Aurora Kinase Inhibitors Research Articles

Combined TRIP13 and Aurora Kinase Inhibition Induces Apoptosis in Human Papillomavirus-Driven Cancers.

Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers. We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression. Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition-induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells' sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition-induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells. Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.

Rosin Derivative IDOAMP Inhibits Prostate Cancer Growth via Activating RIPK1/RIPK3/MLKL Signaling Pathway.

Rosin derivatives such as dehydroabietic acid and dehydroabietic amine belonging to diterpenoids have similar structure with androgen that inhibited the occurrence and development of prostate cancer. In this study, the effects and possible mechanism of the rosin derivative IDOAMP on prostate cancer were investigated. Our results showed that IDOAMP effectively inhibited cell viabilities of LNCaP, PC3, and DU145 prostate cells. After the treatment with IDOAMP, the levels of cleaved-PARP, LC3BII/I, and HMGB1 were increased, whereas the expression of GPX4 was decreased. Interestingly, cell viability was reversed by the supplements of necrostatin-1 and necrosulfonamide. Meanwhile, the IDOAMP downregulated the expression of human Aurora kinase A that was overexpressed in prostate cancer. In addition, co-IP results showed that IDOAMP inhibited the binding of Aurora kinase A to the receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3. However, the binding of RIPK1 to FADD, RIPK3, or MLKL was significantly promoted. Further studies showed that the phosphorylation levels of RIPK1, RIPK, and MLKL were increased in a concentration-dependent manner. In in vivo model, IDOAMP reduced the tumor volumes and weights. In conclusion, IDOAMP directly inhibited Aurora kinase A and promoted the RIPK1/RIPK3/MLKL necrosome activation to inhibit the prostate cancer.

AURKB, in concert with REST, acts as an oxygen-sensitive epigenetic regulator of the hypoxic induction of MDM2

The acute response to hypoxia is mainly driven by hypoxia-inducible factors, but their effects gradually subside with time. Hypoxia-specific histone modifications may be important for the stable maintenance of long-term adaptation to hypoxia. However, little is known about the molecular mechanisms underlying the dynamic alterations of histones under hypoxic conditions. We found that the phosphorylation of histone H3 at Ser-10 (H3S10) was noticeably attenuated after hypoxic challenge, which was mediated by the inhibition of aurora kinase B (AURKB). To understand the role of AURKB in epigenetic regulation, DNA microarray and transcription factor binding site analyses combined with proteomics analysis were performed. Under normoxia, phosphorylated AURKB, in concert with the repressor element-1 silencing transcription factor (REST), phosphorylates H3S10, which allows the AURKB–REST complex to access the MDM2 proto-oncogene. REST then acts as a transcriptional repressor of MDM2 and downregulates its expression. Under hypoxia, AURKB is dephosphorylated and the AURKB–REST complex fails to access MDM2, leading to the upregulation of its expression. In this study, we present a case of hypoxia-specific epigenetic regulation of the oxygen-sensitive AURKB signaling pathway. To better understand the cellular adaptation to hypoxia, it is worthwhile to further investigate the epigenetic regulation of genes under hypoxic conditions.

CHD1 Promotes Sensitivity to Aurora Kinase Inhibitors by Suppressing Interaction of AURKA with Its Coactivator TPX2.

CHD1 plays a critical role in controlling AURKA activation and promoting Aurora kinase inhibitor sensitivity, providing a potential clinical biomarker to guide cancer treatment.

Abstract 1858: Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels

Abstract Human papilloma virus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) is common in young patients (<60 years). All patients experience either permanent toxicity from standard therapy or death from recurrent disease. To address this unmet need for more effective and less toxic therapy, we performed a high throughput drug screen comparing drug efficacy in HPV+ and HPV- cell lines. We tested the drug sensitivity of 864 unique drugs (0-3.1 µM) divided into 51 classes based on their targets in 33 squamous cancer cell lines using a parameter that is independent of cell division. Only one class of drugs, Aurora kinase inhibitors, was more effective in HPV+ than HPV- cell lines. To validate our screening results, we tested two Aurora kinase inhibitors (Alisertib, Barasertib) and observed significant cell death in HPV+ cells, but not in HPV- cells. Alisertib reduced tumor volume in HPV+ PDX mouse models without toxicity. To investigate the underlying mechanism, we compared the expression of ~300 proteins, using RPPA, to drug efficacy and validated the results using immunoblotting. The expression of Rb, pRb(S807/811), and p16 correlated with drug sensitivity. To establish role for Rb, we used a bidirectional approach to manipulate RB1. Reduction in RB1 levels using either shRNA or stable E7 overexpression in HPV- cells resulted in increased Alisertib-induced apoptosis. We also used siRNA to knock down E7 expression in two HPV+ cell lines which resulted in elevated levels of Rb, thereby rescuing the cells from Alisertib-induced apoptosis. Studies have shown Rb-deficient cells have elevated levels of the mitotic-checkpoint protein MAD2, that may impact cell fitness. To test the role of MAD2, we depleted the levels of MAD2 and observed diminishing levels of cyclin B1 accumulation and reduced Alisertib-induced apoptosis. Additionally, we altered the activated levels of MAD2 protein by overexpression of TRIP13 that resulted in partially rescuing cells from undergoing Alisertib-induced apoptosis. These results indicate that Rb- deficient MAD2 overexpressing cells require a finely tuned balance of MAD2 and TRIP13 levels for their mitotic exit. Furthermore, we observed combined inhibition of TRIP13 with Aurora A led to more apoptosis in Rb-deficient cells than the single agents. Our results demonstrate that Rb-deficient cancers are sensitive to Aurora A inhibition due to an imbalance between MAD2 and TRIP13. Based on the above finding, our present study shows that TRIP13 inhibition in combination with Aurora kinase inhibitors leads to more apoptosis and can pave a path for novel therapies in the management of HPV+ tumors. Citation Format: Soma Ghosh, Tuhina Mazumdar, Wei Xu, Reid T. Powell, Clifford Stephan, Li Shen, Curtis R. Pickering, Jing Wang, Faye M. Johnson. Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1858.

