The DNA methyltransferase inhibitor 5-aza-4'-thio-2'-deoxycytidine induces C>G transversions and acute lymphoid leukemia development.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza TdCyd or ATC) is a recently described thiol substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. Here, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole exome sequencing revealed thousands of acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed thousands of acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC.

Transcriptional regulation and therapeutic potential of cyclin-dependent kinase 9 (CDK9) in sarcoma.

Sarcomas include various subtypes comprising two significant groups - soft tissue and bone sarcomas. Although the survival rate for some sarcoma subtypes has improved over time, the current methods of treatment remain efficaciously limited, as recurrent, and metastatic diseases remain a major obstacle. There is a need for better options and therapeutic strategies in treating sarcoma. Cyclin dependent kinase 9 (CDK9) is a transcriptional kinase and has emerged as a promising target for treating various cancers. The aberrant expression and activation of CDK9 have been observed in several sarcoma subtypes, including rhabdomyosarcoma, synovial sarcoma, osteosarcoma, Ewing sarcoma, and chordoma. Enhanced CDK9 expression has also been correlated with poorer prognosis in sarcoma patients. As a master regulator of transcription, CDK9 promotes transcription elongation by phosphorylation and releasing RNA polymerase II (RNAPII) from its promoter proximal pause. Release of RNAPII from this pause induces transcription of critical genes in the tumor cell. Overexpression and activation of CDK9 have been observed to lead to the expression of oncogenes, including MYC and MCL-1, that aid sarcoma development and progression. Inhibition of CDK9 in sarcoma has been proven to reduce these oncogenes' expression and decrease proliferation and growth in different sarcoma cells. Currently, there are several CDK9 inhibitors in preclinical and clinical investigations. This review aims to highlight the recent discovery and results on the transcriptional role and therapeutic potential of CDK9 in sarcoma.

Discoidin Domain Receptor Tyrosine Kinase 1 (DDR1) Is a Novel Therapeutic Target in Liposarcoma: A Tissue Microarray Study.

BACKGROUND: Liposarcoma is the most commonly diagnosed subtype of soft tissue sarcoma. As these tumors often arise near vital organs and neurovascular structures, complete resection can be challenging; consequently, recurrence rates are high. Additionally, available chemotherapeutic agents have shown limited benefit and substantial toxicities. There is, therefore, a clear and unmet need for novel therapeutics for liposarcoma. Discoidin domain receptor tyrosine kinase 1 (DDR1) is involved in adhesion, proliferation, differentiation, migration, and metastasis in several cancers. However, the expression and clinical importance of DDR1 in liposarcoma are unknown. QUESTIONS/PURPOSES: The purposes of this study were to assess (1) the expression, (2) the association between DDR1 and survival, and (3) the functional roles of DDR1 in liposarcoma. METHODS: The correlation between DDR1 expression in tumor tissues and clinicopathological features and survival was assessed via immunohistochemical staining of a liposarcoma tissue microarray. It contained 53 samples from 42 patients with liposarcoma and 11 patients with lipoma. The association between DDR1 and survival in liposarcoma was analyzed by Kaplan-Meier plots and log-rank tests. The DDR1 knockout liposarcoma cell lines were generated by CRISPR-Cas9 technology. The DDR1-specific and highly selective DDR1 inhibitor 7RH was applied to determine the impact of DDR1 expression on liposarcoma cell growth and proliferation. In addition, the effect of DDR1 inhibition on liposarcoma growth was further accessed in a three-dimensional cell culture model to mimic DDR1 effects in vivo. RESULTS: The results demonstrate elevated expression of DDR1 in all liposarcoma subtypes relative to benign lipomas. Specifically, high DDR1 expression was seen in 55% (23 of 42) of liposarcomas and no benign lipomas. However, DDR1 expression was not found to be associated with poor survival in patients with liposarcoma. DDR1 knockout or treatment of 7RH showed decreased liposarcoma cell growth and proliferation. CONCLUSION: DDR1 is aberrantly expressed in liposarcoma, and it contributes to several markers of oncogenesis in these tumors. CLINICAL RELEVANCE: This work supports DDR1 as a promising therapeutic target in liposarcoma.

