Aurora Kinase Pathway Research Articles

Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.

Triple-negative breast cancer (TNBC) poses a therapeutic challenge due to its aggressive nature and lack of targeted therapies. Epigenetic modifications contribute to TNBC tumorigenesis and drug resistance, offering potential therapeutic targets. Recent advancements in three-dimensional (3D) organoid cultures, enabling precise drug screening, hold immense promise for identifying novel compounds targeting TNBC. In this study, we established two patient-derived TNBC organoids and implemented a high-throughput drug screening system using these organoids and two TNBC cell lines. Screening a library of 169 epigenetic compounds, we found that organoid-based systems offer remarkable precision in drug response assessment compared to cell-based models. The top 30 compounds showing the highest drug sensitivity in the initial screening were further assessed in a secondary screen. Four compounds, panobinostat, pacritinib, TAK-901, and JIB-04, targeting histone deacetylase, JAK/STAT, histone demethylases, and aurora kinase pathways, respectively, exhibited potent anti-tumor activity in TNBC organoids, surpassing the effect of paclitaxel. Our study highlights the potential of these novel epigenetic drugs as effective therapeutic agents for TNBC and demonstrates the valuable role of patient-derived organoids in advancing drug discovery.

Abstract A037: TT-00420, a novel kinase inhibitor potentially benefiting c-Myc amplified/overexpressed osteosarcoma

Abstract Osteosarcoma (OS) is the most common bone tumor in pediatric patients, whose effective therapies are of great unmet medical needs, particularly for those of high-risk features. c-Myc, a well-known oncogene, is commonly amplified/overexpressed in OS patients, contributes to dismal prognosis and associates with lower 5-year overall survival rate, which is also confirmed by our retrospective analysis for the correlation between c-Myc gene amplification/overexpression and patient prognosis in our patient cohort. TT-00420, a clinical-stage small molecule targeting Aurora A/B, VEGFRs, FGFRs, JAKs and CSF1R, has demonstrated down-regulation on c-Myc expression in TNBC studies. Moreover, as reported, Aurora kinases and c-Myc regulate each other back and forth, triggering maintenance of the malignant state. In this study, we examined the efficacy of TT-00420 in c-Myc-amplified/overexpressed OS. In pre-clinical clonogenic assay, TT-00420 significantly inhibited the colony formation in c-Myc-overexpressed OS cell lines in a dose-dependent manner. In MNNG/HOS cell line, we demonstrated that TT-00420 dramatically suppressed Aurora kinase pathway and blunted c-Myc protein expression level, indicating TT-00420 might down-regulate c-Myc by Aurora kinases inhibition. In pre-clinical in-vivo efficacy studies, 143B cells were injected into the medullary cavity of the tibia to make the orthotopic OS xenograft model. With 15 mg/kg/day treatment of TT-00420, tumor growth was obviously inhibited as compared to vehicle group. Immunohistochemistry (IHC) analyses in tumors revealed that TT-00420-treated tumors exhibited increased apoptosis with upregulated expression of cleaved caspase 3 (CC3) and H2AX, and significantly decreased proliferations with raised expression of Ki67. Moreover, in a c-Myc-overexpressed OS patient-derived xenograft (PDX) model from a male pediatric patient, 15 mg/kg/day TT-00420 dramatically blunted the tumor growth with a TGI of 87.6%. In China, an investigator-initiated trial is currently ongoing to evaluate the safety and tolerability of TT-00420 in OS patients with c-Myc overexpression (ChiCTR2000036618). Taken together, TT-00420 depicted robust anti-tumor activity in c-Myc overexpressed OS both in vitro and in vivo with downregulation of c-Myc expression, indicating its potential as a novel monotherapy for OS patients with c-Myc overexpression. Future clinical trial result is of great expectation. Citation Format: Yingqi Hua, Xiaoyan Qiang, Kai Tian, Yafei Jiang, Frank Wu, Peng Peng. TT-00420, a novel kinase inhibitor potentially benefiting c-Myc amplified/overexpressed osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A037.

