Alterations In Phosphatidylinositol 3-kinase Research Articles

ER+, HER2- advanced breast cancer treated with taselisib and fulvestrant: genomic landscape and associated clinical outcomes.

Taselisib is a potent β-sparing phosphatidylinositol 3-kinase (PI3K) inhibitor that, with endocrine therapy, improves outcomes in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated (PIK3CAmut) advanced breast cancer. To understand alterations associated with response to PI3K inhibition, we analysed circulating tumour DNA (ctDNA) from participants enrolled in the SANDPIPER trial. Participants were designated as either PIK3CAmut or PIK3CA no mutation was detected (NMD) per baseline ctDNA. The top mutated genes and tumour fraction estimates identified were analysed for their association with outcomes. In participants with PIK3CAmut ctDNA treated with taselisib + fulvestrant, tumour protein p53 (TP53; encoding p53) and fibroblast growth factor receptor 1 (FGFR1) alterations were associated with shorter progression-free survival (PFS) compared to participants with NMD in these genes. Conversely, participants with PIK3CAmut ctDNA harbouring a neurofibromin 1 (NF1)alteration or high baseline tumour fraction estimate experienced improved PFS upon treatment with taselisib + fulvestrant compared to placebo + fulvestrant. Broadly, alterations in oestrogen receptor (ER), PI3K and p53 pathway genes were associated with resistance to taselisib + fulvestrant in participants with PIK3CAmut ctDNA. Altogether, we demonstrated the impact of genomic (co-)alterations on outcomes with one of the largest clinico-genomic datasets of ER+, HER2-, PIK3CAmut breast cancer patients treated with a PI3K inhibitor.

Abstract 2233: Landscape of driver mutations in MAPK/PI3K/AKT signaling pathways reveals insights into therapeutic targeting strategies

Abstract Alterations in the signaling cascade of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways are common mechanisms of cancer initiation and progression. RAS proteins (KRAS/HRAS/NRAS), a crucial component of this cascade, are often found to be activated in cancer cells. Recent advancements in RAS targeting offer new treatment strategies for patients, however studies suggest that the clinical responses may be tissue-dependent. Thus, understanding the landscape of RAS alterations and the concomitant mutations observed in their effector pathways may be crucial in developing appropriate disease-specific therapeutic strategies for effectively addressing this biology. In this study, we utilized comprehensive genomic profiling data from 282,792 cancer patients across 77 tumor types, tested in the course of routine clinical care (FoundationOne) to assess the landscape of gene alterations within this signaling cascade. RAS alterations were identified in 27% of the cohort. Mutations in PI3K and PTEN were also frequent (>10%); alterations in FGFR1-4, EGFR, RAF (ARAF/BRAF/RAF1) and ERBB2 were each identified in over 5% of samples. A closer inspection of non-small cell lung carcinomas (NSCLC, n=55,893), colorectal carcinomas (CRC, n=35,104) and pancreatic cancers (PC, n=17,447), revealed distinct mutational spectrums. RAS alterations, particularly KRAS, exhibited varying prevalence: PC (85%), CRC (54%) and NSCLC (32%). In NSCLC, 35% of the KRAS alterations were identified to be G12C. In contrast, the predominant KRAS alterations in CRC and PC were G12D (29% CRC, 41% PC) and G12V (20% CRC, 30% PC). Further, only CRCs exhibited a high rate of KRAS G13D (16% of all KRAS alterations); KRAS G12R was found exclusively to PC (15%) and KRAS amplifications were most frequently identified in NSCLC (11%). Distinct patterns were also observed in genes upstream and downstream of the RAS proteins. Alterations in ALK, EGFR, STK11 and MET were significantly more prevalent in NSCLC (P < 10−5); whereas gene alterations in PI3K, RAF, MTOR and ERBB2 were more frequently identified in CRC (P < 10−5). In contrast, PC showed a predominant presence of RAS mutations, with limited mutations in other genes of the signaling cascade. Of note, PI3K alterations were identified at a much lower prevalence in PC (4%) compared to NSCLC (11%) and CRC (19%). Our findings suggest a tissue-dependent spectrum of driver mutations exist across the MAPK/PI3K/AKT signaling pathways. The observed mutational profiles may explain some of the efficacy differences observed by targeting RAS across these diseases and have implications on acquired resistance mechanisms in a tissue-specific manner. Additional insights may be gleaned by investigating immunotherapy related biomarkers with these alterations to suggest combination strategies for effectively targeting the RAS signaling cascade. Citation Format: Smruthy Sivakumar, Ethan S. Sokol, Garrett M. Frampton, Priti S. Hegde, David Fabrizio. Landscape of driver mutations in MAPK/PI3K/AKT signaling pathways reveals insights into therapeutic targeting strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2233.

