Zebrafish Model of MLL-Rearranged Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of leukemia. Standard treatment includes chemotherapy as well as stem cell transplantation, but for aging patients and those with impaired immune function these rigorous therapies are not always possible. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) exhibit far worse prognoses than patients without. Given these circumstances new therapies must be developed. Methods: Danio rerio (zebrafish) has emerged as a powerful model organism for investigating human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. We developed a transient transgenic model exhibiting AML characteristics by microinjecting single-cell zebrafish embryos with a tissue specific MLL-ENL expression construct. Results: We found that the expression of MLL-ENL induced a clustered expansion of MLL+ and pu.1+ myeloid cells on the yolk sac at 48 and 72 hours post fertilization (hpf). To characterize our transient AML model, we treated MLL-ENL expressing embryos with either one of or a combination of two drugs that are currently being used in human AML drug trials, Venetoclax and Flavopiridol. We found that treatment with either drug reduced the myeloid expansion induced by the expression of MLL-ENL, and that co-treatment reduced the observed myeloid expansion even further. Conclusions: Although further analysis is required, these data suggest that we successfully developed a transient transgenic AML model in zebrafish. Furthermore, these data suggest that Venetoclax and Flavopiridol co-treatment could yield better outcomes for AML patients than treatment with either drug individually.https://scholarscompass.vcu.edu/gradposters/1112/thumbnail.jp

A dual fluorescent multiprobe assay for prion protein genotyping in sheep

BACKGROUND: Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep. METHODS: A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above. RESULTS: The assay is succesfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep. CONCLUSIONS: We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations

The Influence of Number and Timing of Pregnancies on Breast Cancer Risk for Women With BRCA1 or BRCA2 Mutations

International audienceBACKGROUND:Full-term pregnancy (FTP) is associated with a reduced breast cancer (BC) risk over time, but women are at increased BC risk in the immediate years following an FTP. No large prospective studies, however, have examined whether the number and timing of pregnancies are associated with BC risk for BRCA1 and BRCA2 mutation carriers.METHODS:Using weighted and time-varying Cox proportional hazards models, we investigated whether reproductive events are associated with BC risk for mutation carriers using a retrospective cohort (5707 BRCA1 and 3525 BRCA2 mutation carriers) and a prospective cohort (2276 BRCA1 and 1610 BRCA2 mutation carriers), separately for each cohort and the combined prospective and retrospective cohort.RESULTS:For BRCA1 mutation carriers, there was no overall association with parity compared with nulliparity (combined hazard ratio [HRc] = 0.99, 95% confidence interval [CI] = 0.83 to 1.18). Relative to being uniparous, an increased number of FTPs was associated with decreased BC risk (HRc = 0.79, 95% CI = 0.69 to 0.91; HRc = 0.70, 95% CI = 0.59 to 0.82; HRc = 0.50, 95% CI = 0.40 to 0.63, for 2, 3, and ≥4 FTPs, respectively, P trend < .0001) and increasing duration of breastfeeding was associated with decreased BC risk (combined cohort P trend = .0003). Relative to being nulliparous, uniparous BRCA1 mutation carriers were at increased BC risk in the prospective analysis (prospective hazard ration [HRp] = 1.69, 95% CI = 1.09 to 2.62). For BRCA2 mutation carriers, being parous was associated with a 30% increase in BC risk (HRc = 1.33, 95% CI = 1.05 to 1.69), and there was no apparent decrease in risk associated with multiparity except for having at least 4 FTPs vs. 1 FTP (HRc = 0.72, 95% CI = 0.54 to 0.98).CONCLUSIONS:These findings suggest differential associations with parity between BRCA1 and BRCA2 mutation carriers with higher risk for uniparous BRCA1 carriers and parous BRCA2 carriers

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants—CS-1 and CS-2 (mainly Italy)—which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters

MYELOID SPECIFIC EXPRESSION OF MLL FUSION PROTEINS ASSOCIATED WITH HUMAN ACUTE MYELOID LEUKEMIA (AML) INDUCES AN AML-LIKE PHENOTYPE IN ZEBRAFISH EMBRYOS

