Dylan Conklin

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

IntroductionAlterations 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.MethodsForty-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.ResultsCell 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.ConclusionsThese studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991.

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. Clin Cancer Res; 20(13); 3507–20. ©2014 AACR.

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. Mol Cancer Ther; 12(4); 509–19. ©2013 AACR.

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 IC50 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 G0–G1 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. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

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.

Pre-clinical activity of the PARP inhibitor AZD2281 in human breast cancer cell lines and in combination with DNA damaging agents.

Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast cancer. AZD2281, a potent, oral, PARP inhibitor has been shown to have clinical activity in patients with BRCA mutant breast cancers. Laboratory studies have suggested that non-BRCA mutant breast cancers may also be sensitive to PARP inhibition in tumors as a consequence of deficiencies in other homologous recombination (HR) repair components. Using a large panel of human breast cancer cell lines we tested the hypotheses that (1) there may be a subset of non-BRCA mutant breast cancers that are sensitive to single-agent AZD2281 and (2) AZD2281 would potentiate the cytotoxic effects of the DNA damaging agent cisplatin. Methods: 43 human cell lines representing known molecular subgroups of breast cancer (i.e. ER+, HER2 amplified, “triple-negative”), and 3 immortalized breast lines were treated in duplicate in adherent plates with AZD2281 using two-fold dilutions over 6 concentrations for 6 days. Dose response curves were generated using a cell count assay to calculate the IC 50 of AZD2281. In addition, a subset of cell lines that grow under anchorage independent conditions were grown in triplicate in the presence and absence of 1 µM AZD2281 in soft agar for at least 3 weeks and growth inhibition was calculated as per cent of untreated control. Cell lines (both sensitive and resistant to single agent AZD2281) were also evaluated in combination with cisplatin in a cell count assay to assess the interaction between the two agents.
 Results: The majority of breast cancer cell lines evaluated in the short term 2-D growth assay did not show significant growth inhibition (IC 50 50 Conclusion: The PARP inhibitor AZD2281 has significant pre-clinical activity in human breast cancer cell lines. In a clonogenic assay, cell lines representing the “triple negative” subtype were especially sensitive to AZD2281 supporting clinical development in this population, regardless of BRCA status. In addition, these pre-clinical data support the hypothesis that PARP inhibition may potentiate the effects of chemotherapy induced DNA damage and provide further rationale for clinical development. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1038.

Panobinostat (LBH589), a pan-DAC inhibitor, induces cell death in ER+ and HER2 amplified cell lines in vitro and is synergistic in vivo with trastuzumab.

Abstract #4047 Background: Histone modifications have been investigated as causing altered gene expression resulting in activation of oncogenic pathways. Panobinostat (LBH589) is a potent pan-deacetylase (DAC) inhibitor in clinical development. Given the potentially broad biological effects of this class of agent, we assessed the in vitro effects of panobinostat on a large panel of molecularly profiled human breast cancer cell lines to better define the differential effects of panobinostat across subtypes and to help guide clinical development. In addition, preclinical studies evaluated the effects of panobinostat and the HER2-targeted antibody, trastuzumab.
 Methods: 43 human cell lines representing known molecular subgroups of breast cancer, and 3 immortalized breast lines were treated in duplicate with panobinostat using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate IC 50 and LD 50 of panobinostat. Data were analyzed against baseline gene expression data (Agilent microarray) to identify genes associated with sensitivity (S) and resistance (R) to panobinostat. 12 cell lines (6 S; 6 R) were then treated with clinically relevant concentrations of panobinostat; RNA was isolated after 48 hrs. Agilent expression profiles compared pre-and post-treatment cell lines to identify differentially expressed genes (DEGs) that change in S and R cell lines. In vivo , nude mice bearing HER2+ and pathway-dependent BT-474 xenografts were treated with panobinostat alone and in combination with trastuzumab. Tumor growth inhibition and delay were measured.
 Results: In vitro panobinostat has potent anti-proliferative effects across all breast cancer subtypes with most IC 50 values 50 50nM (Chi-square 50 >1000nM), which suggests some differential activity in malignant cell lines versus those that are more “normal”. When gene expression profiles were analyzed pre-and post-treatment, a remarkable overlap was seen in number and specific genes changing after panobinostat exposure in both S and R lines. However, the degree of gene changes was greater in S lines than R lines. In vivo , panobinostat acted synergistically with trastuzumab to inhibit growth of the HER2 amplified breast cancer cell line, BT-474.
 Conclusion: These studies suggest that ER+ and HER2 amplified breast cancers may be more likely to respond to the pan-DAC inhibitor panobinostat. Profiling studies identified 218 DEGs that were significantly changed between S and R lines. These data support the ongoing development of panobinostat in breast cancer and provide rationale for patient selection. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4047.

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.

