Selective Inhibitor Of Cyclin-dependent Kinases Research Articles

Abstract 2823: Identification of highly selective inhibitors of cyclin-dependent kinase 12

Abstract CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors. To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models. Citation Format: Axel Choidas, Carsten Schultz-Fademrecht, Carsten Degeenhardt, Peter Habenberger, Uwe Koch, Jan E. Eickhoff, Ann Kathrin Greifenberg, Peter Nussbaumer, Matthias Geyer, Bert M. Klebl. Identification of highly selective inhibitors of cyclin-dependent kinase 12. [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 2823.

5-Substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines with anti-proliferative activity as potent and selective inhibitors of cyclin-dependent kinases

A series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine derivatives was synthesized and evaluated for their cyclin-dependent kinase (CDK) inhibition activity. The most potent compounds contained various hydroxyalkylamines at the 5 position and possessed low nanomolar IC50 values for CDK2 and CDK5. Preliminary profiling of one of the most active compounds on a panel of 50 protein kinases revealed its high selectivity for CDKs. The compounds arrested cells in S and G2/M phases, and induced apoptosis in various cancer cell lines. Significant dephosphorylation of the C-terminus of RNA polymerase II and focal adhesion kinase (FAK), well-established substrates of CDKs, has been found in treated cells. Cleavage of PARP-1, down-regulation of Mcl-1 and activation of caspases correlated well with CDK inhibition and confirmed apoptosis as the primary type of cell death induced in cancer cells treated with the compounds in vitro. A comparison of known purine-based CDK inhibitor CR8 with its pyrazolo[4,3-d]pyrimidine bioisosteres confirmed that the novel compounds are more potent in cellular assays than purines. Therefore, pyrazolo[4,3-d]pyrimidine may emerge as a novel scaffold in medicinal chemistry and as a source of potent CDK inhibitors.

Palbociclib for the Treatment of Estrogen Receptor-Positive, HER2-Negative Metastatic Breast Cancer.

Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 that acts by reducing phosphorylation of the tumor suppressor gene retinoblastoma. When added to the aromatase inhibitor letrozole in a randomized phase II trial for first-line therapy of estrogen receptor-positive, HER2-negative metastatic breast cancer, palbociclib significantly increased progression-free survival compared with letrozole alone [palbociclib + letrozole: 20.2 months; 95% confidence interval (CI), 13.8-27.5; letrozole: 10.2 months; 95% CI, 5.7-12.6; HR, 0.49; 95% CI, 0.32-0.75; P = 0.0004]. On the basis of these results, the drug was recently granted accelerated approval by the FDA, and confirmatory studies are ongoing. Because this drug has a rational target in an oncologic pathway, concurrent biomarker development is of interest. In breast cancer, the most useful predictive biomarkers identified thus far are estrogen receptor and HER2 receptor status, although additional studies are ongoing. In this article, we review the development of palbociclib and its use in treatment of hormone receptor-positive metastatic breast cancer in the context of other FDA-approved agents in this setting.

The Role of CDK4/6 Inhibition in Breast Cancer.

Imbalance of the cyclin D and cyclin-dependent kinase (CDK) pathway in cancer cells may result in diversion away from a pathway to senescence and toward a more proliferative phenotype. Cancer cells may increase cyclin D-dependent activity through a variety of mechanisms. Therapeutic inhibition of CDKs in tumors to negate their evasion of growth suppressors has been identified as a key anticancer strategy. In this review, we outline the development of CDK inhibitory therapy in breast cancer, including the initial experience with the pan-CDK inhibitor flavopiridol and the next generation of oral highly selective CDK4 and CDK6 inhibitors PD0332991 (palbociclib), LEE011 (ribociclib), and LY2835219 (abemaciclib). Data from phase I and II studies in estrogen receptor-positive (ER+) breast cancer demonstrate promising efficacy with manageable toxic effects, chiefly neutropenia. We discuss these studies and the phase III studies that are accruing or nearing completion. We describe the application of such therapy to other breast cancer settings, including HER2-positive breast cancer and the adjuvant treatment of early breast cancer. We also discuss potential concerns surrounding the combination of CDK inhibitors with chemotherapy and their effects on repair of double-strand DNA breaks in cancer cells. Oral highly selective CDK inhibitors show great promise in improving the outcomes of patients with ER+ breast cancer, although caution must apply to their combination with other agents and in the early breast cancer setting.

