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All ADCC ratios from Figure 3 and trastuzumab/pertuzumab ADCC comparison

Densitometry analysis for Figure 6B

Supplementary Table 2 from Activated Phosphoinositide 3-Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib

Supplementary Table 1 from Activated Phosphoinositide 3-Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib

Purpose:Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC.Experimental Design:Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array–determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry–based protocols.Results:Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell–mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone.Conclusions:TKIs differentially alter tumor cell phenotype which can impact NK cell–mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Densitometry for Figure 1D

Densitometry analysis for Figure 6D/E

Flow cytometry MFI and % positive cells data for fluorescently labelled trastuzumab and pertuzumab in SKBR3, HCC1954, T47D and MCF-7 treated with TKIs

Rituximab ADCC negative control

Flow cytometry control scatter plots for trastuzumab-488 and pertuzumab-550

MFI comparison at T0 and T6 for T47D/MCF-7

Trastuzumab and lapatinib provide clinical benefit to women with human epidermal growth factor receptor 2 (HER)–positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. Consequently, there is an urgent need to identify new predictive factors for these agents. The aim of this study was to investigate the role of receptor tyrosine kinase signaling and phosphoinositide 3-kinase (PI3K)/AKT pathway activation in conferring resistance to trastuzumab and lapatinib. To address this question, we evaluated response to trastuzumab and lapatinib in a panel of 18 HER2-amplified cell lines, using both two- and three-dimensional culture. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1, and MDA-453 cell lines were found to be innately resistant to trastuzumab, whereas the MDA-361, MDA-453, HCC-1569, UACC-732, JIMT-1, HCC-202, and UACC-893 cells are innately lapatinib resistant. Lapatinib was active in de novo (SUM-225, HCC-1419, and HCC-1954) and in a BT-474 cell line with acquired resistance to trastuzumab. In these cells, trastuzumab had little effect on AKT phosphorylation, whereas lapatinib retained activity through the dephosphorylation of AKT. Increased phosphorylation of HER2, epidermal growth factor receptor, HER3, and insulin-like growth factor IR correlated with response to lapatinib but not trastuzumab. Loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab, but lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway correlates with resistance to trastuzumab, which can be overcome by lapatinib. In conclusion, pharmacologic targeting of the PI3K/AKT pathway may provide benefit to HER2-positive breast cancer patients who are resistant to trastuzumab therapy. Mol Cancer Ther; 9(6); 1489–502. ©2010 AACR.

Densitometry for Figure 2B

Suppl Fig. S3

Suppl Table 2

Suppl Material & Suppl. Table S1

Intrinsic and acquired resistance to HER-targeting drugs occurs in a significant proportion of HER2-overexpressing breast cancers. Thus, there remains a need to identify predictive biomarkers that could improve patient selection and circumvent these types of drug resistance. Here, we report the identification of neuromedin U (NmU) as an extracellular biomarker in cells resistant to HER-targeted drugs. NmU overexpression occurred in cells with acquired or innate resistance to lapatinib, trastuzumab, neratinib, and afatinib, all of which displayed a similar trend upon short-term exposure, suggesting NmU induction may be an early response. An analysis of 3,489 cases of breast cancer showed NmU to be associated with poor patient outcome, particularly those with HER2-overexpressing tumors independent of established prognostic indicators. Ectopic overexpression of NmU in drug-sensitive cells conferred resistance to all HER-targeting drugs, whereas RNAi-mediated attenuation sensitized cells exhibiting acquired or innate drug resistance. Mechanistic investigations suggested that NmU acted through HSP27 as partner protein to stabilize HER2 protein levels. We also obtained evidence of functional NmU receptors on HER2-overexpressing cells, with the addition of exogenous NmU eliciting an elevation in HER2 and EGFR expression along with drug resistance. Finally, we found that NmU seemed to function in cell motility, invasion, and anoikis resistance. In vivo studies revealed that NmU attenuation impaired tumor growth and metastasis. Taken together, our results defined NmU as a candidate drug response biomarker for HER2-overexpressing cancers and as a candidate therapeutic target to limit metastatic progression and improve the efficacy of HER-targeted drugs. Cancer Res; 74(14); 3821–33. ©2014 AACR.

Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin-sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies.

Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2+BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2+BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2+BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2+BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2+breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2+BrCa.

