Hongling Liao

Comprehensive Genomic Analysis of Rhabdomyosarcoma Reveals a Landscape of Alterations Affecting a Common Genetic Axis in Fusion-Positive and Fusion-Negative Tumors

UNLABELLED Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Childrens Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma. SIGNIFICANCE This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.

The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534).

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

Clonality and Evolutionary History of Rhabdomyosarcoma

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification.

Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.

Abstract A25: Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition

PAX-fusion negative rhabdomyosarcoma (RMS) arises from skeletal muscle precursors that have failed to differentiate normally despite the expression of the myogenic master transcription factor, MYOD1. The cure rate for relapsed or refractory fusion negative RMS is poor despite aggressive multi-modality treatment. Novel treatment approaches such as the use of targeted therapies including those that induce skeletal muscle differentiation might improve overall survival for patients with fusion negative RMS. Genetic studies have shown that the most common single nucleotide variant in fusion negative RMS is an oncogenic change in one of the RAS isoforms, namely NRAS, HRAS or KRAS. In this study, we hypothesized that targeting aberrant RAS activity releases the differentiation block in fusion negative RMS and sought to unravel the underlying epigenetic mechanisms through which RAS signaling drives oncogenic transcription in RMS. To achieve this goal, we combined high-throughput drug screening with biochemical, RNAseq and ChIPseq assays across a panel of RMS cell lines driven by oncogenic RAS mutations. Critically, we demonstrated that expression of oncogenic RAS was necessary for survival of these RAS-mutated RMS cells. In addition, overexpression of mutant RAS isoforms in C2C12 myoblasts inhibited myogenic differentiation induced by low-serum conditions. This differentiation block was mediated primarily by engagement of the RAF-MEK-ERK MAP kinase pathway. In corroboration with these observations, an unbiased screen of the ability of small molecules to impact cell viability demonstrated that inhibitors of the MAP kinase pathway were the most potently selective class of molecules for RAS-mutated RMS. In particular, trametinib, an allosteric, non-ATP competitive inhibitor of MEK1/2, was the most consistently potent MEK inhibitor in RAS-mutated RMS cell lines. Trametinib treatment induced G1 arrest and skeletal muscle differentiation in RAS-mutated RMS cell lines. Trametinib also slowed tumor growth and prolonged survival in xenograft models of RAS-mutated RMS. To determine the mechanism by which MEK inhibition induced skeletal muscle differentiation in RAS-mutated RMS, we analyzed changes in gene expression, transcription factor deposition and histone modification in RMS cells treated with trametinib. Trametinib treatment increased expression of myogenic transcription factors, such as MYOG and MEF2C, and decreased expression of transcription factors important for proliferation, such as MYC and ID3, in RAS-mutated RMS cells. ChIPseq experiments demonstrated that this transcriptional reprogramming was driven in part by changes in the active enhancer landscape, since H3K27ac deposition at MYH3, TTNT2 and other muscle-specific loci increased with trametinib treatment. Both MYC and MYOD1 bound the active enhancers induced by trametinib treatment in RAS-mutated RMS, despite an overall decrease in MYC expression. Finally, we found significant ERK2 deposition on the MYOG promoter in the untreated cells. ERK2 is known to recruit the Polycomb repressive machinery at developmental loci in embryonic stem cells and therefore aberrant ERK2 activity may facilitate repression of MYOG expression in RAS-mutated RMS. In summary, our data support a model of RAS-driven RMS in which aberrant ERK activity drives tumor cell proliferation, in part through increased expression and stability of MYC, and prevents myogenic differentiation, in this case through alterations in the enhancer landscape and interactions with the Polycomb repressive machinery. Future work is aimed at identifying rational combinations of trametinib and direct epigenetic modulators that synergistically drive RAS-mutated RMS differentiation with the goal of providing measurable clinical benefit in relapsed or refractory RAS-mutated RMS. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Jack F. Shern, Young K. Song, Hongling Liao, Hsein-Chao Chou, Sivasish Sindiri, Arnulfo Mendoza, Xiaohu Zhang, Rajarashi Guha, Diana C. Haines, James P. Madigan, Jun S. Wei, Marc Ferrer, Craig J. Thomas, Javed Khan. Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A25.

Abstract PR16: Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma

Master transcription factors establish enhancers to regulate cell identity genes by recruiting epigenetic machinery, and are sequentially exchanged during changes in cell identity (ie, differentiation). Commonly, the fusion of transcription factors profoundly alters proper progression of cell identity, serving as the signature oncogenic event in many malignancies. The most common soft tissue cancer of childhood, rhabdomyosarcoma (RMS), is characterized by an inability to exit the proliferative myoblast-like state, presumably by blocking myogenic transcription factors from advancing the active enhancer landscape. This is achieved by either chromosomal translocation resulting in the oncogenic fusion transcription factor PAX3/7-FOXO1 (Fusion-Positive alveolar subtype, FP-RMS) or mutations in the tyrosine kinase/RAS/PIK3C axis (Fusion-Negative embryonal subtype, FN-RMS). Patients who harbor a PAX-fusion typically relapse despite aggressive therapy and have very poor survival. Here we hypothesized that the PAX3-FOXO1 fusion gene causes epigenetic reprogramming resulting in increased proliferation and a failure to terminally differentiate. Furthermore we hypothesized that disrupting the epigenetic machinery recruited by this fusion gene would provide a tractable target for therapy. We mapped the landscape of epigenetic alterations caused by the PAX3-FOXO1 fusion gene using a combination of RNA-seq, DNase hypersensitivity, and ChIP-seq against histone marks and transcription factors in cell lines and models of FP-RMS. We found high expression of several master transcription factors (including MYOD1, MYOG, MYCN, and SOX8) in FP-RMS primary tumors and cell lines, resembling human skeletal muscle myoblasts. ChIP-seq revealed that PAX3-FOXO1 is exclusively bound to distal, active enhancers and the histone modification most enriched surrounding PAX3-FOXO1 was acetylated H3K27. Furthermore we found that the introduction of the fusion gene into fibroblast cells opened up the chromatin at these same sites, completely rewiring the active enhancer landscape, recapitulating a transcriptome locked in a myoblast-like state. Genome-wide profiling of MYOD1, MYOG and MYCN reveals that all three master regulators collaborative bind at nearly every PAX3-FOXO1 driven super enhancer (SE), while typical enhancers (TEs) rarely have more than two of these four. PAX3-FOXO1 has a 7-fold preference for SEs over TEs. We also find that PAX3-FOXO1 bound, myogenic enhancers are decommissioned throughout normal skeletal muscle differentiation. To identify small molecules that would inhibit the PAX3-FOXO1 induced epigenetic machinery we treated a panel of FP-RMS cell lines with 1912 targeted agents and chemical probes at multiple concentrations and measured cell viability. Classes of molecules selectively potent for PAX3-FOXO1 driven cells (as compared to normal fibroblasts) hit connected biologically relevant targets including SE controlled receptor tyrosine kinases (including FGFR4, IGF1R, ALK), and transcriptional cofactors involved in SE complexes (including HDACs and BRD). In an expanded panel of RMS cell lines we confirmed that FP-RMS is selectively sensitive to the BET bromodomain inhibitors with the most potent being JQ1. These inhibitors selectively suppress PAX3-FOXO1 dependent transcription as measured by reporter assays and RNA-seq analysis. Indeed, coactivators of looped chromatin p300, MED1 and BRD4 excessively co-localize with PAX3-FOXO1 genome wide. In vivo , JQ1 selectively ablated PAX3-FOXO1 dependent SE driven transcription, and significantly delayed tumor progression in xenografts of PAX3-FOXO1 driven cell lines. In conclusion we found that PAX3-FOXO1 establishes myogenic super enhancers that are sensitive to BET bromodomain inhibition which constitutes a novel therapeutic strategy for children with PAX-fusion driven rhabdomyosarcoma. This abstract is also presented as Poster A16. Citation Format: Berkley E. Gryder, Marielle E. Yohe, Jack Shern, Hsien-Chao Chou, Young Song, Rajesh Patidar, Sam Li, Sivasish Sindiri, Abigail Cleveland, Hongling Liao, Xinyu Wen, Xiaohu Zhang, Lesley Mathews-Griner, Rajarshi Guha, Paul Shinn, Marc Ferrer, Scott Martin, Madhu Lal, Craig Thomas, Javed Khan. Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR16.