Palbociclib: A new breakthrough for advanced breast cancer

Palbociclib: A new breakthrough for advanced breast cancer

Dinaciclib (SCH 727965) and Ofatumumab for the Treatment of Relapsed and Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL): Results of a Phase 1b/2 Study

BACKGROUND: Despite recent advances in the treatment of relapsed/refractory (R/R) CLL/SLL, patients (pts) with genetically high-risk disease, particularly complex abnormal karyotype and deletion(17p), continue to demonstrate inferior survival. Dinaciclib, a selective inhibitor of cyclin-dependent kinases (CDKs), has demonstrated activity in high-risk CLL/SLL. In an effort to improve response rates and reduce the risk for hyperacute tumor lysis syndrome (TLS), we conducted a NCI-sponsored phase 1b/2 study (NCI 9268) to characterize the safety and efficacy of dinaciclib when given in combination with the anti-CD20 monoclonal antibody ofatumumab.METHODS: Pts with CLL/SLL R/R after ≥1 prior therapy, age ≥ 18 years, ECOG ≤ 2, and retained end-organ function received ofatumumab according to the approved dose and schedule (weekly x 8, monthly x 4) starting on Cycle 1 Day 1. Dinaciclib was administered as a 2-hour infusion on days 1, 8, and 15 of a 28-day cycle beginning on Cycle 2 Day 1 and continued for up to 6 cycles. Pts could then continue single-agent dinaciclib indefinitely at MD discretion. During the phase 1b portion of the study, 3 pts were treated with 7 mg/m2 on Day 1 escalated to 10 mg/m2 on Day 8 (DL1); 3 additional phase 1b pts and all pts enrolled to the phase 2 were escalated to 14 mg/m2on Day 15 (DL2). All pts received aggressive hydration and pre-medication prior to dinaciclib to prevent TLS, peg-filgrastim on Day 16 of Cycles 2-7, and required antimicrobial prophylaxis (val-/acyclovir, ciprofloxacin, and TMP/SMX). Response was assessed with CT imaging according to IWCLL 2008 criteria at the end of cycle 1, after odd-numbered cycles, and every 3 months during follow-up.RESULTS: As of 30 July 2014, 36 pts (DL1 n = 4; DL2 n=32) have been treated: median age 62.5 years (range 35-80), 13 (36%) female, ECOG ≤1 in 32 (89%), and median number prior therapies was 2 (range 1-5). Del (17p) was present in 25 pts (69%), del (11q) in 16 (44%), and complex abnormal karyotype in 15 (42%). No dose limiting toxicities were observed in the phase 1b portion of the study. The most common grade ≥ 3 adverse events (without regard to attribution) included hyperglycemia in 5 pts (16%), hypocalcemia in 4 (13%), hypophosphatemia in 13 (41%), leukopenia in 12 (37%), anemia in 11 (34%), and thrombocytopenia in 9 (28%). Neutropenia was common (18% grade 3, 47% grade 4), but grade ≥ 3 infections were rare (1 pleural, 5 lung, 2 sepsis). Only 1 pt with highly proliferative disease refractory to both ibrutinib and IPI-145 developed TLS, which was further complicated by grade 5 sepsis. Pts have received a median 4 cycles (range 1-12), and 4 pts (11%) remain on treatment. Reasons for discontinuation are listed in the table. Best protocol response of partial response was recorded for 12 pts (33%), and an additional 20 (56%) patients achieved stable disease. Responses were observed in all genetic subgroups and deepened with continued dinaciclib treatment (see table). Estimated median PFS was 10.4 months (95%CI 8.2-12.2 months) and median overall survival (OS) had not been reached (95%CI 13.2-NR).CONCLUSIONS: Dinaciclib can be safely administered with ofatumumab to pts with R/R CLL/SLL. Serious non-hematologic adverse events are infrequent, and supportive measures help limit infectious complications. Initiating treatment with ofatumumab, stepwise dose escalation of dinaciclib, and rigorous prophylaxis abrogate the risk for TLS. These results confirm the activity of dinaciclib in a population enriched for genetically high-risk CLL/SLL. Responses improved with continued therapy, and discontinuation prior to best response likely impacted observed response rates. Further study of dinaciclib in combination with novel monoclonal antibodies and/or other kinase inhibitors is warranted, particularly in genetically high-risk disease.TableReason for Discontinuationn=36 (%)Completed planned therapy13 (41)Adverse event/complication6 (19)Disease progression6 (19)Alternative therapy3 (9)Death on study2 (6)Other2 (6)Best Protocol Response to Daten=36 (%)PR12 (33)SD20 (56)PD2 (6)IE1 (3)Cycle 2 Responsen=34 (%)*PR0SD32 (94%)PD2 (6)Cycle 4 Responsen=23 (%)PR5 (22)SD18 (78)PD0Cycle 6 Responsen=15 (%)PR11 (73)SD4 (27)PD0*1 pt with probable PD discontinued during Cycle 1 DisclosuresOff Label Use: Dinaciclib is an investigational agent made available through NCI/CTEP. Awan:Boehringer Ingelheim: Consultancy; Lymphoma Research Foundation: Research Funding. Maddocks:Pharmacyclics, Seattle Genetics, MorphoSys: Advisory Board Other, Research Funding. Byrd:Pharmacyclics: Research Funding.

