Many pediatric brain tumors, including malignant astrocytomas (MA), are thought to originate from neural stem cells (NSCs), which, due to the occurrence and accumulation of growth-promoting gene alterations, may give rise to various cell subpopulations, including tumor-initiating cells (TICs). TICs are considered to have increased resistance to conventional therapy for MA, and consequently are important contributors to MA recurrence. Whereas NSCs undergo asymmetric cell divisions (ACD) to self-renew and differentiate at a one-to-one ratio, TICs proliferate and self-renew, and fail to generate fully differentiated cells, suggestive of defective ACD. A mutant, activated form of BRAF, BRAFVE, and concomitant homozygous deletion of CDKN2A, encoding p16, have been found in a significant fraction of pediatric MA. BRAFVE is known to promote proliferation while suppressing normal cellular differentiation. Whether increased production of self-renewing cells through increasing symmetric cell divisions (i.e., decreased ACD) is manifested in association with BRAFVE induced cell transformation is currently unknown. To bridge the gap in our understanding of BRAFVE-induced transformation, and its relationship with ACD, we will examine effects of BRAFVE in p16 deficient NSCs as well as in corresponding p16 deficient astrocytes of mouse and human origin. In addition, we will investigate relationships between BRAFVE-p16 deficient tumors and their adaptation to BRAFVE targeted therapy, with our primary focus directed to tumor cell subpopulation and ACD changes resulting from treatment. Related research will be performed in the context of the following specific aims.
Aim 1. Using genetically engineered mouse models (GEMMs), we will determine effects of BRAFVE expression on ACD, proliferation, differentiation, and survival, and association with MA tumorigenesis in NSCs and mature astrocytes.
Aim 2. To complement the GEMMs studies in aim 1, we will suppress p16 expression and force BRAFVE expression in human NSCs and normal human astrocytes, using lentiviral shRNA knockdown and BRAFVE gene transfer, respectively. Modified NCS and NHAs as well as MA cells with BRAFVE expression will be characterized, both in vitro and in vivo, for the same characteristics as for the mouse model tumors in aim 1.
Aim 3. Investigate BRAFVE tumor cells and tumor tissues, in vitro and in vivo, respectively, for molecular changes, TIC composition, and ACD in association with response to BRAFVE targeted therapy. This research will include comparison of effects when tumors are in a responsive phase to therapy, as well as when they have acquired resistance to therapy, and will utilize both human tumor xenograft and mouse allograft models. Our project will: 1) generate new information regarding the cellular origin of BRAFVE induced MA; 2) provide insight about the molecular mechanisms of neoplastic transformation resulting in brain tumor development; 3) increase our understanding of brain tumor cell subpopulations that are responsible for therapy resistance and tumor recurrence, and in so doing, 4) will ultimately lead to improved treatment outcomes for MA patients.
Despite the investigation of numerous novel therapeutics and treatment approaches, the outcome for pediatric malignant astrocytoma (MA) patients has seen little improvement over several decades of basic, preclinical, and clinical research. Our research is expected to 1) inform regarding the cellular origin of MA; 2) provide new insight about the molecular mechanisms of neoplastic transformation, especially those involving asymmetric cell division (ACD), that promote brain tumor development; 3) increase our understanding of brain tumor cell subpopulations responsible for therapy resistance and tumor recurrence, and as such our studies will 4) ultimately facilitate the identification of molecular targets for improved MA treatment.
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