PIK3CA missense mutations promote glioblastoma pathogenesis, but do not enhance targeted PI3K inhibition

Background Glioblastoma (GBM) is the most common adult primary brain tumor. Multimodal treatment is empiric and prognosis remains poor. Recurrent PIK3CA missense mutations (PIK3CAmut) in GBM are restricted to three functional domains: adaptor binding (ABD), helical, and kinase. Defining how these mutations influence gliomagenesis and response to kinase inhibitors may aid in the clinical development of novel targeted therapies in biomarker-stratified patients. Methods We used normal human astrocytes immortalized via expression of hTERT, E6, and E7 (NHA). We selected two PIK3CAmut from each of 3 mutated domains and induced their expression in NHA with (NHARAS) and without mutant RAS using lentiviral vectors. We then examined the role of PIK3CAmut in gliomagenesis in vitro and in mice, as well as response to targeted PI3K (PI3Ki) and MEK (MEKi) inhibitors in vitro. Results PIK3CAmut, particularly helical and kinase domain mutations, potentiated proximal PI3K signaling and migration of NHA and NHARAS in vitro. Only kinase domain mutations promoted NHA colony formation, but both helical and kinase domain mutations promoted NHARAS tumorigenesis in vivo. PIK3CAmut status had minimal effects on PI3Ki and MEKi efficacy. However, PI3Ki/MEKi synergism was pronounced in NHA and NHARAS harboring ABD or helical mutations. Conclusion PIK3CAmut promoted differential gliomagenesis based on the mutated domain. While PIK3CAmut did not influence sensitivity to single agent PI3Ki, they did alter PI3Ki/MEKi synergism. Taken together, our results demonstrate that a subset of PIK3CAmut promote tumorigenesis and suggest that patients with helical domain mutations may be most sensitive to dual PI3Ki/MEKi treatment.

we determined whether these mutations influenced response to single agent and combination PI3K/MEK inhibitors buparlisib and selumetinib, respectively, to elucidate the utility of PIK3-CA mut as a predictive biomarker.
Buparlisib (BKM120) has been proposed as a potential targeted therapy for GBM [30][31][32][33]. It is currently being investigated in a Phase II clinical trial in GBM patients (ClinicalTrials.gov, NCT01339052). While there have been no GBM clinical trials of selumetinib, we and others have shown its efficacy in preclinical models [34,35]. Enriching future clinical trials with likely responders based on mutational profiles promises to improve the chances of clinical success.

Supplement
Supplemental methods, figures, and tables can be found online.

Cell culture
NHA and NHA RAS lines were a kind gift from Russell O. Pieper [25]. Cells were maintained as adherent cultures at 37˚C and 5% CO 2 in DMEM supplemented with 5% FBS and 1% penicillin/streptomycin (complete DMEM). To generate NHA and NHA RAS lines expressing GFP, PIK3CA WT , or PIK3CA mut , 135,000 and 120,000 cells respectively were plated on 60 cm 2 plates. Lentiviruses were added two days after plating, then incubated with cells overnight in complete DMEM containing 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO) at 37˚C and 5% CO 2 . Two days post-infection, transduced cells were selected by culture in complete DMEM plus 300 μg/ml hygromycin B (Gold Biotechnology, St. Louis, MO) for 14 days. Stable gene expression was confirmed by immunoblot for the HA tag on PIK3CA wt and PIK3CA mut . All in vitro experiments were performed in DMEM with 2.5% FBS and 1% penicillin/streptomycin (low serum medium) unless otherwise stated.

Immunoblots
Control (parental, GFP, and PIK3CA WT ) and PIK3CA mut NHA and NHA RAS cells were treated with either vehicle control (DMSO), buparlisib and/or selumetinib, or serum starved for 24 h. Proteins were isolated and immunoblots were performed as described [19,24,35]. Raw immunoblot images are shown in Supplemental Immunoblots. Each blot included both a molecular weight ladder and a reference standard composed of lysates of cultured TRP astrocytes. Quantification was performed using the following formula, where x is an individual blot and i is the target in question: relative intensity x;i ¼ target i loading control x TRP x loading control x [19,24,35]. N = 1-3 blots, mean is denoted in the corresponding figure legend. Bands annotated in red are omitted from final figures.