Highlights of the Latest Advances in Research on CDK Inhibitors.

Uncontrolled proliferation is the hallmark of cancer and other proliferative disorders and abnormal cell cycle regulation is, therefore, common in these diseases. Cyclin-dependent kinases (CDKs) play a crucial role in the control of the cell cycle and proliferation. These kinases are frequently deregulated in various cancers, viral infections, neurodegenerative diseases, ischemia and some proliferative disorders. This led to a rigorous pursuit for small-molecule CDK inhibitors for therapeutic uses. Early efforts to block CDKs with nonselective CDK inhibitors led to little specificity and efficacy but apparent toxicity, but the recent advance of selective CDK inhibitors allowed the first successful efforts to target these kinases for the therapies of several diseases. Major ongoing efforts are to develop CDK inhibitors as monotherapies and rational combinations with chemotherapy and other targeted drugs.

The CDK4/6 inhibitor LY2835219 overcomes vemurafenib resistance resulting from MAPK reactivation and cyclin D1 upregulation.

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma.

Spinal cord injury causes brain inflammation associated with cognitive and affective changes: role of cell cycle pathways.

Experimental spinal cord injury (SCI) causes chronic neuropathic pain associated with inflammatory changes in thalamic pain regulatory sites. Our recent studies examining chronic pain mechanisms after rodent SCI showed chronic inflammatory changes not only in thalamus, but also in other regions including hippocampus and cerebral cortex. Because changes appeared similar to those in our rodent TBI models that are associated with neurodegeneration and neurobehavioral dysfunction, we examined effects of mouse SCI on cognition, depressive-like behavior, and brain inflammation. SCI caused spatial and retention memory impairment and depressive-like behavior, as evidenced by poor performance in the Morris water maze, Y-maze, novel objective recognition, step-down passive avoidance, tail suspension, and sucrose preference tests. SCI caused chronic microglial activation in the hippocampus and cerebral cortex, where microglia with hypertrophic morphologies and M1 phenotype predominated. Stereological analyses showed significant neuronal loss in the hippocampus at 12 weeks but not 8 d after injury. Increased cell-cycle-related gene (cyclins A1, A2, D1, E2F1, and PCNA) and protein (cyclin D1 and CDK4) expression were found chronically in hippocampus and cerebral cortex. Systemic administration of the selective cyclin-dependent kinase inhibitor CR8 after SCI significantly reduced cell cycle gene and protein expression, microglial activation and neurodegeneration in the brain, cognitive decline, and depression. These studies indicate that SCI can initiate a chronic brain neurodegenerative response, likely related to delayed, sustained induction of M1-type microglia and related cell cycle activation, which result in cognitive deficits and physiological depression.

