Abstract 2530: Antimyeloma activity of a small molecule multi-targeted kinase inhibitor, AT9283, via potent aurora kinase and STAT3 inhibition

AT9283, a Small Molecule Multi-Targeted Kinase Inhibitor Induces Antimyeloma Activity Via Potent Aurora Kinase and STAT3 Inhibition.

Aurora kinases (A, B and C) are frequently amplified and overexpressed in a wide variety of cancers, and their up-regulation often correlates with poor prognosis. The potential role of these kinases in tumorigenesis indicates that they could be appealing target for molecular therapy. Although extensive efforts have been done to develop Aurora kinases inhibitors (AKIs), selective for A, B or both kinases, so far, none of them have yet been approved by FDA due to their severe toxicity. The aim of this study has been to identify novel and powerful AKIs, Aurora B kinase inhibitors. In living cells, we tested the inhibition of Aurora B kinase by 7 potential novel AKIs, called MK1-7, which share a common molecular scaffold, by evaluating Serine 10 phosphorylation of Histone H3, a bona fide downstream target of the kinase in HeLa cells. We selected 4 compounds (MK1, 2, 6 and 7), that were able to reduce phosphorylation of Histone H3 at 10 nM dose. Then, we evaluated the effect of each inhibitor on: cell cycle progression, spindle assembly checkpoint (SAC) escape, cell proliferation and viability. Although with different efficacy, these 4 MKs perturb cell cycle progression inducing polyploidy. In addition, cells treated with the 4 MKs escape from mitosis overtaking the SAC checkpoint. These MKs blocked cell growth with an IC50 ranging from 0.37 to 2.27 nM (MK1 IC50=1,2 nM; MK2 IC50=2,27 nM; MK6 IC50=0,44 nM; MK7 IC50=0,37 nM). Based on MK7 most powerful efficacy, its effect on cell division and viability was more extensively investigated. HeLa cells treated with MK7 were positive for apoptotic Annexin V and propidium iodide staining and showed an increase in caspase 3 and PARP cleavages, indicating induction of apoptosis. Additionally, MK7 treatment caused alteration in mitotic spindle formation, chromosome segregation and cytokinesis. Finally, we observed that MK7 is able to inhibit growth of A2780 (human ovarian carcinoma), HL-60 (human promyelocytic leukemia), HCT116 (human colorectal carcinoma) and 8505-C (human undifferentiated thyroid carcinoma) cell lines. In conclusion, our in vitro data have identified in MK7 a powerful compound which could be widely used to inhibit Aurora B kinase in different cancer types.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Aurora kinases, whose expression is linked to genetic instability and cellular proliferation, are being investigated as novel therapeutic targets in multiple myeloma (MM). In this study, we investigated the preclinical activity of a small-molecule multitargeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A, Aurora kinase B, and Janus kinase 2/3. We evaluated the in vitro antimyeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a xenograft mouse model of human MM. Our data showed that AT9283 induced cell-growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both Aurora kinase A and Aurora kinase B inhibition, such as increase of cells with polyploid DNA content, decrease in phospho-histone H3, and decrease in phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, Aurora kinase A, or Aurora kinase B showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies showed decreased MM cell growth and prolonged survival in AT9283-treated mice compared with controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells. Enhanced cytotoxicity was associated with increased inhibition of phosphorylated STAT3 and phosphorylated extracellular signal-regulated kinase. Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination therapy for treating patients with MM.

