Therapeutic Target In Cancer Cells Research Articles

Abstract LT04: Dissecting Mediator Kinase Activity in Normal Tissue Homeostasis and Cancer

Abstract Cyclin-dependent kinase 8 (CDK8) and its paralog CDK19, collectively known as Mediator kinase, are part of the multimeric Mediator complex that fine-tune gene expression programs on multiple levels, most notably by controlling transcription factor (TF) activity at promoter- and enhancer regions as well as organising the chromatin landscape. While Mediator kinases have been predominantly studied as a therapeutic target in cancer cells, their functions in a physiologically relevant context remain poorly understood. To study in vivo functions of Mediator, we developed a comprehensive suite of genetically engineered mouse models (GEMMs), including conditional floxed alleles (Cdk8fl/fl/Cdk19fl/fl) and kinase-dead knock-ins (Cdk8D173A/fl/Cdk19D173A/fl), enabling the analysis of scaffolding and kinase-dependent functions of this critical transcriptional module. Herein, we show that Mediator kinase has distinct kinase-dependent functions in modulating homeostasis and tissue lineage specification in diverse organs. Moreover, using a proteo-genomic approach we show that the Mediator Kinase module functionally interacts with the SWI/SNF complex to mediate transcriptional output. Collectively, our work expands the understanding of how Mediator Kinase regulates tissue homeostasis and provides a framework for considering how Mediator Kinase inhibition may be effectively employed in the clinical setting. Citation Format: Marius Dannappel, Danxi Zhu, Claire Sun, Ron Firestein. Dissecting Mediator Kinase Activity in Normal Tissue Homeostasis and Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT04.

Abstract 5784: Structure-based drug design against caPCNA for selective cancer chemotherapy development

Abstract Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes, and it is also a proliferation biomarker in a variety of human tumors. A unique cancer-associated isoform of the protein, caPCNA, has been previously identified, allowing for rational-based drug discovery studies to develop leads with the potential for selective therapeutic targeting of cancer cells. Several strategies have been employed to develop agents targeting caPCNA, including peptide and small molecule-based inhibitors, but the success in developing therapeutically tractable compounds has been limited so far. Here, we present the crystal structures and corresponding biochemical characterizations of our novel small molecule-based caPCNA inhibitors, which bind into the PIP-box binding pocket of PCNA. One of our lead compounds is now an investigational new drug that we observed selectively kills cancer cells, and it appears to induce replication stress, apoptosis and increase cancer cell sensitivity to genotoxic agents, while these effects are not observed in non-malignant cell controls. This caPCNA inhibitor is orally administrable, metabolic stable and it suppresses tumor growth as a monotherapy or as a combination treatment, and it has entered clinical trials in the United States. PCNA plays a critical role in temporarily dislodging RNA polymerase II from transcription replication conflict sites, to enable the replication fork to proceed. Thus, structure-based drug discovery approaches to target transcription replication conflict resolution machinery, such as PCNA protein, may open a novel therapeutic avenue for exploiting this cancer-selective vulnerability. Citation Format: Jennifer Jossart, Pouya Haratipour, Long Gu, Carone M. li, Robert Lingeman, Yilun Liu, Robert J. Hickey, Linda H. Malkas, Jeff J. Perry. Structure-based drug design against caPCNA for selective cancer chemotherapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5784.

