Citations

Rationale: The generation of animal models for human cardiomyopathy creates opportunities to uncover mechanisms of diseases’ pathogenesis and to identify genes that modify the progression of pathogenesis. In contrast to candidate gene-based approaches, the phenotype-based forward genetic screening approach is less effectively employed in a vertebrate, mainly due to the prohibitively enormous colony management efforts. Objective: To identify genetic modifiers of cardiomyopathy through developing an unbiased forward genetic approach in the vertebrate Danio rerio. Methods and Results: Using an expression-based insertional mutagenesis screening strategy, we identified 49 Zebrafish Insertional Cardiac (ZIC) mutants from a collection of ~300 gene break-transposon (GBT) lines. By applying doxorubicin (DOX) stress to these heterozygous ZIC mutants, we initially identified four GBT lines that either enhanced or suppressed DOX-induced cardiomyopathy phenotypes. Here, we focus on the GBT0411 line which significantly enhances DOX-induced cardiomyopathy phenotypes. We cloned the gene and identified a single transposon insertion that disrupts the long nuclei isoform of dnajb6b in this GBT0411 mutant. Consistent with its cardiomyopathy modifier function, we detected a predominant expression of dnajb6b in the heart. While mutations affecting the cytoplasmic functions of DNAJB6 were recently reported to cause limb-girdle muscular dystrophy type 1D, our study suggested that the impaired balance between Dnajb6b long (nuclei) and short (cytoplasmic) isoform accounted for the cardiomyopathy modifier effect. Conclusions: Our results demonstrated the feasibility of identifying genetic modifiers of cardiomyopathy using a forward genetic approach in a vertebrate. Specifically, our data implicate DNAJB6 as a new genetic risk factor for DOX-induced cardiomyopathy. Without any presumption, this forward genetic strategy opens the door to systematically identify genetic factors that modify the progression of cardiomyopathy, which will facilitate the practice of personalized medicine and the development of novel therapies.

Background: Cardiomyopathy and heart failure affect millions of people worldwide. Because genetic modifiers contribute in large part to the highly variable phenotypic expression of cardiomyopathy in patients even with identical disease-causing mutations, the identification of modifier genes for this disease will greatly improve risk stratification, prognostic test development, and personalized therapy. However, only a rather limited number of modifier genes for cardiomyopathy have been identified sporadically. Objective: To identify genetic modifiers for cardiomyopathy using a novel insertional mutagenesis screening approach in adult zebrafish. Methods and Results: We screened 476 gene break-transposon (GBT) lines and isolated 44 zebrafish insertional cardiac (ZIC) mutants. Employing doxorubicin (DOX) stress to these ZIC mutants, we identified four candidate GBT lines that modified the progression of DOX-induced cardiomyopathy. Here, we report the detailed study of the GBT0411 mutant that exacerbated DOX-induced cardiomyopathy. GBT0411 mutant was tagged to the dnajb6b gene. Mutations in the short (sarcomeric) isoform of its human homologue gene DNAJB6 was recently reported to cause limb-girdle muscular dystrophy type 1D. Interestingly, our data showed that long (nuclei) isoform (dnajb6b[L]) was the major isoform expressed in the heart, and loss-of-function of which deteriorated the progression of DOX-induced cardiomyopathy. We further found that a cardiomyocyte-specific dnajb6b(L) transgene reverted the deleterious modifying effect of GBT0411 mutant, and exerted a cardioprotective function on chronic anemia induced cardiomyopathy. Mechanistically, Dnajb6b(L) could partially localize to endoplasmic reticulum (ER) upon ER stress, and function as an ER stress suppressor. Indeed, inhibition of ER stress by using a chemical chaperon mimics the cardioprotective effect of dnajb6b(L) transgene. Conclusions: By conducting an unbiased mutagenesis screening in adult zebrafish, we identified dnajb6b as a novel genetic modifier for cardiomyopathy. A cardioprotective function was identified by overexpressing its long isoform in cardiomyocytes, which might be conveyed by inhibition of ER stress response.