Conical drill bit for optimized external ventricular drain placement: a proof-of-concept study.

OBJECTIVE: Despite external ventricular drain (EVD) procedures being commonplace in neurosurgical practice, suboptimal placement rates remain high, and complications are not uncommon. The angle of the EVD catheter insertion and the accuracy of the drill hole placement are major factors determining successful EVD placement that are dependent on the drill bit morphology. The standard cylindrical 2-fluted twist drill bit creates a relatively deep and narrow drill hole that requires precise positioning, has limited visibility of the drill hole bottom and restricted catheter angular adjustment range, and poses the risk of inadvertent dural puncture. To overcome the standard problems associated with EVD drill bit morphology, the authors propose novel cone-shaped drill bits for EVD placement. METHODS: Conical drill bits of 30° and 45° were designed, manufactured, and tested in a simulated laboratory setting as well as in three human cadavers with intact skull, dura mater, and brain. Drill bit performance was rated by neurosurgical trainees across various domains using Likert scale-type questions. RESULTS: In the laboratory, maximum drilling temperatures adjacent to the drill hole were recorded and compared for the standard drill bit and the 30° and 45° conical drill bits and were not significantly different (p = 0.631 and p = 0.326, respectively). The maximum temperature recorded directly underneath the drilling site for the 45° drill bit was significantly higher than the temperature of the standard drill bit (p = 0.043). The differences between the standard and 30° drill bits were not significant (p = 0.783). Upon cadaver testing, the drilling times with 30° and 45° conical drill bits were significantly longer than those with the standard drill bit (p = 0.036 and p = 0.002, respectively). Likert scale scores were significantly higher for the conical 30° (median [IQR] 4.7 [3.3-5]) and 45° (4 [2-5]) drill bits than for the standard drill bit (1.7 [1-2.5], p < 0.0001), indicating significantly better performance. Conical drill bits used as a "rescue" strategy allowed for an EVD catheter angular adjustment range 6 to 9 times greater than that for the standard drill bit and resulted in a zero inadvertent dural puncture rate. CONCLUSIONS: The 30° conical drill bit can be safely used on its own or as a rescue tool to potentially achieve improved confidence, visualization, targeting, and precision of EVD placement while essentially eliminating the possibility of unintentional dural puncture with minimal increase in the total procedure time.

Advances in the development of chordoma models for drug discovery and precision medicine.

Chordomas are malignant bone tumors that arise from remnants of the notochord. These tumors are generally slow-growing, locally aggressive, and invasive. Chordomas are typically resistant to conventional chemo- and radiotherapy. The clinical management of this disease is very challenging, usually, treatment is surgical resection, which may be combined with radiotherapy. Although chordomas have undergone histologic and genetic analysis, the molecular mechanisms that drive their pathogenesis and resistance are still largely unknown. For many years this could be attributed to the lack of accurate and reliable in vitro and in vivo tumor models. Yet, over the past decade, many efforts have been made to prioritize the generation of useful chordoma cell lines, and tumor models that have shed more light on this malignancy and have made efficacious drug discovery a greater possibility. This review summarizes and discusses recent enhancements and improvements made to generate useful chordoma models and their applications in drug discovery and precision medicine.

Mcm2 hypomorph leads to acute leukemia or hematopoietic stem cell failure, dependent on genetic context.

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2Cre/Cre Lin- Sca-1+ Kit+ hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes.

Mutant Idh2 Cooperates with a NUP98-HOXD13 Fusion to Induce Early Immature Thymocyte Precursor ALL.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2R140Q transgenic mice to examine the role of the Idh2R140Q mutation in leukemia. No leukemia developed in Idh2R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion (NHD13) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2R140Q/NHD13 double transgenic mice. Idh2R140Q/NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2R140Q/NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2R140Q/NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3, and EZH2 were also found in Idh2R140Q/NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2R140Q/NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2R140Q/NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. SIGNIFICANCE: T-cell leukemia induced in Idh2R140Q/NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

A small protein encoded by a putative lncRNA regulates apoptosis and tumorigenicity in human colorectal cancer cells.

