INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors.

Cyclin-dependent kinase 2 (CDK2) is thought to play an important role in driving proliferation of certain cancers, including those harboring CCNE1 amplification and breast cancers that have acquired resistance to CDK4/6 inhibitors (CDK4/6i). The precise impact of pharmacologic inhibition of CDK2 is not known due to the lack of selective CDK2 inhibitors. Here we describe INX-315, a novel and potent CDK2 inhibitor with high selectivity over other CDK family members. Using cell-based assays, patient-derived xenografts (PDX), and transgenic mouse models, we show that INX-315 (i) promotes retinoblastoma protein hypophosphorylation and therapy-induced senescence (TIS) in CCNE1-amplified tumors, leading to durable control of tumor growth; (ii) overcomes breast cancer resistance to CDK4/6i, restoring cell cycle control while reinstating the chromatin architecture of CDK4/6i-induced TIS; and (iii) delays the onset of CDK4/6i resistance in breast cancer by driving deeper suppression of E2F targets. Our results support the clinical development of selective CDK2 inhibitors. SIGNIFICANCE: INX-315 is a novel, selective inhibitor of CDK2. Our preclinical studies demonstrate activity for INX-315 in both CCNE1-amplified cancers and CDK4/6i-resistant breast cancer. In each case, CDK2 inhibition induces cell cycle arrest and a phenotype resembling cellular senescence. Our data support the development of selective CDK2 inhibitors in clinical trials. See related commentary by Watts and Spencer, p. 386. This article is featured in Selected Articles from This Issue, p. 384.

G1T48, an oral selective estrogen receptor degrader, and the CDK4/6 inhibitor lerociclib inhibit tumor growth in animal models of endocrine-resistant breast cancer.

PURPOSE: The combination of targeting the CDK4/6 and estrogen receptor (ER) signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER-positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here, we report the identification of G1T48, a novel orally bioavailable, non-steroidal small molecule antagonist of ER. METHODS: The pharmacological effects and the antineoplastic mechanism of action of G1T48 on tumors was evaluated using human breast cancer cells (in vitro) and xenograft efficacy models (in vivo). RESULTS: G1T48 is a potent and efficacious inhibitor of estrogen-mediated transcription and proliferation in ER-positive breast cancer cells, similar to the pure antiestrogen fulvestrant. In addition, G1T48 can effectively suppress ER activity in multiple models of endocrine therapy resistance including those harboring ER mutations and growth factor activation. In vivo, G1T48 has robust antitumor activity in a model of estrogen-dependent breast cancer (MCF7) and significantly inhibited the growth of tamoxifen-resistant (TamR), long-term estrogen-deprived (LTED) and patient-derived xenograft tumors with an increased response being observed with the combination of G1T48 and the CDK4/6 inhibitor lerociclib. CONCLUSIONS: These data show that G1T48 has the potential to be an efficacious oral antineoplastic agent in ER-positive breast cancer.

CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.

Transient CDK4/6 inhibition protects hematopoietic stem cells from chemotherapy-induced exhaustion.

Conventional cytotoxic chemotherapy is highly effective in certain cancers but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise ("exhaustion"), which limits the use of chemotherapy and success of cancer therapy. We show that the coadministration of G1T28 (trilaciclib), which is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil treatment model. Consistent with a cell-intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity, and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer.

Preclinical development of G1T38: A novel, potent and selective inhibitor of cyclin dependent kinases 4/6 for use as an oral antineoplastic in patients with CDK4/6 sensitive tumors.

