Inhibitor-resistant Tumors Research Articles

Dissecting Therapeutic Resistance to ERK Inhibition.

The MAPK pathway is frequently activated in many human cancers, particularly melanomas. A single-nucleotide mutation in BRAF resulting in the substitution of glutamic acid for valine (V(600E)) causes constitutive activation of the downstream MAPK pathway. Selective BRAF and MEK inhibitor therapies have demonstrated remarkable antitumor responses in BRAF(V600) (E)-mutant melanoma patients. However, initial tumor shrinkage is transient and the vast majority of patients develop resistance. We previously reported that SCH772984, an ERK 1/2 inhibitor, effectively suppressed MAPK pathway signaling and cell proliferation in BRAF, MEK, and concurrent BRAF/MEK inhibitor-resistant tumor models. ERK inhibitors are currently being evaluated in clinical trials and, in anticipation of the likelihood of clinical resistance, we sought to prospectively model acquired resistance to SCH772984. Our data show that long-term exposure of cells to SCH772984 leads to acquired resistance, attributable to a mutation of glycine to aspartic acid (G(186D)) in the DFG motif of ERK1. Structural and biophysical studies demonstrated specific defects in SCH772984 binding to mutant ERK. Taken together, these studies describe the interaction of SCH772984 with ERK and identify a novel mechanism of ERK inhibitor resistance through mutation of a single residue within the DFG motif. Mol Cancer Ther; 15(4); 548-59. ©2016 AACR.

Abstract LB-A06: Analysis of gene expression and protein expression of the AXL receptor in clinical biospecimen

Abstract Introduction It has become obvious that targeted therapy of patients has to be monitored by taking and analyzing biopsies on a regular basis. Therefore, laboratory methods have to be optimized to be able to handle minute amounts of clinical biospecimen, e.g. biopsies. In this study we are presenting the development of a Simple Western Size assay and a quantitative Real-time PCR assay for the detection and analysis of AXL protein and gene expression in biopsies. Background Targeted therapy in personalized medicine is often affected by resistance mechanisms, such as activating mutations of signaling molecules and signaling pathway bypasses. AXL, a tyrosine kinase receptor, is expressed in a variety of cancers and has been revealed as the most highly expressed gene in preclinical models with acquired resistance, and second most common alteration in EGFR (epidermal growth factor receptor) inhibitor-resistant tumors, behind the T790M mutation. Up-regulation and/or activation of AXL are shown to be predictive for lack of response to ErbB family receptor-targeted inhibitors, e.g. to Her2-targeted agents. Methods Protein expression was examined by Simple Western Size technology and gene expression was analyzed using quantitative Real-time PCR. For assay development, high and low AXL expressing breast cancer cell lines were used to establish “fit for purpose” assays. Subsequently, Her2-positive and Her2-negative samples from breast cancer patients were analyzed by screening for AXL gene and protein expression. Furthermore, correlations of gene and protein expression were conducted. Results In this study, we developed and validated an assay for the detection of AXL protein expression using the Simple Western technology. This highly sensitive technology enables high-throughput screening of extremely small sample amounts such as biopsies or laser capture microdissected material. Our results showed a broad linear dynamic range of AXL protein in both, high and low AXL expressing cell lines as well as a high reproducibility between multiple runs. The protein loading range suitable to detect AXL within whole cell lysates showed detection levels of AXL protein down to 10 ng of total protein. AXL protein expression results were highly comparable to AXL gene expression results in analyzed breast cancer cell lines and breast cancer patient samples. This newly developed assay will allow us to analyze and quantify AXL protein expression profiles in breast cancer tissue and subsequently correlate them with e.g. Her-2 expression status. Conclusion The elucidation of networks and mechanisms underlying drug resistance will greatly improve the development of new compounds and promote personalized therapy. In order to gain scientific knowledge regarding gene and protein expression of resistance relevant marker, e.g. AXL, it is mandatory to establish assays that are very robust, sensitive and furthermore, if not most important, applicable to biospecimen being collected in the clinical setting. Citation Format: Kristina Bernoth, Florian T. Unger, Nicole Lange, Hartmut Juhl, Kerstin A. David. Analysis of gene expression and protein expression of the AXL receptor in clinical biospecimen. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A06.