Cell growth
NHA and NHA RAS were plated in triplicate or quadruplicate in 96-well tissue culture plates and absorbance (cell growth) was assessed using CellTiter 96 Aqueous One Cell Proliferation Assay (MTS, Promega, Madison, WI) according to manufacturer's instructions. Relative absorbance was measured daily as described and fit to an exponential growth equation to calculate rate constants (k) and doubling times [ln(2)/k]. Differences in growth rate constants (k) were compared using the extra-sum-of-squares F test [35].

Cell migration
Migration rate across a cell-free gap was determined using culture inserts according to manufacturer's instructions (Ibidi, Munich, Germany). Briefly, cells were imaged every 2 hours for 12 hours after creation of a cell free gap using a VistaVision inverted microscope equipped with a 4X objective and a DV-300 camera (VWR, Radnor, PA). Gap closure rates were calculated from 2-12 hours using linear regression and compared via ANCOVA.

Colony formation in soft agar
Colony formation was determined as described with minor modifications [25,41,42]. Briefly, cells were suspended in a mixture of DMEM/0.35% agarose (Denville Scientific INC., Holliston, MA) supplemented with 2.5% FBS and 14,000 cells were plated per well in 6-well plates. Cells were maintained for 4 weeks, fixed, and stained with 0.005% crystal violet in 70% ethanol. Plates were imaged on a Typhoon Trio (GE Healthcare) and colonies ! 30 μm 2 were counted.

Mouse use
This study was carried out in strict accordance with the recommendation of the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The Institutional Animal Care and Use Committee of the University of North Carolina (Chapel Hill, NC) approved this study (Protocol #16-112). Animals were housed in a SPF facility in IVC cages with enrichment of nestlets and a shelter on corn cob bedding at a density of 2-5 animals per cage. Animals were kept on a 12-hour light/dark cycle at a temperature of 21 o +/-2 o Celsius and were monitored daily by experienced laboratory staff following experimental initiation.

Orthotopic xenografts
Control and PIK3CA mut NHA RAS lines were harvested by trypsinization, counted, and suspended in serum-free DMEM with 5% methyl cellulose. Male and female adult athymic (Foxn1 nu/nu ) nude mice (Charles River, Wilmington, MA; mean age~3 months; N = 5-10 per group, mean = 9) were anesthetized with Avertin (250 mg/kg) and 2 x 10 5 GFP, PIK3CA WT , PIK3CA R88Q , PIK3CA E542K , or PIK3CA H1047R NHA RAS cells were injected into the right basal ganglia of mice (N = 5-10 per group, mean = 9) using the coordinates 1, -2, and -4 mm (A, L, D) from bregma as previously described [19,24]. Subjects received bupivacaine for local nerve block and a single dose of ketorolac for post-surgical analgesia. Animals were monitored daily for the onset of neurological symptoms (lethargy, hunching, seizures, paralysis, loss of righting reflex) and euthanized via CO 2 asphyxiation within 24 hours of onset, immediately followed by brain tissue harvest. Symptoms were frequently severe at first observation due to rapid tumor progression. Animals did not die without euthanasia. Survival was determined by Kaplan-Meier analyses and was compared by log-rank tests.

Statistics
GraphPad Prism (La Jolla, CA) was used for statistical analyses. Error bars are SEM unless otherwise stated. P 0.05 were considered significant.

Results
PIK3CA mut are frequent in GBM and are heterogeneously distributed across multiple encoded protein domains, including ABD, helical, and kinase (S1A Fig) [15,16]. PTEN deletion or activating AKT mutations cooperate with activated MAPK signaling (hereafter MAPK) to promote tumorigenesis in preclinical glioma models [19,22]. However, the role of PIK3CA mut in has not been examined in these models. To this end, we examined the 2 most frequent (hotspot) helical (E542K, E545K) and kinase (M1043V, H1047R) domain PIK3CA mut found in GBM and other cancer types (S1A-S1C Fig). ABD mutations are less prevalent in most cancers. Of these, R88Q is the most common and only recurrent mutation in GBM [4,15]. We therefore evaluated it, as well as a second, C90Y. We transduced each of 6 PIK3CA mut into NHA and NHA RAS via lentiviral vectors. Parental, GFP, or PIK3CA WT -transduced lines served as controls.

