Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic ...tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.
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•Abemaciclib monotherapy induces intra-tumor T cell inflammatory signature•Treatment causes MHC class I and II upregulation in tumor cells and increased NFAT signaling in T cells•Synergy with PD-L1 checkpoint blockade enhances adaptive and innate immune activation•Combination therapy leads to complete tumor rejection and immunological memory
Schaer, Beckmann et al. describe unique immune-modulating properties of abemaciclib that include upregulation of antigen presentation on tumor cells and increased T cell activation. These activities synergize with anti-PD-L1 therapy to further enhance immune activation, including macrophage and DC antigen presentation, and also lead to a reciprocal increase in abemaciclib-dependent cell cycle gene regulation.
Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising ...clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3′UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.
•A wide range of sensitivity to abemaciclib is observed among Rb+ tumor cells•CDKN2A mutant cancers show only intermediate sensitivity to CDK4/6 inhibition•D-cyclin activating features are associated with highly sensitive cells•About 5% of endometrial cancers bear a stabilizing mutation in the CCND1 3′UTR
Gong et al. identify a subset of cancers highly sensitive to CDK4/6 inhibition, which are characterized by various genomic aberrations known to elevate D-cyclin levels but not by CDKN2A mutations. They also identify a recurrent CCND1 3′UTR mutation associated with increased CCND1 expression in endometrial cancer.
Loss-of-function mutations in the retinoblastoma gene
are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic ...lethal with
mutation (
), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest
association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against
cancer cells and leads to durable regression of
tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between
and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.
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Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of ...microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355.
Summary
LY2603618 is an inhibitor of checkpoint kinase 1 (CHK1), an important regulator of the DNA damage checkpoints. Preclinical experiments analyzed NCI-H2122 and NCI-H441 NSCLC cell lines and in ...vitro/in vivo models treated with pemetrexed and LY2603618 to provide rationale for evaluating this combination in a clinical setting. Combination treatment of LY2603618 with pemetrexed arrested DNA synthesis following initiation of S-phase in cells. Experiments with tumor-bearing mice administered the combination of LY2603618 and pemetrexed demonstrated a significant increase of growth inhibition of NCI-H2122 (H2122) and NCI-H441 (H441) xenograft tumors. These data informed the clinical assessment of LY2603618 in a seamless phase I/II study, which administered pemetrexed (500 mg/m
2
) and cisplatin (75 mg/m
2
) and escalating doses of LY2603618: 130–275 mg. Patients were assessed for safety, toxicity, and pharmacokinetics. In phase I, 14 patients were enrolled, and the most frequently reported adverse events included fatigue, nausea, pyrexia, neutropenia, and vomiting. No DLTs were reported at the tested doses. The systemic exposure of LY2603618 increased in a dose-dependent manner. Pharmacokinetic parameters that correlate with the maximal pharmacodynamic effect in nonclinical xenograft models were achieved at doses ≥240 mg. The pharmacokinetics of LY2603618, pemetrexed, and cisplatin were not altered when used in combination. Two patients achieved a confirmed partial response (both non-small cell lung cancer), and 8 patients had stable disease. LY2603618 administered in combination with pemetrexed and cisplatin demonstrated an acceptable safety profile. The recommended phase II dose of LY2603618 was 275 mg.
Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, ...respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation.
or
mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of
-mutant ER+ breast cancer.
The combined influence of oncogenic drivers, genomic instability, and/or DNA damage repair deficiencies increases replication stress in cancer. Cells with high replication stress rely on the ...upregulation of checkpoints like those governed by CHK1 for survival. Previous studies of the CHK1 inhibitor prexasertib demonstrated activity across multiple cancer types. Therefore, we sought to (1) identify markers of prexasertib sensitivity and (2) define the molecular mechanism(s) of intrinsic and acquired resistance using preclinical models representing multiple tumor types. Our findings indicate that while cyclin E dysregulation is a driving mechanism of prexasertib response, biomarkers associated with this aberration lack sufficient predictive power to render them clinically actionable for patient selection. Transcriptome analysis of a pan-cancer cell line panel and
models revealed an association between expression of E2F target genes and prexasertib sensitivity and identified innate immunity genes associated with prexasertib resistance. Functional RNAi studies supported a causal role of replication fork components as modulators of prexasertib response. Mechanisms that protect cells from oncogene-induced replication stress may safeguard tumors from such stress induced by a CHK1 inhibitor, resulting in acquired drug resistance. Furthermore, resistance to prexasertib may be shaped by innate immunity.