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary, cancers associated with mutations in the neurofibromin 1 gene 1. MPNSTs are often resistant to chemotherapies and have high rates of disease recurrence, highlighting the lack of effective treatment options for this cancer. Aurora kinase A inhibitors (AKIs) have shown promise against MPNST cell lines 2. We expanded this study by testing AKI in human MPNST xenotransplant mice models. Treatment resulted in stabilized disease with tumor cells undergoing senescence and endoreduplication. Aurora kinase A (AURKA) is an emerging target in cancer, however, targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumor response. Therefore, we utilized three MPNST cell lines and profiled them for the expression and activity of AURKA as well as their responses to AKIs. The most proliferative lines, S462 and 2884, express equivalent levels of AURKA, however, S462 cells were more sensitive to kinase inhibition. Both cell lines experienced apoptosis, senescence and endoreduplication in response to AKI treatment. AURKA activity is regulated by a co-activator, the Targeting Protein for XKlp2 (TPX2) and a molecular brake, the Receptor for Hyaluronan Mediated Motility (RHAMM)3. Interestingly, published analysis of copy number variation has identified hemizygous loss of the RHAMM gene in half of the examined high-grade MPNST, but not in benign or low grade tumors 4. We proposed that MPNSTs with RHAMM deletions are oncogene addicted to AURKA activity and are therefore, particularly susceptible to AKI. We profiled our MPNST lines for RHAMM and TPX2 expression and found that S462 cells express significantly more TPX2 and less RHAMM compared to 2884 cells. Furthermore, S462 cells had increased kinase. To determine whether levels of these molecular regulators could affect AKI efficacy we depleted RHAMM and TPX2 in 2884 and S462 cells respectively. While cells with reduced TPX2 have unchanged responses to AKIs, RHAMM depleted cells have a 2 fold reduction in IC-50s. We also looked at the effect of AKI against a population of MPNST tumor-initiating cells (TICs) from the S462 line. Compared to adherent cells, S462 TICs have elevated AURKA activity and their ability to self-renew in vitro is arrested by AKI. Indeed, the altered levels of kinase activity in the RHAMM and TPX2 depletion lines correlated with their ability to form and maintain sphere culture. In addition, we find that AKI treated S462 TICs differentiated into terminal neurons. All in all, these data indicate AURKA as a rational therapy for aggressive MPNSTs with RHAMM serving as a biomarker for AKI efficacy. Citation Format: Pooja Mohan, Joan Castellsague, Jihong Jiang, Kristi Allen, Helen Chen, Oksana Nemirovsky, Melanie Spyra, Kaiji Hu, Lan Kluwe, Miguel Pujana, Alberto Villanueva, Victor Mautner, Sandra Dunn, Jonathan Keats, Conxi Lazaro, Christopher Maxwell. Common genomic alterations in malignant peripheral nerve sheath tumors augment Aurora A activity and sensitize tumors to aurora kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2013-LB-214

Novel Combinatorial Therapeutic Options in NEAT1-Depleted Multiple Myeloma Cells By the Integration of in Vitro and in silico Drug Screening Approaches

Aberrant expression of proteins involved in cell division is a constant feature in multiple myeloma (MM), especially in high-risk disease. Increasingly, therapy of myeloma is moving towards individualization based on underlying genetic abnormalities. Aurora kinases are important mediators of cell cycle and are up regulated in MM. Functional loss of Aurora kinases results in genetic instability and dysregulated division leading to cellular aneuploidy and growth arrest. We investigated the role of Aurora kinase inhibition in MM, using a small molecule inhibitor A1014907. Low nanomolar A1014907 concentrations induced aneuploidy in MM cell lines independent of underlying cytogenetic abnormalities by inhibiting Aurora Kinases. However, A1014907 induced more pronounced and dose dependent apoptosis in cell lines with t(4;14) translocation. Translocation t(4;14) is observed in about 15% of patients with MM leading to constitutive activation of FGFR3 in two-thirds of these patients. Further investigation of the mechanism of action of A1014907 revealed potent FGFR3 pathway inhibition only in the sensitive cell lines. Thus, our results show that aurora kinase inhibition causes cell cycle arrest and aneuploidy with minimal apoptosis whereas inhibiting both aurora kinase and FGFR3 activity induced potent apoptosis in MM cells. These results support clinical evaluation of A1014907 in MM patients with t(4;14) translocation and/or FGFR3 expression.

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.

Chapter 10 - Aurora kinase: An emerging potential target in therapeutics

Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow of myeloma patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken. The human MM cell lines, RPMI8226 or U266, were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents, cyclophosphamide (CY), melphalan (MEL), and bendamustine, the proteasome inhibitor, carfilzomib, the corticosteroid, dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). After 48 hours, cell viability was assessed. Combinations of INCB052793 plus the three alkylating agents or carfilzomib synergistically inhibited the viability of both cell lines in vitro. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1S MM cell line. In vivo, mice bearing the human patient derived MM tumor LAGκ-1A had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at Day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant effect on tumor growth compared to all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through Day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. Studies to further understand the mechanistic effects of these combinations on myeloma signaling and the tumor microenvironment are ongoing. In conclusion, these in vitro and in vivo studies demonstrate that the combination of INCB052793 with a broad spectrum of anti-MM agents is effective, and provide further support for the clinical evaluation of these drug combinations in MM patients. Citation Format: Eric Sanchez, Mingjie Li, Cathy Wang, Puja Mehta, George Tang, Haiming Chen, James R. Berenson. Effects of INCB052793, a selective JAK1 inhibitor, in combination with standard of care agents in human multiple myeloma (MM) cell lines and xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1339.