MiR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in gastric cancer through regulation of the PI3K/AKT/mTOR pathway

Gastric cancer (GC) is a deadly malignancy worldwide. In the past, it has been shown that cellular signaling pathway alterations play a crucial role in the development of GC. In particular, deregulation of the PI3K/AKT/mTOR pathway seems to affect multiple GC functions including growth, proliferation, metabolism, motility and angiogenesis. Targeting alterations in this pathway by microRNAs (miRNAs) represents a potential therapeutic strategy, especially in inhibitor-resistant tumors. The objective of this study was to evaluate the expression of 3 pre-selected miRNAs, miR-101-2, miR-125b-2 and miR-451a, in a series of primary GC tissues and matched non-GC tissues and in several GC-derived cell lines, and to subsequently evaluate the functional role of these miRNAs. Twenty-five primary GC samples, 25 matched non-GC samples and 3 GC-derived cell lines, i.e., AGS, MKN28 and MKN45, were included in this study. miRNA and target gene expression levels were assessed by quantitative RT-PCR and western blotting, respectively. Subsequently, cell viability, clone formation, cell death, migration and invasion assays were performed on AGS cells. miR-101-2, miR-125b-2 and miR-451a were found to be down-regulated in the primary GC tissues and the GC-derived cell lines tested. MiRNA mimic transfections significantly reduced cell viability and colony formation, increased cell death and reduced cell migration and invasion in AGS cells. We also found that exogenous expression of miR-101-2, miR-125b-2 and miR-451a decreased the expression of their putative targets MTOR, PIK3CB and TSC1, respectively. Our expression analyses and in vitro functional assays suggest that miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in primary GCs as well as in GC-derived AGS cells.

Molecular basis underlying resistance to Mps1/TTK inhibitors.

Mps1/TTK is a dual-specificity kinase, with an essential role in mitotic checkpoint signaling, which has emerged as a potential target in cancer therapy. Several Mps1/TTK small-molecule inhibitors have been described that exhibit promising activity in cell culture and xenograft models. Here, we investigated whether cancer cells can develop resistance to these drugs. To this end, we treated various cancer cell lines with sublethal concentrations of a potent Mps1/TTK inhibitor in order to isolate inhibitor-resistant monoclonal cell lines. We identified four point mutations in the catalytic domain of Mps1/TTK that gave rise to inhibitor resistance but retained wild-type catalytic activity. Interestingly, cross-resistance of the identified mutations to other Mps1/TTK inhibitors is limited. Our studies predict that Mps1/TTK inhibitor-resistant tumor cells can arise through the acquisition of mutations in the adenosine triphosphate-binding pocket of the kinase that prevent stable binding of the inhibitors. In addition, our results suggest that combinations of inhibitors could be used to prevent acquisition of drug resistance. Interestingly, cross-resistance seems nonspecific for inhibitor scaffolds, a notion that can be exploited in future drug design to evict possible resistance mutations during clinical treatment.

Abstract 5025: Immune expression profiling of MAPK inhibitor resistant tumors based upon mechanisms of resistance

Abstract Purpose: To evaluate the association between the immunoreactivity of the immune inhibitory ligand PD-L1 with tumour infiltrating lymphocytes (TILs) and known mechanisms of MAPK inhibitor (MAPKi) resistance in tumor biopsies resected at the time of MAPKi disease progression. Experimental Design: 23 patients with disease progression on MAPKi (vemurafenib, dabrafenib, and combination dabrafenib and trametinib) each had a progressing tumor biospied. All tumors underwent MAPKi resistance screening including RNA expression profiling. Morphological features, PD-L1 immunohistochemical immunoreactivity, and RNA expression profile were correlated with mechanisms of resistance (including mutations in NRAS and MEK, and amplification and aberrant splicing of BRAF). Results: PD-L1 immunoreactivity was significantly higher in tumors that harbored an aberrant BRAF splicing variant (n = 8) or MEK mutations (n = 5) than those with BRAF amplification (n = 4), NRAS mutations (n = 2) or those with unknown resistance mechanisms (n = 4) (mean 27% vs 1%, p = 0.049). Additionally, the PD-L1 immunoreactivity in tumors with known MAPK-reactivating mechanisms (n = 19) tended to be higher than those with unknown resistance mechanisms (n = 4) (mean 17% vs 0%, p = 0.283). The immunoreactivity of PDL1 in progressed lesions was independent of BRAFV600 genotype, RECIST response and type of MAPK inhibitor. Correlations with TIL subsets, RNA expression, and response to subsequent anti-PD-1 therapy are underway. Conclusions: The induction of PD-L1 immunoreactivity following MAPKi treatment in human melanoma is variable, however specific mechanisms of MAPKi resistance may confer increased expression of the inhibitory ligand. Further study is underway examining the association of the above with TIL infiltration, the RNA expression profile, and the response to subsequent anti-PD1 therapy. Citation Format: James S. Wilmott, Hojabr Kakavand, Alexander M. Menzies, Ricardo Vilain, Gulietta M. Pupo, John F. Thompson, Richard F. Kefford, Matteo S. Carlino, Helen Rizos, Georgina V. Long, Richard A. Scolyer. Immune expression profiling of MAPK inhibitor resistant tumors based upon mechanisms of resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5025. doi:10.1158/1538-7445.AM2015-5025

