Hallmark Feature Of Cancer Research Articles

8123 Identification Of Glutamine Metabolism As A Druggable Therapeutic Vulnerability For Adrenocortical Carcinoma

Abstract Disclosure: V. Chortis: None. C. Yao: None. C. Ribeiro: None. L. Kelley: None. L. Nagano: None. M. Berber: None. S. Raveenthiraraj: None. P. Vendramini: None. K. Kiseljak-Vassiliades: None. D.L. Carlone: None. M.C. Haigis: None. K.S. Borges: None. D.T. Breault: None. Dysregulation of cellular metabolism is a hallmark feature of cancer that can be targeted therapeutically but remains underexplored in adrenocortical carcinoma (ACC), despite the urgent need for improved treatments. We employed a recently developed transgenic mouse model of ACC (BPCre), driven by the two most common mutations in human ACC (β-catenin gain-of-function and p53 loss-of-function), aiming to characterize in vivo ACC metabolism at a tissue level and to identify metabolic vulnerabilities. We employed metabolomics (liquid chromatography-mass spectrometry) and transcriptomics (bulk RNA-seq) to compare tissue metabolism in spontaneous BPCre tumors with control adrenals. Polar metabolites were analyzed using a library that provides a representative cross-section of major carbon and nitrogen-handling pathways. We identified widespread dysregulation of tumor metabolism (64/175 metabolites significantly dysregulated, FDR<0.05), with the most significantly dysregulated pathways including purine and pyrimidine metabolism, proline and glutamate metabolism, glycolysis, and the pentose phosphate pathway (FDR<0.05). Orthogonal analysis by RNA-seq revealed congruent metabolic pathway alterations. Comparison of the most significantly dysregulated pathways between BPCre mouse tumors and human ACC using publicly accessible human transcriptomic datasets (TCGA and GTEx) revealed extensive overlap. Given the prominent dysregulation of several glutamine (Gln)-dependent metabolic pathways in mouse and human ACC, we hypothesized that Gln catabolism is likely to represent a targetable metabolic vulnerability in ACC. Pharmacological targeting of Gln metabolism using the Gln antagonist 6-Diazo-5-Oxo-L-Norleucine (L-DON) induced marked cytotoxicity in two cell lines derived from BPCre tumors and three human ACC cell lines (NCI-H295R, CU-ACC1-2), as assessed by cell viability and clonogenic assays. Remarkably, this effect was rescued only by nucleoside supplementation, suggesting that Gln’s contribution to de novo purine and pyrimidine biosynthesis is the critical metabolic dependency in ACC cells. Treatment with the L-DON pro-drug JHU-083, in vivo, led to marked growth inhibition of subcutaneous mouse ACC tumor implants in both immunocompetent and immunodeficient mice. As expected, tissue metabolomics of JHU-083-treated tumors confirmed extensive depletion of purine metabolites. Tumor immunophenotyping by flow cytometry revealed significantly increased infiltration with Natural Killer cells, likely potentiating the anti-tumor effect of glutamine antagonism in immunocompetent mice. JHU-083 was also effective against human NCI-H295R xenografts, in vivo. This work reveals important new insights into ACC metabolism and provides pre-clinical evidence supporting Gln antagonism as a new metabolic treatment approach for ACC. Presentation: 6/2/2024

Histone serotonylation regulates ependymoma tumorigenesis.

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.

Abstract IA022: Mechanisms of splicing dysregulation and dependency in cancer

Abstract Dysregulated RNA splicing is a hallmark feature of cancer. Much like mutational signatures in tumors, RNA mis-splicing patterns are widely heterogeneous and segregate tumors into clades, suggesting there are distinct mechanisms underlying these splicing aberrations. However, the mechanisms driving such distinct RNA splicing dysregulation remain largely unknown. Herein, we discovered that copy-number variation of essential splicing factors is a pervasive cause of splicing dysregulation, and in some tumor contexts, confers deep dependencies on RNA-binding proteins (RBPs) that converge on these mechanisms. Using a systematic chemical biology approach, we delineate how splicing RBPs participate in distinct types of RNA quality control in cells, and when disrupted, produce surprisingly unique patterns of RNA mis-splicing. These RBP-specific mis-splicing patters are common in distinct cancer types. Notably, unbiased genome-wide analysis revealed that RBP-specific mis-splicing patterns found in tumors were significantly associated with copy number loss of a network of functionally linked splicing factors. These splicing factors often reside on tumor suppressor loci that are frequently deleted in breast and other cancers, suggesting that copy number loss of essential splicing RBPs is a collateral event with selected loss of tumor suppressors. Importantly, RBP-specific mis-splicing signatures also segregate tumor models that are dependent on the concordant RBP, suggesting that these tumors evolve defects in RBP function that predispose them to further perturbation. Together, our work suggests that deletions of tumor suppressor loci may drive collateral dysregulation of RNA splicing through partial loss of splicing RBPs and provokes the hypothesis that these mis-splicing signatures may predict actionable dependencies in the cancers that harbor them. Citation Format: Kristen Karlin. Mechanisms of splicing dysregulation and dependency in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA022.

