premalignant T-cell clones prior to adult T-cell leukemia/lymphoma diagnosis.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive hematological malignancy caused by human T-cell leukemia virus type-1 (HTLV-1). ATL is preceded by decades of chronic HTLV-1 infection, and the tumors carry both somatic mutations and proviral DNA integrated into the tumor genome. In order to gain insight into the oncogenic process, we used targeted sequencing to track the evolution of the malignant clone in 6 individuals, 2 to 10 years before the diagnosis of ATL. Clones of premalignant HTLV-1-infected cells bearing known driver mutations were detected in the blood up to 10 years before individuals developed acute and lymphoma subtype ATL. Six months before diagnosis, the total number and variant allele fraction of mutations increased in the blood. Peripheral blood mononuclear cells from premalignant cases (1 year prediagnosis) had significantly higher mutational burden in genes frequently mutated in ATL than did high-risk, age-matched HTLV-1 carriers who remained ATL-free after a median of 10 years of follow-up. These data show that HTLV-1-infected T-cell clones carrying key oncogenic driver mutations can be detected in cases of ATL years before the onset of symptoms. Early detection of such mutations may enable earlier and more effective intervention to prevent the development of ATL.

Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.

BACKGROUND: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (TTN) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. METHODS: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z-/-) and A-band (TTN-A-/-) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. RESULTS: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z-/--CMs had sarcomeres and visibly contracted, whereas TTN-A-/--CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z-/--CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z-/--CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. CONCLUSIONS: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.

Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy.

Mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared with gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape segregation to the nuclear lamina and inactivation during cardiogenesis. These regions exhibit up-regulation of multiple noncardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. However, chromatin compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. Thus, global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.

Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory.

Functional changes in spatial genome organization during human development are poorly understood. Here we report a comprehensive profile of nuclear dynamics during human cardiogenesis from pluripotent stem cells by integrating Hi-C, RNA-seq and ATAC-seq. While chromatin accessibility and gene expression show complex on/off dynamics, large-scale genome architecture changes are mostly unidirectional. Many large cardiac genes transition from a repressive to an active compartment during differentiation, coincident with upregulation. We identify a network of such gene loci that increase their association inter-chromosomally, and are targets of the muscle-specific splicing factor RBM20. Genome editing studies show that TTN pre-mRNA, the main RBM20-regulated transcript in the heart, nucleates RBM20 foci that drive spatial proximity between the TTN locus and other inter-chromosomal RBM20 targets such as CACNA1C and CAMK2D. This mechanism promotes RBM20-dependent alternative splicing of the resulting transcripts, indicating the existence of a cardiac-specific trans-interacting chromatin domain (TID) functioning as a splicing factory.