Selective Inhibitor Of Cyclin-dependent Kinases Research Articles

A study of the renal hypofunction and accompanying adverse events in breast cancer patients treated with palbociclb

Abstract Background Palbociclb is an orally available selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) that is indicated for patients with hormone receptor-positive HER2-negative advanced or recurrent breast cancer with 3 weeks on/1 week off schedule. Previous report showed that elevated serum creatinine (Scr) was only 2.0% in frequency for all grade, without cases of grade 3 or more. Anecdotal episode of declined eGFR (implying elevated Scr) seems to be more frequent in daily clinical practice; however, the actual frequency of declined eGFR and its impact on severe toxicity has not been determined yet. Objective To investigate the frequency of declined eGFR in patients treated with palbociclib and to determine the association between declined eGFR and severe toxicity. Methods Consecutive patients treated with palbociclib at National Cancer Center Hospital East between December 1 2017 and May 31 2018 were retrospectively investigated. Patient characteristics, eGFR at cycle 1 day1 (C1D1) and C1D15, C2D1, C2D15, and C3D1 of palbociclib, grade 4 neutropenia, treatment discontinuation or interruption caused by toxicity were retrieved from medical records. The frequency of declined eGFR and its correlation with severe toxicity were analyzed. Results A total of 42 patients were included. Median age was 65 years (range 43-80), and 41 cases were female. Median eGFR at the start of palbociclib was 76 mL/min/1.73m2, which declined 12.5 in median (range 3.0-39.0) on C1D15. At C2D1, eGFR was recovered (change in median -3 mL/min/1.73m2; range -17.0 - +19.0). Declined eGFR was not correlated significantly with either grade 4 neutropenia or treatment discontinuation. Conclusions While eGFR was declined by median of 12.5 mL/min/1.73m2 with palbociclib administration on C1D15, eGFR recovered to the baseline by drug interruption. Declined eGFR was not correlated with severe toxicity such as grade 4 neutropenia or toxicities leading to treatment discontinuation.

344P - A phase I study of XZP-3287, a novel oral CDK4/6 Inhibitor, administered on a continuous dosing schedule, in patients with advanced solid tumours

BackgroundXZP-3287 is a novel selective inhibitor of cyclin-dependent kinases 4/6 (CDK 4/6). Preclinical data suggested a comparable antitumor activity with Palbociclib and Abemaciclib, and a more favourable safety profile of mild myelosupression. A continuous dosing schedule (28days/cycle) is adopted in this first-in-human study to achieve sustained target inhibition. MethodsThe study is designed as an accelerated titration followed by a standard 3+3 dose escalation. Eligible patients are those with locally advanced or metastatic solid tumours, and who have progressed despite of refractory to standard therapy or no standard-of-care therapy is available. Results16 subjects were enrolled in this study by April 1, 2019. The DLT evaluation in 320mg QD dose group was completed. No DLT or drug related serious adverse event (SAE) was observed from 20mg QD to 320mg QD dose groups. The drug related adverse events (AE) were mainly CTCAE grade 1 and 2, and reversible. Drug-related AEs (≥10%) were diarrhea (31.3%), leucopenia (31.3%), blood creatinine increased (25%), anemia (18.8%), neutropenia (18.8%), vomiting (18.8%), ALT increased (12.5%), thrombocytopenia (12.5%), blood alkaline phosphatase increased (12.5%) and hyperuricaemia (12.5%). Myelosuppression was observed from 160mg QD level. A negative correlation was showed between the AUC/Cmax of steady state and the maximum percentage decrease from baseline of neutrophil/platelet counts (r=-0.57 to-0.48), which was consistent with pharmacological effects of CDK4/6 inhibition. Furthermore, clinical activity was observed at low and not yet optimal dose levels. Among 7 efficacy evaluable subjects, 3 achieved SD , and 4 experienced PD. 1 SD patient in 240mg QD dose group had sustained SD for over 8 months and the target lesions was reduced by 23.7% from baseline which previously treated by chemotherapy, The latest data (Apr.30.) showed that, all the 3 patients in 320mg QD dose group achieved SD (2 patients exhibited tumor shrinkage by 10.9% and 8.3% respectively). These data will be update at annual meeting. ConclusionsThis study demonstrated that single-agent XZP-3287 was well tolerated and showed the CDK4/6 inhibition activity in human. Legal entity responsible for the studyXuanzhu Biopharmaceutical Ltd. FundingXuanzhu Biopharmaceutical Ltd. DisclosureAll authors have declared no conflicts of interest.

