Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here we describe the generation of a collection of pediatric RMS tumor organoid (tumoroid) models comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within four to eight weeks, indicating the feasibility of personalized drug screening. Molecular, genetic and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to six months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

Genomic Instability and Replicative Stress in Multiple Myeloma: The Final Curtain?

Multiple Myeloma (MM) is a genetically complex and heterogeneous hematological cancer that remains incurable despite the introduction of novel therapies in the clinic. Sadly, despite efforts spanning several decades, genomic analysis has failed to identify shared genetic aberrations that could be targeted in this disease. Seeking alternative strategies, various efforts have attempted to target and exploit non-oncogene addictions of MM cells, including, for example, proteasome inhibitors. The surprising finding that MM cells present rampant genomic instability has ignited concerted efforts to understand its origin and exploit it for therapeutic purposes. A credible hypothesis, supported by several lines of evidence, suggests that at the root of this phenotype there is intense replicative stress. Here, we review the current understanding of the role of replicative stress in eliciting genomic instability in MM and how MM cells rely on a single protein, Ataxia Telangiectasia-mutated and Rad3-related protein, ATR, to control and survive the ensuing, potentially fatal DNA damage. From this perspective, replicative stress per se represents not only an opportunity for MM cells to increase their evolutionary pool by increasing their genomic heterogeneity, but also a vulnerability that could be leveraged for therapeutic purposes to selectively target MM tumor cells.

Mutant TP53 prevents Telomere Shortening in Acute Myeloid Leukemia

Background High telomerase activity represents a critical feature of hematopoietic stem cells. Excessive shortening of telomere length (TL) due to replicative stress may be - in analogy to many solid tumors - a hallmark of myeloid neoplasia (MN). Also, telomeric footprints in leukemic genomes may vary between various subtypes corresponding to the differentiation arrest at various stages of hematopoietic ontogeny or specific molecular defects. Critical TL shortening has been associated with genomic instability and accelerated acquisition of genomic lesions leading to a more aggressive phenotype. These processes have not been systematically studied in MN, especially AML. Aim By taking advantage of next-generation sequencing to assay both molecular features and TL within large cohorts of patients, we tested the hypothesis that TL shortening is excessive in highly proliferative MN, but that distinct invariant differences characterize genetic subtypes. Methods Our cohort included AML (N=734), MDS (N=701), healthy controls (HC) (N=11) and PNH (N=102) serving as clonal non-malignant controls. All patients were diagnosed according to WHO standards before being subjected to transcriptome (WTS) and genome (WGS, 100x) sequencing. To retrieve TL characteristics and telomere repeat heterogeneity from WGS data, we used TelomereHunter (TH). In parallel, we performed C-Circle assays. Patients were annotated for clinical features and analyzed for genetic/transcriptomic patterns. Results For a subset of patients for whom corresponding benign lymphocyte DNA was available a significant TL shortening in blasts vs control lymphocytes (A; P=.0023) was detected. While age correlation was established in controls, despite a trend, in MN age did not significantly affect TL (B) and thus subsequent comparisons were not adjusted for age. Next, we studied a cohort of patients with AML, MDS, PNH and HC and found that TL shortening was an overarching finding in AML, MDS and PNH as compared to HC (C). Since no matched DNA was available as reference, we examined the distribution of TL across different age cohorts, AML patients divided according to age cohorts harbored TL in a similar range (D, P=.057). Classic morphologic (E) or cytogenetic subtypes AML exhibited no difference. Similarly, no differences were found between high and low risk MDS patients (not shown). The variability of TL ranges suggested that there may be molecular factors which affect individual TL. When we compared TL grouped according to frequent mutations, only TP53 mutations were associated with longer TL (F, P<.0001). A significant positive correlation (G, P=.021) between TL and TP53 clonal burden was found; samples with the longest vs shortest TL showed significantly higher TP53 VAF (H, P=.0229). In analogy, the presence of multiple TP53 mutations (putative biallelic inactivation) showed longer TL than single hits but no association was found between the nature of mutations and TL (I, J, K). Availability of WTS data allowed us to assess the telomerase activity using the EXTEND score (ES) which has been shown to assess telomerase activity. Indeed, the ES was correlated with TL (L) and TP53 mutant status was associated with a higher ES compared to WT samples (M, P<.0001). Similarly, because of the compensatory upregulation of TP53 in mutant cases, we have also found that TP53 mRNA levels correlated with ES (N P<.0001). Another explanation of TL increase could be the occurrence of alternative lengthening (ALT). TH software allows for estimation of the abundance of specific telomeric repeats. Singleton analysis showed that increase in telomere repeats variants (TTTGGG, O, P=.003) was related to mutations in TP53 arguing against the involvement of ALT. The final confirmation that TL extension was not due to ALT was provided by C-Circle assays. When C-Circle assays were performed for samples with a high/low TL and mutant/WT TP53, none of the subgroups was identified as ALT + (P). Conclusion We stipulate that TL measurements using NGS will be helpful to investigate pathophysiological features associated with TL shortening. Availability of therapies targeting the telomere machinery (Imetelstat) may offer an opportunity for personalized therapy beyond MPN, its current indication. It remains to be tested whether long TL associated with TP53 mutations can serve as marker of sensitivity or resistance to these agents. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy; Novartis: Consultancy.

