A Bioinformatics Tool for Identifying Intratumoral Microbes from the ORIEN Dataset.

Evidence supports significant interactions among microbes, immune cells, and tumor cells in at least 10%-20% of human cancers, emphasizing the importance of further investigating these complex relationships. However, the implications and significance of tumor-related microbes remain largely unknown. Studies have demonstrated the critical roles of host microbes in cancer prevention and treatment responses. Understanding interactions between host microbes and cancer can drive cancer diagnosis and microbial therapeutics (bugs as drugs). Computational identification of cancer-specific microbes and their associations is still challenging due to the high dimensionality and high sparsity of intratumoral microbiome data, which requires large datasets containing sufficient event observations to identify relationships, and the interactions within microbial communities, the heterogeneity in microbial composition, and other confounding effects that can lead to spurious associations. To solve these issues, we present a bioinformatics tool, microbial graph attention (MEGA), to identify the microbes most strongly associated with 12 cancer types. We demonstrate its utility on a dataset from a consortium of nine cancer centers in the Oncology Research Information Exchange Network. This package has three unique features: species-sample relations are represented in a heterogeneous graph and learned by a graph attention network; it incorporates metabolic and phylogenetic information to reflect intricate relationships within microbial communities; and it provides multiple functionalities for association interpretations and visualizations. We analyzed 2,704 tumor RNA sequencing samples and MEGA interpreted the tissue-resident microbial signatures of each of 12 cancer types. MEGA can effectively identify cancer-associated microbial signatures and refine their interactions with tumors. SIGNIFICANCE: Studying the tumor microbiome in high-throughput sequencing data is challenging because of the extremely sparse data matrices, heterogeneity, and high likelihood of contamination. We present a new deep learning tool, MEGA, to refine the organisms that interact with tumors.

Discrepancies in indel software resolution with somatic CRISPR/Cas9 tumorigenesis models.

CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.

CDK4/6-MEK Inhibition in MPNSTs Causes Plasma Cell Infiltration, Sensitization to PD-L1 Blockade, and Tumor Regression.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. EXPERIMENTAL DESIGN: Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. RESULTS: Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CONCLUSIONS: CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.

The tumor microbiome as a predictor of outcomes in patients with metastatic melanoma treated with immune checkpoint inhibitors.

Emerging evidence supports the important role of the tumor microbiome in oncogenesis, cancer immune phenotype, cancer progression, and treatment outcomes in many malignancies. In this study, we investigated the metastatic melanoma tumor microbiome and potential roles in association with clinical outcomes, such as survival, in patients with metastatic disease treated with immune checkpoint inhibitors (ICIs). Baseline tumor samples were collected from 71 patients with metastatic melanoma before treatment with ICIs. Bulk RNA-seq was conducted on the formalin-fixed paraffin-embedded (FFPE) tumor samples. Durable clinical benefit (primary clinical endpoint) following ICIs was defined as overall survival ≥24 months and no change to the primary drug regimen (responders). We processed RNA-seq reads to carefully identify exogenous sequences using the {exotic} tool. The 71 patients with metastatic melanoma ranged in age from 24 to 83 years, 59% were male, and 55% survived >24 months following the initiation of ICI treatment. Exogenous taxa were identified in the tumor RNA-seq, including bacteria, fungi, and viruses. We found differences in gene expression and microbe abundances in immunotherapy responsive versus non-responsive tumors. Responders showed significant enrichment of several microbes including Fusobacterium nucleatum, and non-responders showed enrichment of fungi, as well as several bacteria. These microbes correlated with immune-related gene expression signatures. Finally, we found that models for predicting prolonged survival with immunotherapy using both microbe abundances and gene expression outperformed models using either dataset alone. Our findings warrant further investigation and potentially support therapeutic strategies to modify the tumor microbiome in order to improve treatment outcomes with ICIs.

A bioinformatics tool for identifying intratumoral microbes from the ORIEN dataset.

