Aurora Kinase Inhibitors Research Articles

4-[(5-Methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone Inhibits MCPyV T Antigen Expression in Merkel Cell Carcinoma Independent of Aurora Kinase A.

Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV), and MCPyV-positive tumor cells depend on expression of the virus-encoded T antigens (TA). Here, we identify 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone (PHT)-a reported inhibitor of Aurora kinase A-as a compound inhibiting growth of MCC cells by repressing noncoding control region (NCCR)-controlled TA transcription. Surprisingly, we find that TA repression is not caused by inhibition of Aurora kinase A. However, we demonstrate that β-catenin-a transcription factor repressed by active glycogen synthase kinase 3 (GSK3)-is activated by PHT, suggesting that PHT bears a hitherto unreported inhibitory activity against GSK3, a kinase known to function in promoting TA transcription. Indeed, applying an in vitro kinase assay, we demonstrate that PHT directly targets GSK3. Finally, we demonstrate that PHT exhibits in vivo antitumor activity in an MCC xenograft mouse model, suggesting a potential use in future therapeutic settings for MCC.

Interaction between Human Papillomavirus-Encoded E6 Protein and AurB Induces Cell Immortalization and Proliferation-A Potential Target of Intervention.

The human papillomavirus E6 and E7 oncoproteins interact with a different subset of host proteins, leading to dysregulation of the apoptotic, cell cycle, and signaling pathways. In this study, we identified, for the first time, that Aurora kinase B (AurB) is a bona fide interacting partner of E6. We systematically characterized the AurB-E6 complex formation and its consequences in carcinogenesis using a series of in vitro and cell-based assays. We also assessed the efficacy of Aurora kinase inhibitors in halting HPV-mediated carcinogenesis using in vitro and in vivo models. We showed that AurB activity was elevated in HPV-positive cells, and this correlated positively with the E6 protein level. E6 interacted directly with AurB in the nucleus or mitotic cells. A previously unidentified region of E6, located upstream of C-terminal E6-PBM, was important for AurB-E6 complex formation. AurB-E6 complex led to reduced AurB kinase activity. However, the AurB-E6 complex increased the hTERT protein level and its telomerase activity. On the other hand, AurB inhibition led to the inhibition of telomerase activity, cell proliferation, and tumor formation, even though this may occur in an HPV-independent manner. In summary, this study dissected the molecular mechanism of how E6 recruits AurB to induce cell immortalization and proliferation, leading to the eventual cancer development. Our findings revealed that the treatment of AZD1152 exerted a non-specific anti-tumor effect. Hence, a continuous effort to seek a specific and selective inhibitor that can halt HPV-mediated carcinogenesis should be warranted.

Abstract 1565: Targeting of KIF11 and AURKA to improve outcomes in Ewing sarcoma

Abstract Ewing Sarcoma (EWS) is a rare and aggressive pediatric malignancy of the bone and soft tissues. Despite intensive systemic combination chemotherapy and surgery/radiotherapy for local control, patients with metastatic disease have dismal prognosis (<30% overall survival rate). To identify better therapies for patients with refractory, recurrent and metastatic disease, we previously conducted in silico and drug re-purposing screens (PMID: 27863422; 31025088) that identified proteins essential for mitotic spindle formation and cell cycle progression such as Aurora Kinase A (AURKA), Kinesin Family Member 11 (KIF11), Kinesin Family Member 15 (KIF15) and its binding partner; targeting protein for Xklp2 (TPX2) that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, indicating their potential as therapeutic targets. KIF11 and KIF15 are mitotic motor kinesins essential for bipolar spindle formation, whereas AURKA is a mitotic kinase essential for centrosome maturation and mitotic spindle assembly. Though KIF11 and AURKA inhibitors have been extensively used in clinical trials before but have not been tested together in combination for any type of cancer. We hypothesized that targeting these two key proteins essential for mitotic progression provides a therapeutic vulnerability for Ewing Sarcoma by inducing G2/M arrest and mitotic catastrophe. Specific KIF11 (SB-743921) and AURKA (VIC-1911, VITRAC Therapeutics) inhibitors were used for targeting these proteins. We evaluated the in vitro activity of VIC-1911 and SB-743921 and synergy was measured by percentage inhibition of growth via SynergyFinder, that employs reference models (Bliss) to score synergy (scores >10 is synergistic). EWS showed strong synergistic interaction at physiologically relevant doses compared to control cell lines (e.g., mesenchymal stem cells and non-EWS tumor cells). This drug combination was effective in refractory EWS cells established post-chemotherapy (synergy score >24) and at much lower doses in cells established from pre-treatment tumor tissue (synergy score >27) indicating effectiveness at various clinical stages of EWS disease. Importantly, this therapy appeared preferential to EWS cells with an EWS-ETS fusion as compared to non-EWS cells. We observed zero colony formation in vitro with the combination treatment and increased percentage accumulation of cells in G2/M phase upon cell-cycle analysis indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed sustained tumor regression (NED at 30 days after ending therapy). The combination arm showed significant survival versus control (p≤ 0.001, HR=0.42, 95% CI=0.14-1.25), VIC-1911 (p≤ 0.001, HR=0.48, 95% CI=0.16-1.42) or SB-743921 (p ≤ 0.01, p≤ HR=0.71, 95% CI=0.21-2.34) alone. Efforts are underway to translate these preclinical studies into an early phase clinical trial of patients with histologically proven EWS who have failed standard of care therapy. Citation Format: Soumya M. Turaga, Vikalp Vishwakarma, Stacey Hembruff, Rajni Puri, Priya Sabu, Glenson Samuel, Andrew K. Godwin. Targeting of KIF11 and AURKA to improve outcomes in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1565.