Abstract 2592: Aurora kinase is an actionable target in low molecular weight cyclin E induced mammary tumors

Abstract Background: Cyclin E, a key mediator for G1 to S transition, has unique tumor-specific deregulation in multiple malignancies including breast, ovarian, and colorectal cancers. Our group previously reported that the oncogenic roles of cyclin E are primarily a consequence of the accumulation of several truncated forms, collectively termed low-molecular-weight cyclin E (LMW-E). LMW-E isoforms provide a growth advantage by inducing early G1 to S phase transition, genomic instability, increased cell proliferation and metastasis, as shown in breast cancer cell lines and transgenic mouse models. Clinically, assessment of 2,500 breast cancer patients has revealed that LMW-E expression in tumors is associated with a high risk of recurrence across all subtypes. Further, LMW-E expression is an independent predictor of response to neoadjuvant chemotherapy. Current gaps in knowledge in the field that remain pertain to (i) if LMW-E is an early or late event in oncogenesis (ii) if it is required for both the initiation and maintenance of mammary tumors, (iii) the secondary oncogenic events induced by LMW-E and if these events can be targeted therapeutically. Methods: To address the above questions, we generated three tetracycline-inducible LMW-E (TLMW-E) transgenic model systems with expression of mammary specific human LMW-E in different genetic backgrounds: These include a bi-transgenic model (MMTV-rtTA and Tet-O-LMW-E), a tri-transgenic (bi-transgenic in a p53+/- background) and quad-transgenic (tri-transgenic in a cyclin E knockout background). For each model, TLMW-E mediated tumor initiation, progression and metastasis, is reported as a function of time. Molecular analysis of the mammary gland and tumors are performed to identify therapeutic vulnerabilities of LMW-E expressing tumors. Results: TLMW-E dependent alterations in cell cycle are early and irreversible events, leading to increased proliferation starting from the hyperplastic stages of tumor development. Transcriptomic analysis of TLMW-E derived mammary tumors revealed significant alterations in the G2/M transition and E2F signaling. We also interrogated the role of G2/M regulator, Aurora Kinase A (AURKA), that was altered in a TLMW-E dependent fashion. Correlative analysis using publicly available datasets confirmed the linked co-overexpression of AURKA and cyclin E in basal breast cancer as compared to normal breast tissue. In vivo studies using the AURKA inhibitor, alisertib, showed that mammary tumors were sensitive to AURKA inhibition as a single agent and in combination with chemotherapy, as a function of LMW-E expression. Conclusion: Collectively, our data suggest that the induced expression of LMW-E is tumorigenic, independent of p53 or endogenous cyclin E status and responsive to treatment with an AURKA inhibitor. Citation Format: Said Akli, Amriti R. Lulla, Francisco R. Saenz, Fuchenchu Wang, Natalie W. Fowlkes, Tuyen Bui, Yan Wang, Sofia Mastoraki, Min Jin Ha, Kelly K. Hunt, Khandan Keyomarsi. Aurora kinase is an actionable target in low molecular weight cyclin E induced mammary tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2592.

Abstract 1052: Augmentation of standard chemotherapy response by inhibition of Aurora kinase A in preclinical models of pancreatic cancer

