Lloye M. Dillon

Therapeutic targeting of cancers with loss of PTEN function

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is one of the most frequently disrupted tumor suppressors in cancer. The lipid phosphatase activity of PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to repress tumor cell growth and survival. In the nucleus, PTEN promotes chromosome stability and DNA repair. Consequently, loss of PTEN function increases genomic instability. PTEN deficiency is caused by inherited germline mutations, somatic mutations, epigenetic and transcriptional silencing, post-translational modifications, and protein-protein interactions. Given the high frequency of PTEN deficiency across cancer subtypes, therapeutic approaches that exploit PTEN loss-of-function could provide effective treatment strategies. Herein, we discuss therapeutic strategies aimed at cancers with loss of PTEN function, and the challenges involved in treating patients afflicted with such cancers. We review preclinical and clinical findings, and highlight novel strategies under development to target PTENdeficient cancers.

P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT and MEK/ERK signaling in breast cancer

Phosphatidylinositol 3-kinase (PI3K) promotes cancer cell survival, migration, growth and proliferation by generating phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the inner leaflet of the plasma membrane. PIP3 recruits pleckstrin homology domain-containing proteins to the membrane to activate oncogenic signaling cascades. Anticancer therapeutics targeting the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway are in clinical development. In a mass spectrometric screen to identify PIP3-regulated proteins in breast cancer cells, levels of the Rac activator PIP3-dependent Rac exchange factor-1 (P-REX1) increased in response to PI3K inhibition, and decreased upon loss of the PI3K antagonist phosphatase and tensin homolog (PTEN). P-REX1 mRNA and protein levels were positively correlated with ER expression, and inversely correlated with PI3K pathway activation in breast tumors as assessed by gene expression and phosphoproteomic analyses. P-REX1 increased activation of Rac1, PI3K/AKT and MEK/ERK signaling in a PTEN-independent manner, and promoted cell and tumor viability. Loss of P-REX1 or inhibition of Rac suppressed PI3K/AKT and MEK/ERK, and decreased viability. P-REX1 also promoted insulin-like growth factor-1 receptor activation, suggesting that P-REX1 provides positive feedback to activators upstream of PI3K. In support of a model where PIP3-driven P-REX1 promotes both PI3K/AKT and MEK/ERK signaling, high levels of P-REX1 mRNA (but not phospho-AKT or a transcriptomic signature of PI3K activation) were predictive of sensitivity to PI3K inhibitors among breast cancer cell lines. P-REX1 expression was highest in estrogen receptor-positive breast tumors compared with many other cancer subtypes, suggesting that neutralizing the P-REX1/Rac axis may provide a novel therapeutic approach to selectively abrogate oncogenic signaling in breast cancer cells.

Strategically Timing Inhibition of Phosphatidylinositol 3-Kinase to Maximize Therapeutic Index in Estrogen Receptor Alpha–Positive, PIK3CA-Mutant Breast Cancer

Purpose: Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)–positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER+ breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index. Experimental Design: Antiestrogen-sensitive and antiestrogen-resistant ER+ human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. Results: Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone. Conclusions: Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER+, PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens is warranted. Clin Cancer Res; 22(9); 2250–60. ©2016 AACR. See related commentary by Toska and Baselga, p. 2099

Combined inhibition of both p110α and p110β isoforms of phosphatidylinositol 3-kinase is required for sustained therapeutic effect in PTEN-deficient, ER+ breast cancer.

Purpose: Determine the roles of the PI3K isoforms p110α and p110β in PTEN-deficient, estrogen receptor α (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors. Experimental Design: Anti-estrogen-sensitive and -resistant PTEN-deficient, ER+ human breast cancer cell lines, and mice bearing anti-estrogen–resistant xenografts were treated with the anti-estrogen fulvestrant, the p110α inhibitor BYL719, the p110β inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor–initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured. Results: p110β primed cells for response to growth factor stimulation. Although p110β inhibition suppressed cell and tumor growth, dual targeting of p110α/β enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen–sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, whereas long-term treatment only suppressed proliferation to provide durable regression. Conclusions: p110β is the dominant PI3K isoform in PTEN-deficient, ER+ breast cancer cells. Upon p110β inhibition, p110α did not induce significant reactivation of AKT, but combined targeting of p110α/β most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Because apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110α/β inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents. Clin Cancer Res; 23(11); 2795–805. ©2016 AACR.