Results of a phase 1b study of osimertinib plus sapanisertib or alisertib for osimertinib-resistant, EGFR-mutant non–small cell lung cancer (NSCLC).

9105 Background: The aurora kinase and mTOR pathways are implicated in resistance to EGFR inhibitor osimertinib. Here, we investigated the safety and efficacy of the aurora kinase inhibitor alisertib and the mTOR inhibitor sapanisertib in combination with osimertinib. Methods: This is a phase 1b study with dose finding and expansion portions (NCT04479306). The dose finding portion used a Bayesian optimal interval (BOIN) design to assess two arms: osimertinib 80 mg daily in combination with alisertib 20 mg, 30 mg, and 40 mg daily day 1-21 of 28-day cycle (osi-ali arm) and osimertinib 80 mg daily in combination with sapanisertib 2 mg and 3 mg daily (osi-sapa arm). Dose limiting toxicities (DLTs) were predefined in the protocol. Patients with EGFR (L858R/exon 19 deletion) mutant NSCLC whose disease have progressed on osi and up to one additional line of systemic therapy were assigned, at investigator discretion, to either study arm. Tumor biopsy was mandatory at study entry and optional upon progression. Results: As of February 1, 2022, 40 patients are enrolled (20 in each arm). One DLT was observed in each arm: grade 3 nausea in ali-osi arm and grade 3 AST elevation in osi-sapa arm. Grade 3 treatment emergent adverse events (TEAEs) occurred in 10% of each arm, and no grade 4 TEAEs were observed. The most common TEAEs in osi-ali arm was leucopenia (45%) and anemia (35%) while in osi-sapa arm, hyperglycemia (45%) and stomatitis (40%). In osi-ali arm (n = 20), median progression free survival (mPFS) was 1.9 months while objective response rate (ORR) and disease control rate (DCR) were 5% (95% CI: 0.1 ̃ 24.9%) and 40% (95% CI: 19.1 ̃ 63.9%), respectively. In osi-sapa arm (n = 16, evaluated for response to date), mPFS was 4.6 months while ORR and DCR were 12.5% (95% CI: 1.6 ̃ 38.3%) and 68.7% (95% CI: 35.7 ̃ 82.7%), respectively. Conclusions: Osimertinib with alisertib or sapanisertib is well tolerated in osimertinib-resistant, EGFR mutant NSCLC. The sapanisertib combination, but not the alisertib combination, demonstrates antitumor activity suggesting that mTOR inhibition warrants further exploration in this population. Biomarker analysis is ongoing to identify the molecular determinants of response and resistance to sapasertib. Clinical trial information: NCT04479306.

CDK6 Is a Therapeutic Target in Myelofibrosis.

Myelofibrosis (myelofibrosis) is a deadly blood neoplasia with the worst prognosis among myeloproliferative neoplasms (MPN). The JAK2 inhibitors ruxolitinib and fedratinib have been approved for treatment of myelofibrosis, but they do not offer significant improvement of bone marrow fibrosis. CDK6 expression is significantly elevated in MPN/myelofibrosis hematopoietic progenitor cells. In this study, we investigated the efficacy of CDK4/6 inhibitor palbociclib alone or in combination with ruxolitinib in Jak2V617F and MPLW515L murine models of myelofibrosis. Treatment with palbociclib alone significantly reduced leukocytosis and splenomegaly and inhibited bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of myelofibrosis. Combined treatment of palbociclib and ruxolitinib resulted in normalization of peripheral blood leukocyte counts, marked reduction of spleen size, and abrogation of bone marrow fibrosis in murine models of myelofibrosis. Palbociclib treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, treatment with palbociclib or depletion of CDK6 inhibited Aurora kinase, NF-κB, and TGFβ signaling pathways in Jak2V617F mutant hematopoietic cells and attenuated expression of fibrotic markers in the bone marrow. Overall, these data suggest that palbociclib in combination with ruxolitinib may have therapeutic potential for treatment of myelofibrosis and support the clinical investigation of this drug combination in patients with myelofibrosis. SIGNIFICANCE: These findings demonstrate that CDK6 inhibitor palbociclib in combination with ruxolitinib ameliorates myelofibrosis, suggesting this drug combination could be an effective therapeutic strategy against this devastating blood disorder.