Novel cyclin-dependent kinase 9 (CDK9) inhibitor with suppression of cancer stemness activity against non-small-cell lung cancer

A series of novel, highly potent, selective CDK9 inhibitors with cancer stem cells (CSCs) inhibition activity were designed and synthesized for non-small-cell lung cancer (NSCLC) therapy. Structure-activity relationship analysis based on enzymatic and cellular activities led to the discovery of a promising inhibitor 21e. 21e potently inhibited CDK9 with IC50 value of 11 nM and suppressed the stemness properties of NSCLC effectively. It could decrease the stemness phenotypes of NSCLC cells, including tumor sphere formation, side-population and stemness markers abundance. 21e displayed good selectivity over the CDK family kinases and kinase profiling assay against 381 kinases. In addition, 21e inhibited cell proliferation, colony-formation, and cell cycle progression and induced apoptosis in NSCLC. In H1299 xenograft mouse model, a once-daily dose of compound 21e at 20 mg/kg significantly suppressed the tumor growth without obvious toxicity. Studies of mechanisms of action indicated that 21e efficiently inhibited CDK9 signaling pathway and stemness both in vitro and in vivo. Collectively, 21e as a novel CDK9 inhibitor with CSCs inhibition properties could be a promising agent for the treatment of NSCLC.

Palbociclib Promotes Dephosphorylation of NPM/B23 at Threonine 199 and Inhibits Endometrial Cancer Cell Growth.

Endometrial cancer incidence rates are growing, especially in countries with rapid socioeconomic transitions. Despite recent advances in chemotherapy, hormone therapy, and targeted therapy, advanced/recurrent disease remains a clinical challenge. Palbociclib—a selective inhibitor of cyclin-dependent kinases (CDK) 4/6—has therapeutic potential against estrogen receptor (ER)-positive and HER2-negative breast cancer. However, the question as to whether it can be clinically useful in endometrial cancer remains open. Here, we show that combined treatment with palbociclib and megesterol acetate exerts synergistic antiproliferative effects against endometrial cancer cells. Treatment of cancer cells with palbociclib suppressed NPM/B23 phosphorylation at threonine 199 (Thr199). We further demonstrated that CDK6 acts as a NPM/B23 kinase. Palbociclib-induced NPM/B23 dephosphorylation sensitized endometrial cancer cells to megesterol acetate through the upregulation of ERα expression. Immunohistochemistry revealed an overexpression of phospho-NPM/B23 (Thr199) in human endometrial cancer, and phospho-NPM/B23 (Thr199) expression levels were inversely associated with Erα in clinical specimen. In a xenograft tumor model, the combination of palbociclib and megesterol acetate successfully inhibited tumor growth. Taken together, our data indicate that palbociclib promoted NPM/B23 dephosphorylation at Thr199—an effect mediated by disruption of CDK6 kinase activity. We conclude that palbociclib holds promise for the treatment of endometrial cancer when used in combination with megesterol acetate.

Discovery and Characterization of SY-1365, a Selective, Covalent Inhibitor of CDK7.

Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.

Development of a Selective CDK7 Covalent Inhibitor Reveals Predominant Cell-Cycle Phenotype

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of ahighly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibitionof E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may beuseful for treatment of cancers marked by E2F misregulation.

Efficacy and Safety of Ribociclib With Letrozole in US Patients Enrolled in the MONALEESA-2 Study