Estrogen receptor alpha and androgen receptor are commonly expressed in well-differentiated liposarcoma

BackgroundLiposarcoma (LS) is the second-most common type of soft-tissue sarcoma. Despite advances in knowledge and treatment of this disease, there remains a need for more effective LS therapy. Steroid hormone receptors regulate metabolism in adipocytes. Estrogen receptor alpha (ER), progesterone receptor (PR), and androgen receptor (AR) have been implicated in the pathophysiology of other cancer types. We sought to comprehensively determine temporal expression patterns of these receptors in LS.MethodsWe analyzed 561 histologically subtyped LS specimens from 354 patients for expression of ER, PR, and AR by immunohistochemistry (IHC) using diagnostic-grade reagents and protocols. The fractions of positively stained tumor cells were scored within each specimen. IHC scores were compared across LS subtypes using the Kruskal-Wallis test, and subtypes were compared using Dunn’s post-hoc test. Ages of patients with receptor-positive vs. -negative LS were compared by t-test. Genders and races were compared for hormone receptor positivity using Fisher’s exact test and Chi-square analysis, respectively. Recurrence-free survival was compared between receptor-positive and negative patients by log-rank test. p< 0.05 was considered significant.ResultsER and AR were frequently expressed in LS, while few tumors expressed PR. Most of the ER + and AR + samples were of the well-differentiated LS subtype. A smaller fraction of de-differentiated LS expressed ER or AR, but expression was common within well-differentiated regions of tumors histologically classified as de-differentiated LS. In LS specimens from patients who underwent multiple surgeries over time, receptor expression frequently changed over time, which may be attributable in part to intratumor heterogeneity, varying degrees of de-differentiation, and biopsy bias. ER and AR were frequently co-expressed. Receptor status was not significantly associated with gender or race, but AR and PR expression were associated with earlier age at diagnosis. Receptor expression was not associated with altered recurrence-free survival.ConclusionsER and AR are commonly expressed in LS, particularly in well-differentiated tumors. These data warrant further functional study to determine receptor function in LS, and the potential efficacy of anti-hormone therapies for the treatment of patients with LS.

The PREX1/Rac signaling axis: Potential as a biomarker and therapeutic target in breast cancer

PREX1 is a Rac guanine exchange factor that coordinates signaling inputs from G protein-coupled receptors and receptor tyrosine kinases (RTKs). PREX1 creates a positive feedback loop to drive RTK, phosphatidylinositol 3-kinase (PI3K)/AKT, and MEK/ERK signaling. High PREX1 levels predict sensitivity to PI3K inhibitors in breast cancer cells.

Abstract P5-20-03: Understanding pharmacodynamics and consequences of PI3K inhibition in ER+ breast tumors

PI3K inhibitors have shown promise for the treatment of anti-estrogen-resistant breast cancers. Current PI3K inhibitor treatment regimens incompletely and transiently inhibit the pathway in carcinomas, and are accompanied by adverse effects in patients. We found that different periods of PI3K inhibition (12, 24, 36 h) potentiated anti-estrogen-induced apoptosis and inhibition of proliferation to similar extents in cultured ER+ cells. We thus hypothesized that short-term, complete inhibition of PI3K will have a greater anti-tumor effect and reduced systemic toxicity than chronic, partial inhibition. Pharmacokinetic analysis of the orally available pan-PI3K inhibitor GDC-0941 at low (100 mg/kg) and high (800 mg/kg) doses in mice revealed that plasma levels peaked after 15-30 min. (18.6 uM and 20.7 uM, respectively), and decreased to a plateau phase after 1 h that was maintained for 8 h with low dose (6.8-10.7 uM) and 23 h with high dose (7.9-15 uM). We performed MCF-7 tumor pharmacokinetic analyses with low and high doses, and with 2 low doses administered 12 hours apart. Tumor GDC-0941 levels peaked after 9 h (1.6 uM with low-dose; 16.9 uM with high-dose). The second low dose increased tumor drug concentrations to 3.2 uM at 9 h after the second dose, compared to 1.6 uM at 9 h after the first dose. After 48 h, tumor drug concentrations decreased to 0 uM with low dose, and to 4.5 uM with high dose. Mice bearing MCF-7 tumors were treated with fulvestrant (5 mg/wk). Three days later, GDC-0941 was administered to assess pharmacodynamic effects. Phospho-AKT and -S6 levels (markers of PI3K and mTORC1 activities, respectively) were maximally suppressed after 1 h and 3 h of high- and low-dose treatments, respectively, returned to baseline within 16 h after low-dose treatment, and remained suppressed for 36 h following high-dose treatment. PARP cleavage (marker of apoptosis) occurred within 1 h and 3 h of high- and low-dose treatments, and increased over time. Re-treatment of mice with low-dose GDC-0941 after 12 h induced continued inhibition of PI3K and mTORC1 for 9-12 h, suggesting that BID low-dose treatment may be sufficient to continually inhibit PI3K. Comparison of high-dose and low-dose BID tumors showed that these treatments induced similar amounts of PI3K inhibition and PARP cleavage at 21-24 h. Mice bearing MCF-7 or fulvestrant-resistant T47D/FR tumors were treated with vehicle, fulvestrant, GDC-0941 (100 mg/kg QD 5 d/wk; 100 mg/kg BID 3 d/wk, 800 mg/kg QW), or combinations of fulvestrant and GDC-0941. Drug combinations induced tumor regression, fulvestrant did not affect tumor growth, high-dose GDC-0941 QW slowed tumor growth, and low-dose GDC-0941 QD or BID appreciably inhibited tumor growth. However, there was no significant difference among doses and schedules of GDC-0941 in the context of a fulvestrant backbone in either tumor model. These data suggest that transient/metronomic (QD, BID) and chronic/infrequent (QW) PI3K inhibition may provide similar anti-tumor efficacy in combination with an anti-estrogen. However, these tumor growth data conflict with cell fate data indicating that high-dose GDC-0941 induced much more apoptosis than low-dose GDC-0941. Ongoing studies will reveal how different schedules of PI3K inhibition shape tumor biology. Citation Format: Wei Yang, Jennifer R Bean, Lloye Dillon, Laurent Salphati, Jodie Pang, Xiaolin Zhang, Michelle Nannini Pepe, Todd W Miller. Understanding pharmacodynamics and consequences of PI3K inhibition in ER+ breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-20-03.