Evidence supports significant interactions among microbes, immune cells, and tumor cells in at least 10-20% of human cancers, emphasizing the importance of further investigating these complex relationships. However, the implications and significance of tumor-related microbes remain largely unknown. Studies have demonstrated the critical roles of host microbes in cancer prevention and treatment responses. Understanding interactions between host microbes and cancer can drive cancer diagnosis and microbial therapeutics (bugs as drugs). Computational identification of cancer-specific microbes and their associations is still challenging due to the high dimensionality and high sparsity of intratumoral microbiome data, which requires large datasets containing sufficient event observations to identify relationships, and the interactions within microbial communities, the heterogeneity in microbial composition, and other confounding effects that can lead to spurious associations. To solve these issues, we present a bioinformatics tool, MEGA, to identify the microbes most strongly associated with 12 cancer types. We demonstrate its utility on a dataset from a consortium of 9 cancer centers in the Oncology Research Information Exchange Network (ORIEN). This package has 3 unique features: species-sample relations are represented in a heterogeneous graph and learned by a graph attention network; it incorporates metabolic and phylogenetic information to reflect intricate relationships within microbial communities; and it provides multiple functionalities for association interpretations and visualizations. We analyzed 2704 tumor RNA-seq samples and MEGA interpreted the tissue-resident microbial signatures of each of 12 cancer types. MEGA can effectively identify cancer-associated microbial signatures and refine their interactions with tumors.

Loss of Nf1 and Ink4a/Arf Are Associated with Sex-Dependent Growth Differences in a Mouse Model of Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive form of cancer that accounts for half of all pediatric soft tissue sarcomas. Little progress has been made in improving survival outcomes over the past three decades. Mouse models of rhabdomyosarcoma are a critical component of translational research aimed at understanding tumor biology and developing new, improved therapies. Though several models exist, many common mutations found in human rhabdomyosarcoma tumors remain unmodeled and understudied. This study describes a new model of embryonal rhabdomyosarcoma driven by the loss of Nf1 and Ink4a/Arf, two mutations commonly found in patient tumors. We find that this new model is histologically similar to other previously-published rhabdomyosarcoma models, although it substantially differs in the time required for tumor onset and in tumor growth kinetics. We also observe unique sex-dependent phenotypes in both primary and newly-developed orthotopic syngeneic allograft tumors that are not present in previous models. Using in vitro and in vivo studies, we examined the response to vincristine, a component of the standard-of-care chemotherapy for RMS. The findings from this study provide valuable insight into a new mouse model of rhabdomyosarcoma that addresses an ongoing need for patient-relevant animal models to further translational research.

Low-Cost, High-Pressure-Synthesized Oxygen-Entrapping Materials to Improve Treatment of Solid Tumors.

Tumor hypoxia drives resistance to many cancer therapies, including radiotherapy and chemotherapy. Methods that increase tumor oxygen pressures, such as hyperbaric oxygen therapy and microbubble infusion, are utilized to improve the responses to current standard-of-care therapies. However, key obstacles remain, in particular delivery of oxygen at the appropriate dose and with optimal pharmacokinetics. Toward overcoming these hurdles, gas-entrapping materials (GeMs) that are capable of tunable oxygen release are formulated. It is shown that injection or implantation of these materials into tumors can mitigate tumor hypoxia by delivering oxygen locally and that these GeMs enhance responsiveness to radiation and chemotherapy in multiple tumor types. This paper also demonstrates, by comparing an oxygen (O2 )-GeM to a sham GeM, that the former generates an antitumorigenic and immunogenic tumor microenvironment in malignant peripheral nerve sheath tumors. Collectively the results indicate that the use of O2 -GeMs is promising as an adjunctive strategy for the treatment of solid tumors.

Augmenting chemotherapy with low-dose decitabine through an immune-independent mechanism.