Abstract 959: Aromatase inhibition shapes the clonal architecture of estrogen receptor-positive breast cancers

Abstract BACKGROUND: Aromatase inhibitors are routinely used to treat estrogen receptor (ER)-positive breast cancer in postmenopausal women. Yet, one-third of ER positive tumors are resistant to aromatase inhibitors with a further one-third developing resistance over time. The underlying mechanism conferring resistance to endocrine therapy is poorly understood. In the current work, we study the effect of aromatase inhibitor therapy on the clonal evolution of breast cancer and how it relates to therapy response. MATERIALS AND METHODS: We selected 12 aromatase inhibitor sensitive (post-treatment Ki67 < 10%) and 10 aromatase inhibitor resistant tumors (Ki67 > 10%) that had previously been whole genome sequenced (Ellis, et al. 2014). We obtained surgical samples of the same tumors after ∼4 months of aromatase inhibitor treatment, whole-genome sequenced them, and also obtained RNA-sequencing data for 19 of the pre-treatment and 18 of the post-treatment tumors. To better understand spatial heterogeneity we also whole-genome sequenced 11 secondary core biopsies taken from a subset of both the baseline and surgical tumors. RESULTS AND DISCUSSION: We classified each tumor based on its clonal heterogeneity, as inferred with the sciClone R package, and found 3 ‘simple’ tumors, comprised of only a founding clone, and 19 ‘complex’ tumors that contain one or more subclones. We further classified each tumor based on a stability metric, where ‘stable’ tumors (n = 4) showed little or no evidence of evolution between baseline and surgical intervention and ‘complex’ tumors (n = 18) exhibited a spectrum of clonal changes. These changes ranged from moderate shifts in subclonal frequencies to whole-scale remodeling, where minor subclonal populations gained near-complete dominance. In one extreme case, we observed co-occurring tumors of independent origin, with selection against an ER-positive population and relative expansion of an ER-negative tumor. Spatial heterogeneity was widespread as well, with 6 out of 11 secondary cores containing clonal architecture that differed from the primary. We find that the majority of tumors are both ‘complex’ and ‘dynamic’ in that they are made up of multiple subclones at baseline that undergo clonal selection following endocrine therapy. This clonal evolution may provide a path by which a tumor can bypass therapeutic intervention. Further study is needed to identify the genetic aberrations that underlie this differential response. Our results suggest that clonal heterogeneity is widespread in hormone positive breast cancer and has relevance to endocrine therapy response. Citation Format: Christopher A. Miller, Yevgeniy Gindin, Charles Lu, Obi Griffith, Malachi Griffith, Dong Shen, Jeremy Hoog, Mark Watson, Sherri R. Davies, Kelly Hunt, Jacqueline E. Snider, Katherine DeSchryver, Richard K. Wilson, Mathew J. Ellis, Elaine Mardis. Aromatase inhibition shapes the clonal architecture of estrogen receptor-positive breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 959. doi:10.1158/1538-7445.AM2015-959

Highlights of This Issue

Stat5a/b is critical in pathogenesis of prostate cancer and myeloproliferative disorders but only a few pharmacological Stat5a/b inhibitors are currently available. Here, Liao and colleagues identified a new Stat5a/b inhibitor, IST5-002, through structure-based in silico screening and medicinal chemistry by targeting the SH2 domain. IST5-002 blocked both Jak2 and Bcr-Abl–mediated phosphorylation of Stat5a/b and disrupted dimerization, nuclear translocation, DNA binding, and transcriptional activity. IST5-002 potently reduced the expression of Stat5a/b-regulated genes and induced extensive apoptotic cell death in multiple models of prostate cancer and chronic myeloid leukemia. IST5-002 provides a lead structure for further clinical development targeting Stat5a/b-driven solid tumors and hematological malignancies.Notch inhibitors are being tested in the clinic as a therapeutic for multiple cancer indications, but in most cases gastrointestinal toxicities have limited their effectiveness. Proia and colleagues designed a monoclonal antibody, 23814, against the Notch1 ligand-binding domain that cross-reacts with human and mouse Notch1. The antibody blocked Notch1 function in vivo, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity enabled combination treatment of 23814 with the VEGFR inhibitor tivozanib, which significantly inhibited the growth of several VEGFR inhibitor-resistant tumor models. These results suggest specific Notch1 antibodies may be combined with other angiogenesis inhibitors for the treatment of VEGF inhibitor–resistant tumors.TM4SF1 is highly expressed by both tumor cells and the tumor vascular endothelium, and it internalizes from the cell surface, making it an attractive dual-action target for antibody–drug conjugate therapy. Here, Visintin and colleagues show that an anti-human TM4SF1 antibody conjugated to the auristatin mc-3377 effectively inhibited the growth of human tumor xenografts, and an anti-mouse TM4SF1 antibody targeting the mouse tumor vasculature was similarly effective. When combined, tumor regression was even more pronounced. These data validate TM4SF1 as a suitable therapeutic target to selectively deliver cytotoxic drugs to both tumor cells and tumor vasculature.The Melanoma Dream Team, funded by Stand Up To Cancer and the Melanoma Research Alliance, was formed to examine whether therapy selection based on systematic integration of large-scale genomic and pharmacopeia information improves upon the practice of using a physician's empiric choice for treatment of non–V600E BRAF-mutated metastatic melanoma. LoRusso and colleagues describe a nontreatment pilot study demonstrating the feasibility of using next-generation sequencing techniques to identify molecular aberrations and generate an individualized treatment plan in this patient population. The experience gained in the conduct of this pilot trial led directly to the design of a randomized, statistically driven clinical trial.

