Protective effect of ursodeoxycholic acid and resveratrol against tacrolimus induced hepatotoxicity.

Tacrolimus (TAC) is a potent and well-tolerated immunosuppressive drug, but serious side effects including nephrotoxicity and hepatotoxicity have been reported. Ursodeoxycholic acid (UDCA) and resveratrol (RSV) exhibit hepatoprotective effects in liver diseases. We investigated the hepatoprotective effect of UDCA and RSV against TAC induced hepatotoxicity. We divided 40 male rats into five equal groups: A) control group, B) TAC group, C) TAC + UDCA group, D) TAC + RSV group, E) TAC + UDCA + RSV group. We administered 0.5 mg/kg TAC once daily, 25 mg/kg UDCA twice daily and 10 mg/kg RSV once daily. The drugs in the experimental groups were given by gavage from the first day of the study and continued for 21 days. Histopathologic and biochemical analyses were performed on day 22. In group B, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), total oxidative status (TOS) and malondialdehyde (MDA) levels were higher compared to group A, and catalase (CAT), superoxide dismutase (SOD) levels and total antioxidant status (TAS) were lower compared to group A. Severe cellular swelling, degeneration and focal necrosis were more evident in group B than in groups C-E. Histopathological improvement was observed in groups C-E, where UDCA and RSV were combined, compared to group B. We found that UDCA and RSV, together or separately, protected the liver against oxidative stress damage caused by TAC.

Follicular Helper and Regulatory T Cells Drive the Development of Spontaneous Epstein-Barr Virus Lymphoproliferative Disorder.

Epstein-Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a small percentage of immunocompromised patients (~20%) develop EBV-LPD. Engraftment of immunodeficient mice with peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive donors leads to spontaneous, malignant, human B-cell EBV-LPD. Only about 20% of EBV+ donors induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI), while another 20% of donors never generate EBV-LPD (No-Incidence, NI). Here, we report HI donors to have significantly higher basal T follicular helper (Tfh) and regulatory T-cells (Treg), and depletion of these subsets prevents/delays EBV-LPD. Transcriptomic analysis of CD4+ T cells from ex vivo HI donor PBMC revealed amplified cytokine and inflammatory gene signatures. HI vs. NI donors showed a marked reduction in IFNγ production to EBV latent and lytic antigen stimulation. In addition, we observed abundant myeloid-derived suppressor cells in HI donor PBMC that decreased CTL proliferation in co-cultures with autologous EBV+ lymphoblasts. Our findings identify potential biomarkers that may identify individuals at risk for EBV-LPD and suggest possible strategies for prevention.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a potential prognostic factor in patients with gastric adenocarcinoma.

BACKGROUND: Gastric cancer is one of the leading causes of cancer-related death, and many researchers are focused on the discovery and use of different biomarkers in prognosis prediction. The use of Nrf2 as a prognostic marker in patients with gastric cancer remains controversial. In this study, we evaluated the expression of Nrf2 protein in gastric adenocarcinoma. PATIENTS AND METHODS: A total of 86 patients who underwent gastric resection and D2 lymph node dissection due to gastric adenocarcinoma were included. Clinicopathological characteristics, such as age, gender, gastrectomy type, pathologic T (pT) and N (pN) stages, tumor grade, tumor type per Lauren's classification, presence of lymphovascular invasion, and Nrf2 expression were evaluated. RESULTS: While pT, pN, and Nrf-2 expression were found to be negative prognostic predictors for overall survival in one-way analysis of variance, Nrf-2 expression was the only significant negative prognostic predictor in multivariance analysis. pT, pN, diffuse type, high tumor grade, and Nrf-2 expression significantly affected overall survival in Kaplan-Meier survival analyses (p = 0.02, p = 0.03, p < 0.01, p = 0.027, and p = 0.001, respectively). CONCLUSIONS: Our findings support that Nrf2 is a prognostic marker in patients with gastric adenocarcinoma. Anti-Nrf2 therapy options should be investigated to improve prognosis in gastric cancer patients.