The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pretreatment for anticancer therapies. In a variation of this idea, this study explores the concept of adding low-dose decitabine (DAC) following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of DAC following treatment with the chemotherapy agent gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, DAC did not induce a robust antitumor T cell response in sarcoma. Furthermore, DAC synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces biphasic cell cycle arrest and apoptosis. Therapeutic efficacy was sequence dependent, with gemcitabine priming cells for treatment with DAC through inhibition of ribonucleotide reductase. This study identifies an apparently unique application of DAC to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising paradigm for cancer treatment by augmenting chemotherapy through addition of DAC to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.

PRC2 loss drives MPNST metastasis and matrix remodeling.

The histone methyltransferase PRC2 plays a complex role in cancer. Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with frequent loss-of-function mutations in PRC2 that are associated with poor outcome. Here, we identify a critical role for PRC2 loss in driving MPNST metastasis. PRC2-dependent metastatic phenotypes included increased collagen-dependent invasion, upregulation of matrix-remodeling enzymes, and elevated lung metastasis in orthotopic mouse models. Furthermore, clinical sample analysis determined that PRC2 loss correlated with metastatic disease, increased fibrosis, and decreased survival in patients with MPNSTs. These results may have broad implications for PRC2 function across multiple cancers and provide a strong rationale for investigating potential therapies targeting ECM-remodeling enzymes and tumor fibrosis to improve outcomes in patients with MPNSTs.

Utility of CD138/syndecan-1 immunohistochemistry for localization of plasmacytes is tissue-dependent in B6 mice.

OBJECTIVE: Inflammation is present in many diseases and identification of immune cell infiltration is a common assessment. CD138 (syndecan-1) is a recommended immunohistochemical marker for human plasmacytes although it is also expressed in various epithelia and tumors. Similarly, CD138 is a marker for murine plasmacytes, but its tissue immunostaining is not well-defined. Endogenous CD138 expression is an important confounding factor when evaluating plasmacyte infiltration. We studied two plasmacyte markers (CD138 and Kappa light chains) for endogenous immunostaining in five organs and one tumor from B6 mice. RESULTS: Plasmacytes in Peyer's patches were positive for CD138 and Kappa markers without endogenous immunostaining. Endogenous CD138 immunostaining was widespread in liver, kidney, lung and a malignant peripheral nerve sheath tumor (MPNST) versus regionalized immunostaining in skin and small intestine wall. Endogenous Kappa immunostaining was absent in all tissues except for plasmacytes. Tissues with widespread endogenous CD138 immunostaining were contrasted by absence of endogenous Kappa immunostaining. Here, plasmacytes would not be distinguished by CD138, but would be obvious by Kappa immunostaining. Our study suggests that utility of immunostaining for plasmacytes by CD138 is tissue dependent in mice. Additionally, Kappa immunostaining may be a useful alternative in mouse tissues with confounding endogenous CD138 immunostaining.

Oncogenic RABL6A promotes NF1-associated MPNST progression in vivo.

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted along with NF1, a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of MPNSTs is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown. Methods: Using CRISPR-Cas9 targeting of Nf1 + Cdkn2a or Nf1 + Tp53 in the mouse sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Terminal tumors were evaluated by western blot, qRT-PCR, and immunohistochemistry. Results: Mice lacking Rabl6 displayed slower tumor progression and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in Rabl6-null, Nf1 + Cdkn2a lesions whereas loss of RABL6A caused upregulation of the CDK inhibitor, p27, in all tumors. Paradoxically, both models displayed elevated Myc protein and Ki67 staining in terminal tumors lacking RABL6A. In Nf1 + p53 tumors, cellular atypia and polyploidy were evident and increased by RABL6A loss. Conclusions: These findings demonstrate that RABL6A is required for optimal progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for unwanted alterations, including increased Myc, cellular atypia, and polyploidy, that ultimately promote a hyper-proliferative tumor phenotype akin to drug-resistant lesions.