23814, an Inhibitory Antibody of Ligand-Mediated Notch1 Activation, Modulates Angiogenesis and Inhibits Tumor Growth without Gastrointestinal Toxicity.

Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit.

Somatic STK11 and concomitant STK11/KRAS mutational frequency in stage IV lung adenocarcinoma adrenal metastases.

Somatic serine/threonine kinase 11 (STK11) also known as liver kinase B1 (LKB1) is a tumor suppressor gene and ranks as the third most frequently mutated gene in lung adenocarcinoma. However, current molecular testing guidelines recommend evaluating for epidermal growth factor receptor mutations and ALK fusions to guide therapy in all patients with advanced stage adenocarcinoma, regardless of gender, race, or smoking history. Identifying alternative "driver" mutations and using actionable targeted pharmacotherapy is a key approach to providing effective individualized medical care. The analytical sensitivity and parallel multigene approach of targeted next-generation sequencing is an attractive methodology for use for cytology specimens. The presented lung adenocarcinoma study revealed that STK11 mutations alone and concomitant KRAS/STK11 mutations were identified in 18.2% and 4.5% of solitary adrenal metastases, respectively. Molecular profiling of epidermal growth factor receptor tyrosine kinase inhibitor resistant tumors may help to identify patients who would most benefit from alternative single or dual pathway inhibition potentially leading to a revision in current molecular testing guidelines.

Game of isoforms: PI3K β-sparing inhibitor is coming.