HDAC1 regulates the chromatin landscape to control transcriptional dependencies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a quiescent B-cell malignancy that depends on transcriptional dysregulation for survival. The histone deacetylases are transcriptional regulators whose role within the regulatory chromatin and consequence on the CLL transcriptome is poorly characterized. Here, we profiled and integrated the genome-wide occupancy of HDAC1, BRD4, H3K27Ac, and H3K9Ac signals with chromatin accessibility, Pol2 occupancy, and target expression signatures in CLL cells. We identified that when HDAC1 was recruited within super-enhancers (SEs) marked by acetylated H3K27 and BRD4, it functioned as a transcriptional activator that drove the de novo expression of select genes to facilitate survival and progression in CLL. Targeting HDACs reduced BRD4 and Pol2 engagement to downregulate the transcript and proteins levels of specific oncogenic driver genes in CLL such as BLK, a key mediator of the B-cell receptor pathway, core transcription factors such as PAX5 and IKZF3, and the antiapoptotic gene, BCL2. Concurrently, HDAC1, when recruited in the absence of SEs, repressed target gene expression. HDAC inhibition reversed silencing of a defined set of protein-coding and noncoding RNA genes. We focused on a specific set of microRNA genes and showed that their upregulation was inversely correlated with the expression of CLL-specific survival, transcription factor, and signaling genes. Our findings identify that the transcriptional activator and repressor functions of HDACs cooperate within the same tumor to establish the transcriptional dependencies essential for survival in CLL.

Molecular, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia.

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations affect outcomes of patients treated with intensive chemotherapy. We studied 1725 patients newly diagnosed with AML (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (ie, FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes, providing a rationale to study the biology and treatment approaches in this molecular group. This trial was registered at www.clinicaltrials.gov as #NCT00048958 (CALGB 8461), #NCT00899223 (CALGB 9665), and #NCT00900224 (CALGB 20202).

Clinical and molecular relevance of genetic variants in the non-coding transcriptome of patients with cytogenetically normal acute myeloid leukemia.

Expression levels of long non-coding RNA (lncRNA) have been shown to associate with clinical outcome of patients with cytogenetically normal acute myeloid leukemia (CN-AML). However, the frequency and clinical significance of genetic variants in the nucleotide sequences of lncRNA in AML patients is unknown. Herein, we analyzed total RNA sequencing data of 377 younger adults (aged <60 years) with CN-AML, who were comprehensively characterized with regard to clinical outcome. We used available genomic databases and stringent filters to annotate genetic variants unequivocally located in the non-coding transcriptome of AML patients. We detected 981 variants, which are recurrently present in lncRNA that are expressed in leukemic blasts. Among these variants, we identified a cytosine-to-thymidine variant in the lncRNA RP5-1074L1.4 and a cytosine-to-thymidine variant in the lncRNA SNHG15, which independently associated with longer survival of CN-AML patients. The presence of the SNHG15 cytosine-to-thymidine variant was also found to associate with better outcome in an independent dataset of CN-AML patients, despite differences in treatment protocols and RNA sequencing techniques. In order to gain biological insights, we cloned and overexpressed both wild-type and variant versions of the SNHG15 lncRNA. In keeping with its negative prognostic impact, overexpression of the wild-type SNHG15 associated with higher proliferation rate of leukemic blasts when compared with the cytosine-to-thymidine variant. We conclude that recurrent genetic variants of lncRNA that are expressed in the leukemic blasts of CN-AML patients have prognostic and potential biological significance.

Rare t(X;14)(q28;q32) translocation reveals link between MTCP1 and chronic lymphocytic leukemia.

Rare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis.

The Relationship Between HER-2 Expression Levels and 18F-FDG PET/CT Parameters in Gastric Cancer

