The use of broad-spectrum antimycotic therapy, immunosuppressive therapy, and indwelling medical devices has contributed to the increased frequency of mucosal and systemic infections caused by Candida glabrata. A major concern for C. glabrata and other Candida spp. infections is the increase in drug resistance. To address these issues, additional molecular tools for the study of C. glabrata are needed. In this investigation, we developed an Agrobacterium tumefaciens transformation system for C. glabrata. A number of parameters were investigated to determine their effect on transformation frequency, and then an optimized protocol was developed. The optimal conditions for the transformation of C. glabrata were found to be an infection incubation temperature of 26 °C, 0.2 mM acetosyringone in both induction media and co-culture media, 0.7% agar concentration, and a multiplicity of infection of 50:1 A. tumefaciens to C. glabrata. Importantly, the frequency of multiple integrations was low (5%), demonstrating that A. tumefaciens generally integrates at single sites in C. glabrata, which is consistent with other fungal A. tumefaciens transformation systems. The development of this system in C. glabrata adds another tool for the molecular manipulation of this increasingly important fungal pathogen.
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, representing >25% of all cancers in children 0-14 years. Despite major advancements in pediatric ALL treatment, it remains the second most lethal childhood cancer, accounting for ~25% of deaths. The two types of ALL are precursor-B, or B-ALL, and precursor-T, or T-ALL, which have distinct molecular landscapes. Of these types, T-ALL comprises about 15% and 25% of pediatric and adult cases, respectively, and is historically considered more aggressive and treatment-resistant, with an inferior prognosis. In the precision medicine era, it is imperative to identify genetic alterations and aberrant gene expression patterns, to better understand tumor biology and improve treatment outcomes by identifying new therapeutic targets. Our study investigates a novel transcription factor, odd-skipped related transcription factor 2 (OSR2), which we hypothesize is a putative T-ALL tumor suppressor. We are using a zebrafish T-ALL model expressing transgenic human MYC (hMYC) regulated by a lymphoblast-specific promoter, rag2. Prior work in zebrafish and human T-ALL found low OSR2 levels in ~95% of T-ALL. Based on this, we then used RNA-seq to analyze 10 hMYC zebrafish T-ALL, confirming low-to-absent osr2 in all 10 T-ALL relative to wild-type (WT) T cells. We further confirmed decreased osr2 expression by qRT-PCR of additional T-ALL and WT thymocytes. We hypothesized that if OSR2 suppresses T-ALL, impaired zebrafish Osr2 function might increase T-ALL incidence and shorten latency. To test this, we bred osr2-mutant fish to rag2:hMYC transgenic animals to create three genotypes: heterozygous osr2-mutant (osr2het) fish, heterozygous hMYC (hMYChet) fish, and compound-heterozygote (osr2het;hMYChet) fish. We screened these genotypes for T-ALL incidence by serial fluorescence microscopy, with T-ALL subsequently confirmed by fluorescence-based flow cytometry. By 7 months of age, we found 9/18 (50%) of double-heterozygous fish developed T-ALL, compared to 0/7 hMYChet fish (p = 0.026); osr2het fish also did not develop T-ALL. Together, our findings suggest osr2 allelic loss accelerates MYC-driven T-ALL, supporting our hypothesis that osr2 is a T-ALL tumor suppressor. Disclosures No relevant conflicts of interest to declare.
MYCis a key oncogene overexpressed by many cancers, however, its oncogenic mechanisms are poorly understood. MYC is also central to acute lymphoblastic leukemia (ALL), the most common and second most lethal pediatric malignancy. Much of MYC's oncogenicity has been attributed to its transcription factor function, but data suggest MYC also deregulates replication in transcription-independent fashion. As a known master regulator of cancer transcriptomes and epigenomes, we hypothesize that MYC dramatically alters both gene expression and replication timing (non-random spatiotemporal process where some part of the genome replicates early, and other late) in both types of ALL - B-ALL and T-ALL. Conceivably, MYC exerts oncogenic effects upon the ALL transcription and replication programs, with some changes shared by B- and T-ALL, and others unique to only one. We aim to address two novel questions not been investigated before. First, in ALL, do the same genetic loci show aberrant RNA transcriptionandDNA replication? Second, how similar are the affected loci in two closely-related, yet distinct, ALL types driven by the same oncogene? The basis of our project is a unique double-transgenicrag2:hMYC,lck:GFPzebrafish pre-clinical model we established, which is the only animal model proven to develop both B-ALL and T-ALL. We previously showed that gene expression profiles (GEP) differentiating zebrafish B- and T-ALL also distinguish human B- and T-ALL, making this an ideal model system to study human ALL. In this model, B-ALL and T-ALL are induced by human MYC(hMYC) regulated by aD.rerio(zebrafish)rag2promoter.Since B and T lymphoblasts both expressrag2, both lineages