BackgroundIn the Mammary Oncology Assessment of LEE011’s (Ribociclib's) Efficacy and Safety (MONALEESA-2) study, combination treatment with the selective inhibitor of cyclin-dependent kinases 4/6 ribociclib with letrozole significantly improved progression-free survival (PFS) versus letrozole alone in postmenopausal women with hormone receptor-positive HR+/HER2− advanced breast cancer (ABC). Herein we present results from the subset of US patients enrolled in MONALEESA-2. Patients and MethodsPostmenopausal women with HR+/HER2− ABC without previous treatment for advanced disease were randomized (1:1) to ribociclib 600 mg/d (3 weeks on/1 week off) with letrozole 2.5 mg/d (continuous) or placebo with letrozole. The primary end point was locally assessed PFS. ResultsOverall, 213 US patients were enrolled in MONALEESA-2 (ribociclib, n = 100; placebo, n = 113). Baseline characteristics were similar between treatment groups and consistent with the global population. With a median follow-up of 27 months, 38 (38%) and 29 (26%) patients in the ribociclib and placebo groups, respectively, had continued to receive treatment. Median PFS was 27.6 months with ribociclib and 15.0 months with placebo (hazard ratio, 0.53). The most common all-cause adverse events were neutropenia (ribociclib, 72.0% [n = 72]; placebo, 4.6% [n = 5]), nausea (ribociclib, 69.0% [n = 69]; placebo, 44.0% [n = 48]), and fatigue (ribociclib, 60.0% [n = 60]; placebo, 50.5% [n = 55]). Two patients (ribociclib, 2.0%; placebo, 0%) experienced febrile neutropenia. ConclusionIn the US subset of MONALEESA-2, ribociclib with letrozole showed superior efficacy versus letrozole alone. These findings are consistent with the global population and support first-line use of ribociclib with letrozole in patients with HR+/HER2− ABC.

Lysosomal trapping of palbociclib and its functional implications

Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.

Palbociclib and Fulvestrant Act in Synergy to Modulate Central Carbon Metabolism in Breast Cancer Cells.

The aims of this study were to determine whether combination chemotherapeutics exhibit a synergistic effect on breast cancer cell metabolism. Palbociclib, is a selective inhibitor of cyclin-dependent kinases 4 and 6, and when patients are treated in combination with fulvestrant, an estrogen receptor antagonist, they have improved progression-free survival. The mechanisms for this survival advantage are not known. Therefore, we analyzed metabolic and transcriptomic changes in MCF-7 cells following single and combination chemotherapy to determine whether selective metabolic pathways are targeted during these different modes of treatment. Individually, the drugs caused metabolic disruption to the same metabolic pathways, however fulvestrant additionally attenuated the pentose phosphate pathway and the production of important coenzymes. A comprehensive effect was observed when the drugs were applied together, confirming the combinatory therapy’s synergism in the cell model. This study also highlights the power of merging high-dimensional datasets to unravel mechanisms involved in cancer metabolism and therapy.

Development and Use of Clinical Pharmacodynamic Assays to Demonstrate Target Engagement for AZD4573, a Selective and Potent CDK9 Inhibitor Currently in Phase I Clinical Trials

CDK9 (cyclin-dependent kinase 9) regulates RNA transcription through its phosphorylation of RNA polymerase II and subsequent release from a “paused” elongation state, resulting in transcriptional activity. A transient inhibition of CDK9 activity will significantly impact transcripts with a short half-life, which includes the transcript for MCL1, an anti-apoptotic protein. AZD4573 is a highly potent and selective CDK9 inhibitor. We have previously reported that AZD4573 treatment results in dose-dependent inhibition of pSer2 of RNA polymerase II, reductions in MCL1 mRNA and protein, and an increase in cleaved caspase 3, resulting in cell death of hematological tumor cell lines and regressions in AML/DLBCL in vivo models [Cidado et. al., AACR Annual Meeting, Abstract 310 (2018)].AZD4573 is currently being evaluated in a Phase I all-comers relapsed/refractory hematological malignancy trial (NCT03263637). To generate clinical evidence that the compound is inhibiting its target and having the expected downstream effects, we have developed pharmacodynamic biomarker (PD) assays for clinical use to measure the changes in pSer2/RNApol II, MCL1 protein, cleaved Caspase 3 and cleaved PARP. We have chosen a capillary electrophoresis platform which allows quantification and multiplexing while requiring lower sample quantities (<20ng/capillary) than Western blotting. Data from AZD4573-treated MV4,11 AML cells using the clinical assays were similar to what had been shown previously with Western blotting, i.e. similar dose-dependence across 0.1nM to 1uM and >80% inhibition of both pSer2 and MCL1 at 100nM (in 10% FCS). Next, we validated the use of these assays with human peripheral blood mononuclear cells (PBMCs) since we expected that some patients from the trial are likely to have significant numbers of circulating tumor cells (i.e. AML/CLL). After incubation of human whole blood with 1uM AZD4573 for different times, PBMCs were isolated and analyzed using the capillary electrophoresis assays. These results showed at least ~80% inhibition of pSer2 at 2h, consistent with preclinical efficacy modeling and predicted clinically achievable levels. In addition, we optimized the PBMC isolation protocol for the clinical sites and showed that compound washout during the procedure did not diminish AZD4573-mediated effects. In another study, we mimicked potential clinical conditions (e.g. 30 min, 1h, and 2h between blood collection and PBMC isolation, ambient vs 37°C storage conditions) and demonstrated that significant ex vivo apoptosis (i.e. activation of cleaved caspase 3) was unlikely to occur if PBMCs were isolated within 1-2h of blood collection. Finally, by showing that AZD4573 target engagement and pathway inhibition can be measured in normal PBMCs, these assays will be informative for all patients, including those without circulating tumor cells (e.g. many lymphomas).Here we describe the development and characterization of a suite of pharmacodynamic assays for clinical use with blood samples for our CDK9 program. Preliminary data from our Phase I study demonstrate successful implementation of these assays and support that AZD4573 inhibits CDK9 with the expected downstream effects, in the clinical setting. Updated results will be presented at the meeting. DisclosuresYeh:AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Cidado:AstraZeneca: Employment, Equity Ownership. Barrett:AstraZeneca: Employment, Equity Ownership.