Abstract Background: The prognosis of pancreatic ductal adenocarcinoma (PDAC) is very poor with a 5-year survival rate of 8%. Current standard chemotherapeutic treatment nab-paclitaxel (NPT) plus gemcitabine (Gem) has been able to give an average prognosis of 8.5 months after detection. Aurora Kinase A (AURKA), an enzyme responsible for the regulation of cytokinesis, is mutated in over 55% of PDAC. Increased expression of AURKA inhibits metabolic stress-induced autophagy, thus promoting cell survival. Additionally, amplification of AURKA has been shown to lead to taxane resistance. Through inhibition of AURKA signaling with a small-molecule inhibitor alisertib (MLN8237, ALS), we evaluated an enhancement of NPT+Gem chemotherapy response in PDAC. Methods: In vitro cell proliferation was evaluated in PDAC-associated cell lines by WST-1 assay. Protein expression was measured by Immunoblot analysis. Tumor growth and survival studies were performed using human PDAC cells AsPC-1 in NOD/SCID mice. Intratumoral mechanism of action was determined by IHC and Immunoblot analysis. Results: In AsPC-1 subcutaneous xenografts, chemotherapy regimen (NPT+Gem) and alisertib inhibited tumor growth while their combination exhibited an additive effect. The increase in tumor size was 255 mm3 in controls, 105 mm3 after NPT+Gem, 133 mm3 after ALS and 38 mm3 after NPT+Gem+ALS treatment. In peritoneal dissemination xenografts, animal survival was 23 days in controls that was increased by NPT+Gem (41 days, 78% increase), ALS (25 days, 9% increase) and NPT+Gem+ALS exhibited an additive effect (49 days, 113% increase). NPT+Gem and ALS effect on tumor cell proliferation and apoptosis corresponded with subcutaneous tumor growth data. In vitro proliferation analysis of PDAC epithelial cells (AsPC-1, Panc-1), stromal cells and endothelial cells demonstrated that NPT+Gem and ALS inhibited cell proliferation and their combination treatment had an additive effect. Immunoblot analysis of PDAC cells showed increased levels of cleaved caspase-3 and cleaved PARP-1 in combination therapy pointing to increased levels of apoptosis with NPT+Gem+ALS treatment in all cell lines tested. Conclusion: These findings demonstrate that nab-paclitaxel plus gemcitabine standard chemotherapy response can be enhanced through specific inhibition of aurora kinase A signaling by alisertib in PDAC. The data support the potential of this combinatorial therapeutic strategy for clinical PDAC therapy. Citation Format: David Kronenberger, Margaret A. Schwarz, Md Sazzad Hassan, Urs von Holzen, Roderich E. Schwarz, Niranjan Awasthi. Augmentation of standard chemotherapy response by inhibition of Aurora kinase A in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1052.

Abstract 2015: The Aurora kinases are a potential therapeutic target in Ewing sarcoma

Abstract The poor survival rates and limited treatment options for patients with relapsed/metastatic Ewing sarcoma (ES) highlights the need for more personalized targeted therapeutic approaches. In this project, we have investigated the prognostic potential of Aurora kinase expression in ES patients and evaluated the activity of Aurora kinase inhibitors in ES cell lines and patient derived cultures. The prognostic value of Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Aurora kinase C (AURKC) was determined through interrogation of the online GSE17618 dataset using Cox models and Kaplan Meier plots. RNA seq data was obtained from the CCRG dataset for expression analysis (Roundhill et al., 2021, Cell Oncol, 44(5), 1065-85). Viable cell number was determined using the trypan blue exclusion assay on the Vi-CELL XR. Cells were treated with increasing concentrations of inhibitor (Table 1) or DMSO vehicle control for 48h. Protein was extracted after 48h for western blotting. In ES patients, high RNA expression of AURKA and AURKB is associated with a 5- and 3-fold increased risk of death respectively (n=42). Risk of an event was increased 3- or 2-fold with high expression of AURKA or AURKB respectively (n=42). High AURKC expression was not prognostic. Expression of AURKA and AURKB was confirmed at the RNA level in ES cell lines and patient derived cultures, and at the protein level in cell lines. Aurora kinase inhibitors (Table 1) decreased viable cell number of ES cell lines and patient derived cultures. Western blotting confirmed a decrease in AURKA and MYC-C expression with increasing concentration of inhibitor. AMG900 was the most potent inhibitor in TC32 cells (Table 1) and decreased viable cell number of patient derived cultures (n=5, p<0.01). High expression of AURKA or AURKB mRNA predicts poor outcome for ES patients. Inhibitors of these kinases reduce viable cell number of both ES cell lines and patient derived cultures. We are currently investigating the mechanism of action. Table 1. Summary of the effect of Aurora kinase inhibitors in TC32 ES cells. Aurora Kinase Inhibitor Concentration Range Used (nM) Target Kinases(IC50, nM) EC50 TC32 Cells (nM) (n=3) P value (EC50 AMG900 vs EC50 another inhibitor) References AMG900 1.25-50 AurA (5), AurB (4), AurC (1) + 10 kinases 1.57 N/A Payton et al., 2010, Cancer Res, 70(23), 9846-54 MLN8237 5-300 AurA (1.2), AurB (396.5) + 22 kinases 29.48 0.0468 Manfredi et al., 2011, Clin Cancer Res, 17(24), 7614-24 CCT137690 5-300 AurA (15), AurB (25), AurC (19) + 3 kinases 97.56 0.0004 Bavetsias et al., 2010, J Med Chem, 53(14), 5213-28 LY3295668 31.25-1000 AurA (0.8) AurB (1038)AurC (98) + 2 kinases Not able to calculate N/A Gong et al., 2019, Cancer Discov, 9(2), 248-263 EC50 values were calculated by nonlinear regression (GraphPad PRISM). Statistical differences between EC50 of AMG900 and the EC50 of other inhibitors were evaluated using two-way ANOVA and Tukey’s multiple comparisons test. IC50 - half maximal inhibitory concentration, EC50 - half maximal effective concentration Citation Format: Molly McNae, Elizabeth A. Roundhill, Susan A. Burchill, Richard W. Bayliss. The Aurora kinases are a potential therapeutic target in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2015.