Pairing a prognostic target with potential therapeutic strategy for head and neck cancer.

We have previously identified and validated a panel of molecular prognostic markers (ATP13A3, SSR3, and ANO1) for Head and Neck Squamous Cell Carcinoma (HNSCC). The aim of this study was to investigate the consequence of ATP13A3 dysregulation on signaling pathways, to aid in formulating a therapeutic strategy targeting ATP13A3-overexpressing HNSCC. Gene Set Enrichment Analysis (GSEA) was performed on HNSCC microarray expression data (Internal local dataset [n=92], TCGA [n=232], EMBL [n=81]) to identify pathways associated with high expression of ATP13A3. Validation was performed using immunohistochemistry (IHC) on tissue microarrays (TMAs) of head and neck cancers (n=333), staining for ATP13A3 and phosphorylated Aurora kinase A (phospho-T288). Short interfering RNA was used to knockdown ATP13A3 expression in patient derived HNSCC cell lines. Protein expression of ATP13A3 and Aurora kinase A was then assessed by immunoblotting. GSEA identified Aurora kinase pathway to be associated with high expression of ATP13A3 (p=0.026). The Aurora kinase pathway was also associated with a trend towards poor prognosis and tumor aggressiveness (p=0.086, 0.094, respectively). Furthermore, the immunohistochemical staining results revealed a significant association between Aurora kinase activity and high ATP13A3 expression (p<0.001). Knockdown of ATP13A3 in human head and neck cell lines showed decrease in Aurora kinase A levels. Tumors with high ATP13A3 are associated with high Aurora kinase activity. This suggests a potential therapeutic role of Aurora kinase inhibitors in a subset of poor prognosis HNSCC patients with overexpression of ATP13A3.

Abstract 2726: Functional multiomic profiling identifies Aurora kinase inhibition as a therapeutic strategy in RIT1-mutant lung adenocarcinoma

Abstract Treatment of lung cancer is evolving from standard cytotoxic to personalized treatment based on the molecular alterations unique to each patient’s tumor. In our recent work with TCGA and others, we identified somatic mutations in the gene encoding the RAS-related small GTPase, RIT1, in lung adenocarcinomas. In addition, somatic RIT1 mutations have also been identified in myeloid malignancies and germline RIT1 mutations are found in the developmental “RAS-opathy,” Noonan syndrome. In all these diseases, RIT1 mutations are mutually-exclusive with other RAS/MAPK pathway mutations, including mutations in EGFR, KRAS, and ALK. While our previous work demonstrated that gain-of-function RIT1 mutations induce activation of PI3K and MEK signaling, the specific effectors used by RIT1 to promote tumorigenesis are unknown. To genetically dissect the signaling pathways downstream of RIT1, KRAS, EGFR, and PIK3CA, we performed genome-wide CRISPR/Cas9 screens in four isogenic lung adenocarcinoma cell lines in which cell survival is dependent on the expression of the oncogene. Through integrative analysis of essential genes across the four cell lines, we identified synthetic lethal relationships unique and shared in each isogenic background. Among the dependencies identified was the requirement of aurora kinase pathway genes for survival of RIT1-mutant cells, raising the possibility of therapeutic aurora kinase inhibition in RIT1-positive tumors. To evaluate whether the dependence on Aurora kinases extended to small molecule inhibition, we performed a screen of 160 small molecules in clinical development or approved by the FDA. Consistent with our CRISPR/Cas9 results, we find that RIT1-mutant cells depend on Aurora kinase activity for survival. Interestingly, global phosphoproteomic profiling identified RIT1 regulation of an Aurora kinase-Haspin axis. Taken together these results identify a new arm of RIT1 signaling and nominate Aurora kinase inhibition as a potential therapeutic strategy in RIT1-mutated tumors. Citation Format: Athea Vichas, Naomi T. Nkinsi, Alice Berger. Functional multiomic profiling identifies Aurora kinase inhibition as a therapeutic strategy in RIT1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2726.