Background Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. Methods We performed TIL analysis and T-cell analysis by IHC on the pretreatment and ‘On-treatment’ samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. Results In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10–3) but not TILs (p = 0.1) in their ‘On-treatment’ tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p Conclusions The immune system may be ‘primed’ prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Purpose: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. Experimental Design: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array–determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry–based protocols. Results: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell–mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. Conclusions: TKIs differentially alter tumor cell phenotype which can impact NK cell–mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Background An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient’s tumor that can confer clinical efficacy or drug resistance. Methods The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. Results Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. Conclusions Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

Background Junctional adhesion molecule-A (JAM-A) is an adhesion molecule whose overexpression on breast tumor tissue has been associated with aggressive cancer phenotypes, including human epidermal growth factor receptor-2 (HER2)-positive disease. Since JAM-A has been described to regulate HER2 expression in breast cancer cells, we hypothesized that JAM-dependent stabilization of HER2 could participate in resistance to HER2-targeted therapies. Methods Using breast cancer cell line models resistant to anti-HER2 drugs, we investigated JAM-A expression and the effect of JAM-A silencing on biochemical/functional parameters. We also tested whether altered JAM-A expression/processing underpinned differences between drug-sensitive and -resistant cells and acted as a biomarker of patients who developed resistance to HER2-targeted therapies. Results Silencing JAM-A enhanced the anti-proliferative effects of anti-HER2 treatments in trastuzumab- and lapatinib-resistant breast cancer cells and further reduced HER2 protein expression and Akt phosphorylation in drug-treated cells. Increased epidermal growth factor receptor expression observed in drug-resistant models was normalized upon JAM-A silencing. JAM-A was highly expressed in all of a small cohort of HER2-positive patients whose disease recurred following anti-HER2 therapy. High JAM-A expression also correlated with metastatic disease at the time of diagnosis in another patient cohort resistant to trastuzumab therapy. Importantly, cleavage of JAM-A was increased in drug-resistant cell lines in conjunction with increased expression of ADAM-10 and -17 metalloproteases. Pharmacological inhibition or genetic silencing studies suggested a particular role for ADAM-10 in reducing JAM-A cleavage and partially re-sensitizing drug-resistant cells to the anti-proliferative effects of HER2-targeted drugs. Functionally, recombinant cleaved JAM-A enhanced breast cancer cell invasion in vitro and both invasion and proliferation in a semi-in vivo model. Finally, cleaved JAM-A was detectable in the serum of a small cohort of HER2-positive patients and correlated significantly with resistance to HER2-targeted therapy. Conclusions Collectively, our data suggest a novel model whereby increased expression and cleavage of JAM-A drive tumorigenic behavior and act as a biomarker and potential therapeutic target for resistance to HER2-targeted therapies. Electronic supplementary material The online version of this article (10.1186/s13058-018-1064-1) contains supplementary material, which is available to authorized users.

Background Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. Methods We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. Results In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. Conclusions Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies. Electronic supplementary material The online version of this article (10.1186/s12885-018-4852-1) contains supplementary material, which is available to authorized users.

As HER2 is a client protein of the molecular chaperone Hsp90, targeting Hsp90 may be beneficial in HER2-positive breast cancer. In this study, the activity of the Hsp90 inhibitor NVP-AUY922 was assessed in HER2 overexpressing breast cancer cell lines, including two cell line models of acquired trastuzumab-resistance. The seven HER2-positive breast cancer cell lines tested showed significant sensitivity to NVP-AUY922 in vitro, with IC50 values between 6 and 17 nM. Combining NVP-AUY922 with chemotherapy did not improve response. NVP-AUY922 in combination with trastuzumab, significantly enhanced growth inhibition in three of the seven cell lines tested. In conclusion, our data shows that NVP-AUY922 displays potent anti-cancer activity in HER2-positive and trastuzumab-resistant breast cancer cells, and supports further testing of NVP-AUY922 in patients with HER2-positive breast cancer.

The identification of a targeted therapy for patients with triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. The p53 gene is mutated in approximately 80% of patients with TNBC, and is a potential therapeutic target for patients with this form of breast cancer. The 2-sulfonylpyrimidine compound, PK11007, preferentially decreases viability in p53-compromised cancer cell lines. We investigated PK11007 as a potential new treatment for TNBC. IC50 values for inhibition of proliferation in a panel of 17 breast cell lines by PK11007 ranged from 2.3 to 42.2 μM. There were significantly lower IC50 values for TNBC than for non-TNBC cell lines (p = 0.03) and for p53-mutated cell lines compared with p53 WT cells (p = 0.003). Response to PK11007 however, was independent of the estrogen receptor (ER) or HER2 status of the cell lines. In addition to inhibiting cell proliferation, PK11007 induced apoptosis in p53 mutant cell lines. Using RNAseq and gene ontology analysis, we found that PK11007 altered the expression of genes enriched in pathways involved in regulated cell death, regulation of apoptosis, signal transduction, protein refolding and locomotion. The observations that PK11007 inhibited cell proliferation, induced apoptosis and altered genes involved in cell death are all consistent with the ability of PK11007 to reactivate mutant p53. Based on our data, we conclude that targeting mutant p53 with PK11007 is a potential approach for treating p53-mutated breast cancer, including the subgroup with TN disease.

