Multi-Kinase Inhibitors, Aurora Kinases, and Cancer
Abstract
Inhibitors that impact the function of kinases are valuable both for biological research and the therapy of kinase-associated diseases, such as various cancers. There are numerous inhibitors that are quite specific for certain kinases, and several of them are either already approved for cancer therapy or are in clinical studies at various phases. However, this does not mean that every single kinase inhibitor is suitable for targeted therapy. Some are not effective, others might be toxic, or fail other criteria for use in vivo. On the other hand, even in cases of successful therapy, many responders eventually develop resistance to the inhibitors. The limitations of various single kinase inhibitors can be addressed using compounds that target multiple kinases. This strategy can increase the effectiveness of inhibitors through synergistic effects or help diminish the likelihood of drug resistance. To date, several families of kinases are popular targets of inhibition in cancers, such as tyrosine kinases, cyclin-dependent kinases, mitogen-activated protein kinases, phosphoinositide 3-kinases, as well as their pathway “players” and aurora kinases. Aurora kinases play an important role in the control of mitosis and are often altered in diverse human cancers. Here, we describe the most interesting multi-kinase inhibitors that inhibit aurora kinases among other targets and their use in preclinical and clinical cancer studies.
Keywords: Protein kinases, Aurora kinases, Multi-kinase inhibitors, Cancer, Cell cycle
Introduction
Protein kinases are important regulators of protein phosphorylation and constitute one of the largest and most diverse gene families. Protein phosphorylation affects functions such as protein location, interaction with other molecules, or molecular activity. Thus, kinases and the proteins phosphorylated by them participate in almost all cell signaling pathways and coordinate nearly all cellular processes. The human genome encodes 538 protein kinases as well as some pseudo-protein kinase genes. More than 470 of these kinases comprise a single superfamily with related catalytic domains and are grouped into families and subfamilies based on sequence similarity and molecular function. Deregulation of kinases can result in significant changes in cellular processes such as proliferation, cell cycle, differentiation, motility, angiogenesis, and apoptosis. This can lead to various diseases such as cancers, cardiovascular disorders, diabetes, inflammation, immune diseases, neurodegenerative diseases, and others. Almost half of known oncogenes are kinases, and others are either phosphorylated by kinases or activated by kinase-initiated signaling pathways. Similarly, phosphorylation of various kinases, such as ERBB2, AKT, CDK4, p38, ERK, and AURK, is associated with cancer progression.
Tyrosine kinases are essential regulators of normal cell functions, which they achieve by tyrosine phosphorylation of various proteins. More than half of them are receptor tyrosine kinases, while others are cytoplasmic tyrosine kinases. Due to their crucial role in cell biology and numerous aberrations in diseases including cancers, the majority of developed kinase inhibitors target tyrosine kinases. The first inhibitor that provided proof of principle that inhibition of an abnormal kinase can suppress cancer progression was Gleevec (imatinib), an inhibitor of the cytoplasmic tyrosine kinase ABL1. The fusion gene BCR–ABL1, which combines the ABL1 gene and part of the BCR gene, is present in approximately 95% of people with chronic myelogenous leukemia (CML), 25% with acute lymphoblastic leukemia (ALL), and in rare cases of acute myelogenous leukemia (AML). Gleevec is one of the best drugs to treat these leukemias. Following ABL1, inhibitors for other cytoplasmic tyrosine kinases such as SRC, FAK, and JAKs have been developed and approved. Receptor tyrosine kinases function as receptors for growth factors, differentiation factors, cytokines, or hormones and are expressed in specific cell types. Genetic mutations or overexpression of receptor tyrosine kinases have been implicated in cancer; therefore, inhibitors against these proteins are also important. Receptor tyrosine kinases such as EGFR, ERBB2, FGFRs, VEGFRs, IGF1R, FLT3, RET, and PDGFRs have established inhibitors used in targeted therapy.