Abstract 542: Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma

Abstract The purpose of this study was to identify new combination strategies for improving treatment of head and neck squamous cell carcinomas (HNSCCs). HNSCCs often express high levels of Aurora kinase A (AURKA), most studied as a kinase important for mitotic progression in normal cells. Notably the overexpression of AURKA in many cancers allows it to act throughout the cell cycle, acquiring new cancer-promoting activities. While several AURKA inhibitors are in clinical trials, limited efficacy is observed with single treatment, causing us to evaluate potential AURKA combination therapeutic combinations. Particularly relevant to HNSCC, which often overexpress and depend on EGFR, overexpression of AURKA can promote resistance to EGFR inhibitors by mechanisms variously identified as including activation of the EGFR effectors AKT, ERK, and BIM. Using viability and clonogenic assays, we have found that the AURKA inhibitor VIC-1911 synergizes with the EGFR inhibitors afatinib and erlotinib in multiple HNSCC cell lines and retains activity in HNSCC cell models selected for resistance to these EGFR inhibitors. However, AURKA inhibition increases levels of AURKA protein and its protein-stabilizing partner TPX2, counteracting drug effect. We also combined inhibition of AURKA with inhibition of WEE1, a regulator of the G2/M checkpoint, finding this dual inhibition enhanced levels of apoptosis in EGFR inhibitor resistant HNSCC cell lines. Intriguingly, one recent study reported AURKA can also modulate the glycolytic signaling pathway, increasing the cancer-promoting Warburg effect. We have found that inhibition of AURKA can significantly reduce glycolytic flux in HNSCC cell lines and alter expression and phosphorylation of key glycolytic enzymes. Dual inhibition of AURKA in combination with inhibitors of glycolytic enzymes, or enzymes regulating oxidative phosphorylation, showed marked synergy. These and other results investigating AURKA signaling mechanisms in HNSCC demonstrate that the non-canonical functions of AURKA include viable therapeutic targets that can enhance the efficacy of AURKA inhibition in HNSCC. Citation Format: Flaviane N da Silva, Theodore Nguyen, Jong Woo Lee, Barbara Burtness, Erica Golemis. Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 542.

Abstract 1200: Aurora kinase A is a potential therapeutic target regulated by the MYCN oncogene in human retinoblastoma

Abstract Retinoblastoma (RB) is a pediatric intraocular neoplasm characterized by loss of function mutations in the RB1 gene. RB has a near-perfect prognosis in developed countries, but a significant dip in patient survival is observed in low and middle-income nations, with an overall survival rate of around 40%. Chemotherapy is a primary treatment modality in RB. However, the immediate and long-term side effects of conventional and intra-arterial chemotherapies necessitate the need for additional treatment options. Aurora kinases are serine/threonine kinases that play a pivotal role in mitosis. In recent years, Aurora kinase A (AURKA) has gained prominence as a drug target in various tumors and several small molecule inhibitors of AURKA are currently in clinical development. In this study, we have shown that AURKA is overexpressed in RB patient specimens and cell lines using immunohistochemistry and immunoblotting. Moreover, induced expression of AURKA significantly correlated with optic nerve invasion of the tumor suggesting a role of AURKA in RB progression. Inhibition of AURKA in RB cell lines and patient specimens using small molecule inhibitors or selective silencing with lentivirus-mediated shRNA knockdown led to decreased cell viability, increased apoptosis, cell cycle arrest at G2/M phase and subsequent induction of polyploidy. To further investigate the deleterious effects of AURKA inhibition within the tumor microenvironment, chorio-allantoic membrane (CAM) xenografts with RB cells were performed. The in-ovo data revealed a delay in tumor growth when treated with AURKA inhibitors. MYCN is an essential oncogene in RB progression immediately to RB1 mutations. Notably, an in-silico promoter analysis predicted the presence of MYCN binding sites upstream of the transcription start site of AURKA. Chromatin immunoprecipitation - qPCR (ChIP-qPCR) confirmed the binding of MYCN to the promoter region of AURKA. Besides, immunoblots corroborated that AURKA was downregulated in MYCN knockdown RB cells. Overall, our findings show that elevated AURKA levels in RB could be targeted in-vitro and in-ovo, and its expression is transcriptionally regulated by the MYCN oncogene. These data not only shed light on the role of AURKA in RB tumorigenesis but also unravel its potential as a therapeutic target, in isolation or through MYCN-driven downstream pathways. Acknowledgements: Department of Biotechnology - Innovative Young Biotechnologist Award (DBT-IYBA to MMR), Ministry of Science and Technology, Government of India; Hyderabad Eye Research Foundation (HERF), L V Prasad Eye Institute, Bhubaneswar, India; Indian Council of Medical Research - Senior Research Fellowship (ICMR-SRF to NAB), Ministry of Health and Family Welfare, Government of India. Citation Format: Naheed Arfin Borah, Ruchi Mittal, Devjyoti Tripathy, Swathi Kaliki, Suryasnata Rath, Srinivas Patnaik, Mamatha M. Reddy. Aurora kinase A is a potential therapeutic target regulated by the MYCN oncogene in human retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1200.