A series of N -{ para -(ferrocenyl) ethynyl benzoyl} amino acid and dipeptide methyl and ethyl esters 4–18 were prepared by coupling para -(ferrocenyl) ethynyl benzoic acid 3 to the amino acids GABA(OMe), GABA(OEt) and the dipeptide esters GlyGly(OMe), GlyGly(OEt), Gly-L-Ala(OMe), Gly-L-Ala(OEt), Gly-D-Ala(OMe), Gly-D-Ala(OEt), Gly-L-Leu(OEt), Gly-L-Phe(OEt), SarGly(OMe), SarGly(OEt), Sar-L-Ala(OEt), L-ProGly(OEt) and L-Pro-L-Ala(OEt) using the standard N -(3-dimethylaminopropyl)- N’ -ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt) protocol. All the compounds were fully characterized using a combination of I.R., UV-Vis, 1 H NMR, 13 C NMR, DEPT-135, 1 H- 13 C COSY (HMQC) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Selected compounds 5 , 7 , 9, 11, 16, 17 and 18 showed micromolar activity in the H1299 NSCLC cell line, with IC 50 values in the range of 3.8–8.3 μM.

Although HER2 targeted therapies have improved prognosis for HER2 positive breast cancer, HER2 positive cancers which co-express ER have poorer response rates to standard HER2 targeted therapies, combined with chemotherapy, than HER2 positive/ER negative breast cancer. Administration of hormone therapy concurrently with chemotherapy and HER2 targeted therapy is generally not recommended. Using publically available gene expression datasets we found that high expression of IGF1R is associated with shorter disease-free survival in patients whose tumors are ER positive and HER2 positive. IGF1R is frequently expressed in HER2 positive breast cancer and there is significant evidence for crosstalk between IGF1R and both HER2 and ER. Therefore, we evaluated the therapeutic potential of targeting ER and IGF1R in cell line models of HER2/ER/IGF1R positive breast cancer, using tamoxifen and two IGF1R targeted tyrosine kinase inhibitors (NVP-AEW541 and BMS-536924). Dual inhibition of ER and IGF1R enhanced growth inhibition in the four HER2 positive cell lines tested and caused an increase in cell cycle arrest in G1 in BT474 cells. In addition, combined treatment with trastuzumab, tamoxifen and either of the IGF1R TKIs enhanced response compared to dual targeting strategies in three of the four HER2 positive breast cancer cell lines tested. Furthermore, in a cell line model of trastuzumab-resistant HER2 positive breast cancer (BT474/Tr), tamoxifen combined with an IGF1R TKI produced a similar enhanced response as observed in the parental BT474 cells suggesting that this combination may overcome acquired trastuzumab resistance in this model. Combining ER and IGF1R targeting with HER2 targeted therapies may be an alternative to HER2 targeted therapy and chemotherapy for patients with HER2/ER/IGF1R positive breast cancer.

The vitamin D receptor (VDR) is a member of the thyroid-steroid family of nuclear transcription factors. Following binding of the active form of vitamin D, i.e., 1,25(OH)2D3 (also known as calcitriol) and interaction with co-activators and co-repressors, VDR regulates the expression of several different genes. Although relatively little work has been carried out on VDR in human cancers, several epidemiological studies suggest that low circulating levels of vitamin D are associated with both an increased risk of developing specific cancer types and poor outcome in patients with specific diagnosed cancers. These associations apply especially in colorectal and breast cancer. Consistent with these findings, calcitriol as well as several of its synthetic analogues have been shown to inhibit tumor cell growth in vitro and in diverse animal model systems. Indeed, some of these vitamin D analogues with low calcemic inducing activity (e.g., EB1089, inecalcitol, paricalcitol) have progressed to clinical trials in patients with cancer. Preliminary results from these trials suggest that these vitamin D analogues have minimal toxicity, but clear evidence of efficacy remains to be shown. Although evidence of efficacy for mono-treatment with vitamin D analogues is currently lacking, several studies have reported that supplementation with calcitriol or the presence of high endogenous circulating levels of vitamin D enhances response to standard therapies.

