Aparna Kaul

Pediatric glioma-associated KIAA1549:BRAF expression regulates neuroglial cell growth in a cell type-specific and mTOR-dependent manner

Tandem duplications involving the BRAF kinase gene have recently been identified as the most frequent genetic alteration in sporadic pediatric glioma, creating a novel fusion protein (f-BRAF) with increased BRAF activity. To define the role of f-BRAF in gliomagenesis, we demonstrate that f-BRAF regulates neural stem cell (NSC), but not astrocyte, proliferation and is sufficient to induce glioma-like lesions in mice. Moreover, f-BRAF-driven NSC proliferation results from tuberin/Rheb-mediated mammalian target of rapamycin (mTOR) hyperactivation, leading to S6-kinase-dependent degradation of p27. Collectively, these results establish mTOR pathway activation as a key growth regulatory mechanism common to both sporadic and familial low-grade gliomas in children.

Akt- or MEK-mediated mTOR inhibition suppresses Nf1 optic glioma growth

BACKGROUND Children with neurofibromatosis type 1 (NF1) develop optic pathway gliomas, which result from impaired NF1 protein regulation of Ras activity. One obstacle to the implementation of biologically targeted therapies is an incomplete understanding of the individual contributions of the downstream Ras effectors (mitogen-activated protein kinase kinase [MEK], Akt) to optic glioma maintenance. This study was designed to address the importance of MEK and Akt signaling to Nf1 optic glioma growth. METHODS Primary neonatal mouse astrocyte cultures were employed to determine the consequence of phosphatidylinositol-3 kinase (PI3K)/Akt and MEK inhibition on Nf1-deficient astrocyte growth. Nf1 optic glioma-bearing mice were used to assess the effect of Akt and MEK inhibition on tumor volume, proliferation, and retinal ganglion cell dysfunction. RESULTS Both MEK and Akt were hyperactivated in Nf1-deficient astrocytes in vitro and in Nf1 murine optic gliomas in vivo. Pharmacologic PI3K or Akt inhibition reduced Nf1-deficient astrocyte proliferation to wild-type levels, while PI3K inhibition decreased Nf1 optic glioma volume and proliferation. Akt inhibition of Nf1-deficient astrocyte and optic glioma growth reflected Akt-dependent activation of mammalian target of rapamycin (mTOR). Sustained MEK pharmacologic blockade also attenuated Nf1-deficient astrocytes as well as Nf1 optic glioma volume and proliferation. Importantly, these MEK inhibitory effects resulted from p90RSK-mediated, Akt-independent mTOR activation. Finally, both PI3K and MEK inhibition reduced optic glioma-associated retinal ganglion cell loss and nerve fiber layer thinning. CONCLUSION These findings establish that the convergence of 2 distinct Ras effector pathways on mTOR signaling maintains Nf1 mouse optic glioma growth, supporting the evaluation of pharmacologic inhibitors that target mTOR function in future human NF1-optic pathway glioma clinical trials.

Novel BRAF Alteration in a Sporadic Pilocytic Astrocytoma

Pilocytic astrocytoma (PA) is the most frequently encountered glial tumor (glioma or astrocytoma) in children. Recent studies have identified alterations in the BRAF serine/threonine kinase gene as the likely causative mutation in these childhood brain tumors. The majority of these genetic changes involve chromosome 7q34 tandem duplication, resulting in aberrant BRAF fusion transcripts. In this paper, we describe a novel KIAA1549:BRAF fusion transcript in a sporadic PA tumor associated with increased ERK activation and review the spectrum of BRAF genetic alterations in this common pediatric low-grade central nervous system neoplasm.

Conditional KIAA1549:BRAF mice reveal brain region- and cell type-specific effects.

Low‐grade brain tumors (pilocytic astrocytomas) that result from a genomic rearrangement in which the BRAF kinase domain is fused to the amino terminal of the KIAA1549 gene (KIAA1549:BRAF fusion; f‐BRAF) commonly arise in the cerebellum of young children. To model this temporal and spatial specificity in mice, we generated conditional KIAA1549:BRAF strains that coexpresses green fluorescent protein (GFP). Although both primary astrocytes and neural stem cells (NSCs) from these mice express f‐BRAF and GFP as well as exhibit increased MEK activity, only f‐BRAF‐expressing NSCs exhibit increased proliferation in vitro. Using Cre driver lines in which KIAA1549:BRAF expression was directed to NSCs (f‐BRAF; BLBP‐Cre mice), astrocytes (f‐BRAF; GFAP‐Cre mice), and NG2 progenitor cells (f‐BRAF; NG2‐Cre mice), increased glial cell numbers were observed only in the cerebellum of f‐BRAF; BLBP‐Cre mice in vivo. The availability of this unique KIAA1549:BRAF conditional transgenic mouse strain will enable future mechanistic studies aimed at defining the developmentally–regulated temporal and spatial determinants that underlie low‐grade astrocytoma formation in children. genesis 51:708–716.

The impact of coexisting genetic mutations on murine optic glioma biology.

