Payal Jain

MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

CRAF gene fusions in pediatric low-grade gliomas define a distinct drug response based on dimerization profiles.

Pediatric low-grade gliomas (PLGGs) are commonly associated with BRAF gene fusions that aberrantly activate the mitogen-activated protein kinase (MAPK) signaling pathway. This has led to PLGG clinical trials utilizing RAF- and MAPK pathway-targeted therapeutics. Whole-genome profiling of PLGGs has also identified rare gene fusions involving another RAF isoform, CRAF/RAF1, in PLGGs and cancers occuring in adults. Whereas BRAF fusions primarily dysregulate MAPK signaling, the CRAF fusions QKI-RAF1 and SRGAP3-RAF1 aberrantly activate both the MAPK and phosphoinositide-3 kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathways. Although ATP-competitive, first-generation RAF inhibitors (vemurafenib/PLX4720, RAFi) cause paradoxical activation of the MAPK pathway in BRAF-fusion tumors, inhibition can be achieved with ‘paradox breaker’ RAFi, such as PLX8394. Here we report that, unlike BRAF fusions, CRAF fusions are unresponsive to both generations of RAFi, vemurafenib and PLX8394, highlighting a distinct responsiveness of CRAF fusions to clinically relevant RAFi. Whereas PLX8394 decreased BRAF-fusion dimerization, CRAF-fusion dimerization is unaffected primarily because of robust protein–protein interactions mediated by the N-terminal non-kinase fusion partner, such as QKI. The pan-RAF dimer inhibitor, LY3009120, could suppress CRAF-fusion oncogenicity by inhibiting dimer-mediated signaling. In addition, as CRAF fusions activate both the MAPK and PI3K/mTOR signaling pathways, we identify combinatorial inhibition of the MAPK/mTOR pathway as a potential therapeutic strategy for CRAF-fusion-driven tumors. Overall, we define a mechanistic distinction between PLGG-associated BRAF- and CRAF/RAF1 fusions in response to RAFi, highlighting the importance of molecularly classifying PLGG patients for targeted therapy. Furthermore, our study uncovers an important contribution of the non-kinase fusion partner to oncogenesis and potential therapeutic strategies against PLGG-associated CRAF fusions and possibly pan-cancer CRAF fusions.

Shared ACVR1 mutations in FOP and DIPG: Opportunities and challenges in extending biological and clinical implications across rare diseases

Gain-of-function mutations in the Type I Bone Morphogenic Protein (BMP) receptor ACVR1 have been identified in two diseases: Fibrodysplasia Ossificans Progressiva (FOP), a rare autosomal dominant disorder characterized by genetically driven heterotopic ossification, and in 20-25% of Diffuse Intrinsic Pontine Gliomas (DIPGs), a pediatric brain tumor with no effective therapies and dismal median survival. While the ACVR1 mutation is causal for FOP, its role in DIPG tumor biology remains under active investigation. Here, we discuss cross-fertilization between the FOP and DIPG fields, focusing on the biological mechanisms and principles gleaned from FOP that can be applied to DIPG biology. We highlight our current knowledge of ACVR1 in both diseases, and then describe the growing opportunities and barriers to effectively investigate ACVR1 in DIPG. Importantly, learning from other seemingly unrelated diseases harboring similar mutations may uncover novel mechanisms or processes for future investigation.

Overcoming resistance to single-agent therapy for oncogenic BRAF gene fusions via combinatorial targeting of MAPK and PI3K/mTOR signaling pathways

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

MYB-QKI drives childhood brain tumors via tripartite mechanism

Payal Jain and Adam C. Resnick Center for Data Driven Discovery in Biomedicine (D3b), The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA; Division of Neurosurgery, The Children’s Hospital of Philadelphia, PA, USA; Center of Childhood Cancer Research, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

Novel FGFR2-INA fusion identified in two low-grade mixed neuronal-glial tumors drives oncogenesis via MAPK and PI3K/mTOR pathway activation