Background: Combining bevacizumab and chemotherapy produced superior response rates compared with chemotherapy alone in metastatic breast cancer. As bevacizumab may cause hypertension (HTN) and increase the risk of cardiac failure, we performed a pilot study to evaluate the feasibility and toxicity of a non-anthracycline-containing combination of docetaxel with cyclophosphamide and bevacizumab in early stage breast cancer patients. Methods: Treatment consisted of four 3-weekly cycles of docetaxel and cyclophosphamide (75/600 mg/m2). Bevacizumab was administered 15 mg/kg intravenously on day 1, and then every 3 weeks to a total of 18 cycles of treatment. Serum biomarker concentrations of vascular endothelial growth factor (VEGF), cardiac troponin-I (cTnI), myeloperoxidase (MPO), and placental growth factor (PlGF) were quantified using enzyme-linked immunosorbent assay (ELISA) in 62 patients at baseline and whilst on treatment to determine their utility as biomarkers of cardiotoxicity, indicated by left ventricular ejection fraction (LVEF). Results: A total of 106 patients were accrued in nine sites. Median follow up was 65 months (1–72 months). Seventeen protocol-defined relapse events were observed, accounting for an overall disease-free survival (DFS) rate of 84%. The DFS rates for hormone receptor positive (HR+) and triple-negative (TN) patients were 95% versus 43%, respectively. The median time to relapse was 25 (12–54) months in TN patients versus 38 (22–71) months in HR+ patients. There have been 13 deaths related to breast cancer . The overall survival (OS) rate was 88%. The 5-year OS rate in HR+ versus TN was 95% versus 57%. None of the measured biomarkers predicted the development of cardiotoxicity. Conclusions: We observed a low relapse rate in node-positive, HR+ patients; however, results in TN breast cancer were less encouraging. Given the negative results of three large phase III trials, it is unlikely that this approach will be investigated further. Trial Registration ClinicalTrials.gov Identifier: NCT00911716.

In pre-clinical studies, triple-negative breast cancer (TNBC) cells have demonstrated sensitivity to the multi-targeted kinase inhibitor dasatinib, however, clinical trials with single-agent dasatinib showed limited efficacy in unselected populations of breast cancer, including TNBC. To study potential mechanisms of resistance to dasatinib in TNBC, we established a cell line model of acquired dasatinib resistance (231-DasB). Following an approximately three-month exposure to incrementally increasing concentrations of dasatinib (200 nM to 500 nM) dasatinib, 231-DasB cells were resistant to the agent with a dasatinib IC50 value greater than 5 &mu, M compared to 0.04 &plusmn, 0.001 &micro, M in the parental MDA-MB-231 cells. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970. Treatment of 231-DasB cells with dasatinib did not inhibit phosphorylation of Src kinase. The 231-DasB cells also had significantly increased levels of p-Met compared to the parental MDA-MB-231 cells, as measured by luminex, and resistant cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpdA, with an IC50 value of 1.4 &plusmn, 0.5 &micro, M compared to an IC50 of 6.8 &plusmn, 0.2 &micro, M in the parental MDA-MB-231 cells. Treatment with CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Combined treatment with dasatinib and CpdA significantly inhibited the growth of MDA-MB-231 parental cells and prevented the emergence of dasatinib resistance. If these in vitro findings can be extrapolated to human cancer treatment, combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance and improve response rates to dasatinib treatment in TNBC.

Background:Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Epidermal growth factor receptor (EGFR) has been shown to be over-expressed in TNBC and represents a rational treatment target.Methods:We examined single agent and combination effects for afatinib and dasatinib in TNBC. We then determined IC50and combination index values using Calcusyn. Functional analysis of single and combination treatments was performed using reverse phase protein array and cell cycle analysis. Finally, we determined the anticancer effects of the combination in vivo.Results:A total of 14 TNBC cell lines responded to afatinib with IC50values ranging from 0.008 to 5.0 µM. Three cell lines, belonging to the basal-like subtype of TNBC, were sensitive to afatinib. The addition of afatinib enhanced response to the five other targeted therapies in HCC1937 and HDQP1 cells. The combination of afatinib with dasatinib caused the greatest growth inhibition in both cell lines. The afatinib/dasatinib combination was synergistic and/or additive in 13/14 TNBC cell lines. Combined afatinib/dasatinib treatment induced G1 cell cycle arrest. Reverse phase protein array results showed the afatinib/dasatinib combination resulted in efficient inhibition of both pERK(T202/T204) and pAkt(S473) signalling in BT20 cells, which was associated with the greatest antiproliferative effects. High baseline levels of pSrc(Y416) and pMAPK(p38) correlated with sensitivity to afatinib, whereas low levels of B-cell lymphoma 2 (Bcl2) and mammalian target of rapamycin (mTOR) correlated with synergistic growth inhibition by combined afatinib and dasatinib treatment. In vivo, the combination treatment inhibited tumour growth in a HCC1806 xenograft model.Conclusions:We demonstrate that afatinib combined with dasatinib has potential clinical activity in TNBC but warrants further preclinical investigation.

