Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion ...genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 ...germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.
•Germline RPA1 gain-of-function missense mutations result in a telomere biology disorder phenotype.•Somatic rescue events arise in hematopoiesis secondary to germline RPA1 mutation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction ...and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.
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Paulk et al. performed sequential directed evolution screens of AAV capsid libraries in primary human hepatocytes in vivo and against human sera from thousands of patients. Resultant capsid variants AAV-NP40, AAV-NP59, and AAV-NP84 exhibit unique seroreactivity and significantly increase functional human hepatocyte transduction in vivo compared to existing hepatotropic serotypes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk subtype of ALL in children. There are conflicting data on the incidence and prognosis of Ph-like ALL in adults. ...Patients with newly diagnosed B-cell ALL (B-ALL) who received frontline chemotherapy at MD Anderson Cancer Center underwent gene expression profiling of leukemic cells. Of 148 patients, 33.1% had Ph-like, 31.1% had Ph+, and 35.8% had other B-ALL subtypes (B-other). Within the Ph-like ALL cohort, 61% had cytokine receptor-like factor 2 (CRLF2) overexpression. Patients with Ph-like ALL had significantly worse overall survival (OS), and event-free survival compared with B-other with a 5-year survival of 23% (vs 59% for B-other, P = .006). Sixty-eight percent of patients with Ph-like ALL were of Hispanic ethnicity. The following were associated with inferior OS on multivariable analysis: age (hazard ratio HR, 3.299; P < .001), white blood cell count (HR, 1.910; P = .017), platelet count (HR, 7.437; P = .005), and Ph-like ALL (HR, 1.818; P = .03). Next-generation sequencing of the CRLF2+ group identified mutations in the JAK-STAT and Ras pathway in 85% of patients, and 20% had a CRLF2 mutation. Within the CRLF2+ group, JAK2 mutation was associated with inferior outcomes. Our findings show high frequency of Ph-like ALL in adults, an increased frequency of Ph-like ALL in adults of Hispanic ethnicity, significantly inferior outcomes of adult patients with Ph-like ALL, and significantly worse outcomes in the CRLF2+ subset of Ph-like ALL. Novel strategies are needed to improve the outcome of these patients.
•Approximately 20% to 25% of adults with B-ALL have Ph-like ALL with increased frequency of Ph-like ALL in adults with Hispanic ethnicity.•Adult patients with CRLF2+ ALL have poor long-term outcomes; novel strategies are needed to improve the outcomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is ...a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SAMD9 and SAMD9L germline mutations have recently emerged as a new class of predispositions to pediatric myeloid neoplasms. Patients commonly have impaired hematopoiesis, hypocellular marrows, and a ...greater risk of developing clonal chromosome 7 deletions leading to MDS and AML. We recently demonstrated that expressing SAMD9 or SAMD9L mutations in hematopoietic cells suppresses their proliferation and induces cell death. Here, we generated a mouse model that conditionally expresses mutant Samd9l to assess the in vivo impact on hematopoiesis. Using a range of in vivo and ex vivo assays, we showed that cells with heterozygous Samd9l mutations have impaired stemness relative to wild-type counterparts, which was exacerbated by inflammatory stimuli, and ultimately led to bone marrow hypocellularity. Genomic and phenotypic analyses recapitulated many of the hematopoietic cellular phenotypes observed in patients with SAMD9 or SAMD9L mutations, including lymphopenia, and pinpointed TGF-beta as a potential targetable pathway. Further, we observed nonrandom genetic deletion of the mutant Samd9l locus on mouse chromosome 6, mimicking chromosome 7 deletions observed in patients. Collectively, our study has enhanced our understanding of mutant Samd9l hematopoietic phenotypes, emphasized the synergistic role of inflammation in exaggerating the associated hematopoietic defects, and provided insights into potential therapeutic options for patients.
Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, ...particularly to the bone marrow. Advances in therapy and understanding of the metastatic process have been limited due, in part, to the lack of animal models harboring bone marrow disease. The widely used transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here, we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the bone marrow. This model recapitulates 2 frequent alterations in metastatic neuroblastoma, overexpression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. Although overexpression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced bone marrow metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation, and downregulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine TGF-β pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood-borne metastasis and therefore activation of this pathway may explain the enhanced bone marrow metastasis observed in this animal model.
Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in ...neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Cochlear hair cells (HCs) are mechanosensory receptors that transduce sound into electrical signals. HC damage in nonmammalian vertebrates induces surrounding supporting cells (SCs) to divide, ...transdifferentiate and replace lost HCs; however, such spontaneous HC regeneration does not occur in the mammalian cochlea. Here, we acutely ablate the retinoblastoma protein (Rb), a crucial cell cycle regulator, in two subtypes of postmitotic SCs (pillar and Deiters' cells) using an inducible Cre line, Prox1-CreER(T2). Inactivation of Rb in these SCs results in cell cycle reentry of both pillar and Deiters' cells, and completion of cell division with an increase in cell number of pillar cells. Interestingly, nuclei of Rb(-/-) mitotic pillar and Deiters' cells migrate toward the HC layer and divide near the epithelial surface in a manner similar to the SCs in the regenerating avian auditory epithelium. In contrast to postmitotic Rb(-/-) HCs which abort cell division, postmitotic Rb(-/-) pillar cells can proliferate, maintain their SC fate and survive for more than a week. However, no newly formed HCs are detected and SC death followed by HC loss occurs. Our studies accomplish a crucial step toward functional HC regeneration in the mammalian cochlea in vivo, demonstrating the critical role of Rb in maintaining quiescence of postmitotic pillar and Deiters' cells and highlighting the heterogeneity between these two cell types. Therefore, the combination of transient Rb inactivation and further manipulation of transcription factors (i.e., Atoh1 activation) in SCs may represent an effective therapeutic avenue for HC regeneration in the mammalian cochlea.
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