Targeting activated PI3K/mTOR signaling overcomes acquired resistance to CDK4/6-based therapies in preclinical models of hormone receptor-positive breast cancer.

BACKGROUND: Combined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2- breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance. METHODS: In this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance. RESULTS: We show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2- breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2- breast cancer models. CONCLUSIONS: These data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2- breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

In vitro activity of the mTOR inhibitor everolimus, in a large panel of breast cancer cell lines and analysis for predictors of response.

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets.

OBJECTIVE: Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. DESIGN: Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. RESULTS: 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. CONCLUSION: The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

Dovitinib demonstrates antitumor and antimetastatic activities in xenograft models of hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. This study aims at investigating the antitumor, antiangiogenesis and antimetastatic activities of dovitinib in preclinical models of HCC. METHODS: 21-0208 and SK-HEP1 cells as well as patient-derived HCC models were employed to study the antitumor effect of dovitinib. Changes of biomarkers relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. RESULTS: Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced cell migration. Dovitinib inhibited basal expression and FGF-induced phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo, dovitinib potently inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated with inactivation of FGFR/PDGFR-ß/VEGFR-2 signaling pathways. Dovitinib also caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, dovitinib potently inhibited primary tumor growth and lung metastasis and significantly prolonged mouse survival. CONCLUSIONS: Dovitinib demonstrated significant antitumor and antimetastatic activities in HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.

Natural history of rising serum prostate-specific antigen in men with castrate nonmetastatic prostate cancer.

PURPOSE: To describe the natural history of nonmetastatic prostate cancer and rising prostate-specific antigen (PSA) despite androgen deprivation therapy. PATIENTS AND METHODS: The 201 patients in this report were the placebo control group from an aborted randomized controlled trial to evaluate the effects of zoledronic acid on time to first bone metastasis in men with prostate cancer, no bone metastases, and rising PSA despite androgen deprivation therapy. Relationships between baseline covariates and clinical outcomes were assessed by Cox proportional hazard analyses. Covariates in the model were baseline PSA, Gleason sum, history of bilateral orchiectomies, regional lymph node metastases at diagnosis, prior prostatectomy, time from androgen deprivation therapy to random assignment, time from diagnosis to random assignment, and PSA velocity. RESULTS: At 2 years, 33% of patients had developed bone metastases. Median bone metastasis-free survival was 30 months. Median time to first bone metastases and overall survival were not reached. Baseline PSA level greater than 10 ng/mL (relative risk, 3.18; 95% CI, 1.74 to 5.80; P < .001) and PSA velocity (4.34 for each 0.01 increase in PSA velocity; 95% CI, 2.30 to 8.21; P < .001) independently predicted shorter time to first bone metastasis. Baseline PSA and PSA velocity also independently predicted overall survival and metastasis-free survival. Other covariates did not consistently predict clinical outcomes. CONCLUSION: Men with nonmetastatic prostate cancer and rising PSA despite androgen deprivation therapy have a relatively indolent natural history. Baseline PSA and PSA velocity independently predict time to first bone metastasis and survival.

Targeting PI3K/mTOR overcomes resistance to HER2-targeted therapy independent of feedback activation of AKT.

PURPOSE: Altered PI3K/mTOR signaling is implicated in the pathogenesis of a number of breast cancers, including those resistant to hormonal and HER2-targeted therapies. EXPERIMENTAL DESIGN: The activity of four classes of PI3K/mTOR inhibitory molecules, including a pan-PI3K inhibitor (NVP-BKM120), a p110α isoform-specific PI3K inhibitor (NVP-BYL719), an mTORC1-specific inhibitor (NVP-RAD001), and a dual PI3K/mTORC1/2 inhibitor (NVP-BEZ235), was evaluated both in vitro and in vivo against a panel of 48 human breast cell lines. RESULTS: Each agent showed significant antiproliferative activity in vitro, particularly in luminal estrogen receptor-positive and/or HER2(+) cell lines harboring PI3K mutations. In addition, monotherapy with each of the four inhibitors led to significant inhibition of in vivo growth in HER2(+) breast cancer models. The PI3K/mTOR pathway inhibitors were also effective in overcoming both de novo and acquired trastuzumab resistance in vitro and in vivo. Furthermore, combined targeting of HER2 and PI3K/mTOR leads to increased apoptosis in vitro and induction of tumor regression in trastuzumab-resistant xenograft models. Finally, as previously shown, targeting mTORC1 alone with RAD001 leads to consistent feedback activation of AKT both in vitro and in vivo, whereas the dual mTOR1-2/PI3K inhibitor BEZ235 eliminates this feedback loop. However, despite these important signaling differences, both molecules are equally effective in inhibiting tumor cell proliferation both in vitro and in vivo. CONCLUSION: These preclinical data support the findings of the BOLERO 3 trial that shows that targeting of the PI3K/mTOR pathway in combination with trastuzumab is beneficial in trastuzumab-resistant breast cancer.