Loss Of INPP4B Research Articles

Regulation of B-1 cell numbers and B cell-mediated antibody production by Inpp4b.

T and B lymphocytes are crucial players in cellular and humoral immune responses. The development, activation and differentiation of T and B lymphocytes are regulated by the best characterized PI3K-PI (3,4,5) P3-AKT phosphoinositide signalling pathway. As a branch of the phosphoinositide signalling pathway, the lipid phosphatase INPP4B inhibits AKT activation through degrading the phosphoinositide signalling messenger PI (3,4) P2. However, the role of Inpp4b in T and B lymphocytes remains elusive. Here, we reported that Inpp4b was highly expressed in human and murine T- and B-1 lymphocytes. Despite its higher expression in T lymphocytes, neither T cell development and homeostasis nor in vitro T cell activation and CD4+ T cell differentiation were altered upon loss of Inpp4b. Interestingly, combined direct phenotype analysis of Inpp4b conventional knockout mice and adoptive transfer studies revealed that ablation of Inpp4b intrinsically reduced peritoneal B-1 cells rather B-2 cells. Moreover, Inpp4b deficiency led to impaired thymus independent (TI) and thymus dependent (TD) antigens-induced antibody production. Further in vitro analysis revealed that CD40-mediated B cell proliferation was impaired upon ablation of Inpp4b. Our findings reveal that Inpp4b is required in regulating B-1 cell numbers and B cell-mediated antibody production.

Abstract 2497: Inositol Polyphosphate 4-Phosphatase Type-II B protects mice from benign prostatic hyperplasia

Abstract BPH is the most common tumor-like condition that afflicts more than 50% of men between the ages of 51-60 and over 90% in men older than 80. While connection between obesity and BPH is well established, the driving signaling pathways are not clear. BPH is a nonmalignant enlargement of prostate epithelium that requires androgen receptor (AR) signaling. INPP4B and EZH2 are highly expressed in prostate epithelium where they regulate AR signaling and play an important role in prostate cancer etiology. EZH2 is an epigenetic silencer that methylates histone H3 on K9 and K27 residues and INPP4B is a dual specificity phosphatase that dephosphorylates PI(3,4)P2 and PI(4,5)P2, membrane lipids that activate Akt and PKC signaling pathways respectively. We discovered that Inpp4b-/- male mice develop BPH at 3 months old of age and subsequently prostate intraepithelial neoplasia (PIN) when fed high fat diet. While expression of AR in the prostates of these mice is similar, AR activity is reduced. The loss of INPP4B caused reduction in EZH2 protein levels in prostate cancer cell lines and in mouse prostate. Inhibition of Akt and PKC signaling reduced EZH2 levels in ventral and dorsolateral prostate lobes of Inpp4b-/- males, suggesting that INPP4B upregulates EZH2 levels by suppressing Akt and PKC signaling pathways. Consistent with decline in EZH2 levels, Inpp4b-/- males have reduced levels of H3K27me3 and H3K9me2 in the ventral prostate, and high fat diet exacerbated this phenotype. Our data suggests that INPP4B modulates epigenetic profile of prostate epithelium by stabilizing EZH2 levels. Both loss of INPP4B and high fat diet treatment lead to decline in EZH2 levels and activity and contribute to development of BPH in mouse model. Citation Format: Yasemin Ceyhan, Manqi Zhang, Irina U. Agoulnik. Inositol Polyphosphate 4-Phosphatase Type-II B protects mice from benign prostatic hyperplasia [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 2497.

INPP4B protects from metabolic\xa0syndrome and associated disorders

A high fat diet and obesity have been linked to the development of metabolic dysfunction and the promotion of multiple cancers. The causative cellular signals are multifactorial and not yet completely understood. In this report, we show that Inositol Polyphosphate-4-Phosphatase Type II B (INPP4B) signaling protects mice from diet-induced metabolic dysfunction. INPP4B suppresses AKT and PKC signaling in the liver thereby improving insulin sensitivity. INPP4B loss results in the proteolytic cleavage and activation of a key regulator in de novo lipogenesis and lipid storage, SREBP1. In mice fed with the high fat diet, SREBP1 increases expression and activity of PPARG and other lipogenic pathways, leading to obesity and non-alcoholic fatty liver disease (NAFLD). Inpp4b−/− male mice have reduced energy expenditure and respiratory exchange ratio leading to increased adiposity and insulin resistance. When treated with high fat diet, Inpp4b−/− males develop type II diabetes and inflammation of adipose tissue and prostate. In turn, inflammation drives the development of high-grade prostatic intraepithelial neoplasia (PIN). Thus, INPP4B plays a crucial role in maintenance of overall metabolic health and protects from prostate neoplasms associated with metabolic dysfunction.