Isolated spinal cord contusion in rats induces chronic brain neuroinflammation, neurodegeneration, and cognitive impairment

Cognitive dysfunction has been reported in patients with spinal cord injury (SCI), but it has been questioned whether such changes may reflect concurrent head injury, and the issue has not been addressed mechanistically or in a well-controlled experimental model. Our recent rodent studies examining SCI-induced hyperesthesia revealed neuroinflammatory changes not only in supratentorial pain-regulatory sites, but also in other brain regions, suggesting that additional brain functions may be impacted following SCI. Here we examined effects of isolated thoracic SCI in rats on cognition, brain inflammation, and neurodegeneration. We show for the first time that SCI causes widespread microglial activation in the brain, with increased expression of markers for activated microglia/macrophages, including translocator protein and chemokine ligand 21 (C–C motif). Stereological analysis demonstrated significant neuronal loss in the cortex, thalamus, and hippocampus. SCI caused chronic impairment in spatial, retention, contextual, and fear-related emotional memory—evidenced by poor performance in the Morris water maze, novel objective recognition, and passive avoidance tests. Based on our prior work implicating cell cycle activation (CCA) in chronic neuroinflammation after SCI or traumatic brain injury, we evaluated whether CCA contributed to the observed changes. Increased expression of cell cycle-related genes and proteins was found in hippocampus and cortex after SCI. Posttraumatic brain inflammation, neuronal loss, and cognitive changes were attenuated by systemic post-injury administration of a selective cyclin-dependent kinase inhibitor. These studies demonstrate that chronic brain neurodegeneration occurs after isolated SCI, likely related to sustained microglial activation mediated by cell cycle activation.

Dinaciclib (SCH 727965) Is a Novel Cyclin-Dependent Kinase (CDK) Inhibitor That Exhibits Activity In Patients With Relapsed Or Refractory Chronic Lymphocytic Leukemia (CLL)

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults. While the breadth of therapies for this incurable disorder has increased recently, patients afflicted with high risk disease have few and predominantly unsatisfactory options. Cyclin-dependent kinase (CDKs) inhibitors, such as Flavopiridol, have demonstrated clinical activity in CLL, though have been associated with significant therapy associated tumor lysis (TLS). Dinaciclib is a selective inhibitor of CDKs 1, 2, 5, and 9 that exhibits activity in CLL, including high risk subtypes.We performed a phase 1 dose escalation trial in relapsed and refractory CLL in order to determine the recommended phase 2 dose and maximum administered dose of dinaciclib. Patients received treatment as a 2-hour IV infusion on days 1, 8 and 15 of a 28-day cycle. The initial starting dose, determined based on preclinical data, was 5 mg/m2. Other dosing cohorts of 7 mg/m2, 10 mg/m2, 14 mg/m2 and 17mg/m2 were included in the dose escalation schema. An expansion cohort of patients utilizing an intra-patient dose escalation of 7mg/m2 on day 1, 10mg/m2 on day 8 and 14mg/m2 on day 15 during cycle 1 and 14mg/m2 on days 1, 8, and 15 of each subsequent cycle was further employed. Patients continued on treatment until there was disease progression, unacceptable toxicity or subject's refusal to continue.In total, 52 patients have been enrolled on this trial. The median age is 62.4 years (range 43-79), with 46% of patients ≥65 years. The majority of patients have advanced Rai stage (65%) and/or bulky disease (69%) and 46% with del(17p13.1). The median number of prior therapies is 4 (range 1-12) with 92% having received prior fludarabine and 31% having received prior flavopiridol. One episode of a dose limiting toxicity (DLT) was noted at 7mg/m2 (sepsis/death) and two at dose level of 17mg/m2 (hyperacute tumor lysis syndrome and pneumonia). One further DLT of TLS occurred at the recommended phase II dose level of 14mg/m2 during cohort expansion for a total of 18 patients treated at 14mg/m2. The most common serious adverse events included leucopenia, anemia, thrombocytopenia and metabolic evidence of tumor lysis, which occurred in 15% of patients. Clinical response was assessed utilizing the 1996 NCI-WG CLL criteria. The overall response rate of evaluable patients was 58% (28 of 48), while 57% patients with del(17p13.1) and 63% with del(11q22) achieved at least a partial response. The median progression free survival was 481 days (95% CI, 253 – 481). For responders, the median treatment duration was 214.5 days (range, 79 – 470 days).In conclusion, Dinaciclib is clinically active in this population of relapsed and refractory CLL. Importantly, patients with high risk disease, such as del(17p13.1) demonstrated impressive overall responses. Utilizing a dose escalation treatment model, Dinaciclib is well tolerated, safe and associated with typical toxicities which are readily managed. The use of this agent in combination with other efficacious agents, especially in the high risk CLL population is warranted. Disclosures:Jones:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Small:Merck: Employment. Im:Merck: Employment. Zhou:Merck: Employment.