Objectives: Human epidermal growth factor receptor-2 (HER-2) is a protooncogene encoded by ERBB2 on chromosome 17. 18Fluoridefluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) examination is frequently used to detect distant metastasis in gastric cancer imaging. This study aimed to investigate the relationship between the data obtained in the 18F-FDG PET/CT examination and HER-2 expression status in patients with gastric cancer. Methods: A total of 115 patients diagnosed with gastric cancer between 2016 and 2020, with HER-2 immunohistochemical followed by 18F-FDG PET/CT examination for staging purposes were included. Results: HER-2 immunohistochemical examination revealed 71 patients (61.7%) with negative and 44 (38.3%) with positive results. The median maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) values of patients positive with HER-2 were 9.95, 5, 30.44, and 139.16, respectively, whereas patients negative with HER-2 were 9.3,5.4,36.62, and 190.424, respectively (p>0.05). The median cancer antigen 19-9 (CA 19-9) levels of patients positive with HER-2 was 33.52, whereas 11.79 in those who were negative (p=0.016). The mean age was 69.3±9.35 years in patients with distant metastases, whereas 65.2±10.9 in those without distant metastases (p=0.042). Median SUVmax and SUVmean values in patients with distant metastases were 11.1 and 6.3, respectively, and 8.2 and 4.5 in those without distant metastases (p=0.002 and p=0.001, respectively). The median CA 19-9 and carcinoembryonic antigen (CEA) levels in patients with distant metastases were 31.34 and 9.20, respectively, whereas those without distant metastases were 11.55 and 2.26, respectively (p=0.011 and p=0.001, respectively). Conclusion: In our study, no statistically significant difference was found in terms of HER-2 status, SUVmax, SUVmean, MTV, TLG, distant metastasis, presence of lymph node metastasis, age, gender, tumor diameter, grade, and localization, and CEA levels in patients with gastric cancer. A statistically significant difference was found between HER-2 status and CA 19-9 levels. A statistically significant relationship was found between distant metastases in the 18F-FDG PET/CT examination and SUVmax, SUVmean, age, CEA levels, and histopathologic diagnosis; however, the relationship between distant metastasis in the 18F-FDG PET/CT scan and MTV, TLG, tumor diameter, localization, and grade was not statistically significant.

Negative-to-Positive Lymph Node Ratio as an Independent Prognostic Factor for Gastric Adenocarcinoma.

OBJECTIVE: To investigate the association between the ratio of negative/positive lymph nodes (RNP) and other clinic pathological parameters. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY:  Faculty of Medicine, Cumhuriyet University, Sivas, Turkey, from February 2008 to December 2019. METHODOLOGY: Consecutive 119 patients with gastric adenocarcinoma, who underwent gastrectomy and D2 lymph node dissection, were included. RNP, other clinicopathological parameters such as tumour grade, type and lymphovascular invasion (LVI) were analysed, as their prognostic impact was investigated. RESULTS: RNP was an independent prognostic factor for overall survival (p = 0.003) and was significantly associated with poor survival (p <0.001). Advanced pathologic T and N stage, presence of perineural invasion (PNI), presence of LVI, high tumour grade, and diffuse-type as per Louren's classification, and the number of the negative lymph nodes were also significantly associated with poor survival (all p <0.05). Although pathologic N stage (p <0.01), PNI (p <0.01), LVI (p <0.01), tumour type as per Louren's classification (p <0.01), tumour grade (p <0.01) and the number of negative lymph nodes (p <0.01) were significantly associated with overall survival in univariate analyses; only gender (p = 0.025), gastrectomy type (p = 0.037), PNI (p = 0.028), tumour type (p = 0.006), and number of  negative lymph nodes (p = 0.003) were meaningfully associated with survival in a multivariate analysis. CONCLUSION: The ratio of negative/positive lymph nodes can be used as an independent prognostic marker in patients with gastric cancer, who undergo curative resection, as an alternative prognostic marker to the pathologic N stage. Key Words: Stomach neoplasms, Lymph node ratio, Prognosis, Gastrectomy, Lymph nodes.

Validation of protein arginine methyltransferase 5 (PRMT5) as a candidate therapeutic target in the spontaneous canine model of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of blood cancers arising in lymphoid tissues that commonly effects both humans and dogs. Protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the symmetric di-methylation of arginine residues, is frequently overexpressed and dysregulated in both human solid and hematologic malignancies. In human lymphoma, PRMT5 is a known driver of malignant transformation and oncogenesis, however, the expression and role of PRMT5 in canine lymphoma has not been explored. To explore canine lymphoma as a useful comparison to human lymphoma while validating PRMT5 as a rational therapeutic target in both, we characterized expression patterns of PRMT5 in canine lymphoma tissue microarrays, primary lymphoid biopsies, and canine lymphoma-derived cell lines. The inhibition of PRMT5 led to growth suppression and induction of apoptosis, while selectively decreasing global marks of symmetric dimethylarginine (SDMA) and histone H4 arginine 3 symmetric dimethylation. We performed ATAC-sequencing and gene expression microarrays with pathway enrichment analysis to characterize genome-wide changes in chromatin accessibility and whole-transcriptome changes in canine lymphoma cells lines upon PRMT5 inhibition. This work validates PRMT5 as a promising therapeutic target for canine lymphoma and supports the continued use of the spontaneously occurring canine lymphoma model for the preclinical development of PRMT5 inhibitors for the treatment of human NHL.