Abstract PO-18: Comprehensive correlation between genetic alterations in DLBCL and deregulated activation of the PI3K-AKT pathway isolates unique players in lymphoma dissemination and inferior outcome

Abstract Diffuse large B-cell lymphoma (DLBCL) is characterized by deregulation of numerous signaling pathways that direct cell proliferation and survival during B-cell maturation in the germinal center reaction. The subtype of germinal center B-cell DLBCL (GCB-DLBCL) demonstrates addiction to tonic B-cell receptor (BCR) signaling in part through the PI3K-AKT pathway. However, a comprehensive catalogue of the somatic genetic alterations leading to this deregulation is currently lacking and represents a limitation in advancing precision medicine options. In this study, we present a comprehensive analysis in 347 DLBCL biopsies uniformly treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), describing recurrent mutations, copy number alterations, and clinical parameters that are associated with AKT activation (pAKT) as determined by immunohistochemistry. We observed a significantly enhanced pAKT population including players in the Gα13 signaling (GNA13, P2RY8, ARHGEF1, RHOA, and S1PR2) and PI3K activation pathways (NFATC1, USP7, PTEN, INPP4B). Following this correlation, we observed a co-occurrence of BCL2 protein overexpression and mutation in Gα13 signaling associated with advanced clinical stage (P = .008) in 183 GCB-DLBCL cases, as well as activation of cell migration and cell-cell adhesion pathways, indicating their contribution to lymphoma dissemination. This co-occurrence was shown to be associated with shorter time to progression in GCB-DLBCL (P = .005). We furthermore uncovered recurrent deletions of INPP4B, a lipid phosphatase involved in AKT deactivation, to be associated with increased pAKT in GCB-DLBCL. We observed that INPP4B loss is similarly associated with shorter time to progression (P = .036) in GCB-DLBCL, and demonstrated altered AKT phosphorylation in a GCB-DLBCL-derived cell line with a monoallelic INPP4B deletion. We present here a useful comprehensive tool, through a positive correlation to enhanced pAKT staining, to uncover novel prognostic indicators related to genetic alteration status in DLBCL. Our findings provide novel insight into somatic genomic alterations underlying prosurvival signaling of GC-derived lymphomas, and present new avenues for therapeutic targeting. Citation Format: Shannon Healy, Daisuke Ennishi, Elena Viganò, Aixiang Jiang, Anja Mottok, Stacy Hung, Olga Kutovaya, Gerben Duns, Bruce Woolcock, David Scott, Christian Steidl. Comprehensive correlation between genetic alterations in DLBCL and deregulated activation of the PI3K-AKT pathway isolates unique players in lymphoma dissemination and inferior outcome [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-18.

The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple-Negative Breast Cancer.

Inactivation of the tumor suppressor lipid phosphatase INPP4B is common in triple-negative breast cancer (TNBC). We generated a genetically engineered TNBC mouse model deficient in INPP4B. We found a dose-dependent increase in tumor incidence in INPP4B homozygous and heterozygous knockout mice compared with wild-type (WT), supporting a role for INPP4B as a tumor suppressor in TNBC. Tumors derived from INPP4B knockout mice are enriched for AKT and MEK gene signatures. Consequently, mice with INPP4B deficiency are more sensitive to PI3K or MEK inhibitors compared with WT mice. Mechanistically, we found that INPP4B deficiency increases PI(3,4)P2 levels in endocytic vesicles but not at the plasma membrane. Moreover, INPP4B loss delays degradation of EGFR and MET, while promoting recycling of receptor tyrosine kinases (RTK), thus enhancing the duration and amplitude of signaling output upon growth factor stimulation. Therefore, INPP4B inactivation in TNBC promotes tumorigenesis by modulating RTK recycling and signaling duration. SIGNIFICANCE: Inactivation of the lipid phosphatase INPP4B is frequent in TNBC. Using a genetically engineered mouse model, we show that INPP4B functions as a tumor suppressor in TNBC. INPP4B regulates RTK trafficking and degradation, such that loss of INPP4B prolongs both PI3K and ERK activation.This article is highlighted in the In This Issue feature, p. 1079.