Abstract 6583: Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein

Abstract Proliferating cell nuclear antigen (PCNA) is a proliferation biomarker in a variety of human tumors and has critical cellular functions in DNA replication and repair. We previously identified a unique cancer-associated isoform of the protein, termed caPCNA, which allows for structure-based drug discovery studies to develop inhibitors for selective therapeutic targeting of cancer cells. Distinct strategies have been used to develop caPCNA targeting agents, which includes peptide and small molecule-based compounds, although the success in developing therapeutically tractable compounds has been limited so far. Here, we present the crystal structures and corresponding biochemical characterizations of our novel small molecule-based caPCNA inhibitors, which bind into the PIP-box binding pocket of PCNA. Our lead compound selectively kills cancer cells, and it appears to induce replication stress, apoptosis and increase cancer cell sensitivity to genotoxic agents, while these effects are not observed in non-malignant cell controls. This caPCNA inhibitor is orally administrable, metabolic stable and it suppresses tumor growth as a monotherapy or as a combination treatment and is now an investigational new drug in Phase 1 clinical trials. PCNA plays a critical role enabling the replication fork to proceed from transcription replication conflict sites, by temporarily dislodging RNA polymerase II. Thus, structure-based drug discovery against caPCNA may open a novel therapeutic avenue for the development of selective chemotherapeutics, through targeting a cancer cell’s vulnerability to unresolved transcription replication conflicts. Citation Format: Jennifer Jossart, Long Gu, Pouya Haratipour, Caroline Li, Robert Lingeman, Robert Hickey, Linda Malkas, John J. Perry. Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6583.

RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.

RING finger 43 (RNF43), a RING-type E3 ubiquitin ligase, is a key regulator of WNT signaling and is mutated in 6-10% of pancreatic tumors. However, RNF43-mediated effects remain unclear, as only a few in vivo substrates of RNF43 are identified. Here, it is found that RNF43-mutated pancreatic cancer cells exhibit elevated B-RAF/MEK activity and are highly sensitive to MEK inhibitors. The depletion of RNF43 in normal pancreatic ductal cells also enhances MEK activation, suggesting that it is a physiologically regulated process. It is confirmed that RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent degradation, resulting in reduced MEK activity and proliferative ability in cancer cells. In addition, phosphorylation of B-RAF at T491 suppresses B-RAF ubiquitination by decreasing the interaction between RNF43 and B-RAF. Mutations at K499 in B-RAF are identified in various cancer types. MEK and WNT inhibitors synergistically suppress the growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo. Collectively, the research reveals a novel mechanism by which RNF43 inhibits B-RAF/MEK signaling to suppress tumor growth and provide a new strategy for the treatment of RNF43-inactivated pancreatic cancer.

ATR Inhibition in Advanced Urothelial Carcinoma.

The ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase 1 (CHK1) pathway is intricately involved in protecting the integrity of the human genome by suppressing replication stress and repairing DNA damage. ATR is a promising therapeutic target in cancer cells because its inhibition could lead to an accumulation of damaged DNA preventing further replication and division. ATR inhibition is being studied in multiple types of cancer, including advanced urothelial carcinoma where there remains an unmet need for novel therapies to improve outcomes. Herein, we review preclinical and clinical data evaluating 4 ATR inhibitors as monotherapy or in combination with chemotherapy. The scope of this review is focused on contemporary studies evaluating the application of this novel therapy in advanced urothelial carcinoma.

ABCB1 and ABCC1 Function during TGF-β-Induced Epithelial-Mesenchymal Transition: Relationship between Multidrug Resistance and Tumor Progression.

Multidrug resistance (MDR) and induction of metastasis are some of the puzzles encountered during cancer chemotherapy. The MDR phenotype is associated with overexpression of ABC transporters, involved in drug efflux. Metastasis originates from the epithelial-mesenchymal transition (EMT), in which cells acquire a migratory phenotype, invading new tissues. ABC transporters' role during EMT is still elusive, though cells undergoing EMT exhibit enhanced ABCB1 expression. We demonstrated increased ABCB1 expression but no change in activity after TGF-β-induced EMT in A549 cells. Moreover, ABCB1 inhibition by verapamil increased snail and fibronectin expression, an event associated with upregulation of ABCB1, evidencing coincident cell signaling pathways leading to ABCB1 and EMT-related markers transcription, rather than a direct effect of transport. Additionally, for the first time, increased ABCC1 expression and activity was observed after EMT, and use of ABCC1 inhibitors partially inhibited EMT-marker snail, although increased ABCC1 function translated into collateral sensibility to daunorubicin. More investigations must be done to evaluate the real benefits that the gain of ABC transporters might have on the process of metastasis. Considering ABCC1 is involved in the stress response, affecting intracellular GSH content and drug detoxification, this transporter could be used as a therapeutic target in cancer cells undergoing EMT.

