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.

Concise total syntheses of (-)-jorunnamycin A and (-)-jorumycin enabled by asymmetric catalysis.

The bis-tetrahydroisoquinoline (bis-THIQ) natural products have been studied intensively over the past four decades for their exceptionally potent anticancer activity, in addition to strong Gram-positive and Gram-negative antibiotic character. Synthetic strategies toward these complex polycyclic compounds have relied heavily on electrophilic aromatic chemistry, such as the Pictet-Spengler reaction, that mimics their biosynthetic pathways. Herein, we report an approach to two bis-THIQ natural products, jorunnamycin A and jorumycin, that instead harnesses the power of modern transition-metal catalysis for the three major bond-forming events and proceeds with high efficiency (15 and 16 steps, respectively). By breaking from biomimicry, this strategy allows for the preparation of a more diverse set of nonnatural analogs.

Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines.

To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer.

Preclinical Activity of Abemaciclib Alone or in Combination with Antimitotic and Targeted Therapies in Breast Cancer.

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2- and ER+/HER2+ subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2-, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER+/HER2-, HER2+/ER+, and TNBCs. Mol Cancer Ther; 17(5); 897-907. ©2018 AACR.

Proteasome ubiquitin receptor PSMD4 is an amplification target in breast cancer and may predict sensitivity to PARPi.

Poly (ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair under investigation as a chemotherapeutic target. Current randomized phase three trials of PARPi in metastatic breast cancer are limited to patients with documented BRCA1/2 mutations and no biomarker of PARPi beyond BRCA status is available. In an effort to identify novel biomarkers for PARP inhibition, we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array-CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down-regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. Functional studies reveal that knock-down of PSMD4 in amplified breast cancer cells and loss of the PSMD4 amplicon result in knock-down of PARP1 protein. We show that PSMD4 is amplified and overexpressed in breast cancer and its overexpression correlates with poor survival. Knock-down of PSMD4 results in a significant decrease in cell growth. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi. Finally, this study shows not only that PSMD4 copy number correlates with PARPi sensitivity, but also, that it may be a better predictor of sensitivity to PARPi than BRCA1/2 mutation.

Characterization of Human Cancer Cell Lines by Reverse-phase Protein Arrays.

Cancer cell lines are major model systems for mechanistic investigation and drug development. However, protein expression data linked to high-quality DNA, RNA, and drug-screening data have not been available across a large number of cancer cell lines. Using reverse-phase protein arrays, we measured expression levels of ∼230 key cancer-related proteins in >650 independent cell lines, many of which have publically available genomic, transcriptomic, and drug-screening data. Our dataset recapitulates the effects of mutated pathways on protein expression observed in patient samples, and demonstrates that proteins and particularly phosphoproteins provide information for predicting drug sensitivity that is not available from the corresponding mRNAs. We also developed a user-friendly bioinformatic resource, MCLP, to help serve the biomedical research community.

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.

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.

AMG 900, pan-Aurora kinase inhibitor, preferentially inhibits the proliferation of breast cancer cell lines with dysfunctional p53.

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10 nM of AMG 900 >10 %), 19 cell lines as less sensitive to AMG 900 (lethality at 10 nM of AMG 900 <10 %). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio = 2.53, p = 0.09). mRNA expression levels of AURKA, AURKB, and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR = 1.86, p = 0.004) and low baseline p21 protein levels (RR = 2.28, p = 0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53. AMG 900 caused the accumulation of cells with >4 N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

PD-0332991, a potent and selective inhibitor of cyclin-dependent kinase 4/6, demonstrates inhibition of proliferation in renal cell carcinoma at nanomolar concentrations and molecular markers predict for sensitivity.

BACKGROUND: PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines. MATERIALS AND METHODS: Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression. RESULTS: IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041). CONCLUSION: PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.

Inhibition of HSP90 with AUY922 induces synergy in HER2-amplified trastuzumab-resistant breast and gastric cancer.

HSP90 enables the activation of many client proteins of which the most clinically validated is HER2. NVP-AUY922, a potent HSP90 inhibitor, is currently in phase II clinical trials. To explore its potential clinical use in HER2-amplified breast and gastric cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and -resistant models. A panel of 16 human gastric and 45 breast cancer cell lines, including 16 HER2-amplified (3 and 13, respectively) cells, was treated with AUY922 over various concentrations. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. Effects of this combination on downstream markers were analyzed via Western blot analysis. AUY922 exhibited potent antiproliferative activity in the low nanomolar range (<40 nmol/L) for 59 of 61 cell lines. In both histologies, HER2-amplified cells expressed greater sensitivity to AUY than HER2-negative cells. In conditioned trastuzumab-resistant models, AUY922 showed a synergistic effect with trastuzumab. In vitro, the combination induced greater decreases in HER2, a G2 cell-cycle arrest, and increased apoptosis. In a trastuzumab-resistant gastric cancer xenograft model, the combination of AUY922 and trastuzumab showed greater antitumor efficacy than either drug alone. These data suggest that AUY922 in combination with trastuzumab has unique efficacy in trastuzumab-resistant models. The combination of HSP90 inhibition and direct HER2 blockade represents a novel approach to the treatment of HER2-amplified cancers and clinical trials based on the above data are ongoing.

Dacomitinib (PF-00299804), an irreversible Pan-HER inhibitor, inhibits proliferation of HER2-amplified breast cancer cell lines resistant to trastuzumab and lapatinib.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC(50) values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G(0)-G(1) arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib.

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro.

INTRODUCTION: Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines. METHODS: Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response. RESULTS: Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade. CONCLUSIONS: These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991