Abstract P5-20-02: PTEN status dictates the roles of PI3K isoforms p110α and p110β in modulation of AKT/mTOR and response to growth factor signaling in ER+ breast cancer

Class 1A phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, survival, and metabolism. PI3Ks are heterodimeric lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (p110α, p110β, or p110δ). p110α and p110β play distinct roles in PI3K signaling in carcinoma cells. p110α is frequently activated by growth factor receptor kinase signaling. In contrast, p110β was shown to play a role in insulin metabolic action, G protein-coupled receptor (GPCR) and Rac1 signal transduction, and oncogenic transformation. Cancer cells deficient in PTEN, the tumor suppressor phosphatase that antagonizes PI3K signaling, are often sensitized to pharmacological inhibitors of p110β. As a result, early clinical studies with p110β inhibitors are often restricted to patients with PTEN-deficient cancers. However, analysis of data from the Genomics of Drug Sensitivity in Cancer database revealed that genetic lesions in PTEN or PIK3CA (encodes p110α) were significantly and independently associated with increased sensitivity to the p110β inhibitor AZD6284. Among 668 cancer cell lines evaluated, 61 lines had AZD6482 IC50 values ≤2 mM, but only 25 lines harbored an alteration in PTEN and/or PIK3CA. Thus, a significant fraction of cancer cell lines (and tumors) without PTEN/PIK3CA alterations are likely to be sensitive to p110β inhibition. To explore the role of p110β in PI3K signaling in ER+ PTEN-deficient breast cancer, we treated cells with the p110β inhibitor GSK2636771 and the p110α inhibitor BYL-719, alone or in combination, and assessed effects on steady state and growth factor-induced activation of AKT and MEK/ERK activation, and cell growth. p110β inhibition reduced the viability of PTEN-deficient cells; however, combined inhibition of p110α and p110β was more effective at reducing AKT and ERK phosphorylation and increasing apoptosis in ER+ PTEN-deficient cells. Furthermore, anti-estrogen treatment potentiated the anti-proliferative effects of PI3K inhibition. p110β inhibition reduced insulin-like growth factor 1 (IGF-1)-induced pAKT levels, and delayed AKT phosphorylation in both PTEN-deficient and PTEN-wild-type cells. In contrast, p110β inhibition sensitized both PTEN-wild-type and PTEN-deficient cells to heregulin stimulation, and promoted PI3K (p85)/HER3 interaction. These results indicate that p110β inhibition desensitizes cells to IGF-1 stimulation, hypersensitizes cells to heregulin, and modulates downstream AKT and MEK/ERK activation in response to growth factor receptor activation. Our findings suggest that the anti-tumor efficacy of p110β inhibitors may be related to growth factor dependence and PTEN status. Citation Format: Lloye M Dillon, Stephanie J Bouley, Todd W Miller. PTEN status dictates the roles of PI3K isoforms p110α and p110β in modulation of AKT/mTOR and response to growth factor signaling in ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-20-02.