Abstract 1847: Enhancing the efficacy of conventional chemotherapy with novel targeted small molecules to kill Ewing sarcoma cancer stem-like cells

Abstract Since Ewing sarcoma (ES) is a stem cell-like cancer1, improved outcome for some patients will be realised when treatments to eradicate ES stem-like cells (ES-CSCs) are evaluated in clinical trials. Therefore, we have identified small molecules that kill ES-CSCs, and examined their efficacy alone and in combination with chemotherapy. ES patient-derived cultures and paired ES-CSCs established from treatment-naïve tumors were investigated1. The effect of 1293 small molecules (5µM, 48h) on ES-CSC cell number was examined using high content imaging (Operetta). The effect of the most active small molecules was examined in combination with chemotherapies used to treat ES using high content imaging and CalcuSyn (Biosoft). ES-CSCs were more resistant to chemotherapy than paired patient derived cells (p<0.05)1. In the drug screen, 6% (75/1293) of small molecules were more effective at killing ES-CSCs than doxorubicin. Eight percent (6/75) of the most effective small molecules targeted the Ras/MAPK pathway, including inhibitors of aurora kinases (n=3) and RAS GTPases (n=3). The efficacy of aurora kinase inhibitors, CCT137690 and CD532, was confirmed in 6 ES-CSCs. Patient-derived cells were more sensitive to aurora kinase inhibitors than ES-CSCs (p<0.0001). In combination with chemotherapy, CCT137690 and CD532 synergistically killed patient-derived ES cells (Table 1). We have identified small molecules that kill patient-derived ES-CSCs and confirmed they have synergistic activity with classic chemotherapies, supporting our hypothesis that ES-CSC targeted drugs in combination with existing chemotherapies is a promising therapeutic strategy. 1 Roundhill et al. 2021. Cellular Oncology, 44(5):1065-1085 Table 1. Summary of synergy between the aurora kinase inhibitors and chemotherapeutic agents in 4 patient-derived cell cultures (ES1 - ES4). Patient derived cells were treated with increasing concentrations of chemotherapies and small molecules (0.25x, 0.5x, 2x, 4x the EC50). Concentration range of doxorubicin=0.08-6.7µM, vincristine=0.01-1.5µM, etoposide=0.01-33µM, actinomycin-D=0.02-10µM, carboplatin=8.4-135µM, 4-hyperoxyifosfamide=2.6-152µM, CCT137690=0.07-14µM, CD532=0.05-14µM. Where data is available, concentrations did not exceed the maximum achievable concentration in plasma (Liston and Davis. 2017. Clinical Cancer Research, 15(14):3489-3498). Synergy = combination index <0.9. ES1 ES2 ES3 ES4 CCT137690 (EC50, uM) 3 3.6 0.3 0.4 CD532 (EC50, uM) 1.8 3.5 0.2 1.1 Doxorubicin + CCT137690 Synergy Synergy Synergy Synergy Doxorubicin + CD532 Synergy Synergy Vincristine + CCT137690 Synergy Vincristine + CD532 Synergy Synergy Etoposide + CCT137690 Synergy Synergy Etoposide + CD532 Synergy Synergy Actinomycin-D + CCT137690 Synergy Synergy Actinomycin-D + CD532 Synergy Synergy Synergy Carboplatin + CCT137690 Synergy Synergy Carboplatin + CD532 Synergy Synergy 4-Hyperoxyifosfamide + CCT137690 Synergy Synergy 4-Hyperoxyifosfamide + CD532 Synergy Citation Format: Elizabeth A. Roundhill, Lee Jeys, Michael Parry, Kenneth S. Rankin, Richard Foster, Jacquelyn Bond, Susan Burchill. Enhancing the efficacy of conventional chemotherapy with novel targeted small molecules to kill Ewing sarcoma cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1847.