Abstract P2-11-03: Predictive significance of an optimized immunohistochemical panel for basal-like breast cancer on the CCTG MA.12 phase III randomized clinical trial

Abstract Background: Tamoxifen and aromatase inhibitors constitute the backbone of treatment for estrogen receptor positive early stage breast cancers based on immunohistochemical (IHC) results. While these therapies significantly reduce the risk of relapse of breast cancer, they have toxicities and add to costs. Gene expression profiling assays are widely used to quantify risk and support targeted therapies for luminal breast cancers treated with adjuvant endocrine therapy. In a prospective-retrospective analysis of the phase III CCTG MA.12 clinical trial (randomizing premenopausal women treated with adjuvant chemotherapy, to either tamoxifen or placebo for 5 years), luminal subtype by PAM50 was significantly predictive for tamoxifen benefit. However, IHC classification (as estrogen receptor positive) could not demonstrate a statistically significant benefit from tamoxifen over placebo. More accurate IHC biomarkers incorporating nestin positivity or loss of inositol polyphosphate-4-phosphate (INPP4B) have recently been optimized to identify the basal-like intrinsic subtype of breast cancer regardless of ER/PR/HER2 status. In this study, we examined the capacity of these basal biomarkers to identify intrinsic subtype and predict benefit from adjuvant tamoxifen vs. placebo in the CCTG MA.12 clinical trial Methods:492 formalin-fixed paraffin embedded blocks of primary tumor from patients randomized in the CCTG MA.12 trial were used to build tissue microarrays. IHC staining for nestin and INPP4B was done according to published methods, and interpretation was performed by pathologists with no access to molecular data. The performance of the nestin and INPP4B panel was assessed against PAM50 gene expression assay as a standard reference. A prespecified statistical plan was executed independently by the Canadian Cancer Trials Group, testing the primary hypothesis that patients with basal breast cancer, when defined as “positive for nestin or negative for INPP4B,” would not benefit from tamoxifen (primary endpoint: disease-free survival).Results: 366 primary tumor samples from the CCTG MA.12 had a full dataset available for IHCbiomarker evaluation, PAM50 subtype and clinical outcomes. Positive staining of nestin or loss of INPP4B was observed in 47 (13%) of the total cases and was significantly associated with poor prognostic factors including high grade, younger age, ER negativity, triple negative and core basal IHC status (p<0.01). “Nestin+ or INPP4B-” status was significantly associated with basal-like PAM50 gene expression subtype, while the other cases (nestin- and INPP4B+) were significantly associated with PAM50 luminal subtype. Patients assigned as basals by “nestin+ or INPP4B-” IHC status did not demonstrate a benefit from adjuvant tamoxifen vs. placebo (HR=1.39, 95%CI [0.37-5.26], p=0.63), whereas “nestin- and INPP4B+” cases displayed a significantly higher benefit from adjuvant tamoxifen vs. placebo (HR=0.67, 95% CI [0.45-0.98], p=0.04), (p-interaction=0.87). While, in this trial, ER clinical status as determined in community hospitals or centrally by single biomarker IHC was not significantly predictive for tamoxifen benefit, patients classified as IHC non-basal “nestin- and INPP4B+” within the ER+ subgroup did benefit from addition of tamoxifen (HR=0.62, 95%CI [0.38-1.01], p=0.05).Conclusions:The nestin/INPP4B IHC panel offers a feasible and inexpensive methodology to accurately identify intrinsic subtype of breast cancer. Patients assigned as IHC basal by this panel did not display a superior survival when treated with tamoxifen vs. placebo in the adjuvant setting. Within the ER+ subgroup, these cases could potentially be spared the side effects of currently recommended endocrine therapies that do not benefit this group of patients. Citation Format: Karama Asleh, Dongxia Gao, Vivien H Bramwell, Dongsheng Tu, Lois E Shepherd, Torsten O Nielsen. Predictive significance of an optimized immunohistochemical panel for basal-like breast cancer on the CCTG MA.12 phase III randomized clinical trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-03.

