Abemaciclib In Combination Research Articles

Abstract 583: The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade

Abstract Targeting cyclin dependent kinases 4 and 6 (CDK4/6) with inhibitors such as abemaciclib has shown promise in early and late phase clinical trials in both breast cancer and NSCLC. While there is evidence that patients benefit from single-agent abemaciclib, combination strategies leveraging this compound together with immunotherapy are of interest for the treatment of these and other cancers. Consequently, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. However, because most preclinical studies have been performed using xenograft tumors in immune-compromised mice, the potential immunomodulatory effects of abemaciclib have not been adequately ascertained. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with checkpoint immunotherapy in a murine syngeneic tumor model sensitive to abemaciclib using immuno-competent mice. Abemaciclib monotherapy of established murine CT26 tumors, which harbor KRAS G12C mutation and CDKN2A deletion, caused a dose-dependent delay in tumor growth. Surprisingly, gene expression analysis showed that treatment was associated with an increase in intra-tumor immune inflammation without major alteration in immune subset frequencies. Testing of various dosing regimens in this preclinical model found that monotherapy abemaciclib pretreatment followed by combination with anti-PD-L1 antibody therapy, induced an enhanced anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy exhibited superior anti-tumor efficacy, resulting in complete tumor regression (CR) in 50-60% of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice which maintained CRs after cessation of combination therapy were able to resist later CT26 rechallenge, demonstrating that abemaciclib in combination with anti-PD-L1 enabled the generation of an immunologic memory. Examination of intra-tumor gene expression during treatment found that combination therapy further amplified the immune/T cell activation signature compared to both monotherapies. Intra-tumoral suppression of cell cycle genes, which are indicative of inhibition of CDK4/6, was also greater during the combination therapy, suggesting that the effects anti-PD-L1 therapy may augment the cell cycle arrest induced by abemaciclib. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results demonstrate that it is possible to combine CDK4/6 inhibition by abemaciclib with checkpoint immunotherapy to improve tumor efficacy. The synergistic responses observed in terms of tumor efficacy, immune activation, and cell cycle control provides support for the clinical investigation of this combination. Citation Format: Jack Dempsey, Lysiane Huber, Amelie Forest, Jennifer R. Stephens, Thompson N. Doman, Jason Manro, Andrew Capen, Robert S. Flack, Gregory P. Donoho, Sean Buchanan, Alfonso De Dios, Kyla Driscoll, Michael Kalos, Ruslan Novosiadly, Richard P. Beckmann, David A. Schaer. The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2017-583

MONARCH 2: Abemaciclib in combination with fulvestrant in patients with HR+/HER2- advanced breast cancer who progressed on endocrine therapy

1000 Background: Abemaciclib, an oral, selective inhibitor of CDK4 & 6, dosed on a continuous schedule, demonstrated clinical activity as monotherapy in patients (pts) with treatment refractory hormone receptor positive (HR+) metastatic breast cancer (MBC). The tolerability and activity of abemaciclib + fulvestrant (F) supported Phase 3 evaluation. Methods: MONARCH 2 is a double-blind Phase 3 trial of abemaciclib + F vs placebo (P) + F in women with HR+/HER2- advanced breast cancer. Women who progressed on (neo)adjuvant endocrine therapy (ET), ≤12 months from end of adjuvant ET, or on first line ET for MBC and who had not received chemotherapy for metastatic disease were eligible. Pts were randomized 2:1 to receive abemaciclib at 150 mg Q12H (or 200 mg prior to amendment) or P plus F (500 mg, per label) and stratified by metastatic site (visceral, bone only, or other) and resistance to prior ET (primary vs secondary). Pre/perimenopausal pts received a gonadotropin-releasing hormone agonist. The primary objective was investigator-assessed progression-free survival (PFS). Secondary endpoints included objective response rate (ORR) and other efficacy and safety endpoints. Assuming a hazard ratio (HR) of 0.703 in favor of abemaciclib + F, 378 events were needed for 90% power at one sided α=.025. Results: 669 pts were randomized to abemaciclib + F (N=446) and to P + F (N=223). 56% of pts had visceral disease, 72% had measurable disease, 25% had primary ET resistance, and 82% were postmenopausal. In the ITT population 379 PFS events were observed with a median PFS of 16.4 m for abemaciclib + F and 9.3 m for P + F (HR: 0.553; 95% CI: 0.449, 0.681, P<.0000001 by log-rank test). In pts with measurable disease, the ORR was 48.1% (3.5% complete response [CR]) for abemaciclib + F and 21.3% (0% CR) for P + F. The most frequent treatment emergent adverse events for abemaciclib + F vs P + F were diarrhea (86.4% vs 24.7%), neutropenia (46.0% vs 4.0%), nausea (45.1% vs 22.9%), and fatigue (39.9% vs 26.9%). Conclusions: Abemaciclib + fulvestrant was an effective treatment in patients with HR+/HER2- advanced breast cancer who progressed on endocrine therapy with significantly improved PFS and ORR. Clinical trial information: NCT02107703.