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Simple SummaryA remodeling in calcium homeostasis and the protein kinase AKT signaling pathway often promotes tumorigenic traits in cancer cells. Changes in calcium signaling can be mediated through altered expression or activity of calcium channels and pumps, which constitute a class of targetable therapeutic targets. Currently, the interplay between the two signaling pathways in breast cancer cells is unclear. A better understanding of the association between calcium and AKT signaling, and the molecular players involved may identify novel therapeutic strategies for breast cancers with abnormal AKT signaling. Using fluorescence calcium imaging and gene silencing/knockout techniques, we showed that increased AKT activation results in increased calcium entry, and that this is mediated through ORAI1 calcium channels. Future studies exploring therapeutic strategies to target PTEN-deficient or hyperactivated AKT cancers should consider this novel correlation between AKT activation and ORAI1-mediated calcium influx.Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

Apoptosis in Human Papillomavirus-Induced Cervical Cancer Cells By Higher Doses of Ascorbic Acid

Objective: To assess the anti-cancer role of ascorbic acid using high doses against the cervical cancer cell line and to establish a cell culture facility in the laboratory.
 Study Design: In Vitro Study.
 Place and Duration of Study: Department of Biochemistry and Molecular Biology, Army Medical College, Rawalpindi Pakistan, from Aug 2014 to Jul 2015.
 Methodology: HeLa cells were grown in Dulbecco’s modified eagle medium, and upon reaching confluency, cells were challenged with different doses of ascorbic acid. The MTT assay was employed to study the cytotoxic effects of increased doses of ascorbic acid on HeLa cells.
 Results: An increasing dose of ascorbic acid was found to be cytotoxic toward HeLa cells, and its EC50 was found to be between 5-6µM. A high dose of ascorbic acid is selectively cytotoxic to HeLa cell lines of cervical cancer, which may imply the treatment of cervical cancer patients. NF- kB inducing kinase is a salient protein in TNFα-induced NF-kB activation. Ascorbate has been reported to inhibit that activation which has anti-apoptotic roles in tumour progression. CB-Dock was employed to propose the binding sites for NIK-Ascorbate interaction, which might be a therapeutic target for cancer cells.
 Conclusion: The study concludes that high dose ascorbic acid is cytotoxic to cervical cancer cells HeLa and may have important implications in treating cervical cancer patients. Therapeutic intervention of this vitamin may contribute to suppressing cancer development.

NRF2 and Mitochondrial Function in Cancer and Cancer Stem Cells.

The NRF2–KEAP1 system is a fundamental component of the cellular response that controls a great variety of transcriptional targets that are mainly involved in the regulation of redox homeostasis and multiple cytoprotective mechanisms that confer adaptation to the stress conditions. The pleiotropic response orchestrated by NRF2 is particularly relevant in the context of oncogenic activation, wherein this transcription factor acts as a key driver of tumor progression and cancer cells’ resistance to treatment. For this reason, NRF2 has emerged as a promising therapeutic target in cancer cells, stimulating extensive research aimed at the identification of natural, as well as chemical, NRF2 inhibitors. Excitingly, the influence of NRF2 on cancer cells’ biology extends far beyond its mere antioxidant function and rather encompasses a functional crosstalk with the mitochondrial network that can influence crucial aspects of mitochondrial homeostasis, including biogenesis, oxidative phosphorylation, metabolic reprogramming, and mitophagy. In the present review, we summarize the current knowledge of the reciprocal interrelation between NRF2 and mitochondria, with a focus on malignant tumors and cancer stem cells.