SAT-326 INPP4B Suppresses Prostate Inflammation And Protects Mice Fed With High-fat Diet From The Development Of Prostate Intraepithelial Neoplasia

High fat diet leads to obesity, chronic inflammation, accelerated prostate cancer progression, and decreased survival. INPP4B, a dual specificity phosphatase, is a tumor suppressor which opposes Akt and PKC signaling pathways activated in response to obesity and prostate cancer progression. Previous research demonstrated that overexpression of PKC in mouse prostate leads to prostate intraepithelial neoplasia (PIN). Akt pathway stimulates prostate cancer progression in mice and is activated in all advanced prostate cancers in men. Using INPP4B knockout mouse model we tested whether INPP4B regulates prostate physiology in mice on high fat diet. The WT and Inpp4b-/- male mice were fed a low-fat diet (LFD) (11% fat) or high-fat diet (HFD) (58% fat) for 12 weeks. The mouse prostate was dissected into the anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP). We observed that INPP4B and androgen receptor (AR) were expressed primarily in DLP and VP. INPP4B expression was decreased in the DLP of HFD fed mice. In the prostate of mice fed a LFD, we observed similar histological architecture in both WT and Inpp4b-/- mice. All the Inpp4b-/- mice fed a HFD developed PIN, whereas WT mice did not. Cells with polymorphic nuclei were abundant in PIN loci. The staining of α-SMA indicated that prostate epithelial cells invaded the stroma of HFD Inpp4b-/- mice. Consistent with the previous findings, HFD activated PKC, and the loss of INPP4B increased the levels of phospho-PKC βII and ζ in HFD mouse DLP. We previously reported that INPP4B loss changes AR transcriptional output in prostate cancer cells and normal mouse prostate. In HFD group, loss of INPP4B did not affect the level of AR in any of the three prostate lobes but led to an altered AR-dependent transcriptional activity. We show that the pro-inflammatory cytokines Il6, Tnfα and Il1β are expressed at low or undetectable levels in prostates of mice fed LFD. However, all three mediators were strikingly elevated in DLP of HFD Inpp4b-/- mice, where most of the PIN was also observed. Using an IL6 ELISA, we confirmed increases of IL6 protein levels in the prostates of the Inpp4b-/- HFD group. Consistent with elevated levels of inflammatory cytokines in prostates of Inpp4b-/- mice we detected an increase of macrophage specific biomarkers in the DLP of HFD groups, indicating HFD promotes macrophage infiltration in both WT and Inpp4b-/- mice. Importantly, no changes in PTEN were detected at both RNA and protein levels in Inpp4b-/- mice, indicating that increased inflammation and PIN development is due to the loss of INPP4B. Our data suggests that INPP4B suppresses oncogenic signaling pathways and opposes inflammation and neoplastic transformation in prostates of mice maintained on a high fat diet.

Inositol polyphosphate 4-phosphatase type II regulation of androgen receptor activity.

Activation and transcriptional reprogramming of AR in advanced prostate cancer frequently coincides with the loss of two tumor suppressors, INPP4B and PTEN, which are highly expressed in human and mouse prostate epithelium. While regulation of AR signaling by PTEN has been described by multiple groups, it is not known whether the loss of INPP4B affects AR activity. Using prostate cancer cell lines we showed that INPP4B regulates AR transcriptional activity and the oncogenic signaling pathways Akt and PKC. Analysis of gene expression in prostate cancer patient cohorts showed a positive correlation between INPP4B expression and both AR mRNA levels and AR transcriptional output. Using an Inpp4b-/- mouse model, we demonstrated that INPP4B suppresses Akt and PKC signaling pathways and modulates AR transcriptional activity in normal mouse prostate. Remarkably, PTEN protein levels and phosphorylation of S380 were the same in Inpp4b-/- and WT males, suggesting that the observed changes were due exclusively to the loss of INPP4B. Our data show that INPP4B modulates AR activity in normal prostate and its loss contributes to the AR-dependent transcriptional profile in prostate cancer.