Abstract OT2-01-07: A phase 2 study of abemaciclib plus pembrolizumab for patients with hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC)

Abstract Background: Abemaciclib is a small molecule inhibitor of both cyclin-dependent kinase (CDK) 4 and CDK6 administered orally twice daily on a continuous schedule. In I3Y-MC-JPBA, a phase I study, abemaciclib demonstrated acceptable safety, tolerability, and single-agent activity as monotherapy in different tumor types, including HR+ MBC (Patnaik A,et al. Cancer Discov 2016;6:1–14). In MONARCH 1, a phase 2 study, abemaciclib demonstrated a 19.7% objective response rate (ORR) with a median duration of response (DoR) of 8.6 months, median progression-free survival (PFS) of 6.0 months, and clinical benefit rate (CBR) of 42.4% in patients with HR+, HER2- MBC whose disease progressed on or after endocrine therapy and chemotherapy (Dickler, M. et al. American Society of Clinical Oncology (ASCO), abstract #510 (2016).). Pembrolizumab is a humanized monoclonal antibody against the programmed death receptor-1 (PD-1) protein that has shown preliminary efficacy in single-arm monotherapy trials in ER+/HER2- advanced breast cancer. Trial design: This open-label, phase 2 study will evaluate the safety and preliminary efficacy of abemaciclib 150 mg given orally every 12 hours on days 1-21 of a 21-day cycle in combination with intravenous pembrolizumab 200 mg on day 1 of a 21-day cycle in approximately 75 patients with stage IV non-small cell lung cancer or HR+, HER2- MBC (ClinicalTrials.gov NCT02779751). The study will include 3 disease-specific cohorts, each with approximately 25 patients. Only the HR+, HER2- MBC cohort will be presented here. Eligibility criteria: Eligible patients for the MBC cohort include women with confirmed HR+, HER2- MBC who have completed at least 1 but no more than 2 prior chemotherapy regimens in the metastatic setting; will provide tumor tissue prior to and after treatment (cycle 3, day 1); have measurable disease (RECIST v.1.1), adequate organ function, an ECOG performance status ≤1, and a life expectancy ≥12 weeks; are ≥18 yrs of age and able to swallow oral medications; and have not received treatment with any CDK 4 and 6 inhibitors or PD-1 or PD-L1 inhibitors. Specific aims: The primary objective is to characterize the safety profile of the combination of abemaciclib and pembrolizumab. Key secondary objectives include ORR, DoR, disease control rate (DCR), PFS, overall survival (OS), and characterization of pharmacokinetics. Statistical methods: The safety population includes patients who received at least one dose of study drug. ORR, DoR, DCR, and PFS analyses will be evaluated according to RECIST v.1.1 and irRECIST for disease progression. Time-to-event variables, such as DoR, PFS, and OS, will be estimated by Kaplan-Meier methodology. An interim analysis of safety and preliminary efficacy may occur for each cohort after all patients have completed (or discontinued from) approximately 24 weeks of treatment. The final analysis of OS will occur based on data collected for approximately 12 months after the last patient receives treatment. Target accrual: Approximately 75 patients are planned for the trial; 25 patients will comprise the MBC cohort. Contact information: 1-877-CTLILLY (1-877-285-4559). Citation Format: Rugo H, Tolaney S, Dickler M, Kabos P, Ho C-L, Wildiers H, Jerusalem G, Alés-Martínez JE, Hossain A, Johnston E, Gianni L. A phase 2 study of abemaciclib plus pembrolizumab for patients with hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-07.