PI3K inhibition in breast cancer: Identifying and overcoming different flavors of resistance

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is commonly deregulated in many human tumors, including breast cancer. Somatic mutations of the PI3K alpha catalytic subunit (PIK3CA) are the most common cause of pathway hyperactivation. Hence, several PI3K inhibitors have been investigated with one of them, alpelisib, recently approved for the treatment of endocrine sensitive, PIK3CA mutated, metastatic breast cancer. Unfortunately, all patients receiving a PI3K inhibitor eventually develop resistance to these compounds. Mechanisms of resistance include oncogenic PI3K alterations, pathway reactivation through upstream or downstream effectors and enhancement of parallel pro-survival pathways. We review the prognostic and predictive role of PI3K alterations in breast cancer, focusing on resistance to PI3K inhibitors and on biomarkers with potential clinical relevance. We also discuss combination strategies that may overcome resistance to PI3K inhibitors, thus increasing the efficacy of these drugs in breast cancer.

Phosphatidylinositol 3-kinase pathway genomic alterations in 60,991 diverse solid tumors informs targeted therapy opportunities.

BackgroundThe phosphatidylinositol 3‐kinase (PI3K) pathway is frequently altered in cancer. This report describes the landscape of PI3K alterations in solid tumors as well as co‐alterations serving as potential resistance/attenuation mechanisms.MethodsConsecutive samples were analyzed in a commercial Clinical Laboratory Improvement Amendment‐certified laboratory using comprehensive genomic profiling performed by next‐generation sequencing (315 genes). The co‐alterations evaluated included the Erb‐B2 receptor tyrosine kinase 2 (ERBB2), ERBB3, ERBB4, RAS, MET proto‐oncogene tyrosine kinase (MET), and mitogen‐activated protein kinase kinase (MAP2K) genes as well as tumor protein 53 (TP53), estrogen receptor 1 (ESR1), and androgen receptor (AR). ResultsAlterations in any of 18 PI3K‐pathway associated genes were identified in 44% of 60,991 tumors. Although single base and insertions/deletions (indels) were the most frequent alterations, copy number changes and rearrangements were identified in 11% and 0.9% of patients, respectively. Overall, the most frequently altered genes were PIK3 catalytic subunit α (PIK3CA) (13%), phosphatase and tensin homolog (PTEN) (9%), and serine/threonine kinase 11 (STK11) (5%). Tumor types that frequently harbored at least 1 PI3K alteration were uterine (77%), cervical (62%), anal (59%), and breast (58%) cancers. Alterations also were discerned frequently in tumors with carcinosarcoma (89%) and squamous cell carcinoma (62%) histologies. Tumors with a greater likelihood of co‐occurring PI3K pathway and MAPK pathway alterations included colorectal cancers (odds ratio [OR], 1.64; P < .001), mesotheliomas (OR, 2.67; P = .024), anal cancers (OR, 1.98; P = .03), and nonsquamous head and neck cancers (OR, 2.03; P = .019). The co‐occurrence of ESR1 and/or AR alterations with PI3K alterations was statistically significant in bladder, colorectal, uterine, prostate, and unknown primary cancers.ConclusionsComprehensive genomic profiling reveals altered PI3K‐related genes in 44% of solid malignancies, including rare disease and histology types. The frequency of alterations and the co‐occurrence of resistance pathways vary by tumor type, directly affecting opportunities for targeted therapy.

Phosphatidylinositol-3 Kinase Inhibitors, Buparlisib and Alpelisib, Sensitize Estrogen Receptor-positive Breast Cancer Cells to Tamoxifen

Tamoxifen is the standard first-line hormonal therapy for premenopausal women with estrogen receptor (ER)-positive metastatic breast cancer (BC). One of the crucial mechanisms underlying hormonal therapy resistance is the collateral activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We explored whether PI3K inhibitors, buparlisib and alpelisib, enhance the efficacy of tamoxifen against ER-positive BC cells. We have observed a synergism between alpelisib or buparlisib and tamoxifen in the treatment for ER-positive BC cell lines harboring different PI3K alterations. Immunoblotting analysis showed alpelisib, buparlisib, or either drug in combination with tamoxifen downregulated the PI3K downstream targets in the MCF-7 and ZR75-1 cells. In the MCF-7 cells transfected with a constitutive active (myristoylated) AKT1 construct or mutant ER, the synergistic effect between alpelisib and tamoxifen was markedly attenuated, indicating that synergism depends on AKT inhibition or normally functioning ER. Combining alpelisib or buparlisib with tamoxifen also attenuated MCF-7 tumor growth in Balb/c nude mice. Our data suggest that additional PI3K blockade might be effective in enhancing the therapeutic effect of tamoxifen in ER-positive BC and support the rationale combination in clinical trials.

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kβ silencing.

Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kβ, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kβ kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kβ depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kβ inhibition. A small proportion of these tumors was resistant to PI3Kβ downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kβ dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kβ associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN-mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN.