Cell cycle activation contributes to increased neuronal activity in the posterior thalamic nucleus and associated chronic hyperesthesia after rat spinal cord contusion.

Spinal cord injury (SCI) causes not only sensorimotor and cognitive deficits, but frequently also severe chronic pain that is difficult to treat (SCI pain). We previously showed that hyperesthesia, as well as spontaneous pain induced by electrolytic lesions in the rat spinothalamic tract, is associated with increased spontaneous and sensory-evoked activity in the posterior thalamic nucleus (PO). We have also demonstrated that rodent impact SCI increases cell cycle activation (CCA) in the injury region and that post-traumatic treatment with cyclin dependent kinase inhibitors reduces lesion volume and motor dysfunction. Here we examined whether CCA contributes to neuronal hyperexcitability of PO and hyperpathia after rat contusion SCI, as well as to microglial and astroglial activation (gliopathy) that has been implicated in delayed SCI pain. Trauma caused enhanced pain sensitivity, which developed weeks after injury and was correlated with increased PO neuronal activity. Increased CCA was found at the thoracic spinal lesion site, the lumbar dorsal horn, and the PO. Increased microglial activation and cysteine-cysteine chemokine ligand 21 expression was also observed in the PO after SCI. In vitro, neurons co-cultured with activated microglia showed up-regulation of cyclin D1 and cysteine-cysteine chemokine ligand 21 expression. In vivo, post-injury treatment with a selective cyclin dependent kinase inhibitor (CR8) significantly reduced cell cycle protein induction, microglial activation, and neuronal activity in the PO nucleus, as well as limiting chronic SCI-induced hyperpathia. These results suggest a mechanistic role for CCA in the development of SCI pain, through effects mediated in part by the PO nucleus. Moreover, cell cycle modulation may provide an effective therapeutic strategy to improve reduce both hyperpathia and motor dysfunction after SCI.

P-0119 - The Cd4/6 Inhibitor, Pd0332991, Has Activity in Metastatic Hepatocellular Carcinoma: Preliminary Results of a Phase II Trial