P3.02c-102 Safety and Tolerability of Abemaciclib Combined with LY3023414 or with Pembrolizumab in Patients with Stage IV NSCLC: Topic: IT Clinical

Currently, treatment options are limited for patients with advanced and/or metastatic NSCLC particularly after initial treatment. In a prior phase 1 study, abemaciclib, a CDK4 & 6 inhibitor, demonstrated single-agent anti-tumor activity when dosed orally on a continuous schedule, with an acceptable safety profile in patients with previously treated metastatic NSCLC (NCT01394016). PI3kinase is an escape pathway after CDK inhibition in tumor models and aberrant immunity is a hallmark of cancer, providing the rationales to combine abemaciclib with PI3K and with checkpoint inhibitors. An ongoing Phase 1b multicenter, open-label, 3+3 dose-escalation trial with an expansion phase is investigating abemaciclib in combination with multiple single-agent options in metastatic NSCLC (NCT02079636). Here we report preliminary results for two arms of the study. In Part D, abemaciclib was administered orally on a continuous schedule every 12 hours (q12h) in combination with the PI3K/mTOR inhibitor, LY3023414, at 100, 150, or 200 mg q12h. In Part E, abemaciclib was administered in combination with the anti-PD-1 antibody, pembrolizumab (200 mg I.V. infusion q3 weeks). Patients with late stage NSCLC and 1-3 prior therapies without central nervous system metastasis were treated until disease progression or other discontinuation criteria were met. Primary endpoints for each cohort included safety/tolerability and identification of the recommended phase 2 dose. Safety assessments followed the Common Terminology Criteria for Adverse Events (NCI-CTCAE v4.0). Parts D and E began enrolling patients on April 13, 2015 and April 29, 2016, respectively. As of August 24, 2016, Parts D and E escalation included, respectively, 22 [male (64%)/Caucasian (77%)/stage IV (91%)/adenocarcinoma (91%)] and 6 patients [male (33%)/Caucasian (100%)/stage IV (67%)/adenocarcinoma (100%)]. ECOG PS was ≤1 in both cohorts. In Part D, 1 patient on dose level-2 (DL-2) experienced a dose limiting toxicity (DLT) (G4 thrombocytopenia). Evaluation of additional dose levels is ongoing. Seventeen patients (77%) experienced ≥1 treatment-related emergent adverse event (TRAE). Common TRAEs were nausea (50%), diarrhea (50%), vomiting (36%), fatigue (32%), and decreased appetite (27%). In Part E, no DLTs or deaths occurred in the two dosing cohorts evaluated. Four patients (67%) experienced ≥1 TRAE with 75% G1/2. Common TRAEs included fatigue (50%), diarrhea and proteinuria, (33%, each). The majority of previously treated advanced/metastatic NSCLC patients administered abemaciclib with LY3023414 or with pembrolizumab had manageable and tolerable adverse events, similar to those of the single agents.

P3.02b-062 Safety of Necitumumab and Abemaciclib Combination Therapy in Patients with Advanced Non-Small Cell Lung Cancer (NSCLC): Topic: EGFR Clinical