Abstract 1468: MYC-C interacts with Aurora kinase A in Ewing sarcoma cells

Abstract EWSR1-ETS fusion genes result in upregulation and high expression of the oncogene MYC-C in Ewing sarcoma (ES). MYC-C is therefore an attractive therapeutic target in these aggressive cancers with limited treatment options. Unsuccessful efforts to target MYC-C proteins directly have resulted in the development of inhibitors to associated stabilizing proteins, such as Aurora kinase A (AURKA), to induce proteasomal degradation1. In this study we test the hypothesis that MYC-C and AURKA interact in ES. Peptide pull-down assays were performed using Strep-Tactin Sepharose beads and N-biotinyl MYC-C peptides. After incubation with AURKA, eluted proteins were analyzed by SDS-PAGE. Intracellular colocalization of proteins was examined by immunofluorescence of TC-32 and SK-N-MC cell lines using confocal microscopy (Zeiss LSM880) and high content imaging (CD7). Cells were stained with anti-MYC-C and anti-AURKA antibodies, and DAPI used to visualize nuclei. DuoLink® proximity ligation assays (PLA; Sigma) were performed on TC-32 and SK-N-MC cell lines. Images were captured on the confocal microscope and the number of nuclear foci quantified.MYC-C binds AURKA through its transcriptional activation domain (residues 1-155). Residues 10-34 of MYC-C are sufficient for binding with AURKA (residues 122-403) in cell-free peptide assays. Colocalization of MYC-C and AURKA was visualized in mitotic SK-N-MC cells, but not in TC-32 cells. The proteins colocalize at the spindle poles. PLA confirmed an interaction between MYC-C and AURKA in TC-32 and SK-N-MC ES cells. The number of foci per nucleus was significantly higher in the SK-N-MC p53-null cells compared to the wild-type p53 TC-32 cells (p<0.0001). In conclusion, MYC-C and AURKA colocalize and interact in the nucleus of ES cells. The frequency of interactions is greater in p53-null cells compared to cells with wild-type p53. Peptide pull-down assays identified residues 10-34 of MYC-C as the minimal AURKA interacting region. Disruption of this interaction by AURKA inhibitors may provide a mechanism for MYC-C degradation in ES, which could be exploited to develop novel therapeutic combinations to improve outcomes. 1Richards MW, Burgess SG, Poon E, Carstensen A, Eilers M, Chesler L, Bayliss R. Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors. Proc Natl Acad Sci U S A. 2016 ;113(48):13726-13731. Citation Format: Molly McNae, Selena G. Burgess, Richard Bayliss, Susan A. Burchill. MYC-C interacts with Aurora kinase A in Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1468.

Abstract 429: Efficacy of JSI-1187 or VIC-1911 in combination with KRAS G12C inhibitors for the treatment of KRAS G12C-mutated non-small cell lung cancer and colorectal cancer

Abstract KRAS is the predominantly mutated RAS isoform, accounting for 85% of RAS mutations in cancers. KRAS G12C mutation occurs in approximately 14% of lung adenocarcinomas and 3% of colorectal adenocarcinomas. Several drugs have been developed that inhibit the function of KRAS proteins with a “G12C” mutation. The response rate to these drugs in patients with KRAS G12C-mutated NSCLC is approximately 40%. Primary resistance to G12C inhibitors in these patients may be explained in part by a lack of dependency on KRAS signaling. Despite the clinical benefit observed in some patients treated with G12C inhibitors, acquired resistance to single-agent inhibitor therapy eventually develops in most patients. KRAS G12C-mutated NSCLC cell lines treated with G12C- specific inhibitor demonstrated reactivation of the down-stream MAPK pathway as a mechanism of acquired resistance. As a terminal kinase in this pathway, inhibition of ERK1/2 activity could potentially be instrumental in overcoming G12C inhibitor resistance. Moreover, aurora kinase A (AURKA) has been identified as necessary for cancer cell escape from G12C inhibitor-induced quiescence, which may be due to a stabilizing reaction between AURKA, KRAS G12C and the downstream effector CRAF. JSI-1187 is an oral reversible ERK1/2 inhibitor, and VIC-1911 is an oral selective AURKA inhibitor. To evaluate the effects of the concomitant inhibition of JSI-1187 or VIC-1911 with sotorasib, we assessed the response in 4 G12C-mutated cell lines, including two NSCLC and two CRC. Loewe synergy scores calculated showed values between 0.5 to 14.3 in these cell lines, which signified a synergistic effect of JSI-1187 or VIC-1911 combined with sotorasib. JSI-1187 or VIC-1911 combined with sotorasib or adagrasib demonstrated marked enhancement of the antitumor effect compared with single-agent treatment in a H2122 mouse xenograft model. Additionally, the antitumor growth effect of JSI-1187 and sotorasib was significantly better than the combination of the MEK inhibitor, trametinib, and sotorasib. Greater inhibition of tumor growth was also observed for the combination vs. monotherapy in H358 and SW837 CRC- bearing mouse models. Furthermore, KRAS G12C-mutated patient-derived xenografts were used to investigate a synergistic effect. Combined treatment with VIC-1911 and sotorasib demonstrated greater tumor growth inhibition than either monotherapy alone in a NSCLC PDX model. In addition, the combination of JSI-1187 and sotorasib demonstrated a significant increase in tumor growth inhibition in a CRC PDX model. Taken together, our preclinical studies suggest that the combination of an ERK inhibitor, JSI-1187, or an AURKA inhibitor, VIC-1911, may potentially overcome the primary resistance and prevent or delay the acquired resistance to G12C inhibitors. Citation Format: Lei Zhang, Ao Li, Shanzhong Jian, Chengyi Du, Wan Zhu, Yue Tao, Meiwei Wang, Wen Xu, Wenshan Hao, Linda Paradiso, Thomas Myers, Keizo Koya, Jintao Zhang. Efficacy of JSI-1187 or VIC-1911 in combination with KRAS G12C inhibitors for the treatment of KRAS G12C-mutated non-small cell lung cancer and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 429.

