James G. Greger

Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations

Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAFV600E and the YUSIT1 BRAFV600K melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1K59del) or NRAS mutation (NRASQ61K and/or NRASA146T) with and without MEK1P387S in the BRAFV600E background and NRASQ61K in the BRAFV600K background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRASQ61K or NRASA146T in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1K59del, but not MEK1P387S, decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27kip1 protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations. Mol Cancer Ther; 11(4); 909–20. ©2012 AACR.

Pattern of Rash, Diarrhea, and Hepatic Toxicities Secondary to Lapatinib and Their Association With Age and Response to Neoadjuvant Therapy: Analysis From the NeoALTTO Trial

PURPOSE We investigated the pattern of rash, diarrhea, and hepatic adverse events (AEs) secondary to lapatinib and their association with age and pathologic complete response (pCR) in the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (NeoALLTO) phase III trial. PATIENTS AND METHODS Patients with HER2-positive early breast cancer were randomly assigned to receive lapatinib (Arm A), trastuzumab (Arm B), or their combination (Arm C) for 6 weeks followed by the addition of paclitaxel for 12 weeks before surgery. We investigated the frequency and time to developing each AE according to age (≤ 50 v > 50 years) and their association with pCR in a logistic regression model adjusted for age, hormone receptors, tumor size, nodal status, planned breast surgery, completion of lapatinib administration, and treatment arm. RESULTS Only patients randomly assigned to arms A and C were eligible (n = 306). Younger patients (≤ 50 years) experienced significantly more rash compared with older patients (74.4% v 47.9%; P < .0001). Diarrhea and hepatic AEs were observed in 78.8% and 41.2% of patients, respectively, with no differences in rate or severity or time of onset according to age. Early rash (ie, before starting paclitaxel) was independently associated with a higher chance of pCR, mainly in patients older than 50 years (odds ratio [OR] = 3.76; 95% CI, 1.69 to 8.34) but not in those ≤ 50 years (OR = 0.92; 95% CI, 0.45 to 1.88; P for interaction = .01). No significant association was observed between pCR and diarrhea or hepatic AEs. CONCLUSION Our results indicate that the frequency and clinical relevance of lapatinib-related rash is largely dependent on patient age.

Synergistic effects of foretinib with HER-targeted agents in MET and HER1- or HER2-coactivated tumor cells.

The HER and MET receptor tyrosine kinases (RTK) are coactivated in a subset of human tumors. This study characterizes MET and HER expression and signaling in a panel of human tumor cell lines and the differential susceptibility of these cell lines to single agents or combinations of foretinib, a multikinase MET inhibitor, with HER-targeted agents, erlotinib or lapatinib. Most MET-amplified tumor lines without HER1 or HER2 amplification are sensitive to foretinib, whereas MET-amplified lines with HER1 or HER2 amplification are more sensitive to the combination of foretinib with lapatinib or erlotinib. Interestingly, MET-overexpressing tumor cell lines with HER1 or HER2 amplification also exhibited reduced sensitivity to lapatinib or erlotinib in the presence of hepatocyte growth factor (HGF), indicating MET activation can decrease the effectiveness of HER1/2 inhibitors in some cell lines. Consistent with this observation, the effect of HGF on lapatinib or erlotinib sensitivity in these cells was reversed by foretinib, other MET inhibitors, or siRNA to MET. Western blot analyses showed that combining foretinib with erlotinib or lapatinib effectively decreased the phosphorylation of MET, HER1, HER2, HER3, AKT, and ERK in these cells. Furthermore, HER2-positive advanced or metastatic breast cancer patients treated with lapatinib who had higher tumor MET expression showed shorter progression-free survival (19.29 weeks in MET-high patients vs. 28.14 weeks in MET-low patients, P < 0.0225). These data suggest that combination therapy with foretinib and HER-targeted agents should be tested as a treatment option for HER1- or HER2-positive patients with MET-amplified or -overexpressing tumors. Mol Cancer Ther; 10(3); 518–30. ©2011 AACR.