BACKGROUND Children with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome are prone to the development of optic pathway gliomas resulting from biallelic inactivation of the NF1 gene. Recent studies have revealed the presence of other molecular alterations in a small portion of these NF1-associated brain tumors. The purpose of this study was to leverage Nf1 genetically engineered mouse strains to define the functional significance of these changes to optic glioma biology. METHODS Nf1+/- mice were intercrossed with Nf1(flox/flox) mice, which were then crossed with Nf1(flox/flox); GFAP-Cre mice, to generate Nf1(flox/mut); GFAP-Cre (FMC) mice. These mice were additionally mated with conditional KIAA1549:BRAF knock-in or Pten(flox/wt) mice to generate Nf1(flox/mut); f-BRAF; GFAP-Cre (FMBC) mice or Nf1(flox/mut); Pten(flox/wt); GFAP-Cre (FMPC) mice, respectively. The resulting optic gliomas were analyzed for changes in tumor volume, proliferation, and retinal ganglion cell loss. RESULTS While KIAA1549:BRAF conferred no additional biological properties on Nf1 optic glioma, FMPC mice had larger optic gliomas with greater proliferative indices and microglial infiltration. In addition, all 3 Nf1 murine optic glioma strains exhibited reduced retinal ganglion cell survival and numbers; however, FMPC mice had greater retinal nerve fiber layer thinning near the optic head relative to FMC and FMBC mice. CONCLUSIONS Collectively, these experiments demonstrate genetic cooperativity between Nf1 loss and Pten heterozygosity relevant to optic glioma biology and further underscore the value of employing genetically engineered mouse strains to define the contribution of discovered molecular alterations to brain tumor pathogenesis.

Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord

Recent studies have demonstrated that the umbilical cord (UC) is an excellent source of mesenchymal stromal cells (MSCs). However, current protocols for extracting and culturing UC-MSCs do not meet current good manufacturing practice (cGMP) standards, in part due to the use of xenogeneic reagents. To support the development of a cGMP-compliant method, we have examined an enzyme-free isolation method utilizing tissue homogenization (t-H) followed by culture in human platelet lysate (PL) supplemented media. The yield and viability of cells after t-H were comparable to those obtained after collagenase digestion (Col-D). Importantly, kinetic analysis of cultured cells showed logarithmic growth over 10 tested passages, although the rate of cell division was lower for t-H as compared to Col-D. This slower growth of t-H-derived cells was also reflected in their longer population doubling time. Interestingly, there was no difference in the expression of mesenchymal markers and trilineage differentiation potential of cells generated using either method. Finally, t-H-derived cells had greater clonogenic potential compared to Col-D/FBS but not Col-D/PL and were able to maintain CFU-F capacity through P7. This bench scale study demonstrates the possibility of generating therapeutic doses of good quality UC-MSCs within a reasonable length of time using t-H and PL.

Abstract 2322: The KIAA1549:BRAF fusion gene regulates mTOR signaling and gliomagenesis in a cell type- and brain region-specific manner.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The two most common genetic alterations in low-grade pediatric brain tumors (glioma) are NF1 gene inactivation and tandem duplications involving the BRAF kinase gene. In this study, we sought to define the role of KIAA1549:BRAF fusion (fusion BRAF; f-BRAF) gene in glioma development. We demonstrate that f-BRAF regulates cerebellar NSC proliferation by activating the mTOR pathway downstream of MAPK. Also, f-BRAF expression in cerebellar neuroglial progenitors is sufficient to induce glioma-like lesions in mice. Interestingly, f-BRAF expression does not increase the proliferation of NSCs derived from other brain regions, nor does it increase primary astrocyte proliferation. These brain region- and cell type-specific growth promoting effects of f-BRAF are mediated by tuberin/Rheb-dependent modulation of mTOR, leading to S6-kinase activation and p27 degradation. Finally, we demonstrate that mTOR activation is also observed in fusion BRAF-associated human sporadic pilocytic astrocytomas. Our findings highlight the importance of spatial, cellular and molecular heterogeneity in brain tumor development, and provide compelling preclinical evidence for the use of mTOR pathway inhibitors for both sporadic and familial low-grade gliomas. Citation Format: Aparna Kaul, Yi-Hsien Chen, Ryan J. Emnett, Sonika Dahiya, David Gutmann. The KIAA1549:BRAF fusion gene regulates mTOR signaling and gliomagenesis in a cell type- and brain region-specific manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2013-2322

Abstract 2498: Differential rapamycin sensitivity distinguishes two distinct populations of tumor cells in murine glioma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Solid tumors are complex ecosystems, composed of a heterogeneous ensemble of cells that comprise the tumor mass (neoplastic compartment) and the surrounding stroma (non-neoplastic compartment). Currently, research efforts are aimed at defining the individual contributions of each cell type to tumor development and maintenance. Previous studies using a genetically-engineered mouse model of Neurofibromatosis type 1 (NF1)-associated optic glioma have underscored the critical role of one stromal cell type (microglia) in the growth of these tumors. In the current study, we analyze neoplastic cell heterogeneity and demonstrate that two distinct proliferating populations of neoplastic cells exist in the tumor. These distinct cell types, neuroglial progenitors and differentiated astrocytes, are distinguished by their sensitivity to rapamycin. The molecular basis for this differential sensitivity to rapamycin reflects dramatic differences in mammalian target of rapamycin (mTOR) complex protein expression and basal mTOR activation. We leverage this cell type-specific sensitivity to target neuroglial progenitor cells in the tumor and inhibit optic glioma formation in vivo. Collectively, these findings emphasize the importance of cellular diversity in glioma formation and growth, and highlight the importance of developing novel treatment strategies that co-target multiple cell populations for maximal therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2498. doi:1538-7445.AM2012-2498