Combination of AZD4573, a Selective CDK9 Inhibitor, with Other Cell Death Inducing Agents Can Overcome De Novo Venetoclax Resistance in Preclinical Hematologic Tumor Models

Apoptosis is controlled through the dynamic interactions of the Bcl2 protein family, and cancers have evolved mechanisms to hijack this pathway to evade apoptosis, often by upregulating anti-apoptotic proteins (e.g. Mcl1, Bcl2). This survival adaptation creates a dependency that could be exploited therapeutically, which is why considerable effort has been made to develop small molecule inhibitors of the anti-apoptotic Bcl2 family proteins. This class of drug was clinically validated with the approval of venetoclax, a selective Bcl2 inhibitor, for the treatment of CLL. Venetoclax is undergoing evaluation in numerous other clinical trials for predominantly hematologic malignancies.Despite impressive responses observed with venetoclax in CLL (ORR 79%), acquired resistance is beginning to emerge. Likewise, other hematologic indications are more intrinsically resistant to venetoclax, exhibiting much lower response rates in the respective Phase I clinical trials (AML = 38%, NHL = 44%, MM = 12%). Preclinical studies with venetoclax have reported increased levels of other anti-apoptotic proteins as a likely mechanism contributing to both de novo and acquired resistance. Therefore, combining cell death inducing agents that inhibit Mcl1 or BclxL could be a means of combating resistance.Inhibition of cyclin-dependent kinase 9 (CDK9), which regulates transcription elongation, has been reported to reduce protein levels of genes with short-lived transcripts and proteins, such as MCL1. AZD4573 is a novel and selective CDK9 inhibitor that shows potent single agent activity, inducing cell death in vitro and tumor regressions in vivo in a diverse set of hematologic cancers (Cidado et. al., AACR Annual Meeting 2018). AZD4573 is currently being evaluated in a Phase I clinical trial for patients with hematological malignancies (NCT03263637).This study evaluates whether combinations of AZD4573 with other cell death inducing agents could overcome de novo venetoclax monotherapy resistance. A panel of 12 AML and 6 DLBCL cell lines were treated for 6 h with venetoclax, AZD4573, and a selective BclxL probe compound (AZ'3202) either as single agents or in combinations and assayed for caspase activation. The Loewe model was used to calculate synergy scores to assess benefit over monotherapy, and combinations with scores >5 were deemed beneficial. Treatment with AZD4573+venetoclax and AZD4573+AZ'3202 resulted in beneficial combinations for 13/18 and 10/18 cell lines, respectively. On the other hand, venetoclax+AZ'3202 showed significant combination benefit in only two cell lines (NB4, SUDHL4), suggesting a primary dependency upon Mcl1 for most of these hematologic cancer cell lines. Interestingly, cell lines sensitive to single agent AZ'3202 (4 AML, 0 DLBCL) did not show any combination benefit when treated with AZD4573+venetoclax, highlighting the exquisite dependency of those four models upon BclxL and mutual exclusivity with Mcl1.Cell lines benefitting from the AZD4573+venetoclax combination tended to fall into one of two categories: having single agent activity to either agent that is enhanced by the combination or having no single agent activity but the combination shifts the cell line into a responder. SUDHL4 cells were sensitive to AZD4573 (caspase activation EC50 = 18 nM) but not venetoclax (EC50 = 476 nM) while OCI-AML3 was insensitive to both (EC50 > 30 µM). In vitro biomarker kinetic analysis revealed an increase in Mcl1 levels (~2-fold) after 3 h of venetoclax treatment that was abrogated upon combination treatment, providing a mechanistic rationale for the combination benefit. Furthermore, when tested in an OCI-AML3 tumor xenograft study in mice, AZD4573 or venetoclax monotherapy exhibited minimal tumor growth inhibition (44% and 16%, respectively) while the combination led to tumor regressions (64%) with minimal effect on body weight. In a SUDHL4 tumor xenograft study, venetoclax monotherapy displayed minimal tumor growth inhibition (25%), but intermittent dosing of AZD4573 exhibited 94% tumor growth inhibition. Still, combination therapy demonstrated a clear benefit as it led to complete tumor regressions with 6/8 mice remaining tumor-free until the end of study (150 days). Together, this work presents supporting evidence that combining cell death inducing agents would be effective at overcoming de novo or acquired resistance associated with monotherapy treatments. DisclosuresCidado:AstraZeneca: Employment, Equity Ownership. Boiko:AstraZeneca: Employment. Proia:AstraZeneca: Employment. Woods:AstraZeneca: Employment. Tavana:AstraZeneca: Employment. San Martin:AstraZeneca: Employment. Tron:AstraZeneca: Employment. Shao:AstraZeneca: Employment. Drew:AstraZeneca: Employment.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50= 5nM) that has broad anti-cancer activity invitro and is effective in invivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 invivo, making it an excellent target for epigenetic therapy of cancer.