Epigenetic modulation in sensitizing metastatic sarcomas to therapies and overcoming resistance.

Sarcomas are a class of rare malignancies of mesenchymal origin with a heterogeneous histological spectrum. They are classically associated with poor outcomes, especially once metastasized. A path to improving clinical outcomes may be made through modifying the epigenome, where a variety of sarcomas demonstrate changes that contribute to their oncogenic phenotypes. This Perspective article identifies and describes changes in the sarcoma genome, while discussing specific epigenetic changes and their effect on clinical outcomes. Clinical attempts at modulating epigenetics in sarcoma are reviewed, as well as potential implications of these studies. Epigenetic targets to reverse and delay chemotherapy resistance are discussed. Future directions with primary next steps are proposed to invigorate the current understanding of epigenetic biomarkers to enact targeted therapies to epigenetic phenotypes of sarcoma subtypes. Modifications to prior studies, as well as proposed clinical steps, are also addressed.

Divergent immune landscapes of primary and syngeneic Kras-driven mouse tumor models.

Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. Both genetically engineered mouse models (GEMMs) and syngeneic cell transplant approaches use immunocompetent mice to define immune-dependent events in tumor development and progression. Due to their rapid and reproducible nature, there is expanded interest in developing new syngeneic tools from established primary tumor models. However, few studies have examined the extent that syngeneic tumors reflect the immune profile of their originating primary models. Here, we describe comprehensive immunophenotyping of two well-established GEMMs and four new syngeneic models derived from these parental primary tumors. To our knowledge, this is the first systematic analysis comparing immune landscapes between primary and orthotopic syngeneic tumors. These models all use the same well-defined human-relevant driver mutations, arise at identical orthotopic locations, and are generated in mice of the same background strain. This allows for a direct and focused comparison of tumor immune landscapes in carefully controlled mouse models. We identify key differences between the immune infiltrate of GEMM models and their corresponding syngeneic tumors. Most notable is the divergence of T cell populations, with different proportions of CD8+ T cells and regulatory T cells across several models. We also observe immune variation across syngeneic tumors derived from the same primary model. These findings highlight the importance of immune variance across mouse modeling approaches, which has strong implications for the design of rigorous and reproducible translational studies.

Combination therapies for MPNSTs targeting RABL6A-RB1 signaling.

Precision medicine relies on a detailed molecular understanding of disease pathogenesis. Here, we consider urgently needed therapeutic options for malignant peripheral nerve sheath tumors (MPNSTs) based on emerging insights into druggable pathway alterations found to drive this deadly cancer. Recent observations demonstrate an essential role for an oncogenic GTPase, RABL6A, in promoting MPNST progression through hyperactivation of cyclin-dependent kinases (CDKs) and inactivation of the retinoblastoma (RB1) tumor suppressor. Monotherapies with CDK4/6 inhibitors have shown limited efficacy and durability in pre-clinical studies of MPNSTs and in clinical studies of other tumors. Therefore, we discuss the rationale and clinical benefits of inhibiting multiple RABL6A effectors, particularly CDK4/6 and MEK kinases, in targeted combination therapies suitable for MPNSTs and other Ras-driven malignancies.

Locally invasive, castrate-resistant prostate cancer in a Pten/Trp53 double knockout mouse model of prostate cancer monitored with non-invasive bioluminescent imaging.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

Special Issue: "Genomics and Models of Nerve Sheath Tumors".

Nerve sheath tumors arising in the context of neurofibromatosis type 1 (NF1) include benign tumors such as cutaneous, diffuse and plexiform neurofibromas; atypical neurofibromas or atypical neurofibromatosis neoplasms of uncertain biological potential (ANNUBP); and the aggressive soft tissue sarcoma, the malignant peripheral nerve sheath tumor (MPNST) [...].

Tumor Subtype Determines Therapeutic Response to Chimeric Polypeptide Nanoparticle-based Chemotherapy in Pten-deleted Mouse Models of Sarcoma.