Aging Phenotypes and Restoring Functional Deficits in Older Adults With Hematologic Malignancy.

BACKGROUND: Gauging fitness remains a challenge among older adults with hematologic malignancies, and interventions to restore function are lacking. We pilot a structured exercise intervention and novel biologic correlates of aging using epigenetic clocks and markers of immunosenescence to evaluate changes in function and clinical outcomes. METHODS: Older adults (n=30) with hematologic malignancy actively receiving treatment were screened and enrolled in a 6-month exercise intervention, the Otago Exercise Programme (OEP). The impact of the OEP on geriatric assessment metrics and health-related quality of life were captured. Clinical outcomes of overall survival and hospital utilization (inpatient length of stay and emergency department use) in relationship to geriatric deficits were analyzed. RESULTS: Older adults (median age, 75.5 years [range, 62-83 years]) actively receiving treatment were enrolled in the OEP. Instrumental activities of daily living and physical health scores (PHS) increased significantly with the OEP intervention (median PHS: visit 1, 55 [range, 0-100]; visit 2, 70 [range, 30-100]; P<.01). Patient-reported Karnofsky performance status increased significantly, and the improvement was sustained (median [range]: visit 1, 80 [40-100]; visit 3, 90 [50-100]; P=.05). Quality of life (Patient-Reported Outcome Measurement Information System [PROMIS]) improved significantly by the end of the 6-month period (median [range]: visit 1, 32.4 [19.9-47.7]; visit 3, 36.2 [19.9-47.7]; P=.01]. Enhanced measures of gait speed and balance, using the Short Physical Performance Battery scores, were associated with a 20% decrease in risk of death (hazard ratio, 0.80; 95% CI, 0.65-0.97; P=.03) and a shorter hospital length of stay (decrease of 1.29 days; 95% CI, -2.46 to -0.13; P=.03). Peripheral blood immunosenescent markers were analyzed in relationship to clinical frailty and reports of mPhenoAge epigenetic analysis are preliminarily reported. Chronologic age had no relationship to overall survival, length of stay, or emergency department utilization. CONCLUSIONS: The OEP was effective in improving quality of life, and geriatric tools predicted survival and hospital utilization among older adults with hematologic malignancies.

Gene expression signature predicts relapse in adult patients with cytogenetically normal acute myeloid leukemia.

Although ∼80% of adult patients with cytogenetically normal acute myeloid leukemia (CN-AML) achieve a complete remission (CR), more than half of them relapse. Better identification of patients who are likely to relapse can help to inform clinical decisions. We performed RNA sequencing on pretreatment samples from 268 adults with de novo CN-AML who were younger than 60 years of age and achieved a CR after induction treatment with standard "7+3" chemotherapy. After filtering for genes whose expressions were associated with gene mutations known to impact outcome (ie, CEBPA, NPM1, and FLT3-internal tandem duplication [FLT3-ITD]), we identified a 10-gene signature that was strongly predictive of patient relapse (area under the receiver operating characteristics curve [AUC], 0.81). The signature consisted of 7 coding genes (GAS6, PSD3, PLCB4, DEXI, JMY, NRP1, C10orf55) and 3 long noncoding RNAs. In multivariable analysis, the 10-gene signature was strongly associated with relapse (P < .001), after adjustment for the FLT3-ITD, CEBPA, and NPM1 mutational status. Validation of the expression signature in an independent patient set from The Cancer Genome Atlas showed the signature's strong predictive value, with AUC = 0.78. Implementation of the 10-gene signature into clinical prognostic stratification could be useful for identifying patients who are likely to relapse.

A hierarchical and collaborative BRD4/CEBPD partnership governs vascular smooth muscle cell inflammation.