Abstract 976: BRAF inhibitor GSK2118436 - single agent and combination activity with other targeted agents in BRAF V600 mutant melanoma

Although BRAF inhibitors display promising clinical response in BRAF V600 mutant melanoma patients, not all patients respond to these agents and the durability of the response is limited. To improve BRAF targeted therapy, we characterized a panel of BRAF V600 mutant melanoma cell lines and determined their response to cell growth inhibition, apoptosis induction and cell signaling changes by GSK2118436 alone and in combination with the MEK inhibitor GSK1120212 or PI3K/mTOR inhibitor GSK2126458. Nine of 11 BRAF V600E , 4/5 BRAF V600K and 1/1 BRAF V600D mutant lines were sensitive to GSK2118436 (IC 50 P124S or MEK2 K66_K68del mutations. GSK2118436 treatment decreased MEK and ERK phosphorylation and cyclin D1 in both the sensitive and insensitive lines. However, GSK2118436 decreased S6 ribosomal protein phosphorylation (pS6P) only in the sensitive lines. Cell lines sensitive to GSK2118436 were also sensitive to the allosteric MEK inhibitor GSK1120212 (IC 50 50 s, growth of these lines was inhibited less effectively by GSK2118436 compared to the PTEN WT lines evaluated by IC 70 . PTEN NULL cells displayed higher levels of basal AKT phosphorylation (pAKT) than PTEN WT lines. Treatment of PTEN NULL lines with GSK2118436 increased pAKT while similar treatment of PTEN WT lines decreased pAKT and increased PTEN expression. In combination with the PI3K/mTOR inhibitor GSK2126458, GSK2118436 or GSK1120212 effectively inhibited MEK/ERK and PI3K/mTOR signaling, enhanced cell growth inhibition, and induced apoptosis evaluated by caspase 3/7 activation and/or PARP cleavage in both PTEN WT and PTEN NULL lines. In PTEN deficient lines, these combinations were more effective than the combination of GSK2118436 and GSK1120212. These results indicate MEK1/2 mutations associate with lack of sensitivity to single agent BRAF and MEK inhibitors. In addition, PTEN deficiency may reduce the effectiveness of BRAF or MEK inhibitors. The combination of GSK2118436 with GSK1120212 or GSK2126458 may improve the effectiveness of BRAF targeted therapy in BRAF V600 mutant melanoma. Our results support the testing of GSK2118436 with these combinations in the clinic and warrant further investigation of MEK and PTEN mutation status and pS6P as a pharmacodynamic marker to improve treatment outcome in BRAF V600 mutant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 976. doi:1538-7445.AM2012-976

Abstract B71: Acquired resistance to the BRAF inhibitor GSK2118436, mediated by NRAS or MEK mutation, can be overcome with combinations that inhibit BRAF and MEK, BRAF and PI3K/mTOR, or MEK and PI3K/mTOR.