PIK3CA mut induce PI3K in vitro
Expression of PIK3CA WT and all 6 PIK3CA mut was similar in both NHA and NHA RAS (S2 Fig), suggesting that phenotypic differences would be attributable to PIK3CA mut examined. Neither PIK3CA WT nor ABD PIK3CA mut significantly activated proximal (pAKT) or distal (pS6) PI3K in serum-starved NHA (Fig 1A-1C). In contrast, E542K and H1047R significantly induced proximal and all 4 helical/kinase mutations induced distal PI3K.
We previously showed that mutant Kras cooperates with Pten deletion to activate PI3K in immortalized mouse astrocytes [19]. Therefore, we also determined the effects of PIK3CA mut on PI3K in NHA RAS . PIK3CA WT and all PIK3CA mut increased proximal PI3K compared to parental NHA RAS (Fig 1D and 1E). Furthermore, helical and kinase PIK3CA mut potentiated proximal PI3K more than PIK3CA WT . However, distal PI3K was only increased by M1043V in Representative immunoblots (A) and quantification showed that proximal PI3K (pAKT) was increased by helical and kinase mutants in NHA (B). Distal PI3K (pS6) was only increased by H1047R (C) ( Ã , P 0.02). E542K and H1047R increased proximal PI3K and all helical and kinase mutants increased distal PI3Kcompared to PIK3CA WT NHA ( ‡, P 0.04). Representative immunoblots (D) and quantification showed that proximal PI3K was increased by PIK3CA WT and all mutants (E). Helical and kinase mutants also increased proximal PI3K compared to PIK3CA WT NHA RAS ( ‡, P 0.007). Only M1043V increased distal PI3K compared to parental ( Ã , P = 0.03) and PIK3CA WT ( ‡, P = 0.03) NHA RAS (F). Bar graph data are set relative to parental lines (N = 4 biologic replicates). Fold changes in pAKT and pS6 relative to PIK3CA WT are shown as heatmaps.
NHA RAS (Fig 1D and 1F). Taken together, these results suggest that mutant RAS cooperated with ectopic expression of both PIK3CA WT and PIK3CA mut to increase proximal PI3K.
There is extensive cross-talk between PI3K and MAPK pathways [44]. We therefore determined the effects of PIK3CA mut on MAPK. Neither PIK3CA WT nor any of the PIK3CA mut significantly altered MAPK (pERK1/2) in NHA and NHA RAS (S3 Fig). Thus, PIK3CA mut activated PI3K without affecting the MAPK pathway.
PIK3CA mut induce NHA proliferation in vitro PIK3CA mut , particularly helical and kinase mutants, activated PI3K, suggesting that they may also promote cell growth (Fig 1). MTS assays using high-serum (10% FBS) showed that PIK3-CA WT and a subset of PIK3CA mut slightly increased growth rate ( 15%) of rapidly-proliferating NHA (doubling times 1 day; S4 Fig). We therefore hypothesized that growth factor concentrations in high-serum media were masking the effects of PIK3CA mut on NHA growth. Indeed, MTS assays using low-serum (2.5% FBS) revealed increased proliferation of both GFP and PIK3CA WT NHA compared to parental cells (Fig 2A and S5A Fig). While all PIK3CA mut except C90Y increased proliferation compared to parental and PIK3CA WT NHA, proliferation rates were similar in all NHA RAS lines (Fig 2B and S5B Fig). Taken together, these data suggest that PIK3CA mut promote astrocyte growth in the absence, but not presence, of mutant RAS.

PIK3CA mut induce migration in vitro
Complete surgical resection of GBM is precluded by its diffuse brain infiltration [45]. The PI3K pathway has an established role in migration [46]. We previously showed that Pten deletion increased migration of immortalized mouse astrocytes [19]. We therefore determined the effects of PIK3CA mut on migration of NHA and NHA RAS using an in vitro gap closure assay. GFP, PIK3CA WT and all PIK3CA mut showed increased migration compared to parental NHA (Fig 2C and S5C Fig). PIK3CA WT and PIK3CA mut except C90Y migrated faster than GFP NHA (P 0.04). Similarly, PIK3CA mut except C90Y migrated faster than GFP and parental NHA RAS (P 0.006, Fig 2D and S5D Fig). Moreover, E542K and H1047R PIK3CA mut migrated faster than NHA and NHA RAS overexpressing PIK3CA WT (Fig 2C and 2D).

