A transgenic mouse model of Down syndrome acute lymphoblastic leukemia identifies targetable vulnerabilities.

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Interleukin-15-armored GPC3-CAR T cells for patients with solid cancers.

Interleukin-15 (IL15) promotes the survival of T lymphocytes and enhances the antitumor properties of CAR T cells in preclinical models of solid neoplasms in which CAR T cells have limited efficacy1-4. Glypican-3 (GPC3) is expressed in a group of solid cancers5-10, and here we report the first evaluation in humans of the effects of IL15 co-expression on GPC3-CAR T cells. Cohort 1 patients (NCT02905188/NCT02932956) received GPC3-CAR T cells, which were safe but produced no objective antitumor responses and reached peak expansion at two weeks. Cohort 2 patients (NCT05103631/NCT04377932) received GPC3-CAR T cells that co-expressed IL15 (15.CAR), which mediated significantly increased cell expansion and induced a disease control rate of 66% and antitumor response rate of 33%. Infusion of 15.CAR T cells was associated with increased incidence of cytokine release syndrome, which was rapidly ameliorated by activation of the inducible caspase 9 safety switch. Compared to non-responders, tumor-infiltrating 15.CAR T cells from responders showed repression of SWI/SNF epigenetic regulators and upregulation of FOS and JUN family members as well as genes related to type I interferon signaling. Collectively, these results demonstrate that IL15 increases the expansion, intratumoral survival, and antitumor activity of GPC3-CAR T cells in patients.

A novel treatment strategy utilizing panobinostat for high-risk and treatment-refractory hepatoblastoma.

BACKGROUND & AIMS: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To develop new therapeutic strategies for these patients, our laboratory has developed a preclinical testing pipeline. Given that histone deacetylase (HDAC) inhibition has been proposed for HB, we hypothesized that we could find an effective combination treatment strategy utilizing HDAC inhibition. METHODS: RNA sequencing, microarray, NanoString, and immunohistochemistry data of patient HB samples were analyzed for HDAC class expression. Patient-derived spheroids (PDSp) were used to screen combination chemotherapy with an HDAC inhibitor, panobinostat. Patient-derived xenograft (PDX) mouse models were developed and treated with the combination therapy that showed the highest efficacy in the PDSp drug screen. RESULTS: HDAC RNA and protein expression were elevated in HB tumors compared to normal livers. Panobinostat (IC50 of 0.013-0.059 µM) showed strong in vitro effects and was associated with lower cell viability than other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). All four models responded to VIP therapy with a decrease in tumor size compared to placebo. After 6 weeks of treatment, two models demonstrated necrotic cell death, with lower Ki67 expression, decreased serum alpha fetoprotein and reduced tumor burden compared to paired VI- and placebo-treated groups. CONCLUSIONS: Utilizing a preclinical HB pipeline, we demonstrate that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from patients with high-risk, relapsed, and treatment-refractory HB. IMPACT AND IMPLICATIONS: Patients with treatment-refractory hepatoblastoma have limited treatment options with survival rates of less than 50%. Our manuscript demonstrates that combination therapy with vincristine, irinotecan, and panobinostat reduces the size of high-risk, relapsed, and treatment-refractory tumors. With this work we provide preclinical evidence to support utilizing this combination therapy as an arm in future clinical trials.

Molecular Profiling of a Hepatocellular Neoplasm Not Otherwise Specified (HCN-NOS) Demonstrates Distinct Molecular Features in Hepatoblastoma and HCC-Like Components.

Hepatoblastomas (HB) are embryonal tumors with quiet genomes diagnosed mostly in children under 3 years of age and often cured by surgical resection and chemotherapy. However, a subset of HBs behave aggressively, displaying characteristic histologic features and higher genomic instability. Hepatocellular neoplasm-not otherwise specified (HCN-NOS) is a provisional diagnostic category for tumors exhibiting either intermediate or a combination of both HB and hepatocellular carcinoma (HCC) histological features. In this study, we characterized an HCN-NOS diagnosed in a 3-year-old patient presenting with a liver mass, in which both HB and HCC histological components were amendable to macro-dissection and molecular profiling. The spectrum of mutations, copy number changes, mRNA, and protein expression profiles within these 2 histologically distinct tumor areas demonstrate molecular heterogeneity and suggest intratumoral clonal evolution of this hepatocellular CTNNB1-mutant lesion.