Cutaneous melanoma has been at the forefront of targeted therapy breakthroughs over the last 5 years. The FDA approval of BRAF and MEK inhibitors for the treatment of late-stage, mutant V600E/K BRAF-harboring melanoma patients has been heralded due to high response rates and improvements in progression-free survival, but the durable effects of these inhibitors are limited. Alterations in the phosphoinositide 3-kinase (PI3K) pathway including inactivation of PTEN (a tumor suppressor that normally down-regulates PI3K signaling) and, less frequently, mutations in PIK3CA (the gene encoding the α isoform of the PI3K catalytic subunit) have been identified in mutant BRAF melanomas. Furthermore, PI3K pathway signaling is often up-regulated in association with resistance to mutant BRAF and/or MEK targeting agents. Despite the evidence highlighting the importance of the PI3K pathway in melanoma, initial testing of class I PI3K inhibitors in patients has not produced dramatic results. An issue for combinatorial studies is the overlapping toxicities of MAPK pathway inhibitors and PI3K-targeting agents that limits their effective dosing. A recent study from Deuker and colleagues provides important preclinical advances in this area by examining the PI3K isoform dependencies in mutant BRAF melanomas. PI3K functions as a heterodimer with a p110 catalytic subunit and a regulatory subunit. Four isoforms of p110 subunits exist: α, β, δ and γ (Jean and Kiger, 2014; Yuan and Cantley, 2008). p110α and p110β are ubiquitously expressed, whereas δ and γ isoforms display highest expression in immune cells. Importantly, isoform-selective PI3K inhibitors have been developed to target tumor dependencies and minimize toxicities in non-tumor cells. Deuker et al. utilized these selective inhibitors to determine the PI3K isoform dependency of mutationally active PI3Kα and PTEN-deficient subgroups of mutant BRAF melanomas using cell lines derived from mouse models and human patients. In vitro, PTEN-deficient lines were less sensitive to the α-isoform PI3K inhibitor, BYL719, compared to mutant H1047R PI3Kα cells. In contrast to other cancer types, in which PTEN-deficient tumors are often PI3Kβ-dependent (Wee et al., 2008), PTEN-null melanomas did not rely on PI3Kβ. Growth of PTEN-null mutant BRAF melanomas was blocked by a PI3Kα/δ/γ-targeting but β-sparing inhibitor, GDC-0032. In addition, the δ/γ inhibitor, IPI145, enhanced the effects of BYL719 suggesting involvement of the δ/γ isoforms in melanoma. PI3Kα-dependency is also suggested to be present in BRAF V600E/WT PTEN melanomas, which represent a substantial proportion of mutant BRAF melanomas, although confirmation is warranted in a larger panel of BRAF V600E/WT PTEN melanoma models. “More dramatic tumor regressions were observed in vivo with the combination of BRAF-MEK pathway and PI3K inhibitors. ” A long-term limitation to both BRAF and MEK inhibitors in melanoma is acquired drug resistance and patients who develop resistance have few second-line treatment options. In addition to up-front combination, treatment of MEK inhibitor-resistant tumors with the PI3K β-sparing inhibitor resulted in a cytostatic response. These data were generated from a single MEK inhibitor-resistant tumor. As multiple mechanisms of resistance are likely to occur, it will be important to further explore effects in cultured- and patient-derived BRAF and MEK inhibitor-resistant models. Overall, the study presented by Deuker et al., provides an important preclinical advance in targeting the PI3K pathway in both first-line combinations as well as a possible second-line salvage option for BRAF-MEK pathway resistant melanomas. These findings should propel investigation of methods to quantitatively and non-invasively measure PI3K signaling in preclinical in vivo melanoma models and to determine the most effective dosing regimens. Given the high degree of immune reactivity of melanomas and remarkable advances in immune-based therapies, other key issues will be to determine the effect of targeting PI3K on infiltration of immune cells into melanomas and the extent to which PI3K-targeting agents can be combined with immune-based therapies. Advances in mutant BRAF melanomas will lead to studies addressing the importance of PI3K isoform specific targeting in wild-type BRAF subsets of melanoma for which there is an unmet need for new therapeutic strategies.

Abstract 3711: Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors

Abstract The MAPK pathway represents a prime target for therapeutic intervention in cancer. Recent approvals of B-Raf and Mek inhibitors lead to exciting antitumor activities and survival benefits. However the observation of resistance to such Raf and Mek inhibitors has been documented in both clinical and preclinical trials. These phenomena stimulated further research into the regulation of the MAPK pathway and led to the suggestion to target the downstream kinase Erk, as well as to combine its targeting with Raf and Mek inhibitors. Here we report that Erk inhibitors result in efficient growth inhibition of tumor cells that are resistant to Raf and Mek inhibitors. AEZS-134 - a highly potent and selective ATP competitive Erk inhibitor - overcomes the Raf inhibitor induced paradoxical cell activation and the aquired resistance to Mek inhibitors in tumor cells. We demonstrate potent antiproliferative activity of AEZS-134 in B-Raf wildtype, B-RafV600E mutant, Ras wildtype and K-Ras mutant tumor cell lines in comparison to common Raf inhibitors. Furthermore we show that AEZS-134 is efficacious in Mek inhibitor resistant Hct116 and MDA MB231 cells which have been well characterized in terms of Mek F129L allosteric binding pocket mutation, varying degrees of K-Ras amplification, cellular proliferation assays and MAPK pathway phosphorylation studies. Our data provide a rational for new therapeutic opportunities by using Erk inhibitors in oncology. In order to maximize therapeutic benefit for patients and considering that Erk is directly downstream of B-Raf and Mek, targeting Erk may be more effective than targeting Raf or Mek in a variety of MAPK resistance settings. AEZS-134 and related compounds provide potent and selective tools to help address this question. Development of the Erk inhibitors by Æterna Zentaris is integral part of our inhouse kinase research program comprising the investigation of different compounds for single Erk inhibition, single PI3K inhibition and dual kinase inhibitions of Erk and PI3K. All compounds are exclusively synthesized by our medicinal chemistry department. Citation Format: Irene Seipelt, Peter Schmidt, Helene Maerzhaeuser, Matthias Gerlach, Kai Jung, Tilmann Schuster, Michael Teifel. Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2014-3711

Abstract 1337: BH3-mimetic gossypol promotes autophagy to inhibit the proliferation of mutant BRAF melanoma cells with high expression of p21Cip1