Cyclin-dependent kinase 9 as a potential specific molecular target in NK-cell leukemia/lymphoma.

BAY 1143572 is a highly selective inhibitor of cyclin-dependent kinase 9/positive transcription elongation factor b. It has entered phase I clinical studies. Here, we have assessed the utility of BAY 1143572 for treating natural killer (NK) cell leukemias/lymphomas that have a poor prognosis, namely extranodal NK/T-cell lymphoma, nasal type and aggressive NK-cell leukemia, in a preclinical mouse model in vivo as well as in tissue culture models in vitro. Seven NK-cell leukemia/lymphoma lines and primary aggressive NK-cell leukemia cells from two individual patients were treated with BAY 1143572 in vitro. Primary tumor cells from an aggressive NK-cell leukemia patient were used to establish a xenogeneic murine model for testing BAY 1143572 therapy. Cyclin-dependent kinase 9 inhibition by BAY 1143572 resulted in prevention of phosphorylation at the serine 2 site of the C-terminal domain of RNA polymerase II. This resulted in lower c-Myc and Mcl-1 levels in the cell lines, causing growth inhibition and apoptosis. In aggressive NK-cell leukemia primary tumor cells, exposure to BAY 1143572 in vitro resulted in decreased Mcl-1 protein levels resulting from inhibition of RNA polymerase II C-terminal domain phosphorylation at the serine 2 site. Orally administering BAY 1143572 once per day to aggressive NK-cell leukemia-bearing mice resulted in lower tumor cell infiltration into the bone marrow, liver, and spleen, with less export to the periphery relative to control mice. The treated mice also had a survival advantage over the untreated controls. The specific small molecule targeting agent BAY1143572 has potential for treating NK-cell leukemia/lymphoma.

Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

ABSTRACT We recently reported that primary acute lymphoblastic leukemia (ALL) cells are susceptible to the microtubule depolymerizing agent vincristine (VCR) in G1 phase. This finding prompted testing another G1 phase-active compound, palbociclib (PCB), a highly selective inhibitor of cyclin-dependent kinases 4/6 (CDK4/6), alone and in combination with VCR. PCB used alone caused G1 arrest in ALL cells with no effect on cell viability, and similar results were obtained for the retinoblastoma (RB)-proficient T98G glioblastoma cell line. In contrast, HeLa cells failed to arrest in the presence of PCB, consistent with their lack of dependence on the CDK4/6-RB pathway. When ALL cells were pretreated with PCB, they became refractory to death in G1 phase induced by VCR treatment, whereas HeLa cells retained VCR sensitivity after PCB pretreatment. Immunofluorescence microscopy showed that PCB did not disrupt the microtubule network nor prevent VCR from doing so. Furthermore, ALL cells pretreated with PCB retained susceptibility to the Bcl-2/Bcl-xL inhibitor ABT-263, indicating that downstream apoptotic signaling was unaffected. When released from PCB-enforced arrest, ALL cells reinitiated cycling and regained sensitivity to VCR. ALL cells treated with cycloheximide also arrested in G1 phase and became insensitive to VCR, independently reinforcing conclusions derived from PCB-imposed arrest. Thus, primary ALL cells advancing through G1 phase are strictly dependent on functional microtubules for survival whereas microtubules are dispensable for G1-arrested cells. These findings provide novel insight into interphase microtubule function and, from a therapy standpoint, strongly caution against combining microtubule targeting agents and CDK4/6 inhibitors for ALL.

Cycling Toward Progress: Ribociclib, a CDK 4/6 Inhibitor for Breast Cancer.

Ribociclib is an orally active, highly selective inhibitor of cyclin-dependent kinase (CDK) 4 and 6. It is the second CDK 4/6 inhibitor approved for hormone receptor-positive breast cancer. The addition of ribociclib to an aromatase inhibitor has resulted in marked improvements in progression-free survival for patients with metastatic breast cancer. Clin Cancer Res; 24(13); 2981-3. ©2018 AACRSee related article by Shah et al., p. 2999.

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.

A phase 1, open-label, multicenter, non-randomized study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of AZD4573, a potent and selective CDK9 inhibitor, in subjects with relapsed or refractory hematological malignancies.

TPS7588Background: Cyclin-dependent kinase 9 (CDK9) belongs to the group of transcription-regulating CDKs and promotes transcription elongation through phosphorylation of RNA Polymerase II at serin...

Abstract P5-21-27: Efficacy and safety of ribociclib plus letrozole in US patients enrolled in the MONALEESA-2 study