PIK3CA mut potentiate cellular transformation and tumorigenesis
Anchorage-independent growth (colony formation in soft agar) is an established marker of cellular transformation [25,41]. NHA RAS , but not NHA, form colonies in vitro and develop high-grade tumors in orthotopic mouse xenograft models [25]. We therefore first determined whether PIK3CA mut promote NHA colony formation by selecting the most potent mutant in each domain (R88Q, E542K, H1047R) based on their effect on proximal PI3K, proliferation, and migration in NHA (Figs 1 and 2). Only H1047R induced colony formation relative to GFP and parental NHA (Fig 3A). Since this was the only mutation to potentiate NHA colony formation, we also tested its effect in NHA RAS . However, no significant increase in colony formation was evident (Fig 3A).
We next performed orthotopic xenografts of GFP, PIK3CA WT , and PIK3CA mut in NHA RAS to determine whether PIK3CA mut potentiate tumorigenesis in vivo. Mice with R88Q, E542K, or H1047R PIK3CA mut NHA RAS tumors died more quickly than mice with control GFP tumors (P 0.003). Additionally, mice with E542 or H1047R PIK3CA mut tumors succumbed to disease more quickly than mice with tumors that overexpressed either PIK3CA WT (P 0.002) or R88Q PIK3CA mut (P<0.0001; Fig 3B and 3C). Upon histopathological examination, tumor morphology was consistent across genotypes (S6 Fig). Taken together, these results indicate that both the mutated domain and concomitant mutant RAS influence the role of PIK3CA mut in gliomagenesis in vitro and in vivo.

PI3Ki efficacy is similar regardless of PIK3CA mut status
Neuro-oncology is transitioning towards precision medicine, wherein tumor mutation profiles are utilized to tailor targeted treatments [8,47]. Knowing which oncogenic driver mutations influence targeted inhibitor response is a prerequisite. To this end, we determined the effects of PIK3CA mut on efficacy of the PI3Ki buparlisib in vitro. Buparlisib induced a dose-dependent decrease in growth of control and PIK3CA mut NHA and NHA RAS (S7A and S7B Fig). High nanomolar IC 50 were evident regardless of the specific PIK3CA mut (Fig 4A) but tended to be slightly higher in NHA RAS (Fig 4B and S7C Fig)
Mutant RAS cooperated with PIK3CA WT and PIK3CA mut to potentiate activation of proximal PI3K (Fig 1), so we investigated whether RAS status influences PI3Ki-induced changes in PI3K and MAPK signaling. Buparlisib induced dose-dependent PI3K inhibition and MAPK activation in both control and PIK3CA mut NHA RAS (Fig 4F-4H and S9C and S9D and S10 Figs). Proximal PI3K inhibition was least pronounced in helical and kinase PIK3CA mut lines at low buparlisib concentrations, demonstrating that higher PI3Ki doses are required to ablate PI3K in the presence of PIK3CA mut in NHA RAS . These results also indicate that PIK3CA mut status does not influence alternate MAPK activation.

MEKi efficacy is independent of PIK3CA mut in NHA
Because PI3Ki promoted MAPK regardless of PIK3CA/RAS status, we determined efficacy of the MEKi selumetinib in control and PIK3CA mut NHA and NHA RAS lines in vitro. Selumetinib caused gradual, dose-dependent decreases in growth (S11A and S11B Fig) and had similar IC 50 in both parental NHA and NHA RAS . While PIK3CA mut status influenced IC 50 in neither NHA (Fig 5A) nor most NHA RAS lines, C90Y and H1047R were slightly more resistant than parental NHA RAS . (Fig 5B and S11C Fig). Thus, PIK3CA mut and mutant RAS had little to no effect on MEKi sensitivity.