over-express MYC, causing highly-penetrant B- and T-ALL. Differential activity of aD. rerio lckpromoter causes B cells to fluoresce dimly and T cells to fluoresce brightly, allowing us to identify and purify B-ALL and T-ALL by fluorescent microscopy and fluorescence-based flow cytometry, respectively. This unique model enables comparing B- and T-ALL in one genetic background. We have purified >20 zebrafish ALL (both T-ALL and B-ALL) and isolated their RNA and DNA. We are now analyzing RNA-seq gene expression profiles (GEP) and replication timing (RT) profiles via next generation sequencing (NGS). We will compare both ALL types to identify mRNA signatures that are unique to, or shared by, both types. We seek loci that shift DNA replication from early-to-late, or late-to-early, to define the regions that replicate at the same time in both ALL types, versus loci that vary by ALL type. We will also interrogate these data to determine whether GEP and RT profiles correlate with each other, and with known MYC target genes. In conclusion, GEP and RT have never been analyzed in the same cancer sample, or in related cancers driven by the same oncogene. Exploiting our expertise with thehMYCzebrafish model, we are delineating how MYC alters transcription and replication, to ascertain if these affect the same loci and define which loci are unique to one ALL type or shared by both. MYC hyper-activity is seen ~70% of human cancers - making MYC a crucial oncogene in human cancer biology, so our findings are likely to inform not only mechanisms operative in ALL, but also other MYC-driven cancers. Disclosures No relevant conflicts of interest to declare.
MYC is over-expressed by many cancers, yet its oncogenic mechanisms are incompletely understood. MYC is central to acute lymphoblastic leukemia (ALL) - the most common and second most lethal pediatric malignancy, and ALL afflicts even more adults. Much of MYC's oncogenic function is attributed to its role as a transcription factor, but MYC has been shown to deregulate DNA replication independent of transcription. As master regulator of transcriptomes and epigenomes, we predict that MYC impacts both biologic features in both ALL types, B- and T-ALL. We hypothesize that MYC alters both RNA expression and DNA replication (the ordered spatio-temporal process where genomic domains replicate in either early or late S-phase) in B- and T-ALL, and that these perturbations-some shared, others unique to one ALL type-drive leukemogenesis. Our project utilizes a unique double-transgenic rag2:hMYC, lck:GFP zebrafish ALL model that we established, which is the only animal model that develops both highly penetrant B- and T-ALL. In this model, B- and T-ALL are induced by human MYC (hMYC) that is regulated by a zebrafish (Danio rerio) rag2 promoter. Because B and T lymphoblasts each express rag2, both lineages over-express MYC, inducing B- and T-ALL. The differential activity of the D. rerio lck promoter (regulating GFP) causes B cells to fluoresce dimly and T cells to fluoresce brightly, permitting identification of B- vs. T-ALL by fluorescent microscopy and FACS-purification. Thus, we can compare B- and T-ALL in an isogenic background. We have collected 30 ALL samples (18 T-ALL, 12 B-ALL) and completed two types of analyses on 12 T-ALL and 3 B-ALL. Using RNA-seq, we established gene expression profiles (GEP) for both ALL types; principal component analysis and other clustering algorithms demonstrate B- and T-ALL are distinct. Although we analyze the entire transcriptome, we prioritize genes conserved in humans to focus on translatable targets. To assess DNA replication, we generated Replication Timing (RT) profiles by first FAC-sorting ALL cells based on cell cycle phase (G1, S, G2; defined by DNA content) and then performing whole-genome sequencing to generate RT profiles for the same ALL analyzed by RNA-seq. We identified differentially replicating regions by comparing RT of B-vs. T-ALL, revealing many loci where replication reproducibly shifts from early-to-late, or late-to-early, based on ALL type. Overall, despite their shared genetic driver (MYC) , we found RT differences that distinguish B- vs. T-ALL in ~30% of the genome. Most differences occur in large chromosomal domains, suggesting abnormal chromatin structure in ALL. An additional unexpected result was that many ALL G1 samples had read count differences across large chromosomal regions, indicating the presence of aneuploidies/large CNAs. Several were recurrent and lineage-specific (i.e., exclusive to B- or T-ALL). Together, our data demonstrate differences in RNA transcription, DNA replication, and regions of genomic instability that are lineage-specific, despite a shared MYC oncogene that drives both B- and T-ALL. We will next determine which deranged loci are also perturbed in human ALL, with an overarching goal of finding prognostic biomarkers and therapeutic targets. MYC hyper-activity occurs in ~70% of human malignancies. Thus, MYC is crucial to virtually all cancer biology, making our findings likely to inform not only the mechanisms that drive ALL, but also other cancers where MYC is oncogenic. Disclosures No relevant conflicts of interest to declare.
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