Navigating relapsed hepatoblastoma: Predictive factors and surgical treatment strategy.

OBJECTIVE: Hepatoblastoma (HB) is the most common primary hepatic malignancy in childhood. Relapse occurs in more than 50% of high-risk patients with a high mortality due to ineffective salvage therapies. The purpose of this study is to identify risk factors for relapsed HB and predictors of survival in a single tertiary referral center. METHODS: A retrospective chart review showed 129 surgically treated HB patients from October 2004 to July 2020. Of the cohort, 22 patients presented with relapsed HB. Relapse was defined as re-appearance of malignancy after 4 weeks of normalized AFP and disappearance of all tumors on imaging. RESULTS: Patients with relapsed HB had a 5-year overall survival (OS) of 45.4% compared to 93.1% in those without relapse (p = 0.001). When comparing PRETEXT IV, microvascular invasion, metastatic disease, and age on multivariate logistic regression, only PRETEXT IV was an independent risk factor for relapsed HB with an OR of 2.39 (95% CI: 1.16-4.96; p = 0.019). Mixed epithelial and mesenchymal HB (12/19, 63.2%) was the most common histology of primary tumors while pure epithelial HB (13/15, 86.6%) was the most common relapsed histology. Combination of surgical and medical therapy for relapsed disease was predictive of survival with an HR of 16.3 (95% CI: 1.783-149.091; p = 0.013) compared to only chemotherapy. CONCLUSIONS: This study demonstrates that PRETEXT IV staging is an independent predictor of relapsed disease. The most common relapsed histology was epithelial, suggesting a potential selection or resistance of this component. Surgical resection is a critical component of multimodal therapy for relapsed HB.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

Effective methods for bulk RNA-seq deconvolution using scnRNA-seq transcriptomes.

BACKGROUND: RNA profiling technologies at single-cell resolutions, including single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, scnRNA-seq for short), can help characterize the composition of tissues and reveal cells that influence key functions in both healthy and disease tissues. However, the use of these technologies is operationally challenging because of high costs and stringent sample-collection requirements. Computational deconvolution methods that infer the composition of bulk-profiled samples using scnRNA-seq-characterized cell types can broaden scnRNA-seq applications, but their effectiveness remains controversial. RESULTS: We produced the first systematic evaluation of deconvolution methods on datasets with either known or scnRNA-seq-estimated compositions. Our analyses revealed biases that are common to scnRNA-seq 10X Genomics assays and illustrated the importance of accurate and properly controlled data preprocessing and method selection and optimization. Moreover, our results suggested that concurrent RNA-seq and scnRNA-seq profiles can help improve the accuracy of both scnRNA-seq preprocessing and the deconvolution methods that employ them. Indeed, our proposed method, Single-cell RNA Quantity Informed Deconvolution (SQUID), which combines RNA-seq transformation and dampened weighted least-squares deconvolution approaches, consistently outperformed other methods in predicting the composition of cell mixtures and tissue samples. CONCLUSIONS: We showed that analysis of concurrent RNA-seq and scnRNA-seq profiles with SQUID can produce accurate cell-type abundance estimates and that this accuracy improvement was necessary for identifying outcomes-predictive cancer cell subclones in pediatric acute myeloid leukemia and neuroblastoma datasets. These results suggest that deconvolution accuracy improvements are vital to enabling its applications in the life sciences.

MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma.

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Hepatoblastomas with carcinoma features represent a biological spectrum of aggressive neoplasms in children and young adults.

BACKGROUND & AIMS: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS. METHODS: We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (>8). RESULTS: Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (∼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes. CONCLUSIONS: Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms. LAY SUMMARY: We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.

Krüppel-like Factor 4 Supports the Expansion of Leukemia Stem Cells in MLL-AF9-driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

Escalated (Dependent) Oxycodone Self-Administration Is Associated with Cognitive Impairment and Transcriptional Evidence of Neurodegeneration in Human Immunodeficiency Virus (HIV) Transgenic Rats.

