Seropositivity for CMV, EBV, HBV, HCV, and HIV in Leukemia Patients and Its Relationship with Cytogenetic Changes

Introduction: Leukemias are involving the bone marrow and the soft tissues in inner parts of the bones, where new blood cells are formed. This malignancy is the most common pediatric cancer, which its etiologic causes are not well understood. This multifactorial disease is believed to linked with genetic and non-hereditary environmental factors. Cytogenic analyses of chromosomal abnormalities provide diagnostic and prognostic values in leukemia patients. Given the high prevalence of viral diseases and clinical suspicions on the relationship between certain viral infections and leukemia, it is necessary to investigate this possible relationship, especially in third-world countries. The present study recruited 65 children with leukemia (AML, CML, or ALL) who were presented to two tertiary hospitals. At first, all the patients underwent testing for HBV, HCV, CMV, EBV, and HIV. Bone marrow specimens were studied for identifying possible chromosomal abnormalities in cytogenic investigations. According to our findings, there was a relationship between incidence of leukemia, the 12:21 chromosomal translocation and CMV infection. Therefore, preventing CMV infection can lead to a reduced incidence of leukemia. It is expected that the findings of this study enlighten the scientists to conduct more extensive research on the relationship between viral diseases and leukemia in third-world countries.Method:The present study recruited 65 children with leukemia (AML, CML, or ALL) who were presented to two tertiary hospitals. At first, all the patients underwent testing forHBV, HCV, CMV, EBV, and HIV. Bone marrow specimens were studied for identifying possible chromosomal abnormalities in cytogenic investigations.Result:According to our findings,there was a relationship between the incidence of leukemia,the 12:21 chromosomal translocation, and CMV infection.Therefore, preventing CMV infection can lead to a reducedincidence of leukemia.Conclusion:In this study, we demonstrated that leukemia is relevant to the 12:21 chromosomal translocation and CMV virus infections, So the reduction in leukemia prevalence is dependent on the prevention of CMV disease. It is expected that the findings ofthis studyenlighten the scientists to conduct more extensive researchon the relationship between viral diseasesand leukemia in third-world countries.Trial registrations:Clinical trial registration code:IR.AJAUMS.REC.1399.161Evaluated by: AJA UNIVERSITY OF MEDICAL SCIENCESApproval Date:2020-11-15Approval statement: The project was found to be in accordance with the ethical principles and the national norms and standards for conducting Medical Research in Iran.

CXCR4 Mediates Enhanced Cell Migration in CALM-AF10 Leukemia

Leukemia transformed by the CALM-AF10 chromosomal translocation is characterized by a high incidence of extramedullary disease, central nervous system (CNS) relapse, and a poor prognosis. Invasion of the extramedullary compartment and CNS requires leukemia cell migration out of the marrow and adherence to the cells of the local tissue. Cell adhesion and migration are increasingly recognized as contributors to leukemia development and therapeutic response. These processes are mediated by a variety of cytokines, chemokines, and their receptors, forming networks of both secreted and cell surface factors. The cytokines and cytokine receptors that play key roles in CALM-AF10 driven leukemia are unknown. We find high cell surface expression of the cytokine receptor CXCR4 on leukemia cells expressing the CALM-AF10 oncogenic protein, contributing to the migratory nature of this leukemia. Our discovery of altered cytokine receptor expression and function provides valuable insight into the propagation and persistence of CALM-AF10 driven leukemia.

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 +/- 1.28 micromolar. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Horizontal Transfer and Evolution of the Biosynthetic Gene Cluster for Benzoxazinoid