Abstract Background: Endocrine-based therapy is the standard of care for patients with hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2−) advanced breast cancer (ABC), in whom delaying disease progression is the primary goal. In the randomized Phase 3 MONALEESA-2 study (ClinicalTrials.gov identifier, NCT01958021), first-line ribociclib (a selective inhibitor of cyclin-dependent kinases 4 and 6) + letrozole significantly prolonged progression-free survival (PFS) compared with placebo + letrozole in postmenopausal women with HR+, HER2− ABC (hazard ratio, 0.556; 95% confidence interval [CI], 0.429–0.720) at the interim analysis cutoff date. Here, we present safety and efficacy data from US patients who were enrolled in the MONALEESA-2 study. Methods: Postmenopausal women (N=668) with HR+, HER2− ABC who did not receive prior systemic treatment for ABC and had an Eastern Cooperative Oncology Group performance status score of ≤1, adequate bone marrow and organ function, and no history of active cardiac dysfunction were randomized 1:1 to receive either ribociclib (600 mg/d, 3 weeks on/1 week off) + letrozole (2.5 mg/d, continuous) or placebo + letrozole. The primary end point was locally assessed PFS. The data cutoff for this analysis was January 29, 2016. Results: Baseline demographics, patient disposition, and prior therapy were well balanced across treatment groups in the US subset (N=213; ribociclib group, n=100; placebo group, n=113). Median treatment duration was 16.3 months. In US patients, median PFS was significantly prolonged in the ribociclib group (median PFS, not reached [NR]; 95% CI, 17.1 months to NR) versus the placebo group (median PFS, 14.4 months [95% CI, 11.1–18.4 months]; hazard ratio, 0.451 [95% CI, 0.281–0.724]; P=0.000363), with a median follow-up of 11.0 months. In a subsequent preplanned analysis of overall survival, median PFS was 27.6 months in the ribociclib group versus 15.0 months in the placebo group (hazard ratio, 0.527; 95% CI, 0.351–0.793). The overall response rate (ORR) was significantly greater in the ribociclib group (ORR, 38.0%; 95% CI, 28.5–47.5%) versus the placebo group (ORR, 26.5%; 95% CI, 18.4–34.7%; P=0.040). A trend toward benefit was observed with ribociclib in all subgroups (including age, disease burden, prior therapy, and biomarker status), which was consistent with the overall population. No on-treatment deaths occurred. The most common all-grade adverse events, irrespective of causality, were neutropenia (ribociclib, 69.0%; placebo, 3.7%), nausea (ribociclib, 65.0%; placebo, 39.4%), fatigue (ribociclib, 56.0%; placebo, 46.8%), and diarrhea (ribociclib, 41.0%; placebo, 32.1%). The most common grade 3 (≥25% in either group) and grade 4 (≥10% in either group) AE was neutropenia (grade 3: ribociclib, 39.0%; placebo, 0%; grade 4: ribociclib, 14.0%; placebo, 0%). Two patients (2.0%) in the ribociclib group and none in the placebo group experienced febrile neutropenia. Conclusions: The US patients in the MONALEESA-2 study derived significant PFS benefit from ribociclib + letrozole compared with placebo + letrozole, with a 55% reduction in the relative risk of disease progression. The safety profile was consistent with and comparable to the global study population. Citation Format: Yardley DA, Hart L, Favret A, Blau S, Diab S, Richards D, Sparano J, Beck JT, Richards P, Ward P, Ramaswamy B, Tsai M, Pluard T, Tolaney S, Esteva F, Small T, Purkayastha D, Miller M, Hortobagyi G. Efficacy and safety of ribociclib plus letrozole in US patients enrolled in the MONALEESA-2 study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-27.

Abstract OT3-05-05: MonarchE: A randomized, open-label, phase 3 study of abemaciclib combined with standard adjuvant endocrine therapy versus standard adjuvant endocrine therapy alone in patients with high risk, node positive, early stage, HR+, HER2- breast cancer

Abstract Background: Abemaciclib, an oral, selective inhibitor of cyclin-dependent kinases 4 and 6 dosed on a twice daily continuous schedule, has demonstrated clinical efficacy and tolerability in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer when administered as monotherapy (MONARCH 1) and in combination with endocrine therapy (ET) in MONARCH 2 and MONARCH 3. In neoMONARCH, abemaciclib plus anastrozole as neoadjuvant therapy reduced the breast tumor cell proliferation marker Ki67 to a greater extent than anastrozole alone after 2 weeks of treatment. Endocrine monotherapy is the current standard of care in the adjuvant setting. However, a proportion of pts relapse despite this therapy. A population with a higher risk of recurrence (15% at 5 years) may be identified based on the clinical and pathological characteristics of disease. Optimizing adjuvant therapy for these pts is an important need. Trial Design: MonarchE (NCT03155997) is a multicenter, randomized, open-label Phase 3 trial that will evaluate the potential for abemaciclib to enhance adjuvant ET. Pts will be randomized 1:1 to abemaciclib 150 mg twice daily continuous schedule plus standard of care (SOC) adjuvant ET versus SOC adjuvant ET alone and stratified by prior chemotherapy (neoadjuvant, adjuvant, or none), menopausal status (pre- or post-), and region (N. America/Europe, Asia, or other). Pts may have started ET within 8 weeks prior to randomization. Pts will receive abemaciclib for up to 2 years in combination with ET per physician's choice (such as tamoxifen or an aromatase inhibitor, +/- ovarian suppression). ET alone will be continued as clinically indicated. All randomized pts will be followed for a total of 10 years. Eligibility Criteria: Eligible pts (male or female) must have early stage resected HR+, HER2- invasive breast cancer with either ≥ 4 positive pathological axillary lymph nodes (pALNs), or 1 to 3 positive pALNs and at least one of the following high risk markers: primary tumor size ≥5 cm, histological grade 3 tumor, or centrally assessed Ki67 index of ≥20% (in a subset of pts). Pts must have completed definitive locoregional therapy (+/- (neo)adjuvant chemotherapy) and be randomized no more than 12 weeks after completion of last non-ET (surgery, chemotherapy, or radiotherapy). Pts must have tumor tissue available for biomarker analysis prior to randomization. Specific Aims: The primary objective of monarchE is to evaluate invasive disease-free survival (IDFS) per the STEEP System.1 Secondary objectives include evaluation of IDFS in pts with Ki67 index of ≥20%, distant relapse-free survival, overall survival, safety, pharmacokinetics, and pt health outcomes. Statistical Methods: Assuming an IDFS hazard ratio of .73, the study is powered to approximately 80% to test the superiority of abemaciclib plus standard ET at a 1-sided α=0.025 using a stratified log-rank test. Target accrual: Approximately 3580 pts Contact information: 1-877-285-4559 Reference: 1. Hudis et al. J Clin Oncol. 2007;25(15):2127-2132. Citation Format: Rastogi P, Toi M, Harbeck N, Bourayou N, Frenzel M, Johnston S. MonarchE: A randomized, open-label, phase 3 study of abemaciclib combined with standard adjuvant endocrine therapy versus standard adjuvant endocrine therapy alone in patients with high risk, node positive, early stage, HR+, HER2- breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-05-05.