Abstract 1416: Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. The disease-initiating event in most CRC cases is the loss of function (LOF) of the adenomatous polyposis coli (APC) gene, accounting for 70-80% of CRC. Strategies to target cells that harbor the truncation mutation in APC, which leads to its LOF, may provide the opportunity to intercept tumorigenesis in CRC patients. High-risk CRC populations include patients with familial adenomatous polyposis (FAP), the inherited disorder caused by germline APC mutations. Aurora Kinase (AK), a family of serine/threonine kinases, is essential for proper cell division, and overexpression of AK has been associated with multiple heme and solid tumor malignancies, including CRC. Aurora kinases have been well-studied, both preclinically and clinically, as a target for therapeutic intervention across multiple cancers; however, the role of AK in CRC disease progression has not been fully explored. Here we report a synthetic lethal dependency between APC and AK which was identified in a human isogenic colon organoid model containing a genetically engineered APC LOF (APCLOF). Aurora A-specific, Aurora B-specific, and Pan-AK inhibitors were evaluated in isogenic APCLOF or APCWT colon organoids derived from multiple donors. All AK inhibitors demonstrated selective growth inhibition of the APCLOF organoid. The strongest synthetic lethal effect was observed with a pan-AK inhibitor showing greater than 1000-fold selectivity in the APCLOF colon organoids compared to APCWT. Interestingly, this activity diminished as oncogenic driver mutations in KRAS and/or p53 genes were introduced into the APCLOF organoids. These findings suggest that therapeutic benefit of AK inhibitors would be greater in early APCLOF driven adenomas as opposed to advanced CRC. Despite these encouraging results indicating therapeutic opportunity in early adenoma disease, on-target genotoxicity was observed in vitro through increases in micronuclei at pharmacologic doses. Therefore, although there is a high unmet need in FAP for therapeutic options, potential genotoxicity associated with AK inhibitor treatments may limit utility as an intervention strategy. Citation Format: Dana S. Gaffney, Eileen Vesely, David Pocalyko, Kurtis Bachman, Karla Wiehagen, Glenn Cowley, Victoria Wong, Prashanth Gokare, Peggy Guzzie-Peck, Sandy Weiner, Ann De Smedt, Jacqueline Kinyamu-Akunda, Tammy Bush, Zhuming Zhang, Robert Schulingkamp, Sylvia F. Boj, Rene Overmeer, Carla Verissimo, Roshni Nair. Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1416.

Abstract 3251: Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1

Abstract Effective targeted therapies are lacking for head and neck squamous cell carcinoma (HNSCC) that is common and fatal. Developing targeted therapies for HNSCC is challenging as the genomic landscape is dominated by mutations in tumor suppressors, including NOTCH1. To address this need, we previously demonstrated that PI3K inhibition led to apoptosis in NOTCH1 mutant HNSCC cell lines. The underlying mechanism of this sensitivity is unknown and important to elucidate as modest responses and development of acquired resistance are the leading causes of failure for targeted therapies. To address this knowledge gap, we measured the levels of 304 proteins using reverse phase protein array in NOTCH1WT and NOTCH1MUT HNSCC cell lines upon PI3K inhibition and identified Aurora kinase B (AURKB) to be differentially regulated. We also identified AURKB levels to be maintained in the NOTCH1MUT cell lines with acquired resistance to PI3K inhibition. To determine if the maintenance of AURKB expression contributes to PI3K inhibitor resistance, we depleted AURKB in resistant NOTCH1WT HNSCC cells and overexpressed AURKB in sensitive NOTCH1MUT HNSCC cells. This manipulation of AURKB levels led to increased sensitivity and resistance to PI3K inhibition respectively. To use a pharmacologic approach, we combined the pan-Aurora kinase inhibitor danusertib (0-2µM) with PI3K/mTOR inhibitor omipalisib (0-200nM) in 56 HNSCC cell lines for 72h and observed a substantial decrease in cell viability in >80% of NOTCH1WT and >90% of NOTCH1MUT HNSCC lines. Additionally, concurrent Aurora kinase and PI3K inhibition in NOTCH1WT, NOTCH1MUT, and NOTCH1MUT acquired resistant HNSCC cells for 24h resulted in elevated cell death compared to single agents, as measured by the induction of cleaved PARP and cleaved Caspase 3; and Annexin V positive cells. Mice bearing NOTCH1MUT xenografts showed complete tumor regression when treated with a combination of pan-PI3K inhibitor Copanlisib and Aurora inhibitor Alisertib as compared to control groups. AURKB depletion in NOTCH1WT and overexpression in NOTCH1MUT HNSCC cells revealed significant changes in the total protein levels of AKT and PDK1. Similarly, AKT depletion and overexpression in NOTCH1WT and NOTCH1MUT HNSCC cells altered sensitivity to PI3K inhibition. However, manipulation of AKT levels affected PDK1 and not AURKB levels, demonstrating AURKB to be upstream to AKT and PDK1. Therefore, maintenance of AURKB levels mediates resistance to PI3K inhibition in HNSCC through AKT and PDK1. We identified AURKB as a central player governing the sensitivity to PI3K inhibitor-induced apoptosis in the context of NOTCH1 mutation status in HNSCC through its effects on AKT and PDK1. These novel findings may lead to the development of more robust therapeutic approach for NOTCH1 mutant squamous carcinoma as well as patients who develop acquired resistance to targeted therapies. Citation Format: Pooja A. Shah, Anne M. Fernandez, Vaishnavi Sambandam, Hongyun Zhao, Tuhina Mazumdar, Li Shen, Jing Wang, Faye M. Johnson. Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3251.

