BH3 mimetics in combination with nilotinib or ponatinib represent a promising therapeutic strategy in blast phase chronic myeloid leukemia.

Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

Targeting BCR-ABL-Independent TKI Resistance in Chronic Myeloid Leukemia by mTOR and Autophagy Inhibition.

Background: Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods: Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration-approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)-derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4-6 mice per group). All statistical tests were two-sided. Results: We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion: Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

The influence of lead content in drinking water, household dust, soil, and paint on blood lead levels of children in Flin Flon, Manitoba and Creighton, Saskatchewan.

Lead exposure continues to be an important health issue despite the general removal of lead sources in commercial and industrial applications. Low levels of lead exposure have been found to produce adverse neurodevelopmental effects in children with no evidence that a threshold exists for this critical endpoint. Blood lead levels (BLLs) were measured in children (n=118) under the age of 7years in the northern Canadian smelter community of Flin Flon, Manitoba and Creighton, Saskatchewan. An environmental sampling component was included to examine the relationship between lead content in outdoor soil, household dust, tap water, and paint within a given household and the corresponding BLLs in participating children. The geometric mean (GM) BLL for study participants was 1.41µg/dL. Blood lead levels varied slightly by age category with the lowest levels found among the children under age 2 (GM=1.11µg/dL) and the highest levels found among children between 2 and 3years of age (GM=1.98µg/dL). Results from the multivariate modeling indicated that BLLs had a significant positive association with the age of housing (p<0.05), with children living in households constructed prior to 1945 being more likely to have higher levels (p=0.034). Outdoor soil (GM=74.7µg/g), household dust from kitchen floors (GM=1.34µg/ft2), and maximum household lead paint were found to be significantly correlated (p<0.05) to BLLs. Although a statistically significant association between concentrations of lead in these household media and the corresponding BLLs exists, the variability in BLLs was poorly explained by these factors alone (r2=0.07, 0.12 and 0.06 for soil, household dust, and paint, respectively). Lead concentrations in flushed (GM=0.89µg/L) and stagnant (GM=2.07µg/L and 1.18µg/L) tap water samples were not significantly correlated (p>0.05) to BLLs.

CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression.

Targeting the fusion oncoprotein BCR-ABL with tyrosine kinase inhibitors has significantly affected chronic myeloid leukemia (CML) treatment, transforming the life expectancy of patients; however the risk for relapse remains, due to persistence of leukemic stem cells (LSCs). Therefore it is imperative to explore the mechanisms that result in LSC survival and develop new therapeutic approaches. We now show that major histocompatibility complex (MHC)-II and its master regulator class II transactivator (CIITA) are downregulated in CML compared with non-CML stem/progenitor cells in a BCR-ABL kinase-independent manner. Interferon γ (IFN-γ) stimulation resulted in an upregulation of CIITA and MHC-II in CML stem/progenitor cells; however, the extent of IFN-γ-induced MHC-II upregulation was significantly lower than when compared with non-CML CD34+ cells. Interestingly, the expression levels of CIITA and MHC-II significantly increased when CML stem/progenitor cells were treated with the JAK1/2 inhibitor ruxolitinib (RUX). Moreover, mixed lymphocyte reactions revealed that exposure of CD34+ CML cells to IFN-γ or RUX significantly enhanced proliferation of the responder CD4+CD69+ T cells. Taken together, these data suggest that cytokine-driven JAK-mediated signals, provided by CML cells and/or the microenvironment, antagonize MHC-II expression, highlighting the potential for developing novel immunomodulatory-based therapies to enable host-mediated immunity to assist in the detection and eradication of CML stem/progenitor cells.

The bioaccessibility of soil-based mercury as determined by physiological based extraction tests and human biomonitoring in children.

Environmental contaminants associated with soil particles are generally less bioavailable than contaminants associated with other exposure media where chemicals are often found in more soluble forms. In vitro methods, such as Physiological Based Extraction Tests (PBET), can provide estimates of bioaccessibility for soil-based contaminants. The results of these tests can be used to predict exposure to contaminants from soil ingestion pathways within human health risk assessment (HHRA). In the current investigation, an HHRA was conducted to examine the risks associated with elevated concentrations of mercury in soils in the northern Canadian smelter community of Flin Flon, Manitoba. A PBET was completed for residential soils and indicated mean bioaccessibilities of 1.2% and 3.0% for total mercury using gastric phase and gastric+intestinal phase methodologies, respectively. However, as many regulators only allow for the consideration of in vitro results for lead and arsenic in the HHRA process, in vitro bioaccessibility results for mercury were not utilized in the current HHRA. Based on the need to assume 100% bioaccessibility for inorganic mercury in soil, results from the HHRA indicated the need for further assessment of exposure and risk. A biomonitoring study was undertaken for children between 2 and 15 years of age in the community to examine urinary inorganic mercury concentrations. Overall, 375 children provided valid urine samples for analysis. Approximately 50% of urine samples had concentrations of urinary inorganic mercury below the limit of detection (0.1 µg/L), with an average creatinine adjusted concentration of 0.11 µg/g. Despite high variability in mercury soil concentrations within sub-communities, soil concentrations did not appear to influence urinary mercury concentrations. The results of the current investigation indicate that mercury bioaccessibility in residential soils in the Flin Flon area was likely limited and that HHRA estimates would have been better approximated through inclusion of the in vitro study results.

Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.

We characterized leukemia stem cells (LSC) in chronic phase chronic myelogenous leukemia (CML) using a transgenic mouse model. LSC were restricted to cells with long-term hematopoietic stem cell (LTHSC) phenotype. CML LTHSC demonstrated reduced homing and retention in the bone marrow (BM), related to decreased CXCL12 expression in CML BM, resulting from increased G-CSF production by leukemia cells. Altered cytokine expression in CML BM was associated with selective impairment of normal LTHSC growth and a growth advantage to CML LTHSC. Imatinib (IM) treatment partially corrected abnormalities in cytokine levels and LTHSC growth. These results were validated using human CML samples and provide improved understanding of microenvironmental regulation of normal and leukemic LTHSC and their response to IM in CML.

Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity.

Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3' boundary of a heterochromatin region located upstream of the chicken ß-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5' boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and ß-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and ß-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.

VEZF1 elements mediate protection from DNA methylation.

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.

Determination of the chromatin domain structure in arrayed repeat regions: organization of the somatic 5S RNA domain during embryogenesis in Xenopus laevis.

The size of the DNA loop containing the Xenopus laevis somatic 5S RNA gene cluster has been estimated using a simple, precise and sensitive method that we have developed for use on any tandemly arrayed DNA repeat region, and was found to increase during development We have found that after the mid-blastula transition, when transcription is activated in the embryo, a subset of somatic 5S RNA genes becomes specifically associated with the nuclear matrix. This association correlates with the transcriptional activity of the 5S genes.

Chromatin domains and territories: flexibly rigid.

The nucleus is a highly organized solid-state system, rigid and flexible at the same time, where enzymes are organized in complex processing factories. This is achieved by the organization of nuclear DNA into territories and domains, which allow compartmentalization and compaction without sacrificing accessibility. The present review discusses the implications of the organization of chromosomal domains and territories in development and carcinogenesis.

Local or general anesthesia for open hernia repair: a randomized trial.

OBJECTIVE: To compare patient outcome following repair of a primary groin hernia under local (LA) or general anesthesia (GA) in a randomized clinical trial. SUMMARY BACKGROUND DATA: LA hernia repair is thought to be safer for patients, causes less postoperative pain, cost less, and is associated with a more rapid recovery when compared with the same operation performed under GA. METHODS: All patients presenting to three surgeons during the study period with a primary groin hernia were considered eligible. Outcome parameters measured including tests of vigilance, divided attention, sustained attention, memory, cognitive function, pain, return to normal activity, and costs. RESULTS: Two hundred seventy-nine patients were randomized to LA or GA hernia repair; 276 of these had an operation, with 138 participants in each group. At 6, 24, and 72 hours postoperatively there were no differences in vigilance or divided attention between the groups. Similarly, memory, sustained attention, and cognitive function were not impaired in either group. Although physical activity was significantly impaired at 24 hours, this and return to usual social activities were similar in both groups. While patients in the LA group had significantly less pain on moving, at 6 hours they were less likely to recommend the same operation to someone else. GA hernia repair cost 4% more than the same operation under LA. CONCLUSIONS: There are no major differences in patient recovery after LA or GA hernia repair. Patients should be offered a choice of anesthesia, LA or GA, for repair of their groin hernia.

Five-year follow-up of patients undergoing laparoscopic or open groin hernia repair: a randomized controlled trial.

OBJECTIVE: To compare laparoscopic with open hernia repair in a randomized clinical trial at a median follow-up of 5 years. SUMMARY BACKGROUND DATA: Follow-up of patients in clinical trials evaluating laparoscopic hernia repair has been short. METHODS: Of 379 consecutive patients admitted for surgery under the care of one surgeon, 300 were randomized to totally extraperitoneal hernia repair or open repair, with the open operation individualized to the patient's age and hernia type. All patients, both randomized and nonrandomized, were followed up by clinical examination annually by an independent observer. RESULTS: Recurrence rates were similar for both randomized groups. In 1 of the 79 nonrandomized patients, a recurrent hernia developed. Groin or testicular pain was the most common symptom on follow-up of randomized patients. The most common reason for reoperation was development of a contralateral hernia, which was noted in 9% of patients; 11% of all patients died on follow-up, mainly as a result of cardiovascular disease or cancer. CONCLUSIONS: These data show a similar outcome for laparoscopic and open hernia repair, and both procedures have a place in managing this common problem.