Abstract Gossypol is a BH3-mimetic small-molecule pan-inhibitor of Bcl-2. In this study, we described the potential therapeutic effects of gossypol on BRAF inhibitor-resistant melanoma cells. We found that gossypol retained its efficacy in mutant melanoma clones resistant to BRAF inhibitor through a mechanism independent of MEK-ERK inhibition. Gossypol caused G2/M arrest in BRAF mutant A375P cells with high expression of p21Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells, which suggests that apoptosis and autophagy can occur simultaneously and act as cooperative partners for the induction of cell death. Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21Cip1 is a prerequisite for a positive response to its clinical application. Citation Format: Michael Lee, Gun-Hee Jang, Jun-Ho Ahn, Na-Yeon Kim. BH3-mimetic gossypol promotes autophagy to inhibit the proliferation of mutant BRAF melanoma cells with high expression of p21Cip1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1337. doi:10.1158/1538-7445.AM2014-1337

TARGETING RESISTANCE PATHWAYS IN BRAF-MUTANT PEDIATRIC GLIOMAS

BACKGROUND: Mutational activation of BRAF is a common finding in pediatric gliomas. As many as 14% of pediatric high grade glioma and up to 66% of certain subtypes of low grade pediatric glioma contain the BRAFV600E mutation. Small molecule inhibitors that selectively target BRAFV600E, and that are FDA approved for treating melanoma, have shown significant efficacy in treating BRAFV600E glioma in pre-clinical studies. Nonetheless, and despite showing initial efficacy, acquired drug resistance significantly limits the use of BRAFV600E inhibitors in clinical settings. Here, we have identified molecular mechanisms of BRAFV600E inhibitor resistance in glioma, and our detailing of these adaptive mechanisms offer strategies to delay and/or overcome drug resistance. METHODS: To enable our studies, we generated multiple BRAFV600E inhibitor resistant glioma cell lines and tumor models. RESULTS: BRAFV600E inhibitor resistant AM38 and DBTRG-05MG human glioma cells, subjected to extended exposure to PLX4720, show reduced MAP kinase inhibition as well as reduced cell cycle arrest and apoptotic response, relative to corresponding naive cells that have not been previously exposed to inhibitor. In addition, BRAFV600E glioma xenografts, established by implantation with PLX4720-resistant glioma cells, do not respond to inhibitor treatment, resulting in lack of benefit to animal subject survival from treatment. By phospho-kinase array and gene expression analysis, we found that certain receptor tyrosine kinases, including EGF and Axl receptors, and pro-survival kinases involved in Wnt signaling, are hyperactivated in BRAFV600E inhibitor resistant gliomas. Armed with this information, we show that pharmacological and genetic inhibition of those pathways significantly reduces the viability of PLX4720 resistant cells. CONCLUSIONS: In summary, we have identified mechanisms of BRAFV600E inhibitor resistance from sustained BRAF inhibitor treatment of glioma cells, and these mechanisms involve activation of signaling mediators important to tumor cell proliferation and survival. Our results suggest novel therapeutic strategies for treating BRAFV600E -mutant pediatric glioma that should delay acquired resistance, and possibly take advantage of tumor adaptive mechanisms to BRAF inhibitor therapy. SECONDARY CATEGORY: Pediatrics.

Breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-GP/ABCB1) restrict oral availability and brain accumulation of the PARP inhibitor rucaparib (AG-014699).

Rucaparib is a potent, orally available, small-molecule inhibitor of poly ADP-ribose polymerase (PARP) 1 and 2. Ongoing clinical trials are assessing the efficacy of rucaparib alone or in combination with other cytotoxic drugs, mainly in breast and ovarian cancer patients with mutations in the breast cancer associated (BRCA) genes. We aimed to establish whether the multidrug efflux transporters ABCG2 (BCRP) and ABCB1 (P-gp, MDR1) affect the oral availability and brain penetration of rucaparib in mice. In vitro, rucaparib was efficiently transported by both human ABCB1 and ABCG2, and very efficiently by mouse Abcg2. Transport could be inhibited by the small-molecule ABCB1 and ABCG2 inhibitors zosuquidar and Ko143, respectively. In vivo, oral availability (plasma AUC0-1 and AUC0-24) and brain levels of rucaparib at 1 and 24h were increased by the absence of both Abcg2 and Abcb1a/1b after oral administration of rucaparib at 10mg/kg. Our data show to our knowledge for the first time that oral availability and brain accumulation of a PARP inhibitor are markedly and additively restricted by Abcg2 and Abcb1a/1b. This may have clinical relevance for improvement of rucaparib therapy in PARP inhibitor-resistant tumors with ABCB1 and/or ABCG2 expression and in patients with brain (micro)metastases positioned behind a functional blood-brain barrier.