Acute myeloid leukemia (AML) is the most common type of adult acute leukemia and accounting for for 50% of all leukemic deaths and causing more than 11,000 fatal outcomes in the US in 2020. Standard treatment includes chemotherapy and stem cell transplantation, but for aging patients and those with impaired immune function these rigorous therapies are not always possible. Furthermore, AML patients harboring a chromosomal rearrangement involving the mixed lineage leukemia (MLL) gene resulting in the production of an MLL fusion protein (FP) have particularly poor prognoses. Development of treatments for this subclass of AML, termed MLL-r AML, have progressed slowly which highlights the need for new therapies. In recent years, zebrafish have emerged as a powerful model organism for studying human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. In this dissertation transient transgenic zebrafish models were developed with myeloid specific expression of known AML-associated MLL fusion protein genes, MLL fused with eleven-nine leukemia (MLL-ENL) and MLL fused with AF9 (MLL-AF9) were developed. Myeloid specific expression of MLL-ENL or MLL-AF9 (collectively, MLL-FPs) induced an expansion of MLL-FP expressing cells on the yolk from 30 hours post fertilization (hpf) to 7 days post fertilization (dpf), and induced an expansion of myeloperoxidase (mpx), spi-1 proto-oncogene b (spi1b), and macrophage expressed (mpeg) expressing cells on the yolk at 48 hpf. Double colorimetric whole mount in situ hybridization (WISH) revealed that MLL-FP expression colocalized with mpx, spi1b, and mpeg expression at 48 hpf and 72 hpf, which suggests the affected cells are of the myeloid lineage and is indicative of an AML-like phenotype. Furthermore, MLL-FP expression led to an expansion of B-cell lymphoma 2 (bcl2) and cyclin dependent kinase 9 (cdk9) expressing cells on the yolk—both are upregulated in AML patients. Finally, MLL-FP expressing embryos were treated with two drugs that are either approved for AML treatment or are currently being used in human AML trials, venetoclax (BCL2 inhibitor) and flavopiridol (CDK9 inhibitor). While individual treatment with either drug reduced MLL-ENL expression, co-treatment significantly reduced the number of MLL-ENL expressing cells on the yolk compared to individual treatments or controls. Treatment of MLL-AF9 expressing embryos did not yield a significant change in the number of MLL-AF9 expressing cells. Together, these data suggest that a transient transgenic MLL-r zebrafish model exhibiting AML-like characteristics was developed and highlight the effectiveness of using zebrafish for drug screens to identify novel AML therapeutic combinations

Zebrafish Model of MLL-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the second most common type of leukemia and accounts for 80% of adult acute leukemia cases and is characterized by the accumulation of poorly or undifferentiated myeloid blast cells. Standard treatment includes chemotherapy, which if unsuccessful, is followed by more rigorous chemotherapy as well as stem cell transplantation. Considering most patients are over the age of 45, these more rigorous therapies are not always possible, and as such, new therapies must be developed. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) that results in the expression of an MLL fusion protein exhibit far worse prognoses than patients without. In recent years, Danio rerio (zebrafish) has emerged as a powerful model organism for investigating human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. The first objective of this study was to develop a transient transgenic AML model in zebrafish, and the second objective was to determine if co-treatment with two medications currently in human trials for AML, Venetoclax and Flavopiridol, would be more effective than using either drug individually. In order to develop a transient transgenic AML model, we first developed a DNA construct encoding a known mixed lineage leukemia (MLL) fusion protein associated with human AML, MLL-ENL, driven by the zebrafish lysozyme C (lyz) promoter, which drives myeloid specific expression in zebrafish. We then microinjected single-cell zebrafish embryos with DNA encoding lyz driven MLL-ENL along with transposase mRNA to facilitate the genomic integration of MLL-ENL. Injected embryos were first tested for MLL-ENL expression, and subsequently tested for AML phenotypic characteristics, via whole mount in-situ hybridization (WISH) at 72 hours post fertilization (hpf). First, WISH analysis utilizing a human MLL riboprobe verified MLL-ENL expression in injected embryos, and WISH analysis utilizing the same MLL riboprobe revealed an expansion and clustering of MLL positive cells in injected embryos, characteristic of an AML phenotype. Embryos injected with MLL-ENL DNA were then treated with either DMSO (vehicle), 200 nanomolar (nM) Venetoclax, 200 nM Flavopiridol, or 200 nM Venetoclax and 200 nM Flavopiridol from 24 hpf to 72 hpf. MLL WISH analysis of injected and treated embryos revealed a reduction in MLL positive cells in both Venetoclax treated embryos and Flavopiridol treated embryos, and an even greater reduction in MLL positive cells in embryos treated with both Venetoclax and Flavopiridol, compared to controls. Although further analysis is required to be confident, these data suggest that we successfully developed an AML transient transgenic model in zebrafish. Furthermore, these data suggest that Venetoclax and Flavopiridol co-treatment could yield better outcomes for AML patients than treatment with either drug individually

Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) mainly in the central nervous system. Polymorphisms in the PrP gene are linked to differences in susceptibility for prion diseases. The mechanisms underlying these effects are still unknown. Here we describe studies of the influence of sheep PrP polymorphisms on the conversion of PrP(C) into protease-resistant forms. In a cell-free system, sheep PrP(Sc) induced the conversion of sheep PrP(C) into protease-resistant PrP (PrP-res) similar or identical to PrP(Sc). Polymorphisms present in either PrP(C) or PrP(Sc) had dramatic effects on the cell-free conversion efficiencies. The PrP variant associated with a high susceptibility to scrapie and short survival times of scrapie-affected sheep was efficiently converted into PrP-res. The wild-type PrP variant associated with a neutral effect on susceptibility and intermediate survival times was converted with intermediate efficiency. The PrP variant associated with scrapie resistance and long survival times was poorly converted. Thus the in vitro conversion characteristics of the sheep PrP variants reflect their linkage with scrapie susceptibility and survival times of scrapie-affected sheep. The modulating effect of the polymorphisms in PrP(C) and PrP(Sc) on the cell-free conversion characteristics suggests that, besides the species barrier, polymorphism barriers play a significant role in the transmissibility of prion diseases