Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on ...RNA (ADAR)1 gene, occurs in 30-50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse.
T cell acute lymphoblastic leukemia (T-ALL) is a rare but aggressive hematological cancer that occurs primarily in children and adolescents. Here, we present a protocol for in vitro co-culture assay ...that enables robust expansion of primary T-ALL cells. We describe steps for seeding T-ALL and stromal cells in 3D organoids and subsequent flow analysis to capture the T-ALL cell growth for long-term culture. This protocol provides a valuable platform for in vitro functional studies and drug screenings using patient-derived cells.
For complete details on the use and execution of this protocol, please refer to Rivera et al.1
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•A protocol for in vitro propagation of primary T-ALL cells•Procedures for culturing T-ALL cells in artificial thymic organoids•Detailed steps for flow cytometry analysis of expanded T-ALL cells
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
T cell acute lymphoblastic leukemia (T-ALL) is a rare but aggressive hematological cancer that occurs primarily in children and adolescents. Here, we present a protocol for in vitro co-culture assay that enables robust expansion of primary T-ALL cells. We describe steps for seeding T-ALL and stromal cells in 3D organoids and subsequent flow analysis to capture the T-ALL cell growth for long-term culture. This protocol provides a valuable platform for in vitro functional studies and drug screenings using patient-derived cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Highlights • A-to-I editing increases transcriptome diversity through multiple mechanisms. • Aberrant regulation of A-to-I editing promotes cancer and degenerative disease. • The functional impact of ...many RNA editing sites is still not fully understood. • Treatments reverting A-to-I editing may have great potential in several diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that ...promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Leukemia-initiating cells (LICs) are regarded as the origin of leukemia relapse and therapeutic resistance. Identifying direct stemness determinants that fuel LIC self-renewal is critical for ...developing targeted approaches. Here, we show that the RNA-editing enzyme ADAR1 is a crucial stemness factor that promotes LIC self-renewal by attenuating aberrant double-stranded RNA (dsRNA) sensing. Elevated adenosine-to-inosine editing is a common attribute of relapsed T cell acute lymphoblastic leukemia (T-ALL) regardless of molecular subtype. Consequently, knockdown of ADAR1 severely inhibits LIC self-renewal capacity and prolongs survival in T-ALL patient-derived xenograft models. Mechanistically, ADAR1 directs hyper-editing of immunogenic dsRNA to avoid detection by the innate immune sensor melanoma differentiation-associated protein 5 (MDA5). Moreover, we uncover that the cell-intrinsic level of MDA5 dictates the dependency on the ADAR1-MDA5 axis in T-ALL. Collectively, our results show that ADAR1 functions as a self-renewal factor that limits the sensing of endogenous dsRNA. Thus, targeting ADAR1 presents an effective therapeutic strategy for eliminating T-ALL LICs.
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•Elevated A-to-I RNA modifications are associated with increased risk of relapse•Loss of ADAR1 impairs LIC self-renewal partly via ADAR1-MDA5 axis•Cell-intrinsic level of MDA5 dictates the dependence of LICs on ADAR1-MDA5 axis•RNA-editing-independent activity suppresses ISGs
Rivera et al. showed that widespread adenosine-to-inosine RNA editing by the RNA-editing enzyme ADAR1 is associated with leukemia relapse in patients with T-ALL. ADAR1 promotes LIC stemness via regulation of stem cell gene expression and suppression of MDA5-directed dsRNA sensing by RNA hyper-editing and RNA-editing-independent mechanisms.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T‐cell ...phenotype (T‐ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T‐ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA‐sequencing analysis on patient‐derived samples followed by splice isoform‐specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T‐ALL. Moreover, patients with T‐ALL displayed pro‐survival splice isoform switching favoring pro‐survival isoforms compared to normal healthy stem cells. Cumulatively, pro‐survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T‐ALL proliferation and survival, thus serving as a potential therapeutic option.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
In this issue of Cancer Cell, Kleppe et al. describe a combination strategy designed to inhibit BET bromodomain and JAK/STAT signaling as a method for effectively inhibiting NF-κB and cytokine ...production in myeloproliferative neoplasms (MPNs). The results provide a strong rationale for clinical evaluation of dual BET/JAK inhibition in MPNs.
In this issue of Cancer Cell, Kleppe et al. describe a combination strategy designed to inhibit BET bromodomain and JAK/STAT signaling as a method for effectively inhibiting NF-κB and cytokine production in myeloproliferative neoplasms (MPNs). The results provide a strong rationale for clinical evaluation of dual BET/JAK inhibition in MPNs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
DNA−protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an α,β-unsaturated aldehyde, acrolein. DNA−protein cross-links are ...subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA−peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA−peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or a dodecylpeptide was covalently attached at the N 2 position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) κ, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol κ was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol κ would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol κ, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA−peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA−peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N 2-guanine-linked DNA−peptide cross-links.
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IJS, KILJ, NUK, PNG, UL, UM
Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive ...niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.
We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity.
These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cancer stem cells (CSCs) can regenerate all facets of a tumour as a result of their stem cell-like capacity to self-renew, survive and become dormant in protective microenvironments. CSCs evolve ...during tumour progression in a manner that conforms to Charles Darwin's principle of natural selection. Although somatic DNA mutations and epigenetic alterations promote evolution, post-transcriptional RNA modifications together with RNA binding protein activity (the 'epitranscriptome') might also contribute to clonal evolution through dynamic determination of RNA function and gene expression diversity in response to environmental stimuli. Deregulation of these epitranscriptomic events contributes to CSC generation and maintenance, which governs cancer progression and drug resistance. In this Review, we discuss the role of malignant RNA processing in CSC generation and maintenance, including mechanisms of RNA methylation, RNA editing and RNA splicing, and the functional consequences of their aberrant regulation in human malignancies. Finally, we highlight the potential of these events as novel CSC biomarkers as well as therapeutic targets.
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IJS, NUK, SBMB, UL, UM, UPUK
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