Substance use disorder is associated with accelerated disease progression in people with human immunodeficiency virus (HIV; PWH). Problem opioid use, including high-dose opioid therapy, prescription drug misuse, and opioid abuse, is high and increasing in the PWH population. Oxycodone is a broadly prescribed opioid in both the general population and PWH. Here, we allowed HIV transgenic (Tg) rats and wildtype (WT) littermates to intravenously self-administer oxycodone under short-access (ShA) conditions, which led to moderate, stable, "recreational"-like levels of drug intake, or under long-access (LgA) conditions, which led to escalated (dependent) drug intake. HIV Tg rats with histories of oxycodone self-administration under LgA conditions exhibited significant impairment in memory performance in the novel object recognition (NOR) paradigm. RNA-sequencing expression profiling of the medial prefrontal cortex (mPFC) in HIV Tg rats that self-administered oxycodone under ShA conditions exhibited greater transcriptional evidence of inflammation than WT rats that self-administered oxycodone under the same conditions. HIV Tg rats that self-administered oxycodone under LgA conditions exhibited transcriptional evidence of an increase in neuronal injury and neurodegeneration compared with WT rats under the same conditions. Gene expression analysis indicated that glucocorticoid-dependent adaptations contributed to the gene expression effects of oxycodone self-administration. Overall, the present results indicate that a history of opioid intake promotes neuroinflammation and glucocorticoid dysregulation, and excessive opioid intake is associated with neurotoxicity and cognitive impairment in HIV Tg rats.

CRLF2 overexpression results in reduced B-cell differentiation and upregulated E2F signaling in the Dp16 mouse model of Down syndrome.

Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.

CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain-of-function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti-tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.

Restoration of the molecular clock is tumor suppressive in neuroblastoma.

MYCN activation is a hallmark of advanced neuroblastoma (NB) and a known master regulator of metabolic reprogramming, favoring NB adaptation to its microenvironment. We found that the expression of the main regulators of the molecular clock loops is profoundly disrupted in MYCN-amplified NB patients, and this disruption independently predicts poor clinical outcome. MYCN induces the expression of clock repressors and downregulates the one of clock activators by directly binding to their promoters. Ultimately, MYCN attenuates the molecular clock by suppressing BMAL1 expression and oscillation, thereby promoting cell survival. Reestablishment of the activity of the clock activator RORα via its genetic overexpression and its stimulation through the agonist SR1078, restores BMAL1 expression and oscillation, effectively blocks MYCN-mediated tumor growth and de novo lipogenesis, and sensitizes NB tumors to conventional chemotherapy. In conclusion, reactivation of RORα could serve as a therapeutic strategy for MYCN-amplified NBs by blocking the dysregulation of molecular clock and cell metabolism mediated by MYCN.

A human liver chimeric mouse model for non-alcoholic fatty liver disease.

BACKGROUND & AIMS: The accumulation of neutral lipids within hepatocytes underlies non-alcoholic fatty liver disease (NAFLD), which affects a quarter of the world's population and is associated with hepatitis, cirrhosis, and hepatocellular carcinoma. Despite insights gained from both human and animal studies, our understanding of NAFLD pathogenesis remains limited. To better study the molecular changes driving the condition we aimed to generate a humanised NAFLD mouse model. METHODS: We generated TIRF (transgene-free Il2rg -/-/Rag2 -/-/Fah -/-) mice, populated their livers with human hepatocytes, and fed them a Western-type diet for 12 weeks. RESULTS: Within the same chimeric liver, human hepatocytes developed pronounced steatosis whereas murine hepatocytes remained normal. Unbiased metabolomics and lipidomics revealed signatures of clinical NAFLD. Transcriptomic analyses showed that molecular responses diverged sharply between murine and human hepatocytes, demonstrating stark species differences in liver function. Regulatory network analysis indicated close agreement between our model and clinical NAFLD with respect to transcriptional control of cholesterol biosynthesis. CONCLUSIONS: These NAFLD xenograft mice reveal an unexpected degree of evolutionary divergence in food metabolism and offer a physiologically relevant, experimentally tractable model for studying the pathogenic changes invoked by steatosis. LAY SUMMARY: Fatty liver disease is an emerging health problem, and as there are no good experimental animal models, our understanding of the condition is poor. We here describe a novel humanised mouse system and compare it with clinical data. The results reveal that the human cells in the mouse liver develop fatty liver disease upon a Western-style fatty diet, whereas the mouse cells appear normal. The molecular signature (expression profiles) of the human cells are distinct from the mouse cells and metabolic analysis of the humanised livers mimic the ones observed in humans with fatty liver. This novel humanised mouse system can be used to study human fatty liver disease.

Cell lines of the same anatomic site and histologic type show large variability in intrinsic radiosensitivity and relative biological effectiveness to protons and carbon ions.