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

Targeting the methyltransferase SETD8 impairs tumor cell survival and overcomes drug resistance independently of p53 status in multiple myeloma

Background Multiple myeloma (MM) is a malignancy of plasma cells that largely remains incurable. The search for new therapeutic targets is therefore essential. In addition to a wide panel of genetic mutations, epigenetic alterations also appear as important players in the development of this cancer, thereby offering the possibility to reveal novel approaches and targets for effective therapeutic intervention. Results Here, we show that a higher expression of the lysine methyltransferase SETD8, which is responsible for the mono-methylation of histone H4 at lysine 20, is an adverse prognosis factor associated with a poor outcome in two cohorts of newly diagnosed patients. Primary malignant plasma cells are particularly addicted to the activity of this epigenetic enzyme. Indeed, the inhibition of SETD8 by the chemical compound UNC-0379 and the subsequent decrease in histone H4 methylation at lysine 20 are highly toxic in MM cells compared to normal cells from the bone marrow microenvironment. At the molecular level, RNA sequencing and functional studies revealed that SETD8 inhibition induces a mature non-proliferating plasma cell signature and, as observed in other cancers, triggers an activation of the tumor suppressor p53, which together cause an impairment of myeloma cell proliferation and survival. However, a deadly level of replicative stress was also observed in p53-deficient myeloma cells treated with UNC-0379, indicating that the cytotoxicity associated with SETD8 inhibition is not necessarily dependent on p53 activation. Consistent with this, UNC-0379 triggers a p53-independent nucleolar stress characterized by nucleolin delocalization and reduction of nucleolar RNA synthesis. Finally, we showed that SETD8 inhibition is strongly synergistic with melphalan and may overcome resistance to this alkylating agent widely used in MM treatment. Conclusions Altogether, our data indicate that the up-regulation of the epigenetic enzyme SETD8 is associated with a poor outcome and the deregulation of major signaling pathways in MM. Moreover, we provide evidences that myeloma cells are dependent on SETD8 activity and its pharmacological inhibition synergizes with melphalan, which could be beneficial to improve MM treatment in high-risk patients whatever their status for p53.

Actin nucleation safeguards single-stranded DNA and promotes replication fork protection

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress and protect stalled replication forks. All such responses rely on the formation of Replication Protein A (RPA)-ssDNA complexes, yet supra-physiological binding of RPA to ssDNA is toxic. How cells regulate RPA availability to promote fork protection and genome stability is largely unknown. Here we establish that during replication excess RPA is sequestered by monomeric actin and released upon replicative stress through transition to polymeric actin state. Impairment in actin nucleation leads to RPA sequestration, deprotection of ssDNA generated at the stressed forks and consequently, catastrophic fork collapse and hypersensitivity to replication inhibitors. In line with this, we show that increasing RPA load is sufficient to restore efficient fork protection in actin polymerization mutants. Collectively, this work identifies a simple yet robust RPA-buffering mechanism regulating its availability to bind ssDNA and protect replication forks against nucleolytic degradation. Inhibition of this pathway could be of therapeutic interest in treatment of cancers.

A CDK-regulated chromatin segregase promoting chromosome replication

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme—Yta7—entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.