The tetrameric structure of Plasmodium falciparum phosphoglycerate mutase is critical for optimal enzymatic activity

The glycolytic enzyme phosphoglycerate mutase (PGM) is of utmost importance for overall cellular metabolism and has emerged as a novel therapeutic target in cancer cells. This enzyme is also conserved in the rapidly proliferating malarial parasite Plasmodium falciparum, which have a similar metabolic framework as cancer cells and rely on glycolysis as the sole energy-yielding process during intraerythrocytic development. There is no redundancy among the annotated PGM enzymes in Plasmodium, and PfPGM1 is absolutely required for the parasite survival as evidenced by conditional knockdown in our study. A detailed comparison of PfPGM1 with its counterparts followed by in-depth structure-function analysis revealed unique attributes of this parasitic protein. Here, we report for the first time the importance of oligomerization for the optimal functioning of the enzyme in vivo, as earlier studies in eukaryotes only focused on the effects in vitro. We show that single point mutation of the amino acid residue W68 led to complete loss of tetramerization and diminished catalytic activity in vitro. Additionally, ectopic expression of the WT PfPGM1 protein enhanced parasite growth, whereas the monomeric form of PfPGM1 failed to provide growth advantage. Furthermore, mutation of the evolutionarily conserved residue K100 led to a drastic reduction in enzymatic activity. The indispensable nature of this parasite enzyme highlights the potential of PfPGM1 as a therapeutic target against malaria, and targeting the interfacial residues critical for oligomerization can serve as a focal point for promising drug development strategies that may not be restricted to malaria only.

Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer

BackgroundThe activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However,...

Various Aspects of Calcium Signaling in the Regulation of Apoptosis, Autophagy, Cell Proliferation, and Cancer.

Calcium (Ca2+) is a major second messenger in cells and is essential for the fate and survival of all higher organisms. Different Ca2+ channels, pumps, or exchangers regulate variations in the duration and levels of intracellular Ca2+, which may be transient or sustained. These changes are then decoded by an elaborate toolkit of Ca2+-sensors, which translate Ca2+ signal to intracellular operational cell machinery, thereby regulating numerous Ca2+-dependent physiological processes. Alterations to Ca2+ homoeostasis and signaling are often deleterious and are associated with certain pathological states, including cancer. Altered Ca2+ transmission has been implicated in a variety of processes fundamental for the uncontrolled proliferation and invasiveness of tumor cells and other processes important for cancer progression, such as the development of resistance to cancer therapies. Here, we review what is known about Ca2+ signaling and how this fundamental second messenger regulates life and death decisions in the context of cancer, with particular attention directed to cell proliferation, apoptosis, and autophagy. We also explore the intersections of Ca2+ and the therapeutic targeting of cancer cells, summarizing the therapeutic opportunities for Ca2+ signal modulators to improve the effectiveness of current anticancer therapies.

Aryl Hydrocarbon Receptor Connects Inflammation to Breast Cancer.

Aryl hydrocarbon receptor (AhR), an evolutionary conserved transcription factor, is a pleiotropic signal transductor. Thanks to its promiscuous ligand binding domain, during the evolution of eukaryotic cells its developmental functions were integrated with biosensor functions. Its activation by a multitude of endogenous and exogenous molecules stimulates its participation in several pathways, some of which are linked to inflammation and breast cancer (BC). Over time, the study of this malignancy has led to the identification of several therapeutic targets in cancer cells. An intense area of study is dedicated to BC phenotypes lacking adequate targets. In this context, due to its high constitutive activation in BC, AhR is currently gaining more and more attention. In this review, I have considered its interactions with: 1. the immune system, whose dysregulation is a renowned cancer hallmark; 2. interleukin 6 (IL6) which is a pivotal inflammatory marker and is closely correlated to breast cancer risk; 3. NF-kB, another evolutionary conserved transcription factor, which plays a key role in immunoregulatory functions, inflammatory response and breast carcinogenesis; 4. kynurenine, a tryptophan-derived ligand that activates and bridges AhR to chronic inflammation and breast carcinogenesis. Overall, the data here presented form an interesting framework where AhR is an interesting connector between inflammation and BC.

Replisome genes regulation by antitumor miR-101-5p in clear cell renal cell carcinoma.