Targeting the Vav3 oncogene enhances docetaxel-induced apoptosis through the inhibition of androgen receptor phosphorylation in LNCaP prostate cancer cells under chronic hypoxia

BackgroundThe Vav family of Rho/Rac guanosine nucleotide exchange factors comprises three members in mammalian cells. Vav3 enhances androgen receptor (AR) activity during progression to androgen independence in prostate cancer. We examined Vav3 small interfering RNA (siRNA) effects on cell proliferation and apoptosis in docetaxel-treated LNCaP cells under chronic hypoxia (LNCaPH).MethodsWe examined individual and combined effects of Vav3 siRNA (si-Vav3) and docetaxel on cell growth and apoptosis under chronic hypoxia by cell proliferation, flow cytometric, DNA fragmentation, and immunoblot analyses. To clarify the molecular basis of si-Vav3- and docetaxel-induced apoptosis, we analyzed alterations in phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulate kinase (ERK), c-jun N-terminal kinase (JNK), and AR pathways using kinase inhibitors in LNCaPH cells. The effects of si-Vav3/atelocollagen complex alone or in combination with docetaxel were assessed on xenografts in nude mice by tumor growth delay.ResultsVav3 overexpression was observed in LNCaPH compared with the expression under normoxia. Interrupting Vav3 signaling using siRNA enhanced docetaxel-induced cell growth suppression compared with that induced by docetaxel alone by inhibition of Akt and ERK phosphorylation, resulting in AR phosphorylation inhibition. In addition to increased B-cell lymphoma 2 (Bcl-2) phosphorylation through JNK signaling in response to docetaxel, si-Vav3 enhanced docetaxel-induced apoptosis, as characterized by the accumulation of sub-G1 phase cells and DNA fragmentation, through Bcl-xL/Bcl-2-associated death promoter (Bad) dephosphorylation, resulting in increased caspase-9, caspase-3, and cleaved poly(ADP-ribose) polymerase activation. Xenograft tumor growth was slightly inhibited by si-Vav3/atelocollagen complex injection and combined use of si-Vav3/atelocollagen complex and docetaxel produced a greater effect than docetaxel alone.ConclusionsInterrupting Vav3 signaling enhances docetaxel-induced apoptosis in LNCaP cells under chronic hypoxia by inhibiting the PI3K/Akt, ERK, and AR signaling pathways. Therapy targeting Vav3 in combination with docetaxel may have practical implications for managing castration-resistant prostate cancer.

Preclinical study of lithium sensitivity in human breast PTEN knock out cell lines

e14641 Background: Mutations of PIK3CA, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), are found in ∼25% of breast cancers. The tumor suppressor PTEN reverses the effects of PI3K and genetic alterations leading to loss or reduced function of PTEN also occur at high frequency in breast cancers. We have used somatic cell gene targeting to create paired isogenic cell lines with critical genetic alterations as their only differentiating factor. Our lab has introduced two “hotspot” oncogenic PIK3CA mutations into the MCF-10A human breast epithelial cell line. This study led to the discovery that lithium, an FDA approved therapy for bipolar disorders, has selective anti-neoplastic properties against human breast cancer cell lines containing oncogenic PIK3CA mutations. Due to the opposing interactions between PI3K and PTEN, we are now studying whether loss of PTEN also confers sensitivity to lithium using human somatic cell knock outs. Methods: Targeting vectors were created and used to knock out the PTEN gene in MCF-10A. Biochemical and phenotypical characterizations were performed. Paired isogenic cell lines were exposed to lithium to evaluate heterozygous and homozygous loss of PTEN as a predictive biomarker of response. Proteomic and phenotypic changes were measured and assessed. Results: Homozygous loss of PTEN (PTEN-/-) resulted in a transformed phenotype enabling growth in the absence of exogenous growth factors, a hallmark of malignant potential. Preliminary results suggest that treatment of PTEN-/- cells with lithium leads to a decrease in cell proliferation with selectivity for mutant PTEN knock out cell lines, although the effects are not as pronounced as that seen with mutant PIK3CA knock in cells. Conclusions: Our preliminary results suggest that loss of PTEN predicts for sensitivity to lithium in breast cell lines in a manner similar to that seen with oncogenic PIK3CA. We hypothesize that differences in the magnitude of response to lithium may be the result of varied levels of non-PI3K-AKT-mTOR activation between the PIK3CA knock in cell lines and the PTEN knock out cells. No significant financial relationships to disclose.

Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC 50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3Kgamma/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca 2+] i) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca 2+] I and the implication of Ca 2+ on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca 2+] i in a dose-dependent manner (1 pM–100 nM). In cells from aged rats, hormone concentrations higher than physiological (≥1 nM) were required to observe significant increases in [Ca 2+] i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca 2+] i biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca 2+] i phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10 −8 M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85α, the regulatory subunit of PI3K, as well as the phosphorylation on Thr 308 of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca 2+ chelation (BAPTA-AM, 5 μM) affected the tyrosine phosphorylation of p85α and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca 2+ is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca 2+. Taken together, these results suggest that PTH regulation of enterocyte [Ca 2+] i involves Ca 2+ mobilization from IP 3-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr 308 is impaired with ageing.