Network analysis revealed aurora kinase dysregulation in five gynecological types of cancer.

Gene markers are crucial for cancer prognosis and treatment. Previous studies have placed greater emphasis on individual diagnostic genes, thereby ignoring systemic-level attributes across diseases. Female-specific cells namely, breast, endometrium, cervical, ovarian and vulvar cells are highly susceptible to cancer. To date, a limited number of molecular studies have been performed that evaluate common biological processes across gynecological types of cancer. Differentially expressed genes in breast, cervical, endometrial, vulvar and ovarian cancer were utilized to construct protein-protein interaction networks, and to identify a common module across the five cancer types. A single common module with 8 nodes and 26 edges was mined among the five cancer systems. In total, four hub genes were present across the five cancer gene sets. Genes in the common module were enriched for the common pathways and associated diseases. The aurora kinase pathway was revealed to be conserved across the five cancer types surveyed. The present study, therefore, revealed that the aurora kinase pathway has a crucial function in the pathogenesis of the five aforementioned gynecological types of cancer through cross-tumor conservation.

Silver nanoparticle-induced phosphorylation of histone H3 at serine 10 is due to dynamic changes in actin filaments and the activation of Aurora kinases

The phosphorylation of histone H3 at serine 10 (p-H3S10) has been closely correlated with mitotic chromosome condensation. We previously reported that silver nanoparticles (AgNPs) significantly induced p-H3S10 independent of mitosis. In the present study, we examined the mechanisms underlying the induction of p-H3S10 by AgNPs. A treatment with AgNPs markedly induced p-H3S10 in a dose-dependent manner in three types of cell lines, and this was dependent on the cellular incorporation of AgNPs. The immunofluorescent staining of AgNP-induced p-H3S10 was thin and solid throughout the nucleus, and differed from that normally associated with mitosis. AgNPs induced the formation of globular actin in a dose-dependent manner. Latrunculin B (LatB) and phalloidin, inhibitors of actin polymerization and depolymerization, respectively, inhibited p-H3S10, suggesting that dynamic changes in actin filaments are related to AgNP-induced p-H3S10. Furthermore, p-H3S10 was mediated by Aurora kinase (AURK) pathways, which were suppressed by LatB and siRNA for cofilin 1, an actin-depolymerizing protein. AgNO3 (Ag ions) exerted similar effects to those of AgNPs. These results suggest that Ag ions released from AgNPs incorporated into inner cells changed the dynamics of actin filaments, and this was followed by the activation of AURKs, leading to the induction of p-H3S10.

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Defining the Primate T Cell Transcriptome during GvHD: New Data Implicating Sonic Hedgehog and Aurora Kinase Pathways in GvHD Pathogenesis and Prevention

Defining the Primate T Cell Transcriptome during GvHD: New Data Implicating Sonic Hedgehog and Aurora Kinase Pathways in GvHD Pathogenesis and Prevention

Defining the Primate T Cell Transcriptome during Graft Versus Host Disease: New Data Implicating the Hedgehog and Aurora Kinase Pathways in Pathogenesis and Prevention

