PIK3R1 Mutations Research Articles

Abstract 4869: Whole genome sequence analysis of MLL rearranged infant acute lymphoblastic leukemias reveals remarkably few somatic mutations: A Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Abstract Infant acute lymphoblastic leukemia (ALL) is characterized by MLL rearrangements (MLLr) and poor prognosis. To determine the complement of somatic mutations in this high risk leukemia, we performed whole genome sequencing (WGS) on 22 infants with MLL rearranged ALL. An analysis of the structure of the MLLr revealed that over half had complex rearrangements involving either three or more chromosomes, carried cryptic rearrangements, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In three cases, genetic rearrangements were predicted to generate in addition to the MLL-partner gene fusion, novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B, AFF1-RAD51B-MLL; MLLT10-ATP5L-YPEL4 / ATP5L-YPEL4. An analysis of the number of non-silent mutations revealed infant ALL to have the lowest frequency of somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLLr, a mean of only 3.5 SVs and 2.2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case. Despite the paucity of mutations several pathways were recurrently targeted including PI3K/RAS pathway in 45% (KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and ARHGAP32 (p200Rho/GAP)), B cell differentiation in 23% as a result of mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with their matched diagnostic samples revealed a marked increase in the number of mutations at relapse. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low number of mutations detected in infant ALL, we used exome sequencing to determine the frequency of non-silent SNVs in 20 MLLr leukemias (9 ALLs, 10 AMLs and 1 AUL) in older children (7-19 yrs). This analysis revealed that non-infant pediatric MLL leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 7.4/case in older patients vs 2.2/case in infants, p<0.001). Although the higher frequency of mutations may be a reflection of age, the low number of cooperating mutations in infants raises the possibility that the target cell of transformation differs between infants and older children, with the cells present during early development requiring fewer cooperating mutations to induce leukemia. In summary our data demonstrated an exceedingly small number of mutations in infant leukemia. The number of detected somatic mutations may represent the lower limit required to transform a normal human cell into cancer. 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 4869. doi:1538-7445.AM2012-4869

Abstract 111: PIK3R1 gene alterations, insights on its role in lung cancer

Abstract Gene abnormalities are the cause of cancer and efforts are still needed to identify all the genes that promote carcinogenesis, including those altered at low frequencies. The occurrence of homozygous deletions (HD) is one mechanism for tumor suppressor gene inactivation. We scrutinized the SNP arrays at the Sanger Database to search for HD in lung cancer cell lines. Several candidate tumor suppressors, including PIK3R1, were found to be altered by HD. PIK3R1 codifies for p85α, a regulatory subunit of the PI3K, and mutations have been found in different tumor types. The PI3K pathway is highly relevant in cancer since it contains well known oncogens and tumor suppressors. Despite that, the functional significance of PIK3R1 mutations is uncertain, because activating and inactivating alterations have been described in tumors. Here, we aimed to clarify the role of this gene in cancer. We amplified by PCR each individual exon of the gene and confirmed the presence of a HD at the NCI-H2171 cell line. Mutational analysis of PIK3R1 was then extended to a panel of 86 lung cancer cell lines. We identified several changes including an heterozygous nonsense mutation, c.211G>T (p.G71*), an heterozygous missense mutation, c.211G>T (p.D13H) and several intronic changes outside the canonical splice sites. Intriguingly, the levels of p85α protein in the cell line carrying the p.G71* mutation were almost zero, indicating the presence of a second hit. To search for the presence of intragenic small deletions, undetectable by single exon amplification, we studied the cDNA of PIK3R1. No additional alterations were detected in this cell line nor in the other cells carrying PIK3R1 heterozygous missense mutations. Next, we used cancer cell lines that have no p85α protein due to HD to determine whether the lack of p85α impinges the AKT-mTOR pathway. To this end, we tested the levels of pAKT-T308, pAKT-S473 and pS6-S235/236, upon different cell culture conditions (i.e. +/−IGF) in cancer cells carrying alterations at distinct components of the PI3K pathway. We found that cells with alterations at ERBB2 and PTEN had constitutive high levels of pAKT and pS6, in all the conditions tested. Surprisingly, some cells lacking p85α protein due to HD at PIK3R1, readily regulated the activation/inactivation of the pathway, accordingly to the cell culture conditions. This indicates that the inactivation of the p85α protein in cancer does not compromise the appropriate modulation of AKT/mTOR signalling, possibly due to a compensation effect by other regulatory subunits. In conclusion, PIK3R1 is mutated in lung cancer, albeit at low frequencies. Furthermore, PIK3R1 alterations contribute to oncogenesis by mechanisms that require further investigation. 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 111. doi:1538-7445.AM2012-111

High Frequency of PIK3R1 and PIK3R2 Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability

We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function, including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.