Inhibition of the p16INK4a/cyclin D/CDK4/6/RB pathway is an effective therapeutic strategy for the treatment of estrogen receptor positive (ER+) breast cancer. Although efficacious, current treatment regimens require a dosing holiday due to severe neutropenia potentially leading to an increased risk of infections, as well as tumor regrowth and emergence of drug resistance. Therefore, a next generation CDK4/6 inhibitor that can inhibit proliferation of CDK4/6-dependent tumors while minimizing neutropenia could reduce both the need for treatment holidays and the risk of inducing drug resistance.Here, we describe the preclinical characterization and development of G1T38; a novel, potent, selective, and orally bioavailable CDK4/6 inhibitor. In vitro, G1T38 decreased RB1 (RB) phosphorylation, caused a precise G1 arrest, and inhibited cell proliferation in a variety of CDK4/6-dependent tumorigenic cell lines including breast, melanoma, leukemia, and lymphoma cells. In vivo, G1T38 treatment led to equivalent or improved tumor efficacy compared to the first-in-class CDK4/6 inhibitor, palbociclib, in an ER+ breast cancer xenograft model. Furthermore, G1T38 accumulated in mouse xenograft tumors but not plasma, resulting in less inhibition of mouse myeloid progenitors than after palbociclib treatment. In larger mammals, this difference in pharmacokinetics allowed for 28 day continuous dosing of G1T38 in beagle dogs without producing severe neutropenia. These data demonstrate G1T38 has unique pharmacokinetic and pharmacodynamic properties, which result in high efficacy against CDK4/6 dependent tumors while minimizing the undesirable on-target bone marrow activity, thus potentially allowing G1T38 to be used as a continuous, daily oral antineoplastic agent.

Preclinical Characterization of G1T28: A Novel CDK4/6 Inhibitor for Reduction of Chemotherapy-Induced Myelosuppression.

Chemotherapy-induced myelosuppression continues to represent the major dose-limiting toxicity of cytotoxic chemotherapy, which can be manifested as neutropenia, lymphopenia, anemia, and thrombocytopenia. As such, myelosuppression is the source of many of the adverse side effects of cancer treatment including infection, sepsis, bleeding, and fatigue, thus resulting in the need for hospitalizations, hematopoietic growth factor support, and transfusions (red blood cells and/or platelets). Moreover, clinical concerns raised by myelosuppression commonly lead to chemotherapy dose reductions, therefore limiting therapeutic dose intensity, and reducing the antitumor effectiveness of the treatment. Currently, the only course of treatment for myelosuppression is growth factor support which is suboptimal. These treatments are lineage specific, do not protect the bone marrow from the chemotherapy-inducing cytotoxic effects, and the safety and toxicity of each agent is extremely specific. Here, we describe the preclinical development of G1T28, a novel potent and selective CDK4/6 inhibitor that transiently and reversibly regulates the proliferation of murine and canine bone marrow hematopoietic stem and progenitor cells and provides multilineage protection from the hematologic toxicity of chemotherapy. Furthermore, G1T28 does not decrease the efficacy of cytotoxic chemotherapy on RB1-deficient tumors. G1T28 is currently in clinical development for the reduction of chemotherapy-induced myelosuppression in first- and second-line treatment of small-cell lung cancer. Mol Cancer Ther; 15(5); 783-93. ©2016 AACR.

CDK4/6 inhibition induces epithelial cell cycle arrest and ameliorates acute kidney injury.

Acute kidney injury (AKI) is common and urgently requires new preventative therapies. Expression of a cyclin-dependent kinase (CDK) inhibitor transgene protects against AKI, suggesting that manipulating the tubular epithelial cell cycle may be a viable therapeutic strategy. Broad spectrum small molecule CDK inhibitors are protective in some kidney injury models, but these have toxicities and epithelial proliferation is eventually required for renal repair. Here, we tested a well-tolerated, novel and specific small molecule inhibitor of CDK4 and CDK6, PD 0332991, to investigate the effects of transient cell cycle inhibition on epithelial survival in vitro and kidney injury in vivo. We report that CDK4/6 inhibition induced G0/G1 cycle arrest in cultured human renal proximal tubule cells (hRPTC) at baseline and after injury. Induction of transient G0/G1 cycle arrest through CDK4/6 inhibition protected hRPTC from DNA damage and caspase 3/7 activation following exposure to the nephrotoxins cisplatin, etoposide, and antimycin A. In vivo, mice treated with PD 0332991 before ischemia-reperfusion injury (IRI) exhibited dramatically reduced epithelial progression through S phase 24 h after IRI. Despite reduced epithelial proliferation, PD 0332991 ameliorated kidney injury as reflected by improved serum creatinine and blood urea nitrogen levels 24 h after injury. Inflammatory markers and macrophage infiltration were significantly decreased in injured kidneys 3 days following IRI. These results indicate that induction of proximal tubule cell cycle arrest with specific CDK4/6 inhibitors, or "pharmacological quiescence," represents a novel strategy to prevent AKI.