Trials of anti-EGFR necitumumab and the CDK4 and CDK6 inhibitor abemaciclib have demonstrated anti-tumor activity of each agent in patients with NSCLC. In a xenograft model of NSCLC, the addition of necitumumab to abemaciclib improved the anti-tumor efficacy compared to either monotherapy. Single-arm, multicenter Phase 1b study to investigate the combination of necitumumab and abemaciclib in patients with stage IV NSCLC (NCT02411591). The safety interim population includes squamous and non-squamous patients treated with the recommended dose of necitumumab 800mg IV on days 1 and 8, every 21 days in combination with abemaciclib 150mg (dose identified in preceding dose escalation part of study) administered every 12 hours on days 1–21. Major eligibility criteria include: progression after platinum-based chemotherapy regimen and maximum 1 other prior chemotherapy for advanced and/or metastatic disease (prior treatment with EGFR-TKI and ALK inhibitors was mandatory in patients whose tumor has EGFR-activating mutations or ALK translocations, respectively); ECOG PS 0-1; tumor tissue availability for biomarker analysis and measurable disease. Treatment will continue until disease progression, unacceptable toxicity, or withdrawal of consent by the patient, or sponsor/investigator decision. This safety interim includes 16 squamous and non-squamous patients treated at recommended dose (necitumumab 800mg + abemaciclib 150mg) and having completed 2 cycles of study treatment (or otherwise discontinued study treatment). The most common (>15% patients) adverse events (AEs) of any grade are shown in the Table. Grade ≥3 AEs were reported in 6 patients (diarrhea, nausea, vomiting, neutropenia, decreased appetite, hypophosphotaemia, dyspnoea were each reported in 1 patient and fatigue in 2 patients); grade ≥3 AEs were judged to be related to study treatment in 4 patients. No patients have discontinued the study due an AE. The combination of necitumumab and abemaciclib in advanced NSCLC is well tolerated when administered according to recommended dosing schedules.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival.Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor.Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib.Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. DisclosuresIriyama:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

1297TiP - Phase 1b expansion cohort study to evaluate the safety and efficacy of necitumumab and abemaciclib combination therapy in patients with stage IV non-small cell lung cancer (NSCLC)

1297TiP - Phase 1b expansion cohort study to evaluate the safety and efficacy of necitumumab and abemaciclib combination therapy in patients with stage IV non-small cell lung cancer (NSCLC)

Abstract 2836: Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines

Abstract Breast cancer is the second most common cancer worldwide after lung cancer. About 70% of breast cancers express estrogen receptor α (ER+) and/or progesterone receptor (PR+), and these biomarkers are indicative of hormone dependence. However up to 50% acquire resistance to hormone therapy [1, 2]. Estrogen independent ER+ breast cancer depends on CDK4 for tumor growth and CDK4 inhibitors have emerged as a promising approach to treat this type of tumors [3]. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and it is being evaluated in advanced clinical trials for its potential to reduce metastatic ER+ breast cancer growth. We have evaluated combination of abemaciclib with an anti-estrogen therapy in an in vitro breast cancer panel. Phenotypic characterization of sensitive cell lines was carried out by monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches. Using an in vitro panel with a diversity of breast cancer cell lines, a synergistic effect of abemaciclib in combination with the ER down-regulating drug fulvestrant was observed based on Bliss score. This combination treatment demonstrated effective growth inhibition in ER+ cells and exhibited synergism in MCF-7, T47D and ZR-75-1. The mechanistic analyses revealed that the combination of abemaciclib with fulvestrant promoted a decrease in cancer cell proliferation due to G1 phase arrest at doses tested. This growth inhibition was accompanied by increased hallmarks for cell senescence as observed by markers such as SA-β-galactosidase staining or morphological changes. Subsequently, an increase in biomarkers for apoptosis was also observed. These changes occurred in a time dependent manner and were significantly greater with the combination than fulvestrant single agent treatment. We conclude the combination of abemaciclib with fulvestrant better prevented proliferation of breast cancer cell lines by blocking cell proliferation and lead to induction of senescence and apoptosis as compared to fulvestrant treatment alone in ER+ cells. Bibliography [1] American Cancer Society, Cancer Facts & Figures 2014. [2] Dixon J.M. (2014) New Journal of Science. Volume 2014, Article ID 390618. [3] Miller TW et al. (2011) Cancer Discov. Volume 1 (4): 338-51. Citation Format: Raquel Torres, Bruna Calsina, Ana Hermoso, Carmen Baquero, Cecilia Mur, Karsten Boehnke, Joaquín Amat, Alfonso De Dios, Xueqian Gong, Sean Buchanan, Richard Paul Beckmann, Maria Jose Lallena. Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines. [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 2836.