PIK3R1 (p85α) is somatically mutated at high frequency in primary endometrial cancer.

Phosphoinositide 3-kinase (PI3K) is an important therapeutic target. Mutations in PIK3CA, which encodes p110α, the catalytic subunit of PI3K, occur in endometrioid endometrial cancers (EEC) and nonendometrioid endometrial cancers (NEEC). The goal of this study was to determine whether PIK3R1, which encodes p85α, the inhibitory subunit of PI3K, is mutated in endometrial carcinoma. We carried out exonic sequencing of PIK3R1 from 42 EECs and 66 NEECs. The pattern of PIK3R1 mutations was compared with the patterns of PIK3CA, PTEN, and KRAS mutations. The biochemical effect of seven PIK3R1 mutations was examined by stable expression in U2OS cells, followed by coimmunoprecipitation analysis of p110α, and Western blotting of phospho-AKT(Ser473) (p-AKT(Ser473)). We found that PIK3R1 was somatically mutated in 43% of EECs and 12% of NEECs. The majority of mutations (93.3%) were localized to the p85α-nSH2 and -iSH2 domains. Several mutations were recurrent. PIK3R1 mutations were significantly (P = 0.0015) more frequent in PIK3CA-wild type EECs (70%) than in PIK3CA mutant EECs (18%). Introduction of wild-type p85α into U2OS cells reduced the level of p-AKT(Ser473) compared with the vector control. Five p85α mutants, p85αdelH450-E451, p85αdelK459, p85αdelY463-L466, p85αdelR574-T576, and the p85αN564D positive control, were shown to bind p110α and led to increased levels of p-AKT(Ser473). The p85αR348X and p85αK511VfsX2 mutants did not bind p110α and showed no appreciable change in p-AKT(Ser473) levels. In conclusion, our study has revealed a new mode of PI3K alteration in primary endometrial tumors and warrants future studies to determine whether PIK3R1 mutations correlate with clinical outcome to targeted therapies directed against the PI3K pathway in EEC and NEEC.

Abstract LB-247: Mutation and functional analysis of PIK3CA and PIK3R1 in endometrial cancer

Abstract The phosphatidylinositol 3 kinase (PI3K) pathway is the most frequently activated pathway across multiple tumor lineages and this is a potential therapeutic target. As a critical step to accelerate therapeutic development in endometrial cancer, we performed a comprehensive analysis of mutation and function of PI3K pathway members in a set of 243 highly characterized endometrial tumors. Whole gene resequencing revealed the highest frequencies of mutation in the PI3K pathway of any tumor lineage including PTEN (44%), PIK3CA (40%; encoding the p110α catalytic subunit of PI3K) and PIK3R1 (20%; encoding the p85a regulatory subunit). Indeed, when complete protein loss is considered, almost 80% of endometrioid endometrial cancers demonstrate an aberration in the PI3K pathway. Remarkably, mutations in the PI3K pathway were not mutually exclusive and indeed co-existence of PIK3CA or PIK3R1 mutation with heterozygous PTEN mutation occurred at frequencies higher than predicted by the frequency of each lesion alone. High-throughput reverse-phase protein array suggested that the dominant signaling effects occurred with PTEN protein loss and that PIK3CA or PIK3R1 mutation functionally mimic PTEN protein loss as indicated by protein markers including phosphorylated Akt and stathmin. Thus it appears likely that co-mutations in the PI3K pathway are selected to compensate haploinsufficiency of PTEN due to PTEN heterozygous mutation. To determine the functional consequences of mutations in PIK3R1, we demonstrated that several somatic PIK3R1 mutations tested conferred cytokine-independent growth on interleukin-3-dependent Ba/F3 cells and induced Akt phosphorylation in endometrial cancer cell lines indicating that they were gain of function mutations likely acting as oncogenes. Strikingly two of the gain of function mutations (E160* and R348*) lacked the ability to bind to the p110 catalytic subunit and E160* failed to bind to both p110 and PTEN. We demonstrate that E160* disrupts a novel mechanism of pathway regulation wherein p85 binds and stabilizes PTEN. Together, the data indicate that the PI3K pathway is targeted in the vast majority of endometrioid endometrial cancers representing a novel opportunity for implementation of targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2011-LB-247

A Systematic Study of Gene Mutations in Urothelial Carcinoma; Inactivating Mutations in TSC2 and PIK3R1

BackgroundUrothelial carcinoma (UC) is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations.Methodology/Principal FindingsWe performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%.Conclusions/SignificanceOur data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC.