As a group, mixed neuronal-glial tumors (MNGTs) exhibit genetic variability, including stable genomes, whole chromosome gains, BRAF-V600E, and FGFR1 mutations [8, 9, 11, 12]. While histologic criteria are described to distinguish MNGT types ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNT), non-specific features preclude confident classification in a high proportion of cases [2, 8, 10, 12]. Herein, we report the characterization of a novel FGFR2-INA fusion gene identified during clinical genomic profiling in two cases of MNGTs that could not be specifically classified as GG or DNT. Clinical, imaging, histology, and fusion gene characteristics of each case are summarized in suppl. Table 1 (Online Resource 1). Both patients presented with seizures, cortical-based tumors, and one patient’s tumor was recurrent. By histology and immunohistochemistry, both cases consisted of oligodendrocyte-like cells and admixed neurons within microcytic spaces (Fig. 1a). GFAP-positive astrocytes, CD34 expression (MNGT-1), and calcification were observed. Both cases lacked pools of mucin, floating neurons, specific glioneuronal elements, eosinophilic granular bodies, and perivascular inflammation. Features were most similar to DNT; however, both lacked key criteria for this diagnosis. Targeted RNA-sequencing revealed a novel in-frame fusion between FGFR2 exon 17 and INA exon 2 (Fig. 1b) in both cases. Additional DNA sequence and copy number variants of clinical significance were also identified by targeted next-generation sequence panel [suppl. Tables 2, 3, 4 (Online Resource 1)] [7]. FGFR2, a receptor kinase, regulates several growth-related signaling pathways implicated in cancer progression, including RAS-RAF-MAPK and PI3K/AKT/mTOR [3]. INA encodes the alpha-internexin protein involved in cytoskeletal organization and neuronal morphogenesis [6]. The novel fusion retains the extracellular immunoglobin-like and tyrosine kinase domains of FGFR2, suggesting oncogenic activation of downstream signaling, and the truncated coil 2 and tail region of INA, suggesting dimerization (Fig. 1c). Payal Jain and Lea F. Surrey are co-first authors and contributed equally.

Abstract 4375: Pediatric low-grade gliomas with CRAF fusions respond differentially to targeted therapeutics based on their dimerization profiles

INTRODUCTION: Recent studies have identified QKI-RAF1 and SRGAP3-RAF1 as CRAF (or RAF1) fusions in pediatric low-grade gliomas (PLGGs). CRAF fusions, like BRAF fusions are activating mutations driving the mitogen activated protein kinase (MAPK) pathway. Our previous findings suggest effective inhibition of BRAF fusion driven tumors using second-generation RAF inhibitor, PLX8394 and downstream MEK inhibitors (MEKi). We sought to investigate the mechanistic basis of response of CRAF fusions to clinically relevant RAF inhibitors and downstream pathway inhibitors, studying effect on CRAF dimerization profiles. METHODS: Heterologous cell model systems with stable expression of CRAF fusions were generated and used for testing downstream signaling pathways via immunoblotting. Soft agar assays and flank xenografts in immuno-compromised mice were used to characterize the oncogenic properties of CRAF fusions. Pathway activation and oncogenicity were further assessed in the presence of first - and second-generation RAF inhibitors, PLX4720 and PLX8394 respectively, MEKi, and mTOR inhibitors as single agents or in combination. Myc- and Flag-tagged constructs of CRAF fusions were used in co-immunoprecipitation assays to assess dimerization profiles of CRAF fusions with or without inhibitors. RESULTS: CRAF fusions activated the MAPK and PI3K pathways. CRAF fusions can homo-dimerize as well as hetero-dimerize with full-length BRAF, CRAF and the N-terminal fusion partner protein. Compared to BRAF fusions, CRAF fusions were not found to be responsive to any RAF inhibitors tested, including the paradox breaker PLX8394. We found that while PLX8394 decreased BRAF fusion-mediated dimerization, dimerization of CRAF fusion is unaffected. Clinically relevant MEKi AZD6244 and GSK1120212 both suppressed CRAF fusion driven pathways and growth in vitro but in mice flank xenografts, GSK1120212 partially inhibited CRAF fusion driven tumors. Since the CRAF fusions also activate the PI3K pathway, combinatorial targeting using MEKi and mTOR inhibitor, GSK1120212 and RAD001 respectively, was found to show robust tumor inhibition in vivo. CONCLUSIONS: This study demonstrates that CRAF fusions do not respond to RAF inhibitors, show partial response to single-agent MEKi, and respond robustly to combinatorial targeting of both MAPK and PI3K pathways via GSK1120212 and RAD001. Since PLX8394 does not affect CRAF fusion mediated dimerization, this provides a mechanistic basis for unresponsiveness to RAF inhibitors. Additionally, the N-terminal fusion partner also contributes to dimerization of CRAF fusions. Therefore, CRAF fusions are distinct from BRAF fusions in terms of responsiveness to targeted therapies due to dimerization profiles. These findings suggest molecular classification of PLGGs prior to treatment and provide preclinical rationale for combination therapy of CRAF fusion expressing PLGGs. Citation Format: Payal Jain, Tamara Fierst, Amanda Silva, Jake Budlow, Harry Han, Phillip B. Storm, Angela Waanders, Adam Resnick. Pediatric low-grade gliomas with CRAF fusions respond differentially to targeted therapeutics based on their dimerization profiles. [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 4375.