Abstract 2828: Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer

Abstract The cyclin D:CDK-4/6:Rb axis is dysregulated in a number of different cancers and is implicated in resistance to hormonal therapy in breast cancer. Pharmacologically targeting cyclin dependent kinase 4 and 6 (CDK4 and 6) has proven to be a successful therapeutic approach in ER+ breast cancer (BC). This study aimed to identify the molecular subtypes of BC that are sensitive to the novel CDK4 and 6 inhibitor, abemaciclib, and identify the best combination strategies for the clinical development. Growth inhibition activity of abemaciclib was assessed in a panel of 46 BC cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. In vivo activity of abemaciclib was assessed in cell line xenograft models of ER+ and HER2+/ER+ BCs. For ER+ BC, mice were treated daily with clinically achievable doses of abemaciclib (50-75 mg/kg) as a single agent or in combination with tamoxifen or fulvestrant. Combinations with trastuzumab, docetaxel and tamoxifen were assessed in the HER2+/ER+ xenografts. Sensitivity to abemaciclib was observed predominately in multiple luminal BC cell lines and a small subset of triple negative cell lines that had intact Rb signaling. Activating mutations in PIK3CA also marked for abemaciclib sensitivity. Abemaciclib potentiated the anti-proliferative effects of cytotoxic/anti-mitotic agents when given simultaneously or 48 hours prior to treatment in vitro. Significant tumor growth inhibition (TGI) was observed with single agent abemaciclib in the ER+ BC cell line xenografts. In ZR751 xenografts, the addition of either tamoxifen or fulvestrant to abemaciclib induced complete inhibition of tumor growth for the 12 weeks of treatment. In the MCF7 model, treatment was withdrawn after five weeks, which triggered tumor regrowth in each of the single agent arms. However, complete responses were maintained in the combination arms for a further six weeks post drug withdrawal. In HER2 amplified xenografts, abemaciclib single agent treatment induced significant TGI in trastuzumab sensitive and resistant xenografts, and combination with trastuzumab further increased this anti-tumor effect. The addition of tamoxifen to this combination induced a further increment in TGI. Consistent with the in vitro findings, the combination of abemaciclib and the anti-mitotic agent docetaxel was not antagonistic in vivo, and the addition of docetaxel to the triple combination of abemaciclib, trastuzumab, and tamoxifen induced the most efficacy of any of the treatment arms tested. Combinations were well tolerated in animals. These data highlight the potential of abemaciclib to have single agent activity in addition to combined activity with anti-mitotic or targeted therapies for breast cancer. Citation Format: Neil A. O’Brien, Dylan Conklin, Tong Luo, Ondrej Kalous, Erika von Euw, Sara A. Hurvitz, Richard P. Beckmann, Colleen Mockbee, Dennis J. Slamon. Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer. [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 2828.

Abstract 282: Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1

Abstract KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, including melanoma, lung, colorectal, and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor currently in phase I clinical trial, has been shown to inhibit cell proliferation of RAS- or BRAF-mutant tumor cells in vitro and xenograft tumor growth in vivo. An unbiased screen for compounds that synergize with LY3009120 in RAS/BRAF mutant cancers identified inhibitors of CDK4 among the top hits. In this study, we found that combined inhibition of RAF and CDK4 and 6 by LY3009120 and abemaciclib cooperatively reduced viability of tumor cells with KRAS, NRAS or BRAF mutation in vitro. In animal models, the LY3009120 and abemaciclib combination exhibited synergistic regression of tumor growth in multiple xenograft models with KRAS, NRAS, or BRAF mutation. Molecular mechanistic analysis revealed that pan-RAF inhibitor treatment suppressed the cyclin D1 upregulation which was mediated through CDK4 and CDK6 inhibition by abemaciclib, and the combination treatment cooperatively demonstrated more complete inhibition of RB phosphorylation. These results were further verified by CDK4 and CDK6 siRNA knockdown and another CDK4 and CDK6 selective inhibitor palbociclib. Importantly, the more complete phospho-RB inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combinational treatment led to more significant cell cycle G0/G1 arrest and apoptosis of tumor cells. These preclinical findings suggest that the combinational inhibition of RAF and CDK4 and CDK6 signaling by LY3009120 and abemaciclib is synergistic and should be further studied compared to single agents in the treatment of cancer in patients with KRAS, NRAS or BRAF mutations. Citation Format: Shih-Hsun Chen, Youyan Zhang, Robert D. Van Horn, Tinggui Yin, Lysiane Huber, Teresa F. Burke, Xueqian Gong, Wenjuan Wu, Shripad Bhagwat, Sean Buchanan, Richard P. Beckmann, Ramon V. Tiu, Sheng-Bin Peng. Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1. [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 282.