Abstract P2-10-02: Basal biomarkers nestin and INPP4b predict gemcitabine benefit in metastatic breast cancer: Results from the phase III SBG0102 clinical trial

Abstract Background: A growing body of evidence is suggesting that basal-like and triple negative breast cancers may be particularly sensitive to nucleoside analogues (gemcitabine, capecitabine). In a prospective-retrospective analysis of the phase III SBG0102 clinical trial randomizing metastatic breast cancer patients to gemcitabine plus docetaxel (GD) or to higher-dose single agent docetaxel (D), patients with basal-like breast cancer by PAM50 gene expression had significantly better overall survival (OS) in the gemcitabine arm. By immunohistochemistry (IHC), triple negative status was not predictive, but is a poor surrogate for the basal-like intrinsic subtype. More accurate IHC biomarkers have since become available defining basal-like breast cancers by nestin positivity or by loss of inositol polyphosphate-4-phosphate (INPP4b). Methods: Formalin-fixed paraffin embedded blocks of primary tumor tissue corresponding to 270 of the 337 patients participated in the SBG0102 trial were used to build tissue microarrays. IHC staining and interpretation for nestin and INPP4b by pathologists (who had no access to clinical data) followed published methods. A prespecified statistical plan was executed independently by Danish Breast Cancer Group statisticians, testing the primary hypothesis that patients with basal breast cancer – defined as positive for nestin or negative for INPP4b, regardless of ER/PR/HER2 status – would have superior OS on the GD treatment arm when compared to the D treatment arm by interaction test. Secondary outcomes included time to tumor progression (TTP) and response rate. Kaplan-Meier method with log-rank test of nestin and INPP4b status was used to measure OS and TTP. Forest plots were used to visualize predictive capacities relative to IHC markers and treatment effects. Results: Two hundred fifty two cases were evaluable for this study, among which 38 (15%) had been classified as basal-like, 45 (18%) as HER2-Enriched, 74 (29%) as luminal A and 91 (36%) as luminal B by PAM50. Among 241 cases being evaluable for both IHC nestin and INPP4b markers, positive staining of nestin or loss of INPP4b was observed in 43 (17%) of the total cases and was significantly associated with PAM50 basal-like subtype (p<0.0001). Within a median follow up of 13 years, patients assigned as IHC-basal by virtue of being “nestin+ or INPP4b-” demonstrated a significantly lower OS when compared to non-basal cases defined as “nestin- and INPP4b+” (HR=2.45, 95% CI: 1.47-4.07) (p=0.0006). The IHC-basal patients did much better on the GD vs. the D arm (HR=0.36, 95% CI: 0.19-0.68) whereas there was no such difference in outcomes for other patients (HR=0.99). The interaction test was significant (p-interaction<0.005). Conclusions: The nestin/INPP4b IHC panel offers a practical and inexpensive technology to identify basal-like patients. In the metastatic setting, women with IHC-basal breast cancers defined using these markers have superior overall survival when randomized to gemcitabine-containing chemotherapy compared to docetaxel alone. Citation Format: Asleh-Aburaya K, Lyck Carstensen S, Burugu S, Gao D, Tykjær Jørgensen CL, Won JR, Jensen M-B, Balslev E, Lænkholm A-V, Nielsen DL, Ejlertsen B, Nielsen TO. Basal biomarkers nestin and INPP4b predict gemcitabine benefit in metastatic breast cancer: Results from the phase III SBG0102 clinical trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-02.