Background: PD-0332991 is an orally available, pyridopyrimidine-derived, selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), a key regulator of cell growth. Treatment with PD-0332991 results in the inhibition of RB protein phosphorylation & cell cycle arrest. In preclinical studies, PD-0332991 demonstrated potent target specificity and significant inhibition of tumor growth in-vitro and in xenografts. In these systems, it was more effective than the currently approved drug sorafenib. Phase I clinical trials showed an acceptable toxicity profile for the drug. Thus, PD0332991 represents an ideal candidate for treatment of patients with advanced HCC. Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide and the 3rd most frequent cause of cancer-related mortality. Surgical resection and liver trans-plantation are considered the main curative options for HCC. Standard cytotoxic drugs administered singularly or in combination give poor response rates and little survival benefit. The MKI sorafenib has been shown to significantly increase PFS & OS in advanced HCC in randomized Phase 3 trials. However, even with such favorable and landmark studies the response rate to sorafenib is low and overall benefit modest. There is still a critical need for key molecular targets and new drugs for the treatment of advanced HCC. Methods: This is an open-label, nonrandomized single institution trial for subjects with inoperable, recurrent/refractory advanced HCC. Subjects must have failed or be intolerant of standard first line therapy with sorafenib. Eligible subjects are treated with 125 mg PD-0332991 orally per day, Days 1-21 of a 28 day cycle, until disease progression, unacceptable toxicity, withdrawal or death. Tumor response by CT or MRI is assessed using mRECIST v1.1 every 8 w. Toxicity assessments are per NCI CTCAE v4.0.

Inhibition of NF-κB–Mediated Signaling by the Cyclin-Dependent Kinase Inhibitor CR8 Overcomes Prosurvival Stimuli to Induce Apoptosis in Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure. We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry-based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR. CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-κB signaling at the transcriptional level and through inhibition of the inhibitor of IκB kinase (IKK) complex, resulting in stabilization of IκBα expression. CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL.

CDK9-dependent transcriptional elongation in the innate interferon-stimulated gene response to respiratory syncytial virus infection in airway epithelial cells.

Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3(-/-) MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser(2) carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.

Abstract 700: Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells.

Abstract Cyclin-dependent kinases (CDKs) form complexes with partner cyclins to regulate cell cycle progression and transcription. CDK1, CDK2, CDK4 and CDK6 all regulate cell cycle progression and CDK8 and CDK9 are regulators of transcription. CDK7 is a unique member of this family because it can regulate the cell cycle CDKs as part of the CDK-activating kinase (CAK) and also transcription in conjunction with the TFIIH transcription factor by phosphorylating the COOH-terminal domain of RNA polymerase II (Pol II CTD). A hallmark of cancer is deregulated cell cycle progression and many cancers have abnormal CDK activity which makes CDKs a good target for cancer therapy. Here we show BS-194, a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7 and CDK9 (IC50 = 3, 30, 30, 250 and 90nmol/L, respectively). In vitro cell based assays have shown that BS-194 inhibits the phosphorylation of CDK substrates, Rb and RNA Poll II, down regulation of cyclins A, E and D1 and cell cycle arrest in the S and G2/M phases. BS-194 also has potent anti-proliferative activity in 60 cancer cell lines tested (mean GI50 = 28nmol/L). BS-194 is orally bioavailable and shows potent in vivo tumour growth inhibition of xenograft models of MCF7 and HCT116 cells. We have used BS-194 as a potent inhibitor of CDK activity in HCT116 cells and performed gene microarray analysis to determine novel pathways and identify novel biomarkers associated with CDK inhibition. In summary, the gene microarray has identified genes differentially expressed in response to BS-194 treatment that are primarily involved in cell cycle, apoptosis and transcription pathways. Citation Format: Silvia Ottaviani, Sean Delaney, Hetal Patel, Manikandan Periyasamy, Alexander Bondke, Brian Slafer, Richard Starkey, Sebastian H.B. Kroll, Matthew J. Fuchter, Anthony G.M. Barrett, R. Charles Coombes, Simak Ali. Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells. [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 700. doi:10.1158/1538-7445.AM2013-700

Effect of PD-0332991, a potent and selective inhibitor of cyclin-dependent kinase 4/6, on proliferation in renal cell carcinoma at nanomolar concentrations and prediction of sensitivity by molecular markers.