PIK3CA WT and PIK3CA mut influence MEKi-induced PI3K activation in NHA RAS
Selumetinib inhibited MAPK in both control and PIK3CA mut NHA (Fig 5C) and induced dose-dependent decreases in pERK regardless of PIK3CA mut status (Fig 5C and 5D). We and others have shown that MEKi induces alternate PI3K activation in preclinical GBM models [35,44,49]. We extended these findings here, showing that selumetinib potentiated proximal PI3K 1.4-5-fold in control and PIK3CA mut NHA (Fig 5C and 5E). Induction in NHA was least pronounced with E542K and H1047R, the mutations that most potentiated tumorigenesis in NHA RAS (Fig 3B and 3C).
Mutant RAS cooperated with PIK3CA WT and PIK3CA mut to promote activation of proximal PI3K (Fig 1). We therefore investigated whether PIK3CA mut influence MEKi-induced changes in MAPK and PI3K in NHA RAS . PIK3CA mut status did not affect MAPK inhibition in selumetinib-treated NHA RAS lines (Fig 5F and 5G and S12A and S12B Fig). Selumetinib induced alternate activation of proximal PI3K in GFP and parental NHA RAS but ablated it in PI3K-CA WT and all PIK3CA mut NHA RAS (Fig 5F and 5H and S12A and S12C Fig). Taken together, these results indicate that ectopic PIK3CA expression in combination with mutant RAS prevents MEKi-induced alternate PI3K activation. Dual PI3Ki/MEKi treatment is synergistic in PIK3CA mut NHA and NHA RAS We and others have shown that dual PI3Ki/MEKi efficacy is increased relative to treatment with either alone [35,44,48,49]. However, the effects of GBM-associated mutations on PI3Ki/MEKi synergism remain unclear. To this end, we determined whether PIK3CA mut influence the effects of dual PI3Ki/MEKi treatment on NHA and NHA RAS growth in vitro. Buparlisib plus selumetinib synergistically inhibited growth in control and PIK3CA mut NHA (Fig 6A). Synergy was highest in R88Q and E542K NHA relative to PIK3CA WT and H1047R.
PIK3CA WT and PIK3CA mut marginally decreased MEKi efficacy in NHA RAS (Fig 5B and  S11B and S11C Fig). They also cooperated with mutant RAS to prevent MEKi-induced potentiation of proximal PI3K (Fig 5F-5H and S12 Fig). Therefore, both PIK3CA WT and PIK3CA mut may alter PI3Ki/MEKi synergism when combined with mutant RAS. Dual buparlisib/selumetinib treatment synergistically inhibited growth of all NHA RAS lines (Fig 6B). However, synergism was most pronounced at higher drug concentrations in NHA RAS relative to NHA lines. Furthermore, synergy was highest in R88Q, E542K, and M1043V mutant NHA RAS (S13 Fig). Taken together, these data suggest that mutant RAS and PIK3CA alter PI3Ki/MEKi synergism.
We and others have shown that PI3K activation via Pten deletion or constitutively active AKT cooperates with MAPK activation to potentiate gliomagenesis [19,[22][23][24]. We extended these findings here by demonstrating that mutant RAS promoted PIK3CA WT -and PIK3CA mutinduced proximal PI3K (Fig 1). Unlike in NHA, PIK3CA mut did not increase proliferation of NHA RAS , likely due to the rapid proliferation rate of parental cells (Fig 2A and 2B). H1047R PIK3CA mut also did not potentiate colony formation of NHA RAS , likely because NHA RAS cells are more aggressive and form colonies more readily and at higher density than NHA cells (Fig 3A). However, the E542K and H1047R PIK3CA mut potentiated malignancy of NHA RAS in vivo compared to GFP and PIK3CA WT (Fig 3B and 3C). These results are consistent with previous findings that constitutively active AKT does not enhance proliferation or colony formation of NHA RAS in vitro but promotes tumorigenesis in vivo [22]. These data suggest that E542K and H1047R PIK3-CA mut promote in vivo gliomagenesis equally in the presence, but not absence, of mutant RAS.
In contrast to helical and kinase PIK3CA mutations, ABD PIK3CA mut did not increase PI3K, migration, or colony formation of NHA more than PIK3CA WT (Figs 1-3). Similar results were obtained with ABD PIK3CA mut in NHA RAS . Moreover, R88Q PIK3CA mut did not promote tumorigenesis of NHA RAS more than PIK3CA WT in vivo (Fig 3B and 3C). Taken together, these results demonstrate that the phenotypic consequences of ABD PIK3CA mut and ectopic over-expression of PIK3CA WT are similar. Furthermore, they suggest that ABD PIK3-CA mut may be passenger mutations in GBM. However, ectopic expression of PIK3CA WT and PIK3CA mut may not fully recapitulate the effects of PIK3CA mut when expressed under its endogenous promoter. Furthermore, other cooperating mutations and/or cellular origin may influence the role of PIK3CA mut in gliomagenesis. Future work will be required to investigate the role of PIK3CA mut in other genetic and cellular contexts.