Prolonged treatment of HER2 positive breast cancer cells with tyrosine kinase inhibitors (TKIs) leads to the emergence of acquired resistance. However, the effects of continuous TKI exposure on cell fate, and the steps leading to the acquisition of a resistant phenotype are poorly understood. To explore this, we exposed five HER2 positive cells lines to HER2 targeted therapies for periods of up to 4 weeks and examined senescence associated &beta, galactosidase (SA-&beta, gal) activity together with additional markers of senescence. We found that lapatinib treatment resulted in phenotypic alterations consistent with a senescent phenotype and strong SA-&beta, gal activity in HER2-positive cell lines. Lapatinib-induced senescence was associated with elevated levels of p15 and p27 but was not dependent on the expression of p16 or p21. Restoring wild type p53 activity either by transfection or by treatment with APR-246, a molecule which reactivates mutant p53, blocked lapatinib-induced senescence and caused increased cell death. In contrast to lapatinib, SA-&beta, gal activity was not induced by exposing the cells to trastuzumab as a single agent but co-administration of lapatinib and trastuzumab induced senescence, as did treatment of the cells with the irreversible HER2 TKIs neratinib and afatinib. Neratinib- and afatinib-induced senescence was not reversed by removing the drug whereas lapatinib-induced senescence was reversible. In summary, therapy-induced senescence represents a novel mechanism of action of HER2 targeting agents and may be a potential pathway for the emergence of resistance.

Recent DNA sequencing and gene expression studies have shown that at a molecular level, almost every case of breast cancer is unique and different from other breast cancers. For optimum management therefore, every patient should receive treatment that is guided by the molecular composition of their tumor, i.e., precision treatment. While such a scenario is still some distance into the future, biomarkers are beginning to play an important role in preparing the way for precision treatment. In particular, biomarkers are increasingly being used for predicting patient outcome and informing as to the most appropriate type of systemic therapy to be administered. Mandatory biomarkers for every newly diagnosed case of breast cancer are estrogen receptors and progesterone receptors in selecting patients for endocrine treatment and HER2 for identifying patients likely to benefit from anti-HER2 therapy. Amongst the best validated prognostic biomarker tests are uPA/PAI-1, MammaPrint and Oncotype DX. Although currently, there are no biomarkers available for predicting response to specific forms of chemotherapy, uPA/PAI-1 and Oncotype DX can aid the identification of lymph node-negative patients that are most likely to benefit from adjuvant chemotherapy, in general. In order to accelerate progress towards precision treatment for women with breast cancer, we need additional predictive biomarkers, especially for enhancing the positive predictive value for endocrine and anti-HER2 therapies, as well as biomarkers for predicting response to specific forms of chemotherapy. The ultimate biomarker test for achieving the goal of precision treatment for patients with breast cancer will likely require a combination of gene sequencing and transcriptomic analysis of every patient's tumor.

The identification and validation of a targeted therapy for patients with triple-negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti-cancer activity of PRIMA-1 and PRIMA-1MET (APR-246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild-type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA-1 and PRIMA-1MET was found to be cell-line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA-1MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC50 value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r=-0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA-1MET induced apoptosis and inhibited migration in a p53 mutant-dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA-1MET is a potential new approach for treating p53-mutated breast cancer, including the subgroup with triple-negative (TN) disease.