Abstract 3342: The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status

The class 1a phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, survival, and metabolism. These PI3Ks are heterodimeric lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (p110α, p110β, or p110δ). p110α and p110β play distinct roles in PI3K signaling in carcinoma cells. p110α is activated by growth factor receptor kinase signaling. In contrast, p110β was shown to play a role in insulin metabolic action, G protein-coupled receptor (GPCR) signaling, and oncogenic transformation. Cancer cells deficient in PTEN, the tumor suppressor phosphatase that antagonizes PI3K signaling, have been shown to be sensitized to pharmacological inhibitors of p110β. As a result, early clinical studies with p110β inhibitors are restricted to patients with PTEN-deficient cancers. However, analysis of data from the Genomics of Drug Sensitivity in Cancer database revealed that genetic lesions in PTEN or PIK3CA (encodes p110α) were significantly and independently associated with increased sensitivity to the p110β inhibitor AZD6284. Among 668 cancer cell lines, 96, 57, and 5 lines harbored alterations in PTEN, PIK3CA, or both, respectively. Among 61 cell lines with IC50 values ≤2 μM, only 25 harbored an alteration in PTEN and/or PIK3CA. Thus, a significant fraction of cancer cell lines (and tumors) without PTEN alterations are likely to be sensitive to p110β inhibition. To explore the role of p110β in PI3K signaling in ER+ breast cancer, we treated cells with the p110β inhibitor GSK2636771 and monitored effects on growth factor-induced activation of AKT and MEK/ERK. We found that p110β inhibition reduced basal pAKT and pERK levels in PTEN-deficient cells. Furthermore, p110β inhibition reduced insulin-like growth factor 1 (IGF-1)-induced pAKT levels and delayed the onset of AKT phosphorylation in both PTEN-deficient and PTEN-wild-type cells. In contrast, p110β inhibition sensitized both PTEN-wildtype and PTEN-deficient cells to heregulin stimulation. These results indicate that p110β desensitizes cells to IGF-1 stimulation and hyper-sensitizes them to heregulin stimulation and suggests that p110β regulates downstream AKT and MEK/ERK activation in response to growth factor receptor activation. Our findings advocate for the testing of p110β inhibitors in cell lines (and tumors) selected based on growth factor dependence and PTEN status. Citation Format: Stephanie J. Bouley, Lloye M. Dillon, Todd W. Miller. The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2014-3342

Abstract 5512: Pharmacodynamic analysis of PI3K inhibition in breast tumors: A model to improve early clinical investigation of novel agents

PI3K inhibitors have shown promise for the treatment of anti-estrogen-resistant breast cancers. Dose determination in clinical studies is often performed in a dose-escalation fashion until toxicities are observed; however, this approach does not provide information on target inhibition or allow optimization of scheduling for maximal anti-cancer effects. Current PI3K inhibitor treatment regimens incompletely and transiently inhibit the pathway in carcinomas, and are accompanied by adverse effects in patients. We hypothesize that short-term, complete inhibition of PI3K will have a greater anti-tumor effect and reduced adverse effects than chronic, partial inhibition. Through detailed analysis of the timing and magnitude of drug effects on tumor biology, drug doses and schedules may be optimized during early clinical development to improve anti-tumor effects in subsequent trials. Pharmacokinetic analysis of the PI3K inhibitor GDC-0941 at low (100 mg/kg) and high (800 mg/kg) doses in mice revealed that plasma levels peaked after 15-30 min (18.6 uM and 20.7 uM, respectively), and decreased to a plateau phase after 1 h that was maintained for 8 h with low-dose GDC-0941 (6.8-10.7 uM) and for 23 h with high-dose GDC-0941 (7.9-15 uM). Thus, GDC-0941 appears to be retained in a tissue compartment and slowly released back into plasma over time. Mice bearing MCF-7 tumors were treated with the anti-estrogen fulvestrant (5 mg) for three days, and then low- or high-dose GDC-0941, or 2 low doses of GDC-0941 12 hours apart, to assess pharmacodynamic effects and tumor cell response. PARP cleavage (marker of apoptosis) occurred within 1 h and 3 h of high- and low-dose treatments, respectively, and apoptosis increased over time. Phosphorylated AKT and S6 levels (markers of PI3K and mTORC1 activities) were maximally suppressed after 1 h and 3 h of high- and low-dose treatments, respectively, and returned to baseline within 16 h after low-dose treatment. In vitro analysis revealed that PI3K inhibition induced upregulation of growth factor receptors upstream of PI3K, indicating a requirement for sustained and robust PI3K inhibition. Re-treatment of mice with low-dose GDC-0941 after 12 h induced continued inhibition of PI3K and mTORC1 for >9 h, suggesting that twice daily low-dose treatment may be sufficient to continually inhibit PI3K. In contrast, high-dose GDC-0941 suppressed PI3K and mTORC1 for >24 h. Analysis of these doses and schedules on tumor growth is ongoing. Citation Format: Wei Yang, Jennifer R. Bean, Lloye Dillon, Laurent Salphati, Michelle Nannini, Todd W. Miller. Pharmacodynamic analysis of PI3K inhibition in breast tumors: A model to improve early clinical investigation of novel agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5512. doi:10.1158/1538-7445.AM2014-5512