The role of serine/threonine kinases in cancer is also of interest to researchers in both academia and industry. Many of these molecules have inhibitors that are under investigation or already approved for targeted cancer therapy. Cyclin-dependent kinases (CDKs) are protein kinases of the CMGC group that play an essential role in controlling critical cellular processes such as the cell cycle and transcription regulation. The human genome contains 21 genes encoding cyclin-dependent kinases, and at least nine of them are targeted in cancer therapy. The first inhibitors to target the cell cycle were CDK inhibitors, and to date, more than 30 inhibitors have been developed and tested in clinical trials. Palbociclib (PD-0332991, Ibrance), a CDK4/6 inhibitor, has been approved by the FDA. Aurora kinases, another family of protein kinases, are important cell cycle regulators. The family consists of three members (A, B, and C) involved in somewhat different but overlapping tasks such as mitotic entry, centrosome maturation, chromosome segregation, cytokinesis, spindle assembly, and meiosis. Many small molecule inhibitors have been developed for aurora kinases; some inhibit all three isoforms and are called pan-Aurora inhibitors, while others are selective for one or two isoforms. Although many inhibitors are specific for single kinases or kinase families, not all are suitable for every cancer type. The effectiveness of most inhibitors depends on tumor type, expressed biomarkers, and the kinases themselves. Additionally, many patients who initially respond to inhibitors eventually develop resistance. To reduce drug resistance, increase effectiveness, or achieve synergistic effects, drugs targeting multiple kinases can be used. This article discusses multi-kinase inhibitors that combine inhibition of aurora kinases with other molecules such as tyrosine kinases or cyclin-dependent kinases.
Multi-Kinase Inhibitors and Aurora Kinases
AT9283 is a potent inhibitor with IC50 values of 1.1 nM for JAK3, 1.2 nM for JAK2, 3.0 nM for AURKA and AURKB, and 4 nM for ABL1 (T315I mutant). AT9283 induces a polyploid phenotype and inhibits colony formation by targeting AURKB (IC50 of 30 nM). Treatment with AT9283 at doses of 15 and 20 mg/kg resulted in substantial tumor growth reduction of 67% and 76%, respectively, in an HCT116 colon carcinoma xenograft mouse model. AT9283 effectively inhibited proliferation of JAK2-dependent cell lines and the formation of erythroid colonies from hematopoietic progenitors isolated from patients with myeloproliferative disorders carrying JAK2 mutations. It also suppressed proliferation in a JAK2-dependent murine leukemia model. In imatinib-resistant BCR–ABL-positive leukemia cells, AT9283 induced apoptosis and altered the cell cycle via ABL1 inhibition. Cells derived from CML patients were more sensitive to colony formation inhibition by AT9283 than cells from healthy donors. AT9283 treatment also reduced proliferation of leukemic cells and extended survival in SCID mice xenografted with cells from patients harboring BCR–ABL E255K or BCR–ABL T315I mutations. Apoptosis and cell-growth inhibition were also induced by AT9283 in RPMI8226 and U266 multiple myeloma cell lines. Decreased tumor growth and prolonged survival were observed in multiple myeloma xenograft SCID mice treated with AT9283. Interestingly, the combination of AT9283 with lenalidomide increased apoptosis by 56%. The inhibitor was also investigated in aggressive B-non-Hodgkin’s lymphoma cell lines. In DAOY and D556 human multiple myeloma cell lines, proliferation and cell viability were enhanced by the combination of dasatinib and AT9283. Treatment with less than 1 µM induced polyploidy, apoptosis, and inhibited cell proliferation. Combination with docetaxel doubled apoptosis compared to either AT9283 or docetaxel alone (23% and 10%, respectively). Additionally, AT9283 alone and in combination with docetaxel revealed substantial tumor growth inhibition and higher survival rates than docetaxel alone or lower concentration AT9283. AT9283 was also found to considerably inhibit growth and survival of cell lines derived from pediatric leukemia patients via Flt-3 dephosphorylation. Combinations with drugs such as apicidin, 17-AAG, and doxorubicin were found to synergize with AT9283.