Abstract 1563: Concomitant inhibition of Aurora kinase A and WEE1 kinases results in synergistic tumor control and heightens DNA replication stress in head and neck and lung carcinomas

Abstract Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) and lung cancer mainly harbor disruptive mutations in TP53 and/or CDKN2A tumor suppressor genes, which are associated with refractoriness to treatment as well as poor prognosis and survival. Mutational loss of TP53 function fosters elevated expression of Aurora kinase A (AURKA) that plays a crucial role in mitotic progression at G2/M and stabilizes DNA replication forks at S phase. We have previously demonstrated that combined inhibition of AURKA and WEE1, a G2/M checkpoint kinase induced by replication stress, results in synergistic anti-tumor effects in HNSCC in vitro and in vivo. We have now further confirmed synergy using the highly selective AURKA inhibitor VIC1911 in HNSCC and lung cancers harboring TP53 mutations and explored mechanisms of cell death. VIC1911 and adavosertib combination synergistically suppressed cell growth and survival in both 2D- and 3D-culture systems relative to vehicle or single-agent treatment in TP53-mutated HNSCC FaDu and CAL27, and lung cancer A549 and NCI-H358 cells, with no observable toxicity in normal cells, predicting a favorable therapeutic index. Furthermore, combination treatment accelerated mitotic entry and resulted in accumulation of mitotic catastrophe, as demonstrated by time-lapse imaging, and apoptotic cell death as evidenced by cleaved PARP assays. Notably, we found that combination VIC1911 and adavosertib in HNSCC cells drastically slowed and stalled progression of replication forks in DNA fiber analysis (Mean: DMSO=3.117; adavosertib=1.495; VIC1911=0.9757; combination=0.5548 kb/min; P <0.0001) and amplified DNA damage as measured by γH2AX, indicating induction of severe replication stress. In vivo, this combination resulted in significant tumor regression in both HNSCC and lung adenocarcinoma patient-derived and cancer cell-derived xenografted mice compared with either vehicle or single-agent treatment. Taken together, these results suggest that AURKA inhibition increases dependency on WEE1 by enhancing replication stress and mitotic catastrophe, and support clinical evaluation of combined AURKA and WEE1 inhibition as a novel and effective treatment for HNSCC and lung cancer patients with elevated AURKA expression. Citation Format: Jong Woo Lee, Sundong Kim, Sebastian Cruz-Gomez, Jackie Shi, Cindy Yang, Barbara Burtness. Concomitant inhibition of Aurora kinase A and WEE1 kinases results in synergistic tumor control and heightens DNA replication stress in head and neck and lung carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1563.

Abstract 523: Targeting myc-amplified cancers with a novel prodrug inhibiting aurora A kinase

Abstract Upregulation of Aurora kinases has been associated with increased tumor progression, and thus they are appealing targets for the development of anti-cancer therapies. In addition to have a critical role in cell cycle regulation, Aurora kinases have been shown to stabilize MYC-family oncoproteins for the maintenance of malignancy. Aurora kinase inhibitors such as Alisertib has demonstrated compelling anti-tumor efficacies; however, the reported side effects including serious haematological disturbances have limited its risk-benefit ratio in clinical use. In this study we discovered a novel pyrimidine-based Aurora kinase inhibitor compound A that degraded MYC- and MYCN- oncoproteins with better potency than Alisertib. The acyl-based prodrug design leads to the discovery of compound B which was able to regress MYC- or MYCN- overexpressing tumor xenografts including small cell lung cancer, neuroblastoma, hepatocellular carcinoma and medulloblastoma using a once-a-week (QW) oral dosing regimen. Pharmacokinetic studies revealed that the tumor/plasma ratio of compound B was about 20 at 24 h post drug administration, and the active compound remained detectable in the tumors after 7 days. No significant haematological or liver/kidney biochemical aberration was observed in mice treated with up to 500 mg/kg of DBPR728 on a QW dosing regimen in a 21-day cycle. The unique pharmacokinetic and molecular properties of compound B hold potential clinical promise for treating MYC- and MYCN- amplified tumors with manageable on-target haematological adverse effects caused by Aurora kinase inhibition. Citation Format: Ya-Hui Chi, Chun-Ping Chang, Teng-Kuang Yeh, Wan-Ping Wang, Yen-Ting Chen, Chia-Hua Tsai, Yan-Fu Chen, Yi-Yu Ke, Jing-Ya Wang, Ching-Ping Chen, Tsung-Chih Hsieh, Mine-Hsine Wu, Chiung-Tong Chen. Targeting myc-amplified cancers with a novel prodrug inhibiting aurora A kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 523.