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21Cip1

AimsThe aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors. Main methodsThe IC50 values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1. Key findingsGossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G2/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G2/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis. SignificanceTaken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21Cip1 is a prerequisite for a positive response to its clinical application.

Microtubule Motors: A New Hope for Kinesin-5 Inhibitors?

A new study demonstrates that two microtubule plus end-directed kinesins can oppose each other. The cause of this apparent contradiction is the specific orientation of microtubules on which each motor exerts its force.

Abstract 2342: Inhibition of protein kinase C delta induces apoptosis through JNK-H2AX pathway in melanoma.

Abstract Metastatic melanoma is the major cause of skin cancer death, and the annual incidence of melanoma continues to increase. Despite the impressively high rates of response to BRAF inhibitors in patients with melanomas harboring BRAF mutations, most of these patients eventually relapse after developing resistance to the drug, due in part to secondary mutations in NRAS. Although NRAS mutation is the second most common genetic mutation in melanoma patients (after BRAF mutation), there is currently no treatment option that targets NRAS-mutated melanomas. New targeted therapies are urgently needed. Our previous reports have demonstrated the sensitivity of pancreatic cancer cell lines carrying oncogenic KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCd), suggesting that PKCd inhibitors might be effective in melanomas with primary or acquired NRAS mutations. PKCd is not required for normal mammalian development, making it a potential tumor-specific target. In this study, we investigated the effect of PKCd inhibition, and the efficacy of a new PKCd inhibitor BJE6-106 (B106), in melanoma. Inhibition of PKCd by B106 (at nano-molar concentrations), or by siRNA, inhibited the growth of multiple melanoma cell lines carrying NRAS mutations, and induced apoptosis mediated by terminal caspase activation. Analysis of the molecular mechanisms involved in this NRAS signaling-targeted approach demonstrated activation of the JNK pathway after PKCd inhibition, leading to the activation (phosphorylation) of H2AX, a histone H2A variant. Activation of H2AX was attenuated when JNK1/2 levels were repressed, indicating that H2AX activation is mediated by the JNK pathway in response to PKCd inhibition. Although the phosphorylation of H2AX has in the past been primarily known for its role in repair of DNA double strand breakage, more recent studies have proposed an active role for phospho-H2AX in the induction of apoptosis. Consistent with these reports, knockdown of H2AX prior to inhibition of PKCd mitigated the induction of caspase-dependent apoptosis in NRAS mutant melanoma cell lines. To explore the potential activity of new PKCd inhibitors in inhibitor-resistant BRAF-mutant tumors, we derived melanoma cell lines harboring BRAF mutations that evolved resistance to a BRAF inhibitor PLX4032 (vemurafenib). B106 effectively induced cytotoxicity in these cells, suggesting the potential clinical application of targeting PKCd in patients who have relapsed following treatment with PLX4032. Taken together, our present study suggests that inhibition of PKCd causes caspase-dependent apoptosis in melanomas with NRAS mutations and in PLX4032-resistant BRAF mutant melanomas. This apoptosis is mediated via activation of the JNK-H2AX pathway, which involves a novel role for phospho-H2AX in the execution of apoptosis. Citation Format: Asami Takashima, Douglas V. Faller. Inhibition of protein kinase C delta induces apoptosis through JNK-H2AX pathway in melanoma. [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 2342. doi:10.1158/1538-7445.AM2013-2342

Abstract 1619: Combination of VEGFR inhibitor lenvatinib (E7080) and Met/EphB4 inhibitor golvatinib (E7050) overcomes VEGFR inhibitor-resistant tumor vascular.