Background: The addition of H to chemotherapy improved disease outcome in HER2+ MBC. The combination of H and L with chemotherapy improved event free survival (EFS) in the neo-adjuvant setting but not in the adjuvant setting. However, improved EFS did not translate to overall survival benefit. This international trial (ICORG (CTRIAL-IE) 11-10/NCT01526369) compared the efficacy of TH vs. THL in first line treatment of HER2+ MBC. The primary aim of this study was to assess any benefit for the addition of L to TH in an advanced HER2+ breast cancer patient population. Target accrual was not met due to changes to standard of care mid-study. Methods: Patients (Pts) (n= 75 enrolled, randomized 1:1) received weekly T (80mg/m², for 3 weeks of a 4 week cycle) + H (8mg/kg loading dose on cycle 1 day 1 and 4mg/kg every 2 weeks) + L (1,000 mg daily)) until disease progression, unacceptable toxicity or consent withdrawal. The primary outcome measure was progression free survival (PFS). Secondary outcome measure was overall survival (OS). Eligibility criteria included: Female > 18 years, invasive MBC, measurable disease by RECIST criteria V1.1, HER2+ by testing of the primary tumour and if available the biopsied metastatic lesion. No prior systemic therapy for metastatic disease (except one line of hormonal therapy for metastatic disease without H). No recurrence within 12 months (mo) from completion of adjuvant chemotherapy to the development of metastatic disease. No recurrence within 6 mo from completion of adjuvant H to the development of metastatic disease and no prior L treatment. Results: Of the 75 pts enrolled, n=65 (TH n=34, THL n=31) were included in the PFS analysis. Median PFS (95% CI) was numerically higher, but not statistically significant, in THL (24.0 mo (9.5, 34.5)) vs TH (19.3 mo (8.7, 22.7)), HR 0.827, p= 0.601. For OS analysis, n=69 (TH n=35 and THL n=34) were included. 41/69 (59.4%) had no prior therapy. No statistically significant, clinically meaningful improvement in OS (Median (95% CI)) was observed (TH 53.8 mo (41.8, -) vs. THL 45.6 mo (29.7, -), HR 1.048, p=0.891). Analysis of the safety set (n=70 (TH n= 36, THL n=34)) revealed a higher incidence of Grade 3/4 AEs in THL (61.8%) vs. TH (50%), p=0.322. There were 2 on-treatment fatalities due to adverse events (AEs) in the THL arm, one due to respiratory failure, the other due to central nervous system metastasis. There were no on-treatment fatalities in the TH arm. GI disorders were the most prevalent AE, with the incidence of diarrhea significantly higher in the THL arm (88% vs. 42%, p Citation Format: John Crown, William Jacot, Denis M. Collins, Linda Coate, Anna Saetersdal, M John Kennedy, Catherine Kelly, Blanca Cantos Sanchez De Ibarguen, Raquel Andrés Conejero, Luis Costa, Margarida Brito, Maccon Keane, Pedro Sanchez Rovira, Miguel Martin, Miriam O'Connor, Manuel Ramos Vazquez, Elena Alvarez, Seamus O'Reilly, Johanna Mattson, Laura Jolis Lopez, Alvaro Rodriguez Lescure, Esperanza Blanco, Coralia Bueno Muino, Brian Moulton, Norma O'Donovan, Andres Hernando, Marc Nolan, Imelda Parker, Giuseppe Gullo, Bryan Hennessy. A phase III randomized study of paclitaxel (T) and trastuzumab (H) versus T, H and lapatinib (L) in first line treatment of HER2+ metastatic breast cancer (MBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT212.