The success of preclinical studies using AT9283 initiated several clinical trials in different cancers. A Phase I dose-escalation study in 40 patients with advanced solid malignancies used AT9283 as a continuous central venous infusion starting at 1.5 mg/day for 3 days (4.5 mg/72 h). Main toxic effects were myelosuppression, gastrointestinal disturbance, fatigue, and alopecia, with a maximum tolerated dose established at 27 mg/72 h. Four patients with esophageal, non-small-cell lung cancer, and colorectal cancer demonstrated stable disease for approximately six months. Another Phase I study in patients with advanced solid tumors or non-Hodgkin’s lymphoma administered AT9283 as a 24-hour infusion on days 1 and 8 of a 21-day cycle. Main toxicities were fatigue, gastrointestinal disturbance, anemia, lymphocytopenia, and neutropenia; dose-limiting toxicity was neutropenia. The maximum tolerated dose was 47 mg/day, with a recommended Phase II dose of 40 mg/day. A Phase I study in 48 patients with relapsed/refractory leukemia or myelofibrosis administered AT9283 as a continuous 72-hour infusion every 21 days; the maximum tolerated dose was 324 mg/72 h. Major dose-limiting toxicities included myocardial infarction, hypertension, cardiomyopathy, tumor lysis syndrome, pneumonia, and multiorgan failure. Two patients with chronic myeloid leukemia showed evidence of benefit. Another Phase I study in children and adolescents with solid tumors used the same 72-hour infusion schedule every 21 days. Common toxicities were neutropenia, anemia, thrombocytopenia, fatigue, infections, febrile neutropenia, and ALT elevation. One patient with a neuroectodermal tumor achieved a partial response after 16 cycles, and three cases were stable for at least four cycles. A Phase II study in eight patients with relapsed or refractory multiple myeloma was closed due to no observed responses and considerable toxicity, including myelosuppression, neutropenia, skin ulceration, and infections.
CYC116 is an inhibitor with IC50 values of 8.0 nM for AURKA, 9.2 nM for AURKB, and 44 nM for VEGFR2 and FLT3. It inhibited proliferation and induced cell death in the AML cell line MV4-11 at an IC50 of 34 nM. Tumor growth delay was observed in mice with subcutaneous NCI-H460 xenografts treated orally with 75–100 mg/kg of CYC116. The HCT116 colon cancer cell line was shown to be either sensitive or resistant to CYC116 depending on serine hydroxymethyltransferase expression. Combination of CYC116 with matrine had a stronger cytotoxic effect on RPMI8226 multiple myeloma cells than either drug alone. The mode of action of CYC116 was also studied in P388 mouse leukemia grown subcutaneously as a solid tumor, where a decrease in tumor volume, tumor neovascularization, and leukemia bone marrow infiltration was observed. A Phase I pharmacologic study of CYC116 in patients with advanced solid tumors was initiated but terminated without published results.
ENMD-2076 is an inhibitor with IC50 values of 1.86 nM for FLT3, 10.4 nM for RET, 14 nM for AURKA, and 15.9 nM for VEGFR3 and FLT4. It demonstrated antiproliferative activity at concentrations of 0.025–0.53 µM in 10 human leukemia cell lines and at 0.012–0.7 µM in seven solid tumor cell lines and human umbilical vein endothelial cells (HUVEC). ENMD-2076 also inhibited tumor growth in nude mice xenografted with colon carcinoma, melanoma, breast cancer, multiple myeloma, AML, and acute promyelocytic leukemia cell lines. It showed significant cytotoxicity against multiple myeloma cell lines and primary cells but not hematopoietic progenitors, as well as inhibition of tumor growth in H929 cell xenograft mice. HT-29 human colorectal cancer cell line xenograft models treated orally with 100–200 mg ENMD-2076 daily for 28 days showed initial tumor growth inhibition. Proliferation inhibition by ENMD-2076 in AML was confirmed by another study, which showed significant growth arrest and apoptosis induction in THP-1 and Kasumi-1 cell lines. ENMD-2076 also displayed antiproliferative activity against 29 breast cancer cell lines, with the most sensitive being cells lacking estrogen receptor and ERBB2 overexpression or triple-negative breast cancer cells with increased TP53 expression or mutation. A Phase I safety, pharmacokinetic, and pharmacodynamic study was performed in 67 patients with advanced solid tumors, evaluating doses of 60–200 mg. Two patients at the highest dose experienced hypertension and neutropenia, suggesting 160 mg as the maximum tolerated dose. Two patients with platinum-resistant ovarian cancer had partial responses, indicating encouraging antitumor activity in ovarian cancer. CASI Pharmaceuticals is currently conducting multiple Phase II studies of ENMD-2076 in triple-negative breast cancer, advanced/metastatic soft tissue sarcoma, and advanced ovarian clear cell carcinomas.