Abstract 5021: Preclinical evaluation of immunomodulatory effects of aurora kinase inhibition in HPV+ cancers

Abstract Human papillomavirus (HPV) is the causative agent of cervical cancer and some cancers of the penis, vulva, vagina, anus, and oropharynx. In an earlier study, we identified Aurora Kinase (AK) inhibitors from a large-scale drug screening as a unique class of reagents to induce selective apoptosis in HPV+ human tumor cells in vitro and in vivo (using HPV+ PDX mouse models). We tested the in vivo efficacy of alisertib, an Aurora Kinase A inhibitor, in an immunocompetent mouse model (mEER) for HPV+ oropharyngeal cancers. In preparation for this, we first tested 300 nM alisertib on 3 HPV+ murine cancer cell lines: TC-1, C3.43, and mEER for 48h. We observed that when compared to the vehicle (DMSO) control group, the level of phospho Aurora Kinase A was completely inhibited. Furthermore, in multiple HPV+ murine tumor cell lines we observed significant apoptosis as measured by annexin V 7-AAD staining along with higher cell surface expression of calreticulin (CRT), and high mobility group box 1 protein (HMGB1) which are markers for immunogenic cell death (ICD). Both CRT and HMGB1 serve as “eat me” signals that in turn trigger APC-mediated dead-cell antigen uptake, a crucial event for priming the responses from the innate immune system. In support of this, we observed that in a co-culture setting, the conditioned medium from alisertib-treated tumor cells increased the frequencies of CD8+ cells expressing GnzB as well as a significant decrease in the population of T regulatory cells (Tregs) (CD4+ FoxP3+). Additionally, we observed an increase in the innate immune natural killer cell subset. Future studies will explore the in vivo efficacy of Aurora Kinase inhibition along with immune checkpoint blockade and/or therapeutic vaccination. Our proposed research will have a positive impact because we will gain insight into the immune responses underlying the co-dependency of HPV+ cancers on Aurora kinase. Our research may lead to an increase in the rates of long-term, durable clinical responses or may allow for therapy de-escalation. Citation Format: Pragya Sinha, Soma Ghosh, Faye M. Johnson, Jagannadha K. Sastry. Preclinical evaluation of immunomodulatory effects of aurora kinase inhibition in HPV+ cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5021.

Abstract 1572: Antiproliferative activity of enzalutamide and alisertib in prostate cancer cells overexpressing HMGA2

Abstract Prostate cancer is one of the common types of cancer and remains the second leading cause of cancer-related deaths in men in the United States. High Mobility Group Protein AT-hook 2 (HMGA2), a DNA binding protein acts as a transcriptional regulating factor in gene transcription and facilitates epithelial-mesenchymal transition (EMT), which is usually the onset of prostate cancer progression and metastasis. HMGA2 isoforms include wild-type or full length that induces EMT, while the truncated isoform is associated with elevated proliferation and migration. Enzalutamide is a second-generation antiandrogen drug known to inhibit androgen receptor (AR) translocation into the nucleus by competitively binding AR and preventing androgen binding. Alisertib (MLN8237) is a small molecule inhibitor of Aurora kinase A has been utilized in clinical trials for neuroendocrine cancers and can also inhibit EMT. We aim to investigate which of the two drugs is effective in reducing proliferation in prostate cells overexpressing HMGA2 isoforms (wild-type and truncated). In this study, we treated LNCaP cells overexpressing HMGA2 with enzalutamide (1-30 µM) and alisertib (2.5-40 µM). Treatment with enzalutamide and alisertib indicated a dose-dependent decrease in cell proliferation with optimal dose at 20 µM for both drugs. LNCaP cells (LNCaP Neo) expressing low level of HMGA2 were observed to respond better to enzalutamide treatment, while those overexpressing HMGA2 (WT and TR) respond better to alisertib treatment. Interestingly, treatment with 20 µM enzalutamide and alisertib followed by Western blot indicated enzalutamide to have no effects on protein expression of HMGA2 isoforms, but greatly decreased the expression of AR in LNCaP cell overexpressing truncated HMGA2. On the other hand, alisertib increased the expression of wild-type HMGA2 whilst having no effects on protein expression of AR. Both drugs at 20 µM did not have any significant effect on the expression of EMT markers (snail and vimentin).In conclusion, alisertib is more potent than enzalutamide in decreasing cell proliferation in prostate cancer cells expressing HMGA2. Further studies need to be conducted to understand the role of HMGA2 in promoting resistance to enzalutamide and whether alisertib may be a better drug for patients that express HMGA2. Acknowledgements: These studies were supported by NIH/NIMHD 2U54MD007590 and 5U54MD013376-8281. Citation Format: Yusuf Mansur Liadi, Valerie Odero-Marah. Antiproliferative activity of enzalutamide and alisertib in prostate cancer cells overexpressing HMGA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1572.