Abstract 6303: Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models

Abstract Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin lymphoma (NHL) and the most common form of adult lymphoma. Despite efforts to develop new therapies, the standard of care for DLBCL remains chemotherapy based. The current standard of care is comprised of a regimen of 4 drugs (cyclophosphamide, doxorubicin, vincristine, and prednisone) combined with immunotherapy using a chimeric mAb against the protein CD20 (rituximab), R-CHOP. Unfortunately only 50 to 60% of patients with DLBCL achieve and maintain complete remission after first-line therapy and 40 to 50 % relapse or have refractory disease. In addition, the average age at the time of diagnosis for DLBCL is 64 which makes intensive chemotherapy challenging. Therefore, the development of new treatment options is critical. We explored preclinical activity of AZD2811 nanoparticle, a potent and selective inhibitor of Aurora B kinase combined with venetoclax, an inhibitor of BCL2 currently used as standard of care in treatment of Haematological cancers, in B-Cell non-Hodgkin lymphoma models. Twenty five B-cell NHL cell lines including 11 Diffuse Large B-cell Lymphoma (DLBCL) cell lines were exposed to a 7 x 7 matrix of AZD2811 and venetoclax for 72 hrs. Cell viability was assessed using Celltiter-glo. Strong combination activity (HSA > 0.1 and combination Emax > 0.5) was observed in 6 cell lines including 2 DLBCL cell lines (WSU-DLCL2 and KARPAS_422). Moreover, pre-treatment with pan caspase inhibitor Q-VD-OPH, increased cell viability in cells treated with the combination. Next we explored the significant gene expression and mutation biomarkers (effect size > 1, p < 0.05) for this combination associated with Bliss and combination Emax in B-cell NHL cell lines. We found downregulation of 4 genes (FLT4, TIE1, PRKN) and mutations in 2 genes (EP300 and PTEN) to be associated with Bliss score and upregulation of one gene (FGR) associated with combination Emax. In vivo, antitumor activity of AZD2811 combined with venetoclax was assessed in mice bearing WSU-DLCL2luc xenografts. Once weekly intravenous administration of 25 mg/kg AZD2811 resulted in a statistically significant tumor growth inhibition (TGI) of 74%, while daily oral gavage of 100 mg/kg venetoclax resulted in 49% TGI but failed to reach statistical significance. While both monotherapies were unable to prevent progressive tumor growth, a combination dosing regimen of AZD2811 and venetoclax drove progressive tumor regression resulting in statistically significant complete regression (98% regression) by the third week of dosing. These results suggest combining AZD2811 with venetoclax has potential to be active in DLBCL patients. Citation Format: Azadeh Cheraghchi-Bashi, Brandon Willis, Kevin. Mongeon, Jacob Gordon, Krishna Bulusu, Elizabeth Coker, Patricia Jaaks, Mathew Garnett, Jerome Mettetal. Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6303.

Identification and Characterization of Natural and Semisynthetic Quinones as Aurora Kinase Inhibitors.

Aurora kinases (Aurora A, B, and C) are a family of serine/threonine kinases that play critical roles during mitotic initiation and progression. Aurora A and B kinases are ubiquitously expressed, and their overexpression and/or amplification in many cancers have been associated with poor prognosis. Several inhibitors that target Aurora kinases A, B, or both have been developed during the past decade with efficacy in different in vitro and in vivo models for a variety of cancers. Recent studies have also identified Aurora A as a synthetic lethal target for different tumor suppressors, including RB1, SMARCA4, and ARID1A, which signifies the need for Aurora-A-selective inhibitors. Here, we report the screening of a small library of quinones (nine naphthoquinones, one orthoquinone, and one anthraquinone) in a biochemical assay for Aurora A kinase that resulted in the identification of several quinones as inhibitors. IC50 determination against Aurora A and B kinases revealed the inhibition of both kinases with selectivity toward Aurora A. Two of the compounds, natural quinone naphthazarin (1) and a pseudo anthraquinone, 2-(chloromethyl)quinizarin (11), potently inhibited the proliferation of various cancer cell lines with IC50 values ranging from 0.16 ± 0.15 to 1.7 ± 0.06 and 0.15 ± 0.04 to 6.3 ± 1.8 μM, respectively. Treatment of cancer cells with these compounds for 24 h resulted in abrogated mitosis and apoptotic cell death. Direct binding of both the compounds with Aurora A kinase was also confirmed through STD NMR analysis. Docking studies predicted the binding of both compounds to the ATP binding pocket of Aurora A kinase. We have, therefore, identified quinones as Aurora kinase inhibitors that can serve as a lead for future drug discovery endeavors.