Genetically predicted 486 blood metabolites in relation to risk of colorectal cancer: A Mendelian randomization study.

Metabolic disorders are a hallmark feature of cancer. However, the evidence for the causality of circulating metabolites to promote or prevent colorectal cancer (CRC) is still lacking. We performed a two-sample Mendelian randomization (MR) analysis to assess the causality from genetically proxied 486 blood metabolites to CRC. Genome-wide association study (GWAS) data for exposures were extracted from 7824 Europeans GWAS on metabolite levels. GWAS data for CRC from the GWAS catalog database GCST012879 were used for the preliminary analysis. The random inverse variance weighted (IVW) is the primary analysis for causality analysis while MR-Egger and weighted median as complementary analyses. Cochran Q test, MR-Egger intercept test, MR-PRESSO, Radial MR, and leave-one-out analysis were used for sensitivity analyses. For significant associations, additional independent CRC GWAS data GCST012880 were used for replication analysis and meta-analysis. For the final identification of metabolites, Steiger test, linkage disequilibrium score regression, and colocalization analysis were performed for further evaluation. Multivariable MR was performed to assess the direct effect of metabolites on CRC. The results of this study indicated significant associations between six metabolites pyruvate (odds ratio [OR]: 0.49, 95% confidence interval [CI]: 0.32-0.77, p = 0.002), 1,6-anhydroglucose (OR: 1.33, 95% CI: 1.11-1.59, p = 0.002), nonadecanoate (19:0) (OR: 0.40, 95% C I:0.4-0.68, p = 0.0008), 1-linoleoylglycerophosphoethanolamine (OR: 0.47, 95% CI: 0.30-0.75, p = 0.001), 2-hydroxystearate (OR: 0.39, 95% CI: 0.23-0.67, p = 0.0007), gamma-glutamylthreonine (OR: 2.14, 95% CI: 1.02-4.50, p = 0.040) and CRC. MVMR analysis revealed that genetically predicted pyruvate, 1-linoleoylglycerophosphoethanolamine and gamma-glutamylthreonine can directly influence CRC independently of other metabolites. The current work provides evidence to support the causality of the six circulating metabolites on CRC and a new perspective on the exploration of the biological mechanisms of CRC by combining genomics and metabolomics. These findings contribute to the screening, prevention and treatment of CRC.

Too much and never enough: Synthetic excess and metabolic inefficiency of aneuploidy in tumorigenesis

Aneuploidy is a hallmark feature of cancer. Relative to normal diploid tissue, aneuploid tumors are often net copy number increased, in a range from hyper‐diploid to tetraploid. This results in widespread replication stress and proteotoxic stress that affects diverse cellular complexes. Utilizing multi‐omics approaches in novel models of cancer‐associated copy number alterations derived from diploid human mammary epithelial cells, we uncovered an aneuploidy stress response driven in part by Myc that transcriptionally upregulates biomass production capacity and stresses metabolic capacity. Basal and peak glycolytic and oxidative rates are increased in aneuploid cells compared to diploids, however free nucleotide pools and metabolic intermediates, particularly pyrimidines, are depleted. CRISPR screens revealed a universally increased dependence on de novo pyrimidine synthesis genes in aneuploid cells with diverse CNAs, suggesting a struggle to match pyrimidine synthesis capacity with need. The biosynthetic demand and inefficiency driven by aneuploidy may partially explain the switch to aerobic glycolysis in tumors and present vulnerabilities to be therapeutically exploited.

Association of VEGF haplotypes with breast cancer risk in North-West Indians

BackgroundAngiogenesis is a complex and coordinated process regulated by different growth factors and is one of the hallmark features of cancer. VEGF is one of the most important endothelial cell mitogen and has a critical role in normal physiological and tumor angiogenesis. The objective of this study was to investigate the potential association of haplotypes of six VEGF polymorphisms with breast cancer risk in North-West Indians.MethodsSamples of 250 breast cancer patients and 250 age and sex matched controls were genotyped for VEGF −2578C/A, −2549I/D, −460T/C, +405C/G, −7C/T and +936C/T polymorphisms. Haplotypes were generated to determine the better contribution of VEGF polymorphisms to breast cancer risk.ResultsHaplotypes CDTCCC (OR = 0.56, 95%CI, 0.38–0.81; p = 0.003) and CDTGCC (OR = 0.63, 95%CI, 0.44–0.92; p = 0.018) of VEGF −2578C/A, −2549I/D, −460T/C, +405C/G, −7C/T and +936C/T polymorphisms were significantly associated with decreased risk of breast cancer. CDTCCC haplotype was also significantly associated with reduced risk of breast cancer in pre and post menopausal as well as both obese and non obese patients. Haplotype CDTGCC was marginally associated (p = 0.07) with reduced risk of breast cancer in non-obese patients as compared with non-obese controls where as haplotype AICGTC was marginally associated (p = 0.09) with reduced risk of breast cancer in obese patients when compared with non-obese patients. The CDTGCC haplotype was significantly associated with increased risk of breast cancer in premenopausal obese patients (OR = 1.98, 95%CI, 1.10–3.56; p = 0.02).ConclusionsOur data indicated that CDTCCC and CDTGCC haplotypes of VEGF −2578C/A, −2549I/D, −460T/C, +405C/G, −7C/T and +936C/T polymorphisms were significantly associated with breast cancer risk in North-West Indians. Further studies on multiethnic groups with larger sample size are required to confirm our results.

Abstract 2807: Variations in glycolytic activity of HCC subtypes as monitored by in vivo h 13C MRI

Abstract Hepatocellular carcinoma (HCC) is the 4th leading cause of cancer-related death worldwide. Despite therapeutic advancements, a significant proportion of patients progress, highlighting the need to improve diagnostic approaches to monitor therapy. Radiofrequency ablation (RFA), an accepted locoregional therapy for early HCC, can stimulate more aggressive tumor biology within under-treated or distant tumors. Increased glycolytic activity is a hallmark feature of cancer, but variations in metabolism and its link to tumor aggression can impact the sensitivity of HCC to therapies affecting these pathways. Tumor metabolic phenotypes can be characterized by hyperpolarized 13C MRI (h13C MRI), which can also be used to assess treatment response. In this study, we used orthotopic rat HCC models with two different cell lines N1S1 and MCA-RH7777, and analyzed their mRNA expression of glycolysis-related genes and in vivo glycolytic activity with h13C MRI. We then partially-treated tumors with radiofrequency ablation (RFA) and used h13C MRI to measure in vivo tumor metabolism before and 3 days after RFA to compare them to histologic and phenotypical measures of disease progression. N1S1 tumors grew only as single lesions, while MCA-RH7777 rapidly produced intra-hepatic and distal metastases. Glycolysis-related genes including HK2, HK3, PFKFB3, and MCT4 were highly expressed in N1S1, but not in MCA-RH7777, while GPT1 was highly expressed in MCA-RH7777 but not in N1S1. In vivo h13C MRI demonstrated significantly increased 13C lactate flux in N1S1 tumor compared to the background normal liver, while 13C lactate flux in MCA-RH7777 rat model did not, consistent with mRNA expression patterns. Tumor 13C lactate flux was significantly increased and 13C alanine flux was significantly decreased following partial RFA treatment to N1S1 tumors when compared to untreated tumors, which was associated with an increase in tumor Ki67 expression. However, no change was seen on h13C MRI between the RFA and control arms in MCA-RH7777 tumors, even though growth and total tumor burden progressed significantly more following RFA. In contrast to N1S1, there was also no difference between 13C lactate flux and 13C alanine flux within MCA-RH7777 tumors and the neighboring normal hepatic parenchyma, suggesting that glycolytic activity in these tumors did not drive RFA-stimulated progression. Targeting of glycolytic pathways as a potential therapy for HCC may therefore be limited to certain subtypes, and h13C MRI may be used to select for sensitivity. Citation Format: Qianhui Dou, Aaron K. Grant, Cody Callahan, Muneeb Ahmed, Leo L. Tsai. Variations in glycolytic activity of HCC subtypes as monitored by in vivo h 13C MRI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2807.

Fibrinogen-Albumin Ratio as a New Promising Preoperative Biochemical Marker for Predicting Oncological Outcomes in Gastric Cancer: A Multi-institutional Study.

The systemic inflammatory response caused by host-tumor interactions is currently recognized as a hallmark feature of cancer. No study has confirmed which systemic inflammatory factors can accurately predict the progression and long-term prognosis of gastric cancer (GC). Through the analysis of receiver operating characteristic curve (ROC), in the discovery cohort, a variety of indicators composed of usual inflammatory factors were compared. Fibrinogen-albumin ratio (FAR), which can accurately predict the long-term survival of GC patients was selected and was further verified in the test cohort and the external validation cohort. The ROC curve analysis showed that the area under curve (AUC) value of FAR on the overall survival (OS) of GC patients was higher than that of other combined markers (P < 0.01). Patients in the high FAR group showed more advanced pathological stages, larger tumor diameters, and more poorly differentiated pathological type than those in the low FAR group (P < 0.05). Logistic regression analysis elucidated that, FAR was an independent risk factor for LN metastasis and tumor invasion of GC. High FAR was an independent risk factor for poor prognosis of GC patients. The relationship between FAR and pathological stage of GC and long-term prognosis of patients was verified in the test cohort and the external validation cohort with the same FAR cutoff value. The results are consistent with those of the discovery cohort. As a new developed inflammation-related marker, FAR can independently and effectively predict the tumor burden and long-term prognosis of patients with advanced GC.

Computational Prediction of Chemical Tools for Identification and Validation of Synthetic Lethal Interaction Networks.

Cancer is one of the leading causes of death and chromosomal instability (CIN) is a hallmark feature of cancer. CIN, a source of genetic variation in either altered chromosome number or structure contributes to tumor heterogeneity and has become a hot topic in recent years prominently for its role in therapeutic responses. Synthetic lethality and synthetic rescue based approaches, for example, advancing CRISPR-Cas9 platform, are emerging as a powerful strategy to identify new potential targets to selectively eradicate cancer cells. Unfortunately, only few of them are further explored therapeutically due to the difficulty in linking these targets to small molecules for pharmacological intervention. This, however, can be alleviated by the efforts to bring chemical, bioactivity, and genomic data together, as well as established computational approaches. In this chapter, we will discuss some of these advances, including established databases and in silico target-ligand prediction, with the aim to navigate through the synthetically available chemical space to the biologically targetable landscape, and eventually, to the chemical modeling of synthetic lethality and synthetic rescue interactions, that are of great clinical and pharmaceutical relevance and significance.

Exploiting aneuploidy-imposed stresses and coping mechanisms to battle cancer.

Aneuploidy, an irregular number of chromosomes in cells, is a hallmark feature of cancer. Aneuploidy results from chromosomal instability (CIN) and occurs in almost 90% of all tumours. While many cancers display an ongoing CIN phenotype, cells can also be aneuploid without displaying CIN. CIN drives tumour evolution as ongoing chromosomal missegregation will yield a progeny of cells with variable aneuploid karyotypes. The resulting aneuploidy is initially toxic to cells because it leads to proteotoxic and metabolic stress, cell cycle arrest, cell death, immune cell activation and further genomic instability. In order to overcome these aneuploidy-imposed stresses and adopt a malignant fate, aneuploid cancer cells must develop aneuploidy-tolerating mechanisms to cope with CIN. Aneuploidy-coping mechanisms can thus be considered as promising therapeutic targets. However, before such therapies can make it into the clinic, we first need to better understand the molecular mechanisms that are activated upon aneuploidization and the coping mechanisms that are selected for in aneuploid cancer cells. In this review, we discuss the key biological responses to aneuploidization, some of the recently uncovered aneuploidy-coping mechanisms and some strategies to exploit these in cancer therapy.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Assessment of the Antineoplastic Effects of the Investigational Agent PAX2M as C-MYC Onco-Protein Blocking Agent in Human Colorectal Cancer Cells Model Sw480 Cell Line

MYC modifies the expression of many genes, and the deregulated expression of C-MYC represents a hallmark feature of cancer because most, if not all, human cancers have a deregulation in C-MYC and associated with poor prognoses. The Primary human colorectal carcinomas were enormously associated with C-MYC overexpression. This study involved an assessment of the cytotoxic effects of PAX2M, a direct C-MYC oncoprotein blocking agent, in a colorectal cancer cell line, and the evaluation of its safety in the normal tissue cell line. The results indicate a significant reduction in the cells viability at the dose-dependent fashion moreover, PAX2M displayed a lower toxicity in normal cells The investigational agent PAX2M described in this study that directly targets C-MYC oncoprotein is likely to be a promising therapeutic agent for colorectal cancer treatment.

Mitochondrial membrane depolarization enhances TRAIL-induced cell death in adult human granulosa tumor cells, KGN, through inhibition of BIRC5

BackgroundCellular metabolic changes that accompany malignant transformation have been heralded as hallmark features of cancer. However, metabolic signatures between neoplasms can be unique, allowing for distinctions in malignancy, invasion and chemoresistance between cancer types and subtypes. Mitochondria are central metabolic mediators, as cellular bioenergetics veers from oxidative phosphorylation to glycolysis. Herein, we evaluate the role of mitochondria in maintenance of cellular metabolism, proliferation, and survival in the adult granulosa tumor cell line, KGN, as well as three epithelial ovarian cancer cell lines to determine distinctions in specific features.ResultsNotably, KGN cells were susceptible to TRAIL- and cisplatin-induced death following pretreatment with the metabolic inhibitor FCCP, but not oligomycin A. Collapse of mitochondrial membrane potential was found concomitant with cell death via apoptosis, independent from extrinsic canonical apoptotic routes. Rather, treatment with FCCP resulted in elevated cytochrome c release from mitochondria and decreased responsiveness to BIRC5. Following knockdown of BIRC5, mitochondrial membrane depolarization further sensitized KGN cells to induction of apoptosis via TRAIL.ConclusionsThese results indicate an essential role, distinct from metabolism, for mitochondrial membrane potential in KGN cells to sense and respond to external mediators of apoptotic induction.

Abstract 1301: Using the evolutionary ages of genes to understand hallmark features of cancer

Abstract The evolutionary age of a gene is closely tied to its role in cancer. As organisms evolved into multicellular forms, new pathways emerged to control core cellular processes formed in unicellular ancestors such as cell division, DNA replication and energy metabolism. Tumors rely on many of these core processes, at the same time as they suppress key pathways linked to the emergence of multicellularity. This suggests the guiding principles behind many hallmarks of cancer could be better understood in an evolutionary context, by investigating interactions between unicellular and multicellular genes and how they are disrupted in cancer. We have found the evolutionary histories of genes are associated with their expression and regulation (Trigos et al., PNAS 2017), the selection and response to genetic alterations, as well as the rewiring of transcriptional networks across 7 tumor types. Our results reveal different transcriptional and mutational patterns in cancer between genes with different evolutionary ages, with selection for genetic alterations in early metazoan genes leading to the transcriptional rewiring of the regulation between unicellular and multicellular genes, driving carcinogenesis. Examples of how this approach can help prioritize genes for drug treatment and inform novel treatment strategies will be given. Citation Format: David L. Goode, Anna S. Trigos, Anthony T. Papenfuss, Richard B. Pearson. Using the evolutionary ages of genes to understand hallmark features of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1301.

Phase I study of procaspase activating compound-1 (PAC-1) for treatment of advanced malignancies.

TPS2621 Background: Dysregulation in apoptotic pathways is a hallmark feature of cancer, offering opportunities for pharmacologic intervention in its treatment. Caspases, a family of proteases, play several roles in apoptosis. Caspase-3 catalyzes the intra-cellular proteolysis that characterizes part of the apoptotic process. Caspase-3 levels are low in many tumors including: glioblastoma; breast, colon, lung, and liver cancers: lymphoma: neuroblastoma; and melanoma. In contrast, procaspase-3, the precursor of caspase-3 is elevated in these tumors. We describe a phase I study of PAC-1, a drug that catalyzes the conversion of procaspase -3 to caspase-3. It induces apoptosis in tumor cell lines in vitro. In vivo activity has also been shown, when combined with alkylating chemotherapy, in rodent glioma models and in canines diagnosed with malignant glioma. Methods: This Phase I dose escalation study has two components: the first (C1) to determine the maximum tolerated dose (MTD) of PAC-1 in advanced malignancies; the second (C2) to determine the MTD of PAC-1 when combined with temozolomide (TMZ) in patients with recurrent anaplastic astrocytoma (AA) or glioblastoma (GBM). A modified Fibonacci 3 + 3 design is used, expanding to nine subjects at the MTD level in each component. PAC-1 pharmacokinetics is assessed in all subjects during the first cycle. Secondary objectives include pharmacodynamics and correlations of PAC-1 activity with procaspase-3 expression in tumor tissue. Neurologic toxicity is closely monitored throughout the study. Inclusion criteria: diagnoses of advanced malignancies (C1) and recurrent AA or GBM (C2), ECOG PS 0-2, adequate organ function. Exclusion criteria: prior cytotoxic therapy in the last 3-6 weeks (varying with drug class) or uncontrolled chronic illness. Administration and design: For C1, PAC-1 (orally administered) is dosed at 75-1,000 mg daily on days 1-21 of each 28 day cycle. For C 2, the first PAC-1 dose is 375 mg daily with potential for escalation to 1,000 mg daily. TMZ, PO, is dosed at 150 mg/m2 daily, days 8-12, of each cycle. Enrollment to date for C1 is 27, (dose level 6); and for C2 is 4 (dose level 1). The study is open to accrual. Clinical trial information: NCT02355535.

γ-Secretase inhibitor reduces immunosuppressive cells and enhances tumour immunity in head and neck squamous cell carcinoma.

Immune evasion is a hallmark feature of cancer, and it plays an important role in tumour initiation and progression. In addition, tumour immune evasion severely hampers the desired antitumour effect in multiple cancers. In this study, we aimed to investigate the role of the Notch pathway in immune evasion in the head and neck squamous cell carcinoma (HNSCC) microenvironment. We first demonstrated that Notch1 signaling was activated in a Tgfbr1/Pten-knockout HNSCC mouse model. Notch signaling inhibition using a γ-secretase inhibitor (GSI-IX, DAPT) decreased tumour burden in the mouse model after prophylactic treatment. In addition, flow cytometry analysis indicated that Notch signaling inhibition reduced the sub-population of myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs) and regulatory T cells (Tregs), as well as immune checkpoint molecules (PD1, CTLA4, TIM3 and LAG3), in the circulation and in the tumour. Immunohistochemistry (IHC) of human HNSCC tissues demonstrated that elevation of the Notch1 downstream target HES1 was correlated with MDSC, TAM and Treg markers and with immune checkpoint molecules. These results suggest that modulating the Notch signaling pathway may decrease MDSCs, TAMs, Tregs and immune checkpoint molecules in HNSCC.

Inhibitors of cyclin-dependent kinases as cancer therapeutics.

Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies.

Abstract 4607: Expression of Bcl-2 pro-survival family proteins predicts pharmacological responses to ABT-199, a novel and selective Bcl-2 antagonist, in multiple myeloma models.

Abstract A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues mediated, in part, by the the Bcl-2 family of pro-apoptotic and pro-survival proteins. ABT-199 is a potent BH3-only mimetic that selectively antagonizes the pro-survival function of Bcl-2. The work presented describes the efficacy of ABT-199 as a single agent and in combination with bortezomib in preclinical models of Multiple Myeloma (MM). Expression of Bcl-2 protein was detected in 95% of MM cell lines (n=21) evaluated and ABT-199 reduced cell viability in a sub-set of these lines (7/21) with EC50 values less than 0.5 μM. Expression of Bcl-xL and Mcl-1 at the mRNA and protein levels were also evaluated to determine predictors of drug sensitivity and resistance to ABT-199. MM lines that expressed higher levels of Bcl-2 relative to Bcl-xL and Mcl-1 were the most sensitive to ABT-199 treatment while those that were less sensitive expressed higher levels of Mcl-1 or Bcl-xL. Other predictors of sensitivity to ABT-199 included higher levels of Bcl-2/Bim complexes and t(11;14) status. Ex vivo, 75% of the MM bone marrow biopsies and aspirates (n=27) had high Bcl-2 levels whereas 50% had high Bcl-xL expression, demonstrating that a subset of patient samples (33%) have a favorable biomarker profile (Bcl-2-high/Bcl-xL-low) that may be predictive of ABT-199 activity. Efficacy of ABT-199 as a single agent and in combination with bortezomib was evaluated in vivo in MM xenograft models that expressed Bcl-2 (OPM-2, KMS-11, RPMI-8226, H929 and MM.1s). Bortezomib treatment alone at maximum tolerated doses resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at maximum tolerated doses was efficacious as a single agent in xenograft models that expressed higher protein levels of Bcl-2 but relatively lower levels of Bcl-xL. The combination of ABT-199 with bortezomib increased overall response rates, durability of anti-tumor activity and levels of apoptotic markers such as cleaved caspase 3 and PARP when compared to bortezomib alone even in MM xenograft models that also express relatively high levels of Mcl-1. Treatment with bortezomib in vivo increased levels of Noxa; a pro-apoptotic BH3-only protein that selectively antagonizes Mcl-1. Therefore, greater efficacy may be achieved when ABT-199 is combined with bortezomib due to inhibition of Bcl-2 by ABT-199 and neutralization of Mcl-1 by Noxa in tumors that express both pro-survival proteins. Our preclinical data supports the evaluation of ABT-199 as a single agent and in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Citation Format: Deepak Sampath, Elizabeth Punnoose, Erwin Boghaert, Lisa Belmont, Franklin Peale, Nguyen Tan, Jun Chen, Walter Darbonne, Peng Yue, Jason Oeh, Leslie Lee, Wayne J. Fairbrother, Andrew J. Souers, Steven W. Elmore, Joel D. Leverson. Expression of Bcl-2 pro-survival family proteins predicts pharmacological responses to ABT-199, a novel and selective Bcl-2 antagonist, in multiple myeloma models. [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 4607. doi:10.1158/1538-7445.AM2013-4607

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

AbstractAbstract 5028Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM.A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity.ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures:Sampath:Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

Abstract B44: Probing the effects of linker flexibility in second mitochondria-derived activator of caspases mimetic dimers and assessing their ability to induce apoptosis in a MDA-MB-231 cell line

Abstract Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (lAPs). X-linked inhibitor of apoptosis protein (XIAP) is one of the best characterized lAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), in particular caspase −3, −7, and −9 through one of its baculovirus IAP repeat (BIR) domains. Second mitochondria-derived activator of caspases (SMAC) is an antagonist protein released from the mitochondria and into the cytosol, to disrupt the complexes produced between XIAP and caspase. SMAC binds to the BIR domains in XIAP through its tetra-peptide motif, AVPI. Studies have demonstrated that the first and third amino acid positions of this tetra-peptide motif are important for binding, while positions two and four can be modified to increase binding affinity (AXPX). It has also been shown that dimers of SMAC mimetics show greater potency than their monomer counter parts. Previously, our laboratory has synthesized and tested SMAC dimers using three glycine amino acids to link AXPX sequences. These compounds showed very little activity. We also synthesized and tested SMAC dimers comprised of a lysine amino acid, linking two AXPX sequences; these mimetics did display significant activity. In particular an (AKPF)2K sequence showed 50% cell death at 10 uM when combined with death ligand TRAIL. Synthesis of these compounds was conducted using solid phase peptide synthesis (SPPS) on a polystyrene resin. We hypothesized that flexibility in the linker joining the two AXPX sequences was necessary to increase binding affinity. Consequently several SMAC mimetic dimers built on two different linkers (gamma-amino butyric acid and ornithine), both of which have a four carbon spacer between each terminus, have been prepared and evaluated for activity using a MDA-MB-231 breast cancer cell line. The first series of SMAC mimetic compounds consists of MeAXPX-GABA-MeAXPX amino acid sequence while the second series of SMAC mimetics are comprised of a MeAXPX-Ornithine-XPXMeA amino acid sequence. All data and results, including synthesis, biological assays, and the testing of these compounds will be the major focus of this presentation. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B44.