Optimising the combination dosing strategy of abemaciclib and vemurafenib in BRAF-mutated melanoma xenograft tumours.

Background:Resistance to BRAF inhibition is a major cause of treatment failure for BRAF-mutated metastatic melanoma patients. Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, overcomes this resistance in xenograft tumours and offers a promising drug combination. The present work aims to characterise the quantitative pharmacology of the abemaciclib/vemurafenib combination using a semimechanistic pharmacokinetic/pharmacodynamic modelling approach and to identify an optimum dosing regimen for potential clinical evaluation.Methods:A PK/biomarker model was developed to connect abemaciclib/vemurafenib concentrations to changes in MAPK and cell cycle pathway biomarkers in A375 BRAF-mutated melanoma xenografts. Resultant tumour growth inhibition was described by relating (i) MAPK pathway inhibition to apoptosis, (ii) mitotic cell density to tumour growth and, under resistant conditions, (iii) retinoblastoma protein inhibition to cell survival.Results:The model successfully described vemurafenib/abemaciclib-mediated changes in MAPK pathway and cell cycle biomarkers. Initial tumour shrinkage by vemurafenib, acquisition of resistance and subsequent abemaciclib-mediated efficacy were successfully captured and externally validated. Model simulations illustrate the benefit of intermittent vemurafenib therapy over continuous treatment, and indicate that continuous abemaciclib in combination with intermittent vemurafenib offers the potential for considerable tumour regression.Conclusions:The quantitative pharmacology of the abemaciclib/vemurafenib combination was successfully characterised and an optimised, clinically-relevant dosing strategy was identified.

Abstract P4-13-25: Abemaciclib, an inhibitor of CDK4 and CDK6, combined with endocrine and HER2-targeted therapies for women with metastatic breast cancer

Abstract Background: Abemaciclib, a small molecule inhibitor of CDK4 and CDK6, induces G1 cell cycle arrest in Rb-proficient human cancers.1 The clinical safety profile of abemaciclib enables continuous oral dosing to achieve sustained target inhibition, resulting in single-agent antitumor activity against multiple human cancers. The drug also reaches relevant concentrations in the central nervous system and, in patients taking the drug orally, can be detected in the cerebrospinal fluid.2 For women with previously treated hormone receptor positive (HR+) metastatic breast cancer (MBC), abemaciclib as a single agent achieved a six-month clinical benefit rate of 61.1% and an objective response rate of 33.3%.3 Clinical trials investigating abemaciclib combined with fulvestrant4 or aromatase inhibitors5 have led to randomized Phase 3 studies for women with HR+ breast cancer.6,7 Methods: This Phase 1b study (NCT02057133) with multiple cohorts evaluates safety and tolerability of abemaciclib combined with endocrine or HER2-targeted therapies for MBC. Secondary objectives include pharmacokinetics (PK) and antitumor activity of abemaciclib when given in combination with other therapies. Cohorts were opened to enrollment sequentially. Patients with HR+ HER2 negative MBC received abemaciclib orally every 12 hours (Q12H) in combination with the following standard therapies daily until progression: letrozole (Part A), anastrozole (Part B), tamoxifen (Part C), exemestane (Part D), or exemestane plus everolimus (Part E). Patients with HER2 positive MBC received abemaciclib orally Q12H in combination with trastuzumab every 21 days until progression (Part F). Adverse events (AEs) were graded by NCI CTCAE v4.0 and tumor response was assessed radiographically using RECIST v1.1. Results: Abemaciclib has been combined with multiple targeted therapies for the treatment of women with MBC. We previously reported safety and early efficacy results for the combinations of abemaciclib with letrozole, anastrozole, tamoxifen, exemestane, and exemestane plus everolimus.5 Due to limited follow-up at that time, the efficacy results were not mature. Safety, PK, and efficacy results with approximately 6 months of additional follow-up will be reported across all parts of the study. The most common treatment-emergent AEs include effects on the gastrointestinal and hematopoietic systems. Consistent with previously reported results for both single-agent abemaciclib and the combination of abemaciclib with fulvestrant, tumor responses have been observed among women receiving abemaciclib in combination with targeted therapies for MBC. Conclusions: This study for women with MBC demonstrates the potential for abemaciclib to be combined with therapies targeting specific signaling pathways.