PURPOSE: To show that intrinsic radiosensitivity varies greatly for protons and carbon (C) ions in addition to photons, and that DNA repair capacity remains important in governing this variability. METHODS: We measured or obtained from the literature clonogenic survival data for a number of human cancer cell lines exposed to photons, protons (9.9 keV/µm), and C-ions (13.3-77.1 keV/µm). We characterized their intrinsic radiosensitivity by the dose for 10% or 50% survival (D10% or D50% ), and quantified the variability at each radiation quality by the coefficient of variation (COV) in D10% and D50% . We also treated cells with DNA repair inhibitors prior to irradiation to assess how DNA repair capacity affects their variability. RESULTS: We found no statistically significant differences in the COVs of D10% or D50% between any of the radiation qualities investigated. The same was true regardless of whether the cells were treated with DNA repair inhibitors, or whether they were stratified into histologic subsets. Even within histologic subsets, we found remarkable differences in radiosensitivity for high LET C-ions that were often greater than the variations in RBE, with brain cancer cells varying in D10% (D50% ) up to 100% (131%) for 77.1 keV/µm C-ions, and non-small cell lung cancer and pancreatic cancer cell lines varying up to 55% (76%) and 51% (78%), respectively, for 60.5 keV/µm C-ions. The cell lines with modulated DNA repair capacity had greater variability in intrinsic radiosensitivity across all radiation qualities. CONCLUSIONS: Even for cell lines of the same histologic type, there are remarkable variations in intrinsic radiosensitivity, and these variations do not differ significantly between photon, proton or C-ion radiation. The importance of DNA repair capacity in governing the variability in intrinsic radiosensitivity is not significantly diminished for higher LET radiation.

MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma.

Hepatoblastoma (HB) is the most common pediatric liver malignancy. High-risk patients have poor survival, and current chemotherapies are associated with significant toxicities. Targeted therapies are needed to improve outcomes and patient quality of life. Most HB cases are TP53 wild-type; therefore, we hypothesized that targeting the p53 regulator Murine double minute 4 (MDM4) to reactivate p53 signaling may show efficacy. MDM4 expression was elevated in HB patient samples, and increased expression was strongly correlated with decreased expression of p53 target genes. Treatment with NSC207895 (XI-006), which inhibits MDM4 expression, or ATSP-7041, a stapled peptide dual inhibitor of MDM2 and MDM4, showed significant cytotoxic and antiproliferative effects in HB cells. Similar phenotypes were seen with short hairpin RNA (shRNA)-mediated inhibition of MDM4. Both NSC207895 and ATSP-7041 caused significant upregulation of p53 targets in HB cells. Knocking-down TP53 with shRNA or overexpressing MDM4 led to resistance to NSC207895-mediated cytotoxicity, suggesting that this phenotype is dependent on the MDM4-p53 axis. MDM4 inhibition also showed efficacy in a murine model of HB with significantly decreased tumor weight and increased apoptosis observed in the treatment group. This study demonstrates that inhibition of MDM4 is efficacious in HB by upregulating p53 tumor suppressor signaling.

Inferring clonal composition from multiple tumor biopsies.

Knowledge about the clonal evolution of a tumor can help to interpret the function of its genetic alterations by identifying initiating events and events that contribute to the selective advantage of proliferative, metastatic, and drug-resistant subclones. Clonal evolution can be reconstructed from estimates of the relative abundance (frequency) of subclone-specific alterations in tumor biopsies, which, in turn, inform on its composition. However, estimating these frequencies is complicated by the high genetic instability that characterizes many cancers. Models for genetic instability suggest that copy number alterations (CNAs) can influence mutation-frequency estimates and thus impede efforts to reconstruct tumor phylogenies. Our analysis suggested that accurate mutation frequency estimates require accounting for CNAs-a challenging endeavour using the genetic profile of a single tumor biopsy. Instead, we propose an optimization algorithm, Chimæra, to account for the effects of CNAs using profiles of multiple biopsies per tumor. Analyses of simulated data and tumor profiles suggested that Chimæra estimates are consistently more accurate than those of previously proposed methods and resulted in improved phylogeny reconstructions and subclone characterizations. Our analyses inferred recurrent initiating mutations in hepatocellular carcinomas, resolved the clonal composition of Wilms' tumors, and characterized the acquisition of mutations in drug-resistant prostate cancers.