Abstract 1645: JAB-2485: A potent, highly selective small-molecule Aurora kinase A inhibitor that targets cell division

Abstract Background: Aurora kinase A (AURKA), a crucial mitotic regulator, is frequently dysregulated in a wide range of cancers and contributes to clinical aggressiveness and poor patient survival. Abnormally activated AURKA promotes tumorigenesis through enhancing cancer cell proliferation, epithelial-mesenchymal transition, and cancer stem cell self-renewal, rendering it an attractive therapeutic target. We have developed JAB-2485, a highly selective small-molecule AURKA inhibitor. Methods: Biochemical and cell-based assays were performed to determine the IC50 of JAB-2485 on Aurora kinase family members. Fluorescence activated cell sorting (FACS) and caspase 3/7 assay were performed to detect JAB-2485-induced cell cycle arrest and apoptosis, respectively. The CellTiter-Glo assay was performed to evaluate the effect of JAB-2485 in multiple cancer cell lines. Finally, the antitumor effect of JAB-2485 either as a single agent or in combinations was investigated in tumor cell line-derived xenografts. Results: JAB-2485 is potent and highly selective on AURKA, with IC50 of 0.33nM and around 1700-fold selectivity over AURKB. Following treatment of JAB-2485, the level of AURKA phosphorylation as a pharmacodynamic marker was significantly decreased in a dose-dependent manner. As a single agent, JAB-2485 inhibited in vitro proliferation of small cell lung cancer (SCLC), triple-negative breast cancer (TNBC) and neuroblastoma cell lines along with induced G2/M cell cycle arrest and apoptosis, and significantly inhibited in vivo tumor growth on SCLC and neuroblastoma xenografts. Furthermore, JAB-2485 showed strong synergy with cisplatin and paclitaxel in SCLC and TNBC animal models, respectively. Conclusions: Taken together, preclinical results provide robust evidence supporting further evaluation of JAB-2485 both as a single agent and in combinations for treatment of various types of cancers. A Phase 1/2a clinical trial of evaluating JAB-2485 in adult patients with advanced solid tumors is enrolling patients in the US (NCT05490472). Citation Format: Guiqun Yang, Yanping Wang, Haijun Li, Mingming Chen, Yiwei Lin, Xin Sun. JAB-2485: A potent, highly selective small-molecule Aurora kinase A inhibitor that targets cell division [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1645.

Abstract 4763: Two epigenetically distinct cellular states in osteosarcoma are regulated by a state-specific set of transcription factors driving differential drug response

Abstract Osteosarcoma is a highly aggressive cancer seen mostly in children and young adults which is characterized by aneuploidy and dramatic structural rearrangements associated with large copy number gain and widespread chromosomal loss. Osteosarcoma therapy has not change in over forty years, highlighting the need for deeper molecular understanding of this disease which could in turn identify new therapeutic opportunities. While DNA-level heterogeneity between tumors has been well-described, not much is known regarding epigenetic heterogeneity within osteosarcoma and how this may impact critical phenotypes such as therapy response and metastasis. To characterize the osteosarcoma epigenome, we integrated ATAC-seq and RNA-seq across a unique set of patient samples, PDXs and PDX-derived cell lines obtained across the disease continuum including biopsies, resections, and metastases. ATAC-seq analysis revealed the presence of at least two distinct epigenetic cell states, epigenetic cluster 1 (EC1) and epigenetic cluster 2 (EC2). We used the GREAT algorithm to identify enriched pathways for the genes linked to these differential open regions (~2000 unique peaks per EC). EC1 is defined by genes related to mesenchymal cell proliferation and osteoblast differentiation, whereas EC2 is enriched in genes related to extracellular matrix organization and regulation of kinase activity. Accessible chromatin peaks in EC1 showed transcription factor binding motifs related to development including RUNX2/3, MEOX2, HOXA2/5 and DLX; whereas members of the AP1 complex including FOSL1/2, c-FOS, JUN were enriched in open regions in EC2. This is correlated with high protein expression of these transcription factors in a cluster-specific manner and differential binding to chromatin. We defined a gene-signature of 343 genes by integrating differential open chromatin with differential gene expression of EC1 and EC2. This gene-signature is able to separate gene expression data of patient samples into 2 clusters, suggesting these epigenetic cell states are found in patient samples. We assigned the patient samples to one EC and performed a survival analysis using only primary patient samples. EC1 shows a lower survival probability than EC2. To identify possible therapeutic implications of these EC, we evaluated a drug response dataset generated with ~40 targeted agents for the same panel of PDX-cell lines. Whereas EC1 cell lines were highly sensitive to a selective inhibitor of Aurora B, cell lines in EC2 were inhibited by a MEK inhibitor. PDX-cell lines injected in a subcutaneous model recapitulated the differential drug response in vivo. In summary, we have identified two epigenetically cell states regulated by a state-specific set of transcription factors defining a gene signature that could help to define osteosarcoma subclasses and predict drug response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Leanne Sayles, Maria Pons Ventura, Alex Lee, Vijay Ramani, E. Alejandro Sweet-Cordero. Two epigenetically distinct cellular states in osteosarcoma are regulated by a state-specific set of transcription factors driving differential drug response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4763.