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.

Cathepsin K is a potent disaggregase of α-synuclein fibrils.

The intracellular accumulation of α-synuclein (α-syn) amyloid fibrils is a hallmark of Parkinson's disease. Because lysosomes are responsible for degrading aggregated species, enhancing lysosomal function could alleviate the overburden of α-syn. Previously, we showed that cysteine cathepsins (Cts) is the main class of lysosomal proteases that degrade α-syn, and in particular, CtsL was found to be capable of digesting α-syn fibrils. Here, we report that CtsK is a more potent protease for degrading α-syn amyloids. Using peptide mapping by liquid chromatography with mass spectrometry, critical cleavage sites involved in destabilizing fibril structure are identified. CtsK is only able to devour the internal regions after the removal of both N- and C-termini, indicating their protective role of the amyloid core from proteolytic attack. Our results suggest that if overexpressed in lysosomes, CtsK has the potential to ameliorate α-syn pathology.

Genome-Wide Analysis of the FOXA1 Transcriptional Network Identifies Novel Protein-Coding and Long Noncoding RNA Targets in Colorectal Cancer Cells.

Differentiation status of tumors is correlated with metastatic potential and malignancy. FOXA1 (forkhead box A1) is a transcription factor known to regulate differentiation in certain tissues. Here, we investigate FOXA1 function in human colorectal cancer (CRC). We found that FOXA1 is robustly expressed in the normal human colon but significantly downregulated in colon adenocarcinoma. Applying FOXA1 chromatin immunoprecipitation coupled with deep sequencing and transcriptome analysis upon FOXA1 knockdown in well-differentiated CRC cells and FOXA1 overexpression in poorly differentiated CRC cells, we identified novel protein-coding and lncRNA genes regulated by FOXA1. Among the numerous novel FOXA1 targets we identified, we focused on CEACAM5, a tumor marker and facilitator of cell adhesion. We show that FOXA1 binds to a distal enhancer downstream of CEACAM5 and strongly activates its expression. Consistent with these data, we show that FOXA1 inhibits anoikis in CRC cells. Collectively, our results uncover novel protein-coding and noncoding targets of FOXA1 and suggest a vital role of FOXA1 in enhancing CEACAM5 expression and anoikis resistance in CRC cells.

Distinct DNA Methylation Signatures in Neuroendocrine Tumors Specific for Primary Site and Inherited Predisposition.

PURPOSE: To compare the deoxyribonucleic acid (DNA) methylation signature of neuroendocrine tumors (NETs) by primary tumor site and inherited predisposition syndromes von Hippel-Lindau disease (VHL) and multiple endocrine neoplasia type 1 (MEN1). METHODS: Genome-wide DNA methylation (835 424 CpGs) of 96 NET samples. Principal components analysis (PCA) and unsupervised hierarchical clustering analyses were used to determine DNA methylome signatures. RESULTS: Hypomethylated CpGs were significantly more common in VHL-related versus sporadic and MEN1-related NETs (P < .001 for both comparisons). Small-intestinal NETs (SINETs) had the most differentially methylated CpGs, either hyper- or hypomethylated, followed by duodenal NETs (DNETs) and pancreatic NETs (PNETs, P < .001 for all comparisons). There was complete separation of SINETs on PCA, and 3 NETs of unknown origin clustered with the SINET samples. Sporadic, VHL-related, and MEN1-related PNETs formed distinct groups on PCA, and VHL clustered separately, showing pronounced DNA hypomethylation, while sporadic and MEN1-related NETs clustered together. MEN1-related PNETs, DNETs, and gastric NETs each had a distinct DNA methylome signature, with complete separation by PCA and unsupervised clustering. Finally, we identified 12 hypermethylated CpGs in the 1A promoter of the APC (adenomatous polyposis coli) gene, with higher methylation levels in MEN1-related NETs versus VHL-related and sporadic NETs (P < .001 for both comparisons). CONCLUSIONS: DNA CpG methylation profiles are unique in different primary NET types even when occurring in MEN1-related NETs. This tumor DNA methylome signature may be utilized for noninvasive molecular characterization of NETs, through DNA methylation profiling of biopsy samples or even circulating tumor DNA in the near future.

Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation.

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4 Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1∆, slx5∆, and hir2∆ strains, GAL-cse4K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation.

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast.

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.

Thymic precursor cells generate acute myeloid leukemia in NUP98-PHF23/NUP98-HOXD13 double transgenic mice.

Transgenic mice that express either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment develop a wide spectrum of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at age 9-14 months. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Remarkably, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within three months, characterized by replacement of the thymus with leukemic myeloblasts. The marked infiltration of thymus led to the intriguing hypothesis that AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow (BM). Transplantation of CD4-CD8- double negative (DN) thymocytes (which were also negative for Mac1 and Gr1) from leukemic NHD13/NP23 mice demonstrated that DN thymocytes could transmit AML, and limiting dilution studies showed that leukemia initiating cells were increased 14-fold in the thymus compared to BM. Further thymocyte fractionation demonstrated that DN1 and DN2, but not DN3 or DN4 fractions transmitted AML, and a marked expansion (100-fold) of Lineage-Sca1 + Kit + (LSK) cells in the thymus of the NP23-NHD13 mice. Taken together, these results show that the thymus of NP23-NHD13 mice acts as a reservoir for AML initiating cells and that thymic progenitors can transmit AML.

A unique mutator phenotype reveals complementary oncogenic lesions leading to acute leukemia.

Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4-32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2cre/cre NHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.

HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction.

The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS-FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1 We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma.

Engineered Bcor mutations lead to acute leukemia of progenitor B-1 lymphocyte origin in a sensitized background.

Approximately 10% of NUP98-PHF23 (NP23) mice develop an aggressive acute lymphoblastic leukemia of B-1 lymphocyte progenitor origin (pro-B1 ALL), accompanied by somatic frameshift mutations of the BCL6 interacting corepressor (Bcor) gene, most commonly within a 9-bp "hotspot" in Bcor exon 8. To determine whether experimentally engineered Bcor mutations would lead to pro-B1 ALL, we used clustered, regularly interspaced, short palindromic repeats-associated protein 9 to introduce a Bcor frameshift mutation into NP23 hematopoietic stem and progenitor cells through the use of Bcor small guide RNAs (Bcor sgRNAs). Recipient mice transplanted with NP23 bone marrow or fetal liver cells that had been transduced with a Bcor sgRNA developed pro-B1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. Similar to a subset of human B-cell precursor ALL, the murine pro-B1 ALL had acquired somatic mutations in Jak kinase genes. JAK inhibitors (ruxolitinib and tofacitinib) inhibited the growth of pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentrations (100 nM). Our results demonstrate that Bcor mutations collaborate with NP23 to induce pro-B1 ALL, and that JAK inhibitors are potential therapies for pro-B1 ALL.

Rapid and reversible suppression of ALT by DAXX in osteosarcoma cells.

Many tumors maintain chromosome-ends through a telomerase-independent, DNA-templated mechanism called alternative lengthening of telomeres (ALT). While ALT occurs in only a subset of tumors, it is strongly associated with mutations in the genes ATRX and DAXX, which encode components of an H3.3 histone chaperone complex. The role of ATRX and DAXX mutations in potentiating the mechanism of ALT remains incompletely understood. Here we characterize an osteosarcoma cell line, G292, with wild-type ATRX but a unique chromosome translocation resulting in loss of DAXX function. While ATRX and DAXX form a complex in G292, this complex fails to localize to nuclear PML bodies. We demonstrate that introduction of wild type DAXX suppresses the ALT phenotype and restores the localization of ATRX/DAXX to PML bodies. Using an inducible system, we show that ALT-associated PML bodies are disrupted rapidly following DAXX induction and that ALT is again restored following withdrawal of DAXX.