413 Background: Cell cycle dysregulation is prevalent in renal cell carcinoma (RCC). PD-0332991 is an orally active, potent, and selective inhibitor of cyclin-dependent kinases (CDK) 4 and 6, blocking retinoblastoma (Rb) phosphorylation at nanomolar concentrations. Methods: 28 RCC and immortalized kidney cell lines were used to examine the effects of PD-0332991 on proliferation to determine the half maximal inhibitory concentration (IC50). Effects of PD-0332991 on cell-cycle, apoptosis, and Rb phosphorylation were assessed with flow cytometry and western blot analysis for five of the cell lines: RCC-HB and SW 156 (sensitive/malignant), R444 and Hs 891.T (resistant/malignant), and CCD 1103 (resistant/immortalized non-malignant). Molecular markers for response prediction were studied using array CGH and gene expression profiling. Results: Concentration-dependent inhibition of proliferation was identified in response to PD-0332991, with IC50values ranging from 25.0nM up to 700nM; five cell lines were identified as completely resistant at 1000nM. PD-0332991 induced G0/G1 cell cycle arrest, as well as induction of late apoptosis in SW 156, and Rb phosphorylation was blocked in a time-dependent fashion in both sensitive cell lines, while resistant lines were unaffected. Genotype and expression data of CDKN2A and CDKN2B were combined and a consensus was made regarding p16 and p15 status; significant association between loss and sensitivity to PD-0332991 was identified for p16 (p = 0.027). For CCND1, CCNE1, E2F1, Rb, CDK4, and CDK6 no amplifications or homozygous deletions were identified by array CGH; cell lines were then classified as having high or low expression for each of these markers. E2F1 had low expression levels significantly associated with response to PD-0332991 (p = 0.041). Conclusions: PD-0332991 shows anti-proliferative activity in RCC through blockade of the cell cycle. The decreased expression of molecular markers p16 and E2F1 predict for sensitivity to PD-0332991 in RCC.

SNS-032 inhibits mTORC1/mTORC2 activity in acute myeloid leukemia cells and has synergistic activity with perifosine against Akt

BackgroundAcute myeloid leukemia (AML) is a heterogeneous disorder with aberrant regulation of a variety of signal pathways. Therefore, simultaneous targeting of two or even more deregulated signal transduction pathways is needed to overcome drug resistance. Previously, it was reported that SNS-032, a selective cyclin-dependent kinase inhibitor, is an effective agent for treatment of AML; however, the molecular mechanisms of SNS-032-induced cell death of AML cells are not yet fully understood. The aim of the study was to characterize the effects in vitro of SNS-032, used alone and in combination with an Akt inhibitor perifosine, against AML cells and to identify the mechanism involved.ResultsSNS-032 significantly induced cytotoxicity in human AML cell lines and blasts from patients with newly diagnosed or relapsed AML. However, Kasumi-1 cells and some of leukemic samples (14.9%) from AML patients were resistant to SNS-032-mediated cell death. Western blot analysis showed that SNS-032 strongly inhibited the phosphorylation of mammalian target of rapamycin (mTOR) on Ser 2448 and Ser2481, and that removal of SNS-032 resulted in partial recovery of cell death and reactivation of phosphorylation of mTOR. Moreover, exogenous insulin-like growth factor-1 (IGF-1) did not reverse SNS-032-induced cell growth inhibition and downregualtion of phosphor-mTOR at Ser2448 and Ser2481 although slight suppression of IGF-1R expression was triggered by the agent. Furthermore, SNS-032 at a lower concentration (60–80 nM) enhanced AML cell cytotoxicity induced by perifosine, an Akt inhibitor. Importantly, SNS-032 treatment reduced colony formation ability of AML cells, which was significantly increased when two agents were combined. This combination therapy led to almost complete inhibition of Akt activity.ConclusionWe conclude that SNS-032 might directly target mammalian target of rapamycin complex 1 (mTORC1)/mTORC2. Our results further provide a rationale for combining SNS-032 with perifosine for the treatment of AML.