Antiproliferative Activity of a New Quinazolin-4(3H)-One Derivative via Targeting Aurora Kinase A in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common lung cancer subtype. Although chemotherapy and targeted therapy are used for the treatment of patients with NSCLC, the survival rate remains very low. Recent findings suggested that aurora kinase A (AKA), a cell cycle regulator, is a potential target for NSCLC therapy. Previously, we reported that a chemical entity of quinazolin-4(3H)-one represents a new template for AKA inhibitors, with antiproliferative activity against cancer cells. A quinazolin-4(3H)-one derivative was further designed and synthesized in order to improve the pharmacokinetic properties and antiproliferation activity against NSCLC cell lines. The derivative, BIQO-19 (Ethyl 6-(4-oxo-3-(pyrimidin-2-ylmethyl)-3,4-dihydroquinazolin-6-yl)imidazo [1,2-a]pyridine-2-carboxylate), exhibited improved solubility and antiproliferative activity in NSCLC cells, including epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI)-resistant NSCLC cells. BIQO-19 effectively inhibited the growth of the EGFR-TKI-resistant H1975 NSCLC cells, with the suppression of activated AKA (p-AKA) expression in these cells. The inhibition of AKA by BIQO-19 significantly induced G2/M phase arrest and subsequently evoked apoptosis in H1975 cells. In addition, the combination of gefitinib and BIQO-19 exhibited synergistic antiproliferative activity in NSCLC cells. These findings suggest the potential of BIQO-19 as a novel therapeutic agent for restoring the sensitivity of gefitinib in EGFR-TKI-resistant NSCLC cells.

First-in-human, phase I study of TT-00420, a multiple kinase inhibitor, as a single agent in advanced solid tumors.

3013 Background: TT-00420 is a spectrum-selective multi-kinase inhibitor that targets cell proliferation, angiogenesis, and immune-oncology pathways by inhibiting Aurora kinases A/B and Janus kinases (JAK) involved in cytokine signaling and receptor tyrosine kinases (FGFRs and VEGFRs) involved in the tumor microenvironment. TT-00420 has demonstrated anti-tumor activity in both in vitro and in vivo preclinical models of solid tumors, including triple-negative breast cancer (TNBC) and cholangiocarcinoma (CCA). Methods: This phase I, first-in-human, dose escalation and expansion study of TT-00420 ( NCT03654547 ) enrolled adult patients with advanced or metastatic solid tumors. Capsules in 1 mg or 5 mg formulation were administered orally once daily in 28-day cycles. Dose escalation was guided by Bayesian modeling with overdose control. The primary safety endpoints were to determine dose limiting toxicities (DLTs) and a dose recommended for dose expansion (DRDE). Secondary endpoints included pharmacokinetics (PK) and preliminary efficacy evaluated per RECIST v1.1 criterion. Results: As of February 7, 2022, 48 advanced solid tumor patients were enrolled in the study, and received at least one dose of TT-00420 in 7 dose levels: 1 mg q.d. (N = 1), 3 mg q.d. (N = 1), 5 mg q.d. (N = 4), 8 mg q.d. (N = 10), 10 mg q.d. (N = 6), 12 mg q.d. (N = 20), and 15 mg q.d. (N = 6). DLTs were observed in 3 out of 40 DLT-evaluable patients, including 1 patient at 8 mg q.d. who had Grade (Gr) 3 palmar-plantar erythrodysaesthesia syndrome and 2 patients at 15 mg q.d. who both had Gr 3 hypertension. Among the twenty (20) safety evaluable patients treated at 12 mg, the DRDE, drug-related TEAEs included hypertension (n = 11, 55.0%; Gr 3: n = 6, 30%); diarrhea (n = 7, 35%, Gr 3: n = 1, 5%); mucosal inflammation (n = 7, 35%; Gr 3: n = 1, 5%); palmar-plantar erythrodysaesthesia syndrome (n = 6, 30%; Gr 3: n = 0, 0%); and vomiting (n = 4, 20%; Gr 3: n = 0, 0%). No grade 4 suspected adverse events were reported. Out of 42 patients who had at least one post-baseline scan, 7 (16.7%) had a best response of partial response (PR) and 22 (52.4%) had stable disease (SD). Among 7 PRs, 3 were CCA patients (one for each treated at 8 mg, 10 mg, or 12 mg), 2 were TNBC patients (one for each at 10 mg, or 12 mg), 1 was HER2-negative BC patient at 12 mg, and 1 was CRPC patient at 12 mg. Sustainable stable disease for six months or longer was observed in patients with colon cancer (n = 1), head and neck cancer (n = 1), and peritoneal mesothelioma (n = 1). Conclusions: TT-00420 monotherapy was well tolerated and had favorable PK characteristics. The TEAEs observed in dose escalation and dose expansion cohorts were manageable with concomitant treatment and/or dose interruptions of TT-00420 and reversible upon the discontinuation of TT-00420 treatment. Taking safety, efficacy and clinical PK into consideration, 10 mg p.o. q.d. was recommended for phase II study of TT-00420 in patients with advanced CCA. Clinical trial information: NCT03654547.

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.

A new imaging platform (iScreen) allows for the concurrent assessment of micronucleus induction and genotoxic mode of action in human A375 cells.