Uncommon GNAQ, MMP8, AKT3, EGFR, and PIK3R1 Mutations in Thyroid Cancers

Frequent mutations in the GNAQ, MMP8, Akt3, EGFR, and PIK3R1 genes have been reported in human cancers but mostly have not been well examined in thyroid cancer. Selected exons of GNAQ, MMP8, AKT3, EGFR, and PIK3R1 genes were sequenced in various thyroid cancers. We found a G2203A EGFR mutation, resulting in a G735S amino acid change, in one of 21 (5%) papillary thyroid cancer samples. We did not find any mutation in the MMP8 gene, but observed a frequent SNP A259G (K87E) genotype switch in various types of thyroid cancer samples. We did not find any mutation in the GNAQ, AKT3, and PIK3R1 genes in various types of thyroid cancer. No mutation in these genes was found in 12 cell lines derived from various types of thyroid cancer. Therefore, unlike in other cancers, mutations in these genes are uncommon in thyroid cancer.

E17K substitution in AKT1 in prostate cancer

Background:The phosphatidylinositol 3-kinase (PI3K)–AKT pathway is activated in many cancers. Mutational hotspots in AKT1 and in the regulatory and catalytic subunits of PI3K have been detected in multiple tumour types. In AKT1, the E17K substitution leads to a PI3K-independent activation of AKT1.Methods:A mutational profiling of AKT1 and of the mutational hotspots in PIK3CA and PIK3R1 was carried out in samples from primary and recurrent prostate tumours.Results:We show that, in prostate cancer, AKT1(E17K) had a prevalence of 1.4%. The mutation seemed to be associated with a favourable clinical course but it was not associated with a specific tumour growth pattern. Activating mutations in PIK3CA or PIK3R1 were not found in prostate cancer.Conclusion:The E17K substitution in AKT1 is rare in prostate cancer. It seems associated with a favourable clinical outcome but not with a specific histology of the tumour.

Abstract 305: Frequent PIK3R1 somatic mutations promote oncogenic signaling

Abstract We performed a systematic analysis of human fresh frozen tumors for somatic mutations in the RAS-effector pathways and have identified frequent mutations (>8%) in PIK3R1 (p85α) regulatory subunit of PI3K (p110) enzyme complex. Mutations in other members of the PI3K regulatory subunits, along with mutations in additional components of the pathway were also identified in this study. An integrated analysis of somatic mutations and genomic copy number variations identified by comparative genome hybridization revealed distinct mutation patterns across tumor types. In order to understand the functional consequence of p85α mutations, we generated mutant p85α constructs and tested them for activity in biochemical and cellular assays. In this studies, we found that all the mutants that contain an intact p110-binding domain were able to interact with p110α, p110β and p110δ. Also, all these mutants stabilized expression of p110 class IA members when tested in pan-p85 null fibroblasts, indicating that the p110-stabilizing activity of p85 mutants was still intact. However, in un-stimulated cells, some of the p85α mutants were not as effective as wild-type p85α in inhibiting the activity of p110. Furthermore, these p85α somatic mutants activated AKT signaling leading to cell survival. Additionally, cells expressing the p85α mutants when transplanted in vivo promote a leukemia-like disease leading to decreased overall survival of the mice compared to wild-type p85α. These data demonstrate an alternate mechanism for activation of class IA PI3Ks in tumors through somatic mutations in p85α that abrogates its p110 regulatory activity while maintaining its p110-stabilizing activity. The frequent occurrence of p85α mutations and its ability to promote survival signaling suggest that tumors with such mutations are likely candidates for inhibitors that target the components of the PI3K pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 305.

Identification of a CpG Island Methylator Phenotype that Defines a Distinct Subgroup of Glioma

We have profiled promoter DNA methylation alterations in 272 glioblastoma tumors in the context of The Cancer Genome Atlas (TCGA). We found that a distinct subset of samples displays concerted hypermethylation at a large number of loci, indicating the existence of a glioma-CpG island methylator phenotype (G-CIMP). We validated G-CIMP in a set of non-TCGA glioblastomas and low-grade gliomas. G-CIMP tumors belong to the proneural subgroup, are more prevalent among lower-grade gliomas, display distinct copy-number alterations, and are tightly associated with IDH1 somatic mutations. Patients with G-CIMP tumors are younger at the time of diagnosis and experience significantly improved outcome. These findings identify G-CIMP as a distinct subset of human gliomas on molecular and clinical grounds.