The Novel CDK4/6-Inhibitor Abemaciclib Induces Early G1-Arrest in MCL Cell Lines, Sensitizes Cells to Cytarabine Treatment and Is Additive with Ibrutinib

Introduction: Mantle cell lymphoma (MCL) is characterized by t(11;14) resulting in a constitutive cyclin D1 overexpression. The cyclin D1-CDK4/6 complex inactivates Rb through phosphorylation, leading to G1/S-phase transition. Therefore, inhibition of CDK4/6 is an efficient and rational approach to overcome cell cycle dysregulation in MCL. We evaluated the efficiency of the novel CDK4/6 inhibitor abemaciclib in various MCL cell lines and in primary MCL cells in combination with cytarabine (AraC) and ibrutinib.Material & Methods: MCL cell lines (Granta 519, JeKo-1, Maver-1, Mino) and primary MCL cells were exposed to abemaciclib alone and combined with AraC or ibrutinib. Cells were pretreated with abemaciclib and exposed to AraC or ibrutinib with or without consecutive wash-out of the CDK4/6 inhibitor. Proliferation and viability were measured by tryptan blue staining and Cell Titer Glo assay. Flow cytometry was used for cell-cycle (PI-staining) and apoptosis analysis (Annexin V PE/7AAD-staining). Western Blot analysis showed protein expression and phosphorylation status of various downstream proteins.Results: Abemaciclib inhibited cell proliferation by induction of early G1-arrest. Western Blot analysis revealed reduced phosphorylation of Rb on serine 795 without changes in CDK 4 and cyclin D1 expression, in line with reversible cell cycle arrest. IC50-values of sensitive cell lines (JeKo-1, Maver-1, Mino) were <30 nM after 72 h. We observed an almost complete and reversible G1-arrest in all sensitive cell lines by FACS analysis (JeKo-1: G1-phase +51,7 %; S/G2-phase -51,7 % at 31,25 nM after 24 h; G1-phase +35,4 %; S/G2-phase -34,8 % after 72 h), whereas cell viability was not reduced.Wash-out of abemaciclib after 24 h resulted in synchronized S-phase entry in all sensitive cell lines (e.g. Mino: G1-phase -20,4 %; S-phase +30,5 %). The sequential combination of abemaciclib followed by AraC showed strong synergy in Mino cells (CI=0,22 for 31,25 nM abemaciclib and 3,33 µM cytarabine). In contrast, simultaneous exposure to abemaciclib had a protective effect against AraC treatment in all sensitive cell lines, due to an ongoing G1-arrest (Mino: CI=-0,19 for 31,25 nM abemaciclib and 3,33 µM AraC).In primary MCL cells, 31,25 nM of abemaciclib had no impact on cell death. Moreover, no sensitization to AraC was observed as all cells were resting in G0-phase.The combination of abemaciclib induced G1 arrest and ibrutinib had additive or synergistic effects in sensitive cell lines (JeKo-1, Mino and Maver).Conclusion: The novel CDK4/6 inhibitor abemaciclib causes reversible G1 cell cycle arrest without loss of viability at low nanomolar doses. Rationale drug combinations exploiting the sequential effect may achieve major benefits, but drug interactions are complex: Pretreatment with abemaciclib sensitizes MCL cell line cells to AraC whereas simultaneous application protects them from AraC treatment. Further analyses explore the interaction with other targeted approaches (inhibitors of the B-cell receptor pathway) to better understand the underlying molecular mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Brain Exposure of Two Selective Dual CDK4 and CDK6 Inhibitors and the Antitumor Activity of CDK4 and CDK6 Inhibition in Combination with Temozolomide in an Intracranial Glioblastoma Xenograft.