Abstract 2770: Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform

Abstract Aurora kinases (Aur) are a family of serine/threonine kinases essential for genetic stability and cell division during mitosis. In humans, activity of the three paralogs (AurA, AurB, and AurC) is regulated throughout the cell cycle, with peak activity during mitosis. Each paralog shares a conserved C-terminal catalytic domain, but differs in substrate specificity, sub-cellular localization, and function. AurA mainly controls centrosome maturation and bipolar spindle assembly; AurB phosphorylates histone H3 (Ser10), which aids in chromatin condensation and separation; AurC, whose expression is restricted to germ cells of both genders, shares similar characteristics to AurB including substrate localization, specificity, and function during mitosis. Aberrant Aur expression leads to genomic instability or aneuploidy, and overexpression of the three paralogs is found in several cancer types, including lung, colon, and gliomas. Aur inhibition induces growth arrest and apoptosis in a variety of cancer cell types. Thus, design and development of Aur inhibitors as anti-tumor agents has been widely explored in recent years, and many are under preclinical and clinical investigation. We used the Eurofins Discovery OncoPanel™ cell-based profiling platform to investigate the specific effects of AurA, AurB, and AurC inhibitors against a select panel of human cancer cell lines. To determine the mechanistic effect of Aur inhibition in cells, we measured phospho-histone H3 (Ser10) by high-content analysis. OncoPanel human tumor cell lines arrested with nocodazole were treated with Aur inhibitors for 2 hours. Phospho-histone H3 (Ser10) levels were measured and the percentage of the DMSO vehicle-treated control cell signal was calculated for each test point. Ten-point dose-response curves were fitted using a 4-parameter log-logistic model with custom curve-fitting software to determine IC50 values. The inhibitors were also evaluated for their effect on tumor cell proliferation, apoptosis, and cell cycle arrest over the same dose-response range, after a 10-day incubation. Following exposure, the cells were fixed, stained with DAPI, anti-cleaved caspase-3, and anti-phospho-histone H3 (Ser28), and imaged. Cell proliferation dose-response curves were also fitted using the same model as above. Correlations were then made between observed mechanistic and functional activity. Activity of the AurA-selective inhibitor, MLN8054, was also evaluated in the Eurofins Discovery BioMAP® Diversity PLUS human primary cell phenotypic profiling service to give a broader view of its biological activity. These studies demonstrate the combined value of using the OncoPanel and BioMAP platforms to determine mechanistic, functional, and broader biological activity of therapeutic candidates in relevant cellular models for a better translational understanding of potential therapeutic capabilities and accelerate decision-making. Citation Format: Luciano Galdieri, Justin Lipner, Steven M. Garner, Jillian Krings, Emily Hoehn, Brendan Lahm, Kaleb Collver, Kaitlyne Powers, Alastair J. King. Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2770.

The heterogeneous transition state of resistance to RET kinase inhibitors converges on ERK1/2-driven Aurora A/B kinases

AimWhile most patients with RET-altered cancer responded to the RET protein tyrosine kinase inhibitors (TKIs) pralsetinib (BLU667) and selpercatinib (LOXO292), few achieved a complete response. Heterogeneity in residual tumors makes it difficult to target their diverse genetic alterations individually. The aim of this study is to characterize the cancer cells that persist under continuous RET TKI treatment and identify the shared vulnerability of these cells. MethodsWe analyzed residual RET-altered cancer cells under prolonged RET TKI treatment by whole exome sequencing (WES), RNA-seq analysis, and drug-sensitivity screening. These were followed by tumor xenograft experiments of mono- and combinational drug treatments. ResultsBLU667- and LOXO292-tolerated persisters were cellularly heterogeneous, contained slowly proliferating cells, regained low levels of active ERK1/2, and displayed plasticity in growth rate, which we designated as in the transition state of resistance (TSR). TSR cells were genetically heterogeneous. Aurora A/B kinases were among the most significantly upregulated genes and that the MAPK pathway activity had significantly higher transcript footprints. MEK1/2 and Aurora kinase inhibitors were the most effective drugs when combined with a RET kinase inhibitor. In a TSR tumor model, combination of BLU667 with an Aurora kinase or a MEK1/2 kinase inhibitor caused TSR tumor regression. ConclusionOur experiments reveal that the heterogeneous TSR cancer cells under continuous RET TKI treatment converge on the targetable ERK1/2-driven Aurora A/B kinases. The discovery of the targetable convergent point in the genetically heterogeneous TSR points to an effective combination therapy approach to eliminate the residual tumors.

Design, synthesis, and biological evaluation of 6-(imidazo[1,2-a] pyridin-6-yl) quinazolin-4(3H)-one derivatives as potent anticancer agents by dual targeting Aurora kinase and ROR1

ROR1 and Aurora kinase were overexpressed in various cancers and essential for cell proliferation, survive and metastasis. Pharmaceutical inhibition of ROR1 and Aurora kinase abrogated the activation of downstream signaling and induced cancer cell apoptosis. Hence, ROR1 and Aurora kinase considered as attractive therapeutic targets for the development of anticancer drugs. In the present work, three series of novel 6-(imidazo[1,2-a] pyridin-6-yl)-quinazolin-4(3H)-one derivatives were designed and synthesized via bioisosterism and scaffold-hopping strategies guided by FLF-13, an Aurora kinase inhibitor we discovered earlier. Most of compounds in series 2 and series 3 showed submicromolar to nanomolar inhibitory activity against multiple cancer cell lines. More importantly, compounds 12d and 12f in series 3 showed nanomolar inhibitory activity against all test cancer cells. The most promising compound 12d exhibited potent inhibitory activity against Aurora A and Aurora B with IC50 values of 84.41 nM and 14.09 nM, respectively. Accordingly, compounds 12d induced G2/M phase cell cycle arrest at 24 h and polyploidy at 48 h. It's worth noting that 12d also displayed inhibitory activity against ROR1 and induce cell apoptosis. Furthermore, 12d could significantly inhibit the tumor growth in SH-SY5Y xenograft model with tumor growth inhibitory rate (IR) up to 46.31 % at 10 mg/kg and 52.66 % at 20 mg/kg. Overall, our data suggested that 12d might serve as a promising candidate for the development of therapeutic agents for cancers with aberrant expression of ROR1 and Aurora kinases by simultaneously targeting ROR1 and Aurora kinase.