Graft versus host disease (GvHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate targeted strategies for prevention and treatment. To address this, we determined the gene expression profile of non-human primate (NHP) T cells during active and partially controlled acute GvHD (aGvHD), in order to accomplish two goals: 1) uncover important genetic drivers of aGvHD and 2) identify novel, targetable pathways for optimal aGvHD prevention.Utilizing microarray technology, we measured the gene expression profiles of flow cytometrically purified CD3+ T cells from NHP recipients of MHC partially-matched HCT in three treatment cohorts resulting in increasing degrees of survival: 1) no immunoprophylaxis (No Rx, MST = 7.5); 2) sirolimus monotherapy (MST = 17) tacrolimus-methotrexate (Tac-Mtx) dual prophylaxis (MST = 49). Arrays were performed on T cells purified on Day +14 post-transplant (unless terminal analysis occurred earlier due to severe disease). This comparison allowed us to determine the impact of both mTOR and calcineurin inhibition on the molecular pathways dysregulated during GvHD, and to determine which genes and pathways remained dysregulated despite prophylaxis.Pathways identified by this strategy may contain new therapeutic targets unaffected by current immunoprophylactic approaches. We found that the transcriptional profile of donor T cells from HCT recipients with unprophylaxed GvHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function and cytokine synthesis (Figure 1a). By identifying pathways unaffected by sirolimus or tac-mtx therapy (Figure 1b), we discovered multiple potentially druggable targets not previously implicated in the pathophysiology of aGvHD. These targets prominently included the hedgehog and the aurora kinase A pathways. Utilizing a murine aGvHD model, we demonstrated that pharmacologic inhibition of these pathways could mitigate disease and improve survival (Figure 2a,b).These data provide the first identification of the T cell transcriptome of primate acute GvHD and the hedgehog and aurora kinase A pathways as novel potential targets for prevention of this disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deep sequencing of sarcomatoid renal cell carcinoma (sRCC) and identification of aurora kinases and mTOR as potential therapeutic targets.

411 Background: The presence of sarc elements in pts with RCC portends a poor prognosis. Some studies have focused on epithelial-to-mesenchymal transition (EMT) as a driver of sarc ccRCC; however, the potential role of other targetable entities is unknown. Methods: From an institutional database, we identified 13 pts with metastatic sarc ccRCC and 13 pts with metastatic ccRCC lacking sarc elements matched by age and risk group. All patients had available FFPE tissue derived from nephrectomy. For a limited sample of 5 pts with sarc ccRCC, RNA sequencing was performed on microdissected cc, sarc, and benign renal cortical sections from FFPE blocks. Reads were aligned to hg19 genome with Tophat v2.0. IHC was used to explore expression of implicated genes in the entire matched cohort of 26 pts. Results: With respect to RNA sequencing analysis for 5 pts with sarc ccRCC, reads had between 4% and 24% exon coverage. Hierarchical clustering demonstrated that most samples were clustered by patient, not by disease state (i.e., sarc vs non-sarc), suggesting that inter-patient variation is large. However, comparisons between cc and sarc components for these 5 pts demonstrated a significant increase in cell growth pathways, with the most highly enriched gene ontology pathways being M-phase, cell cycle, mitosis and aurora kinase pathways. No EMT-related pathways were found to be enriched. IHC assessments of VEGF and mTOR pathways were performed, given their purported role in RCC cell growth. While no differences were noted in pVEGFR2, higher pS6K staining was noted in both the sarc and non-sarc components of pts with sarc ccRCC relative to pS6K staining in tumor tissue derived from ccRCC pts lacking sarc elements (p&lt;0.05 for both). No differences in EMT marker expression were noted (specifically, E-cad, N-cad and Twist); IHC assessments for aurora kinases A and B will be reported. Conclusions: Although cytotoxic agents and VEGF-directed therapies have been explored in sarc ccRCC, the finding of mTOR pathway activity underscores the need to examine mTOR inhibitors in this disease. Furthermore, emerging agents targeting aurora kinases may have a role in sarc ccRCC.

Abstract 2049: Preclinical profile of AMG900 in combination with HDACIs in prostate cancer