Abstract Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several types of tumors, however, some tumor shows intrinsic resistance to VEGFR inhibitors. In addition, patients initially responding develop acquired resistance to VEGFR inhibitors after varying periods of time. Thus it is urgent to develop strategy to overcome anti-VEGF therapy resistance. Hepatocyte growth factor (HGF)/Met signaling is activated in various types of tumors. Recently, it is reported that HGF/Met signaling contributes to resistance to tyrosine kinase inhibitors such as EGFR, BRAF, ALK inhibitors. This suggests that Met inhibitors may be promising in treating HGF/Met activated anti-VEGF therapy-resistant tumors. We have identified lenvatinib (E7080) as a highly-potent VEGFR inhibitor and phase III trial is ongoing in thyroid cancer. Lenvatinib inhibited the growth of HUVEC induced by VEGF, however, addition of HGF to HUVEC conferred resistance to lenvatinib. This resistance was cancelled by the addition of golvatinib (E7050) which is identified as a potent Met/EphB4 inhibitor and phase I trial of the lenvatinib/golvatinib combination therapy is ongoing. In mouse models bearing various tumor cell lines expressing VEGF and HGF, treatment with lenvatinib alone demonstrated only moderate antitumor activity, however, combination with golvatinib demonstrated synergistic antitumor activity with decrease of tumor vessel density. Furthermore, it is reported that EphB4 has critical roles in tumor vessel development and maturation. Our unique in vitro assay systems, endothelial cells/pericyte 2D co-culture assay and endothelial cells/pericyte 3D co-culture sprouting assay, suggested that EphB4 inhibitory activity of golvatinib disrupted anti-VEGF therapy-resistant tumor vascular in the combination with lenvatinib. Together, these data demonstrated that combination therapy with golvatinib may be promising to overcome anti-VEGF therapy resistance. Citation Format: Satoshi Kawano, Takayuki Nakagawa, Yuya Nakazawa, Yoshiaki Sato, Tomohiro Matsushima, Yasuhiro Funahashi. Combination of VEGFR inhibitor lenvatinib (E7080) and Met/EphB4 inhibitor golvatinib (E7050) overcomes VEGFR inhibitor-resistant tumor vascular. [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 1619. doi:10.1158/1538-7445.AM2013-1619

Abstract LB-196: Anti-DLL4 reduces tumor growth and tumorigenicity in B-RAF V600E melanomas including those with acquired resistance to B-RAF inhibitors

Abstract Melanoma is frequently driven by oncogenic mutation in the B-RAF gene (V600E) and this mutation is found in approximately 60% of patients. These patients are highly sensitive to kinase inhibitors targeting B-RAF. However, recent clinical experience has shown that after an initial response, a high percentage of patients develop resistance to B-RAF inhibitors through various mechanisms. In our studies, we evaluated whether inhibition of DLL4-Notch signaling might be effective in B-RAF mutant melanoma. We utilized two patient-derived melanoma xenografts containing the B-RAFV600E mutation, OMP-M2 and OMP-M8. The OMP-M8 xenograft was derived from a patient who was treated with the B-RAF inhibitor Vemurafenib (PLX-4032) and showed an early response to treatment, but later became resistant. We performed combination efficacy experiments with the B-RAF inhibitor PLX-4720 (doses 20-80 mg/kg, oral gavage, qdx5) and anti-DLL4 antibodies (30 mg/kg, IP once a week), and found that PLX-4720 combined with anti-DLL4 treatment caused a significant inhibition of tumor growth in OMP-M2 tumors. In serial transplant limiting dilution assay (LDA) studies with the OMP-M2 model, PLX-4720 treatment induced a significant enrichment of tumor initiating cells. In contrast, anti-DLL4 reduced the frequency of tumor initiating cells compared to controls, as a single agent and in combination with PLX-4720. We also found that the OMP-M8 tumor xenografted in mice retained the lack of response to the B-RAF kinase inhibitor, showing that the xenografts responded to treatment similarly as the patient in the clinic. Interestingly, the B-RAF inhibitor resistant tumor OMP-M8 was sensitive to anti-DLL4, which inhibited the growth of the primary tumor and significantly reduced the frequency of tumor initiating cells as a single agent and in combination with a chemotherapeutic agent. These findings suggest that DLL4-Notch inhibition can be effective in B-RAF V600E mutant melanoma including patients with acquired resistance to B-RAF kinase inhibitors. 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 LB-196. doi:1538-7445.AM2012-LB-196

RETRACTED ARTICLE: Semaphorin 4D cooperates with VEGF to promote angiogenesis and tumor progression

The semaphorins and plexins comprise a family of cysteine-rich proteins implicated in control of nerve growth and development and regulation of the immune response. Our group and others have found that Semaphorin 4D (SEMA4D) and its receptor, Plexin-B1, play an important role in tumor-induced angiogenesis, with some neoplasms producing SEMA4D in a manner analogous to vascular endothelial growth factor (VEGF) in order to attract Plexin-B1-expressing endothelial cells into the tumor for the purpose of promoting growth and vascularity. While anti-VEGF strategies have been the focus of most angiogenesis inhibition research, such treatment can lead to upregulation of pro-angiogenic factors that can compensate for the loss of VEGF, eventually leading to failure of therapy. Here, we demonstrate that SEMA4D cooperates with VEGF to promote angiogenesis in malignancies and can perform the same function in a setting of VEGF blockade. We also show the potential value of inhibiting SEMA4D/Plexin-B1 signaling as a complementary mechanism to anti-VEGF treatment, particularly in VEGF inhibitor-resistant tumors, suggesting that this may represent a novel treatment for some cancers.