Recent results from clinical trials with the BRAF inhibitors PLX4032 and GSK2118436 have shown encouraging clinical response rates; however, acquired resistance has limited response duration. In an effort to identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated nine GSK2118436 resistant clones derived from A375 BRAFV600E melanoma cell line (IC50=0.028μM). In three day cell growth assays, the resistant clones were insensitive to the BRAF inhibitors GSK2118436 (IC50>10μM) and PLX4032 (IC50>10μM). In addition, they displayed greater than 10-fold less sensitivity to an allosteric inhibitor of MEK1/2, GSK1120212 (IC50>0.06μM), as compared to the parental A375 cells (IC50=0.005μM). Genetic characterization of these clones by Sanger sequencing identified an in-frame deletion in MEK1 (MEK1K59del), or NRAS mutations (NRASQ61K and/or NRASA146T) with and without a MEK1P387S mutation. In addition, all clones retained the BRAFV600E mutation and no additional mutations were identified in BRAF, KRAS, HRAS, ARAF, CRAF or PTEN. Stable knockdown of NRAS by shRNA partially restored GSK2118436 sensitivity in the NRAS mutant clones. Expression of mutant NRAS in the parental cells decreased sensitivity to GSK2118436 similar to that observed in the NRAS mutant clones. Similarly, expression of MEK1K59del, but not MEK1P387S, decreased sensitivity of the parental cells to GSK2118436. In three day growth assays, treatment of the resistant clones with GSK2118436 in combination with GSK1120212 enhanced cell growth inhibition 19 to 50% at the IC50 of the combination and was ≥3-fold more potent than the most active single agent, GSK1120212. Sustained treatment of greater than ten days with this combination inhibited >90% of cell growth compared to 30–70% with GSK1120212 alone. By western blot analysis, the combination of GSK2118436 and GSK1120212 effectively decreased ERK phosphorylation and cyclin D1 protein, and increased p27kip1 protein levels in all resistant clones. S6 ribosomal protein phosphorylation was not completely inhibited in all clones with this combination. In contrast, the combination of GSK2118436 or GSK1120212 with the PI3K/mTOR inhibitor GSK2126458 effectively blocked S6 ribosomal protein phosphorylation in the resistant clones. Decreases in ERK phosphorylation and cyclin D1 protein were similar to the combination of GSK2118436 and GSK1120212. The addition of GSK2118436 to GSK2126458 was slightly more potent (∼2-fold) than GSK2126458 alone and enhanced cell growth inhibition 12 to 35% at the IC50 for the combination in 5/7 clones with NRAS mutation and both MEK1K59del clones. The combination of GSK1120212 and GSK2126458 was >2-fold more potent than the most active single agent and enhanced cell growth inhibition by 23 to 33% in all the resistant clones. This combination was synergistic (combination index = 0.24–0.75) in 8/9 clones. Taken together these results demonstrate that mutation within NRAS or MEK contribute to resistance to BRAF inhibitors in BRAFV600E mutant melanoma. These resistant cells are responsive to the combination of BRAF and MEK inhibitors, as well as BRAF or MEK inhibitors with PI3K/mTOR inhibitors. Clinical trials are ongoing or planned to test these combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B71.

Abstract A238: The effects of foretinib on MET phosphorylation, gene expression, cell proliferation and tumor growth in MET‐amplified tumor cells in vitro and in vivo

The MET receptor tyrosine kinase (RTK) is frequently deregulated in many types of cancer and is a promising target for cancer drug development. Foretinib is a multikinase inhibitor with potent activity against MET and is currently in phase II clinical trials. In these studies, we determined the anti‐tumor activity of foretinib in MET‐amplified tumor cells and the MKN45 gastric xenograft tumor model in nude mice. We also measured the effect of foretinib on MET phosphorylation by immunoprecipitation and western blot and evaluated gene expression by Affymetrix microarrays in these models. In vitro foretinib demonstrated the most potent cell growth inhibitory activity in MET‐amplified tumor cell lines MKN45 (IC50 = 9 ± 2 nM), HS746T (IC50 = 8 ± 2 nM), SNU5 (IC50 = 2 ± 0.2 nM) and NCI‐H1993 (IC50 = 69 ± 5 nM). Foretinib inhibited the phosphorylation of MET, suppressed the phosphorylation of AKT and ERK, and reduced the expression of cyclin D1 in a dose dependent manner in these cells. Furthermore, foretinib given orally showed durable and near complete inhibition of MET phosphorylation (>90%) and dose dependent anti‐tumor activity against MKN45 MET‐amplified gastric xenograft tumors in nude mice. Tumor stasis and significant tumor growth delay were observed for foretinib dosed at 10 mg/kg and PF‐02341066, a MET selective inhibitor, dosed at 50 mg/kg in the same study. In addition, foretinib or PF‐02341066 showed similar gene alterations on both MKN45 cells and xenograft tumors. These genes are involved in RTK cross‐talk and MET signaling pathways (e.g., HER2, HER3, MET, PIK3IP1, DUSP4, DUSP6), cell motility (e.g., CDH1, TIMP2, CLDN3, MMP1), cell proliferation (e.g., CCND1, TP53INP1, TYMS, CCNB1) and apoptosis (e.g., TNFSF10, CASP8). These results demonstrate that foretinib is a potent MET inhibitor with associated changes in gene expression which correlate with anti‐tumor activity in MET‐amplified tumor cells both in vitro and in vivo. This gene signature warrants further investigation for its relevance as a MET‐inhibitor mediated pharmacodynamic marker. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A238.