Abstract 3008: Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer

BACKGROUND: Aurora kinases (AK) A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including ovarian cancer (OC). AMG 900, a potent and highly selective small molecule inhibitor of AKs, showed promising single-agent activity in heavily pretreated patients with advanced OC in a Phase 1b clinical trial. In this study, we report the preclinical effects of AMG 900 in a panel of well-characterized human cancer cell lines representing clinically-relevant OC subtypes. METHODS: The anti-proliferative effects of AMG 900 were evaluated using a 5-day cell count assay. Cell lines were classified as sensitive to AMG 900 when lethality was > 15% at 10 nM. Molecular markers were profiled including TP53 mutation status, AURKA, CCNE1, MYC copy number, and p53, p21 and cyclin E1 protein by reverse phase protein array. Flow cytometry and imaging methods were used to evaluate the mechanism of action of AMG 900 alone and in combination with chemotherapy. The combination of AMG 900 plus docetaxel was evaluated in an IGROV-1 ovarian endometrioid carcinoma xenograft model. RESULTS: One third of the cell lines (11 of 35) were classified as sensitive to AMG 900 and showed enrichment for TP53 mutations and serous OC subtype. However, 10 of 24 resistant cell lines harbored TP53 mutations, indicating that TP53 mutational status alone was not sufficient for predicting AMG 900 sensitivity. Inhibition of AK activity by AMG 900 in OC cells resulted in aborted cell division leading to polyploidy and cell death (suggestive of aurora-B dominant phenotype). Re-plating of remnant cells after AMG 900 treatment showed an attenuation of cell regrowth, where TP53 mut IGROV-1 cells showed minimal regrowth compared to TP53 wt OVCAR-5 cells. AMG 900 inhibited proliferation at low nanomolar concentrations in the majority of OC cell lines and enhanced the effects of paclitaxel, carboplatin, and doxorubicin in IGROV-1 cells. In tumor-bearing mice, administration of AMG 900 at 7.5 mg/kg (PO) for two days per week or docetaxel at 10 mg/kg (IP) weekly for four cycles significantly inhibited the growth of IGROV-1 tumor xenografts (P Citation Format: Ondrej Kalous, Dylan Conklin, Kanthinh Manivong, William Wayne, Kelly Hanestad, Jude Canon, Robert Loberg, Gregory Friberg, Erick Gamelin, Florian D. Vogl, Gloria Juan, Angela Coxon, Dennis Slamon, Richard Finn, Marc Payton. Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3008.

Abstract 2437: MEK162 (ARRY 438162), a MEK1/2 inhibitor, has activity in melanoma cells independent of BRAF and NRAS mutation status.

Background. Activation of the Ras/Raf/MEK/MAP kinase pathway is implicated in uncontrolled cell proliferation and tumor growth. Mutated, oncogenic forms of Ras are found in colon, pancreatic, and lung cancers; BRAF mutations have been identified in more than 60% of malignant melanomas and from 40-60% of papillary thyroid cancers. MEK, a dual specific kinase, is a key player in this pathway; it is downstream of both Ras and Raf and activates ERK1/2 through phosphorylation of key tyrosine and threonine residues. MEK162 (ARRY 438162) is a novel small molecule ATP-uncompetitive inhibitor of the kinases MEK1 and MEK2. MEK162 showed promising data in an ongoing Phase 2 Clinical Trial in patients with BRAF and NRAS mutated advanced melanoma. This is the first targeted therapy to show activity in patients with NRAS mutated melanoma. Methods and Results. The melanoma cell line panel was the most sensitive after investigating the growth inhibitory effect of MEK162 on 328 cancer cell lines from diverse histologies including melanoma, head and neck, colon, pancreas, lung, ovarian, liver, kidney, breast and endometrial. When a cutoff of IC50 70% Inhibition at 1uM after 6 days of culture was used 83% out of 47 melanoma cell lines were sensitive to the treatment with the MEK inhibitor. Sensitivity to MEK162 was independent of BRAFV600E andNRASQ61mutation status in this cell line panel. Cell cycle arrest and apoptosis was assessed upon exposure to MEK162 using flow cytometry. MEK162 led to a G1 arrest and marked increase in apoptotic cells in the majority of the sensitive melanoma cell lines regardless of their origin and oncogenic driver mutations. Western blots were used to characterize the changes induced by exposure to MEK162 in the MAPK and PI3K/mTOR pathways. MEK1/2 inhibition resulted in a decrease in pERK in all the cell lines tested regardless of their mutational status and the in vitro sensitivity. We observed an increase in pMEK more prominently in NRASQ61L mutant and wild type for NRAS and BRAF mutations cell lines than in BRAFV600Emutant cell lines. We found pAKT and pS6 decreased in the NRAS and BRAF mutant cell lines after treatment, suggesting that the inhibition of the mTOR pathway by MEK162 may be crucial for the sensitivity to the drug. Conclusion. These data provide evidence for supporting the use of MEK162 in the treatment of patients with melanoma. Citation Format: Erika M. Von Euw, Hong-Mei Rong, Neil O9Brien, Dylan Conklin, Veerauo Konkankit, Ke-Wei Gong, Angela Zubel, Ronald Linnartz, Richard Finn, Bartosz Chmielowski, Dennis Slamon. MEK162 (ARRY 438162), a MEK1/2 inhibitor, has activity in melanoma cells independent of BRAF and NRAS mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2013-2437