JNJ-7706621 is an inhibitor with IC50 values of 4 nM for CDK2, 9 nM for CDK1, 11 nM for AURKA, and 15 nM for AURKB. The IC50 for additional CDKs or other kinases is at least tenfold higher. It displayed effective growth inhibition of cell lines derived from cervical, colon, ovarian, prostate, breast, and uterine cancers, as well as melanoma, with IC50 values ranging from 112 to 514 nM. The inhibitor also induced apoptosis in HeLa cells.
JNJ-7706621 is an inhibitor with IC50 values of 4 nM for CDK2, 9 nM for CDK1, 11 nM for AURKA, and 15 nM for AURKB. The IC50 values for additional CDKs or other kinases are at least tenfold higher. It displayed effective growth inhibition of cell lines derived from cervical, colon, ovarian, prostate, breast, and uterine cancers, as well as melanoma, with IC50 values ranging from 112 to 514 nM. The inhibitor also induced apoptosis in HeLa cells.
PHA-739358 (danusertib) is a multi-kinase inhibitor targeting ABL1, AURKA, AURKB, AURKC, FLT3, and RET kinases. It inhibits AURKA with an IC50 of 13 nM, AURKB at 79 nM, and ABL1 at 25 nM. PHA-739358 has demonstrated potent antitumor activity in various cancer cell lines and xenograft models. It induces polyploidy and apoptosis by inhibiting aurora kinases and suppresses tumor growth in mouse models of leukemia and solid tumors. Clinical trials have evaluated its safety and efficacy in patients with advanced solid tumors and hematologic malignancies.
VX-680 (tozasertib) is a pan-aurora kinase inhibitor that targets AURKA, AURKB, and AURKC with IC50 values in the low nanomolar range. It also inhibits FLT3 and BCR-ABL kinases. VX-680 induces polyploidy and apoptosis in cancer cells and has shown antitumor activity in preclinical models of leukemia and solid tumors. Despite promising preclinical results, clinical development faced challenges due to toxicity and limited efficacy in some trials.
AMG 900 is a potent pan-aurora kinase inhibitor with IC50 values of 5 nM for AURKA and 4 nM for AURKB. It inhibits multiple kinases including FLT3 and RET. AMG 900 induces mitotic arrest, polyploidy, and apoptosis in cancer cells and has demonstrated antitumor activity in xenograft models of various cancers. Clinical trials are ongoing to assess its safety and efficacy in patients with advanced solid tumors and hematologic malignancies.
Danusertib, VX-680, and AMG 900 exemplify multi-kinase inhibitors that target aurora kinases alongside other kinases implicated in cancer progression. These inhibitors exhibit the potential to overcome resistance mechanisms associated with single kinase inhibitors by simultaneously targeting multiple signaling pathways critical for tumor growth and survival.
In summary, multi-kinase inhibitors that include aurora kinases among their targets represent a promising therapeutic strategy in cancer treatment. They combine the inhibition of cell cycle regulators with other oncogenic kinases, potentially enhancing antitumor efficacy and reducing the likelihood of drug resistance.LY3295668 Ongoing clinical trials continue to evaluate the safety and effectiveness of these compounds across various cancer types.