Anna Cordeiro

Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3. In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20–30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1.

Inherited bone marrow failure syndromes (IBMFS) are a group of rare inherited genetic disorders defined by impaired hematopoiesis and multi- or unilineage cytopenias. Dyskeratosis Congenita (DC) is characterized by pancytopenia, abnormal skin pigmentation and leukoplakia and is caused by mutations in genes involved in telomere biogenesis and/or RNA processing such as TERT, TERC, DKC1 and PARN. Shwachman-Diamond syndrome (SDS) is characterized by neutropenia and exocrine pancreatic insufficiency and occurs as a result of mutations in genes required for ribosome subunit maturation such as SBDS, DNAJC21 and EFL1. How the disruption of such ubiquitous cellular processes leads to distinct cytopenic phenotypes is not fully understood. Further, IBMFS patients have a high risk of developing myelodysplastic syndrome and/or acute myeloid leukemia with a cumulative incidence of 36% by 30 years of age for SDS patients and 13% for DC patients who do not undergo a stem cell transplant. As IBMFS are rare and large numbers of primary human samples are not readily available for mechanistic studies, we employed zebrafish (Danio rerio) given their highly conserved hematopoietic program and ease of genetic manipulation. Using CRISPR-Cas9 genomic editing, we generated deletion mutations predicted to introduce premature stop codons in the zebrafish orthologs, parn and dnajc21, two more recently identified causes of DC and SDS, respectively. Homozygous zygotic parn mutants had normal morphology and were viable to adulthood. However, fertile homozygous adult females were not recoverable in subsequent generations, implicating parn as an essential factor in oocyte specification. In contrast, homozygous dnajc21 mutants showed normal development to adulthood. Using whole mount in situ hybridization (WISH), we found increased expression of hematopoietic precursor markers (erythroid - gata1 and myeloid - lcp1) and concurrently decreased expression of mature hematopoietic markers (erythroid - hbbe3, myeloid - mpx and thrombocyte - CD41) in parn mutants at 24- and 48-hours post-fertilization (hpf). Further, o-dianisidine staining showed reduced hemoglobinized erythrocytes at 48 hpf. These data indicate that multilineage embryonic hematopoiesis is compromised in parn mutants, recapitulating the pancytopenia observed in patients with DC. WISH for mpx in dnajc21 mutants revealed reduced expression at 24 and 48 hpf, recapitulating the neutropenia seen in SDS. Activation of the TP53 tumor suppressor pathway has been suggested to mediate marrow failure and leukemic progression in some types of IBMFS. Using quantitative PCR to measure the expression of tp53 and its downstream effector p21 at 48 hpf, we observed no significant changes in parn mutants but significantly upregulated tp53 expression in dnajc21 mutants. To further study the role of tp53 in dnajc21-mutant SDS, we crossed dnajc21 mutants with a zebrafish line carrying a tp53 R217H point mutation that confers anti-apoptotic phenotypes. Heterozygous tp53 loss in a dnajc21-/- or +/- background resulted in reduced overall larval growth and abnormal yolk sac development, suggesting defective lipid metabolism. Using the lipophilic dye, Oil Red O, we observed reduced lipid distribution in the vasculature and caudal hematopoietic tissue region (equivalent to mammalian fetal liver) of dnajc21 mutants at 48 hpf. In summary, these findings support a role for PARN in hematopoietic lineage specification through mechanisms that are predominantly TP53-independent. By contrast, DNAJC21 is required for neutrophil specification and normal lipid metabolism and may function in a TP53-dependent manner. These zebrafish models provide new insights into the unique biology underlying these IBMFS and can serve as an in vivo platform for identifying therapeutic compounds that restore normal hematopoiesis and prevent leukemic transformation. Disclosures: Dror: Alexion Canada: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021; RepaetDiagnostic Laboratory: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression by binding to the 3’-UTR of their target genes, can act as oncogenes or tumor suppressors. Recently, other types of non-coding RNAs—piwiRNAs and long non-coding RNAs—have also been identified. Hodgkin lymphoma (HL) is a B cell origin disease characterized by the presence of only 1% of tumor cells, known as Hodgkin and Reed-Stenberg (HRS) cells, which interact with the microenvironment to evade apoptosis. Several studies have reported specific miRNA signatures that can differentiate HL lymph nodes from reactive lymph nodes, identify histologic groups within classical HL, and distinguish HRS cells from germinal center B cells. Moreover, some signatures are associated with survival or response to chemotherapy. Most of the miRNAs in the signatures regulate genes related to apoptosis, cell cycle arrest, or signaling pathways. Here we review findings on miRNAs in HL, as well as on other non-coding RNAs.

PiwiRNA-651 as marker of treatment response and survival in classical Hodgkin lymphoma

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (⩾60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P=0.0025), shorter leukemia-free survival (P=0.026) and higher cumulative incidence of relapse (P=0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P=0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML.

The lincRNA HOTAIRM1 , located in the HOXA genomic region, is expressed in acute myeloid leukemia, impacts prognosis in patients in the intermediate-risk cytogenetic category, and is associated with a distinctive microRNA signature The lincRNA HOTAIRM1, located in the HOXA genomic region, is expressed in acute myeloid leukemia, impacts prognosis in patients in the intermediate-risk cytogenetic category, and is associated with a distinctive microRNA signature

Epigenetic mechanisms are crucial for the inactivation of key genes related to the survival of Hodgkin lymphoma (HL) cells, and methylation is a frequent epigenetic mechanism of microRNA silencing. We have examined the methylation-induced silencing of tumor suppressor microRNAs in HL cell lines and confirmed our results in patient lymph nodes. In addition, we evaluated the in vitro effectiveness of 5-aza-2-deoxycytidine (5-Aza-dC) in HL cell lines. Ten microRNAs containing CpG islands in their promoter region were re-expressed in both the L-428 and L-1236 cell lines. Interestingly, miR-34a and miR-203, both known tumor suppressor microRNAs, were found to be methylated in cell lines and in patient samples. 5-Aza-dC treatment resulted in a dose-dependent antiproliferative effect at 72 h in all the HL cell lines. In summary, 5-Aza-dC treatment of HL cell lines inhibits proliferation at high doses and produces re-expression of the tumor suppressor microRNAs at low-intermediate doses.

The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer