Chromosome 8 gain is associated with high-grade transformation in MPNST.

One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma that commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven successful in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient-derived xenografts (PDX). These PDX were compared with the primary tumors from which they were derived using copy number analysis, whole exome sequencing, and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single-cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft-tissue sarcomas. These data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis and may account for the aggressive nature and poor outcomes in this sarcoma subtype.

Whole exome sequencing reveals the maintained polyclonal nature from primary to metastatic malignant peripheral nerve sheath tumor in two patients with NF1.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas with high metastatic rates and poor overall patient survival. There are currently no effective therapies, underscoring the pressing need to define the molecular etiologies that underlie MPNST progression. The aim of this study was to examine clonal progression and identify the molecular events critical for MPNST spread. METHODS: In two patients with temporally and spatially distinct metastatic lesions, we employed whole exome sequencing (WES) to elucidate the genetic events of clonal progression, thus identifying the molecular events critical for MPNST spread. RESULTS: First, we demonstrated shared clonal origins for the metastatic lesions relative to the primary tumors, which were maintained throughout the course of MPNST progression, supporting the conclusion that cancer cells with metastatic potential already exist in the primary neoplasm. Second, we discovered TRIM23, a member of the Tripartite Motif family of proteins, as a regulator of MPNST lung metastatic spread in vivo. CONCLUSIONS: The ability to track the genomic evolution from primary to metastatic MPNST offers new insights into the sequence of genetic events required for tumor progression and has identified TRIM23 as a novel target for future study in this rare cancer.

Unmasking Intra-tumoral Heterogeneity and Clonal Evolution in NF1-MPNST.

Sarcomas are highly aggressive cancers that have a high propensity for metastasis, fail to respond to conventional therapies, and carry a poor 5-year survival rate. This is particularly true for patients with neurofibromatosis type 1 (NF1), in which 8%-13% of affected individuals will develop a malignant peripheral nerve sheath tumor (MPNST). Despite continued research, no effective therapies have emerged from recent clinical trials based on preclinical work. One explanation for these failures could be the lack of attention to intra-tumoral heterogeneity. Prior studies have relied on a single sample from these tumors, which may not be representative of all subclones present within the tumor. In the current study, samples were taken from three distinct areas within a single tumor from a patient with an NF1-MPNST. Whole exome sequencing, RNA sequencing, and copy number analysis were performed on each sample. A blood sample was obtained as a germline DNA control. Distinct mutational signatures were identified in different areas of the tumor as well as significant differences in gene expression among the spatially distinct areas, leading to an understanding of the clonal evolution within this patient. These data suggest that multi-regional sampling may be important for driver gene identification and biomarker development in the future.

TYK2 promotes malignant peripheral nerve sheath tumor progression through inhibition of cell death.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that arise most commonly in the setting of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome. Despite aggressive multimodality therapy, outcomes are dismal and most patients die within 5 years of diagnosis. Prior genomic studies in our laboratory identified tyrosine kinase 2 (TYK2) as a frequently mutated gene in MPNST. Herein, we explored the function of TYK2 in MPNST pathogenesis. METHODS: Immunohistochemistry was utilized to examine expression of TYK2 in MPNSTs and other sarcomas. To establish a role for TYK2 in MPNST pathogenesis, murine and human TYK2 knockdown and knockout cells were established using shRNA and CRISPR/Cas9 systems, respectively. RESULTS: We have demonstrated that TYK2 was highly expressed in the majority of human MPNSTs examined. Additionally, we demonstrated that knockdown of Tyk2/TYK2 in murine and human MPNST cells significantly increased cell death in vitro. These effects were accompanied by a decrease in the levels of activated Stats and Bcl-2 as well as an increase in the levels of Cleaved Caspase-3. In addition, Tyk2-KD cells demonstrated impaired growth in subcutaneous and metastasis models in vivo. CONCLUSION: Taken together, these data illustrate the importance of TYK2 in MPNST pathogenesis and suggest that the TYK2 pathway may be a potential therapeutic target for these deadly cancers.

Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors.

PURPOSE: The response to acute and long-term arginine starvation results in a conditional adaptive metabolic reprogramming that can be harnessed for therapeutic opportunities in ASS1-negative tumors. Here, we investigate the underlying biology of priming ASS1- tumors with arginine deiminase (ADI-PEG20) before treatment with gemcitabine (GEM) and docetaxel (DTX) in sarcoma, pancreatic cancer, and melanoma cell lines. EXPERIMENTAL DESIGN: ASS1- tumor cell lines were treated to create LTAT (long-term ADI treated) cell lines (ASS1+) and used for drug combination studies. Protein expression of ASS1, dCK, RRM2, E2F1, c-MYC, and hENT1 was measured. c-MYC activity was determined, live-cell immunofluorescent studies for hENT1, uptake assays of FITC-cytosine probe, and rescue studies with a c-MYC inhibitor were all determined in the presence or absence of the ADI-PEG20:GEM:DTX. RESULTS: In examining modulations within the pyrimidine pathway, we identified that the addition of DTX to cells treated with ADI-PEG20 resulted in translocation of stabilized c-Myc to the nucleus. This resulted in an increase of hENT1 cell-surface expression and rendered the cells susceptible to GEM. In vivo studies demonstrate that the combination of ADI-PEG20:GEM:DTX was optimal for tumor growth inhibition, providing the preclinical mechanism and justification for the ongoing clinical trial of ADI-PEG20, GEM, and DTX in sarcoma. CONCLUSIONS: The priming of tumors with ADI-PEG20 and DTX results in the stabilization of c-MYC potentiating the effect of GEM treatment via an increase in hENT1 expression. This finding is applicable to ASS1-deficient cancers that are currently treated with GEM.

The Transcriptional Regulator SnoN Promotes the Proliferation of Cerebellar Granule Neuron Precursors in the Postnatal Mouse Brain.

Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.