PURPOSE: Nanoparticle-encapsulated drug formulations can improve responses to conventional chemotherapy by increasing drug retention within the tumor and by promoting a more effective antitumor immune response than free drug. New drug delivery modalities are needed in sarcomas because they are often chemoresistant cancers, but the rarity of sarcomas and the complexity of diverse subtypes makes it challenging to investigate novel drug formulations. EXPERIMENTAL DESIGN: New drug formulations can be tested in animal models of sarcomas where the therapeutic response of different formulations can be compared using mice with identical tumor-initiating mutations. Here, using Cre/loxP and CRISPR/Cas9 techniques, we generated two distinct mouse models of Pten-deleted soft-tissue sarcoma: malignant peripheral nerve sheath tumor (MPNST) and undifferentiated pleomorphic sarcoma (UPS). We used these models to test the efficacy of chimeric polypeptide doxorubicin (CP-Dox), a nanoscale micelle formulation, in comparison with free doxorubicin. RESULTS: The CP-Dox formulation was superior to free doxorubicin in MPNST models. However, in UPS tumors, CP-Dox did not improve survival in comparison with free doxorubicin. While CP-Dox treatment resulted in elevated intratumoral doxorubicin concentrations in MPNSTs, this increase was absent in UPS tumors. In addition, elevation of CD8+ T cells was observed exclusively in CP-Dox-treated MPNSTs, although these cells were not required for full efficacy of the CP nanoparticle-based chemotherapy. CONCLUSIONS: These results have important implications for treating sarcomas with nanoparticle-encapsulated chemotherapy by highlighting the tumor subtype-dependent nature of therapeutic response.

Distinct Tumor Microenvironments Are a Defining Feature of Strain-Specific CRISPR/Cas9-Induced MPNSTs.

The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype-phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is observed in C57BL/6, 129X1 and 129/SvJae mice. Indel pattern analysis demonstrates that indel frequency, type and size are similar across all genetic backgrounds. Gene expression and IHC analysis identify multiple strain-dependent differences in CD4+ T cell infiltration and myeloid cell populations, including M2 macrophages and mast cells. These data highlight important strain-specific phenotypes of genomically-matched MPNSTs that have implications for the design of future studies using similar in vivo gene editing approaches.

CDKs in Sarcoma: Mediators of Disease and Emerging Therapeutic Targets.

Sarcomas represent one of the most challenging tumor types to treat due to their diverse nature and our incomplete understanding of their underlying biology. Recent work suggests cyclin-dependent kinase (CDK) pathway activation is a powerful driver of sarcomagenesis. CDK proteins participate in numerous cellular processes required for normal cell function, but their dysregulation is a hallmark of many pathologies including cancer. The contributions and significance of aberrant CDK activity to sarcoma development, however, is only partly understood. Here, we describe what is known about CDK-related alterations in the most common subtypes of sarcoma and highlight areas that warrant further investigation. As disruptions in CDK pathways appear in most, if not all, subtypes of sarcoma, we discuss the history and value of pharmacologically targeting CDKs to combat these tumors. The goals of this review are to (1) assess the prevalence and importance of CDK pathway alterations in sarcomas, (2) highlight the gap in knowledge for certain CDKs in these tumors, and (3) provide insight into studies focused on CDK inhibition for sarcoma treatment. Overall, growing evidence demonstrates a crucial role for activated CDKs in sarcoma development and as important targets for sarcoma therapy.

RABL6A Is an Essential Driver of MPNSTs that Negatively Regulates the RB1 Pathway and Sensitizes Tumor Cells to CDK4/6 Inhibitors.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs. EXPERIMENTAL DESIGN: We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors. RESULTS: RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G1 arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in vitro in an RABL6A-dependent manner and suppressed MPNST growth in vivo. Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation. CONCLUSIONS: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.