Analysis of microRNA (miRNA) regulatory networks is useful for exploring novel biomarkers and therapeutic targets in cancer cells. The Cancer Genome Atlas dataset shows that low expression of both strands of pre‐miR‐101 (miR‐101‐5p and miR‐101‐3p) significantly predicted poor prognosis in clear cell renal cell carcinoma (ccRCC). The functional significance of miR‐101‐5p in cancer cells is poorly understood. Here, we focused on miR‐101‐5p to investigate the antitumor function and its regulatory networks in ccRCC cells. Ectopic expression of mature miRNAs or siRNAs was investigated in cancer cell lines to characterize cell function, ie, proliferation, apoptosis, migration, and invasion. Genome‐wide gene expression and in silico database analyses were undertaken to predict miRNA regulatory networks. Expression of miR‐101‐5p caused cell cycle arrest and apoptosis in ccRCC cells. Downstream neighbor of son (DONSON) was directly regulated by miR‐101‐5p, and its aberrant expression was significantly associated with shorter survival in propensity score‐matched analysis (P = .0001). Knockdown of DONSON attenuated ccRCC cell aggressiveness. Several replisome genes controlled by DONSON and their expression were closely associated with ccRCC pathogenesis. The antitumor miR‐101‐5p/DONSON axis and its modulated replisome genes might be a novel diagnostic and therapeutic target for ccRCC.

Fuzzy modeling and global optimization to predict novel therapeutic targets in cancer cells.

MotivationThe elucidation of dysfunctional cellular processes that can induce the onset of a disease is a challenging issue from both the experimental and computational perspectives. Here we introduce a novel computational method based on the coupling between fuzzy logic modeling and a global optimization algorithm, whose aims are to (1) predict the emergent dynamical behaviors of highly heterogeneous systems in unperturbed and perturbed conditions, regardless of the availability of quantitative parameters, and (2) determine a minimal set of system components whose perturbation can lead to a desired system response, therefore facilitating the design of a more appropriate experimental strategy.ResultsWe applied this method to investigate what drives K-ras-induced cancer cells, displaying the typical Warburg effect, to death or survival upon progressive glucose depletion. The optimization analysis allowed to identify new combinations of stimuli that maximize pro-apoptotic processes. Namely, our results provide different evidences of an important protective role for protein kinase A in cancer cells under several cellular stress conditions mimicking tumor behavior. The predictive power of this method could facilitate the assessment of the response of other complex heterogeneous systems to drugs or mutations in fields as medicine and pharmacology, therefore paving the way for the development of novel therapeutic treatments.Availability and implementationThe source code of FUMOSO is available under the GPL 2.0 license on GitHub at the following URL: https://github.com/aresio/FUMOSOSupplementary information Supplementary data are available at Bioinformatics online.

Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells

ABSTRACTMyeloid cell leukemia-1 (Mcl −1) is one of the most frequently amplified genes in cancer, and its overexpression is associated with poor prognosis and drug resistance. As a member of the Bcl-2 family it is involved in the control of the mitochondrial (intrinsic) cell death pathway. Alternative splicing of the (Mcl-1) gene results in the expression of two functionally distinct proteins, the anti-apoptotic Mcl-1L (exon 2 included) and the pro-apoptotic Mcl-1S (exon 2 skipped). Our data shows that transfecting siRNAs that target hnRNP K and the hnRNP F/H family result in a switch in splicing towards the pro-apoptotic Mcl-1S. Specific binding sites for these and other Mcl-1 splicing factors were investigated and identified by RNA immunoprecipitation and through construction of a Mcl-1 minigene construct. Moreover, this study shows up to a 30 fold change in the levels of Mcl-1S can be achieved through double and triple knockdowns of the most significant RNA binding proteins involved in Mcl-1 splicing, as well as activation of the mitochondrial cell death pathway. Targeting the splicing process of Mcl-1 along with other apoptotic regulators provides an exciting new therapeutic target in cancer cells, and may provide a way to overcome therapy resistance.