In contrast to the wide application of zebrafish embryos in developmental genetic studies, the value of an adult zebrafish to dissect signaling pathways in human diseases such as cardiomyopathy remains largely elusive. Previously, we have established anemic tr265/tr265 fish as the first adult zebrafish model of cardiomyopathy. Here, we generate the second adult zebrafish cardiomyopathy model induced by injection of a single bolus of doxorubicin (DOX). Despite their different pathogenesis, cardiac enlargement induced by either anemia or DOX can be effectively attenuated by inhibition of target of rapamycin (TOR) signaling via rapamycin treatment. However, along the progression of both models, we have also detected dynamic TOR activity and distinct effects of TOR signaling inhibition at different stages of pathogenesis. To assess the long term effects of TOR haploinsufficiency, we utilized a zebrafish target of rapamycin (ztor) mutant that was identified from a mutagenesis screen. We show that sustained TOR inhibition in ztor/+ improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the anti-hypertrophy, anti-apoptosis, and pro-autophagy function of TOR signaling inhibition. Together, our results prove adult zebrafish as a novel vertebrate model for human cardiomyopathies and provide genetic evidences for a cardioprotective function of TOR signaling inhibition in different forms of cardiomyopathies.

The present forward mutagenesis screen strategies in vertebrates are difficult to identify genes and mutants affecting a specific organ of interest. Here, we report a strategy to enrich cardiac mutants by leveraging the transparency of zebrafish embryos and the high efficiency of pGBT-RP2 (RP2), a gene-breaking transposon-based in vivo protein trap cassette. By selectively raising up founder fish with Red Fluorescence Protein (RFP) reporter expression in the heart, we successfully generated 15 cardiac mutant lines. All tagged loci exhibit cardiac expression and the overall knockdown efficiency at transcript level is greater than 95%. One of the cardiac lines, RP2_ #135, is a homozygous embryonic lethal line that is caused by a RP2 insertion in methionine adenosyltransferase 2a (mat2a) . Taking advantage of the LoxP sites inside the RP2 vector, we further demonstrated that the cardiac phenotypes in RP2_ #135 is due to a disrupted expression of mat2a in cardiomyocytes, as indicated by a conditional rescue assay using Tg(cmlc2:Cre-YFP) , a myocardium specific Cre transgenic line. Finally, we show that the reduced cardiomyocyte number, but not size, in the mat2a mutant is conveyed by the p53-mediated apoptosis pathway. Together, our data prove the feasibility of a facile approach to generate a vertebrate cardiac mutant collection with the following two unique features. First, the expression dynamics of each tagged gene is reported by RFP. Second, the causality between the tissue-specific gene expression and the resulted cardiac phenotypes can be attested genetically.

Abstract Clinical practice guidelines recommend that individuals with HC MBL are evaluated annually for evidence of progression as it has been shown that they progress to develop CLL requiring therapy at a rate of 1-5%/year. Next-generation sequencing identified 59 genes recurrently mutated in CLL, and we previously showed that the tumor mutational load (TML), i.e., the total number of these genes mutated, is associated with a shorter time to first treatment (TTT) among 112 HC MBLs independent of the CLL international prognostic index (CLL-IPI), an established prognostic score. Herein, we continue to support our finding in additional HC MBLs and report the pooled results across the two cohorts. From the Mayo Clinic CLL Resource, we sequenced a total 167 HC MBL (112 from the initial round and 55 from additional sequencing). We extracted DNA from CD5+/CD19+ B-cells and sequenced the coding regions of 59 driver genes. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies >0.01% and by those identified in public databases, unless present in known CLL mutation hotspots. After filtering, we computed the TML based on the number of genes with high impact (nonsense, frameshift and splicing) and hotspot mutations. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for sex and CLL-IPI. Stratified analyses were conducted by low/intermediate risk and high/very high CLL-IPI. Of the 167 HC MBL, low risk CLL-IPI comprised 60%, intermediate risk 28%, high risk 12%; and none were very-high risk. The most commonly mutated genes were SF3B1 (11%), NOTCH1 (9%), TP53 (7%), BIRC3 (7%), and MYD88 (6%); and 52 (31%) individuals had mutations in at least one gene. The median follow-up was 4.4 years, and 26 of the 167 individuals progressed to CLL requiring therapy. HC MBL with ≥2 mutated genes had a 28% 5-year risk of needing therapy compared to 6% among those with no mutated genes. Similar to our initial finding (HR=1.53, CI:1.12-2.07; P=0.007), we found a shorter TTT in the additional HC MBLs (HR=4.63, CI:1.57-13.7, P=0.006), and in the pooled cohort (HR=1.72; CI:1.29-2.28, P=0.0002). When we stratified by CLL-IPI risk, TML remained associated with TTT among individuals with low/intermediate CLL-IPI risk in the additional HC MBLs (HR=3.52, CI:1.11-11.2, P=0.03) and in the pooled cohort (HR=2.25, CI:1.34-3.77, P=0.002). A non-significant 2.8 fold association was observed with high risk CLL-IPI in the pooled cohort. In conclusion, the TML along with the CLL-IPI provide clinicians with a more accurate prognostic information regarding disease progression requiring therapy, especially for those MBLs with low to intermediate risk based on CLL-IPI. If validated, these findings may modify current practice guidelines for individuals with HC MBL. Citation Format: Geffen Kleinstern, Cecilia Bonolo de Campos, Nicholas J. Boddicker, Daniel R. O'Brien, Shulan Tian, Chantel Barney, Kari G. Rabe, Cristine Allmer, Laura Bruins, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Huihuang Yan, James R. Cerhan, Neil E. Kay, Celine Vachon, Esteban Braggio, Susan L. Slager. Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 884.