Multiple roles of cyclin-dependent kinase 4/6 inhibitors in cancer therapy.

BACKGROUND: Cyclin-dependent kinases (CDKs) regulate cell proliferation and coordinate the cell cycle checkpoint response to DNA damage. Although inhibitors with varying selectivity to specific CDK family members have been developed, selective CDK4/6 inhibitors have emerged as the most attractive antineoplastic agents because of the importance of CDK4/6 activity in regulating cell proliferation and the toxic effects associated with inhibition of other CDKs (eg, CDK1 and CDK2). METHODS: FVB/N wild-type mice (n = 13) were used to evaluate carboplatin-induced myelosuppression in bone marrow by complete blood cell counts after treatment with the CDK4/6 inhibitor PD0332991. Genetically engineered murine models of retinoblastoma (Rb)-competent (MMTV-c-neu) and Rb-incompetent (C3-TAg) breast cancer (n = 16 MMTV-c-neu mice in the carboplatin plus vehicle control group, n = 17 MMTV-c-neu mice in the carboplatin plus PD0332991 group, n = 17 C3-TAg mice in the carboplatin plus vehicle control group, and n = 14 C3-TAg mice in the carboplatin plus PD0332991 group) were used to investigate the antitumor activity of PD0332991 alone or in combination with chemotherapy. All statistical tests were two-sided. RESULTS: Coadministration of PD0332991 with carboplatin compared with carboplatin alone in FVB/N wild-type mice increased hematocrit (51.2% vs 33.5%, difference = 17.7%, 95% confidence interval [CI] = -26.7% to -8.6%, P < .001), platelet counts (1321 vs 758.5 thousand cells per µL, difference = 562.5 thousand cells per µL, 95% CI = -902.8 to -222.6, P = .002), myeloid cells (granulocytes and monocytes; 3.1 vs 1.6 thousand cells per µL, difference = 1.5 thousand cells per µL, 95% CI = -2.23 to -0.67, P < .001), and lymphocytes (7.9 vs 5.4 thousand cells per µL, difference = 2.5 thousand cells per µL, 95% CI = -4.75 to -0.18, P = .02). Daily administration of PD0332991 exhibited antitumor activity in MMTV-c-neu mice as a single agent. However, the combination of carboplatin plus PD0332991 decreased antitumor activity compared with carboplatin alone in Rb-competent mice (mean percent change in tumor volume at day 21 = -52.6% vs 3.7% for carboplatin and carboplatin plus PD0332991, respectively, difference = 56.3%, 95% CI = -109.0% to -3.6%, P = .04). In contrast, Rb-deficient tumors in C3-Tag mice were resistant to PD0332991, and coadministration of PD0332991 plus carboplatin had no effect on in vivo tumor growth (mean percent change in tumor volume at day 21 = 118.8% and 109.1% for carboplatin and carboplatin plus PD0332991, respectively, difference = 9.7%, 95% CI = -183.5% to 202.9%, P = .92). Finally, in tumor-bearing mice, coadministration of PD0332991 with carboplatin provided statistically significant protection of platelets (P = .04). CONCLUSION: We believe that the present data support a possible role for CDK4/6 inhibitors in a majority of patients with advanced cancer: to either inhibit tumor growth in CDK4/6-dependent tumors or ameliorate the dose-limiting toxicities of chemotherapy in CDK4/6-indepdendent tumors. Our data also suggest CDK4/6 inhibitors should not be combined with DNA-damaging therapies, such as carboplatin, to treat tumors that require CDK4/6 activity for proliferation.