Abstract POSTER-BIOL-1313: Loss of INPP4B causes a DNA repair defect and upregulation of EGFR expression and can be targeted with PARP inhibitor treatment

Abstract Treatment options for ovarian cancer patients remain limited and overall survival is less than 50 percent despite recent clinical advances. This study aimed to identify treatment options for ovarian tumors deficient in the lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B). INPP4B has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. To identify further synthetic-lethal gene interactions in INPP4B-deficient ovarian cancer cells we employed the GlaxoSmithKline small compound library in a high-throughput screen. We successfully validated 9 out of initial 19 hit compounds which target Akt, VEGFR (Vascular Endothelial Growth Factor Receptor), p38α, LCK (Lymphocyte-specific protein Kinase) and TGFRβ1 (Transforming Growth Factor Receptor beta 1). Interestingly, we found that INPP4B loss resulted in increased efficacy of small compound inhibitors directed against EGFR (Epidermal Growth Factor Receptor), contrary to expectation from the literature given the documented activation of Akt in these cells. Cancer cells with high phosphorylation levels of Akt through PTEN loss or PI3-kinase mutation have been shown to be resistant to EGFR inhibitors. Using cell proliferation, clonogenic and xenograft assays we demonstrated that loss of INPP4B in the human ovarian cancer cell line OVCA433 and Hey resulted in significant sensitivity towards EGFR inhibition using Gefitinib, Erlotinib and Lapatinib compared to PTEN knockdown pools. Inhibition of ErbB2 using Herceptin did not result in synthetic lethality in Ovca433 INPP4B knockdown pools. As anticipated, Ovca429 INPP4B knockdown cell pools showed resistance to Gefitinib treatment due to an inherent PIK3CA mutation in Ovca429 ovarian cancer cells. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP and EGFR inhibitor response, and consequently offering novel therapeutic options for a significant patient subgroup. Citation Format: Christina Anja Gewinner. Loss of INPP4B causes a DNA repair defect and upregulation of EGFR expression and can be targeted with PARP inhibitor treatment [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1313.

PIPPing on AKT1: How Many Phosphatases Does It Take to Turn off PI3K?

In this issue of Cancer Cell, Ooms and colleagues show that the lipid phosphatase PIPP/INPP5J, frequently inactivated in triple-negative breast cancers, functions as a tumor suppressor by specifically modulating the activity of AKT1 in the context of oncogenic PI3K signaling, leading to inhibition of metastatic dissemination.

Abstract 2650: PI 3-Kinase inhibitors enhance the synthetic lethality of Parp inhibitors

Abstract Introduction: Clinical trials have shown promising responses of BRCA-linked breast and ovarian cancers to PARP inhibitor therapy, but remissions are often short-lived and incomplete. Frequent loss of INPP4B and PTEN leads to activation in the PI3K pathway in TNBC, making the PI3K pathway an attractive treatment target. Here we present mechanistic data that explain how PI3K- and PARP-inhibition enhance each other. Methods: We examined the effects of PI3K-inhibitors BKM120 or BYL719 and PARP-inhibitor Olaparib on the metabolism of HCC1937 cells (BRCA1 5382C mutation and homozygous deletion of PTEN and p53) measuring lactic acid production, via seahorse analysis, carbon metabolism using targeted Mass Spectrometry (LC-MS/MS) and DNA synthesis. PI3K- but not Akt- or SGK-inhibition induced a DNA damage response in this BRCA1 mutant cell line as well as in a mouse model of BRCA1-related breast cancer (K14-Cre BRCA1f/f p53f/f) mouse model. Summary: Metabolic profiling showed that PI3K-inhibition decreased flux through glycolysis and specifically through the non-oxidative pentose-phosphate pathway, the main source of ribose-5-phosphate required for the de novo synthesis of nucleotides in BRCA1-mutant breast cancer cells. Nucleotide shortage led to replication stress with the appearance of γH2AX, increased poly-ADP-ribosylation and an acute drop in DNA synthesis in cell cultures as well as in vivo. PI3K inhibition led to a reduced and error-prone S-phase due to a decrease in nucleotide biosynthesis which could be rescued upon exogenous supply of nucleosides. In a mouse model (K14-Cre BRCA1f/fp53f/f induced breast cancers) we could confirm that both BKM120 and BYL719 enhanced the efficacy of Parp-inhibitor Olaparib (median PFS for Olaparib 50 days, for BKM120 4 days, for BYL719 6 days and for both combinations, BKM120+Olaparib and BYL719+Olaparib > 120 days). Conclusion: Preceding cell cycle arrest, PI3K-inhibition leads to a decrease in DNA synthesis due to decreased Ribose-5-phosphate production required for nucleotide synthesis. PI3K-inhibitors lower nucleotide pools, lead to impaired DNA damage repair and S-phase progression and further augment the synthetic lethality of Parp-inhibitors in BRCA1-related breast cancer. Citation Format: Ashish P. Juvekar, Sina Yadegarynia, Hai Hu, Costas A. Lyssiotis, Hui Liu, John M. Asara, Ralph Scully, Lewis C. Cantley, Gerburg M. Wulf. PI 3-Kinase inhibitors enhance the synthetic lethality of Parp inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2015-2650