Abstract Chronic lymphocytic leukemia (CLL) is a neoplastic disease of mature B-cells with a highly heterogeneous clinical course. While European ancestry (EA) populations present an increased incidence of CLL, African ancestry (AA) populations have a younger median age of onset, higher frequency of adverse prognostic factors, and inferior clinical outcomes. Despite the considerable effort to characterize the genetic landscape of CLL, AA are overwhelmingly underrepresented. Our hypothesis is that the clinical differences observed between AA and EA populations are, in part, explained by underlying genetic features. To address this imbalance, we identified 90 AA patients diagnosed with CLL, 64% of which were untreated at sample collection. RNA and DNA were extracted from CD5+/CD19+ clonal B-cells. We performed mRNA-seq and targeted sequencing in 59 recurrently mutated somatic CLL driver genes. Differentially expressed genes were identified using edgeR. Data was compared to our previously analyzed EA CLL cohort (N=445). The median age at diagnosis was 59 years for AA and 66 for EA and 74% of AA and 50% of EA had unmutated IGHV (u-IGHV) status. When evaluating the entire AA and EA cohorts, there was a significant increased frequency of mutations in TP53, SF3B1, and NFKBIE, identified in 29%, 24%, and 20% of AA CLLs, compared to 5%, 9%, and 9%, respectively, in EA CLLs (p<0.01). When exclusively evaluating the untreated and u-IGHV cases, AA CLLs showed greater proportion of TP53 (30% vs 12%; p=0.0145), NFKBIE (30% vs 15%; p=0.0492), BIRC3 (21% vs 10%; p=0.0697), and KRAS (15% vs 7%; p=0.1683). Furthermore, there was an increase in mutations targeting relevant molecular pathways, such as NF-κB (42% vs 15%) and MAPK (18% vs 8%). Upregulation MAPK pathway was also confirmed by mRNA-seq analysis in the AA u-IGHV CLLs. Because of the high prevalence of TP53 mutations in the AA cohort, we further evaluated differential gene expression in the DNA Damage/Telomere Stress-Induced Senescence pathway. AA CLLs presented a significant downregulation of multiple genes associated with genome stability and cellular DNA damage response - DDR (including TP53 and ATM), double strand break repair (H2AFX and RAD50), telomere maintenance (POT1 and ACD), and cell cycle regulation (RB1, CCNA1, and CCNE2) (FDR<0.05). DDR is responsible for DNA repair or induction of apoptosis, with its deficiency resulting in the accumulation of chromosomal aberrations, negatively impacting clinical outcome in CLL. Disparities in cancer are influenced by numerous factors that affect disease risk, screening and diagnosis, access to treatment, and survival. We identified an increased number of genomic alterations in the AA CLL cohort, primarily inducing activation of NF-κB and MAPK pathways and DDR impairment, with the increased frequency of mutations, notably in TP53 and BIRC3, expected to negatively impact prognosis. Citation Format: Cecilia Bonolo de Campos, Daniel R. O'Brien, Chantal E. McCabe, Huihuang Yan, Geffen Kleinstern, Zhiquan Wang, Laura A. Bruins, Cristine Allmer, Nicholas J. Boddicker, Charla R. Secreto, Aaron D. Norman, Shulan Tian, Kari G. Rabe, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Richard Furman, J Brice Weinberg, James R. Cerhan, Celine M. Vachon, Neil E. Kay, Susan L. Slager, Esteban Braggio. Characterization of underlying genomic features among African ancestry populations diagnosed with chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2209.