PPARgamma agonists inhibit vasopressin-mediated anion transport in the MDCK-C7 cell line.

PPARgamma agonists are synthetic ligands for the peroxisome proliferator-activated receptor-gamma (PPARgamma). These agents have insulin-sensitizing properties but can cause fluid retention, thereby limiting their usefulness in patients at risk for cardiovascular disease. The side effect etiology is unknown, but the nature of presentation suggests modulation of renal salt and water homeostasis. In a well-characterized cell culture model of the principal cell type [Madin-Darby canine kidney (MDCK)-C7], PPARgamma agonists inhibit vasopressin-stimulated Cl(-) secretion with agonist dose-response relationships that mirror receptor transactivation profiles. Analyses of the components of the vasopressin-stimulated intracellular signaling pathway indicated no PPARgamma agonist-induced changes in basolateral membrane conductances, intracellular cAMP, protein kinase A, or total cellular adenine nucleotides. The PPARgamma agonist-induced decrease in anion secretion is the result of decreased mRNA of the final effector in the pathway, the apically located cystic fibrosis transmembrane regulator (CFTR). These data showing that CFTR is a target for PPARgamma agonists may provide new insights into the physiology of PPARgamma agonist-induced fluid retention.

Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers.

In breast cancer, overexpression of ErbB2 or aberrant regulation of survivin, a member of the inhibitor of apoptosis family, is associated with resistance to chemo/hormone therapy and predicts for a poor clinical outcome. A functional link between the two predictive factors has not been previously shown. Here, using genetic and pharmacologic approaches to block ErbB2 signaling, we show that ErbB2 regulates survivin protein expression in ErbB2-overexpressing breast cancer cells. Selective knockdown of ErbB2 using small interfering RNA markedly reduced survivin protein, resulting in apoptosis of ErbB2-overexpressing breast cancer cell lines such as BT474. Alternatively, inhibition of ErbB2 signaling using lapatinib (GW572016), a reversible small-molecule inhibitor of ErbB1/ErbB2 tyrosine kinases, at pharmacologically relevant concentrations, leads to marked inhibition of survivin protein with subsequent apoptosis. The effect of lapatinib on survivin seems to be predominantly posttranslational, mediated by ubiquitin-proteosome degradation as lactacystin, a proteosome inhibitor, reverses these effects. Furthermore, lapatinib down-regulated the expression of His-tagged survivin, which was under the transcriptional control of a heterologous promoter, providing additional evidence supporting a posttranslational mechanism of regulation. In contrast, trastuzumab and gefitinib failed to down-regulate survivin in ErbB2-overexpressing breast cancer cells. Importantly, the clinical relevance of these findings was illustrated in patients with ErbB2-overexpressing breast cancer whose clinical response to lapatinib was associated with marked inhibition of survivin in their tumors. These findings shed new light on the mechanism by which ErbB2 overexpression protects against apoptotic stimuli in breast cancer and identifies therapeutic interventions to improve clinical outcomes in these aggressive tumors.

Identification of liver receptor homolog-1 as a novel regulator of apolipoprotein AI gene transcription.