Bromodomain protein BRD4 reads histone acetylation (H3K27ac), an epigenomic mark of transcription enhancers. CCAAT enhancer binding protein delta (CEBPD) is a transcription factor typically studied in metabolism. While both are potent effectors and potential therapeutic targets, their relationship was previously unknown. Here we investigated their interplay in vascular smooth muscle cell (SMC) inflammation. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed H3K27ac/BRD4 enrichment at Cebpd in injured rat carotid arteries. While genomic deletion of BRD4-associated enhancer in SMCs in vitro decreased Cebpd transcripts, BRD4 gene silencing also diminished Cebpd mRNA and protein, indicative of a BRD4 control over CEBPD expression. Bromodomain-1, but not bromodomain-2, accounted for this BRD4 function. Moreover, endogenous BRD4 protein co-immunoprecipitated with CEBPD, and both proteins co-immunoprecipitated the Cebpd promoter and enhancer DNA fragments. These co-immunoprecipitations (coIPs) were all abolished by the BRD4-bromodomain blocker JQ1, suggesting a BRD4/CEBPD /promoter/enhancer complex. While BRD4 and CEBPD were both upregulated upon tumor necrosis factor alpha (TNF-α) stimulation of SMC inflammation (increased interleukin [IL]-1b, IL-6, and MCP-1), they mediated this stimulation via preferentially elevated expression of platelet-derived growth factor receptor alpha (PDGFRα, versus PDGFRß), as indicated by loss- and gain-of-function experiments. Taken together, our study unravels a hierarchical yet collaborative BRD4/CEBPD relationship, a previously unrecognized mechanism that prompts SMC inflammation and may underlie other pathophysiological processes as well.

ADAM10 expression in gastric adenocarcinoma: Results of a curative gastrectomy cohort.

OBJECTIVE: Gastric cancer is among the most common human cancers with high mortality rates. ADAM10, a member of the ADAM (a disintegrin and metalloproteinase) family has also been found to be associated with gastric carcinoma and has been suggested as a potential therapeutic target. Here, we investigated the association of ADAM10 expression with prognosis in gastric adenocarcinoma patients that underwent gastric resection with D2 lymph node dissection. METHODS: Total 86 consecutive patients that underwent resection for gastric adenocarcinoma were included. Immunohistochemical ADAM10 expression and its association with clinicopathological parameters were analyzed. Univariate and multivariate analyses and survival analyses were performed using SPSS ver.22. RESULTS: High grade tumors, advanced stage tumors and diffuse type tumors showed significantly worse prognosis. A statistically significant association between ADAM10 expression and overall survival (OS) was observed in the univariate analysis, however, this association did not maintain its significance in the multivariate analysis. No statistically significant association was found ADAM-10 expression and clinicopathological parameters. CONCLUSION: Immunohistochemical ADAM10 expression may be used as a prognostic marker in gastric adenocarcinoma, however, introduction of a standardized immunohistochemical scoring system seems to be necessary for evaluation of ADAM10 staining.

Recurrent XPO1 mutations alter pathogenesis of chronic lymphocytic leukemia.

BACKGROUND: Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. METHODS: We performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor). RESULTS: We report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL. CONCLUSIONS: These findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells.

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

Transducin ß-like protein 1 controls multiple oncogenic networks in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin ß-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signalling pathway by binding to ß-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B-cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, ß-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

Effects of DNA topoisomerase IIα splice variants on acquired drug resistance.

DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.

LC-FACSeq is a method for detecting rare clones in leukemia.

Detecting, characterizing, and monitoring rare populations of cells can increase testing sensitivity, give insight into disease mechanism, and inform clinical decision making. One area that can benefit from increased resolution is management of cancers in clinical remission but with measurable residual disease (MRD) by multicolor FACS. Detecting and monitoring genomic clonal resistance to treatment in the setting of MRD is technically difficult and resource intensive due to the limited amounts of disease cells. Here, we describe limited-cell FACS sequencing (LC-FACSeq), a reproducible, highly sensitive method of characterizing clonal evolution in rare cells relevant to different types of acute and chronic leukemias. We demonstrate the utility of LC-FACSeq for broad multigene gene panels and its application for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treatment of patients with chronic lymphocytic leukemia. This technique is generalizable for monitoring of other blood and marrow infiltrating cancers.