PIK3CA mut do not influence PI3Ki efficacy
The precision medicine initiative seeks to direct treatment with targeted inhibitors based on tumor mutation profiles [8,47]. However, this requires an understanding of how oncogenic mutations influence drug response. Mutational activation of kinases can cause oncogene addiction, in which tumor cells become reliant upon the activated signaling pathway(s), and are thus highly sensitive to their inhibition [50]. Additionally, kinase mutations can alter drug affinity, thereby altering efficacy [51]. Buparlisib inhibits purified PIK3CA WT and the most common PIK3CA mut , E542K, E545K, and H1047R, with similar IC 50 [52,53]. Because these helical and kinase domain PIK3CA mut activated PI3K and promoted gliomagenesis, we hypothesized that they would also increase PI3Ki efficacy. However, higher buparlisib doses were required to ablate PI3K in cells expressing PIK3CA mut , particularly those in the helical and kinase domains, and these mutations did not influence PI3Ki efficacy in vitro (Fig 4). These results suggest that PIK3CA mut neither induce oncogene addiction nor enhance PI3Ki sensitivity. Whether they influence efficacy of isoform-specific PI3Ki or inhibitors of downstream kinases, such as AKT and mTOR, remains to be determined.

PIK3CA mut influence MEKi-induced PI3K activation and PI3Ki/MEKi synergism
We previously found that buparlisib induced widespread kinome changes, including MAPK activation, in immortalized murine astrocytes with Pten deletion and mutant Kras [35]. We expanded these findings here by demonstrating that buparlisib potentiated MAPK regardless of RAS/PIK3CA mutation status (Fig 4C-4H). PIK3CA mut also had minimal to no effect on sensitivity of NHA and NHA RAS to MEKi in vitro (Fig 5A and 5B).
We and others have shown that MEKi promote PI3K in preclinical GBM models [35,44,49]. We found that selumetinib increased proximal PI3K in control and PIK3CA mut NHA, and in GFP and parental NHA RAS (Fig 5C-5H). Interestingly, this increase was not apparent in PIK3CA WT and PIK3CA mut NHA RAS (Fig 5F and 5H and S12A and S12C Fig). The mechanism by which ectopic PIK3CA expression in combination with mutant RAS alters MEKi response is unclear. A mutually inhibitory crosstalk between PI3K and MAPK is mediated by p70S6K in glioma stem cells [44]. MAPK inhibition induces PI3K in non-GBM cell lines via removal of a negative feedback loop on RTK [54]. Similarly, selumetinib induces widespread kinome changes in breast cancer models, including increased expression and activity of multiple RTK [55]. Taken together, these results suggest that PIK3CA WT and PIK3CA mut may cooperate with mutant RAS to alter MEKi-induced dynamic kinome changes, particularly as it pertains to PI3K activation.
Dual PI3Ki/MEKi treatment is effective in multiple preclinical GBM models [35,44,48,49]. It remains unclear whether the underlying genetics of these models influence drug synergism. We therefore determined if PIK3CA mut affected PI3Ki/MEKi synergism in the presence and absence of mutant RAS. Consistent with other GBM models, we found that dual buparlisib/selumetinib treatment was synergistic in NHA and NHA RAS lines (Fig 6). However, RAS/ PIK3CA mut status influenced drug response. Higher concentrations of buparlisib and selumetinib were required to maximize synergism in NHA RAS lines compared to NHA. Furthermore, synergy was generally greater in R88Q and E542K NHA and NHA RAS compared to those with either PIK3CA WT or H1047R. Taken together, these results suggest that GBM patients with helical PIK3CA mut may be most sensitive to dual PI3Ki/MEKi treatment. Given that ABD PIK3CA mut showed no significant tumorigenic effects in vitro and in vivo, their utility in predicting PI3Ki/MEKi synergy remains questionable.

Conclusion
Defining the role of frequently occurring mutations in GBM pathogenesis and drug response can aid identification of predictive biomarkers. Our results demonstrate that PIK3CA mut differentially promote gliomagenesis and do not predict PI3Ki sensitivity but do impact PI3Ki/ MEKi synergism.  (Fig 2A and 2B). Growth was determined by assessing changes in relative absorbance daily by MTS. Migration of control and PIK3CA mutant NHA (C) and NHA RAS (D) across a gap (Fig 2C and 2D).