Genotoxicity testing guidelines require the assessment of the clastogenic and aneugenic potential of compounds. While in vitro micronucleus assays detect both types of endpoints, it requires labor-intensive microscopic scoring and does not discriminate between the two modes of actions. Here, we present a novel high-content imaging platform in A375 human cells that addresses the need for rapid scoring while providing additional mechanistic information. We evaluated the new platform with 12 compounds, three compounds from each mechanistic class (clastogen, aneugen tubulin binder, aneugen aurora inhibitor, and nongenotoxicant) following 4- and 24-h compound treatments. The approach we developed is first discriminating between genotoxicant and nongenotoxicant using an image analysis algorithm to quantify micronucleus induction below a 60% cytotoxicity cutoff. Then it uses centromere protein A (CENPA) staining for the genotoxic compounds to discriminate between aneugens and clastogens. Lastly, we use phosphorylated histone H2AX Ser139 (γH2AX) staining to confirm clastogenicity and changes in phosphorylated histone 3 Ser10 (pH3) and increases in polyploidy in mitotic cells to discriminate between aneugens that bind tubulin from those that affect aurora kinases. All compounds were correctly classified, and we showed by using benchmark dose-response analysis that the imaging platform in A375 cells is at least as sensitive as the MicroFlow® assay in TK6 cells for genotoxicant but appears to be more specific for the nongenotoxicants. A detailed comparison of the cell lines and a more comprehensive validation with a much larger compound set, predictive and dose-response modeling will be presented in the future.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Abstract B041: Mimicking tumor acidic and hypoxic microenvironment in vitro towards generation of more predictive screening platform for solid tumors

Abstract The drug discovery process in oncology relies mainly on screening and preclinical validation platforms that use human cancer cells that are grown in standard monolayer (2D) cultures. However, 2D monolayer cell culture methods poorly reflect the tumor microenvironment (TME) and the transcriptome and epigenome attributes of the cancer cells in vivo, leading to inaccurate estimate of drug efficacy in clinic. The TME of solid tumors is complex yet it consist of generic hallmarks such as hypoxia and extracellular acidosis that are common across tumor types and directly modulate gene expression and epigenome. Here we hypothesized that in vitro culture systems that recapitulate the acidic and hypoxic features of the solid TME, more accurately reflect the gene expression profile of cells in in vivo tumors, and thus serve as improved drug screening platform that reliably determine drug efficacies. Here we surveyed systematically the impact on transcriptome and epigenome hallmarks of exposure to hypoxia and acidosis on 2D and 3D cultures of A549 lung adenocarcinoma cell line as a model for solid tumor. To recapitulate naturally occurring hypoxia in vitro, A549 cells were adapted over several passages to growth in 3D. Transcriptome data derived from cells adapted to 3D, when compared to 2D and patient tumor datasets suggested that time spent in 3D increased the concordance of transcriptome profile with tumors from patients. Furthermore adaptation to 3D revealed emergence of hypoxia hallmarks when compared to 2D. To model the combined effect of hypoxia and acidosis, the 3D adapted A549 spheroids were additionally exposed to low pH conditions (6.4) for a duration of 5 days. The additional exposure to low pH led to enhanced tumor progressive phenotypes as evidenced by enrichment in genes associated with hallmarks of inflammation, epithelial mesenchymal transition (EMT), cell invasion and drug resistance. To determine the impact of hypoxic and acidic TME on cytotoxic response to anti-cancer drugs, the 2D and 3D adapted spheroids with or without additional exposure to low pH were screened against a library consisting of 184 anti-cancer drugs. Additional exposure to low pH led to an overall increase in drug resistance. However the combined hypoxic and acidic conditions in 3D revealed novel susceptibilities for compounds that belong to the family of Aurora kinase inhibitors. These results provide indications that accurate modeling of hypoxic and acidic TME in vitro could reveal expected resistance mechanisms and specific drug susceptibilities. This study introduces a 3D screening platform that recapitulates the acidosis and hypoxia of solid TME and captures the transcriptomic profile in vitro that is highly concordant with tumors in patients. This model allows the study of resistance mechanisms and unravel novel susceptibilities that is otherwise undiscovered in standard 2D conditions and could result in the identification of anticancer drugs that are more clinically relevant. Citation Format: Nazanin Rohani Larijani, Marielle Huot, Anne Lenferink, Noël R. Raynal. Mimicking tumor acidic and hypoxic microenvironment in vitro towards generation of more predictive screening platform for solid tumors [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B041.

AURKA and PLK1 inhibition selectively and synergistically block cell cycle progression in diffuse midline glioma

SummaryDiffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. In this study, we show that Aurora kinase A (AURKA) is overexpressed in DMG and can be used as a therapeutic target. Additionally, AURKA inhibition combined with CRISPR/Cas9 screening in DMG cells, revealed polo-like kinase 1 (PLK1) as a synergistic target with AURKA. Using a panel of patient-derived DMG culture models, we demonstrate that treatment with volasertib, a clinically relevant and selective PLK1 inhibitor, synergizes with different AURKA inhibitors, supporting the CRISPR screen results. Mechanistically, our results show that combined loss of PLK1 and AURKA causes a G2/M cell cycle arrest which blocks vital parts of DNA-damage repair and induces apoptosis, solely in DMG cells. Altogether, our findings highlight the importance of AURKA and PLK1 for DMG propagation and demonstrate the potential of concurrently targeting these proteins as a therapeutic strategy for these devastating pediatric brain tumors.