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites, first identified in maize (Zea mays) and subsequently shown to be encoded by a biosynthetic gene cluster (BGC), the Bx cluster. Data mining of mining 40 high-quality grass genomes identified complete Bx clusters (containing genes Bx1 to Bx5 and Bx8) in three genera (Zea, Echinochloa and Dichanthelium) in the Panicoideae and partial clusters in the Triticeae. The Bx cluster originated from gene duplication of native analogues of Bx genes and chromosomal translocation. An ancient Bx cluster including additional Bx genes (e.g., Bx6) is found in ancestral Panicoideae. The ancient Bx cluster was gained by the Triticeae ancestor via a horizontal transfer (HT) event from the ancestral Panicoideae and later separated into three parts on different chromosomes. Bx6 appears to have been under less constrained selection during evolution of the Panicoideae as evidenced by the fact that was translocated ∼1.31-Mb away from the Bx cluster in Z. mays, moved to other chromosomes in Echinochloa, and even lost in Dichanthelium. Further investigation indicated that intense selection and polyploidization shaped the evolutionary trajectory of the Bx cluster in the grass family. This study provides the first case of HT of BGCs among plants and sheds new insights on the evolution of BGCs.SignificanceBiosynthetic gene clustering and horizontal gene transfer are two evolutionary inventions for rapid adaption by organisms. Horizontal transfer of a gene cluster has been reported in fungi and bacteria, but not in plants up to now. By mining the genomes of 40 monocot species, we deciphered the organization of Bx gene cluster, a biosynthetic gene cluster for benzoxazinoids in grasses. We found that the Bx cluster was formed by gene duplication of native analogues of individual Bx genes and directional translocation. More importantly, the Bx cluster in Triticeae was inherited from the Panicoideae via horizontal transfer. Compared with the native analogues, Bx clusters in grasses show constrained purifying selection underscoring their significance in environmental adaption.

A Case of Inherited t(4;10)(q26;q26.2) Chromosomal Translocation Elucidated by Multiple Chromosomal and Molecular Analyses. Case Report and Review of the Literature

We present a complex chromosomal anomaly identified using cytogenetic and molecular methods. The child was diagnosed during the neonatal period with a multiple congenital anomalies syndrome characterized by: flattened occipital region; slight turricephaly; tall and broad forehead; hypertelorism; deep-set eyes; down slanting and short palpebral fissures; epicanthic folds; prominent nose with wide root and bulbous tip; microstomia; micro-retrognathia, large, short philtrum with prominent reliefs; low set, prominent ears; and congenital heart disease. The GTG banding karyotype showed a 46,XY,der(10)(10pter→10q26.2::4q26→4qter) chromosomal formula and his mother presented an apparently balanced reciprocal translocation: 46,XX,t(4;10)(q26;q26.2). The chromosomal anomalies of the child were confirmed by MLPA, and supplementary investigation discovered a quadruplication of the 4q35.2 region. The mother has a triplication of the same chromosomal fragment (4q35.2). Using array-CGH, we described the anomalies completely. Thus, the boy has a 71,057 kb triplication of the 4q26–q35.2 region, a 562 kb microdeletion in the 10q26.3 region, and a 795 kb quadruplication of the 4q35.2 region, while the mother presents a 795 kb triplication of the 4q35.2 region. Analyzing these data, we consider that the boy’s phenotype is influenced only by the 4q partial trisomy. We compare our case with similar cases, and we review the literature data.

EWSR1-WT1 Target Genes and Therapeutic Options Identified in a Novel DSRCT In Vitro Model

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft tissue sarcoma with a lack of effective treatment options and a poor prognosis. DSRCT is characterized by a chromosomal translocation, resulting in the EWSR1-WT1 gene fusion. The molecular mechanisms driving DSRCT are poorly understood, and a paucity of preclinical models hampers DSRCT research. Here, we establish a novel primary patient-derived DSRCT in vitro model, recapitulating the original tumor. We find that EWSR1-WT1 expression affects cell shape and cell survival, and we identify downstream target genes of the EWSR1-WT1 fusion. Additionally, this preclinical in vitro model allows for medium-throughput drug screening. We discover sensitivity to several drugs, including compounds targeting RTKs. MERTK, which has been described as a therapeutic target for several malignancies, correlates with EWSR1-WT1 expression. Inhibition of MERTK with the small-molecule inhibitor UNC2025 results in reduced proliferation of DSRCT cells in vitro, suggesting MERTK as a therapeutic target in DSRCT. This study underscores the usefulness of preclinical in vitro models for studying molecular mechanisms and potential therapeutic options.