Abstract Background: Histone deacetylase (HDAC) enzymes maintain the structural conformation of chromatin. Inhibition of HDACs induces G1 arrest and differentiation. Previous microarray analysis demonstrated downregulation of the aurora kinase-pathway in prostate cancer cell lines treated with HDAC-inhibitors (HDACIs) vorinostat and valproic acid (VPA). Hence we sought to combine these HDACIs with AMG900, a pan-Aurora kinase inhibitor, to assess potential additive or synergistic effects, hypothesizing that low-dose combinations will have a proapoptotic effect comparable to a higher dose of AMG900 as a single agent. Methods: We used MTS assays, clonogenic assays, beta-galactosidase staining and western blot analyses to investigate differences in proliferation and molecular markers with combinations of AMG900 and HDACIs compared to AMG900 alone. Results: In vitro treatment of a panel of prostate cancer cell lines with low-dose combinations of AMG900 with HDACIs revealed enhanced antiproliferative responses compared to single agent use. Short term MTS assays only demonstrated moderate synergism in DU-145 and LNCaP cells co-treated with VPA and synergism in PC3 cells co-treated with vorinostat, based on the Chou-Talalay-method (CalcuSyn). However, all cell lines demonstrated significant decreased long term survival when combining low-dose AMG900 (1nM) with HDACIs. In DU-145 and PC3 cells lines, the low-dose AMG900 (1nM) combinations demonstrated equivalent clonogenic survival to high dose AMG900 treatment (5nM), either alone or in combination with HDACIs. We noted morphologic changes in treated cell lines and explored senescence with beta-galactosidase staining. DU-145 and PC3 cells, when treated with a combination of AMG900 and HDACIs, demonstrated a significant increase in beta-galactosidase positive cells, suggesting cell senescence (10-20% in single agent vs 50% in low-dose combinations). We also explored the mechanisms associated with decreased clonogenic survival at low doses and found a significant increase in cleaved PARP in DU-145 cells with both combinations, and with Vorinostat in LNCaP cells, but not PC3 cells which have a baseline level of ongoing apoptosis. In addition, western blot analysis showed significant differences in ACH3, pH3 and Cyclin B1 in combination treatment compared to single agent use. Conclusions: A combination of low-dose AMG900 with HDACIs in prostate cancer cell lines demonstrates synergy and efficacy equivalent to high doses of AMG900 alone, confirming our hypothesis. 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 2049. doi:1538-7445.AM2012-2049

Combined Inhibition of Janus and Aurora Kinases by the Novel Small Molecule Inhibitor AZD1480 Induces Cell Death and Downregulates PD-L1 and PD-L2 Expression In Hodgkin Lymphoma

AbstractAbstract 2848Hodgkin Lymphoma (HL) cell proliferation and survival is sustained by a complex network of cytokine signaling, involving the Hodgkin and Reed-Sternberg cells and tumor microenvironment. Following cytokine stimulation, JAK-STAT activation promotes the transcription of target genes involved in proliferation, survival, and immune escape. Programmed Death-ligands 1 and 2 (PD-L1 and PD-L2) and the Th2 chemokine TARC are immune-modulators involved in immune evasion, respectively through inhibition of effector T cell function (PD-L1, PD-L2) and attraction and homing of Th2 cells (TARC). Aurora kinases are frequently overexpressed in human cancers and play essential functions in chromosome alignment and cytokinesis. The role of Aurora kinases in Hodgkin lymphomagenesis is not defined yet. In this study we report the activity profile of the JAK2 inhibitor AZD1480 in HL cell lines (HD-LM2, L-428, KM-H2, L-540).To assess the effect of AZD1480 on cell proliferation, cells were incubated with increasing concentrations of AZD1480 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs). A significant growth inhibition was evident after 72 hrs of incubation, specially using the high doses of AZD1480 (5μM). The L-540 cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with AZD1480 1μM. Inhibition of STAT3, STAT5 and STAT6 phosphorylation in the L-540, L-428 and HD-LM2 cell lines was observed with concentrations equal to 0.1 μM or higher. Using Annexin V- propidium iodide staining, we found that AZD1480 induced cell death by apoptosis in a dose dependent manner after 72 hrs of incubation when a high concentration (5μM) of the drug was used. Lower concentrations of AZD1480 (1μM) promoted a statistically significant increase in cell death only in the L-540 and to a lesser extent in the L-428 cell line. Consistent with this data, also caspase 9, 3 and PARP cleavage was observed in all the cell lines exposed to AZD1480 5 μM. AZD1480 5μM promoted a marked increase in the G2/M fraction in all the cell lines as soon as 24 hrs after incubation, especially in the HD-LM2 and L-428 cell lines. Treatment with lower doses (1μM) did not affect significantly the cell cycle. Since AZD1480 was also reported to inhibit Aurora A kinase at nanomolar concentrations in enzymatic assays, we assessed if the significant increase in the G2/M fraction was related to the inhibition of the Aurora A kinase. We evaluated the levels of autophosphorylation on Thr-288 by western blotting. Cells were pretreated with Nocodazole 400 ng/ml for 18 hrs in order to achieve a mitotic block, and then exposed to AZD1480 (1-5μM) and/or the proteasome inhibitor MG132 (20μM) (in order to prevent the potential overriding of the Nocodazole induced mitotic block), for 3 hours. A dose-dependent inhibition of Aurora A was detected in all the cell lines, with a complete abrogation when higher doses of AZD1480 were used (5μM). These findings are consistent with the analysis of the cell cycle fractions, showing dose-dependent changes of the cell cycle at 24 hrs following incubation with AZD1480. AZD1480 also decreased the secretion of key cytokines involved autocrine and paracrine survival loops and immune escape. Following incubation with AZD1480 1μM for 72 hrs cell culture supernatants were analyzed by ELISA: decreased levels of IL-6, IL-13, TARC, and IL-21 were observed in HD-LM2, L-428 and L-540 cells. Moreover we assessed the expression of PD-L1 and PD-L2 by flow cytometry and observed significant downregulation in the PD-L1/PD-L2 overexpressing cell lines (L-540 and HD-LM2). These data suggest that AZD1480 has a pleiotropic mechanism of action in HL by targeting the JAK-STAT and the Aurora kinase pathway, and by altering the pattern of cytokine and chemokine secretion and the expression of factors involved in immune escape. Our study provides the rationale for further clinical investigation of AZD1480 in HL. Disclosures:No relevant conflicts of interest to declare.