Hormone Therapy plus mTOR Inhibitors in the Treatment of Endometrial Carcinoma.

Hormonal therapies such as progestins have only modest activity in the treatment of advanced endometrial cancer. Mechanisms of resistance to progestin therapy are not well understood. However, activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway has been associated with resistance to hormonal therapy and alterations in components of the PI3K/AKT/mTOR pathway, including inactivating mutations in PTEN, activating mutations in PIK3CA and mutations in PIK3R1, are very common in endometrial carcinomas. mTOR inhibitors, including temsirolimus, everolimus and ridaforolimus, are also known to be active against endometrial cancer, and interest has been stimulated in combinations of hormonal treatment with mTOR inhibitors, as both therapies have single-agent activity, and it is hypothesised that mTOR inhibition would enhance sensitivity to hormonal therapy.

241 INVITED Novel therapeutic targets within the autophagic pathway

Rapamycin analogs have reproducible but modest efficacy in endometrial cancer (EC). Identification of molecular biomarkers that predict benefit could guide clinical development.Fixed primary tissue and whole blood were collected prospectively from patients enrolled on GOG 248. DNA was isolated from macro-dissected tumors and blood; next–generation sequence analysis was performed on a panel of cancer related genes. Associations between clinical outcomes [response rate (RR) 20%; progression-free survival (PFS) median 4.9 months] and mutations (PTEN, PIK3CA, PIK3R1, KRAS, CTNNB1, AKT1, TSC1, TSC2, NF1, FBXW7) were explored.Sequencing data was obtained from tumors of 55 of the 73 enrolled pts. Mutation rates were consistent with published reports: mutations in PTEN (45%), PIK3CA (29%), PIK3R1 (24%), K-RAS (16%), CTNNB1 (18%) were common and mutations in AKT1 (4%), TSC1 (2%), TSC2 (2%), NF1 (9%) and FBXW7 (4%) were less common. Increased PFS (HR 0.16; 95% CI 0.01–0.78) and RR (response difference 0.83; 95% CI 0.03–0.99) were noted for AKT1 mutation. An increase in PFS (HR 0.46; 95% CI 0.20–0.97) but not RR (response difference 0.00, 95% CI − 0.34–0.34) was identified for CTNNB1 mutation. Both patients with TSC mutations had an objective response. There were no statistically significant associations between mutations in PIK3CA, PTEN, PIK3R1, or KRAS and PFS or RR.Mutations in AKT1, TSC1 and TSC2 are rare, but may predict clinical benefit from temsirolimus. CTNNB1 mutations were associated with longer PFS on temsirolimus.

Genetic Alterations of Phosphoinositide 3‐kinase Subunit Genes in Human Glioblastomas

Genetic alterations of PI3K (phosphoinositide 3-kinase) subunits have been documented in a number of tumor types, with increased PI3K activity linked to gene amplification and mutation of catalytic subunits, as well as mutations of regulatory subunits. Among high grade gliomas, activation of the PI3K-AKT signaling pathway through loss of PTEN function is common. We therefore investigated whether genetic alteration of class IA PI3Ks might provide a mechanism for deregulation of this pathway in glioblastomas. We studied a series of glioblastomas with FISH to assess copy number of catalytic subunits (PIK3CA and PIK3CD) and with PCR-SSCP to screen for somatic mutations of conserved regions of both catalytic and regulatory subunits. FISH revealed frequent balanced copy number increases of both PIK3CA and PIK3CD, and one case showed an extra copy limited to PIK3CA. One glioblastoma exhibited a 9-bp deletion that encompassed the exon-intron junction of exon 12 of PIK3R1, documenting for the first time a mutation within a PI3K regulatory subunit in human glioblastoma. This deletion would be predicted to yield a truncated protein that lacks the inhibitory domain, resulting in increased PI3K activity. Furthermore, the case with selected PIK3CA copy number gain and the case with a truncating PIK3R1 mutation both featured AKT activation without PTEN mutation. These results suggest that genetic alterations of class IA PI3K subunit genes can occasionally play a role in human glioblastoma by activating the PI3K-AKT signaling pathway independently of PTEN mutation.