TK216 targets microtubules in Ewing sarcoma cells

Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11;22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for EWS patients. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding α-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

Convergent adaptation of Saccharomyces uvarum to sulfite, an antimicrobial preservative widely used in human-driven fermentations

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VIIXVI and XIXVI chromosomal translocation generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.

Adenylate Kinase 2 Is a Selective Multiple Myeloma Cell Dependency That Is Preferentially Essential in NSD2-Overexpressing Cells

Background: Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable. Advances in MM therapy have come about due to therapies that target vulnerabilities of the plasma cell such as high protein load (proteasome inhibitors; PIs), dependence on specific transcription factors such as IKZF1 and IKZF3 which are degraded by immunomodulatory drugs (IMiDs), the susceptibility of B cells to glucocorticoids and the presence of specific B cell markers that can serve as targets for monoclonal antibodies and CAR-T cells. Gene editing screens offer a way to identify novel MM therapeutic targets. Objectives: The molecular heterogeneity of MM imposes challenges to discovering generalized therapeutic targets. Therefore, identification of selective dependencies associated with a particular recurrent genetic lesion is a promising strategy to personalize therapy. Here, we aim to identify vulnerabilities linked to the chromosomal translocation t(4;14), a recurrent rearrangement in MM characterized by overexpression of the histone methyltransferase NSD2. Methods: Genome-wide CRISPR-based loss-of-function screens were performed in NSD2-high and low isogenic cells derived from the t(4;14) MM cell line KMS11 to define selective dependencies associated with NSD2 overexpression. High-confidence hits were corroborated by in vitro competitive growth assays where individual candidates are genetically knocked out or suppressed or chemically inhibited. Detailed investigation was performed for selected candidates using various molecular and biochemical assays to elucidate mechanisms by which these genes contribute to MM cell fitness. Results: A fitness screen in NSD2-high and low isogenic MM cells identified 1118 essential genes which are common between the cell pair. We further revealed 282 genes whose loss is more detrimental to cells overexpressing NSD2 and 139 genes that are preferentially essential when NSD2 levels are low. Pathway analysis of NSD2-high selectively essential genes indicated that these cells are more dependent on mitochondrial processes including oxidative phosphorylation. Although proteasomal degradation is essential for all MM cells, our screens indicated that NSD2-high cells are more dependent on the proteasome, which was validated by increased sensitivity to the PI bortezomib. One of the high-confidence selective NSD2-high hits was the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). Analyzing the dependence of hundreds of human cell lines on AK2 using the cancer dependency map portal (depmap.org/portal/), we found that AK2 is not a common essential gene. The top enriched linages with AK2 dependency included MM with notable representation of t(4;14)-positive cell lines. Analysis of the multiple myeloma research foundation (MMRF)-CoMMPass data demonstrated that MM patients with high NSD2 expression, despite poor prognosis, display enhanced overall survival when AK2 levels are low. In vitro competitive growth assays in NSD2-high and low MM cells confirmed the increased dependence of NSD2-overexpressing cells on AK2. In addition, NSD2-high MM cells displayed elevated sensitivity to AK2 inhibitors. Moreover, AK2 knockdown in t(4;14) MM cell lines increased sensitivity to the PI bortezomib. Mechanistically, we showed that AK2 disruption activates apoptotic unfolded protein response (UPR) signaling in MM cells. Metabolomic profiling in NSD2-high and low MM cells revealed accumulation of purine metabolites and reduction of pyrimidine metabolites upon NSD2 overexpression. Intriguingly, purine supplementation rescued MM cell depletion due to AK2 loss. These observations suggested that MM cells, especially those with NSD2 overexpression, are addicted to elevated purine levels and that lethality of MM cells upon AK2 loss is due to perturbed purine metabolism. How impaired purine metabolism activates UPR signaling is currently under investigation. Conclusions: Our work indicated that NSD2 overexpression resulting from chromosomal translocation t(4;14), despite its oncogenic role, generates metabolic dependencies in MM cells. Our findings further suggest that inhibition of AK2, a mitochondrial enzyme involved in purine metabolism, can induce UPR-mediated apoptosis in MM cells and could be used in combination with PI therapy to treat MM patients with t(4;14) translocations. Disclosures Licht: Epizyme: Research Funding.