Pediatric Acute Lymphoblastic Leukemia Emerging Therapies—From Pathway to Target

Over the past 40 years, the 5-years-overall survival rate of pediatric cancer reached 75-80%, and for acute lymphoblastic leukemia (ALL), exceeded 90%. Leukemia continues to be a major cause of mortality and morbidity for specific patient populations, including infants, adolescents, and patients with high-risk genetic abnormalities. The future of leukemia treatment needs to count better on molecular therapies as well as immune and cellular therapy. Advances in the scientific interface have led naturally to advances in the treatment of childhood cancer. These discoveries have involved the recognition of the importance of chromosomal abnormalities, the amplification of the oncogenes, the aberration of tumor suppressor genes, as well as the dysregulation of cellular signaling and cell cycle control. Lately, novel therapies that have already proven efficient on relapsed/refractory ALL in adults are being evaluated in clinical trials for young patients. Tirosine kinase inhibitors are, by now, part of the standardized treatment of Ph+ALL pediatric patients, and Blinatumomab, with promising results in clinical trials, received both FDA and EMA approval for use in children. Moreover, other targeted therapies such as aurora-kinase inhibitors, MEK-inhibitors, and proteasome-inhibitors are involved in clinical trials that include pediatric patients. This is an overview of the novel leukemia therapies that have been developed starting from the molecular discoveries and those that have been applied in pediatric populations.

Aurora Kinase A Regulation by Cysteine Oxidative Modification

Aurora kinase A (AURKA), which is a member of serine/threonine kinase family, plays a critical role in regulating mitosis. AURKA has drawn much attention as its dysregulation is critically associated with various cancers, leading to the development of AURKA inhibitors, a new class of anticancer drugs. As the spatiotemporal activity of AURKA critically depends on diverse intra- and inter-molecular factors, including its interaction with various protein cofactors and post-translational modifications, each of these pathways should be exploited for the development of a novel class of AURKA inhibitors other than ATP-competitive inhibitors. Several lines of evidence have recently shown that redox-active molecules can modify the cysteine residues located on the kinase domain of AURKA, thereby regulating its activity. In this review, we present the current understanding of how oxidative modifications of cysteine residues of AURKA, induced by redox-active molecules, structurally and functionally regulate AURKA and discuss their implications in the discovery of novel AURKA inhibitors.

Evolution of Antiretroviral Drug Rilpivirine and Approach to Oncology.

Rilpivirine is an antiretroviral drug used to treat AIDS worldwide. The drug is a non-nucleoside reverse transcriptase inhibitor that halts the cDNA elongation process and, thus, the capacity of the HIV-1 virus to replicate. With the new wave of drug repurposing in recent years, rilpivirine has been studied in this regard. This drug is useful in Zika virus treatment, with in vivo results indicating regression in neuronal effects often associated with this infection. Several cancer types have also been researched, from breast to leukemia and pancreatic cancer, and rilpivirine has proved to have inhibitory effects in various cell lines with low concentrations, causing cellular death, apoptosis, and cell cycle arrest. The pathways are not yet established, but some works have hypothesized and demonstrated that rilpivirine causes inhibition of Aurora A kinase and has effects on the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway and the vascular endothelial growth factors-receptors (VEGFs-VEGFRs) pathway, which are known to be altered in cancer and tumors and can be targeted for cancer treatment. Further testing and clinical trials are needed, but this review demonstrates the potential of rilpivirine's repurposing for cancer treatment.

Structure optimization, synthesis, and biological evaluation of 6-(2-amino-1H-benzo[d]imidazole-6-yl)-quinazolin-4(3H)-one derivatives as potential multi-targeted anticancer agents via Aurora A/ PI3K/BRD4 inhibition

Aurora A (Aurora kinase A), a critical regulator of cell mitosis, is frequently overexpressed in many malignant cancers, and has been considered as a promising drug target for cancer therapy. Likewise, Phosphatidylinositol 3-kinase alpha (PI3Kα) is also regarded as one of the most important targets in cancer therapy by mediating the cell growth and angiogenesis of various human cancers. In addition, Bromodomain-containing protein 4 (BRD4) modulates oncogene expressions of Myc, Aurora kinase and various RTKs. Recently, accumulating evidences indicated that hyperactivated or abnormally expressed Aurora A, PI3Kα or BRD4 are closely associated with drug resistance and poor prognosis of non-small cell lung cancer (NSCLC). Hence, simultaneous inhibition of Aurora A, PI3Kα, and BRD4 is expected to be a new strategy for NSCLC therapy. In this study, we performed further structure optimization of 6-(2-amino-1H-benzo[d]imidazole-6-yl)-quinazolin-4(3H) -one based on previous study to obtain a series of derivatives for discovering potential Aurora A, PI3Kα and BRD4 multi-targeted inhibitors. MTT assay showed that most of the newly synthesized compounds exhibited an evident anticancer activity against the NSCLC cells. Among them, the IC50 values of the most potent compound 9a were 0.83, 0.26 and 1.02 μM against A549, HCC827 and H1975 cells, respectively. In addition, 9a markedly inhibited the Aurora A and PI3Kα kinase activities with IC50 values of 10.19 nM and 13.12 nM. Compound 9a induced G2/M phase arrests and apoptosis of HCC827 cells by simultaneous inhibition of Aurora A/PI3K/ BRD4 signaling pathways. Collectively, our studies suggested that 9a might be a potential multi-targeted inhibitor for NSCLC therapy.