Abstract 4372: MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism

Diffuse pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children. While BRAF mutations and MYBL1 rearrangements have recently been identified as diagnostic and treatment related oncogenic drivers in pediatric gangliogliomas and diffuse astrocytomas, respectively, for the majority of diffuse PLGGs the oncogenic driver(s) remain unknown. Recent efforts to identify new oncogenic drivers lack sufficient power to determine the true frequency of alterations in genes or to associate specific alterations with rare histological subtypes, such as Angiocentric gliomas (AGs). To address this issue, we performed genomic analysis of new and published data from 249 PLGGs including 19 AGs. We identified MYB-QKI fusions as a specific, recurrent, single candidate driver event in AGs. Although MYB is expressed during early brain development in a subset of progenitor cells, it is not known to play a role in normal cortical brain where AGs frequently occur. In vitro functional studies show MYB-QKI rearrangements promote tumorigenesis in mouse neural stem cells (mNSCs) through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression, and hemizygous loss of the tumor suppressor QKI. Expression of the MYB-QKI fusion increases mNSC proliferation and activates known MYB target genes including KIT and CDK6. H3K27ac enhancer profiling of AG tumors demonstrate active enhancer elements are translocated proximally to the MYB promoter while in vitro promoter assays confirmed that QKI enhancer sequences activate the MYB promoter. Together with functional studies demonstrating that MYB-QKI can also bind and activate the MYB promoter, these data support a positive auto-regulatory feed-back loop model in which active MYB-QKI is able to drive its own expression through enhanced MYB-promoter-activation. Furthermore, when injected orthotopically into immunodeficient mice, expression of truncated MYB or MYB-QKI is sufficient to induce tumor formation that recapitulate histologic and immunophenotypic characteristics of AGs. Analysis of AG specific MYB-QKI fusion represents the first example of how a single driver rearrangement simultaneously transforms cells via three genetic and epigenetic mechanisms in a cancer. Citation Format: Lori Ramkissoon, Pratiti Bandopadhayay, Payal Jain, Guillaume Bergthold, Adam Resnick, Rameen Beroukhim, Keith Ligon. MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism. [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 4372.

Abstract A138: Targeting activated CRAF fusions in pediatric low-grade gliomas

This abstract has been withheld from publication due to its inclusion in the AACR-NCI-EORTC Molecular Targets Conference 2015 Official Press Program. It will be posted online at the time of its presentation in a press conference or in a session: 10:00 AM ET Friday, November 6. Citation Format: Payal Jain, Tamara Fierst, Amanda Silva, Jake Budlow, Katie Boucher, Harry Han, Phillip B. Storm, Angela Waanders, Adam C. Resnick. Targeting activated CRAF fusions in pediatric low-grade gliomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A138.