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain Kp,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. Kp,uu increased 2.8- and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) Kp,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 Ki values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time.

A phase Ib study of abemaciclib in combination with multiple single agents in stage IV NSCLC.

8047 Background: Abemaciclib, a cell cycle kinase inhibitor of CDK4/CDK6, demonstrated acceptable safety and early clinical activity in metastatic NSCLC, given orally as monotherapy on a continuous...

Abstract P5-19-13: Clinical activity of abemaciclib, an oral cell cycle inhibitor, in metastatic breast cancer

Abstract Background: Abemaciclib, a small molecule inhibitor with selectivity against cyclin-dependent kinases 4 and 6 (CDK4/6), induces G1 arrest in Rb-proficient human breast cancers. In an early phase clinical trial, the safety and antitumor activity of abemaciclib (LY2835219) were evaluated in 2 cohorts of patients with metastatic breast cancer (mBC). One cohort evaluated single-agent abemaciclib in an unselected population of patients with mBC [Part D], while the combination of abemaciclib plus fulvestrant was evaluated in patients with hormone receptor positive (HR+) mBC [Part G]. We previously reported early results for these 2 cohorts of patients with mBC treated with either single-agent abemaciclib or the combination of abemaciclib plus fulvestrant (Patnaik et al, ASCO 2014). In the single-agent cohort, 47 patients with previously treated mBC were enrolled (36 HR+). All patients with &amp;gt;30% tumor reduction had HR+ mBC (13 of 36 patients). In this group of 13 patients with HR+ mBC, 9 patients had confirmed response for an objective response rate of 25%, and 4 patients had unconfirmed response. This study was ongoing with 14 of 36 HR+ mBC patients on treatment at time of analysis (range 238-471 days). Patients continuing on single-agent abemaciclib included 4 patients with unconfirmed response and 6 patients with confirmed response. For the combination of abemaciclib plus fulvestrant, 18 patients with HR+ mBC enrolled and 13 patients (72%) were still on treatment (range 31-143 days) at the time of analysis. Methods: In the single-agent cohort, patients with mBC were treated with abemaciclib at 150 or 200mg orally every 12 hours on a continuous schedule. In the combination cohort, patients with HR+ mBC (n=18) were treated with the combination of abemaciclib plus fulvestrant. Patients received abemaciclib at 200mg orally every 12 hours on a continuous schedule. Patients also received fulvestrant at 500mg intramuscularly every month. NCI CTCAE v4.0 was used to grade adverse events (AEs) and RECIST v1.1 was used to assess tumor response. Results: In the single-agent cohort, patients began enrolling in May 2012 with the last patient enrolled in March 2013. Patients had a median of 7 prior systemic therapies and 81% of patients had ≥2 metastatic sites. In the combination cohort, patients began enrolling in September 2013 with the last patient enrolled in January 2014. Patients in the combination cohort had a median of 4 prior systemic therapies and 67% of patients had ≥2 metastatic sites. An updated analysis will be presented for objective response rate, duration of treatment and clinical benefit rate and will include an additional 6 months of information for both the single-agent and combination cohorts. New analyses will include time to response, duration of response, change in tumor size over time, and characteristics of responders. In addition, safety data will include longer term follow-up through approximately September 2014. Conclusions: Abemaciclib is an oral cell cycle inhibitor that demonstrates single-agent activity against mBC, especially for HR+ disease. Based on its safety and efficacy profile, abemaciclib warrants further clinical investigation in confirmatory studies, both as a single agent and in combination with endocrine therapy. Citation Format: Sara M Tolaney, Lee S Rosen, Muralidhar Beeram, Jonathan W Goldman, Leena Gandhi, Anthony W Tolcher, Kyriakos P Papadopoulos, Drew W Rasco, Scott P Myrand, Palaniappan Kulanthaivel, Joan M Andrews, Martin Frenzel, Damien M Cronier, Edward M Chan, Keith T Flaherty, Patrick Y Wen, Geoffrey I Shapiro, Amita Patnaik. Clinical activity of abemaciclib, an oral cell cycle inhibitor, in metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-13.