Abstract S1-6: Results of a randomized phase 2 study of PD 0332991, a cyclin-dependent kinase (CDK) 4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2− advanced breast cancer (BC)

Abstract Background: PD 0332991, a selective inhibitor of CDK 4/6, prevents cellular DNA synthesis by blocking cell cycle progression. Preclinical studies in a BC cell line panel identified the luminal ER subtype, elevated expression of cyclin D1 and Rb protein, and reduced p16 expression as being associated with sensitivity to PD 0332991 (Finn et al. 2009). Synergistic activity was also observed in vitro when combined with tamoxifen. After determination of the recommended phase 2 dose in combination with letrozole (letrozole 2.5 mg QD plus PD 0332991 125 mg QD on Schedule 3/1), a randomized phase 2 study comparing letrozole alone (L) to letrozole plus PD 0332991 (L+P) was initiated. Methods: The phase 2 portion of the study was designed as a two-part study; Part 1 enrolled post- menopausal women with ER+/HER2− advanced BC; Part 2 in addition to ER+/HER2− as eligibility criteria, screened for CCND1 amplification and/or loss of p16 by FISH. The primary endpoint is progression-free survival (PFS); secondary endpoints include response rate, overall survival, safety, and correlative biomarker studies. In both parts, post-menopausal women with ER+/HER2− advanced BC were randomized 1:1 to receive letrozole either with or without PD 0332991. Pts continue on assigned study treatment until disease progression, unacceptable toxicity, or consent withdrawal, and are followed for tumor assessments every 2 months. Results: 66 pts were randomized in Part 1 and 99 pts in Part 2. Preliminary results from Part 1 of this study have been previously reported (IMPAKT Breast Cancer Conference, Abstract #292, Finn et al. May 2012) demonstrating a significant improvement in median PFS in the L+P vs. L arm (HR = 0.35; 95% CI, 0.17 to 0.72; p = 0.006). With the additional 99 pts randomized in Part 2 (N = 165), the statistically significant improvement in median PFS (26.2 vs. 7.5 months, respectively) continues to be observed with a HR=0.32 (95% CI, 0.19 to 0.56) with p <0.001. The response rate for the L+P arm (n = 84) was 31% vs. 26% for the L arm (n = 81) and the clinical benefit rate was 68% vs. 44%, respectively. The most commonly reported treatment-related AEs in the combination arm were neutropenia, leukopenia, anemia, and fatigue. The updated results from the combined Part 1 and Part 2 group will be presented in December 2012. Conclusions: The combination of PD 0332991 and letrozole is well tolerated and shows encouraging clinical benefit, confirming the sensitivity of ER+ BC to PD 0332991 observed in preclinical models. A phase 3 trial in this setting will commence in 2013. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S1-6.

Effect of aprepitant on the pharmacokinetics of the cyclin-dependent kinase inhibitor dinaciclib in patients with advanced malignancies

Dinaciclib, a selective inhibitor of cyclin-dependent kinase (CDK) 1, CDK2, CDK5, and CDK9, is metabolized via CYP3A4. Aprepitant, a neurokinin-1 receptor antagonist for the prevention of chemotherapy-induced nausea and vomiting, is an inhibitor and inducer of CYP3A4. We conducted a randomized, crossover study to investigate the effects of single oral doses of aprepitant when coadministered with dinaciclib. As part of a phase 1 dose-escalation trial, subjects with advanced malignancies were randomized into a 2-period, multi-cycle, crossover study to investigate the effect of single doses of oral aprepitant on the pharmacokinetics of 29.6 mg/m(2) dinaciclib administered by 2-h intravenous infusion. During cycle 1 and cycle 2, subjects received dinaciclib with aprepitant in one cycle and dinaciclib without aprepitant in the other cycle; aprepitant was administered at a dose of 125 mg orally on day 1 and 80 mg orally on days 2 and 3, along with standard dosing regimens of ondansetron and dexamethasone. Twelve patients completed the study; T (max) occurred approximately 2 h after the initiation of the infusion. The percent geometric mean ratio (dinaciclib + aprepitant vs. dinaciclib alone) was 106 % (90 % confidence interval [CI] 89-126 %) and 111 % (90 % CI 93-132 %) for dinaciclib C(max) and AUC([I]), respectively. The half-life and clearance of dinaciclib were similar, with or without aprepitant. Coadministration of dinaciclib with aprepitant resulted in no clinically significant effect on the pharmacokinetics and did not alter the safety profile of dinaciclib in patients with advanced malignancies.