Introduction: The phase II neoadjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy (docetaxel, carboplatin) in combination with trastuzumab, trastuzumab and lapatinib or lapatinib alone in HER2+ breast cancer patients. Lapatinib is a dual HER2/EGFR tyrosine kinase inhibitor. Trastuzumab is a monoclonal antibody which targets HER2 and is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune effector cells. Studies have shown that tumour infiltrating lymphocyte (TIL) levels and the cytotoxic capacity of circulating immune cells can correlate with response to trastuzumab. Less is known regarding the effect of chemotherapy on the immune response to trastuzumab. This study examines the effects of neo-adjuvant chemotherapy on the phenotype and cytotoxic capacity of peripheral blood mononuclear cells (PBMCs) of HER2+ breast cancer patients. Methods: Matched blood samples were taken pre- and post-neoadjuvant treatment. PBMCs were isolated by density centrifugation and frozen. Direct PBMC-mediated cytotoxicity and trastuzumab-ADCC levels were assessed against the HER2+ breast cancer cell line (SKBR3) and non-MHC class I-restricted leukemic cell line (K562) using a FACS based assay (n=19 matched pre-and post-treatment samples). The immunophenotype of 17 pre-treatment and 13 post-treatment samples was also determined using the DURAClone IM antibody panel (CD16, CD56, CD19, CD14, CD4, CD8, CD3, CD45) on the CytoFLEX platform. Analysis of both data sets was performed using FCS Express and MedCalc. Results: The cytotoxic capacity of PBMCs was reduced following neo-adjuvant chemotherapy. Direct cytotoxicity elicited against the K562 cell line was reduced post-treatment (p=0.04). ADCC elicited against the SKBR3 cell line was also decreased in post-treatment samples (p=0.009). A decrease in cytotoxic capacity was associated with alterations in the immunophenotype of the post-treatment PBMC samples. The proportion of CD56+ NK cells (p=0.001), CD19+ B cells (p=0.001) and CD14+ monocytes (p=0.03) decreased significantly post-treatment. CD56+ NK cells are hypothesised to be the main ADCC effector cell population in peripheral blood. Interestingly, the proportion of CD3+ T cells (p=0.002), including CD4+ (p=0.007) and CD8+ (p=0.035) T cell populations, were increased post-treatment. Conclusion: These results indicate that neo-adjuvant chemotherapy reduces the cytotoxic capacity of circulating immune cells and alters the proportion of adaptive and innate immune cell subsets. These effects warrant further investigation particularly with the emergence of immunotherapies in HER2+ breast cancer. Citation Format: Nicola Gaynor, Alfonso Blanco, Alexandra Canonici, Alexander J. Eustace, Martina McDermott, Barry Moran, Jean M. Fletcher, Norma O'Donovan, John Crown, Denis M. Collins. The effect of neo-adjuvant chemotherapy on immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 951.

e12646 Background: HER2+ BC patients (pts) achieving a pathological complete response (pCR) to neo-adjuvant therapy have better long-term survival outcomes. Alternative treatment strategies may be more beneficial for pts that will not achieve a pCR but there are no predictive biomarkers for stratification of pts to appropriate clinical studies. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune cells like NK cells can be elicited by therapeutic antibody therapies including the HER2-targeting trastuzumab (T). We hypothesised that the presence of immune checkpoint-inhibited ADCC-mediating immune cells in the blood may identify pts with an immune-suppressed tumour microenvironment who will not achieve a pCR. We used an in vitro functional assay to assess the PD-1-inhibited ADCC response of pre-treatment peripheral blood mononuclear cells (PBMCs) from HER2+ BC pts as a potential biomarker of pCR. Methods: Pre-treatment PBMC samples (n = 21) were obtained from HER2+ BC pts who received neo-adjuvant chemotherapy (docetaxel, carboplatin) with a HER2-targeted therapy (T n = 5, T and lapatinib (L) n = 13, or L alone n = 3), ICORG 10-05 (NCT01485926). Pt response was determined as pCR (n = 7), partial response (PR, n = 10) or non-response (NR, n = 4). The immune function assay used a flow cytometry-based method to determine the T (10µg/µl)-mediated ADCC (T-ADCC) capacity of the PBMCs +/- the anti-PD-1 therapy pembrolizumab (P) (10µg/µl). The HER2+ BC cell line SKBR3 was used as the target cell line. Direct PBMC-mediated cytotoxicity against the non-MHC Class I-restricted leukemic K562 cell line was also determined. Statistical analysis was carried out using the MedCalc program. Results: There was no significant difference in direct cytotoxicity against K562 (pCR vs NR p = 0.13, pCR vs PR p = 0.52, PR vs NR p = 0.40), or in T-ADCC against SKBR3 (pCR vs NR p = 0.71, pCR vs PR p = 0.52, PR vs NR p = 0.32), between response cohorts in the absence of P. When T-ADCC was examined in individual pt samples, the addition of P resulted in significant increases in T-ADCC vs T-ADCC in the absence of P in 3/4 NR (p = 0.03, p = 0.01, p = 0.002) and 4/10 PR (p = 0.003, p = 0.007, p = 0.009, p = 0.01). No significant increase in T-ADCC was observed in the pCR cohort (0/7) as a result of the addition of P. Using pre-treatment PBMCs, the assay detected 7/14 (50%) of HER2+ BC pts who did not achieve a pCR. Conclusions: PD-1-inhibited ADCC-capable immune cells may identify a subset of HER2+ BC pts who will not achieve a pCR. Further investigation is warranted using a larger patient cohort.