Abstract 1058: Synergistic activity of p97 inhibitors with histone deacetylase 6 inhibitors in mantle cell lymphoma

Abstract p97, or valosin-containing protein (VCP), is an ATPase whose function is essential to restore protein homeostasis in cells. Working in concert with the ubiquitin proteasome system, p97 promotes the retrotranslocation of misfolded proteins from the endoplasmic reticulum and/or their degradation. Consequently, p97 inhibition has emerged as a novel therapeutic target in cancer cells, especially those that depend on high protein turnover and/or ER function. p97 is also present in a complex with many protein complexes including the HSP90, histone deacetylase (HDAC) 6 and heat shock factor 1 (HSF1)-also called the repressive complex. p97 participates in the disaggregation of the repressive complex to regulate HSP90 and HSF1 function under stressed conditions. Accumulation of misfolded polyubiquitylated proteins in the cytosol or on damaged organelles promotes the binding of p97 to polyubiquitylated proteins in an HDAC6-dependent manner and promotes their turnover by autophagic degradation. Given that perturbation of protein homeostasis and ER function induces apoptosis as well as autophagy in B cell malignancies such as multiple myeloma and mantle cell lymphoma (MCL), we hypothesized that inhibition of p97 function in combination with HDAC6 inhibitors would induce proteotoxic stress and/or apoptotic cell death in MCL cells. In this study, we report that the p97 inhibitors DBeQ, ML240 and NMS-873 induce a dose-dependent loss of cell viability in cultured and primary MCL cells. Treatment of MCL cells with ML240 induces the accumulation of polyubiquitylated proteins and induces markers of ER stress such as glucose regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation factor 2 (eIF2) α as well as the autophagic markers LC3-B and p62 (protein whose accumulation is suggestive of reduced autophagic clearance of misfolded proteins). Co-treatment with ML240 and the HDAC6 inhibitor ACY-1215 induces more accumulation of polyubiquitylated proteins and markers of enhanced ER stress as well as autophagy, than either agent alone. Mechanistically we determined that co-treatment with ML240 and ACY-1215 inhibits the binding of p97 to polyubiquitylated proteins and HDAC6, resulting in the reduced clearance of cytotoxic protein aggregates in the cells. Co-treatment with ML240 and ACY-1215 also induces the accumulation of cytosolic polyubiquitylated proteins and their co-localization with LC-3B puncta as demonstrated by immunofluorescent microscopy. These observations are suggestive of enhanced initiation of autophagy but not its completion. Consequently, treatment of MCL cells with ML240 and ACY-1215 resulted in enhanced proteotoxic stress and synergistic apoptotic cell death in MCL cells. Collectively our studies create a strong rationale to test efficacy of the combination of p97 inhibitors in combination with HDAC6 inhibitors in MCL. Citation Format: Rekha M. Rao, Pratikkumar H. Vekaria, Trisha Home, Anusha Vallurupalli, Abdulraheem Yacoub, Frank Schoenen, Joseph McGuirk. Synergistic activity of p97 inhibitors with histone deacetylase 6 inhibitors in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1058. doi:10.1158/1538-7445.AM2017-1058