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease showing a rapid clinical course, with a median survival of <1 year. Whole-genome and whole-exome sequencing have identified key genetic lesions in T-PLL, including structural alterations such as inversions, translocations, and large copy number variants, as well as recurrent somatic mutations of genes encoding chromatin regulators and of those in the JAK-STAT signaling pathway. Epigenetic alteration is implicated in the susceptibility and progression of both cancer and non-cancerous diseases. However, genome-wide epigenetic data has not been generated for T-PLL, which prohibits the mechanistic studies of transcriptional misregulation in T-PLL. This study used micrococcal nuclease digestion and sequencing (MNase-seq) to profile the chromatin accessible regions, i.e., gene regulatory regions such as promoters, enhancers and insulators, in both T-PLL patients and healthy individuals. Our analysis revealed the prevalence of lost open chromatin regions in T-PLL. We also generated histone H3 lysine 4 monomethylation (H3K4me1) and lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation and sequencing (ChIP-seq) data from the same samples. We identified 5,462 and 2,833 active enhancers (>=2.5 kb away from transcription start sites) that are lost and gained in T-PLL, respectively, compared to normal. The differential H3K27ac peaks are preferentially associated with the nearest genes differentially expressed between T-PLL and normal (chi-square test p <2.2e-16), indicating a role of epigenetic alteration in transcriptional misregulation. In addition, we identified alterations of active enhancers that target oncogenes with key roles in T-PLL, such as TCL1Aand MYC. Together, our analysis has provided insights into the epigenetic mechanisms that drive oncogenic activation in T-PLL. Citation Format: Shulan Tian, Henan Zhang, Eric Klee, Huihuang Yan, Wei Ding. Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2119.

Abstract Richter's transformation (RT) is a clinically challenging and aggressive transformation of CLL (Chronic lymphocytic leukemia) to lymphoma. Immune-checkpoint therapy has improved outcomes in selective lymphoma types. Nearly 40% of RT patients respond to anti-PD1, while the underlying CLL do not show any response, and 10% of CLL patients progress post-anti-PD1 treatment. Previous studies mostly focused on T-cell responses elicited by anti-PD1, however, intrinsic changes in CLL and RT B-cells induced by the treatment remains elusive. We hypothesized that pre-existing active transcriptional circuitry and distinct B-cell subsets in the CLL and RT B-cells may determine the response to anti-PD1 treatment. Peripheral blood mononuclear cells (PBMCs) from 3 patients who received pembrolizumab(anti-PD1) as a single agent. Samples were obtained at baseline and 3 months post-treatment initiation. Among these patients, 2 are progressors while 1 is a responder. We performed single-cell RNAseq (10X Genomics) on B-cells enriched from PBMCs derived from 3 samples (1 Responder: RS2, 2 progressors: CLL14 and CLL17) before and after treatment using chromium single cell 3' gene expression library. A total of ~10000 cells were captured with an average detection of 1000 genes/sample. To delineate the transcriptional changes upon the anti-PD1 treatment, we first integrated the single-cell data from 6 samples (before and after Pembro) and performed unsupervised clustering analysis using UMAP (Uniform Manifold Approximation and projection). We identified 8 different clusters based on highly expressed marker genes. Cluster 2(C2) was increased in the responder as compared to the progressors post-pem treatment. To determine the activated pathways in each cluster, we performed gene-set enrichment analysis (GSEA) using the Enrichr method. This analysis revealed upregulation of distinct pathways related to IFNG/MYC(C1), IFNG(C2), ER stress(C3), B-cell dependent T cell activation related pathway(C4), MYC targets/TNFα(C5,6,7) indicating intra-tumor transcriptional heterogeneity. To decipher the intratumor cell differentiation changes, we performed pseudotime trajectory analysis using Monocle 2 coupled with B cell developmental stage markers. We identified 7 different states, with distinct changes of state in responder sample RS2 (State 4,5, 6, and 7 were increased in RS2 post-treatment) while CLL samples(progressors) did not have distinct changes. Integration with B cell development revealed that aggressive B cells with CD38 expression were highly induced upon anti-PD1 in responder RS2 while regulatory (CD1d) and memory B cells (CD27) remained the same, suggesting PD1 induced intrinsic changes contributing to the disease response. We identified distinct transcriptional states in B cells within responders and progressors which may govern the intra-tumor and inter-tumor heterogeneity to the anti-PD1 response. Citation Format: Prajish Sundaram Iyer, Lu Yang, Yang Zhi Zhang, Hyo Jin Kim, Charla Secreto, Sutapa Sinha, Neil Kay, Stephen Ansell, Wei Ding, Lili Wang. Tumor intrinsic changes determine immune-checkpoint response in Richter's transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1612.