Abstract GS6-02: The benefit of abemaciclib in prognostic subgroups: An exploratory analysis of combined data from the MONARCH 2 and 3 studies

Abstract Background: Abemaciclib is an orally administered, selective inhibitor of cyclin-dependent kinases 4 &amp; 6 that is dosed on a twice daily continuous schedule. Abemaciclib has demonstrated clinical efficacy with a generally tolerable safety profile in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer in combination with fulvestrant in MONARCH 2 (NCT02107703) and in combination with non-steroidal aromatase inhibitors (NSAI) in MONARCH 3 (NCT02246621). These analyses were conducted to evaluate if patient and disease characteristics may better inform in whom and when abemaciclib should be initiated to define optimal treatment strategies. Methods: MONARCH 2 and 3 enrolled patients with HR+, HER2- advanced breast cancer. In MONARCH 2, patients whose disease had progressed while receiving endocrine therapy were treated with abemaciclib/placebo plus fulvestrant. In MONARCH 3, patients were treated with abemaciclib/placebo plus NSAI as initial therapy for advanced disease. An exploratory pooled analysis of the two studies was performed to determine significant prognostic factors. Efficacy results (progression-free survival [PFS] and objective response rate [ORR] in patients with measurable disease) were examined for patient subgroups corresponding to each of the identified significant prognostic factors. Subpopulation treatment effect pattern plot (STEPP) methodology was performed to examine the association between treatment-free interval (TFI) following adjuvant endocrine therapy and outcomes of endocrine therapy alone or in combination with abemaciclib in MONARCH 3. Results: Analyses of clinical factors in over 1000 patients confirmed the following to have prognostic value: bone-only disease, liver metastases, tumor grade, progesterone receptor (PgR) status, and ECOG performance status. Prognosis was poor in patients with liver metastases, PgR-negative tumors, and high-grade tumors. While all subpopulations benefited from the addition of abemaciclib to endocrine therapy regardless of prognosis, substantial benefit of abemaciclib was observed in poor prognosis subgroups, characterized by large increases in PFS (hazard ratios = 0.4 to 0.5) and ORR (over 30%). In addition, STEPP analysis of TFI on a subset of the MONARCH 3 population showed that patients with the shortest TFI appeared to have a poorer prognosis and received more benefit from the addition of abemaciclib compared to patients with longer TFI. Conclusions: This exploratory analysis has provided data that could help optimize treatment strategies by identifying that patients with poor prognostic factors may receive greater benefit from the addition of abemaciclib to endocrine therapy. Citation Format: Goetz MP, O'Shaughnessy J, Sledge Jr. GW, Martin M, Lin Y, Forrester T, Mockbee C, Smith IC, Di Leo A, Johnston S. The benefit of abemaciclib in prognostic subgroups: An exploratory analysis of combined data from the MONARCH 2 and 3 studies [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS6-02.