Abstract
Many cancers are under replicative stress (RS) arising from the combined influence of oncogenic drivers (i.e. Cyclin E, E2F, Myc), genomic instability and/or deficiencies in DNA Damage ...Repair (DDR). Tumors survive RS by upregulating checkpoints such as those driven by the ATR/CHK1 pathway. Dependence of tumors under high RS on the Chk1 pathway is currently being explored in the clinic with the Chk1 kinase inhibitor prexasertib. Preclinical and clinical studies have demonstrated evidence of efficacy in various cancers supporting the notion that this drug has the potential to improve the standard of care for patients with these malignancies. Two pivotal steps to ultimately fulfill the promise of a novel therapeutic in the clinic are, 1) to enable a patient selection strategy via predictive biomarkers of drug response or de novo resistance; 2) to gain insight into mechanisms underlying the development of acquired resistance, a common phenomenon of cancer therapeutics that contributes to non-durable response. Two experimental objectives to address these challenges for prexasertib were set, 1) to profile a large pan-cancer tumor cell line panel for prexasertib response and explore the association between drug response and baseline molecular profile via transcriptomic profiling, (2) to establish an in vitro tumor line with acquired resistance to prexasertib and characterize it via transcriptomic and targeted proteomic profiling to probe the mechanism of acquired resistance to prexasertib. Key observations from this study: A) pathways corresponding to E2F targets, G2M checkpoint and the Spindle Assembly Checkpoint (SAC), with a distinct enrichment for replication fork genes emerged across three different studies, (a) across a pan-cancer tumor line panel associated with prexasertib sensitivity, (b) associated with resistance in a tumor cell line with acquired resistance to prexasertib and (c) as top hits in an unbiased functional shRNA screen aimed at identifying genes that upon knockdown sensitize the resistant tumor cell line to prexasertib. B)knockdown of specific replication fork components in the drug-resistant tumor line was associated with marked sensitization to prexasertib and concomitant evidence of RS and DNA damage.To explain these paradoxical observations, we postulate that tumors may upregulate the expression of E2F target/G2M/SAC genes to resist oncogene-induced RS and that they may rely on similar genes to survive prexasertib-induced RS. Based on this data, transcriptomic profiling may prove of utility in the identification of patient tailoring biomarkers for prexasertib and as tool to unravel mechanisms of acquired resistance.
Citation Format: Wayne D. Blosser, Jack A. Dempsey, Ann M. McNulty, Wenjuan Wu, Philip J. Ebert, Jason C. Ting, Yue W. Webster, Trent R. Stewart, Xueqian Gong, Farhana F. Merzoug, Sean Buchanan, Ricardo Martinez. Enhanced gene expression of replication fork and other E2F targets genes is associated with sensitivity and, paradoxically, also with acquired drug resistance, to the Chk1 inhibitor prexasertib abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2535.