Abstract Purpose The multiple energy extraction (MEE) delivery technique for synchrotron‐based proton delivery systems reduces beam delivery time by decelerating the beam multiple times during one accelerator spill, but this might cause additional plan quality degradation due to intrafractional motion. We seek to determine whether MEE causes significantly different plan quality degradation compared to single energy extraction (SEE) for lung cancer treatments due to the interplay effect. Methods Ten lung cancer patients treated with IMPT at our institution were nonrandomly sampled based on a representative range of tumor motion amplitudes, tumor volumes, and respiratory periods. Dose‐volume histogram (DVH) indices from single‐fraction SEE and MEE four‐dimensional (4D) dynamic dose distributions were compared using the Wilcoxon signed‐rank test. Distributions of monitor units (MU) to breathing phases were investigated for features associated with plan quality degradation. SEE and MEE DVH indices were compared in fractionated deliveries of the worst‐case patient treatment scenario to evaluate the impact of fractionation. Results There were no clinically significant differences in target mean dose, target dose conformity, or dose to organs‐at‐risk between SEE and MEE in single‐fraction delivery. Three patients had significantly worse dose homogeneity with MEE compared to SEE (single‐fraction mean D 5% ‐D 95% increased by up to 9.6% of prescription dose), and plots of MU distribution to breathing phases showed synchronization patterns with MEE but not SEE. However, after 30 fractions the patient in the worst‐case scenario had clinically acceptable target dose homogeneity and coverage with MEE (mean D 5% –D 95% increased by 1% compared to SEE). Conclusions For some patients with breathing periods close to the mean spill duration, MEE resulted in significantly worse single‐fraction target dose homogeneity compared to SEE due to the interplay effect. However, this was mitigated by fractionation, and target dose homogeneity and coverage were clinically acceptable after 30 fractions with MEE.

Purpose: Endovascular occlusion of aneurysms with the use of detachable platinum coils has proved to be a safe and durable alternative to the traditional surgical clipping. The aim of the present study is to understand the mechanism of apoptosis in aneurysm embolized with platinum coils using rabbit elastase‐induced aneurysm model. Materials & Methods: Elastase‐induced saccular aneurysms were created at the origin of right common carotid artery in thirty rabbits. Aneurysms were allowed to mature for 8 weeks then 20 aneurysms were embolized using platinum coils by endovascular means. Ten uncoiled aneurysms were used as controls. After 2 weeks (n=10) and 4 weeks (n=10) of implantation, aneurysm samples were harvested for apoptotic studies. Results: Expression of pro‐caspases‐3, ‐8 and ‐9 were elevated in coiled aneurysm embolized with platinum coils at both time points when compared with uncoiled aneurysm and control left carotid artery. Cleaved caspases‐3, ‐8 and ‐9 were found to be expressed only at 4 weeks after embolization. Cells within aneurysm cavity were TUNEL positive at 4 weeks only. Expression of tumor necrosis‐α (TNF‐α) was high in coiled aneurysm when compared with controls. There was no significant difference in the expression of Fas‐ligand among groups. Decreased expression of antiapoptotic protein, Bcl‐2, and increased expression of proapoptotic proteins, Bad, Bax and Bid, were observed in coiled aneurysms at both time points. Conclusion: Activation apoptosis in aneurysms after embolization with platinum coils is mediated by both TNF‐α mediated‐extrinsic and Bcl‐2 mediated‐intrinsic pathway.