Abstract 2335: Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing

Abstract MCL-1 is a a pro-survival BCL2 protein family member which is over-expressed in drug resistant cancer cells. Our purpose was to assess whether MCL-1 increases genomic instability along with its anti-apoptotic function in cancer cells. Depletion of MCL-1 by treating human small cell lung cancer (SCLC) lines with immunotoxin sensitizes these cells to increased killing by BH3 mimetic - Navitoclax (ABT-263) both in vitro and in mice xenograft models. These SCLC cell lines are resistant to both single agent Immunotoxin or ABT-263. In addition, inhibition of NANOG or NANOGP8 by lentivrus delivered shRNA depletes MCL-1 in human colorectal cancer cells and sensitizes these cells to increased cell killing by venetaclox (ABT-199). This increased killing depended on loss of MCL-1, was caspase-dependent and reversed by re-expression of MCL-1. Depletion of MCL-1 in cancer cells , increases cell sensitivity to ionizing radiation (IR) induced death, which was reversed by expression of MCL-1. In response to IR, MCL-1 depleted cells showed reduced survival even at radiation doses less than 6 Gy, however an increase in caspases 3/7 activity was observed at 6 Gy and above, suggesting that apoptosis is activated only when MCL-1 depleted cells are irradiated with higher IR doses. In order to explain the decreased survival post- irradiation the genomic instability and DNA repair pathways were analyzed in MCL-1 depleted cells. Post-irradiation MCL-1 depleted cells exhibited increased genomic instability as measured by increase in chromosome aberrations at different phases of the cell cycle. The increase in aberrations were significantly higher in G2 and S- phases of the cell cycle suggesting defect in Homologous recombination repair pathway (HR). Moreover, the MCL-1 depleted cells show decreased gamma-H2AX foci at earlier time points (30 and 90 minutes) post-irradiation indicating defect in DNA Damage response (DDR) and higher residual foci at 360 minutes, indicating defective DNA repair. These observations were further confirmed by decreased phosphorylation of ATR suggesting defect in DNA Damage response and higher levels of residual 53BP1 and RIF1 foci in MCL-1 depleted cells , confirming DNA DSB repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1 depleted cells exhibited a reduced frequency of IR-induced MRE11, BRCA1, RPA and Rad51 foci formation, decreased DNA end resection and decreased HR repair in the DR-GFP DSB repair model. Similarly, after Hydoxyurea (HU) induction of stalled replication forks in MCL-1 depleted cells there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR mediated resolution of collapsed forks. In summary, MCL-1 is an important therapeutic target in cancer cells and its depletion increases cell killing by either increase in apoptosis or suppression of HR and increased replication stress. Citation Format: Abid Mattoo, J Milburn Milburn Jessup, Tej K. Pandita. Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2017-2335

TRTH-28. HIGH THROUGHPUT SCREENING OF NOVEL HISTONE DEACETYLASE INHIBITORS FOR EPIGENETIC THERAPY OF PRIMARY BRAIN TUMORS

Abstract Histone deacetylases (HDACs) are crucial regulators of epigenetic and posttranslational modifications and therefore represent a promising therapeutic target in cancer cells that harbor distinct epigenomes from normal cells. To exploit the therapeutic potential of HDAC inhibitors (HDACi) we synthesized a unique in-house library of more than 200 inhibitors. For the evaluation of the antitumor effects of the compound library in brain tumor cell lines, we successfully established an optimal screening workflow. The screening procedure was streamlined by automated dispensing of cell lines, reagents and inhibitors using state of the art equipment. The drugs were evaluated for their effect on tumor cell viability in a panel of cell lines derived from different brain tumor entities (8 glioblastoma, 10 medulloblastoma and 6 atypical teratoid/rhabdoid tumor cell lines) and compared to 5 normal control tissues. Corresponding dose-response profiles were generated using an optimized bioinformatics workflow. In addition to our in-house library commercially available and clinically used HDACi (e.g. Vorinostat) were included. The semi-automated setup enabled the miniaturization of the assay format to 384-well plates. In combination with additional modifications, a remarkable increase of the overall throughput was realized, while generating accurate and reproducible results. Based on this workflow, we created a unique and comprehensive data set and could thereby identify various HDACi acting universally across brain tumors or being specifically active in distinct tumor entities. Promising candidates are currently being further characterized, e.g. Panobinostat showed activity in the majority of brain tumor models at low nanomolar concentrations. The growing importance of HDAC inhibitors is reflected by an increasing number of HDACi in clinical trials, with four HDACi already approved by the FDA for treating lymphomas and multiple myelomas. Our study supports the finding that HDACi are valid therapeutic agents and that selected inhibitors are promising candidates for future epigenetic therapy of primary brain tumors.

Super-Enhancer-Driven Transcriptional Dependencies in Cancer

Transcriptional deregulation is one of the core tenets of cancer biology and is underpinned by alterations in both protein-coding genes and noncoding regulatory elements. Large regulatory elements, so-called super-enhancers (SEs), are central to the maintenance of cancer cell identity and promote oncogenic transcription to which cancer cells become highly addicted. Such dependence on SE-driven transcription for proliferation and survival offers an Achilles heel for the therapeutic targeting of cancer cells. Indeed, inhibition of the cellular machinery required for the assembly and maintenance of SEs dampens oncogenic transcription and inhibits tumor growth. In this article, we review the organization, function, and regulation of oncogenic SEs and their contribution to the cancer cell state.