Loss of INPP4B causes a DNA repair defect through loss of BRCA1, ATM and ATR and can be targeted with PARP inhibitor treatment.

Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.

Prognostic value of INPP4B protein immunohistochemistry in ovarian cancer.

Ovarian cancer is associated with poor prognosis and altered protein expression patterns may be useful for identifying patients likely to have poor disease outcomes. The impact of altered INPP4B protein expression on prognosis is unclear. The aim of this study was to evaluate the implication of INPP4B expression changes in a large series of ovarian cancer tissue samples. Tissue microarrays were constructed from 599 epithelial ovarian tumors and stained with antibodies for INPP4B, p53, and PTEN. Proportional hazard models were used to estimate survival hazard ratios (HRs) associated with altered protein expression. Seventy-nine percent of the ovarian cancers demonstrated loss of INPP4B, whereas 53% showed aberrant p53 expression (i.e., complete loss of p53 or over-expression of p53) and 8% showed loss of PTEN. INPP4B was frequently lost in serous and endometrioid cancer subtypes, aberrant p53 expression was most common among serous subtype, and loss of PTEN was most common among endometrioid tumors (p for all three proteins across histologic subtypes ≤ 0.0001). INPP4B loss or aberrant p53 expression were both associated with increased mortality (HR = 1.84; 95% CI 1.27 - 2.68 and HR = 3.10; 95% CI 2.33 - 4.11, respectively); however, in multivariate models, only the relationship with p53 achieved statistical significance (HR = 1.20; 95% CI 0.82 - 1.76 for INPP4B and HR = 1.73; 95% CI 1.28 - 2.34 for p53). Conclusion: The INPP4B protein is frequently lost in serous and endometrioid subtypes of ovarian cancer. A possible prognostic role of INPP4B for endometrioid ovarian tumors requires further evaluation.

PI3K pathway activation in high-grade ductal carcinoma in situ--implications for progression to invasive breast carcinoma.

To assess the prevalence of phosphoinositide 3-kinase (PI3K) pathway alterations in pure high-grade ductal carcinoma in situ (DCIS) and DCIS associated with invasive breast cancer (IBC), and to determine whether DCIS and adjacent IBCs harbor distinct PI3K pathway aberrations. Eighty-nine cases of pure high-grade DCIS and 119 cases of high-grade DCIS associated with IBC were characterized according to estrogen receptor (ER) and HER2 status, subjected to immunohistochemical analysis of PTEN, INPP4B, phosphorylated (p)AKT and pS6 expression, and to microdissection followed by Sequenom genotyping of PIK3CA and AKT1 hotspot mutations. Alterations affecting the PI3K pathway were found in a subset of pure DCIS and DCIS adjacent to IBC. A subtype-matched comparison of pure DCIS and DCIS adjacent to IBC revealed that PIK3CA hotspot mutations and pAKT expression were significantly more prevalent in ER-positive/HER2-negative DCIS adjacent to IBC (P values, 0.005 and 0.043, respectively), and that in ER-negative/HER2-positive cases INPP4B loss of expression was more frequently observed in pure DCIS (a P value of 0.013). No differences in the parameters analyzed were observed in a pairwise comparison of the in situ and invasive components of cases of DCIS and adjacent IBC. Analysis of the PIK3CA-mutant allelic frequencies in DCIS and synchronous IBC revealed cases in which PIK3CA mutations were either restricted to the DCIS or to the invasive components. Molecular aberrations affecting the PI3K pathway may play a role in the progression from high-grade DCIS to IBC in a subset of cases (e.g., a subgroup of ER-positive/HER2-negative lesions).