Abstract
Abemaciclib (LY2835219) is an ATP-competitive inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), which is currently undergoing clinical evaluation as a single-agent treatment and in ...combination with the anti-hormonal therapy (SERD) fulvestrant in estrogen receptor positive (ER+) breast cancer (BrCa). Breast cancer cell line (15 lines) sensitivity to treatment with abemaciclib was assessed using multiple approaches including EdU incorporation, phosphorylation of retinoblastoma protein at serine 807/811 (pRb_s807/811) and RNA transcriptional profiling. We identified molecular features including ER-positivity (ER+), and luminal histology, as key to greater sensitivity while loss of Rb was associated with lower sensitivity. Changes in the Modaplex based RNA transcriptional array profiles of 29 cell cycle related target genes across a panel of 15 human breast cancer cell lines further characterized sensitivity to abemaciclib and highlighted potential targets of response. A sub-group of targets including MKi67, E2F1, MCM7, FOXM1, RRM2 and TOPIIα were significantly inhibited in highly sensitive cell lines previously characterized with EC50 < 50nM (EdU, pRb_serine 807/811). Additionally, we looked at abemaciclib induced transcriptional changes in vivo treating nude mice bearing human, ER+ breast cancer (ER+/HER2-) tumor xenografts and found that the inhibition of expression of this same group of transcriptional targets plus CCNE1 and CDKN2C correlated with the concentration-dependent inhibition of CDK4/CDK6. Abemaciclib washout studies following longer-term exposure in T47-D (ER+/HER2-) demonstrated durable cell cycle inhibition resulting in significant inhibition of pRb_s807/811 even after the removal (washout) of abemaciclib while longer exposure also resulted in inhibition of estrogen receptor-α (ERα) expression in a panel of ER+ breast cancer cell lines that were evaluated over a 8 day time course.
Using taqman low density RNA array (TLDA) cards designed with known ERα driven transcriptional targets, we investigated the impact of abemaciclib single agent and in combination with fulvestrant on changes in RNA expression in four day treated breast cancer cells (ER+/HER2-). Single agent abemaciclib appeared to inhibit a subset of ERα driven targets including TFF1, GREB1, ESR1, E2F1, CDC45, FOXM1 and IGFBP4 (inhibition >%50). Interestingly, comparing single agent (% inhibition) abemaciclib to fulvestrant monotherapy in MCF-7 cells, we found that abemaciclib inhibited CDC45, E2F1, ESR1 and FOXM1 to a greater extent than that observed for these same targets following monotherapy with fulvestrant. Combining abemaciclib with fulvestrant identified an additional sub-group of ERα driven targets whereby the combination provided greater inhibition than that observed with any of the single agent treatments (IGFBP4, E2F1, FOXM1 and CDC45). Additionally, the combination treatment as compared to single-agent treatments also induced greater inhibition of various cell cycle regulatory genes including MCM7, CDKN2C, TOP2a, RRM2, and MKi67.
Citation Format: Ann M. McNulty, Teresa Burke, Jack A. Dempsey, Christophe C. Marchal, Andrew E. Schade, Hadrian P. Szpurka, Michele S. Dowless, Jennifer Stephens, Jennifer Stephens, Denise Edmondson, Keith Stayrook, William C. Caldwell, Sean Buchanan, Farhana F. Merzoug, Richard P. Beckmann. The CDK4/CDK6 inhibitor abemaciclib inhibits transcriptional targets which facilitate growth in ER+ breast cancer cells. abstract. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A12.
Dysregulation of Wnt signaling is common in a variety of human malignancies. Activation of the canonical Wnt or beta-catenin pathway has been especially well documented in cancer, although other ...non-canonical Wnt signaling pathways also have been implicated in neoplasia. In most instances, constitutive signaling through the beta-catenin pathway involves activation of effector molecules or loss of tumor suppressor function downstream of Wnt binding to its cell surface receptors. Nonetheless, in recent years increasing evidence suggests that secreted Wnt antagonists act as tumor suppressors, with their expression often silenced by promoter hypermethylation. This implies that maximal constitutive signaling in cancer requires unimpaired Wnt stimulation at the cell surface as well as enhanced signal propagation within the cell. However, an understanding of the role secreted Wnt antagonists may play in cancer is complicated by the multiplicity of these proteins, their potential Wnt-independent activities and observations indicating that sometimes they may promote tumor growth. Just as the particular function of Wnt signaling in development and homeostasis varies with the setting, the impact of secreted Wnt antagonists on neoplasia depends on the molecular, cellular and tissue context.
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