The orphan nuclear receptor liver receptor homolog-1 (LRH-1) has been reported to play a role in bile acid biosynthesis and reverse cholesterol transport. In this study, we examined the role of LRH-1 in the regulation of the apolipoprotein AI (APOAI) gene. Using RNA interference and adenovirus-mediated overexpression, we show that LRH-1 directly regulates APOAI gene transcription. Transient transfection experiments and EMSAs revealed that LRH-1 directly regulates APOAI transcription by binding to an LRH-1 response element located in the proximal APOAI promoter region. Chromatin immunoprecipitation experiments revealed that LRH-1 binds to the human APO AI promoter in vivo. Finally, we show that the transcriptional repressor SHP (small heterodimer partner) suppressed APOAI gene expression by inhibiting LRH-1 transcriptional activity. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOAI transcription and underscore the role of this receptor in cholesterol homeostasis.

Impact of PPARgamma overexpression and activation on pancreatic islet gene expression profile analyzed with oligonucleotide microarrays.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) serves as a target for the thiazolidinedione class of antidiabetic drugs and is an important regulator of adipose tissue differentiation. By contrast, the principal target genes for PPARgamma in the pancreatic islet and the impact of their induction on insulin secretion are largely undefined. Here, we show that mRNAs encoding both isoforms of rodent PPARgamma, gamma1 and gamma2, are expressed in primary rat islets and are upregulated by overexpresssion of the lipogenic transcription factor sterol response element-binding protein 1c. Unexpectedly, however, oligonucleotide microarray analysis demonstrates that graded activation of PPARgamma achieved with 1) the thiazolidinedione GW-347845, 2) transduction with adenoviral PPARgamma1, or 3) a combination of both treatments progressively enhances the expression of genes involved in fatty acid oxidation and transport. Moreover, maximal activation of PPARgamma1 reduces islet triglyceride levels and enhances the oxidation of exogenous palmitate while decreasing glucose oxidation, cellular ATP content, and glucose-, but not depolarization-stimulated, insulin secretion. We conclude that, in the context of the pancreatic islet, the principal response to PPARgamma expression and activation is the activation of genes involved in the disposal, rather than the synthesis, of fatty acids. Although fatty acid oxidation may have beneficial effects on beta-cell function in the longer term by countering beta-cell "lipotoxicity," the acute response to this metabolic shift is a marked inhibition of insulin secretion.

Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis.

The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression of CYP7A1 in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.

PML-dependent apoptosis after DNA damage is regulated by the checkpoint kinase hCds1/Chk2.

The promyelocytic leukaemia (PML) gene is translocated in most acute promyelocytic leukaemias and encodes a tumour suppressor protein. PML is involved in multiple apoptotic pathways and is thought to be pivotal in gamma irradiation-induced apoptosis. The DNA damage checkpoint kinase hCds1/Chk2 is necessary for p53-dependent apoptosis after gamma irradiation. In addition, gamma irradiation-induced apoptosis also occurs through p53-independent mechanisms, although the molecular mechanism remains largely unknown. Here, we report that hCds1/Chk2 mediates gamma irradiation-induced apoptosis in a p53-independent manner through an ataxia telangiectasia-mutated (ATM)-hCds1/Chk2-PML pathway. Our results provide the first evidence of a functional relationship between PML and a checkpoint kinase in gamma irradiation-induced apoptosis.

Human tumor suppressor ARF impedes S-phase progression independent of p53.

Using alternative reading frames, the human ARF-INK4a locus encodes two unrelated proteins that both function in tumor suppression. p16(INK4a) maintains the retinoblastoma protein in its growth-suppressive state through inhibition of cyclin D-dependent kinase activity, whereas ARF binds with MDM2 and stabilizes p53. The majority of the activity of ARF to date is ascribed to its ability to activate p53, resulting in a G(1) cell cycle arrest or apoptosis. We show here that ARF colocalizes with DNA replication protein A (RPA32) and that overexpression of ARF reduces the rate of DNA synthesis resulting in accumulation of an S-phase cell population. Impediment of DNA synthesis by ARF can occur and becomes more evident in the absence of p53. Hence, the biological consequence of ARF induction varies dependent on cellular p53 status, inducing predominantly a G(1) arrest or apoptosis in p53-positive cells or causing S-phase retardation when p53 function is comprised.