Esophageal Adenocarcinoma: Treatment Modalities in the Era of Targeted Therapy

Esophageal adenocarcinoma is an aggressive malignancy with a poor outcome, and its incidence continues to rise at an alarming rate. Current treatment strategies combining chemotherapy, radiation, and surgery are plagued with high rates of recurrence and metastasis. Multiple molecular pathways including the epidermal growth factor receptor, vascular endothelial growth factor, v-erb-b2 erythroblastic leukemia viral oncogene homolog (ERBB2), and Aurora kinase pathways are activated in many esophageal adenocarcinomas. In many cases, these pathways have critical roles in tumor progression. Research on the mechanisms by which these pathways contribute to disease progression has resulted in numerous biologic agents and small molecules with the potential to improve outcome. The promise of targeted therapy and personalized medicine in improving the clinical outcome is now closer than it has ever been.

Gene expression-signature of belinostat in cell lines is specific for histone deacetylase inhibitor treatment, with a corresponding signature in xenografts

Belinostat is a hydroxamate-type histone deactylase inhibitor (HDACi), which has recently entered phase I and II clinical trials. Microarray-based analysis of belinostat-treated cell lines showed an impact on genes associated with the G2/M phase of the cell cycle and downregulation of the aurora kinase pathway. Expression of 25 dysregulated genes was measured in eight differentially sensitive cell lines using a novel high-throughput assay that combines multiplex reverse transcriptase-PCR and fluorescence capillary electrophoresis. Sensitivity to belinostat and the magnitude of changes in overall gene modulation were significantly correlated. A belinostat-gene profile was specific for HDACi in three cell lines when compared with equipotent concentrations of four mechanistically different chemotherapeutic agents: 5-fluorouracil, cisplatin, paclitaxel, and thiotepa. Belinostat- and trichostatin A (HDACi)-induced gene responses were highly correlated with each other, but not with the limited changes in response to the other non-HDACi agents. Moreover, belinostat treatment of mice bearing human xenografts showed that the preponderance of selected genes were also modulated in vivo, more extensively in a drug-sensitive tumor than a more resistant model. We have demonstrated a gene signature that is selectively regulated by HDACi when compared with other clinical agents allowing us to distinguish HDACi responses from those related to other mechanisms.