<div>Abstract<p>The aurora kinases constitute one family of serine/threonine kinases whose activity is essential for mitotic progression. The aurora kinases are frequently upregulated in human cancers and are associated with sensitivity to chemotherapy in certain ones. In the present study, we investigated whether aurora kinases could be a target to overcome radioresistance or enhance the radiosensitivity of lung cancer. For that purpose, we determined the therapeutic potential of daurinol, an investigational topoisomerase inhibitor, alone and in combination with radiation, by observing its effect on aurora kinases. Daurinol decreased cell viability and proliferation in human colon and lung cancer cells. Gene expression in daurinol-treated human colon cancer cells was evaluated using RNA microarray. The mRNA expression of 18 genes involved in the mitotic spindle check point, including aurora kinase A (AURKA) and aurora kinase B (AURKB), was decreased in daurinol-treated human colon cancer cells as compared with vehicle-treated cells. As expected, radiation increased expression levels of AURKA and AURKB. This increase was effectively attenuated by siRNAs against AURKA and AURKB, which suppressed cell growth and increased apoptosis under radiation. Furthermore, the expression of AURKA and AURKB was suppressed by daurinol in the presence or absence of radiation in colon and lung cancer cells. Daurinol alone or in combination with radiation decreased lung cancer growth in xenograft mouse models. Our data clearly confirm the antitumor and radiosensitizing activity of daurinol in human lung cancer cells through the inhibition of AURKA and AURKB. <i>Mol Cancer Ther; 14(7); 1693–704. ©2015 AACR</i>.</p></div>

<div>Abstract<p>The aurora kinases constitute one family of serine/threonine kinases whose activity is essential for mitotic progression. The aurora kinases are frequently upregulated in human cancers and are associated with sensitivity to chemotherapy in certain ones. In the present study, we investigated whether aurora kinases could be a target to overcome radioresistance or enhance the radiosensitivity of lung cancer. For that purpose, we determined the therapeutic potential of daurinol, an investigational topoisomerase inhibitor, alone and in combination with radiation, by observing its effect on aurora kinases. Daurinol decreased cell viability and proliferation in human colon and lung cancer cells. Gene expression in daurinol-treated human colon cancer cells was evaluated using RNA microarray. The mRNA expression of 18 genes involved in the mitotic spindle check point, including aurora kinase A (AURKA) and aurora kinase B (AURKB), was decreased in daurinol-treated human colon cancer cells as compared with vehicle-treated cells. As expected, radiation increased expression levels of AURKA and AURKB. This increase was effectively attenuated by siRNAs against AURKA and AURKB, which suppressed cell growth and increased apoptosis under radiation. Furthermore, the expression of AURKA and AURKB was suppressed by daurinol in the presence or absence of radiation in colon and lung cancer cells. Daurinol alone or in combination with radiation decreased lung cancer growth in xenograft mouse models. Our data clearly confirm the antitumor and radiosensitizing activity of daurinol in human lung cancer cells through the inhibition of AURKA and AURKB. <i>Mol Cancer Ther; 14(7); 1693–704. ©2015 AACR</i>.</p></div>

Targeting Aurora Kinases as Therapy in Multiple Myeloma.

A Novel Aurora-a Kinase Inhibitor MLN8237 Induces Cytotoxicity and Cell Cycle Arrest in Experimental Multiple Myeloma Models.

The aurora kinases constitute one family of serine/threonine kinases whose activity is essential for mitotic progression. The aurora kinases are frequently upregulated in human cancers and are associated with sensitivity to chemotherapy in certain ones. In the present study, we investigated whether aurora kinases could be a target to overcome radioresistance or enhance the radiosensitivity of lung cancer. For that purpose, we determined the therapeutic potential of daurinol, an investigational topoisomerase inhibitor, alone and in combination with radiation, by observing its effect on aurora kinases. Daurinol decreased cell viability and proliferation in human colon and lung cancer cells. Gene expression in daurinol-treated human colon cancer cells was evaluated using RNA microarray. The mRNA expression of 18 genes involved in the mitotic spindle check point, including aurora kinase A (AURKA) and aurora kinase B (AURKB), was decreased in daurinol-treated human colon cancer cells as compared with vehicle-treated cells. As expected, radiation increased expression levels of AURKA and AURKB. This increase was effectively attenuated by siRNAs against AURKA and AURKB, which suppressed cell growth and increased apoptosis under radiation. Furthermore, the expression of AURKA and AURKB was suppressed by daurinol in the presence or absence of radiation in colon and lung cancer cells. Daurinol alone or in combination with radiation decreased lung cancer growth in xenograft mouse models. Our data clearly confirm the antitumor and radiosensitizing activity of daurinol in human lung cancer cells through the inhibition of AURKA and AURKB.