A survey of immunohistochemical biomarkers for basal-like breast cancer against a gene expression profile gold standard

Gene expression profiling of breast cancer delineates a particularly aggressive subtype referred to as ‘basal-like’, which comprises ∼15% of all breast cancers, afflicts younger women and is refractory to endocrine and anti-HER2 therapies. Immunohistochemical surrogate definitions for basal-like breast cancer, such as the clinical ER/PR/HER2 triple-negative phenotype and models incorporating positive expression for CK5 (CK5/6) and/or EGFR are heavily cited. However, many additional biomarkers for basal-like breast cancer have been described in the literature. A parallel comparison of 46 proposed immunohistochemical biomarkers of basal-like breast cancer was performed against a gene expression profile gold standard on a tissue microarray containing 42 basal-like and 80 non-basal-like breast cancer cases. Ki67 and PPH3 were the most sensitive biomarkers (both 92%) positively expressed in the basal-like subtype, whereas CK14, IMP3 and NGFR were the most specific (100%). Among biomarkers surveyed, loss of INPP4B (a negative regulator of phosphatidylinositol signaling) was 61% sensitive and 99% specific with the highest odds ratio (OR) at 108, indicating the strongest association with basal-like breast cancer. Expression of nestin, a common marker of neural progenitor cells that is also associated with the triple-negative/basal-like phenotype and poor breast cancer prognosis, possessed the second highest OR at 29 among the 46 biomarkers surveyed, as well as 54% sensitivity and 96% specificity. As a positively expressed biomarker, nestin possesses technical advantages over INPP4B that make it a more ideal biomarker for identification of basal-like breast cancer. The comprehensive immunohistochemical biomarker survey presented in this study is a necessary step for determining an optimized surrogate immunopanel that best defines basal-like breast cancer in a practical and clinically accessible way.

Abstract 1041: Alterations of INPP4B, PIK3CA and pAkt of the PI3K pathway are associated with squamous cell carcinoma of the lung

Abstract The aim of the study was to investigate how alterations in the PI3K pathway correlate with the non-small cell lung cancer subtypes squamous cell carcinoma (SSC) and adenocarcinoma (ADCA). We analyzed copy number variation (CNV) and protein expression of the newly identified phosphatase INPP4B, protein expression of pAkt, PDPK1 and PTEN as well as mutational status of PIK3CA and PTEN in 180 lung cancer cases. Nineteen % of all cases displayed loss of INPP4B copy whereas 47% lacked expression. Both CNV and expression analyses showed a correlation between loss of INPP4B and the squamous subtype (RR = 1.44, p = 0.06; RR = 1.86, p = 0.00003). Elevated level of pAkt expression was seen in 63% of all cases and also correlated specifically to SCC (RR = 1.71, p = 0.001). PDPK1 was expressed in 70% of all cases, more in male than female patients, but no difference between the tumor subtypes. Regarding PTEN, 50% of all cases displayed loss of expression, of which seven cases were identified with mutations in the phosphatase or C2 domains. We detected nine cases (5%) of PIK3CA mutations, all identified as the E545K hot spot mutation in the helical domain, all except one in SCC (RR = 1.69, p = 0.05). When analyzing all PI3K pathway components together, we show that patients with at least one alteration in the PI3K pathway are twice as likely to have SCC, than ADCA (RR = 2.01, p = 0.02). Interestingly, we also found a strong correlation between high pAkt expression and PTEN expression, which we speculate can be explained by a feedback-loop via EGR1 and ARF recently proposed by Yu et al, 2009. As comparison, we also analyzed MAPK pathway genes, where we identified fifteen KRAS mutations (8%) and one BRAF mutation (1%), significantly associated to ADCA. The intermediate insulin receptor substrate 1 (IRS-1) is involved in activation of both the PI3K and MAPK pathways and we were interested in studying the Gly972Arg polymorphism previously shown to be associated with prostate, colon and breast cancer. However, we could not detect any associations to any subgroup. In conclusion, we show here that several components of the PI3K pathway, alone and in combination, are correlated to development of squamous cell carcinoma of the lung. 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 1041. doi:10.1158/1538-7445.AM2011-1041