Brian B. Haines

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

BackgroundHyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence.MethodsWe used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets.ResultsThe RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer.ConclusionsThese data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

Gauging NOTCH1 Activation in Cancer Using Immunohistochemistry

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

Discovery of biomarkers predictive of GSI response in triple negative breast cancer and adenoid cystic carcinoma

UNLABELLED Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. SIGNIFICANCE NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.

SAR profiles of spirocyclic nicotinamide derived selective HDAC1/HDAC2 inhibitors (SHI-1:2)

A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.

Combination of the mTOR Inhibitor Ridaforolimus and the Anti-IGF1R Monoclonal Antibody Dalotuzumab: Preclinical Characterization and Phase I Clinical Trial

Purpose: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin–like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination. Experimental Design: In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10–40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored. Results: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER+/high-proliferative breast cancer showed antitumor activity. Conclusions: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER+/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379). Clin Cancer Res; 21(1); 49–59. ©2014 AACR.

Divergent Kinetics Differentiate the Mechanism of Action of Two HDAC Inhibitors

Histone deacetylases (HDACs) play diverse roles in many diseases including cancer, sarcopenia, and Alzheimers. Different isoforms of HDACs appear to play disparate roles in the cell and are associated with specific diseases; as such, a substantial effort has been made to develop isoform-selective HDAC inhibitors. Our group focused on developing HDAC1/HDAC2-specific inhibitors as a cancer therapeutic. In the course of characterizing the mechanism of inhibition of a novel HDAC1/2-selective inhibitor, it was determined that it did not exhibit classical Michaelis-Menten kinetic behavior; this result is in contrast to the seminal HDAC inhibitor SAHA. Enzymatic assays, along with a newly developed binding assay, were used to determine the rates of binding and the affinities of both the HDAC1/2-selective inhibitor and SAHA. The mechanism of action studies identified a potential conformational change required for optimal binding by the selective inhibitor. A model of this putative conformational change is proposed.

Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2).

We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.

An Unbiased Oncology Compound Screen to Identify Novel Combination Strategies

Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo. This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155–62. ©2016 AACR.

Delayed and Prolonged Histone Hyperacetylation with a Selective HDAC1/HDAC2 Inhibitor.

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.

Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer.

BackgroundMammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.MethodsUsing hormone-sensitive breast cancer cells stably transfected with the aromatase gene (MCF-7/AC-1), works presented herein describe the in vitro and in vivo antitumor efficacy of the following compounds: dalotuzumab (DALO; “MK-0646”; anti-IGF-1R antibody), ridaforolimus (RIDA; “MK-8669”; mTORC1 small molecule inhibitor) and letrozole (“LET”, aromatase inhibitor).ResultsWith the exception of MK-0646, all single agent and combination treatment arms effectively inhibited xenograft tumor growth, albeit to varying degrees. Correlative tissue analyses revealed MK-0646 alone and in combination with LET induced insulin receptor alpha A (InsR-A) isoform upregulation (both mRNA and protein expression), thereby further supporting a triple therapy approach.ConclusionThese data provide preclinical rationalization towards the combined triple therapy of LET plus MK-0646 plus MK-8669 as an efficacious anti-tumor strategy for ER-positive breast tumors.