Abstract 4457: Chromatin remodeling as a potential new strategy for fusion positive rhabdomyosarcoma therapy

Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric tumor driven by an oncogenic fusion protein, PAX3-FOXO1, which acts as a transcription factor. Conventional chemotherapy is effective for low risk patients who have a 5-year overall survival greater than 65%, while high risk patients, including those with metastatic disease, have less than 40% survival. Consequently, we hypothesize that targeting the fusion protein or its collaborators in transcription regulation will provide novel therapies for this aggressive subtype of RMS. To identify new druggable PAX3-FOXO1 interactors, we performed a combined proteomic and genetic screen which led to the discovery of the NuRD complex (Nucleosome Remodelling and Deacetylase) as a major PAX3-FOXO1 co-regulator. The NuRD complex is unique among the chromatin remodelling complexes due to its dual enzymatic activity. It can act by histone deacetylation through HDAC1/2 (histone deacetylases) or influence nucleosome positioning through CHD4 (chromodomain-DNA-binding protein 4). Intriguingly, it has been associated with both activating and repressive activities in gene expression and its role in cancer development is not fully understood yet. We found that in FP-RMS, silencing of CHD4 affected the expression of approximately 50% of PAX3-FOXO1 regulated target genes. These were mainly genes which are usually upregulated, suggesting an activating role for NuRD. Consistent with CHD4 activation activity, ChIP-seq experiments demonstrated that CHD4 and HDAC2 co-localize with the fusion protein in cis-regulatory sites of a subset of its target genes. Interestingly, gene expression analysis showed that both CHD4 and HDAC2 are highly expressed in tumor tissue and myoblasts when compared to normal skeletal muscle, inferring a potential role of the NuRD complex in maintaining the undifferentiated phenotype observed in FP-RMS. Importantly, CHD4 silencing had no effect on myoblasts proliferation whereas a profound growth reduction was seen in FP-RMS cell lines, suggesting a unique tumour dependency on this chromatin remodeler. In addition, depletion of CHD4 caused a complete regression of xenograft tumours in mice.In summary, we have identified the NuRD complex as an essential positive co-regulator of PAX3-FOXO1 transcriptional activity. Our data propose a critival role of one of the NuRD core component CHD4 in FP-RMS cell viability, making CHD4 an attractive new target for therapy. To our knowledge, CHD4 is the first chromatin remodeler identified to associate with PAX3-FOXO1 transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumour development. Collectively, our findings suggest CHD4 as a potential novel therapeutic target in this childhood malignancy.Ongoing work is currently underway to identify first-in-class small molecules to inhibit CHD4 protein. Citation Format: Joana G. Marques, Berkley Gryder, Maria Boehm, Marco Wachtel, Young Song, Hsien-Chao Chou, Rajesh Patidar, Hongling Liao, Javed Khan, Beat W. Schaefer. Chromatin remodeling as a potential new strategy for fusion positive rhabdomyosarcoma therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4457.

Abstract B31: Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed a siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of NB cell lines, those with MYCN amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of its known target histone H3, and upregulation of p53 pathway in MYCN-amplified NB cells with wild-type TP53. Both wild-type and p53-mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. Citation Format: Dominik Bogen, Jun S. Wei, David O. Azorsa, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Jonathan M. Keller, Lesley A. Mathews Griner, Marc Ferrer, Young K. Song, Hongling Liao, Arnulfo Mendoza, Berkley E. Gryder, Sivasish Sindri, Jianbin He, Xinyu Wen, Xinyu Wen, Shile Zhang, John F. Shern, Marielle E. Yohe, Sabine Taschner-Mandl, Jason Shohet, Craig J. Thomas, Scott E. Martin, Peter F. Ambros, Javed Khan. Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B31.