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer which accounts for 15–20% of this disease and is currently treated with genotoxic chemotherapy. The BCL2 (B-cell lymphoma 2) family of proteins controls the process of mitochondrial outer membrane permeabilization (MOMP), which is required for the activation of the mitochondrial apoptosis pathway in response to genotoxic agents. We previously developed a deterministic systems model of BCL2 protein interactions, DR_MOMP that calculates the sensitivity of cells to undergo mitochondrial apoptosis. Here we determined whether DR_MOMP predicts responses of TNBC cells to genotoxic agents and the re-sensitization of resistant cells by BCL2 inhibitors. Using absolute protein levels of BAX, BAK, BCL2, BCL(X)L and MCL1 as input for DR_MOMP, we found a strong correlation between model predictions and responses of a panel of TNBC cells to 24 and 48 h cisplatin (R2 = 0.96 and 0.95, respectively) and paclitaxel treatments (R2 = 0.94 and 0.95, respectively). This outperformed single protein correlations (best performer BCL(X)L with R2 of 0.69 and 0.50 for cisplatin and paclitaxel treatments, respectively) and BCL2 proteins ratio (R2 of 0.50 for cisplatin and 0.49 for paclitaxel). Next we performed synergy studies using the BCL2 selective antagonist Venetoclax /ABT199, the BCL(X)L selective antagonist WEHI-539, or the MCL1 selective antagonist A-1210477 in combination with cisplatin. In silico predictions by DR_MOMP revealed substantial differences in treatment responses of BCL(X)L, BCL2 or MCL1 inhibitors combinations with cisplatin that were successfully validated in cell lines. Our findings provide evidence that DR_MOMP predicts responses of TNBC cells to genotoxic therapy, and can aid in the choice of the optimal BCL2 protein antagonist for combination treatments of resistant cells.

Background Insulin Receptor (INSR) signalling may play a role in resistance to insulin-like growth factor receptor (IGF1R) therapy. Although IGF1R is frequently expressed in triple negative breast cancer (TNBC), we found that an IGF1R monoclonal antibody did not inhibit the growth of TNBC cells. Thus, we hypothesised that dual targeting of IGF1R and INSR may be more effective in TNBC. Methods Relative mRNA expression of INSR and IGFIR were analysed across different subtypes of breast cancer using the BreastMark database. INSR-A, INSR-B, IGF1R and insulin like growth factor 2 (IGFII) was quantified by qRT-PCR in a panel of 11 TNBC cell lines. Proliferation assays were performed on TNBC cell lines treated with the dual IR/INSR inhibitor Linsitinib (OSI-906). Combinations were performed by testing Linsitinib with Xentuzumab (BI-836845), cisplatin, docetaxel, or the PI3K inhibitor Dactolisib (NVP-BEZ235). We also tested the anti-proliferative effect of Linsitinib in combination with Xentuzumab when stimulated with IGF-I and IGF-II following serum-starvation for 24 hours. Finally, Linsitinib was tested in HCC-1143 xenografts to assess the effect of low-dose treatment on tumour formation. Results No significant differences were observed between the basal-like and non-basal-like cell lines for IR or IGF1R mRNA expression. However, IGF-II mRNA was more frequently detected in non-basal-like (80%, 4/5) compared to basal-like cell lines (20%, 1/5). Only three of the 12 cell lines tested showed sensitivity to Linsitinib with IC50 values less than 10 µM. The two most sensitive cell lines, HDQ-P1 and HCC1143, expressed detectable levels of IGF1R, INSR-A and INSR-B mRNAs. Although Linsitinib blocked IGF-I and IGF-II stimulated proliferation in both HCC1143 and HDQ-P1 cells, addition of Linsitinib to either chemotherapy or Dactolisib did not enhance therapeutic response. Linisitinib did not reduce tumour formation or tumour growth in an in vivo TNBC cell-line xenograft model. Conclusions Although IGF1R has been shown to be frequently expressed in TNBC, our in vitro and in vivo data suggest that it may not be a good therapeutic target in the TNBC subtype. Legal entity responsible for the study The authors. Funding Boehringer Ingleheim. Disclosure N. O’Donovan: Research grant / Funding (institution): Boehringer Ingleheim. J. Crown: Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution): Boehringer Ingleheim; Shareholder / Stockholder / Stock options, Full / Part-time employment: OncoMark Limited; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Puma Biotechnology; Honoraria (self), Advisory / Consultancy: Seattle Genetics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy: Vertex; Honoraria (self), Speaker Bureau / Expert testimony: Genomic Health; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Travel / Accommodation / Expenses: MSD Oncology; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Abbvie. All other authors have declared no conflicts of interest.

Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma.