Target In Other Cancers Research Articles

TMIC-35. TUMOR CELL-DERIVED SPERMIDINE ACCELERATES GLIOBLASTOMA PROGRESSION VIA LYMPHOCYTE DEPLETION

Abstract Therapeutic resistance in glioblastoma (GBM) is partly due to a highly immunosuppressive environment resulting in ineffective anti-tumor immune response. This likely underlies the lack of success of immunotherapies that are successful in other cancers; novel ways to target this environment are needed. Naturally occurring polyamines (PAs) have been identified as a putative therapeutic target in other cancers based on their increased presence and correlation to poorer prognosis. Under normal conditions, spermidine (SPD) – a member of the PA family – is critical for cell growth. While PAs are increased in GBM patients, little is known about their impact on GBM growth. In syngeneic immunocompetent mouse glioma models, mass spectrometry revealed that SPD increases in the tumor microenvironment and circulation as compared to non-tumor-bearing mice. We treated the same models with exogenous SPD, which significantly decreased survival. However, in immunocompromised mice, no survival difference was observed, suggesting SPD drives GBM progression through immune interaction. Immune profiling the mouse models treated with SPD showed peripheral reduction of CD4+ and CD8+ T cells. Due to the observed T cell changes, we repeated the exogenous SPD paradigm in RAG1 knockout mice that do not have circulating B/ T cells. Similar to NSG mice, we see no survival difference, indicating that SPD drives GBM by affecting lymphoid cells. To investigate the origin of SPD in our models, we knocked down the rate limiting enzyme of the PA biosynthesis pathway in glioma cells in immunocompetent mice. Mice implanted with knockdown cells showed a robust survival extension, suggesting that tumor cells produce SPD which affects the tumor microenvironment. Ongoing studies are evaluating uptake from the environment on the progression of GBM using pharmacological inhibitors. Understanding the interactions of PAs and the immune response as a whole will allow us to alter the environment to enhance immunotherapeutic efficacy.

Pathway2Targets: an open-source pathway-based approach to repurpose therapeutic drugs and prioritize human targets.

Recent efforts to repurpose existing drugs to different indications have been accompanied by a number of computational methods, which incorporate protein-protein interaction networks and signaling pathways, to aid with prioritizing existing targets and/or drugs. However, many of these existing methods are focused on integrating additional data that are only available for a small subset of diseases or conditions. We have designed and implemented a new R-based open-source target prioritization and repurposing method that integrates both canonical intracellular signaling information from five public pathway databases and target information from public sources including OpenTargets.org. The Pathway2Targets algorithm takes a list of significant pathways as input, then retrieves and integrates public data for all targets within those pathways for a given condition. It also incorporates a weighting scheme that is customizable by the user to support a variety of use cases including target prioritization, drug repurposing, and identifying novel targets that are biologically relevant for a different indication. As a proof of concept, we applied this algorithm to a public colorectal cancer RNA-sequencing dataset with 144 case and control samples. Our analysis identified 430 targets and ~700 unique drugs based on differential gene expression and signaling pathway enrichment. We found that our highest-ranked predicted targets were significantly enriched in targets with FDA-approved therapeutics for colorectal cancer (p-value < 0.025) that included EGFR, VEGFA, and PTGS2. Interestingly, there was no statistically significant enrichment of targets for other cancers in this same list suggesting high specificity of the results. We also adjusted the weighting scheme to prioritize more novel targets for CRC. This second analysis revealed epidermal growth factor receptor (EGFR), phosphoinositide-3-kinase (PI3K), and two mitogen-activated protein kinases (MAPK14 and MAPK3). These observations suggest that our open-source method with a customizable weighting scheme can accurately prioritize targets that are specific and relevant to the disease or condition of interest, as well as targets that are at earlier stages of development. We anticipate that this method will complement other approaches to repurpose drugs for a variety of indications, which can contribute to the improvement of the quality of life and overall health of such patients.

Causal AI in silico Patient Model Identifies Minichromosome Maintenance (MCM) Family Genes As Novel Predictors for Overall Survival in Multiple Myeloma

The minichromosome maintenance (MCM) complex is among the best-known protein families involved in eukaryotic replication initiation. The six conserved proteins of the MCM complex, namely MCM2-MCM7, have been well studied and form a DNA helicase. Dysregulation of members of the MCM complex has been demonstrated in multiple cancer types, including gastric cancer, liver cancer, cervical cancer, and prostate cancer, among others. However, comparatively little is currently known about the role of the MCM complex in hematological oncology. We show here the first evidence of a causal link between members of the MCM complex, specifically MCM2, MCM3, and MCM4, and overall survival of multiple myeloma patients. This result was further validated in a separate, externally collected cohort of patients. In this study, an in-silico patient model of Multiple Myeloma Research Foundation (MMRF) IA19 CoMMpass data was developed using GNS Healthcare's Causal AI technology, REFSTM. REFSTM has been previously used to analyze MMRF data to identify the epigenetic regulator PHF19 as a marker of aggressive disease. Simulated genetic perturbations performed on the in-silico model identified changes in the expression of DNA replication genes MCM2, MCM3, and MCM4 as strong causal drivers of overall survival (OS) in MM patients (MCM2 and MCM4 shown in figure 1). These targets were validated using a Cox proportional-hazards model on an out-of-cohort Dana-Farber Cancer Institute MM dataset (DFCI). We report Cox hazard ratios of 1.62 (1.41, 1.85), 1.72 (1.42, 2.10), and 1.75 (1.50, 2.05) respectively, for MCM2, MCM3 and MCM4 in the MMRF IA19 dataset. Among patients in the DFCI dataset, we report Cox hazard ratios of 1.40 (1.09, 1.79), 1.50 (1.14, 1.99), and 1.56 (1.17, 2.08), respectively for MCM2, MCM3 and MCM4. In addition, causal modeling indicated large negative prognostic median effect sizes for overall survival. In summary, this work demonstrates MCM2, MCM3, and MCM4 as candidate genes involved in MM pathology. These genes are known targets in other cancers due to their role in DNA replication and cell cycle pathways. We could not identify any existing Open Targets literature related to these genes as drug targets or predictive markers in MM. Our results, validated out-of-cohort, suggest that MCM2, MCM3, and MCM4 are newly discovered predictors of overall survival among multiple myeloma patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-32. SPERMIDINE DRIVES GLIOBLASTOMA PROGRESSION VIA SELECTIVE MODULATION OF THE IMMUNE SYSTEM

Abstract Glioblastoma (GBM) is incurable despite aggressive standard of care treatments (maximal safe surgical resection, radiation, chemotherapy). GBM therapeutic resistance is due to multiple factors, including tumor heterogeneity and a highly immunosuppressive environment. Naturally occurring polyamines have been identified as a putative therapeutic target in other cancers based on their increased presence and function in normal conditions; they are critical for cell growth and proliferation and cellular functions including autophagy and apoptosis. While polyamines are increased in GBM patients, little is known about their impact on GBM growth. In syngeneic immune competent mouse glioma models (GL261, SB28), mass spectrometry data revealed that spermidine (SPD) – a member of the polyamine family – is increased in tumor tissue as compared to non-neoplastic control brain tissue (sham implanted animals). To test the impact of SPD on tumor growth, we treated mouse glioma models with exogenous SPD and found treatment significantly decreased survival. However, in immunocompromised host mice, no such difference was observed, indicating the mechanism through which SPD is driving GBM progression likely involves immune system alterations. Depletion of myeloid derived suppressor cells in vivo via anti-Gr-1 antibody rescues the decrease in survival caused by exogenous SPD, indicating that SPD drives GBM by affecting immune-suppressive cell subsets, namely MDSCs. To assess if SPD is associated with more aggressive GBM growth in human patients, we are currently analyzing polyamine levels in human GBM samples of long vs short term survivors. We are also exploring the effect of dietary polyamines on GBM via the gut-brain-microbiome axis, as polyamines are enriched in many foods and produced by a subset of commensal gut microbes. Understanding the interactions between polyamines, the tumor microenvironment, and the immune response provide a new mechanism for GBM regulation and identify opportunities to alter the environment in the body to enhance immunotherapeutic efficacy.

Preclinical evaluation of proteolytic targeting of LCK as a therapeutic approach in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and there is an unmet need for targeted therapies, especially for patients with relapsed disease. We have recently identified pre-T cell receptor and lymphocyte-specific protein tyrosine kinase (LCK) signaling as a common therapeutic vulnerability in T-ALL. LCK inhibitor dasatinib showed efficacy against T-ALL in preclinical studies and in patients with T-ALL; however, this is transient in most cases. Leveraging the proteolysis targeting chimera (PROTAC) approach, we developed a series of LCK degraders using dasatinib as an LCK ligand and phenyl-glutarimide as a cereblon-directing moiety. Our lead compound SJ11646 exhibited marked efficiency in cereblon-mediated LCK degradation in T-ALL cells. Relative to dasatinib, SJ11646 showed up to three orders of magnitude higher cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples in vitro, with drastically prolonged suppression of LCK signaling. In vivo pharmacokinetic and pharmacodynamic profiling indicated a 630% increase in the duration of LCK suppression by SJ11646 over dasatinib in patient-derived xenograft models of T-ALL, which translated into its extended leukemia-free survival over dasatinib in vivo. Last, SJ11646 retained a high binding affinity to 51 human kinases, particularly ABL1, KIT, and DDR1, all of which are known drug targets in other cancers. Together, our dasatinib-based phenyl-glutarimide PROTACs are promising therapeutic agents in T-ALL and valuable tools for developing degradation-based therapeutics for other cancers.

Dysregulation of Translation Factors EIF2S1, EIF5A and EIF6 in Intestinal-Type Adenocarcinoma (ITAC).

Simple SummaryIntestinal-type adenocarcinoma (ITAC) belongs to the group of sinonasal cancers which are a rare and heterogenous group of malignant neoplasms. Within this group, intestinal-type adenocarcinoma (ITAC) represents the most frequently occurring tumour, especially in Europe, and has been associated with exposure to occupational hazards, such as wood dust and leather. Eukaryotic translation initiation factors have been described as promising targets for novel cancer treatments, but hardly anything is known about these factors in ITAC. Here we performed in silico analyses, evaluated the protein levels of EIF2S1, EIF5A and EIF6 in tumour samples and non-neoplastic tissue controls obtained from 145 patients, and correlated these results with clinical outcome data, including tumour site, stage, adjuvant radiotherapy and survival. In silico analyses revealed significant upregulation of the translation factors EIF6 (ITGB4BP), EIF5, EIF2S1 and EIF2S2 (p < 0.05) with a higher arithmetic mean expression in ITAC compared to non-neoplastic tissue (NNT). Immunohistochemical analyses using antibodies against EIF2S1 and EIF6 confirmed a significantly different expression at the protein level (p < 0.05). In conclusion, this work identifies the eukaryotic translation initiation factors EIF2S1 and EIF6 to be significantly upregulated in ITAC. As these factors have been described as promising therapeutic targets in other cancers, this work identifies candidate therapeutic targets in this rare but often deadly cancer.Intestinal-type adenocarcinoma (ITAC) is a rare cancer of the nasal cavity and paranasal sinuses that occurs sporadically or secondary to exposure to occupational hazards, such as wood dust and leather. Eukaryotic translation initiation factors have been described as promising targets for novel cancer treatments in many cancers, but hardly anything is known about these factors in ITAC. Here we performed in silico analyses, evaluated the protein levels of EIF2S1, EIF5A and EIF6 in tumour samples and non-neoplastic tissue controls obtained from 145 patients, and correlated these results with clinical outcome data, including tumour site, stage, adjuvant radiotherapy and survival. In silico analyses revealed significant upregulation of the translation factors EIF6 (ITGB4BP), EIF5, EIF2S1 and EIF2S2 (p < 0.05) with a higher arithmetic mean expression in ITAC compared to non-neoplastic tissue (NNT). Immunohistochemical analyses using antibodies against EIF2S1 and EIF6 confirmed a significantly different expression at the protein level (p < 0.05). In conclusion, this work identifies the eukaryotic translation initiation factors EIF2S1 and EIF6 to be significantly upregulated in ITAC. As these factors have been described as promising therapeutic targets in other cancers, this work identifies candidate therapeutic targets in this rare but often deadly cancer.

MR1-restricted T cells: the new dawn of cancer immunotherapy.

Cancer immunotherapy has recently undergone rapid development into a validated therapy for clinical use. The adoptive transfer of engineered autologous T cells, such as chimeric antigen receptor (CAR) T cells, has been remarkably successful in patients with leukemia and lymphoma with cluster of differentiation (CD)19 expression. Because of the higher number of antigen choices and reduced incidence of cytokine release syndrome (CRS) than CAR-T cells, T cell receptor (TCR)-T cells are also considered a promising immunotherapy. More therapeutic targets for other cancers need to be explored due to the human leukocyte antigen (HLA)-restricted recognition of TCR-T. Major histocompatibility complex (MHC), class I-related (MR1)-restricted T cells can recognize metabolites presented by MR1 in the context of host cells infected with pathogens. MR1 is expressed by all types of human cells. Recent studies have shown that one clone of a MR1-restricted T (MR1-T) cell can recognize many types of cancer cells without HLA-restriction. These studies provide additional information on MR1-T cells for cancer immunotherapy. This review describes the complexity of MR1-T cell TCR in diseases and the future of cancer immunotherapy.

Targeting Oncoproteins with a Positive Selection Assay for Protein Degraders

Cancer genome sequencing studies have identified frequent driver mutations that alter the function of transcription factors (TFs). However, most TFs, are classically "undruggable". The discovery that IMiDs [immunomodulatory drugs e.g.Lenalidomide (LEN) and Pomalidomide (POM)] exert powerful antimyeloma effects by triggering the proteasomal degradation of the critical TFsIKZF1 and IKZF3has led togreat interest in the discovery of"degraders"in other cancers. Existing "down" assays to identify destabilizers (e.g.measuring the loss of the protein of interest [POI]) often have(1) poor signal/noise ratios and narrow dynamic ranges and (2) generate uninterestinghits e.g.drugs thatinhibit transcription or translation. We present a novel positive-selection "up" assay, compatible with high throughput screening, for identifying drugs or sgRNAs that destabilize a POI. We have used this platform to identify novel destabilizers of IKZF1. Our assay is based on a previously published "suicide gene", a variant of the nucleotide salvage gene deoxycytidine kinase (DCK). This variant (DCK*) has mutations that increase its specificity for the synthetic substrate 2-Bromovinyldeoxyuridine (BVdU). Cells that express DCK* are killed by BVdU. We made mammalian expression vectors co-expressing GFP and DCK*, IKZF1, or a DCK*-IKZF1 fusion protein as a single bicistronic mRNA and stably introduced each into 293T cells. In the absence of POM, cells expressing DCK* or DCK*-IKZF1 are killed by BVdU . However, in the presence of an IKZF1 destabilizer, e.g. POM, the DCK*-IKZF1 protein is degraded, making the cells resistant to BVdU. Cells expressing DCK* alone are unaffected by POM and serve as a specificity control for the assay (Panel A). We did a pilot chemical "up" screen with a ~ 2000 bioactive compound library (that included LEN and POM) using 293T cells expressing DCK*-IKZF1. In parallel, we conducted a "down" screen, using 293T cells co-expressing an IKZF1-Firefly Luciferase (Fluc) fusion protein and Renilla luciferase (Rluc) from a single bicistronic mRNA. Compounds that decreased the ratio of Fluc/Rluc activity were scored as hits. As expected, LEN and POM scored in both assays,but there was considerably more noise in the down assay. Lastly, we used the DCK*-IKZF1 cells to screen a library of uncharacterized IMiD derivatives. The screen correctly identified a novel IMiD derivative (MI-2-61) and a known next-generation IMiD (Avadomide) with greater potency against IKZF1 than POM. To identify novel degraders of IKZF1, we used the DCK*-IKZF1 cells and DCK* control cells to screena metabolic inhibitor/anticancer library of ~600 compounds. We identified Spautin-1 as a compound that rescues DCK*-IKZF1 cells, but not DCK* control cells from BVdU toxicity. Spautin-1 downregulates exogenous IKZF1 in 293T cells (Panels B and C) and endogenous IKZF1 in KMS11 myeloma cells. Spautin-1 reportedly suppresses autophagy through inhibition of USP10 and USP13. However, siRNA mediated knockdown of USP10 and USP10 neither altered IKZF1 protein levels, nor blocked downregulation of IKZF1 by Spautin-1. Moreover, Spautin-1 downregulated IKZF1 in 293FT cells in which autophagy was disabled by CRISPR/Cas9-mediated disruption of genes crucial to the autophagy pathway suggesting that the downregulation of IKZF1 by Spautin-1 occurs via a novel mechanism. Unlike IMiDs, the downregulation of IKZF1 by Spautin-1 does not require the CRBN E3 ligase. However, it is blocked by inhibitors of the E1 ubiquitin activating enzyme or the proteasome, but not by neddylation inhibitors required for Cullin-dependent E3 ligases. These data suggest that Spautin-1 triggers the proteasomal degradation of IKZF1 using a non-Cullen E3 ligase. The downregulation of exogenous IKZF1 by Spautin-1 requires the N-terminus of IKZF1, but not the zinc finger domain (ZF2) targeted by IMiDs. Preliminary structure-activity relationship (SAR) studies identified both active and inactive Spautin-1 derivatives, suggesting that downregulation of IKZF1 by Spautin-1 reflects a specific protein binding event and that Spautin-1's potency and specificity can be optimized further. We are undertaking studies to identify the mechanism by which Spautin-1 degrades IKZF1 in the hope that it may represent a novel druggable pathway to therapeutically degrade IKZF1, and validate the use of this platform to discover degraders of "undruggable" targets in other cancers. Figure Disclosures Koduri: Cedilla Therapeutics: Consultancy. Paulk:Novartis: Current Employment. Harris:ONO Pharmaceutical: Consultancy. Buhrlage:Adenoid Cystic Carcinoma Foundation: Other: Scientific Advisory Board. Kaelin:Cedilla Therapeutics: Other: Scientific Founder; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; Fibrogen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Tango Therapeutics: Other: Founder; Tracon Therapeutics: Membership on an entity's Board of Directors or advisory committees.

HER2 expression patterns in paired primary and metastatic endometrial cancer lesions

Background:Despite successful implementation of drugs targeting the human epidermal growth factor receptor 2 (HER2) receptor in breast and gastric cancers, the potential of HER2 as a therapeutic target in other cancers has been less studied, including endometrial cancer. We investigated expression levels of HER2 (ERBB2) in a large cohort of endometrial cancer lesions, also including complex atypical hyperplasia and metastatic lesions.Methods:67 precursor lesions, 790 primary endometrial cancers and 383 metastatic lesions were investigated for HER2 expression in relation to clinicopathologic features and outcome. Protein levels were assessed by immunohistochemistry (using the HercepTest and staining index (SI) criteria), mRNA levels by microarrays and amplification status by chromogenic in situ hybridisation.Results:High HER2 protein levels were significantly associated with features of aggressive disease and increased mRNA ERBB2 levels. HER2 expression defined by the SI proved to be a better predictor of survival compared with the HercepTest. A discordant HER2 expression pattern between paired primary and metastatic lesions was detected, revealing substantial reduction in HER2 expression from primary to metastatic disease.Conclusions:Loss of HER2 expression is common in metastatic endometrial cancer lesions and assessment of HER2 levels in the metastatic lesions may be important to define the potential benefit of anti-HER2 treatments in endometrial cancer patients.

Mutation of the DNA damage gene signature to predict for better outcome in malignant melanoma.

e21036 Background: DNA damage response is a fundamental cellular pathway with an important role in maintaining genomic integrity. As Malignant Melanoma (MM) is associated with high mutation rates, and we postulated that mm is likely to have abnormal DNA repair pathway aberrations that could have prognostic and/or therapeutic significance. Methods: We conducted an unbiased assessment of genes involved in the DNA damage response through a comprehensive survey of established public data repositories and published literature and abstracts in all cancers. This survey was used to generate a 6-gene DNA Damage Signature (DDS). The DDS was then applied to 287 mm samples in The Cancer Genome Atlas. Results: The DDS was altered in 25% (72/287) of patients with mutations being present in all 6 genes including ATM (6%), TP53BP1 (7%), MDC1 (7%), BRCA1 (5%), PRKDC (8%) and RAD50 (1.4%). The DDS was more commonly altered in mm harboring alterations in BRAF (78%, 112/143), NRAS (89%, 73/82) or NF1 (75%, 5/20) than in triple wild type tumors (60%,19/32). The correlation between alteration in BRAF/NRAS/NF1 and DDS (p = 0.02) reached significance, as did the tendency for DDS and NF1 altered subtype to co-occur (p &lt; 0.05). Importantly, alteration in the DDS was associated with significantly longer disease free (88 vs. 44 months, p = 0.02) and overall survival (152 vs. 66 months, P = 0.02). The mutations in ATM and BRCA1, shown to be targets in other cancers for PARP inhibition were seen in these patients as well as other oncogenic or hot-spot mutations. Conclusions: The improved survival associated with alteration of the DDS implicate DNA damage response signaling as favorably modulating the immune system, and propose a possible role for PARP inhibition as a therapeutic strategy in melanoma.

Genetic aspects of pituitary carcinoma: A systematic review.

Background:Pituitary carcinoma (PC) is a rare type of malignant intracranial neoplasm defined as distant metastasis of pituitary adenoma (PA). Although PC incidence is low because only 0.1% to 0.2% of PAs ultimately develop into PCs, the prognosis is poor and 66% of patients die within the first year. Existing therapeutic measures, including surgical removal, chemotherapy, and radiotherapy, have limited effectiveness. The lack of efficacy of current treatments is largely caused by the limited understanding of the molecular pathogenesis of PA and the malignant transformation to PC. Therefore, the aim of this systematic review was to summarize published research regarding gene and protein expression in PC to clarify the molecular mechanisms underlying PC genesis and development and identify new candidate diagnostic biomarkers and therapeutic targets for potential use in personalized treatment of PC.Methods:We followed the PRISMA guidelines to plan and conduct this systematic review. PubMed, Embase, and Web of Science databases were searched for relevant studies conducted before December 16, 2015 describing the association of PC with gene expression at the mRNA and protein levels. MeSH terms combined with free terms were used to retrieve the references.Results:In total, 207 records were obtained by primary search, and 32 were included in the systematic review. Compared with normal pituitary gland and/or PA, 30 and 18 genes were found to have higher or lower expression, respectively, in PCs using different analytical methods. Among them, we selected 9 upregulated and 7 downregulated genes for further analysis based on their identification as candidate treatment targets in other cancers, potential clinical application, or further research value.Conclusion:Previous studies demonstrated that many genes promote PC malignant transformation, angiogenesis, invasion, metastasis, and recurrence. Although most of these genes and proteins have not been fully analyzed with regard to their downstream mechanisms or potential diagnostic and therapeutic application, they have the potential to become candidate PC biomarkers and/or molecular targets for guiding personalized treatment. Modern advanced technologies should be utilized in future research to identify more candidate genes for PC pathogenesis, as precisely targeted gene therapies against PC are urgently required.

Prognostic and Predictive Values of Subcellular Localisation of RET in Renal Clear-Cell Carcinoma.

Metastatic renal cell carcinoma (RCC) presents a poor prognosis and an unpredictable course. To date, no validated biomarkers can predict the outcome of RCC. Ongoing efforts are conducted to identify the molecular markers of RCC progression, as well as the targets for novel therapeutic approaches. RET is a tyrosine kinase receptor which has been investigated as a possible target in other cancers because it is involved in oncogenic activation. To evaluate the predictive and prognostic functions of RET in ccRCC, a tissue microarray study was conducted on 273 ccRCC patients. Results showed that both RET cytoplasmic and nuclear expression were independently associated with PFS and OS, and the combined RET cytoplasmic and nuclear statuses demonstrated that the ratio of high nuclear RET and cytoplasmic RET was the strongest predictor of both PFS and OS. The high cytoplasmic RET expression retained its independent poor prognostic value in targeted drug treated patients. The RET nuclear expression was associated with distant metastasis. Moreover, the RET nuclear expression was an independent predictor of ccRCC postoperative metastasis. In conclusion, RET may be useful in prognostication and can be used at initial diagnosis to identify patients with high potential to develop metastasis.

Abstract CT211: IMMU-130, an SN-38 antibody-drug conjugate (ADC) targeting CEACAM5, is therapeutically active in metastatic colorectal cancer (mCRC): Initial clinical results of two Phase I studies

Abstract IMMU-130, an ADC of the active metabolite of CPT-11, SN-38, conjugated by a pH-sensitive linker (7.6 average drug-antibody ratio) to the humanized anti-CEACAM5 antibody (labetuzumab), is completing two Phase I trials. In both, eligible patients with advanced mCRC were required to have failed/relapsed standard treatments, one being the topoisomerase-I inhibiting drug, CPT-11 (irinotecan), and an elevated plasma CEA (&amp;gt;5 ng/mL). IMMU-130 was administered every 14 days (EOW) at doses starting from 2.0 mg/kg in the first protocol (IMMU-130-01). Febrile neutropenia occurred in 2 of 3 patients at 24 mg/kg; otherwise at ≤16 mg/kg, neutropenia (≥ Grade 2) was observed in 7 patients, with one also experiencing thrombocytopenia. One patient [of 8 who received &amp;gt; 4 doses (2 cycles)] showed a 40.6% decrease in liver (starting at 7 cm) and lung target lesions (PR by RECIST) for 4.7 months, with no major toxicity, tolerating a total of 18 doses at 16 mg/kg. The study is continuing at 12 mg/kg EOW. Since SN-38 is most effective in S-phase cells, a more protracted exposure could improve efficacy. Thus, in a second Phase I trial (IMMU-130-02), dosing was intensified to twice-weekly, starting at 6 mg/kg/dose for 2 weeks (4 doses) with 1 week off, as a treatment cycle, in a 3+3 trial design. Neutropenia and manageable diarrhea were the major side effects, until dose reduction to 4.0 mg/kg twice-weekly, with early results indicating multiple cycles are well-tolerated. Currently, tumor shrinkage occurred in 3 patients, with 1 in continuing PR (-46%) by RECIST, among 6 patients who completed &amp;gt;4 doses (1 cycle). In both trials, CEA blood titers correlated with tumor response, and high levels did not interfere with therapy. There have been no anti-antibody or anti-SN-38 antibody reactions, based on ELISA tests. In each study, the ADC was cleared by 50% within the first 24 h, which is much longer exposure than with typical doses of the parental molecule, CPT-11. These results indicate that this novel ADC, given in different regimens averaging ∼16-24 mg/kg/cycle, shows therapeutic activity in advanced mCRC patients. Since CEACAM5 has elevated expression in breast and lung cancers, as well as other epithelial tumors, it may be a useful target in other cancers as well. A Phase II trial evaluating twice-weekly dosing and a new regimen, once-weekly x 2 every 21 days, is ongoing in mCRC. Citation Format: Neil H. Segal, Efrat Dotan, Jordan D. Berlin, Alexander N. Starodub, Michael J. Guarino, Leonard B. Saltz, Pius P. Maliakal, Serengulam V. Govindan, William A. Wegener, Robert M. Sharkey, David M. Goldenberg. IMMU-130, an SN-38 antibody-drug conjugate (ADC) targeting CEACAM5, is therapeutically active in metastatic colorectal cancer (mCRC): Initial clinical results of two Phase I studies. [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 CT211. doi:10.1158/1538-7445.AM2014-CT211

Abstract 1277: Utilizing uterine cancer models to study malignant transformation driven by Lkb1 loss.

Abstract The serine-threonine kinase LKB1 is an upstream component of the AMPK-mTOR pathway and regulates cell survival and growth. Inherited germline mutations in LKB1 predispose women to endocervical malignancies whereas somatic mutations in LKB1 promote cervical cancer progression. Our lab has shown that tissue specific knockout of Lkb1 in the endometrial epithelium of mice facilitates growth of highly invasive, lethal tumors that are well differentiated and retain structural polarity. Additionally, stable shRNA mediated knockdown of LKB1 in human immortalized endocervical cells drives anchorage-independent growth. To investigate potential pathways involved in LKB1 mediated oncogenic transformation, we have employed a microarray based approach comparing hits from human immortalized endocervical cell lines and mouse tissue to identify specific genes that are differentially regulated when LKB1 function is absent. Preliminary data suggests that pro-angiogenic and inflammatory mediators are upregulated in LKB1 deficient cells. We are currently assessing the overall contribution of these factors to growth and invasiveness in our model systems and the mechanisms by which Lkb1 loss facilitates their activation. These pathways can be potential therapeutic targets in other cancers characterized by Lkb1 loss of function mutations. Citation Format: Christopher G. Peña, Yuji Nakada, Diego Castrillon. Utilizing uterine cancer models to study malignant transformation driven by Lkb1 loss. [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 1277. doi:10.1158/1538-7445.AM2013-1277

Transcriptome Sequencing of Tumor Subpopulations Reveals a Spectrum of Therapeutic Options for Squamous Cell Lung Cancer

BackgroundThe only therapeutic options that exist for squamous cell lung carcinoma (SCC) are standard radiation and cytotoxic chemotherapy. Cancer stem cells (CSCs) are hypothesized to account for therapeutic resistance, suggesting that CSCs must be specifically targeted. Here, we analyze the transcriptome of CSC and non-CSC subpopulations by RNA-seq to identify new potential therapeutic strategies for SCC.MethodsWe sorted a SCC into CD133− and CD133+ subpopulations and then examined both by copy number analysis (CNA) and whole genome and transcriptome sequencing. We analyzed The Cancer Genome Atlas (TCGA) transcriptome data of 221 SCCs to determine the generality of our observations.ResultsBoth subpopulations highly expressed numerous mRNA isoforms whose protein products are active drug targets for other cancers; 31 (25%) correspond to 18 genes under active investigation as mAb targets and an additional 4 (3%) are of therapeutic interest. Moreover, we found evidence that both subpopulations were proliferatively driven by very high levels of c-Myc and the TRAIL long isoform (TRAILL) and that normal apoptotic responses to high expression of these genes was prevented through high levels of Mcl-1L and Bcl-xL and c-FlipL—isoforms for which drugs are now in clinical development. SCC RNA-seq data (n = 221) from TCGA supported our findings. Our analysis is inconsistent with the CSC concept that most cells in a cancer have lost their proliferative potential. Furthermore, our study suggests how to target both the CSC and non-CSC subpopulations with one treatment strategy.ConclusionsOur study is relevant to SCC in particular for it presents numerous potential options to standard therapy that target the entire tumor. In so doing, it demonstrates how transcriptome sequencing provides insights into the molecular underpinnings of cancer propagating cells that, importantly, can be leveraged to identify new potential therapeutic options for cancers beyond what is possible with DNA sequencing.

MiR-152 and miR-185 co-contribute to ovarian cancer cells cisplatin sensitivity by targeting DNMT1 directly: a novel epigenetic therapy independent of decitabine

Epithelial ovarian cancer (EOC) is a highly lethal gynaecological malignancy. Cisplatin is the basal chemotherapeutic agent used to treat EOC, but resistance to cisplatin leads to chemotherapy failure. MicroRNAs are a novel class of regulators that function by controlling gene expression at the post-transcriptional level. Several recent reports have identified some microRNAs that are related to chemotherapy sensitivity. In this study, we found two microRNAs miR-152 and miR-185 that were significantly downregulated in the cisplatin-resistant ovarian cell lines SKOV3/DDP and A2780/DDP, compared with their sensitive parent line SKOV3 and A2780, respectively. Subsequently, the roles of miR-152 and miR-185 were evaluated in vitro and in vivo. The overexpression of miR-152 or miR-185 increased cisplatin sensitivity of SKOV3/DDP and A2780/DDP cells by inhibiting proliferation and promoting apoptosis, then we further confirmed that these miRNAs functioned through suppressing DNA methyltransferase 1 (DNMT1) directly. Concordantly, CD-1/CD-1 nude mice that were injected intraperitoneally with SKOV3/DDP cells transfected with miR-152 mimics exhibited upregulated cisplatin sensitivity in vivo. Interestingly, we found that there were no significant changes in the expression of these two microRNAs after treatment with decitabine (DAC), a traditional epigenetic therapeutic agent, suggesting these miRNAs represented two new regulators independent of DAC. Finally, the survival assay in A549 and HepG2 cells revealed that the two microRNAs involved in cisplatin sensitivity were related to cell types. Our results indicated that miR-152 and miR-185 were involved in ovarian cancer cisplatin resistance in vitro and in vivo by targeting DNMT1 directly. These molecules may serve as potential epigenetic therapeutic targets in other cancers.

Warburg Effect’s Manifestation in Aggressive Pheochromocytomas and Paragangliomas: Insights from a Mouse Cell Model Applied to Human Tumor Tissue

A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.

Abstract LB-6: Exploring nicotinamide-N-methyltransferase kinetics through targeted metabolomic profiling for its prognostic value in glioblastoma

Abstract Purpose: Glioblastoma (GBM) is the most aggressive form of glioma. With average patient survival of about 14 months, largely due to its resistance to conventional therapies, there is opportunity for significant improvements to care. Nicotinamide-N-methyltransferase (NNMT) is an enzyme responsible for the methylation of Nicotinamide and other xenobiotic compounds. Although NNMT's exact role in the malignant phenotype remains poorly understood, it has been investigated for its potential use as a prognostic marker or therapeutic target in other cancers. It is detected at very low levels in normal brain tissue but at significantly higher levels in GBM. Additionally, Kaplan-Meier plots from publically available data sets suggest that higher NNMT expression is correlated with adverse patient outcome. In addition to gene interference and various functional assays, we employed a targeted metabolomic approach, using LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to study intracellular levels of Nicotinamide and N-methylnicotinamide to gain a better understanding of NNMT's role in GBM. Methods: We determined the expression profile of NNMT in established (ATCC) and in our panel of patient derived, primary GBM cell lines. By generating an NNMT isogenic model in U87 cells, we were able to study the functional consequences of differential NNMT expression using in vitro assays such as MTS Assay, Clonogenic Survival Assay, Annexin V, ATP Assay, Comet Assay, and Western Blotting. We intracranially injected NOD-SCID mice with U87 NNMT cells to assess differences in tumorgenicity in vivo. We have also used LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to measure intracellular levels of the metabolites relevant to NNMT enzymatic function. Results and Conclusions: Results indicate that NNMT protein is highly expressed in primary cell lines. Encouragingly, U87 NNMT Knockdown cells are less proliferative, more sensitive to radiation in vitro, and are less tumorgenic in vivo. Recapitulating the clinical observations, these results could justify why clinically patients with lower expression of NNMT enjoyed increased overall survival. These results also suggest that finding ways to decrease NNMT in patient's tumors may improve response to radiation. Additional preliminary results indicate that intracellular N-methylnicotinamide levels correlate directly with NNMT protein expression and could be used a surrogate biomarker for intracellular NNMT levels, potentially predicting not only overall survival but perhaps response to radiation. 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-6. doi:1538-7445.AM2012-LB-6

Silencing of the COPS3 Gene by siRNA Reduces Proliferation of Lung Cancer Cells Most Likely via induction of Cell Cycle Arrest and Apoptosis

The COPS3 gene has stimulating effect on cell proliferation and progression of osteosarcomas and related cells. However, the features of COPS3 and its potential application as a therapeutic target in other cancers has not yet been studied. In this study, therefore, the effect of COPS3 silencing via COPS3 siRNA on lung cancer cell proliferation was examined. Expression levels of COPS3 gene in COPS3 siRNA infected cells and control siRNA infected cells were compared with real time PCR and Western blot analysis. Cell proliferation levels were comprehensively analyzed by MTT, BrdU incorporationy, and colony formation assays. For mechanistic assessment the effects of COPS3 silencing on cell cycle and apoptosis were analyzed using flow cytometry. Results showed that successful silencing of the COPS3 gene at both translational and transcriptional levels significantly reduced the proliferation and colony formation by lung cancer cells (p<0.01). Flow cytometry showed cell cycle arrest in the G0/G1 phase after COPS3 silencing, and more importantly, apoptosis was induced as a result of COPS3 knockdown, which negatively affected cell survival. Therefore, these results provide another piece of important evidence that the COPS3 gene expressed in lung cancer cells may play a critical role in stimulating proliferation. Down-regulation of COPS3 could significantly inhibit lung cancer cell growth, which was most likely mediated via induction of cell cycle arrest in G0/G1 phase and apoptosis.

Abstract 122: Downregulated miR-375 in tongue cancer and its putative role as a tumor suppressor microRNA

Abstract MicroRNAs are approximately 22-nt RNAs involved in posttranscriptional regulation of target gene expression. Oral cancers are ranked as the sixth most prevalent cancer worldwide, with squamous cell carcinoma accounting for the most common type (83% – 90%). Evolution and progression of oral cancers is thought to result from the accumulation of genetic alterations in molecular pathways. In order to identify aberrantly expressed miRNAs in oral squamous cell carcinomas (OSCCs), miRNA microarray was performed using 3 healthy controls and 17 OSCC tissue samples all derived from human tongue. Clustering expression patterns of miRNAs in the 17 OSCC samples using Pearson correlation coefficient demonstrated TNM stage-dependent miRNA expression pattern in OSCC. In the expression profile, 134 miRNAs (105 upregulated miRNAs and 29 downregulated miRNAs) were identified to be aberrantly expressed in OSCCs (p &amp;lt; 0.05). The expression of miRNAs was validated by quantitative RT-PCR. MiR-375, the most underexpressed miRNA, was 7.2-fold (p=0.004) downregulated in OSCC samples compared to 5 healthy tongue samples. When compared to the control tongue samples, 88% (15/17) of OSCC samples showed lower miR-375 levels. Furthermore, the expression of miR-375 was inversely proportional to the TNM stage score (stage 2: 32.7-fold, p=0.008; stage 4: 38.4-fold, p=0.002). MTS assay showed that transfected miR-375 mimic inhibited cell proliferation of human oral cancer cell line CAL 27 in dose dependent manner. YWHAZ (14-3-3ζ), PDK1, JAK2, and ELAVL4 (HuD) were reported as miR-375 targets in other cancers. These 4 putative miR-375 targets were examined in CAL 27 cells. JAK2 and ELAVL4 were barely detectable in CAL 27 and thus not significantly regulated by miR-375. In contrast, YWHAZ and PDK1 were repressed both at the mRNA (54% and 19%, respectively) and protein levels (∼30% for both) after transfection of miR-375 mimic into CAL 27 cells. Since both YWHAZ and PDK1 genes are known to play important roles in proliferation and possibly in apoptosis, miR-375 may be the gatekeeper for the balance of the two cellular processes. The absence of miR-375 expression and its inhibitory role in oral cancer cells implicates miR-375 as a tumor suppressor miRNA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 122. doi:10.1158/1538-7445.AM2011-122